article
stringlengths 0
682k
| abstract
stringlengths 146
3.69k
|
|---|---|
to gain insight into the experience of family caregivers of demented people with the decision - making regarding institutionalisation, and to develop a guideline for professional caregivers how to ease the transition from home to nursing - home. the (possible) institutionalisation of a relative with dementia has high impact on both the person with dementia and his relatives. as far as we know, qualitative interviews were held with 15 family caregivers (eight children, six partners and one other relative) of a person with dementia., the decision - making may be hindered by a negative view of nursing - home care, reluctant relatives, or feelings of guilt. family caregivers often experience mixed feelings and more or less lack support from relatives and friends or from health care professionals throughout the process. after institutionalisation, family caregivers have to cope with aversive and aggressive behaviours of their relative, loneliness, taking on a new social role, and discussing their concerns about nursing - home care with nursing - home staff. a transition programme for people with dementia and their relatives should include timely discussion of the possibility of moving into a nursing - home or a small - scale living facility. professionals should discuss the choices and support the decision - making of people with dementia and their relatives. after institutionalisation, the client - relative dyad should be supported to give new meaning to their lives, both as a couple and individually. | purposeto gain insight into the experience of family caregivers of demented people with the decision - making regarding institutionalisation, and to develop a guideline for professional caregivers how to ease the transition from home to nursing-home.theorythe (possible) institutionalisation of a relative with dementia has high impact on both the person with dementia and his relatives. as far as we know, there are no evidence - based guidelines for professionals involved in this difficult time.methodsqualitative interviews were held with 15 family caregivers (eight children, six partners and one other relative) of a person with dementia. interviews focused on their expectations and needs regarding decision - making and institutionalisation. the audio - recorded interview transcripts were analysed using atlas-ti.resultspreliminary analyses reveal practical and emotional problems. before institutionalisation, the decision - making may be hindered by a negative view of nursing - home care, reluctant relatives, or feelings of guilt. family caregivers often experience mixed feelings and more or less lack support from relatives and friends or from health care professionals throughout the process. difficulty communicating the institutionalisation to their relative with dementia is another problem. some relatives totally deny the possibility of institutionalisation. after institutionalisation, family caregivers have to cope with aversive and aggressive behaviours of their relative, loneliness, taking on a new social role, and discussing their concerns about nursing - home care with nursing - home staff.conclusionsa transition programme for people with dementia and their relatives should include timely discussion of the possibility of moving into a nursing - home or a small - scale living facility. professionals should discuss the choices and support the decision - making of people with dementia and their relatives. after institutionalisation, the client - relative dyad should be supported to give new meaning to their lives, both as a couple and individually. |
drugs are an important cause of liver injury and manifestations may range from asymptomatic elevation of liver enzymes to fulminant hepatic failure and may account for 20 to 40% of all instances of fulminant hepatic failure. more than 900 drugs, toxins, and herbs have been reported to cause liver injury. in the united states drugs are the most common cause of acute liver failure and 39% are due to acetaminofen and 13% are idiosyncratic reaction due to other medications. knowledge of commonly implicated agents and high degree of suspicion are essential for an accurate diagnosis. a seventy six years old male developed anorexia, nausea and right upper quadrant pain following intake of azithral (azithromycin) 500 mg per day for three days along with rantac (ranitidine-300 mg / day) and calpol (acetaminophen-500 mg) for upper respiratory tract infection. it was followed by passage of high color urine, jaundice, pruritis and gradual sleep disturbance and after four days he was admitted through emergency with altered sensorium and restlessness. no history of dyspnea, orthopnea, paroxysmal nocturnal dyspnea or edema could be elicited. he was non - alcoholic and was not suffering from any hepatic, cardiovascular or respiratory diseases and he took no other drugs within last three months. at presentation he was restless, confused and blood pressure was 110/70, pulse 90/min and respiratory rate was 24/min. jugular venous pulse was not elevated. laboratory testing revealed normal serum urea, creatinine, fasting blood sugar, electrolytes and blood counts. liver function test (lft) showed total/ conjugated hyperbilirubinemia (7.0/4.1 mg%), markedly elevated alanine aminotransferase (alt -1640 iu / l ; normal range 9 to 40 iu / l), aspartate aminotransferase (ast-930 iu / l ; normal range 10 to 35 iu / l), alkaline phosphatase (alp-1894 iu / l ; normal range 30 to 120 iu / l) and a normal lactate dehydrogenase (ldh-330 iu / l ; range 105 - 333 iu / l) level. prothrombine time (inr-2.2) and serum ammonia (83 mol / l ; normal range 10 - 40 on chest x - ray, the cardiothoracic index was augmented with clear lung fields. the patient was treated conservatively and showed improvement of signs and symptoms and lft within 14 days. acute hepatocellular injury, whether due to viral hepatitis, hepatic ischemia, or drug hepatotoxicity, results in elevated levels of serum aminotransferases (ast and alt). in viral and drug - induced hepatitis, the ast and alt levels steadily increase and peak in the low thousands range within seven to 14 days. in ischemic hepatitis serum ldh is reported to be markedly elevated and a ratio of serum alt to ldh of < 1.5, and serum alp remains normal. drug induced hepatotoxicity is mainly due to intrinsic/ predictable drug reactions or idiosyncratic and the risk factors are race, older age, female sex, pre existing liver disease, genetic factors, other co - morbidities and long acting drug formulation. azithromycin has a long half - life and has been demonstrated to reach high liver concentrations, exceeding the serum levels by 50 folds. the early liver injury could be hypersensitivity - mediated and rare cases of cholestatic hepatitis, and severe iatrogenic hepatitis requiring hepatic transplantation, has been reported. our patient developed features of hepatocellular toxicity and hepatic encephalopathy one week after the last dose of azithromycin with markedly elevated alt, ast, alp and serum ammonia levels. elevated aminotransferase, conjugated hyperbilirubinemia and prolonged p- time are suggestive of subfulminant or fulminant necrosis. at presentation the patient was haemodynamically stable with a normal serum ldh level and there was no evidence of other organ dysfunction. absence of clinical or radiological evidence of decompensated heart failure raises the possibility of an ischemic hepatitis unlikely. the diagnosis of azithromycin induced hepatotoxicity was considered because of the temporal relationship between drug intake and onset of clinical signs and symptoms and absence of other causes of liver disease. the naranjo probability scale (score - six) and who causality assessment scale, indicated azithromycin as the probable / likely cause of the adverse drug associated hepatocellular toxicity in this case. awareness and early reconition of azithromycin induced hepatocellular toxicity could be life saving and rewarding while prescribing this commonly used macrolide derivative. | azithromycin is a widely used macrolide derivative and has generally been considered to be a very safe medication. though gastrointestinal symptoms and reversible hearing loss are common, potentially serious side effects including angioedema and cholestatic jaundice occurred in less than one percent of patients. we report a case of asymptomatic dilated cardiomyopathy with azithromycin induced severe hepatocellular toxicity and hepatic encephalopathy. |
the local anesthetic (1% lidocaine with epinephrine, 1:100,000 for tendon surgery in 1 or 2 digits or in the palm) is injected to the digits of the patient in the waiting area starting 30 minutes before surgery for anesthesia. injection of such mixture to the hand was found to be safe.123456) waiting after injection allows the epinephrine to take effect and provide an adequately dry working field. 1). we keep the total dose of infiltration less than 7 mg / kg. usually, less than 50 ml is required for tendon surgery in the hand (including digits and palm areas), and we use premixed 1% lidocaine with 1:100,000 epinephrine. occasionally, 50 to 100 ml is required when surgery is involved in 3 or 4 digits or in the forearm ; in these cases, we dilute with saline to a concentration of 0.5% lidocaine with 1:200,000 epinephrine. the injection starts proximally with a fine needle (25 or 27 gauge) (fig. is injected in the most proximal part of the likely dissection area to block the nerves distally. this site is located in the distal part of the palm for a zone 2 tendon repair. in this way, the large volume of the first injection will bathe all of the nerves leading to the area of the next injections. for a zone 1 tendon repair, we usually inject the solution similarly as for a zone 2 tendon repair. in making the first injection, we insert the needle perpendicular to the skin into the subcutaneous fat to reduce pain of injection.6) then the syringe is stabilized with fingers propped on the skin to avoid needle wobble. wait 15 to 45 seconds until the patient tells us that the sting is gone. then, we proceed to inject the rest of infiltration very slowly (more than 5 minutes) without moving the needle (fig. sometimes, i make this first injection in a quicker fashion than i described above and inject continuously in the way that we all use for local anesthesia, if the patient tolerates well. about 10 to 15 minutes after the first injection, the whole distal area of dissection is almost or totally numb. the next three injections are used mainly for the epinephrine vasoconstriction effect. between both digital nerves, 2 ml of infiltration is injected subcutaneously at the palmar digital crease, so the bevel of the needle does not lacerate the nerve fascicles (fig. another 2 ml of infiltration is also injected subcutaneously at the digital crease of the proximal interphalangeal (pip) joint between both digital nerves (fig. the final 1 ml of infiltration is then injected subcutaneously at the digital crease of the distal interphalangeal (dip) joint between both digital nerves (fig. the surgery starts 30 minutes after the first injection or 15 minutes after completion of all injections. the sheath is opened through a small incision along the midline, about 1 to 1.5 cm usually. retraction of the tendon ends, especially the proximal tendon end, is very common. flexion of the distal finger joints helps bring the distal end into the operating field. if the tendon is cut in zone 2, flexion of the metacarpophalangeal (mp) joint sometimes helps the proximal stump advance to the sheath opening area, but very often a separate incision at the distal palm is needed to find the proximal stump and it is advanced distally through the preserved sheath to approximate the distal tendon. if the tendon is cut in zone 1, flexion of the pip joint usually brings the proximal end back to the operative field. during the surgery, some mild bleeding may be seen when the skin incision is made, but after wiping the sites with gauzes, the bleeding usually stops. we no longer use cautery but occasionally use a hemostat on bigger vessels for a few minutes to achieve hemostasis. the flexor digitorum profundus (fdp) tendon is repaired with a 6-strand core suture repair (4 - 0 suture, m - tang method using looped suture (fig. alexandria, va, usa), or a triple kessler method with asymmetric core suture purchases at two tendon stumps8)) plus a simple running peripheral suture (6 - 0 suture) (fig. core suture purchases should be at least 7 mm, ideally around 10 cm (fig. 5). this is a very important requirement for the core suture.910) the core suture is performed with some extent of tension to make the two tendon ends approximate tightly to increase gapping resistance, but not too tight to avoid marked tendon bulkiness. it is important to make the repair a bit tensed, rather than being loose or tension - free. my method is to tension the core suture lines to the degree that makes the tendon segments within the core sutures shortened slightly, by approximately 10% (fig. 5).1112) the lacerated flexor digitorum superficialis (fds) tendon is repaired when the wound is clean and the repair is easy and the cut is not in the area of zone under the a2 pulley. in many cases, i do not repair the fds tendon, especially when at delayed primary repair, and when in the a2 pulley area, or when fds tendon retracted proximally. repair of damaged synovial sheath is usually not necessary and whether the sheath is closed does not affect outcomes. however, it is necessary to take care to preserve most or most parts of the annular pulleys if possible. when other annular pulleys are intact, we can vent the a4 pulley entirely if the tendon ends can pass under this narrow pulley or the pulley restricts tendon gliding (which is often the case).7) if the injury site is at or around the a2 pulley, a part (1/2 to 2/3) of the a2 pulley can be vented.6) my method of venting is a direct cut along the volar midline using scissors. pulley venting may be done together with cutting of a part of adjacent synovial sheath. the total length of sheath - pulley release in the site of repair surgery is 1.5 cm or 2 cm depending on the finger length.6) our studies show that this length of release allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact. i have performed such venting for over 10 years and have not seen functional damage to the finger after properly doing the venting. we ask the patient to actively extend and flex the surgically repaired digits. this test verifies that the repair is strong and is able to tolerate early active digital motion during rehabilitation.1314) this test is called " digital extension - flexion test."14) there are thee needed parts of this test : (1) full active extension to verify no gapping between the tendon ends (fig. 6) ; (2) smooth active flexion to verify smooth gliding of the tendon and its repair site ; and (3) active flexion to almost totally flex the digit to verify that the no pulley prevents tendon gliding. if the examination creates a visible gap between the two tendon ends during finger extension, the suture is usually not tight enough and needs to be replaced with a tighter suture with proper tension to avoid rupture. here, i strongly stress that the repeated total active movement examination may be needed to verify that the repair is strong and pulley release is sufficient. the allowable length of the pulley release for the a2 is 2/3, and the entire a4 pulley can be vented with a part of the synovial sheath when all other annular pulleys are well preserved. usually this venting allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact. repair of flexor tendon in proximal zone 1 can use similar approaches because only fdp tendon is repaired. to find or pass the retracted proximal end, the a4 pulley may need entire venting if the repair is very close to that pulley. a separate incision in the proximal part of the digit or distal palm may be necessary to find the retracted fdp tendon and a4 may need total venting to pass the proximal stump distally. the patient is protected with a very short forearm based splint, from about 6 - 8 cm above the wrist to the tip of the finger, with the wrist at 20 to 30 flexion, mp joint at slight flexion, and the interphalangeal joints in extension. 15) i sometimes put the patient 's wrist in neutral position.715) i think, with a strong repair method and validation of good repair using the intraoperative extension - flexion test, we have a greater room to place the wrist at a position between a wrist neutral position and slight flexion, and may even slight extension. the early active motion that i use is a combined passive - active motion, starting from 3 to 5 days after the surgery.15) i do not move the operated digits in the first 3 days after surgery because edema is prominent, pain is severe, and adhesions do not form in this period. motion starts from day 4 or 5. in the initial 1 to 2 weeks after surgery, we keep the active finger flexion within only one - third of the total range of finger motion (fig. the partial range of active motion is progressively increased to two - thirds in week 3 or 4 after surgery. the patient should avoid full range of active flexion in the first 2 to 3 weeks after surgery because the final extreme digital flexion produces the greatest bending force to the tendon and tendon is prone to disruption. i sometimes start full active motion even a little later in the patient who had tendon repairs in multiple fingers, or when the tendon is repaired 3 to 5 weeks after trauma or after repair of ruptured primary repair. in performing these motion exercises, the full range of passive digital motion (10 to 30 repetitions) the number of exercise sessions can range from 5 to 6 times per day and hourly in daytime, depending on the need of motion to prevent or correct digital joint stiffness and patient 's desire or availability for the exercise. four to 5 sessions are minimal each day and 10 to 30 repetitions of passive motion and 20 to 30 repetitions of active motion are minimal for each session in our practice. i do not order patients to move hourly, but some patients prefer to do exercise whenever he or she has free time. i encourage full active extension of the interphalangeal joints in all the postsurgical weeks and encourage differential pip and dip joint motions. many patients need further extension exercises because of some extension or flexion lags, which can be corrected in subsequent exercises. 8), and persistent exercises of up to 5 or 6 month may still show improvement of function. the local anesthetic (1% lidocaine with epinephrine, 1:100,000 for tendon surgery in 1 or 2 digits or in the palm) is injected to the digits of the patient in the waiting area starting 30 minutes before surgery for anesthesia. injection of such mixture to the hand was found to be safe.123456) waiting after injection allows the epinephrine to take effect and provide an adequately dry working field. 1). we keep the total dose of infiltration less than 7 mg / kg. usually, less than 50 ml is required for tendon surgery in the hand (including digits and palm areas), and we use premixed 1% lidocaine with 1:100,000 epinephrine. occasionally, 50 to 100 ml is required when surgery is involved in 3 or 4 digits or in the forearm ; in these cases, we dilute with saline to a concentration of 0.5% lidocaine with 1:200,000 epinephrine. the injection starts proximally with a fine needle (25 or 27 gauge) (fig. is injected in the most proximal part of the likely dissection area to block the nerves distally. this site is located in the distal part of the palm for a zone 2 tendon repair. in this way, the large volume of the first injection will bathe all of the nerves leading to the area of the next injections. for a zone 1 tendon repair, we usually inject the solution similarly as for a zone 2 tendon repair. in making the first injection, we insert the needle perpendicular to the skin into the subcutaneous fat to reduce pain of injection.6) then the syringe is stabilized with fingers propped on the skin to avoid needle wobble. wait 15 to 45 seconds until the patient tells us that the sting is gone. then, we proceed to inject the rest of infiltration very slowly (more than 5 minutes) without moving the needle (fig. sometimes, i make this first injection in a quicker fashion than i described above and inject continuously in the way that we all use for local anesthesia, if the patient tolerates well. about 10 to 15 minutes after the first injection, the whole distal area of dissection is almost or totally numb. the next three injections are used mainly for the epinephrine vasoconstriction effect. between both digital nerves, 2 ml of infiltration is injected subcutaneously at the palmar digital crease, so the bevel of the needle does not lacerate the nerve fascicles (fig. another 2 ml of infiltration is also injected subcutaneously at the digital crease of the proximal interphalangeal (pip) joint between both digital nerves (fig. the final 1 ml of infiltration is then injected subcutaneously at the digital crease of the distal interphalangeal (dip) joint between both digital nerves (fig. the surgery starts 30 minutes after the first injection or 15 minutes after completion of all injections. the sheath is opened through a small incision along the midline, about 1 to 1.5 cm usually. retraction of the tendon ends, especially the proximal tendon end, is very common. flexion of the distal finger joints helps bring the distal end into the operating field. if the tendon is cut in zone 2, flexion of the metacarpophalangeal (mp) joint sometimes helps the proximal stump advance to the sheath opening area, but very often a separate incision at the distal palm is needed to find the proximal stump and it is advanced distally through the preserved sheath to approximate the distal tendon. if the tendon is cut in zone 1, flexion of the pip joint usually brings the proximal end back to the operative field. during the surgery, some mild bleeding may be seen when the skin incision is made, but after wiping the sites with gauzes, the bleeding usually stops. we no longer use cautery but occasionally use a hemostat on bigger vessels for a few minutes to achieve hemostasis. the flexor digitorum profundus (fdp) tendon is repaired with a 6-strand core suture repair (4 - 0 suture, m - tang method using looped suture (fig. alexandria, va, usa), or a triple kessler method with asymmetric core suture purchases at two tendon stumps8)) plus a simple running peripheral suture (6 - 0 suture) (fig. 4). core suture purchases should be at least 7 mm, ideally around 10 cm (fig. this is a very important requirement for the core suture.910) the core suture is performed with some extent of tension to make the two tendon ends approximate tightly to increase gapping resistance, but not too tight to avoid marked tendon bulkiness. it is important to make the repair a bit tensed, rather than being loose or tension - free. my method is to tension the core suture lines to the degree that makes the tendon segments within the core sutures shortened slightly, by approximately 10% (fig. 5).1112) the lacerated flexor digitorum superficialis (fds) tendon is repaired when the wound is clean and the repair is easy and the cut is not in the area of zone under the a2 pulley. in many cases, i do not repair the fds tendon, especially when at delayed primary repair, and when in the a2 pulley area, or when fds tendon retracted proximally. repair of damaged synovial sheath is usually not necessary and whether the sheath is closed does not affect outcomes. however, it is necessary to take care to preserve most or most parts of the annular pulleys if possible. when other annular pulleys are intact, we can vent the a4 pulley entirely if the tendon ends can pass under this narrow pulley or the pulley restricts tendon gliding (which is often the case).7) if the injury site is at or around the a2 pulley, a part (1/2 to 2/3) of the a2 pulley can be vented.6) my method of venting is a direct cut along the volar midline using scissors. pulley venting may be done together with cutting of a part of adjacent synovial sheath. the total length of sheath - pulley release in the site of repair surgery is 1.5 cm or 2 cm depending on the finger length.6) our studies show that this length of release allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact. i have performed such venting for over 10 years and have not seen functional damage to the finger after properly doing the venting. we ask the patient to actively extend and flex the surgically repaired digits. this test verifies that the repair is strong and is able to tolerate early active digital motion during rehabilitation.1314) this test is called " digital extension - flexion test."14) there are thee needed parts of this test : (1) full active extension to verify no gapping between the tendon ends (fig. 6) ; (2) smooth active flexion to verify smooth gliding of the tendon and its repair site ; and (3) active flexion to almost totally flex the digit to verify that the no pulley prevents tendon gliding. if the examination creates a visible gap between the two tendon ends during finger extension, the suture is usually not tight enough and needs to be replaced with a tighter suture with proper tension to avoid rupture. here, i strongly stress that the repeated total active movement examination may be needed to verify that the repair is strong and pulley release is sufficient. the allowable length of the pulley release for the a2 is 2/3, and the entire a4 pulley can be vented with a part of the synovial sheath when all other annular pulleys are well preserved. usually this venting allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact. repair of flexor tendon in proximal zone 1 can use similar approaches because only fdp tendon is repaired. to find or pass the retracted proximal end, the a4 pulley may need entire venting if the repair is very close to that pulley. a separate incision in the proximal part of the digit or distal palm may be necessary to find the retracted fdp tendon and a4 may need total venting to pass the proximal stump distally. the patient is protected with a very short forearm based splint, from about 6 - 8 cm above the wrist to the tip of the finger, with the wrist at 20 to 30 flexion, mp joint at slight flexion, and the interphalangeal joints in extension. 15) i sometimes put the patient 's wrist in neutral position.715) i think, with a strong repair method and validation of good repair using the intraoperative extension - flexion test, we have a greater room to place the wrist at a position between a wrist neutral position and slight flexion, and may even slight extension. the early active motion that i use is a combined passive - active motion, starting from 3 to 5 days after the surgery.15) i do not move the operated digits in the first 3 days after surgery because edema is prominent, pain is severe, and adhesions do not form in this period. motion starts from day 4 or 5. in the initial 1 to 2 weeks after surgery, we keep the active finger flexion within only one - third of the total range of finger motion (fig. 7). the partial range of active motion is progressively increased to two - thirds in week 3 or 4 after surgery. the patient should avoid full range of active flexion in the first 2 to 3 weeks after surgery because the final extreme digital flexion produces the greatest bending force to the tendon and tendon is prone to disruption. i sometimes start full active motion even a little later in the patient who had tendon repairs in multiple fingers, or when the tendon is repaired 3 to 5 weeks after trauma or after repair of ruptured primary repair. in performing these motion exercises, the full range of passive digital motion (10 to 30 repetitions) the number of exercise sessions can range from 5 to 6 times per day and hourly in daytime, depending on the need of motion to prevent or correct digital joint stiffness and patient 's desire or availability for the exercise. four to 5 sessions are minimal each day and 10 to 30 repetitions of passive motion and 20 to 30 repetitions of active motion are minimal for each session in our practice. i do not order patients to move hourly, but some patients prefer to do exercise whenever he or she has free time. i encourage full active extension of the interphalangeal joints in all the postsurgical weeks and encourage differential pip and dip joint motions. many patients need further extension exercises because of some extension or flexion lags, which can be corrected in subsequent exercises. 8), and persistent exercises of up to 5 or 6 month may still show improvement of function. therefore, the patient can actively move the digits or the hand to ensure that tenolysis is adequate and the tendon is strong enough to move the tendon. the method of anesthesia is the same as we described earlier for primary repair, and surgical incisions for tenolysis are the same with those used traditionally. the only difference is that the surgeons can ask the patient to actively flex or extend the digit or the hand to see active gliding of the tendon. if the tenolysis is sufficient, the surgery is complete. if the tenolysis is insufficient, as indicated by insufficient digit or hand motion, further release of scar around the tendon is necessary such active motions help the surgeon to test the strength of these tendons. if the tendon is remarkably elongated or is broken when the digits or the hand actively moved, tendon reconstruction using a tendon grafting should be considered. during active motion, strength of the pulley can be tested as well and whether the pulleys restrict tendon gliding can be assessed. if the pulleys are broken during active tendon motion, important annular pulleys may need reconstruction. restriction of the tendon gliding from the pulleys may need further release of adhesions or a pulley plasty or pulley shortening procedure. tendon transfer is actually best indicated for such wide - awake surgery, because adjusting tension of the transfer has always been difficult. with the patient being awake, the digits or the hand can move actively to determine correct tension of the transfer. the patient should be asked to perform the desired action after transferring the tendon before the surgeon places the suture to fix the transferred tendon to the damaged tendon. this practice has been a routine for some hand surgeons now, and has been used in most commonly performed tendon transfers such as extensor indicis proprius transfer to the extensor pollicis longus, flexor carpi radialis transfer to the extensor digitorum communis. the anesthesia method is the same as described for the flexor tendon repair in the palm. if a lengthy incision or dissection over an extensive area is necessary in the forearm, 0.5% lidocaine with 1:200,000 epinephrine is used to decrease the total amount of injected epinephrine. strength and gliding of the tendon suture site (usually pulvertaft weave suture) are also possible with the patient actively moving the digits or the hand. besides tendon surgery, similar wide - awake surgery has been used in carpal tunnel release, cubital tunnel release, wrist arthroscopy, and some benign tumor resection.16171819) these uses have greatly reduced the cost of the surgery and diminished the use of supplies during surgery.16171819) many patients now can be operated in a minor procedure room. in our department, we established a wide - awake surgical room right in the ward. many patients do not need to go to the main operating theatre to have surgery. such a wide - wake surgical room reduces surgical waiting time and save the time of patient transfer and recovery, representing a huge increase in efficiency of surgical treatment as compared to previous surgical settings. | tendon surgery is unique because it should ensure tendon gliding after surgery. tendon surgery now can be performed under local anesthesia without tourniquet, by injecting epinephrine mixed with lidocaine, to achieve vasoconstriction in the area of surgery. this method allows the tendon to move actively during surgery to test tendon function intraoperatively and to ensure the tendon is properly repaired before leaving the operating table. i applied this method to primary flexor tendon repair in zone 1 or 2, tenolysis, and tendon transfer, and found this approach makes tendon surgery easier and more reliable. this article describes the method that i have used for tendon surgery. |
obesity quantified as body mass index (bmi) is beginning to replace undernutrition and infectious disease as the most significant contributor to ill health. obesity can lead to serious health problems, including diabetes, hypertension, and coronary artery disease. in addition, it may also affect the surgical and clinical outcomes of patients undergoing various surgical procedures. given the increasing incidence of obesity in men, it is imperative to understand the impact of bmi on surgical outcomes for patients with prostate cancer. in general, radical prostatectomy is the treatment of choice for patients with clinically localized prostate cancer and a life expectancy > 10 years. however, obesity may be a complicating factor in retropubic radical prostatectomy (rrp), resulting in higher complication rates, greater blood loss, higher transfusion rates, and worse functional results [5 - 8 ]. robot - assisted laparoscopic radical prostatectomy (ralp) is gaining in popularity for the treatment of clinically localized prostate cancer. ralp offers several advantages, compared with traditional open methods, including decreased blood loss, shorter hospital stay, and less perioperative morbidity [10 - 13 ]. however, the impact of obesity on surgical outcomes in ralp has not been well defined. several previous studies have attempted to characterize this effect, with somewhat conflicting results [14 - 16 ]. in this study we evaluated the impact of obesity on perioperative outcomes, including operative time, estimated blood loss (ebl), complications, and time to oral intake in patients undergoing ralp compared with rrp. from april 2008 to may 2011, 181 patients with prostate cancer underwent a radical prostatectomy performed by a single surgeon at our center. standard preoperative assessment included age, height, weight, baseline serum prostate - specific antigen (psa), gleason score, clinical stage, and prostate volume. all patients had adenocarcinoma of the prostate proved by prostate needle biopsy. upon admission, total operative time was regarded as the time between initiation of the skin incision and end of wound closure according to operative records. all complications occurring during the perioperative period were classified according to the modified clavien classification system (css). according to the modified css, low - grade complications (grade i or ii) were regarded as minor complications and high - grade complications (grade iii to v) as major complications. patients were subdivided into two bmi groups according to the world health organization (who) recommendation for asians : patients with bmi of 25 kg / m or less were considered nonobese and those with bmi greater than 25 kg / m were considered obese. all of the 111 ralp procedures were performed by using the transperitoneal approach, as described by menon. six ports, one 12-mm port for the scope, three 8-mm ports for the instrument arms, and 10-mm and 5-mm ports for the assistant, were placed. a transverse inverted u peritoneal incision was made, followed by entry into the space anterior to the peritoneum and space of rezius. the deep dorsal venous complex was ligated and bladder neck and seminal vesicle dissection, posterior dissection, and urethral transection were sequentially performed. chi - square test and fisher exact test for proportions and student 's t - test for continuous variables were used for statistical comparison of the groups. for all results, we calculated the p - value for whether the differences between the groups were statistically significant ; p25 kg / m) might have had less fat tissue in the abdominal cavity or abdominal wall than western obese patients (bmi>30 kg / m) and similar fat tissue to overweight patients. we suggest that ralp is a feasible procedure in obese korean patients with localized prostate cancer. the advantages of ralp compared with rrp and the disadvantages of obesity in the surgical treatment of prostate cancer are already well known and well established. few published studies, however, have dealt with the benefit of ralp in obese patients. in our study, ralp was a more effective and safer procedure in obese patients than was traditional open radical prostatectomy. in the management of obese patients with localized prostate cancer, ralp should be considered as a primary choice for treatment. | purposeobesity has been suggested as a risk factor for worse perioperative outcomes, especially in radical prostatectomy, in several studies. however, the impact of obesity on perioperative outcomes has not yet been well elucidated for robot - assisted laparoscopic radical prostatectomy (ralp). we evaluated whether obesity had an adverse effect on outcomes following ralp compared with retropubic radical prostatectomy (rrp).materials and methodsfrom april 2008 to may 2011, 181 patients underwent radical prostatectomy (ralp, 111 ; rrp, 70). these patients were subdivided into two groups according to body mass index (bmi) : the nonobese group (bmi, 25 kg / m2 or less) and the obese group (bmi, greater than 25 kg / m2). perioperative outcomes in ralp and rrp were retrospectively compared between the two groups.resultsin rrp, patients in the obese group (n=20) showed greater blood loss and a higher complication rate than did those in the nonobese group (n=50). however, in ralp, no statistically significant differences in perioperative outcomes were observed between the obese (n=37) and the nonobese (n=74) groups. ralp showed less blood loss and a lower complication rate in both the obese and nonobese groups than did rrp.conclusionsralp is thought to be a more effective and safer procedure in obese patients compared with traditional open radical prostatectomy. in the management of obese patients with localized prostate cancer, ralp should be considered as a primary choice for treatment. |
a 74 year old male presented to his family physician with intermittent right sided headaches of 2 months duration. there was no history of trauma, migraine and no associated neck pain. otherwise, his general health was very good. clinical examination was unremarkable, therefore, no further investigations were instigated and the patient was discharged with simple analgesia. four months later, he returned to his family physician with worsening symptoms, namely, the headaches became more intense, persistent and were associated with slurred speech. clinical examination revealed atrophy of the right side of the tongue and deviation to the right on protrusion - right hypoglossal nerve palsy (fig. his baseline blood tests which included a whole blood count, urea and electrolytes, serum calcium and glucose were all normal. a computed tomography of the brain showed skull base sclerotic metastases, subsequently, a magnetic resonance imaging of the brain revealed a focal abnormality of the right petrous bone with involvement of the hypoglossal nerve (fig. bone metastases are commonly associated with thyroid, lung, breast, prostate and renal cancer, prostate cancer differs from the others by nature of its ' sclerotic bone metastases. therefore, in addition to the chest x - ray, ultra sound scan of the renal tract, a serum prostate specific antigen (psa) was requested, this was found to be elevated at 101 ng / ml. he was then referred to a urologist with suspected prostate cancer. he denied suffering from lower urinary tract symptoms and there was no history of back pain. subsequently, prostate gland biopsies confirmed a well - differentiated adenocarcinoma, gleason score 3 + 3=6. although, there has been no recovery of the hypoglossal nerve palsy following a bi - lateral scrotal orchidectomy, his recent psa level remains low at 3.2 ng / ml. at 32 months of follow - up skull base metastatic cancer is often silent ; however, as the disease advances it commonly results in cranial nerve palsies. a combination of computed tomography and magnetic resonance imaging of the head is used to diagnose skull base metastases. although rare, cranial nerve palsies are associated with advanced prostate, breast, lymphoma and lung cancers. collet - sicard syndrome is caused by a lesion that involves the lower 4 cranial nerves. it results in the paralysis of vocal cords, palate, trapezius and sternocleidomastoid muscles. clinically, it results in anaesthesia of the larynx, pharynx and soft palate. as described in our patient, unilateral occipital headache with ipsilateral hypoglossal nerve palsy is termed occipital - condyle syndrome. although, sclerotic bone metastases are commonly associated with prostate cancer, other cancers with bone metastases must be excluded ; such as thyroid, lung, breast and renal cancer. skull base involvement of metastatic prostate cancer is almost invariably associated with known hormone - refractory disease. cranial neuropathy as the presenting feature is extremely rare, and although, there is one reported case of hypoglossal nerve involvement as the presenting feature, this was associated with involvement of multiple cranial nerves at the time of presentation. to the best of our knowledge, we report the first case of solitary hypoglossal nerve palsy as the presenting feature of advanced prostate cancer. neurologists, neurosurgeons and otolaryngologists may be the first clinicians to see such a patient ; therefore, prostate cancer should be amongst the differential diagnoses considered in middle - aged and elderly men presenting with a cranial neuropathy and evidence of skull metastasis. furthermore, if a psa test is elevated then the patient should be referred to the urologist. | prostate cancer is the most frequently diagnosed solid organ cancer in men and is the second leading cause of cancer - related deaths in men in the united kingdom. commonly, it metastasizes to bones and lymph nodes, however, in advanced hormonerefractory disease it may involve the skull base leading to associated cranial nerve palsies. cranial nerve palsy as the presenting feature of advanced hormone - sensitive prostate cancer is extremely rare. to the best of our knowledge, we report the first case of solitary hypoglossal nerve palsy as the presenting feature of advanced prostate cancer. neurologists, neurosurgeons and otolaryngologists may be the first clinicians to see such a patient ; therefore, prostate cancer should be amongst the differential diagnoses considered in middle - aged and elderly men presenting with a cranial neuropathy and evidence of skull metastasis. |
traumatic injuries generally disrupt the pulpal blood supply causing pulp necrosis and leading to anaerobic conditions favorable for the growth of opportunistic microorganisms, which may subsequently result in the development of periapical lesions. periapical lesions generally represent an inflammatory response to invasion of the root canal system by microorganisms and their by - products. such lesions grow via a variety of mechanisms, including osmotic fluid accumulation in the lumen, epithelial proliferation, and molecular mechanisms (nair, 1998). thus, if the lesion is effectively evacuated of the inflammatory exudates, so as to reduce the hydrostatic pressure, and if the microbiological etiology is removed by nonsurgical root canal treatment, these lesions may regress by the mechanisms of apoptosis. various techniques have been proposed for reducing hydrostatic pressure, such as, the decompression technique (loushine., 1991, martin., 2007), aspiration - irrigation technique (hoen., 1990), and aspiration through the root canal system, which may result in a shrinkage of the lesion. elimination of the microorganisms, in case of large periapical lesions, has been a challenge for the clinician., the endodontic treatment may fail if the root canal treatment has not adequately eliminated or reduced the intraradicular burden, leading to persistent infections. therefore, some form of chemical irrigation and disinfection is necessary to optimally disinfect the root canal system. various medicaments have been widely advocated to help eliminate bacteria, reduce periapical inflammation and pain, and induce healing. calcium hydroxide has been commonly used as an intracanal medicament, however, it has been reported that it is not effective in disinfecting the root canal system associated with persistent endodontic infections. various combinations of antibiotics have also been used and among them a mixture of ciprofloxacin, metronidazole, and minocycline has been shown to be very effective in eliminating endodontic pathogens in vitro and in situ. this combination is commonly referred to as triple antibiotic paste. ozan and er have found that a combination of antibiotic drugs (metronidazole, ciprofloxacin, and minocycline), when used as antibacterial dressing, is successful in healing large cyst - like periradicular lesions. this case report describes the nonsurgical management of a large persistent periapical lesion by decompression and aspiration of fluid through the root canal space, with subsequent application of triple antibiotic paste. a 21-year - old male patient, with no relevant medical history, presented with a painful recurrent swelling, since six months, on the front right side of the upper jaw. the patient gave a history of trauma to the maxillary anterior teeth 10 years prior. he had undergone root canal treatment twice, at an interval of two years, for the same. intraoral examination revealed a painful swelling of the palatal mucosa, adjacent to teeth 11 and 12. there was moderate pain on palpation in relation to the labial side of tooth 11. there was a large radiolucent lesion with a uniform radiolucency and well - defined margins involving the apices of these teeth [figure 1 ]. preoperative radiograph taking into account the medical and dental history of the patient, a presumptive diagnosis of periapical cyst was established. nonsurgical endodontic retreatment was planned, to treat the involved teeth. following access cavity preparation and gutta - percha removal, there was drainage of straw - colored fluid from tooth 11. when the drainage ceased, the apical foramen for both teeth was gauged using hand k - files and the apical width was found to be equivalent to size 45 for tooth 11 and size 30 for tooth 12 [figure 2 ]. the root canals were prepared using k - files, until the final preparation sizes of 60 and 40, respectively, for 11 and 12, were achieved. the canals were thoroughly irrigated with 3% sodium hypochlorite (naocl) and normal saline. the final irrigation was performed with 2% chlorhexidine (chx) and the access cavity was temporarily sealed with cavit g. the patient was prescribed analgesics for three days and was advised warm saline rinses. working length radiograph the next day, when the patient came, the swelling had decreased. the canals were again copiously irrigated and filled with a paste of calcium hydroxide ca(oh)2 mixed with 2% chx. the intracanal dressing was to be reviewed after one week, but the patient reported back in two days with a recurrence of swelling. after irrigating the canals, the intracanal ca (oh)2 dressing was again repeated for one week. there was no decrease in the swelling or pain even after the second change of dressing. a 25-guage needle attached to a 5 ml syringe was inserted through the root canal, past the apical foramen, into the bony cavity. approximately 5 ml of the fluid was aspirated while simultaneous digital pressure was applied on the labial and palatal cortical plates. after irrigating and drying the cavity, ca (oh)2 paste was again placed in the canal. at the next appointment after one week, there was considerable decrease in the swelling, but the pain in the labial vestibule still remained on palpation. the canals were again debrided, irrigated, aspirated, and it was decided to fill triple antibiotic paste in the canal. after two days, the patient reported with absence of pain and swelling, but a slight discomfort on palpation on the labial side. the dressing was left undisturbed for another one week, after which the canals were obturated with gutta - percha and zinc oxide eugenol cement sealer [figure 3 ]. immediate postoperative radiograph the patient was recalled after a three - month interval. however, the patient turned up only after one year. the clinical and radiographic examination demonstrated that the patient was asymptomatic and exhibited proper integrity of the periodontal tissues after one year [figures 4 and 5 ]. there is clinical evidence that as the periapical lesions increase in size, the proportion of the radicular cysts increases. however, some large lesions may be granulomas or may have a direct communication with the root canal system (apical pocket cyst or bay cyst) and respond favorably to nonsurgical treatment. on the other hand, true cysts are completely enclosed by the lining epithelium and may be attached to the root apex by a cord of epithelium. in the past, it was considered that true cysts would not respond to root canal treatment alone and that surgery was always required. however, in recent years, there is greater awareness of the root canal morphology, and the development of newer instruments, techniques, and materials has greatly enhanced the clinician 's abilities. of late, it has been proved that if the lesion is effectively evacuated of the inflammatory exudates and the microbial load is reduced with an effective intracanal medicament, it is possible to stimulate the immunological system to induce repair, even in cystic lesions. considering all this, nonsurgical root canal treatment should always be the first choice in cases of non - vital teeth with infected root canals. elimination of bacteria from the root canal is the most important factor for the successful treatment of periapical lesions, and the lack of regression of such lesions is generally assigned to the persistence of bacteria inside the root canal. in the present case, failure of the root canal treatment twice before the patient presented to our clinic could be attributed to the improper debridement and disinfection. moreover, poor obturation of the root canals allowed leakage and more bacterial contamination. a presumptive diagnosis of a periapical cyst was made on the basis of the radiographic features and the presence of straw - colored fluid. aspiration through the root canal system is a simple technique that reduces the hydrostatic pressure without causing additional discomfort to the patient. the aspiration technique was used in this case by inserting a 25 gauge needle just beyond the root apex in tooth 11, so as to reduce the hydrostatic pressure. during aspiration, digital pressure was applied on the labial and palatal cortical plates that aided in reducing the size of the swelling. it is important to apply digital pressure throughout the procedure so as to avoid entrapment of air in the bony cavity, which can result in increased intra - bony pressure. the role of intracanal medicament can not be underestimated in the eradication of the bacteria, as chemomechanical preparation alone is not enough to predictably eliminate all the bacteria, and a small portion of the flora survives. to date, calcium hydroxide has been considered the gold standard for optimally disinfecting root canals. it has been proved to be a reliable and effective means of canal disinfection in cases of primary apical periodontitis. however, its role in the elimination of the bacteria associated with persistent apical infections is controversial. bacteria associated with persistent apical infections have the ability to invade the dentinal tubules and buffer the high ph produced by calcium hydroxide. in addition, dentin has also been found to have a buffering effect, further compromising the antimicrobial effect of calcium hydroxide. all these may be the possible reasons for persistent swelling and pus discharge from the root canals after calcium hydroxide dressing. use of antimicrobial agents has been suggested by various authors for the eradication of bacteria associated with persistent endodontic infections. due to the potential risk of adverse effects following systemic application, and the ineffectiveness of systemic antibiotics in necrotic pulpless teeth and periradicular tissues, the local application of antibiotics application of a single antibiotic may be ineffective in the sterilization of the canal due to the vast microflora harboring the root canal system, consisting of both aerobic and anerobic bacteria. to overcome these obstacles, the concept of lesion sterilization and tissue repair (lstr) by the cariology research unit of the niigata university school of dentistry, was developed. this uses a triple antibiotic paste of ciprofloxacin, metronidazole, and minocycline, for disinfection. it has been shown that this combination of drugs can kill any bacteria in the carious lesions, necrotic pulp, infected root dentin, and periapical lesions. even in the present case, there was a remarkable reduction in the symptoms after the use of triple antibiotic paste. the swelling did not recur in the 14-day course of the dressing and when the dressing was removed, no pus discharge was observed. even though a very low dose of the drug combination is required for topical application, one should consider that unnecessary, inadequate, and lengthy application of antibacterial drugs may induce drug - resistant bacteria. other case reports have also demonstrated healing with this combination of antibiotic paste, but they have used the drug for one to three months. contradictory to this, in our case, the patient was comfortable within one week of placement of the triple antibiotic paste, although we allowed it to remain for two weeks. early resolution of symptoms after placement of the triple antibiotic paste, in our case, could be attributed to additional use of the aspiration technique. however, if the topical application of the drug is preceded by the noninvasive technique of decompression and aspiration through the root canal system in order to reduce the hydrostatic pressure of the confined fluid, it may help to decrease the application time of the drug mixture. in this case report, it is seen that a large periapical cyst - like lesion has healed by nonsurgical root canal treatment, using intracanal aspiration and triple antibiotic paste. this confirms that even large periapical lesions can respond favorably to nonsurgical treatment, and therefore, it must always be the first treatment of choice. | a patient with a large periapical lesion in relation to the maxillary right central and lateral incisors is presented here. during the conservative root canal treatment, aspiration of the fluid was done through the root canal, followed by placement of triple antibiotic paste for two weeks. complete periapical healing was observed at the 24-month recall. this report confirms that for treatment of a large periapical lesion it is not always necessary to do surgical treatment and even cyst - like periapical lesions heal following conservative endodontic therapy. |
renal transplantation is the optimal mode of treatment for the patients with end stage renal failure. one of the major problems for transplantation is the discrepancy between the donor and recipient numbers with far less donor than recipients. as a consequence, patients with renal failure have to wait for a long time before they can be offered an allograft. this situation is especially worse in some countries like japan, with small cadaver programme where the average waiting time is 16 years. a significant number of patients die from the complications of chronic renal insufficiency on long - term dialysis before they get a transplant. this situation is more important especially in cases where chronic kidney disease has lead to other medical problems and patient either die of the complications or become too unwell for a transplant. various measures including the use of marginal donors and use of kidneys from maastricht category ii non - heart - beating donors (nhbd) have been utilized to increase the donor pool along with measures to improve and prolong graft function and survival. in addition, increasingly elderly donors are used, therefore increasing the risk of renal malignancy. one potential area, first described by penn has been to transplant kidneys after ex vivo resection of small tumours. this was a very radical idea, because firstly, there has been evidence of transmission of donor - derived malignancy into recipient from the very early days of transplantation. secondly as a general rule, organs from donors with malignancies have not been used for the same fear with some exceptions such as central nervous system tumours. surprisingly - outcomes of the patients described in penn 's series were not as bad as could have been anticipated. the contemporary experience with partial nephrectomy and its success for the treatment of small renal cell cancers has lead to extrapolation of similar technique for the management of allograft malignancy albeit sporadically. the purpose of this paper is to summarise the current evidence with regards to the utilization of kidneys with tumours for transplant and the use of conservative surgery for allografts where possible. pubmed, medline, embase and cinhal were linked searched for renal tumour / tumor, kidney tumour / tumor, allograft tumour / tumor, nephron sparing surgery, partial nephrectomy, and transplant to indentify potentially relevant articles. articles concerning the use of kidneys after resection of renal tumour for transplant and partial nephrectomy of allograft for renal tumours were selected. from the above - mentioned criteria of the literature search the following different types of case reports / case series were identified which are discussed separately. normal practice when confronted with a tumour of kidney on procurement is to return it to the donor and not use any other organs. in cases of deceased donors it meant that the contralateral kidney can not be used as well because of the concerns of micro metastasis and bilaterality of some of the renal cell carcinomas (rcc). penn, reviewing the cincinnati transplant tumour registry (cttr), described a total of 14 cases of ex vivo resection of small renal cell cancers detected incidentally followed by transplantation. frozen section was employed, and where margins were clear, kidneys were used although it is not clear whether all of the tumour bearing kidneys underwent frozen section. of the cadaveric donors, the contralateral kidneys, all of which appeared healthy, were transplanted as well. apart from these cases of renal carcinomas, there was one case of oncocytoma within the kidney which was transplanted after resection. of all the cases where the tumour was adequately resected before transplantation there was no recurrence in a followup ranging up to 210 months. buell. presented 14 cases of transplantation after renal tumour resection from the same database as used by penn. median tumour size was 2.0 cm (range 0.54.0 cm) and all were of low histological grade. they have described two further cases since the initial data review with no recurrence and good graft function. a similar case series from australia only included elderly recipients or those with significant co morbidities and high chance of death without transplantation. furthermore the recipients had high levels of hla mismatching with the donors and were selected on the basis that if there was a recurrence to occur, stopping immunosuppression may help in tumour lysis by recipient 's immune response. 41 patients received kidneys after ex vivo resection of tumour of which 10 were reported as benign lesion on histopathology. there was only one recurrence noted 9 years after transplantation out of the remaining 30 patients. notably this tumour recurrence was at a distance from the initial resection site, this therefore may not be a tumour recurrence but another primary within a field change renal tissue. the patient refused any further treatment, and the lesion has grown 0.2 cm in 18 months since diagnosis. in a followup study on these patients this group has recently published long - term outcomes which are significantly better than wait - listed patients on dialysis and are comparable to the live unrelated transplants. mannami. from japan published a series of 42 restored kidneys from live donors. eight donors with small renal cell carcinoma (< 3.5 cm) underwent donor nephrectomy and ex vivo resection of the tumour followed by transplantation of the kidney. five patients were alive, three with functioning grafts, two died with functioning grafts from unrelated caused, and one was lost to followup. no tumour recurrence has been noted in any of these patients. another 8 patients had donor nephrectomies which had benign diseases of which 5 had partial resection and kidney used for transplantation. three recipients are alive with functioning grafts, while four have gone back to dialysis (after 3,18,51,73 months). there have also been 6 case reports [1116 ] of live related kidney donation when a tumour was detected incidentally in or ex vivo and the kidney was transplanted after resection. no recurrence has been noted in any of these cases with a followup of up to more than 10 years. renal cell carcinoma represents around 4.6% of all the tumours in allograft recipients with only 10% of these occurring in the allograft itself. again the standard practice here has been to perform transplant nephrectomy with the patient invariably returning to dialysis and normally being put on a waiting list for another transplant if feasible. until now, more than 50 cases of allograft renal cell tumours have been described in the literature of which at least 35 cases have had nephron sparing surgery (nss) for their allograft tumour [6, 11, 1836 ]. tumour sizes have range, from 0.5 to 4.0 cm although there have been two case reports of larger (68 cm) tumours all being successfully treated with nss [18, 19 ]. postoperative followup is from one month to more than 10 years with one recurrence 5 years after nss in renal allograft. these kidneys again are normally not used as rcc can be bilateral especially the papillary subtype. penn has described 14 cases in which the contralateral kidney was transplanted from patients with renal tumour. this patient died 75 months after transplantation from a de novo cancer of one of his own kidneys. the remaining patients did not have any recurrence with a followup ranging from 0.5 to 153 months. has described a case of two allograft recipients from a single donor with tubulopapillary tumour (17 mm) in the right kidney ; only the left kidney was utilized for transplantation. shortly after transplantation, the recipient underwent an ultrasound (us) examination of the allograft which did not reveal any tumour. 3 months later a biopsy was done for rejection which revealed a poorly differentiated tumour and the patient underwent radical allograft nephrectomy. no additional chemotherapy was given apart from discontinuation of immunosuppression (prednisolone and azathioprine). lymph nodes that had been noted to be enlarged on ct scan disappeared two month after nephrectomy. the patient underwent re transplantation two years later and was disease free and dialysis independent at 3 year followup. another patient received the heart transplant from the same donor but died from bony metastasis from the renal cell carcinoma. in at least 4 cases [3, 13, 20, 39 ] there have been accidental transplantation of rcc mistaken as a benign pathology on procurement. partial nephrectomy / enucleation in all these cases was performed before transplantation with adequate resection margins. all recipients retained the allograft because of complete excision of the tumour and were kept under close follow - up with no recurrence so far. the cases where there have been transplantation of tumour, either partially resected or unrecognized at the time of transplant have resulted in disastrous outcomes [3, 38 ]. reported a series of 8 patients who underwent nephrectomy for a distal ureteric transitional cell carcinoma (tcc). one patient had a recurrence of tcc after 15 months and was offered graft nephrectomy but opted for partial resection of ureteric tumour to prevent returning to dialysis. he died three years after partial resection from a squamous cell carcinoma of lung with liver metastasis. his tcc also had squamous metaplasia and a dna study to determine exact origin of primary tumour could not be established because of inadequate tissue samples. transplantation of kidneys with cancers is a novel idea not only among patients but also among the transplant community. to be able to exploit this potential donor pool it is of utmost importance that both the health care specalists ; transplants surgeons and nephrologists and the patients both donors and recipients are comfortable with the idea of using such kidneys. to determine this, structured questionnaires were sent to focus group of patients on the north east renal transplant waiting list, postnephrectomy patients for small renal cancer, nephrologists and transplant surgeons in the uk. those respondents that had lost their kidney, removed for tumour, had the highest consent rate and patients potentially receiving such kidney the lowest. the transplant surgeon and this survey was done in uk from where there have been no case reports of using organs after removal of tumour and but still the response was largely favourable. given that since this survey there has been an increase in total number of such organs being utilized, one can extrapolate that current belief may be more favourable. one of the worries about transplantation of tumour affected kidneys is the potential of tumour recurrence and growth in state of potential immune inattention due the immunosuppressive therapy. renal cell carcinoma is known to be an immunogenic tumour but in the presence of immunosuppression, if there was any transplantation of tumour cells in the host, then the potential of continued growth will be higher in a host with a compromised immune system. furthermore, immunosuppression in itself has been known to increase the incidence of de novo malignancy [41, 42 ]. because of these concerns, an immunosuppressive agent with no potential to increase de novo malignancy and better still to have antitumour activities would probably be ideal. rapamycin has shown some promise as being a protective agent against rcc progression [21, 43, 44 ]. incidence of rcc has increased in western countries in the last few years owing to the widespread use of us and ct scanning [45, 46 ]. most rcc are now picked up at an early stage on investigations done for other reasons. furthermore the incidence of rcc in allografts will continue to increase as older people donate organs and graft survival is improved by better immunosuppression. longitudinal studies have shown that many small tumours have a slow growth pattern with low metastatic spread in tumours of <3 cm [48, 49 ]. autopsy studies have shown that rcc are present in 1%20% of patients dying from unrelated cases, meaning that many of the tumours will not prove to be clinically significant in the course of patient 's life [50, 51 ]. recent evidence has changed this practice dramatically as survival after radical nephrectomy (rn) and partial nephrectomy (pn) has shown to be comparable. favourable outcomes have been observed after nss for < 4 cm rccs and rn has been described as surgical overkill for these tumours. furthermore, local recurrence after nss has been reported to be < 5% with recurrences mostly associated with large and multifocal tumours. a significant risk of dying in patients on dialysis particularly in older patient has been one of the driving forces to increase the number of kidney donors. renal transplantation seems to confer a substantial survival advantage over dialysis in patients with end - stage renal failure. a significant number of patient accepted for dialysis are older patients, who have a mortality risk of 25%. with longer waiting times for a transplant, it is inevitable that many of the patients will die before they can receive a transplant which would have improved their quality of life and longevity. furthermore 16% to 23% of suspicious lesion resected from kidneys are either benign or of low malignant potential [5355 ] and not using these kidneys with small tumours after partial nephrectomy for transplantation seems wastage of precious organs when one considers the benefits of transplantation over dialysis. a suspicious lesion found at multiorgan retrieval should have an excision biopsy and histological confirmation of clear margins before any of the organs can be transplanted. a malignant lesion in the kidney when unrecognized and transplanted continues to grow under the immunosuppression carries high risk of metastasis and can result in fatal outcome. if the biopsy confirms clear margins with favourable histology then these organs could be used for transplantation as risk of recurrence is very low. situation is more complex when it comes to using restored organs from live (related / unrelated) renal cell carcinoma patients. major difference being that these are live cancer patients first and therefore must never be treated primarily as potential organ donors to prevent any bias in treating their primary problem which may lead to provision of less than optimal treatment and ultimately harm to these patients [8, ed ].. series concerning ureteric carcinoma patient, where adherence to standard practice for treating these tumours was not practiced with disastrous consequences. with changing trends, radical nephrectomy is now regarded as an alternate standard of care to partial nephrectomy for t1a tumours when partial nephrectomy is not technically feasible. this is due to the comparable oncological outcomes after partial nephrectomy and evidence that radical nephrectomy is an independent predictor of low gfr. a positive outcome for a recipient can never justify harm to a live donor ; on the contrary, for a transplant with a live donor to be regarded as a success means that both the recipient and the donor have done well. series were given the options of observation, radical or partial nephrectomy without any mention of the possibility of use of organs for transplantation. only after the patients had decided to opt for radical nephrectomy possibility of domino donation this approach has the benefit of making sure that patients make their own decisions without any pressure from clinicians. other important factor is to make sure that beliefs of the clinician do not affect patient 's treatment choices. importance of detailed informed consenting can not be over emphasised for the recipients of such restored organs. all the relevant information especially of the origin of the organ and potential of recurrence and associated risk must be discussed fully and patients understanding checked. routine followup of the patients with annual us have been suggested to make sure any recurrence is diagnosed as early as possible. if one kidney is found to have a tumour it is important that the other kidney is closely followed up. it is easier in the live donor setting when the donor can be carefully followed up but in cadaveric donation there has to be a central database for tracking the contralateral kidney which might be transplanted into a recipient in a different unit. immunosuppression is essential after transplant and unfortunately this has been associated with the higher incidence of cancers in recipients as opposed to the general population. certain newer immunosuppressive agents have anti tumour activity and their use can, in theory not only reduce the chances of recurrence but they can also be used to treat patient should a recurrence occur. furthermore human tissue act 2004 that covers the use of organs for transplant in the uk allows anyone to be a donor including live related and unrelated (altruistic donor) provided there is adequate consenting. this means that donation can also occur from patients suffering from small renal cell carcinoma who have radical nephrectomy as primary treatment provided measures are taken to ensure that these patients are treated appropriately in the first place and both donor and recipients had given informed consent. there are several important issues in using such marginal and potentially dangerous organs ; patients should have complete understanding of the implications of the type of organ they are donating and receiving, good surgical technique and rigorous pathological testing of the resected tissue to make sure there is no tumour left behind, regular followup with adequate investigations, and a reliable organ tracking system to investigate the recipient of contralateral organ should one organ develop a recurrence. on top of this, transplant surgeons and nephrologists should be comfortable in using such organs. usage of such organs is still in its infancy, and for a much wider acceptance of this source to occur, there is need for more research. one interesting area will be to explore the new immunosuppressive agents with antiproliferative properties on such recipients with the potential to reduce recurrence rate or better still to prevent it altogether while either replacing standard immunosuppressive agents or reducing their required dose thereby reducing side effects. | renal transplantation confers improvement in quality of life and survival when compared to patients on dialysis. there is a universal shortage of organs, and efforts have been made to overcome this shortage by exploring new sources. one such area is the use of kidneys containing small tumours after resection of the neoplasm. this paper looks at the current evidence in the literature and reviews the feasibility of utilizing such a source. |
the proportion of candidemia cases caused by non - albicans candida species has been increasing (1). some non - albicans candida species are associated with resistance to the usual antifungal azoles (2). c. haemulonii, a non - albicans candida species, microbiologically, the susceptibility profile of c. haemulonii shows that it is resistant to amphotericin b and other antifungal agents such as azoles (4), which have often been associated with clinical treatment failure (5, 6). no treatment regimen for invasive c. haemulonii infections has been clearly established. we investigated a catheter - related candidemia infection due to c. haemulonii and report on resolution of the infection using caspofungin, an echinocandin antifungal agent. on june 25, 2009, a 67-yr - old man with a cerebral infarction was hospitalized in the department of rehabilitation medicine for physical rehabilitation. prior to admission, he experienced cerebral infarction and was hospitalized for two months, beginning in march, 2009. a physical examination did not reveal any signs of infection, including at the tracheostomy site. the patient received a right subclavian venous catheter for total parenteral nutrition on the second day of hospitalization because of the risk of aspiration when receiving nasogastric tube feedings. on august 15 (the 50th day of hospitalization), the patient had a temperature of 37.8, a white cell count of 10.4 10/liter, and a c - reactive protein level of 10.8 mg / liter. the mild fever continued, and the level of crp and white cell count continued to increase. on august 29 (the 64th day of hospitalization), a blood culture collected 72 hr previously showed yeast growth, and the subclavian venous catheter was removed. intravenous fluconazole (400 mg once a day) was administered as empirical therapy, pending identification of the yeast. a qualitative culture of the catheter tip also produced a yeast culture. the yeast from the blood culture and catheter tip, designated as lys-1, was identified as candida sp. on the seventh day of fluconazole therapy. the blood cultures performed on the fourth and 14th days of fluconazole therapy were still positive for yeast, and the mild fever persisted. on september 15 (the 18th day of fluconazole treatment), the infectious disease department was consulted and recommended an echocardiography, a bone scan, an abdominal ct, and doppler sonography for venous thrombosis. the bone scan, abdominal ct, and doppler sonography revealed no significant findings. on september 17 (the 84th day of hospitalization), the treatment regimen was changed to caspofungin (50 mg daily after a 70 mg loading dose). on the second day of the caspofungin treatment, the patient became afebrile and showed clinical improvement. in resolution, the patient underwent a 17-day course of caspofungin and remained stable after discontinuation of the antifungal agent. conventional automated methods in the clinical microbiology laboratory identified the lsy-1 isolate as candida sp. thus, we attempted to identify the culture at the species level using a molecular method. to identify the isolate lsy-1, a portion of the large subunit (lsu) rrna gene was amplified using the primers lsu - f (5'-gcatatcaataagcggaggaaaag-3 ') and lsu - r (5'-ggtccgtgtttcaagacg-3 '). template dna and 20 pm of each primer were added to a pcr mixture tube (accu - power pcr premix ; bioneer, daejeon, korea). each cycle consisted of 30 sec at 95, 30 sec at 50, and 1 min at 72, followed by a final extension at 72 for 1 min. the purified pcr product was sequenced directly using the same primers as those used in the pcr amplification. the determined sequences (519 bp) were compared with the gen - bank public database using the blastn program (http://blast.ncbi.nlm.nih.gov/blast.cgi). the lsu rrna gene sequence of the isolate lsy-1 was a complete match with the corresponding sequence of the reference strain, c. haemulonii strain tjy2d (genbank accession numbers eu359820). in addition, the isolate in question showed many similarities to the sequences of the c. haemulonii strains and similarities of less than 85% with other candida species (fig. conventional automated methods in the clinical microbiology laboratory identified the lsy-1 isolate as candida sp. thus, we attempted to identify the culture at the species level using a molecular method. to identify the isolate lsy-1, a portion of the large subunit (lsu) rrna gene was amplified using the primers lsu - f (5'-gcatatcaataagcggaggaaaag-3 ') and lsu - r (5'-ggtccgtgtttcaagacg-3 '). template dna and 20 pm of each primer were added to a pcr mixture tube (accu - power pcr premix ; bioneer, daejeon, korea). each cycle consisted of 30 sec at 95, 30 sec at 50, and 1 min at 72, followed by a final extension at 72 for 1 min. the purified pcr product was sequenced directly using the same primers as those used in the pcr amplification. the determined sequences (519 bp) were compared with the gen - bank public database using the blastn program (http://blast.ncbi.nlm.nih.gov/blast.cgi). the lsu rrna gene sequence of the isolate lsy-1 was a complete match with the corresponding sequence of the reference strain, c. haemulonii strain tjy2d (genbank accession numbers eu359820). in addition, the isolate in question showed many similarities to the sequences of the c. haemulonii strains and similarities of less than 85% with other candida species (fig. c. haemulonii was originally described from a sample taken from the gut of a blue - striped grunt (haemulon scirus) in 1962 (7), and lavarde. (8) reported the first clinical isolation of this fungus from a human in 1984. antifungal resistance is the focus of attention in the management of invasive candidiasis. in previous cases, most c. haemulonii were resistant to both fluconazole and amphotericin b (5, 9). clinically, when amphotericin b was administered empirically, it failed to eradicate c. haemulonii candidemia (5). the majority of cases in which fluconazole was administered empirically also failed to eradicate candidemia (5, 6). although fluconazole administration has been shown to resolve candidemia, these cases could be considered transient candidemia combined with the removal of the intravenous catheter. therefore, the resistance of c. haemulonii poses a therapeutic challenge for the treatment of invasive candidiasis. microbiologically, previous reports and our case show that c. haemulonii is susceptible to echinocandins such as caspofungin or micafungin (5, 6, 10), and that it was not resistant to new triazoles such as voriconazole. however, it is not certain that echinocandins or voriconazole are actually efficacious in the treatment of c. haemulonii candidemia. there have been only two cases of clinical success in the eradication of c. haemulonii candidemia via echinocandins administration (5, 6). one case used micafungin, and the other used caspofungin. of those, in the treatment with caspofungin of a neonate, echinocandin was combined with amphotericin b. therefore, our case could demonstrate clinical success with caspofungin administration in the eradication of c. haemulonii candidemia in an adult patient. in our case, the vitek ii (biomrieux sa) clinical yeast identification system was used for initial identification of c. haemulonii, and sequence analysis of the partial 26s rrna gene (519 bp) was performed for confirmation. the 519-bp sequences of the isolate matched completely with the corresponding sequences of the reference strain, c. haemulonii strain tjy2d, available in the genbank database (accession number eu359820). in recent studies, the identification results of the vitek 2 system have closely corresponded with those of molecular methods for the identification of c. haemulonii, while the vitek 1 system and the api 32c system usually fail to identify c. haemulonii (5, 10). known risk factors for candidemia are use of central - venous catheterization, total parenteral nutrition, previous multiple antibiotics, previous steroid therapy, previous abdominal surgery, and an immunocompromised status (11, 12). in our case, long - term central - venous catheterization for hyperalimentation was the associated risk factor for candidemia, and no other source of infection suggested the possibility of catheter - related candidemia. in previous case reports, the majority of patients also received an intravenous central line and were in an immunocompromised status. considering the recent case series of invasive c. haemulonii infections, c. haemulonii is considered to be an emerging yeast pathogen for which the optimal strategy of patient management has yet to be elucidated. in conclusion, as found in the previous case reports, fluconazole and amphotericin b are not reliable empirical antifungal agents for the treatment of c. haemulonii candidemia. echinocandins, such as caspofungin or micafungin, may be an appropriate empirical choice of antifungal agent for invasive c. haemulonii infections. | candida haemulonii, one of the non - albicans candida species, is an emerging yeast pathogen that is known to be resistant to amphotericin b and other antifungal agents such as azoles. these anti - fungal agents have often been associated with clinical treatment failure, so no treatment regimen has been clearly established for invasive c. haemulonii infections. we investigated a catheter - related infection of c. haemulonii candidemia in an adult patient in long - term hospital care. in the early stages, the candidemia remained persistent despite treatment with fluconazole. however, after changing the antifungal agent to caspofungin, the candidemia was resolved. fluconazole and amphotericin b are not reliable empirical antifungal agents for invasive c. haemulonii infections, as shown in previous case reports. an echinocandin such as caspofungin may be an appropriate empirical choice of antifungal agent for an invasive c. haemulonii infection. |
carcinoma of eac is a very rare malignancy with an incidence of 1 - 6 cases per million population per year. squamous carcinoma is the most frequent neoplasm which is about four times more common than basal carcinomas. these tumors have an aggressive nature and usually present in advanced stages due to a lack of clear anatomical barriers. depending on the stage of disease and treatment protocols, 5-year survival rates range from 10% for advanced disease to 83% for early disease. those tumors which are found to be unresectable and treated with nonsurgical modalities have a very dismal outcome. logically in such patients, reduction in size by the use of neoadjuvant chemotherapy might result in successful surgical resection. based on the preliminary experience with induction chemotherapy in a selected population of technically unresectable tumors at our center, we performed a retrospective analysis of 4 patients evaluating the role of neoadjuvant chemotherapy in technically unresectable cancers. we present a retrospective analysis of 4 patients with carcinoma eac, who received neoadjuvant chemotherapy (nact) at our center between 2010 and 2014. these data were retrieved from a prospectively maintained database, electronic medical records, and the case files of the patients. these 4 patients belonged to a wide age group ranging from 28 to 60 years. one of them was a tobacco chewer, and one was a cigarette smoker and the rest 2 patients had no addictions. all 4 patients presented with swelling over temporal region along with serosanguineous discharge from the affected ear. two of them had pain and decreased hearing in the affected ear each, and one had facial asymmetry. one out of these 4 patients had a past history of chronic supporative otitis media for which he was operated twice by modified radical mastoidectomy. they were discussed in the multidisciplinary skull base clinic at our center and were considered technically unresectable and planned for induction chemotherapy in view of the following : occipital bone and soft tissue infiltration (n = 2).temporal dura, temporalis muscle and infratemporal fossa (n = 1) andleft temporal lobe and middle cranial fossa (n = 1). temporal dura, temporalis muscle and infratemporal fossa (n = 1) and left temporal lobe and middle cranial fossa (n = 1). these patients received neoadjuvant chemotherapy with 2 or 3 drugs consisting of combinations of a taxane (paclitaxel or docetaxel) and platinum (cisplatin or carboplatin) with or without 5-fluorouracil. the choice of regimens was decided on the patients performance status, creatinine clearance (as calculated by the cockcroft - gault formula), patient 's preference, logistic, and financial constraints. the chemotherapy protocol were as follows : docetaxel, cisplatin and 5 fluorouracil (dcf) - docetaxel 75 mg / m (d1), cisplatin 75 mg / m (d1) and 5 fluorouracil 1000 mg / m (d1-d5) every 21 days with primary granulocyte colony - stimulating factor prophylaxis.paclitaxel 175 mg / m and cisplatin 75 mg / m every 3 weekly. docetaxel, cisplatin and 5 fluorouracil (dcf) - docetaxel 75 mg / m (d1), cisplatin 75 mg / m (d1) and 5 fluorouracil 1000 mg / m (d1-d5) every 21 days with primary granulocyte colony - stimulating factor prophylaxis. after 2 cycles of chemotherapy, the patients were re - evaluated in the multidisciplinary skull base clinic. the response was assessed clinically by the surgeons for resectability and radiologically according to the response evaluation criteria in solid tumors (recist 1.1) criteria. this assessment was carried out by the same group of surgeons who had initially assessed the patients. three of these 4 patients received 3 drug chemotherapy with tpf and one of them received 2 drug chemotherapy with paclitaxel and cisplatin. all the 4 patients received 2 cycles of chemotherapy after which all of them had a clinical response in form of decrease in pain, ear discharge, and swelling over temporal region. radiologically, the response evaluation scan showed a partial response (pr) in 3 patients and stable disease (sd) in 1 patient by recist criteria. figure 1 shows the magnetic resonance imaging (mri) picture of a patient with an enhancing mass in the right external auditory canal and right temporal bone extending to the mastoid region before chemotherapy which decreased in size and extent after chemotherapy as depicted in figure 2. figure 3 shows the mri picture of a mass in the right external auditory canal and middle ear cavity involving high infratemporal fossa (itf) making it inoperable. as depicted in figure 4, post chemotherapy all 3 patients who received 3 drug chemotherapy had a pr while 1 patient who received 2 drug chemotherapy had an sd. one of the 3 patients, who achieved pr underwent surgical resection, however the other 2 patients still remained unresectable in view of persistent intradural extension and infratemporal fossa involvement though there was regression o the external auditory component of the disease. the 1 patient who had sd after 2 drug chemotherapy could undergo surgical resection as there was the clearance of disease in the infratemporal fossa. magnetic resonance image before chemotherapy an enhancing mass in the right external auditory canal and right temporal bone involving the mastoid magnetic resonance image after chemotherapy decrease in size and extent of the mass in right external auditory canal and right temporal bone involving the mastoid magnetic resonance image showing a mass in right external auditory canal and middle ear cavity involving high infratemporal fossa magnetic resonance image showing decrease in size of mass in right external auditory canal and middle ear cavity with no disease appreciable in high infratemporal fossa recent follow - up shows that 3 out of these 4 patients are alive. two of these patients have clinically and radiologically controlled disease. both these patients had a pr after 3 drug chemotherapy, however, remained unresectable and underwent concurrent chemoradiation in view of the persistent intradural extension and infratemporal fossa involvement, respectively. one of these who had an sd after 2 drug chemotherapy underwent surgical resection but a right frontal region recurrence at 3 months follow - up and is presently undergoing palliative radiotherapy for the same. the other patient who had a pr after 3 drug chemotherapy underwent surgical resection followed by adjuvant chemoradiation but developed a left posterior auricular swelling at 6 months follow - up and died due to the progression of disease. to the best of our knowledge, this is the first data documenting response to chemotherapy in carcinoma of eac and the use of neoadjuvant chemotherapy in technically unresectable carcinoma eac. carcinoma of external auditory canal (eac) is a very rare malignancy with a global incidence of 1 - 6 cases per million per year. these tumors have an aggressive nature with a tendency to spread early along preformed vascular and neural pathways, invading adjacent structures. due to this reason and also because the initial symptoms are similar to those of benign diseases, these tumors are usually diagnosed in advanced stages. depending on the stage of disease and treatment protocols, 5-year survival rates range from 10% for advanced disease to 83% for early disease. nonsurgical modalities like definitive radiation have a 3-year disease - free survival as low as 15% and the mean survival in the palliatively irradiated group was 3.6 months. it may be useful to make unresectable patients resectable thereby improving outcome in such patients. squamous cell carcinoma of head and neck cancer responds very well to chemotherapy, but the differential response has also been noted for different sites of disease. the retrospective analysis of the prospectively collected data at our hospital from 2008 to 2012 suggested that out of 862 patients referred for neoadjuvant chemotherapy, the sites were oral cavity in 83.6%, maxilla in 4.8%, larynx in 3.8%, laryngopharynx in 0.9%, and hypopharynx in 8.2%. we have used neoadjuvant chemotherapy previously in the technically unresectable head and neck cancers at our hospital which led to successful resection and improved overall survival (os). a retrospective analysis of 721 patients with stage iv technically unresectable oral cavity cancer who received neoadjuvant chemotherapy showed that 310 patients (43%) had sufficient reduction in tumor size to undergo surgical resection. the locoregional control rate at 24 months (32% vs. 15%) and the median estimated os (19.6 months vs. 8.16 months) significantly improved in patients undergoing surgery as compared to those treated with nonsurgical treatment. similarly, in another study of 41 patients with locally advanced technically unresectable maxillary carcinoma who were treated with induction chemotherapy, the response evaluation depicted sd in 43.9% and pr in 39%. after induction, surgery could be performed in 29.3% of patients. overall, the median progression - free survival was 10 months and os at 36 months was 35%. based on these results, we used neoadjuvant chemotherapy in patients with carcinoma of eac to patients who were considered unresectable in view of disease involving occipital bone, temporal dura, temporalis muscle, infratemporal fossa, left temporal lobe, and extensive soft tissue infiltration. licitra. also showed that the use of chemotherapy in resectable oral cavity cancers reduced the amount of demolitive surgery (31% vs. 52% in the control group) without detecting any increased number of positive surgical margins, and lessened postoperative radiotherapy (33% vs. 46%) possibly due to the presence of new safe revised margin with downstaging of the tumor. hence, the use of induction chemotherapy definitely seems to make a proportion of these patients with borderline unresectable tumors resectable which may improve the overall outcome and lead to an increase in the os. all 3 patients who received 3 drug chemotherapy achieved a pr while the 1 patient who received 2 drug chemotherapy had an sd. two of these 4 patients (1 after 3 drug chemotherapy and 1 after 2 drug chemotherapy) underwent successful resection after induction chemotherapy. those who were still unresectable after induction chemotherapy received the concurrent chemoradiation. in light of these studies and the results of the 4 patients in our case series, our approach of using induction chemotherapy could convert potentially unresectable tumors to a resectable disease that could produce better outcomes. the results of these 4 patients suggest that carcinoma eac responds to chemotherapy. the use of induction chemotherapy in carcinoma eac may have a role in making a proportion of borderline unresectable patients operable. | background : carcinoma of external auditory canal (eac) is a very rare malignancy with surgical resection as the main modality of treatment. the outcomes with nonsurgical modalities are very dismal. we present a retrospective analysis of 4 patients evaluating the role of neoadjuvant chemotherapy in technically unresectable cancers.materials and methods : this is a retrospective analysis of 4 patients from our institute from 2010 to 2014 with carcinoma eac who were deemed unfit for surgery due to extensive disease involving occipital bone with soft tissue infiltration (n = 2), temporal dura (n = 1), left temporal lobe, and extensive soft tissue involvement (n = 1). all these patients received neoadjuvant chemotherapy with docetaxel, cisplatin and 5 fluorouracil (n = 3) and paclitaxel and cisplatin (n = 1).results : response evaluation showed a partial response (pr) in 3 and stable disease (sd) in 1 patient by response evaluation criteria in solid tumors criteria. all 3 patients who received 3 drug chemotherapy had pr while 1 patient who received 2 drug chemotherapy had sd. two of these patients underwent surgery, and other 2 underwent definitive chemoradiation. one of 3 patients who achieved pr underwent surgical resection ; the other 2 remained unresectable in view of the persistent intradural extension and infratemporal fossa involvement. one patient who had sd could undergo surgery in view of clearance of infraatemporal fossa. recent follow - up shows that 3 out of these 4 patients are alive.conclusion:this indicates that there may be a role of induction chemotherapy in converting potentially unresectable tumors to resectable disease that could produce better outcomes in carcinoma eac. |
both transcription factors (tfs) and micrornas (mirnas) are key players in gene regulation in multicellular organisms (1). based on pairing between mirnas and mrnas, mirna targets are predicted by searching for matches with the mirna seed regions (2). on the other hand, the use of a position weight matrix (pwm) is the leading model for detection of tf binding sites (tfbss). a pwm represents the sequence motif and depicts the dna binding preferences of the tf. traditionally, regulation of genes by tfs is predicted by analyzing promoter regions and determined experimentally by dnase - foot - printing assays or electrophoretic mobility shift assays (emsa). nowadays, functional protein dna binding sites are increasingly studied on a genomic scale by using chip - seq. these studies indicate that only some of the functional tfbs are located in promoter regions ; introns and untranslated regions (utrs) also contain a substantial number of functional sites (35). for example, regulatory sites in the first intron might interact with sites in the promoter region due to dna looping (6,7). of the estimated 2000 human tfs, 300 are thought to bind to the core promoter and to play a role in the general transcription machinery, whereas the rest bind more specifically and regulate a fraction of genes (8). the latter tfs are expressed in almost all tissues or only in a few tissues, depending on whether their function is broad or more specific. over half of the human genes are believed to have alternative promoters (9) and consequently one should investigate the promoters, utrs and intronic regions of each individual transcript. in this update, we describe the new features and expansions of the contra webserver. in this tool, for any genomic region tf binding sites can be detected and visualized of the known transcripts of a gene of interest. starting from one of nine reference organisms, a scientist can easily investigate regulation at the transcription level using the latest ucsc multiz alignments, which are accessible through the contra interface. alternatively, sequence files and pwms can be uploaded for analysis of the user 's own data. similar web tools with their pros and cons compared to contra v2 are listed in supplementary table s1. the first version of contra provided users with a flexible way to analyze promoter alignments (10). users were able to visualize or explore tfbss in the promoter region of a gene of interest. pwm libraries from the jaspar core database and transfac database were used to identify tfbss in a multi - species alignment with human as reference species. even though the human genome is one of the most widely used reference genomes, the lack of other reference species and alignments was regarded as one of the most important shortcomings in the first version of contra. in addition to the promoter region, users can now look for tfbss in 5-utr, 3-utr and introns. evidence is rising that these regions are at least as important in transcriptional regulation as the promoter region itself (35,11). (3) demonstrated that many (3540%) of the tcf4 binding sites are intronic. furthermore, considerable fractions of znf-263-, ctcf-, nrsf- and stat1 binding sites are located in 5-utr, 3-utr and intronic regions. a detailed overview of the relative importance of the aforementioned genomic regions is given in supplementary table s2. in the first edition of contra, searching for tfbss was only possible in multiple alignments in relation to the human genome, which left many users empty handed. in contra v2, multiple alignments with mouse, chicken, cow, frog, zebrafish, fruitfly, worm and yeast as reference species have been added. a detailed overview of the different genome assemblies, genes and multiz alignments available in contra v2 is presented in table 1. although the human genome is the most widely studied genome, other model organisms should not be ignored. the importance of the different model organisms is illustrated in supplementary figure s1, in which the popularity of the different organisms is compared in terms of pubmed hits. table 1.summary of the number of genes, non - coding genes and transcripts for each reference organism that can be analyzed in contra v2reference speciescommon nameassemblygenesrefseq transcriptscoding (nm _) (%) non - coding (nr _) (%) ensembl transcriptsmultiple sequence alignmenthomo sapienshumanhg1922 16737 47486.313.7151 222multiz46way of 46 vertebrate genomes (hg19)mus musculusmousemm921 78627 62193.36.788 186multiz30way of 30 vertebrate genomesbos tauruscowbostau411 55912 42797.72.331 598multiz5way : cow, dog, human, mouse, platypusgallus galluschickengalgal34905517690.19.923 392multiz7way : chicken, human, mouse, rat, opossum, frog, zebrafishxenopus tropicalisfrogxentro28358969599.80.228 937multiz7way : frog, chicken, opossum, human, mouse, rat, zebrafishdanio reriozebrafishdanrer613 81215 77695.64.432 992multiz6way : zebrafish, tetraodon, stickleback, frog, mouse, humandrosophila melanogasterfruit flydm314 23023 55094.15.923 017multiz15way of 15 insectscaenorhabditis eleganswormce619 90324 89297.12.935 019multiz6way of 6 wormssaccharomyces cerevisiaeyeastsaccer27130nanana7130multiz7way of 7 yeast speciesfor each species, a specific ucsc multiz alignment is used. summary of the number of genes, non - coding genes and transcripts for each reference organism that can be analyzed in contra v2 for each species, a specific ucsc multiz alignment is used. in contra v2, transcripts can be searched for using the official hgnc gene name, hgnc symbol, alias, ensembl gene i d (ensg), the entrez gene i d, the refseq mrna i d (nm_/nr _) or the ensembl transcript i d (enst). for every species, the most recent alignments are then automatically fetched from ucsc and processed. users can select binding motifs from different sources, including the latest versions of the transfac database (update 2010.4) (12), the jaspar core database update 2010 (13), the phylofacts database (14) and a collection of homeodomain tf pwms derived from a protein binding microarray (pbm) (15). creating a custom pwm is as easy as uploading a fasta file containing aligned sequences. the contra v2 web interface automatically converts the data into the right format. in contra v2, tfs and mirnas often work together in what is termed a feed - forward loop (ffl). these ffls regulate many important biological processes, such as those in development and tumor formation (16). non - coding transcripts are treated as regular transcripts in contra, and they can be analyzed in the same way. to verify whether the results on non - coding genes are meaningful, we looked for binding sites in the promoter region of mirna-223 (hsa - mir-223 or mir223) with refseq accession number nr_029637. (17) have shown that mir233 is regulated by a wide range of tfs, such as nfat, c / ebp, gata1 and pu.1. analysis in contra v2 not only supports the presence of the binding sites for these tfs but also shows that they have been strongly conserved during evolution (figure 1). figure 1.visualization of the evolutionarily conserved mechanism for mirna-223 regulation in the promoter region, as described by fukao. the c / ebp tf, predicted using the jaspar positional weight matrix ma0102.2 ; in blue, the nfat tf (transfac m00935) ; in green ; the gata1 tf (jaspar ma0035.2) ; and in pink, the pu.1 tf (jaspar ma0080.2). the figure was created with the free multiple alignment editor jalview using the contra fasta and fc file on the results page. (b) region of (a) was mapped using blat on the mir-223 promoter in the ucsc genome browser (black box). visualization of the evolutionarily conserved mechanism for mirna-223 regulation in the promoter region, as described by fukao. the c / ebp tf, predicted using the jaspar positional weight matrix ma0102.2 ; in blue, the nfat tf (transfac m00935) ; in green ; the gata1 tf (jaspar ma0035.2) ; and in pink, the pu.1 tf (jaspar ma0080.2). the figure was created with the free multiple alignment editor jalview using the contra fasta and fc file on the results page. (b) region of (a) was mapped using blat on the mir-223 promoter in the ucsc genome browser (black box).. a wide variety of examples on the use of contra v2 can be found in online supplementary data. supplementary figures s2s6 show results of contra v2 analyses on different genomic regions, using the ucsc multiz46way alignment based on the human hg19 reference sequence and illustrating experimentally validated binding sites from literature. supplementary figure s7 depicts an evolutionarily conserved binding site in the second intron of the mus musculus nestin gene, as described by jin. (18). in supplementary figure s8, two sine oculis (so) binding sites are conserved in the second intron of the drosophila lz gene, which confirms the study of yan. finally, the promoter of the s. cerevisiae phd1 (flo11) gene in supplementary figure s9 shows two conserved tea tfbss, which supports the regulatory mechanism proposed by heise. if the genomic region of interest, for example, from another reference organism or for a new transcript, is not available in contra, alignment files in either the ucsc multiple alignment format (maf), in multi - fasta format or in clustal format can be uploaded. on the help page of the web site are demos showing how to obtain such a maf file in the ucsc genome browser, how to upload and analyze this file, and how to use the feature color (fc) file and fasta file on the result page to produce publication - quality figures similar to those in the online supplementary data of this article. if a pwm model for a particular tf is not present in the available collections, uploading one 's own pwm is also possible. this can be either in the pwm format, but less experienced users can simply upload an alignment file in multi - fasta format. contra v2 runs on a centos 5 server configured with an apache web server (version 2.2.3), mysql server (5.0.77), php 5.1.6 and perl 5.8.8. the interface is programmed in php, and alignments are fetched from ucsc using perl scripts. an overview picture of these hits, created with jalview, is embedded in the overview page with the help of the highslide thumbnail viewer (http://www.highslide.com). different tfs on the result page are visualized dynamically using javascript. for each alignment block, both a file with pwm scores and a file containing a phylogenetic conservation score for each tf is provided (see file 1 in supplementary data for more details). scores in the contra v2 exploration part are calculated in the same way as in the previous version of contra, with the exception that due to the inclusion of other genomic regions, we no longer take into account the distance to the transcription start site. first, users have to choose whether they want to visualize or explore a gene of interest. in this step, it is also necessary to indicate the reference species and the gene of interest. the second step lists a group of available transcripts for genes matching the search terms, from which one can be selected. for every gene, all possible refseq and ensembl transcript variants are listed with a link to the genomic location in the respective genome browser. this way, genes with alternative promoters, utrs or alternative intronic regions can be analyzed for regulatory differences. in step three, different genomic regions of the selected transcript can be chosen (upstream, introns, 5-utr and 3-utr). the final step offers users an extensive choice of pwm motifs : up to 20 pwm motifs can be simultaneously taken into account for analysis. for the visualization part these blocks consist of local alignments produced by the tba program (threaded) (21). in the exploration part agency for innovation through science and technology in flanders (grant number 091213). funding for open access charge : department for molecular biomedical research, vib, ghent, belgium. | transcription factors are important gene regulators with distinctive roles in development, cell signaling and cell cycling, and they have been associated with many diseases. the contra v2 web server allows easy visualization and exploration of predicted transcription factor binding sites in any genomic region surrounding coding or non - coding genes. in this new version, users can choose from nine reference organisms ranging from human to yeast. contra v2 can analyze promoter regions, 5-utrs, 3-utrs and introns or any other genomic region of interest. hundreds of position weight matrices are available to choose from, but the user can also upload any other matrices for detecting specific binding sites. a typical analysis is run in four simple steps of choosing the gene, the transcript, the region of interest and then selecting one or more transcription factor binding sites. the contra v2 web server is freely available at http://bioit.dmbr.ugent.be/contrav2/index.php. |
denture stomatitis is an erythematous pathogenic condition of denture bearing mucosa, caused mainly by microbial factors, especially candida albicans.1 its prevalence has been reported at 11 - 67% in complete denture wearers.2 the main reservoir of c. albicans and related candida species has been shown to be the tissue surface of maxillary complete dentures.1,3 recently, it has been pointed out that continuous swallowing or aspiration of microorganisms from denture plaque can cause unexpected infections to immunocompromised host or medicated old person.4 two methods have been proposed for routine denture biofilm removal including mechanical and chemical cleansing.5 the chemical method is considered to be the most effective for inhibiting c. albicans infection and denture biofilm formation.5,6,7 however, the routine use of denture cleansers has been known to cause adverse effects on physical characteristics of denture materials and resilient liners. goll.8 reported that resilient liners increased discoloration, porosity, surface and size changes, and solubility by the use of denture cleanser for 30 days, harrison.7 reported that resilient liners were damaged by alkaline peroxide type of denture cleanser, and nikawa.9 reported that peroxide type of denture cleanser caused damage to resilient liners, and was not related to the quantity. there have been numerous studies to investigate compatibility between resilient liners and denture cleansers, primarily focusing on changes of physical properties such as surface roughness, viscoelastic properties, and color.7,8,9 however, there is not much information available about c. albicans biofilm formation on resilient liners which are immersed in denture cleansers within a given period. therefore, this study was aimed to analyze the effect of denture cleansers on c. albicans biofilm formation over resilient liners and to evaluate compatibility between resilient liners and denture cleansers. the null hypothesis was that there would be no significant differences in the degree of ra, ph and the binding level of c. albicans among groups with immersing in different denture cleansers. three commercially available resilient liners were investigated, acrylic resin using as control group (table 1). each specimen was processed according to the manufacturer 's directions and prepared to be a uniform size (14 mm diameter 1 mm thickness) with a smooth surface by placing glass slides. two commercial denture cleansers were studied using distilled water (dw) as control solution (table 2). candida albicans atcc 10231 was purchased from american type culture collection and used in this study. c. albicans was cultured using trypicase soy broth (tsb ; beckton dickinson, sparks, md, usa) at 37 in aerobic condition. to investigate adhesion assay, the yeast was incubated with specimens in tsb aerobically at 37 for 18 hours. unstimulated whole saliva (uws) was collected by spitting method from non - smoking healthy persons in glass bottle on ice. for saliva collection, the institutional review board at korea university guro hospital approved this study protocol (md11006). saliva was centrifugated at 6,500 g for 5 min at 4, and then the supernatant was diluted to a ratio of 1:1 with phosphate buffered saline and filtered by polyvinylidene difluoride (pvdf) membrane with pore size of 0.22. filtered - saliva was stored at 4. the specimens was incubated serially in denture cleanser for 8 hours at room temperature, washed three times with autoclaved pbs and in uws for 16 hours at 37. the soaking stage was repeatedly carried out every day for 60 days. fifteen of seventy - five specimens were used for the measurements of average surface roughness (ra), and the remainder was used for biofilm assay. denture cleanser - treated specimens were washed three times with distilled water and dried using airbrush until removing distilled water. the average surface roughness of specimen was measured three times with a profilometer (surtronic 3p ; taylor - hobson, leicester, uk). the cultured medium was centrifuged at 3,000 g for 10 min, and the supernatant was placed to a new tube. the ph of the cultured medium was measured at indicated times (12, 24, 36, and 48 hours). radio - labeling was performed by anaerobically incubating c. albicans in 10 ml of tsb containing 1 ci / ml methyl-[3h ] thymidine (tsbh ; amersham pharmacia biotech, nj, usa) for 18 hours at 37. in order to assay adhesion, 5 ml of the culture suspension was inoculated in 500 ml of tsbh, and then the tsbh containing the isotope - labeled c. albicans was dispensed into 85 mm - diameter dish. after 15 samples of specimen were placed on 85 mm - diameter dish, 20 ml of tsbh which contained radiolabeled c. albicans was added. the dish was then incubated for 48 hours at 37, and the samples were washed three times with phosphate buffered saline for removing unbound c. albicans. in order to detach c. albicans, the dish was vortexed in 4 ml of lysis buffer [0.5 m tris (ph 9.0), 10 mm nacl, 20 mm edta, 1% sds ], and 3 ml of each the suspension was mixed with 3 ml of aqueous cocktail solution (packard, ct, usa), and the radioactivity was counted with -counter (bd, nj, usa). the statistical significances were evaluated by two - way or three - way anova, and mann - whitney test. the statistical significance of the relation between ra and adhesion was evaluated by correlation analysis. the degree of ra was decreased in the following order : coe - soft, acr ylic resin, gc reline and sofreliner tough (p.05). changes with time the binding levels of c. albicans were decreased in the following order : coe - soft, gc reline, sofreliner tough, and acrylic resin (p<.05, table 4, fig. 3). biofilm formation on acrylic resin was greater when immersed in denture cleansers than in d.w. also, biofilm formation on coe - soft was significantly increased in denture cleansers than in d.w.. interestingly, however, biofilm formation on polysiloxanes was significantly increased in polident, however, decreased in cleadent. the statistical significance of relationship between ra and biofilm formation of c. albicans was evaluated with correlation analysis, however, there was only a very low interrelationship (r=0.184 ; pearson correlation coefficients). this study aimed to analyze the effect of denture cleansers on c. albicans biofilm formation over resilient liners by evaluating ra, ph change, and c. albicans binding level. there are several reports to suggest the relationship between surface roughness and c. albicans adherence to denture materials.10,11,12 verran.10 reported that significantly higher number of c. albicans was observed on roughened than on smooth surfaces, and radford.12 reported significantly greater adhesion of c. albicans to rough than smooth surfaces. if repetitive immersion in denture cleanser roughens the surface of resilient liners, c. albicans adhesion would be expected to increase. therefore, we evaluated ra in this study, and presented the average surface roughness (ra) of soft relining materials immersed in denture cleansers is shown in table 1. pmma (coe - soft) showed the greatest, whereas polysiloxanes the least ra. the plasticizer component of pmma (for example, dibutylphthalate) was leached out in the thermocycling process more than polysiloxane, therefore, ra of pmma may have been increased. this may be due to deterioration of pmma components by denture cleansers during the thermocycling process, regardless of capacity of denture cleanser to remove c. albicans biofilm. the major pathology of denture stomatitis is the growth and acid production of c. albicans on denture fitting surface.13 acid production of c. albicans induces cytotoxin. furthermore, c. albicans activates acid proteinase and phospholipase, and aggregates candida.14 nikawa.15 reported that resilient liners inhibits c. albicans growth, colonization and biofilm formation. the inhibitory effects of resilient liners on fungal growth were observed in the following three manners : delay in the beginning of rapid decline in ph, decreases in the rate of ph change and increases in minimum ph.16,17 an expected, therefore, we observed changes of medium ph by resilient liners when immersed in denture cleanser. the ph changes in culture medium varied according to resilient liners and denture cleansers, however statistically significant difference was not observed between groups immersed in d.w and denture cleansers for 60 days (fig. goll.8 reported that the physical characteristics of soft relining materials are deteriorated by using denture cleanser for 30 days, and harrison.7 and nikawa.9 showed that peroxide type of denture cleansers damages soft relining materials. moreover, nikawa.13 reported that mismatched use of denture liners and denture cleanser increases biofilm formation. in the present study, however, daily use of denture cleansers did not always aggravate candida biofilm formation on resilient liners. although biofilm formation of c. albicans on acrylic resin and pmma (coe - soft) was increased by using denture cleansers, it was decreased on polysiloxanes (gc - reline and sofreliner tough) by using cleadent (fig. the biofilm formation level of c. albicans on pmma was the highest when two denture cleansers were used : polident and cleadent. further researches are needed to investigate the biofilm formation level of c. albicans on other pmma - based resilient liner. in polysiloxane groups, cleadent was more effective to remove biofilm of c. albicans than polident. polident consisted of sodium perborate, oxone and everase while cleadent consisted of oxybleach, proteinase, flabonoid, anion detergent, carbonate, and organic acid. in this study, we evaluated the relationship between biofilm formation and ra, and the result showed low correlation (r=0.184), thus implying that ra of each specimen is not the essential factor to form biofilm of c. albicans. c. albicans biofilm formation was found to increase on polysiloxane groups when polident was used, and to decrease by using cleadent. as there is little overlap among ingredients which manufacturers indicated, it is not possible at present to find out which ingredient of denture cleansers caused this opposite result. additional study is needed to find out which ingredient of cleansers has adverse effects on denture liners. based on the c. albicans binding levels results, it is not recommended to immerse coe - soft in denture cleansers, and gc reline and sofreliner tough should be immersed in cleadent. | purposethe purpose of this study was to analyze the effect of denture cleansers on candida albicans biofilm formation over resilient liners and to evaluate compatibility between resilient liners and denture cleansers.materials and methodsacrylic resin (lucitone 199) and 3 resilient liners (coe - soft, gc reline and sofreliner tough tough) were incubated in denture cleansers (polident and cleadent) for 8 hours a day and in unstimulated saliva for 16 hours a day (n=25/gp) for 60 days. two - way and three - way repeated measures anova were performed to compare the surface roughness (ra), ph and c. albicans binding level by radioisotope (=0.05). the statistical significance of the relation between ra and adhesion was evaluated by correlation analysis.resultsthe degree of ra was significantly decreased in the following order : coe - soft, acrylic resin, gc reline and sofreliner tough. the immersion in denture cleansers significantly increased ra of resilient liners, except for sofreliner tough in cleadent. no significant differences in ph curves were observed among groups immersed in distilled water and denture cleansers. the binding levels of c. albicans were significantly decreased in the following order : coe - soft, gc reline, sofreliner tough, and acrylic resin. the immersion in cleadent seemed to decrease c. albicans binding level on gc reline and sofreliner tough.conclusionbased on the c. albicans binding levels results, it is not recommended to immerse coe - soft in denture cleansers, and gc reline and sofreliner tough should be immersed in cleadent. |
subjects with metabolic syndrome (national cholesterol education program criteria) participated in a double - blind, randomized, and placebo - controlled crossover study. each subject was studied four times (see supplementary fig. participants were then randomized to one of four 3-week treatments (placebo plus placebo, ramipril [10 mg / day ] plus placebo, tadalafil [10 mg o.d. ] plus placebo, and ramipril plus tadalafil) separated by a 1-week washout period. during the last week of treatment, subjects ate a nitrate-, sodium-, and calorie - controlled diet. on the last day, they collected a 24-h urine sample and fasted overnight. at 0730 h, supine blood pressure and heart rate were measured thrice, 2 min apart. blood was drawn via venous catheter for plasma renin activity (pra), ace activity, angiotensin ii, aldosterone, fibrinolytic parameters, no metabolites, l - citrulline, l - arginine, and cgmp. at 0800 h, subjects underwent a frequently sampled intravenous glucose tolerance test (additional information available in the online appendix). insulin sensitivity index, glucose effectiveness index, homeostasis model assessment of insulin resistance, and -cell function were calculated using a modified version of minimal model (minmod) formulas. acute insulin response to glucose (airg) was assessed from the area under the insulin curve for the first 10 min following dextrose infusion. because airg disregards changes in insulin sensitivity, we used disposition index, calculated from insulin sensitivity and airg, as a more reliable indicator of -cell function (2). ramipril significantly increased pra and decreased ace activity and angiotensin ii (supplementary table a3). tadalafil did not affect the renin - angiotensin - aldosterone system or alter effects of ramipril. ramipril reduced systolic (p = 0.01) (fig. 1) and diastolic blood pressure (p < 0.001). tadalafil did not affect blood pressure but tended to enhance the ramipril effect on diastolic blood pressure (p = 0.06 for interaction, controlling for sex and race). effect of treatment on systolic blood pressure (bp), diastolic blood pressure, insulin sensitivity, and -cell function. 1). no treatments altered glucose effectiveness, a measure of insulin - independent glucose disposal (0.017 0.006, 0.017 0.008, 0.017 0.009, and 0.015 0.007 min for placebo, tadalafil, ramipril, and ramipril plus tadalafil, respectively). in contrast, tadalafil significantly improved -cell function after controlling for sex (p = 0.01) (fig. 1) or baseline fasting glucose (p = 0.05). in a subgroup analysis, tadalafil improved -cell function in women (154.4 48.0, 331.9 209.3, 229.1 202.1, and 259.7 95.8 mmol l during placebo, tadalafil, ramipril, and ramipril plus tadalafil treatment, respectively ; p = 0.01 for tadalafil effect) but not in men. there was a trend toward improved -cell function during tadalafil treatment in individuals with baseline fasting hyperglycemia (195.3 103.1, 278.7 114.0, 157.2 52.6, and 210.6 72.3 mmol l during placebo, tadalafil, ramipril, and ramipril plus tadalafil treatment, respectively ; p = 0.06 for tadalafil) but not in subjects with normal fasting glucose. there was no effect of race and no interactive effect of ramipril or tadalafil on -cell function. ramipril (p = 0.02) and tadalafil (p = 0.02) improved the disposition index in women but not in men after controlling for fasting glucose. this was attributable to a synergistic effect of ramipril and tadalafil on the disposition index (1,001.8 909.5, 977.8 728.5, 1,308.8 976.2, and 1,982.2 1,982.2 units during placebo, tadalafil, ramipril, and ramipril plus tadalafil, respectively ; p = 0.05 for ramipril plus tadalafil). ramipril significantly increased pra and decreased ace activity and angiotensin ii (supplementary table a3). tadalafil did not affect the renin - angiotensin - aldosterone system or alter effects of ramipril. ramipril reduced systolic (p = 0.01) (fig. 1) and diastolic blood pressure (p < 0.001). tadalafil did not affect blood pressure but tended to enhance the ramipril effect on diastolic blood pressure (p = 0.06 for interaction, controlling for sex and race). effect of treatment on systolic blood pressure (bp), diastolic blood pressure, insulin sensitivity, and -cell function. 1). no treatments altered glucose effectiveness, a measure of insulin - independent glucose disposal (0.017 0.006, 0.017 0.008, 0.017 0.009, and 0.015 0.007 min for placebo, tadalafil, ramipril, and ramipril plus tadalafil, respectively). in contrast, tadalafil significantly improved -cell function after controlling for sex (p = 0.01) (fig. 1) or baseline fasting glucose (p = 0.05). in a subgroup analysis, tadalafil improved -cell function in women (154.4 48.0, 331.9 209.3, 229.1 202.1, and 259.7 95.8 mmol l during placebo, tadalafil, ramipril, and ramipril plus tadalafil treatment, respectively ; p = 0.01 for tadalafil effect) but not in men. there was a trend toward improved -cell function during tadalafil treatment in individuals with baseline fasting hyperglycemia (195.3 103.1, 278.7 114.0, 157.2 52.6, and 210.6 72.3 mmol l during placebo, tadalafil, ramipril, and ramipril plus tadalafil treatment, respectively ; p = 0.06 for tadalafil) but not in subjects with normal fasting glucose. there was no effect of race and no interactive effect of ramipril or tadalafil on -cell function. ramipril (p = 0.02) and tadalafil (p = 0.02) improved the disposition index in women but not in men after controlling for fasting glucose. this was attributable to a synergistic effect of ramipril and tadalafil on the disposition index (1,001.8 909.5, 977.8 728.5, 1,308.8 976.2, and 1,982.2 1,982.2 units during placebo, tadalafil, ramipril, and ramipril plus tadalafil, respectively ; p = 0.05 for ramipril plus tadalafil). the phosphodiesterase 5 inhibitor tadalafil, alone or in combination with ramipril, improved basal and glucose - stimulated -cell function. the latter effect was independent of insulin sensitivity, as indicated by improvement in the disposition index (2). metabolic syndrome, an insulin - resistant state, frequently progresses to type 2 diabetes. loss of -cell function and impaired insulin sensitivity both contribute to the development of diabetes (2). -cell dysfunction may play a greater role than previously appreciated in that pancreatic -cell apoptosis precedes overt diabetes in high - risk individuals (10) and surgical reduction of pancreatic mass causes impaired glucose tolerance and diabetes (11). to our knowledge, no prior human or animal studies have reported an effect of phosphodiesterase 5 inhibition on -cell function. previous studies provide conflicting data, however, regarding the effect of no on -cell function, with some suggesting that no suppresses insulin secretion (13,14) and others indicating that no enhances insulin secretion (12,15). a higher frequency of fasting hyperglycemia among women with metabolic syndrome may have confounded this sex difference. alternatively, women may be more sensitive than men to decreased cgmp degradation. in support of this possibility, three of six women studied, but no men, reported muscle aches during tadalafil treatment. although aceis and arbs improve glucose uptake and/or insulin secretion in vitro and in rodents, studies in humans provide mixed data regarding their effects on insulin resistance (4). we did not detect an effect of ramipril on insulin sensitivity or -cell function but did detect an effect of ramipril on disposition index in women. studies are needed to determine whether the effect of tadalafil is limited to women or related to the magnitude of hyperglycemia. given the increasing role attributed to -cell dysfunction in the pathogenesis of type 2 diabetes, these data suggest a novel therapeutic intervention in a high - risk population. | objectivethis study tested the hypothesis that phosphodiesterase 5 inhibition alone or in combination with ace inhibition improves glucose homeostasis and fibrinolysis in individuals with metabolic syndrome.research design and methodsinsulin sensitivity, -cell function, and fibrinolytic parameters were measured in 18 adults with metabolic syndrome on 4 separate days after a randomized, crossover, double - blind, 3-week treatment with placebo, ramipril (10 mg / day), tadalafil (10 mg o.d.), and ramipril plus tadalafil.resultsramipril decreased systolic and diastolic blood pressure, ace activity, and angiotensin ii and increased plasma renin activity. ramipril did not affect insulin sensitivity or -cell function. in contrast, tadalafil improved -cell function (p = 0.01). this effect was observed in women (331.9 209.3 vs. 154.4 48.0 32 mmol1 l1, respectively, for tadalafil treatment vs. placebo ; p = 0.01) but not in men. there was no effect of any treatment on fibrinolysis.conclusionsphosphodiesterase 5 inhibition may represent a novel strategy for improving -cell function in metabolic syndrome. |
all participants were relatives of patients with type 1 diabetes. there were 97,273 serum samples collected and tested for ica at the initial screening. (14,15), eligibility for the trials was further assessed on the basis of metabolic abnormalities (parenteral insulin trial) and the presence of iaa (oral insulin trial). of the screening samples, 84% were later tested for the presence of gad65, ica512, and iaa measured by the micro method (miaa). those who were ica did not meet the criteria for trial entry or chose not to enter the trials. those who had all autoantibody determinations and sufficiently complete data were included in the analyses (n = 29,035). participants were asked whether they were informed by a physician that they had developed type 1 diabetes. the follow - up interval was the time between the date of the response to the questionnaire and the date of the initial screen for autoantibodies (those who did not develop type 1 diabetes) or between the date of diagnosis as indicated on the questionnaire and the date of the initial screen (those who developed type 1 diabetes). the mean sd age of the individuals in the questionnaire cohort (n = 28,507) was 17.9 13.0 years (55% female). the procedures for the dpt-1 trials have been described elsewhere (14,15). in both the parenteral and oral insulin trials, blood samples were obtained for plasma glucose and c - peptide measurements in the fasting state and at 30, 60, 90, and 120 min. those with glucose values in the diabetic range (fasting glucose 126 mg / dl and/or 2-h glucose 200 mg / dl) were asked to return for confirmation at a follow - up visit. the follow - up interval was the time between the date of last contact and the date of the first screen (those who did not develop type 1 diabetes) or the time between the date of diagnosis and the date of the first screen (those who developed type 1 diabetes). in 61% of those with type 1 diabetes in the dpt-1 trials, the trials cohort was significantly younger and had a lower proportion of female participants than the questionnaire cohort (p < 0.001 for both). ica values were determined by an immunofluorescence assay on frozen sections of blood type o human pancreas in the dpt-1 ica core laboratory (gainesville, fl, february 1994september 1997 and january 1999october 2003 ; new orleans, la, september 1997january 1999). ica values of 10 juvenile diabetes foundation (jdf) units were considered positive. in the 1995 immunology of diabetes society workshop (18), this ica assay had a specificity of 100% and a sensitivity of 74.4% for patients with new - onset type 1 diabetes who were aged < 30 years. based on a receiver operating characteristic curve, in this dataset with a positive jdf value of 10, the assay sensitivity was 75.0% with a 95.7% specificity (no age influence on the values). autoantibodies against gad65 and ica512bdc (ica512) were determined at the barbara davis center (denver, co). iaa (using the micro - volume requiring assay) values were determined at the barbara davis center or the joslin diabetes center (boston, ma). as described previously, a combined gad65 and ica512 radioassay was performed (19). labeled recombinant gad65 and ica512 autoantibodies were produced by in vitro transcription / translation with differential labeling ([h]gad65 and [s]ica512) (8,13). the upper limits of normal (0.032 for gad65 and 0.049 for ica512) were established as the 99th percentile for gad65 and for ica512 from receiver operating characteristic curves in 198 healthy control subjects and 50 patients with new - onset diabetes. in this dataset, a gad65 index of 0.032 (used for the analysis) provided a 41.8% sensitivity and a 98.3% specificity (no difference by age - group), and for ica512 an index of 0.049 (used for the analysis) resulted in a sensitivity of 57.5% and specificity of 98.5% (no difference by age - group). in the 2000, 2002, and the 2003 diabetes antibody standardization program (dasp) for proficiency testing, the sensitivity / specificity results for the gad65 assay were 84%/96%, 90%/93%, and 84%/98% and for the ica512 assay were 52%/100%, 62%/99%, and 58%/100%, respectively. the interassay coefficients of variation for ica512 and gad65 were 8 and 10%, respectively. the miaa assay (20) was performed as described and expressed as an index with the upper limits of 0.02 and 0.01 (boston and denver laboratories, respectively) based on the 99th percentile of healthy control values. in the combined dataset, determination of miaa on samples began later than for gad65 and ica512 (assay development needed). in the 2003 dasp proficiency testing, the sensitivity for the miaa assay was 74 and 56% and the specificity was 90 and 98%, respectively, for the denver and boston laboratories. the correlation coefficient between both laboratories was r = 0.90 (p < 0.0001). the log - rank test was used to compare the distributions of event times between groups. cox proportional hazards regression models were used to examine effects on type 1 diabetes risk over time. the kaplan - meier estimate of the survival function was used to obtain estimates of type 1 diabetes occurrence. participants were asked whether they were informed by a physician that they had developed type 1 diabetes. the follow - up interval was the time between the date of the response to the questionnaire and the date of the initial screen for autoantibodies (those who did not develop type 1 diabetes) or between the date of diagnosis as indicated on the questionnaire and the date of the initial screen (those who developed type 1 diabetes). the mean sd age of the individuals in the questionnaire cohort (n = 28,507) was 17.9 13.0 years (55% female). the procedures for the dpt-1 trials have been described elsewhere (14,15). in both the parenteral and oral insulin trials, blood samples were obtained for plasma glucose and c - peptide measurements in the fasting state and at 30, 60, 90, and 120 min. those with glucose values in the diabetic range (fasting glucose 126 mg / dl and/or 2-h glucose 200 mg / dl) were asked to return for confirmation at a follow - up visit. the follow - up interval was the time between the date of last contact and the date of the first screen (those who did not develop type 1 diabetes) or the time between the date of diagnosis and the date of the first screen (those who developed type 1 diabetes). in 61% of those with type 1 diabetes in the dpt-1 trials, the trials cohort was significantly younger and had a lower proportion of female participants than the questionnaire cohort (p < 0.001 for both). participants were asked whether they were informed by a physician that they had developed type 1 diabetes. the follow - up interval was the time between the date of the response to the questionnaire and the date of the initial screen for autoantibodies (those who did not develop type 1 diabetes) or between the date of diagnosis as indicated on the questionnaire and the date of the initial screen (those who developed type 1 diabetes). the mean sd age of the individuals in the questionnaire cohort (n = 28,507) was 17.9 13.0 years (55% female). the procedures for the dpt-1 trials have been described elsewhere (14,15). in both the parenteral and oral insulin trials, blood samples were obtained for plasma glucose and c - peptide measurements in the fasting state and at 30, 60, 90, and 120 min. those with glucose values in the diabetic range (fasting glucose 126 mg / dl and/or 2-h glucose 200 mg / dl) were asked to return for confirmation at a follow - up visit. the follow - up interval was the time between the date of last contact and the date of the first screen (those who did not develop type 1 diabetes) or the time between the date of diagnosis and the date of the first screen (those who developed type 1 diabetes). in 61% of those with type 1 diabetes in the dpt-1 trials, the trials cohort was significantly younger and had a lower proportion of female participants than the questionnaire cohort (p < 0.001 for both). ica values were determined by an immunofluorescence assay on frozen sections of blood type o human pancreas in the dpt-1 ica core laboratory (gainesville, fl, february 1994september 1997 and january 1999october 2003 ; new orleans, la, september 1997january 1999). ica values of 10 juvenile diabetes foundation (jdf) units were considered positive. in the 1995 immunology of diabetes society workshop (18), this ica assay had a specificity of 100% and a sensitivity of 74.4% for patients with new - onset type 1 diabetes who were aged < 30 years. based on a receiver operating characteristic curve, in this dataset with a positive jdf value of 10, the assay sensitivity was 75.0% with a 95.7% specificity (no age influence on the values). autoantibodies against gad65 and ica512bdc (ica512) were determined at the barbara davis center (denver, co). iaa (using the micro - volume requiring assay) values were determined at the barbara davis center or the joslin diabetes center (boston, ma). as described previously, a combined gad65 and ica512 radioassay was performed (19). labeled recombinant gad65 and ica512 autoantibodies were produced by in vitro transcription / translation with differential labeling ([h]gad65 and [s]ica512) (8,13). the upper limits of normal (0.032 for gad65 and 0.049 for ica512) were established as the 99th percentile for gad65 and for ica512 from receiver operating characteristic curves in 198 healthy control subjects and 50 patients with new - onset diabetes. in this dataset, a gad65 index of 0.032 (used for the analysis) provided a 41.8% sensitivity and a 98.3% specificity (no difference by age - group), and for ica512 an index of 0.049 (used for the analysis) resulted in a sensitivity of 57.5% and specificity of 98.5% (no difference by age - group). in the 2000, 2002, and the 2003 diabetes antibody standardization program (dasp) for proficiency testing, the sensitivity / specificity results for the gad65 assay were 84%/96%, 90%/93%, and 84%/98% and for the ica512 assay were 52%/100%, 62%/99%, and 58%/100%, respectively. the interassay coefficients of variation for ica512 and gad65 were 8 and 10%, respectively. the miaa assay (20) was performed as described and expressed as an index with the upper limits of 0.02 and 0.01 (boston and denver laboratories, respectively) based on the 99th percentile of healthy control values. in the combined dataset, determination of miaa on samples began later than for gad65 and ica512 (assay development needed). in the 2003 dasp proficiency testing, the sensitivity for the miaa assay was 74 and 56% and the specificity was 90 and 98%, respectively, for the denver and boston laboratories. the correlation coefficient between both laboratories was r = 0.90 (p < 0.0001). ica values were determined by an immunofluorescence assay on frozen sections of blood type o human pancreas in the dpt-1 ica core laboratory (gainesville, fl, february 1994september 1997 and january 1999october 2003 ; new orleans, la, september 1997january 1999). ica values of 10 juvenile diabetes foundation (jdf) units were considered positive. in the 1995 immunology of diabetes society workshop (18), this ica assay had a specificity of 100% and a sensitivity of 74.4% for patients with new - onset type 1 diabetes who were aged < 30 years. based on a receiver operating characteristic curve, in this dataset with a positive jdf value of 10, the assay sensitivity was 75.0% with a 95.7% specificity (no age influence on the values). autoantibodies against gad65 and ica512bdc (ica512) were determined at the barbara davis center (denver, co). iaa (using the micro - volume requiring assay) values were determined at the barbara davis center or the joslin diabetes center (boston, ma). as described previously, a combined gad65 and ica512 radioassay was performed (19). labeled recombinant gad65 and ica512 autoantibodies were produced by in vitro transcription / translation with differential labeling ([h]gad65 and [s]ica512) (8,13). the upper limits of normal (0.032 for gad65 and 0.049 for ica512) were established as the 99th percentile for gad65 and for ica512 from receiver operating characteristic curves in 198 healthy control subjects and 50 patients with new - onset diabetes. in this dataset, a gad65 index of 0.032 (used for the analysis) provided a 41.8% sensitivity and a 98.3% specificity (no difference by age - group), and for ica512 an index of 0.049 (used for the analysis) resulted in a sensitivity of 57.5% and specificity of 98.5% (no difference by age - group). in the 2000, 2002, and the 2003 diabetes antibody standardization program (dasp) for proficiency testing, the sensitivity / specificity results for the gad65 assay were 84%/96%, 90%/93%, and 84%/98% and for the ica512 assay were 52%/100%, 62%/99%, and 58%/100%, respectively. the interassay coefficients of variation for ica512 and gad65 were 8 and 10%, respectively. the miaa assay (20) was performed as described and expressed as an index with the upper limits of 0.02 and 0.01 (boston and denver laboratories, respectively) based on the 99th percentile of healthy control values. in the combined dataset, determination of miaa on samples began later than for gad65 and ica512 (assay development needed). in the 2003 dasp proficiency testing, the sensitivity for the miaa assay was 74 and 56% and the specificity was 90 and 98%, respectively, for the denver and boston laboratories. the correlation coefficient between both laboratories was r = 0.90 (p < 0.0001). the log - rank test was used to compare the distributions of event times between groups. cox proportional hazards regression models were used to examine effects on type 1 diabetes risk over time. the kaplan - meier estimate of the survival function was used to obtain estimates of type 1 diabetes occurrence. the prevalence of each autoantibody at the initial screening is shown in table 1 for both cohorts. autoantibody prevalence was much higher in the trials cohort, which is attributable to the selection for ica positivity and for the additional trial entry criteria. although some of those in the trials cohort were ica at the initial screening, prevalence of positive autoantibodies at initial screening in the questionnaire cohort, ica512 positivity was most commonly associated with one or more autoantibodies (65%), whereas miaa positivity was least commonly associated with other autoantibodies (22%). the percentages of gad65 and ica positivity with associated autoantibodies were 37 and 39%, respectively. the occurrence of type 1 diabetes according to the number of biochemical autoantibodies is shown separately for the questionnaire and trials cohorts and in the aggregate in fig., there tended to be an increasing occurrence of type 1 diabetes as the number of autoantibodies increased (p < 0.001 for both). curves indicate the occurrence of type 1 diabetes (t1d) over follow - up according to the number of autoantibodies present at the initial screening in the questionnaire cohort (a), in the trials cohort (b), and in the cohorts combined (c). in all three panels, there were significant trends among the groups of an increasing occurrence of type 1 diabetes with increasing autoantibody number. (the fraction in parentheses indicates the number who developed type 1 diabetes among the number in the group at baseline.) the occurrence of type 1 diabetes among those with a single autoantibody is shown in table 2 for the questionnaire cohort. none of the 407 individuals with the presence of only miaa developed type 1 diabetes. the occurrence of type 1 diabetes was similar for those with gad65 alone (4.4%), ica512 alone (4.6%), and ica alone (3.9%). in proportional hazards models, there were significant associations between the occurrence of type 1 diabetes and each of those autoantibodies occurring singly (p < 0.001 for all). when age was added as a covariate associations of type 1 diabetes occurrence with the presence of single autoantibodies in the questionnaire cohort at initial screening reference group : negative for all autoantibodies (type 1 diabetes / total = 41 of 26,651). supplemental table a1 (available at http://care.diabetesjournals.org/cgi/content/full/dc09-0934/dc1) shows the occurrence of type 1 diabetes among those who were single autoantibody the occurrence of type 1 diabetes for those with ica alone was somewhat higher than the occurrence for the other autoantibodies alone. the distribution of miaa values was examined to determine whether the lack of occurrence of type 1 diabetes in those with miaa alone could be the result of a preponderance of low titers. because miaas were measured in two laboratories (positive results : n = 123 for boston and n = 284 for denver) and the threshold was higher for an abnormal value in boston than in denver (0.02 vs. 0.01), the distributions were examined for each laboratory. among those with abnormal values, the median values for boston and denver were 0.108 and 0.024, respectively. figure 2 shows the effect of adding each autoantibody as a second autoantibody to the presence of a single autoantibody (any of the other three). when ica, ica512, or gad65 was each included as a second autoantibody, the occurrence of type 1 diabetes was significantly greater (p < 0.001 for each) than when there was single autoantibody positivity. however, with miaa as a second autoantibody there was no significant difference from the presence of one autoantibody. (there was little difference in risk increment between gad65 and ica when either was the first autoantibody and the other was added [data not shown ].) when each of the autoantibodies was present additionally as a third autoantibody (supplemental fig. a1, available in an online appendix), the risk increased appreciably with ica and ica512 (both p < 0.001) but did not increase significantly with gad65 and miaa. curves indicate the occurrence of type 1 diabetes (t1d) in the questionnaire cohort over follow - up for the presence of one autoantibody and for the additional presence of a second autoantibody at the initial screening. thus, in each panel the groups that included a specific second positive autoantibody were compared with the group that had one positive autoantibody from any of the others. the panels show that when ica (a), gad65 (b), or ica512 (c) each was present as a second autoantibody, there were significant and similar increases in the occurrence of type 1 diabetes ; however, there was no increase when miaa was present as a second autoantibody (d). the number shown for each curve (1, 2) indicates the number of autoantibodies. (the fraction in parentheses indicates the number who developed type 1 diabetes among the number in the group at baseline.) the trials cohort was used to assess the influence of a second autoantibody besides ica. there was an increase in the percentage of those developing type 1 diabetes when gad65 and ica512 each was present besides ica (ica alone : 13 of 65 [20% ], ica with gad65 : 30 of 87 [34% ], and ica with ica512 : 11 of 22 [50% ]). however, when age was included as a covariate in proportional hazards models, neither the additional presence of gad65 nor that of ica512 was significant. the numbers for the additional presence of miaa as a second autoantibody were too small for a meaningful analysis ; however, 3 of 6 (50%) developed type 1 diabetes. associations of autoantibody titers were examined in the combined cohorts. of those who did not develop type 1 diabetes (n = 28,652), ica512 and gad65 titers were much more strongly correlated (r = 0.31) than the titers of any other autoantibody pair (r ranged from 0.03 to 0.13). however, among those who developed type 1 diabetes (n = 383), although the ica512-gad65 correlation remained similar (r = 0.30), the correlations of other autoantibody pairs tended to increase, especially when the pair included an ica titer (with ica titer : r = 0.39 for gad65 titer, r = 0.51 for ica512 titer, and r = 0.34 for miaa titer). in this large dataset, the association between the development of type 1 diabetes and titer (log - transformed) was examined among those who were positive for single autoantibodies in the combined cohorts. because of the lack of cases of type 1 diabetes for those with miaa positivity alone, the analysis was not performed for that autoantibody. (type 1 diabetes / total = 27 of 582) and ica titer (29 of 472) were each predictive of type 1 diabetes (p < 0.01 for both, with and without age as a covariate). there was borderline significance (p = 0.04 and p = 0.07 with age added) for the ica512 titer (6 of 113), but the number for that analysis was small. the analyses presented above were designed specifically to discern the extent to which positivity for a single autoantibody predicts the occurrence of type 1 diabetes. they showed that among those in the questionnaire cohort, ica, ica512, and gad65, as single positive autoantibodies, were similarly predictive of type 1 diabetes. in addition, each of those autoantibodies appeared to add significant increments of risk when they were included as second autoantibodies in the questionnaire cohort. however, type 1 diabetes was not associated with miaa as a single autoantibody, and the inclusion of miaa as a second or a third autoantibody appeared to have little effect. the addition of single biochemical autoantibodies to ica in the trials cohort did not significantly increase type 1 diabetes risk with age included as a covariate. this finding could be related to the selection criteria for that cohort and to the small numbers. the findings from this study are better understood by considering them in the context of the characteristics of each cohort. the vast majority of those in the questionnaire cohort were ica at the initial screening, whereas those in the trials cohort were mostly ica at the initial screening, and eventually all became positive before randomization. moreover, the trials cohort was selected for additional characteristics, including metabolic impairment and iaa positivity. these factors, together with glucose tolerance test surveillance in that cohort, could explain the relatively stronger association of type 1 diabetes with single positivity of ica when the cohorts were combined. the finding that autoantibody number predicts type 1 diabetes is consistent with other studies (9,15). the lack of a miaa effect was similar to a previous finding of little effect when iaa was included as a third autoantibody (21). however, there have been other studies that showed a higher risk associated with iaa positivity (15,22,23). other autoantibodies, either accounted for or unaccounted for, could have explained these associations. ica individuals who were also ia-2a have been observed to have a high risk for type 1 diabetes (11). the addition of ica to other autoantibodies has been shown to substantially increase the risk of type 1 diabetes in first - degree relatives (24). an autoantibody to zinc transporter 8 has recently been found to be predictive of type 1 diabetes in children (12). the percentages of common positivity with at least one other autoantibody ranged from 22% for miaa to 65% for ica512. in addition, the correlations of titers between autoantibody pairs differed, and the correlations varied according to the subsequent development of type 1 diabetes. it is possible that associations among autoantibodies according to positivity and titer are a function of the stage of progression to type 1 diabetes in the study population. if so, the serial follow - up of these associations could provide insight into both the prediction and pathogenesis of type 1 diabetes. among those with single positivity of gad65 and ica, thus, titers can provide useful information, even within the positive range. in a previous report, among those with positive autoantibodies, the titers of ia-2 antigen and iaa were both predictive of type 1 diabetes (25). although a cutoff is set at the 99th percentile for each of the autoantibodies, the strength of signal for iaa of patients with new - onset type 1 diabetes is much closer to the range of signal for normal control subjects compared with that for either gad65 or ica512 autoantibodies. thus, it is possible that a sizable proportion of miaa values represents false - positive results or low - affinity iaa associated with lower risk (26). because confirmation and persistence were not determined in the current study, these factors will be important to evaluate in future studies. the findings in this study might not be fully generalizable because they were derived from relatives of patients with type 1 diabetes. as discussed above, the trials cohort was selected on the basis of certain criteria. in addition, the composition of the questionnaire cohort could have been influenced by the willingness to respond to the questionnaire and even to some extent by the absence of the qualifying criteria for entry into the trials. as discussed above, the increased occurrence of type 1 diabetes in the trials cohort is probably attributable to selection factors for trial entry and to oral glucose tolerance test surveillance. our findings show that although autoantibody number is a predictor of type 1 diabetes, the particular type and titer of an autoantibody can influence prediction. moreover, it is evident that the frequencies and the associations of autoantibodies with each other can vary to a great extent. | objectivethere is limited information from large - scale prospective studies regarding the prediction of type 1 diabetes by specific types of pancreatic islet autoantibodies, either alone or in combination. thus, we studied the extent to which specific autoantibodies are predictive of type 1 diabetes.research design and methodstwo cohorts were derived from the first screening for islet cell autoantibodies (icas) in the diabetes prevention trial type 1 (dpt-1). autoantibodies to gad 65 (gad65), insulinoma - associated antigen-2 (ica512), and insulin (micro - iaa [miaa ]) were also measured. participants were followed for the occurrence of type 1 diabetes. one cohort (questionnaire) included those who did not enter the dpt-1 trials, but responded to questionnaires (n = 28,507, 2.4% ica+). the other cohort (trials) included dpt-1 participants (n = 528, 83.3% ica+).resultsin both cohorts autoantibody number was highly predictive of type 1 diabetes (p < 0.001). the questionnaire cohort was used to assess prediction according to the type of autoantibody. as single autoantibodies, ica (3.9%), gad65 (4.4%), and ica512 (4.6%) were similarly predictive of type 1 diabetes in proportional hazards models (p < 0.001 for all). however, no subjects with miaa as single autoantibodies developed type 1 diabetes. as second autoantibodies, all except miaa added significantly (p < 0.001) to the prediction of type 1 diabetes. within the positive range, gad65 and ica autoantibody titers were predictive of type 1 diabetes.conclusionsthe data indicate that the number of autoantibodies is predictive of type 1 diabetes. however, miaa is less predictive of type 1 diabetes than other autoantibodies. autoantibody number, type of autoantibody, and autoantibody titer must be carefully considered in planning prevention trials for type 1 diabetes. |
the vision and change deliberations on transforming undergraduate biology education recently articulated a need to engage students in the process of science and to present science as a vibrant and active field (american association for the advancement of science, 2010). extensive pedagogic research concludes that participation in open - ended research endeavors fosters a sense of ownership over a biological subject, and enhances teaching and learning in biological sciences (teagle foundation, 2007). developing innovative cross - disciplinary approaches and empowering faculty with the tools to implement novel strategies remains a challenge at all levels of undergraduate education. in the last 5 yr, the rise of next - generation (nextgen) sequencing approaches in addressing biological problems has been spectacular, but incorporating nextgen sequencing data for active teaching in the undergraduate curriculum remains a major challenge. for faculty at small and medium - sized institutions of higher education, high teaching loads, lack of access to state - of - the - art equipment, and budgetary constraints typically conspire to inhibit faculty from considering nextgen sequencing in their own experiments. high capital costs, extraordinarily high rates of technological change, and daunting computational and analytical requirements make the technology exceptionally challenging to assimilate into the undergraduate curriculum. the genome consortium for active teaching (gcat ; campbell., 2006) was developed a decade ago to meet similar challenges in relation to the use of microarrays in undergraduate biology education. gcat offers highly discounted microarray chips and array scanning and a supporting network of faculty expertise to educators working with undergraduates. in one decade, the effort has trained over 360 faculty and 24,000 undergraduates in the use and interpretation of microarray data. the newly trained students were enrolled in primarily undergraduate institutions, including those that historically serve underrepresented populations. gcat has met many of the goals of the bio2010 report (campbell., 2007), and recently expanded its focus into synthetic biology (wolyniak., 2010). now that nextgen sequencing is rapidly superseding microarray technology for a variety of technical and economic considerations, gcat and others recognized the need to find cost - effective and innovative strategies to facilitate active teaching of nextgen technology at the undergraduate level. understanding the advantages and limitations of continually evolving transformative technologies like nextgen sequencing is essential preparation for future life scientists, medical professionals, and, indeed, a scientifically literate citizenry, as the age of personalized medicine moves toward becoming reality. in addition, analyzing raw sequence data provides students with learning opportunities that underscore interdisciplinary concepts central and relevant to studies of all forms of life. in this paper, we report proceedings of a workshop from a national science foundation (nsf)funded incubator grant for research coordination networks for undergraduate biology education (rcn / ube). the network aimed to collaborate with a centrally located genome - sequencing core facility at the pennsylvania state university (psu) to enable undergraduate students and faculty in the mid - atlantic region to access state - of - the - art sequencing technology. initial network participants included juniata college, susquehanna university, duquesne university, hampton university, morgan state university, ramapo college of new jersey, gettysburg college, lycoming college, lock haven university, mount aloysius college, bucknell university, and hood college, with the genome - sequencing facility at psu supporting the data acquisition and dissemination aspects of the initiative. the meeting was held july 1114, 2011, at juniata college and psu and included the individuals who helped write the incubator grant and invited speakers malcolm campbell (davidson college), anton nekrutenko (psu), istvan albert (psu), and bill morgan (college of wooster). through presentations and periodic whole - group discussions, we worked together to exchange ideas to develop a structure to approach the problem of introducing nextgen sequencing to undergraduates. during the course of the meeting, a number of parallels emerged between the thinking of the participants and the philosophy of gcat. members of both groups valued the academic freedom provided by their ability to choose and direct their own research and scholarly activities. both groups recognized the value of communal support from colleagues at similar small institutions to help compensate for lack of a critical mass of peers on each campus. both groups recognized the strategic need to partner with other groups, like the microarray manufacturers in gcat 's initial plan, or genome - sequencing facilities, such as psu. all of these considerations suggested that the mid - atlantic network for nextgen sequencing could operate more effectively and enable the approach to be replicated elsewhere if it partnered with gcat as a new arm of that consortium. gcat has established an efficient dissemination strategy through its website and listserv, and many of the members currently using microarrays will be poised to transition to nextgen sequencing as it replaces gene chip technology. our shared values and the success of the rcn / ube grant led to an agreement with malcolm campbell for our rcn / ube to become gcat - seek(quence) and to complement another gcat initiative in the emerging field of synthetic biology (gcat - synbio ; wolyniak., 2010). at our network meeting, we formed a nascent community of biologists from distinct areas (e.g., molecular biology, environmental science, plant biology, microbiology) aiming to develop parallel research studies in the scholarship of teaching and learning and discovery science. the specific goals of the workshop were to : 1) learn lessons from the gcat model ; 2) learn the scope of nextgen sequencing technology, applications, and analysis ; 3) develop common learning goals for students using this technology and develop appropriate methods of assessment and ; 4) develop goals and an administration plan for the network. malcolm campbell presented the keynote address on gcat, describing lessons learned from administration of the consortium. in particular, he emphasized the importance of an undergraduate focus, inclusion of minority - serving institutions, assessment of educational activities, advertisement of the network, faculty development, and taking on the most difficult problems related to a technology to make the network valuable. the gcat model also recognized the importance of the investigator retaining ownership in the direction of his or her research. this was one of the key reasons for adopting a model in which an investigator requested the raw sequence data related to their research expertise and passion. deb grove and craig praul (codirectors of the psu genome core facility) detailed the latest sequencing technologies, applications, and costs associated with their ion torrent pgm, roche 454flx, and solid 5500xl platforms. these are massively parallel dna - sequencing machines capable of providing hundreds of millions to tens of billions of nucleotides of dna sequence data in about a week. the resulting data must be processed using specialized bioinformatics techniques that may require high - powered computers. it was determined that up to 50% of costs could be cut when individual researchers cooperate to share sequencing runs. unique dna sequence adapters (bar codes) may be ligated onto an investigator 's dna fragments before sequencing. this process allows each investigator 's samples to be individually labeled, pooled with other samples, and automatically separated after bulk sequencing. psu biochemistry and molecular biology faculty anton nekrutenko and istvan albert framed challenges and approaches to nextgen data analysis. anton nekrutenko described the galaxy bioinformatics analysis framework that he and his colleagues developed (blankenberg., 2010 ; he emphasized the importance of the evolutionary underpinnings of bioinformatics analysis, and how comparison is key to understanding genomes. istvan albert, director of the bioinformatics consulting center at psu, suggested that bioinformatics analysis is challenging, because it is a highly interdisciplinary science incorporating information technology to manage data, computer science to analyze data, statistics to find meaningful patterns, and biology to form relevant hypotheses. he stressed that bioinformatics can not be learned passively from a book but requires active - learning approaches and student commitment. the challenge facing undergraduate faculty in introducing bioinformatics was addressed by ash stuart (ramapo college of new jersey), eric sakk (morgan state university), and bill morgan (college of wooster), who is working on a related initiative in genomics education. the effectiveness of interdisciplinary approaches, open - ended inquiry, case studies, online student learning communities, undergraduate conferences, exchange of students between schools, and interaction among students in different disciplines on a single campus all had the potential to improve student communication, collaboration, and leadership skills. it was stressed that one of the desired skills students should acquire was adaptability, because the field of bioinformatics changes so rapidly. having a forum to discuss the impact of genomic sciences and bioinformatics analysis using examples from the daily news bill morgan discussed his progress toward a free, online genomics textbook focused on interactive case studies with mathematical sidebars and modeled after the text, discovering genomics, proteomics, and bioinformatics, by campbell and heyer (2006). he has assembled a group of faculty members from throughout the united states with expertise in genomics to help coordinate the development of learning modules in 10 topic areas. the workshop participants were particularly interested in reviewing the learning objectives for the genome - sequencing topic. it was suggested that a template with learning objectives, protocols, and assessment instruments be developed for the next - generation sequencing module that would encourge faculty adoption because it would allow customization of its activities to the datasets of individual investigators. no innovation in education can be considered successful if it is not subjected to rigorous evaluation and assessment in the context of defined learning objectives. the workshop participants discussed the learning objectives the network should have, and formed an assessment leadership team chaired by tammy tobin (susquehanna university) and jay hosler (juniata college). this team will guide development of appropriate instruments to monitor student outcomes for network participants, as well as to support individual faculty in assessing the impact of students working with raw sequence data in individual classes. core learning outcomes proposed for the gcat - seek network were the ability for instructors and students to do the following : explain each step in the generation and analysis of nextgen sequence data.discuss the basic biology assumptions that underlie sequence analysis (e.g., evolution, structure, and function).evaluate the strengths and weaknesses of the methods used in nextgen sequencing, including the impact that data quality has on bioinformatics analysis.construct a testable hypothesis and experimental design that uses nextgen sequencing and bioinformatics tools.choose and justify the appropriate methods for a specific nextgen sequencing application. discuss the basic biology assumptions that underlie sequence analysis (e.g., evolution, structure, and function). evaluate the strengths and weaknesses of the methods used in nextgen sequencing, including the impact that data quality has on bioinformatics analysis. construct a testable hypothesis and experimental design that uses nextgen sequencing and bioinformatics tools the goal is to have best teaching practices established and validated through appropriate assessment and distributed to all of the network participants and their colleagues. following whole - group discussion, it was determined the agreed purpose of gcat - seek is to 1) bring functional genomic methods into the undergraduate curriculum, primarily through independent and classroom - based student research using centralized core facilities to make nextgen sequence data accessible to undergraduates ; 2) create a clearinghouse of information for educators to use when teaching nextgen sequencing and related topics ; 3) create a large database of raw data and analyzed results for pedagogical use by gcat - seek members ; and 4) develop a global network of educators who are using functional genomics and nextgen sequencing in the undergraduate curriculum. gcat - seek specifically aims to obtain group discounts at regional research - intensive core facilities, to negotiate software discounts, and to garner support for mini - grants to help cover the cost of the initial sequencing runs for network participants. the network aims to support its members through online listservs, periodic workshops, and meetings, following an approach similar to that successfully used by other gcat groups (campbell., 2006). the network approach will add efficiency by coordinating projects and partnering investigators with appropriate sequencing platforms. given that even the smallest purchasable unit of nextgen sequence will often contain a great excess of information for any given project and that many projects can be combined using bar codes (as discussed in the introductory paragraph), an organized staging for related samples from different investigators was envisioned. this may reduce cost of data acquisition to a few thousand dollars from departmental budgets or mini - grant programs. furthermore, additional cost efficiency for network participants can be achieved through coordination of the synthesis and maintenance of a database of highly purified bar - coded primers for metagenomic analysis. at a time of severe budget scrutiny, the efficiency and cost - effectiveness of the proposed approach is apparent. sequencing cores are not running at capacity, and technological advances are lowering sequencing costs. bioinformatics programs, databases, and computing requirements for many types of projects are all either already in the public domain or well within the budgets of even the smallest undergraduate colleges. given sufficient interest, regional replication of some elements of gcat - seek in the future should be considered as a means of lowering travel costs for meetings and workshops for participants and allowing students to more easily visit genome core facilities. thus, with a modest investment, this program can start to meet the challenges of training the next generation of life scientists by engaging undergraduates in the process of science presented in the context of modern technology. | to transform undergraduate biology education, faculty need to provide opportunities for students to engage in the process of science. the rise of research approaches using next - generation (nextgen) sequencing has been impressive, but incorporation of such approaches into the undergraduate curriculum remains a major challenge. in this paper, we report proceedings of a national science foundation funded workshop held july 1114, 2011, at juniata college. the purpose of the workshop was to develop a regional research coordination network for undergraduate biology education (rcn / ube). the network is collaborating with a genome - sequencing core facility located at pennsylvania state university (university park) to enable undergraduate students and faculty at small colleges to access state - of - the - art sequencing technology. we aim to create a database of references, protocols, and raw data related to nextgen sequencing, and to find innovative ways to reduce costs related to sequencing and bioinformatics analysis. it was agreed that our regional network for nextgen sequencing could operate more effectively if it were partnered with the genome consortium for active teaching (gcat) as a new arm of that consortium, entitled gcat - seek(quence). this step would also permit the approach to be replicated elsewhere. |
drug administration errors appear to be a major source of iatrogenic harm to the hospitalized patients. a study has estimated that drug - related errors occur in one out of five doses given to patients in hospitals. many drug errors have been reported in the literature involving labor and delivery, with some resulting in fatalities. a 28-year - old primigravida was admitted to the antenatal ward for close fetal and maternal observation with dichorionic diamniotic twin pregnancy complicated by severe preeclampsia as well as abnormal doppler studies. the 24-h urine protein was elevated to 6 g, requiring labetalol 400 mg t.i.d., to control her blood pressure. induction of labor was planned as there was persistence in the abnormality of doppler studies of twin babies. physical examination revealed an easy looking airway and unremarkable cardiovascular and respiratory exam. induction was carried out with amniotomy, oxytocin infusion and labor epidural used for pain control. her initial magnesium sulfate levels were 1.4 on the first day evening and 1.97 on the next day morning. there was a small second - degree perineal tear, which was repaired, but bleeding per vagina persisted. b y that time, she had already lost approximately 1 l of blood, but she was adequately resuscitated and her vitals were stable. the uterus contracted post misoprostol, but shortly afterwards was filled with clots and blood. therefore, one dose of hemabate (carboprost tromethamine) was given intramuscularly. at this stage, she complained of increased shortness of breath. a second intravascular (iv) line was started, as there was persistent excessive vaginal bleed. the bleeding settled with administration of hemabate, but her level of consciousness continued to decrease. the patient was found to be having difficulty in responding verbally, but did continue to breathe unsupported. the bleeding had stopped, but her respiratory efforts continued to decrease to such an extent that she required intubation. a rapid sequence induction and intubation was performed with 40 mg of propofol and 100 mg succinylcholine and her oxygen saturation was maintained at 100%. blood samples were sent for blood gas analysis, serum electrolytes including calcium and magnesium levels, coagulation profile, and hemoglobin level. the working diagnosis at the time was magnesium toxicity versus possibility of pulmonary, amniotic fluid embolus, and brain edema secondary to preeclampsia maternal stroke or seizure. a trial of 1 g of calcium gluconate was given because the last magnesium level had been within the normal therapeutic range. during the physical examination, her pupils were non - dilated, but they were asymmetrical and reacting to light. she was promptly taken for a head computed tomography (ct) scan, which was negative. chest ct conducted to rule out pulmonary embolism was inconclusive. in the meantime, magnesium levels had returned to be within the toxic range at 8.6 mmol / l. magnesium toxicity was perceived to be the etiology behind her respiratory arrest and neurologic suppression. she was admitted to the intensive care unit (icu) for respiratory support and correction of magnesium levels. an additional 2 g of calcium was given and planned for forced diuresis with lasix. the team visited the labor and delivery room, as the increase in magnesium level had occurred within a short period of time, a drug error was suspected. the duration and dose of the magnesium infusion that was started the night before matched with the amount of magnesium remaining in the bag. the garbage from the delivery room was checked for discarded bags and an empty bag labeled with magnesium sulfate was found in the garbage. the working diagnosis was inadvertent magnesium sulfate toxicity secondary to misplacement of magnesium sulfate iv bag for oxytocin. therefore, the patient had received 40 g of magnesium sulfate as an iv push. by early evening of the day of delivery, her magnesium levels had returned to within therapeutic range. after extubation, she was alert, oriented, and asymptomatic and required no supplemental oxygen. she was observed in the icu overnight and transferred to the postpartum floor in a stable condition. magnesium is a crucial physiologic element that plays an important clinical role under many conditions. it is commonly used in obstetric practice for the treatment of preeclampsia, eclampsia, and preterm labor. occasionally, the administration of magnesium sulfate results in an accidental overdose and harm to the patient. a report in the american journal of maternal child nursing in 2004 reported 52 cases of accidental magnesium sulfate overdosing in labor and delivery settings. many of these errors resulted from the unintended rapid infusion of the entire contents of a bag containing magnesium sulfate. hypermagnesemia, though rare, is often iatrogenic due to inadvertent overdose of iv magnesium, and even with normal renal function the associated hypermagnesemia is clinically significant. elevated serum magnesium levels can result in a variety of clinical signs and symptoms that are dose - dependent and predominantly affect the cardiovascular and neuromuscular systems causing loss of deep tendon reflexes (dtrs) and progressive muscle weakness, including the diaphragm and other respiratory muscles, leading to acute respiratory failure. in addition, an overdose of the same can lead to hypotension, complete heart block, and cardiac arrest. it is important to remember that an untreated respiratory arrest leads to cardiac arrest as the cardiac muscle becomes hypoxic and ischemic. in the present case, the patient had postpartum bleeding due to uterine atony with hypoventilation following the accidental infusion of 40 mg of magnesium sulphate (mgso4). the patient was managed with iv calcium, fluids, and forced diuresis with lasix to assist excretion of magnesium sulfate. the patient was on ventilatory support until the magnesium levels returned to normal and there was improvement in the conscious levels. the presented scenario illustrates a medical error where a wrong medication was administered ; this was primarily due to the similar appearance of mgso4 and oxytocin infusion bags manufactured by hospital pharmacy. it is available as 40 g mgso4 in 1-l bags, while the oxytocin infusions are 20 units in 1-l bags. the labels were not much different in appearance, and there was no color coding either for easy differentiation. review of literature in fields other than healthcare reveals that an error is often not the result of a single act, but precipitated by the co - occurrence of a number of factors that include physical, social, and ambient environments. during critical situations, a person tends to see what they expect to see, and words are not usually recognized by what is written but by their shapes (the poggenorf effect). the incorrect drug administration was thought to be a chance occurrence due to simple human error. therefore, we would like to suggest the development of hospital policies for the proper storage of high - risk medications and avoiding look - alike or similar appearance of packaging and labeling. the lines of authority and areas of responsibility should be clearly defined between the workers, and there should be adequate communication among the personnel involved in the medication use. high - risk medications should always be administered via an infusion pump and should be double checked before injecting to the patient. however, despite years of use and provider familiarity, the administration of mgso4 occasionally results in accidental overdose and patient harm. color coding of premixed bags, use of color - coded tags on lines, and vigilance in its use is required for safe care of mothers and babies. | during labor and child delivery, a wide range of drugs are administered. most of these medications are high - alert medications, which can cause significant harm to the patient due to its inadvertent use. errors could be caused due to unfamiliarity with safe dosage ranges, confusion between similar looking drugs, mislabeling of drugs, equipment misuse, or malfunction and communication errors. we report a case of inadvertent infusion of a large dose of magnesium sulphate in a pregnant woman. |
despite breast - conserving surgery is widely accepted as the main surgical procedure in treatment modalities, approximately 2030% breast cancer patients still require or request mastectomy. for these patients, this result highlights the importance of the nipple - areola complex (nac) in the cosmetic outcome. however, there are problems with reconstructed nipples, including lack of projection, colour mismatch, shape, size, texture and position. in 1962, nipple - sparing mastectomy (nsm) or subcutaneous mastectomy was first described by freeman. nsm is a surgical procedure that allows the preservation of the skin and nac in mastectomy. when the nac was preserved during mastectomy, patients reported improved satisfaction, body image, and psychological adjustment. since there is limited evidence and no consensus regarding its oncological safety, nsm has been recommended only in carefully selected patients with experienced multidisciplinary teams by national comprehensive cancer network clinical practice guidelines. the prevailing argument is that if the nac is left in place, there is a chance of leaving either occult tumour or a certain amount of breast tissue that is at risk of developing subsequent cancer. while the incidence of local recurrence (lr) after nsm has been reported to be as high as 24% of cases, recurrence specifically at the nipple areola region has been reported in only 2% of cases. therefore, selection criteria for nsm in breast cancer patients are urgently needed. in clinical practice, appropriate standard for selecting patients with low risk of nac involvement, we highlighted oncologic safety of nsm from the following perspectives : preoperative patient selection, surgical approach / pathology evaluation during operation, and patient outcome. the incidences of nipple involvement were variable, ranging from 9.5% to 24.6% in recent studies. incidence of nac involvement in breast cancer in recent years (2008 - 2012) nac : nipple - areola complex ; lcis : lobular carcinoma in situ ; nr : not reported. first of all, some studies included clinically involved nac while others did not. according to mallon., who compared occult nac malignancy with overall nac malignancy, the incidence of the former was significantly lower. in wang however, after excluding 21 cases with clinically abnormal nac, the nac involvement rate fell to 7%. secondly, studies that included paget 's disease, ductal carcinoma in situ and invasive ductal carcinoma (idc) reported higher nac involvement rates, since these lesions were the most common types of lesions that involve the nac. some studies also included the presence of lobular carcinoma in situ (lcis) in the nipple as evidence of malignancy involvement. because lcis is regarded as a marker of risk rather than a precursor lesion, the reported incidence of nipple involvement was increased. for instance, in studies that included lcis, the incidence of malignant nac involvement (3058%) was significantly higher than average. finally, the pathological protocol used for nac evaluation had a major role in determining the incidence of nac involvement. variation in methodology existed between studies regarding the depth of sampling from nipple skin, slicing orientation, slice width, and number of sections per block. above all, the distance from the base of the nipple examined was ill defined, and a low rate of nac positivity was associated with longer distance. pathological protocols that did not require serial coronal sections of the nipple also yielded a lower rate of nac involvement. furthermore, a review of pathology records alone might not be appropriate for the evaluation of nac involvement. according to schecter. 's study, only 4 of 13 cases of nac involvement were identified based on a review of medical records alone, the others were identified by the pathologist upon re - examination of the nac sections from all study patients. preoperative patient selection is essential for nsm oncological safety. careful patient selection could decrease the incidence of nac involvement, thus decreasing lr rates. possible factors that should be considered for evaluation before nsm include : clinical evaluations of nipple involvement, tumour - nipple distance (tnd), tumour size, axillary lymph node involvement, disease stage, histological grade, human epidermal growth factor receptor-2 (her-2) overexpression, and hormone receptor status. one study showed that clinical nac involvement, as determined by patient symptoms or physical examination (e.g., nipple retraction, palpable mass in the nipple, nipple bleeding or nipple discharge), was present in 61% of nac - positive but only 14% of nac - negative cases. another study demonstrated that, the sensitivity of detecting nac involvement was 61% with clinical evaluation (history and/or physical examination) and 56% with magnetic resonance imaging (mri), indicating the importance of clinical evaluation in preoperative patient selection. tumour - nipple distance tumour - nipple distance was the most notable factor associated with nac involvement among all the investigated factors. tnd was the minimum distance from the base of the nac to the nearest lesion margin. data showed that the mean tnd was 2.0 cm for nac - positive tumours and 4.7 cm for nac - negative tumours. most authors suggested that the cut - off value should be set at 2 cm. d'alonzo. supported a 1 cm cut - off value according to their reports. billar. showed that mammography was the best imaging option for identifying nac involvement, followed by mri and ultrasound imaging. friedman. reported that mri of the breast could reveal nipple involvement even when it was clinically unsuspected. an increase in the diameter of the primary tumour was associated with an increased risk for nipple involvement. in a previous study, nac - positive tumours were larger, with a mean size of 3.3 cm versus 2.5 cm for nac - negative tumours. a more recent study showed that only a tumour size > 2 cm and a distance from the tumour edge to the nac 2, positive lymph node and her-2 (+). risk factors with low evidence were indicated by individual studies and therefore needed more evidence. this group included negative er and pr status, possibly, certain histological types (invasive cancer with eic). risk factors for nac involvement categorized by evidence level nac : nipple - areola complex ; tnd : tumour - nipple distance ; ln : lymph node ; eic : extensive intraductal component ; pr : progesterone receptor ; er : estrogen receptors ; her-2 : human epidermal growth factor receptor 2. although all of the pathology information could be obtained preoperatively by core needle biopsy or open biopsy before the final surgery, there was no data regarding the relationship between breast biopsy pathology results and nipple involvement in breast cancer. some authors also computed a nac involvement score based on mammographic tnd, pathological stage, and tumour size to distinguish between the presence and absence of nac involvement. however, the study was based on only 31 patients. after careful preoperative evaluations, frozen section pathology during surgery can determine the surgical margins, while final pathology provides definite nac status. however, there are no available standard protocols for surgical approaches or pathological examination. surgical nsm techniques could affect both the oncological safety and aesthetic outcome of patients. although the lack of available published data precluded the recommendation of any specific surgical approach, a lateral, radial, lateral mammary fold, or inframammary fold incision appears to provide excellent access to the glandular breast tissue in all four quadrants, permits axillary exploration (and removal of axillary breast tissue), and preserves skin flap sensation. with flap elevation, the entire breast tissue is excised, leaving 45 mm thickness of skin flap. some support removing all ductal tissue of the nipple core, while some believe leaving 5 mm of glandular tissue behind nac. the employed reconstruction strategy depends on a general assessment of patient preference, as well as of the risks and benefits. however, the authors were not yet ready to offer nsm with immediate autologous breast reconstruction as their standard of care. frozen section analysis frozen section analysis serves as the standard to rule out nac involvement. if frozen section analysis is positive, traditional mastectomy or skin sparing mastectomy (ssm) otherwise, the surgeon proceeds with nsm. other patients will undergo permanent section evaluation and nac will be ultimately removed only if the final pathology is positive due to potential false - negative results from frozen section analysis. wagner. used surgical clips or sutures placed on the circumference of the areolar margin at the 3, 6, 9, and 12 o'clock positions and a fifth marking clip or suture immediately underneath the nipple to evaluate perioperative pathology. noted a 4.63% false - negative rate of frozen section histology of the nac base compared with definitive histology. another study showed that 11 of 157 (7%) cases exhibited nac involvement, all of which were identified with intraoperative frozen section analysis with subsequent removal of the nac. moreover, nipple core needle biopsies had also been performed to evaluate possible occult nac involvement intraoperatively. final pathological evaluation final evaluation protocols of paraffin - embedded tissue also varied among studies. the main concern was the definition of nac involvement, that is, the amount of tissue associated with the nac was different. as mentioned before, some studies emphasized the importance of removing all ductal tissue of the nipple core to ensure oncologic completeness by sharp dissection or point diathermy. other studies suggested that leaving 5 mm of glandular tissue behind nac was necessary to preserve its blood supply and decrease the nac necrosis rate, and accepted that leaving breast tissue might result in a higher risk of lr or development of new disease. however, the mean thickness of the skin flaps in mastectomy was 45 mm, which was at the level of the superficial fascia dividing the subcutaneous fat from the breast glandular tissue. recent data suggested that when performing a nsm, the dissection plane could be even closer to the base of the nipple, including the entire duct bundle, with a reasonably low risk of necrosis. multiple prospective and retrospective studies investigating lr in nsm have been conducted to address oncologic safety, which are summarized in table 3. nac could only be preserved when no malignant cells were identified at pathology evaluation, otherwise nac was re - excised. however, due to various patient selection standards, treatment protocols, and follow - up time, the oncological outcomes of nsm patients were difficult to compare among studies. nac : nipple - areola complex ; nr : not reported ; na : not applicable ; eliot : intraoperative radiotherapy with electrons ; nsm : nipple - sparing mastectomy ; lr : local recurrence ; na : not available. local recurrence in nipple - sparing mastectomy most studies have demonstrated that there was no significant difference in lr, distant metastasis (dm), and overall survival (os) between traditional mastectomy and nsm to treat primary breast cancer. sakurai. conducted a cohort study with a median follow - up time of 78 months. the probability of lr was slightly higher in the nsm cohort than in the mastectomy cohort, but no significant difference was found (8.2% vs. 7.6%, p = 0.81). gerber. did not observe any difference between the lr, dm, and breast cancer - specific death between modified mastectomy, ssm, and nsm after a mean follow - up of 101 months. on the other hand, some researchers found a lr that was different between radical mastectomy and subcutaneous mastectomy (1.3% vs. 3.8% at 5 years). local recurrence in the nsm cohort often involved the nipple and/or areola, skin flap, and local lymph nodes, with nac recurrence rates between 0% and 3.7%. the disease - free survival after nac removal in the nac recurrence cases was 93% at the 5-year follow - up, demonstrating that nsm was indeed an oncologically sound treatment for breast cancer. however, os after primary surgery was significantly worse in patients who suffered an early lr (2 cm and a distance from the tumour edge to the nac 2, positive lymph node and her-2 (+). risk factors with low evidence were indicated by individual studies and therefore needed more evidence. this group included negative er and pr status, possibly, certain histological types (invasive cancer with eic). risk factors for nac involvement categorized by evidence level nac : nipple - areola complex ; tnd : tumour - nipple distance ; ln : lymph node ; eic : extensive intraductal component ; pr : progesterone receptor ; er : estrogen receptors ; her-2 : human epidermal growth factor receptor 2. although all of the pathology information could be obtained preoperatively by core needle biopsy or open biopsy before the final surgery, there was no data regarding the relationship between breast biopsy pathology results and nipple involvement in breast cancer. some authors also computed a nac involvement score based on mammographic tnd, pathological stage, and tumour size to distinguish between the presence and absence of nac involvement. frozen section pathology during surgery can determine the surgical margins, while final pathology provides definite nac status. however, there are no available standard protocols for surgical approaches or pathological examination. surgical nsm techniques could affect both the oncological safety and aesthetic outcome of patients. although the lack of available published data precluded the recommendation of any specific surgical approach, a lateral, radial, lateral mammary fold, or inframammary fold incision appears to provide excellent access to the glandular breast tissue in all four quadrants, permits axillary exploration (and removal of axillary breast tissue), and preserves skin flap sensation. with flap elevation, the entire breast tissue is excised, leaving 45 mm thickness of skin flap. some support removing all ductal tissue of the nipple core, while some believe leaving 5 mm of glandular tissue behind nac. the employed reconstruction strategy depends on a general assessment of patient preference, as well as of the risks and benefits. however, the authors were not yet ready to offer nsm with immediate autologous breast reconstruction as their standard of care. frozen section analysis frozen section analysis serves as the standard to rule out nac involvement. if frozen section analysis is positive, traditional mastectomy or skin sparing mastectomy (ssm) otherwise, the surgeon proceeds with nsm. other patients will undergo permanent section evaluation and nac will be ultimately removed only if the final pathology is positive due to potential false - negative results from frozen section analysis. wagner. used surgical clips or sutures placed on the circumference of the areolar margin at the 3, 6, 9, and 12 o'clock positions and a fifth marking clip or suture immediately underneath the nipple to evaluate perioperative pathology. noted a 4.63% false - negative rate of frozen section histology of the nac base compared with definitive histology. another study showed that 11 of 157 (7%) cases exhibited nac involvement, all of which were identified with intraoperative frozen section analysis with subsequent removal of the nac. moreover, nipple core needle biopsies had also been performed to evaluate possible occult nac involvement intraoperatively. final pathological evaluation final evaluation protocols of paraffin - embedded tissue also varied among studies. the main concern was the definition of nac involvement, that is, the amount of tissue associated with the nac was different. as mentioned before, some studies emphasized the importance of removing all ductal tissue of the nipple core to ensure oncologic completeness by sharp dissection or point diathermy. other studies suggested that leaving 5 mm of glandular tissue behind nac was necessary to preserve its blood supply and decrease the nac necrosis rate, and accepted that leaving breast tissue might result in a higher risk of lr or development of new disease. however, the mean thickness of the skin flaps in mastectomy was 45 mm, which was at the level of the superficial fascia dividing the subcutaneous fat from the breast glandular tissue. recent data suggested that when performing a nsm, the dissection plane could be even closer to the base of the nipple, including the entire duct bundle, with a reasonably low risk of necrosis. multiple prospective and retrospective studies investigating lr in nsm have been conducted to address oncologic safety, which are summarized in table 3. nac could only be preserved when no malignant cells were identified at pathology evaluation, otherwise nac was re - excised. however, due to various patient selection standards, treatment protocols, and follow - up time, the oncological outcomes of nsm patients were difficult to compare among studies. nac : nipple - areola complex ; nr : not reported ; na : not applicable ; eliot : intraoperative radiotherapy with electrons ; nsm : nipple - sparing mastectomy ; lr : local recurrence ; na : not available. local recurrence in nipple - sparing mastectomy most studies have demonstrated that there was no significant difference in lr, distant metastasis (dm), and overall survival (os) between traditional mastectomy and nsm to treat primary breast cancer. sakurai. conducted a cohort study with a median follow - up time of 78 months. the probability of lr was slightly higher in the nsm cohort than in the mastectomy cohort, but no significant difference was found (8.2% vs. 7.6%, p = 0.81). gerber. did not observe any difference between the lr, dm, and breast cancer - specific death between modified mastectomy, ssm, and nsm after a mean follow - up of 101 months. on the other hand, some researchers found a lr that was different between radical mastectomy and subcutaneous mastectomy (1.3% vs. 3.8% at 5 years). local recurrence in the nsm cohort often involved the nipple and/or areola, skin flap, and local lymph nodes, with nac recurrence rates between 0% and 3.7%. the disease - free survival after nac removal in the nac recurrence cases was 93% at the 5-year follow - up, demonstrating that nsm was indeed an oncologically sound treatment for breast cancer. however, os after primary surgery was significantly worse in patients who suffered an early lr (<3 years after primary surgery) than in those who suffered a late lr (68% and 86%, respectively, p = 0.03). role of radiotherapy in nipple - sparing mastectomy some authors have proposed that additional radiotherapy should play the same role as in breast conservative treatment, that is, reducing the lr risk in the remaining breast tissue. suggested the use of an electron intraoperative radiotherapy treatment (eliot) when the nsm technique was employed. in their study, a total dose of 16 gy of eliot was delivered intraoperatively in the region of the nac. they also reported that in another eliot series of 516 cases, final histology revealed foci of carcinoma in 63 cases. while 7 of these 63 cases underwent secondary nac removal, 56 cases in which the areolas were preserved did not develop lr after 19 months of follow - up. in addition, benediktsson and perbeck reported that radiotherapy effectively reduced lr, with a lr of 8.5% among patients who underwent radiation therapy versus 28.4% among patients who did not undergo radiation therapy over a median follow - up period of 156 months. in another study, a comparison between 800 patients receiving eliot and 201 patients receiving delayed irradiation was conducted, and no difference in survival was detected between groups. some studies questioned the necessity of radiotherapy due to the lack of difference in lr between their studies (which employed neither intra - operative nor postoperative radiotherapy) and other published studies. based on current studies, nsm appears to be oncologically safe after careful patient selection and assessment of margins. although many studies presented in this review reported acceptable levels of lr, the lack of retrospective long - term studies makes nsm a controversial option for breast cancer treatment. currently, many issues associated with nsm remain unresolved, including the lack of standardised patient selection criteria and consensus regarding the operative approach, pathology protocols, and the role of radiotherapy in nsm. heterogeneity of the results between studies means that additional well - designed prospective cohort studies are essential to answer these questions. | objective : to evaluate the oncologic safety of nipple - sparing mastectomy (nsm) for breast cancer patients based on current literature.data sources : a comprehensive literature search of medline, embase databases was conducted for studies published through march 2014.study selection : our search criteria included english - language studies that focused on nsm at nipple - areola complex (nac) involvement, patient selection, and recurrence. prophylaxis nsm, case series or reports that based on very small population were excluded. in the end, 42 studies concerning nsm and oncological safety were included into the review.results:nsm is a surgical procedure that allows the preservation of the skin and nac in breast cancer patients or in patients with prophylactic mastectomy. however, the oncologic safety and patient selection criteria associated with nsm are still under debate. the incidence of nac involvement of breast cancer in recent studies ranges from 9.5% to 24.6%, which can be decreased through careful patient selection. tumour - nipple distance, tumour size, lymph node involvement and molecular characteristics can be evaluated preoperatively by clinical examinations, imaging studies and biopsies to predict the risk of nac involvement. currently, there is no available standard protocol for surgical approaches to nsm or pathological examination of nsm specimens. the local recurrence (ranges from 0% to 24%) of nsm is not significantly higher than that of traditional mastectomy in selected patients based on long - term follow - up. the role of radiotherapy in nsm is still controversial and is not universally accepted.conclusions:nsm appears to be oncologically safe following careful patient selection and assessment of margins. |
despite the technological progress aimed at improving success and reducing complication rates during cardiac resynchronization therapy (crt) device implantation, in a proportion of cases, the delivery of a left ventricular (lv) pacing lead through the coronary sinus (cs) still fails. as recently demonstrated, transvenous implantation of a lv pacing lead is safe and feasible using remote magnetic navigation of a guidewire. however, we encountered certain limitations in order to reach the ultimate goal of reproducibly implanting the lv lead remotely. among these limitations, one of them was the lack of having a real - time three - dimensional (3d) cs model to facilitate more accurate navigation of the guidewire., haifa, israel) is becoming an established technique for the 3d reconstruction of vessels using data obtained from standard vascular angiography. the reconstructed vessel can be subsequently imported into the niobe (stereotaxis, st louis, mo, usa) magnetic navigation system, and magnetic vectors can be selected based on the virtual model of the vessel. our purpose was to test the feasibility of reconstructing the anatomy of the cs using this software and evaluate the accuracy of navigating within the cs based on this 3d model. these patients met the standard criteria for crt comprising advanced heart failure refractory to medical therapy, low ejection fraction (< 35%), and a broad qrs (120 ms) on the electrocardiogram (ecg) with a left bundle branch block (lbbb)-like morphology. all procedures were performed in the room equipped with magnetic navigation (niobe ii, stereotaxis). in all patients, the left cephalic vein was dissected. a right ventricular (rv) shock lead (model 1580 riata, st jude medical, sylman, ca minneapolis, mn, usa) was introduced and actively fixed to the rv apical wall. after a double left subclavian venous puncture, a right atrial active fixation lead (model 5076, medtronic, inc.) a 9 fr long guiding sheath (model attain 6216, medtronic, inc.) was introduced in order to cannulate the cs. angiograms of the cs were performed using 30 left anterior oblique (lao), antero - posterior (ap), and 30 right anterior oblique (rao) projections (projections allowed when the magnets are in the navigate position). these angiograms were imported into the cardiop - b system (paeion inc.). an automatic algorithm detects the vessel edges in each of the projections used for the reconstruction. in case edge, detection was inaccurate (not exactly superimposed onto the angiographic edge), and manual adjustments could be made so as to obtain the best reconstruction possible. on the basis of the traced edges and using a minimum of two complementary angiographic views, a 3d reconstruction of the vessel was performed. the system allows reconstructing the body of the vessel and one bifurcation or side branch (sb) at a time. the reconstructed 3d model of the cs and its sb can then be imported into niobe (stereotaxis), where, after alignment to the real - time fluoroscopic views, flythrough images of the model can be used to navigate (figure 1). the quality of the reconstruction was assessed by two operators performing the procedure and graded as (i) not possible to be reconstructed, (ii) poor, or (iii) good, after superimposing and aligning it to the real - time angiography in the lao, ap, and rao projections (figure 2). the need of manual adjustment of the traced edges in order to improve the quality of the reconstruction was also evaluated. (top) at the left, three - dimensional reconstruction of the coronary sinus with one side branch in an antero - posterior view. note the marked angulation of the lateral side branch. at the right, a flythrough image of the reconstruction. (bottom) reconstructed model aligned to the fluoroscopic view in left anterior oblique projection (left panel) and antero - posterior projection (right panel). four panels, as seen in real - time, showing the different automatically selected vectors (green arrows) chosen to navigate within the reconstructed coronary sinus model. in each panel, at the top left : three - dimensional reconstruction of the coronary sinus ; top right : endovascular view ; bottom left : right anterior oblique projection and not aligned three - dimensional model to facilitate the visualization of the side branch ; bottom right : left anterior oblique projection with aligned three - dimensional model. note how the vector changes according to the portion of the vessel it is aiming at. magnetic navigation is performed by positioning two external magnets (stereotaxis) at each side of the table in order to generate a 0.08 t magnetic field within patients. the orientation of the magnetic field is established by the interaction of the two external magnets with each other and can be automatically determined or specified by an operator working remotely in the viewing room. a single 2 mm neodymium iron boron magnet attached at the tip of a 0.014 guidewire (cronus soft support endovascular guide wire, stereotaxis), aligns itself to the direction of the magnetic vector. in this way, by changing the orientation of the magnets, the resultant magnetic vector changes and the tip of the guidewire aligns itself in the direction of the newly applied vector. a dedicated software package, the navigant (stereotaxis) allows the full integration of the niobe system and the imported cardioop 3d reconstruction (paeion). in this way, the vectors required to navigate within the reconstructed vessels and to access the desired portion of the vessel can be automatically determined using the system (figure 2). if the vectors suggested by the system are not accurate enough to allow access to the desired sb, manual adjustments can be made. in this study, the guidewire was manually advanced after each modification of the magnetic field (every 35 mm steps), in order to access the ideal sb (as previously defined by the operators). once the target sb was engaged, an over - the - wire lv pacing lead (medtronic attain, medtronic, inc. or quicksite, st jude medical, sylmar, ca, usa) was introduced and lodged in the vessel. feasibility of (i) deploying the guidewire and lv lead into the selected sb using the automatically determined vectors, (ii) need for manual adjustments of these vectors, (iii) fluoroscopy time (ft) required for cannulation of the target sb, (iv) total ft, and (v) procedure time were assessed. these patients met the standard criteria for crt comprising advanced heart failure refractory to medical therapy, low ejection fraction (< 35%), and a broad qrs (120 ms) on the electrocardiogram (ecg) with a left bundle branch block (lbbb)-like morphology. all procedures were performed in the room equipped with magnetic navigation (niobe ii, stereotaxis). in all patients, the left cephalic vein was dissected. a right ventricular (rv) shock lead (model 1580 riata, st jude medical, sylman, ca minneapolis, mn, usa) was introduced and actively fixed to the rv apical wall. after a double left subclavian venous puncture, a right atrial active fixation lead (model 5076, medtronic, inc.) a 9 fr long guiding sheath (model attain 6216, medtronic, inc.) angiograms of the cs were performed using 30 left anterior oblique (lao), antero - posterior (ap), and 30 right anterior oblique (rao) projections (projections allowed when the magnets are in the navigate position). these angiograms were imported into the cardiop - b system (paeion inc.). an automatic algorithm detects the vessel edges in each of the projections used for the reconstruction. in case edge, detection was inaccurate (not exactly superimposed onto the angiographic edge), and manual adjustments could be made so as to obtain the best reconstruction possible. on the basis of the traced edges and using a minimum of two complementary angiographic views, a 3d reconstruction of the vessel was performed. the system allows reconstructing the body of the vessel and one bifurcation or side branch (sb) at a time. the reconstructed 3d model of the cs and its sb can then be imported into niobe (stereotaxis), where, after alignment to the real - time fluoroscopic views, flythrough images of the model can be used to navigate (figure 1). the quality of the reconstruction was assessed by two operators performing the procedure and graded as (i) not possible to be reconstructed, (ii) poor, or (iii) good, after superimposing and aligning it to the real - time angiography in the lao, ap, and rao projections (figure 2). the need of manual adjustment of the traced edges in order to improve the quality of the reconstruction was also evaluated. (top) at the left, three - dimensional reconstruction of the coronary sinus with one side branch in an antero - posterior view. note the marked angulation of the lateral side branch. at the right, a flythrough image of the reconstruction. (bottom) reconstructed model aligned to the fluoroscopic view in left anterior oblique projection (left panel) and antero - posterior projection (right panel). four panels, as seen in real - time, showing the different automatically selected vectors (green arrows) chosen to navigate within the reconstructed coronary sinus model. in each panel, at the top left : three - dimensional reconstruction of the coronary sinus ; top right : endovascular view ; bottom left : right anterior oblique projection and not aligned three - dimensional model to facilitate the visualization of the side branch ; bottom right : left anterior oblique projection with aligned three - dimensional model. note how the vector changes according to the portion of the vessel it is aiming at. magnetic navigation is performed by positioning two external magnets (stereotaxis) at each side of the table in order to generate a 0.08 t magnetic field within patients. the orientation of the magnetic field is established by the interaction of the two external magnets with each other and can be automatically determined or specified by an operator working remotely in the viewing room. a single 2 mm neodymium iron boron magnet attached at the tip of a 0.014 guidewire (cronus soft support endovascular guide wire, stereotaxis), aligns itself to the direction of the magnetic vector. in this way, by changing the orientation of the magnets, the resultant magnetic vector changes and the tip of the guidewire aligns itself in the direction of the newly applied vector. a dedicated software package, the navigant (stereotaxis) allows the full integration of the niobe system and the imported cardioop 3d reconstruction (paeion). in this way, the vectors required to navigate within the reconstructed vessels and to access the desired portion of the vessel can be automatically determined using the system (figure 2). if the vectors suggested by the system are not accurate enough to allow access to the desired sb, manual adjustments can be made. in this study, the guidewire was manually advanced after each modification of the magnetic field (every 35 mm steps), in order to access the ideal sb (as previously defined by the operators). once the target sb was engaged, an over - the - wire lv pacing lead (medtronic attain, medtronic, inc. or quicksite, st jude medical, sylmar, ca, usa) was introduced and lodged in the vessel. feasibility of (i) deploying the guidewire and lv lead into the selected sb using the automatically determined vectors, (ii) need for manual adjustments of these vectors, (iii) fluoroscopy time (ft) required for cannulation of the target sb, (iv) total ft, and (v) procedure time were assessed. the procedural outcome of the 16 consecutive patients included in the study is detailed in table 1. all had an lbbb - like broad qrs complex on the ecg, a severely depressed left ventricular ejection fraction, and advanced symptoms of heart failure (new york heart association functional class 3 1). the mean procedure time was 99 26 min, the mean ft 23 14 min, and the mean ft required to position the lv lead into the target cs sb was 1.7 1.3 min. procedure characteristics and left ventricular lead position al, antero - lateral ; ant, anterior ; lvl, left ventricular lead ; l, lateral ; pl, postero - lateral side ; sb, coronary sinus side branch ; sd, standard deviation. in 15 patients (93%), the software was unable to reconstruct the cs and consequently, the lv lead implantation was conventionally performed. in 10 cases, minor manual adjustments to the traced edges of the cs was required in order to obtain a 3d reconstruction of good (in 8 cases) or poor (2 cases) quality. in the remaining five patients, no manual adjustments were made in order to obtain a good reconstruction (figure 1). overall, the quality of the reconstruction was considered to be good in 13 cases (81%) and poor in 2 (12%) (table 2). quality of reconstruction and navigation in all 15 patients, in whom magnetic navigation based on the reconstructed cs was performed, the target sb was successfully engaged with the magnetically steered guidewire. in 13 patients (87%), this was possible based only on the automatically selected vectors ; in 2 cases (13%), manual modification of the automatically selected vectors was required in order to navigate distally in the target vessel. in one case, the lead had to be definitively deployed in an anterior sb due to the lack of acceptable pacing thresholds in other locations. however, navigation through the cs was feasible in this patient using the automatically selected vectors. in two other cases, the lv lead had to be deployed in a different sb than that initially targeted because they could not be engaged with the selected leads. it is interesting to note that in patient 1, after cs angiography, no further fluoroscopy was required to engage the target sb and the position of the lead. one patient suffered from diaphragmatic stimulation at high pacing output (10 v) but the lead required no repositioning because of the lack of diaphragmatic capture with lower output (7.5 v). in 15 patients (93%), the 3d reconstruction of the cs was successfully performed. in case 4, the software was unable to reconstruct the cs and consequently, the lv lead implantation was conventionally performed. in 10 cases, minor manual adjustments to the traced edges of the cs was required in order to obtain a 3d reconstruction of good (in 8 cases) or poor (2 cases) quality. in the remaining five patients, no manual adjustments were made in order to obtain a good reconstruction (figure 1). overall, the quality of the reconstruction was considered to be good in 13 cases (81%) and poor in 2 (12%) (table 2). in all 15 patients, in whom magnetic navigation based on the reconstructed cs was performed, the target sb was successfully engaged with the magnetically steered guidewire. in 13 patients (87%), this was possible based only on the automatically selected vectors ; in 2 cases (13%), manual modification of the automatically selected vectors was required in order to navigate distally in the target vessel. in one case, the lead had to be definitively deployed in an anterior sb due to the lack of acceptable pacing thresholds in other locations. however, navigation through the cs was feasible in this patient using the automatically selected vectors. in two other cases, the lv lead had to be deployed in a different sb than that initially targeted because they could not be engaged with the selected leads. it is interesting to note that in patient 1, after cs angiography, no further fluoroscopy was required to engage the target sb and the position of the lead. one patient suffered from diaphragmatic stimulation at high pacing output (10 v) but the lead required no repositioning because of the lack of diaphragmatic capture with lower output (7.5 v). to the best of our knowledge, this is the first reported use of 3d reconstruction software to create an accurate real - time virtual map of the cs. we demonstrate that this virtual 3d model can be integrated in the magnetic navigation system in order to allow reliable navigation within the cs. on the basis of the reconstructed image, the system allows automatic vector selection to guide a magnetically enabled wire to its desired position within the cs in order to later advance the lv pacing lead. transvenous lv lead implantation is sometimes time - consuming ; it may require prolonged fts and can eventually fail. local complications generated by the delivery system and the occlusive angiogram of the cs (dissection of the vessel, spasm, or even rupture) may lead to implantation failure. however, the main reason for implantation failure remains sometimes challenging anatomical properties of the cs. valvular structures, stenosis, lack of sbs, highly angulated sbs, and tortuous vessels are common findings during crt implantation. despite continuous innovations and technological improvement of the delivery systems, guidewires, and lv leads, 10% of the implantation attempts still fail. implantation procedures are conventionally guided by the use of fluoroscopy, which offer a two - dimensional view of the vessel of interest. in this way, tortuous sbs can appear foreshortened or overlapped, resulting in inaccurate interpretations of the anatomy and making decisions regarding the selection of the appropriate material and manoeuvres required to reach certain vessels more difficult. magnetic navigation of guidewires was meant to help overcome these difficulties and allows access to places in which the use of conventional technology is very challenging. the possibility of performing an accurate 3d reconstruction of a vessel that is integrated into the magnetic navigation system allows more reliable and effective navigation when compared with that guided only by fluoroscopy. the advantage of this reconstruction is that it is performed with the patient in the same position and under the same circumstances (heart rate, rhythm, haemodynamic status, etc.) this should allow more precise navigation than using imported pre - operative 3d images obtained under different circumstances than during implantation. in our experience, it was not possible to obtain a 3d model of the cs, in only one case, probably due to the fact that the diameter of this particular vessel was larger than conventionally encountered. this software was developed to reproduce the anatomy of coronary arteries, which are narrower and less tortuous than the cs, and its side - branches. in two other cases, the sizes of the vessels were no different than those that were more accurately reconstructed. probably, the particular anatomy of these vessels required other fluoroscopic projections than the ones used (lao 30, ap, and rao 30) to create the model. nevertheless, these reconstructions were good enough to allow navigation and deployment of the guidewire into the desired sb. in the remaining 13 cases, a good quality reconstruction was obtained. in 10 cases, the automatically traced contour of the vessel had to be manually modified in order to correct for inaccuracies in the interpretation of the vessel s edge. however, in most of them, the end result of the reconstruction was satisfactory. performing the 3d reconstruction, importing it to the magnetic navigation system, and aligning it to the fluoroscopic views required only a few minutes and did not delay the procedure significantly. the reconstruction of the cs was performed while the implanting physician was selecting the appropriate lead, preparing it, and introducing it into the guiding sheath. this software, and its integration into the magnetic navigation system, offers a reliable 3d view of the cs and its sbs allowing precise navigation within the vessel. in all patients, reconstruction of the cs was feasible and the target sb was successfully engaged by the guidewire. in 87% of these cases, this was achieved only using the automatically selected vectors and, in two cases, manual modifications were required. using the 3d reconstruction as a model for navigation provides much more information than the fluoroscopic views and allows the system (and the operator) to more precisely interpret the sb location, direction, angulation, and length. it is the model that is used to guide the implant, consequently, potentially reducing the use of fluoroscopy and limiting the amount of projections (and contrast injections) required to interpret the angiograms. in all patients, in whom the cs was successfully reconstructed, navigation within the cs was only based on the reconstruction irrespective of whether vectors were automatically selected or manually adjusted. one limitation of this technique is that the 3d model of the vessel is obtained from static images of a beating heart. although the fluoroscopic images used to perform the reconstruction were ecg - gated, it remains a static model that is being used to guide an implantation performed in a beating heart. also, respirator movements are not compensated by this system. consequently, inaccuracies of magnetic navigation based on this 3d reconstructed model may also be due to a lack of compensation for respiratory movements and cardiac cycle. whether developments as rotational angiography will serve as better models for magnetic navigation is another step to investigate. however, nowadays, it is not possible to integrate rotational angiography into the magnetic navigation system. another limitation of the present version of the software is that it allows reconstruction of the body of the vessel and only one sb or bifurcation. in this way, in order to navigate to a different sb than the initially selected, a new reconstruction using the second sb was required. in our view, once the guiding sheath and the guidewire can be remotely advanced using an advancer system similar to the one used for radiofrequency ablation catheters, this software will allow remote lv lead implantations with minimal fluoroscopic exposure. one example is the patient in whom navigation using the reconstructed cs image was enough to engage the desired sb without the use of fluoroscopy. transvenous lv lead implantation is sometimes time - consuming ; it may require prolonged fts and can eventually fail. local complications generated by the delivery system and the occlusive angiogram of the cs (dissection of the vessel, spasm, or even rupture) may lead to implantation failure. however, the main reason for implantation failure remains sometimes challenging anatomical properties of the cs. valvular structures, stenosis, lack of sbs, highly angulated sbs, and tortuous vessels are common findings during crt implantation. despite continuous innovations and technological improvement of the delivery systems, guidewires, and lv leads, 10% of the implantation attempts still fail. implantation procedures are conventionally guided by the use of fluoroscopy, which offer a two - dimensional view of the vessel of interest. in this way, tortuous sbs can appear foreshortened or overlapped, resulting in inaccurate interpretations of the anatomy and making decisions regarding the selection of the appropriate material and manoeuvres required to reach certain vessels more difficult. magnetic navigation of guidewires was meant to help overcome these difficulties and allows access to places in which the use of conventional technology is very challenging. the possibility of performing an accurate 3d reconstruction of a vessel that is integrated into the magnetic navigation system allows more reliable and effective navigation when compared with that guided only by fluoroscopy. the advantage of this reconstruction is that it is performed with the patient in the same position and under the same circumstances (heart rate, rhythm, haemodynamic status, etc.) this should allow more precise navigation than using imported pre - operative 3d images obtained under different circumstances than during implantation. in our experience, it was not possible to obtain a 3d model of the cs, in only one case, probably due to the fact that the diameter of this particular vessel was larger than conventionally encountered. this software was developed to reproduce the anatomy of coronary arteries, which are narrower and less tortuous than the cs, and its side - branches. in two other cases, the sizes of the vessels were no different than those that were more accurately reconstructed. probably, the particular anatomy of these vessels required other fluoroscopic projections than the ones used (lao 30, ap, and rao 30) to create the model. nevertheless, these reconstructions were good enough to allow navigation and deployment of the guidewire into the desired sb. in the remaining 13 cases, a good quality reconstruction was obtained. in 10 cases, the automatically traced contour of the vessel had to be manually modified in order to correct for inaccuracies in the interpretation of the vessel s edge. however, in most of them, the end result of the reconstruction was satisfactory. performing the 3d reconstruction, importing it to the magnetic navigation system, and aligning it to the fluoroscopic views required only a few minutes and did not delay the procedure significantly. the reconstruction of the cs was performed while the implanting physician was selecting the appropriate lead, preparing it, and introducing it into the guiding sheath. this software, and its integration into the magnetic navigation system, offers a reliable 3d view of the cs and its sbs allowing precise navigation within the vessel. in all patients, reconstruction of the cs was feasible and the target sb was successfully engaged by the guidewire. in 87% of these cases, this was achieved only using the automatically selected vectors and, in two cases, manual modifications were required. using the 3d reconstruction as a model for navigation provides much more information than the fluoroscopic views and allows the system (and the operator) to more precisely interpret the sb location, direction, angulation, and length. it is the model that is used to guide the implant, consequently, potentially reducing the use of fluoroscopy and limiting the amount of projections (and contrast injections) required to interpret the angiograms. in all patients, in whom the cs was successfully reconstructed, navigation within the cs was only based on the reconstruction irrespective of whether vectors were automatically selected or manually adjusted. one limitation of this technique is that the 3d model of the vessel is obtained from static images of a beating heart. although the fluoroscopic images used to perform the reconstruction were ecg - gated, it remains a static model that is being used to guide an implantation performed in a beating heart. also, respirator movements are not compensated by this system. consequently, inaccuracies of magnetic navigation based on this 3d reconstructed model may also be due to a lack of compensation for respiratory movements and cardiac cycle. whether developments as rotational angiography will serve as better models for magnetic navigation is another step to investigate. however, nowadays, it is not possible to integrate rotational angiography into the magnetic navigation system. another limitation of the present version of the software is that it allows reconstruction of the body of the vessel and only one sb or bifurcation. in this way, in order to navigate to a different sb than the initially selected, a new reconstruction using the second sb was required. in our view, once the guiding sheath and the guidewire can be remotely advanced using an advancer system similar to the one used for radiofrequency ablation catheters, this software will allow remote lv lead implantations with minimal fluoroscopic exposure. one example is the patient in whom navigation using the reconstructed cs image was enough to engage the desired sb without the use of fluoroscopy. a reliable 3d reconstruction of the cs can be performed using at least two complementary angiographic views. this reconstruction, when integrated into the magnetic navigation system, allows accurate navigation within the vessel of a magnetically steered guidewire to perform lv lead implantations. | aimsleft ventricular (lv) lead implantation is feasible using remote magnetic navigation of a guidewire (stereotaxis, st louis, mo, usa). a novel software that performs a three - dimensional (3d) reconstruction of vessels based on two or more angiographic views has been developed recently (cardiop - b system, paeion inc., haifa, israel). the objective of this paper is to evaluate : (i) the performance of the 3d reconstruction software which reproduce the anatomy of the coronary sinus (cs) and (ii) the efficacy of remotely navigating a magnetic guidewire within the cs based on this reconstruction.methods and resultsin patients undergoing cardiac resynchronization therapy implantation, a 3d reconstruction of the cs was performed using the cardiop - b system. accuracy of the reconstruction was evaluated by comparing with the cs angiogram. this reconstruction was imported into the stereotaxis system. on the basis of the reconstruction, magnetic vectors were automatically selected to navigate within the cs and manually adjusted if required. feasibility of deploying the guidewire and lv lead into the selected side branch (sb), fluoroscopy time (ft) required for cannulation of the target sb, and total ft were also evaluated. sixteen patients were included. in one case, the software could not reconstruct the cs. the quality of the reconstruction was graded as good in 13 and poor in 2. in 10 cases, manual adjustments to the traced edges of the cs were required to perform the 3d reconstruction, and in 5, no adjustments were required. in 13 patients, the target sb was engaged on the basis of the automatically selected vectors. in two cases, manual modification of the vector was required. mean total ft was 23 14 min and the ft required to cannulate the target sb was 1.7 1.3 min.conclusiona 3d reconstruction of the cs can be accurately performed using two angiographic views. this reconstruction allows precise magnetic navigation of a guidewire within the cs. |
substance abuse or drug addiction is one of the most important health issues in every society, which can lead to physical and mental problems (1). detoxification is the first step after a patient is willing to go for the treatment and it has to be done in the most efficient and convenient way. the detoxification method has to overcome the withdrawal syndrome and help the patient to reduce the signs and symptoms as much as possible through the process (2, 3). opioid withdrawal syndrome is a set of symptoms that occurs 6 - 8 hours after discontinuation of opioid class drugs. different methods have been introduced for the treatment of opioid withdrawal syndrome, which include the opioid and non - opioid medications. they consist of opioid antagonists (naloxone, naltrexone) ; complete and partial opioid agonists (buprenorphine, methadone, levo--acetylmethadol), and 2 adrenergic agonists (clonidine, lofexidine) (3, 4). also, their efficacy and success rate has been reported from 10% to 90% (5, 6). tramadol is an atypical analgesic with a dual mechanism of action : serotonin and norepinephrine reuptake inhibition and modest -opioid agonist. a prospective human laboratory study demonstrated that oral tramadol doses of 200 or 400 mg produced modest opioid withdrawal suppression among opioid - dependent research volunteers. these participants were maintained on subcutaneous morphine (60 mg / d) who experienced opioid withdrawal (3). another drug is methadone, which is a synthetic narcotic analgesic compound developed in germany just prior to world war ii. after the war, methadone was studied and found to have effects similar to those of morphine but with longer duration (7). different studies have shown that gabapentin is effective in the reduction of withdrawal syndrome (8, 9). another study suggests that gabapentin reduces opioid use during a 10-day buprenorphine detoxification procedure (10). rapid detoxification, which usually takes less than 5 days is more appropriate for patients using heroin (6). it is recommended that we avoid using anesthesia - assisted rapid opioid detoxification (aarod) because of its mortality and morbidity in favor of evidence - based options for opioid dependence treatment (11). transcutaneous electrical acupoint stimulation (teas) is an acceptable, inexpensive adjunctive treatment that is feasible to implement on an inpatient unit and may be a beneficial adjunct to pharmacological treatments for opioid detoxification (12). a retrospective cohort control study suggests that tramadol may be comparable to buprenorphine in the management of mild to moderately severe heroin withdrawal (13). another study indicated a few clinical differences between parenteral buprenorphine and oral tramadol protocols when used in the management of acute heroin withdrawal. as a consequence, tramadol shows some promise as an opioid withdrawal management medication (14). however, in another research, tramadol was found to have limited detoxification efficacy in moderate to severe opioid withdrawal and substantial risk of seizures as compared to buprenorphine (15). in another study, tramadol compared to clonidine (in the management of heroin withdrawal) was more effective in preventing most withdrawal symptoms (16). a study that used the objective opioid withdrawal scale (oows) to compare the efficacy and safety of tramadol versus methadone showed that tramadol may be as effective as methadone in the control of withdrawal syndrome (17). buprenorphine and methadone appear to be the most effective detoxification treatment compared to 2-adrenergic agonists (18). this study aimed to compare the efficacy of tramadol plus gabapentin versus methadone in treatment of the opiate withdrawal. this clinical trial was conducted on male opiate dependent subjects admitted to shahid beheshti hospital in kerman (iran) in 2013. inclusion criteria consisted of opioid dependency diagnosis based on dsm - iv - tr, substance abuse for at least a year, and age range from 18 to 65 years. the eligible patients were enrolled consecutively. and the exclusion criteria were consumption of other medications such as bzd, tca, corticosteroids, clonidine and codeine, iatrogenic dependency, critical medical conditions like diabetes, acute hepatitis, and other liver diseases. the study was approved by the ethics committee of kerman university of medical with the registration number of k90/530. written informed consent was signed by all participants after a thorough explanation of the study by the physician. they were assured that their information would be kept confidential and they can leave the study whenever they want. a demographic questionnaire was then filled out at the first day of admission and the participants were randomly assigned (using random digit numbers) in two groups by a nurse. the sample size was calculated to find out a 20% difference in withdrawal symptoms considering type i error of 0.05 and 80% power. the control group received 10 to 30 mg of methadone (syrup) in the first day and it was reduced 2.5 to 5 mg each day from the second day. the other group received 200 to 300 mg of tramadol (tablet) at the first and second day for patients using opium and residue and 300 to 400 mg for patients using heroin divided in 3 doses, 100 mg was added in the third day and was reduced to 50 mg each day from the fourth day. nine hundred miligram of gabapentin (capsule) was given to them each day divided in three doses. the patients were visited every day and the withdrawal syndrome and craving were assessed at the first, second, fourth, sixth, and eight day of admission using the cows, arsw and vas (in persian language), which their reliability were confirmed (19). however, the patient was aware of the treatment and all 59 subjects were enrolled in the study. the independent t test and chi - square were used to compare nominal and quantitative variables (mann - whitney u for non - parametric data). 2-way repeated measure analysis of variance (anova) was used to compare the cows, arsw and vas (which was converted to a number from 0 to 10). out of 59 subjects, 29 patients took the methadone treatment and 30 patients received the tramadol and gabapentin treatment protocol. as presented in table 1, the mean age of the patients (33.9 years in methadone group and 32.4 years in tramadol and gabapentin group) and years of dependency (5.5 years in methadone group and 5.9 years in tramadol and gabapentin group) were comparable in both groups ; however, the pre - intervention drug consumption was higher in tramadol and gabapentin treatment group (p = 0.02). marital status, degree of education and occupation were not significantly different in the two groups. as demonstrated in figure 1, the square of arsw score has been compared between two treatment groups at the first, second, fourth, sixth, and eight day of admission. the differences in the arsw score between two groups were not significant (p value = 0.263). as demonstrated in figure 2, the square of cows score has been compared between two treatment groups at the first, second, fourth, sixth, and eight day of admission. the differences in the cows score between two groups were not significant (p value = 0.862). as demonstrated in figure 3, the square of vas score has been compared between two treatment groups at the first, second, fourth, sixth, and eight day of admission. the differences in the vas score between two groups were nearly significant (p = 0.057). in methadone group an upsurge of clinical signs was seen in the third day of treatment, whilst in tramadol and gabapentin treatment group a smooth decrease in severity of clinical signs was seen from the onset of treatment. this clinical trial was conducted on 59 male opiate dependent subjects, which were treated in two different groups. twenty - nine subjects received methadone and 30 subjects received tramadol and gabapentin for treatment. all the patients were aware of their medications. although the usage amount (which was much higher in the tramadol plus gabapentin group, p value = 0.02) and type of drug abused (p value = 0.003) was significantly different in the two groups but the differences in the overall arsw (p value = 0.263) and cows (p value = 0.862) scores between the groups were not significant. the mean square of arsw and cows score at days 1 to 8 were similar. these data suggest that the severity of withdrawal syndrome in the two groups was not significantly different. a similar study, which used the oows instrument for assessment of withdrawal syndrome to compare the efficacy and safety of tramadol versus methadone for treatment of opiate withdrawal showed that tramadol may be as effective as methadone in the control of withdrawal syndrome (17). the differences in the vas score between two the groups were near significant (p value = 0.057). and the craving is higher in the group receiving methadone from the second day of admission even though the usage amount was higher in the tramadol and gabapentin group. the highest craving was at the third day of admission in the methadone group (figure 3). in this clinical trial, we added gabapentin to tramadol to reduce the risk of seizure by tramadol and to reduce the withdrawal syndrome intensity as been noticed in different studies (8, 9). another study suggests that gabapentin reduces opioid use during a 10-day buprenorphine detoxification procedure (10). most of the studies that compared tramadol with buprenorphine suggested that tramadol may be as effective as buprenorphine in the management of opioid withdrawal syndrome (13 - 15). in another study, tramadol has been compared to clonidine in the management of heroin withdrawal and tramadol was more effective in preventing most withdrawal symptoms (16). the findings of our study suggest that the combination of tramadol with gabapentin is an efficient method for opioid detoxification. the strong point of the study is the use of 3 different assessments (arsw, cows, and vas) in the two groups. the weak point of the study is the significant differences in respect to the types and amount of drug abuse in two groups. it is recommended for further studies to assess the patient 's long - term substance use after the detoxification period. the limitation of the study was that the two groups had significant differences in respect to the types and amount of drug abuse and the variables were not adjusted in the final analysis. | background : substance abuse or drug addiction is one of the most important health issues in every society, which can lead to physical and mental problems.objectives:this study aimed to compare the efficacy of tramadol plus gabapentin versus methadone use in the treatment of opiate withdrawal.patients and methods : consenting male subjects who fulfilled the dsm-4 criteria for opiate dependence syndrome (opium, residue, and heroin) were randomly assigned in two groups to receive tramadol plus gabapentin or methadone. assessment tools were adjective rating scale for withdrawal (arsw), clinical opiate withdrawal scale (cows) and visual analogue craving scale (vas). fifty - nine subjects were enrolled and evaluated on days 1, 2, 3, 4, 6, and 8 during their 10 days of admission. twenty - nine participants received methadone and the other 30 received tramadol plus gabapentin for their treatment.results:mean (sd) age of the patients in methadone group and tramadol plus gabapentin group were 33.9 (7.1) and 32.4. (8.1), respectively (p = 0.462). the overall arsw (p value = 0.263) and cows (p = 0.862) scores between the two groups were comparable. the differences in the vas score for craving between the two groups was marginally significant (p = 0.057). the highest vas score was at the third day of admission in both groups and it was generally higher in methadone group.conclusions:the severity of withdrawal syndrome in two groups was not significantly different. the craving was higher in the group receiving methadone from the second day of admission even though the usage amount was higher in the tramadol plus gabapentin group. the findings of this study suggest that the combination of tramadol plus gabapentin is an efficient method for opioid detoxification. |
for many years, the size and complexity of the triticeae genomes, namely, wheat (17 gb, hexaploid), barley (5 gb, diploid), and rye (8 gb, diploid), have hampered the development of genomics and its application to breed crops with improved composition and characteristics designed to satisfy the needs of consumers, processors, and producers. despite the recognition that a reference genome sequence is key to accelerating crop improvement, the triticeae are the last major crops for which a complete genome sequence is not available (feuillet and eversole 2007). thus, the establishment of genome sequence enabled technology platforms for the triticeae has lagged behind advances in other cereal crops such as corn and rice. in the past decade, however, extensive efforts to develop whole - genome and chromosome - specific bacterial artificial chromosome (bac) libraries (allouis. 2003 ; safar. 2004), extensive est collections (itec http://avena.pw.usda.gov/genome/ ; lazo. 2004 ; zhang. 2004), transformation systems, wild germplasm and mutant collections, as well as dna chips have permitted the establishment of large - scale genomics resources and research programs aimed at enabling high - quality sequencing of the triticeae genomes. given their complexity, in particular that of the hexaploid wheat genome, physical maps must be established as a scaffold for sequence assembly as it is simply not possible to achieve a reference genome sequence using whole genome shotgun approaches (feuillet. 2011 ; ariyadasa and stein 2012). in the past 4 years, a number of initiatives such as the international wheat genome sequencing consortium (www.wheatgenome.org ; feuillet and eversole 2007), the international barley sequencing consortium (www.barleygenome.org), the uk wheat consortium (http://www.wheatbp.net/wheatbp/documents/doc_research.php), and the european triticeaegenome fp7 project (www.triticeaegenome.eu) have developed a suite of genomic resources and knowledge to provide the foundation for physical mapping and sequencing the wheat and barley genomes. before the development of these resources, map - based cloning was quite laborious and time - consuming, and consequently, only a few genes have been isolated in the triticeae (bschges. 2002 ; 2008 ; huang. 2003 ; yan. 2003 ; charles. 2009 ; fu. 2009 ; turner. 2005 ; komatsuda. 2007 ; 2008 ; nair. 2010 ; faris. 2010 ; breen. this was due in part to the challenge of walking efficiently along the chromosomes in repetitive sequence regions with the difficulty of identifying unique probes for screening bac libraries. with more than 80 % of the sequence identified as transposable elements, the issues are acute in the triticeae genomes, although the deployment of markers that cross the often unique boundaries between repetitive elements has reduced the problem to some degree (flavell. chromosome walking is no longer necessary and, assuming the genetic resolution is high enough, physical maps enable direct landing at the target site thereby enabling more efficient gene cloning. in the absence of complete genome sequences and given the relatively high gene order conservation (collinearity) observed in the grass genomes, genomics studies in the triticeae have utilized comparative genomics approaches with other grass genomes. to date, five genomes relevant to the triticeae have been sequenced, namely rice, brachypodium, sorghum, maize, and foxtail millet (irgsp 2005 ; ibi 2010 ; paterson. comparisons between these different genomes enable the identification of conserved gene regions that can support molecular marker design and identify candidate genes for traits that are well conserved between species. hence, the high - quality rice genome sequence (irgsp 2005) combined with the sequences of a number of genomes from the other grasses (brachypodium, sorghum, maize) was used to develop molecular markers such as the conserved orthologous set (cos) molecular markers (bertin. 2005 ; quraishi. 2009) and to accelerate discovery of wheat and barley genes (fu. however, genes such as very recently duplicated genes and those involved in end use quality (for example bread, pasta), rapidly evolving genes such as disease resistance genes, as well as genes involved in large regulatory networks are more species - specific, and thus, it is crucial to have access to genomic resources and a reference sequence of the target genome. while for barley, whole genome bac libraries have been produced (schulte. 2011), the generation of bac clones from flow - sorted chromosomes (safar. 2004 ; af. 2010) and chromosome arms (reviewed in dolezel. 2012) has been key to reducing the complexity of the hexaploid wheat genome analyses. typically, libraries of 30,00090,000 bac clones are generated from the flow sorting of approximately a million chromosomes (http://olomouc.ueb.cas.cz/dna-libraries/cereals) to give a coverage of over 10 for the predicted chromosome size (dolezel. the bac libraries are then fingerprinted using snapshot labelling and analysis (luo. 2003), using five restriction endonucleases, and bac contigs are assembled generally using the fingerprinted contig (fpc) software (soderlund. 2010) and good results were obtained in wheat, as exemplified by the construction of the first physical map of the 1 gb wheat chromosome 3b (paux. 2008), the length of the physical contigs was slightly lower than in small model genomes. sequencing bac contigs (choulet. in fact, due to the high level of repetitive dna in the triticeae genomes, very stringent criteria must be used to ensure a reliable assembly with the fpc software. this, in turn, often results in short contig lengths as well as an unreliable assembly in some difficult regions. to address these problems, frenkel. the ltc algorithm reduces the rate of false connections and q - clones by systematically exploring the topological contig structure and performing iterative clone clustering and ordering so that highly reliable and contigs longer than in fpc are recovered. the ltc detects weak connections in contigs obtained by fpc and through iterative steps undertakes their repair. a further improvement in the reliability of physical map construction can be obtained using the power of next - generation sequencing technologies. keygene (www.keygene.com) recently developed a new approach called whole genome profiling (wgp, van oeveren. 2011) that is based on the sequencing of the ends of restriction endonuclease fragments after digestion of bac pools. the application of wgp to a subset of the wheat chromosome 3b physical map (philippe. 2012) demonstrated that this is a viable approach for a complex genome such as wheat and that it reduces the amount of chimeric and misassembled clones compared to the snapshot method while providing sequence tag information that can be used to support pooling strategies for sequencing. thus, robust methodologies and protocols are now in place for assembling reliable physical maps in the triticeae genomes. the full potential of physical maps in supporting map - based cloning and marker development for breeding can only be achieved when the bac contigs are linked sufficiently to genetic and phenotypic maps. thus, once physical maps have been assembled, it is essential that the bac contigs are anchored at high density with molecular markers. this requires the development of thousands of markers followed by their assignation to the genetic and physical maps. in the triticeae, large collaborative efforts have been deployed in the past 15 years to develop est and ssr markers for genetic mapping enabling the construction of genetic maps that carry several hundreds of markers (wheat.pw.usda.gov/gg2/index.shtml). however, it is only with the advent of ngs technologies that high throughput development and genotyping of snp markers progressed to a significant degree in wheat and barley. the deployment of kaspar - based assays for snps in wheat provided the basis for a map of avalon cadenza with 2,923 snps (allen. recently, large transcriptome sequencing and resequencing efforts in australia and the usa enabled the development of a 9k infinium wheat snp - chip (akunov, personal communication). in barley, comprehensive sets of several thousands of snp markers have been successfully developed and mapped in numerous populations (close. 2012). with additional gene - based snp datasets originating from programs in the uk (http://www.wheatisp.org ; allen. 2011) and from 5,000 cos - snps designed in the triticeaegenome project, a 90k snp - chip is currently under design and should be available in the near future (akhunov, personal communication). focusing on the transcriptome enables a reduction in complexity that also can be achieved by the digestion of genomic dna with restriction enzymes and sequencing of selected fragments. genotyping by sequencing (gbs) technologies, developed originally in maize, have been applied to wheat and barley (elshire. 2011 2012) thereby providing another platform for dna sequence - based ordering of reference points along the triticeae genomes. with this approach, 34,396 snps were mapped by gbs in the oregon wolfe barley reference population, while 19,720 snps were mapped in the synthetic w97846 opata85 reference wheat population. because they rely on sequence information, the snp- and gbs - based approaches provide thousands of sequence tags that can be integrated into physical maps in silico thereby increasing the efficiency of anchoring strategies. this more direct integration of bac sequencing and anchoring to genetic maps was carried out successfully in the rice genome sequencing program using bac - end sequences (reviewed in ariyadasa and stein 2012), and it is currently being applied in wheat and barley (ibsc 2012). the in silico, sequence - based anchoring complements the well - established process of identifying known molecular markers in bac pools using pcr (paux. 2008) as well as the broad range of hybridization array technologies that are available (reviewed in ariyadasa and stein 2012). genetic mapping in the triticeae genomes is difficult partly as a result of the very low rate of recombination observed in the centromeric and pericentromeric regions that can represent up to one half of an entire chromosome (birchler. 2009 ; kanisay and dawe 2009). on wheat chromosome 3b, it was estimated that 90 % of crossing over occur in only 40 % of the chromosome, mostly in the telomeric regions, whereas 27 % of the chromosome did not show any crossing over (saintenac. sufficient resolution in ordering physical contigs to genetic maps using molecular markers can be obtained when utilizing mapping populations with high recombination frequencies. this can be achieved using recombinant inbred line (ril) populations of several thousand individuals. two populations of 2,600 and 4,000 rils have been produced for this purpose in wheat (renan chinese spring) and barley (barke morex), respectively, in the triticeaegenome project with the rationale of using parents that are the references for genome sequencing and physical mapping, i.e., chinese spring in wheat and morex in barley. this complements the continued efforts of the international triticeae mapping initiative to develop a reference population for genetic mapping in wheat using a cross between the synthetic wheat w7984 opata m85 population. a new population of 2,039 rils has been established recently with a core set of 42 ssr markers used to genotype the lines and facilitate a community - base effort to build a detailed map (sorrells. (2011) reported 215 doubled haploid lines that were genotyped with 1,446 molecular markers. furthermore, multi - parent advanced generation intercross populations originating from multiple founder lines (typically 8 to 16) and therefore relying on a larger diversity and amount of recombination events than bi - parental populations have been established in wheat (mackay and powell 2007 ; huang. these are currently being tested for anchoring physical maps in different projects. in the regions near the centromeres or within large blocks of heterochromatin where little if any recombination occurs, it is not possible to reliably anchor and order a physical map. targeting these regions is important, however, as it is now clear that genes are present all along the physical maps (choulet. 2011), including in bac clones assigned to the centromeres and to retrotransposable element - rich regions of chromosomes. genes, including the important vernalisation gene vrn - d4, have been mapped to the centromere region (yoshida. 2010). in another study, the characterization of a 0.8-mb dna segment from chromosome 3b that was composed almost entirely of transposable elements except for a small gene island of three conserved genes (breen. thus, an alternative strategy for targeting centromeres and other repetitive sequence - rich regions of the chromosomes where recombination is low has been established following radiation hybrid mapping approaches that are widely used in animal genetics. radiation hybrid mapping relies on assaying radiation - induced chromosomal fragments with molecular markers defined by the physical map under study (riera - lizarazu. the frequency of markers remaining together on the same chromosome fragment defines a measure of how physically close the markers are to each other. rh mapping was evaluated during the construction of the 3b physical map using a panel of 184 rh lines tested with 65 isbp markers. a resolution of approximately 263 kb per break was observed (paux. in particular, for the terminal bin of chromosome 3bl (3bl7 - 0.63 - 1.00), 35 loci corresponding to 32 loci were ordered and confirmed the physical map established using markers ordered by standard recombination - based mapping (paux. 2008). more recently, the same panel was used to establish a high density rh map with 541 marker loci anchored to chromosome 3b spanning a total distance of 1871.9 cr (kumar. the observations on the existence of a core set of coding genes that is conserved among the grass genomes (salse. 2009) have also provided a basis for anchoring chromosome (arm) specific dna sequences to genetic and physical maps (mayer. the so - called chromosome zipper approach was developed originally in barley to determine a virtual order of genes using inference of synteny information along the grass genomes (mayer. incorporation of chromosome arm - specific microarray hybridization information is providing an important cross - reference for the positioning of genes in this framework. in addition, cross - referencing to bac clones that contain particular genes identified by dna hybridisation helps in ordering bac and fpc contigs. typically 4,0009,000 genic sequences per chromosome are found for wheat chromosomes, with some likely to represent gene fragments and pseudogenes (choulet. following their identification, genes conserved between wheat, brachypodium, rice, sorghum, and barley (hernandez. 2012) can then be clustered into syntenic groups and, along with dense genetic marker information, used to define an estimated gene order in wheat and barley. the analysis also identifies predicted genes that may be unique to wheat and barley and thus might be significant in accounting for the specific agronomic attributes of these crops. high throughput hybridization platforms can be used for anchoring physical maps if arrays of mapped markers are hybridized to labelled pools of bacs (rustenholz. the hybridization of dna from complex genomes to microarrays with long oligonucleotides that assay different clusters of multiple snps, small deletion / insertion differences, copy number variants, and presence / absence variation in genomic dna rather than single snps have also been used to assay polymorphisms for genotyping (fu. were hybridised to a barley agilent 15 k expression microarray and allowed 738 barley orthologous genes to be located to their respective bac clones (rustenholz. the study showed that 68 % of the genes identified in the study were syntenic between wheat chromosome 3b and barley 3h. early analyses of ribosomal dna loci in the triticeae (dubcovsky and dvorak 1995) indicated that despite a good conservation between the genomes, specific rearrangements occurred. indications for this conclusion were already evident in bac sequencing and comparison during map - based cloning projects. more recently, the construction of physical maps and whole chromosome analyses demonstrated that 3040 % of the gene complements in wheat and barley do not reside in the conserved syntenic gene order space (choulet. rearrangements including deletions and expansions in the genomes of wheat have been frequent (dubcovsky and dvorak 2007 ; wicker. 2011) and are considered to be the result of waves of retrotransposable elements movements assumed to have occurred in the recent evolutionary history of wheat (charles. 2008 ; choulet. in the case of the rdna loci in the triticeae, a further variable includes amplification events that increase the number of tandem copies of the genes at a given locus (dubcovsky and dvorak 1995). (2011) include the repair of double - strand breaks in dna which can include a dna segment (plus or minus a gene sequence) from elsewhere in the genome. (2011) argued that pseudogenes mostly arise from genome rearrangements, unique gene - domain fusions have been reported to be important in generating a gene that confers temperature - dependent resistance to wheat stripe rust wheat (fu. protein kinase domains were fused with domains that could be recognized as being important in disease resistance gene networks. (2011) in an analysis of the short arm of chromosome 1d (1ds) which exists as a ditelosomic chromosome in a standard genetic stock of wheat used for chromosome sorting. gene sequences for 1ds were assigned to two regions of brachypodium chromosome 2, syntenic regions 1s and 1l, even though these brachypodium regions are clearly differentiated when alignments with gene sequences from the ditelosomics 1as, 1bs and 1al, 1bl are carried out. in addition, the wheat ests mapping to the proximal region of 1ds in independent studies were also missing from the ditelo 1ds sequence dataset (wicker. 2011). in addition to providing the basis for analyzing conserved gene orders on a large scale (as described earlier), comparative genomics has been important in wheat and barley to define conserved features of genes, and several examples are now available for the identification of candidate genes for the phenotypes being studied. the characterization of the vrn genes in wheat and barley (yan. 2003, 2004, 2006 ; yoshida. 2010) was facilitated by comparative analyses across wheat, barley, and rice. the importance of modifications in intron 1 for variation in expression of the vrn genes was established through the analysis of allelic variants. a comparison to variants in lolium perenne (asp. 2011) showed that indels in several different locations within intron 1 could modify vrn1 expression. additional examples include the identification of the bract suppression gene trd1, a gata transcription factor, on barley 1h through fine mapping and anchoring of the phenotype to a syntenic region in rice (houston. 2012), the boron toxicity tolerance gene on barley 4h (sutton. 2007), the rht - d1 gene region on 4d (duan. 2012), and the nac transcription factor on wheat 6b (distelfeld., the functional attributes appeared to be quite different in rice and wheat (distelfeld. 2012) and indicated extrapolating function based on shared dna sequence structure may not always be possible. the unique features of the triticeae genomes emphasize the importance of high - resolution mapping populations and physical maps developed in the target species to enable the de novo identification of genes that are unique to wheat and barley biology, as well as genes underlying qtls. as genes and gene regions of interest 2012) provides a valuable methodology for identifying new alleles in related varieties or wild relatives. the recent progress in high throughput marker development combined with new association genetics panels, physical maps, and survey sequences represent a breakthrough in map - based cloning in the triticeae. in wheat, physical maps of chromosome 1a, 1b, 3a, 3b, and 3d (http://urgi.versailles.inra.fr/gb2/gbrowse/wheat_phys_pub/) as well as survey sequences of all 21 individual bread wheat chromosomes are already available on line (http://urgi.versailles.inra.fr/species/wheat/sequence-repository). in barley, a physical map, survey sequences of sorted chromosomes, several thousand of shotgun sequenced bac clones and whole genome shotgun sequence assemblies were integrated to a physical / genetic genome scaffold providing an excellent template for accelerated map - based cloning and comparative genome - based candidate gene identification (ibsc 2012) (http://mips.helmholtz-muenchen.de/plant/triticeae/barleydisclaimer.jsp ; http://webblast.ipk-gatersleben.de/barley). as an example, in the triticeae more than 15 gene and qtl projects are benefiting already from access to these resources (table 1) and, since the reference sequence of chromosome 3b is underway, 343 scaffolds accounting for 29 mb targeting 74 bac - contigs sequences have already been provided to laboratories worldwide. once candidate genes are identified, functional validation is needed to ascertain the function of the candidate. both reverse and forward genetics approaches are now well established for wheat and barley. stable (agrobacterium, biolistic) and transient (vigs) transformation systems are performed routinely in many laboratories worldwide, and mutagenized collections have been produced in both wheat and barley. in wheat, several mutant populations have been produced in diploid, tetraploid, and hexaploid genetics backgrounds, and projects are underway to establish tilling by sequencing (tsai. tilling resources have been established (caldwell. 2004 ; talam. 2009) that show a wide range in phenotype diversity for the discovery of genes contributing to the complex networks that underpin plant phenotypes. in parallel, natural allelic variation can also be explored by ecotilling approaches and associated to phenotypic variation (xia. 2012).table 1examples of gene and qtl projects benefiting from the access to wheat and barley physical maps and sequenceslocustraitchromosomespeciesorganizationpiuse of physical map / sequence informationyrh52stripe rust resistance1bsemmer wheatuniversity of haifa- israelt. fahima2 physical non - overlapping contigs identified (total of 3.7 mb with estimated gap of 0.4 mb)pv - qtlfiber content (qtl)1blbread wheatinra - francej. salse1 physical map contig (645 kb) anchored to co - segregating cos cg markerqsng.sfrglume blotch resistance (qtl)3bsbread wheatuniversity of zurich switzerlandb. tuberosaregion < 200 kb in the physical map containing 4 candidate genessr2stem rust resistance3bsbread wheatcsiro and murdoch universityw. spielmeyer1 sequenced contig (1.26 mb) spanning the sr2 genetic intervalsv2leaf rust resistance3bsbread wheatinta - argentinamj. dieguez2 sequenced contigs (1, 2, and 3 mb) identified with flanking markers used for marker development, screening of bac library from resistant cultivar and candidate gene identificationsst1solid stem (saw fly resistance)3bdurum wheatuniversity of saskatchewan canadac. pozniak17 sequenced contigs identified for marker development, 8 new cosegrating markers developedqft-3bflowering time (qtl)3bsbread wheatieb - czech republicj. safar38 sequenced contigs identified for marker developmentqcrs.cpi-3bcrown rot (qtl)3bsbread wheatcsiro australiac. feuillet9 sequence contigs identified for marker development and candidate gene identificationyr49yellow rust resistance3dsbread wheatcsiro australiaw. spielmeyercontig at the proximal side identified, contig at distal side not identified yet, and cosegregating contig identifiedcul4tillering3hbarleyuniversity of milan italyl. rossinione bac spanning the cul4 genetic interval and candidate gene identified and sequenced examples of gene and qtl projects benefiting from the access to wheat and barley physical maps and sequences in table 1, a summary is provided for traits and their respective genes that have been defined in wheat and barley. the table focuses on a list of genes on the group 1 and 3 chromosomes in wheat and barley. at a broader level, in addition to identifying the genes for disease resistance, grain quality traits have also been well defined at the dna level. examples include the low and high molecular weight glutenin subunit loci on the group 1 chromosomes important for flour processing quality, and the ha locus on 5d defining the soft / hard attributes of the grain (chantret. increased density of markers along genetic and physical maps using array - based techniques for genes and insertion sequence - based polymorphism markers as well as gbs in wheat and barley enable breeders to perform accurate association mapping studies (heffner. association genetics has only been recently applied to crop plants, (mackay and powell 2007) yet it is an exciting alternative to conventional qtl mapping in bi - parental crosses. it draws on the principle that linkage disequilibrium (ld) tends to be maintained over many generations between loci which are physically linked to one another. panels of non - related lines represent many cycles of historical recombinations compared to typical qtl mapping populations. ld will likely decay very rapidly with genetic distance if the panel used consists of very diverse lines ; thus, correlations between marker and qtl will be identified only if the marker is tightly linked to the qtl (paux. the number of markers needed to accomplish such studies therefore increases with the diversity of the panel, and a balance must be found between the resolution of the panel and the power to detect associations. barley and wheat can now rely on the genomics tools described above to develop large quantities of molecular markers. however, to take the full advantage of these resources, it is essential that adapted association panels are developed. in the past years, several elite panels have been developed for wheat and barley, notably for european germplasm. (2011) described a panel of 195 french - grown elite winter wheat varieties. 2011) used a 455 central european winter wheat varieties panel for association mapping of qtl for grain yield, heading date and fusarium head blight resistance, while cockram. (2010) studied a 500 barley cultivar panel for 15 morphological traits. in the triticeaegenome project, breeders from france, the uk, and germany established a panel of 376 varieties based on (1) general phenotypic levels of adaptation to field conditions, (2) phenotypic homogeneity, and (3) genotypic diversity. this panel has been used for genotyping and phenotyping adaptive traits such as yield, heading date, and plant height. it has been distributed to colleagues in india and argentina who are interested in identifying new alleles in european winter wheat and is accessible for further collaboration. thus, most of the material currently grown in europe for wheat and barley is represented in one panel or another. (2011) are complementary resources to investigate traits in western and central european wheat germplasm that could be used to cross - validate qtl regions. 2011) identified qtl underlying grain yield on chromosomes 1b, 1d, 2a, 4a, 4d, 5a, 5b, and 7a and heading date on chromosomes 1b, 2b, 4b, 4d, 5a, 5d, and 7d. moreover, the potential favorable alleles identified in these panels are more likely to be taken up by breeders as they will be already available in elite germplasm and more amenable for introduction into marker - assisted recurrent selection. finally, genomic selection (gs) has been proposed as an alternative to use marker data in breeding that could correct some of the deficiencies in classical marker - assisted selection (meuwissen 2009) and is the subject of further development in crop plants (heffner. the triticeaegenome panel (376 individuals) represents a good example of the diversity in west european wheat, and it could be used to study gs in wheat. indeed, the size of the triticeaegenome panel is sufficient to be divided into a training set and a validation set, and phenotypic data have already been gathered over 2 years in a total of seven locations. the panel itself is not linked to any commercial breeding programme, so it can be envisaged that both researchers and breeders use the resource. the triticeae community established a unique database, graingenes (http://wheat.pw.usda.gov/), to provide a suite of services for the triticeae and oat communities, including databases, documents, tools, data files, announcements, curation, and community assistance. to date, graingenes stores 76 wheat genetic maps, more than 100,000 genetic markers, and approximately 271,000 wheat ests. these sequences can be searched through a blast server or by using queries to get additional information on genetic mapping data. graingenes also hosts the triticeae repeat databank that comprises 1,717 sequences of wheat transposable elements. another database, harvest (http://harvest.ucr.edu/) that is highly frequented by users of the triticeae community provides access to curated est assemblies of wheat and barley as well as meta data linking to marker resources and orthologs of related grasses (close. 2008). with the developments in physical mapping and sequencing activities, for example, the inra urgi wheat database (http://urgi.versailles.inra.fr/species/wheat) provides access to the physical maps of the triticeaegenome chromosome 1bl, 1as, 3b, and 3d as well as to the 3a physical map which is hosted at the wggrc. chinese spring chromosomes that are constructed under the framework of the iwgsc are being integrated regularly into the database (http://www.wheatgenome.org/projects/iwgsc-bread-wheat-projects/physical-mapping/). a gbrowse displays the physical maps in relation with other datasets (e.g., genetic markers, reference sequences, qtls, and snps). to date, the database stores 26 wheat genetic maps, 19,029 markers, 324 qtls, 10,819 snps, and 544,529 ests. in addition, urgi hosts the iwgsc sequence repository that provides access to the survey sequence assemblies of the 21 chromosomes of chinese spring (http://urgi.versailles.inra.fr/species/wheat/sequence-repository). the urgi database is mirrored at the mips which host equivalent datasets for the barley genome (http://mips.helmholtz-muenchen.de/plant/barley/index.jsp). in support of the international efforts to obtain reference sequences from rice (ricegaas ; http://ricegaas.dna.affrc.go.jp/) and the triticeae, a versatile, easy - to - use online automated tool for annotation, a semi - automated annotation pipeline called triannot has been developed (leroy. 2012). its modular architecture allows for the annotation and masking of transposable elements, the structural and functional annotation of protein - coding genes with an evidence - based quality indexing, and the identification of conserved non - coding sequences and molecular markers. to date, the triannot pipeline is parallelized on a 712 cpu computing cluster that can run a 1-gb sequence annotation in less than 26 h. when evaluated on rice and wheat data sets, triannot systematically showed a higher level of reliability than other annotation pipelines that are not improved for wheat. as it is easily adaptable to the annotation of other plant genomes, triannot should become a useful resource for the annotation of large and complex genomes in the future. the rapid advances in wheat and barley provide the basis for the next steps in the efficient exploitation of the newly available resources for research and breeding. the international consortia discussed in this review have been successful in establishing an interface of cooperation to facilitate the exchange of genome sequence data and to increase the impact of advances on growers and breeding programs. emerging technological advances in physical and genetic mapping, marker development and association genetics, bioinformatics and map - based cloning provide a foundation for step changes in germplasm development that would otherwise take a much longer period of time. | the genomic resources of small grain cereals that include some of the most important crop species such as wheat, barley, and rye are attaining a level of completion that now is contributing to new structural and functional studies as well as refining molecular marker development and mapping strategies for increasing the efficiency of breeding processes. the integration of new efforts to obtain reference sequences in bread wheat and barley, in particular, is accelerating the acquisition and interpretation of genome - level analyses in both of these major crops. |
studies on the causes and effects of allergic diseases have become the topic of several papers recently. allergy is a polygenic disease and its occurrence is affected by environmental factors, most of all those related to civilization progress : air and water pollution, adding preservatives, artificial colorants and other additives to food, exposure to tobacco smoke, mass application of antibiotics. a significant role is also attributed to factors related to life style, such as low physical activity, frequent staying indoors with closed windows or air - conditioning, and diet based on highly processed food. allergy undoubtedly is a civilization disease and the growth of its frequency is mostly observed in countries of high life standard [3 - 6 ]. it is difficult, however, to assess the correlation between allergic diseases and physical growth, when we are not in possession of complete data until the end of growth process. acceleration or retardation of the development observed during childhood not necessarily will continue throughout the whole childhood and adolescence. persons who grow fast in their childhood often mature earlier and earlier finish the growth process, in consequence remaining short as adults. the catch - up phenomenon is also observed, thanks to which the child makes up for developmental delays caused by bad living conditions or diseases. in the present study, therefore, correlation between the occurrence of allergy and adult body size has been analyzed. firstly, it was checked whether the prevalence of allergy is higher in persons from families with high social - economic status. the study was approved by an institutional ethics committee and was performed in accordance with guidelines of the declaration of helsinki of 1975 for human research. the data to be analyzed were obtained from students of the jagiellonian university in krakow and the opole university in opole, poland. information on the socioeconomic status (ses) and allergy occurrence was collected by means of a questionnaire. the socioeconomic status was determined by variables commonly used in poland : place of residence in childhood and parents ' education. in addition, the respondents were asked for self - assessment of the financial standing of their families. to determine the allergy occurrence, the following question was asked : were you diagnosed with allergy during medical examination and what factors are you allergic to ? particular attention was paid to the fact that the responses should concern the cases of allergy found by physicians only. to determine the significance of differences in the prevalence of allergy, depending on the social variables, the chi2 test was applied. the differences in anthropometric parameters, depending on allergy occurrence were assessed by means of a multi - factor analysis of variance. the prevalence of allergy in the study group was 14.6%, (14.6% in women and 14.4% in men). due to the lack of significant differences, the analysis was continued disregarding the division into gender in the further part of the paper (table 1). the prevalence of allergy among children in relation to ses and lifestyle. allergic rhinitis was found in most of the persons surveyed (47), food allergy in 5 students, both types of allergy occurred in 6. the most frequent allergens were house dust mites, pollens of trees, shrubs and grass, dog and/or cat fur and molds. foods included strawberries, cocoa and chocolate and preservatives contained in foods. additionally, 3 people, 2 of whom with allergic rhinitis declared allergies to drugs (penicillin, sulphamidin), 2 people were allergic to apitoxin. the prevalence of allergy in the students who lived in the city in their childhood was much higher in comparison with the students who lived in the country (table 1). a significantly larger number of cases of allergies were recorded in students from high ses families than in those from low ses families (table 1). the prevalence of allergy in the person surveyed was varied, depending on the complex assessment of the socioeconomic status, but not on the detailed elements contained in this indicator, such as parents ' education. body height and relative body mass their values are largely affected by the nutrition method, both quantitatively and qualitatively ; physical activity and incidence of diseases in childhood. this is the reason for considering another factor - the complex social - economic status assessment index - while determining the influence of allergy occurrence on the adult height and bmi. the results of the two - factor analysis of variance did not show any significant differences in anthropometric parameters between people with and without allergies (table 2). however, women and men diagnosed with allergies were, on average, shorter than people without allergies (table 2). as for the equal status groups, the people with allergies were always shorter than those without allergies. in case of the bmi, among students with allergy bmi values were, on average, slightly lower than among students without allergy (table 2). variation in mean height and bmi in relation to social background and the occurrence of allergic diseases. allergy disorders have a multiorgan character, characterized by various symptoms occurring with different intensity. allergy disorders may manifest at various stages of personal development. according to debor 's theory, the ' allergy march ' can be observed. at infant age and in early childhood, the symptoms of food allergy are most often observed, due to immature alimentary system and introducing particular foods for the first time. these are the periods when skin symptoms also appear. at later age, dermal allergy, allergic conjunctivitis, rhinitis, and asthma predominate. however, the fact that many of the surveyed suffered from food allergies in their childhood, can not be excluded. these findings accord with the results of other studies and can not be explained just by a higher ability to detect the disease in persons of high socioeconomic status [9 - 11 ]. a higher socioeconomic status is correlated with a lifestyle not necessarily beneficial for an allergic person. the allergic disease incidence is enhanced by some elements related to the conditions and lifestyle characteristic for this group, such as air conditioning, closed rooms, or little time spent outdoors. people of high status more often use finished foods, most of them highly processed. it has been proven that the socioeconomic status not only bears on the diagnosis of allergic diseases, but also on history of allergy. although allergies are detected in people of high socioeconomic position, the disease usually has a milder history. people of a good financial standing can afford more expensive medicines, holiday travels to regions of low air pollution, the use of vitamins and minerals or special food products. the last two factors are significant in case of food allergies, where the application of the elimination diet is necessary. in case of the elimination of numerous foods, this may cause deficiency of some vitamins and minerals, necessary for the normal development. also, the medicines applied to cure the allergy may affect the biological development of allergic children. most of the studies do not indicate the statistical occurrence of significant differences in the level of development between children with or without allergies. a slightly higher body mass, however, was observed in the former, which probably is a consequence of taking medicaments. slightly higher values of height were found in allergic children from the city poznan in poland. most of the allergic children were from families of high socioeconomic status and this is the group in which the development acceleration is most often observed in. some scientists posit that the allergy occurrence can be a consequence of rapid development and short period of maturing of the immunological system. the hypothesis has been confirmed by a significantly earlier age of sexual maturing noted in girls with allergies and asthma compared with healthy girls. most of the studies, however, indicate the lack of differences in the development, depending on allergic disease occurrence. it presumably is correlated with the status. parents with high earnings and extensive knowledge on proper child care and pro - health prophylactics are able to provide the child with appropriate conditions for development that allow the child to realize its genetic potential, despite the occurrence of the disease. their growing process was finished and body height value is a result of life conditions and style throughout the entire process of childhood and youth. the results, compliant with the studies of other authors, did not present any significant differences in body height, between the allergic and non - allergic people. interesting is the fact that the size of differences is much alike, regardless of whether we consider them in the group of high or low status.. shorter stature in allergic people may also be a consequence of their faster maturing. in case of the bmi, no differences depending on allergy occurrence were observed. this characteristic is much more dependent on the current living conditions and style than on the living conditions of the preceding development periods. the study concerned a selected group - students. in poland, the majority of secondary school leavers continue education at universities, but not all of them. the socioeconomic characteristics of the subjects show that, although they represent groups of various positions, the people of the lowest income were not among them. summarizing, the results of the tests presented in this study indicate that allergic diseases may affect the growing process. however, upon providing appropriate conditions, the growth of children with allergies is not different from the control population. | the aim of the paper was to asses the relationship between socioeconomic status, the prevalence of allergy and physical development. the data were obtained from 478 female students and 195 male students aged 19 - 24. the prevalence of allergy in the group surveyed was 14.6% (14.6% in women and 14.4% in men). allergic diseases were more frequent in students of high socioeconomic status. the results of the analysis of variance did not show any significant differences in anthropometric characteristics between students with and without allergies. however, women and men with allergies diagnosed are on average shorter than people without allergies. the dependency is also visible after adjusting for socioeconomic status. as for the equal status groups, the people with allergies are always shorter than those without allergies. summarizing, the results of the tests presented indicate that allergic diseases may affect the growing process. however, upon providing appropriate conditions, the growth of children with allergy is not different from the control population. |
paediatric obesity is at epidemic proportions, and is associated with significant short- and long - term medical, psychological and social morbidities. although, the global advocacy for physical activity recommend sports participation as a key strategy to address the growing burden of childhood inactivity it is currently unclear whether sports participation can be an effective tool for the prevention of paediatric obesity. significant limitations in this body of literature must be addressed before evidence - based policies can be articulated under the assumption that sports participation provides a buffer against the onset of paediatric obesity. a recent systematic review has highlighted the substantial limitations that are present within the research examining the relationships between sports participation, physical activity and obesity. an over - reliance on cross - sectional designs makes causal inferences impossible. in the few longitudinal studies that have been reported, none have used either objective measures of physical activity or body composition, relying on simple proxies for body composition such as the body mass index (bmi). the use of self - reported measures of physical activity is likely to inflate the observed relationships between sports participation and physical activity, particularly if reported concurrently. further, while bmi is an adequate measure of adiposity in childhood, it can potentially bias results towards findings of no difference in sporting contexts due to an inability to observe the differences between overweight non - participants and muscular participants. in those longitudinal studies that have also used objective measures of physical activity, none have addressed the issue of bi - directionality specifically between sports participation and adiposity / physical activity. furthermore, issues such as these have been articulated as important limitations beyond the literature pertaining to sports participation, and are also pertinent to research in physical activity. the purpose of this study was to address the limitations of previous research by investigating longitudinal associations between sports club participation, objectively measured physical activity, and adiposity [as measured by both bmi and fat mass index (fmi) ]. specifically, several important research questions (rq) have been addressed : (1) what are the cross - sectional associations between sports club participation, objectively measured physical activity, and adiposity ? (2) do measures of physical activity and adiposity predict subsequent sports club participation ? (3) does sports club participation predict subsequent measures of physical activity and adiposity ? and (4) do changes in sports club participation predict changes in objective measures of physical activity and adiposity ? as a secondary objective, this study also compared the strength of the associations between sports participation and bmi / fmi. the gateshead millennium study (gms) is a birth cohort of adolescents born between may 1999 and june 2000, described in detail elsewhere. briefly, all children born to gateshead - resident mothers in pre - specified recruiting weeks were invited to participate. data for the current analyses were collected at three separate data sweeps : in 20062007, 20082009 and 2012, corresponding to age 68, 810 and 1113 years, hereafter referred to as 7, 9 and 12y. for each phase, all families who had not previously opted - out from the cohort were sent a letter and information leaflet inviting them to take part. informed written consent was obtained from the main carer of each child, and children provided assent to their participation. ethical approval for the study was granted by gateshead and south tyneside lrec (for the 7y data sweep) and newcastle university ethics committee (9 and 12y). at each timepoint, height was measured to 0.1 cm with a leicester portable height measure (chasmors, london, uk) and weight measured to 0.1 kg in light indoor clothing, and bmi and bmi z - score according to uk 1990 data were derived. the fat mass index (fmi) was the measure of body fatness used in the present study. we have shown previously that having a fat mass outcome greatly increased the ability to detect associations compared to having only a proxy for fat mass as the outcome measure between ages 7 and 9y. fat mass was estimated from tanita bioelectrical impedance (tbf-300ma) by applying constants for the hydration of fat - free mass having first estimated total body water using validated sex and age - specific prediction equations. fmi was then calculated by dividing fat mass by height squared. at 7, 9 and 12y children were asked to wear an actigraph gt1 m accelerometer on the right hip for 7 days, removing it only for bed and water - based activities. they were also given a time sheet to log when the monitor had been worn. data were collected in 15 s sampling intervals (epochs) but collapsed to 60 s epochs when summarised. three constructs of physical activity (total volume of physical activity in mean counts per minute, cpm ; amount of time spent in moderate - vigorous intensity physical activity (mvpa) (minutes, and proportion of time spent in mvpa) ; and one of sedentary behaviour (proportion of time spent in sb)) were used in the present study. the cut - point of 3200 cpm was used to define the threshold for mvpa, and 1100 cpm for sedentary behaviour (defined as no trunk movement as measured by accelerometry). the actigraph gt1 m model has been shown to have a consistent bias of 9% relative to model 7164 which has been used widely in previous research. retest reliabilities of approximately 70% for each of the constructs with a minimum of 6 h recording per day. at 9 and 12y children self - completed the youth sports survey questionnaire (adapted from godin and shepherd 1985) assessing which school- and outside - school sports clubs they had participated in recently, time spent at each club and how many times per week they attended. total time spent in each club per week was calculated, and times for all clubs summed and used in analyses. linear regression analyses were used to test for both cross - sectional and longitudinal associations ; coefficients and 95% ci are reported, with r and p value. socio - economic status (ses) was described using townsend scores, an area - based measure derived from the uk census in 2001, and divided into quintiles. sports club participation was the initial dependent variable, before being one of the independent variables in the analyses with 12y fmi, 12y bmi and 12y bmi z - score as dependent variables. the adjusted models are presented with each independent variable included one at time with adjustment for ses and sex, due to their role as likely confounders of all the associations tested, but not including the other independent variables due to likely collinearity. a total of 609 participants were involved in data collection at 7y, 585 at 9y and 525 at 12y. of these, 209 (50%) children took part in a school - sports club, and 342 (82%) in an outside - school sports club. one hundred and thirty - five (32%) children participated in both a school- and an outside - school club. at 12y, 512 children answered the question, 324 participated in any club (63%) ; 208 (64%) took part in a school - sports club and 252 (78%) in an outside - school sports club. one hundred and thirty - six (42%) children participated in both a school- and an outside - school sports club. two hundred and thirty - six children participated in a sports club at both 9y and 12y. in univariate analyses, 12y sports club participation was significantly associated with all accelerometry variables. after adjustment for sex and ses, 12y sports club participation was positively associated with total cpm and negatively associated with % sb, but no longer associated with mvpa (table 2). at 9y, sports club participation was not associated with 9y accelerometer measured physical activity or sedentary behaviour (data not shown). in univariate regression analyses, 12y fmi was significantly inversely associated with 12y sports club participation, and mvpa and total activity as measured by accelerometry. neither 12y bmi nor bmi z score were associated with 12y sports club participation, although both were significantly inversely associated with mvpa min, and bmi was also associated with total activity. sports club participation at 9y was highly predictive of participation at 12y, even after adjustment for sex and ses (p = 0.001). no other measure of either accelerometry or body composition at 9y predicted sports club participation at 12y. none of the 7y variables were associated with 9y sports club participation, although fmi (p = 0.089) and bmi z score (p = 0.065) approached significance (data not shown). at 9y, sports club participation predicted all 12y body composition measures. including 12y sports club participation as a covariate introduced co - linearity issues so analyses were performed separately (table 3). sports club participation at 9y did not predict physical activity as measured by accelerometry at 12y. there were no significant associations between percentage change in sports club participation and percentage change in fmi or accelerometry from 9y to 12y. percentage change in sports club participation from 9y to 12y did not predict 12y body composition. longitudinal associations between sports club participation, body composition and physical activity from childhood to adolescence. this study addressed important limitations from previous research in order to provide evidence on the associations between sports club participation, objectively measured physical activity, and adiposity in childhood and adolescence. firstly, we found that sports club participation at the age of 12 years was not associated with bmi or bmi z - score at the same age, but was associated with fmi. this highlights the benefits of our study relative to previous work, as despite its prevalence as a measure of adiposity in paediatric sports participants, simple proxies of adiposity (bmi) have been unable to detect associations convincingly in the way that our body composition measure has. regular physical activity in childhood, including sports participation, is associated with increases in bone density and skeletal muscle power. such benefits render bmi as potentially ineffective as a measure of adiposity, and this may underpin the lack of association between sports participation and paediatric obesity that is currently evident in the literature. in contrast, this study found no cross - sectional association between sports club participation and either bmi, bmi z score, or fmi at 9 years of age. given that the contribution of sports club participation to improved levels of adiposity is achieved directly through its contribution to physical activity, this finding may reflect greater levels of active free play outside of sports at age 9, or increased levels of sedentary behaviours outside of sports at age 12, although in our study there was no association between sports club participation and accelerometry at 9y. in a us sample, at age 9 children engaged in approximately 54 min of mvpa on every day of the week, however, by the age of 12 this dropped to approximately 36 min per day. the recorded levels of mvpa in the current sample were even lower, probably due to the use of different cutpoints. thus, sports participation may become an increasingly significant source of physical activity over late childhood and early adolescence when physical activity is likely to decline dramatically and rates of obesity incidence are highest. we found that physical activity and body composition at age 9 did not predict sports club participation at age 12. this provides some evidence that sports club participation is the source of increased physical activity and decreased levels of adiposity, rather than a consequence of these variables. this finding reinforces that sports can be an appropriate avenue to promote health for all children, regardless of weight status or physical activity levels. national studies conducted in the united states have concluded that more than half of all youth who are obese participate in organised sports, and more than one quarter of organised sport participants are overweight or obese. sports club participation at age 9 was strongly associated with sports club participation at age 12. this finding shows that sports club participation in childhood tracks into late childhood and early adolescence. this is unsurprising given typically low levels of dropout from organised sports in this age group. it remains unclear however, whether sports club participation would track far beyond early adolescence because adolescence is a period of rapid acceleration in the dropout rate from organised sports. evidence shows that there are low to moderate levels of tracking of physical activity from childhood to adolescence. however, more research is needed to assess the level of tracking of organised sports participation and its associated health trajectories through this period and into adulthood. importantly, we found that sports club participation at age 9 predicts measures of body composition at age 12. given the degree of tracking from 9y to 12y, sports club participation predicts body composition longitudinally in part because sports club participation tends to track from ages 9 to 12. the long - term health benefits of sports club participation are most likely evident because children maintain their participation in sports clubs, rather than sports club participation providing any long - term protective effects. although participation in sports clubs may contribute to concurrent levels of physical activity, they are unlikely to provide children with the skills or motivation to maintain healthy lifestyles. for example, we found that sports club participation at age 9 did not predict levels of physical activity at age 12. therefore, it is imperative that if organised sports are to be used to promote physical activity and reduce the burden of adiposity in childhood the focus of policy and intervention should be on preventing drop out from organised sports, and for encouraging uptake in children from poorer areas. the time period of late childhood / early adolescence appears to be critical for both declining mvpa, increasing incidence of new cases of obesity, and increased excess weight gain, so preventing the decline in physical activity in late childhood should be a priority. this is particularly so in areas such as britain where the participation rate in organised sports is already high and while the impetus of the olympic legacy is still strong. it is important to also consider non - sport mvpa as this should make an important contribution to overall mvpa, even for those children and adolescents involved in sports clubs. firstly, the use of objectively measured physical activity removes potential biases in the concurrent self - reporting of sports club participation and physical activity. the beta coefficients presented in the results are also likely to be conservative estimates ; differences in intra - individual variation in physical activity produce an intraclass correlation coefficient of approximately 0.5, and this value can be used to correct measurement error (coefficient / icc), assuming that all measurement error stems from intra - individual variability. secondly, the use of fmi, in addition to bmi, as a measure of body composition allows a greater sensitivity to detect the health benefits of sports club participation. one limitation of the present study was that sedentary behaviour was defined as no movement of the trunk and based on a cutpoint derived from a calibration study of preschool children. lower cutpoints are more popular now, but the optimum cutpoint is unclear at present, and one recent study found that the cutpoint used in the present study provided stronger associations with cardiometabolic health than alternative cutpoint. further limitations include the necessity of a self - report measure of sports club participation, with the potential problems of recall bias. however, as we are asking the children about regular events we believe the questionnaire is robust enough to deal with this issue. the increased associations observed at 12y over 9y may reflect greater accuracy of recall when the children are older, or an increase in the intensity and focus of their sports club participation. the conservative choice of accelerometer cutpoints may have reduced the levels of mvpa observed, but this is unlikely to have affected the associations ; rather there would be a different dose response. present study findings are tentative, as the use of objective measures of physical activity and a measure of adiposity will need to be replicated in other populations / settings. this study suggests that that sports club participation may be associated with decreased levels of adiposity. furthermore, the health benefits of sports club participation in childhood are likely generated from continuous participation in sports clubs, rather than any long - term or protective effects that may be provided by participation. participation in sports clubs tracks strongly from childhood to early adolescence.participation in sports clubs at age 12 years was associated with lower body fat.children's participation in sports clubs could be encouraged as a way to minimise an increase in body fat.participation in sports clubs should be encouraged in young children at risk of overweight or obesity, particularly those from more deprived areas. children 's participation in sports clubs could be encouraged as a way to minimise an increase in body fat. participation in sports clubs should be encouraged in young children at risk of overweight or obesity, particularly those from more deprived areas. | objectivesseveral important research questions have been addressed : (1) what are the cross - sectional associations between sports club participation, objectively measured physical activity, and adiposity ? (2) do measures of physical activity and adiposity predict subsequent sports club participation ? (3) does sports club participation predict subsequent measures of physical activity and adiposity ? and (4) do changes in sports club participation predict changes in objective measures of physical activity and adiposity?designlongitudinal and cross-sectional.methodsdata from the gateshead millennium study birth cohort (n = 609 at age 7 years) were analysed for associations between adiposity, sports club participation and accelerometer - measured physical activity from ages 7y to 9y to 12y.resultsseventy-two per cent of 9 year olds and 63% of 12 year olds took part in a sports club. sports club participation was significantly associated with overall accelerometer - measured physical activity at 12y (coefficient = 0.0.09 ; 95% ci : 0.010.16) but not 9y. an inverse relationship between fat mass (estimated from bioelectric impedance) and sport club participation, and between fat mass and accelerometer - measured physical activity was observed at 12y, but not 9y. sports club participation at 9y was highly predictive of participation at 12y. sports club participation was significantly associated with socioeconomic status ; fewer children from poorer areas took part.conclusionssports club participation in adolescence may be associated with decreased levels of adiposity. furthermore, the potential benefits of sports club participation for adiposity are likely generated from continuous participation in sports, rather than any long - term protective effects. |
the tomato belongs to the solanaceae family that includes more than 3,000 species. in particular, the section of the lycopersicon genus solanum consists of 13 species or subspecies : the cultivated tomato, solanum lycopersicum, which is the only domesticated species, and 12 wild species (such as s. chmielewskii, s. habrochaites, s. pennellii, and s. pimpinellifolium). its world production is estimated to be around 159 million tons, while the average annual fresh tomato consumption is 18 kg per european and 8 kg per capita in the us. in the last few years, tomato consumption has further increased since tomato fruits supply both fresh market and processing products such as soups, juices, purees, and sauces. the economic research service of the usda estimates that 35% of raw tomatoes are processed into sauces, 18% into tomato paste, 17% into canned tomatoes, 15% into juices, and 15% into catsup. the tomato fruits, like those of many other plant species that are part of our diet, are an important source of substances with known beneficial effects on health, including vitamins, minerals, and antioxidants. indeed, tomato fruit consumption has been associated with a reduced risk of inflammatory processes, cancer, and chronic noncommunicable diseases (cncd) including cardiovascular diseases (cvd) such as coronary heart disease, hypertension, diabetes, and obesity. antioxidant metabolites are a group of vitamins, carotenoids, phenolic compounds, and phenolic acid, with health - enhancing effects on our body [2, 3 ]. the total antioxidant activity of tomato fruits is commonly classified into hydrophilic and lipophilic ones. the first is conferred mainly by soluble phenolic compounds and vitamin c and shows a significant impact on total antioxidant activity (83%), while the latter is conferred by carotenoids, vitamin e, and lipophilic phenols (17%). many factors such as genetics (cultivar or variety), environment (light, temperature, mineral nutrition, and air composition), and cultural practices (ripening stage at harvest and irrigation system) affect the chemical composition of tomatoes [5, 6 ]. in this paper, a global point on tomatoes nutritional importance and mechanisms of action of different phytochemicals against inflammation processes according to the last discoveries is discussed. recently, significant progresses have been made in order to improve the levels of human health promoting compounds in tomato fruits through metabolic engineering and/or breeding [7, 8 ]. however, breeding work is complicated by the complex nature of many of these characters, which often requires quantitative genetic approaches for the identification of genes and qtls (quantitative trait loci) involved in their regulation. in fact, the synthesis of many of the compounds responsible for the tomato nutritional quality is the result of coordinated activities that involve many of the primary and secondary metabolism pathways regulated by developmental, physiological, and environmental signals. therefore, in this paper, we will summarize the recent progress made to improve the nutritional quality of tomato fruits according to current state of the art. regular consumption of tomato fruit and its products is associated with lower risk of cncd and several types of cancer and inflammation because of interaction of phytochemicals with metabolic pathways that are related to inflammatory response and oxidative stress. some evidences support a role for tomato products in the prevention of lipid peroxidation that is a risk factor of atherosclerosis and cardiovascular disease. high dietary intake of tomato products can reduce ldl cholesterol levels and increase ldl resistance to oxidation. reported that tomato consumption during a meal attenuated postprandial lipemia - induced oxidative stress and associated inflammatory response. an important role of tomatoes has been also attributed to the conservation of dna stability. daily intake of a tomato drink called lyc - o - mato significantly reduced (by about 42%) dna damage in lymphocytes subjected to oxidative stress. these effects are related to the presence of several phytochemicals in raw tomatoes and their products. table 1 shows the general effects of tomato bioactive compounds on health, while table 2 summarizes the main evidences about anti - inflammatory and antioxidant activities. they include provitamin a carotenoids, such as -carotene and -cryptoxanthin, and non - provitamin a carotenoids, such as lutein and lycopene. more than 600 carotenoids were identified in nature, among which around 40 are present in food normally included in human diet.. for example, carotenoid content of various tomato varieties grown in ireland differed from those grown in spain. several studies have reported the health benefits of carotenoids related to their antioxidant power, such as immune system stimulation and antitumor activity [18, 45, 70 ]. dietary supplements of carotenoids may act as moderate hypocholesterolemic agents, as a consequence of their inhibitory effect on macrophage 3-hydroxy-3-methyl glutaryl coenzyme a (hmgcoa) reductase, the rate - limiting enzyme in cholesterol synthesis. carotenoids cause changes in the expression of many proteins participating in cell proliferation and signaling pathways. for example, lycopene is associated with reduction of cyclin d1 protein, a known oncogene overexpressed in many primary tumors. in addition, lycopene can increase expression of several differentiation - related proteins, such as cell surface antigen (cd14), oxygen burst oxidase, and chemotactic peptide receptors. according to scientific evidences, the carotenoid intake from tomatoes is the most important nutritional contribution of this fruit, and therefore, the highest weight in the index of antioxidant nutritional quality is attributed to carotenoids content. tomatoes contain 840 g per gram fresh weight of lycopene, about 80% of total dietary intake of this carotenoid [73, 74 ]. field - grown tomatoes appear to contain higher levels of lycopene, ranging from 5.2 to 23.6 mg/100 g fresh weight (fw) than greenhouse - grown tomatoes (0.1 and 10.8 mg/100 g fw). accumulation during fruit ripening results from the downregulation of the lycopene cyclase gene (crtl), a gene conserved through evolution from the time of cyanogenic bacteria. lycopene is a polyunsatured molecule containing 13 double bonds that can exist in trans- and cis - configurations. in fresh tomatoes, lycopene is mainly found in trans - conformation, while thermal treatments, light, acids, oxygen, and digestion can cause transformation into the more bioactive cis - form. lycopene is the main phytochemical in tomato fruits for its strong antioxidative role associated with its ability to act as free radical scavengers from reactive oxygen species (ros), generated by partial reduction of oxygen. they are produced either from normal cell metabolisms in situ or from external sources (pollution, radiation, and cigarette smoke). radical accumulation in the body generates a phenomenon called oxidative stress that results from an imbalance between formation and neutralization of ros / rns in cells. one of the most undesirable effects of ros is lipid peroxidation with consequent formation of radicals. these processes play a major role in the aging and development of chronic and degenerative illness such as cancer, atherosclerosis, autoimmune disorders, cataract, rheumatoid arthritis, and neurodegenerative and cardiovascular diseases. lycopene can transform the high reactive free electron on dna to a more stable free radical by delocalization along its conjugate 13 double bonds. ros can activate the transcription nuclear factor kappa b (nfkb) that gives rise to the expression of genes of pro- and anti - inflammatory cytokines and their subsequent production. anti - inflammatory cytokines such as il-10 are produced to control inflammation, while proinflammatory cytokines including tumor necrosis factor - alpha (tnf-), il-6, and il-8 increased the inflammatory response. doses of 10 mg lycopene / day administered for 3 months decreased psa (prostate specific antigen) level, tumor grade, bone pain, and urinary tract symptoms in patients with metastatic prostate cancer, interfering with growth factor receptor signaling and cell cycle progression. in addition, lycopene can inhibit other cancer types (breast, colorectal, endometrial, lung, oral, and pancreatic). nevertheless, according to some studies, lycopene is not potent enough to be clinically useful as a drug for prostate cancer because high concentrations (above 1 mol / l) are needed to achieve significant response in humans [8688 ]. in presence of diseases such as cancer and cardiovascular diseases, higher levels of lycopene ranging from 35 to 75 mg per day may be required. lycopene can modulate cyclooxygenase pathways and xenobiotic metabolism because -carotene and lycopene supplementation interact with the metabolism of linoleic acid, resulting in either an increase (-carotene) or decrease (lycopene) in plasma concentration. an 83% reduction in prostate cancer risk was observed in a group of men enrolled in the physician 's health study, with the highest plasma lycopene concentration (0.40 many chronic diseases and increasing of mortality are directly related to the global obesity epidemic (body mass index 30 kg / m), which started in the 1980s. serum concentrations of c - reactive protein (crp), tumor necrosis factor (tnf-), and interleukin (il)-6 are significantly correlated with weight, body mass index (bmi), and waist circumference. these aspects assume a major importance considering that a link between skeletal muscle and adipose tissue has been reported and related with the control of body weight, muscle mass, and fat mass. in particular interleukin (il)-15 and tnf- play an important role in crosstalk between adipose tissue and skeletal muscle. il-6 and tnf- contribute to liver production of crp and atherosclerosis by inducing insulin resistance and upregulating the expression of other inflammatory mediators. di renzo. reported that, in italian caucasian females, the fat mass percentage (fm%) is a main factor of an increase in il-6 production and insulin resistance. in particular, 174 g / c polymorphism in il-6 promoter has been recognized as a marker which could contribute to identify vulnerable individuals at risk of age and obesity - related diseases. there is now strong evidence for the benefits of tomatoes in terms of protection against diseases associated with metabolic syndrome and obesity. in particular, lycopene reduces transcript levels of proinflammatory cytokines with downregulation of il-6 expression [16, 17 ] reported the effect of tomato - derived lycopene consumption on markers of inflammation and oxidative stress in a group of patients with extreme obesity. obese patients showed abnormally higher markers of inflammation and oxidation products and lower plasma carotenoids compared to control subjects (0.54 versus 0.87 g / ml). following lycopene treatment, a significant elevation of plasma carotenoids, specifically lycopene, occurred in the treatment versus the placebo group. kuopio ischemic heart disease risk factor study examined the relation between serum antioxidant and intima - mediated thickness of the common carotid artery, a marker related to the risk of having an acute coronary event. lower levels of plasma lycopene were showed in men who had a coronary event compared with men who did not. several studies demonstrated that anti - inflammatory effects derived by tomato products consumption were superior to that of lycopene delivered as a single compound [27, 95 ]. -carotene is considered a provitamin because it can be converted into retinol, a compound essential for vision. it is known as a strong antioxidant and the best quencher of singlet oxygen. at low partial pressures of oxygen, such as those found in most tissues under physiological conditions, -carotene was found to inhibit oxidation. in tomatoes grown in a field located in the s. marzano area, in commercial cherry tomato varieties, -carotene amount reached 1.2 mg/100 gr fw. a study found that both -carotene and lycopene (2.5 mmol / l, 2 h) totally abolished tnf--induced ros levels (1 ng / ml, 16 h) in tnf--treated human umbilical vein endothelial cells. this was due to the redox balance protection and to the maintenance of nitric oxide (no) bioavailability. erythema formation was significantly diminished when -carotene was applied on human skin or with a dietary intervention alone or in combination with -tocopherol for 12 weeks. this could be due to stereospecific interactions with retinoic acid receptors in the artery wall.. found that low concentrations of serum -carotene may be associated with an increased risk of chf (congestive heart failure) and cardiac death in men. the initial antioxidant activity of -carotene is followed by a prooxidant action at high oxygen tension. this mechanism may be related to adverse effects observed under the supplementation of high doses of -carotene. a study reported that supplements in doses of 20 mg daily for 58 years had no protective effect on macrovascular outcomes or total mortality of diabetic male smokers. reported that there was no significant difference between risks of lethal prostate cancer with the use of -carotene during radiation therapy compared with that of placebo. other trials about -carotene supplementation will be required in order to clarify the protection role associated with -carotene. lutein is a yellow carotenoid synthetized in chloroplasts and chromoplasts and found in high quantities in leaves whose main function is to contribute to photosynthesis [102, 103 ]. it is one of the most widely found carotenoid xanthophyll pigments in fruits and vegetables normally consumed. average lutein concentration in raw tomato was reported up to 32 g/100 g fw, while guil - guerrero and rebolloso - fuentes reported a content up to 800 g/100 g fw in cherry variety. increasing interest around this compound was due to its important role in preserving eye health in association with zeaxanthin. the improvement of visual function and symptoms was also reported in atrophic age - related macular degeneration (armd), a condition including genetic, cardiovascular, nutritional, and environmental factors [22, 23 ]. nevertheless, up to date, the relationship between the consumption of lutein and maintenance of normal vision has not been clearly demonstrated. epidemiological studies in vitro and in mouse model demonstrated that increased dietary intake of lutein is protective against the development of early atherosclerosis in human and animals through the reduction of inflammation and oxidative stress in the artery wall. reported that there was an inverse correlation between the risk of developing a myocardial infarction, adipose tissue lutein content, and dietary lutein intake. furthermore, carotid artery intima - media thickness appears to be inversely correlated with serum lutein concentration. armoza. studied the mechanisms involved in the action of tomato products in endothelial cells and demonstrated that inhibition of nf-b signaling may be one of the main mechanisms employed by lutein and lycopene to reduce inflammatory leukocyte adhesion to endothelium. reported that the regular consumption of lutein is associated with an increase of the capacity of dna repair in lymphocytes and a major resistance of dna to endogenous damage. nevertheless, a real correlation between the consumption of lutein and protection of dna, proteins, and lipids from oxidative damage has not been demonstrated yet. however, tomato products are also sources of other compounds such as vitamins a, b, and e. also these compounds have important effects on human health that will be described below. vitamin e family includes eight molecules : -, -, -, and -tocopherol ; and -, -, -, and -tocotrienol. this classification is based on the nature of the isoprenoid chain ; tocopherols contain a phytyl chain while tocotrienols contain a geranylgeranyl chain. tocopherols are the most abundant vitamins in tomato plant and contribute to maintain optimal plant photosynthesis rate under high levels of stress. reported a vitamin e level in tomatoes between 0.17 and 0.62 mg/100 gr fw. they demonstrated a high synergistic effect of -tocopherol - lycopene mixtures, and also for lycopene--carotene, lycopene - lutein, and lutein--carotene mixtures. this effect could be due to the fact that electrons can transfer from the carotenoid to the -tocopherolxyl radical to regenerate -tocopherol. an analogous mechanism has been suggested for the interactions between vitamin c, carotenoids, and -tocopherol. a synergistic interaction between ascorbic acid and -tocopherol during the process of lipid peroxidation is well known. decreasing risk of advanced prostate cancer was associated with increasing dose of supplemental vitamin e uptake among men in the screening arm of the prostate, lung, colorectal, and ovarian cancer screening trial. in a study carried out on finnish men and women, those who ingested higher intake of dietary vitamin e showed a decreased incidence of type 2 diabetes. on the contrary, in a trial with a 10-year followup, with alternate - day doses of 600 iu (international units) vitamin e, similar results were reported for the cardiovascular events and mortality in healthy women that ingested 600 iu of natural - source vitamin e. however, a study that evaluated the effect of adding tomato extract to the treatment regime of moderate hypertensives with uncontrolled blood pressure levels indicated a significant correlation between systolic blood pressure values and level of antioxidant activity. l - ascorbic acid and dehydroascorbic acid are the main dietary forms of vitamin c, a labile molecule with reducing property. it is a water - soluble compound easily absorbed but it is not stored in the body. reported that salad tomatoes grown in field conditions contained between 15 and 21 mg/100 g fw, while a range of industrial grades of tomatoes had a mean value of 19 mg/100 g fw. reported a vitamin c content between 8.0 and 16.3 mg/100 g fw. ascorbic acid (asa) content in fresh tomatoes depends on genotype, climatic conditions, fruit development, maturation, senescence, and time of storage. asa content in tomato fruit increases reaching a maximum and then began to decline with ripening. reported a maximum level of 94.9 mg/100 g after 74 days from fruit set and slow reducing of its content with the color change. this decrease is concomitant with the increase in the activity of ascorbate oxidase, a cu - containing enzyme that catalyses the oxidation reaction of ascorbate to dha with the reduction of molecular oxygen to water. adults have a mean body pool of 1.22.0 g of ascorbic acid that may be maintained with 75 mg / day of ascorbic acid. insufficient intakes of ascorbic acid lead to scurvy, a disease characterized by dry skin, open sores on the skin, weariness, impaired wound healing, and depression [119, 120 ]. most of the known functions of ascorbic acid are correlated with its ability as electron donor and potent antioxidant in humans. in fact, vitamin c protects against oxidation of ldl by different types of oxidative stresses and inhibits ldl oxidation by vascular endothelial cells.. demonstrated that the combination of ferulic acid and ascorbic acid offered a protection to the myocardium. this was due to the attenuation of the alterations in the levels of lipids, lipid peroxidation, lipoprotein profile, and lipid metabolizing enzymes. ascorbic acid with vitamin e prevents oxldl - induced overexpression of vascular endothelial growth factor (vegf) and its receptor is responsible for atherosclerotic plaque formation. in addition, vitamin c decreases plasma vascular cell adhesion molecule-1 responsible for monocyte adhesion and inflammation [31, 32 ]. vitamin c in tomato is highly bioavailable, so a regular intake of small amounts of tomato products can increase cell protection from dna damage induced by oxidant species. this effect may originate from the synergism of vitamin c with lycopene. some studies reported that ascorbic acid can prevent cancer by neutralizing free radicals before they can damage dna and initiate tumor growth or may act as a prooxidant helping body to destroy tumors in their early stages. other researchers affirmed that there was no sufficient evidence that vitamin c and vitamin e can help prevent cancer. jacob. discovered that the consumption of tomato juice (500 ml) for 2 weeks reduced total cholesterol and crp (c - reactive protein) levels which is a marker of inflammation. this study confirmed that a synergic effect between vitamin c and lycopene is required for the beneficial effects of tomato juice on oxidative stress and inflammation. folates represent all forms of vitamin b found in biological systems, while folic acid is the synthetic form found in dietary supplements and fortified foods. investigated the level of 5-methyltetrahydrofolate in commercial raw tomato cultivar harvested in murcia (spain). they found the maximum level in ronaldo cultivar, equal to 31.5 g/100 g fw. folate metabolism is involved in several physiological mechanisms in the field of andrology and gynecology. in particular, folates have a role in various one - carbon transfer reactions, including purine and pyrimidine biosynthesis, amino acid metabolism, methylation of nucleic acids, proteins, and lipids. for these reasons, folic acid supplementation (200400 g / day) is recommended for pregnant women to reduce pregnancy - induced folate deficiency, which can lead to megaloblastic anemia. homocysteine metabolism is regulated by the nutritional status of folate, vitamin b-12, and vitamin b-6. some researchers consider hyperhomocysteinemia a marker for cardiovascular disease or a risk factor [35, 131, 132 ]. phenolic compounds are widespread phytochemicals composed of an aromatic ring and one or more hydroxyl substituents. they can be constituted by simple phenolic molecules or polymerised compounds [133, 134 ]. in tomato fruit, phenolics include flavonoids, phenolic acids (hydroxybenzoic and hydroxycinnamic acids), and tannins.. phenolics may modulate cellular signaling processes during inflammation or may serve as signaling agents themselves [135, 136 ]. the level and the composition of phenolic compounds in tomato fruits depend greatly on genotype, environmental, and storage conditions. luthria. demonstrated that the spectral quality of solar uv radiation significantly affects phenolic content. polyphenolic compounds are associated with therapeutic tools in inflammatory diseases including cardiovascular diseases, obesity and type ii diabetes, neurodegenerative diseases, cancer, and aging. these effects are due to the phenolic ability to interact with a wide spectrum of molecular targets central to the cell - signaling machinery. main molecular mechanisms include (a) the inhibition of proinflammatory enzymes, such as cyclooxygenase (cox-2), lipoxygenase (lox), and inducible nitric oxide synthase (inos) ; (b) the inhibition of phosphoinositide 3-kinase (pi 3-kinase), tyrosine kinases, and nuclear factor - kappa b (nf-b) ; (c) the activation of peroxisome proliferators - activated receptor gamma (ppar) ; and (d) the activation of mitogen - activated protein kinase (mapk), protein kinase c (pkc), and the modulation of several cell survival / cell - cycle genes [48, 49 ]. studies reported that resveratrol increases the survival of mice fed with a high caloric diet producing changes including improvement in insulin sensitivity and decreased fat accumulation and body weight. these changes are associated with induction of mitochondrial - related transcription factors such as estrogen - related receptor (err-), nuclear respiratory factor 1 (nrf-1), and transcription mitochondrial factor a (tfam). potential beneficial effects of polyphenols have also been obtained for quercetin, able to reduce ffa levels, total cholesterol, and body weight in rats. finally, in 2007 shen. conducted a human clinical trial to examine plasma antioxidation and the levels of blood lipids, after ingestion of tomato products. the authors demonstrated that triglyceride levels and low - density lipoprotein cholesterol were decreased, while high - density lipoprotein cholesterol was increased. flavonoids constitute the largest group of naturally occurring phenolics in tomatoes and contribute to the determination of aroma, fragrance, and colour [141, 142 ]. main classes in tomatoes include flavonols (such as quercetin and kaempferol), flavanols (such as catechins), flavanones (such as naringerin), anthocyanidins, and stilbenes (such as resveratrol). they are usually located in the skin and only in small quantities in the other parts of the fruit. tomatoes of the spanish cherry variety paloma reached levels of 203 g quercetin / g fw. detected levels of total flavonoids in tomatoes from commercial greenhouses in close proximity to srheim research center were between 2.6 and 25.6 mg/100 fw. some flavonoids such as rutin (a flavonol glycoside), quercetin, resveratrol, and catechin are active in rheumatoid arthritis. the mechanisms of action include inhibition of osteoclast / macrophage differentiation and function and estrogen modulation [50, 53 ]. rutin, quercetin, glycosides of quercetin, and resveratrol have been shown to exert intestinal anti - inflammatory activity [37, 38 ]. in particular, rutin and quercetin have been proposed to act as quercetin prodrugs, preventing premature absorption of the aglycone in the small intestine and releasing it in the colon [51, 52 ]. quercetin exerts significant anti - inflammatory effects on il-1 activated human astrocytes, reducing cytokines and chemokines and oxidative stress. resveratrol acts on blood lipid levels and also at the atherosclerotic plaque and reduces cellular infiltration, fibrosis, and expression of inflammatory cytokines in a model of autoimmune myocarditis [54, 55 ]. rossi. conducted an epidemiological study in italy that demonstrated a favorable role of dietary proanthocyanidins on gastric cancer risk, while nishiumi. reported that flavonoids modulate signal transduction pathways at each stage of carcinogenesis. after absorption, flavonoids are transported to target organs where they exert their anticarcinogenic activity. anthocyanins are one of the flavonoid phytopigments ; they may act on adipocytes and modulate the expression levels of adipocytokines. in particular, c3 g (cyanidin 3-glucoside) was reported to upregulate the expression of adiponectin that can increase insulin sensitivity at the level of human adipocytes controlling obesity [56, 57 ]. in human endothelium in animal models, intake of anthocyanin fruit extracts improves cognitive and motor performance [146, 147 ]. hydroxybenzoic acids are gallic, p - hydroxybenzoic, protocatechuic, syringic, and vanillic acids, while ferulic, caffeic, p - coumaric, and sinapic acids belong to hydroxycinnamic acids. they reported amounts of chlorogenic acid between 14.31 (in daniella variety) and 32.84 mg / kg fw (in senor variety), caffeic acid between 1.39 (in senor variety) and 13.00 (in ramillete variety), p - coumaric acid between values under limit of detection (in liso and remate varieties) and 5.77 mg / kg (in daniella variety), and ferulic acid between 1.60 (in riso variety) and 5.38 (in durina variety) mg / kg fw. luthria. identified amounts of p - coumaric acid between 3.5 and 5.5 mg/100 g fw, while the content of ferulic acid was between 0.9 and 1.5 mg/100 g fw. [150, 151 ] reported that half of the ingested chlorogenic acid was metabolized to hippuric acid. however, phenolic acids are studied mainly for their role as antioxidants [62, 63 ]. some studies reported a protective effect of caffeic acid and its related catechols against hydroxyl radical formation in vitro. they found that these compounds protected at low concentration in vitro against dna oxidation induced by iron. other studies reported a correlation between a series of phenolic acids with the inhibition of ap-1 transcriptional activity, implicated in the processes that control inflammation, cell differentiation, and proliferation. tannins include compounds of hydrolysable tannins that are polymers of ellagic acid, or gallic and ellagic acids, with glucose and condensed tannins (proanthocyanidins), which derive from the condensation of monomers of flavanol units [152, 153 ]. they play an essential role in sensory properties of fruits and fruit products such as taste and color. their antioxidant ability is exerted by scavenging free radicals, chelating trace metals, and binding proteins. some studies showed that tannins can enhance glucose uptake and inhibit adipogenesis, acting like potential drugs for the treatment of noninsulin dependent diabetes mellitus. reported that tannins, in addition to anti - inflammatory effects, have a role in the mechanisms of action of antibacterial, antiviral, anticarcinogenic, and cardiovascular system preventing. tomatoes are consumed fresh or are used to manufacture a wide range of processed products that can show a very different composition compared to fresh fruit. for example, significant losses of ascorbic acid can occur during postharvest storage period and during preparation and cooking of foods. this is due to oxidation and leaching into the water used for cooking [75, 154 ]. for the production of tomato paste from fresh tomatoes, heat treatment and/or homogenization can disrupt the cellular matrix of tomatoes determining the bioavailability of different nutrients. lycopene bioavailability was found increased after heat - processed tomatoes compared to fresh tomatoes [157, 158 ]. other studies demonstrated that thermal processing of tomato pulp at 130c improved the in vitro bioaccessibility of lycopene [159, 160 ]. however, as bound antioxidants are released by processing, labile antioxidant compounds are destroyed simultaneously. veronica. observed loss of vitamin c in heat - processed tomatoes at 88c with an estimated d88c value (the time taken for 90% reduction of the initial vitamin c content at 88c) of 276 min. in addition, they found an antioxidant activity of raw tomatoes equal to 4.13 0.36 mol of vitamin c equiv / g of tomato. after heat treatment at 88c for 2, 15, and 30 min, the total antioxidant activity significantly increased to 5.29 0.26, 5.53 0.24, and 6.70 0.25 mol of vitamin c equiv / g of tomato, respectively (p < 0.01). this effect could be explained by the increased amount of lycopene, the major phytochemical in tomatoes. gahler. investigated how heat treatments affect the contents of vitamin c and polyphenols as well as the hydrophilic antioxidant capacity. tomato juice was produced under industrial - like conditions ; baked tomatoes as well as tomato sauce and tomato soup were prepared under household conditions. vitamin c contents decreased during the thermal processing, while the total phenolics concentration and the water - soluble antioxidant capacity increased. seybold. investigated the content of carotenoids and vitamin e in samples of tomato sauce, tomato soup, baked tomato slices, and tomato juice taken at different times of heating. -carotene amount decreased or was stable while -tocopherol content significantly rose during short - term heating. chang. studied the effects of hot - air - drying (ad) treatment on tomato. they found that this process could enhance the nutritional value of tomatoes by increasing parts of the total flavonoids, total phenolics, and lycopene contents. a combination of unit operations involving heat such as blanching, pasteurization, and duration affected the anthocyanin content of fruits and vegetables. cyanidin-3-glucoside and pelargonidin-3-glucoside in fruit puree were significantly affected by thermal process treatments of 70c during holding times of 2 min. as a whole, these evidences could have a significant impact on consumer 's food selection, increasing their awareness of the health benefits of processed tomatoes in the prevention of chronic diseases and inflammation. the ability of various tomato phytonutrients to be positively correlated to prevent or ameliorate chronic disease is appealing. the studies carried out on the effects of phytochemicals on human health prompted researchers to find novel ways to get biofortified tomato genotypes with enhanced levels of phytonutrients such as anthocyanins, lycopene, ascorbic acid, and folate. in the last few years, crop biofortification gave an enormous contribution to understand the relationship between diet and health, reduce the risk of chronic disease, and better understand the regulatory systems in plant species. as described below, attempts to create novel tomato plant lines that carry genes for the accumulation of essential nutrients in tomato fruit can be achieved both through metabolic engineering and conventional breeding [165, 166 ] (table 3). plant metabolic engineering adopted different biochemical approaches to improve the amount of various phytonutrients in tomato fruits. one strategy is based on modification of key - rate limiting steps in metabolic pathways. for example, the expression in tomato of two isoforms of the gdp - mannose-3,5-epimerase, a key enzyme of the ascorbic acid biosynthesis pathway, resulted in a modest increase of ascorbate (1.6-fold) in the fruits of transformed plant. in another study, the cytosolic - targeted tomato mdhar (monodehydroascorbate reductase) and dhar (dehydroascorbate reductase) genes were overexpressed in tomato var., there was a 1.6-fold increase of asa in fruits of plants grown in low light condition, while mdhar transformants showed a reduced level of asa by 0.7-fold in mature green fruits. another approach in metabolic engineering is the expression of genes of a specific biosynthetic pathway belonging to other organisms. for example, attempts to increase asa accumulation in tomato fruits were carried out by expression of the genes phytoene desaturase (crti) of erwinia uredovora and the yeast - derived gdp mannose pyrophosphorylase (gmpase) and arabinono-1,4-lactone oxidase (alo) [169, 170 ]. the synthesis of tetrahydrofolate in mitochondria requires hydroxymethyl dihydropteridine (hmdhp), synthetized in the cytosol by gtp, and p - aminobenzoate (paba), synthetized in chloroplasts from chorismate. the first studies aimed to increase folate in tomato involved the overexpression of the bacterial gtp cyclohydrolase i (gchi) to increase the supply of pteridines from cytosol. the transformed plants showed a low increase of folates (2- to 4-fold) in fruit probably because the supply of paba became limiting [165, 172, 173 ]. the enhanced expression of a gene encoding for the aminodeoxychorismate synthase from arabidopsis (atadcs), aimed at increasing the supply of paba, did not result in any change of folate levels in tomato fruit. on the contrary, the expression of gchi and atadcs resulted in 20-fold higher levels of folate compared to the control. using an alternative strategy, apel and bock introduced the lycopene -cyclase gene from the eubacterium erwinia herbicola and the higher plant narcissus pseudonarcissus into the tomato plastid genome and obtained a 50% increase in total carotenoid accumulation in genotypes expressing the plant enzyme. other studies involve the accumulation of novel metabolites in tomato, such as resveratrol, using the expression of a gene encoding for a grape stilbene synthase. another approach to improve the levels of phytonutrient in plant organs consists in modulating regulatory genes whose products control flux through several biosynthetic pathways. for example, davuluri. enhanced the amount of anthocyans by suppressing an endogenous photomorphogenesis regulatory gene, det1 (de - etiolated 1), using fruit - specific promoters combined with the rna interference (rnai) technology. the last approach to increase levels of phytonutrients involves an enhancing expression of transcription factors that regulate specific biosynthetic pathways. this system does not work well in a complex branched pathway like those of folate biosynthesis but seems to be very efficient to enhance the levels of secondary metabolites such as anthocyanins and flavonols. the anthocyanin pathway was modified in tomato using the transcription factors delia (del) and rosea1 (ros1) from snapdragon antirrhinum majus. the fruit of transformed plants accumulated high level of anthocyanins with concentrations similar to those found in blackberries and blueberries. another transcription factor (atmyb12), which regulates the caffeoylquinic acid and flavonol synthesis, proved to be a good candidate for the production of tomato fruits with high levels of polyphenolic antioxidants compounds. drawbacks of this strategy are that not all the transcription factors regulating pathway have been identified and that transcriptional regulators could have pleiotropic effects when their expression is enhanced [165, 177, 185 ]. one way to produce biofortified plants is to take advantage of the natural variation of plant genomes. for example, recently a new genetic combination was obtained through conventional breeding, which produced a phenotype with deep purple fruit pigmentation, due to an accumulation of anthocyanins on the peels. such a genotype was named sun black. tomato fruit quality usually exhibits quantitative variation controlled by several genes and influenced by environment. for these reasons one main focus of genetic studies has been the identification of genes and quantitative trait loci (qtl) that control the accumulation in tomato fruit of phytonutrients, such as lycopene [165, 178, 179 ]. as for other crops, however, the germplasm of cultivated tomato shows a reduced genetic variability, both for its natural reproduction systems (autogamy) and as a consequence of domestication and breeding, the latter often based on intercrossing advanced lines. in contrast, wild species (such as s. pimpinellifolium, s. neorickii, s. habrochaites, s. chmielewskii, and s. pennellii) and heirlooms tomatoes have a rich genetic variability ; therefore, one goal of plant breeding has become to screen wild genetic resources for valuable traits that could be introduced into modern varieties to improve specific traits [180, 186, 187 ]. in this regard,, in order to identify components of fruit metabolic composition, has phenotyped tomato introgression lines (il) in which marker - defined genomic regions of the wild species solanum pennellii were replaced with homologous intervals of the cultivated variety m82. in our laboratory, two introgression lines of solanum pennellii were identified harboring qtls that increase the content of ascorbic acid, phenols, and soluble solids [181, 182 ]. in a subsequent work, the selected qtls were pyramided into cultivated varieties to increase antioxidant content in tomato fruits. although the conventional breeding is a good strategy to improve the quality of tomato fruit, this technique has some limitations due to sexual incompatibility and requires long breeding programs, whereas genetic engineering has no such limitations and novel genes can be introduced directly into local cultivars. today, several genomic resources are available for tomato, such as high - density genetic maps, genomic and cdna libraries, chip illumina, germplasm collections, and populations of introgression lines. in addition, the sequence of the tomato genome has recently been completed (http://solgenomics.net/organism/solanum_lycopersicum/genome) by an international consortium. using the existing genetic resources and genomic tools, it is now possible to integrate and apply the information of the genome sequence to discover new genes and allelic variants for genetic traits important for consumers, such as fruit quality. one of the most likely outcomes of the availability of the tomato genome sequence will be the development of high - throughput molecular markers to be used for genetic analysis and breeding programs and the discovery of novel genes. moreover, by combining the sequence of tomato genome with the possibility of massive resequencing, it is now possible to investigate the genetic variability in large populations of individuals with the aim of identifying polymorphisms (snps / indels) in a panel of candidate genes. finally, the use of approaches, such as rna - seq, also called whole transcriptome shotgun sequencing (wtss), provides a new gateway to identify the level of gene expression, new transcripts, splice variants, and expressed snps. in table 4, there are many evidences supporting the anti - inflammatory and anticancer action of tomato fruit bioactive compounds. the present review reports a brief description, even though not exhaustive, of bioactive compounds available in tomato fruit and of their beneficial properties on human health. the content of these compounds might be increased to obtain biofortified food taking into account, among other things, the great influence of processing transformation that is required for some derived tomato foods. therefore, the final destination of biofortified tomatoes needs to be considered when selecting new genotypes for fresh market or processing. it is necessary to dissect tomatoes complex genetic control to identify key genes of biochemical pathways underlying their biosynthesis. the best strategy to transfer them into improved genotypes might be chosen between metabolic engineering via genetic transformation and precision breeding by the aid of molecular markers. in both cases, the richness of genetic and genomic resources today available for tomato might highly enhance the success of tomato breeding aimed to obtain new biofortified genotypes. | consumption of tomato fruits, like those of many other plant species that are part of the human diet, is considered to be associated with several positive effects on health. indeed, tomato fruits are an important source of bioactive compounds with known beneficial effects including vitamins, antioxidants, and anticancer substances. in particular, antioxidant metabolites are a group of vitamins, carotenoids, phenolic compounds, and phenolic acid that can provide effective protection by neutralizing free radicals, which are unstable molecules linked to the development of a number of degenerative diseases and conditions. in this review, we will summarize the recent progress on tomatoes nutritional importance and mechanisms of action of different phytochemicals against inflammation processes and prevention of chronic noncommunicable diseases (e.g., obesity, diabetes, coronary heart disease, and hypertension). in addition, we will summarize the significant progress recently made to improve the nutritional quality of tomato fruits through metabolic engineering and/or breeding. |
bcc, the most commonly diagnosed skin cancer in persons of fair complexion, has become the focus of intensified translational debate lately. following the circumstantial evidence that ewes gave birth to cyclopic and malformed lambs after nibbling on veratrum californicum, a corn lily, the causative teratogenic compound, cyclopamine, was discovered [1, 2 ]. increased research on this agenda and the understanding of its functioning led to the discovery of the hedgehog signalling pathway (hh) as an essential cascade in embryonic development. proof of a specific mutation in bcc 's hedgehog pathway showed for the first time that an aberrant hh signalling is also strongly implicated in cancerogenesis of skin tumors. though a wide range of efficient therapeutic options are well established in the treatment of sporadic bcc, the newly developed hh inhibitors and first study results give rise to a curative or even secondary - prophylactic approach in hereditary, advanced, or even metastatic variants. this paper summarizes the current knowledge of clinical aspects and the molecular pathogenesis of this form of skin cancer. moreover, we discuss current and future therapies that are needed in order to allow efficient treatment of bcc in complicated localization, in patients with multiple tumors or genetic disease predisposing for bcc development, or patients that are not eligible for surgery. carcinoma epitheliale adenoides and named after its morphological affinity to the normal cell of the basal layer, bcc is the most common keratinocyte skin cancer (ksc) in persons of caucasian ancestry. although it presumably develops from epidermal stem cells of the outer root sheat of the hair follikel, the precise origin of bcc is still unknown thus far [6, 7 ]. its incidence is estimated up to 100 cases per 100,000 and even higher depending on geographical or complexion disparities. hence, bcc as well as other kscs are often excluded from cancer - registry statistics, thereby underestimating the socioeconomic burden of this form of cancer [810 ]. more common in men than in women, bcc usually arises at an average age of 60 years. apart from the environmental exposure to arsenic, ionizing radiation, oral methoxsalen (psoralen), and immunosuppressive therapy such as in organ transplant recipients [11, 12 ], persons with a fair skin type - i complexion (including red or blonde hair, light coloured eyes, freckling) and people with a history of intermittent sun exposure and severe sunburn during childhood are at highest risk. in particular ultraviolet (uv) irradiation in inverse correlation with reduced or impaired skin pigmentation is generally considered to be the major risk factor of basal cell carcinoma [14, 15 ]. depending on timing (childhood, adolescence), pattern (intermittent, continuous), source (natural, artificial), and amount (cumulative sun exposure), its impact on bcc development is, however, far more complex and needs further detailed study. though the rates are still highest for the naturally sun exposed skin of elderly man, the trend over the past decade is clearly towards an increasing incidence of bcc in younger women due to excessive tanning and sunbed use (figure 1). the majority of sporadically occurring bccs arise in sun - exposed areas with over 80% of all cases developing on the head and neck. unlike squamous cell carcinoma (scc), bccs do not have detectable precursor lesions and usually present themselves de novo as a palpable, localised, translucent tumour with overlying teleangiectasias. for hitherto unknown reasons, they differ in three main clinical as well as histological phenotypes : the nodular bcc exhibiting a pearly rolled border at times with central crusting and ulceration, the superficial subtype with its scaly erythematous patch or plaque - like appearance and the sclerosing, infiltrative, or morpheaform variant that clinically presents as a scar - like, centrally atrophic, whitish, indurate tumour with indistinct margins. frequently, those three histological subtypes are mixed. in addition to aggressive bccs such as the infiltrative, micronodular, or basosquamous subtypes, uncommon bcc variants include the clear - cell, granular - cell, or adamantinoid variants, and adnexal differentiation. pigmented tumours, known to carry p53 mutations, may mimic several differential diagnoses including melanoma and therefore need to be confirmed by biopsy. although erosion and ulceration can develop quite early, especially in the nodular variant, sporadic bcc is in general a slow growing, delayed infiltrating, or destructive tumour that, even in view of other risk factors in terms of a large diameter > 2 cm, incomplete incision and perivascular involvement, metastases only occur after years of existence in 0.55% of all cases. once metastasised in regional lymph nodes followed by bone, liver, and lung, the prognosis is poor with a mean survival of at most 3.6 years after diagnosis [19, 20 ]. in contrast to the sporadic variant of bcc, a hereditary disorder, also known as gorlin syndrome or basal cell nevus syndrome (bcns), exhibits a marked propensity to develop numerous bccs already during adolescence and occasionally even in childhood. as an autosomal dominant inherited genodermatosis with an estimated incidence of 1 : 150 000 in the general population, bcns is very rare. it is characterized by a range of developmental anomalies most notably in the head and neck area that allowed the oral pathologist and dentist robert gorlin to describe it first - and a predisposition to various other forms of cancers. apart from skeletal abnormalities such as splayed ribs, sprengel and pectus deformity, these patients suffer from ectopic calcification, odontogenic keratocysts, facial dismorphism with macrocephaly, palmoplantar pits and tumours in terms of cardiac and ovarian fibroma, meningeoma, medulloblastoma, rhabdomyosarcoma, mesenteric cysts, and other neuroectodermal tumours. most prominent among these clinical findings is the early and very strong disposition to develop several, occasionally hundreds of bccs, especially after radiation given for treatment of progressive bcc or medulloblastoma. it was, however, the intensified research on bcns with proof of its cause, a mutated ptch1 gene in the majority of cases, that linked cancer to the hh signalling pathway for the first time in 1996 [4, 22 ]. the hedgehog (hh) family of intercellular signalling proteins play a pivotal role in many fundamental processes of embryogenic development. they are central to differentiation, growth, pattering, morphogenesis, and function of different cells and organs as well as epithelial and mesenchymal tissue interactions in vertebrates and invertebrates alike [23, 24 ]. malfunction or mutation of these proteins lead to substantial impairment as already shown by the prickly, hedgehog - like appearing of mutant flies of drosophila melanogaster after which the family of proteins was named. probably by duplication of a single - ancestral gene, mammalians, in contrast to invertebrates with just one hh gene, develop three different types of homologs : the sonic, the desert, and the indian type. the hh pathway is initiated whenever one of these ligands binds and thereby inactivates the transmembrane tumour - suppressor protein patched homologue 1 (ptch-1). as a consequence, ptch-1 then permits its receptor smoothened (smo), another transmembrane protein, to transmit signals to downstream targets by means of the gli family of transcription factors. under normal conditions, and mostly in adults, the hedgehog pathway is ligand dependent and actively repressed because ptch-1 constantly inhibits smo, the key activator of the gli pathway. especially sonic hedgehog (shh), as the most widely characterized signalling pathway of the three types, provides a unique example of how the same molecular cascade leads to different pattering in different tissue types solely by distinct transcriptional programs based upon its local concentration. inappropriate activation due to mutations within this cascade however was clearly identified by a growing body of evidence to be a pivotal cause of carcinogenesis, in particular, in bcns - associated bccs and medulloblastoma. according to scales de sauvage, so far three different model systems are proposed on how the hh pathway is involved in the generation of different types of cancer (figure 2). independent of the underlying oncogenic mutation, in nearly all sporadic as well as bcns - linked bccs, uncontrolled stimulations of the hedgehog signalling are found [27, 28 ]. due to relatively stable genomes when compared to other extracutaneous cancer, bccs routinely carry mutations in 30% to 50% of the tumors in p53 or ptch-1 [2931 ]. the latter either looses thereby its function (loss of function mutation) or less commonly activates smo (gain of function mutation) [4, 22, 32 ]. continuously stimulated by smo, a variety of cell - specific target genes then interfere with the physiological function via endothelial growth factor and angiopoetin (resulting in angiogenesis, cell proliferation, metastasis, and cell survival), ultimately leading to cancer [26, 33 ]. a few other alternations of the hh pathway, for example, in shh or gli, have been tried to be identified but could not be confirmed so far [29, 34 ]. in view of the known complex interplay of genes and epigenetic and environmental influences in carcinogenesis, the development of bcc and cancer in general is, however, certainly far more complex and can not possibly be reduced to three somatic mutations within the hedgehog pathway. with the focus on the downstream target genes and effects of hh signalling, the bcc carcinogenesis probably constitutes an intricate mechanism of several interacting pathways and mutated genes that regulate pigmentation, dna repair, and apoptosis. especially the sequence of downstream mediators in hh seems to differ in various tissues. several, such as cd95, bcl-2, pdgfr, or cflip, are currently under investigation. furthermore, contributions of the fox gene family, in particular foxm1 and foxe1, appear to be involved in downstream signalling [3538 ]. as hh target genes, both fox proteins control for a normal mitosis and are overexpressed in bcc in comparison to normal keratinocytes [39, 40 ]. but it is not yet understood which changes are crucial in bcc and therefore represent drivers but not passengers during tumorigenesis of bcc [14, 41 ]. a similar lack of knowledge still exists for the interaction of the gli signalling pathway with other cellular signals. the phosphoinositol-3-kinase (pi3k) cascade interacts with shh in at least two ways. while it inhibits protein kinase a (pka-) mediated phosphorylation, it also stabilizes gli2. on the other hand but up to now, no proof for pi3k involvement in bcc carcinogenesis could be given. the relationship of the ras / raf signalling pathway and bcc is less well defined. in comparison, the obvious requirement of wnt signalling in the downstream activation of hh for these tumours hints at novel possibilities to the therapeutic approach in bcc in addition to hh inhibitors (described below). from a clinical point of view, research focuses on the association of bcc with pigmentation and dna repair genes, respectively. at least for sporadic bccs as the classic uv - induced variant and those that arise in patients with xeroderma pigmentosum (xp), a frequent type of ptch1 and p53 mutations could be identified. the clinical presumption that the increased incidence of bcc in elderly could be a consequence of diminished dna repair due to aging seems therefore not so farfetched. hence, the repair of uv - induced damage should reduce bcc development [44, 46 ], although the use of sunscreens failed to lower the risk of bcc to date. for several dna repair gene variants such as xrcc1, xrcc3, xpa, and xpd, a significant association with bcc risk the polymorphism of those mutants involved is unfortunately reflected by diverse and often contradictory results [4952 ]. a variant once proven to be significant was refuted in another study or was, in part, not bcc - specific at all. similarly unpersuasive are the results on melanocortin 1 receptor gene (mcir), the major known genetic variant influencing the degree of skin pigmentation. although it was clearly shown that the nonfunctional variant of mcir had a dose - dependent impact on the incidence of bcc and melanoma, the consecutive lack in pigmentation itself did not influence the result [54, 55 ]. a different mechanism in terms of a paracrine role or distant modulation of proliferation and differentiation of keratinocytes by mcir has also been suggested [56, 57 ]. in general, the functioning of pigmentation and dna repair in healthy individuals, in skin cancer, is so far too little, or at best partially, understood in order to pave the way for prevention or treatment of bcc. a wide range of several effective therapeutic options are available for the therapy of bcc. intended to be curative or at least locally controlling, the treatment can either be surgical or nonsurgical depending on several tumour- or patient - related factors. especially tumour size, location, histological subtype, patient 's health and wishes, possible complications, and aesthetic results should be taken into account. as there is still no preoperative method for the detection of subclinical spread, surgical therapy with 3d histology is the gold standard even in bccs of the head and neck area. in order to ascertain the complete and hereby curative excision, several equally effective techniques are at disposal. with mohs micrographic surgery, the histological confirmed bcc is removed in a bowl - like fashion, immediately frozen, and examined for residual tumour cells in the lateral and basal margins as long as the bcc is totally excised. 5-year recurrence rates for mohs surgery are reported as 1%3% for primary bcc and 3%7% for recurrent tumours similar results are achieved with other less known histological methods such as the la galette technique. conventional surgery with tumour - adapted margins of safety uses a bread loaf horizontal cutting to control for complete excision. depending on the safety margin, a higher rate of residual tumour cells and thus increased recurrence rate of 4%34% is reported. curettage, electrodesiccation, and cryosurgery are further surgical approaches that are easily applied in low - risk lesions with nonaggressive histological features such as superficial bcc of the trunk. the disadvantage is, however, that the complete removal of the bcc can not be histological proven and delayed wound healing due to thermal destruction or impairment of the basal layer may lead to unsatisfactory results. certainly, none of these three techniques is appropriate for recurrent or morpheaform bccs, although in general cure rates of up to 95% and higher are stated. non - surgical treatment options include radiotherapy, photodynamic therapy, and topical application of imiquimod and 5-fluorouracil. all of the proposed procedures comprise, however, the disadvantage that no treatment success can be histologically validated and thus higher recurrence rates have to be taken into account. nonetheless, elderly patients with multiple comorbidities and inoperable tumours profit. the indication for radiotherapy given the multitude of therapeutic options is more limited and rather confined to postoperative recurrences or if a complete resection appears unlikely. since there is a high risk of secondary tumors developing on the radiation side, patients with bcns, xp, epidermodysplasia verruciformis, and iatrogenic immunosuppression should be excluded from radiotherapy. it is also not recommended for patients younger than 60 years, given its potential for carcinogenesis [61, 62 ]. photodynamic therapy requires the application of a photosensitizing agent such as 5-aminolevulinic acid or its ester 3 - 4 hours before the protoporphyrin ix - enriched tumour cells are destroyed. superior with regards to cosmetic outcome when compared to many other treatment options, pdt of superficial bcc showed a 1-year recurrence rate of 9.3% and is not recommended for the nodal subtype due to 5-year relapse rates of 76%. although its precise mechanism is still unknown, the once - daily application of imiquimod 5 days per week for 6 weeks resulted in a histological clearance rate of up to 89.6% in superficial bcc [6567 ]. a clear trend towards improved rates with increased frequencies of application is limited by intensified local and systemic reactions. residual tumours after therapy are nonetheless often difficult to assess, and subtypes other than superficial bcc are no general indication for imiquimod since multiple recurrent lesions can occur. 5-fluorouracil, a topical cytostatic agent, is considered as a therapeutic alternative in patients with multiple, superficial multicentric bccs, for example, in bcns. as a consequence of painful inflammatory and erosive reactions, the patient 's compliance is often limited for this treatment option. given that metastasis and invasion of vital structures by bcc apart from surgical procedures and an additional radiotherapy, an assortment of different chemotherapies such as doxorubicin, paclitaxel, and/or carboplatin with differing response rates have been applied up to now in order to control the tumour load and extend the patient 's life expectancy [8, 69 ]. given the rare nature of metastasis, larger clinical studies have been lacking until recently. the more detailed understanding, achieved as of late, of the molecular pathogenesis of bcc and its causative aberrant pathway has rendered a new therapeutic targeted approach possible. smo inhibitor cyclopamine, the teratogenic steroidal plant alkaloid, which was topically applied, succeeded already to induce regression of four sporadic bccs, since then, a series of small - molecule hh inhibitors have been developed and are currently in clinical development. furthest along is gdc-0449, a more specific and potent smo inhibitor than cyclopamine. administered in different doses of 150, 270, and 540 mg per day as part of a phase i study in metastatic or locally advanced bcc, an objective response in 18 of 33 patients side effects such as hyponatraemia, fatigue, weight loss, and dyspnoea were mild to moderate. several ongoing phase ii trials investigate its efficacy in advanced bcc, medulloblastoma, and breast cancer but also in addition to other chemotherapeutic agents in pancreatic, lung, colorectal, and gastrointestinal cancer. four other new hh inhibitors including lde-225, bms-833823, ipi-926, and pf-04449913 are currently under investigation in phase i trials. all of the tested components target so far smo as the key regulator of the hh pathway. an equally effective inhibition could succeed, however, in targeting the hh downstream signalling. two tested candidates gant58 and gant61, inhibitors of the gli transcription, possibly provide a therapeutic alternative in case of a resistance to smo inhibitors. also interfering with hh target gene transcription and therefore of potential therapeutic interest in bcc while one single microrna regulates hundreds of target genes, the task is to focus its efficacy on the essential target and thus minimize its side effects, which still needs to be mastered (table 1). as discussed by epstein, the use of tyrosine kinase inhibitors, sorafenib or imatinib, seems sensible because pdgfr is supposed to mediate downstream effects in hh signalling. the assortment of further new potential agents for prevention as well as treatment in bcc is plentiful, ranging from vitamin a and d derivates, nsaid, and dna repair enzymes up to melanocortin peptides therapeutics. certainly due to the lack of knowledge of their precise functioning and how, or if at all, they interact with hh pathway, results are so far promising but inconclusive. it may therefore not be surprising that even the widely recognized and in its molecular effects initially well understood standard therapy with systemic retinoids fails to prevent the recurrence of sporadic bcc. in view of those remarkable oncogenomic achievements in the understanding of bcc carcinogenesis, a curative breakthrough, especially in hereditary, locally advanced or metastatic cases, seems imminent. first promising data are yet too scarce to obtain detailed pieces of information about dosage, side effects, response, recurrence, and survival rates or possible medical interactions. not sufficiently known but crucial is certainly the impact of the type of hh mutations and their combinations for the various clinical subtypes of bcc. the change from a phenotype - correlated diagnosis to a genotype analysis, but is bcc in all its clinical and histological variations to be reclassified according to its genotype ? the open intruiging question remains if there is a link between hh mutation, histology, and its clinical aspects that would simplify the indication for a targeted therapy. of equal importance in this complex interplay are without doubt epigenetic phenomena and environmental factors that are at best only initially recognized so far. definitely many more future studies are needed to answer the large number of interesting questions that the discovery of aberrant hh pathway for bcc raised. | due to intensified research over the past decade, the hedgehog (hh) pathway has been identified as a pivotal defect implicated in roughly 25% of all cancers. as one of the most frequent cancer worldwide, the development of basal cell carcinoma (bcc) due to activation of the hh pathway has been convincingly demonstrated. thus the discovery of this central tumor - promoting signalling pathway has not only revolutionized the understanding of bcc carcinogenesis but has also enabled the development of a completely novel therapeutic approach. targeting just a few of several potential mutations, hh inhibitors such as gdc-0449 achieved already the first promising results in metastatic or locally advanced bcc. this paper summarizes the current understanding of bcc carcinogenesis and describes the current mechanism - based therapeutic strategies. |
leucine rich repeat kinase 2 (lrrk2) mutations are a common cause of parkinson s disease (pd). here, we identify inhibitors of lrrk2 kinase, which are protective in in vitro and in vivo models of lrrk2-induced neurodegeneration. these results establish that lrrk2-induced degeneration of neurons in vivo is kinase dependent and that lrrk2 kinase inhibition provides a potential new neuroprotective paradigm for the treatment of pd. |
|
retroperitoneal sarcomas are relatively uncommon tumors, constituting only 1015% of all soft tissue sarcomas (sts) [1, 2 ]. patients usually present in their fifth decade of life, although the age range is wide. males and females are equally affected [4, 5, 6 ]. the most common histological types of retroperitoneal sts are liposarcomas and leiomyosarcomas followed by pleomorphic undifferentiated sarcoma and malignant fibrous histiocytoma. a variety of other histological types exist but they are much less common in the retroperitoneum than in other primary sites., we describe a patient who presented with a large mesenteric root leiomyosarcoma that was treated surgically with favorable outcome. a 65-year - old male presented to our department complaining of recurrent epigastric and upper right abdominal pain radiating to the back for the last 3 months. his family history was consistent with a father who suffered lung malignancy and a mother who expired due to hepatoma. an ultrasound performed prior to his admission revealed a hypoechoic 4 4 cm lesion close to the pancreatic head. on admission, abdominal examination revealed a soft, nondistended abdomen with local tenderness in the upper right quadrant. abdominal tomography revealed a 5 5 cm, rounded, soft density lesion adjacent to the root of the superior mesenteric artery (sma) and pancreatic head (fig. the patient underwent endoscopic ultrasound examination that demonstrated a hypoechoic - heterogeneous solid lesion behind and adjacent to the sma and superior mesenteric vein. 2). pathology obtained by fine needle aspiration from the lesion revealed fragments of mesenchymal tissue that was positively stained for c - kit and actin. laparotomy revealed a large, 7-cm, hard tumor adherent to the medial border of the pancreatic uncinate process laterally, to sma medially, posterior to the portal vein and anterior to the inferior vena cava and left renal vein (fig. he received radiotherapy, and at 4 years of follow - up, the patient is healthy and has no recurrence. local recurrence is the primary cause of mortality after resection in these pa - tients. low tumor grade and smaller tumor size are both well - established prognostic factors and are significantly associated with improved survival. the therapeutic aim of surgery in patients with sts is complete macroscopic resection, ideally with negative microscopic margins. this may oblige the decision to resect the surrounding tissue by the surgeon involved or adhere to the tumor. however, because of the large size of these tumors and the intraoperative difficulty in accessing all involved margins, this goal is difficult to achieve, often resulting in the presence of positive microscopic margins. tumors that are located in a strategic anatomical location, as presented here, are difficult to manage, and the surgeon needs to decide whether the surrounding vital structures are involved in the tumor or can easily and safely be dissected away from the tumor. the pseudocapsule consisting of surrounding tissue to the sarcomatous tumor dictates occasionally unblock resection. in proximity to strategic and vital organs, | high mesenteric root sarcomas are difficult to manage due to their proximity to the superior mesenteric vessels. resection of these tumors along with the blood vessels may lead to a complicated and protracted convalescence for the patient. resection remains the main treatment modality for these tumors. during operation on high mesenteric root sarcomas, sound clinical judgment is needed for the decision not to sacrifice vital blood vessels. |
peer - assisted learning (pal) has been defined as the development of knowledge and skill through active help and support among status equals or matched companions. this methodology has been in use for medical education in the developed countries and shown to improve both knowledge and skills of medical students at par to what is achieved by teacher - student learning. pal can also be seen as a type of interactive teaching - learning. in india, 50250 students are admitted each year in the mbbs course. pal sessions, conducted, as small group discussion (sgd) is perhaps one such method. the current project was undertaken with the objective to assess the effectiveness of pal on improvement in cognitive assessment scores, and it 's acceptance among undergraduate medical students in one public teaching medical university in north india. participants were 9 semester mbbs students posted in pediatrics long clinic in the month of october to november 2014. all the students had attended the lectures on the topics to be covered in pal sessions. in addition, they had also seen cases related to the topic in the previous as well as current posting in pediatrics. each group identified a student leader (sl). the faculty facilitator (ff) then oriented the sls on how to initiate, facilitate and encourage participation from all the members, called the peer - learners (pl) and summarize, the sgds. in each sgd thereafter, the first pl on the right to sl spoke for about 3 min on anyone intended learning objective (ilo) of the topic for the day. each participant then wrote on a diary, self - assessment of their knowledge and areas where further reading was needed. they also noted their impressions on the group dynamics, what went well and what could have been better with reasons for both. the diaries were shown to the ff at the end of the session. before each pal session, the topic to be covered was given on day 1. thereafter, a pretest was taken on the topic by administering multiple - choice questions (mcqs). then, the students were given the ilos in the cognitive domain and given the list of reference books to read. the group re - convened on day 4 and the pal session was conducted as sgds for about 45 min. then, focus group discussion (fgd) was held to collect information on their experience and expectations, what did they specifically like and their suggestions for the future. over a period of 15 days, three pal sessions were carried out on growth and development, lower respiratory tract diseases and malnutrition (undernutrition) in children. at the time of recruitment of students information was collected on their gender, present age, medium of schooling, whether they studied in groups, and the marks they had obtained in first, second and third professional examinations. we planned to give 15 mcqs as a pretest and another 15 as posttest per topic. for sample size calculation for a continuous outcome, we assumed that the mean pretest score would be 8 and after pal session, the improvement would be 3 (37.5%) with a standard deviation of 3. therefore for a significance level of 5%, 17 students would be needed for pretest and 17 for posttest by using formula n = f(,) 2s/. data was collected using predesigned data collection instruments and entered in ms - excel. the univariate descriptive analysis was done for the baseline variables and reported frequencies, percentage and with standard deviation, as applicable. for a comparison of categorical variables chi - square test was used and for continuous variable student 's t - test used. the pre- and post - test difficulty index was compared using unpaired student 's t - test. using a two - tailed distribution, a p < 0.05 was taken as statistically significant. students of the upper one - third were considered as high achievers and lower one - third as low achievers. the difficulty index of each of the mcqs used in pre- and post - test was computed using the formula : difficulty index (p) = ((h+l) /n) 100 where h = number of students who got the correct response in the top one - third (high achievers), l = number of students who got the correct response in the bottom one - third (low achievers), n = number of students in the top + bottom one - third (high + low achievers). average difficulty index of pre- and post - test mcqs was compared using unpaired student 's t - test. the frequency of codes across thematic areas in all fgds was semi - quantitatively ascertained and reported. in october to november 2014, 26 students were posted in pediatric long clinics and invited to participate in pal sessions. the mean age of males and females was similar being 24.3 2.4 years versus 23.9 1.3 years. they reportedly spent approximately an average of 4 h / day studying and only one - fifth said they studied with a colleague sometimes. however, 22 students (84.6%) participated in the pal session and the posttests. data from 22 students who did all the three pre- and post - tests was analyzed. in each posttest, one additional student joined who was not in the pretest and hence this student 's data was not analyzed. the mean difficulty index of pretest versus posttest was 51.7 28.6 and 64.8 26.6 (p = 0.02). the correlation coefficient between pre- and post - test scores was 0.48 (p < 0.0001). mean pretest versus posttest scores was 8.44 2.86 versus 10.48 2.95, and the improvement was statistically significant (p < 0.0001). analysis of nine fgds revealed that most felt that everyone got an opportunity to express ; however, in some groups and in earlier pal sessions the group leader tended to dominate. the group dynamics improved with time, and each member developed a sense of responsibility. most said that the topic was prepared better and they could clarify their doubts on the discussion. they liked the method of pals where getting ilos gave a direction to their studies and subsequent discussions imparted clarity and improved their verbal expression skills. almost all were of the opinion that pal was a very useful activity, more so because its marks of pre- and post - test would not be added to any assessment. pal sessions provided in - depth reading in contrast to the usual lectures, which were boring, and there was limited retention of knowledge. those topics, which were unique to a subject, could be identified for pal sessions. this was a feasibility study of the acceptance and effectiveness of pal conducted for three topics on 26 final semester mbbs students posted in the pediatric long clinics in king george 's medical university over a period of 2 weeks. it showed that pal was well accepted and improved learning as evident by a 24.2% improvement in the posttest mcq scores. similar findings have been reported from ireland where pal was used to train 5 year medical students on the communication skills module. qualitative data analysis was done which revealed a high level of acceptability among tutors and learners as well as reciprocity of educational exchange within the pal setting. the current study also did qualitative analysis, which revealed similar findings. in a review, which included 19 studies of these, 10 studies utilized randomized allocation but most of the study participants were self - selected volunteers. certain studies also assessed by observed clinical evaluations. in most, the student - teachers had been trained formally. the authors concluded that pal or teaching in specific situations did achieve short - term learner outcomes comparable with those produced by faculty - based teaching. furthermore, pal has beneficial effects on student - teacher learning outcomes, as found in the current study. in another review of 40 studies where pal was used in the fields of nursing, physical therapy, occupational therapy, medicine, athletic training, and higher education. overall the strategy was found to be beneficial for the students as it had elements of peer leadership, peer feedback as well as peer mentoring. however, pal had to be viewed from the perspective of program administrators, clinical instructors, and students. also, further research is needed on planned versus unplanned pal. in the current study, pal methodology promoted healthy interaction between students as has been mentioned by other workers in this area. since the pre- and post - test scores did not form a part of the routine assessment, the students found the environment nonthreatening and conducive to learning. the method also promoted close and longer interaction with the ff, which is not usually there. often the students are not aware of what they must know within a topic ; hence the distribution of ilo was welcomed and gave them a direction to learning. yet the improvement in posttest performance indicates that pal methodology does improve learning, which is reflected in assessment scores. in the future, the pre- and post - test mcqs should be randomly drawn from a larger pool of questions. pal was tested only on one batch of students of one semester and posted in pediatrics only. however since these students were in the final semester, their observations could be generalized to other students and to other subjects also. it was not possible to have a comparator group with no intervention, as it would not have been ethical. since the intervention was found beneficial, it should have been given to the entire batch of students. however, clinical posting of students had finished, as the examinations were to begin in the month of december. it is concluded that since pal was well accepted and improved assessment scores this can be adopted for teaching in selected topics across all subjects of mbbs. | background : peer - assisted learning (pal) is the development of knowledge and skill through active help and support of equals. however, this has not been tested in medical education in india.objectives:to assess the effectiveness of pal on improvement in cognitive assessment scores and its acceptance among undergraduate medical students in one public teaching medical university in north india.methodology:after approval from the institutional ethics committee, three pal sessions, 1 per week, each on specific topic, were conducted using small group discussion methodology with a faculty contact and student leader and 46 peer - learners, in 9th semester mbbs students. a pretest with multiple choice questions (mcqs) was followed by distribution of learning objectives and list of resource material. pal session was conducted after 72 h, followed by posttest by mcqs and then focus group discussion (fgd) on students experiences.results:of the 26 students enrolled, three pal sessions was completed by 22 (84.6%) students. the correlation coefficient between pre- and post - test scores was 0.48 (p < 0.0001), with a 24.2% improvement in posttest scores. in the nine fgds most said that pals helped in the better preparation of the topic, clarifying doubts, lessened examination anxiety, improved communication skills, and increased self-confidence.conclusion:pal was well accepted, and it improved assessment scores. therefore, it can be adopted for teaching selected topics across all subjects of mbbs course. |
the analysis of cardiocyte mechanics has historically proven an excellent tool in providing relevant information on the excitation - contraction coupling of the heart. it has provided useful insights toward the proper handling and treatment of many cardiovascular diseases. furthermore, the study of cardiocyte contractility has helped unveil the fundamental processes underlying heart function in health and disease [2, 3 ]. the relevance of this study has created a need for analysis tools in this area of research. the purpose of this paper is to propose a tool for the analysis of neonatal cardiocytes. many inotropic factors modulate the contractile behavior of the heart which can be conveniently studied in isolated cardiocytes [1, 35 ]. adult cardiac ventricular myocytes have been studied in cardiovascular research for almost thirty years, and the popularity of this approach is constantly reinforced by the numerous studies published every year. however, during the last decade, the majority of researchers performing long - term (longer than 1 week) studies have favored the use of embryonic and neonatal cardiocytes. this is due to their versatility and ability to withstand and survive harsher experimental conditions than adult cardiocytes. adult, neonatal, and embryonic cardiocytes are in different states of maturation and development. as the cardiac system of an organism matures, structural changes at the cellular level and in the myocardial anatomy occur to increase contractility and the development of force during contraction. it would be improper to perform studies on a myocyte at a given developmental level and make a direct correlation to another developmental stage, therefore ignoring the marked growth and structural differences that exist. dramatic differences in action potential, physiology, gene expression, and molecular interactions in the neonatal and adult cardiocytes indicate that there is a need for different treatment of adult and newborn hearts. it is therefore necessary to be able to perform equivalent studies on both adult and neonatal cardiocytes in both research and clinical applications. being the most representative of the functions of the cardiocyte must be fully studied both quantitatively and qualitatively in all stages of development. it is important to identify and explore the differences in the adult and neonatal cardiocytes. it is thus desirable to have analysis methods that can be applied in a simple and practical manner to cardiocytes at all levels of development that will allow researchers to accomplish the aforementioned goals. this is particularly so in the case of the neonatal cardiocyte, in which a practical and generally applicable method is still missing. because of this need, we propose a computational framework based on well - established image processing techniques for the assessment of contractility of isolated neonatal cardiac myocytes. the proposed methodology is easy to understand and implement, yet provides a robust account of the neonatal cardiocyte contraction process without the need for expensive and sophisticated equipment. in contrast to the adult cardiocyte, the neonatal cardiocyte is round in shape when first isolated from the heart. once the cells are plated, over time, they begin to form pseudopodia and spread out on the substrate, displaying a myriad of shapes (see figure 1). these developments occur at different rates as they are highly dependent upon the composition of the culture media, isolation procedure, extracellular matrix (ecm), and species of the animal [5, 810 ]. unlike adult cardiocytes which are highly organized and quite similar in morphology, the neonatal cardiocyte is in the process of developing their contractile machinery. the neonatal cardiocyte is generally unable to retract its cell boundary during contraction, and noticeable changes occur only within the cell perimeter. for these reasons, it is difficult to perform contractile measurements on this cell type in a manner similar to that of the adult cardiocyte, in which changes in cell boundary are quantified during contraction. there are several methodologies for assessing the contractility of adult cardiocytes ; however, very few methods have been proposed for the study of neonatal myocytes. the reported methods for quantifying or assessing neonatal cell contraction have required the use of elaborate equipment, such as a proximity detector or an atomic force microscope to measure the increase in cell elevation as a cardiocyte contracts. such contraction quantification and assessment methods are expensive due to the equipment needed. the application of the cell boundary video tracking method and the use of cellular force measurements using single - spaced polymeric microstructures to detect amplitude of contraction and beating rate in neonatal cardiocytes have also been reported in the literature [1216 ]. these have often been found to be laborious, complicated, erratic, and expensive in laboratory studies. we have recently explored the application of fourier transform methods and image analysis techniques in the assessment of contractility in adult cardiocytes with satisfactory results. given our recent experience with this approach, the possibility of applying similar methods to the assessment of neonatal cardiocytes was obvious. image analysis allows the application of investigation methods that are noninvasive and that can adapt to the different shapes and forms that a neonatal cardiocyte might take. additionally, both the image analysis techniques and the fourier transform are methods that can be applied without the need for expensive equipment or sophisticated mechanisms. this approach could potentially open the door to the possibility of applying similar methods for the assessment of contractility of cardiocytes in all stages of development, ranging from embryonic to adult hearts. the correlation between myocyte contractile activities and the changes in intracellular calcium provide an insight into the mechanisms of calcium - dependent contraction. it is well known that ca is essential in cardiac electrical activity and that it is the direct activator of myofilaments in the cardiocyte, resulting in a contraction. in adult ventricular myocytes, contractile activity is known to depend on the release of ca from the sarcoplasmic reticulum (sr) through ryanodine receptor channels (ryr), whereas in the fetal and neonatal myocytes, sr plays a much smaller role in ca regulation. this decreases the capability of these myocytes to load ca as compared to those isolated from mature hearts. fetal and neonatal contraction depends largely on trans - sarcolemmal ca influx rather than on ca released by the sr. this distinction translates into a slower upstroke and decay of ca in comparison with those of adult ventricular myocytes. this behavior is confirmed by a major difference between action potentials (ap) of adult and neonatal rat myocytes, with a significantly longer repolarization phase in neonatal cells as compared to those of adult myocytes. we therefore anticipate neonatal cardiocyte contractility records to exhibit slower activation and relaxation periods as compared to those of the adult cardiac myocyte. given the relationship that exists between ca and mechanical contractility, we also anticipate that the contractile behavior of neonatal cardiocytes would have strong correlation with the adult ca transient behavior. given that a practical and accessible method for quantifying neonatal contractility has not been reported in the literature, we have decided to validate our results by comparing them to the contractile data of adult cardiocytes recently published in. ventricular myocytes were isolated from 13-day - old harlan sprague - dawley rats (rattus norvegicus albinus), and cultured as described by sprenkle.. all animal procedures were in accordance with the san diego state university animal subjects committee (uasc) and nih animal welfare assurance a3728 - 01. after preplating myocytes to remove fibroblasts, pooled myocytes were centrifuged and resuspended in dmem / f-12 (gibco) containing 10% fetal bovine serum (irvine scientific). myocytes were then plated overnight on fibronectin (gibco)-coated 100 mm dishes to allow for recovery. neonatal rat ventricular myocytes were then washed twice with 1 : 1 medium consisting of dmem / f-12, kanamycin, ampicillin, and fungizone on the following day. cells were then incubated overnight in a minimal medium consisting of 1 : 1 medium, supplemented with 1 mg / ml bsa (sigma). on the third day, cells were washed two more times with 1 : 1 medium and replaced with an its medium consisting of minimal medium, 1 x its (insulin - transferrin - selenium, gibco), 0.4 x mem nonessential amino acids mixture (sigma), and 0.1 x mem vitamins medium (gibco). images depicting contracting myocytes in this study were acquired using an inverted phase contrast microscope (nikon number elwd, nikon corporation, tokyo, japan). the cells were field stimulated with a suprathreshold (50%) voltage at a frequency of 0.3 hz, for a 3 msec duration. the stimulation was performed using a pair of platinum wires placed on opposite sides of the chamber connected to an electrical stimulator (hugo sachs elektronik - harvard apparatus, type 223, germany). the polarity of the stimulatory electrodes was reversed automatically every 10 stimuli to prevent electrode polarization. myocyte motion was digitally recorded with a camera (pulnix tm-1327, jai pulnix inc., san jose, ca, usa) mounted on the microscope, at a rate of 30 fps. two popular shape descriptor methodologies used in image processing are contour - based and region - based descriptors. we have applied contour - based descriptors for the assessment of contractile responses of isolated adult cardiac myocytes. such descriptors assume the knowledge of shape boundary information and are usually suitable for characterizing contour shapes without sophisticated boundaries such as those of the adult cardiocyte. region - based shape descriptors specifically the intensity - based descriptors can be applied to more general cases where the contractile responses do not necessarily provoke changes in the overall geometry of the cell, but rather the rearrangement of its interior structures. we describe a method for assessing the contractions of neonatal cardiocytes by analyzing fourier descriptors obtained through (discrete) polar fourier transform (pft). fourier descriptors have been extensively proposed for the purpose of shape recognition, retrieval, classification, and analysis [2230 ]. one of the main advantages of analyzing images in the spectral domain rather than analyzing them in the spatial domain is that it is easier to overcome the noise problem common to digital images. furthermore, the spectral features of an image are usually more concise than the features extracted from the spatial domain. although, the direct application of the (discrete) fourier transform (ft) to an image can provide useful information about its content, it has the disadvantage of lacking rotation invariance. the pft generates rotation - invariant data particularly well suited for effective extraction of orientation features. the following description of the pft is based on the works by averbuch. they proposed a polar fast fourier transform (pfft) method by using a special type of unequally spaced fast fourier transform (usfft), where a different starting grid is employed, instead of the regular cartesian grid. their methodology cleverly decomposes the problem into two stages : first, a pseudo - polar sampling set is used to apply a pseudo - polar fft, and second, a conversion from pseudo - polar to polar ft is performed. this approach provides improved performance as compared to the alternative cartesian - based usfft - based counterparts. the main reason for this improvement is in the ability of the pseudo - polar grid to provide a spatial - varying sampling of the frequency domain, which is closer in density to the final polar grid. furthermore, the pseudo - polar grid gives a denser sampling near the origin, allowing for a more accurate interpolation. as mentioned above, the pseudo - polar fourier transform based on the definition of a polar - like 2d grid provides a fast fourier computation (see figure 2). (and more recently in [34, 35 ]) built their methodology upon their work on radon transform for data in a cartesian grid, based on the summation along lines of absolute slope less than one, with values at non - cartesian locations which are defined using trigonometric interpolation on a zero - padded grid. their implementation of the polar fft starts by defining the pseudo - polar grid points in the frequency domain. there are two types of points on the grid : the basically vertical (bv) subnet and the basically horizontal (bh) subnet. these are expressed as follows : (1)bv={y=lnfornl < n,x=2mln2 forn2m < n2 }, (2)bh={x=lnfornl < n,y=2mln2 forn2<mn2}. the pseudo - polar grid is shown in figure 2 where the bv points are marked with the filled circles, while the bh points are marked with hollow circles. have shown that given the pseudo - polar grid points bv and bh, in order to compute the fourier transform values, a simple 1d - fft can be satisfactorily employed. based on the aforementioned pseudo - polar coordinates system, the polar coordinate system is defined by manipulating the layout through appropriate interpolations. the two operations involved in this transformation are to rotate the rays to obtain an angularly - uniform ray sampling and circle the squares to obtain concentric circles as required in the polar coordinate system. to rotate the rays, the term 2m / n is replaced with tan(m/2n) in x and y in (1) and (2), respectively. that will lead to the new grid points : (3)bvnew={y=lnfornl < n,x=lntan(m2n) forn2m < n2},bhnew={x=lnfornl < n,y=lntan(m2n) forn2<mn2}. the new points are still organized in concentric squares, but the rays are now equispaced based on angle as opposed to slope (see figure 3). to circle the squares, both x and y are divided by a constant along each ray, based on its angle, and thus a function of the parameter m : (4)r(m)=(1+tan2(m2n))1/2. the resulting grid is expressed as follows : (5)y=lnr(m), fornl <. the new points are located along the same line and are equispaced with a different spacing (see figure 4). the final grid is shown in figure 5. in order to prevent wrap around and thus to get geometrically faithful lines, the pseudo - polar grid must be oversampled both radially and angularly. in their implementation, apply the methodology to general images of size n n and obtained a resulting polar ft array of 2n + 1 2n + 1 (the authors thank reviewer for pointing this out). they argued that, if higher oversampling is desired in the destination grid, an easy way of accomplishing this is to modify the initial value n by zero - padding the input array. for measuring the contractile responses in neonatal cardiocytes, we zero - pad each frame with a mask that isolates the cell from the background, while at the same time, it turns each frame into an n n image suitable for applying the above methodology. figure 6 shows a frame of a neonatal cardiocyte that has been cropped to size n n. figure 7 shows the n n mask that is used to isolate the region of interest for the analysis. cross - correlation of the pft from each image (frame) is used to calculate a statistical estimation of the similarities between image components around an interest point of the cardiocyte. this provides an account of the contractile responses of the cardiocyte to the electrical stimulus. cross - correlation is a very simple method which, if used in the proposed manner, provides a robust measurement of the changes manifesting as contractions occur. it is important to note the regularity of the contraction signal as well as the great signal - to - noise ratio that is obtained using this method. in algorithm 1, we present a simplified algorithm for the assessment of contractile responses of neonatal cardiocytes (for details on the implementation of the polar fft the reader is referred to. results obtained by applying the proposed image processing techniques for assessing contractility in isolated neonatal cardiac myocytes proved to be satisfactory. we observe a contractile response of the neonatal cardiocyte that is in accordance with structural and functional differences known to exist between adult and neonatal cardiac myocytes. given the relationship that exists between ca transient levels and contractility, we expected the neonatal contractile and ca transient behavior to have a correlation similar to the one that exists between the adult ca transient and its contractile behavior. adult cardiac myocytes are known to exhibit ca transients that have a faster activation and recovery phase than that of the neonatal cardiocyte. this could be attributed to the fact that the contractile activity of adult ventricular myocytes is known to depend on the release of ca from the sr, whereas in the fetal and neonatal myocytes the sr plays a smaller role in ca regulation, decreasing the capability of these myocytes to load ca. excitation - contraction mechanisms of the fetal and neonatal myocytes are therefore more dependent upon extracellular ca to activate the contractile machinery, which is a slow - acting mechanism compared to that of the sr. the contractile proteins, cellular structures, and myofibrils of the adult cell are highly organized and developed. these factors result in a well - coordinated contractile process that translates into fast contraction and relaxation phases during contractility. in contrary, the neonatal cardiocyte has contractile proteins, cellular structures, and myofibrils in the developmental process, with an excitation - contraction coupling mechanism that is highly dependent on extracellular calcium. based on these facts we anticipated the neonatal cardiocyte would exhibit signals with slower contraction and relaxation phases and a total contraction signal considerably weaker than that of the adult cardiocyte. our results corroborate the anticipated outcome by exhibiting a clear correlation between the known ca transient of a neonatal cardiocyte and the results of our contractility assessment. the activation (contraction) phase is noticeably slower than that of the adult cardiocyte (see figure 9), yet the most significant difference occurs in the relaxation phase, where there is a dramatic difference in the rate at which the neonatal cardiocyte mechanisms return to their basal conditions (see figure 10). we also register a considerably long plateau at the peak of contraction, which can be attributed to the disorganization in the developing myofibrils contracting in an asynchronous fashion with their peaks of contraction occurring at different times (see figure 11). the resulting contractile signals from our preliminary results are in accordance with our expectations, exhibiting slower activation and relaxation rates as compared to those of the adult cardiac myocyte (see figures 9 and 10). the total contraction exhibited by the neonatal cardiocyte is considerably less than the contractility exhibited by the adult cardiocyte (see figure 11). we described a computational framework for the assessment of contractile responses of isolated neonatal cardiac myocytes. the assessment stages are based on mathematically sound and computationally robust algorithms very well established in the scientific community. image analysis provides an opportunity to analyze cellular dynamics without disrupting the cellular environment in vitro. our intent was to establish an accessible approach to assess neonatal contractility by making use of the methods available in the field of image analysis. we feel that the development of this methodology will provide scientists working in the field of cellular cardiology with a practical yet robust option to assess the contractility of neonatal cardiocytes. to our knowledge this is the first practical and easy to implement methodology that has been reported in the literature. the method captures the full extent of the contractile signal, which represents an important contribution to the analysis of cardiocytes and potential to cardiovascular research. our future work entails the use of this methodology in a real - time application, which can be used to analyze the contractility of neonatal cardiocytes under different conditions. we have recently published a similar image analysis - based application for the assessment of adult cardiac myocytes. our intent is the creation of an image analysis - based methodology that can be used for assessing the contractility in cardiocytes in all stages of development and that can provide a reliable alternative to the commercially available contractility analysis tools. the availability of a similar protocol for measuring both neonatal and adult cardiocyte contractions would allow a consistent approach to characterize inotropic effects of drugs on cell types in all stages of development. | we describe a computational framework for the quantitative assessment of contractile responses of isolated neonatal cardiac myocytes. to the best of our knowledge, this is the first report on a practical and accessible method for the assessment of contractility in neonatal cardiocytes. the proposed methodology is comprised of digital video recording of the contracting cell, signal preparation, representation by polar fourier descriptors, and contractility assessment. the different processing stages are variants of mathematically sound and computationally robust algorithms very well established in the scientific community. the described computational approach provides a comprehensive assessment of the neonatal cardiac myocyte contraction without the need of elaborate instrumentation. the versatility of the methodology allows it to be employed in determining myocyte contractility almost simultaneously with the acquisition of the ca2 + transient and other correlates of cell contraction. the proposed methodology can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease models, transgeneity, or other common applications of neonatal cardiocytes. |
ameloblastoma, is derived from the english word amel which means enamel and the greek word blastos which means the germ. it arises from the epithelium of the dental lamina, and it is characterized by its local aggressive behavior and a high recurrence rate. adamantinoma, which is presently used to illustrate a rare form of bone cancer described by fisher in 1913. the term ameloblastoma was coined by ivey and churchill in 1930, a currently accepted term. it is considered as a true neoplasm as the name implies it mimics the cells of the enamel - forming organ. it was described by robinson in 1937, as a benign tumor that is usually unicentric, nonfunctional, intermittent in growth, anatomically benign and clinically persistent. the world health organization (who) (1991) defined ameloblastoma as a benign but locally aggressive tumor with a high tendency to recur, consisting of proliferating odontogenic epithelium lying in a fibrous stroma. ameloblastoma is classified, according to who and the international agency for research on cancer, 2003, as a benign tumor with odontogenic epithelium, mature fibrous stroma and without odontogenic ectomesenchyme. it is slow - growing locally aggressive and accounts for about 10% of all odontogenic tumors in the jaw. solid multicystic ameloblastoma (sma) occur as growths arising from remnants of odontogenic epithelium, exclusively from rests of the dental lamina. smas may also arise as a result of neoplastic changes in the lining or wall of a nonneoplastic odontogenic cyst, in particular dentigerous and odontogenic keratocysts. signaling pathway such as wnt, akt and growth factors like fibroblast growth factor play a pivotal role in the pathogenesis of solid type of ameloblastoma. proteins mainly bone morphogenic protein ameloblastin, enamel matrix proteins calretinin, syndecan-1 and matrix metalloproteinases also play an important contribution in the etiopathogenesis. tumor suppressor genes p53, p63 and p73 bring about molecular changes in the pathogenesis of ameloblastoma. matrix metalloproteinases, triggers mitogens to be released, leading to the proliferation of ameloblastoma cells. mostly this type is diagnosed in young adults, with a median age of 35 years and no gender predilection. the lesions more often progresses slowly, but are locally invasive and infiltrates through the medullary spaces and erodes cortical bone. if left untreated, they resorb the cortical plate and extend into adjacent tissue. crepitation or eggshell crackling might be elicited posterior maxillary tumors might obliterate the maxillary sinus and consequently extend intracranial. radiographically smas show an expansile, radiolucent, multiloculated cystic lesion, with a characteristic soap bubble - like appearance. other findings include cystic areas of low attenuation with scattered regions representing soft tissue components. thinning and expansion of the cortical plate with erosion through the cortex is elicited, with the associated unerupted tooth displaced and resorption of the roots of adjacent teeth common. six histopathologic subtypes of solid ameloblastoma includes follicular, plexiform, acanthomatous, basal cell, granular and da. mixtures of different histological patterns are commonly observed, and the lesions are frequently classified based on the predominant pattern present. the follicular pattern type has the highest recurrence rate of 29.5% and acanthomatous type having the least recurrence rate of 4.5%, and the rate of recurrence depends on the histologic subtypes. the epithelial component of the neoplasm proliferates in the form of islands, strands and cords within the moderately to densely collagenized connective tissue stroma. a prominent budding growth pattern with small, rounded extensions of epithelium projecting from larger islands, recapitulates the various stages of enamel organ formation. the classical histological pattern of ameloblastoma described by vickers and gorlin is characterized by peripheral layer of tall columnar cells with hyperchromasia, reverse polarity of the nuclei and sub - nuclear vacuole formation. follicular type is composed of many small islands of peripheral layer of cuboidal or columnar calls with reversely polarized nucleus. the term plexiform refers to the appearance of anastomosing islands of odontogenic epithelium, with double rows of columnar cells in back to back arrangement. in acanthomatous type, the cells occupying the position of stellate reticulum undergo squamous metaplasia, with keratin pearl formation in the center of tumor islands. in granular cell ameloblastoma, cytoplasm of stellate reticulum - like cells appear coarse granular and eosinophilic. basal cell type, the epithelial tumor cells are less columnar and arranged in sheets. desmoplastic variant is composed of the dense collagen stroma, which appears hypocellular and hyalinized. other histological types are papilliferous - keratotic type, clear cell type, and mucous cell differentiation type. smas contain clear, periodic - acid schiff positive cells most often localized to the stellate reticulum - like areas of follicular sma. mucous cell type of ameloblastoma shows focal mucous cell differentiation, with vacuolated mucous cells. the main modality of treatment is surgery, with wide resection recommended due to the high recurrence rate of solid / multicystic ameloblastomas. the recurrence rate after resection is 13 - 15%, as opposed to 90 - 100% after curettage. recommend a margin of 1.5 - 2 cm beyond the radiological limit is implicated to ensure all microcysts are removed. the peripheral ameloblastoma (pa) is defined as an ameloblastoma that is confined to the gingival or alveolar mucosa. it infiltrates the surrounding tissues, mostly the gingival connective tissue, but it does not involve the underlying bone. the pa arises from remnants of the dental lamina, the so - called glands of serres, odontogenic remnants of the vestibular lamina, pluripotent cells in the basal cell layer of the mucosal epithelium and pluripotent cells from minor salivary glands. the pa is an exophytic growth restricted to the soft tissues overlying the tooth - bearing areas of the jaws, the initial diagnosis often mistaken for fibrous epulis. in the majority of cases, there is no radiological evidence of bone involvement, but a superficial bone erosion known as cupping or saucerization may be detected at surgery. the overall average age is 52.1 years, slightly higher for males than for females. the male / female ratio is 1.9 : 1, as opposed to 1.2 : 1 for the solid type. histologically same patterns are as in solid type, with a common type being acanthomatous. differential includes peripheral reactive lesions such as pyogenic granuloma, epulis, papilloma, fibroma, peripheral giant - cell granuloma, peripheral odontogenic fibroma, peripheral - ossifying fibroma, baden 's odontogenic gingival epithelial hamartoma, and basal cell carcinoma. 9% of recurrence following treatment has been reported, though malignant transformation is rare, metastasis has also been reported. desmoplastic ameloblastoma was first reported by eversole. in 1984 and was recently included in the who 's classification of head and neck tumors (who-2005). this tumor is characterized by an unusual histomorphology, including extensive stromal collagenization or desmoplasia, leading to the proposed term ameloblastoma with pronounced desmoplasia or da. radiographically it produces mixed radiolucent - radioopaque lesion with diffuse border that indicates that the tumor is more aggressive than other variants of ameloblastoma. mixed radiologic appearance expresses the infiltrative pattern of the tumor and when the da infiltrates the bone marrow spaces, remnants of the original nonmetaplastic or nonneoplastic bone were found to remain in the tumor tissue. the infiltrative behavior of the da explains one of the characteristic features of the tumor, the ill - defined border. the da also appears as a poorly defined, mixed, radiolucent - radiopaque lesion mimicking a benign fibro - osseous lesion, especially when evaluating panoramic and periapical radiographs. histologically da appears as irregularly shaped odontogenic epithelial islands surrounded by a narrow zone of loose - structured connective tissue embedded in desmoplastic stroma. about 15.9% rate of recurrence has been reported in da cases treated by enucleation and/or curettage, with an average recurrence period of 36.9 months. the majority of da cases reported treated by resection, most likely due to ill - defined borders, consequently suggesting an infiltration process and aggressive biological behavior. unicystic ameloblastoma (ua) represents an ameloblastoma variant, presenting as a cyst that show clinical and radiologic characteristics of an odontogenic cyst. in histologic examination shows a typical ameloblastomatous epithelium lining part of the cyst cavity, with or without luminal and/or mural tumor proliferation. in 1977, ua, but it was also named in the second edition of the international histologic classification of odontogenic tumors by the who as cystogenic ameloblastoma. ua with an unerupted tooth occurs with a mean age of 16 years as opposed to 35 years in the absence of an unerupted tooth. the mean age is considerably lower than that for solid / multicystic ameloblastoma with no gender predilection. ua is a prognostically distinct entity with a recurrence rate of 6.7 - 35.7%, and the average interval for recurrence is approximately 7 years. three pathogenic mechanisms for the evolution of ua : reduced enamel epithelium, from dentigerous cyst and due to cystic degeneration of solid ameloblastoma. six radiographic patterns are identified for ua, ranging from well - defined unilocular to multilocular ones. comparing unilocular and multilocular variants, there is an apparent predominance of a unilocular configuration in all studies of ua, especially in cases associated with impacted teeth. histopathological classification of uas are : luminal ualuminal and intraluminal uasluminal, intraluminal, and intramural uasluminal and intramural uas. luminal and intraluminal uas luminal, intraluminal, and intramural uas luminal and intramural uas. treatment of ua includes both radical and conservative surgical excision, curettage, chemical and electrocautery, radiation therapy or combination of surgery and radiation. the ameloblastoma is usually of late diagnosis because of its poor symptoms and low prevalence. routine histological classification of the ameloblastoma is mandatory for its morphological characterization and, thus, a better treatment definition. the main success factor associated with the treatment is the early diagnosis and to correlate the histopathologic findings with clinical and radiographic features to achieve at a correct definitive diagnosis as all such lesions might have prognostically different biologic behaviors and the final diagnosis may alter the therapeutic decision significantly. | ameloblastoma is a benign odontogenic tumor generally present in the jaw bone. the tumor originates from the residual epithelium of the tooth germ, epithelium of odontogenic cysts stratified squamous epithelium and epithelium of the enamel organ. it represents approximately 1% of oral tumors. about 80% of ameloblastomas occur in the mandible mainly the third molar region and the remaining 20% in the upper jaw. ameloblastoma clinically appears as an aggressive odontogenic tumor, often asymptomatic and slow - growing, with no evidence of swelling. this article deals with a complete review on ameloblastoma. |
operative site infection is one of the most common complications of obstetrical and gynecological surgeries such as hysterectomy and cesarean section. the use of prophylactic antibiotics in hysterectomy and cesarean section in our hospital is not standardized and is determined by the consultant in charge of the unit. the most commonly used regimens are ciprofloxacin - metronidazole (cip - met) and ceftriaxone - metronidazole (cef - met). considering this background, this study was performed to compare the effectiveness of the cip - met regimen versus the cef - met regimen in preventing infection following various obstetrical and gynecological surgeries. this retrospective study was conducted at the obstetrics and gynecology department, guru gobind singh hospital, jamnagar. data of six months (june 2010 to december 2010) were collected. with prior permission, records of 1084 patients who had undergone vaginal or abdominal hysterectomy, laparoscopy, laparotomy, elective or emergency cesarean section were noted. we excluded case records of those patients who had any associated medical illness like diabetes mellitus, chronic obstructive pulmonary disease, etc. we divided the patients into two groups, who were given one of the two regimens : group 1 (n=534) was given ciprofloxacin 200 mg intravenous infusion 12-hourly plus metronidazole 500 mg intravenous infusion 8-hourly for 48 h or till oral intake by patients, followed by tablet (ciprofloxacin 500 mg 12-hourly plus metronidazole 400 mg 8-hourly) to complete five days treatment. group 2 (n=550) was given injection ceftriaxone 1 g intravenously daily plus injection metronidazole 500 mg intravenous infusion 8-hourly for 48 h or till oral intake by patients, followed by tablet (cefixime 200 mg 12-hourly plus metronidazole 400 mg 8-hourly) to complete five days treatment. the records of patients were checked for occurrence of wound infection in the postoperative period. wound infection rates were analyzed statistically by fisher 's exact test and yates continuity corrected chi - square test using graphpad prism version 5.01 software. out of the total 1084 case records, the distribution of patients according to type of surgery such as cesarean section, laparoscopy, hysterectomy and laparotomy is listed in table 1. the age for obstetrics patients in our study ranged from 18 - 40 years, while for gynecology patients the age was up to 60 years. incidence of postoperative wound infections in obstetric and gynecological surgeries is recorded in table 3. while the overall infection rate of all the surgical procedures in both the groups was 3.6% (39 infections in 1084 patients), the infection rates in group 1 and group 2 were 5.81% (31 infections in 534 patients) and 0.7% (eight infections in 550 patients) respectively (p=0.0001, confidence interval (ci) 95% 1.909.17, odds ratio 4.18). comparative analysis of the total duration, cost and adverse events of antimicrobial therapy is summarized in table 4. distribution of patients according to the type of surgery analysis of risk factors for infections (n=1084) incidence of postoperative wound infections in obstetric and gynecological surgeries comparative analysis of the total duration, cost and adverse events of antimicrobial therapy a number of studies indicate that chemoprophylaxis can be justified in dirty or contaminated surgical procedures, where the incidence of wound infection is high and these include less than 10% of all surgical procedures. postoperative infection in surgical sites in obstetric and gynecological settings has been higher as compared to other specialties because of the contaminated nature of most obstetric and gynecological procedures. consideration of prophylactic antibiotics has been suggested for all elective cesarean deliveries in which the combined incidence of endometritis and wound infection exceeds 5%. a recent review of the literature concluded that a narrow spectrum agent prior to incision or an extended spectrum regimen (with metronidazole) after cord clamp offers the best outcome for patients in cesarean deliveries. nowadays ciprofloxacin - metronidazole and cephalosporin - metronidazole combinations are chosen by surgeons to prevent wound infection following major abdominal surgical procedures. in our study, it was observed that the rate of postoperative infection was significantly higher in group 1 than group 2 (p=0.0001). this might be due to development of resistance to ciprofloxacin by anaerobes which are the potential pathogens in the vaginal flora. on the other hand, low incidence of postoperative infection seen in group 2 in our study might be attributed to the effectiveness of third - generation cephalosporins in mixed aerobic - anaerobic infections. postoperative infection is not only dependent on antibiotic use but also on many other factors, such as age, nutritional status, hygienic condition, anemic status, duration of operation, blood loss during operation and amount of blood transfusion. in the present study a greater number of infections (26 out of 39) were observed in those operations in which the average duration of surgery was between 30 - 120 min (p<0.0001). the surgeries in which all operative wound infections were recorded (i.e. 39), were performed within 120 min. it was also observed that the majority of the infections (34 out of 39) were found in patients of low socioeconomic status (p=0.043). similarly, a higher rate of infections (33 out 39) were noted in patients who were anemic (p=0.023) [table 2 ]. therefore, it could be surmised that prolonged duration of surgery, lower socioeconomic status of the patient and anemia were also responsible for development of infections in our study. thirteen and nine cases of mild nausea and vomiting were reported in group 1 and group 2 respectively. these adverse events found in our study most likely could be due to metronidazole therapy. adopting a cost - effective regimen would save hospital time and days of bed occupancy. for a patient, this would prevent pain and anxiety associated with infection. it will also prevent the loss of time and the loss of daily earnings by reducing the hospital stay of the patient. this retrospective study indicates that though the cip - met regimen was slightly more cost - effective (difference of indian rupees 10.00), the rate of postoperative infection which was the primary concern was significantly higher with this regimen. this suggests that the cef - met regimen was better than the cip - met regimen to control operative site wound infection rates in obstetrical and gynecological surgeries. hence, ceftriaxone should be a part of the essential medicine formulary of the hospital. | objective : to compare the effectiveness of the ciprofloxacin - metronidazole (cip - met) regimen with the ceftriaxone - metronidazole (cef - met) regimen for operative site infection control in women undergoing obstetrical and gynecological surgeries.materials and methods : one thousand and eighty - four case records of women who had undergone various obstetrical and gynecological surgeries who were given cip - met regimen and cef - met regimen were analyzed in predesigned and pretested proforma. patients who were given cip - met regimen and cef - met regimen were classified as group 1 and group 2 respectively. the mode of administration of both the regimens was noted. numbers of wound infections were recorded in the respective groups. socioeconomic status and hemoglobin level of the patients were noted. other data such as hospital stay, duration of operation were also noted.results:out of a total of 1084 case records, 31 (5.8%) and eight (0.7%) patients contracted wound infections in group 1 and group 2 respectively (p = 0.0001).conclusion : the cef - met regimen was found superior to the cip - met regimen to control operative site infection in obstetrical and gynecological surgeries. |
this report evaluated the efficacy of three brushes and one biofilm disclosing agent in complete denture cleansing. methods : twenty - seven wearers of maxillary dentures were distributed into three groups and received different brushes : oral b40, conventional toothbrush (oral b) ; denture, denture - specific brush (condor) ; johnson & johnson, denture - specific brush (johnson & johnson). the 60-day experimental period was divided into two techniques : i - brushing (brush associated with a paste - dentu creme, dentco) three times a day ; ii - brushing and daily application of 1% neutral red on the denture internal surface. biofilm quantification was carried out weekly and the areas with dye biofilm were obtained by means of image tool 2.02 software. biofilm removal was more effective during technique ii (wilcoxon test : p=0.01) for the three groups of brushes. when the brushes were compared in technique i, the kruskal wallis test indicated statistical difference between denture x johnson & johnson and denture x oral b40, in which the denture was more efficient. for technique the disclosed application promoted more efficacy on biofilm removal, regardless of the brush used. although many reports evaluate the efficacy of denture cleansers, surveys point out that removal of denture biofilm by denture wearers is precarious13,5,6,23. deficient patient orientation, inadequate divulgation of specific denture cleanser materials, intrinsic characteristics of prosthetic appliances and deficient manual dexterity of elderly people have been indicated as causes of poor denture hygiene14,20,22,26. biofilm removal can be obtained by means of mechanical methods (brushing and ultrasonic devices) associated with chemical methods (alkaline peroxide and hypochlorite, acids, enzymes and disinfectants) ; among them, brushing is the most common method applied for routine denture biofilm control1,2,17,19,35. knowledge on materials and methods for quantification of denture biofilm is an important factor to be considered. while biofilm quantification on natural teeth is significantly studied and published in the literature, its quantification on complete dentures is poorly known, due to the small number of papers published on this matter and because the procedure is not routinely employed by dentists16,24,27,28. with respect to natural teeth, efficient hygiene control in complete dentures can be obtained by an orientation program, correct use of materials and methods available for denture cleansing and by utilization of a biofilm disclosing agent, allowing quantification and localization of biofilm on dentures, which could allow its removal more effectively5,8,12,16,24,26,28,31,35. the aim of this study was to evaluate the efficacy of a biofilm disclosing agent associated with three brushes on hygiene control and maintenance in complete dentures. after research approval by the institutional review board of ribeiro preto dental school - university of so paulo (no. 2000 - 136358.0), 27 healthy men and women wearing complete denture (maxillary and mandibular complete dentures) were selected, with approximate mean ages of 50 years, and with no motor deficiency according to the discipline of complete denture of forp - usp. patients ' maxillary complete dentures were constructed with heat- cured acrylic resin, acrylic teeth, none of them with any break or repair, wearing time ranging from 1 to 3 years and score of biofilm degree of at least 1 (additive index 2). patients were included in the research after being verbally informed, when they read and signed the consent term. patients were divided into three groups : toothbrush (oral b40 - service industry and commerce ltd., so paulo, so paulo, brazil) ; denture brush (johnson & johnson - johnson & johnson, so paulo, brazil) ; and denture brush (denture - condor s.a., santa catarina, brazil). for all groups, a specific denture paste (dentu - creme, dentco, inc. two hygiene techniques were indicated for all groups ; technique i : the subjects were instructed to brush their dentures three times a day, rinsing their mouth with water after brushing and keeping the denture immersed in water overnight ; technique ii : the instruction was to brush the denture 3 times a day associated with the use of a disclosing agent (1% neutral red) to the last brushing of the day ; in this technique, patients were also instructed to rinse their mouth with water after brushing and to keep the denture immersed in water overnight. after the patients had received instructions and hygiene materials, they were assessed by the investigators, who performed disclosure of the internal surface of maxillary dentures with 1% neutral red solution. the investigator brushed the disclosed dentures until complete removal of the disclosed biofilm (biofilm - free). at first, technique i was employed by all patients for 3 weeks ; after this period, the investigator accomplished complete denture biofilm removal by brushing (biofilm - free) and then technique ii was employed by all patients for further 3 weeks. during each technique, the patients attended weekly returns when the maxillary denture was removed, rinsed with running water (5 seconds) and air - dried (10 seconds). afterwards, the disclosing agent (1% neutral red) was applied on the internal surface with a cotton swab and the denture was rinsed and dried again. the disclosed surfaces were photographed (digital camera, coolpix, nikon, melville, n.y., the camera was fixed on a stand (cs-4 copy stand testrite, newark, nj, usa) at 90 to the internal denture surface. after photographing, dentures were brushed with denture brush, in order to perform full biofilm removal, and then returned to the patients. biofilm quantification was performed at the end of each hygiene technique by a computerized method. by this method, the total internal surface of the denture and biofilm - covered areas were measured using the image tool software (windows, version 2.02, uthsc, san antonio). the percentages of biofilm coverage areas were calculated as the ratio between disclosed areas and the area of denture 's internal surface multiplied by 100. three measurements were performed (one for each visit) of biofilm degree for each treatment (i and ii). preliminary normality and homogeneity tests applied to the results showed no normal distribution ; therefore, the wilcoxon test was employed to compare the effectiveness of both techniques for each brush group ; the kruskal wallis test was employed to compare the effectiveness of brushes. these tests were applied to the average of biofilm percentage for each denture in each treatment (3 measurements). table 1 shows the mean of three measurements of biofilm percentage on the internal surface of the evaluated complete dentures and the sum of means. table 2 shows the statistical significance between techniques i and ii (wilcoxon test). table 3 shows the results of kruskal wallis test applied to the means (%) shown in table i ; this test performs comparisons between brush groups with both techniques. (ns) : non - significant figure 1 shows the comparison between the sum of means obtained for three brushes in each technique, and compares the techniques with each other the relationship between denture biofilm, mucosal inflammation and atrophic chronic candidiasis has been discussed along the years ; there is confirmation that cleansing can help to control or solve the inflammatory conditions of the mucosa by reducing the degree of denture biofilm. nevertheless, several studies call attention to the precarious oral health of complete denture wearers1,5,11,16,17,19,23,34. the toothbrush is the most important element in any hygiene program and in complete denture hygiene control ; brushing is the most common and routine method employed in association with paste as an auxiliary agent. some studies have shown that toothbrushes (conventional brushes) can cause wear on the denture materials and do not provide adequate hygiene, since their design is inappropriate to reach the entire denture surfaces (internal and external)7,9,32,33. such materials are customarily found in other countries, but in the brazilian market there is only a limited number of specific denture products ; as a result, denture wearers make use of conventional brushes to clean their dentures. studies regarding natural teeth hygiene emphasize the importance of incentive programs on the control of biofilm formation and many of these indicate home use of disclosing agents as additional means to brushing14,16,25. in the reviews, complete denture studies indicate the use of these solutions only to quantitatively and qualitatively evaluate the denture biofilm2,3,4,12,17,22,24,28 as a mean to evaluate hygiene products. therefore, it is necessary compare the effectiveness of brushes that are available in the brazilian market, looking for their viable association with alternative methods, as well as using a disclosing agent to make biofilm removal easier and more effective. oral b toothbrush (conventional) has established good acceptance and regularity in the brazilian the years. it is composed of 41 soft bristles positioned perpendicularly at the same side of brush 's head, which has an oval design. the johnson & johnson brush was designed by paranhos,.30 (2000) to evaluate a specific dentifrice on the control of atrophic chronic candidiasis in denture wearers. it has 26 tufts with 16-mm long bristles positioned at the same side of the brush 's head. the longer length makes the bristles become extra - soft and provides a better range to reach the denture areas. the denture brush has a long handle, the head is elongated and has two groups of soft bristles positioned at each side of the head. one of them is more compact with a wedge - like form and is positioned on the extremity of the head. this bristle displays an anterior angulation and is designed to perform brushing on the internal surface of denture. the other group has a larger number of bristles, positioned perpendicular to the head covering its full extension and is designed to perform brushing on the denture 's external surface. with regard to the biofilm quantification method on complete dentures, disclosure is the most employed method in the literature and is customarily associated with the score attribution method (index). nevertheless, some components of this methodology allow variations in results when utilized by different examiners. in this study, the biofilm quantification methodology was based on utilization of disclosed surfaces and a computerized method (image toll 2.02) to quantify biofilm. the image tool 2.02 program was employed to obtain final results, since this method supplies numerical data that avoid percentage calculations of the biofilm coverage area. assessment was performed on the internal denture surface, since it is the area most often used to evaluate cleanser efficacy. lovato,.21 (2000), studying clinical manipulation of a disclosing agent, highlighted the fact that this agent must have the ability to disclose biofilm, be easy to remove from the denture, and not stain the denture after removal. these authors indicated 1% neutral red, 1% sodium fluorescein, replak and 1% monosulphate proflavim as disclosing agent. the 60-day experimental period was divided between technique i (brushing) and technique ii (brushing and disclose home application). before starting technique i, the denture was disclosed and the biofilm was fully removed by brushing (denture - brush - condor ; dentu - crme, dentco) by the investigator. at this moment, the biofilm was photographed, because it was not the purpose of this study to evaluate the improvement of denture hygiene related to the patients ' hygiene habits, but rather to evaluate the efficacy of techniques i and ii used in this study in association with different brushes. all 3 brush groups started the experiment with technique i, which was employed for 3 weeks. after the end of technique i, the patients were instructed to return to their habitual hygiene for 15 days so that the investigator could repeat disclosing and brushing of the maxillary complete dentures, to provide full biofilm removal (biofilm - free) before initiation of technique ii (3 weeks). this 15-day interval was important to reduce the influence from one technique on the other. throughout the entire experimental period, the patients returned weekly for biofilm quantification, since 7 days is an appropriate period for new biofilm deposition and to allow for good patient control. the 1% neutral red was selected due to its good affinity with biofilm, removal efficacy from denture surface and because it does not damage the denture components (teeth and base). regarding the efficacy of brushes during technique ii, no significant difference was found among the three groups. these results indicate that it is not necessary to use a specific brush when an auxiliary agent for biofilm identification was employed prior to brushing. by using disclosing this can be explained by the fact that disclosure promoted biofilm visualization by the patient 5,18,20,25. in agreement with kipot,.18 (1984), it is essential that patients be instructed, trained and motivated to continue adequate oral hygiene. the increase in life expectancy of the brazilian population further increases the needs of edentate patients. this study aimed at contributing with maintenance of patients ' health, since there is direct association between denture biofilm control and associated pathologies as chronic atrophic candidiasis. home use of a disclosing agent improved biofilm control in dentures for all 3 groups of brushes. denture brush was more effective than the others in technique i, while there was no difference among brushes in technique ii. therefore, without the use of a disclosing biofilm agent, a specific brush for denture hygiene should be indicated. on the other hand, any type of brush can be used if associated with home use of a disclosing agent. | objective : this report evaluated the efficacy of three brushes and one biofilm disclosing agent in complete denture cleansing. methods : twenty - seven wearers of maxillary dentures were distributed into three groups and received different brushes : oral b40, conventional toothbrush (oral b) ; denture, denture - specific brush (condor) ; johnson & johnson, denture - specific brush (johnson & johnson). the 60-day experimental period was divided into two techniques : i - brushing (brush associated with a paste - dentu creme, dentco) three times a day ; ii - brushing and daily application of 1% neutral red on the denture internal surface. biofilm quantification was carried out weekly and the areas with dye biofilm were obtained by means of image tool 2.02 software.results:biofilm removal was more effective during technique ii (wilcoxon test : p=0.01) for the three groups of brushes. when the brushes were compared in technique i, the kruskal wallis test indicated statistical difference between denture x johnson & johnson and denture x oral b40, in which the denture was more efficient. for technique ii, there was no statistical difference between brushes (p>0.05).conclusion : the disclosed application promoted more efficacy on biofilm removal, regardless of the brush used. denture (condor) was more efficient than the other brushes during technique i. |
animals : all animals were maintained in accordance with the recommendations of the guide for the care and use of laboratory animals approved by the faculty of agriculture, tokyo university of agriculture and technology. five clinically healthy male shiba goats, weighing 2543 kg and aged 23 years, were used in this study. animals were fed hey cubes (# 1a cubes, eckenberg farms inc., mattawa, wa, u.s.a.) at 600 g / head twice a day, and water was available ad libitum. reagents and chemicals : the sodium salt of df and flufenamic acid (fa) were obtained from sigma - aldrich corporation (st. louis, mo, u.s.a.). smm and sulfadimethoxine were obtained as a sodium salt from daiichi pharmaceutical company (tokyo, japan). all other reagents and chemicals used in this study were of hplc or analytical grade. pharmacokinetic study : df or smm was dissolved in sterilized distilled water for injection and administered either into the left jugular vein or orally to 5 male shiba goats at doses of 1.0 and 10 mg / kg, respectively, using a crossover design with at least a 3-week washout period. in case of the oral administration of these drugs, drug solutions were given with 3 hay cubes. blood (3 ml) was collected from the right jugular vein immediately prior to the treatment and 0.5, 1, 2, 3, 4, 6, 9, 12 and 24 hr after dosing. plasma was separated by the centrifugation of blood at 1,600 g for 10 min and stored at 20c until analysis. stability of df and smm in the rumen juice : two male shiba goats were restrained, and nasal catheters were passed into the rumen. thereafter, 40 ml of rumen fluid was aspirated through the catheter and processed for incubation immediately after its collection. fifty microliters of df or smm solutions (200 g / ml) was added to 950 l of the rumen fluid to give a final concentration of 10 g / ml of the incubation mixture. five samples of each drug were prepared and incubated in a thermostatic shaking water bath at 39c for 24 hr under anaerobic conditions. after the incubation, the concentrations of df and smm were measured by hplc. diclofenac : df concentrations in the plasma and rumen juice were determined by hplc with uv - detection, as described previously with some modifications. briefly, 100 l of fa solution (10 g / ml) was added as an internal standard to 500 l of the plasma or rumen juice sample, followed by the addition of 200 l of phosphoric acid (0.15 m). subsequently, 4 ml of diethyl ether was added to the mixture and shaken for 3 min. the obtained supernatant (organic layer) was transferred into a pear shaped flask and evaporated to dryness by an evaporator (rotavapor r-114, shibata scientific technology, ltd., the residue was reconstituted in 200 l of the mobile phase and filtered using a 0.45-m hplc filter (chromatodisc, 4p, kurabo biomedical industries, ltd., fifty microliters of the filtrate was injected into the hplc column. the hplc system (shimadzu corporation, kyoto, japan) consisted of a pump (lc-10ad), a uv detector (spd-6a), an integrator (chromatopac c - r7a plus) and a loop injector (model 7125). the mobile phase was a mixture of 0.1 m acetate buffer (ph 6.3) and acetonitrile (65:35, v / v). analytical separation was accomplished by using a reversed - phase ods column (tsk - gel ods-120 t, 4.6 m 250 mm, tosoh co., tokyo, japan). the recovery from plasma samples was 106.1 2.8% at 1 g / ml (n=5), while that from rumen juice samples was 110.3 8.5% at 10 g / ml (n=5). the inter - day cv values ranged from 0.83 to 3.72% for plasma samples and from 3.11 to 14.1% for rumen juice samples (n=5, 3 times). sulfamonomethoxine : smm concentrations were determined in the plasma and rumen juice samples by hplc with uv - detection. two hundred microliters of perchloric acid (0.5 m) was added to 200 l of the plasma sample. the mixture was vortexed for 30 sec and then centrifuged at 20,000 g for 5 min at 5c. fifty microliters of the filtrate was injected into the hplc column. in the case of rumen juice samples, smm concentrations were determined after extraction with ethyl acetate. after being incubated for 24 hr, 100 l of sulfadimethoxine solution (10 g / ml) the mixture was vortexed for 30 sec and then centrifuged at 3,000 g for 10 min at 5c. the obtained supernatant was transferred into a pear shaped flask and evaporated to dryness at 30c. the residue was reconstituted in 200 l of the mobile phase and filtered using the 0.45-m hplc filter. the mobile phase was a mixture of 50 mm acetate buffer (ph 5) and acetonitrile (75:25, v / v). analytical separation was accomplished using a reversed - phase c8 column (mightysil rp-8 gp, 4.6 m 250 mm, kanto chemical co., tokyo, japan). the recovery from plasma samples was 101.7 4.34% at 1 g / ml (n=5), while that from rumen juice samples was 99.4 4.2% at 10 g / ml. the inter - day cv values ranged from 3.23 to 5.82% for plasma samples and from 3.39 to 4.67% for rumen juice samples (n=5, 3 times). pharmacokinetic analysis : the plasma concentration - time curves of df after the intravenous injection fit well with the two compartment model. therefore, the curves obtained after the intravenous injection (cpiv (t)) and oral administration (cppo (t)) were described by eqs. 1 and 2, respectively. equations 1 and 2 were simultaneously fit to the plasma concentration - time curves of df after it was intravenously and orally administered to the same goats, respectively, in order to calculate pharmacokinetic parameters by the nonlinear least squares method using the curve fitting program, multi. on the other hand, the plasma concentration - time curves of smm after it was intravenously administered fit well with the one compartment model. therefore, the curves obtained after the intravenous injection (cpiv (t)) and those after the oral administration (cppo (t)) were described by eqs. 3 and 4, respectively. (eq.3) (eq.4) equations 3 and 4 were simultaneously fit to the plasma concentration - time curves after the intravenous injection and oral administration to the same goats, respectively. the area under the concentration versus time curve (auc) was calculated by the trapezoidal method (from time zero to the last sampling time) and integration (from the last sampling time to infinity). total body clearance (cltot), bioavailability, mean residence time (mrt), mean absorption time (mat) and the distribution volume at a steady state (vdss) were calculated by conventional methods. pharmacokinetic study : df or smm was dissolved in sterilized distilled water for injection and administered either into the left jugular vein or orally to 5 male shiba goats at doses of 1.0 and 10 mg / kg, respectively, using a crossover design with at least a 3-week washout period. in case of the oral administration of these drugs, blood (3 ml) was collected from the right jugular vein immediately prior to the treatment and 0.5, 1, 2, 3, 4, 6, 9, 12 and 24 hr after dosing. plasma was separated by the centrifugation of blood at 1,600 g for 10 min and stored at 20c until analysis. stability of df and smm in the rumen juice : two male shiba goats were restrained, and nasal catheters were passed into the rumen. thereafter, 40 ml of rumen fluid was aspirated through the catheter and processed for incubation immediately after its collection. fifty microliters of df or smm solutions (200 g / ml) was added to 950 l of the rumen fluid to give a final concentration of 10 g / ml of the incubation mixture. five samples of each drug were prepared and incubated in a thermostatic shaking water bath at 39c for 24 hr under anaerobic conditions. after the incubation, the concentrations of df and smm were measured by hplc. diclofenac : df concentrations in the plasma and rumen juice were determined by hplc with uv - detection, as described previously with some modifications. briefly, 100 l of fa solution (10 g / ml) was added as an internal standard to 500 l of the plasma or rumen juice sample, followed by the addition of 200 l of phosphoric acid (0.15 m). subsequently, 4 ml of diethyl ether was added to the mixture and shaken for 3 min. the obtained supernatant (organic layer) was transferred into a pear shaped flask and evaporated to dryness by an evaporator (rotavapor r-114, shibata scientific technology, ltd., the residue was reconstituted in 200 l of the mobile phase and filtered using a 0.45-m hplc filter (chromatodisc, 4p, kurabo biomedical industries, ltd., fifty microliters of the filtrate was injected into the hplc column. the hplc system (shimadzu corporation, kyoto, japan) consisted of a pump (lc-10ad), a uv detector (spd-6a), an integrator (chromatopac c - r7a plus) and a loop injector (model 7125). the mobile phase was a mixture of 0.1 m acetate buffer (ph 6.3) and acetonitrile (65:35, v / v). analytical separation was accomplished by using a reversed - phase ods column (tsk - gel ods-120 t, 4.6 m 250 mm, tosoh co., tokyo, japan). the recovery from plasma samples was 106.1 2.8% at 1 g / ml (n=5), while that from rumen juice samples was 110.3 8.5% at 10 g / ml (n=5). the inter - day cv values ranged from 0.83 to 3.72% for plasma samples and from 3.11 to 14.1% for rumen juice samples (n=5, 3 times). sulfamonomethoxine : smm concentrations were determined in the plasma and rumen juice samples by hplc with uv - detection. two hundred microliters of perchloric acid (0.5 m) was added to 200 l of the plasma sample. the mixture was vortexed for 30 sec and then centrifuged at 20,000 g for 5 min at 5c. fifty microliters of the filtrate was injected into the hplc column. in the case of rumen juice samples, smm concentrations were determined after extraction with ethyl acetate. after being incubated for 24 hr, 100 l of sulfadimethoxine solution (10 g / ml) was added as an internal standard to the rumen juice samples. the mixture was vortexed for 30 sec and then centrifuged at 3,000 g for 10 min at 5c. the obtained supernatant was transferred into a pear shaped flask and evaporated to dryness at 30c. the residue was reconstituted in 200 l of the mobile phase and filtered using the 0.45-m hplc filter. the mobile phase was a mixture of 50 mm acetate buffer (ph 5) and acetonitrile (75:25, v / v). analytical separation was accomplished using a reversed - phase c8 column (mightysil rp-8 gp, 4.6 m 250 mm, kanto chemical co., tokyo, japan). the recovery from plasma samples was 101.7 4.34% at 1 g / ml (n=5), while that from rumen juice samples was 99.4 4.2% at 10 g / ml. the inter - day cv values ranged from 3.23 to 5.82% for plasma samples and from 3.39 to 4.67% for rumen juice samples (n=5, 3 times). pharmacokinetic analysis : the plasma concentration - time curves of df after the intravenous injection fit well with the two compartment model. therefore, the curves obtained after the intravenous injection (cpiv (t)) and oral administration (cppo (t)) were described by eqs. 1 and 2, respectively. equations 1 and 2 were simultaneously fit to the plasma concentration - time curves of df after it was intravenously and orally administered to the same goats, respectively, in order to calculate pharmacokinetic parameters by the nonlinear least squares method using the curve fitting program, multi. on the other hand, the plasma concentration - time curves of smm after it was intravenously administered fit well with the one compartment model. therefore, the curves obtained after the intravenous injection (cpiv (t)) and those after the oral administration (cppo (t)) were described by eqs. 3 and 4, respectively. (eq.3) (eq.4) equations 3 and 4 were simultaneously fit to the plasma concentration - time curves after the intravenous injection and oral administration to the same goats, respectively. the area under the concentration versus time curve (auc) was calculated by the trapezoidal method (from time zero to the last sampling time) and integration (from the last sampling time to infinity). total body clearance (cltot), bioavailability, mean residence time (mrt), mean absorption time (mat) and the distribution volume at a steady state (vdss) were calculated by conventional methods. the plasma concentrations of df and smm rapidly increased and peaked 12 hr and 56 hr after being orally administered, respectively, followed by their slow elimination. on the other hand, plasma concentrations decreased rapidly after the intravenous injection with relatively short half - lives (3.05 1.13 hr for df and 1.00 0.11 hr for smm), indicating flip - flop phenomena after the oral administration of both drugs (figs. 1.plasma concentration - time curves of diclofenac (1.0 mg / kg body weight) after its single intravenous (opened circle) and oral administration (closed circle) to male shiba goats. each point and vertical bar represent the mean and standard deviation, respectively (n=5). each line was calculated by eqs. 1 or 2 using pharmacokinetic parameters in table 1. and 2.plasma concentration - time curves of sulfamonomethoxine (10 mg / kg body weight) after its single intravenous (opened circle) and oral administration (closed circle) to male shiba goats. each point and vertical bar represent the mean and standard deviation, respectively (n=5). each line was calculated by eqs. 3 or 4 using pharmacokinetic parameters in table 2.). as shown in tables 1table 1.pharmacokinetic parameters of df in male shiba goats determined after single intravenous and oral administration of 1 mg / kg body weightparametermean sd (n=5)ka (hr)0.194 0.073cmax (g / ml)1.12 0.58tmax (hr)1.51 1.41 (hr)2.09 0.97 (hr)0.250 0.078 t 1/2ka (hr)4.13 1.94 t 1/2 (hr)3.05 1.13auc i.v. (ghr / ml)14.7 6.2auc p.o. (ghr / ml)10.4 4.0cl (l / hr / kg)0.0784 0.0309f (%) 75.4 24.0f (%) 73.9 20.2mrt i.v. (hr)8.42 2.15mat (hr)6.05 2.74vdss (l / kg)0.181 0.102ka = absorption rate constant ; cmax = maximum plasma concentration ; tmax = time to maximum plasma concentration ; = first - order rate constant associated with the distribution phase ; = first - order rate constant associated with the elimination phase ; t 1/2ka = absorption half - life ; t 1/2 = elimination half - life ; auci.v. = area under the plasma concentration time curve after oral administration ; cl = total body clearance ; f = bioavailability calculated by compartmental analysis ; f = bioavailability calculated by non - compartmental analysis ; mrti.v. = mean residence time after p.o administration ; mat = mean absorption time ; vdss = volume of distribution at a steady state. and 2table 2.pharmacokinetic parameters of smm in male shiba goats determined after single intravenous and oral administration of 10 mg / kg body weightparametermean sd (n=5)ka (hr)0.0737 0.0296cmax (g / ml)2.15 0. 29tmax (hr)5.60 2.30kel (hr)0.703 0.084 t 1/2ka (hr)10.5 3.6 t 1/2 (hr)1.00 0.11auc i.v. (ghr / ml)49.9 11.3auc p.o. (ghr / ml)37.5 6.7cl (l / hr / kg)0.212 0.067f (%) 79.3 16.5f (%) 77.1 14.8mrt i.v. (hr)16.6 4.6mat (hr)15.1 4.7vdss (l / kg)0.321 0.134ka = absorption rate constant ; cmax = maximum plasma concentration ; tmax = time to maximum plasma concentration ; kel = elimination rate constant ; t 1/2ka = absorption half - life ; t1/2kel = elimination half - life ; auci.v. cl = total body clearance ; f = bioavailability calculated by compartmental analysis ; f = bioavailability calculated by non - compartmental analysis ; mrti.v. = mean residence time after p.o administration ; mat= mean absorption time ; vdss = volume of distribution at a steady state., a pharmacokinetic analysis indicated the slow absorption of both drugs in male shiba goats. the calculated mats of df and smm were approximately 6 and 15 hr, respectively. the absorption half - life (t1/2ka) of df was slightly longer than its elimination half - life (t1/2). on the other hand, the t1/2ka of smm was markedly longer than its t1/2kel (approximately 10 times). the bioavailabilities of both drugs were more than 70%, as listed in tables 1 and 2. plasma concentration - time curves of diclofenac (1.0 mg / kg body weight) after its single intravenous (opened circle) and oral administration (closed circle) to male shiba goats. each point and vertical bar represent the mean and standard deviation, respectively (n=5). each line was calculated by eqs. 1 or 2 using pharmacokinetic parameters in table 1. plasma concentration - time curves of sulfamonomethoxine (10 mg / kg body weight) after its single intravenous (opened circle) and oral administration (closed circle) to male shiba goats. each point and vertical bar represent the mean and standard deviation, respectively (n=5). each line was calculated by eqs. 3 or 4 using pharmacokinetic parameters in table 2. ka = absorption rate constant ; cmax = maximum plasma concentration ; tmax = time to maximum plasma concentration ; = first - order rate constant associated with the distribution phase ; = first - order rate constant associated with the elimination phase ; t 1/2ka = absorption half - life ; t 1/2 = elimination half - life ; auci.v. = area under the plasma concentration time curve after oral administration ; cl = total body clearance ; f = bioavailability calculated by compartmental analysis ; f = bioavailability calculated by non - compartmental analysis ; mrti.v. = mean residence time after p.o administration ; mat = mean absorption time ; vdss = volume of distribution at a steady state. ka = absorption rate constant ; cmax = maximum plasma concentration ; tmax = time to maximum plasma concentration ; kel = elimination rate constant ; t 1/2ka = absorption half - life ; t1/2kel = elimination half - life ; auci.v. = area under the plasma concentration time curve after oral administration ; cl = total body clearance ; f = bioavailability calculated by compartmental analysis ; f = bioavailability calculated by non - compartmental analysis ; mrti.v. = mean residence time after p.o administration ; mat= mean absorption time ; vdss = volume of distribution at a steady state. since the bioavailabilities of df and smm were incomplete, we evaluated the stability of both drugs in the rumen juice collected from male shiba goats. the recovery from rumen juice samples was completed after a 24-hr incubation and was 104.8 11.9% for df and 99.4 2.85% for smm. in the present study, we examined the absorption profiles of df and smm after their oral administration to male shiba goats. the mat values obtained were long (6 hr for df and 15 hr for smm). the oral pharmacokinetic profiles of df and smm have been clarified in several animal species. the absorption rate constant values for df were previously shown to be 0.51.2 hr in pigs, 0.5 hr in rabbits and 0.38 min1 in rats. these values were markedly higher than those obtained from the male shiba goats in the present study (0.19 hr). the absorption of smm was shown to be fast in pigs as well as horses and humans. the obtained ka values (1.8 hr in pigs and 0.023 min in horses) were markedly higher than those obtained from the male shiba goats in the present study (0.07 hr). since the absorption of drugs from the small intestines is generally fast, gastric emptying is the determining factor for drug absorption after the oral administration of drugs [10, 21 ]. markedly higher ka values were obtained for smm in pigs and df in rats after their intraduodenal administration than after their oral administration [11, 18 ]. this may also have been the case for the male shiba goats used in the present study. therefore, the slow absorption rate of df and smm in the male shiba goats may be due to their long residence time in the forestomach. although a pharmacokinetic analysis indicated the slow absorption of df and smm after their oral administration to goats, the cmax of both drugs achieved rapidly (tmax of df and smm were 1.5 and 5.6 hr, respectively). in addition, the plasma concentration - time curves shown in figs. 1 and 2 revealed the flip - flop phenomena. these phenomena occur when the absorption rate constant is smaller than the elimination rate constant ; therefore, the slope of the terminal log - linear phase after the oral administration of a drug reflects the absorption rate constant. when oral pharmacokinetics exhibits these phenomena, the determining factor of tmax is function of the drug elimination rate constant, and the faster elimination results in the shorter tmax. the elimination half - lives (t1/2 or t1/2kel) obtained for both df and smm were relatively shorter (tables 1 and 2). therefore, the elimination of df and smm in male shiba goats may have been fast enough to achieve cmax rapidly after their oral administration. this result suggests that, even in ruminants, an oral route may be suitable for drugs that have a fast elimination if they are not subjected to an extensive first - pass effect in the liver. the mat of df was less than half that of smm in the present study. this result suggests that absorption of df from the forestomach of male shiba goats may have been markedly high. the ph value of the rumen juice in this study was 6.4, as has been reported previously. furthermore, the pka of df is 4, suggesting that negligible df molecules exist as an unionized form (0.11%) in the contents of the rumen. on the other hand, the pka of smm is 6 [14, 16, 23 ], which suggests that 1050% of smm molecules exist as an unionized form. these findings indicate that smm is more suitable for absorption from the forestomach of goats. however, the partition coefficient between octanol and water (ph 7) is different. that of df is approximately 8, whereas that of smm is less than 1. therefore, df may have been absorbed from the forestomach, because of its extremely high lipid solubility. in the present study, eqs. 1 and 2 or eqs. 3 and 4 were simultaneously fit to intravenous and oral plasma concentration - time data from the same goats, respectively, in order to calculate pharmacokinetic parameters. therefore, it is not uncommon to obtain different values for the same parameter, such as the elimination rate constant, even though both data are obtained from the same individuals. this difference may result in inaccuracies in the absorption rate constants obtained. in order to avoid this problem, we obtained a good fit between the observed points and theoretical curves in the cases of df and smm, as shown in figs. 1 and 2. therefore, we considered the absorption rate constants obtained to be reliable. although the oral bioavailabilities of df and smm were incomplete (tables 1 and 2), both drugs were stable in the rumen juice in the in vitro spiked test, which indicated that both drugs were subjected to the first - pass effect in the liver. previous studies demonstrated that df had good gastrointestinal tolerability and underwent first - pass metabolism [7, 12, 22 ]. weijkamp. reported that sulfamethoxydiazine, sulfathiazole, sulfadimidine and sulfamoxole were stable in the rumen juice of dwarf goats during anaerobic incubation at 39c. a previous study also suggested that the low bioavailability of sulfamethoxazole after its oral administration to goats was most likely due to the first - pass effect in the liver.. suggested that female dwarf goats have higher hydroxylation capacity for sulfamethazine than males. they found that cl values of the sulfonamide in females were 3.5 times higher than males after intravenous injection. they also indicated that this higher capacity is due to lower testosterone levels in females. these facts may suggest that female shiba goats have higher hydroxylation capacity for smm than males. since acetylated metabolites of smm were not found in plasma, smm may be biotransformed mainly into hydroxylated metabolites in shiba goats, like sulfamethazine in dwarf goats. female shiba goats, therefore, may show lower bioavailability due to higher first - pass effect in the liver and shorter tmax due to faster elimination after its oral administration, compared with males in the present study. in conclusion, gastric emptying may be the determining factor for drug absorption after the oral administration of drugs to male shiba goats. the absorption of highly lipophilic drugs from the forestomach may be markedly high in ruminants. in the case of drugs whose elimination is fast, their efficacies may appear from the early stage after their oral administration, even in ruminants, because the elimination rate from the body is the determining factor for tmax in flip - flop phenomena. | in the present study, we examined the oral pharmacokinetics of the acidic drugs, diclofenac (df) and sulfamonomethoxine (smm), which have different physicochemical properties, in shiba goats. df and smm were intravenously and orally administered to 5 male goats using a crossover design. the tmax of df and smm were reached 1.5 and 5.6 hr after they have been orally administered, respectively, and this was followed by their slow elimination. the elimination of both drugs was markedly faster after being intravenously rather than orally administered, which indicated flip - flop phenomena after the oral administration. the mean absorption times (mats) of df and smm were 6 and 15 hr, respectively. this slow absorption may have been due to slow gastric emptying in goats. the large difference observed in mats between df and smm may have been because df, which is more lipophilic than smm, was partly absorbed from the forestomach. therefore, these results suggest that the absorption of highly lipophilic drugs from the forestomach may be markedly high in shiba goats. in case of drugs whose elimination is quite fast, their efficacies may appear from the early stage after oral administration even in ruminants, because elimination rate is the determinant factor of tmax in flip - flop phenomena. such drugs may be used orally even in ruminants. |
it was nt until 1980 however, that autism was formally recognized in the diagnostic and statistical manual of mental disorders (dsm - iii), and included as part of a new class, the pervasive developmental disorders (pdd). at the same time, early psychodynamic theories of the etiology of autism were being abandoned in favor of genetic ones. as early as 1975, case reports of monozygotic twins concordant for autism, followed by several systematic twin studies [510 ] substantiated the strong heritability of autism [1113 ]. standardization of diagnostic criteria, and improvements in our ability to reliably detect chromosomal abnormalities allowed for the identification in the early 1980 s of the first genetic cause of autism fragile x syndrome (fxs) [1517 ]. subsequently, the fragile x gene (fmr1) was discovered, and by 1994 the first animal model became available. this genetically engineered fmr1 knockout mouse (fmr1 ko), has been validated for fxs, and is currently one of the leading animal models of autism. using this mutant mouse, we have been able to address the role of the fmr1 gene and the protein it encodes (fragile x mental retardation protein, fmrp) in brain development. now, over 25 years since fxs was identified as a cause of autism, a new putative therapy has been proposed based on our understanding of the function of fmrp. inherited mutations have the potential to disrupt brain development from the moment of fertilization onward ; however, a genetic etiology does not preclude pathogenesis involving regulated processes later in development. symptoms of autism typically present during the early postnatal period, usually between ages 13 years. this epoch, the so - called critical period, corresponds to a dynamic phase of brain development in which neurite outgrowth, maturation of inhibition and signaling, axon myelination, and synaptic plasticity are set in motion by the complex interplay of molecular genetic programs and experience. disruption of any of one of these processes could hypothetically lead to the characteristic symptoms of autism, which include abnormal social interaction and communication, stereotyped repetitive behaviors, often with co - morbid mental retardation, epilepsy, sleep disturbances, attention deficit and hyperactivity. thus, it has been tempting to speculate that the pathogenesis of autism involves a derailment of at least one of these developmental processes [2426 ]. given this framework, studies of synaptic plasticity in the fmr1 ko mouse have been an obvious priority. a potential breakthrough in understanding the pathogenesis of fragile x came from studies of group 1 metabotropic glutamate receptors (gp1 mglur) [2731 ]. gp1 mglurs (which are further subdivided into mglur1 and mglur5 subtypes) couple to postsynaptic gq - like g - proteins and phospholipase c (plc) as well as to extracellular signal - regulated kinase (erk) transduction pathways [33, 34 ]. their activation leads to the synthesis of new protein at the synapse [28, 35, 36 ], likely through the erk signaling cascade [37, 38 ]. a functional consequence of gp 1 mglur - dependent protein synthesis in the hippocampus is long - term depression (ltd), a form of synaptic plasticity. in the fmr1 ko mouse, this mglur - ltd is exaggerated and no longer protein synthesis - dependent [31, 39 ]. meanwhile, studies of fmrp revealed that the expression of the protein is developmentally regulated [40, 41 ], such that in the post - natal brain it is largely cytoplasmic [42, 43 ], predominantly expressed in neurons [44, 45 ] and enriched postsynaptically at glutamatergic synapses. furthermore, fmrp is an rna binding protein that co - localizes with polyribosomes [44, 4755 ] which are found at the base of dendritic spines where they are thought to mediate local translational control of the synapse. indeed, both in vitro and in vivo metabolic labeling studies have now directly shown that fmrp functions as a repressor of protein synthesis [5760 ]. taken together, these findings led to the hypothesis that gp1 mglurs and fmrp might work in functional opposition to regulate mrna translation at the synapse, and that in the absence of fmrp, unchecked mglur - dependent protein synthesis leads to the pathogenesis of the disease (fig. mglur theory and shown that increased levels of protein synthesis in the fmr1 ko mouse [59, 60 ], are restored to wild type (wt) levels by selective reduction of mglur5 signaling. this manipulation also significantly decreases the magnitude of gp1 mglur - ltd in fmr1 ko mice, confirming the role of mglur5 in producing the exaggerated synaptic plasticity phenotype. furthermore, many of the long - term consequences of gp1 mglur activation are protein synthesis dependent. the mglur theory posits that in the absence of fmrp, as is the case in fragile x syndrome, this balance between fmrp and gp1 mglurs is lost, and unchecked protein synthesis at the synapse leads to the characteristic features of the disease. furthermore, this balance could be restored by reducing gp1 mglur activity at the synapse, by either knockdown or pharmacological blockade of the receptor. the therapeutic implication of the theory is that symptoms of fxs syndrome could be corrected by appropriate modulation of gpi mglur signaling opponent regulation of protein synthesis by fmrp and gpi mglurs. furthermore, many of the long - term consequences of gp1 mglur activation are protein synthesis dependent. the mglur theory posits that in the absence of fmrp, as is the case in fragile x syndrome, this balance between fmrp and gp1 mglurs is lost, and unchecked protein synthesis at the synapse leads to the characteristic features of the disease. furthermore, this balance could be restored by reducing gp1 mglur activity at the synapse, by either knockdown or pharmacological blockade of the receptor. the therapeutic implication of the theory is that symptoms of fxs syndrome could be corrected by appropriate modulation of gpi mglur signaling the synapse is too small to be directly visualized by light microscopy. however, dendritic spines (the postsynaptic half of an excitatory synapse) can be visualized, and are used to estimate the number of excitatory synapses in the brain. dendritic spines are highly modifiable structures, and changes in spine density and morphology have been correlated with synaptic plasticity. furthermore, abnormalities in dendritic spine morphology have long been associated with human mental retardation of unknown etiology, as well as with xlmr (x - linked mental retardation), down, patau, rett and fragile x syndromes [67, 68 ]. application of the selective mglur5 agonist, dhpg, to cultured hippocampal neurons induces a protein synthesis dependent increase in the density of long thin spines. because dhpg application in cell culture also induces rapid protein synthesis dependent internalization of ampa and nmda receptors, receptor internalization may be the prelude to morphologic remodeling in response to plasticity inducing stimuli. this response to stimulation with dhpg parallels spine changes seen in the fmr1 ko mice, which lent support to the theory that exaggerated signaling through mglur5 in the absence of fmrp could account for this morphologic correlate of synaptic plasticity. consistent with this idea, recent studies have shown that that ampa receptor internalization is exaggerated in the absence of fmrp and both this and the increased spine density phenotype seen in fmr1 ko mice [60, 7278 ] are rescued by selective reduction in mglur5 signaling [60, 71 ]. while these in vitro and ex vivo demonstrations of opponent regulation by fmrp and mglur5 provided the necessary foundation for identifying and correcting synaptic abnormalities, we also wanted to determine whether these interactions regulate circuit - level responses in the intact animal. landmark studies of in vivo ocular dominance plasticity (odp) in monkeys and cats [7981 ] established a role for experience dependent plasticity in shaping the circuitry of the brain during the critical period. moreover, because odp occurs on the biologically relevant timescale, in response to perturbations of environmental stimuli using intrinsic patterns of neuronal activity, this paradigm is more readily translated to future studies in human patients (e.g. using visually evoked potentials or transcranial magnetic stimulation). the development of transgenic technologies [19, 84, 85 ] and adaptation of the odp paradigm to rodents [8691 ] has allowed us to answer mechanistic questions about experience dependent plasticity in vivo. for example, odp is in - part mglur5 dependent, requires protein synthesis, and signals through erk transduction. in the fmr1 ko mouse, this plasticity is exaggerated, such that bidirectional modifications that require 7 days of monocular deprivation (md) in wt mice, occur after only 3 days in the absence of fmrp. significantly this hyper - plastic response is reminiscent of the exaggerated synaptic plasticity phenotype seen in the hippocampal slice, and is likewise restored to wt levels by 50% reduction of mglur5 signaling. as mentioned above, epilepsy and mental retardation are both co - morbid features of autism an estimated 538% of autistic patients have seizure or subclinical epileptiform activity while 70% have cognitive impairment [95, 96](but see,). thus, an important goal for modeling the disease is to establish behavioral tasks that recapitulate these symptoms in the fmr1 ko mouse. an estimated 20% of human patients with fxs have epileptiform activity or generalized seizure [99, 100 ]. audiogenic seizure (ags) is a robust paradigm for inducing seizure in the fmr1 ko [60, 101105 ] and recapitulates this neurologic feature of fxs and autism. previous studies have not been able to account for increased epileptiform activity in fmr1-ko mice by any of the anticipated mechanisms. for example, no differences have been observed between wt and fmr1-ko mice in basal synaptic transmission, excitability, paired pulse facilitation, and long - term potentiation in the ca1 region of the hippocampus [106, 107 ]. interestingly, it has been shown that agonists of group i mglurs act as convulsants in rodents [32, 108 ] while selective gp i mglur antagonists block seizures in a range of rodent models of epilepsy [105, 109, 110 ]. increases in epileptiform activity in response to mglur5 stimulation are protein synthesis dependent [111, 112 ], suggesting that in addition to synapse specific changes, circuit level modulation of excitability is sensitive to the state of mglur5 dependent protein synthesis. consistent with this idea, ags seen in the fmr1 ko is attenuated by 50% reduction of mglur5 signaling. despite the moderate to severe mental retardation seen in human patients with fxs, cognitive phenotypes in the fmr1 ko mice have been difficult to model [107, 115117 ]. inhibitory avoidance (ia) is a contextual (fear) conditioning paradigm used in animals to test hippocampus - based associative learning and memory. ia extinction (iae) is a paradigm that tests those conditioned responses in the face of contradictory contextual (safe) conditioning. while ia learning is normal in fmr1 ko mice on the c57-bl6 background [19, 60 ], we have recently identified an iae phenotype in the fmr1 ko. although the synaptic mechanisms underlying iae are not currently known, this behavior, like mglur5-ltd, is protein synthesis dependent. furthermore, since both mglur5-ltd and iae are exaggerated in the fmr1 ko mice and rescued by reduction of mglur5 signaling, one interesting possibility is that mglur5 ltd is the cellular mechanism subserving iae learning. this mechanism is likely distinct from that which subserves ia, since ia training induces nmda - ltp and neither ia nor nmda - ltp [106, 107 ] is disrupted in the fmr1 ko on the c57-bl6 background. in summary, we have discovered that fmrp is a protein that acts to regulate protein synthesis and synaptic plasticity triggered by gp1 mglurs. understanding this balance between fmrp and mglur-5 has allowed us to restore normal function in the fmr1 ko model of autism metabolic, morphologic, synaptic, circuit, and behavioral disruptions can all be corrected by reducing mglur5 signaling by 50%. currently clinical trials based these and related findings are under way to determine safety and efficacy of mglur modifying drugs in human patients with fxs and autism. to put these findings in context, it is important to remember that mglurs and fmrp do not exist in isolation at the synapse. as shown in fig. 2, a number of other synaptic proteins that interact with the mglurs either by direct physical contact or biochemical cascades, have also been identified as autism candidate genes [121126 ] or single gene disorders associated with autism [127134 ]. some of these have been identified as autism candidate genes (shown in purple ; homer, shank, neuroligin, neurexin) ; others are proteins associated with single gene causes of autism (shown in red : fmrp / fxs, tsc / tuberous sclerosis, pten/ hamartoma syndrome, mecp2/rett syndrome, e3a / angelman s syndrome) autism as a synapsopathy. mglur5 some of these have been identified as autism candidate genes (shown in purple ; homer, shank, neuroligin, neurexin) ; others are proteins associated with single gene causes of autism (shown in red : fmrp / fxs, tsc / tuberous sclerosis, pten/ hamartoma syndrome, mecp2/rett syndrome, e3a / angelman s syndrome) for example, gp1 mglur signaling converges on transduction cascades also implicated in pten hamartoma syndrome and tuberous sclerosis complex (tsc), which are other single gene causes of autism. pten inhibits pi3k - dependent signaling, which couples gq signaling to the mtor / s6k pathway for protein synthesis. tsc 1/2 inhibits this same mtor pathway, by acting as a gtpase - activating protein for the ras - related small g protein rheb. for example, both shank and homer proteins crosslink mglur5 to the postsynaptic density, and misregulated homer1b and psd-95 have been implicated in the pathogenesis of fxs [137, 138 ]. the neuroligin / neurexin complex, important for synapse formation and implicated in autism, is in turn tethered to the synapse via its interaction with psd-95. alphacamkii, a major regulatory protein in synaptic plasticity is also tethered to the synapse by psd-95 ; absence of inhibitory phosphorylation of alphacamkii by ube3a, has been implicated in angelman syndrome. interestingly, mglur5 stimulated protein synthesis of alphacamkii and psd-95 are impaired in synaptoneurosomes from fmr1 ko mice. furthermore, camkii dependent phosphorylation of mecp2 links these synaptic proteins to rett syndrome, another single gene disorder associated with autism, and transcriptional regulation of brain derived nerve growth factor (bdnf). in turn, trkb mediated bdnf signals through erk, regulates dendritic spine formation, and has also been implicated in the pathogenesis of fxs. together, these results suggest it may be useful to think of autism as a synapsopathy a disease where disruption of the synapse during development produces a common clinical picture, despite a heterogeneity of interconnected causes. it also raises the interesting possibility that treatments for one cause, such as fragile x, may have efficacy in treating other causes of autism. | autism is an umbrella diagnosis with several different etiologies. fragile x syndrome (fxs), one of the first identified and leading causes of autism, has been modeled in mice using molecular genetic manipulation. these fmr1 knockout mice have recently been used to identify a new putative therapeutic target, the metabotropic glutamate receptor 5 (mglur5), for the treatment of fxs. moreover, mglur5 signaling cascades interact with a number of synaptic proteins, many of which have been implicated in autism, raising the possibility that therapeutic targets identified for fxs may have efficacy in treating multiple other causes of autism. |
the term oncocyte, first used by hamperl in 1950, describes large, highly eosinophilic, granular cells associated with a hurthle cell tumor of the thyroid gland. oncocytoma of the adrenal gland is defined as a neoplasm composed exclusively or predominantly of oncocytes. these oncocytes are large polygonal cells with eosinophilic cytoplasm that result from the abnormal accumulation of mitochondria. oncocytomas can arise in several organs including the kidney, thyroid, salivary glands, parathyroid, lung, pituitary gland, and ovary. most of these adrenal neoplasms are benign, usually nonfunctional, and hence, accidentally detected. only 10 cases of functioning adrenal oncocytomas are reported so far in literature and only three cases manifesting with virilization were seen in children. a 16-year - old girl presented with a 6-month history of primary amenorrhea and pain in the abdomen. the total testosterone level was raised and value was 435 ng / dl (normal range, 14 - 76 ng / dl). dehydroepiandrosteronesulfate (dhea - s) level was > 1500 g / dl (normal range, 61 - 493 g / dl). vanillylmandelic acid (vma) level was 10.10 mg/24 h (normal range, 5 mitoses per 50 high power fields), atypical mitoses, or venous invasion. the four minor criteria are : tumor size > 10 cm or weight > 200 g, tumor necrosis, capsular or sinusoidal invasion. the presence of any one or more major criteria categorizes the lesion as a malignant oncocytoma. the presence of at least one minor criterion yields a diagnosis of uncertain malignant potential or borderline malignancy. adrenal oncocytomas are heterogeneous in appearance on imaging studies and the hypoechoic areas seen on sonography and hypodense areas on ct scans represent necrosis detected on pathological examination. a characteristic finding for adrenal oncocytomas is well - defined fibrous capsule and this is found in both benign and malignant tumors. given the large size, heterogeneous appearance and the presence of necrosis in the tumor, adrenocortical carcinoma was obviously the most likely preoperative diagnostic consideration in our case. ct appearance of adrenocortical carcinoma can be similar to oncocytoma with heterogeneous enhancement and central areas of necrosis. thus unfortunately, the imaging features of adrenocortical oncocytoma does not allow its differentiation from adrenocortical carcinoma. adrenal oncocytoma can be included in the differential diagnosis of especially large, well - defined, and nonfunctioning adrenal tumors. however, nonspecific imaging features do not warrant a definitive preoperative diagnosis and unfortunately do not change the patient`s management. | adrenal oncocytoma is a rare adrenal neoplasm with only 57 cases reported in literature. adrenal oncocytomas can achieve large sizes and are usually nonfunctioning. they are detected accidentally during abdominal scans. most of these adrenal neoplasms are benign. a functioning adrenal oncocytoma manifested with virilization in a 16-year - old female child. there seems to be little benefit in biopsying these tumors and surgery remains the optimum management. |
cells for human brain tumor xenografts can be sourced either from tumors propagated as subcutaneous growths in athymic mice, or from cell culture. utilization of both cell sources is discussed below, along with demonstration of a method for cell implantation. to prepare cells from subcutaneous tumors for transfer to the intracranial compartment, excised flank tumors are placed in culture dishes, where the tissue is initially minced with a scalpel and then mechanically disrupted by repetitive pipetting to create a cell aggregate suspension. the cell aggregate suspension is then passed through a 70 m nylon mesh filter to produce a single cell suspension suitable for intracranial injection. the cell suspension is centrifuged at 1000 rpm for 10 minutes at 4c, and the supernatant aspirated before resuspending the cell pellet in an appropriate volume of serum - free media to obtain a final working concentration (see below). for preparing established cell lines for intracranial implantation, cells are harvested by trypsinizing monolayers, or by collecting neurosphere suspension cultures, then centrifuging and resuspending the cells as indicated above. the number of cells injected is variable dependent on neuroanatomical location of injection. for supratentorial injections we routinely inject 3 - 5 x 10 cells in 3 l of serum - free media (dmem), whereas for brainstem injections, as few as 5 x 10 cells are injected in 0.5 l. injecting larger volumes than recommended can result in tumor cell reflux through the needle tract, with resultant exophytic (figure 1), rather than intracranial tumor growth. after withdrawing sample for intracranial injection, the remaining cell suspension should be placed in ice, with contents mixed frequently to maintain appropriate concentration while completing intracranial tumor establishment among the members of an injection series. note, all procedures described below have been reviewed and approved by the institutional animal use and care committee at university of california san francisco. the surgical area should be prepared by spraying all surfaces with a disinfectant, such as 2% chlorhexidine solution. after using the disinfectant, the following supplies should be placed in the surgical area : heating pad to maintain mouse body temperaturetwo small petri dishes ; one containing 3% hydrogen peroxide, and one containing 2% chlorhexidinesterile gauze and cotton swabssterile disposable scalpelsautoclaved surgical stapler heating pad to maintain mouse body temperature two small petri dishes ; one containing 3% hydrogen peroxide, and one containing 2% chlorhexidine sterile gauze and cotton swabs sterile disposable scalpels autoclaved surgical stapler for anesthesia an injected anesthetic should be used ; typically a ketamine - xylazine mixture. once a mouse is anesthetized, the scalp is prepared by swabbing several times with a piece of sterile gauze dipped in the chlorhexidine solution. eye ointment should be applied to maintain adequate moisture during the procedure. using a sterile scalpel, complete a sagittal incision over the parieto - occipital bone, approximately 1 cm long. the exposed skull surface is then cleaned using a cotton swab soaked in a 3% hydrogen peroxide solution. the coordinates for injection of tumor cells will vary according to the desired site for tumor establishment. the following represents the procedure we use for intracerebral tumor establishment in a neuroanatomical location at which many brain tumor patients experience tumor development. other locations of interest in brain tumor research include the pons, for anatomic modeling of brainstem tumors, and subdural injections for modeling the location of meningeal tumors. prior to tumor cell injection, use a sterile 25 gauge sharp needle to puncture the skull at 2 mm to the right of the bregma and 1 mm anterior to the coronal suture, thereby creating an opening for the injection of tumor cells (see video). this procedure works well for both mice and rats (22 gauge needle for rats). prior to drawing cells into the syringe, mix the contents of the cell suspension by tapping with your finger. load the syringe with the desired amount of cell suspension, being careful to avoid creating air bubbles. the outside of syringe should then be cleaned with an alcohol swab to wipe the exterior free of any adherent cells, which will help prevent extracranial tumor establishment and growth (figure 1a). to ensure that the appropriate injection depth is achieved, use a scalpel to cut 3 mm off the pointed end of a p20 pipetteman tip. this section of the tip can be fitted over the syringe and will act to limit the injection depth, and will additionally ensure that the tip of the syringe needle is 3 mm from the underside of the skull. place the syringe perpendicular to the skull and in the hole previously created, and slowly inject the cell suspension (a 3l suspension should be injected over a 1 minute period). an inappropriate angle of syringe insertion can result in intraventricular injection of cells and subsequent spinal dissemination (fig. 1b : right) upon completing injection, leave the needle in place for another minute, then slowly withdraw (see video) : these steps will help reduce tumor cell reflux. as an alternative to the unassisted or free - hand approach to intracranial tumor cell implantation, one can use a small animal stereotactic frame (panel f of schematic overview), which generally promotes more consistent injection location, but at the expense of substantial amounts of procedural time. in our experience, two surgical staff can inject as many as 60 mice / hour when using the free - hand approach, whereas maximal injection rate with a small animal stereotactic frame is approximately 15 mice / hour. procedural expediency is an important consideration when injecting large series of mice, and helps reduce the time in which tumor cells, to be injected, are left on ice. clean the skull with 3% hydrogen peroxide and dry using a sterile dry cotton swab. using a forceps, draw the scalp together over the skull and staple to close. for optimal healing, the scalp should be stapled with the dermis of each side of the scalp against each other (underside against underside). the stapled scalp should be cleaned using chlorhexidine solution, and buprenorphine then administered by subcutaneous injection for post - operative pain relief. bioluminescence imaging (bli) is based on the oxidation of luciferin [d-(-)-2-(60-hydroxy-20-benzothiazolyl)-thiazone-4-carboxylic acid ] in the presence of oxygen and adenosine triphosphate (atp). this reaction is catalyzed by the enzyme luciferase, which converts chemical energy into photons with resultant emission of light. human cells can be modified to express luciferase (see below), with only luciferase - expressing cells capable of emitting light in the presence of the luciferin substrate. there is minimal background luminescence from animals hosts treated with luciferin, such that there is a very favorable signal - to - noise ratio for detecting luminescence emissions from luciferase - modified tumor cells, allowing highly sensitive monitoring of tumor growth and response to therapy in vivo. moreover, luciferase and its substrate, luciferin, have been shown to be non - toxic to mammalian cells, and we have observed no functional differences between cells expressing luciferase relative to corresponding unmodified cells. luciferin readily crosses cell membranes and the blood - brain barrier after intraperitoneal (i.p.) or intravenous (i.v.) injection in mice, thereby allowing imaging of every compartment that contains luciferase - modified cells. the level of photon emission and the spectrum of emitted light from luciferase - modified cells is adequate to penetrate tissues of small research animals, such as mice and rats, and can be detected externally with low - light imaging cameras. the noninvasive nature of bioluminescence imaging allows repeated monitoring of tumor growth and response to therapy in individual animal subjects. implanted cell sources should be stably modified for firefly luciferase expression such cell sources can be purchased, or produced in individual laboratories using lentivirus that have been constructed for constitutive expression of luciferase. we strongly recommend the use of cells modified with luciferase encoding lentivirus, rather than plasmid, to ensure stable cellular expression of luciferase in vivo, which is necessary for quantitative luminescence imaging to provide accurate indication of changes in tumor burden for individual animal subjects that are repeatedly imaged during the course of an experiment. we recommend conducting quantitative bioluminescence imaging (qbli) 1x-2x weekly, beginning one week following tumor cell injection. our qbli is conducted using the ivis lumina imaging station (caliper life sciences), and our results indicate similar data are obtained in using the ivis lumina, the ivis 150, or the ivis 200 imaging station. in preparation for imaging, mice are simultaneously anesthetized with ketamine / xylazine and administered luciferin (d - luciferin potassium salt, 150 mg / kg, caliper life sciences) via intraperitoneal injection, with mice imaged 12 minutes after injection. the pharmacokinetics of luciferin indicate that the time between administration of luciferin and determination of cell luminescence should be between 10 - 15 minutes post injection of luciferin, in order to obtain maximum luminescence emission and greatest sensitivity of detection. the length of time selected within the 10 - 15 minute time interval should be maintained as constant among the animals that are being imaged. it is extremely important to maintain consistency in length of time between injection of luciferin and obtaining bli readings. regions of interest encompassing the intracranial area of signal are defined using living image software (figure 2), and the total photons / s / sr / cm2 (photons per second per steradian per square cm) are recorded (see video). whereas tumor growth and therapy response are monitored in individual animals, we highly recommend treatment groups of at least 8 for increasing the statistical certainty of conclusions regarding tumor response, or lack thereof, to therapy. with regard to presenting qbli results, luminescence readings are converted to normalized values by dividing each mouse s luminescence, obtained during and subsequent to completion of therapeutic regimen, with its corresponding maximal pretreatment luminescence reading. for survival analysis, the kaplan - meier estimator is used, and from which survival curves are generated, and median survival values determined. retrieval of brain for subsequent analysis of treated and untreated tumor. upon animal euthanasia, the brain of the mouse should be quickly excised (see video), and either placed in formalin for subsequent analysis of tumor morphology and immunohistochemistry, or should be mounted in oct for specimen freezing. in the example shown in figure 3a, mice receiving intracranial tumor cell injection were monitored for intracranial luminescence until successive mean luminescence values indicated progressive tumor growth, and at which time therapy was initiated (gray arrow beginning at day 34 : erlotinib administered daily at 150 mg / kg until required euthanasia). luminescence values for each mouse are set to a normalized value of 1 at time of initiating therapy, with subsequent luminescence readings for each mouse normalized to its final pretreatment imaging value. as an example, a mouse with a final pre - treatment luminescence reading of 2.0 x 10 photons / sec at day 34, whose luminescence had increased to 6.0 x 10 photons / sec at day 38, would have a day 38 normalized luminescence value of 3.0. mean normalized bioluminescence and corresponding standard error for control and treatment groups are plotted for each imaging time point. in this example, a significant difference in mean normalized luminescence is apparent at the first imaging time point subsequent to the initiation of therapy (day 38), with the difference in mean group luminescence showing further increase at subsequent time points. in most instances, anti - tumor activity of therapy, as indicated by qbli, is accompanied by a corresponding significant difference in survival (i.e., p < 0.05), as is the case here (figure 3b). panels 3c and 3d show adjacent hematoxylin & eosin and anti - egfr stained sections of mouse brain obtained at time of euthanasia, following placement of resected brain in formalin and subsequent paraffin - embedding for sectioning. a) exophytic (extracranial) tumor growth (red circle) can be caused by too large an injection volume, residual cell suspension attached to the syringe, or from withdrawing the syringe too quickly after injecting the tumor cells. b) injecting tumor cells into the ventricles can cause spinal dissemination of tumor (mouse to the right), in contrast to properly injected tumor cells, the signal for which stays localized to the injection site (mouse to the left). heat map image representations of bioluminescence intensity for representative mice from control (left) and treatment (right) groups of a therapy response experiment. the living image software can be set to define regions of interest (red circles), or instrument operators can define regions of interest manually. for using images such as these for figure construction, we recommend that the instrument operator shows heat map images using the same bioluminescence heat map range (upper portion of figure), to provide visual representation of extent of bioluminescence difference between animal subjects. figure 3. bioluminescence, survival, and tumor tissue analysis from an experiment in which therapeutic response is evident. a) plot of mean bioluminescence readings for control and treatment group mice, with standard error indicated for each imaging point. b) survival plot for same mice ; p - value determined through use of log - rank test. e and f) magnifications of indicated areas from panels c and d, respectively, with panel e showing negative staining for tumor suppressor protein pten. cells for human brain tumor xenografts can be sourced either from tumors propagated as subcutaneous growths in athymic mice, or from cell culture. utilization of both cell sources is discussed below, along with demonstration of a method for cell implantation. to prepare cells from subcutaneous tumors for transfer to the intracranial compartment, excised flank tumors are placed in culture dishes, where the tissue is initially minced with a scalpel and then mechanically disrupted by repetitive pipetting to create a cell aggregate suspension. the cell aggregate suspension is then passed through a 70 m nylon mesh filter to produce a single cell suspension suitable for intracranial injection. the cell suspension is centrifuged at 1000 rpm for 10 minutes at 4c, and the supernatant aspirated before resuspending the cell pellet in an appropriate volume of serum - free media to obtain a final working concentration (see below). for preparing established cell lines for intracranial implantation, cells are harvested by trypsinizing monolayers, or by collecting neurosphere suspension cultures, then centrifuging and resuspending the cells as indicated above. the number of cells injected is variable dependent on neuroanatomical location of injection. for supratentorial injections we routinely inject 3 - 5 x 10 cells in 3 l of serum - free media (dmem), whereas for brainstem injections, as few as 5 x 10 cells are injected in 0.5 l. injecting larger volumes than recommended can result in tumor cell reflux through the needle tract, with resultant exophytic (figure 1), rather than intracranial tumor growth. after withdrawing sample for intracranial injection, the remaining cell suspension should be placed in ice, with contents mixed frequently to maintain appropriate concentration while completing intracranial tumor establishment among the members of an injection series. note, all procedures described below have been reviewed and approved by the institutional animal use and care committee at university of california san francisco. the surgical area should be prepared by spraying all surfaces with a disinfectant, such as 2% chlorhexidine solution. after using the disinfectant, the following supplies should be placed in the surgical area : heating pad to maintain mouse body temperaturetwo small petri dishes ; one containing 3% hydrogen peroxide, and one containing 2% chlorhexidinesterile gauze and cotton swabssterile disposable scalpelsautoclaved surgical stapler heating pad to maintain mouse body temperature two small petri dishes ; one containing 3% hydrogen peroxide, and one containing 2% chlorhexidine sterile gauze and cotton swabs sterile disposable scalpels autoclaved surgical stapler for anesthesia an injected anesthetic should be used ; typically a ketamine - xylazine mixture. once a mouse is anesthetized, the scalp is prepared by swabbing several times with a piece of sterile gauze dipped in the chlorhexidine solution. eye ointment should be applied to maintain adequate moisture during the procedure. using a sterile scalpel, complete a sagittal incision over the parieto - occipital bone, approximately 1 cm long. the exposed skull surface is then cleaned using a cotton swab soaked in a 3% hydrogen peroxide solution. the coordinates for injection of tumor cells will vary according to the desired site for tumor establishment. the following represents the procedure we use for intracerebral tumor establishment in a neuroanatomical location at which many brain tumor patients experience tumor development. other locations of interest in brain tumor research include the pons, for anatomic modeling of brainstem tumors, and subdural injections for modeling the location of meningeal tumors. prior to tumor cell injection, use a sterile 25 gauge sharp needle to puncture the skull at 2 mm to the right of the bregma and 1 mm anterior to the coronal suture, thereby creating an opening for the injection of tumor cells (see video). this procedure works well for both mice and rats (22 gauge needle for rats). prior to drawing cells into the syringe, mix the contents of the cell suspension by tapping with your finger. load the syringe with the desired amount of cell suspension, being careful to avoid creating air bubbles. the outside of syringe should then be cleaned with an alcohol swab to wipe the exterior free of any adherent cells, which will help prevent extracranial tumor establishment and growth (figure 1a). to ensure that the appropriate injection depth is achieved, use a scalpel to cut 3 mm off the pointed end of a p20 pipetteman tip. this section of the tip can be fitted over the syringe and will act to limit the injection depth, and will additionally ensure that the tip of the syringe needle is 3 mm from the underside of the skull. place the syringe perpendicular to the skull and in the hole previously created, and slowly inject the cell suspension (a 3l suspension should be injected over a 1 minute period). an inappropriate angle of syringe insertion can result in intraventricular injection of cells and subsequent spinal dissemination (fig. 1b : right) upon completing injection, leave the needle in place for another minute, then slowly withdraw (see video) : these steps will help reduce tumor cell reflux. as an alternative to the unassisted or free - hand approach to intracranial tumor cell implantation, one can use a small animal stereotactic frame (panel f of schematic overview), which generally promotes more consistent injection location, but at the expense of substantial amounts of procedural time. in our experience, two surgical staff can inject as many as 60 mice / hour when using the free - hand approach, whereas maximal injection rate with a small animal stereotactic frame is approximately 15 mice / hour. procedural expediency is an important consideration when injecting large series of mice, and helps reduce the time in which tumor cells, to be injected, are left on ice. clean the skull with 3% hydrogen peroxide and dry using a sterile dry cotton swab. apply sterile bone wax to the hole. using a forceps, draw the scalp together over the skull and staple to close. for optimal healing, the scalp should be stapled with the dermis of each side of the scalp against each other (underside against underside). the stapled scalp should be cleaned using chlorhexidine solution, and buprenorphine then administered by subcutaneous injection for post - operative pain relief. background. bioluminescence imaging (bli) is based on the oxidation of luciferin [d-(-)-2-(60-hydroxy-20-benzothiazolyl)-thiazone-4-carboxylic acid ] in the presence of oxygen and adenosine triphosphate (atp). this reaction is catalyzed by the enzyme luciferase, which converts chemical energy into photons with resultant emission of light. human cells can be modified to express luciferase (see below), with only luciferase - expressing cells capable of emitting light in the presence of the luciferin substrate. there is minimal background luminescence from animals hosts treated with luciferin, such that there is a very favorable signal - to - noise ratio for detecting luminescence emissions from luciferase - modified tumor cells, allowing highly sensitive monitoring of tumor growth and response to therapy in vivo. moreover, luciferase and its substrate, luciferin, have been shown to be non - toxic to mammalian cells, and we have observed no functional differences between cells expressing luciferase relative to corresponding unmodified cells. luciferin readily crosses cell membranes and the blood - brain barrier after intraperitoneal (i.p.) or intravenous (i.v.) injection in mice, thereby allowing imaging of every compartment that contains luciferase - modified cells. the level of photon emission and the spectrum of emitted light from luciferase - modified cells is adequate to penetrate tissues of small research animals, such as mice and rats, and can be detected externally with low - light imaging cameras. the noninvasive nature of bioluminescence imaging allows repeated monitoring of tumor growth and response to therapy in individual animal subjects. implanted cell sources should be stably modified for firefly luciferase expression such cell sources can be purchased, or produced in individual laboratories using lentivirus that have been constructed for constitutive expression of luciferase. we strongly recommend the use of cells modified with luciferase encoding lentivirus, rather than plasmid, to ensure stable cellular expression of luciferase in vivo, which is necessary for quantitative luminescence imaging to provide accurate indication of changes in tumor burden for individual animal subjects that are repeatedly imaged during the course of an experiment. we recommend conducting quantitative bioluminescence imaging (qbli) 1x-2x weekly, beginning one week following tumor cell injection. our qbli is conducted using the ivis lumina imaging station (caliper life sciences), and our results indicate similar data are obtained in using the ivis lumina, the ivis 150, or the ivis 200 imaging station. in preparation for imaging, mice are simultaneously anesthetized with ketamine / xylazine and administered luciferin (d - luciferin potassium salt, 150 mg / kg, caliper life sciences) via intraperitoneal injection, with mice imaged 12 minutes after injection. the pharmacokinetics of luciferin indicate that the time between administration of luciferin and determination of cell luminescence should be between 10 - 15 minutes post injection of luciferin, in order to obtain maximum luminescence emission and greatest sensitivity of detection. the length of time selected within the 10 - 15 minute time interval should be maintained as constant among the animals that are being imaged. it is extremely important to maintain consistency in length of time between injection of luciferin and obtaining bli readings. regions of interest encompassing the intracranial area of signal are defined using living image software (figure 2), and the total photons / s / sr / cm2 (photons per second per steradian per square cm) are recorded (see video). whereas tumor growth and therapy response are monitored in individual animals, we highly recommend treatment groups of at least 8 for increasing the statistical certainty of conclusions regarding tumor response, or lack thereof, to therapy. with regard to presenting qbli results, luminescence readings are converted to normalized values by dividing each mouse s luminescence, obtained during and subsequent to completion of therapeutic regimen, with its corresponding maximal pretreatment luminescence reading. for survival analysis, the kaplan - meier estimator is used, and from which survival curves are generated, and median survival values determined. retrieval of brain for subsequent analysis of treated and untreated tumor. upon animal euthanasia, the brain of the mouse should be quickly excised (see video), and either placed in formalin for subsequent analysis of tumor morphology and immunohistochemistry, or should be mounted in oct for specimen freezing. in the example shown in figure 3a, mice receiving intracranial tumor cell injection were monitored for intracranial luminescence until successive mean luminescence values indicated progressive tumor growth, and at which time therapy was initiated (gray arrow beginning at day 34 : erlotinib administered daily at 150 mg / kg until required euthanasia). luminescence values for each mouse are set to a normalized value of 1 at time of initiating therapy, with subsequent luminescence readings for each mouse normalized to its final pretreatment imaging value. as an example, a mouse with a final pre - treatment luminescence reading of 2.0 x 10 photons / sec at day 34, whose luminescence had increased to 6.0 x 10 photons / sec at day 38, would have a day 38 normalized luminescence value of 3.0. mean normalized bioluminescence and corresponding standard error for control and treatment groups are plotted for each imaging time point. in this example, a significant difference in mean normalized luminescence is apparent at the first imaging time point subsequent to the initiation of therapy (day 38), with the difference in mean group luminescence showing further increase at subsequent time points. in most instances, anti - tumor activity of therapy, as indicated by qbli, is accompanied by a corresponding significant difference in survival (i.e., p < 0.05), as is the case here (figure 3b). panels 3c and 3d show adjacent hematoxylin & eosin and anti - egfr stained sections of mouse brain obtained at time of euthanasia, following placement of resected brain in formalin and subsequent paraffin - embedding for sectioning. a) exophytic (extracranial) tumor growth (red circle) can be caused by too large an injection volume, residual cell suspension attached to the syringe, or from withdrawing the syringe too quickly after injecting the tumor cells. b) injecting tumor cells into the ventricles can cause spinal dissemination of tumor (mouse to the right), in contrast to properly injected tumor cells, the signal for which stays localized to the injection site (mouse to the left). heat map image representations of bioluminescence intensity for representative mice from control (left) and treatment (right) groups of a therapy response experiment. the living image software can be set to define regions of interest (red circles), or instrument operators can define regions of interest manually. for using images such as these for figure construction, we recommend that the instrument operator shows heat map images using the same bioluminescence heat map range (upper portion of figure), to provide visual representation of extent of bioluminescence difference between animal subjects. bioluminescence, survival, and tumor tissue analysis from an experiment in which therapeutic response is evident. a) plot of mean bioluminescence readings for control and treatment group mice, with standard error indicated for each imaging point. b) survival plot for same mice ; p - value determined through use of log - rank test. e and f) magnifications of indicated areas from panels c and d, respectively, with panel e showing negative staining for tumor suppressor protein pten. orthotopic (intracranial) brain tumor xenograft establishment provides an appropriate microenvironment for modeling cns cancer to be tested for therapeutic response. this type of modeling additionally provides information regarding therapeutic access to brain and brain tumor, which is critically important to determining whether an experimental agent should be advanced to clinical trial evaluation in patients. because the amount of intracranial xenograft tumor can not be directly measured, such as by calipers, longitudinal monitoring of intracranial tumor growth and response to therapy requires non - invasive imaging, with our experience indicating bioluminescence imaging as the most practical approach for experiments whose primary objective is assessing extent of tumor response to therapy. when the results of bioluminescence imaging are combined with animal subject survival analysis, the two data sets provide a powerful and reliable approach for evaluating experimental therapeutic efficacy. finally, it is critically important that intracranial brain tumor xenografts are harvested from euthanized animal subjects in order to assess morphologic and molecular effects of therapy, and for this we prefer whole brain resection at time of euthanasia, with preservation of resected brain for subsequent analysis. whereas the preceding presentation has been made specific to brain tumor research, the concepts are certainly generalizeable to other human cancers that are amenable to orthotopic modeling in rodents. | transplantation models using human brain tumor cells have served an essential function in neuro - oncology research for many years. in the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non - invasive imaging for assessing tumor burden in host animals. of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies. |
we enrolled the patients who had unergone elective laparoscopic cholecystectomy with the indication of cholecystolithiasis between 2011 and 2013 in the study. then the abdomen was inflated using averess needle and 10-mm trocar was introduced into abdomen. patients who underwent trocar incision using open surgery and those whose incisions were enlarged to extract the gallbladder were excluded from the study. trocars with a caliber of 10-mm were introduced through the epigastric area and 5-mm trocars were inserted through the right upper and lower quadrants. age, gender and comorbidity status of the patients and the presence of obesity were recorded. the study protocol was approved by the local ethics committee. for statistical analyses, graphpad prism (v6.0, graphpad software, inc., study data were evaluated using descriptive statistics (mean, standard deviation, frequency, rate) whereas intergroup comparisons of the parameters that show normal distribution and the evaluation of the mean values of two groups were performed using student s t test. results were evaluated within 95% confidence interval and p 40 kg / m) and 83% had obesity (body mass index > 30 kg / m). obesity was an important risk factor in some uni- and multivariate studies and, as suggested as a hypothesis in the publications supported by some studies, increased intra - abdominal pressure which creates difficulties for complete closure of the defect play an important role in the development of tsh. among four tsh cases presented in the present study, three were obese and the presence of obesity was statistically significant in the subjects who developed tsh (p=0.008). in some previously published multivariate analyses, the correlation between tsh and the age, which is among the risk factors, was not found to be significant. although age of 70 years or more was not significantly relevant, as a hypothesis, weak fascia and decreased volume of abdominal muscles presented herein, evaluation of the age of the patients with tsh yielded significant results and the incidence of tsh was more commonly seen in the advanced age group (p=0.007). there are some conflicting insights about the potential role of the gender of the patients in the development of tsh. while some reports suggested that the incidence was higher in male patients, another study demonstrated a higher incidence rate in female patients. although the role of the gender in tsh is conflicting in the literature, the factors that account for tsh appear to be multifactorial rather than being limited to the gender. the fact that the majority of the cases are female may explain the prejudice about the female gender. in our study, the patients with tsh were all female but any statistically significant difference was not detected between genders (p=1.000). although the effect of pre - existing diabetes on wound healing is well known, it is difficult to explain this correlation., two of four patients with tsh had diabetes but this did not reach statistical significance (p=0.055). it was demonstrated that copd and abdominal wall infections increased the predisposition to the development of incisional hernia. we think that copd leads to the development of hernia by delaying the wound healing and by causing cough episodes that increase the intra - abdominal pressure. in one of the tsh cases presented in the study, copd was present but the effect of copd on tsh development was not statistically significant (p=0.223). in the literature, some studies reported pre - existing umbilical hernia as a risk factor. reported the pre - operative presence of umbilical or paraumbilical hernia in 12% of the patients (84% of them were asymptomatic) who had undergone laparoscopic cholecystectomy. it was reported that, despite primary closure of these defects, 1.8% of them had developed tsh. again, azurin. reported similar results in their study. in nine of their ten patients with tsh trocar site hernia may emerge as an important problem following laparoscopic surgery. therefore, the trocar entry site defects greater than 10 mm should be routinely closed and the defects with a size of 10 mm that can not be enlarged should be absolutely closed in the presence of any risk factor, such as age and obesity. the present study does not allow for any comment on the closure of the trocar defects with a size of 5 mm, but some studies recommend their closures. especially the people with risk factors should be informed about the likelihood of hernia development and an earlier diagnosis should be ensured. development of trocar site hernia after laparoscopic surgery may be prevented by repair of the trocar site in patients who have risk factors such as advanced age and obesity. | objective : the aim of this study is to evalute risk factors which trigger the development of trocar site hernia after elective laparoscopic cholecystectomy operation and to discuss what needs to be done to prevent it.methods:patients operated with laparoscopic cholecystectomy between 2011 and 2013 were evaluated. patients were called back for follow - up visit at 12 month after operation. physical and ultrasonographic examinations were performed at follow - up. factors that facilitate development of trocar site hernia were investigated.results:one hundred and ninty patients were operated during the study period. one hundred and thirty - two patients who had been examined at follow - up period were included in the study. mean age of the patients was 50.6411.86 (1876) years and female / male ratio was five. trocar site hernia was detected in four patients at umblical trocar site. one of these patients had chronic obstructive lung disease, two of them had diabetes and three of them had obesity. advanced age and obesity were found to be statistically significant in patients having trocar site hernia (p value : 0.007, and 0.008, respectively).conclusion : development of trocar site hernia after laparoscopic surgery may be prevented by repair of trocar site in patients taken into consideration risk factors such as advanced age and obesity. |
since more than a decade the topoisomerase i inhibitor irinotecan has been one of the most important drugs in the treatment of metastatic crc although its single agent activity in second line is only 20% and its toxicity is considerable. especially in the pre - cetuximab / panitumab and bevacizumab era new camptotecin analogues with improved activity and less toxicity were therefore warranted. bay 56 - 3722 (formerly bay 38 - 3441) is a camptothecin glycoconjugate that generates camptothecin upon cleavage. bay 56 - 3722 consists of a carbohydrate (fucose) moiety attached to the camptothecin toxophore by a peptide spacer. the camptothecin delivered from bay 56 - 3722 acts by binding to and stabilizing the topoisomerase - i - dna complex, leading to an accumulation of double - stranded dna breaks upon replication, ultimately causing cell death. the lactone form is associated with its antitumor activity, whereas the carboxylate form is inactive [2, 3 ]. first, there were in vitro data suggesting the utility of bay 56 - 3722 in a variety of crc lines. secondly, the two main body tissues with highest levels of radioactivity after administration of bay 56 - 3722 were liver (3.0%) and the large intestine (3.6%). this could provide a potential advantage for bay 56 - 3722 over other chemotherapy agents in patients with metastatic tumors in the liver. bay 56 - 3722 was evaluated in vivo in a panel of human tumor xenografts in nude mice. in most of these experiments, bay 56 - 3722 was tested in comparison with doses of topotecan and not with irinotecan, which would have been more appropriate. bay 56 - 3722 was more efficacious at maximum tolerated dose than topotecan and exhibited less gastrointestinal toxicity and myelosuppression. in patients bay 56 - 3722 has been studied on three schedules, once every 21 days, daily for 3 days every 21 days and daily for 5 days every 21 days [3, 5, 6 ]. in the phase i study where a daily 5 schedule is explored, there appears to be a 4-fold increase in the camptothecin auc comparing day 1 to day 5 suggesting that this schedule might be the most likely schedule to have antitumor activity. the present phase ii study was designed in the beginning of this century to study the antitumor activity, safety and tolerability of bay 56 - 3722 using a daily schedule for 3 days every 3 weeks. the study was conducted at 13 centers in canada, the usa and the netherlands. patients received bay 56 - 3722 iv over 30 min daily for 3 days every 3 weeks until objective evidence of tumor progression, unacceptable toxicity, consent withdrawn or until the investigator deemed that continuation of treatment adds no more benefit for the patient. tumor response measurements were made according to who criteria at baseline and every 6 weeks for the entire duration of treatment. a three stage enrolment procedure would be used (null hypothesis : underlying response rate is less than or equal to 10% ; alternative hypothesis : true response rate is more than or equal to 20% ; one - sided alpha of 0.025 ; power of 90%). a futility analysis was planned when 20 evaluable patients were treated and followed for tumor response for a maximum of six cycles. if none of these patients responded (no pr or cr) to therapy termination of the study if at least one patient responded (5%), an additional 60 patients were planned to be enrolled. the second futility analysis would count the number of responders out of the 80 patients at the end of maximum six cycles : if the number of responders would be less than 10% the likelihood of success would be sufficiently low to warrant discontinuation of the study. if the number of responders would be more than 20% the regimen would be considered active and the study might be closed in preparation for phase iii. nevertheless, if 915 responders were obtained, additional 60 patients would be enrolled and response rate would be evaluated at the end of cycle 6 to determine if the drug was active enough to start phase iii. adverse events were graded by the national cancer institute (nci) common toxicity criteria (ctc) version 2.0. informed consent and protocol were reviewed and approved by the appropriate local ethics or review boards before study initiation. patients were considered eligible if they had histologically confirmed recurrent or metastatic colorectal cancer with documented progression during or within 6 months after treatment with irinotecan. required were adequate bone marrow, renal and liver functions and signed informed consent. twenty - four patients received at least one dose of study treatment and were therefore included in the safety evaluation. one patient did not qualify to receive study medication due to a protocol inclusion criteria violation. of the 24 patients in the safety population, 18 (75%) discontinued study treatment because of disease progression, 4 (17%) because of consent withdrawn, and 2 (8%) because of study termination by the sponsor. of the four patients that withdrew consent, one withdrew it after only one dose of study drug, another one after cycle 1, a third patient due to opting for treatment with capecitabine, and the last patient due to clinical deterioration. the futility analysis that was planned for this study after the first 20 eligible patients were enrolled could not be completed due to an initial clinical hold as well as later discontinuation of the bay 56 - 3722 development program. this study was put on a clinical hold while the safety data were reviewed for the entire bay 56 - 3722 development program. once this review was completed, the clinical hold was removed (after 5 weeks). at the time of the clinical hold, only two patients were taken off study because of lack of the essential irb approval to go through. at the time when the clinical hold was removed, patients had to undergo a new tumor assessment and show no disease progression in order to continue study drug treatment. at least one treatment - emergent event was reported by 23 of the 24 patients (96%). one patient with non - insulin dependent diabetes and coagulant use experienced one episode each of grade 4 rectal bleeding and hypoglycemia. two of these events, grade 2 creatinine elevation and grade 3 renal / genitourinary - other (bilateral hydronephrosis), were considered possibly drug related. bay 56 - 3722, selected for this phase ii study, was a promising drug in diseases that were resistant to other topisomerase i inhibitors because of the enhanced stability of the active lactone moiety of the drug with enhanced pre - clinical antitumor activity and a favorable toxicity profile. based on three phase i studies further phase ii studies in several tumor types were undertaken with the preferred bay-56 - 3722 regimen. none of these studies have been published and we felt that this was an omission. therefore we decided to share our results and the fate of this drug in the current publication. this study was put on a clinical hold while the safety data were reviewed for the entire program, because of excessive toxicity in three patients with hepatocellular carcinoma in two studies in the program, this study not being one of them. since, after review, this toxicity appeared to be disease related, patients were allowed to continue treatment after 4 weeks provided that there was no disease progression in our study. during the clinical hold for toxicity reasons bayer undertook a voluntary action to withdraw camptothecin glycoconjugate (bay 56 - 3722, formerly bay 38 - 3441) from further clinical development due to observed safety issues, lack of therapeutic benefit, and poor enrolment in other studies. due to this decision we were not able to draw conclusions whether this drug is active or not in colorectal cancer. prematurely stopped studies as a result of a decision of the sponsor not to further develop a study drug (based on results in other studies) are extremely rare and the (temporary) withdrawal of the drug during the study puts the patient and the treating physician / local study team in a difficult position. the clinical hold was undertaken for safety reasons in the first place which is easier to accept than for economic reasons. we felt it was our obligation to share this interrupted phase ii study for two reasons : to report the fate of camptothecin glycoconjugate and to report the unique situation of a clinical hold during a phase ii study. | summaryintroduction bay 56 - 3722 (formerly bay 38 - 3441) is a glycoconjugated campthotecin, which was considered an attractive drug to assess in colorectal cancer (crc). patient and methods phase ii study design evaluating the antitumor activity of bay 56 - 3722 iv 320 mg / m2 daily for 3 days every 3 weeks in patients with recurrent or metastatic inoperable crc resistant to irinotecan. results twenty - four patients received the study treatment. triggered by adverse events in two other studies with this compound the study was put on a clinical hold while the safety data were reviewed for the entire program. after the review bayer decided to withdraw bay 56 - 3722 from all clinical investigations. discussion we felt it was our obligation to share this interrupted phase ii study for two reasons : to report the fate of camptothecin glycoconjugate and to report the unique situation of a clinical hold during a phase ii study. |
we first decided to investigate b7 - 1 expression in vitro. toward this aim, we isolated mouse podocytes. we confirmed that after 14 days in culture, the podocytes were still in a differentiated state with suggestive podocyte morphology and that they expressed typical podocyte markers, such as nephrin, podocin, and nestin (figure 1a). then, we exposed the podocytes to lps, a molecule reported to induce b7 - 1 mrna expression in podocytes.4, 5 at baseline, we observed a very small amount of b7 - 1 mrna in our primary culture of podocytes. after lps treatment, we did not see b7 - 1 mrna induction (figure 1b), whereas we could detect il-6 and il-1 mrna induction confirming tlr-4 engagement in primary cultured cells (figure 1b). next, we explored whether b7 - 1 was detectable at the protein level in our differentiated podocytes. a western blot analysis revealed that b7 - 1 was not present at the protein level in podocytes, neither at baseline nor after lps treatment (figure 1c). the costaining experiments did not confirm b7 - 1 expression in primary culture of podocytes either at baseline or after lps treatment (figure 1d). to ensure that the anti - b7 - 1 antibody was able to detect the murine form of b7 - 1, we transfected hela cells with a vector encoding mouse b7 - 1 cdna as positive controls. additionally, we tested 2 other anti - b7 - 1 antibodies that were commercially available. we confirmed that the antibodies were able to detect the b7 - 1 protein in hela transfected cells (figure 1e) but failed to detect b7 - 1 in podocytes exposed either to vehicle or lps. because a primary culture of glomerular cells contain podocytes but also a few endothelial and mesangial cells, we explored b7 - 1 expression in immortalized mouse podocytes. at baseline, we observed a very small amount of b7 - 1 mrna in cultured podocytes, but we did not detect b7 - 1 mrna induction (figure 2a) or b7 - 1 protein after lps treatment (figure 2b). il-6 mrna induction was detected, confirming tlr-4 engagement in podocytes (figure 2a). costaining experiments using 3 commercially available anti - b7 - 1 antibodies confirmed the absence of b7 - 1 expression in immortalized mouse podocytes at baseline or after lps treatment (figure 2c). indeed, in our hands, we did not confirm b7 - 1 expression in stressed podocytes. we next investigated b7 - 1 expression in different mouse models of podocyte injury, including lps, adriamycin (sigma aldrich, st. quentin fallavier, france) nephropathy, lupus prone mice (nzb / w f1), and subtotal nephrectomy. we first validated that the lps (200 g), adriamycin, lupus prone mouse, and nephrectomy models produced albuminuria (figure 3a). as expected, we decided to test 2 additional dosages of lps (300 and 400 g) and failed to induce more proteinuria. using 300 and 400 g, the mice died within 24 hours of injection due to sepsis shock condition (not shown). histological analyses revealed that glomeruli appeared grossly normal in lps - injected mice, whereas glomeruli were severely injured in mice injected with adriamycin, in lupus prone mice, and after nephrectomy (figure 3b). however, electron microscopy revealed that lps induced foot process effacement with cytoskeleton reorganization (figure 3c). we then performed colocalization studies in paraffin - embedded kidneys between nestin, a podocyte marker, and b7 - 1 in all models of injuries. careful examination of glomeruli showed that b7 - 1 was not detectable in podocytes (figure 3d). importantly, we could detect b7 - 1 expression in some infiltrating cells, confirming that anti - b7 - 1 antibodies were efficient in detecting the b7 - 1 epitope in our sections. lastly, we performed costaining between anti - b7 - 1 antibody and nestin on frozen kidney sections. similarly, using the 3 commercially available anti - b7 - 1 antibodies, we could not find b7 - 1 expression in podocytes, whereas we could find b7 - 1 expression in some infiltrating cells (figure 3e). of note, because the anti - b7 - 1 antibodies gave similar results, only the af140, goat antibody from r&d systems europe (lille, france) is shown in figure 3e. we therefore conclude that b7 - 1 is not detectable in these proteinuric mouse models. in this study, we did not observe b7 - 1 expression in mouse podocytes at the protein level at baseline or after injury. we could detect only a very small amount of mrna, which did not increase after lps stimulation, a condition that was reported to induce its expression. our in vivo data using several mouse models of proteinuric diseases corroborate our in vitro findings. importantly, our experimental findings are in line with accumulating evidence from independent groups showing that b7 - 1 blockers are not associated with albuminuria improvement in patients.7, 8, 13 b7 - 1, a costimulatory molecule, was unexpectedly observed in injured podocytes. indeed, it was shown that injured podocytes expressed b7 - 1 leading to cytoskeletal modification in vitro. furthermore, these in vitro findings were also observed in several models of proteinuria including activation of innate immune signaling via tlr-4 by bacterial endotoxin (lps). more strikingly, mice with severe combined immunodeficiency who were exposed to lps rapidly upregulate b7 - 1 in podocytes and develop proteinuria, whereas mice lacking b7 - 1 were protected from lps - induced nephrotic syndrome, suggesting a link between podocyte b7 - 1 expression and proteinuria. importantly, the mice used in the study were knockouts for b7 - 1 but not for podocyte - specific b7 - 1 knockouts, meaning that the beneficial effect that was observed could be related to an effect on immune cells rather than a direct effect on podocytes. yu. then reported the successful treatment of 4 patients with recurrent fsgs on the allografted kidney using a b7 - 1 blocker. however, others did not confirm these clinical results,7, 8, 13 and b7 - 1 expression in podocytes in vivo has been controversial.14, 15 using appropriate controls, we could not confirm that b7 - 1 is expressed at the protein level or induced at the mrna level in injured murine podocytes. we observed that lps engaged the tlr-4 pathway in podocytes as assessed by the mrna induction of il-6 and il-1 but did not induce b7 - 1 expression. in all these models, we could not detect b7 - 1 expression in podocytes. importantly, we could detect infiltrating cells stained for b7 - 1, an internal control of the validity of the different antibodies used. all sample kidneys were fixed, processed in the same way, and stained in the same time to avoid any bias. in fact, it is possible that in human kidney diseases, b7 - 1 plays a role in podocytes, a condition that was not explored here. additionally, abatacept was efficient at inducing proteinuria remission in a few patients. however, it is possible that the effect that was observed with abatacept in fsgs patients was not directly related to a podocyte effect but rather, was due to an action on immune cells or an off - target effect of the drug. many reports did not observe such an effect of abatacept on proteinuria in recurrent fsgs after transplantation, but we know that the disease is extremely heterogeneous, and not all patients will benefit from abatacept treatment. the identification of such responders will remain an important challenge. in conclusion, using the appropriate tools, we did not confirm that b7 - 1 was expressed in murine podocytes under pathological conditions, and further studies are recommended before using b7 - 1 blockers in patients with proteinuric diseases. the mouse strains that were used in these studies included fvb / n, c57bl/6, and balb / c (charles river laboratories, wilmington, ma). the animals were fed ad libitum and housed at a constant ambient temperature under a 12-hour light cycle. all animal procedures were approved by the departmental director of services vtrinaires de la prfecture de police de paris and by the ethical committee of paris descartes university. administered either 200 g of lipopolysaccharide (ultrapure lps ; 1 mg / ml in sterile lps - free phosphate - buffered saline, n = 6) in a total volume of 200 l or an equal volume of sterile lps - free phosphate - buffered saline (n = 6) (ultrapure lps from e. coli o111:b4, invivogen, toulouse, france). urine samples were collected and analyzed 24 hours post injection and immediately prior to sacrifice. eight - week - old male balb / c mice (n = 12) were administered either 17 g / g body weight adriamycin (sigma aldrich) diluted in 0.9% saline or an equal volume of 0.9% saline (n = 12) into the retrobulbar plexus. twenty - four hours before sacrifice, urine was collected for determination of proteinuria. the mice were sacrificed 7 days after injection, and their kidneys were removed for morphological and protein studies. eight - week - old female fvb / n mice were subjected to 75% nephrectomies (n = 10) or sham - operations (n = 10) and were sacrificed 2 months after surgery. after surgery, the mice were fed a defined diet containing 30% casein and 0.5% sodium. twenty - four hours before sacrifice, urine samples were collected for determination of proteinuria. at the time of sacrifice forty - six - week - old mice (n = 6) from jackson laboratories (bar harbor, me) were used as a model of lupus nephritis. twenty - four hours before they were killed, urine samples were collected for determination of proteinuria. at the time they were killed, their kidneys were removed for morphological and protein studies. for urine samples, albumin and creatinine concentrations were measured using an olympus multiparametric analyzer (instrumentation laboratory, bedford, ma). mouse kidneys were fixed in 4% paraformaldehyde and embedded with paraffin, and 4-m sections were stained with masson trichrome. four - micrometer, paraffin - embedded sections were incubated with 3 different anti - b7 - 1 antibodies : (i) anti - b7 - 1 antibody from santa cruz biotechnology inc. (heidelberg, germany ; (ii) anti - b7 - 1 antibody (mouse, 2a2) from abcam (paris, france) ; (iii) anti - b7 - 1 antibody (af140, goat) from r&d systems europe. we also used anti - nephrin (progen biotechnik gmbh, heidelberg, deutschland), anti - podocin (h208, rabbit ; sigma aldrich), and anti - nestin (rat-401 ; developmental studies hybridoma bank, university of iowa, iowa city, ia) antibodies after appropriate antigen retrieval. the sections were deparaffinized and rehydrated, then the slides were brought to boil in tris ethylenediamine tetraacetic acid buffer (10 mm tris base, 1 mm ethylenediamine tetraacetic acid solution, 0.05% tween 20, ph 9.0) and maintained at a subboiling temperature (95 c) for 40 minutes. then, each section was blocked with tris - buffered saline tween-20/5% normal goat serum for 30 minutes. anti - b7 - 1 antibodies were used at a dilution of 1:50 and incubated on the sections overnight at 4 c. the primary antibodies were revealed with the appropriate alexa 488-, 555-, or 647-conjugated secondary antibodies (molecular probes, san diego, ca) used at a dilution of 1:200 for 2 hours at room temperature. immunofluorescence staining was visualized using a zeiss lsm 700 confocal microscope (jena, germany). for each sample, we evaluated all glomeruli per section (a minimum of 50 glomeruli, 2 to 4 sections per mouse, were evaluated per animal). kidneys frozen in optimal cutting temperature compound were cut in 4-m sections, fixed in acetone, and then incubated with the anti - b7 - 1 antibody from santa cruz biotechnology the anti - b7 - 1 antibody (mouse, 2a2) from abcam, or the anti - b7 - 1 antibody (af140, goat) from r&d systems europe, and anti - nestin (rat-401, developmental studies hybridoma bank) antibodies. mouse podocytes were isolated and cultured as previously described. briefly, primary cultures of podocytes were maintained in roswell park memorial institute 1640 medium supplemented with 10% fetal calf serum, 100 u / ml penicillin, 100 g / ml streptomycin, and insulin - transferrin - sodium selenite media (sigma aldrich) for 14 days. for lps experiments, the cells were treated with 50 g / ml of ultrapure e. coli (lps from e. coli o111:b4 ; invivogen) for 24 hours before performing immunofluorescence, quantitative polymerase chain reaction, and a western blot analysis. briefly, cells were maintained in roswell park memorial institute 1640 medium supplemented with 10% fetal calf serum, 100 u / ml penicillin, and 100 g / ml streptomycin. to propagate podocytes, cells were cultivated at 33 c on type i collagen (permissive conditions) in culture medium supplemented with 10 ui / ml recombinant interferon-. to induce differentiation, podocytes were maintained on type i collagen at 37 c without supplementation with interferon- (nonpermissive conditions). for lps experiments, the cells were treated with 50 g / ml of ultrapure e. coli (lps from e. coli o111:b4, invivogen) for 24 hours before performing immunofluorescence, quantitative polymerase chain reaction, and a western blot analysis. hela cells were transfected with pcdna3.1-c-(k)dyk (nm_009855,orf sequence ; genscript hk limited, hong kong, china) plasmids using lipofectamine 2000 according to the supplier s instructions (invitrogen, carlsbad, ca). primary cultures of podocytes and differentiated podocytes were fixed in 4% paraformaldehyde for 20 minutes, permeabilized in phosphate - buffered saline containing 0.2% triton x-100, and then incubated with anti - nestin (rat-401, developmental studies hybridoma bank), anti - nephrin (progen biotechnik), anti - podocin (sigma aldrich), and the 3 different anti - b7 - 1 (h-208, rabbit from santa cruz biotechnology inc. ; 2a2, mouse from abcam ; af140, goat from r&d systems europe) antibodies. the primary antibodies were revealed with the appropriate alexa 488-, 555-, or 647-conjugated secondary antibodies (molecular probes). b7 - 1 mrna was detected in podocytes by real - time reverse transcriptase polymerase chain reaction using an abi prism 7700 sequence detection system (applied biosystems, foster city, ca). the primers (eurogentec, lige, belgium) used were as follows : b7 - 1 forward 5-tgtatgcccaggaaacaggt-3 and reverse 5-agcccgatcaccac - tgatta-3, podocin forward 5-gct - gtc - tgc - tac - tac - cgc - at-3 and reverse 5-ctc - tcc - act - ttg - atg - ccc - ca-3, il-6 forward 5-cca - gag - tcc - ttc - aga - gag - ata - ca-3 and reverse 5-cct - tct - gtg - act - cca - gct - tat - c-3 and il-1 forward 5-tca - ttg - tgg - ctg - tgg - aga - ag-3 and reverse 5-gcc - tgt - agt - gca - gtt - gtc - taa-3. rpl13 and hprt were used as a normalization control. polyacrylamide gel electrophoresis before being transferred onto the appropriate membranes and incubated with the 3 different anti - b7 - 1 (h-208, rabbit from santa cruz biotechnology ; 2a2, mouse from abcam ; af140, goat from r&d systems europe), anti - podocin (sigma aldrich) ; and anti- actin (sigma aldrich) antibodies, followed by the appropriate peroxidase - conjugated secondary antibodies. chemiluminescence was acquired using a fusion fx7 camera (vilber lourmat, eberhardzell, germany), and densitometry was performed using imagej software (national institutes of health, bethesda, md). differences between the experimental groups were evaluated using analyses of variance followed by the tukey - kramer test when significant (p < 0.05). | recent research on podocytes has proposed b7 - 1 as an important player in podocyte biology and as a potential new therapeutic target. b7 - 1 was upregulated in injured podocytes and described as a biomarker to identify patients who may benefit from abatacept, a b7 - 1 blocker. however, after this initial enthusiasm, several reports have not confirmed the efficiency of abatacept at inducing proteinuria remission in patients. in order to resolve these discrepancies, we explored the role of b7 - 1 in the injured podocyte. both primary cultured and immortalized podocytes were exposed to lipopolysaccharides, but this failed to induce b7 - 1 expression at the mrna and protein levels. importantly, tlr-4 engagement confirmed lipopolysaccharide efficacy. we then evaluated b7 - 1 expression in several mouse models of podocyte injury including treatment with lipopolysaccharide or adriamycin, a lupus prone model (nzb / w f1) and subtotal nephrectomy. using 3 commercially available anti - b7 - 1 antibodies and appropriate controls, we could not find b7 - 1 expression in podocytes, whereas some infiltrating cells were positive. thus, our findings do not support a role for b7 - 1 in podocyte biology. hence, further studies are mandatory before treating proteinuric patients with b7 - 1 blockers. |
the study began in december 2004 and terminated on february 17, 2007. a notice sent to all physicians in maritime canada described the study and asked the physicians to submit serum samples from patients with suspected q fever (febrile illness or pneumonia after exposure to parturient cats or other animals ; outbreaks of pneumonia in a family). all samples were sent to the nova scotia public health laboratory for testing for antibodies to coxiella burnetii. physicians of patients with a positive test result were contacted, and they in turn contacted their patients to ask if they would participate in the study. this study was approved by the university of alberta research ethics review board and the capital health research ethics board. during the study period, serum samples from 210 patients suspected of having c. burnetii infection were tested by using commercially available immunoglobulin (ig) m and igg elisas (panbio, brisbane, queensland, australia). convalescent - phase samples were collected from those who agreed ; further testing for igg antibodies to phase i and phase ii c. burnetii antigen was conducted by using an indirect immunofluorescence test as previously described (3). of the 210 patients, 35 had antibodies to c. burnetii and 13 met the criteria for acute q fever (positive igm elisa result and a > 4-fold rise in antibody to phase ii antigen between the acute- and convalescent - phase samples). phase i titers > 512, suggestive of chronic q fever, were found for 3 patients. of the 13 patients who fit the case definition of acute infection, 11 agreed to participate in the study, 1 declined, and 1 moved to another country. of the 11 participating patients, 7 were from nova scotia, 2 were from new brunswick, and 2 were from prince edward island ; 6 were male ; and mean age was 54.6 years (table). one case occurred in december 2004 ; 6 in 2005 ; 4 in 2006, and no cases in the first 6 weeks of 2007. cases occurred in every month except august, september, october, and february. within 2 weeks of onset of symptoms, this patient had occupied a small space (automatic bank teller area) with 2 farmers who smelled of manure. one patient (patient 8) was a sheep farmer who had recently had 60 lambs born on his farm, several of which were stillborn in the 2 weeks before the farmer became ill ; 7 patients had > 1 cat as a pet ; and only 2 (patients 5 and 8) had no pets. in terms of clinical signs, all 11 patients had sweats, fever, and myalgia ; 9 had chills ; 8 had a cough ; 7 were short of breath ; 5 each had nausea, diarrhea, sputum production, and confusion ; 4 had chest pain, which was pleuritic for 2 ; and 2 had abdominal pain and vomiting. of the 7 patients for whom chest radiographs were taken, 6 had acute opacities compatible with pneumonia. patient 11 had diffuse bilateral pneumonia, which required him to be admitted to an intensive care unit to receive ventilatory support (figure). chest radiograph of patient 11 at time of admission to hospital, before intubation, demonstrating extensive bilateral airspace disease. only 4 received initial empiric therapy that would be considered effective against c. burnetii, e.g., doxycycline (n = 2), ciprofloxacin (n = 1), or levofloxacin (n = 1). four other patients received azithromycin, which may have been effective but has suboptimal in vitro activity against c. burnetii (4). acute q fever is still present in maritime canada ; however, the number of cases has diminished considerably from the 1980s and early 1990s. since 2004, only 45 cases have been reported each year. the passive design of our study may have underestimated the number of cases. however, in the 1980s, a number of q fever outbreaks involved entire families. a typical scenario was exposure to the parturient family cat and her newborn kittens, after which everyone in the family became ill (2). some outbreaks involved poker players (5) or most of the employees of a factory (6). for our study, we carefully asked whether family members were ill ; only 2 patients mentioned such illness, and for each, it was a spouse. pneumonia seems to still be the dominant form of acute q fever in maritime canada. major differences in the manifestations of q fever occur in different regions. in maritime canada and in the basque region of spain, the predominant manifestation is pneumonia (7,8) ; in newfoundland and australia, fever with no apparent localization of infection (9,10) ; in the canary islands, fever and hepatitis ; and in southern france, hepatitis and pneumonia, although hepatitis is more frequent than pneumonia (11,12). the factors responsible for these disparate manifestations are not known. when isolates of c. burnetii from different geographic areas were typed by using multisequence typing, all 7 isolates from nova scotia were identical and shared this type with 2 isolates from france and 1 from the united states (13). the reservoirs for human infection with c. burnetii in nova scotia have likely spread from cats and dogs (14) to the more traditional reservoirs of sheep and cattle (12). patient 8, a sheep farmer, had pneumonia that appeared on radiographs as a rounded opacity in the right middle lobe. rounded opacities are very common in cat - associated cases of q fever but may not be specific for cat - associated infection (15). our findings indicate that after c. burnetii is established in an area, it is likely to persist, although the incidence may fluctuate. | since the 1990s, reports of q fever in nova scotia, canada, have declined. passive surveillance for q fever in nova scotia and its neighboring provinces in eastern canada indicates that the clinical manifestation of q fever in the maritime provinces is pneumonia and that incidence of the disease may fluctuate. |
acquired hemophilia is a severe bleeding diathesis occurring with the incidence of approx. 1 case per 1 million individuals per year. it is equally common in both sexes and this potentially fatal disorder is caused by the activity of autoantibodies impairing the function of blood coagulation factors, mainly factor viii (acquired hemophilia a) [13 ]. in approx.. it may also be associated with autoimmune diseases (systemic lupus, rheumatoid arthritis, myasthenia, multiple sclerosis, non - specific inflammatory bowel disease), cancer, dermatological diseases (psoriasis, pemphigus), infections (hepatitis b and c), chronic respiratory diseases (asthma, copd), or diabetes. about 10% of hemophilia cases the first sign of acquired hemophilia is usually sudden, massive, life - threatening bleeding occurring in a patient without previous coagulation disorders. the bleeding events may be spontaneous or follow a surgical procedure or injury. they include hemorrhages into the hypodermis, digestive tract, genitourinary system, retroperitoneal space, as well as into the respiratory system and pleural cavity [1, 3 ]. mortalities have been reported as early as in the first week after the occurrence of bleeding from the digestive tract and lungs. the mortalities that occurred later were associated with soft tissue, intracranial, and retroperitoneal bleeding [1, 4 ]. 9% for the last decade, while earlier reports estimated it at 22 - 31% [9, 10 ]. without any treatment, the mortality rate has been reported at 41%. therefore, spreading the knowledge about this disease may further contribute to lowering the percentage of deaths. the characteristic features of acquired hemophilia include isolated prolonged aptt, low activity of the coagulation factor targeted by the antibodies, and the presence of antibody titer [2, 3, 11 ]. the present study discusses a case of a young man with recurrent, massive bleeding into the pleural cavity caused by acquired hemophilia a and presents an analysis of literature devoted to acquired hemophilia. the patient was a 32-year - old man with hypertension, who had smoked 20 cigarettes a day for 15 years. four years earlier, he had undergone a partial gastrectomy due to a perforated pyloric ulcer with peritonitis and septic shock. he was treated at the pulmonary disease department, where acute interstitial pneumonitis was suspected on the basis of chest x - ray, chest computed tomography (ct), bronchofiberoscopy, abdominal cavity and heart ultrasonography, laboratory tests, and medical history (nicotinism, occupational exposure to wood dust and resins). after the implementation of steroid therapy, the patient was transferred to a center with a higher referral level. cough, fever up to 39c, respiratory failure, and anemia occurred during hospitalization. chest x - ray showed opacities in the lower and middle lung fields on the left side. laboratory tests provided the following results : hemoglobin (hgb) level 9.3 g / dl, hematocrit (hct) 31.9%, red blood cells (rbc) 3.30 10/l, white blood cells (wbc) 17.75 10/l, platelets (plt) 980 10/l (table i). ultrasonography revealed the presence of fluid in the left pleural cavity ; it was aspirated twice, yielding 1400 ml and 150 ml of bloody fluid. he experienced weakness, drenching sweats, blood pressure reduction to 80/40 mmhg, heart rate elevation to 120/min, and hemoglobin level reduction to 6.5 g / dl. the signs of shock were associated with a rapidly growing hematoma in the left pleural cavity, which most likely appeared due to an intercostal artery injury sustained during the aspiration of the pleural cavity. chest x - ray showed an opacity in the entire left lung field and mediastinal shift to the right side (fig. 1). drainage of the left pleural cavity was urgently performed, and the patient was transferred to the thoracic surgery department. suction drainage of the left pleural cavity was continued ; fluids, 4 units of packed red blood cells (prbc), and plasma were administered intravenously. during the first 24 hours laboratory tests revealed the following : hgb 7.5 g / dl, hct 24.6%, rbc 2.63 10/l, wbc 35.37 10/l, plt 331 10/l, features of renal failure, hyperpotassemia, features of liver damage, unstable blood coagulation, and reduced levels of total protein and albumins. the opacity in the left lung field was still present in follow - up chest x - ray. 2000 ml of organized hematoma was evacuated, while the stiff and hepatized basal segments and the lingula of the left lung were resected. thorough hemostasis was performed, and two drains were inserted into the left pleural cavity. the patient was transferred directly from the operating theater to the intensive care unit, from where he was further moved to the thoracic surgery department after 2 days. the results of laboratory tests performed on the first postoperative day were as follows : hgb 8.6 g / dl, hct 25.9%, rbc 3.07 10/l, wbc 20.38 10/l, plt 311 10/l ; 500 ml of bloody content was evacuated through the drains. the patient received 2 units of prbc and 2 units of fresh frozen plasma. during the second postoperative day, the patient 's hemoglobin level dropped to 7.4 g / dl (hct 21.8%, rbc 2.59 10/l, wbc 20.1 10/l, plt 257 10/l) ; the drainage of bloody content from the pleural cavity increased in volume (800 ml + 150 ml). in light of this, 2 units of prbc and 3 units of plasma were transfused. on the third postoperative day, the drained volume was 600 ml + 150 ml ; laboratory test results were as follows : hgb 8.5 g%, hct 25.8%, rbc 2.99 10/l, wbc 20.12 10/l, plt 318 10/l. during the next 2 days, the amount of drained content dropped to 100 ml + 250 ml and 50 ml + 50 ml, respectively, while the level of hemoglobin increased. on the 6 postoperative day, the amount of drained fluid increased once again to 200 ml + 50 ml, and the level of hemoglobin dropped to 7.9 g / dl (hct 25.3%, rbc 2.8 10/l, wbc 31.55 10/l, plt 498 10/l). chest x - ray revealed a massive opacity in the lower field of the left lung, partially in the middle field, as well as in the peripheral sections of the upper field. chest ct showed massive left - sided pulmonary - pleural lesions, an entirely airless lower left lobe, infiltrative lesions in the majority of the upper left lobe, and pleural fluid collections (fig. 2). a decision was made to perform left - sided posterolateral rethoracotomy. during the operation, it was revealed that the entire left pleural cavity was filled with thrombi and blood, which were subsequently removed. in the supradiaphragmatic area, the parietal pleura was bleeding from its entire surface ; no pinpoint bleeding was found. the patient was transferred from the operating theater directly to the icu with signs of respiratory failure ; mechanical ventilation, antibiotics, steroids, prbc preparations, and fresh frozen plasma were applied. laboratory test results after the operation were as follows : hgb 8.2 g%, hct 25%, rbc 2.96 10/l, wbc 22.7 10/l, plt 372 10/l. the amount of drained fluid was 400 ml + 550 ml. on the next day, 4 units of prbc, 2 units of plasma, and recombinant activated factor vii were transfused. it needs to be added that bleeding around the drains occurred on the first day after reoperation and was stopped with the use of hemostatic sutures. on the third postoperative day, the patient started bleeding from the site of the removed arterial line ; the hemorrhage was stopped with the use of a pressure dressing, etamsylate, and tranexamic acid. activated partial thromboplastin time remained at the level of 74 - 84 seconds, while the daily amount of fluid drained from the left pleural cavity was 100 - 300 ml. in total, the patient received 24 units of prbc and 19 units of fresh frozen plasma. coagulopathy was suspected due to the delayed arterial bleeding (4 hours after removal of the catheter from the radial artery). hematological consultations were performed, and blood samples were taken in order to establish the activity of coagulation factors. the function impairment of one of the factors was suspected to be the cause of the continuous bleeding. the performed tests established the activity of factor viii at the level of 3.04% (normal : 70 - 150%), factor ix 76.75% (normal : 70 - 120%), and factor xii 29.9% (normal : 70 - 150%) ; the presence of antibodies against factor viii (circulating anticoagulant) was determined qualitatively. the patient was transferred to the hematology department, where he remained in a moderately severe condition with resting inspiratory and expiratory dyspnea, numerous auscultatory changes over the lungs, features of right ventricular heart failure (hepatomegaly, ascites, lower extremity swelling), and renal failure (oliguria). high levels of inflammatory parameters were observed (esr, crp), indicating liver damage. factor viii supplementation, factor viia (novoseven), prbc (4 units), immunosuppressants (cyclophosphamide and steroids), low - molecular - weight heparin, antibiotics, as well as antihypertensive and antiarrhythmic agents were administered. gradual improvement of the patient 's condition and laboratory parameters was achieved (eradication of the factor viii inhibitor, normalization of the coagulation system and the parameters of heart and liver function). pleural drains were removed on the 10 and 14 postoperative days. during the hospitalization, the patient experienced an episode of paroxysmal atrial fibrillation. in order to specify the etiology of the factor inducing the process of autoimmunization, the applied diagnostic methods were supplemented with imaging examinations (ct of the chest, abdominal cavity, and pelvis), digestive tract endoscopy, as well as serological and virological testing. based on the entire clinical picture, acute interstitial pneumonitis was considered to be the most likely cause of the observed coagulopathy. after being discharged from the hospital, the patient returned to the hematology clinic for a single follow - up examination. he did not attend any subsequent examinations. follow - up image of the chest after the second aspiration of the left pleural cavity. large opacity visible in the entire left lung field, mediastinal shift to the right side follow - up ct of the chest on the sixth postoperative day. extensive, massive left - sided pulmonary - pleural lesions. almost entirely airless lower lobe, enhancing heterogeneously after the use of contrast. expansion of the right lung without infiltrates laboratory test results from each stage of treatment hgb hemoglobin, inr international normalized ratio, pt regardless of damage location, massive, sudden, and life - threatening bleeding frequently constitutes an indication for prompt surgical intervention in any patient, even without previous hemostatic disorders in the medical history. in the course of this disease, intracranial bleeding as well as bleeding into the digestive tract, genitourinary system, retropharyngeal and retroperitoneal space, pleural cavity, and peritoneal cavity were observed [10, 1215 ]. 1% of cases), but is usually potentially fatal and may appear after surgical procedures performed within the area of the chest [3, 5, 16 ]. bleeding within the area of the chest had been described in women with acquired hemophilia after esophageal resection due to cancer. other authors reported a hemorrhage into the pleural cavity in one male in a group of 15 acquired hemophilia patients. such patients may also experience other, less dangerous intramuscular and subcutaneous bleeding, nosebleeds, or bleeding after intravenous punctures and intramuscular injections [15, 12, 13, 18, 19 ]. according to forsyth, bleeding into the patient described in this publication exhibited massive, recurrent bleeding into the pleural cavity as well as other, less dangerous bleeding around the inserted pleural drains and from the arterial line removal site. the patient was initially treated with suction drainage of the left pleural cavity and underwent surgery twice later on. neither the first nor the second operation revealed any pinpoint bleeding ; the patient was bleeding from the entire parietal pleura in the supradiaphragmatic area. embolization of the bleeding vessels and videothoracoscopic procedures due to similar indications have also been described. in acquired hemophilia, the bleeding is often recurrent in nature and is very difficult to stop with the use of surgical methods [16, 21 ]. with regard to bleeding in different locations, the use of minimally invasive surgical methods, such as laparoscopy in the case of bleeding into the peritoneal cavity, has also been reported. however, these methods may prove to be insufficient for achieving effective hemostasis in the case of extensive bleeding. other bleeding episodes, occurring in the presented patient, were stopped with the use of pressure dressings and drugs (etamsylate, tranexamic acid). some authors describe an effective combination therapy with feiba (an activated prothrombin complex concentrate) and tranexamic acid. according to the literature, the average time between the occurrence of the first signs of a bleeding diathesis to the diagnosis of acquired hemophilia is 1.5 months (from 3 days to 9 months). in the case of the described patient, the disease was diagnosed after about 10 days following the occurrence of the first signs. the suspicion was based on medical history, clinical signs, and prolonged aptt in laboratory tests. significantly reduced activity of factor viii (3.04%) and the presence of antibodies against factor viii with prolonged aptt were also revealed. however, factor viii activity is not completely eliminated in acquired hemophilia, as opposed to congenital hemophilia. the patient described in the article was diagnosed with hemophilia at the age of 32. the majority of authors describe much older patients, especially male (60 or older on average), with diagnosed acquired hemophilia [22, 2427 ]. the mean age of patients registered in each2 (the european acquired hemophilia registry) is approx. the goal of acquired hemophilia therapy is the treatment of coagulation disorders and signs of active coagulopathy, bleeding prevention, and antibody eradication [4, 9 ]. in certain cases, persistent anemia caused by recurrent massive or smaller the described patient received 24 units of prbc and 19 units of fresh frozen plasma in total before the cause of the bleeding was found. some patients have required the postoperative transfusion of over 20 units of prbc and 15 units of fresh frozen plasma before acquired hemophilia was diagnosed [2, 16, 22 ]. the described patient received recombinant factor vii a. such treatment is also mentioned by other authors [25, 9, 12 ]. the use of recombinant factor vii a or an activated prothrombin complex concentrate (feiba) constitutes first - line therapy in acquired hemophilia [4, 1315, 23, 26, 29, 30 ]. oral glucocorticosteroids (gcs), e.g. prednisone alone or combined with cytotoxic drugs, mainly cyclophosphamide, are typically used for eradication treatment [1, 2, 4, 9, 31 ]. in the presence of contraindications, rituximab (mabthera) or, in certain cases, other cytotoxic and immunosuppressive drugs, such as azathioprine, vincristine, mycophenolate, or cyclosporine, are administered [4, 6, 32 ]. it was observed that spontaneous remission of acquired hemophilia may occur at different times after diagnosis [1, 26 ]. spontaneous elimination of antibodies against factor viii is estimated at about 14 - 35% within a period of time lasting from 3 weeks to 18 months, or even several years [13, 17, 26 ]. there are reports of extensive, elective surgical and orthopedic procedures performed without any significant hemorrhagic complications in patients with acquired hemophilia ; their success was dependent on appropriate perioperative conduct [26, 29 ]. on the basis of the presented case, an analysis of the literature concerning acquired hemophilia from the last 10 years was performed with special attention to surgical treatment and bleeding in the area of the chest during the course of the disease. selected studies are helpful in comprehensively illustrating the issue of acquired hemophilia in the context of the described case. the literature includes an analysis of the causes of death of 121 patients with acquired hemophilia within 10 years in france. the mean age of these patients was 80.7 years (ranging from 47 to 99). the main cause of death was hemorrhagic shock (52.9%) ; the source of bleeding could not be specifically established in 75% of patients. other authors analyzed the cases of acquired hemophilia in the united kingdom in the years 2001 - 2003. the mean age of the patients was 78 years, with men constituting 43% of this group. another publication described a group of 13 patients (females) treated in the years 1999 - 2011 in a single center. the mean age of the entire group was 60.6 years (from 28 to 84). these patients suffered from bleeding into the hypodermis and muscles as well as postoperative bleeding, often recurring multiple times. in this group, the reasons for delayed diagnosis were also analyzed, with emphasis put on the diagnostic difficulties associated with this rare disease. the establishment of a correct diagnosis required 1.5 months on average (from 3 days to 9 months). the eradication treatment employed the so - called budapest protocol or a modified vad protocol. other authors described 10 cases of acquired hemophilia diagnosed in the years 1988 - 1997 in patients aged 28 - 76, who experienced such signs of hemostasis disorders as petechiae, subcutaneous hematomas, and intramuscular hemorrhages (in approx. german authors presented a group of 67 patients treated in the years 1998 - 2008 in a single center. the group consisted of individuals at the mean age of 62 years and included 50 women. all patients experienced bleeding into the muscles ; other hemorrhages included bleeding into the extraperitoneal space (16 patients), bleeding into the retropharyngeal space (5 patients), hematuria (3 patients), and bleeding into the digestive tract (1 patient). five patients died in total, four of them after surgical procedures. in 27 individuals, recombinant activated factor vii patients with severe, life - threatening bleeding were treated using the so - called modified bonn - malmo protocol. moreover, 3 cases of acquired hemophilia diagnosed in the years 2003 - 2006 have also been described. the first of these patients was a 51-year - old man who underwent a decompression of the fascial compartments due to a massive, post - traumatic hematoma of the arm. hematomas on both legs appeared in another 68-year - old patient treated with warfarin and suffering from bronchial asthma, atrial fibrillation, and a complex heart - valve disorder. the third patient, a 66-year - old man with acute bronchial asthma, suffered from spontaneously appearing hematomas on both lower extremities. other authors described a case of an 82-year - old man with post - traumatic bleeding into the retroperitoneal space. after exploratory laparotomy, the patient experienced surgical site bleeding, which was difficult to stop. another work describes a case of a 26-year - old woman with a large, spontaneous hematoma in the thigh muscles. the authors of the study emphasize the importance of prompt diagnosis and treatment of this disease. it needs to be emphasized that acquired hemophilia always has to be taken into consideration in the presence of sudden bleeding into the skin, mucous membranes, or body cavities (in congenital hemophilia delayed hemorrhages into the muscles or joints, mostly in males) when prolonged aptt is the only abnormality in the coagulation system. in such cases, it is crucial to detect the factor viii inhibitor : perform a normal plasma mixing study and establish aptt, and establish the titer of the antibodies against factor viii with the bethesda test. the titer of the antibodies against factor viii and the level of factor viii do not correspond to the severity of the diathesis. it is essential to begin a dual treatment consisting in the eradication of the antibodies and the administration of factors bypassing the hemostatic defect. | acquired hemophilia a is a coagulation disorder caused by autoantibodies against blood coagulation factor viii. the first sign of this disease is often massive bleeding, which can affect patients after routine procedures. the parameter which indicates the presence of this condition is isolated prolonged activated partial thromboplastin time (aptt). the present article describes a case of a 32-year - old man with acute interstitial pneumonia and pleural effusion, in whom a massive hemothorax appeared after thoracocentesis ; active bleeding was observed after the introduction of a chest tube. the patient was operated on, and no pinpoint bleeding was discovered during the procedure. active bleeding was still taking place postoperatively. the patient underwent another operation after 6 days. once more, no pinpoint bleeding was found. prolonged aptt was observed. the activity of blood coagulation factor viii was 3.04%. the presence of antibodies against factor viii was confirmed, and acquired hemophilia was diagnosed. the article also includes an analysis of the literature on acquired hemophilia. |
a 29-year - old female was under follow up observation at a local clinic for a mass in her left breast palpated 6 months prior to admission. gestational age on admission was 8 weeks. in her presurgical breast ultrasonography, in the area 4 cm from the ten o'clock direction of the left nipple an approximately 4 3 cm sized mass without any distinct boundaries thought to be the mass was deeply located close to the fascia of the pectoralis muscle, thus difficult to operate by local anesthesia. in the operating room, it was decided that tpvb be performed because it would affect the fetus less than general anesthesia. the patient took a sitting position with an assistant was positioned in front of her. she was asked to lower her head and lean against the chest of the assistant. after marking the c7 spinous process (sp) to the t1 sp with a skin marker, each mark of the t1 sp to the t5 sp was marked again horizontally 2.5 cm left of the initial mark. after marking the areas, her skin was prepared with povidone iodide solution and subsequently, 1.5 ml of 2% lidocaine was injected into each marked point subcutaneously. afterwards, using five 20 gauge tuohy needles, the skin of each point was pricked vertically. if the tip of the needle touched the transverse process, the needle was retreated and advanced by changing directions toward the head. the paravertebral space was assessed through a loss of resistance technique, and then the site was fixed. each of the needles was installed in the marked site from the t1 sp to the t5 sp where a total of 20 ml of 0.5% ropiacaine, 4 ml each, was injected. the patient then took the supine position, and blood pressure and pulse rate were 100/55 mmhg and 100, respectively. other specific findings were undetected and the patient did not portray any particular symptoms. the blockage range evaluated by an ice test of the area from t1 to t10. during surgery, the patient presented with severe anxiety and requested to be put asleep, and so propofol (fresofol, fresenius kabi, austria), a pregnancy risk category b drug, was injected using a syringe pump (pilote anesthesie is, fresenius vial s.a, france) at the rate of 90 g / kg / min. after surgery, she was transferred to a recovery room for 45 minutes in order to detect any pain or other special findings she might experience. after transferring to a ward, where she also did not experience any pain, further analgesics were not administered. in the ultrasonography performed the day after surgery, fetal heart beat was 180 bpm, and gestational age measured by crown - to - rump length was 8 weeks + 5 days. the patient was treated with cefotiam 1 g / day through intravenous infusion for 4 days and was discharged without any complications. in cases performing surgery for non - obstetrical problems during pregnancy, maternal death, miscarriages, elective termination, and delivery induced by surgical procedure may be induced. for the fetus, fetal death, prematurity, and major birth defects may be induced. particularly in cases undergoing surgery during the 1st trimester, however, during the entire pregnancy, the incidence of miscarriage or fetal death has been reported to be 5.8%, and the risk raised to 10.5% during the 1st trimester. it has been reported that cases of selective termination due to anxiety of the birth of deformed babies were approximately 1.3%. in cases which the patient must receive surgery unavoidably, safety of the mother and the fetus associated with anesthesia and surgery becomes an important issue. according to the standard labeling of drugs used in pregnancy which was organized in 1979, they are classified in 5 categories. based on the research data conducted on animals and humans, l a, controlled studies have shown no risk ; l b, no evidence of risk found in human beings ; l c, a risk can not be ruled out ; l d, positive evidence of risk exists ; l x, the drug is contraindicated in pregnancy. in animal experiments, nitrous oxide induces resorption and anomaly of the fetus. in another experiments, inhalation anesthesia excluding nitrous oxide did not exert teratogenic effects. studies reported that in humans exposed to sevoflurane at 0.2 ppm concentration for 8 hours, sister chromatid exchange occurred more than in cases without exposure by 30%. intravenous anesthetics like meperidine, propofol, and ketamine belong to category b. fentanyl has been reported to lack teratogenicity ; nonetheless, it exerts embryocidal effects and is classified as category c. it is reported that diazepam rapidly penetrates the placenta and accumulates in the fetus, and exposure to it during pregnancy is associated with cleft palate. different from diazepam, however, midazolam has been reported to lack teratogenicity but their pharmacological reaction is similar with diazepam and thus classified as category d and not recommended during the 1st trimester. a prospectively controlled study can not be conducted on humans due to ethical issues, and so it is indeed impossible to assess drug effects on the human fetus. less systemic absorption of drugs occur than general anesthesia and local anesthetics is classified as category b. thus, if surgical area or methods are appropriate, regional anesthesia can be performed safely. nevertheless, during ra, rapid hemodynamic changes caused by sympathetic block may induce adverse effects on the fetus, and so appropriate precautions must be taken. ra performed during breast surgery include thoracic epidural block (teb), thoracic paravertebral block (tpvb), breast block, and intercostal nerve block. tpvb is carried out by administering local anesthetics to the paravertebral space in the thoracic vertebra and inducing ipsilateral somatic and sympathetic nerve blockade of several levels of the thoracic dermatome. the thoracic paravertebral space (tpvs) is a wedge - shaped space, its base formed by the posterolateral aspect of the vertebral body, intervertebral disc, and intervertebral foramen. the anterolateral wall is formed by the parietal pleura, and the posterior wall is formed by the superior costotransverse ligament. the lateral wall consists of the intercostal space and the intervertebral foramen, and the medial wall is consisted of the epidural space. techniques including the loss of resistance technique utilizing air in assessing the location, and also pressure measurement techniques and the methods using fluoroscopy are known. recently, it was performed using a nerve stimulator or ultrasonography. according to the direction of the accessed needle, the classical approach is to vertically come in 2.5 to 3 cm lateral to the midline. the medial approach is to come in 1 cm laterally, and paravertebral - peridural block is to approach 45 on the coronal plane, in concern with the frequency of injection, satisfactory results were obtained in more than 90% by a single injection, and it has been also reported that in comparison to single injection, success rate was noticeably high in multilevel injection. in a study conducted on patients undergoing thoracotomy, regarding postoperative pain control during the postoperative period, tpvb was superior to teb. pulmonary function tests utilizing the peak expiratory flow rate and oximetry similarly showed better results. however, teb performed in the epidural space can result in catastrophic injuries such as spinal cord injury, and so tpvb may be a safer procedure. hypotension or urinary retention due to the blocking of the sympathetic chain may occur in both teb and tpvb, but the incidence is higher in teb. postoperative nausea, vomiting, and respiratory depression are more frequent in teb than tpvb. when comparing bupivacaine with ropivacaine use in breast surgery, cases using ropivacaine showed better results regarding the satisfaction of the patient after surgery, and the continued analgesic period. the authors performed tpvb that is hemodynamically safer and produce fewer complications in comparison with teb. to raise blockage success rates, multilevel injection was performed, and ropivacaine, which is known for its excellent effects in the aspect of longer analgesic time, was used. in summary, the authors performed tpvb in a woman 8 weeks pregnant considering the risk / benefit ratio and the effects of anesthetics on the mother and the fetus. without complications, | non - obstetrical surgery during the first trimester is stressful to both the mother and the fetus. anesthesiologists are also stressed, not only because of the effects of surgery itself, but also because of the uncertain influences of anesthesia thrown upon on the fetus. the authors present a case of breast surgery successfully performed on a woman 8 weeks pregnant requiring removal of breast abscess by the application of thoracic paravertebral block without any complications. thoracic paravertebral block may be a safe anesthetic method for non - obstetric surgery during early pregnancy. |
one of the very common chronic diseases is chronic renal failure, which may lead to end - stage renal disease (esrd) as a part of that. its yearly incidence has been reported 330 cases out of 1 million in the us and 253 cases out of 1 million people in iran. based on the last statistics in 2007, there are 16,600 hemodialysis patients in iran. esrd patients need renal replacement therapy to survive, and hemodialysis is the most prevalent conventional renal replacement therapy in iran. this sign is a complicated concept and its diagnosis and assessment is difficult for the nurses. in esrd patients, untreated fatigue may highly affect the quality of life and lead to patients increased dependency on others, weakness, loss of physical and psychological energy, social isolation, and depression. the elements that can affect the level of fatigue include depression, anemia, sleep disorders, and restless leg syndrome. medicational interventions such as consumption of growth hormone, anti - depressant and anti - anxiety medications, and levocarnitine and erythropoietin stimulating hormone, as well as non - medicational interventions such as nutrition therapy, sleep disorder treatment, stress management, sport, yoga, depression treatment, drug abuse, and acupressure are used to lower hemodialysis patients fatigue. because of medications side effects and incomplete relief of fatigue after taking anti - fatigue medications, the patients are driven to complementary medicine methods such as acupressure medicine. based on the reported researches, acupressure medicine is one of the interventions with the highest application by nurses in clinical settings, and is considered as a clinical and comprehensive nursing intervention. this is also among the manual treatments for which the nurses have an excellent position to use. acupressure medicine, as a healing art, is beautifully integrated with nursing as it is non - invasive and has advantages such as cost efficacy, lack of complications, no need for special tools, and the ease of learning for the patients and their accompanying persons. the only study conducted on hemodialysis patients fatigue was cho and tsay 's study (2004) in taiwan, which showed that acupressure medicine can lower the level of fatigue in patients of experimental group compared to control group. (2007) in a study on the association between the effect of acupressure medicine and sleep quality of hemodialysis patients showed a significant difference in the sleep quality of experimental and control groups based on pittsburgh sleep scale. previous studies include a systematic review on the effect of acupressure medicine on hemodialysis patients signs including fatigue, and meta - analysis studies on the effect of acupressure medicine on labor pain management in labor unit as well as its effect on post - operation vomiting and nausea in adults. their results showed that despite poor methodology, high risk of bias, and low number of subjects, it can not be absolutely indicated that acupressure medicine is effective on patient 's signs. tsay (2004) in a study on the effect of acupressure medicine on hemodialysis patients fatigue stated that with regard to cultural and lifestyle differences of the patients in various countries, the results of these studies can not be generalized for the patients in other geographic areas. therefore, with regard to the difference between the iranian culture and the culture in other countries, and as no study has been conducted on the effect of acupressure medicine on hemodialysis patients fatigue on the one hand and due to the high application of acupressure medicine in clinical nursing interventions on the other hand (as this type of medicine can provide the patients with a cost - effective and safe nursing intervention), this study was conducted as there was no need of any special tools but just trained finger tips and it could be conducted within a shorter time. therefore, the researcher decided to conduct a study with the goal of defining the effect of acupressure on hemodialysis patients fatigue. study population included all esrd patients undergoing chronic hemodialysis in three hemodialysis centers of nour, alzahra, and shariati, hospitals. inclusion criteria were age of over 18, diagnosis of ersd, undergoing hemodialysis at least for 3 months, the patients with chief complaint of fatigue and having fatigue score 5 based on fatigue severity visual analogue scale, lack of any wound or fracture, being in complete psychological and mental health to attend the study and fill the questionnaire, and not having undergone complementary medicine treatment in the past 3 months of the study. exclusion criteria were patient 's absence for two sessions of acupressure intervention and lack of interest to continue the study. after random subjects allocation through minimization method, 32 subjects were assigned to each group of the study, placebo and control. the data were collected by a three - section questionnaire whose first section contained demographic characteristics including age, sex, education level, marital status, occupation status, and disease etiology. the second section contained fatigue severity scale (fss) with a visual analogue scale ranked between 0 and 10, which was scored and commented as fatigue (0), minor fatigue (1 - 3), moderate fatigue (4 - 6), and severe fatigue (7 - 10). fss is a standard scale whose validity and reliability have been confirmed by gift, and has been frequently used in various studies. the third section included piper fatigue scale with 27 items on four dimensions of mental fatigue including behavioral, emotional, sensory, and cognitive dimensions. each item was scored from 0 to 10, with score zero given for lack of fatigue, 1 - 3 for minor fatigue, 4 - 6 for moderate fatigue, and 7 - 10 for severe fatigue. piper fatigue scale is a standard scale whose validity (content and concurrent) and reliability have been confirmed. in the study of masoodi, its scientific validity was confirmed by the professors of tarbiat modares, tehran, and iran universities. its reliability was confirmed in the study of masoodi. by a correlation coefficient of r = 0.89. in the present study, its internal validity was = 0.77 and its reliability was calculated as 81.2% by test retest. this scale was distributed among the patients once before the intervention and once after the intervention to score the subjects fatigue severity. intervention was conducted in the first 2 h of hemodialysis in the experimental and placebo groups. this intervention was carried out among the subjects regularly undergoing hemodialysis, in both legs, hands, and the waist in three weekly sessions for 4 weeks. in the experimental group, the intervention was conducted on the major acupoints k1, gb 34, st 36, sp 6, bl 23, and ht7. in the placebo group, it was carried out with 1 cm distance from the major above - mentioned acupoints. each session lasted for 20 min, of which 2 min were devoted for primary superficial stroking of the acupoints and the rest of the time (18 min) was for acupressure of the determined six acupoints (3 min for each acupoint). the researcher and a male co - researcher were trained under the supervision of the second supervisor, and then the intervention started by her approval. the researcher (female) and her male co - researcher conducted the intervention separately for the female and male subjects, respectively. determination of acupoints was made based on the second supervisor 's guidance on the acupoints standard location. the amount of needed pressure was practiced by standard scales of 20 g to 6 kg, and the reliability of pressure level was confirmed as 100% after 40 times of practice with mean of 3 - 4 kg on the scales for both researchers hands. the details about the process of research, goals, and conditions were given to the patients, and consequently, those interested to join entered the study after filling the written consent form. sampling and intervention were conducted after obtaining approval from ethical consideration committee of nursing and midwifery school of isfahan university of medical sciences. the data were analyzed by chi - square, kruskal - wallis, paired t - test, one - way analysis of variance (anova), and duncan 's test through spss version 19. mean age of the subjects in the experimental group was 53.4 (13.9) years, in the placebo group 55.4 (11.5) years, and in the control group 54.3 (13.4) years. in the experimental, placebo, and control groups, there were 14 female (43.75%) and 18 male (56.25%) subjects, respectively. the most prevalent etiology of the disease was diabetes in 11 subjects (34.4%) in the study group, hypertension in 14 subjects (43.8%) in the placebo group, and diabetes in 12 subjects (37.5%) in the control group. before intervention, subjects matching was conducted concerning demographic characteristics in the three groups of study, placebo, and control through minimization program, and there was no significant difference in the demographic characteristics between the groups (p > 0.05). one - way anova showed that there was no significant difference between the total mean score of fatigue and the mean scores of piper fatigue scale dimensions before intervention in all the three groups, but these means showed a significant difference after intervention in the three groups (p 0.05). mean score of fatigue in the sensory dimension in the study group was significantly less than in the placebo group (p 0.05). as observed in table 2, there was a significant difference in the total mean score change of fatigue and the mean score change of fatigue severity in all dimensions in the three groups after intervention (p < 0.001). post - hoc duncan test showed that reduction of mean fatigue score in behavioral and emotional dimensions was more in the study group compared to the other two groups of placebo and control (p < 0.05), but it showed no significant difference in the two groups of control and placebo (p = 0.3). post - hoc duncan test showed that mean reduction of total fatigue score and mean reduction of fatigue scores in sensory and cognitive dimensions in the study group were more than in the placebo group, and they were more in the placebo group compared to the control group (p < 0.05). comparison of total mean scores of fatigue, behavioral, emotional, sensory, and cognitive dimensions before and after intervention in the three groups of study, placebo, and control comparison of fatigue score changes and dimensions of piper fatigue scale after intervention in the three groups of study, placebo, and control the results of acupressure medicine on severity of fatigue in hemodialysis patients showed that the study group experienced less fatigue severity compared to the placebo and control groups. as the subjects mean age in the present study was over 50 years, it is concluded that esrd seems to be more prevalent at higher ages. the higher number of male subjects in the present study reveal that esrd prevalence seems to be more prevalent among men, which concords with the findings of nasiri. diabetes and hypertension are the major etiologies for esrd, which is consistent with the finding of the present study. stimulation of acupoints results in improvement of vital energy circulation in the body and increase of opioids like endorphin and enkephalin, which may play a role in the reduction of patients fatigue in all behavioral, sensory, cognitive, and emotional dimensions. the obtained results showed that there was a significant reduction in the total mean score of fatigue in the study group after intervention compared to the placebo and control groups. although a similar improvement was observed in the placebo group based on duncan test, the improvement was more in the study group. it seems that in the placebo group, in addition to verbal and psychological communication between the researcher and the patients, due to the physiologic and psychological effects of acupressure, the patients expect the acupressure to have a positive effect on their body, named as hawthorne phenomenon. (2007) in a study aimed to define the effect of transcutaneous electrical acupoint stimulation (teas) on improvement of muscular fatigue among athletes and showed a significant difference between teas and placebo groups in return of muscular power. this study is in line with the present study except for the lack of a significant difference in the placebo groups after intervention. the difference between these two studies seems to be as a result of the study population, intervention method and the number of subjects. the results showed that the mean score of fatigue in emotional dimension significantly diminished in the study group after intervention compared to the placebo and control groups. from psychological aspect, acupressure induces alpha brain wave stimulation that consequently leads to relaxation and reduction of fatigue and anxiety. this medicine has a positive effect on the body and mind and brings about mental joyfulness and health through stimulation of opioid endogen systems such as endorphin and enkephalin. (2007) reported the significant effect of acupuncture and acupressure on cancer patients fatigue in three groups of acupressure, acupuncture, and placebo in the dimensions of general fatigue, activity, and physical fatigue, although it had no significant effect on the dimension of motivation and psychological fatigue. the difference, observed even in the control group, seems to be as a result of the effect of researcher 's psychological factors. their results showed that acupuncture was more effective than acupressure and acupressure was more effective than placebo. this finding is not consistent with our study, possibly due to the difference in the study population, the scale used, and the length of intervention. our obtained results showed a significant reduction in the mean score of fatigue in sensory dimension in the study group after intervention compared to placebo and control groups. the study results of tsay. and the results of the present study are consistent due to use of similar acupoints and length of intervention. the only difference is that there was no placebo group in their study, and in the control group, the mean score of sensory dimension had a reduction, possibly as a result of psychological and mental elements. our results showed a significant reduction in the mean score of fatigue in cognitive dimension in the study group after intervention compared to the placebo and control groups, which can be due to the effects to acupressure mechanism and researcher 's psychological and mental factors during the study. in the study of harris. in which the effect of the two methods of stimulating acupressure medicine and relaxation on students consciousness and fatigue was studied, their consistent results with the present study may be due to the effect of acupressure mechanism, while there was no difference in the placebo group in this study. our results showed a significant reduction in the mean score of fatigue in behavioral dimension after intervention in the study group compared to the placebo and control groups, as the acupoints used in the present study could have had a synergic effect to reduce fatigue. reported that acupressure was effective on the daily function of pittsburgh sleep quality scale in the elderly in the study group, and there was a significant difference between the study and placebo groups, which is not consistent with our study possibly due to the difference in study population. the results showed a significant reduction in the mean score of fatigue severity in the study group after intervention compared to the placebo and control groups. in a study of molassiotis., the mean reduction score of fatigue severity in acupuncture group was more than in acupressure and placebo groups, which is not consistent with the results of the present study. this difference seems to be as a result of the type of intervention, length of time, frequency of intervention, the acupoints used, and/or the measurement scale used. based on most of the experts ideas, sham acupoints also have a treatment effect as most of the time, positive effects also occur in the placebo groups. with regard to the inefficiency of the used acupoint in the placebo group, patients reduction of signs can be as a result of their expectation concerning a positive effect from the intervention domination and/or they felt secure by the presence of the researcher as well as hawthorne effect. the results of the present study with regard to consideration of fatigue as a problem in hemodialysis patients and application of acupressure medicine by nurses in most of their clinical interventions showed that this non - invasive, cost - effective, learnable, and complication - free method can be used to lower the fatigue in hemodialysis patients. therefore, this caring method of complementary medicine is suggested to be used by nurses in hemodialysis wards to lower the patients fatigue. | background : fatigue is considered as a major problem in hemodialysis patients and can impair their quality of life. the purpose of this study was to investigate the effectiveness of acupressure on fatigue in hemodialysis patients.materials and methods : this is a clinical trial study in which 96 hemodialysis patients participated. patients were randomly assigned into acupressure, placebo, and control groups (32 subjects fulfilling the inclusion criteria assigned to each group). the measures included the form of demographic characteristics, visual analog scale of fatigue, and piper fatigue scale. patients in the acupressure and placebo groups received acupressure intervention during the early 2 h of dialysis on six acupoints with massage for 20 min / day, 3 days per week for 4 weeks. in the placebo group, acupressure intervention was performed as mentioned above with a distance of 1 cm away from the actual intervention site. patients in the control group received routine unit care only. chi- quare test, kruskal - wallis, paired t - test, one - way analysis of variance (anova), and duncan test were used for data analysis.results:one-way anova tests showed significant differences in the total mean score of fatigue and fatigue mean scores in the behavioral, emotional, sensory, and cognitive dimensions in the acupressure, placebo, and control groups.conclusion:the results of this study showed that acupressure may reduce fatigue in hemodialysis patients, and use of this non - pharmacologic technique for hemodialysis nurses is suggested. |
axillary lymph node status is still considered the most important prognostic factor in patients with breast cancer. with the ongoing improvement of breast cancer screening programs, sentinel lymph node biopsy (slnb) has been established as a reliable method to evaluate the lymph node status of the axilla, making standard axillary lymph node dissection (alnd) unnecessary. intraoperative analysis of the sentinel node by frozen section (fs) allows for immediate alnd when a metastasis is found in the sentinel node, thus avoiding a second procedure. however, among the drawbacks of fs are (1) the possibility of false negative and false positive results and (2) increase in operation time, because extra time is scheduled in advance in case the fs turns out to be positive. the sensitivity of fs has been reported to range from 58% to 76%, depending on tumour characteristics (e.g., tumour size) and the method of pathological examination [16 ]. this study was designed to evaluate the benefit of fs in our hospital, with regard to the false negative rate (fnr) and true positive results, as well as the additional operation times. by comparing the operation times of the different procedures we evaluated if intraoperative fs of the slnb is either time saving or time consuming. from a prospectively collected database of breast cancer patients, 628 patients with invasive breast cancer who underwent slnb with fs between january 1st, 2005 and october 1st, 2009 were selected. operation times were retrospectively collected from a separate database, which is kept by the department of anaesthesiology. sentinel lymph node (sln) mapping was performed by using lymphoscintigraphy with or without patent blue dye. on the day of operation, lymphoscintigraphy was performed by injecting 77 mbq 99-tc nanocolloid in different depots (total amount 0.8 ml) peritumoural or periareolar. scans of the involved breast and axilla were first acquired 30 minutes after injection. in case of no visible activity, scans were repeated 2 hours after tracer injection. all blue and/or radioactive nodes counting 10-fold ex vivo relative to the background were regarded as slns and sent for fs. slns with a diameter of more than 5 mm were bisected longitudinally and frozen. frozen sections were taken with a microtome setting of 4 m. until january 1st, 2007, a pathologist was present in our hospital for fs analysis, after this date, telepathology was used. a digital image of the fs was made and sent to the pathologist who examined this image via a screen. during the period of telepathology macrometastases were defined as a diameter > 2 mm, micrometastases as a diameter between 0.2 and 2 mm, and isolated tumour cells (itcs) as single tumour cells or small clusters of cells (diameter < 0.2 mm). statistical analyses were performed by using spss 15.0. diagnostic performance was described in sensitivity and false negative rate (fnr) and p values were calculated for sensitivity and fnr. fs of the sln was performed on 628 patients (of which 2 were males). the mean age was 60.3 years (median 59.9 years, range 3088 years). most of the patients were diagnosed with a ductal carcinoma (472 patients ; 75.2%) or lobular carcinoma (80 patients ; 12.7%). in 399 patients (63.4%), wide local excision (wle) the mean tumour size was 1.62 cm (median 1.5 cm, range 16.3 cm). fs accurately diagnosed the status of the sln in 83.6% of the patients (table 1). fs was negative in 548 cases (87.3%) and positive in 80 cases (12.7%). definitive pathology revealed a negative sentinel node in 449 cases (71.5%) and metastatic disease in 179 cases (27.6%). fs was false negative in 101 cases (16.1%) and false positive in 2 cases (0.3%). in the group of patients with a false negative fs, 69 patients (68.3%) underwent subsequent alnd ; the remaining 32 patients received adjuvant radiation therapy of the axilla or adjuvant systemic therapy. decisions on alternative treatment were based on multidisciplinary breast cancer team consultation and based on individual discussions between patient and surgeon. of all 628 patients who underwent intraoperative fs, the sensitivity of fs, defined as (true positive)/(true positive + false negative), was 43.6%. when separated by t - status, sensitivity was 41.4% for t1-tumours and 47.1% for t2/t3 tumours false negative rate (fnr), defined as (false negative)/(true positive + false negative), was 56.4%. separated by t - status, fnr was, respectively, 58.6% for t1 and 52.9% for t2/t3. in the group of patients with a positive slnb, the percentages of itcs, micro- and macrometastases were 25 (14.0%), 52 (29.1%), and 101 (56.4%), respectively. in one patient, this specific information was missing. fs was less sensitive for detecting micrometastases and itcs (sensitivity 21.1% and 4.0%, resp.) than for detecting macrometastases (sensitivity 65.3%) (p value macro- versus micrometastases 0.001). micrometastases and itcs are more often seen in t1 staged tumours (47.7% versus 38.2%, table 2). results were also analysed by the changes in fs examination techniques during the study period (table 3). since the introduction of telepathology, the sensitivity and fnr worsened. a further deterioration is seen after the change in equipment. when compared to fs examination by a pathologist at site, the results of this second period of telepathology were significantly worse (p = 0.025). operation time was reported as the time from incision to closure. wide local excision (wle) combined with a slnb and a negative fs has a mean operation time of 57.3 minutes (range 25162). if fs was positive and alnd was performed immediately, the mean operation time was 102.7 minutes (range 52178). a mastectomy with slnb takes 72.6 minutes (range 23140) ; when completed with an alnd, the mean operation time was 100.2 minutes (range 51178). in comparison, intraoperative analysis of the sln by fs allows for immediate alnd in case of metastatic disease. in this population of 628 patients who underwent slnb, sensitivity was 43.6% overall ; when divided by t - status, sensitivity varies from 41.4% for t1-tumours to 47.8% for t2-tumours. they also demonstrated a higher sensitivity of fs when the slnb contained macrometastases compared to micrometastases or itcs (65.3% versus 21.1% or 11.1%). a similar correlation between tumour and metastasis size table 4 presents an overview of recent studies on sensitivity and false negative rate of fs in the literature. the sensitivity in these studies ranges from 55.6% to 83.6% ; the false negative rates range from 16.3% to 44.4%. despite high volumes of breast surgery, our clinic does not have an in - hospital pathology department, and therefore telepathology was introduced in 2007. the difference in outcome of sensitivity and fnr however, some authors (weiser., van de vrande., and wada.) also had few t3-tumours in their population and still had a lower fnr [2, 3, 7 ]. due to the correlation between tumour diameter and positive intraoperative examination of the sentinel node, fortunato finally, the very low sensitivity and high false negative rates for intraoperative fs of sentinel nodes are worse than what the literature suggests, may be due to publication bias. however, this may also be due to an increasing recognition of micrometastases. the difference in operation time is 17.2 (in case of wle) and 35 minutes (in case of mastectomy) in favour of alnd in the same procedure, so slnb does not seem to be time consuming. however, this applies to the patients with a positive sentinel node. in this study, 28.5% of the slnbs were positive, which means that 71.5% had a negative sentinel node. for these patients, extra operation time was scheduled because of the possibility of a positive fs and thus subsequent alnd. this means that the fs procedure causes an element of uncertainty in the operation room schedule and consumed unnecessary scheduled operation time because additional operation time is scheduled in every patient undergoing breast surgery with slnb. patients with a true positive fs benefit from this procedure, because they undergo immediate alnd, thereby avoiding a second operation. disadvantages of a second procedure are the increased costs, the potential additional morbidity, and the negative emotional impact on the patient [1113 ]. showed that, with respect to hospital costs, fs analysis seems to be worthwhile as long as the false negative rate does not exceed 35%. this is due to the lower costs of a shorter hospital stay and the association with a decrease in long - term postoperative morbidity. performed a cost analysis and concluded that intraoperative examination of the snb by immunohistochemical staining gave an overall cost saving : the cost saved by avoiding reoperations exceeded the added cost of examination. the cost / benefit balance of fs examination is still being debated, and zavagno. opted to reserve intraoperative histology only for patients with larger tumours, who have a higher risk of nodal metastases. in selecting those women, a nomogram can be useful as a decision aid. compared delayed alnd with immediate alnd and observed an increased axillary operation time and total hospital stay in case of the two - step procedure. it is still unclear whether intraoperative assessment techniques will be cost - effective compared to secondary surgery as both involve extra costs. this argument covers other described methods of intraoperative analysis of the slnb, like touch imprint cytology and cytokeratin immunostain, as well. al together, when the benefit of avoiding reoperative axillary dissection is doubtful, routine intraoperative assessment by fs may be indicated only for a selected group of patients, for example, patients with larger tumours or higher age. another option could be to perform slnb in a separate procedure, perhaps under local anaesthesia. after pathologic results are known, definitive surgery for breast and axilla can be performed. however, nowadays, there is lively discussion about the need of treating the axilla in case of a positive sln. the literature shows a very low axillary recurrence rate in case alnd is omitted after a positive sln. in this light fs was associated with a higher fnr in our population, compared with the literature, and telepathology caused a decrease in sensitivity and a consequent increase in fnr. the sln procedure itself is not time consuming, but since the benefit of avoiding reoperative axillary dissection is only 12.4%, routine fs may be indicated only for a selected group of patients, for example, patients with larger tumours. | aims. intraoperative analysis of the sentinel lymph node (sln) by frozen section (fs) allows for immediate axillary lymph node dissection (alnd) in case of metastatic disease in patients with breast cancer. the aim of this study is to evaluate the benefit of intraoperative fs, with regard to false negative rate (fnr) and influence on operation time. materials and methods. intraoperative analysis of the sln by fs was performed on 628 patients between january 2005 and october 2009. patients were retrospectively studied. results. fs accurately predicted axillary status in 525 patients (83.6%). there were 78 true positive findings (12.4%), of which there are 66 macrometastases (84.6%), 2 false positive findings (0.3%), and 101 false negative findings (16.1%), of which there are 65 micrometastases and isolated tumour cells (64.4%) resulting in an fnr of 56.4%. additional operation time of a secondary alnd after wide local excision and slnb is 17 minutes, in case of ablative surgery 35 minutes. the sln was negative in 449 patients (71.5%), making their scheduled operation time unnecessary. conclusions. fs was associated with a high false negative rate (fnr) in our population, and the use of telepathology caused an increase in this rate. only 12.4% of the patients benefited from intraoperative fs, as secondary alnd could be avoided, so fs may be indicated for a selected group of patients. |
a 70-year - old man presented in january 2011 to our opd with poor vision in both eyes (be). he had extracapsular cataract extraction with implantation of a polymethylmethacrylate intraocular lens (pmma iol) in left eye (le) in june 2007 for nuclear cataract. there was no record of pxf noted on the lens surface or pupillary ruff before cataract surgery. on examination, the best - corrected visual acuity (bcva) was 20/200 in right eye (re) and no perception of light in le. the intraocular pressure (iop) was 42 and 17 mm hg in re and le, respectively. examination of re revealed dense nuclear cataract with no pxf and a cup / disc ratio of 0.9. gonioscopy showed open angles with patchy peripheral anterior synechiae (pas). in le, the pupil was dilated and fixed with frosted granular material similar to pxf over the anterior surface of iol and new vessels on iris were present [fig magnified view showing frosted appearance of pxf on the anterior surface of iol in case 1 a 65-year - old male presented in may 2008 with a 1-year history of decreased vision in be. this was distributed in a concentric ring as radial striations and on the posterior surface of the iol where it appeared as multiple granular spots [figs. gonioscopy of le showed open angles with patchy pas in the inferior angle and darkly pigmented trabecular meshwork. the iop was 18 mmhg and the cupping was 0.8. re had edematous cornea, visual acuity of light perception, and the iop was 28 mmhg. (a) radial distribution of pxf on the anterior surface of iol in case 2. (b) granular deposits of pxf on posterior surface of iol in case 2 a 62-year - old male with air - borne contact dermatitis had cataract surgery 10 years ago in be in our hospital. on examination, bcva was 20/30 in be, pxf was noted in le in pupillary ruff, and on iol placed in sulcus. pxf was radially distributed nasally on the iol and as granular dots on the posterior surface [figs. fundus examination revealed a cupping of 0.8 and 0.6 in re and le, respectively. (b) dot - like deposits of pxf on posterior surface of iol in case 3 a 70-year - old man presented in january 2011 to our opd with poor vision in both eyes (be). he had extracapsular cataract extraction with implantation of a polymethylmethacrylate intraocular lens (pmma iol) in left eye (le) in june 2007 for nuclear cataract. there was no record of pxf noted on the lens surface or pupillary ruff before cataract surgery. on examination, the best - corrected visual acuity (bcva) was 20/200 in right eye (re) and no perception of light in le. the intraocular pressure (iop) was 42 and 17 mm hg in re and le, respectively. examination of re revealed dense nuclear cataract with no pxf and a cup / disc ratio of 0.9. gonioscopy showed open angles with patchy peripheral anterior synechiae (pas). in le, the pupil was dilated and fixed with frosted granular material similar to pxf over the anterior surface of iol and new vessels on iris were present [fig magnified view showing frosted appearance of pxf on the anterior surface of iol in case 1 a 65-year - old male presented in may 2008 with a 1-year history of decreased vision in be. this was distributed in a concentric ring as radial striations and on the posterior surface of the iol where it appeared as multiple granular spots [figs. gonioscopy of le showed open angles with patchy pas in the inferior angle and darkly pigmented trabecular meshwork. re had edematous cornea, visual acuity of light perception, and the iop was 28 mmhg. (a) radial distribution of pxf on the anterior surface of iol in case 2. a 62-year - old male with air - borne contact dermatitis had cataract surgery 10 years ago in be in our hospital. there was no pxf noted at the time of extracapsular cataract extraction in be. on examination, bcva was 20/30 in be, pxf was noted in le in pupillary ruff, and on iol placed in sulcus. pxf was radially distributed nasally on the iol and as granular dots on the posterior surface [figs. fundus examination revealed a cupping of 0.8 and 0.6 in re and le, respectively. (b) dot - like deposits of pxf on posterior surface of iol in case 3 in the three patients that we report, pxf was not noted before the cataract surgery. nor did the fellow eyes of the patients have pxf. in case 1, pxf was noted on the anterior surface of the iol whereas in cases 2 and 3, on the anterior and posterior surfaces. though a rare finding, there are at least five published reports of pxf on iol in the literature. the number may be much more as this entity is generally not looked for in a pseudophakic eye or it could be under - reported. chen. described three cases in which exfoliative material was noted on the posterior surface of iol. all three cases had an open posterior capsule before pxf was first seen. in all our patients, posterior capsule was intact. in case 1, the pxf had a frosted appearance with little granularity but cases 2 and 3 showed radial distribution on iol similar to that seen on the crystalline lens. pxf on posterior surface of iol in two cases appeared as granular dots as described by park. in two of their cases. incidence of glaucoma in eyes with pxf in indian patients has been shown to range between 22.6% and 24.1%. in our patients pxf can be clinically evident some years after cataract surgery ; it is unknown if a subclinical form could have contributed to the development of the glaucoma. reported deposition of pxf on the anterior surface of an acrylic iol. in our patients, park. had hypothesized that iol placed in the sulcus stimulates ciliary body surface and production of pxf. pseudoexfoliative material produced by one intraocular tissue can be deposited on other structures including a pmma iol. the presence of pxf anywhere in only one eye should alert the ophthalmologist in the aggressive management of the glaucoma. to conclude, this case series suggest that pxf could have a role in many more cases of presumed poag without obvious pxf material on lens surface or pupillary ruff. follow - up of patients of poag should include slit - lamp examination even in a pseudophakic eye to look for pxf as it can clinically make its appearance later. | we report three patients with deposition of pseudoexfoliation (pxf) material on intraocular lens years after uneventful cataract surgery. pxf was not present before the cataract surgery. the pxf material was found on the anterior surface of the intraocular lens in the first patient and on the posterior surface as well in the other two patients. all the patients had a polymethylmethacrylate intraocular lens placed in the sulcus. the fellow eyes did not have pxf. all the patients had open angle glaucoma in both eyes. |
the electrical and optical properties of noble metal nanoparticles have attracted considerable scientific interest for many decades. over a century ago, for example, mie building on even earlier work by lorenz and possibly others attributed the colors of colloidal suspensions of au nanoparticles to the nanoparticles visible - wavelength optical scattering properties. interest in the optical properties of noble metal nanoparticles has risen dramatically in recent years with the recognition that these properties, if understood in sufficient detail, can be harnessed to create nanoscale photonic devices and sensors. the discrete dipole approximation (also called the coupled dipole approximation) is one of several numerical methods that have been developed to simulate the response of a small particle to an incident electromagnetic (em) field. in simulations based on the discrete dipole approximation (dda), a nanoparticle is modeled as a regular (typically cubic) lattice of polarizable subunits. the incident em field induces dipole moments in each subunit ; these dipole moments in turn generate local fields that further polarize nearby subunits. once the subunits induced dipole moments are mutually self - consistent, the electromagnetic and optical properties of the dipole lattice are taken to mimic those of the real nanoparticle. the assumption that only dipolar interactions among subunits and between the subunits and the external field need be considered, an assumption that is implicit in dda - based simulations, is generally thought to be a reasonable one provided that the subunits are small enough so that the electric field is nearly constant across an individual subunit ; this assumption is frequently tested by comparing the results obtained from simulations at two or more levels of discretization. the connection between the lattice of dipoles and the real nanoparticle is made through the choice of the polarizability tensor of the polarizable subunits. in the original formulation of the dda approach, was assumed to be an isotropic, diagonal tensor defined by the clausius mossotti (cm) relation where is the number density of the polarizable subunits and is the nanoparticle s dielectric constant ; this relation is exact for an infinite cubic lattice of subunits in a zero - frequency external electric field. for finite (nonzero) frequency external em fields, a radiative reaction correction to the zero - frequency polarizability tensor defined by eq. 1 can be derived from an analysis of the dispersion relation for electromagnetic waves propagating along a lattice of polarizable points. real nanoparticles, of course, have surfaces, and hence can not be represented as infinite lattices ; consequently, the use of polarizabilities defined by eq. 1 in dda - based simulations of nanoparticles represents an additional approximation, one which persists even when the subunits are very small, which is not remediated by radiative reaction corrections or other finite frequency corrections, and which seems to be especially severe for materials whose dielectric constant has a large imaginary component. recent work suggests that the use of subunit polarizabilities that properly account for the anisotropic local environment of dda subunits near surfaces can increase substantially the accuracy with which highly averaged far - field quantities, such as absorption and scattering cross - sections, can be computed using dda - based methods. in this letter, we employ these corrected polarizabilities in dda - based simulations of nanoscale ag prolate spheroids in homogeneous static electric fields ; we find that the new polarizabilities, which include a local environmental correction (lec) to the cm polarizabilities, also substantially improve the description of spatially resolved near - field quantities, such as localized electric field enhancement factors, computed in these simulations. we begin by summarizing some exact results obtained by solving laplace s equation for a homogeneous prolate spheroid in a uniform static external field ; these are the benchmarks against which we assess the dda - based simulations. we consider a prolate spheroid with major semiaxis c (henceforth assumed to coincide with the space - fixed z axis) and minor semiaxis a. the surface of the spheroid is one member of a family of confocal surfaces defined by the parameter. these surfaces satisfy the equation the surface of the spheroid corresponds to = 0. if such a spheroid, with dielectric constant, is immersed in a medium with dielectric constant m and exposed to a uniform static electric field parallel to the space - fixed z axis, the electrical potential at any point outside the spheroid is given by where s = (m)/m and lz () is the dimensionless integral the integral lz(0) can be computed analytically : where the spheroid s eccentricitythe potential inside the spheroid is given by it is clear from this equation that the field inside the spheroid is uniform and parallel to the external field. in addition, the polarization p (dipole moment per unit volume) inside the spheroid is uniform and is given by where 0 is the absolute permittivity of free space. the electric field outside the spheroid isfor points on the z axis, the quantity in square brackets in this equation is the on - axis electric field enhancement factor, which we henceforth denote as f. it has the value ftip = (1 + s)/[1 + slz(0) ] at the spheroid s tip (x, y, z) = (0, 0, c), and approaches f = 1 as z. large ftip values can be achieved when the quantity 1 + slz (0), which is controlled by the spheroid s aspect ratio c / a and dielectric constant, is small in magnitude. the technical aspects of these simulations have been extensively reviewed ; we therefore report only those computational details that are specific to the simulations presented here. we model a spheroid as a collection of n contiguous cubic subunits, with edges of length d, centered at the positions (xyz) = (nxdnydnzd) ; here, (nxnynz) is an integer triple that satisfies where nmax is an integer that determines the discretization level of the spheroid. the edge length d is chosen so that the volume enclosed by the collection of cubic subunits is equal to the spheroid volume. the linear algebraic equations that determine the dipole moments mj of the individual subunits (here j is an index that distinguishes individual subunits) are solved using the complex - arithmetic implementation of the gmres algorithm described by frayss. ; we terminate the algorithm and record the dipole moments mj once the normwise backward error drops below 10. we obtain the wavelength - dependent dielectric function of ag via linear interpolation of the data points compiled by lynch and hunter ; as our main goal in the present work is not to provide results for comparison with experiment, but to compare the accuracy of the results obtained in simulations with and without the local environmental correction to the polarizabilities, we neglect finite - size corrections to the dielectric constant that arise from electronic scattering from the spheroid surface. henceforth, we set m = 1 (corresponding to vacuum as the medium surrounding the spheroid) and e0 = 1 au ; all of the results we report are scaled by 1/e0, so the numerical value of e0 is ultimately irrelevant. first, we examine the polarization p induced in a metallic nanoparticle by a uniform static external electric field. we consider a prolate spheroid with a = 10 nm, c = 40 nm, and dielectric constant = 12.26 + 0.84i (corresponding to an excitation wavelength of 570 nm). for this aspect ratio and dielectric constant, the quantity 1 + slz (0) is purely imaginary and small in magnitude : 1 + slz (0) 0.0633i. we use the dda to simulate this spheroid at several levels of discretization, ranging from n = 6041 subunits (d = 1.405 nm) to n = 24679 subunits (d = 0.879 nm). we divide the dipole moment mj of each subunit by the subunit volume d to obtain the polarization pj for each subunit ; we then divide the magnitude of this vector by the magnitude of the exact polarization vector defined in eq. 7 to obtain a dimensionless relative polarizationfor each subunit. this quantity has the valuewhen the magnitude of a subunit s dipole moment mj is consistent with the exact uniform polarization given by eq. shows how the mean and standard deviation ofevaluated over the n subunits in a given spheroid, depend on the subunit edge length d. we see that for all of the spheroids considered here, the meanvalue differs considerably from the value. for each spheroid, the standard deviation of thevalues is about 0.2, indicating that the polarization within the spheroid is rather nonuniform in contrast to the exact result given by eq. 7and does not become more uniform as the subunits become smaller ; for two of the spheroids, fig. 2 depicts graphically the large subunit - to - subunit variations inthat are observed using cm polarizabilities. by comparison, when we use the subunit polarizabilities of that include the lec, our dda - based simulations produce subunit dipole moments that give for each subunit in the spheroid, indicating that the magnitudes of the dipole moments are in exact agreement with eq. this is not much of a surprise, because the corrected polarizabilities given by rahmani. are defined so that, when used in dda - based simulations, they reproduce exactly the position - dependent polarization inside an object immersed in a static external electric field. what figs. 1 and 2 show is that dda - based simulations that use cm polarizabilities may fail in this regard, and that this failure is not simply a result of the discretization that necessarily accompanies the dda. mean (boxes) and sd (circles) of the relative subunit polarizationsas a function of subunit edge length d, for dda - based simulations of a prolate ag spheroid with semiaxes a = 10 nm and c = 40 nm ; the simulations employ uncorrected cm polarizabilities relative polarizationsderived from dda - based simulations of a prolate ag spheroid with semiaxes a = 10 nm and c = 40 nm ; the simulations employ uncorrected cm polarizabilities. each subunit is represented by a square colored according to the relative polarization scale shown at the bottom of the figure. the upper panel gives the results for a spheroid modeled using n = 6041 subunits of edge length d = 1.405 nm ; the lower panel gives the results for n = 24679 subunits of edge length d = 0.879 nm. only subunits with x = 0, y 0, and z 0 are shown we now examine the near - field properties of the ag nanoparticle, focusing on the localized enhancement of the applied electric field near the nanoparticle s surface. figure 3a compares the magnitude |f| of the exact on - axis electric field enhancement factor f [which is a complex quantity because is complex ; see eq. 8 ] near the spheroid s sharp tip with the dda - based results obtained for n = 24679 subunits using both cm and lec polarizabilities. the enhancement factor computed using lec polarizabilities is in good agreement with the exact result, even at points within 0.4 nm (which is less than one - half of the subunit separation d) of the spheroid s surface (the electric field varies discontinuously across the spheroid s surface, and no dda - based simulation will be able to model this discontinuous change ; it is therefore unreasonable to expect these simulations to give accurate |f| values just outside the spheroid s surface). by contrast, the simulation that employs cm polarizabilities substantially underestimates |f|. in fig. 3b, we show how the values of |f| computed at z = 41 nm (1 nm away from the sharp tip) vary with d over the range of discretizations considered here ; although the |f| values computed using cm polarizabilities vary slightly as d decreases, it appears that very small subunits will be needed before the cm result approaches the exact one. on the other hand, the |f| values computed using lec polarizabilities are within a few percent of the exact result at all levels of discretization. magnitude of f, the on - axis electric field enhancement factor, for a prolate ag spheroid with semiaxes a = 10 nm and c = 40 nm. a dependence of |f| on position z ; the point z = 40 nm is at the spheroid s sharp tip. 8 ; boxes and circles give the results of dda - based simulations with n = 24679 subunits employing lec and cm polarizabilities, respectively. b dependence of |f| at z = 41 nm on the edge length d of the dda subunits. 8 ; boxes and circles give the results of dda - based simulations employing lec and cm polarizabilities, respectively to gain more insight into the relative performance of dda - based simulations employing cm and lec polarizabilities, we use the simulations to compute the electric field enhancement factor in the vicinity of the spheroid s sharp tip, and compare these enhancement factors to reference results obtained by numerically differentiating the exact electrical potential out defined in eq. 3. to partially mitigate the discretization effects that are inherent in dda - based simulations, we rotationally average the field enhancement factor obtained from these simulations by computing it on ten evenly spaced dihedral planes containing the space - fixed z axis and then averaging the enhancement factors obtained for each dihedral plane. fig. 4 shows, for n = 24679 subunits, how the magnitudes of the enhancement factors computed using dda - based simulations compare with the reference results derived from eq. although neither of the dda - based simulations can predict accurately the field enhancement factors at the surface of the spheroid (because the electric field varies discontinuously across the spheroid s surface, as previously noted), the shape, size, and internal structure of the spheroid s near - field hot spot are modeled fairly well by the simulations that employ lec polarizabilities. the dda - based simulations that employ cm polarizabilities, by contrast, yield a hot spot that is too small and whose peak intensity is too low. magnitude of the electric field enhancement factor near the sharp tip of a prolate ag spheroid with semiaxes a = 10 nm and c = 40 nm.. 3 ; cm and lec indicate the results of dda - based simulations employing cm and lec polarizabilities, respectively. the dda - based simulations use n = 24679 subunits of edge length d = 0.879 nm to model the spheroid ; the colored squares in the legend at the bottom of the figure are scaled to have the same edge length in summary, we have modeled the response of a nanoscale ag prolate spheroid to an external electric field using dda - based simulations that employ subunit polarizabilities that either include or omit a local environmental correction. we invoke the electrostatic approximation, in which the incident field is assumed to be spatially uniform and static, but the spheroid s dielectric constants is taken from the wavelength - dependent dielectric function of bulk ag ; this allows us to compare the predictions of the dda - based simulations to exact results obtained by solving laplace s equation for prolate spheroids in a uniform static external field. we have chosen a dielectric constant for the spheroid that maximizes the electric field enhancement factor at the spheroid s sharp tip. the predictions of dda - based simulations that employ lec polarizabilities are much closer to the exact results than are those of dda - based simulations that employ cm polarizabilities ; simulations using cm polarizabilities yield a near - field hot spot that is too small and field enhancement factors that are too low. we therefore conclude that dda - based simulations of metallic nanoparticles that employ uncorrected cm polarizabilities may give inaccurate predictions of the particle s spatially resolved near - field properties, even at locations some distance away from the particle s surface. this work was supported by grants from the university of tennessee honors program (a.e.d.) and the us department of energy (r.j.h.). r.j.h. thanks l. blocker, l. dixon, and e. read (university of tennessee libraries) for bibliographic assistance. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. | we model the response of nanoscale ag prolate spheroids to an external uniform static electric field using simulations based on the discrete dipole approximation, in which the spheroid is represented as a collection of polarizable subunits. we compare the results of simulations that employ subunit polarizabilities derived from the clausius mossotti relation with those of simulations that employ polarizabilities that include a local environmental correction for subunits near the spheroid s surface [rahmani. opt lett 27 : 2118 (2002) ]. the simulations that employ corrected polarizabilities give predictions in very good agreement with exact results obtained by solving laplace s equation. in contrast, simulations that employ uncorrected clausius mossotti polarizabilities substantially underestimate the extent of the electric field hot spot near the spheroid s sharp tip, and give predictions for the field enhancement factor near the tip that are 30 to 50% too small. |
dislocation remains one of the most common complications after primary and especially revision hip arthroplasty. the rate of dislocation is influenced by many different factors and ranges between 0.3 and 10% in primary arthroplasty [15 ] and between 4 and 28% after revision arthroplasty [14 ]. the incidence varies greatly in different studies with a much higher risk for patients with neuromuscular disease or lack of compliance resulting from dementia or substance abuse [1, 2 ]. however, the rate of recurring dislocations has also been associated with surgical approach (including soft tissue repair), surgical volume, and choice of implant. many different methods have been used to solve the problem, both nonoperative and surgical methods. surgically by repositioning malpositioned components, inserting jumbo or bipolar heads, or longer necks and last but not least by using a constrained liner [13 ]. the use of different kinds / brands of constrained liners has been reported with mixed results. we report on the use of the trilogy constrained liner (trilogy and trilogy longevity) in a consecutive series of patients operated on because of recurrent dislocations. we performed a retrospective review of all patients treated with a trilogy constrained liner in the hip clinic hrsholm hospital, denmark, in the period 20052009. the cohort comprised 38 patients all treated with a constrained acetabular insert because of recurrent dislocations (average 4.6 dislocations ; range (110)), one was treated twice. all these patients were reviewed and the ones who were revised using a constrained liner were included in the present study. of these patients, a number had to be excluded. five patients were fitted with another brand of constrained liner and one emigrated and was lost to followup. that left 32 patients all with bimetric femoral components and the trilogy acetabular cup. the constrained liners used were either trilogy (tc) (21 hips) or trilogy longevity (tl) (12 hips) (see figures 1 and 2). the 32 patients included 24 females and 8 males, 20 right hips (1 hip counts twice), and 13 left hips. the primary diagnosis in the majority of cases was arthrosis (88%) ; the rest had avascular necrosis following internal fixation of a femoral neck fracture (12%). (for data on the groups divided by type of liner, see table 1). in addition to the hip problems, 3 patients suffered from dementia and 2 had an ongoing problem with alcohol abuse. no patients with neuromuscular disease the patients were all operated through the posterolateral approach by one of the department 's 4 senior surgeons. during surgery the patients were mobilized using standard precautions (no adduction, no inward rotation, and no flexion past 90 degrees for the first 3 months). at the time of followup, 7 patients had died. three further patients had had additional surgery and had their constrained liner removed for different reasons (they were included in the study but not seen at followup). the rest of the patients were invited to a clinical examination including radiographs of the relevant hip. the radiographs were evaluated for loosening defined as migration of the components or progressive radiolucent lines. the average followup was 26 and 15 months for the trilogy (range 263) and trilogy longevity (range 426), respectively. of the patients still alive, 3 were not seen for followup due to other health issues and personal obligations. the first two patients were interviewed over the phone ; the third patient was not available for interviewing. twenty - three patients had only one previous surgical procedure (tha), 8 had been operated twice, and a single patient had 3 previous procedures. in addition to the primary tha 's, the previous procedures were osteosynthesis of a femoral neck fracture (13%), revisions to treat dislocations (repositioning of cup and lengthening of the neck) (13%), one periprosthetic fracture (3%), one revision because of infection (3%), and one patient had the constrained liner replaced because of failure and another constrained liner inserted (3%) (for data on the groups divided by type of liner see tables 2 and 3). four patients had suffered from one to four further dislocations (12%), three patients with a trilogy constrained liner, and 1 patient with a trilogy longevity constrained liner. one patient had the acetabular shell revised because of loosening (tc), and 2 other patients had debridement with head and liner change because of infection. thus a total of 7 (21%) patients had 15 additional procedures including closed reductions. 18 patients were seen for the followup including radiographs and one only for additional radiographs. no migration was seen in any patients ; one patient had a slight progression of radiolucent lines at the femoral component. one patient had a small cyst in the lateral part of the acetabulum suggesting osteolysis. the patients who for various reasons were not seen for the present followup had been seen previously 215 months postoperatively. apart from the already noted failures no migration or progression of radiolucent lines was found. the patients that were seen for followup had an average harris hip score (hhs) of 81. in the group of patients who suffered further dislocations despite the constrained liner, one suffered from dementia and one had a problem with alcohol abuse. no difference between the tc liner and the tl in any of parameters investigated could be demonstrated. the number of different constrained liners on the market is abundant : bipolar as well as tripolar [7, 8 ] (to match the even larger number of different shells, head, and stem types). published data is currently only available for a few of the constrained liners : osteonics and omnifit (osteonics corp./stryker howmedica) [921 ], trident (stryker) [22, 23 ], s - rom (depuy) [2426 ], duraloc (depuy) [27, 28 ], ringloc (biomet) [20, 29 ], and trilogy (zimmer) [20, 28, 29 ]. some of the studies include patients with more than one brand of liner [20, 24, 25, 28, 29 ]. in addition to different kinds of liners, the indication for insertion of a constrained liner differs as well. we inserted constrained liners as a measure to prevent further dislocations in patients with repeated dislocations, and the constrained liner has not been used in primary surgery in the present series. some of the published papers deal with a similar material [13, 14, 17, 22, 29 ], the rest of the published data are on a more diverse group of patients with indications varying from repeated dislocations or intraoperative instability (both primary procedures and revisions) to neurologic impairment and revision procedures on patients with girdlestone status [911, 15, 16, 1821, 2325, 27, 28 ]. as the type of constrained insert (as well as how it is inserted), indication, surgical approach, number of patients, and follow - up time are widely different, so are the results. (110 hips), mccarthy and lee (39 hips), and stanton. (13 hips) had no redislocations at followup after 3.2, 4.6, and 3.5 years, respectively, although 2 cases of subluxation (2%) have been noted. anderson. (21 hips), pattyn. (46 hips), and berend. (755 hips) published a dislocation rate of 29%, 21.7%, and 17.5% at a followup of 2.5, 2.4, and 10.7 years, respectively. looking at studies of patients with recurring dislocations (including dissociations), the success rate also varies. (40 hips) found redislocation rates of 5.3%, 2.3%, 2.9%, and 7.5% with a followup between 2 and 7 years. this includes not only the dislocations and dissociations, and the breakage of different parts of the liner, but also shells that are pulled out because of increased stress, and revisions because of loosening of the acetabular shell. failure rates of 5.3%, 5.8%, 14.7%, and 10% have been described [14, 17, 22, 29 ]. in our material in addition to the dislocations, one locking ring failed (without dislocation), and one acetabular shell was loose. no other failures were registered, probably because the constrained liner only had been used in well - fixed acetabular shells. the rate of redislocation was 12% and the total failure rate (including loosening of the acetabular shell and loosening of the ring) was 18%. the constrained liner has in the present material been used only as a salvage procedure in a population of patients with recurrent dislocations, which might explain the rather high rate of failure. furthermore, patients with conditions predisposing to hip - dislocation also seemed to be at a higher risk for subsequent failure of the constrained liner. however, 88% of the patients had no further dislocations or loosening of the implant, and no better solution in this group of patients seems available at present. we continue to use this method as a salvage measure for patients with recurring dislocations and well - positioned and well - fixed components. | 32 patients received a trilogy- or trilogy - longevity - constrained acetabular liner for recurrent dislocations after total hip replacement. the constrained liner was inserted into a well - fixed trilogy acetabular shell with snap fit. at 1.8-year followup (range 363 months), 4 patients had suffered further dislocation(s) (12%), and one patient had revision surgery for a loosened acetabular shell. radiologic evaluation detected no definitively loose components, but one patient with progressing radiolucent lines around the femoral component and one patient with an acetabular cyst were found, as well as a patient with a loose locking ring (but otherwise no failure). the nineteen patients who were available for the present followup had a mean harris hip score of 81. the constrained liner is an effective method of dealing with recurrent dislocations in well - fixed components. |
herpes simplex virus type-1 (hsv-1) is a ubiquitous pathogen associated with facial cold sores [1, 2 ], but severe complications especially in neonates and immunocompromised patients may result in corneal blindness, retinitis, and encephalitis [3, 4 ]. the virus can also cause severe opportunistic infections in the patients following allogeneic stem cell transplant [58 ]. such infections could be donor derived or due to the reactivation of an endogenous latent virus. in addition, the drug - resistant hsv-1 is also an emerging concern to promote viral infections after allogeneic stem cell transplantation. it has been proposed that human mesenchymal stem cells (hmscs) may be susceptible to infection if infused in patients with hsv-1 viremia and may compromise the host 's defense against infectious agents [7, 11 ]. hsv-1 infects host cells through initial attachment to cell surface heparan sulfate (hs) followed by fusion of the virion envelope with the plasma membrane of the host cells [12, 13 ]. the current model suggests that entry of virus requires four hsv glycoproteins (gb, gd, gh, and gl) [1419 ] and at least one cellular receptor for glycoprotein d (gd) [2022 ]. the receptors for hsv-1 gd include a member of the tnf - receptor family named hvem [23, 24 ], a member of the immunoglobulin superfamily commonly known as nectin-1 [25, 26 ] and 3-o sulfated heparan sulfate (3-os hs). the later form of hs is enzymatically modified by multiple d - glucosaminyl 3-o - sulfotransferase (3ost) isoforms. among the known 3-osts isoforms all mediate hsv-1 entry [2731 ] and cell - to - cell fusion [22, 32 ] except 3-ost-1 isoform. although hsv-1 infections lead to significant morbidity and mortality in transplant recipient [6, 10, 11 ], the susceptibility of human mesenchymal stems cells (hmscs) to hsv-1 entry and key entry mediator that allows hsv-1 infection is poorly understood. in this study, we investigated the hsv-1 infection of hmscs and the role of hsv-1 entry receptors in this process. the finding that hsv-1 can induce cytopathic effect and productively replicate in hmscs in high titer provides new information on the susceptible nature of target cell population present within the host. we also demonstrated that hs and modified form of hs (3-o - sulfated heparan sulfate ; 3-os hs) is a major determinant of hsv-1 entry and spread into hmscs. wild - type chinese hamster ovarian - k1 (chok1) cells were grown in ham 's f-12 medium (gibco / brl, carlsbad, calif, usa) supplemented with 10% fetal bovine serum (fbs, invitrogen), and penicillin and streptomycin (gibco / brl). hela cells were grown in dmem media supplemented with 10% fetal bovine serum (invitrogen), and penicillin and streptomycin (gibco / brl). human mesenchymal stem cells (hmscs) were purchased from fisher scientific (sv3010201) grown in hyclone advance stem expansion media (fisher scientific, sh30875kt). the hsv-1 (kos) glycoprotein expressing plasmids used were ppep98 (gb), ppep99 (gd), ppep100 (gh), and ppep101 (gl). other plasmids used in this study include pcalifgt7 (t7 rna polymerase) and pt7emcluc (luciferase gene). cells (hmscs, hela) were plated at 2 10 per well in 96-well plates at least 16 h prior to infection. after 6-h postinfection, -galactosidase assays were performed using a soluble substrate o - nitrophenyl--d - galactopyranoside (onpg at 3.0 mg / ml) assay. the enzymatic activity was measured at 410 nm using a microplate reader (biotek instruments inc. viral entry was further confirmed by 5-bromo-4-chloro-3-indolyl--d - galactopyranoside (x - gal) staining. after 6 hr of infection with the reporter virus, the cells were washed with pbs and fixed with 2% formaldehyde and 0.2% glutaradehyde at room temperature for 15 min. the cells were then washed with pbs and permeabilized with 2 mm mgcl2, 0.01% deoxycholate and 0.02% nonidet np-40 for 15 min. after rinsing with pbs, 1.5 ml of 1.0 mg / ml x - gal in ferricyanide buffer was added to each well, and the blue color developed in the cells was examined. cultured monolayers of hmscs were grown overnight in media containing 10% fbs on chamber slides (lab - tek). the cells were either mock infected or infected with green fluorescent protein (gfp)-tagged hsv-1 (vp26-gfp) at 50 pfu / cell in serum - free media optimem (invitrogen). the gfp was fused with the hsv-1 ul35 open reading frame (orf) which encodes a 12-kda capsid protein designated vp26. vp26 is located on the outer surface of the capsid, specifically on the tips of the hexons that constitute the capsid shell. at 90 min post - infection, cells were fixed and stained with 10 nm rhodamine - conjugated phalloidin for f - actin. images of immunofluorescent labeled cells were acquired using a olympus ix50 inverted fluorescence microscope. in order to demonstrate the significance of actin cytoskeleton network during hsv-1 entry into hmscs, monolayer cultures of hmscs (approximately 10 cells) cultured in a 96-well plate were pretreated with the indicated concentrations of agents for 1 hr at room temperature with cytochalasin d (cyto d) and latrunculin b (lat b) (sigma) or mock - treated as a control. cells were infected with the lacz hsv-1 kos (gl86) at 50 pfu / cell for 6 h at 37c. onpg entry assay was performed to estimate the enzymatic activity at 410 nm by spectrophotometer. confluent layers of hmscs (approximately 10) in six - well dishes were infected with hsv-1 (804) strain at 0.01 pfu / cell or mock infected for 2 hrs at 37c. after removal of inoculum, monolayers were overlaid with dmem containing 2.5% heat inactivated calf serum and incubated at 37c. at different time points : 0, 12, 24, and 36 hr, the cells were fixed by using fixative buffer at room temperature for 20 min, followed by giemsa staining for 45 min. the cells were again washed five times in pbs, and the numbers of plaques were counted. a monolayer of cultured hmscs (approximately 4 10 cells per 25-ml flask) was infected (moi, 0.01) with hsv-1(kos) or mock infected with pbs alone for 2 h at 37c. vero cells mock infected of infected with the hsv-1 kos under similar conditions were used as negative and positive controls, respectively. after removal of the inoculum, monolayers were overlaid with dmem containing 2.5% heat - inactivated calf serum and incubated at 37c until the time of harvest (12 to 48 h). infectious virus titers were determined on vero cells cultured in triplicate by using an overlay of medium containing methylcellulose. in order to block secondary plaque formation the cells were washed with pbs buffer, fixed in alcohol, and stained with giemsa stain. virus attachment was determined by using olympus ix50 inverted fluorescence microscope cultured hmscs were transfected with lipofectamine 2000 (invitrogen) with an hsv-1 gd expression plasmid (ppep99) or a control plasmid (pcdna3) in six - well dishes (1.0 g of plasmid dna total per well). the interference assay was performed as described previously. total rna was isolated from cultured hmscs using a qiagen rneasy kit with dnaase in column treatment (qiagen corp., superscript ii reverse transcriptase (invitrogen corp.) was used for rt to generate cdnas. pcr amplification of cdnas was done with primers 5-ggagacaataccctcattca-3 and 5-tatttacatcctcccacagc-3 for hvem, and primers 5-tccttcaccgatggcactatcc-3 and 5-tcaacaccagcaggatgctc-3 for nectin-1. the 3-ost-3 sequences were amplified with primers 5-caggccatcatcatcgg-3 and 5-ccggtcatctggtagaa-3. pcr was performed using thermostart dna polymerase (fisher cat no. ab0908) under the following conditions : 95c 30, 52c 30, 72c 30, and 32 cycles for hvem ; 95c 1, 62c 1, 72c 2, and 35 cycles for nectin-1 ; 95c 1, 55c 1, 72c 2, and 32 cycles for 3-ost. the expected sizes of the pcr products were 582 bps for hvem, 738 bps for nectin-1, and 736 bps for 3-ost-3. the products were separated by electrophoresis on an agarose gel soaked in gel green solution and imaged using bio - rad molecular imager gel doc xr+ system. hmscs plated in 96-well plate were preincubated at room temperature with twofold dilutions of anti - hvem, anti - nectin-1 antibody (prr1), and a control antibody (-tgfbr ii) for 90 min. cells were then challenged with identical doses of hsv-1 (gl86) at 5 10 pfu per well at 37c. after 6 hours, the cells were washed twice with pbs and treated for 1 min with 0.1 m citrate buffer (ph 3.0). the substrate, immunopure onpg was prepared in pbs buffer with nonionic detergent, and -galactosidase activity was read at 410 nm. cultured hmscs plated on 96-well plates were washed with mg- and ca - free pbs and incubated at room temperature for 90 min with heparinase i (1.5 u ml ; sigma) or pbs alone and incubated at room temperature. the cells were then washed with pbs and used for hsv-1 entry. to detect the binding of hsv-1 to the surface of hmscs, cultured monolayers of cells (approximately 10) were grown overnight on glass bottom culture dishes (mat tek corp.). the cells were then treated with heparinases i (1.5 u ml) or with pbs buffer for 45 min, exposed to gfp - tagged hsv-1 k26gfp for 30 min at 4c, and fixed with acetone. the cells were then stained with phalloidin (f - actin) and mounted on glass slides before examination under an olympus ix50 inverted fluorescence microscope. cultured hmscs were plated overnight into 96-well dishes (approximately 4 10 cells / well). they were treated with heparinases i or with pbs buffer as described above for 2 h. this was followed by incubation with gd - fc (1 g / ml) for 1 h and fixation at room temperature with 2% formaldehyde and 0.02% glutaraldehyde for 15 min. the cells were washed with pbs containing 3% bsa (sigma) and then incubated at room temperature sequentially with a biotinylated secondary antibody against rabbit igg (sigma) at a 1 : 500 dilution in pbs-3% bsa for 45 min and amdex streptavidin - conjugated horseradish peroxidase (amersham) at a 1 : 20,000 dilution for 30 min. following washing in pbs, the cells were incubated with 50 l 3,3,5,5-tetramethylbenzidine (sigma) in 50 mm phosphate - citrate buffer. el808 absorbance microplate reader, vt, usa) to measure optical density at 650 nm (od650). in this experiment, the cho - k1 cells designated effector cells were cotransfected with plasmids expressing four hsv-1(kos) glycoproteins : ppep98 (gb), ppep99 (gd), ppep100 (gh), and ppep101 (gl), along with plasmid pt7emcluc, which expresses the firefly luciferase gene under the control of the t7 promoter. cultured hmscs considered target cells were cotransfected with pcalifgt7, which expresses t7 rna polymerase with the chicken actin promoter and the cmv enhancer. effector cells expressing pt7emcluc and pcdna3 (devoid of any glycoproteins) and target hmscs transfected with t7 rna polymerase alone were used as negative controls. in either case, the total amount of dna used for transfection was kept constant. after 16 h, target and effector cells were mixed in a 1 : 1 ratio and then replated in 24-well dishes. activation of the reporter luciferase gene, as a measurement of cell fusion, was examined by reporter lysis assay (promega) at 24 h postmixing by using a sirius luminometer (berthold detection systems). cell fusion was also visualized for multinucleated giant cells or polykaryocytes by microscopy following giemsa (fluka) staining at 24 h postmixing. to determine hsv-1 entry, confluent monolayers of cultured hmscs were infected with serial dilutions of recombinant hsv-1(kos) gl86, which expresses -galactosidase upon entry into cells. hela cells that are naturally susceptible to hsv-1 entry were used as a positive control. entry of hsv-1 was measured after 6 h of viral infection. as shown in figure 1(a), similar to hela cells, hsv-1 entered hmscs in a dose - dependent manner. by contrast, cho - k1 cells without any receptor remained resistant to hsv-1 entry. no -gal activity was observed in mock infected hmscs (figure 1(b), panel a), while a dosage of virus sufficient to infect 100% of hmscs at 10 pfu / cell (figure 1(b), panel (b)) also led to a nearly complete infection of hmscs. in order to obtain more direct and visual evidence of hsv-1 entry, gfp - tagged hsv-1(k26gfp) was used to infect cultured hmscs, and fluorescence microscopy was used to visualize the virions inside the hmscs stained with rhodamine phalloidin, which specifically labels f - actin (figure 1(c), panel (b)). the gfp colocalization with f - actin was seen all over the cell (figure 1(c), panel (b)). this result correlated with the observed susceptibility of hmscs to hsv-1 entry during x - gal assay. our previous findings have shown that hsv-1 entry into natural target cells leads to change in actin cytoskeleton [3638 ]. we therefore decided to examine whether cytoskeleton changes played any significant role in hsv-1 entry into hmscs. to address this issue, fluorescent microscopy of hmscs exposed to gfp - tagged hsv-1 virions at 30 quite noticeably, some gfp - tagged virus particles were found to attach at filopodia - like structures in rhodamine - phalloidin stained hmscs (figures 2(a) and 2(b)). in order to demonstrate the significance of actin network and filopodia during hsv-1 entry into hmscs, we used the actin inhibitors latrunculin b (lat b) or cytochalasin d (cyto d). both agents prevent cytoskeleton changes by blocking actin polymerization [39, 40 ]. as shown in figures 2(c) and 2(d), pretreatment of hmscs with lat b and cyto d resulted in significant inhibition of hsv-1 entry, suggesting that cytoskeleton modification may be needed during the initial phase of hsv-1 infection. because hsv-1 was able to enter cultured hmscs, we next evaluated whether this entry led to a productive virus replication. initially, microscopy was used to obtain visual evidence of hsv-1 replication. syncytial plaque forming hsv-1 (kos) 804 virus was used for infecting cultured hmscs, and the virus was allowed to replicate. cells were fixed at different time points and giemsa stained. the cytopathic effect in the form of plaque formation increased significantly overtime in virus - infected hmscs as seen in figures 3(a) and 3(b). furthermore, to assess viral replication, the infectious yields of virus were determined by plaque assays with vero cells. as shown in figure 3(c), inoculum harvested from infected hmscs produced a larger number of plaques overtime. in contrast, cho - k1 cells infected with identical doses of the same virus failed to produce infectious virions. these results, together with those of the entry assay, show that entry of hsv-1 into cultured hmscs leads to productive infection. in order to determine if the gd receptors play a role in human hmscs infection by hsv-1, a gd - mediated interference assay was used. this assay is based on the principle that cells that are normally susceptible to viral entry become resistant upon expression of viral gd because of sequestration of gd receptors by cell - expressed gd. to carry out the assay, hmscs were transiently transfected with an hsv-1 gd - expressing plasmid (or an equal amount of the empty vector, pcdna3, as a control), followed by infection with serial dilutions of -galactosidase - expressing hsv-1(kos) gl86. as shown in figure 4, hsv-1 entry into gd - expressing hmscs thus, entry into hmscs is likely mediated by gd receptors expressed on the cell surface in a gd - dependent manner. reverse transcriptase (rt)-pcr analysis was performed to determine the identity of gd receptors potentially expressed in hmscs. shown in figure 5(a), products of the expected sizes for cdnas encoding nectin-1, 3-ost-3 and hvem were all detected, indicating that all three genes are normally expressed in hmscs. to determine which receptor were important for hsv-1 entry into hmscs, previously established receptor specific antibodies were used [33, 36 ]. as shown in figure 5(b), antibody against nectin-1 affected hsv-1 entry by 20%, while hvem had no significant effect on hsv-1 entry suggesting that neither of these receptors plays a critical role in mediating hsv-1 infection of hmscs. expression of 3-ost-3 raised the possibility that 3-os hs could be a mediator of hsv-1 entry into hmscs. since cells that use 3-os hs as the major entry receptor become significantly more resistant to hsv-1 upon removal of hs from the surface [37, 38 ], we examined the effect of hs - degrading enzyme heparinase - i (1.5 u ml) on hsv-1k26gfp binding and entry into hmscs. this enzyme is known to cleave off both heparin and hs chains containing 14 linkages between glucosamine and o - sulfated iduronic acid residues. as shown in figure 5(c), heparinase - treated hmscs (h+) were resistant to hsv-1 binding (panel b) compared to the mock - treated cells (h-, panel a). similar resistance to hsv-1 kos gl86 entry was also observed as a result of heparinase treatment (figure 5(d)). next, we tested whether a soluble recombinant form of hsv-1 gd or gd - fc [29, 35 ] would bind cultured hmscs and whether this binding could be adversely affected by removal of hs from the surface using cell enzyme - linked immunosorbent assay [26, 28, 33 ]. cells either treated with heparinases or mock - treated were allowed to bind equal amount of gd, and binding was detected with a spectrophotometer. figure 5(e) shows that gd binding to mock - treated hmscs was not affected ; however, the binding of gd to heparinase - treated hmscs was reduced by about 75%. this finding strongly supports that gd - binding receptors were specifically removed upon heparinase treatment in hmscs, indicating that hs and 3-ost-3 play critical roles in mediating hsv-1 entry through gd binding to cultured hmscs. to further verify that 3-os hs is indeed the mediator of the membrane fusion that occurs during hsv-1 entry into hmscs, we used a quantitative and efficient cell - to - cell fusion assay [14, 22 ]. it has been shown that, similar to the membrane fusion that occurs during entry, cells expressing hsv-1 glycoproteins and gl fuse with cells expressing gd receptors, and thus cell - to - cell fusion mimics the minimum requirement for entry. first, we studied the significance of hs in the fusion of hmscs with hsv-1 glycoproteins expressing effector cho - k1 cells. hs dependence is a way to determine whether 3-os hs is the principal mediator of membrane fusion since, unlike nectin-1 and hvem, which do not require hs for membrane fusion, 3-os hs - mediated cell fusion is highly dependent on the expression of hs on the cell surface. as expected, fusion of hmscs with effector cells expressing the hsv-1 glycoproteins was significantly reduced upon heparinase treatment (figure 6(a)), and a similar result was seen with cho - k1 expressing 3-ost-3 (data not shown), suggesting that expression of hs and 3-os hs in hmscs are required for effective fusion with glycoprotein expressing - cho - k1 cells. in a separate set of experiments, heparinase treatment also reduced multinucleated giant cell formation or polykaryons in hmscs cocultured with effector cho - k1 cells expressing hsv-1 glycoproteins (figures 6(b) and 6(c)). taken together, our results demonstrate that hs and 3-os hs are required for hsv-1 glycoprotein - induced cell to cell fusion in cultured hmscs, further implying their significant role in mediating hsv-1 entry and spread into hmscs. understanding viral entry into hmscs in a healthy donor or in a recipient host is important for two reasons. first, productive viral infection into hmscs may trigger immune response during or after stem cell transplant and, second, the infected population of hmscs may become large reservoir of virions which may constantly expand both immune response and the viral transmission to the localized cells or tissues or may find the new host cells and tissues in the recipient. therefore, viral receptor on these cells could determine the type of the infection, nature of immune response, and viral transmission. we cultured hmscs derived from the adipose tissue of healthy donors to develop an in vitro model for studies of hsv-1 entry and replication and for identification of cellular mediators of entry. although all the three hsv-1 gd - receptors (nectin-1, 3-ost-3 and hvem) were detected, however, antibodies against nectin-1 and hvem failed to block significantly hsv-1 entry (figures 5(a) and 5(b)). adding further strength to this possibility, heparinase enzymes completely abolished both hsv-1 attachment (figure 5(c)) and entry into cultured hmscs (figure 5(d)). in addition, the gd binding exhibited sensitivity to enzyme treatment (figure 5(e)) and the effect of heparinase to viral spread during cell to cell fusion assay was also significantly inhibited in cultured hmscs (figure 6). similar effect of heparinase treatment on hsv-1 entry and fusion has been shown in primary cultures of corneal fibroblasts obtained from human eye donors or in the cells exclusively expressing 3-ost isoforms [28, 29, 33 ]. taken together, we speculate one interesting possibility that although all the three known gd - receptors are expressed in hmscs but hsv-1 preferentially uses hs/3-os - hs to gain entry could be related for two main reasons. first hs and 3-os hs being larger in size second they are also easily accessible to hsv-1 unlike nectin-1 which is localized in cell junctions. this is also possible that nectin-1 and hvem receptors are involved in other complex interactions that do not support hsv-1 entry into hmscs. in summary, we conclude hs and 3-os hs play a key role during hsv-1 infection into hmscs. the finding provides new information on susceptible nature of hmscs population within the host cell and contributes the broader significance of hs and 3-os hs receptor in hsv-1 infection. because hs also play a key role in signaling and immune activation, it is possible that they might play a dual role allowing both hsv-1 infection and facilitating the inflammation to further damage hmscs. this unique feature of hs/3-os hs in hmscs can be exploited to develop the agent that blocks hsv-1 gd interaction with 3-os hs receptor to suppress both viral entry / spread and immune response. finally, hmscs have potential to differentiate into many other cell - types including neurons.. therefore it will be interesting to evaluate the expression pattern of gd - receptors in differentiated stem cells including neuronal stem cells where hsv-1 can easily infect and establish latency. similarly the strategic development to isolate population of stem cell lines that does not support hsv-1 infection may further suppress the hsv-1-induced morbidity and mortality and bring enhanced success of stem cell transplantation. | hematopoietic stem cells recipients remain susceptible to opportunistic viral infections including herpes simplex virus type-1 (hsv-1). the purpose of this investigation was to analyze susceptibility of human mesenchymal stem cells (hmscs) to hsv-1 infection and identify the major entry receptor. productive virus infection in hmscs was confirmed by replication and plaque formation assays using a syncytial hsv-1 kos (804) virus. to examine the significance of entry receptors, rt - pcr and antibody - blocking assays were performed. rt - pcr data showed the expression of gd receptors : nectin-1, 3-o sulfotransferase-3 (3-ost-3), and hvem. antibody - blocking assay together with heparinase treatment suggested an important role for hs and 3-o - sulfated heparan sulfate (3-os hs), but not nectin-1 or hvem, in mediating hsv-1 entry and spread in hmscs. taken together, our results provide strong evidence demonstrating that hsv-1 is capable of infecting hmscs and hs and 3-os hs serve as its entry receptors during this process. |
the alzheimer disease genetics consortium (adgc) performed a genome - wide association study (gwas) of late - disease (load) using a 3 stage design consisting of a discovery stage (stage 1) and two replication stages (stages 2 and 3). both joint and meta - analysis analysis approaches were used. we obtained genome - wide significant results at ms4a4a [rs4938933 ; stages 1 + 2, meta - analysis (pm) = 1.7 109, joint analysis (pj) = 1.7 109 ; stages 13, pm = 8.2 1012 ], cd2ap (rs9349407 ; stages 13, pm = 8.6 109), epha1 (rs11767557 ; stages 13 pm = 6.0 1010), and cd33 (rs3865444 ; stages 13, pm = 1.6 109). we confirmed that cr1 (rs6701713 ; pm = 4.61010, pj = 5.21011), clu (rs1532278 ; pm = 8.3 108, pj = 1.9108), bin1 (rs7561528 ; pm = 4.01014 ; pj = 5.21014), and picalm (rs561655 ; pm = 7.0 1011, pj = 1.01010) but not exoc3l2 are load risk loci13. |
|
fast dissolving drug delivery systems like oral dissolvable films (odfs) and fast disintegrating tablets (fdts) were introduced in the late 1970s as alternative dosage forms for paediatric and geriatric patients who have difficulties in swallowing conventional oral solid dosage forms such as tablets and capsules. odfs are presented as thin flexible solid dosage forms and come in different sizes and shapes. odfs are administered by placing the dosage form on the tongue where they hydrate and break up releasing the active agent into the buccal cavity for oromucosal absorption or swallowing. odfs emerged from the confectionery and oral care department in the form of breath strips and soon grew into a contemporary and widely accepted dosage form by end users for delivering vitamins and personal care products. they are composed basically of the active pharmaceutical ingredient (api), hydrophilic polymers, plasticizers, sweeteners, flavours, colours, surfactants, saliva stimulating agents, and so forth [4, 5 ]. odfs are easy to transport and offer fast, safe, and accurate dosing without the need for water or any measuring device. also, dysphagic, schizophrenic, and dementia patients are able to use odfs with little or no difficulty.. the main disadvantages of odfs are the difficulty in attaining uniform dosage and achieving high drug loadings in the films. also, there is a challenge with packaging of the films which may demand the use of special packaging machines. many drugs have been formulated as odfs ; these include nsaids, antihistamines, antiulcer drugs, nicotine replacements, antiemetics, antipsychotics, anti - alzheimer drugs, antiepileptic drugs, and drugs for sleeping disorders [911 ]. gums are pathological compounds produced by certain plants as a result of mechanical injury or due to undesirable climatic conditions, such as drought. examples include acacia, tragacanth, albizia, khaya, xanthan, and guar gums. gums find wide applications in the pharmaceutical industry where they are used as viscosity enhancers in some suspensions, as emulsifying agents in stabilizing emulsions, as binding agents in tablets and capsules, and so forth. gums have also shown great potential as gelling agents, film forming agents in odf technology, coating agents in tablet cores intended for colonic drug delivery [14, 15 ], and also vehicles in the fabrication of modified release dosage forms. albizia gum is obtained as natural exudates from the incised trunk of albizia zygia (dc.) j. f. macbr. the plant is widely distributed in tropical africa, and it is found in senegal through to kenya, northern angola, and tanzania. khaya gum is obtained from the incised trunk of khaya grandifoliola cdc, family meliaceae. the tree is found in benin, democratic republic of congo, cte d'ivoire, ghana, togo, nigeria, sudan, guinea, and uganda. albizia and khaya gums have been evaluated as binding agents, thickening agents, nonfunctional film coatings [19, 20 ], and compression coatings for colonic drug delivery. osteoarthritis is the third leading diagnosis in the aged population and causes substantial amount of pain leading to impairment and reduction in the quality of life of patients above 65 years. nsaids such as diclofenac, aceclofenac, ibuprofen, and celecoxib are widely used to alleviate the pain of osteoarthritis. these drugs relieve inflammatory pain and slow down the inflammatory process and its associated tenderness and joint constraints. one of the challenges facing geriatric patients is the difficulty in swallowing conventional tablets which affects medication compliance and hinders good therapeutic outcomes. the formulation of active pharmaceutical ingredients (apis) as odf formats can help to resolve medication noncompliance in the elderly. this study was aimed at developing oral dissolvable films of diclofenac sodium using albizia and khaya gums as natural hydrophilic film forming polymers. diclofenac sodium was received as a gift from trade winds chemists (kumasi, ghana). hydroxypropyl methylcellulose (hpmc) e15 (viscosity of 2% solution at 15 cps) was obtained as a gift from uk chemicals ltd. glycerol, tween 80, and citric acid were obtained from the chemical stores of the departments of pharmaceutics and pharmaceutical chemistry, knust (kumasi, ghana). crude albizia and khaya gums were obtained from forest areas around kwahu in the eastern region of ghana, as natural exudates from the incised trunks of the trees albizia zygia (dc.) j. f. macbr., the gums were authenticated, collected, and supplied by a technician at the department of herbal medicine, faculty of pharmacy and pharmaceutical sciences, knust, ghana. crude albizia and khaya gums were freed from extraneous materials and purified using methods described elsewhere [23, 24 ], with minor modifications. gums were oven - dried at 50c for 48 h and sorted into light and dark coloured grades. eight hundred grams of the light coloured grades was separately milled and hydrated with 2 l double strength chloroform for 48 h, with occasional stirring. the gum mucilage substances were filtered with calico and the filtrates were precipitated with three times their volumes of ethanol, refiltered, and washed with diethyl ether. the resultant gums were oven - dried at 40c for 24 h, milled, and screened through 250 m sieves, packed in airtight plastic pouches, and stored in a desiccator ready for use. the moisture content and insoluble matter in the gums were determined using british pharmacopoeia methods. one gram of the gum was dispersed in sufficient distilled water, with occasional agitation, to form 1% w / v gum mucilage. the ph of the resultant mucilage was determined with a calibrated eutech ph meter (ph 510, ph / mv/c meter, singapore) at 25c. the total ash, acid insoluble ash, and water soluble ash of the gums were determined using official methods. the true density of the gums was determined by liquid displacement method at 25c and calculated as the weight of gum divided by the weight of the liquid it displaces. chloroform was used as the displacement liquid as the gums are practically insoluble in the solvent. the solubility of the purified gums was determined in cold and warm water, acetone, chloroform, and ethanol (96%). one gram of the gum was added to 50 ml of the solvent and left overnight at 25c. twenty - five - milliliter portions of the clear supernatant were placed in preweighed glass petri dishes and evaporated to dryness over a water bath. the mass of the residues was weighed with an analytical balance (adam equipment, uk) and expressed as the percentage solubility of the gum in the solvent. one gram of the gum powder was weighed into a 10 ml measuring cylinder. the initial volume of the gum was noted after which distilled water was added to the 10 ml mark. the cylinder was stoppered, mixed lightly, and allowed to stand for 24 h and the final volume occupied by the gum sediment was noted. the swelling index was calculated as follows : swelling index = (xt xo / xo) 100, where xt is volume of gum after 24 h and xo = initial volume of gum. the charring temperature was determined by the open capillary method using the stuart melting point apparatus (bibby scientific ltd., uk). the tube was packed by pressing the open end gently into a sample of the dry powder gum. the gum was transferred from the open end to the bottom of the tube by gently tapping the bottom on the bench. the sample tube was then inserted into the melting point apparatus and the temperature at which the gum sample changed colour was determined. the viscosity of 5, 10, 20, 30, and 40% gum mucilage was measured with a brookfield viscometer (brookfield engineering laboratories, inc. the effect of temperature on the viscosity of 40% gum mucilage was also studied at 25, 50, 65, 75, and 85c with the brookfield viscometer at a shear rate of 30 rpm. the mineral and toxic ion contents of the gums, namely, iron (fe), copper (cu), zinc (zn), manganese (mn), cadmium (cd), lead (pb), mercury (hg), and arsenic (as), were determined with an atomic absorption spectrophotometer (aas) (buck scientific model 210v gp). the gums were subjected to dry ash digestion and the clear supernatant digest after centrifugation was used for the analysis. the file for the aas analysis and hollow cathode lamps were set as follows : fe at 248.3 nm, cu at 324.8 nm, zn at 213.9 nm, mn at 279.5 nm, cd at 228.9 nm, pb at 283.3 nm, hg at 253.7 nm, and ar at 193.7 nm. a calibration curve was plotted for each of the elements to be analyzed from the stock standards. the stock standard followed by the sample solutions the contents of potassium and sodium in the gums were determined by flame photometry (jenway flame photometer pfp7) at wavelengths of 766 nm and 589 nm, respectively. the content of phosphorus was evaluated using the phosphovanadomolybdate method for colour development and absorbance was determined spectrophotometrically (jenway 6051 colorimeter) at 430 nm. the nitrogen and organic carbon contents of the gums were determined by the kjeldahl method and the modified walkley - black wet oxidation method, respectively. diclofenac sodium odfs were prepared using the solvent casting method [7, 29 ], with minor modifications. the polymers were immersed in half the volume of distilled water overnight to obtain a homogeneous dispersion. the specified amount of glycerol was added to the aqueous dispersion and mixed until being homogeneous. the aqueous solution was allowed to stand for 1 h to take out all entrapped air bubbles. another aqueous solution was made by dissolving the specified amounts of diclofenac sodium, aspartame, titanium dioxide, flavour, tween 80, and citric acid in the remainder of the distilled water. both aqueous solutions were put together, stirred, and sonicated for 30 min. twenty - milliliter portions of the resultant aqueous dispersion were cast onto glass petri dishes of diameter 90 mm and oven - dried at 60c for 24. the dried films were meticulously taken out of the petri dishes, inspected for any imperfections, and cut into 2 cm 2 cm sizes with a scalpel. the cut films were packaged singly in aluminium foils and kept in a desiccator pending assessment. all prepared films were checked for their appearances, whether uniform or not, and for the presence or absence of air bubbles, and so forth. the thickness of five randomly selected 2 cm 2 cm films from each odf formulation was determined using a digital caliper (powerfix, milomex ltd., uk). the measurements were taken along various planes of the films and the mean and standard deviation were calculated. the individual weights of ten randomly selected 2 cm 2 cm films were determined using an analytical balance (adam equipment, uk). the ph of the films was determined by dissolving a 2 cm 2 cm film in 10 ml of distilled water. the ph of the resulting solution was measured using a standardized eutech ph meter (ph 510, ph / mv/c meter, singapore). the mean of five determinations of each film was calculated. a bruker ftir spectrophotometer (alpha - platinum atr, jos hansen & soehne gmbh, hamburg, germany) run by the opus software (version 7.2 build 7.2. 139. 1294) was set to baseline to format previous entries that may interfere with the determination. about 0.1 g of diclofenac sodium was loaded onto the stage directly on top of the platinum. when the setup was ready, the opus software generated the spectrum of the loaded sample on the monitor of a computer. the ftir spectra of both albizia (f4) and khaya (f6) films (each containing diclofenac sodium as api) were also determined to assess possible interaction between diclofenac sodium and the excipients in the formulation. the spectra of all three samples were superimposed using the opus software to make comparison of the spectra easier. the spectra were compared on the basis of whether or not the principal bands present in the pure diclofenac sodium were still present in the formulated diclofenac sodium odfs. the petri dish method was employed in the determination of the disintegration time of the odfs. five randomly selected 2 cm 2 cm films were placed into 25 ml distilled water in a petri dish at 37 0.5c. the petri dish and its contents were swirled gently every 10 seconds till the film started to break up. the time taken by the film to break up completely five randomly selected 2 cm 2 cm diclofenac sodium odfs each containing ~50 mg diclofenac sodium were placed in separate conical flasks containing 70 ml of 0.1 m sodium hydroxide. sufficient quantities of 0.1 m naoh were added to produce 100 ml in each flask. the resultant solutions were filtered and 2 ml of each filtrate was diluted to 100 ml with 0.1 m naoh. the drug concentrations were evaluated spectrophotometrically (t90 uv / vis spectrometer, pg instruments ltd., uk) at a wavelength of 276 nm using the regression data of the calibration curve (y = 336.9x 0.0715, r = 0.9971). the mechanical properties of 2 cm 2 cm odfs were evaluated using a brookfield texture analyzer ct3 - 100 with texturepro ct software (brookfield engineering lab. middleboro, ma, usa), fitted with ta - de and ta - dga probes and equipped with a 10 kg load cell. the required test parameters were entered into the texture loader software and the appropriate mode was chosen. in the measurement of tensile strength and elastic modulus, a randomly selected film was held between two clamps of probe ta - dga using low pressure clips, allowing a distance of 3 cm between the sample surface and the base of the probe. for the elongation at break, a film was clamped between the accessory fixtures of probe ta - de. during measurement, the force at break and elongation were shown on the ct3 display when the film broke. with an attached computer and texturepro ct software, the mechanical parameters, namely the experiment was carried out in triplicate for each odf formulation and the average and standard deviations were calculated. the folding endurance of 2 cm 2 cm films was determined by folding the film of uniform cross - sectional area and thickness in the same place until it broke. the number of times the film was folded in the same plane (before breaking) was recorded as the folding endurance of that film. ten randomly sampled 2 cm 2 cm films of uniform thickness per formulation were used and the average of the determinations was recorded as the folding endurance. in vitro drug release studies on the odfs were conducted with an erweka dissolution apparatus (type dt6, gmbh, heusenstamm, germany). a film was placed with the help of forceps into a vessel containing 300 ml phosphate buffer ph 6.8, maintained at 37 0.5c with a stirring rate of 100 rpm. at 0, 7, 14, 21, and 28 min, 10 ml aliquots of the dissolution medium were withdrawn and replaced with an equal volume of fresh medium maintained at 37 0.5c. 5) and 5 ml of the filtrates was diluted ten times with phosphate buffer ph 6.8. the absorbance of the diluted filtrates was determined with a uv - visible spectrophotometer (t90 uv / vis spectrometer, pg instruments ltd., uk) at a wavelength of 276 nm. using the regression data obtained from the calibration curve (y = 333.97x 0.0686, r = 0.9981), the amount of diclofenac sodium in the samples was calculated. the percentage of diclofenac sodium released with reference to the expected content in each film was then calculated. a graph of cumulative mean percentage content of drug dissolved against the respective time points was plotted for each formulation to obtain the release profiles using graphpad prism version 5.00 (graphpad software, inc. dissolution efficiency (de), difference (f1), and similarity (f2) factors and one - way analysis of variance (anova) were used to analyze the drug release data. the dissolution efficiency was calculated using the equation(1)de=0ty dty100t2t1100,where (0ty dt) is area under the dissolution curve (auc) ; y is the percentage dissolved at t2 ; t2 is time for all active ingredient to dissolve ; t1 is time at which first sample was withdrawn. the difference and similarity factors were calculated using the following equations:(2)f1=st = lnrtttst = lnrt100f2=50log1 + 1nst = lnrttt20.5100,where n is time points ; rt is cumulative percentage dissolved at time t for the reference ; and tt is cumulative percentage dissolved at time t for the test. the drug release data were also subjected to one - way anova followed by dunnett 's multiple comparison test using graphpad prism version 5.00 (graphpad software, inc. the drug release data were fitted into the zero - order, first - order, higuchi, and hixson - crowell kinetic models to determine the release mechanism. the model that produced good linearity indicated by a high r value was considered the best model to describe the release kinetics of the formulated films. albizia and khaya gums were collected, purified, and employed in the preparation of the diclofenac sodium odfs. the physicochemical properties of the purified albizia and khaya gums studied are presented in table 2. khaya gum gave a higher purification yield, true density, moisture content, and insoluble matter compared to albizia gum. albizia gum also exhibited higher ash values and greater solubility in four of the solvents tested than khaya gum. in addition, albizia gum produced greater swelling capacity and charring temperature than khaya gum. the moisture content and insoluble matter of the two gums were less than 15% and 0.5%, respectively, and hence complied with the official specifications for natural gums. this is because high moisture content enhances microbial growth and causes some powders to clump and form hard aggregates. the insoluble matter in gums is the result of the mode of gum collection or the foreign materials collected in the gum exudates as they remain on the bark. khaya gum mucilage was more acidic than albizia mucilage and confirms previous findings about the acidity of the two gums. the ph of the gums determines whether they can cause oromucosal irritation when administered as odfs. also, the pharmaceutical applications of natural gums such as thickening and suspending agents which are dependent on their viscosity also tend to be ph - dependent. the ash values provide valuable information about the presence of inorganic and other extraneous materials in a natural product and are helpful in aiding the detection of product adulteration. the acid insoluble ash values of 0.05) was observed in the comparisons of the reference formulation (f1) and formulations f2, f3, f4, f5, and f6. however, there was a significant difference (p < 0.001) between f1 and f7. the kinetic data for the diclofenac sodium odfs are shown in table 8. in drug release studies, mathematical models are currently employed to better understand the mechanism of drug release from dosage forms. advancement in this area has made it possible to predict the release kinetics of drugs based on which mathematical model the dissolution data best fits. the kinetic models employed in the present study were zero - order, first - order, higuchi, and hixson - crowell models. the data from the dissolution studies conducted were fitted into the individual kinetic models to determine their linearity using the coefficient of regressions. in all the film formulations the highest r values were recorded for the higuchi model which describes drug release by fickian diffusion. the higuchi model is mostly applied to describe drug dissolution from various modified release pharmaceutical dosage forms especially in transdermal systems and matrix tablets with water soluble drugs. in can be concluded from the study that albizia and khaya gums possess the requisite physicochemical properties for use as film formers for the development of oral dissolvable films. the odfs produced generally exhibited satisfactory physicomechanical properties with most of the formulations attaining over 75% drug release in phosphate buffer ph 6.8 within 7 min. the study has demonstrated the potential of albizia and khaya gums as vehicles for the fabrication of diclofenac sodium odfs and with film properties enhanced through addition of hpmc in the formulation. | oral dissolvable films (odfs) of diclofenac sodium intended for osteoarthritis were prepared using albizia and khaya gums as hydrophilic film formers. the physicochemical properties of the gums were characterized and the gums were used to prepare diclofenac sodium odfs (~50 mg/4 cm2 film) by solvent casting. the two gums showed satisfactory film forming properties. the physicomechanical properties, drug - excipient compatibility, and in vitro drug release of the films in phosphate buffer ph 6.8 were studied. khaya gum had higher extraction yield, moisture content, insoluble matter and true density while albizia gum showed greater swelling capacity, solubility, and minerals content. the odfs were thin, soft, and flexible with smooth glossy surfaces and possessed satisfactory physicomechanical properties. ftir studies showed that no interaction occurred between the drug and the gums. the odfs disintegrated in 75% drug release within 7 min with dissolution efficiencies of ~8396%. drug releases from f2, f3, f4, f5, and f6 were similar to f1 (p > 0.05 ; f1 15 and f2 < 50). drug release followed the higuchi kinetic model which is indicative of fickian drug diffusion. |
ear, nose, throat (ent) and allergy - immunology specialists frequently encounter with diagnosis of migraine headache in their patients. according to the high prevalence of migraine and allergic rhinitis (ar) in populations, it is obvious that a noticeable proportion of the patients suffer from both conditions. migraine is the second most common type of primary headaches that affects 18% of females and 6% of males (totally 12% of general population). migraine is accompanied with a mixture of neurologic, gastrointestinal, and autonomic signs and symptoms and because of lack of any standard biologic marker for diagnosis of migraine, its diagnosis is based on clinical characteristics of the attacks. on the other hand, allergic diseases in the upper aerodigestive tract are common, and allergy is the primary or secondary cause of complaints in 50% of patients who refer to ent clinics and majority of patients referred to allergy clinics. ar has been observed in 10%30% of adults and up to 40% of pediatric population of united states. in a study conducted in 2005 by use of standard questionnaire for ar, the prevalence of this disease was 14.3% in 6 - 7 years old and 28.2% in 12 - 13 years old children of guilan province of iran. in daily practice, we often encounter with patients who suffer from headaches assumed to have paranasal sinuses origin because of promotion with temperature changes, bilateral location, exposure to allergens, and their incidence in frontal regions ; but in further investigations, a noticeable proportion of self - diagnosed and doctor - diagnosed sinus headaches meet the international headache society (ihs) criteria for migraines, and those migraines misdiagnosed as sinus headaches respond to sumatriptan better than placebo. obviously, many inflammatory mediators with vasoactive functions participate in these diseases, and it seems that there are some pathphysiologic similarities between these two common conditions. many studies have paid to the relationship between these two diseases from different perspectives, including statistical witnesses, clinical similarities, similar mechanisms and mediators, and response to similar treatments.[916 ] in this study, we aimed to determine the prevalence of migraine, with and without aura, in ar patients compared with subjects without this. after approving the proposal of the research in research office and ethical committee of guilan university of medical sciences (research project no. 1443), a comparative, cross - sectional study was performed in a referral- university hospital. with a significance level of 5%, the test power of 80%, and clinically difference equal to 30% (p1 = 34%, p2 = 4%), the sample size was determined to be 46 cases and 46 control subjects. from the patients referred to the ent clinic of amiralmomenin hospital in iran - rasht with and without clinical symptoms and signs of ar, we took written informed consents to do skin prick test (spt) in a standard safe setting. inclusion criteria for cases group were : positive skin test, at least one positive allergic symptom (nasal congestion, rhinorrhea, sneezing, snoring or mouth breathing, throat drainage, and itchy / watery eyes), and one aspect of the physical examination consistent with ar (allergic shiners, nasal crease, pale or boggy turbinate, mucous discharge in the nasal passage or postnasal area, or hypertrophy of posterior nasopharyngeal / oropharyngeal wall lymphoid tissues). those cases under 15 years old, pregnant cases, immunodeficient patients, and cases with known skin diseases such as psoriasis or lichen planus were not included in the study. the subjects must discontinue medications such as steroids, antihistaminic medications, and tricyclic antidepressants for a defined period before entering the study. none of cases and controls was referred specifically for work - up of headache in ent clinic. the spt was done by use of standard criteria and manufacturer recommendations (allergopharma, reinbek, germany) in emergency ward of the hospital by an expert immunologist (rjs) who was blinded about clinical condition of the subjects. after cleaning the forearm of the subjects, one drop from the commercial solution of eight common mixtures of allergens including grasses, trees i, trees ii, weed, d. farinae, d. pteronysinus, cladosporium, cat and dog epithelia (totally 23 allergens along with positive and negative controls i.e., histamine and distinct water, respectively) were put on the skin of forearm, separately. then the skin was pricked through the drops by use of a sterile lancet for entering a little of the allergens into the epidermis of the skin. the responses were measured 15 min later and dermal reaction above 3 mm was assumed as positive response according to the manufacturer guidelines. those subjects who did not react to histamine drop, were assumed as anergic subjects, and those subjects who reacted positively to distinct water drop, were assumed as dermographism and both of these two situations were of exclusion criteria of the study. those subjects who had clinical symptoms and signs of ar and positive spt were grouped as cases, and those who had not clinical symptoms and signs of ar with negative spt were enrolled in control group. a neurologist (as) who was blinded for clinic and spt results of the subjects, visited the subjects of two groups and investigated the symptoms and signs of migraine (with and without aura) in them according to ihs criteria. finally, the data were analyzed by chi - square and fischer exact test in spss version 16 and the prevalence of migraine in case and control groups was determined. the odd ratio was estimated for determining the chance of migraine and its subtypes in ar. from more than 80 subjects with clinical signs and symptoms of ar, 47 subjects had positive skin test, and from 65 persons without history or symptoms and signs of ar, 62 persons had negative skin test. one case from the first group and 2 subjects from the second group were excluded from the study because of occurrence of dermographism. finally, a total of 46 and 60 persons were enrolled in case group and control group, respectively. the former consisted of 14 (30.4%) men and 32 (69.6%) women and the latter 23 (38.3%) men and 37 (61.7%) women (p = 0.419). the mean (sd) of ages in cases was 31.17 8.31 years (1847 years old) and of controls was 37.58 12.63 years (20 - 65 years old). in case group, a total of 17 cases (37%) of the patients fulfilled the criteria of migraine (13 cases without aura and 4 cases with aura) but in control group, only 3 subjects (5%) had the criteria of migraine (2 without and 1 with aura). according to chi - square test, the statistical difference between these two groups was significant in the prevalence of migraine as a whole, and migraine without aura (p < 0.001 in both conditions), but fisher 's exact test did not show significant difference in frequency of migraine with aura (p = 0.164) [tables 1 and 2 ]. odd ratio for involving by migraine without aura in a population with ar was 11.42 (95% ci : 2.43 - 53.76) and for migraine with aura was 5.619 (0.61 - 52.09). comparing the prevalence of migraine without aura in patients with allergic rhinitis and nonallergic group comparing the prevalence of migraine with aura in patients with allergic rhinitis and nonallergic group the data were analyzed in male and female groups separately, and the results were similar to the whole subjects [table 3 ]. also analysis of data was done in separate age groups (i.e., age below 30, 30 - 39 years old, and above 40 years old). especially, in group above 40 years old, the difference of migraine frequency in cases and controls was significant (p < 0.001) [table 4 ]. comparing the prevalence of migraine in patients with allergic rhinitis and nonallergic group according to sex comparing the prevalence of migraine in patients with allergic rhinitis and nonallergic group according to age for omitting the effects of age and sex parameters, the mantel - haenszel common odd ratio and p value for the occurrence of migraine in ar were estimated (or : 8.227, 95% ci : 2.381 - 28.424, p = 0.001). in our study, 46 patients were in ar group and 60 subjects were included in control group. a total of 37% of the cases and 5% of the controls were affected by migraine (p = 0.001). the majority of migraines were without aura, and the incidence of migraine without aura between the cases and controls was different significantly (p < 0.05). the results of analysis in male and female groups were similar to the whole subjects, but considering the age, only among patients older than 40 years the difference of migraine prevalence was statistically significant. by omitting the effects of age and sex parameters the chance of migraine in allergic group was 8.227 folds (95% ci : 2.38128.424, p = 0.001). it was seen that among patients with sinus headache referred to otolaryngology clinics, up to 75% may have histories compatible with the migraine criterion. even in a tertiary care rhinology setting, perry. found that radiography - normal and endoscopy - normal headache patients had a 58% incidence of migraine. similarly, eross. reported a series of 100 consecutive self - diagnosed sinus headache patients and found that 86% were diagnosed as migraine or probable migraine using ihs criteria, and only 3% were found to have rhinosinusitis - attributable headaches. they also found that many migraine or probable migraine patients reported headache triggers such as weather changes, seasonal variation, and exposure to allergens. different studies introduced immunologic mediators such as ige (immunoglobulin e), histamine, tumor necrosis factor- (tnf-), calcitonin gene related peptide (cgrp), vasoactive intestinal peptide (vip), d2 and f2 prostaglandins, interleukin1 (il-1), tryptase, and also activation of mast cells and secondary release of nitric oxide in both conditions.[2171923 ] in a comparative study that was performed by ku and colleagues in cases older than 4 years, in ar group, 26 patients (34%) had headaches that met the ihs criteria for migraines, whereas only 2 patients (4%) in the non - ar group had headaches that met the criteria. fisher 's exact test showed p < 0.0001, the odds ratio was calculated and found to be 14.39 (95%ci : 3.23 - 63.34). they also performed a parallel study in all of the patients in the pediatric and internal medicine clinics without skin testing. jackson and dial have reported that 49 of 100 patients referred to their otolaryngology office for sinus headache had previously unrecognized migraine. among them, only 13% had migraine alone, 19% (of the patients with migraine) had ar as well, 11% had sinusitis, and 6% had both ar and sinusitis. cady and schreiber reported objective endoscopic evidence of nasal congestion and rhinorrhea during migraine attack. in one study that was performed in iran in 2009, among 104 patients who have sinus headaches, either self - ascribed or physician diagnosed, 72 patients (69.2%) did not have any positive sino - nasal findings in the nasal endoscopy and the computed tomography scanning. the response rate to the treatment with sodium valproate for 3 months in these patients was as follows : significant improvement in 44 patients (61.1%), partial response (9.7%), no response (15.3%), and 10 patients (13.9%) withdrew or failed to follow - up. according to wilcoxon test, the patients response rate to sodium valproate was statistically significant (p = 0.001). in another study that was performed by vincent. in 2010, they investigated the prevalence of migraine headache in ar patients (but not compared with a control group) and also investigated the relation between the severity of allergy / atopy with severity and frequency of migraine and the effect of immunotherapy on these parameters. patients were categorized into high (more than 45% positive allergy tests) and low (less than 45% positive allergy tests) atopic groups based on the number of allergy tests that were positive for the frequency and disability analyses. their study suggested that the association of allergy with migraine headaches depended upon age, degree of allergic sensitization, administration of immunotherapy, and the type of headache outcome measure that were studied. the administration of immunotherapy was associated with a decreased prevalence, frequency, and disability of migraine headache in younger subjects, while in our study the correlation between migraine and ar was more significant in patients older than 40 years old. this result may be due to either the small sample size in this age group, or the increased prevalence of both disorders with increasing age, although according to the evidences, the incidence of migraine decreases with increasing age. albeit, because of many differences in study design and the variables in two studies, the results of our study are not comparable with results of this one, and it is better to consider severity of migraine and ar in future studies. the characteristic of our study is not only performing the spt as a standard test for ar diagnosis but also the blindness of immunologist and neurologist of the study to the clinical situation and spt results of the cases and controls. finally, there is a noticeable restriction in our study that our control subjects with negative spt would be positive for other allergens that were not included in our kit (false negative). the prevalence and chance of migraine, especially without aura, are more in patients with ar compared with control subjects. it seems that performing more studies (e.g. interventional studies, clinical trial, and studies in specific age groups) are needed in future. | background : migraine and allergic rhinitis (ar) are two common causes of headache and facial pain that inflammatory mediators with vasoactive function play important roles in both of them. the aim of this research was to determine the prevalence of migraine in ar patients.materials and methods : in a cross - sectional comparative study performed from june to december 2010 in patients with ar sign and symptoms referred to ear, nose, throat (ent) clinic of a university hospital in iran - rasht, 46 patients with positive skin prick test were compared with 60 subject without ar signs and symptoms and with negative skin test. in both the groups, history of migraine according to ihs (international headache society) criteria were investigated. analysis of data was performed by chi - sqaure and fisher exact test by using spss16. odds ratio were estimated for determining the chance of migraine in ar.results:in case group (14 male, 37 female ; mean age : 31.17 8.31 years) and control group (23 male, 32 female ; mean age : 37.58 12.63 years), the prevalence of migraine was 37% and 5%, respectively. the differences in prevalence of migraine and migraine without aura between cases and controls were significant (p = 0.001). the chance of migraine in ar was 8.227 folds (95% ci : 2.38 - 28.42). in subjects older than 40 years old, the difference of prevalence of migraine was significant, contrary to subjects younger than 30 years old and between 30 and 39 years old.conclusions:there is a correlation between migraine especially without aura and ar and this correlation is more powerful with increasing age. |
hypercontractile esophagus, previously referred to as nutcracker esophagus, was defined manometrically by a mean distal contraction amplitude of 180 mmhg. this definition was plagued by a lack of specificity and poor symptom correction 1 2. with the introduction of high - resolution manometry (hrm) in 2000, the most recent definition is a distal contractile integer (dci) of 8000 mmhg.cm.s in 20 % of swallows and has been coined with this definition, there is improved specificity and symptom correlation compared with the previous criteria with standard manometry 3. jackhammer esophagus is rare, occurring in approximately 4 % of cases referred to a tertiary esophageal center 4. the treatment of jackhammer esophagus has included oral nitrates, balloon dilation, and surgical myotomy 5. surgical myotomy has not been widely performed due to the usual requirement for a long myotomy to achieve clinical success, which generally necessitates a combined abdominal and thoracic approach if a complete myotomy of the les is to be performed 6 7 8. with peroral endoscopic myotomy (poem), however, experience with poem for jackhammer esophagus has been limited due to the rarity of the disorder. in addition, the inclusion / exclusion of the lower esophageal sphincter (les) in the myotomy is debated and variably performed, perhaps contributing to inconsistent clinical outcomes in jackhammer esophagus 9 10 11. between january 2014 and july 2015, four patients underwent poem for treatment of jackhammer esophagus at our center. written informed consent for poem all patients had undergone a trial of at least 2 weeks on a proton pump inhibitor (ppi) without improvement in symptoms. manometry was performed using the starlet system (star medical, tokyo, japan) (normal integrated relaxation pressure [irp ] for starlet system 25 mmhg 12) prior to and 2 months after poem. all patients met the criteria for jackhammer esophagus and received a barium esophagram and endoscopic examination prior to poem. furthermore, one patient (patient 3) received a computed tomography (ct) scan because of elevated irp in addition to a distal contractile integral (dci) 8000 mmhg.cm.s in 20 % of swallows. follow - up was performed every 3 months for the first year then annually thereafter or sooner if issues developed. the point at which the tunnel was started was based on the manometry, barium esophagram, and endoscopic examination. the objective was to start the myotomy at the most proximal extent of the hypertensive contractions. a 2- to 3-cm longitudinal mucosal incision was made, submucosal entry achieved, and the submucosal tunnel was created. when the les was included in the myotomy, the tunnel was advanced 2 to 3 cm into the gastric cardia. the myotomy was advanced from 1 to 2 cm distal to the mucosal incision to the distal end of the tunnel. however, when the les was not included the tunnel the myotomy was only advanced to the distal esophagus. after prophylactic antibiotics were instilled, the mucosal entry site was closed with hemostatic clips. on day 1 post - procedure, all patients received a barium esophagram and endoscopy to confirm the mucosal integrity. the diet was advanced over 4 days and patients were discharged on day 4 post - procedure. the manometric and clinical results are summarized in table 1 and details about each patient are described below. esophageal manometry, upper endoscopy, and clinical (eckardt score, gastroesophageal reflux symptoms) examinations were performed 2 months post - poem. iem - ineffective esophageal motility after poem = 50 % ineffective swallows (failed or weak contraction vigor [dci < 450 mmhg.cm.s ]) a 74-year - old man presented with a 15-year history of weekly to daily severe noncardiac chest pain associated with swallowing. manometry exhibited multi - peaked contractions with a mean dci of 12 516.5 mmhg.cm.s and median irp of 19.5 mmhg (fig. 1). a 20-cm myotomy was performed, sparing the les due to lack of its involvement in the abnormal contractions. the patient s chest pain completely resolved, but he developed frequent dysphagia and regurgitation (eckardt score 6). six months later, the patient underwent a second poem with a 10-cm myotomy that included the les, resulting in the resolution of dysphagia and regurgitation (the patient chose to forego repeat manometry after the second poem). clinical follow - up was achieved for a total of 18 and 6 months after the first and second poem s, respectively. 1) patient 1 hrm pre- and post - poem # 1. a multi - peaked contractions with dci of 12 516.5 mmhg.cm.s and median irp of 19.5 mmhg. b post first poem showing a lack of abnormal contractions with failed contraction vigor with a mean dci of 84.2 mmhg.cm.s and median irp of 23.2 mmhg. a 68-year - old man with a 33-year history of regurgitation and dysphagia presented with progression of symptoms and weight loss (eckardt score 5). manometry demonstrated a mean dci of 18 332.4 mmhg.cm.s and a median irp of 16.4 mmhg with inclusion of the les in the hypercontractile segment (fig. 2). after poem, the patient s symptoms completely resolved (eckardt score 0). the follow - up hrm demonstrated a dci of 137.7 mmhg.cm.s and a median irp of 10.5 mmhg. 2) patient 2 hrm pre and post - poem. a hypercontractile contractions with a mean dci of 18 332.4 mmhg.cm.s and median irp of 16.4 mmhg b post - poem with no abnormal contractions with a weak contraction vigor with a mean dci of 137.7 mmhg.cm.s and median irp of 10.5 mmhg. an 87-year - old man with a 40-year history of dysphagia presented with symptom progression leading to weight loss (eckardt score 5). manometry demonstrated a mean dci of 46 700 mmhg.cm.s with a median irp 33.8 mmhg with the les included in the hypercontractile segment (fig. a ct scan was also performed in light of the elevated irp, which did not reveal any infiltrative, neoplastic or vascular obstruction at the distal esophagus. the patient received a 12-cm myotomy that included the les, which resulted in complete symptom resolution (eckardt score 0). clinical follow - up was achieved for a total of 12 months after poem (fig. 3) patient 3 hrm pre- and post - poem. a hypercontractile contractions with a mean dci of 46 700 mmhg.cm.s and median irp 33.8 mmhg b post - poem showing no abnormal contractions and a normal contraction vigor with mean dci of 2019.6 mmhg.cm.s and median irp 16.2 mmhg. a 37-year - old man with a 6-year history of dysphagia, regurgitation, chest pain presented with deterioration of symptoms resulting in weight loss (eckardt score 11). hrm showed dci of 15 388.7 mmhg.cm.s and median irp 7.3 mmhg (fig. after poem, the patient regained the weight he had lost and his chest pain completely resolved with only occasional dysphagia and regurgitation (eckardt score 2). clinical follow - up was achieved for a total of 12 months after poem (fig. 4) (table 1). patient 4 hrm pre- and post - poem. a hypercontractile contractions with a mean dci of 15 388.7 mmhg.cm.s and median irp 7.3 mmhg b post - poem showing no abnormal contractions with a weak contraction vigor with a mean dci of 234 mmhg.cm.s and median irp 12.4 mmhg. a 74-year - old man presented with a 15-year history of weekly to daily severe noncardiac chest pain associated with swallowing. manometry exhibited multi - peaked contractions with a mean dci of 12 516.5 mmhg.cm.s and median irp of 19.5 mmhg (fig. 1). a 20-cm myotomy was performed, sparing the les due to lack of its involvement in the abnormal contractions. the patient s chest pain completely resolved, but he developed frequent dysphagia and regurgitation (eckardt score 6). six months later, the patient underwent a second poem with a 10-cm myotomy that included the les, resulting in the resolution of dysphagia and regurgitation (the patient chose to forego repeat manometry after the second poem). clinical follow - up was achieved for a total of 18 and 6 months after the first and second poem s, respectively. 1) patient 1 hrm pre- and post - poem # 1. a multi - peaked contractions with dci of 12 516.5 mmhg.cm.s and median irp of 19.5 mmhg. b post first poem showing a lack of abnormal contractions with failed contraction vigor with a mean dci of 84.2 mmhg.cm.s and median irp of 23.2 mmhg. a 68-year - old man with a 33-year history of regurgitation and dysphagia presented with progression of symptoms and weight loss (eckardt score 5). manometry demonstrated a mean dci of 18 332.4 mmhg.cm.s and a median irp of 16.4 mmhg with inclusion of the les in the hypercontractile segment (fig. 2). after poem, the patient s symptoms completely resolved (eckardt score 0). the follow - up hrm demonstrated a dci of 137.7 mmhg.cm.s and a median irp of 10.5 mmhg. 2) patient 2 hrm pre and post - poem. a hypercontractile contractions with a mean dci of 18 332.4 mmhg.cm.s and median irp of 16.4 mmhg b post - poem with no abnormal contractions with a weak contraction vigor with a mean dci of 137.7 mmhg.cm.s and median irp of 10.5 mmhg. an 87-year - old man with a 40-year history of dysphagia presented with symptom progression leading to weight loss (eckardt score 5). manometry demonstrated a mean dci of 46 700 mmhg.cm.s with a median irp 33.8 mmhg with the les included in the hypercontractile segment (fig. 3). a ct scan was also performed in light of the elevated irp, which did not reveal any infiltrative, neoplastic or vascular obstruction at the distal esophagus. the patient received a 12-cm myotomy that included the les, which resulted in complete symptom resolution (eckardt score 0). clinical follow - up was achieved for a total of 12 months after poem (fig. 3) patient 3 hrm pre- and post - poem. a hypercontractile contractions with a mean dci of 46 700 mmhg.cm.s and median irp 33.8 mmhg b post - poem showing no abnormal contractions and a normal contraction vigor with mean dci of 2019.6 mmhg.cm.s and median irp 16.2 mmhg. a 37-year - old man with a 6-year history of dysphagia, regurgitation, chest pain presented with deterioration of symptoms resulting in weight loss (eckardt score 11). hrm showed dci of 15 388.7 mmhg.cm.s and median irp 7.3 mmhg (fig. after poem, the patient regained the weight he had lost and his chest pain completely resolved with only occasional dysphagia and regurgitation (eckardt score 2). clinical follow - up was achieved for a total of 12 months after poem (fig. 4) (table 1). patient 4 hrm pre- and post - poem. a hypercontractile contractions with a mean dci of 15 388.7 mmhg.cm.s and median irp 7.3 mmhg b post - poem showing no abnormal contractions with a weak contraction vigor with a mean dci of 234 mmhg.cm.s and median irp 12.4 mmhg. with inclusion of the les in poem for jackhammer esophagus, patients 2, 3, and 4 had excellent clinical results. in contrast, patient 1 in whom les was not included in the myotomy developed regurgitation and dysphagia. however, after the second poem that included the les, his symptoms of dysphagia and regurgitation resolved. with patient 1, the hrm was consistent with jackhammer esophagus, with a mean dci of 12 516.5 mmhg.cm.s and a normal irp. however, the irp was in the upper range of normal and one could speculate that the patient was progressing to achalasia and had a variant of incompletely expressed or early achalasia. the progression from hypercontractile esophagus or diffuse esophageal spasm (des) to achalasia has previously been described, suggesting that the spastic esophageal motor disorders may represent a spectrum of a single disease entity 14 15. interestingly, there have been no reports of progression from one spastic disorder to another demonstrated with hrm. this may be due to the previous misdiagnosis with standard manometry, the superior sensitivity and specificity of hrm, and the new chicago classification. that, on the other hand, would support the notion that the spastic esophageal motor disorders are separate entities rather than a spectrum of a single pathology. the successful treatment of jackhammer esophagus usually requires a long myotomy, often two - thirds or more the length of the esophagus, which can often result in iatrogenic ineffective esophageal motility 16. this was demonstrated in our series by the fact that all patients with a myotomy 20 cm or longer developed ineffective esophageal motility (50 % ineffective swallows), while the patient with a 12-cm myotomy had persevered contraction vigor. without involvement of the les in the hypercontractile segment, inclusion of les in the myotomy has been a contentious topic. however, we postulate that routine inclusion of the les in poem for jackhammer esophagus should be performed for the following reasons : patients with jackhammer esophagus typically require a long myotomy, which results in diminished contraction vigor and in many cases iatrogenic ineffective esophageal motility. in some patients, gravity and the remaining propulsive force is inadequate to propagate the food bolus across the preserved les. without the inclusion of the les in the myotomy to further reduce outflow resistance, symptoms analogous to achalasia may develop (regurgitation, dysphagia, and chest pain). thus, there is a critical (currently unknown) length of esophageal myotomy that once exceeded, results in ineffective esophageal motility, inadequate food bolus propulsion, and symptom development. the importance of les inclusion was demonstrated in a case report by badillo., in which a 50-year - old woman received poem for jackhammer esophagus, resulting in worsening symptoms post - poem. she subsequently presented with continued deterioration in symptoms and was found to have an 8-cm anterolateral diverticulum with moderate narrowing of the gastroesophageal junction. 17 based on her immediate post - poem symptom deterioration and subsequent development of the diverticulum, it is highly likely the les was not included in the myotomy analogous to, although more dramatic, than our patient 1. there is evidence that the non - achalasia spastic esophageal motility disorders can progress to achalasia. albeit rare, if there is progression to achalasia and the les is preserved, symptom development would occur and the patient would require additional treatment. although there is indeed a significant risk of reflux after poem (up to 50 %), there are no cases of reflux refractory to ppi, and only one report of a peptic stricture. 18 19 20 in conclusion, poem is a suitable treatment for patients with jackhammer esophagus. based on our clinical experience and physiologic and manometric observations, we speculate that the obligatory inclusion of the les is justified. inclusion of the les minimizes the risk of symptom development from iatrogenic ineffective esophageal motility or subsequent progression to achalasia. furthermore, from our experience, in addition to the thousands of cases of poem published, the risk of reflux - related complications has been shown to be marginal. thus, it appears that the risks associated with les exclusion are far greater than the risks of reflux - associated complications of les inclusion. however, given the low incidence of jackhammer esophagus, an international, multicenter randomized trial is required in order to obtain a definitive evidence - based answer to whether routine inclusion / exclusion of the les in the application of poem for jackhammer esophagus is warranted. | background and study aims : with the success of peroral endoscopic myotomy (poem) in treatment of achalasia, its successful application to other spastic esophageal motility disorders such as jackhammer esophagus has been noted. the question of whether the lower esophageal sphincter (les) should be included in the myotomy for jackhammer esophagus is a topic of current debate. here, we report our experience and results with four patients with jackhammer esophagus treated with poem. the clinical and manometric results are presented and their potential implications are discussed. patients and methods : between january 2014 and july 2015, four patients underwent poem for treatment of jackhammer esophagus at our center. manometry was performed prior to and after poem. all patients met the chicago classification criteria for jackhammer esophagus and received a barium esophagram and endoscopic examination before having poem. results : all patients had uneventful procedures without any intraoperative or post - procedure complications. patients in which the les was included during poem had resolution or significant improvement in symptoms. one patient in whom the les was preserved had resolution of chest pain but developed significant dysphagia and regurgitation. subsequently this individual received a repeat poem which included the les, resulting in symptom resolution. conclusions : poem is a suitable treatment for patients with jackhammer esophagus. until there are larger - scale randomized studies, we speculate that based on our clinical experience and physiologic and manometric observations, obligatory inclusion of the les is justified to reduce the risk of symptom development from iatrogenic ineffective esophageal motility or subsequent progression to achalasia. |
hemangiomas are tumors characterized by increased numbers of normal or abnormal vessels filled with blood. occasionally, hemangiomas can occur internally, and nearly one - third of these internal lesions are found in the liver. pancreatic hemangiomas are especially rare ; pancreatic vascular neoplasms collectively account for only 0.1% of all pancreatic tumors.1 these tumors are usually diagnosed fortuitously by laparotomies performed to diagnose a large, palpable abdominal mass.2345 we presented a very rare case in which a woman without specific symptoms was found with a cavernous hemangioma in the pancreas tail that mimicked metastatic tumor. a 68-year - old woman was found with a mass in her left kidney on medical checkup. she had no significant past medical history except hypertension and no symptoms (e.g., hematuria, abdominal pain, or abdominal discomfort). an axial contrast - enhanced computed tomography (ct) scan showed a heterogeneous solid mass in the left kidney, suggesting the presence of renal cell carcinoma (rcc). there was a strongly enhancing tiny nodule in the tail of the pancreas that was most likely either a neuroendocrine tumor or a rcc metastasis (fig. because she had no specific symptoms or abnormal laboratory findings, the possibility of rcc metastasizing into the pancreas could not be ruled out. there were no significant postoperative events, and the patient was discharged home on postoperative day 7 without any morbidity or complications. gross pathologic examination revealed a 0.60.5 cm - sized hemangioma confined to the pancreas, and the tissue had a tumor - free margin (fig. immunohistochemical analysis showed that the tumor was positive for cd31, cd34, and factor viii - related antigen, and negative for d2 - 40, rcc, and cd56 (sclc), supporting the diagnosis of hemangioma (fig. hemangiomas rarely occur in the pancreas and often are not suspected clinically ; only 14 patients are reported in the literature since 1939. review of the previous literature reports on pancreatic hemangioma indicated that most hemangiomas occur in females (12/15 patients, including our patient) and are symptomatic (9/15 patients had abdominal pain, and one patient each had melena, thrombocytopenia, abdominal distension, and palpitation, suggesting bleeding). only 1 tumor was found incidentally, in 1939 upon autopsy.234567 the hemangioma was found incidentally at a preoperative evaluation for rcc. unlike previous studies, we found no symptoms suggesting pancreatic hemangioma, likely because it was a tiny pancreatic tail mass. typically, hemangiomas are strongly contrast enhancing in the arterial phase of conventional contrast - enhanced ct imaging;8 however, our case did not present these findings, likely because of the small lesion size. the pancreatic hemangioma thus mimicked metastatic cancer originating from the rcc. to our knowledge, this case was the first report of pancreatic hemangioma without a symptomatic event, and the tumor is the smallest of the reported cases. microscopic findings revealed a typical feature of hemangioma i.e., blood - filled spaces separated by fibrous connective tissue. for a definite diagnosis, immunohistochemistry is required to assess the presence of the factor viii - related antigen, a marker for vascular endothelium that was reported by chang and colleagues.9 subsequently, mundinger and colleagues reported that neoplastic cells in hemangioma also express the endothelial markers cd31 and cd34.5 in our patient, immunohistochemical findings were positive for all 3 markers ; whereas, d2 - 40, a marker for lymphatic endothelium, and cd56, a marker for neural cell, were both negative, further indicating that the tumor mass was a hemangioma. because of its rarity, there is no standard treatment for pancreatic hemangioma. reviewing the previous literature on pancreatic hemangioma, we found that multiple different treatments were administered, from observation to surgical resection.234567 furthermore, the possibility of abdominal pain or hemorrhagic events is typically increased in patients with larger hemangioma masses. however, other clinicians suggested that if the patient 's symptoms are minimal, observation is a possible treatment option, because pancreatic hemangiomas are benign. the location of the pancreatic hemangioma is variable, and may be important for determining the best treatment option. upon reviewing previous literature, we found that pancreatic hemangiomas were located at the head (8/15 patients), neck (1/15 patients), or body / tail (6/15 patients).234567 when the tumor is located at the body / tail, distal pancreatectomy is an option. however, if the tumor is located at the proximal site of the pancreas, pylorus - preserving pancreaticoduodenectomy is indicated. patients who underwent pylorus - preserving pancreaticoduodenectomy had a higher rate of morbidity than patients who underwent distal pancreatectomy (34.7% vs. 27.8%, p<0.05).10 therefore, if a patient has a pancreas head hemangioma with minimal symptoms that can be controlled, close observation and regular follow - up can be one of the treatment options according to risk - benefit analysis. because our case was confined to a tiny mass at the distal pancreas, and we could not rule out distant metastasis from the rcc tumor, we decided to perform a distal pancreatectomy. the literature review indicated that treatment decisions require assessment of the severity of symptoms and location of the tumor. when all the cases were collectively considered, determining the timing of surgery based on comparison of surgical risk - benefit analysis emerged as an important factor. | adult pancreatic hemangioma is a rare disease. we presented a case of a woman with pancreatic tail mass mimicking a distant metastasis from the kidney. a 68-year - old woman was found with a left kidney mass on medical checkup. computed tomography scan showed a 4.3 cm - sized mass in the left kidney, suggesting renal cell carcinoma (rcc), and a strongly enhancing tiny nodule in the pancreatic tail. we could not rule the possibility of rcc metastasis, hence, surgical resection of the pancreatic mass simultaneously with radical nephrectomy for rcc was conducted. gross pathologic examination revealed hemangioma. immunohistochemistry revealed that the tumor was positive for cd34, cd31 and factor viii - related antigen. there were no significant postoperative events, and the patient was discharged on postoperative day 7 without any complications. treatment strategies for pancreatic hemangioma have not been established. to our knowledge, this was the first case report of asymptomatic pancreatic hemangioma. in previous literature, treatment differed on a case - by - case basis, ranging from observation to surgical resection. the most important factor in deciding whether to perform surgery is possibly risk - benefit effectiveness ; however, tumor location, patient symptoms, and other factors are also important. |
acute thermal injuries to the esophagus are uncommon causes of esophageal disease. a candy - cane appearance is an endoscopic feature of thermal injury to the esophagus from hot liquid (1, 2). however, the clinical course of such a finding from solid food is not well documented. we treated a patient that presented with a 2-day history of odynophagia and foreign body sensation after an acute thermal injury of the esophagus from solid food (prawn). here a 53-yr - old man was admitted to the hospital with a two - day history of odynophagia and a foreign body sensation. he had no medical history, and previously denied any esophageal symptoms such as dysphagia, odynophagia, or chest discomfort. two days before admission, the patient began to experience odynophagia and a foreign body sensation in the chest after swallowing several extremely hot pieces of prawn in haste. on arrival, endoscopy revealed a huge longitudinal ulcer, typical of friable hyperemic mucosa with necrotic debris with a tendency for easy to touch bleeding along the full length of the esophagus (fig. the patient was treated with a parenteral nutrition regimen and intravenous pantoprazole to prevent injury from gastroesophageal reflux. the symptoms gradually improved over eight days after the initial event. at eight days after the initial event the patient was started on liquids and progressed to a soft diet, and was then discharged on oral pantoprazole for 1 months. eight weeks after the event, endoscopy showed normal esophageal mucosa with the rim of the previous ulcer scar (fig. here we present the clinical course of serial endoscopy of an acute thermal injury of the esophagus caused by extremely hot solid food. there is only one prior similar report with the endoscopic and histological characteristics of an injured esophagus due to hot liquids (3). however, there is no same case due to solid foods reported in the medical literature. although candy cane esophagus with alternating pink and white linear bands of esophageal mucosa is well known feature of esophageal thermal injury, most of the prior caustic agents were hot liquids. in our case, the typical candy cane appearance was not observed during the clinical course. most early endoscopic findings of the initial injury during the first week showed a predominant whitish pseudomembrane (3). however, our findings, 2 days after the initial event, showed hyperemic areas with marginal necrotic debris, which may have resulted from rupture of bullae. we assume that the appearance of the candy cane esophagus was due to the flow of hot liquid whereas that of burn or ulcer was due to the pressure effect of hot solid. therefore the different causes of thermal injury might make the different endoscopic findings and the endoscopic image of a candy cane appearance may not be characteristic of an acute esophageal thermal injury due to hot solid foods. although most reported series are case reports, conservative management with anti antisecretory treatment such as a proton pump inhibitor (ppi) or histamine2-receptor antagonist (h2ra) to prevent further injuries from gastric acid was underwent without severe complications (3 - 6). most cases were treated for 1 months except for only one case for 14 days (6). in conclusion, with the history of hot food ingestion, esophageal symptoms, a foul odor on examination, and deep linear ulcers on the posterior aspect of the esophagus the diagnosis of an acute thermal injury of esophagus should be considered. | a 53-yr - old man presented with a two - day history of odynophagia and a foreign body sensation. two days before admission, the patient began to experience odynophagia and a foreign body sensation in the chest after swallowing several extremely hot pieces of solid food (prawn) in haste. endoscopy revealed a huge longitudinal ulcer, typical of friable hyperemic mucosa with necrotic debris along the full length of the esophagus in the posterolateral region. here we present the clinical course of serial endoscopy of an acute thermal injury of the esophagus caused by solid food. |
pam4, a new monoclonal antibody (mab) also known as clivatuzumab, is absent from the normal tissues, as well as breast cancer, liver cancer, prostate cancer, and renal cancer. it is reactive with greater than 80% of pancreatic cancer and has a limited reactivity with ovarian cancer, stomach cancer, colon adenocarcinoma, and lung cancer [13 ]. in addition, pam4 is also expressed in its precursor lesions, pancreatic intraepithelial neoplasia (panin), and intraductal papillary mucinous neoplasia (ipmn) in pancreatic cancer. pancreatic cancer is one of the deadliest of the solid malignancies with a 5-year survival rate of 35% [4, 5 ]. it is the fourth commonest cause of cancer - related death among men and women in the united states. in 2013, an estimated 45,220 people in the usa were diagnosed with pancreatic cancer, and 38,460 died of the disease. most cases of pancreatic cancers have advanced stage at time of diagnosis with a median survival of less than 1 year. the dismal prognosis can be partly attributed to the absence of early symptoms, late diagnosis, and the poor response to radio- and chemotherapy. how to establish a methodology to define benign pancreatitis form pancreatic malignancy or metastatic carcinomas remains to be investigated. although ca19 - 9 is the most widely investigated and evaluated marker for testing pancreatic cancer diagnosis, the sensitivity and specificity are not optimal. with rapid advances in imaging technology, ultrasound, computerized tomography (ct), magnetic resonance imaging (mri), positron emission tomography (pet), and pet - ct technologies play an important role in the diagnosis of pancreatic cancer. surgical resection has been the only modality curative treatment for pancreatic cancer, but the majority of patients present at a late stage when the disease does not respond to surgical therapy. radiation and/or chemical therapy have a limited impact on the control of pancreatic cancer, resulting in the rapid regrowth of the tumor [7, 11 ]. thus, there is an urgent need to develop new means for early diagnosis and new therapeutic approaches to improve the clinical outcome of the deadly disease., pam4 is discussed with a focus on its potential as a serum marker for diagnosis and as a target of both radioimmunodiagnostic and radioimmunotherapeutic agents in pancreatic cancer due to its limited distribution on normal tissues and other solid tumors. pam4, a monoclonal antibody to muc1, is an lgg1 immunoglobulin produced by immunization of mice with mucin purified from the xenografted ripi human pancreatic cancer originally a mucinous, moderately differentiated tumor in the head of the pancreas. muc1 is a transmembrane glycoprotein associated with cell transformation, invasion, migration, apoptosis, cellular interactions, immune regulation, and drug resistance [1216 ]. pam4 recognizes a unique and novel epitope which is not reactive with the peptide core of mucin and distinct from that of b72.3, ca19 - 9, dupan2, span1, nd2, cea, and lewis antigens [2, 17 ]. recent studies show that pam4 is reactive with the c - terminal region of the muc5ac. actually the carbohydrate structures are not the part of the pam4 epitope but are necessary to maintain the correct peptide conformation. furthermore, the pam4 epitope was found to be highly sensitive to heat, reduction of disulfide bonds, proteolytic digestion, or deglycosylation. although the detailed characteristic of the pam4 epitope is unknown, it is thought to be dependent on muc1 glycosylation status in recent studies. there is a growing body of evidence that pam4 is highly reactive with pancreatic cancer. in the original study by gold and coworkers, the immunoreactivity of pam4 with pancreatic cancer was evaluated by immunohistochemistry using frozen section tissues of patients. all but four pancreatic cancers (21/25, 23 primary and 2 metastatic) were immunoreactive with pam4. pam4 reactivity showed weak positive staining of 40% (10 of 26) of colorectal cancer, 20% (1 of 5) of gastric cancer, and 6.6% (1 of 15) of lung cancer. interestingly, staining was restricted to the ductules ; minor staining of a few scattered ductule cells was observed. none of the breast cancer, ovarian cancer, liver cancer, prostate cancer, and renal cancer was stained. however, compared to its weak staining in the goblet cells along the gastrointestinal tract, strong staining was present in pancreatic cancer. this was confirmed in a large study by gold. who evaluated the immunoreactivity with pam4 in 320 invasive cancer specimens by tissue microarrays. in the study, pam4 expression was present in 48 of 55 (87%) pancreatic cancers, 6 of 40 (15%) stomach cancers, 7 of 76 (9%) colon adenocarcinomas, 4 of 24 (17%) ovarian cancers, 4 of 40 (10%) lung cancers, 0 of 50 (0%) breast cancers, and 0 of 35 (0%) hepatocellular cancers. invasive pancreatic cancer can arise from three different noninvasive precursor lesions, including pancreatic intraepithelial neoplasias (panin), intraductal papillary mucinous neoplasms (ipmn), and mucinous cystic neoplasms (mcns) [18, 19 ]. a hypothesis that has been proposed is that pam4 may be present during precursor lesions transition to invasive pancreatic adenocarcinoma. assessed the expression of the pam4-reactive muc1 by immunohistology in a study cohort of 55 invasive adenocarcinomas, 63 pancreatic intraepithelial neoplasias (panin), 36 intraductal papillary mucinous neoplasms (ipmn), and 11 normal pancreases. pam4-reactive muc1 was absent from normal pancreas, but it was identified in 87% of invasive cancers with no striking correlation with the clinical stage of disease. there was a trend for those tumors that were better differentiated to show the higher expression of the pam4-reactive muc1. most importantly, pam4 is abundantly present in the earliest stages of pancreatic adenocarcinoma and its expression remains high in all stages of panin patients. for example, pam4 labeled 94% (44 of 47) of the panin patients tissues in the earliest stage (stages 1a and 1b), 91% (10 of 11) of stage ii, 40% (2 of 5) of stage iii, and 86% (31 of 36) of ipmn. on the basis of these studies, it is concluded that pam4 is highly restricted to pancreatic adenocarcinoma and its precursor lesions, but it is also expressed to a lesser degree in other solid tumors. it is not useful in detecting early cancers and defining pancreatic cancer from pancreatitis and other benign lesions due to its poor specificity and sensitivity. pam4 can be shed from tumors and detected in serum. in recent years, the role of serum pam4 in the diagnosis of pancreatic cancer has been evaluated. for pam4 in a well - defined study group of 68 carcinomas, 29 chronic pancreatitides, and 19 healthy volunteers. apart from overall diagnostic performance of pam4 in the study, stage - dependent evaluation showed increasing sensitivities at advanced tumor stages with a sensitivity of 62% at stage i (n = 21), 86% at stage ii (n = 14), and 91% at advanced stages 3 and 4 (n = 33). another article by gold. examined the presence of pam4-reactive muc1 as a serum marker for pancreatic cancer with a sensitivity of 77% and a specificity of 95%. a total of 283 subjects were evaluated, including 53 pancreatic cancer patients, 87 pancreatitis patients, 100 other cancer patients, and 43 healthy volunteers. as with the pam4 immunoassay, none of the healthy specimens and only four of 87 pancreatitis patients (5%) were positive above a cut off of 10.2 units / ml. however, of the 87 pancreatitis samples, the positive rate of ca19 - 9 was 37%. a direct pairwise comparison of pam4 and ca19 - 9 immunoassays for discrimination of pancreatic cancer and pancreatitis resulted in a significant difference, with the pam4 immunoassay demonstrating superior sensitivity and specificity. approximately seven years later, gold tested the pam4 in a large study group of 298 pancreatic ductal adenocarcinomas (pdac), 99 other cancers, 120 benign pancreases, and 79 healthy controls reaching 76% sensitivity and 96% specificity. the specificity was significantly greater for the pam4 assays than ca19 - 9 assays, particularly with regard to chronic pancreatitis (86% and 68%, resp.). besides good overall high diagnostic performance, the detection rate for patients with respect to stage i disease was 64% and was considerably higher for patients with advanced disease (85%). pam4 antigen levels were significantly higher in patients with pdac than in other patient groups. at the same time, they evaluated the combination of pam4 and ca19 - 9 in serial testing and obtained an improved sensitivity (84%) for the overall detection of pdac without a significant loss of specificity (82%) compared with either assay alone in 474 specimens. some reported that both pam4 and ca19 - 9 were present simultaneously in the same serum, but others reported that they were independent of each other. for the most part, sera level from patients with pancreatic cancers arising from other tissues of origin did not have detectable levels of the pam4 antigen. from these studies, serum pam4 was reactive with a higher percentage of pancreatic cancer and gave a greater overall intensity of reaction at equivalent concentrations compared to serum ca19 - 9. it also appeared that pam4 showed a superior sensitivity and specificity for discrimination of pancreatic cancer from pancreatitis than did ca19 - 9. combining pam4 and ca19 - 9 can lead to an improvement in diagnostic accuracy for discriminating pancreatic cancer from pancreatitis and other solid tumors. in the near future on the other hand, it is obvious that a serum - based biomarker would provide a clinically more valuable and cost - effective tool for the early detection and diagnosis of pancreatic cancer. modern imaging modalities, like ultrasound, ct, mri, pet, and pet - ct, provide essential information for detection, diagnosis, and management of pancreatic cancer [8, 9 ]. however, there are many limitations with respect to the detection of small lesions, as well as for discriminating pancreatic cancer and precursor lesions from pancreatitis. detection of small, early stage pancreatic adenocarcinoma in the asymptomatic patient is crucial for the increased survival rates. monoclonal antibody (mab) based imaging holds the potential to impact these problems. taking into account the high specificity of pam4 in pancreatic cancer, several lines of work support the use of pam4-based radioimmunotargeting agents for pancreatic cancer imaging. there are several radiolabeled pam4 agents that have been developed and some are being evaluated in preclinical and/or clinical studies, such as i - pam4, in - pam4, tc - pam4, and y - pam4. more than a decade ago, gold and colleagues used postadministration imaging of i - labeled murine pam4 (i - mpam4) to assess tumor targeting in the four different human pancreatic cancer models (aspc1, bxpc3, hs766 t, and capan1) that represent the range of expected differentiation of this tumor type. after intravenous administration of i - mpam4 there was preferential localization of radioactivity in each tumor line as assessed by the tumor : nontumor ratios and tumor : blood ratios. tumor / nontumor ratios for pam4 were always greater than for nonspecific, isotype - matched ag8. at the same time, the blood activity of i - mpam4 was significantly lower than ag8 over the period of observation. the specific tumor concentration of pam4 increased levels of pam4 protein (from 10 g to 100 g). there was no evidence of pam4 targeting to nontumor tissues except for splenic uptake in capan1 tumor, which may be due to that pam4 released from the tumor became entrapped in the spleen or that circulating antigen - antibody complexes were deposited in the spleen. other studies also showed similar results [24, 25 ]. in these studies, radiolabeled pam4 showed specific localization of the primary orthotopic and metastatic tumors without significant accumulation in noncancer sites. of further note, microautoradiography was performed on 5 m sections through the tumor. in initial clinical trials by mariani., i - mpam4 was injected into five patients with suspected pancreatic cancer (postoperation pathology confirmed pancreatic cancer in four of the five patients, whereas the fifth patient was diagnosed with chronic pancreatitis). they found a quite satisfactory pattern of distribution of the i - mpam4 with good radioactivity accumulation in primary pancreatic cancer and metastatic pancreatic cancer. furthermore, they found no severe adverse clinical reactions and no abnormal results of blood chemistry tests, whereas low nonspecific radioactivity accumulation in the liver, spleen, and bone marrow was due to the blood pool. immunoscintigraphy showed clear tumor uptake in all four pancreatic cancer patients, but failed to find in the pancreatitis patients. significant uptake of i - mpam4 in the tumor lesions was observed at relatively late times, starting about 7296 hours after tracer injection. the tumor lesions detected by immunoscintigraphy ranged in size from bulky lesions to liver metastases about 1 - 2 cm. in another study of five metastatic pancreatic cancer patients who received i - mpam4 igg (n = 2) or tc - mpam4 fab ' (n = 3), gold. observed definitive tumor localization in four of five patients ; the fifth had no staining with mpam4 by immunohistology. consistent with previous findings, mpam4 specifically targetd not only can primary tumors but also metastatic lesions in pancreatic cancer patients. these data indicate the favorable tumor - targeting potential of radiolabeled mpam4 for diagnostic in primary and metastatic pancreatic cancer patients by immunoscintigraphy. murine mabs have a short survival time and induce a human anti - mouse antibody (hama) response. thus humanized pam4 (hpam4) and chimeric pam4 (cpam4) based on mpam4 are being evaluated in preclinical and/or clinical studies. the specificity and biodistribution characteristics of i - labeled cpam4 (i - cpam4) and in - labeled cpam4 (in - cpam4) were shown to be similar to that of i - mpam4 as described above [24, 26 ]. in these studies, accumulation of cpam4 within the capan1 tumor - bearing mice was 2.8-fold higher than nonspecific hll2 (anti - cd22 antibody), with peak levels of cpam4 occurring on day 4 after injection. tumor / blood radiation dose ratios were 3.6 and 0.6 for yttrium-1, 4, 7, 10-tetraazacyclododecane - n, n, n, n-tetra - acetic acid (dota) cpam4 (y - dota - cpam4) and y - dota - hll2, respectively. gulec. investigated the biodistribution of in - labeled humanized pam4 (in - hpam4) in patients suffering from pancreatic cancer in a phase i clinical trial [2729 ]. pancreatic cancers were clearly visible at 24 hours after injection and the visibility was progressively prominent on the subsequent images. there was no qualitative difference apparent in the biodistribution of in - hpam4 at the two doses (10 mg and 100 mg total hpam4). thus, the hpam4 was suitable for the scintigraphic visualization of patients with pancreatic cancer. the group of cardillo developed bspam4, a divalent and bispecific f(ab')2 mab, that was generated from chimeric pam4 fab ' and murine 734 fab ' fragments and then used in conjunction with 2 peptide haptens, in - labeled ac - phe - lys (dtpa)-tyr - lys (dtpa)-nh2 (in - imp-156) and tc - labeled ac - lys (dtpa)-tyr - lys (dtpa)-lys (thiosemicarbazonyl - glyoxyl - cysteinyl-)-nh2 (tc - imp-192) [3033 ]. to confirm the tumor targeting specificity and delivery of bspam4 to the tumor, the biodistribution of this bspam4 was investigated in capan1 tumor - bearing mice using i - labeled bspam4 (i - bspam4). they found significantly higher amount of radioactivity in the tumor with i - bspam4 as compared with nontargeting i - labeled bsrit antibody and significantly greater tumor : nontumor ratios with i - bspam4 than directly radiolabeled pam4 f(ab')2 or pam4 whole igg. furthermore, it is demonstrated that both in - imp-156 and tc - imp-192 were suitable to detect pancreatic adenocarcinoma xenograft tumors pretargeted with bspam4. approximately 4 years later, gold and colleagues developed a novel humanized tri - fab bispecific antibody. the bispecific antibody, tf10, was divalent form mab - pam4 and monovalent for mab-679 and can react against the histamine - succinyl - glycine hapten [3436 ]. biodistribution studies and nuclear imaging of the radiolabeled tf10 and/or tf10-pretargeted hapten - peptide (imp-288) were conducted in nude mice bearing capan1 human pancreatic cancer xenografts. they found greater tumor : nontumor ratios for tf10-pretargeted in - imp-288 as compared with in - imp-288 alone and in - hpam4 alone and superior images for tf10-pretargeted in - imp-288 compared with in - imp-288 alone. moreover, tf10 cleared rapidly from the blood and nontumor tissues, while maintaining high signal strength at the tumor site. of special note, the majority of these tumors were 0.5 cm in diameter. these studies demonstrated the feasibility of the pretargeted bspam4 and tf10 for nuclear imaging of human pancreatic cancer xenograft tumors in nude mice. thus, pam4-based radioimmunoimaging is not only used for determining focal, size, range, and location of pancreatic cancer, but also for early detecting of the small lesions which can not be detected by conventional imaging modalities. besides the usefulness of pam4 as a target of radioimmunodiagnostic agents, radiolabeled pam4 has also been tested for selective treatment of pancreatic cancer. hpam4 radiolabelled with -emitting radioisotopes, such as yttrium-90 (y), has been used for the radioimmunotherapy (rait) of pancreatic cancer in clinical trials. animal studies consistently show that administration of i - pam4 to orthotopic transplants of capan1 tumors exhibited marked regression of tumors and significantly (p 50% and 27% of patients had a decrease of > 75% from baseline after the first cycle at all dose levels. the results of this study showed that y - hpam4 is safe and had antitumor activity in pancreatic cancer patients. pam4 is an lgg1 immunoglobulin that has limited reactivity with nonpancreatic cancers and is absent from the normal pancreas. pam4 is highly reactive with pancreatic adenocarcinoma and its precursor lesions, which makes it a good candidate for pancreatic cancer detection and therapy. in serum analysis, pam4 has a superior sensitivity and specificity for pancreatic cancer compared to ca19 - 9. combining pam4 and ca19 - 9 can lead to an improvement in diagnostic accuracy for discriminating pancreatic cancer from pancreatitis and pancreatic cancer from other solid tumors. taken together, pam4 not only is a good biomarker for pancreatic cancer diagnosis, but also might be a supplementary marker for pancreatic cancer diagnosis. thus far, preclinical and clinical trials of radiolabeled pam4 as a target of both immunodiagnostic and immunotherapeutic agents have shown great potential in the imaging and therapy of pancreatic adenocarcinoma. overall, pam4 is a promising new means to explore. in the near future, pam4 may be implemented into clinical routine for diagnosis, radioimmunodetection, radioimmunotherapy, and management of pancreatic adenocarcinomaand, possibly, may be seen in the field of radioimmunoguided surgery. | pam4, a new monoclonal antibody (mab) known as clivatuzumab, is highly reactive with pancreatic cancer and precursor lesions. it is absent from the normal tissues and has limited reactivity with nonpancreatic cancer. the detailed characteristic of the pam4 epitope is unknown but recent studies have shown that it is dependent on muc1 glycosylation status. the limited pam4 expression pattern makes it an attractive candidate for management of pancreatic adenocarcinoma. in addition, pam4 is a serum biomarker for diagnosis of pancreatic cancer. several different radiolabeled immunodiagnostic and immunotherapeutic agents of pam4 have been developed and some are being evaluated in preclinical and/or clinical studies. the review will focus on pam4 and its potential utility for the diagnosis, radioimmunodetection, and radioimmunotherapy of pancreatic cancer. |
cap polyposis was first described by williams. (1) in 1985. thereafter, about 60 sporadic cases of cap polyposis have been reported in the literature, but it is rarely observed in children. a case series showed that patients ' median age was 52 years and the age range was from 12 years to 76 years (2). (3) reported the youngest case of cap polyposis in a 5-year - old male in 2013. the most common site of polyposis is the rectosigmoid colon, but lesions have also been detected proximal to the colon and in the stomach (4). macroscopic findings include sessile and erosive polyps, dark red in color with fibrous, purulent, and mucoid discharge. an inflammatory and fibrinopurulent mucous cap of granulation tissue is a special histopathological feature of this disease. an epidermal nevus is a hamartoma which is defined as hyperplasia of the epidermis in any part of the body. an epidermal nevus manifests as a yellowish, discrete papillomatous plaque or papule, and is classified according to the cellular component. it may be classified into apocrine, eccrine, sebaceous, follicular, or keratinocytic type. epidermal nevus syndrome is a sporadic neurocutaneous disease with associated ectodermal defects in the brain, eyes, and skin (5). herein, we present an extremely rare case of cap polyposis in an infant with an epidermal nevus and provide a review of the literature. an 11-month - old male patient visited our pediatric department because of rectal prolapse and intermittent bloody stools, in september 2014. he presented with eventration of a reddish mass with intermittent fibrous and purulent exudation from the anus. he was born through cesarean section at 38 weeks of gestation, and his birth weight was 3,890 g. he had received all scheduled vaccinations. at 10 months of age, he was diagnosed with an epidermal nevus which was confirmed through biopsy at our hospital. 1). the patient 's parents reported no familial history of gastrointestinal polyps or carcinomas. this picture shows asymmetric, bright, brown colored, diffuse groups of verrucous plaque, and an elevated macular epidermal nevus located in the lower back and sacral area. laboratory test results were as follows : hemoglobin, 12.7 g / dl ; hematocrit, 37.6% ; platelets, 227,000/l ; protein, 6.8 g / dl ; albumin, 4.7 g / dl. the serum levels of tumor markers such as carcinoembryonic antigen (cea), -fetoprotein (afp), and ca 19 - 9 were within the normal range. plain radiographs of the abdomen and the chest showed normal findings. in the abdominal and pelvic computed tomography (ct) scan, well - enhanced multiple polypoid lesions, each about 1 cm in size, were seen at 4 to 6 cm above the anal verge. enlargement of regional lymph nodes and lymph nodes of both iliac chain areas was noted. ct images suggested the possibility of rectal cancer (t3, n1) with metastasis (fig. ct image shows well - enhanced, multiple polypoid lesions about 1 cm in size located at 5 cm above the anal verge. multiple lymph node enlargement was noted. further evaluation with barium enema study showed several polypoid lesions without stalks in the anus (fig. 3). a barium swallow study revealed normality of the stomach or the small bowel. 4). histological findings revealed reactive lymphoid hyperplasia and multiple polyps with hyperplastic crypts covered with granulation tissue, and showing dystrophic calcification (fig. he had no evidence of helicobacter pylori infection in the campylobacter - like organism test (clo test), a serologic antibody test. three multilobulated polyps were located 1 cm above the anal verge at the 7 o'clock position, 3 cm above the anal verge at the 5 o'clock position, and 3.5 cm above the anal verge at the 4 o'clock position, respectively. under general anesthesia, barium enema study shows multilobulated mass - like mucosal lesions in the recto - anal area. there was no evidence of mucosal destruction or obstruction from the rectum to the ascending colon. multilobulated, 46 cm in size polypoid groups were seen from the anus to the proximal rectum on colonoscopic view. hyperplastic crypts covered with calcified granulation tissue were revealed on histologic examination (h & e, 100). after mesalazine enema was administered, his clinical symptoms improved, and he no longer had bloody stools. at two months after recurrence about 1-cm in size small remnant mucosal polypoid lesions were seen at the inner anus on follow - up endoscopy. there are several case reports of cap polyposis in adults, but only few cases have been reported in children. in particular, a case of cap polyposis in an infant has not been reported. we herein described a case of an 11-month - old infant with localized cap polyposis in the rectoanal area causing hematochezia. he may be the youngest patient diagnosed with cap polyposis in the literature to date. in infants, more common causes of bloody and mucoid stools are infectious gastroenteritis, intussusception, anal fissure, and allergic proctocolitis. however, he had moderate amount of hematochezia and mucoid stools. suspected causes of cap polyposis include abnormal motility of the colon, chronic straining at stool, mucosal prolapse, infectious agents such as h. pylori and immune disorders. naturally, the treatment of polyposis also varies according to the suspected cause, and the optimal management has not been established. the mucosal lesions in some patients were healed with metronidazole, suggesting that an infectious pathogen might play a role in focal inflammation (6). but suzuki. (7) reported that the efficacy rate of metronidazole treatment was only 28.6% (6/21 cases). although the results were from only small reports, the effectiveness of metronidazole as single therapy seemed unsatisfactory. in our case, there was no evidence of systemic inflammation such as leukocytosis or elevated c - reactive protein level. furthermore, no infectious organism was detected in the stool culture or multiplex pcr. h. pylori infection has been implicated in the etiology of cap polyposis (89). the researchers suggested that h. pylori eradication indicates successful management of these patients. in 2002, oiya. a 63-year - old man was diagnosed with cap polyposis and intestinal metaplasia associated with h. pylori by gastroscopy. after h. pylori eradication, follow - up colonoscopy revealed that the elevated mucosal cap lesions had disappeared. this indicated that the immune response to persistent h. pylori infection may be the cause of cap polyposis (8). in 2004, akamatsu. (9) reported three cases of cap polyposis that were treated successfully with eradication therapy. they reported that mntrier 's disease is related to h. pylori infection, and it has histological findings that are similar to cap polyposis (9). in 2014, suzuki. (7) reported that the efficacy of h. pylori eradication therapy in treating cap polyposis was 100% (14/14 cases), and they strongly recommended it. but in our case, the patient showed no evidence of h. pylori infection in gastric mucosal biopsy. mucosal prolapse syndrome is a group of benign chronic inflammatory diseases that includes rectal prolapse, solitary rectal ulcer syndrome, inflammatory polyps, and proctitis cystica profunda (10). these diseases are caused by an increase in anal pressure during defecation and chronic excessive straining due to constipation. cystica profunda of the rectum is characterized by fibromuscular obliteration, hyperplastic glands, and mucin cystic change in the submucosal layer (11). because of the histological similarity between mucosal prolapse syndrome and cap polyposis, chronic mechanical injury to the mucosa due to abnormal motility has been suggested as a crucial etiological factor in the pathogenesis of cap polyposis. the avoidance of excessive straining during defecation has been reported as a successful treatment strategy in some cases (12). on the other hand, some authors reported that chronic straining or constipation was noted in only 7 out of the 11 patients (64%) (13). also, the infant in our case report showed normal results in the anorectal motility study, no constipation, and no evidence of abnormal motility of the colon. steroids, aminosalicylates such as 5-asa, and infliximab have been used to treat cap polyposis. the author suggested that helper t - cell mediated inflammation is the mechanism for explaining the pharmacodynamic effectiveness of steroids or infliximab (14). however, there are many reports on the failure of steroids or infliximab (1315). even close observation without medication has resulted in spontaneous regression of polyposis (7). we speculate that the disease may have a self - limiting course or it may have many deteriorating causes. if patients have refractory or recurrent symptoms despite medical treatment, the attending surgeon should consider total colectomy (613). after complete polypectomy in patients with a few polyps (less than 10 polyps), patients are generally symptom - free for several years (13). in some patients, polyps regrow even after surgical removal. preoperative colonoscopy should be performed for identifying the current status of the disease and for planning the extent of surgical resection. if a patient has persistent symptoms for three months in spite of medical treatment, surgical removal should be performed (16). to the best of our knowledge, the relationship between cap polyposis and epidermal nevus has not been explored in the relevant literature. hence, some skin lesions are associated with intestinal diseases. for example, proteus syndrome is characterized by epidermal nevus and colonic hamartomas (17). a case of gardner syndrome with epidermal nevus reported by romiti. (18) is another example. gardner syndrome is a type of colonic adenomatous polyposis with extracolonic benign lesions that include cutaneous epidermal cysts, fibromas, osteomas, and dental abnormalities. in our case, there may be an association between sacral epidermal nevus and cap polyposis or there was simple incidental coexistence of the two conditions. because cap polyposis is a very rare disease, no large - scale study has been conducted, a case of cap polyposis in an infant. the infant in our case report remains in remission after 5-asa enema that was tried in response to postsurgical recurrence. | cap polyposis is extremely rare in children. we report a case of an 11-month - old male infant who visited our hospital because of rectal prolapse and small amount of hematochezia lasting several days. he also had an epidermal nevus in the sacral area. colonoscopy showed erythematous, multilobulated, circumferential, polypoid lesions with mucoid discharge from the rectum. he was diagnosed with cap polyposis by endoscopy and histologic examination. he was treated with surgical resection, and was closely followed up. in the relevant literature, there is no report of cap polyposis in an infant. we report the first case of cap polyposis in the youngest infant. |
proteus syndrome (ps) is a rare postnatal overgrowth disorder affecting multiple tissue including bone, soft - tissue, and skin. it is a complex hamartomatous condition characterized by partial gigantism of hands, feet or both, plantar hyperplasia, hemangiomas, lipomas, lymphangiomas, varicosities, verrucous epidermal nevi, macrocephaly ; cranial exostosis, and asymmetry of the limbs because of long bone overgrowth. until recently approximately 200 cases of ps has been reported in literature. so we thought it would be relevant to report this case series as several aspects of this condition are important for pediatricians, as pediatricians are the first person to encounter such cases. over a period of 3 years between january 2008 and december 2010, we retrospectively reviewed the case profile of nine patients who presented with localized over - growth and/or hemihypertrophy in the out - patient clinic of genetic and metabolic unit of department of pediatrics. three cases were excluded from the study because their clinical features were consistent with other diagnosis. detailed clinical examination was done for extent of overgrowth, other associated manifestations, anthropometric profile, and complications. the diagnosis of ps was kept on the basis of clinical features, and radiological findings as per the criteria recommended by biesecker. among the nine short - listed cases, one patient had hemi - hypertrophy and he had history of hypoglycemia and omphalocele at birth, so he was diagnosed as a case of " beckwith weidemann syndrome " (bws) and excluded from analysis. two cases were diagnosed as " macrocephaly - capillary malformation syndrome " (m - cm) as per diagnostic criteria described by martnez - glez. of the six patients included in final analysis ; two patients were male and four were female. most common presenting features which brought them to notice of the clinician were focal over - growth and asymmetry. hemihypertrophy was noticed in one patient at birth, and focal over - growths in rest of other patients were noticed later in life. the clinical profiles of each of these cases are presented in [table 1 ]. three patients had dysmorphic facies, one patient had strawberry hemangioma on face and one patient had lipoma over scapular area. cerebriform connective tissue nevi (cctn) were seen in two patients at palmar aspect. two patients had developmental delay ; and among them magnetic resonance imaging (mri) was suggestive of megalencephaly in one and lissencephaly in other (case 2 and 5, respectively) [table 1 ]. sonography of abdomen for internal organ visceromegaly was done in all patients but visceromegaly or internal hemangiomas were not detected. a 10-year - old girl presented with facial deformity, swelling over forehead, hemihypertrophy involving lower limbs, macrodactyly [figure 1a ] and pigmentation anomalies and her x - ray showed macrodactyly [figure 1b ], tibial bowing, and hyperostosis in skull [figure 2 ]. (a) asymmetric and disproportionate over growth of right hand fingers with macrodactyly in a patient with proteus syndrome ; (b) x - ray of both the hands showing bony over growth in the same patient x - ray skull showing hyperostotic lesion over vertex (arrow) in a girl with proteus syndrome clinical and demographic profile of six patients with proteus and proteus like syndromes hallmark of this disorder is the random or mosaic distribution of its manifestations throughout the body. klippel - trnaunay syndrome and hemi hyperplasia - multiple lipomatosis syndromes are the entities most commonly confused with ps. other disorders like bws, banayan - zonana, neurofibromatosis type 1, encephalocraniocutaneous lipomatosis and m - cm syndrome also have similarities to ps. signs of this sporadic syndrome may include over - growth of the long bones, asymmetric macrocephaly, asymmetric over - growth, vertebral anomalies, hyperostosis, partial gigantism of hands or feet, limb asymmetry, connective - tissue nevi, lipomas, and vascular malformations. visceral anomalies such as splenomegaly, asymmetric megalencephaly, white - matter abnormalities, and nephromegaly, as well as malformations involving fatty, muscular, and vascular tissue are seen. category a criteria was fulfilled by only two patients who had cctn at palmar aspect. cctn is hall - mark of the disease, and it can occur on plantar surface of foot, palmar aspect of hand, or rarely on chest, abdomen, back, lateral and dorsal aspect of fingers, and on nose. cctn is never reported at birth, usually appears later in life and it is progressive throughout childhood. dermatological manifestations of ps as reported by beachkofsky., cctn was present among 94% of their 36 patients. among category b, although epidermal nevus was not seen in any of our patients, pigmentation abnormality was present in all : caf - au - lait spots and areas of patchy hyperpigmentation with hypopigmentation. other dermatological manifestation reported are epidermal nevi, cutaneous vascular malformations including capillary, venous, lymphatic or combined malformation, lipoma, partial lipohypoplasia and patchy dermal hyperplasia. extracutaneous manifestations include skeletal and visceral over - growth, tumors, cyst, vascular abnormality, deformity, and hypoplasia or maldevelopment. all patients usually have disproportionate overgrowth of hands and feet and it may involve fingers and toes in the form of macrodactyly. overgrowth in ps is not only disproportionate and asymmetric, but also progressive and distorting. four patients had hemihypertrophy and four had macrodactyly involving fingers as well as toes. in two patients, underlying bony overgrowths disproportionate overgrowth was present at birth in one patient and among rest it was observed in later life. abnormalities of bony cortex with deficiency of overlying soft tissue can be seen in radiograph. one girl in present series had hyperostosis on vertex of skull [figure 2 ]. other tumors which are specific of ps like bilateral ovarian cystadenoma and parotid monomorphic adenoma usually seen in second decade of life. other tumors reported are meningioma, testicular tumor, astrocytoma, optic nerve tumor, breast intraductal papilloma and they also can have simultaneously multiple tumors. one had coarse facies, prominent forehead, widely spaced eyes, depressed nasal bridge, and open mouth. two other patients had asymmetrical facies with large head, large ears and low set ear on the side of hemimhypertophy. she also had kyphoscoliosis, right sided mammary hyperplasia as well as enlargement of right sided labia (case 2). one more patient who presented at 2 month of age had mri changes suggestive of lissencephaly. up to 40% of patients can have central nervous system (cns) manifestations including mental retardation and 71% can have kyphoscoliosis and chest wall asymmetry. when the features of ps are fully established, the diagnosis is not difficult, however, in early childhood, tissue over - growth, epidermal nevi or vascular malformation may not be evident. characteristic lesions may not be seen at birth, only they can have subtle facial asymmetry or mild hemihyperplasia. the characteristic asymmetrical disproportionate over - growth is seen in 17% patients and 43% have epidermal nevi or vascular malformation at birth. therefore, diagnosis is usually delayed until the lesions are fully expressed in later childhood or adolescence. a somatic mutation arises in a somatic cell and is thus present only in that cell and the lineages to which it gives rise, rather than being present in the conceptus and thus constitutively present in every cell of the body. patients with limb length discrepancy, joint immobility and macrodactyly are best managed by team consisting of pediatrician, orthopedic surgeon, plastic surgeon and occupational therapist. ps is characterized by progressive mosaic over - growth of skin, bone, muscle, and fatty tissue. a multidisciplinary approach is needed with follow - up for detection and management of complications. | objective : proteus syndrome (ps) is characterized by patchy or segmental overgrowth and hyperplasia of multiple tissues and organs, along with susceptibility to development of tumors. very few cases are reported in literature from developing countries. due to certain overlapping features with other overgrowth syndromes, diagnosis is usually delayed. our aim was to describe clinical profile of this rare condition in six patients.materials and methods : retrospective case sheet review of patients followed in a pediatric genetic and metabolic clinic at a tertiary care institute of north india with a diagnosis of hemihypertrophy / overgrowth syndrome.results:six cases presented with asymmetric overgrowth and peculiar features suggestive of ps were included in this study. age at presentation was 2 months to 10 years ; two were males and four were females. hemihypertrophy was noticed in only one case at birth, and focal overgrowths in rest of other patients were seen later during childhood.conclusion:due to certain overlapping features with other overgrowth syndromes, diagnosis of ps is usually delayed. pediatricians are the first persons who come across such patients and they should be aware about this rare condition. |
in previous work, extending over several years, we found that x - ray fluorescence analysis (xrf) of high - purity copper (99.98%wt) subjected to hot metal tailoring typically indicates appreciable amounts (up to 1%wt) of sensitized elements (ses), including na, s, al, cl, k, and ca, not present at such significant levels in the untailored material. the tailoring appears to markedly enhance the intensity of xrf emissions, thereby making minor impurities seem far larger. the tailoring also induces substantial changes in properties such as melting point, hardness, color, resistivity, hall effect, specific heat, dc magnetization, and ac susceptibility, examined over a wide temperature range. these have in common adding small amounts of graphite to molten cu, cycling the melt over temperature ranges extending below and above carbon saturation, flushing the melt with inert gases (n2, he, ne, ar, kr), and cooling to solidify. the protocols differ in cycle and overall durations, gas mixture compositions and flow rates, and use or not of irradiating light sources. although some of the property changes induced by tailoring resemble those familiar for impure copper, some features differ markedly. moreover, over a span of time ranging from minutes to years some se concentrations in a given sample were found to decay by factors varying from 2 to 10. the evidence that tailoring does introduce ses and change properties is now extensive, but how this comes about is not understood. recently, we have obtained much superior xrf instrumentation which has enabled more incisive analysis. this paper reports results obtained thereby for copper treated by a more rudimentary version of tailoring, which sufficed to produce in the ingot surface many small regions that are magnetically active. we find the xrf spectra of these regions exhibit novel features, including a contiguous series of ses extending from v, cr, mn, fe, to co. auxiliary tests show this series can not be attributed to bragg peaks or mere aggregation of impurities. among the novel properties are time - dependence of the positions and se content of the magnetic regions and correlations between increases in fluorescence from ses with decreases in the fluorescence from copper. these observations provide more compelling evidence for ses but how tailoring introduces such sensitization effects awaits theoretical explanation. we first outline our previous tailoring protocols, which also produced copper ingots with magnetically active regions, in order to place in context the simplified version employed in the work reported in this paper. figure 1 provides a flowchart, displaying operations involved in three versions of tailoring. in each, copper was heated to its melting point and above using an induction furnace lined with a high - purity (99.68%wt) alumina crucible. small amounts of high - purity graphite (99.9995%wt) were added via a lance. the molten metal was then sequentially subjected to temperature sweeps (t - sweeps), designed to vary the carbon concentration in a periodic manner, typically sinusoidal, to create subsaturated (under) and supersaturated (over) solutions. typically, the initial t - sweeps are subsaturation, after which the melt is held at equilibrium corresponding to carbon saturation for a specified time (hold), and further sweeps then made under supersaturated conditions. finally, cooling to solidification occurs via a specified time and temperature profile. the protocols differ in sub - events imposed during the warm - up, t - sweeps, hold, and cooling stages ; the variants involve injecting inert gases, often mixtures, or varying the direct current (via voltage), power, or frequency supplied to the material within the crucible enclosed by the induction furnace. in most of the experiments reported here, no graphite was added to the copper, and gas injections, as well as current, power, or frequency variations were likewise not used. the reactor was a 100-lb induction furnace (inductotherm), fitted with a 73 - 30r powertrak power supply with an attached induction coil (38.1 cm o.d. the active zone of the coil comprised 8 active wraps (total height 31.1 cm). there is a single inactive cooling wrap above (by 5.7 cm) the top active wrap and another inactive cooling wrap below (by 6.0 cm) the bottom active wrap. the furnace had a high - purity (99.99%wt) graphite cap and a ceramic liner, a cylindrical alumina - based crucible (99.68%wt al2o3, 0.07%wt sio2, 0.08%wt fe203, 0.04%wt cao, 0.12%wt na20 ; 4.5 in. the reactor was loaded with 9080 g of copper (99.98%wt) through its charging port. a slight positive pressure (at 3.45 kpa) of high - purity ar (99.9997%vol) was maintained in the reactor using a continuous backspace purge. the induction furnace was programmed to heat the crucible to 1623 k over a 16.5 h interval, at a rate no greater than 83 c / h, as limited by the integrity of the crucible. the furnace operated at a frequency (in the range 0 3000 hz) determined by a temperature controlled feedback loop governed by a control unit (omega model cn300). after the 16.5 h heat up, a 3 h hold was programmed. for the runs used in this work (designated 14 - 02 - 05 and 14 - 08 - 04, a replicate), the programmed crucible temperature (1623 k) was not attained, even after applying full power (40 kw) for 3 h. the resultant power ramp up imposed via the induction furnace control loop resulted in asymmetric thermal cycling of the material similar to the thermal sweeps used in the usual tailoring.(3) the final temperature achieved in runs 14 - 02 - 05 and 14 - 08 - 04 was 1503 and 1578 k, respectively. all sample cleaning and preparation conformed to industry - established standards. depending on the nature of the surface, some materials are flycut (flatness defined by a maximum elevation change of 10 m) and then cleaned with isopropyl alcohol. for xrf and some other analytical methods, samples were prepared by cutting a cube shape (3.0 cm) from the center of the cooled ingot. an axial and a radial edge are then denoted according to the cylindrical geometry of the crucible. to provide a smooth surface for analysis, the axial and radial faces are either flycut according the nist xrf standards or in rare cases, sequentially polished to obtain an even flatter surface. in the latter case, the sample faces are sequentially polished first with 80, 120, and 340 alumina grit polishing compounds, then followed by 400 and 600 silica polishing compounds, and finished with a 15 m diamond paste. in all cases, the sample is cleaned after surface preparation with isopropyl alcohol and then placed in a cassette / holder. sample handling is performed using clean gloves and integrity is maintained in sample cassette history for subsequent experiments. these experimental protocols have been experimentally and statistically validated to maintain an appropriate level of cleanliness. our previous work used an arl 8410 xrf instrument to analyze the elemental composition of the sample ingots. the arl uses a standardless uniquant algorithm to detect and quantify the presence of various elements, chiefly based on the k line of each element, although secondary and tertiary lines are used as necessary. the arl detects elements from na to u, with a lower detection limit of about 20 ppm. this instrument is widely used for routine analysis due to its excellent energy resolution (1 ev), but its poor spatial resolution, defined by a 29 mm elliptical mask, and lack of scanning capability limited its value for our work. most spectra reported in this paper were obtained using a portable micro - x - ray fluorescence energy dispersive spectrometer (bruker axs artax), mounted on a mobile head capable of scanning along x and y axes. this enables automatic measuring cycles at a single point or along a specified line or over an area of 45 45 mm. the fluorescence excited by a collimated or focused x - ray beam is monitored by a cooled semiconductor detector, which converts the emission into current pulses that are amplified and digitized in a preamplifier and an x - ray digital signal processor, xspv. the xspv is the primary processor for measurement, control, regulation, and communication. a spectroscopic signal amplifier and processor (xdsp) transfers the digital signal to a computer which stores the data. the computer also evaluates the relative elemental composition of the sample, taking account of the excitation energy, detector calibration, and spectral intensity distribution of the fluorescence lines. the artax instrument employs alternate excitation x - ray tubes (w, cr, and mo) ; these provide means to more precisely characterize spectral regions of interest and eliminate false readings that otherwise can arise from the detector picking up radiation from the excitation element. filters made of metal foils (e.g., al, ni, mo, zr, etc.) are used to absorb x - rays coming from the x - ray tube to eliminate those energies from the backscattered radiation from the sample. the filters, mounted on a slide which permits easy exchange and precise positioning, are matched to the interactions of interest. when using the arl, care is taken to ensure the sample is tight fit in the holder to maintain reproducibility of the scanning surface. the orientation of the detector crystal with respect to the sample and the photon detector is controlled synchronously such that characteristic x - ray lines can be measured. a sequential measurement consists of positioning the diffraction crystal at a given angle and the detector at 2 and counting for a given period of time. the crystal and detector are then rotated to a different angle for the next characteristic x - ray line. uniquant version 2 software (omega data systems) controls the crystal and detector placement and reduces the data to a list of elemental composition with the associated concentrations. the artax instrument is much more versatile by virtue of its ability to scan continuously small spatial locations on the samples and the energy of fluorescence photons.(4) samples are placed flat under the artax head and then the area scanned. the artax unit has a method for focusing to allow for optimum detection by setting the proper distance between the sample and the x - ray tube / detector. this also means that all readings are taken with the same geometry, resulting in more consistent results from sample to sample. samples can be analyzed by measuring a single point, a line of points, or an area of points. the artax head moves to the correct position and waits a predetermined time for head stabilization, then detects the x - ray emissions coming from the sample for a preset interval. the operator can set the desired spacing between points and the amount of time that the unit records x - ray emissions at each point. the x - ray spectra can be viewed in real time and output into microsoft excel. figure 2 shows the excitation spectra as generated by the artax ed - xrf using a tungsten (w) tube (shown in red) and a molybdenum (mo) tube (shown in green), both with a 315 m al filter directed at a polyethylene target in air. for the region most pertinent to our experiments, 59 kev, the background is extremely low. all x - rays below 8 kev were filtered by the 315 m al filter. artax ed - xrf excitation spectra taken with a polyethylene base in air with a 0 angle of incidence. mo tube (green) used a 60m polycapillary lens (70 m average observation region), 40 kv, 700 a, 120 s. scans. w tube (red) used a 650 m collimator, 40 kv, 1000 a, 120 s scans. sensitized elements as observed in our previous work using the arl instrument.(2) xrf results for copper, after treatment by the tailoring protocols of figure 1, are compared with those for an untreated sample. the lower detection limit (ldl) the concentrations indicated for most impurities in the untreated copper are below or not much above the ldl. in contrast, for the tailored samples, several elements, among them na, al, s, cl, mn, and fe, appear to be present at levels 1 or 2 orders of magnitude above those in the untreated copper. for both the tailored and untailored cases, the samples were cut from the interior of ingots (as specified above) to ensure that the striking differences seen in the tailored samples do not arise from concentration of impurities near the ingot surface. sensitized elements, from comparison of arl wd - xrf elemental analysis results for copper tailored by variations of the three protocols shown in figure 1. symbols denote data for protocols 1, ; 2, ; 3, ; obtained in runs designated 03 - 99 - 013 ; 03 - 99 - 021a ; 03 - 99 - 021b, respectively. untailored sample () designated 14 - 08 - 01 is also plotted. the latter sample was held at temperatures comparable to the tailored runs for 3 days in the standard high alumina crucible. reported concentrations are the average of all detections above the lower detection limit (20 ppm) obtained from 10 different arl wd - xrf analyses of the same sample. both the tailored samples and the untreated copper were also analyzed by high - resolution glow discharge mass spectrometry (gd - ms). this found contaminant levels consistent with the untreated copper and, for the tailored samples (14 - 00 - 01, subsequently referenced as ingot a), much lower than indicated by the arl wd - xrf results (figure 4). gd - ms measures bulk composition whereas arl wd - xrf probes only to modest depths (e.g., 60 m in high - purity copper). thus, there was concern that tailoring might foster concentration of impurities near the surface or otherwise somehow amplify their apparent concentrations as measured by arl wd - xrf. therefore, as in figure 3, all data for figure 4 were obtained from samples cut from the interior of ingots. as a test, the untailored (natural) copper was melted and held in the same system reactor (high alumina crucible liner) in order to have a blank that should have been subjected to the same potential sources of contamination and contaminant distribution profile as the tailored materials. ten arl analyses of this blank (14 - 08 - 01) were performed and averaged. the results were consistent with the gd - ms, as well as the known impurities in the natural copper, but as seen in figures 3 and 4, indeed indicated concentrations well below what xrf finds in the tailored samples. comparison of wd arl xrf elemental analysis and gd - ms elemental analysis results for tailored copper showing significantly different results. symbols denote data for gd - ms analysis of 14 - 00 - 01 (axial,) and (radial,) ; xrf analysis of 14 - 00 - 01 (axial,) and (radial,) ; and () denotes the average composition of the untailored starting material as determined by gd - ms. for ga, the average was taken to be the ldl as none was detected in the starting material. by comparison to other melted systems, ga in 14 - 08 - 01 of figure 3, was identified as 0.14 ppm. protocol 1 of figure 1 was used to prepare 14 - 00 - 01. in this paper, we shall consider almost solely properties of magnetically active regions (spots) introduced by tailoring copper. figure 5 displays a striking example, in which such spots appear as discrete, roughly evenly spaced points, along an approximately sinusoidal curve. the spots (typically 2.5 mm dia) are not visible to the eye or on microscopic examination but are found, at room temperature, to exhibit equivalent attraction to both poles of a 1/8 in. some spots attract iron filings ; others do not. also, a few spots show an appreciable but partial meissner effect, tilting the probe magnet disk by 3545. gauss meter measurements give an essentially zero reading for all spots, however. on cooling the ingot via liquid nitrogen (to 77 k), the attraction for filings and the partial meissner effect increases for some spots and weakens for others, but these variations do not exhibit a regular pattern. a further curious property is the metastable character of the spots : both their attractive strength and location on the surface of the ingot change with time. for instance, over 8 years, several seemingly typical spots were observed to either disappear completely and then reappear at a later time or to migrate away from their original positions by distances of 25 mm in most cases and in others by distances exceeding 1 cm. distribution of magnetically active spots that appeared in tailored copper (run 14 - 00 - 01, using protocol designated tailored-1 in figure 1). 360 view shown. in close - up view, arrows point to five spots that are holding 1/8 in. spots of magnetic attraction are displayed for tailored copper ingot 14 - 00 - 01 (see figure 5) for the years 2000 and 2008. red dots denote 2000 data, black dots denote 2008 data. some spots (enclosed in red boxes) appear identical (i.e., moved less than 1 mm). some clustered spots seen in 2000 (purple oval) had disappeared by 2008, while three new clusters appeared (orange ovals). the grid height is 2.625 in., and the grid length is 23.25 in. 360 maps, made in 2000 and 2008, of the spots showing magnetic attraction on the surface of the ingot pictured in figure 5. over this 8-year span, some spots appeared to stay in place (moved less than 1 mm) ; there were about six such spots (in overlay enclosed in red boxes : two between 225240, two at 255, one near 275, and one near 115). about 14 other spots remained very close (within 3 mm) ; the displacements did not exhibit any systematic shift. a cluster of points seen in 2000 (purple oval, near 345) had by 2008 disappeared, while three new clusters appeared (orange ovals, near 30, 45, and 150). figure 5) had become less evident in 2008, although some patterning persists (e.g., series of spots between 15 and 45 and between 75 and 105, both marked with blue arrows). in view of the potential for contaminants in the system to aggregate at the surface (cf. appendix b) or accumulate at grain boundaries during cooling, we carried out an artax ed - xrf elemental analysis across multiple grain boundaries on the ingot surface at four randomly chosen locations. each of these line scans consisted of 26 sequential measurements taken in series with 50% overlap spanning the clearly visible grain boundaries. no signs of agglomeration or accumulation of known ferromagnetic contaminants was found to occur along or within the grain boundaries. we also tested for contamination effects by deliberately adding 3 g of fe to a molten cu sample (7 kg), thereby introducing fe at a level (400 ppm) comparable to the nominal levels of sensitized fe seen in tailored ingots (cf. figures 3 and 4). samples cut from either the surface or the interior of the resulting ingot did not exhibit magnetic spots or enhanced intensity or variation with time of xrf emissions. until obtaining the state - of - the - art artax instrumentation, we were unable to examine closely, with high spatial resolution, the magnetic spots by means of xrf spectroscopy. we now report results of artax analysis that exemplify a variety of properties of such spots. some appear on the ingot pictured in figure 5 (designated, for brevity, as ingot a), others on another ingot tailored by the same protocol (ingot b). also, we report xrf spectra and magnetically active regions in copper treated by our simplest version of tailoring (ingots c and d). indeed, that simplest protocol produces a higher density of magnetic spots with a richer content of sensitized elements. for the a ingot (14 - 00 - 01), we carried out an xrf analysis of two spots : one, denoted a1, attracted both our nd / fe / b probe magnet and fe filings ; the other, denoted a2, attracted the probe magnet more strongly but did not attract fe filings. these were compared with a nearby null point on the ingot that did not have a magnetic response. the xrf spectra showed the nominal concentration of iron in both a1 and a2 was much higher than the null point, but with a dramatic time variation, as seen in figure 7. initially, the fe k line (actually a superposition of lines denoted k1 and k2) was about twice as strong for spot a2 as a1 ; 22 h later, this ratio was reversed and 250 h later the ratio had become unity. xrf spectra of a ingot (14 - 00 - 01) in the region of fe k and k lines. note, a si escape peak is visible at 6.3 kev in the uncorrected null spectra shown. the odd changes with time led us to perform a remelt experiment to ascertain the behavior of the magnetism. an ingot designated a (14 - 08 - 10) was tailored via the simplest protocol and found to have magnetic spots, with density comparable to the a ingot. after solidification, magnetic spots reappeared, with similar density. for the b ingot (14 - 00 - 03), the high spatial resolution of the artax instrument made it feasible to survey the fe k and k lines at four points within each of two closely neighboring magnetic spots, denoted b1 and b2. the ratio k/k for a given atom is determined by relative transition probabilities. in actual environments, the ratio can be affected (20%) by the x - ray excitation process (e.g., x - ray tube voltage and exciting x - ray beam filter), self - absorption within the sample, wavelength - dependent response, and efficiency of the crystal and detector. also, accurate fitting of line shapes is important and difficult. theoretical k/k values of 7.91 and 7.25, respectively, were obtained from relativistic hartreefock calculations.(5) experimental values of the k/k ratios for pure elemental fe and cu were found by han,. to be 5.62 and 6.91, respectively.(6) fe k/k ranges between 5.6 and 6.3 for a variety of xrf reference systems with variable excitation sources and detector types.(7) we confirmed agreement with those ratios for systems comparable to our systems, e.g., fe seeded in high - purity copper and the high - purity copper nist standards c1122 and c1251a. variations in the measured k/k ratio are found to be very small (40 ppm, for co 10 ppm, and for ni 60 ppm can be achieved with a properly calibrated quantification routine using known standards of similar composition. however, for normal artax operating conditions, the ldls are much higher. using the same conditions employed to obtain the spectra of figure 8 (and others in this paper), we found that the artax did not detect fe in a nist standard (c1251) that contained 285 ppm fe. these observations reinforce many others we have made that for ses tailoring greatly enhances xrf emission intensities. another exceptional aspect is the large variation of the intensity of the cu k peak at different points of the grid, despite the great preponderance of copper over fe and the other ses. decreases in the cu intensity appear to be correlated, with increases in the intensity of the fe k peak (and less clearly the other ses). similar plots made using only the counts for each individual se are also fairly linear, but with modestly different slopes. (a) profile map showing plot of fe k intensity over the ed - xrf scan grid for a magnetic spot region on ingot c (14 - 02 - 05) ; the location of the spot is near the edge of the bottom of the ingot (within yellow circle in figure 11, upper right). arrows indicate direction in which the artax head moves in scanning, left to right, bottom to top. (b) plot showing approximately linear correlation between decrease in xrf photon counts for cu k emissions and increase in fe k emissions. the correlation coefficient is r = 0.90. the data points are from scans made at each of the 60 vertices of the scanning grid. for eight points (denoted by open circles,) large zr emissions (> 1000 counts) were observed. these points were not included in evaluating r. since zr is present in the reactor containment system (in insulation above the lid of the crucible), its appearance indicates contamination by a speck of refractory material. figure 10 contrasts the striking inverse coordination of the xrf emissions from cu and fe in the tailored c ingot with the totally smooth and indifferent behavior obtained for a nist berylliumcopper standard sample (srm c1122). it contains 97.4% copper and less than 0.16% iron but much more fe than the 99.98% copper used as our starting material. thus, the results for the standard indicate what might be expected from a nontailored ingot, even one with far more iron than our ingots have. the very steady results exhibited by this standard are indeed usual with the artax instrument (electronic stability < 1%) when applied to a flat material (surface fluctuation < such a large modulation of the cu xrf intensity as seen in figures 9 and 10 is unprecedented.(8) we therefore obtained further spectra and considered other potential explanations. (left) ratio of xrf photon counts to total spectrum counts for cu (green points) and for fe (red points), compiled from data of figure 8 for a magnetic spot on ingot c. each pair of points (green and red) represents a single spectrum from a point on the scanning grid of figure 9a. 17, 27, 37, and 47) the cu emission drops while the corresponding fe emission climbs. (right) analogous data for cu (green points) and fe (red points) from a nist c1122 standard sample. all artax analysis parameters were identical for runs with ingot c and the nist standard : w tube, 650 m collimator, 40 kv, 1000 a, 315 m al filter, 40 s / point scans. figure 11 shows five xrf spectra from another magnetic spot on ingot c. the sample preparation and artax analysis were conducted in the same way as for figure 8 and the scanning grid is similar. the emission peaks correspond to the k transitions for v, cr, mn, fe, and co, contiguous elements in the periodic table. here we use a normalization scheme that adjusts the ordinate scale for each xrf feature to the highest signal pulse level it attains in scanning the five spectra. this brings out the remarkably parallel variation of the cu k and k intensities and the opposite variation of that for the emissions from fe and its contiguous ses. in ordinary displays of spectra, using linear ordinate scales, (a) xrf spectra for ingot c for a magnetically active spot located (upper right, yellow circle) near the edge of the bottom of the ingot. (b) grid pattern in format like figure 8 ; white dots mark start (no. 175) and end (no. 179) of grid positions for the five spectra scans (color - coded). for each element, the ordinate scale has been normalized to the intensity observed at no. 177, in order to exhibit most clearly that the cu k and k intensities vary the same way but opposite to the se intensities. we also examined ingot d (14 - 08 - 04), which likewise was produced by the simplest tailoring protocol. figure 12 shows xrf spectra obtained from six distinct grid points within a rather large magnetically active region. again, emissions corresponding to the contiguous series v through co are seen ; in this case, however, the cr k is more prominent than that for fe and co appears distinctly. xrf spectra for copper ingot d (14 - 08 - 04) for a magnetically active spot located (as pictured at upper center, yellow circle) near the edge of the ingot bottom. (b) plot showing fe k signal pulses detected across grid pattern for scanning over spot area. a broader xrf census of the magnetic spots, on ingots c and d, as well as those subjected to other tailoring procedures, finds wide variation in the elemental composition. some spots appear to contain only fe ; others just fe + co or fe + cr. the progression of several contiguous elements seen in figures 8, 11, and especially 12 is unusual but not unique. in xrf spectra, such a regular progression of emission peaks arouses suspicion because bragg scattering is a familiar source of extraneous emission peaks that arise from the crystal structure. x - ray diffraction studies performed by the evans analytical group (austin, tx) on samples cut from ingots c and d showed these have polycrystalline character with a strongly preferred orientation. accordingly, as described in appendix a, we carried out three standard tests for the presence of bragg scattering. is the remarkable attenuation of 1040% in the characteristic xrf emission of the cu k and k lines (as exhibited in figure 812), correlated with increase in emissions from sfe and contiguous ses. the suspicion is that agglomeration of impurity fe into the magnetic spots, due to azeotropic cooling occurring in tailoring, might filter the cu emissions, thus introducing an inverse correlation with the local fe concentration. as a further experimental test, we repeated the xrf analysis of the magnetic spot examined in figure 11, using the mo tube rather than the w tube and thereby improving the spatial resolution 10-fold, to about 70 m. as seen in figure 13, the results are similar to those obtained with the w tube, but even larger attenuations of the cu k and k emissions were observed, up to about 90%, with a concomitant growth in the sfe emissions. figure 14 compares the variation in pulse counts for the cu k emissions with those for sfe, recorded throughout the scanning grid, as shown in figure 13b. again, this plot exhibits an approximately quantitative and remarkably strong inverse correlation between the cu and sfe emissions. 379, 431, 483, 535, and 587) taken from the same magnetically active spot on ingot c as shown in figures 911 but obtained using a mo excitation tube, with parameters 40 kv, 700 a, 315 m al filter, 60 m polycapillary lens (70 m observation region), 120 s scan / spot, 50% overlap. note here that the ordinate scale displays the raw pulse counts so that the relative intensities of emissions from different species can be compared directly. (b) scanning grid pattern akin to that shown in figure 11 showing higher resolution obtained with mo vs w tube. spectra were obtained at points marked by aqua lines / white circles along the vertical path indicated. (c) overlay of mo scanned area (shown in green) on the spectral surface map obtained using the w tube (cf. artax ed - xrf pulse counts for cu k emissions (green) and corresponding sfe k emissions (red). each pair of points represents a single spectrum like those shown in figure 13, recorded at successive positions along four paths on the scanning grid (cf. the cu emissions drop (near scanning points no. 16, 27, 38, and 48) by as much as 90% whereas the sfe emissions climb in an approximately converse pattern. there is extensive and accurate information about attenuation of xrf spectra by thin metal filters, which influence both the penetration depth of the bombarding x - rays and escape of the fluorescence photons. as described in appendix b, those data indicate that a thickness of about 10 m of fe, located within 60 m of the surface, would be required to reduce the cu k emission by 90%. such a high concentration of iron in the magnetic spots would be clearly visible to the naked eye, but in fact the spots are not visible. moreover, if filtering is involved, the inverse correlation between cu and fe emissions should be an exponential rather than linear relation. the gd - ms analysis, as seen in figure 4, confirms that the impurity fe content of samples from the interior of our copper ingots, tailored or not, is only a few ppm. thus, an extremely efficient agglomeration process would be required to create local concentrations of fe several orders of magnitude higher in the magnetic spots as needed to appreciably attenuate xrf from copper. other phenomena familiar in xrf spectroscopy also appear untenable as explanations of the observed drastic attenuation of cu emission. to attain a 90% attenuation by simply diluting copper with iron in the magnetic spots secondary processes, in which a fluorescence photon from cu ejects a k - shell electron from fe would induce attenuation of the cu signal accompanied by enhanced fe fluorescence. such effects, however, are ordinarily insignificant and would require a much greater fe concentration to become a major factor. we know of no previous observation of thermal operations on copper that induce magnetic spots. probing with xrf the elemental composition of those spots, as well as that for samples from the interior of tailored ingots, has revealed very unusual features that invite the designation of sensitized elements. at present, this is merely a convenient shorthand term. it simply indicates that for those elements, xrf emissions appear with intensity far higher than would be expected from the amount present of that element, as determined by other reliable analytical means. we have relied chiefly (but not solely) on high - resolution gd - ms (lower detection limit typically < 0.05 ppm, much lower than for xrf) to monitor elemental composition of the bulk copper samples. multiple gd - ms analyses were obtained for raw chop material, melted raw materials (in graphite, alumina, and zirconia crucibles) and tailored samples. all these analyses (e.g., figure 3) show no appreciable differences between the untreated starting and tailored copper samples. moreover, xrf elemental analysis agrees (within its sensitivity) with the gd - ms results for untreated starting materials, as well as for a blank ingot that had been melted and held in the reactor for the same total time but not subjected to the thermal cycling used in tailoring runs. the thermal cycling involved in tailoring appears to somehow markedly enhance xrf emissions from ses. our experimental results pertaining to magnetic spots on copper are in accord with, but more striking than, evidence for ses found previously in our laboratory for other tailored materials, including aluminum, iron, cobalt, and silicon. the most startling aspect of the xrf from the spots is the drastic attenuation of cu emissions, inversely correlated with se emissions., we consider the most telling aspect is that for tailored samples, from ingot interiors rather than the surface, the arl xrf spectra indicate much larger amounts of the putative ses than the impurity content determined by gd - ms. this suggests that tailoring may strongly enhance the transition probabilities for emissions from the impurities (by amplifying x - ray ejection of k - shell electrons or the fluorescent refilling or both). if so, (e.g., much less fe might produce filtering and attenuation of cu emissions than would be required for untailored samples), a possibly important role of tailoring may be to foster chemical interactions of impurities with copper. however, e.g., copper could become directly involved in magnetic spots if such interactions introduce cupric ions, which have a (3d)(9) valence electron configuration. in current work this includes details of temporal variations that can now be examined because the new xrf tools enable us to track ses in real time. also, we have found that ses often are hypersensitive to sequenced irradiation by light that in untailored material has no effect. | when high - purity copper (99.98%wt) is melted, held in its liquid state for a few hours with iterative thermal cycling, then allowed to resolidify, the ingot surface is found to have many small regions that are magnetically active. x - ray fluorescence analysis of these regions exhibit remarkably intense lines from sensitized elements (se), including in part or fully the contiguous series v, cr, mn, fe, and co. the xrf emissions from se are far more intense than expected from known impurity levels. comparison with blanks and standards show that the thermal tailoring also introduces strongly enhanced se emissions in samples taken from the interior of the copper ingots. for some magnetic regions, the location as well as the se emissions, although persistent, vary irregularly with time. also, for some regions extraordinarily intense sensitized iron (sfe) emissions occur, accompanied by drastic attenuation of cu emissions. |
the world health organization (who) has defined osteoporosis as a skeletal disorder characterized by diminished bone strength resulting in increased fracture risk. bone strength is determined by interacting somatic and genetic factors. reported somatic factors include aging [25 ], menopause [5, 6 ], and body mass index (bmi) [4, 5, 7, 8 ]. to identify the genetic determinants of osteoporosis, an earlier study by the first author of this paper investigated how the incidence of low bone mineral density (bmd) in taiwanese women is affected by interactions among eleven single nucleotide polymorphisms (snps) in nine genes known to be involved in osteoporosis [1016 ], including tumor necrosis factor - alpha (tnf), transforming growth factor - beta 1 (tgfb1 ; tgf1), osteocalcin, parathyroid hormone (pth), interleukin 1 receptor antagonist (il1_ra), heat shock 70 kda protein 1-like (hspa1l ; hsp70 hom), heat shock 70 kda protein 1b (hspa1b ; hsp70 - 2), calcitonin receptor (ctr), and bone morphogenetic protein-4 (bmp-4). generally, several hormones, cytokines, and cell signaling - related proteins were chosen. for example, ctr, which is a receptor for the linear polypeptide hormone calcitonin, reduces blood calcium and suppresses the effects of pth. the cytokine family includes tnf, tgf-, bmp4 (protein of tgf-superfamily), and il-1ra (protein of interleukin 1cytokinefamily) whereas cell - signaling proteins include hsp70 hom and hsp70 - 2. studies of the interactions among these hormones (e.g., and references therein) indicate that osteoporosis is an endocrinological problem. accumulating evidence reveals that snps are potential genetic markers for predicting osteoporosis outcome in taiwanese women. chang. also proposed a novel odds ratio - based genetic algorithm (or - ga) method of using odds ratios for quantitatively measuring the disease risk associated with various snp combinations to determine the susceptibility to osteoporosis in taiwanese women. taiwanese women who are carriers of risk alleles in two or more of these snps are likely to be at increased risk of osteoporosis because several partial deficiencies in these pathways may severely diminish bone density. therefore, snps may indicate risk of osteoporosis in taiwanese women and may be useful in clinical association studies to determine the genetic basis of disease susceptibility. the risk of osteoporosis is likely to be higher than normal in carriers of risk alleles in two or more of these snps because several partial deficiencies in these pathways may substantially decrease bone density. therefore, interacting polymorphisms may affect osteoporosis risk. in, the effects of age, bmi, and eleven interacting polymorphisms in nine genes were studied in terms of their effects on the incidence of low bmd (table 1). the findings showed that specific snp combinations may be risk factors for postmenopausal osteoporosis in taiwanese women. in addition to these specific snp combinations, bmi and age also showed independent associations with bmd in postmenopausal taiwanese women. although an apparent association between snps and osteoporosis has been identified in taiwanese women, a continuing challenge in genomics studies of taiwanese women populations lies in identifying significant genes. exhaustive computation over the model space is infeasible if the model space is very large, as there are 2 models with p snps [20, 21 ]. feature selection techniques are designed to find responsible genes and snps for certain diseases. by selecting a small number of snps with significantly larger effects compared to other snps and by disregarding snps of lesser significance, researchers can focus on the most promising candidate genes and snps for use in diagnosis and therapy [21, 22 ]. in, combined polymorphisms in different genomic regions the findings showed that a combination of several gene polymorphisms contributes to the development of osteoporosis in taiwanese women. however, that study did not report a subset of snps that can be used to predict osteoporosis outcome in this population. therefore, the current study used the same dataset used in to elucidate the relationship between osteoporosis and snps in taiwanese women in a performance comparison of three different classification algorithms with wrapper - based feature selection : multilayer feedforward neural network (mfnn) [2428 ], naive bayes, and logistic regression. the mfnns have proven particularly effective for nonlinear mapping based on human knowledge and are now attracting interest for use in solving complex classification problems. an mfnn containing layers of simple computing nodes, which is analogous to brain neural networks, has proven effective for approximating nonlinear continuous functions and for revealing previously unknown relationships between given input and output variables [25, 26 ]. the unique structure of mfnns enables them to learn by using algorithms such as backpropagation and evolutionary algorithms [31, 32 ]. potential medical applications of mfnns include solving problems in which the relationship between independent variables and clinical outcome are poorly understood. because mfnns are capable of self - training with minimal human intervention, many studies of large epidemiology databases have, in addition to conventional statistical methods, used mfnns for further insight into the interrelationships among variables. a naive bayes classifier assumes that the presence (or absence) of a particular feature of a class is unrelated to the presence (or absence) of any other feature, given the class variable. depending on the precise nature of the probability model, naive bayes classifiers can be trained very efficiently in a supervised learning setting. the classifier obtained by using this set of discriminant functions and by estimating the relevant probabilities from the training set is often called the naive bayesian classifier because, if the the attributes are naively assumed to be independent given the class, direct application of the bayes theorem easily confirms that this classifier is optimal in terms of minimizing the misclassification rate or zero - one loss [34, 35 ]. logistic regression is a statistical method of predicting the outcome of a variable that is categorical (i.e., it can have several different categories) and is dependent on one or more predictor variables. a logistic function can be used to model the probabilities describing the possible outcome of a single trial as a function of explanatory variables. logistic regression is typically used to measure the relationship between a categorical dependent variable and one or more continuous independent variables by converting the dependent variable to probability scores. the wrapper - based feature selection method, in which the feature selection algorithm acts as a wrapper around the classification algorithm, was also used to identify an snp subset with sufficient predictive power to distinguish between high- and low - risk alleles. in the wrapper - based approach, the function used to evaluate feature subsets uses the classification algorithm itself to perform a best - first search for a good subset. starting from an empty feature set, it searches forward for potential feature subsets by performing greedy hillclimbing augmented with a backtracking technique.. showed that it may be superior to hybrid approaches combining chi - square and information - gain methods reported in the literature. a comprehensive literature review shows no attempts to predict osteoporosis outcome in taiwanese women using genetic factors (snps) and the three above mentioned classification algorithms with wrapper - based feature selection method. this study therefore compared performance in three classification algorithms : mfnn, naive bayes, and logistic regression, with and without wrapper - based feature selection techniques. identifying the genes and snps associated with taiwan population of women with osteoporosis would enable researchers to focus on the candidate genes and snps that are most promising for use in diagnosis and therapy. the results of our studies could be generalized to snp searches in genetic studies of human disorders and to development of new molecular diagnostic / prognostic tools. however, before routine application of genomic analysis in clinical practice, genetic markers must be validated in prospective clinical trials. the dataset in this study, which included snps, age, menopause, and bmi, was the same dataset used in a previous study by the first author of this paper. the t - score was calculated according to who classifications using a locally derived reference range provided by the manufacturer. the subjects were divided into two bmd groups according to t - score [3840 ]. subjects with t - score > 1 were enrolled in the high bmd group, and those with t - scores 1 were enrolled in the low bmd group. the overall dataset was derived from 295 cases, including (i) 247 postmenopausal cases (83.73%) and 48 prepremenopausal cases (16.27%) ; (ii) 112 high bmd cases (37.97%) and 183 low bmd cases (62.03%). post - menopause was defined as the absence of menstruation for > 6 months or age 50 years. clinical data used for diagnosis were further converted into numerical form, that is, 1 for high bmd and 0 for low bmd. table 1 shows the 22 snps analyzed in this study, which were the same as those analyzed previously by the first author of this paper. table 1 shows that the nine candidate genes included tnf, transforming growth factor - beta 1 (tgf1), osteocalcin, parathyroid hormone (pth), interleukin 1 receptor antagonist (il1_ra), hsp, calcitonin receptor (ctr), bone morphogenetic protein-4 (bmp-4), and three genotypes per locus. the three families of classification algorithms used as the basis for comparisons in this study were mfnn, naive bayes, and logistic regression. these classifiers were implemented using the waikato environment for knowledge analysis (weka) software. an mfnn is an artificial neural network (ann) model in which connections between the units do not form a directed cycle [2428, 30 ]. from an algorithmic perspective, the underlying process of an mfnn can be divided into retrieving and learning phases. assume an l - layer feedforward neural network with nl units at the lth layer. in the retrieving phase, the mfnn iterates through all layers to produce the retrieval response { ai(l), i = 1, 2,, nl } at the output layer based on test pattern inputs { ai(0), i = 1, 2,, n0 }, the known weights wij of the network, and the nonlinear activation function fi (e.g., sigmoid function). in the learning phase of this mfnn, the backpropagation algorithm and evolutionary algorithms [31, 32 ] are used in the learning scheme. the weight updating mechanism is a backpropagation of corrective signals from the output layer to the hidden layers. the goal is iteratively selecting a set of weights wij(l) for all layers such that the squared error function e can be minimized by a pair of input training patterns { ai(0), i = 1, 2,, n0 } and target training patterns { tj, j = 1, 2,, nl}. mathematically, the iterative gradient descent formulation for updating each specific weight wij(l) can be expressed by the following equation : (1)wij(l)wij(l)ewij(l), where is the learning rate and e/wij(l) can be effectively calculated through a numerical chain rule by backpropagating the error signal from the output layer to the input layer. structurally, however, an mfnn is a spatial and iterative neural network with several layers of hidden neuron units between the input and output neuron layers. the basic function of each neuron is the linear basis function, and activation is modeled with a non - decreasing and differentiable sigmoid function. inputs contain the information about clinical factors, for example, snps, that are needed for the database. the mfnn is trained first by repeatedly providing input - output training pairs and by executing the backpropagation learning algorithm. after this training process is complete, the mfnn is tested by sending testing data inputs (i.e., snps) to the network. the forward propagation of the mfnn reveals the osteoporosis outcome for a specific case so that causes can be inferred from effects. here, the default weka parameters were used, that is, hidden layer neurons = 6, learning rate = 0.3, momentum variable = 0.2, and training time = 500. second, all features in naive bayes, which is the simplest bayesian network, are assumed to be conditionally independent. let (x1, x2,, xp) be features (i.e., snps) used to predict class c (i.e., disease status, 1 = high bmd or 0 = low bmd). given a data instance with genotype (x1, x2,, xp), the best prediction of the disease class is given by class c, which maximizes the conditional probability pr(c = c | x1 = x1, x2 = x2,, xp = xp). bayes theorem is used to estimate the conditional probability pr(c = c | x1 = x1, x2 = x2,, xp = xp), which is decomposed into a product of conditional probabilities. third, the logistic regression generates the coefficients for the following formula used for logit transformation of the probability of a patient having a characteristic of interest : logit(p) = b0 + b1x1 + b2x2 + +bkxk. the formula used to calculate the probability of the characteristic of interest in this study is p = 1/(1 + e), where 1 = high bmd and 0 = low bmd. the wrapper - based feature selection approach, in which a feature selection algorithm acts as a wrapper around a classification algorithm, was used to find a subset of snps that maximizes the performance of the prediction model. figure 1 shows that, in the wrapper approach, the feature subset is selected by using a black box classification algorithm (i.e., selection is performed using the interface alone and does not require knowledge of the algorithm). to search for a good subset, the search space is organized such that each state represents a feature subset. for n features, each state has n bits, and each bit indicates whether a feature is present (1) or absent (0). to determine the connectivity between the states, this study used operators that add or delete a single feature from each state, where the states correspond to the search space commonly used in stepwise method. figure 2 shows an example of the state space and operators obtained by stepwise method in a four - feature problem. the classification algorithms are used to calculate a performance measure for each of 16 different subsets. therefore, the wrapper - based approach conducts a best - first search for a good subset by including the classification algorithm itself (mfnn, naive bayes, or logistic regression) in the feature subset evaluation. to search for potential feature subsets, the best - first search starts from an empty feature set and searches forward by greedy hillclimbing augmented with a backtracking technique. figure 3 shows how mfnn, naive bayes, and logistic regression were applied in the wrapper - based approach. the performance of the prediction models was measured in terms of receiver operating characteristic (roc) and area under the roc curve (auc). the auc of a classifier can be interpreted as the probability of the classifier ranking a randomly chosen positive example higher than a randomly chosen negative one. most researchers have now adopted auc for evaluating the predictive capability of classifiers since auc is a better performance metric compared to accuracy. this study used the auc value for performance comparison of different prediction models using the same dataset. the higher the auc, the better the learning performance. other calculations included sensitivity, the proportion of correctly predicted responders out of all tested responders, and specificity, the proportion of correctly predicted nonresponders out of all tested nonresponders. to investigate the generalization of the prediction models produced by the above algorithms, the repeated 10-fold cross - validation method was used the model was then trained with nine - tenths of the data and tested by the remaining tenth of data to estimate its predictive performance. each time, a different tenth of the data was used as testing data, and a different nine - tenths of the data were used as training data. finally, the average estimate over all runs was reported by running the above regular 10-fold cross - validation 100 times with different splits of data. in repeated 10-fold cross - validation testing, the performance of all models was evaluated with and without feature selection. tables 3 and 4 summarize the results of the repeated 10-fold cross - validation experiments for mfnn, naive bayes, and logistic regression using snps with and without feature selection. first, the auc, sensitivity, and specificity were calculated for the three predictive models without wrapper - based feature selection. table 3 shows that the average auc values for the mfnn, the naive bayes, and the logistic regression prediction models were 0.489, 0.462 and 0.485, respectively. in terms of auc, the the mfnn model (auc = 0.489) outperformed the naive bayes (auc = 0.462) and logistic regression (auc = 0.485) models. a repeated 10-fold cross - validation experiment was performed to compare performance in the three wrapper - based predictive algorithms. table 4 shows that the mfnn, the naive bayes, and the logistic regression models had average auc values of 0.631, 0.569, and 0.620, respectively. in terms of auc, the mfnn model (auc = 0.631) outperformed both the naive bayes model (auc = 0.569) and the logistic regression model (auc = 0.620). out of 11 snps, the wrapper - based mfnn model identified only 4 : rs1800469 (tgf1 - 509), vntr (il1_ra), rs2227956 (hsp70 hom), and rs1801197 (ctr). feature selection using the wrapper - based approach clearly improved performance in the mfnn, the naive bayes, and the logistic regression. overall, the mfnn classifier with the wrapper - based approach demonstrated superior prediction performance (auc = 0.631) compared to the other models. additionally, the mfnn classifier with wrapper - based feature selection required fewer snps (n = 4) compared to the mfnn classifier without feature selection (n = 11). table 4 shows that the aucs did not significantly differ between the mfnn model with wrapper - based feature selection (auc = 0.631) and the logistic regression model with wrapper - based feature selection (auc = 0.620). however, the mfnn classifier with wrapper - based feature selection required fewer snps (n = 4) compared to the logistic regression classifier with wrapper - based feature selection (n = 8), that is, by selecting a small number of snps with significantly larger effects compared to other snps and by disregarding relatively insignificant snps, the mfnn model with wrapper - based feature selection successfully identified a subset of four major snps that could be used to predict osteoporosis outcome in the study population (rs1800469 (tgf1 - 509), vntr (il1_ra), rs2227956 (hsp70 hom), and rs1801197 (ctr)). after confirming that the mfnn model outperforms the logistic regression model, the next objective was finding the candidate genes and snps that are most promising for diagnosing osteoporosis, designing therapies, and predicting outcome in the studied population of taiwanese women with osteoporosis. this study compared three classification algorithms, including mfnn, naive bayes, and logistic regression with and without feature selection in terms of accuracy in predicting osteoporosis outcome in a population of taiwanese women. accounting for models is not a trivial task because even a relatively small set of candidate genes obtains a large number of possible models. the three classifiers were chosen for comparison because they cover varying techniques with different representational models such as probabilistic mfnn, naive bayes, and logistic regression models. the proposed procedures can also be implemented using the publicly available software weka and are thus easily applicable in genomic studies. to the best of our knowledge, this study is the first to propose the use of three classification algorithms, including mfnn, naive bayes, and logistic regression, and wrapper - based feature selection method for modeling osteoporosis outcome in taiwanese women based on genetic factors such as snps. in this paper, the wrapper - based feature selection approach was used to find a subset of snps that maximizes the performance of the prediction model according to how feature selection search is incorporated in the classification algorithms. the results showed that the mfnn classifier with wrapper - based approach was superior to the other tested algorithms and achieved the greatest auc with the smallest number of snps when distinguishing between high and low bmd in taiwanese women. these results suggest that mfnn model is a good method of modeling complex nonlinear relationships among clinical factors and the responsiveness of osteoporosis outcome in taiwanese women. the wrapper - based approach does not require knowledge of the classification algorithm used in the feature selection process, in which features are optimized by using the classification algorithm as part of the evaluation function [21, 23 ]. another advantage of the wrapper - based method is its inclusion of the interaction between feature subset search and the classification model. however, the risk of over - fitting is high when using the wrapper - based method [21, 45 ]. in the current study, use of the wrapper - based feature selection approach to assess high and low bmd individuals revealed a panel of genetic markers, including tgf1 - 509, il1_ra, hsp70 hom, and ctr, which were more prominent compared to other markers observed in the examined taiwanese women population with osteoporosis. a noted limitation of this study is that, due to the small sample size, the auc values were too low (< 0.7) to obtain good dataset classifications. therefore, further prospective clinical trials are recommended to determine whether the observed outcome associations with these candidate genes are reproducible in a larger population of taiwanese women with osteoporosis. this study used an mfnn methodology with wrapper - based feature selection method to predict osteoporosis outcome in taiwanese women based on clinical factors such as snps. the findings suggest that patients and doctors can use the proposed tool to enhance decision making based on clinical factors such as snp genotyping data. however, genetic markers require validation in further prospective clinical trials before routine clinical use of genomic analysis for predicting osteoporosis outcome. | an essential task in a genomic analysis of a human disease is limiting the number of strongly associated genes when studying susceptibility to the disease. the goal of this study was to compare computational tools with and without feature selection for predicting osteoporosis outcome in taiwanese women based on genetic factors such as single nucleotide polymorphisms (snps). to elucidate relationships between osteoporosis and snps in this population, three classification algorithms were applied : multilayer feedforward neural network (mfnn), naive bayes, and logistic regression. a wrapper - based feature selection method was also used to identify a subset of major snps. experimental results showed that the mfnn model with the wrapper - based approach was the best predictive model for inferring disease susceptibility based on the complex relationship between osteoporosis and snps in taiwanese women. the findings suggest that patients and doctors can use the proposed tool to enhance decision making based on clinical factors such as snp genotyping data. |
it is associated with decreased rates of pulmonary complications and decreased use of hospital resources. though many investigations have elucidated the value of early extubation after cardiac surgery, the optimal timing has not been determined. this propensity - matched study was designed to evaluate optimal timing of early extubation and correlate timing of extubation with early and late outcomes. the division of cardiothoracic surgery at carolinas heart and vascular institute computerized database was utilized to identify all patients who had coronary artery bypass (cabg), isolated valve surgery, or valve / cabg combination at carolinas medical center between january 2002 to december 2006 ; 2735 patients were thus identified. of those, 1164 were extubated within six hours (early extubation group) and 1571 were extubated six hours or greater after surgery (conventional extubation group). data including baseline demographics, procedural data, and perioperative outcomes were entered prospectively in a pre - specified database by a dedicated data - coordinating center. standard research approval was obtained via our institutional review board before data identification, analysis, and study approval was initiated. health insurance portability and accountability act of 1996 regulations were followed at all times to maintain personal patient information confidentiality. the society of thoracic surgeons (sts) national cardiac database definitions were used for the purposes of the study. early extubation is defined as removal of breathing tube 9 days). cerebrovascular accident is defined as a central neurological deficit persisting for greater than 72 hours. hemorrhage - related re - exploration is defined as operative re - intervention required for bleeding or tamponade. renal failure is defined as an increase in serum creatinine greater than 2.0 mg / dl, and a doubling of creatinine over baseline preoperative value, and/or a new requirement for dialysis / hemofiltration. monitoring included electrocardiography, pulse oximetry, and end - tidal co2 as well as routine use of pulmonary arterial and radial arterial catheters. anesthetic induction and maintenance routinely included intravenous fentanyl (10 - 20 micrograms / kg), midazolam (5 - 10 mg), propofol as necessary, and pavulon (10 - 20 mg). additionally, inhalational anesthesia was provided with isoflurane. anticoagulation with intravenous heparin was achieved and maintained with the aid of the hepcon hemostasis management system (medtronic). patients were brought to the icu immediately after surgery and supported with mechanical ventilation, with goals of peak inspiratory pressures less than 30 cm h2o, ph 7.35 - 7.45, po2 > 80 and pco2 2.2, systemic venous oxygen saturations > 65%, and systolic blood pressures from 100 - 120 mmhg (unless patient specific characteristics dictated otherwise). post - operative volume of chest tube drainage, hemodynamics, temperature, urine output, blood products transfusion, and analgesics / sedation were recorded hourly and discussed between team members with early extubation being continuously considered. patients were extubated when consistently hemodynamically stable, tolerating spontaneous breathing trial, hemostatic, and neurologically appropriate. contraindications to extubation, without physician approval, included cardiac index 160 mm hg, heart rate > 130, po2 50 chest tube drainage > 100 ml / hour, urine output 6 hours) extubation. early extubation patients were more likely to be males and have multi - vessel coronary artery disease compared to conventional extubation group patients. conventional extubation group patients were more likely to have hypertension, chronic renal failure, previous cerebrovascular accident, and reoperation. additional characteristics of this group include unstable angina, depressed left ventricular ejection fraction, advanced age (greater than 75 years), be in class iii / iv by new york heart association (nyha) functional classification, and have urgent or emergent operation. in addition, the sts risk scores for mortality were significantly higher in the conventional extubation group. patients ' operative and postoperative characteristics in patients with early (6 hours) extubation. early extubation patients were more likely to have undergone isolated cabg compared to conventional extubation group patients. pump, cross clamp and operation times were longer on average for the conventional extubation group. conventional extubation group patients had a higher rate of operative mortality, history of cerebrovascular accident, sepsis, renal failure, atrial fibrillation, hemodialysis, cardiac arrest, hemorrhage - related re - exploration, cardiac tamponade, and blood transfusion. conventional extubation group patients were also more likely to be readmitted to the icu, had higher reintuba - tion rates, and had more prolonged icu and hospital length of stay. propensity score adjustment and multivariable modeling. propensity scores were calculated and shown to balance out the differences at baseline between the early extubation and the conventional extubation group patients (online appendix 1 www.itacta.org) with p < 0.05 being statistically significant. early extubation was associated with decreased rates of pneumonia (p<0.001), stroke (p=0.007), acute renal failure (p<0.001), hemorrhage - related re - exploration (p<0.001), icu length of stay (p<0.001) and hospital length of stay (p<0.001), icu readmission (p<0.001), reintubation (p=0.009), and operative mortality (p=0.043). multivariable models were used as a supplementary analysis and confirmed these findings with the exception of sepsis (online appendix 2 www.itacta.org). specifically, pneumonia (or=0.35), prolonged icu los (or=0.43), prolonged hospital los (or=0.37), readmission to icu (or=0.50), reintubation (or=0.48), cerebrovascular accident (or=0.21), renal failure (or=0.24), re - operation for bleeding (or=0.13), and mortality (or=0.46) were all decreased within the early extubation group. late mortality was assessed out to 16 months after cardiac surgery (figure 1). risk factors for late mortality after cardiac surgery. with a hazard ratio (hr) of 0.45, early extubation was associated with decreased late mortality (p<0.001) when adjusting for other significant covariates. a sub - analysis was conducted to determine the optimal timing of extubation to predict improved postoperative outcomes. extubation within 9 hours emerged as the best predictor of improved postoperative outcomes including morbidity, mortality and reintubation (table 5). specificity, sensitivity and accuracy of extubation < 9 hours to predict postoperative outcomes detailed analysis is presented within online appendix 3 www.itacta.org. mechanical ventilation after cardiac surgery is used to improve oxygenation and ventilation as well as reduce cardiac workload in the hemodynamically unstable patient. early extubation has been correlated with decreased rates of mortality, morbidity, and resource utilization and may function as a predictor of an uncomplicated hospital course. with this study, we evaluated timing of early extubation and its relation to post - operative complications, death, and resource utilization. additionally, we examined late mortality in the early versus conventional extubation groups. despite the society of thoracic surgeons definition for early extubation being extubation within six hours after surgery, an important finding of our study is that extubation up to nine hours after surgery appears to be a better predictor of post - operative outcomes (online appendix 3 www.itacta.org). operative mortality and cardiac cause of death were lower in the early extubation group (p<0.001) as were rates of sepsis, cerebrovascular accident, renal failure, hemodialysis, atrial fibrillation, cardiac arrest, hemorrhage - related re - exploration, and blood transfusion (p<0.001), (table 2). prolonged intubation and continued hemodynamic instability after cardiac surgery leads to significantly increased morbidity and mortality. mechanical ventilatory support for 16 hours or more demonstrated a trend towards worse post - operative outcomes (online appendix 3 www.itacta.org). this mirrors the findings from previous studies where poorer post - operative results occurred in those extubated in greater than 24 hours. early extubation was associated with improved survival up to 16 months after cardiac surgery (p<0.001) while chronic renal failure, congestive heart failure, unstable angina, and advanced age diminished late survival (table 4). similar findings were demonstrated by cheng where there were no deaths in the one year follow - up in those patients extubated early. this study is among the first to demonstrate early extubation as a predictor of improved early and late outcomes after cardiac surgery. limitations. limitations of this study include all those inherent to any retrospective single - institution analysis. all data elements, however, were prospectively entered into a cardiac surgery research database with strict definitions, and the data analysis was performed using appropriately risk - adjusted statistical models to adjust for differences in preoperative risk factors. the accuracy of predicting improved outcomes in those patients extubated in < 9 hours is 65%. early extubation is among the earliest post - operative predictors for those patients who are more likely to have a smooth post - operative course and decreased complications after cardiac surgery. further studies are recom - mended to confirm the findings of this study and implement changes in clinical practice. | introductionearly tracheal extubation is a common goal after cardiac surgery. our study aims to examine whether timing of tracheal extubation predicts improved postoperative outcomes and late survival after cardiac surgery. we also evaluated the optimal timing of extubation and its association with better postoperative outcomes.methodsbetween 2002 and 2006, 1164 patients underwent early tracheal extubation (6 hours after surgery). propensity score adjustment and multivariable logistic regression analysis were used to adjust for imbalances in the patients preoperative characteristics. receiver operating characteristic curves (roc) were used to identify the best timing of extubation and improved postoperative outcomes. cox regression analysis was used to identify whether early extubation is a risk factor for decreased late mortality.resultsresults - early extubation was associated with lower propensity score - adjusted rate of operative mortality (odds ratio = 0.55, 95% confidence intervals = 0.31 - 0.98, p=0.043). extubation within 9 hours emerged as the best predictor of improved postoperative morbidity and mortality (sensitivity = 85.5%, specificity = 52.7%, accuracy = 64.5%). early extubation also predicted decreased late mortality (hazard ratio = 0.45, 95% confidence intervals 0.31 - 0.67, p<0.001).conclusionsearly extubation may predict improved outcomes after cardiac surgery. extubation within 9 hours after surgery was the best predictor of uncomplicated recovery after cardiac surgery. those patients intubated longer than 16 hours have a poorer postoperative prognosis. early extubation predicts prolonged survival up to 16 months after surgery. |
pigmented villonodular synovitis (pvns) of knee joint is a rare disorder of synovium. 25 years male presented to us with painless swelling of left knee joint of 3 months duration. we want to emphasize that early diagnosis and well done arthroscopic synovectomy gives good clinical outcome with low recurrence rate. very few cases have been reported in india. in the original description of the disease, the term pigmented villonodular synovitis was applied to a lesion that occurred in the synovial membrane of joints and tendon sheaths and was characterized by fibrous stroma, hemosiderin deposition, histiocytic infiltrate and giant cells. subsequently, two forms of the disease were identified : a localized subtype characterized by a pedunculated lesion and a subtype with diffuse joint involvement. the most widely held theory is that the disease is an inflammatory reaction of the synovium. now the localized form is suggested to be granulomatous hyperthrophy of synovium and the diffuse form as benign neoplastic process. high rate of recurrence were observed after synovectomy in patients with diffuse form of disease. arthroscopic picture showing orange colored hypertrophied synovium. arthroscopic picture showing orange colored hypertrophied synovium. this case of p.v.n.s is reported for its rarity of incidence and a good result obtained with minimal open intervention. a 25 yr old male presented with chronic painless swelling of left knee joint of three month duration, to our orthopedic department. m.r.i of knee joint showed effusion, low signal intensity on both t1 and t1 weighed images with diagnosis of hyperplastic synovium. arthroscopic synovectomy was done using four anterior and two posterior portals to ensure maximum removal of affected synovium. even with present day imaging modalities the diagnosis is often delayed [7, 8, 9 ]. extra articular involvement and bone erosions are seen in many cases on mri in early cases. the gradient - echo pulse sequences confirms the presence of hemosiderin, which is manifested by the presence of a prominent low signal - intensity blooming artefacts. it identifies the extent of synovial disease in patients with diffuse intra - articular involvement (pvns), for demonstrating the relationship to the tendon sheath in pvns, and for revealing its bursal involvement in pvnb. definition of disease location and extension is important for diagnosis and for treatment planning knee and hip joints are most commonly involved but bursa, tendon sheath may be involved. coutinho el al found study they delay between the onset of symptoms and diagnosis of pvns was 24 months. various treatment modalities were tried with recurrence rate of 25% at the end of 60 months. recurrence is usually seen in first year but they can be seen as late as seventeen years after the initial treatment. excision of synovium in patients with localized form has shown good results with no incidence of recurrence, whereas with diffuse form high rate of recurrence of disease is documented. for diffuse pvns in knee joint open anterior synovectomy fallowed by second stage posterior open synovectomy is recommended for ensuring complete removal of synovium. clen did simultaneous anterior and posterior arthroscopic synovectomies with postoperative radiotherapy found the rates of residual or recurrent tumor and knee function recovery comparable to that with staged synovectomies reported in the literature. well done arthroscopic synovectomy gave equally good results as open synovetomy. in this particular patient, they also found that even though postoperative mri was showing residual synovium in 5 cases the recurrence rate was very low. recurrence can be seen in cases where complete removal of diseased synovium has not been achieved. in post operative period, some inflammatory tissue may be seen within the joint, this inflammation usually subsides on follow up studies. zhongguo in review of 97 cases of pvns of knee also had similar results with arthroscopic synovectomies. radha selectively used radiotherapy with intraarticular yttrium in cases with recurrent disease only. in this case due to unavailability of radiotherapy at our place we offered this patient arthroscopic synovectomy followed by radiotherapy if recurrence occurs. radiotherapy was not given in this case as no recurrence of knee swelling was noted in two years. we still need to follow this patient as recurrence may be noted many years after synovectomy13. currently for knee diffuse pvns arthroscopic simultaneous anterior and posterior synovectomies with post operative radiotherapy is recommended as treatment of choice. joint replacement is reserved for advanced cases of joint destruction. at the time of joint replacement, arthroscopic synovectomy is the modality of treatment of pvns of kneejoint. in this particular patient mri is useful for diagnosis of recurrent disease and can be treated with radio synovectomy. although rare, pigmented villonodular synovitis must be kept as differential diagnosis of patients with chronic synovitis of knee. | introduction : pigmented villonodular synovitis (pvns) of knee joint is a rare disorder of synovium. hip and knee joint are commonly affected joints. the knee pvns presents as a localized or diffuse form. diagnosis if often delayed and permanent joint damage occurs with advanced disease. ultrasound examination shows fluid collection and synovial hypertrophy. magnetic resonance imaging helps in clinching the diagnosis. final confirmation of pvns is done with histopathological examination of synovial tissue removed. post operative radiation has shown to reduce the rate of recurrent disease.case report:25 years male presented to us with painless swelling of left knee joint of 3 months duration. radiographs were normal. mri showed synovial hypertrophy with changes suggestive of pvns. we did arthroscopic six portal synovectomy. the patient regained his function and was asymptomatic at 2 year follow up.conclusion:we want to emphasize that early diagnosis and well done arthroscopic synovectomy gives good clinical outcome with low recurrence rate. radiotherapy should be reserved for recurrent disease. |
bristol has long been acknowledged by historians as an early pioneer in the organisation of poor relief based around a centralised institution. great confinement of people unwilling or unable to work, highlighted the significance of the first english workhouse in bristol. under the bristol poor act of 1696 the key principle was that able - bodied paupers should be set to work, with only young children, and incapacitated sick, elderly and disabled people being granted relief. in 1698 a large workhouse was opened in adapted industrial buildings, on the banks of the river avon adjoining st peter s church, from which it acquired its name. st peter s hospital quickly developed into an archetypal general purpose poor law institution, comprising elements of both punitive workhouse for the idle and poorhouse for the destitute. it incorporated some characteristics of a hospital, with provision for frail elderly and sick people, who included those with mental disorders. although bristol had been one of the earliest regional centres to initiate a voluntary general hospital, in 1737, this was never followed by the establishment of a lunatic hospital as later occurred in several provincial cities. the development of designated facilities for lunatics and idiots in st peter s hospital presumably removed any perceived need for more specialist public or charitable provision. surviving records tell us relatively little about what was actually provided during the eighteenth century. the indications are that conditions for insane people were as spartan and punitive as for other inmates. it was observed in february 1767 that the stone floors in the lunatic s wards made them very injurious and they were ordered to be floored with planks. in april 1768 the corporation of the poor directed that, at least once a week, the physicians and surgeons attached to the house should visit the frenzy objects and also all such objects as shall be from time to time be brought in by warrants of lunacy. the medical men were to report on their state of health and give directions to the master and mistress of the house as to what provisions they should receive. other treatment arrangements were established. in 1769, on the physician s recommendation, a cold bath was installed for the lunatics benefit, though it unfortunately fell into the river, along with a wall and part of the airing court, in october 1771. ward for lunatics, although ideots were evidently accommodated among the other inmates. a picture can be assembled of st peter s hospital in the early nineteenth century, largely from the early writings of dr james cowles prichard, who later gained prominence both as an alienist and an ethnologist. he subsequently enjoyed a distinguished career, being appointed as one of the commissioners in lunacy in 1845. in 1820 its construction was as awkward and inconvenient as possible, being a confused heap of buildings, appended one to the other, without symmetry or plan. many parts were impossible by any care sufficiently to ventilate, or to render decent and comfortable. until recently, some wards had been fitter receptacles for wild beasts than for human beings. the average number of inmates was 420, whom prichard divided into three classes. the most numerous comprised the aged and infirm, orphan children, and others unable to maintain themselves due to sickness or other circumstances. the second included all the vagrants and beggars who are found in bristol, apart from those expelled by the magistrates. the third class were the idiots and lunatics of the lower orders, sent under warrants from the city and the surrounding area. prichard was greatly concerned about the sanitary state of st peter s hospital, which had been subjected to severe epidemic fevers between 1817 and 1819. as a result of its deficient means of separation, seven male lunatics and idiots had died due to contagion. its location was particularly significant, as the clifton physician dr chisholm identified in 1817, when attributing the greatly increased incidence of fevers to conversion of part of the river avon into a this effectively transformed the river into a large pool of stagnant water, chisholm observing that the house stands immediately on the northern bank of this river, so that what formerly contributed to its general salubrity, by facilitating a perpetual drainage of filth, has now become a source of evil to it. prichard carefully depicted st peter s hospital s accommodation for pauper lunatics. although female patients had the benefit of their own for the more problematic among the afflicted beings there was : a row of pens for the temporary confinement of those patients who are violent and intractable, not unlike the domiciles of the royal lions and tigers in the menagerie of the tower, though by no means so respectable in their appearance, or so commodious. these pens are now used only when the state of the patient requires strict confinement, or when by their noise and violence other invalids are molested. a row of pens for the temporary confinement of those patients who are violent and intractable, not unlike the domiciles of the royal lions and tigers in the menagerie of the tower, though by no means so respectable in their appearance, or so commodious. these pens are now used only when the state of the patient requires strict confinement, or when by their noise and violence other invalids are molested. henry alexander, a reform - minded ipswich banker, told the 1815 parliamentary select committee on madhouses that he had observed incurable patients confined underground in partitioned strong wooden cells with small barred windows that admitted little light. peter s from 1822 likewise confirms the presence of a large women s bedlam ward, but no designated men s insane ward. by most standards, the nature of provision was distinctly inferior to that prevailing in public lunatic asylums, even allowing for the exposures in 1815 of poor conditions and abuses at the york lunatic asylum and bethlem hospital. in these unlikely surroundings, his early writings show that, like most of his medical contemporaries, he approached mental illnesses very much as bodily diseases and treated patients accordingly. he came from the heroic treatment school, and became known for his strong reliance on bleeding, purging and blistering. his treatment practices and serious demeanour informed a bristol infirmary patient s poetic lampoon : dr prichard do appear, with his attendance and his care, he fills his patients full of sorrow, you must be bled to - day and cupped tomorrow. dr prichard do appear, with his attendance and his care, he fills his patients full of sorrow, you must be bled to - day and cupped tomorrow. a series of published case histories relating to insane patients admitted to st peter s hospital between 1812 and 1820, some under a frenzy warrant, confirm prichard s reliance on drastic physical methods. many were bled, using methods that included cupping, opening veins or arteries, and the application of leeches. other frequent remedies included shaving the head before application of wet cloths or a large blister, cold shower baths, and warm baths. he deployed a formidable array of medicines, primarily emetics and purgatives, but also opiates, cathartics and digitalis. he was certainly not averse to coercive methods, including use of the strait waistcoat and confinement in the moral treatment or management approaches that were increasingly in vogue among specialist mad - doctors. as an ambitious physician prichard s orientation was essentially medical, but even if he had been inclined toward more progressive methods these would have been almost impossible to implement in the constricted surroundings of st peter s hospital. despite the acknowledged inadequacies and defects of st peter s hospital, a significant change occurred in its legal status in 1823. local legislation secured its formal designation as a county lunatic asylum under the provisions of wynn s act, or the county asylums act, of 1808. that act had empowered county authorities to provide a pauper lunatic asylum, or to join with voluntary subscribers to cater also for charitable and private patients. it was permissive rather than mandatory, and eight counties had opened an asylum by 1823. the act laid down basic standards for the new asylums, in regard to location, building design, medical involvement and curative intent. it also required that, in counties that had established an asylum, all their lunatics and an absence of records precludes any certainty about the circumstances leading to bristol s adoption of the county asylum legislation in 1823. the corporation of the poor had apparently never paid for placement of any lunatics in private madhouses, in contrast to birmingham which also had no public asylum provision. the imposing, expensively constructed edifice in a neighbouring city presented a visible challenge to bristol s leading citizens, exposing their lack of comparable progressive, munificent activity. on a practical level, bristol s northern suburbs fell within gloucestershire, rendering their pauper lunatics eligible for placement in its asylum. a re - designation of st peter s hospital potentially protected bristol from any need to construct an expensive county asylum or from pressures to pay for placements at gloucester or elsewhere. the necessary arrangements were inserted into an obscure clause of the bristol poor act of 1822. it provided that, from march 1823, the hospital or workhouse belonging to the corporation of the poor was deemed an asylum for the reception of lunatics under the provisions of the act of 1808 and supplementary legislation of 1811, 1815 and 1819, as if the city and county of bristol had been named and included in the said several acts. furthermore, there was specific exemption from any requirement to erect a new asylum. thus, the corporation of the poor had secured the status of county lunatic asylum for st peter s hospital, despite the lunatic wards forming merely one part of the institution and its demonstrating none of the locational, spatial or other attributes stipulated in the lunacy legislation. in particular, they had effectively avoided the expectation placed on newly built asylums that they should be sited in an airy and healthy situation. this proved to be a key factor in the critical attention to which it was subsequently exposed. in the 1820s st peter s hospital was a chaotic, rambling, and increasingly overcrowded institution. the numbers of paupers housed rose steadily from 435 in 1821 to 532 in 1826, and 600 in 1832. there is some difficulty in ascertaining the true numbers of mentally disordered people confined, but they made up a relatively small proportion. lunatic asylum, compiled in october 1825, lists only 26 women and 8 men. writing in 1826, james johnson, a former governor of the corporation of the poor, noted that the numbers of bristol s insane poor had increased greatly in recent years and there were seldom less than 30 male and female patients, labouring under this complaint in st peter s. some people, particularly males, were still accommodated elsewhere in the workhouse, which would account for the considerably larger numbers recorded by john brady, the hospital s surgeon and apothecary, in 1828. brady s figures showed 45 female and 24 male lunatics and dangerous idiots. st peter s hospital s unsuitability as a public asylum was increasingly evident. separate wards and nurses, were shown every degree of tenderness consistent with safety, and no coercive measures were ever applied. iron chains, manacles and bolts were not permitted, though he conceded that the pens remained in use for individuals suffering under the highest state of excitement, and leather straps and strait waistcoats were routinely deployed. in june 1827, after a young woman confined in a strait waistcoat managed suicide by climbing onto a table and jumping through a window into the river, the asylum was criticised in the local press as conveniences. in 1830, amid mounting concerns about the inadequate facilities for lunatics and extreme overcrowding in the workhouse, the corporation of the poor agreed to spend 3100 on acquiring a building known as the armoury for use as a pauper lunatic asylum. however, the plan foundered due to ratepayer resistance to the expenditure. treatment approaches in st peter s hospital in the early 1830s can be gleaned from prichard s writings. his firm belief in the close relationship between physical and mental disorders underpinned the continuing emphasis on medical remedies. prichard insisted that the use of purgative medicines is one of the most important and generally available means for the cure of maniacal patients, and advocated the use of emetics, including antimony in nauseating doses to control maniacal excitement. his eclectic approach was in the tradition of earlier provincial insanity physicians like john ferriar and james currie, and similar to contemporary asylum - based alienists like william ellis and paul slade knight. tellingly, however, prichard still had little to say about moral treatment approaches, other than to acknowledge the restorative aspects of fresh air and exercise and advocate that every asylum should provide open air employment for their patients. he made no mention of classification, increasingly regarded as a central element of patient management in public asylums. st peter s hospital s acquired status as a county asylum exempted it from the clauses of the 1834 poor law amendment act requiring removal of dangerous lunatics and idiots from the workhouse to an asylum. its shortcomings were nevertheless highlighted by the investigating assistant poor law commissioner, captain chapman. the inmates were lodged in large rooms, with no classification other than keeping the least violent separate, while those under paroxysms were confined in cells of a very inferior description. however, local apologists challenged the criticisms, arguing that at no asylum could these unhappy creatures find better treatment, and that control and inspection by officers elected by the great body of rate - payers prevented any possibility of abuse. notwithstanding that perspective, the institution s deficiencies remained apparent, regularly illustrated through suicides and escapes. in 1839 the corporation of the poor reluctantly accepted the need for action, after strong criticisms by assistant poor law commissioner neale. the workhouse s deputy governor, mr goldney, conceded that the men s insane ward was most inconvenient because violent lunatics, and such as are imbecile, are mixed together without distinction. their miserable sleeping apartment was rather calculated to engender lunacy than to cure it. major alterations were then carried out at a cost of 2000. in april 1841, after completion of further work, the lunatic wards were claimed to be in a state of perfection. the favourable evaluation of the insane wards state was overtaken by a scathing visiting magistrates report in august 1841. there was neither dining room nor day room and the women were confined to their sleeping area one fire was always kept for culinary purposes. the exercise yard, catering for up to fifty patients, was small and confined, serving also as the only thoroughfare for all carts bringing coal and other materials into st peter s hospital. the new and old cases, the convalescent and incurable, the idiot and melancholic, the noisy and outrageous were all intermingled. the dark, unventilated, underground pens still used for refractory patients were really horrible, and far worse than any cell in the gaol or bridewell. mechanical coercion was utilised extensively, with noisy and maniacal patients almost constantly confined with cuffs or strait waistcoats, or strapped to their seats. those prone to biting were placed in a mask made on precisely the same principle as a muzzle for a dog. the visiting magistrates strongly recommended construction of a new asylum, providing scope for outdoor exercise and employment, close to where a new workhouse had been established at stapleton. the damning report reached the home secretary, the marquis of normanby, who called for remedial action. the corporation of the poor responded by recommending major alterations to the female lunatic ward. st peter s hospital s surgeon insisted that, despite the lunatic wards unfavourable appearance, the patients recovered as frequently, and even more frequently than in most institutions. that defence would again be employed in the future. meanwhile, the interest of central government authorities had been aroused, and the metropolitan commissioners in lunacy ensured inclusion of st peter s hospital in their nation - wide inspection visits beginning in 1842. predictably, when published in 1844, the commissioners report singled it out for almost unqualified censure. reproducing parts of the visiting magistrates report of 1841, they pronounced bristol s centrally located workhouse totally unfit for an asylum for the insane, there being no means of classification, of exercise, or employment. whilst other towns workhouse facilities were also severely criticised, most notably the lunatic wards in birmingham workhouse, they had not attracted the additional opprobrium associated with unwarranted designation as a county asylum. source : post card, early twentieth century, in author s possession (no publisher stated). source : post card, early twentieth century, in author s possession (no publisher stated). the mass of national evidence collated in the metropolitan commissioners in lunacy s 1844 report led directly to the key legislation of 1845, which rendered provision of county lunatic asylums compulsory and created a new national inspectorate, the commissioners in lunacy. for the bristol authorities this heralded the prospect of a sustained onslaught by powerful adversaries, wedded to principles and practices completely at variance with those operating within st peter s hospital. the lunacy reformers, personified by lord shaftesbury and the other commissioners in lunacy, retained a vision of well - designed, purpose - built, curatively orientated public lunatic asylums, located in healthy rural or semi - rural settings, with ample land attached. these asylums were expected to operate on a moral management system, comprising three essential features that were being widely disseminated non - restraint ; classification and separation of patients according to symptomatology and behavioural presentation ; and, organised work or other form of occupation. in this conception, there was no place for a cramped, ill - constructed, dilapidated institution situated in a crowded and unhealthy city centre. the perceived unsuitability of bristol s accommodation for lunatics was undoubtedly increased by its location within a large urban workhouse. the commissioners in lunacy s growing disapproval of provision for the insane inside workhouses was made explicit in a lengthy, detailed supplement to their annual report in 1859. however, as bartlett and others have shown, before the late 1850s the commissioners were prepared to countenance lunatic wards in workhouses in certain circumstances. their concerns had related largely to the presence of acutely mentally ill patients, as distinct from people with congenital or chronic conditions. given this period of relative tolerance of workhouse provision, it can be surmised that the degree of hostility directed toward st peter s hospital s was due mainly to its acquired legal status as a county asylum, which brought it firmly within the lunacy commissioners remit. historians such as david mellett and nick hervey have taken the view that the commissioners in lunacy were a body whose effectiveness was limited, their efforts focused mostly on collecting statistics, confronting the worst excesses of the private lunatic asylum sector, and providing detailed guidance on the construction of new county asylums. a similar position was adopted by bartlett, who argued that the commissioners did not deal particularly strongly with powerful local interest groups or parsimonious authorities, and that their interventions on the issue of inappropriate retention of pauper lunatics in workhouse accommodation were less effective than those of the poor law commissioners. however, melling and his colleagues, based on their devon evidence, credited the commissioners in lunacy with a much more influential role in relation to the county asylum authorities and the borough magistrates of exeter and plymouth. philo has endorsed that perspective in considering the commissioners particular efforts to pressurise county boroughs to accept their responsibility for providing a lunatic asylum under the 1845 act and subsequent legislation in 1853. the bristol experience bears out the determination and ultimate effectiveness of the commissioners when faced with what they perceived as recalcitrant opponents. the commissioners in lunacy s firm conviction of the need for borough asylums had considerable ramifications for the bristol authorities. the relentless hostile scrutiny of provision within st peter s hospital placed the city at the forefront of those boroughs expected to construct a new, well - appointed lunatic asylum that conformed to the expected standards regarding location and facilities. it became the first borough to provide its own lunatic asylum under the 1845 act, in the wake of severe criticism of its workhouse insane wards by the metropolitan commissioners in lunacy and by dr samuel hitch in his special report to the poor law commission in 1844. this can only have accentuated the pressure placed on bristol to follow suit. in bristol, responsibility for management of the existing lunatic asylum and care of its patients was confusingly shared between several bodies. the corporation of the poor (referred to sometimes as the board of guardians after 1834) owned st peter s hospital and managed the workhouse that was still its predominating element. the borough magistrates were given an inspectorial role in relation to the lunatic asylum under a local act passed in 1837, which was delegated to a committee of visitors. the 1845 legislation extended their powers to include some management responsibilities, including staff appointments. it also gave the borough council the option to take over the role but they declined, thus leaving the powers with the magistrates. the act of 1853 reinforced the powers of borough councils and, after demurring for several months, bristol council took over nominal responsibility for the asylum in january 1854. whilst this provided some clarification, there was nevertheless continuing scope for disputes and disagreements between the authorities concerned, with periodic instances of the corporation of the poor, visiting magistrates and town council blaming one another and seeking to shift responsibility when difficulties arose. they could only be relied upon to reach common accord on one subject the need to protect the ratepayers of bristol from the prospective heavy burden of expenditure involved in replacing the asylum in st peter s hospital with a new borough lunatic asylum. for more than a decade after 1845 st peter s hospital was the focus of protracted conflict between central government, as represented primarily by the commissioners in lunacy, and the bristol authorities. the visiting magistrates were routinely referring to the bristol lunatic asylum, but the commissioners steadfastly persisted in describing it as st peter s hospital. the latter s opening salvo was fired in the published report of 1847, following a visit the previous november. although conceding that some improvements had been made to the lunatic wards since 1844, the commissioners considered the present arrangements utterly discreditable and threatened the intervention of some higher authority if the bristol authorities did not take drastic measures. the visiting magistrates acknowledged that the building was entirely unfit for a lunatic asylum and could never be made suitable, particularly because of a want of proper classification that led to frequent violence among male patients. one outspoken member, dr green, maintained that the lunatic establishment was healthy, well arranged, well ventilated, and efficiently conducted. apart from having no gardens, it was equal to any in the kingdom. a superficially conciliatory tone was adopted by the commissioners in lunacy in 1848. whilst lamenting the amount of mechanical restraint employed, they complimented the cleanliness of the wards and the neat appearance and tranquillity of the patients. somewhat reassured, the visiting magistrates proposed building alterations to allow more separation of those who were violent, refractory or filthy in their habits, as well as measures to reduce mechanical restraint, including a night nurse. the commissioners nevertheless advised the home secretary, sir george grey, of the continuing unfitness of st peter s hospital, insisting they had used every opportunity to enforce the duty to make suitable provision. they called on him to exercise his powers under the 1845 act to require the bristol authorities to provide a proper asylum, in a suitable locality near the city. in september 1849 a letter requiring construction of a new asylum went out from the home office. in response the bristol magistrates, while accepting that the present asylum had inconveniences, questioned the commissioners portrayal and saw no pressing necessity for the great expense of building a new asylum at a time when the city was financially straitened. the corporation of the poor concurred, maintaining that st peter s hospital had no defect sufficient to justify the expenditure ; its large number of recoveries belied claims of its inadequacies and unsuitable site. their pleadings secured some temporary respite. in january 1850 the commissioners commended the employment of female patients and accommodation that was generally clean, well ventilated and in good order. they praised the patients tranquil state and the reduced use of restraint, concluding that they had good reason to be satisfied, notwithstanding the inherent & incurable defects of the buildings. encouraged by the commissioners comments, the visitors and workhouse authorities implemented further improvements in the accommodation and amenities and spent money on new clothing and activities for the patients. however, any false hopes that they could fend off the pressures from london were soon dashed. in august 1850 the commissioners in lunacy advised the home secretary that bristol s returning prosperity removed any extenuating circumstances. although satisfied with the care and assiduity demonstrated toward the patients and efforts to rectify the asylum s more prominent evils, the fundamental defects remained : its position, in the centre of a crowded and populous city, and the consequent want of space, involving as that does an almost total absence of courts and open ground for air and exercise, as well as of proper means of classifying and separating the patients, and of employing them in healthful labor out of doors, are objections which nothing short of the acquisition or erection of a new asylum beyond the bounds of the town can effectually remedy. its position, in the centre of a crowded and populous city, and the consequent want of space, involving as that does an almost total absence of courts and open ground for air and exercise, as well as of proper means of classifying and separating the patients, and of employing them in healthful labor out of doors, are objections which nothing short of the acquisition or erection of a new asylum beyond the bounds of the town can effectually remedy. temporary palliatives or postponements, while unwisely proposing interim arrangements for bristol to place its lunatics in a county asylum or private licensed house. a letter from the home office reiterated the demand for provision of a new asylum. they took counsel s advice, which confirmed that the secretary of state had no power to discontinue st peter s hospital s legal status as bristol s county lunatic asylum and consequently could not order removal of its pauper lunatics. having gained a partial victory, they pressed home their advantage by proposing an exchange of rooms and outdoor areas between the asylum and the workhouse, thereby creating additional sleeping rooms and exercise areas. plans were submitted in may 1851. the discomfited commissioners in lunacy conceded that they would effect a considerable improvement and recommended approval, though insisting that these were no more than temporary expedients. their climb - down engendered a misplaced optimism in bristol. entirely do away with the idea of a new building, or at least defer it for a long time. by october 1851 the patients had moved into their new accommodation, and the visiting magistrates observed marked improvements in the demeanour and appearance of the women. amply sufficient for its purposes, offering as fair a chance of recovery as might be expected in a much larger establishment, and anticipated that the commissioners in lunacy would hesitate before inflicting a further burden on the city. however, the asylum s re - modelling bought less than two years grace. it quickly became evident that the buildings retained serious limitations, especially regarding the ability to contain dangerous or risky patients. throughout 1852 such developments aroused the hostile interest of the commissioners in lunacy, at a period when non - restraint principles were strongly influencing ideas on public asylum management, with the elimination of restraint being interpreted as a key indicator of best practice. during 1853, it was being applied with increasing frequency and for longer periods in st peter s hospital, in response to continuing violence among patients and between staff and patients. visiting two weeks after the dismissal of a male attendant for violent conduct, the commissioners attributed the large amount of instrumental coercion to the very imperfect means of separating and classifying the patients, lack of seclusion facilities and single rooms, and the absence of opportunities for outdoor activities. the numerous instances of wounds & bruises inflicted by patients on each other had resulted in at least one death. in one particularly unfortunate incident, a long - standing patient, who had previously been under restraint, committed suicide by setting fire to her clothes in the water closet. the mechanical restraint issue further heightened the central authorities perception that a new asylum was a necessity. overcrowding was also becoming an increasing problem, as at many better appointed county asylums, due to a continuing growth in admissions relative to discharges. the pressures on bristol s visiting magistrates were compounded when the commissioners in lunacy ordered transfer of several lunatics from the city s other workhouse at stapleton in august 1853. at the beginning of september it was noted that, with seventy - seven patients, the house is now becoming very full. furthermore, extensions to the borough boundaries brought clifton within bristol. clifton s lunatics had hitherto been accommodated in the gloucestershire county asylum, but its overcrowding problems caused notice to be given to the clifton board of guardians to remove their patients. a similar eventuality was looming in relation to patients from the bedminster district, currently placed in the somerset asylum at wells. as the potentially responsible county asylum, st peter s hospital plainly did not have capacity to accommodate the potential influxes. faced with the prospect of having to relocate their pauper lunatics, the clifton guardians initially looked toward st peter s hospital. they made representations to the home office, describing it as unfit and inadequate for a county lunatic asylum, severely criticising its means of classification, and defective accommodation for air and exercise. in october 1853 the inevitable order went out from viscount palmerston, the home secretary, requiring the bristol justices to erect or provide a fit and proper asylum for pauper lunatics, and citing the new legislation empowering borough councils to take on the responsibilities. in january 1854 a special meeting of the town council confirmed its distinct unwillingness to comply with the government directives. medico - moral locational discourse that underlay the building of public asylums in rural settings. they were not convinced that outdoor employment was as necessary for patients belonging to crowded city districts, and whose callings were chiefly artistic or mechanical as for those accustomed to rural occupations, contending that work at their own trades would be more conducive to recovery. the need for air and exercise could be met by walking parties and outings, such as those currently provided for some patients. they advocated either incorporating additional space at st peter s hospital or transferring all the lunatics to the more suburban stapleton workhouse. the corporation of the poor, however, by now acknowledged that the asylum s defects were too great to overcome, and relations between them and the borough council became increasingly strained. in early february 1854 a new committee of visitors was appointed, although its key personnel remained largely the same. over the following months attempts were made to improve facilities, including provision of a new workshop for the men and the introduction of two rug looms. the commissioners in lunacy noted the beneficial effects of the measures taken but remained generally unimpressed, particularly in view of the numbers of injuries sustained by patients and the incidence of mechanical restraint. in late december 1854 palmerston s missive further galvanised local opposition. in january 1855 the mayor, j.g. shaw, called for one other grand effort, leaving no stone unturned to avert the threatened burden of taxation and poorer ratepayers being driven mad. he insisted that the necessary classification of patients could be implemented in st peter s hospital, and contended that it was inappropriate to remove pauper lunatics from the crowded courts, lanes, and alleys of the city to place them in unfamiliar fields, groves, and gardens. proposals were made to incorporate more of the workhouse into the lunatic asylum, to increase capacity to 150. the corporation of the poor, although equally anxious to resist the monstrous anticipated expenditure of 40 000 on a new asylum, doubted that the plan would be sanctioned by the commissioners in lunacy, especially as increased noise and factory smoke had materially prejudiced the locality as a receptacle for insane patients. they renewed the suggestion to transfer the lunatics to stapleton workhouse, which had five acres of land attached. however, following an inspection the commissioners declared the site unsuitable, and a further formal letter from the home secretary followed in march. undaunted by the continuing pressure, the borough council and corporation of the poor set aside their differences to act jointly. following yet another order from the home secretary in september the campaign became increasingly vocal, incited by local politicians. in the autumn of 1855, public meetings of irate ratepayers were held all over bristol. in october, at the guildhall, the mayor compared pauper lunatic asylums to royal palaces, surrounded by princely domains. whilst recognising st peter s hospital s shortcomings, he claimed that nineteen - twentieths of those afflicted inmates, if cured and sent back to their homes would return to far worse circumstances. despite objections from william herepath, the long - serving chairman of the visitors, the two bodies renewed their efforts to gain agreement for the asylum s relocation to the stapleton workhouse site. having conferred with the commissioners in lunacy, the poor law commissioners formally refused consent. in bristol the decision brought condemnation of both sets of commissioners and all centralised direction. late 1856 a new site at stapleton had been acquired and the commissioners in lunacy s consent obtained. a building of a simple but cheerful character for two hundred patients was designed by a local architect, j.r. 1857 william herepath proudly told a council meeting that they had taken care not to adopt the most beautiful plan, but had chosen one which was neat but not gaudy, and the projected cost of 30 000 was significantly less than feared. dramatic improvements in their health and demeanour were reported following the move to the new asylum in its semi - rural setting. the last years of st peter s hospital lunatic asylum s operation proved extremely problematic. its various deficiencies, the subject of so much hostile attention, became increasingly apparent as escalating admissions brought increased overcrowding. patient numbers rose from 67 in may 1856 to 88 by the end of the year, and beds had to be moved closer together. the difficulties continued through 1857, accentuated by transfers of patients from the somerset, wiltshire and gloucester asylums. the most disorderly patients. in mid - july 1858, with 98 people, the house was very full. beds in the female wards were moved together to an inconvenient extent, and another workhouse ward was acquired to give space for 15 additional beds. in july 1859 clifton union sought admission of several females, and by december the asylum housed 106 patients. people were sent on leave to create space, despite a report in may 1860 that 10 women out on trial were really not fit persons to be discharged. it was not merely a question of numbers. in terms that became familiar in many public asylums, the visitors complained bitterly about the actual patients they had to manage. in december 1856 they noted the many utterly hopeless cases, especially amongst the women. responding in may 1857 to the commissioners in lunacy s contention that conditions in the house were responsible for very high mortality rates, the visitors countered that it contained about 90 of the most desperate & for the most part incurable cases who demonstrated all the worst features to which insanity is exposed. in october they claimed many cases presented more marked features of insanity than we have generally observed, most being incurable. in early 1860, with overcrowding acute, the asylum was receiving only dangerous cases of the most unfavourable character. according to the visitors, the house never before contained so many deplorable objects. these circumstances contributed to the numerous incidents of patient violence, some serious in nature, both between patients and directed toward nurses, attendants and more senior staff. mechanical restraint and seclusion in a strong room were being deployed with increasing frequency to contain dangerous and threatening behaviours, sometimes for extended periods. whilst the commissioners in lunacy condemned the extent of their use, they largely accepted arguments by the visitors that the buildings limitations made it difficult to manage patients otherwise. the commissioners alluded to this in 1859, identifying poor pay as a significant determinant. wage levels in the asylum had always been low, particularly for females, corresponding more to those in workhouses than in county lunatic asylums. several of those recruited to the female lunatic wards were previously workhouse inmates, a common phenomenon in many large urban workhouses. during the 1850s several nurses, both male and female, were dismissed for misconduct ranging from drunkenness and verbal abuse to assaults on patients. in early 1857 the appointment of a former police constable, with a good character for humanity & general good conduct, was perhaps a sign of raised standards preparatory to the new borough asylum. in recent years there has been an evolving discourse within the history of medicine and related disciplines surrounding questions of place and locality. medical historians have been reminded that the national narrative is determined to a large degree by a summation of events that occurred in numerous cities, towns and villages and by the contributions of a diverse range of actors within those places. indeed, frequently these did not conform to the overall picture and localised studies have become increasingly necessary to generate more comprehensive representations of historical developments. a growing focus on place has been particularly evident within the history of psychiatry and of institutions to accommodate people deemed insane, particularly through the work of chris philo. such a perspective seems particularly apposite in considering the history of st peter s hospital and its provision for the insane. the institution s constricted riverside location in rambling, crowded buildings in the centre of a teeming port city, surrounded by industrial facilities, was perceived as a serious impediment both to inmates basic physical health and to the implementation of practices consistent with the emergent tenets of moral management. bristol s early decision to make public provision for the insane poor within its workhouse deflected any potential impetus toward the voluntary lunatic hospital approach adopted elsewhere in the eighteenth century. it meant, however, that the city was for a long time saddled with a facility far inferior to those available to many of its contemporaries. the highly unusual re - designation of st peter s hospital as a county lunatic asylum, by means of what constituted legislative sleight of hand, characterised bristol s singularly local approach to addressing the growing problem of pauper insanity. it also ensured the perpetuation of outmoded practices and the avoidance of any requirements to provide suitable facilities in a more favourable location. the lunatic asylum contained within st peter s hospital consequently represented key elements that the reform lobby, in the guise of the commissioners in lunacy, sought to confront. it comprised, in reality, little more than the crowded insane wards of an urban workhouse, bereft of any of the attributes regarded as essential for the curative treatment of mentally disordered people. its peculiar legal status, in conjunction with bristol s regional and national prominence, made it subject to the commissioners particular attentions. their determined efforts to force the city s authorities into providing a replacement borough asylum were noticeably earlier and greater than their endeavours in relation to other large towns or cities. in bristol the adherence to an entrenched policy of determined opposition to any additional burden on the city s ratepayers was strengthened by a deep resentment of all manifestations of central government direction in matters regarded as within the purview of the city and its people. these perspectives informed an approach which overlooked, rationalised or tolerated extremely poor conditions, a high level of institutional violence and the extensive use of mechanical restraint. however, the ultimate consequence of assertions of local independence and stubborn resistance to state intervention was to make commissioners and ministers even more determined to enforce their will. in the end nevertheless, despite all the criticisms and prevarications, bristol could still claim to be only the second english borough to construct its own pauper lunatic asylum under the 1845 legislation. the historical significance of the lunatic asylum in st peter s hospital is rather more than its persistence as an anomalous institution in a major city, or of a conflict between local autonomy and central authority, important as those were. beyond them the earl of shaftesbury and his fellow lunacy reformers, through the legislation of 1845 and the endeavours of the commissioners in lunacy, had successfully captured the intellectual as well as the practical initiative. by the early 1850s, the public asylum based on moral management had become the accepted model of provision for most poor people with a mental disorder. in contrast, the bristol authorities defensive position was motivated partly by a conception that urban pauper lunatics could neither appreciate nor benefit from a relatively lavish semi - rural institution. rather, they were construed as being entitled only to something more basic, within the workhouse. the improvements and adaptations made there, designed to offer standards of comfort and care somewhat superior to those experienced by other inmates, were part of a real attempt to uphold a workhouse - based model of provision for the full range of mentally disordered people. in these circumstances, henceforth, the purpose - built county or borough asylum was confirmed as the normal place of confinement and curative treatment, while urban workhouses were to provide primarily for the residual groups of largely harmless people, with immanent or chronic disorders, for whom asylum treatment was deemed inappropriate. | in recent years there has been growing acknowledgement of the place of workhouses within the range of institutional provision for mentally disordered people in nineteenth - century england. this article explores the situation in bristol, where an entrenched workhouse - based model was retained for an extended period in the face of mounting external ideological and political pressures to provide a proper lunatic asylum. it signified a contest between the modernising, reformist inclinations of central state agencies and local bodies seeking to retain their freedom of action. the conflict exposed contrasting conceptions regarding the nature of services to which the insane poor were entitled.bristol pioneered establishment of a central workhouse under the old poor law ; st peter s hospital was opened in 1698. as a multi - purpose welfare institution its clientele included lunatics and idiots, for whom there was specific accommodation from before the 1760s. despite an unhealthy city centre location and crowded, dilapidated buildings, the enterprising bristol authorities secured st peter s hospital s designation as a county lunatic asylum in 1823. its many deficiencies brought condemnation in the national survey of provision for the insane in 1844. in the period following the key lunacy legislation of 1845, the home office and commissioners in lunacy demanded the replacement of the putative lunatic asylum within bristol s workhouse by a new borough asylum outside the city. the bristol authorities resisted stoutly for several years, but were eventually forced to succumb and adopt the prescribed model of institutional care for the pauper insane. |
the first transplantation of mechanical heart valve performed in august 1960 has saved the lives of thousands of heart patients. since that time, each year, the success in valve replacement depends on the myocardial function, health condition, technical skills of the surgical team, and the quality of postoperative care. kortke reports that patients recovery process after heart replacement is slower than in other heart surgeries. in addition, nassar, in his descriptive study, revealed that the mother mortality rate in mechanical valve transplantation was 3 - 4%. during the long years of waiting for the valve replacement, these patients had catastrophic experiences. postoperative complications (e.g. long - term hospitalization, social isolation, and the outcomes of valve replacement) are of upmost significance. hence, protecting the replaced valve and taking care of it are of greatest importance and require the mutual support of family and the patient. after the valve replacement, these patients need long - life care and are at risk for thromboembolism, which obligate them to use anticoagulants. the annual thromboembolic risk among warfarin users and non - users is 1% and 4%, respectively. international normalized ratio (inr) for the patients with mechanical aortic and mitral prosthesis is 2 - 3 and 2.5 - 3.5, respectively. according to menendez, the late - onset complications of valve replacement are : anticoagulant - induced bleeding, prosthesis malfunction, infectious endocarditis, recurrent thrombosis, and valvular insufficiency, which represent the outcomes of failure in the care system. more than the half of the patients did not use the right dose of anticoagulants and had no information on the drug side effects and on the relevant lifestyle. in the present condition, christensen points out that the rate of complications and problems related to treatment with anticoagulants due to self - care and conventional management method are 2.5% and 7.3%, respectively. warfarin use in pregnancy results in some complications (e.g. abortion and physical anomalies). there is no agreement on the right dose of warfarin during pregnancy for supporting both mother and fetus. the doses of warfarin prescribed for valve - replaced patients depend on different factors, which by themselves have different effects on inr. the administration of herbal drugs along with warfarin has different effects on inr. in deccache and aujoulat 's report on hospitalized patients, 20% of the cases had received adequate information on their disease / health condition through advising, 20% of them were not satisfied with their care condition, and the remaining 60% were required to receive more education. replaced patients, due to their susceptibility and the risky outcomes of the procedures, have different levels of worry and anxiety, and continue to live with anxiety, doubt, and worry. the preoperative physical problems and also the postoperative physical complications in valve - replaced patients are associated with a feeling of worry. hence, identification of the needs of such patients from their own experiences is necessary for providing nursing care and lessening the fear and anxiety in them. it seems that the worries, experiences, and attitudes of the patients are ignored by the nursing staff as they are classified as their least important priorities and usually are not considered in routine care planning. assessing the potential sources for fear, worry, and emotional status (doubt, denial, aggressiveness, and isolation, often as a general reaction to illness or self - awareness) is of importance. moreover, to our knowledge, till the present time, no qualitative study has been reported on the experiences of these patients while facing the problems following heart valve replacement. given the special cultural, social, and religious contexts in which the heart valve replaced patients are cared for, the present study was done with a qualitative approach to describe the stressful experiences of heart valve in this qualitative study, qualitative purposeful sampling was performed to obtain the perceived experiences of the heart valve subjects consisted of cases with experiences of valve replacement and the tendency to cooperate and report their experiences. data collection was done by conducting a deep, semi - structured interview using an mp4 voice recorder. each interview was started with an open question, what were your experiences in facing and living with the reality of valve replacement ? in addition, to receive further information and for a deep perception of words, some explorative questions like can you give more explanations ? and data collection was done until saturation was reached. using the graneheim and lundman content analysis for qualitative data, the synchronous description and analysis of the interviews were done as follows : (1) typing the entire interview script immediately after each session ; (2) reading through the whole text for a general perception of its content ; (3) determining the meaning units and the initial codes ; (4) classifying similar initial codes into more comprehensive categories ; and (5) determining the main themes of categories. the explanation and analysis were done in the following sequence : immediately after each interview session, its content was re - written in the word software, imported to maxqda - v7, and then the scripts were read several times to find general perceptions of the patients based on the aim of the research. then the meaning units / initial codes were derived, merged, and classified based on similarities. all efforts were taken to keep maximal homogeneity within the categories and also maximal heterogeneity between categories, and avoid the inclusion of the same data in two categories. ranking was done for new category based on aims and the main concepts were taken for the validity and reliability of results. based on polit and beck 's approval, for the accuracy of research findings in addition, for the acceptance of the data, all academic members with an experience of qualitative research were requested to evaluate the codes derived. through reviewing the handwritten scripts by the participants and peer - reviewing it by the research team colleagues, the validity of data was checked. for transferability, the participants were requested to declare some items for completing and correcting the codes. the written informed consent and the signed agreement of having the right to withdraw from the study or continue with the study whenever they wanted to were obtained from all participants. their demographic data are shown in table 1. from the rich and informative description of participants, then, based on similarities, they were classified and summarized into 14 subcategories. based on the conceptual and abstract exploration of the themes and using analysis and comparison, five subsidiary themes and a main theme of a life associated with fear and worry were extracted. most therapeutic centers in iran lack the facilities for valve replacement surgery, and this is also the case regarding experiences of the care team. these patients were often referred to the main centers. for removing my womb, i was hospitalized for 2 months. i was referred to one of them (doctors) for surgery most of the doctors rejected me due to the lack of therapeutic faccilities. (participant # 3, 32 years female, mitral valve replacement). in case of need for treatment or surgery the second source of worry for participants is the carelessness and failure of the therapeutic team. so, the patients do not refer to the doctors unless they are assured about them the reason for their worry is that if these patients face a problem, what do they do ? they desperately refer to other doctors and therapeutic centers which gives them a sort of worry. i fear everyone and everything and i told myself about the failure in surgery and its bad outcomes, i.e. dying for nothing. (participant # 2, 47-year - old female, mitral valve replacement done twice). for a cheeper pt, i often refer to hospitals, i always have the anxiety of a wrong pt. these worries are : worry of unexpected events, worry of the outcome of warfarin use, worry of the functioning of prosthetic valve, and worry of coagulatory test results. all the time the patients are worried about an unexpected event happening for different reasons, e.g. shortage of drugs and the discontinuty of valve function. sometimes i no longer bear it and have anxiety. for instance, when i have no warfarin, i think if the valves were non - functional, what can i do for it, how long really it works for me. these worries are intensified with a high risk of endocarditis, angiography, tooth extraction, or less education. i have a hard job, i must lift heavy loads of construction materials as a constitutional worker. (participant # 12, 34-year - old male, both mitral and aortic valve replacement). (participant # 2, 47-year - old male, mitral valve replaced twice). sometimes these worries result in the patient not following the treatment. when they performed angiography for me, i feared of dying for no reasons. despite the instructions of the doctor, i left the hospital for home, but desperately i came here again. (participant # 8, 50-year - old male, both mitral and aortic valve replacement). these patients should continue using warfarin until the end of life and all team members emphasize on this. so, patients are worried about forgetting the drug or its overdose, especially in some conditions like pregnancy and/or surgery. now, i fear everything, even going to a dentist and a bad luck. when patients face a new situation (a change in weather, travel and food regimen, using new drugs, and change in body metabolism, all of which have an effect on inr), they have to repeat the test and report its results to their doctors. the facilities are not available all the time and in case of inadequte education, it results in recurrent referrals and longer hospitalizations. disturbances in daily living and transmission of the disease to offsprings are one of the worries. for a long time during their life, these patients are involved in rheumatic heart disease, valvular involvement and replacement, and its outcomes to the extent that the condition afffects their routine life which results in a busy mind and constant worry. there was a great fear and threat in my mind on living with this sort of valve. another source of worry among the valve - replaced patients, especially affecting the offspring, is the repetitive nature of the exacerbation of the disease and the results of lab reports. however, in the present time, majority of the society members and their families, for different reasons, do not provide the required support. one thing that made me have more fear on operation and reject its proposal was my children 's fear of losing me and dying, and hence i bore the pain and rejected the operation., (participant # 8, 50-year - old male, both aortic and mitral valve replacement). for instance, during pregnancy, a change in the anticoagulant regimen (e.g. warfarin, heparin) should be made. another stressor that worsened my conditions was that everyday i heard words on the condition of my newborn baby. will it be healthy or have some problems in its heart ? (participant # 4, 40-year - old female, mitral valve replacement). when these pateints use the teratogenic drug warfarin, especially in the initial stages of the pregnancy, they have a troublesome life. because all the time, they hear about the teratogenicity of these agents and are worried about the defects it causes in their babies. fear of hospital is one of the themes that consisted of the following : fear of re - operation, fear of hospital milieu and the facilities of the hospital. the patients are not aware of most of the therapeutic / diagnostic methods used for valve - replaced pateints during their life period to the extent that they think that most of their problems could be solved with some ordinary lab tests. sometimes the unfavorable outcomes of these tests made them reject the diagnostic procedures or re - operation. my fear 's origin is in having no information, forgetting the instructions and not obeying the orders. sometimes the patients fear originated from their previous experiences or, in most cases, the information they received in relation to dying or related to the matter that they thought that they died because of their inadequate search for a better hospital and hurry in operation. so, they are much worried about the facilities and the capability of the surgeons. i was so feared of signing the informed consent and took the responsibility of discharging from hospital. (participant # 8, 50-year - old male, both aortic and mitral valve replacement). these pateints are asking and searching much for their obligatory operation valve surgery and also the alternatives, especially the non - surgical ones. one of their concerns in this regard is the carelessness of the surgeons, their insufficient experience, or the lack of facilities of the care team. the common source of fear for these patients is the unfamiliar environment and pepole. among the multiple sources of fear, one important thing is the pateints worry on the conflict and disagreement between the therapeutic team, mostly on diagnostic and treatment issues. the patients minds are occupied with problems on the follow - up and post - treatment risks and outcomes, which mostly begin after the valve replacement. in such big cities, like tehran, you can find the tactful doctors, only a few of them are here and they are busy all the time. (participant # 11, 45-year - old male, mitral valve replacement). because of their worry, pateints are trying to communicate with the nurses and doctors and ask them about the disease, treatment, and lifestyle. but because of inadequate education and the lack of opportunities to communicate with the therapeutic team, the pateints receive information from other sources which results in having doubts about the treatment or the fundamental errors that, by itself, aggravates the their anxiety. the theme of fear for unknowns consists of fear of the unexpected and unfavorable events and fear of the establishment of new condition the cause of fear is often similar previous experience of the patients, i.e. the risk of infection, disturbances in inr, bleeding, and clotting, that the probability of their occurrence increases in exposure to the new situation. in addition, often in therapeutic settings, the newly arrived non - professional personnel lack the required capability to care for the pateints. these situations are fearful for patients ; especially on hospitalization and during the diagnostic / therapeutic procedures, the pateints are worried about the occurrence of unknown events. i am afraid of being hospitalized, i am afraid of the doctors act, they do in a way that my valve be occluded and instruct me to the operation. in this case, it would be my third operation. when the pateints are worried, they try not to get hospitalized as much as possible ; otherwise, they try to get discharged earlier, all of which signify the lack of confidence in the therapeutic / health system that definitely has some negative effects on the patients treatment. knowing nothing about the problems, their reasons, and problem - solving strategies spontaneously, i felt a sort of post - operative anxiety in myself, i thought maybe my condition got worse, i am feared of the noise in my valves, it causes me not to have a comfortable sleep overnight, all the time it occupies my mind. (participant # 7, 67-year - old female, mitral valve replacement). sometimes fear following the valve replacement is productive in nature and acts as a motive for following the lab results and the efficacy of treatment and education. for example, it results in a feeling of lack of confidence on the therapeutic team, which causes worry, injury, and anxiety. most therapeutic centers in iran lack the facilities for valve replacement surgery, and this is also the case regarding experiences of the care team. these patients were often referred to the main centers. for removing my womb, i was hospitalized for 2 months. i was referred to one of them (doctors) for surgery most of the doctors rejected me due to the lack of therapeutic faccilities. (participant # 3, 32 years female, mitral valve replacement). in case of need for treatment or surgery the second source of worry for participants is the carelessness and failure of the therapeutic team. so, the patients do not refer to the doctors unless they are assured about them. the reason for their worry is that if these patients face a problem, what do they do ? they desperately refer to other doctors and therapeutic centers which gives them a sort of worry. i fear everyone and everything and i told myself about the failure in surgery and its bad outcomes, i.e. dying for nothing. (participant # 2, 47-year - old female, mitral valve replacement done twice). (participant # 7, 67-year - old female, mitral valve replacement). for a cheeper pt, i often refer to hospitals, i always have the anxiety of a wrong pt. these worries are : worry of unexpected events, worry of the outcome of warfarin use, worry of the functioning of prosthetic valve, and worry of coagulatory test results. all the time the patients are worried about an unexpected event happening for different reasons, e.g. shortage of drugs and the discontinuty of valve function. sometimes i no longer bear it and have anxiety. for instance, when i have no warfarin, i think if the valves were non - functional, what can i do for it, how long really it works for me. these worries are intensified with a high risk of endocarditis, angiography, tooth extraction, or less education. i have a hard job, i must lift heavy loads of construction materials as a constitutional worker. (participant # 12, 34-year - old male, both mitral and aortic valve replacement). (participant # 2, 47-year - old male, mitral valve replaced twice). sometimes these worries result in the patient not following the treatment. when they performed angiography for me, i feared of dying for no reasons. despite the instructions of the doctor, i left the hospital for home, but desperately i came here again. (participant # 8, 50-year - old male, both mitral and aortic valve replacement). these patients should continue using warfarin until the end of life and all team members emphasize on this. so, patients are worried about forgetting the drug or its overdose, especially in some conditions like pregnancy and/or surgery. now, i fear everything, even going to a dentist and a bad luck. when patients face a new situation (a change in weather, travel and food regimen, using new drugs, and change in body metabolism, all of which have an effect on inr), they have to repeat the test and report its results to their doctors. the facilities are not available all the time and in case of inadequte education, it results in recurrent referrals and longer hospitalizations. disturbances in daily living and transmission of the disease to offsprings are one of the worries. for a long time during their life, these patients are involved in rheumatic heart disease, valvular involvement and replacement, and its outcomes to the extent that the condition afffects their routine life which results in a busy mind and constant worry. there was a great fear and threat in my mind on living with this sort of valve. another source of worry among the valve - replaced patients, especially affecting the offspring, is the repetitive nature of the exacerbation of the disease and the results of lab reports. however, in the present time, majority of the society members and their families, for different reasons, do not provide the required support. one thing that made me have more fear on operation and reject its proposal was my children 's fear of losing me and dying, and hence i bore the pain and rejected the operation., (participant # 8, 50-year - old male, both aortic and mitral valve replacement). the other source of worry is the transmission of disease to the children. for instance, during pregnancy, a change in the anticoagulant regimen (e.g. warfarin, heparin) should be made. another stressor that worsened my conditions was that everyday i heard words on the condition of my newborn baby. will it be healthy or have some problems in its heart ? (participant # 4, 40-year - old female, mitral valve replacement). when these pateints use the teratogenic drug warfarin, especially in the initial stages of the pregnancy, they have a troublesome life. because all the time, they hear about the teratogenicity of these agents and are worried about the defects it causes in their babies. fear of hospital is one of the themes that consisted of the following : fear of re - operation, fear of hospital milieu and the facilities of the hospital. the patients are not aware of most of the therapeutic / diagnostic methods used for valve - replaced pateints during their life period to the extent that they think that most of their problems could be solved with some ordinary lab tests. sometimes the unfavorable outcomes of these tests made them reject the diagnostic procedures or re - operation. my fear 's origin is in having no information, forgetting the instructions and not obeying the orders. sometimes the patients fear originated from their previous experiences or, in most cases, the information they received in relation to dying or related to the matter that they thought that they died because of their inadequate search for a better hospital and hurry in operation. so, they are much worried about the facilities and the capability of the surgeons. it took 6 months that i decided for the operation. i was so feared of signing the informed consent and took the responsibility of discharging from hospital. (participant # 8, 50-year - old male, both aortic and mitral valve replacement). these pateints are asking and searching much for their obligatory operation valve surgery and also the alternatives, especially the non - surgical ones. one of their concerns in this regard is the carelessness of the surgeons, their insufficient experience, or the lack of facilities of the care team. the common source of fear for these patients is the unfamiliar environment and pepole. among the multiple sources of fear, one important thing is the pateints worry on the conflict and disagreement between the therapeutic team, mostly on diagnostic and treatment issues. the patients minds are occupied with problems on the follow - up and post - treatment risks and outcomes, which mostly begin after the valve replacement. in such big cities, like tehran, you can find the tactful doctors, only a few of them are here and they are busy all the time. (participant # 11, 45-year - old male, mitral valve replacement). because of their worry, pateints are trying to communicate with the nurses and doctors and ask them about the disease, treatment, and lifestyle. but because of inadequate education and the lack of opportunities to communicate with the therapeutic team, the pateints receive information from other sources which results in having doubts about the treatment or the fundamental errors that, by itself, aggravates the their anxiety. the theme of fear for unknowns consists of fear of the unexpected and unfavorable events and fear of the establishment of new condition, the cause of fear is often similar previous experience of the patients, i.e. the risk of infection, disturbances in inr, bleeding, and clotting, that the probability of their occurrence increases in exposure to the new situation. in addition, often in therapeutic settings, the newly arrived non - professional personnel lack the required capability to care for the pateints. these situations are fearful for patients ; especially on hospitalization and during the diagnostic / therapeutic procedures, the pateints are worried about the occurrence of unknown events. i am afraid of being hospitalized, i am afraid of the doctors act, they do in a way that my valve be occluded and instruct me to the operation. in this case when the pateints are worried, they try not to get hospitalized as much as possible ; otherwise, they try to get discharged earlier, all of which signify the lack of confidence in the therapeutic / health system that definitely has some negative effects on the patients treatment. knowing nothing about the problems, their reasons, and problem - solving strategies spontaneously, i felt a sort of post - operative anxiety in myself, i thought maybe my condition got worse, i am feared of the noise in my valves, it causes me not to have a comfortable sleep overnight, all the time it occupies my mind. (participant # 7, 67-year - old female, mitral valve replacement). sometimes fear following the valve replacement is productive in nature and acts as a motive for following the lab results and the efficacy of treatment and education. for example, it results in a feeling of lack of confidence on the therapeutic team, which causes worry, injury, and anxiety. findings from the main themes of fear and worry signified the actual experience of the patients. analysis of the participants experiences showed that the patients got their fear and worry from the following sources : worry on care condition, worry of the specified conditions of the patients, worry on the instability in life, fear of hospital, and fear of unknowns. of these fear sources, three sources are related to human sources and the remaining two sources to non - human (environmental and facilities) sources. these novel findings signify the role of therapeutic team, especially nurses, its presence, quality, and communication with patients for eliminating their fear and worry. the findings of our study reveal that when the participants are unable to communicate with the therapeutic team, they do have a feeling of fear and worry. on facing the stressful conditions, patients require nursing care followed by their understanding of the condition. in situations where the nurses had no intimate and emotional relations with their clients, the patients feared and were worried. these findings are in line with the studies of hawley who revealed that a positive conversation between the emergency ward nurses and patients resulted in confidence, sympathy, insurance, and relieving the worry on the unknowns of the disease. baldursdottir and jonsdottir pointed out that the patients reported capability as the most important care behavior of the emergency ward nurses. in our study, we found from the participants experiences that worry on the therapeutic team and worry on the carelessness of the team are the sources of unexpected events. revealed that the most important worries among chinese patients were about delay in administering the drug, no on - time treatment, and the non - constant sitting on patient 's bedside. also, jooybari. showed that being in the same room, patients could have an intimate relationship among them and in the absence of the nurse, this relationship develops a sort of robust motivation for cooperation and social interactions. badir stated that family members have a significant role in patients health and recovery process. tensions in valve - replaced patients are the psychosocial effects of the problem (e.g. waiting for the result of clinical test, fear of losing the valve, etc.). these sources of tension in everyday life require adaptation and, in some cases, are among the routine problems, like using warfarin or not using it and also its effects on the body. these usual events, by themselves, are not catastrophic and ailing, but due to their cumulative effects on the individual 's life, they could result in some somatic problems for the patient. other stressors with regard to patient 's condition are the ones that have an effect on his / her life, e.g. life events that, in some cases, cause social / psychological crises like physical disability, permanent disability, or problematic life with a prosthetic valve. revealed that some factors (e.g. physical inactivity, low salary due to inactivity, lack of entertainment, and lower satisfaction in emotional support) are among the factors causing post - heart transplantation disappointment in patients. the constant noise of prosthetic valve, the financial problems, and the physical disability result in more anxiety. losing the role function or perceived aim in life can cause tension and discomfort. each of these specified variables or the extra needs of the patients results in ineffective adaptation which can be resolved with post - replacement recovery and rehabilitation. in line with these findings,, in their study on heart recipients, revealed that the group of recipients who followed a regular training program and received emotional support postoperatively had a significant improvement in psychological function. gross and coworkers reported that anxiety is an inseparable problem of chronic disease and these patients have no favorable quality of life. hence, their anxiety must be assessed and managed because of its effect on treatment and in following the prescriptions. in their study, corruble. emphasized on anxiety as a leading issue which, especially in post - transplantation period, could be due to the drug misuse and other factors and results in an increased mortality rate. overall, the actual assessment of the stressful situation and its sources can improve the care condition of such patients. the worry and fear after heart valve replacement originate from two main sources, namely human sources or the personnel involved in care and the non - human sources which relate to the environment and logistic facilities. being aware of these fears and worry sources and stressors helps the caregivers in providing care, essential education, and promoting the quality of life and survival. identifying the actual problems of the heart valve replaced patients and detection of the unknowns of these patients signify their needs for support, advice, and psychological rehabilitation. | background : few attempts were made for alleviating the physical / psychological problems among the cardiac valve replaced patients and no comprehensive study was done based on the experiences of such patients. this study was undertaken to describe the stressful experiences of the heart valve - replaced patients.materials and methods : in this qualitative study performed during 2012 - 2013 with a content analysis approach, 13 patients from tehran and kashan therapeutic centers participated. the study sampling was accomplished with purposeful sampling using a semi - structured interview that continued until data saturat ion. all interviews were recorded, and were immediately handwritten word by word and finally typewritten. description and analysis of the data were done by graneheim and lundman content analysis.results:one hundred and seventy - five primary codes were derived among the 680 codes taken from the participants interviewed. using abstract and deep perception of the categories, 14 subcategories and 5 themes were derived. the themes are as follows : worry of care conditions, worry of life with the ongoing condition of having prosthetic cardiac valve, worry regarding the instability in life, fear of hospital, and fear of unknown factors. each theme consisted of special subsidiary themes with specific functions.conclusions:the main themes of fear and worry about on losing the valve were identified and introduced in the cardiac valve - replaced patients. as the nature and function of these themes are different in different societies, recognition and discrete definition of them are necessary for care planning and promotion. |
a large body of the recent psychiatric literature is questioning the empirical value of current prescribing habits as well as the psychiatrists choice of medication selection as increasing amounts of polypharmacy and risk of medications creep into patients regimes.112 without better evidence - based research, often medication choices are made by trial and error, leading to significant delays to effective treatment. there is little empirical evidence supporting the benefits of polypharmacy, and continuing a trial - and - error approach to the implementation of psychotropic medications. this is discussed in a recently published paper1 in which polypharmacy is compared to washing patients out of their current medication as a possible next step in medication management. in the newly reported combining medication to enhance depression outcomes study,13 a national institutes of health (nih)-funded research to determine whether starting several antidepressants at the same time would be associated with increased efficacy, no significant difference between the response or remission rates were observed. this large, well - designed study supports the contention that psychiatry is still in need of evidence - based tools to orient psychotropic treatment selection. this same conclusion was highlighted by recent programmatic documents of the national institute of mental health (nimh), which highlighted the need for objective evidence - based neuroscience1417 instruments in addition to diagnoses based on symptom clusters in selecting the most effective treatments. additionally, reports from the large nih - funded sequence treatment alternatives to relieve depression (stard) study,18 as well as other publications,19,20 have revealed the lack of biomarkers and the limitations of relying on symptom - based prescribing followed by quantitative electroencephalography (qeeg) which involves computerized spectral analysis of electroencephalography (eeg) signals provides information that can not be extracted through visual inspection of eeg alone and has been previously used to predict the outcome of antidepressant treatment. some studies suggest that baseline qeeg parameters may also serve to predict the total burden of treatment - emergent side effects or more specifically to predict treatment - emergent suicidal ideation.21,22 there is ample previous evidence for the qeeg - based treatment outcomes in the literature. suffin and emory,23 through referenced - eeg (now called psychiatric eeg evaluation registry or peer report), initially examined attentional and affective disorders and their successful association with pharmacotherapeutic outcomes. other smaller preliminary studies have suggested a potential role in using this information for medication selection for depression,2426 eating disorders,27 and substance abuse25 with similar promising results. another pilot study28 was conducted to compare this same methodology with the texas medication algorithm project (tmap) used for patients with treatment - resistant depression. the data in that study resulted in statistically greater change from baseline outcome scores than those treated with tmap - guided therapy. in a larger, multicenter, randomized trial, debattista29 compared the referenced - eeg database treatment group (experimental) with an optimized treatment based on the stard study guidelines (control) in patients with treatment - refractory major depressive disorder.18 the experimental group s selection led to statistically better outcomes compared with the control group. a recent retrospective chart review in the treatment of depression in eating disorders30 reported on 22 patients with a 2-year history prior to using the peer report to guide treatment and followed them for 25 years. patients demonstrated significant decrease in depressive symptoms hamilton depression rating scale, severity of illness clinical global impression severity (cgi - s), and overall clinical global impression improvement (cgi - i). the peer report information utilizing the referenced - eeg database can assist clinicians treating nonpsychotic psychiatric patients with objective choices that offer (1) caution against medications potentially associated with adverse events for a given patient, while at the same time (2) giving evidence - based knowledge from other patients with similar brainwave patterns associated with positive responses to specific medications in the growing database. the methodology for determining the medication ratings in the database has been previously published.30 an uncontrolled retrospective chart review of clinical cases having received a qeeg utilizing the referenced - eeg database (now called peer report) was performed. the objective was to determine if peer information would improve overall global ratings and quality of life in nonpsychotic patients in a typical psychiatric outpatient clinical setting. in addition, we hypothesized that the report might help caution the prescriber about the potential of severe adverse events and reduce the number of such negative outcomes. an uncontrolled retrospective chart review of clinical cases having received a qeeg utilizing the referenced - eeg database (now called peer report) was performed. the objective was to determine if peer information would improve overall global ratings and quality of life in nonpsychotic patients in a typical psychiatric outpatient clinical setting. in addition, we hypothesized that the report might help caution the prescriber about the potential of severe adverse events and reduce the number of such negative outcomes. we reviewed the charts of 435 patients who elected to undergo qeeg assessment between 2003 through mid-2011 in an outpatient psychiatric clinic with three prescribers. the patient population consisted of any nonpsychotic diagnosis typically seen in a private outpatient clinical setting that agreed to follow the medication plan suggested by the referenced - eeg database and peer report, along with any comorbid conditions. the option for using the report was given to patients who could safely washout of medication for at least 5 half - lives and desired to add evidenced - based data to their treatment decisions. most of them were treatment - resistant (having failed at least two previous medication trials). the patients had to pay for the test, but a compassionate - use policy allowed for a sliding scale. all patients receiving an eeg had signed two different informed consents stating that their data could be used for purposes of research or publication in the future and that all health insurance portability and accountability act standards and personal health information would not be included or divulged in any way. they were also given choices of other treatments or allowed to cease testing at any time without affecting their treatment options, as per standard informed consent language. abstracted data from the patient s chart included primary and secondary diagnoses, age, history of failed medication trials, adverse events to prior medications, severe adverse events (eg, agitation, hostility, aggressiveness, suicidality, homicidality, mania, hypomania), severity of symptoms, and off - label use. in our clinic (neuro - therapy clinic, inc, denver, co) depression and anxiety rating scales are administered as part of the initial assessment for each new patient, regardless of diagnosis. two different depression scales were used because the clinic changed assessment tools within the time period of the review. including all anxiety and depression scores for the population (even on patients without an anxiety or depression diagnosis) skews the group depression / anxiety statistics toward being less severe since many of the patients did not have depressive or anxious symptoms, thus causing lower scores than might be anticipated. postdepression / anxiety scores were not available because outcomes were measured by global scores, such as cgi - i and quality of life enjoyment and satisfaction short form (q - les - q - sf), in agreement with the diagnostic agnostic fundamentals of the peer data. clinical chart data was analyzed from the point of medication implementation (guided by the peer report) to the point where the prescriber determined patients were at maximum medical improvement (mmi) the term traditionally used, in medical disability, for example, to define the point at which they were thought to be stable and at which no further medication changes would alter their outcome. the outpatient clinic included three individual prescribers who rated patients at each session according to the cgi - i rating scale31,32 at all visits since january 2009. patients also filled out q - les - q - sf ratings.33 data pertaining to all known prior medications, along with known severe side effects, were also collected. raw data was recorded on spreadsheets, and changes in scores were calculated by subtracting baseline scores from the mmi scores. the cgi - i31 is a well - accepted seven - point likert scale used regularly in both clinical practice and research. a score of 4 means no change, compared to baseline. very much improved, two is much improved, and three is improved. similarly, scores of 57 represent worsening. a score of 2 or 1 (much or very much improved) is typically used as the definition of a good clinical response. the q - les - q - sf33 is a quality - of - life questionnaire assessing 14 areas of a patient s life circumstances. while the q - les - q - sf has never been normed into typical bands of mild, moderate, or severe, psychiatric patients in treatment who are considered responders tend to score in the mid-60s or above. schechter, among others, have written that psychiatric patients successfully treated for depression and anxiety score in the mid- to upper-60s, while patients with moderate symptoms or who function with only some difficulty tend to score ~64%.3438 descriptive statistics (mean, range, standard deviations, percentage change) were used to characterize study subjects on demographic and clinical measures at baseline, including age, severity of illness, history of failed medication trials, and previous severe adverse events prior to medications prescribed after initiating the peer report. for the primary study outcomes, we performed student s t - tests to compare the mean number of medications used by subjects, their mean improvement as measured by the cgi - i, and their mean quality of life scores as measured by the q - les - q - sf, before and after starting peer guided treatment. due to the relative rarity of events related to suicidality and the difficulty in determining exact pre - period time windows for specification of incidence rates per unit of time, we report suicidal occurrences as counts (number of events observed) before and after subjects started peer - guided treatment. no statistical analyses were applied to the counts of suicide - related occurrences that were observed in this study. rates of severe adverse events before and after subjects started peer - guided treatment and rates of pre - off - label medication use were also reported. no statistical analyses were applied to the adverse event rates or off - label use rates that were measured in this study. where there were no statistical analyses applied, it was either due to the fact that some measures had uncertainty about equal measurement intervals or we erred on the side of caution by not making statistical comparisons of pre versus post data since the rates of some measurements were so low that we did not have much statistical power to make adequate comparisons. statistical analyses were performed using sas version 10.0 (sas institute inc, cary, nc). all tests were two - sided, with an a priori alpha - level of 0.05 for all comparisons. we reviewed the charts of 435 patients who elected to undergo qeeg assessment between 2003 through mid-2011 in an outpatient psychiatric clinic with three prescribers. the patient population consisted of any nonpsychotic diagnosis typically seen in a private outpatient clinical setting that agreed to follow the medication plan suggested by the referenced - eeg database and peer report, along with any comorbid conditions. the option for using the report was given to patients who could safely washout of medication for at least 5 half - lives and desired to add evidenced - based data to their treatment decisions. most of them were treatment - resistant (having failed at least two previous medication trials). the patients had to pay for the test, but a compassionate - use policy allowed for a sliding scale. all patients receiving an eeg had signed two different informed consents stating that their data could be used for purposes of research or publication in the future and that all health insurance portability and accountability act standards and personal health information would not be included or divulged in any way. they were also given choices of other treatments or allowed to cease testing at any time without affecting their treatment options, as per standard informed consent language. abstracted data from the patient s chart included primary and secondary diagnoses, age, history of failed medication trials, adverse events to prior medications, severe adverse events (eg, agitation, hostility, aggressiveness, suicidality, homicidality, mania, hypomania), severity of symptoms, and off - label use. in our clinic (neuro - therapy clinic, inc, denver, co) depression and anxiety rating scales are administered as part of the initial assessment for each new patient, regardless of diagnosis. two different depression scales were used because the clinic changed assessment tools within the time period of the review. including all anxiety and depression scores for the population (even on patients without an anxiety or depression diagnosis) skews the group depression / anxiety statistics toward being less severe since many of the patients did not have depressive or anxious symptoms, thus causing lower scores than might be anticipated. postdepression / anxiety scores were not available because outcomes were measured by global scores, such as cgi - i and quality of life enjoyment and satisfaction short form (q - les - q - sf), in agreement with the diagnostic agnostic clinical chart data was analyzed from the point of medication implementation (guided by the peer report) to the point where the prescriber determined patients were at maximum medical improvement (mmi) the term traditionally used, in medical disability, for example, to define the point at which they were thought to be stable and at which no further medication changes would alter their outcome. the outpatient clinic included three individual prescribers who rated patients at each session according to the cgi - i rating scale31,32 at all visits since january 2009. patients also filled out q - les - q - sf ratings.33 data pertaining to all known prior medications, along with known severe side effects, were also collected. raw data was recorded on spreadsheets, and changes in scores were calculated by subtracting baseline scores from the mmi scores. the cgi - i31 is a well - accepted seven - point likert scale used regularly in both clinical practice and research. a score of 4 means no change, compared to baseline. very much improved, two is much improved, and three is improved. similarly, scores of 57 represent worsening. a score of 2 or 1 (much or very much improved) is typically used as the definition of a good clinical response. the q - les - q - sf33 is a quality - of - life questionnaire assessing 14 areas of a patient s life circumstances. while the q - les - q - sf has never been normed into typical bands of mild, moderate, or severe, psychiatric patients in treatment who are considered responders tend to score in the mid-60s or above. schechter, among others, have written that psychiatric patients successfully treated for depression and anxiety score in the mid- to upper-60s, while patients with moderate symptoms or who function with only some difficulty tend to score ~64%.3438 descriptive statistics (mean, range, standard deviations, percentage change) were used to characterize study subjects on demographic and clinical measures at baseline, including age, severity of illness, history of failed medication trials, and previous severe adverse events prior to medications prescribed after initiating the peer report. for the primary study outcomes, we performed student s t - tests to compare the mean number of medications used by subjects, their mean improvement as measured by the cgi - i, and their mean quality of life scores as measured by the q - les - q - sf, before and after starting peer guided treatment. due to the relative rarity of events related to suicidality and the difficulty in determining exact pre - period time windows for specification of incidence rates per unit of time, we report suicidal occurrences as counts (number of events observed) before and after subjects started peer - guided treatment. no statistical analyses were applied to the counts of suicide - related occurrences that were observed in this study. rates of severe adverse events before and after subjects started peer - guided treatment and rates of pre - off - label medication use were also reported. no statistical analyses were applied to the adverse event rates or off - label use rates that were measured in this study. where there were no statistical analyses applied, it was either due to the fact that some measures had uncertainty about equal measurement intervals or we erred on the side of caution by not making statistical comparisons of pre versus post data since the rates of some measurements were so low that we did not have much statistical power to make adequate comparisons. statistical analyses were performed using sas version 10.0 (sas institute inc, cary, nc). all tests were two - sided, with an a priori alpha - level of 0.05 for all comparisons. clinical chart data was analyzed from the point of medication implementation (guided by the peer report) to the point where the prescriber determined patients were at maximum medical improvement (mmi) the term traditionally used, in medical disability, for example, to define the point at which they were thought to be stable and at which no further medication changes would alter their outcome. the outpatient clinic included three individual prescribers who rated patients at each session according to the cgi - i rating scale31,32 at all visits since january 2009. patients also filled out q - les - q - sf ratings.33 data pertaining to all known prior medications, along with known severe side effects, were also collected. raw data was recorded on spreadsheets, and changes in scores were calculated by subtracting baseline scores from the mmi scores. the cgi - i31 is a well - accepted seven - point likert scale used regularly in both clinical practice and research. very much improved, two is much improved, and three is improved. similarly, scores of 57 represent worsening. a score of 2 or 1 (much or very much improved) is typically used as the definition of a good clinical response. the q - les - q - sf33 is a quality - of - life questionnaire assessing 14 areas of a patient s life circumstances. while the q - les - q - sf has never been normed into typical bands of mild, moderate, or severe, psychiatric patients in treatment who are considered responders tend to score in the mid-60s or above. schechter, among others, have written that psychiatric patients successfully treated for depression and anxiety score in the mid- to upper-60s, while patients with moderate symptoms or who function with only some difficulty tend to score ~64%.3438 descriptive statistics (mean, range, standard deviations, percentage change) were used to characterize study subjects on demographic and clinical measures at baseline, including age, severity of illness, history of failed medication trials, and previous severe adverse events prior to medications prescribed after initiating the peer report. for the primary study outcomes, we performed student s t - tests to compare the mean number of medications used by subjects, their mean improvement as measured by the cgi - i, and their mean quality of life scores as measured by the q - les - q - sf, before and after starting peer guided treatment. due to the relative rarity of events related to suicidality and the difficulty in determining exact pre - period time windows for specification of incidence rates per unit of time, we report suicidal occurrences as counts (number of events observed) before and after subjects started peer - guided treatment. no statistical analyses were applied to the counts of suicide - related occurrences that were observed in this study. rates of severe adverse events before and after subjects started peer - guided treatment and rates of pre - off - label medication use were also reported. no statistical analyses were applied to the adverse event rates or off - label use rates that were measured in this study. where there were no statistical analyses applied, it was either due to the fact that some measures had uncertainty about equal measurement intervals or we erred on the side of caution by not making statistical comparisons of pre versus post data since the rates of some measurements were so low that we did not have much statistical power to make adequate comparisons. statistical analyses were performed using sas version 10.0 (sas institute inc, cary, nc). all tests were two - sided, with an a priori alpha - level of 0.05 for all comparisons. charts of 435 psychiatric patients who elected to undergo qeeg assessment between 2003 through mid-2011 were reviewed. a total of 178 patients were excluded from the analysis for the following reasons : lost to follow - up, 127 ; noncompliance, 29 ; insufficient data, 16 ; other, 6. there were an additional 27 patients who, after their peer assessment, did not need medication for their treatment. abstracted data from the patient s chart included primary and secondary diagnoses, age, history of failed medication trials, adverse events to prior medications, severe adverse events (as mentioned previously), severity of symptoms, and off - label use. the 230 patients who received and followed peer averaged 36.4 (767.1 ; standard deviation [sd ] 13.9) years of age. the average number of previous medication treatment failures was 5.9, dropping to 2.4 after treatment guided by the peer report (table 1). these included either medications prescribed at the same time or failed attempts in both pre- and post - peer guidance. the percent of patients lost to follow - up was similar to different comparable control populations. these included other patients seen in the clinic that did not get a qeeg, and published studies utilizing the referenced - eeg database for depression efficacy29 in treatment - resistant patients, and a retrospective chart review in the eating - disorder population30 (table 2). also noted in table 2 is the additional benefit for 10.5% of those patients who required no pharmacological treatment after washout. the first were statistics relating to average number of drugs used and time to mmi (table 1). the second measurements were clinical global scores that would not be dependent on any diagnostic category but addressed overall functioning. table 1 consists of the cgi - i and the q - les - q - sf. eighty - seven percent of patients achieved significant improvement (cgi - i of 1 or 2), and this was achieved within four visits for 68.7% of patients. previous suicidality was determined by reviewing the chart for patients suicidal thoughts, ideations, plans, or attempts discovered in the intake and past history. if the same or different patient had any of these identical symptoms after starting the peer - guided treatment, it was recorded as an occurrence. for those patients the review charting was followed throughout their association with the clinic, which averaged 721.9 (543591) days (table 1). for comparison, 7% of subjects in stard level 1 who experienced treatment - emergent suicidal ideation experienced a new onset of suicidal ideation. 39 also, between 11.1% and 34.8% of patients in the nimh stard study who discontinued treatment in the second treatment level described severe adverse events as the reason.40 in the current version of the peer report, medication classes as well as specific medications are rated in three categories based on the historical use from other providers data in the registry for success (similar to an antibiotic sensitivity report). the three categories are s (sensitive or > 85% chance of treatment success), i (intermediate or 35%85%), and (resistant or 85% chance of treatment success), i (intermediate or 35%85%), and (resistant or < 35% chance of success). reviewing all past medications and patient reported responses, severe adverse events (eg, agitation, hostility, aggressiveness, suicidality, homicidality, mania, hypomania) were noted and compared with the rating score of their current peer report. r on the peer report were associated with rates of severe adverse effects 55% of the time. r ratings of medications can be considered as a potential cautionary flag in that, had the information been available at the time of giving the medication, the adverse response may have been prevented. the same analysis was performed in the dataset of a previously published multicenter depression efficacy study29 (des) testing peer efficacy in major depressive disorder. in that dataset, r - rated medications were associated with severe adverse effects 50% of the time. table 3 shows both studies along with the rating on the drug causing the significant adverse side effect. one drug in the chart review had insufficient information in the database to be included. the pooled results were statistically significant assuming an equal distribution across r / i / s categories as the null hypothesis. the medications prescribed based on review of the qeeg data contained in the current version of the referenced - eeg database and peer report were from four different classes, ie, anticonvulsants, antidepressants, stimulants (which included monoamine oxidase inhibitors due to their stimulating effect on the eeg and the way the database classifies them), and beta - blockers. patients were treated with either monotherapy or combinations of medications guided by the four categories listed in the peer report. clinical judgment always superseded data from the report as the peer report offers more data to incorporate into the clinical decisionmaking process and is not meant as a stand - alone cookbook for psychotropic medications. since not all medications are part of the current version of the database, substitutions were allowed in the following instances : duloxetine for venlafaxine ; oxcarbazepine for carbamazepine ; and lisdexamphetamine for dextroamphetamine. our chart review was not designed to define us food and drug administration (fda) labeling, nor was it intended to encourage such prescribing habits. due to this concern we examined the amount of off - label prior drug - use in this population as well as the literature and found a paucity of evidence for most off - label prescribing. when having to decide on changing or adding a medication, factoring in polypharmacy risks and benefits unfortunately is done with inadequate information. the large degree of off - label prescribing should be done with as much data as is available to the prescriber in order to optimize safety and outcome. this secondary analysis was performed to help address this possible criticism of off - label prescribing since it is done with such frequency and insufficient evidence. peer has the potential to enhance the information available to the prescriber when making these difficult decisions, thus offering some objectivity to off - label usage. radley wrote about off - label medication - prescribing, placing psychiatric medications as one of the most frequently prescribed at 31%. yury found that there is no published data for 40% of the most popular augmentation strategies and 55% of frequent combination of medications for augmentation. additionally, an agency for healthcare research and quality (ahrq) report43 reviewing the top off - label use of medications according to such factors as risk, cost, side effects, and drug interactions, revealed 17 of them to be psychotropic. of those studied, 65% had inadequate evidence for off - label prescribing (ie, quetapine, clonazepam, escitalopram, lorazepam, trazodone, zolpidem, sertraline, bupropion, venlafaxine, duloxetine, aripiprazole). the remaining 35% (ie, gabapentin, risperidone, amitriptyline, olanzapine, devalproex, lamotrigine) averaged 12.5% adequate evidence, 33.2% uncertain evidence, and 54.3% inadequate evidence. in another ahrq report on off - label use of atypical antipsychotic medications, it concluded that, with few exceptions, there was not enough evidence to consider the off - label use of these drugs and that the increased risk of adverse events was concerning.44 we used our own data to perform a review of all the drugs labeling and dates of approval, using the fda s website for each medication and approval date for each diagnosis. the date used for the prescribing date of previous medications was prior to 2003, the beginning date of the review, since patient history of medications start / stop dates is frequently unreliable. this analysis revealed that 62% of previously prescribed psychotropic medications were offlabel at the time of their prescription date. this assessment leaves many variables not accounted for, such as what previous diagnosis the patient had received at the time the drug was prescribed, which might be different than the diagnosis determined in our clinic or what the patient reported. we are not implying any relationship between off - label prescribing and the medications used by peer guidance. rather, since there is an abundance of off - label psychotropic medication - prescribing, such prescribing, with more evidenced - based information, can decrease risks and increase success, according to the results of this review. despite the evidence of increased safety and efficacy in this and previous studies,2130 there are several limitations inherent in this retrospective chart review that may limit the conclusions one can draw from a case series. first, the peer report was initially established as a predictor of future treatment success. at this point we are not able to separate the role of peer in selecting the most efficacious treatments from the selection of better - tolerated treatments (since, many times, poorly tolerated treatments are abandoned in clinical practice and ultimately not efficacious). however, it is comforting to see that peer reports appear to be associated with effective and well - tolerated medication choices. second, while this review did not systematically benefit from research - ready data, our analyses suggest overall improvement on both cgi - i and q - les - q - sf global scales after peer. third, there was no comparison group in this study so it is not clear what the effects of treatment would have been in a parallel cohort of subjects not utilizing the peer analysis. however, some information is provided by comparing our results with the patients pre - peer experience (ie, treatment failure) and historic data, making each patient s pre / post results their own control. fourth, this is a cohort of persons who could afford the costs (despite the compassionate sliding scale offered) associated with the eeg test, and thus these patients may not be representative of all similar patients. fifth, not all available medications are in the current version of the database, leading to potential limitations. however, this would reduce our ability to detect a benefit with peer reports compared to standard practice (and increases our confidence in our positive results). our chart review was not designed to define us food and drug administration (fda) labeling, nor was it intended to encourage such prescribing habits. due to this concern we examined the amount of off - label prior drug - use in this population as well as the literature and found a paucity of evidence for most off - label prescribing. when having to decide on changing or adding a medication, factoring in polypharmacy risks and benefits unfortunately is done with inadequate information. the large degree of off - label prescribing should be done with as much data as is available to the prescriber in order to optimize safety and outcome. this secondary analysis was performed to help address this possible criticism of off - label prescribing since it is done with such frequency and insufficient evidence. peer has the potential to enhance the information available to the prescriber when making these difficult decisions, thus offering some objectivity to off - label usage. radley wrote about off - label medication - prescribing, placing psychiatric medications as one of the most frequently prescribed at 31%. yury found that there is no published data for 40% of the most popular augmentation strategies and 55% of frequent combination of medications for augmentation. additionally, an agency for healthcare research and quality (ahrq) report43 reviewing the top off - label use of medications according to such factors as risk, cost, side effects, and drug interactions, revealed 17 of them to be psychotropic. of those studied, 65% had inadequate evidence for off - label prescribing (ie, quetapine, clonazepam, escitalopram, lorazepam, trazodone, zolpidem, sertraline, bupropion, venlafaxine, duloxetine, aripiprazole). the remaining 35% (ie, gabapentin, risperidone, amitriptyline, olanzapine, devalproex, lamotrigine) averaged 12.5% adequate evidence, 33.2% uncertain evidence, and 54.3% inadequate evidence. in another ahrq report on off - label use of atypical antipsychotic medications, it concluded that, with few exceptions, there was not enough evidence to consider the off - label use of these drugs and that the increased risk of adverse events was concerning.44 we used our own data to perform a review of all the drugs labeling and dates of approval, using the fda s website for each medication and approval date for each diagnosis. the date used for the prescribing date of previous medications was prior to 2003, the beginning date of the review, since patient history of medications start / stop dates is frequently unreliable. this analysis revealed that 62% of previously prescribed psychotropic medications were offlabel at the time of their prescription date. this assessment leaves many variables not accounted for, such as what previous diagnosis the patient had received at the time the drug was prescribed, which might be different than the diagnosis determined in our clinic or what the patient reported. we are not implying any relationship between off - label prescribing and the medications used by peer guidance. rather, since there is an abundance of off - label psychotropic medication - prescribing, such prescribing, with more evidenced - based information, can decrease risks and increase success, according to the results of this review. despite the evidence of increased safety and efficacy in this and previous studies,2130 there are several limitations inherent in this retrospective chart review that may limit the conclusions one can draw from a case series. first, the peer report was initially established as a predictor of future treatment success. at this point we are not able to separate the role of peer in selecting the most efficacious treatments from the selection of better - tolerated treatments (since, many times, poorly tolerated treatments are abandoned in clinical practice and ultimately not efficacious). however, it is comforting to see that peer reports appear to be associated with effective and well - tolerated medication choices. second, while this review did not systematically benefit from research - ready data, our analyses suggest overall improvement on both cgi - i and q - les - q - sf global scales after peer. third, there was no comparison group in this study so it is not clear what the effects of treatment would have been in a parallel cohort of subjects not utilizing the peer analysis. however, some information is provided by comparing our results with the patients pre - peer experience (ie, treatment failure) and historic data, making each patient s pre / post results their own control. fourth, this is a cohort of persons who could afford the costs (despite the compassionate sliding scale offered) associated with the eeg test, and thus these patients may not be representative of all similar patients. fifth, not all available medications are in the current version of the database, leading to potential limitations. however, this would reduce our ability to detect a benefit with peer reports compared to standard practice (and increases our confidence in our positive results). our retrospective chart analysis of clinical cases indicates that peer report using the referenced - eeg database may be a useful metric tool for clinicians making medication recommendations for refractory nonpsychotic patients. as this data indicates, peer report may be useful as a negative marker to potentially avoid some risk of severe adverse events and in selecting more efficacious agents in treatmentresistant patients. the results of this review are encouraging and indicate that treating patients with the additional information conveyed by the referenced - eeg database and peer report may result in better treatment responses in a group of patients who had not previously responded to trial - and - error medication selection (currently considered standard practice). for example, the use of stimulants in some patients with obsessive - compulsive disorder, anxiety, or eating disorders without the use of an antidepressant would not be a traditional medication choice, yet in these cases appeared to be what was needed for their particular neurophysiology (given positive clinical outcomes). the potential cost savings as a result of an effective medication regimen suggest that peer analysis may be cost - effective. the durability of response with medications selected according to data provided by this tool and the broader options of medication combinations suggested by data in the report portends well for treatment compliance, number of medication trials, and treatment efficiency. getting a patient on the correct medicine with improvement within a few sessions not only reduces the cost of trial - and - error prescribing, but, more importantly, reduces patient suffering. it is also noted that at this point in the development of the database, almost all the medications are generic. similarly, the use of atypical antipsychotics for the nonpsychotic patient in our clinic is estimated to be significantly lower than the use in practices treating similar patient populations. future directions for the use of this technology include development of additional drugs and including comparisons with other neuroimaging techniques to address neuroanatomy in addition to clinical correlations. more studies need to be done on the ability to prevent serious adverse events, suicidality, as well as targeting the best medication option for the individualized patient. finally, a separate database is being completed for predicting outcomes of transcranial magnetic stimulation therapy as a way of deciding the probability of success when the decision to spend health care dollars needs to be considered. further research in these areas need to confirm the value of qeeg as a simple, inexpensive, and noninvasive outpatient clinical tool for accuracy, safety, and cost savings. | we previously reported on an objective new tool that uses quantitative electroencephalography (qeeg) normative- and referenced - electroencephalography sampling databases (currently called psychiatric eeg evaluation registry [peer ]), which may assist physicians in determining medication selection for optimal efficacy to overcome trial - and - error prescribing. the peer test compares drug - free qeeg features for individual patients to a database of patients with similar eeg patterns and known outcomes after pharmacological interventions. based on specific eeg data elements and historical outcomes, the peer report may also serve as a marker of future severe adverse events (eg, agitation, hostility, aggressiveness, suicidality, homicidality, mania, hypomania) with specific medications. we used a retrospective chart review to investigate the clinical utility of such a registry in a naturalistic environment.resultsthis chart review demonstrated significant improvement on the global assessment scales clinical global impression improvement and quality of life enjoyment and satisfaction short form as well as time to maximum medical improvement and decreased suicidality occurrences. the review also showed that 54.5% of previous medications causing a severe adverse event would have been raised as a caution had the peer report been available at the time the drug was prescribed. finally, due to the significant amount of off - label prescribing of psychotropic medications, additional, objective, evidence - based data aided the prescriber toward better choices.conclusionthe peer report may be useful, particularly in treatment - resistant patients, in helping to guide medication selection. based on the preliminary data obtained from this chart review, additional studies are warranted to establish the safety and efficacy of adding peer data when making medication decisions. |
in the theories of traditional chinese medicine (tcm), health of human body is considered as a particular state of dynamic equilibrium that could be maintained within a certain range by bodies themselves and be disturbed by the influences of multiple factors. when the disequilibrium goes up to a certain extent, the bodies would develop morbidity. zheng hou in chinese, a summary of pathogeny, location and pathology of one stage in the development of disease, is a comprehensive response to the internal and external action of body and environment, and also changes with the development of diseases. tcm grasps the pathogeny, location and pathology of the disease through making a comprehensive analysis of the patient s symptoms and physical characteristics. the tcm syndrome is built on the bases of long - term and substantial clinical practice. during the process of recognition the diseases, doctors of tcm obtain the information about physical characteristics and symptoms of patients firstly by diagnostic means such as observing, smelling, consulting and pulse - taking, and then extrapolate the pathogeny, location, characteristic, the possible trend of conversion, etc. common tcm aetiology and diagnostic criteria tcm syndromes and similar diseases in western medicine refer to the different understanding and description of a specific disease respectively according to the diagnostic criteria of tcm and western medicine. it is not a one - to - one correspondence between them because of the close association with a co - existing difference. one certain tcm syndrome may correspond to different diseases in western medicine ; a kind of disease in western medicine may also have a variety of relevant tcm syndrome. qi is the energy produced by the original material of the life, the function of which is to promote body growth, maintain organs function, metabolism and excretion. the concept of blood in tcm is very different from in modern medicine, the former means all the nutrients from digested food. qi and blood are closely linked, qi is evolved from the blood, meanwhile, qi can promote blood production. in tcm, wind, cold, wet, dry and fire are five major factors to cause illness. diseases caused by fire usually show symptoms of thirsty and upset, but wet patients often show poor appetite and feeling as if being wrapped around the head. what s more, it can also lead other factors to cause disease. such as wind - cold illness wind is not only come from the external environment, but also can be caused by the disorder of the body functions. for example, when the lung - yang was too exuberant, the wind form lung would blows eyes and cause the increase of red blood in eyes. syndrome has an important position in the tcm system, which is the key to recognize diseases and the foundation to treat them. because of the complexity of the concept and non - professional description of it, the research is hard to go further. one single symptom involves multiple anatomical organizations or systems. however, there was not yet a breakthrough in realization of the hypostasis of tcm syndrome, as the previous investigations limited to a local tissues or organs. proteomics is the systematic study of the expressed proteins of a tissue or cell type in a parallel manner to provide detailed properties, such as the structure and function, of biological systems in health and disease [3, 4 ]. the three most important tasks of proteomics are protein separation, protein identification and characterization and protein bioinformatics (fig. 1). the important steps in the proteomics work flow. two - dimensional gel electrophoresis is the central tool for displaying the proteome. proteins are separated on the basis of charge in the first dimension and molecular mass in the second. the emerging technology of mass spectrometry - based quantitative proteomics provides a powerful tool to systematically and quantitatively assess quantitative differences in protein profiles of different samples and is become an important component of biomedical and clinical research. proteins are separated on the basis of charge in the first dimension and molecular mass in the second. with the help of specialized software, analysis of gel images allows comparisons of multiple gels to comprehensive proteome databases on the internet, and the difference between healthy and diseased samples can be revealed by a process of subtraction. being digested with trypsin, the proteins were cleaved at specific amino acid sequences and were broken into mixture of peptides. the masses of the peptides were measured by mass spectrometry to produce a peptide mass fingerprint. by compared with the peptide masses predicated from theoretical digestion of protein sequences currently contained within databases, the protein can be identified. ultimately, protein bioinformatics were employed to find the relevant biochemical pathways or disease implications highlighted which were displayed from the complex experimental details [68 ]. with the two - dimensional gel electrophoresis and mass spectrometric detection technology matured and widely used, it becomes much easy to rapid separate and identify of multiple proteins. thus, it provided the new perspectives for biology studies. since being proposed in 1995, proteomic has gained rapid developments. in 1997, just two articles could be searched on the topic of proteomic through the web of science database in the isi web of knowledge. five years later, in 2002, the number of published articles themed by proteomics has raise to about 1000. over the last 5 years, the number maintains at a high level (approximately, 3000 per year). as the new perspectives it provided, since its birth 2) and been placed great expectation on, specially received extensive attention in the area of medical diagnosis and drug development [915 ]. as can be seen from fig. 2, proteomics technology is closely associated with many fields of medicine, such as oncology, haematology, pathology, immunology, neurosciences, etc. today proteomics was applied to foster an improved understanding of the pathogenesis, develop new biomarkers for diagnosis and search potential targets for development of drugs. a total of 21,111 articles were retrieved on 22 june 2010 by searching the theme of proteomics through the web of science database in the isi web of knowledge, then analysed by subject area and sorted by the number of articles. the top 21 names of disciplines and the number of related articles are listed here. the essence of tcm syndrome is a comprehension of the regularity of disease occurrence and development and its performance of symptoms. because disease is a regular performance of the body functions, there must be materials, based on which to keep our bodies working. proteins are the embodiment and implementation of life activities, influenced and decided by genetic regulation and environmental factors. clinical studies have shown that most diseases are related to changes in proteins, including protein abundance, structure, function and interaction. there will be a corresponding performance in the coated tongue for almost all of the diseases (table 1). zhang. separated the proteome of fur organization in four groups by two - dimensional gel electrophoresis, scanned them into the computer after silver staining, then analysed and compared the electrophoretogram by image master 2d platinum software. in their experiments, there were some differential protein spots found between different tongue groups (table 2). therefore, proteomics technology can be utilized in tcm syndromes research and bring its essence to light, providing a scientific basis for diagnosis, prevention and treatment. the differences of coated tongue at protein level in addition, in the holistic and systemic context, proteomics and tcm have a convergence not only taking care of individual genes and proteins, but also paying more attention to the relationship between them. the overcoming of one - sidedness and singleness of tcm (study) and the avoidance of complication and tedious processes resulting from that would enhance the accuracy and efficiency during the study. with the further development, mature of proteomics and its increasing cross - penetration between various disciplines, it will serve as a strong research platform for the study of tcm syndromes and help promoting the disclosure of the nature of the discipline. at present, there have been researchers applying proteomics in the determination of tcm syndrome to discover the relevant proteins to provide evidences for tcm by comparing the differences (expression, structure, function and interaction) of proteins between healthy persons and patients, eventually to disclose the material foundation and mechanism of diseases (table 3). as an example, tan qx,. exerted serum proteomic techniques to prepare protein map, combing disease differentiation and dialectic, taking as samples the serum of healthy persons and patients with liver depression diseases, and separating proteins by two - dimensional gel electrophoresis. after the comparison between the normal groups and liver depression groups, the differential protein group was found and 12 differential protein spots were identified. the further analysis demonstrated these differential proteins were mainly related to immunization, neuroendocrine and nutrient metabolism, consistent with the existing research findings. application of proteomics in the studies of tcm syndrome the corresponding concepts of chinese medicine please refer to footnote indicators in table 1. meanwhile, proteomics was also applied to study the pharmacology of tcm (table 4). the most popular pathway is the combined use of 2d gel electrophoresis, biological mass spectrometry and other proteomics technology to isolate, identify and characterize proteins, the expressions of which were changed [2630 ]. furthermore, the further characterization ranging from the basic physicochemical properties to the prediction of potential post - translational modifications, from three - dimensional structures to potential physiological function, was performed to find the relationship between the changes of physiological and proteome. these differentially expressed proteins (group) provide clues to search for biomarkers and drug targets and are advantageous to standardizing tcm diagnosis and improving the scientificity of disease treatment. application of proteomics in the studies of tcm guizhi decoction is one of the most famous prescription, first recorded in the treatise on cold - induced febride diseases. it was containing ramulus cinnamomi, paeonia lactiflora, radix glycyrrhiza, rhizome zingiberis recens and ziziphus jujube mill. tianma gouteng decoction contains gastrodia elata blume, gardenia jasminoides ellis, scutellaria baicalensis georgi, eucommia ulmoides oliver, leonurus japonicus houtt, chinese taxillus twing, caulis polygoni multiflori, cyathula officinalis kuan and ramulus uncariae cumuncis. being constrained by research tools, levels of recognition, analytical methods and other factors, the endeavour to introduce proteomics into the studies of tcm symptoms is still confronted with many difficulties. although abundant proteomics responsible for tcm syndrome have been discovered, the problem what the role these proteomics play in the generation and development has not yet been resolved. despite of the rapid advancements acquired by proteomics in the past years, there are also some disadvantages : low reproducibility, difficult separation of proteins of big or small isoelectric point. moreover, some hydrophobic proteins, insoluble membrane proteins and large molecular weight, low - abundance proteins may be ignored in the process of examination. the scope of tcm symptoms commonly covers multiple organs, organization or functional system throughout the body, whereas proteomics explore and recognize the regularity of vital movement from the perspective of proteins. how to resolve the discrepancies between micro and macro cognition is one pending problem demanding the continuous exploration, the other one is how to appropriately process and analyse the thousands of dates derived from related experiments. nevertheless, the combination of proteomic studies with the tcm symptoms gives us a great temptation. the history of development of modern biology takes on the features that the direction has changed from part to whole and from linear thinking to complexity. the overall dynamic development of proteomics more reflects its growing unity with the thinking methodology of tcm. both proteomics and tcm answer the same question what is the nature of disease from two different angles. proceeding with the microcosmic, the former reveals the essence of the generation, development and healing of diseases by investigating proteins, whereas the latter grasp the disease regularity through awareness of symptoms on the base of the long - term clinical practice from a macrocosmic perspective. chinese medicine has a wealth of experience, meanwhile proteomics has a substantial research potential. the organic integration of the two aspects will bring a great enhancement of our knowledge of disease, not at the microscopic, but also at macroscopic. on one hand, proteomics introduce new thoughts and give a big impetus to tcm ; on the other hand, the unique theory and perspective of tcm offer flesh consideration for the development of proteomics. | abstractsyndrome of chinese medicine is an understanding of the regularity of disease occurrence and development and its performance of symptoms. syndrome is the key to recognize diseases and the foundation to treat them. however, because of the complexity of the concept and the limitation of present investigations, the research of syndrome is hard to go further. proteomics has been received extensive attention in the area of medical diagnosis and drug development. in the holistic and systemic context, proteomics have a convergence with traditional chinese medicine (tcm) syndrome, which could overcome the one - sidedness and singleness of tcm and avoid the complication and tedious processes. chinese medicine has a wealth of experience and proteomics has a substantial research potential, the integration of the two aspects will bring a great enhancement of our knowledge of disease. |
transition metal - mediated reactions in the cool zone of the combustors account for the majority of polychlorinated dibenzo - p - dioxins and polychlorinated dibenzofurans (pcdd / f) formation in combustion systems. both copper and iron ions, which are present in combustion generated particulate matter, are considered the most active metals in promoting pcdd / f formation. these transition metal - mediated reactions typically occur in the low temperature, postcombustion zone and cool zone in air pollution control devices. previous research on surface catalyzed reactions (precursor model of pcdd / f formation) has shown pcdd / fs are formed primarily via two general schemes : pcdfs by a langmuir hinshelwood (l h) mechanism and pcdds is via an eley rideal (e r) mechanism. h mechanism involves the reaction of two adsorbed surface species, and the e r mechanism is via reaction of one gas phase species with a surface - bound adsorbed species. these mechanisms are initiated by chemisorption of substituted aromatic species to metal oxide or hydroxide surface sites to form phenoxyl - type, environmentaly persistent free radicals (epfrs), which subsequently react to form pcdd / fs and other toxic air pollutants. chlorinated phenols have been demonstrated to be key intermediates in essentially all pathways of pcdd / f formation. more recently, chlorinated benzenes have been experimentally demonstrated to be potent precursors of pcdd / fs and are among the most abundant aromatic compounds in incinerator exhaust. despite large laboratory efforts, a significant discrepancy has been observed between the field and laboratory measurements of pcdd / pcdf ratios in the surface precursor model. in general, laboratory experiments result in ratios of > 1, meaning more pcdds being formed, while incinerators measurements usually yield ratios of < 1. these discrepancies are strong arguments for the de novo mechanism as the main source of pcdd / f formation. this is despite the fact that pcdd / f emissions correlate very well with the concentration of chloro - benzenes and sometimes chloro - phenols in the combustors exhaust. addressing this discrepancy these results were in stark contrast to our earlier experiments performed at the same conditions, but in the presence of chlorophenol only (similar to most of the laboratory study focusing on precursor model). in that case these differences only emphasize the need of a more inclusive studies of the different precursors, especially since chlorobenzenes concentrations are usually much higher in the incinerator exhaust. a key question is how do chlorinated benzenes and phenols react to form pcdd / fs when both are present as a mixture ? this is surprising as studies of the pcdd / d formation in a complex feed streams have indicated the competition for the adsorption sites among feed constituents as one of the critical factors. this manuscript reports the first systematic analysis on the effect of chlorobenzene / chlorophenol ratio on pcdd / pcdf ratio. specifically, we report the formation of pcdd / fs from the pyrolysis and oxidation of a mixture of 2-mcp and 1,2-dcbz in the molar ratio of 1:10, 1:1 and 10:1 over 5% copper(ii) oxide on silica fly ash surrogate and compare it with earlier results for pure 2-mcp and 1,2-dcbz. it has to be noted, however that during the reaction some formation of metal chlorides can occur, due to the hcl elimination reaction, and part of the activity may result from metal chloride phase. the surface - mediated reactions of a mixture of 2-mcp and 1,2-dcbz over 5% cuo / silica surfaces were investigated using the system of thermal diagnostic studies (stds), which is described in detail elsewhere. briefly, the system is composed of a thermal reactor located in a high - temperature furnace housed within a gas chromatographic oven that facilitates precise temperature control as well as sample introduction. a computer - interfaced control module is used to set and monitor all experimental parameters. a gc - ms system is interfaced in - line with the thermal reactor for analysis of the reactor effluents. the method of incipient wetness was used to prepare the catalytic material that served as a surrogate for combustion - generated, copper rich fly ash. silica gel powder (aldrich, grade 923 100200 mesh size) was introduced into a water solution of copper(ii) nitrate (aldrich) in the amount for incipient wetness to occur and proportion to produce 5% cuo on silica by weight. the sample was allowed to age for 24 h at room temperature and dried at 120 c for 12 h before calcination in air for 5 h at 450 c. the samples were then ground and sieved to a mesh size of 100120 corresponding to a particle size of 120150 m. 50 mg of catalytic material was placed between quartz wool plugs in a 0.3 cm i.d. fused silica reactor in the stds. to avoid condensation of the reaction products, prior to each experiment, the catalytic material was oxidized in situ at 450 c for 1 h at an air flow - rate of 5 cc / min to activate the surface of the sample. the reagent mixture samples of 2-mcp (aldrich) and 1,2-dcbz (aldrich) were introduced separately into the flow stream using a digital syringe pump (kd scientific, model-100) through a vaporizer maintained at 180 c. 20% oxygen in helium for oxidation experiments and pure helium for pyrolytic experiments were used as a carrier gas. the rate of injection was selected to maintain a constant gas phase concentration of 50 ppm of 2-mcp and 1,2-dcbz mixtures for temperatures ranging from 200 to 550 c. the overall flow rate of the reaction gas stream was maintained at 5 cc / min and a contact time of 0.02s. the composition of the reagent mixture is described by the 1,2-dcbz/2-mcp concentration ratio in the gas phase r (cf. conversion of pure 2-mcp and 1,2-dcbz over exact surfaces were studied previously, and their yields are also provided with assigned r = () (due to division by 0) or 0 for comparison, respectively. products from the reaction were analyzed using an in - line agilent 6890 gc - msd system after 60 min of the collection of reaction products at the headspace of capillary column at 60 c. products were identified based on both mass spectra of the standards, comparisons to the nist mass spectra library when available, and gas - chromatographic retention times. the yields of the products were calculated by use of the expression : yield = ([product]/[2-mcp+1,2-dcbz]o) 100, where [product ] is the concentration of specific product formed (in moles) and [2-mcp+1,2-dcbz]o is the initial concentration of 2-mcp+1,2-dcbz mixtures (in moles) injected into the reactor. each experimental data point presented in the manuscript is an average of three experimental runs. the reactor was periodically cleaned by heating in air at 800 c, and blank runs were routinely performed to ensure no deposits or carryover of reaction products from run to run. our previous results have shown the surface mediated pcdd / f forming reactions of chlorinated phenols and chlorinated benzenes proceed according to the mars - van krevelen (mvk) mechanism. accordingly, the oxidation process does not require gas phase oxygen for the reaction, and surface and lattice -o species or oh groups are actively involved in the process. gas phase oxygen, if present, ensures reoxidation of the surface and resupplies the consumed surface species. figure 1 presents the decomposition / oxidation profile of the 1,2-dcbz/2-mcp mixtures over cuo / silica system under pyrolytic conditions. despite the absence of oxygen in the gas phase, a significant fraction of the reagents were decomposed / oxidized below 350 c, with over 80% decomposition at temperatures of 350500 c. other products were also detected e.g., polychlorinated phenols, dihydroxybenzenes, polychlorinated benzenes, naphthalenes, (cf. supporting information for detailed product tables). decomposition / oxidation profiles of 1,2-dcbz/2-mcp mixtures over a cuo / silica surface under pyrolytic conditions. the graph presents percent 1,2-dcbz and 2-mcp unreacted or formed as a reaction product. the presence of the co - component in the reacting mixture affects the decomposition of 1,2-dcbz, while the oxidation / degradation of 2-mcp is affected much less (cf. in fact, introducing even small amounts of 2-mcp into the reacting stream (r=10, 5 ppm 2-mcp) results in much higher breakthrough of 1,2-dcbz (the decomposition of 1,2-dcbz shifts toward higher temperatures). these data indicate a competitive adsorption between the 1,2-dcbz and 2-mcp, with a faster rate of 2-mcp adsorption. the presence of gas phase o2 under oxidative conditions enhanced the overall decomposition of each reagent by 1020% below 350 c. this further supports the mvk type reaction, with the competitive adsorption determining the relative decomposition of each component.. pcdd / f yields from mixtures of 1,2-dcbz/2-mcp over cuo / silica at oxygen rich conditions at various reaction feed ratios. the inhibited adsorption of 1,2-dcbz in the copresence of 2-mcp may result from a different interaction of those molecules with the metal oxide surfaces. we have previously shown 2-mcp interacts with a surface oh predominantly by h2o elimination, forming a monodentate surface chlorophenoxy species. this is not the case for 1,2-dcbz which predominantly adsorbs on to two sites forming a bidentate o - dihydroxybenzene species (i.e., chemisorbed catechol). in fact, rapid acceleration of the 1,2-dcbz decomposition above 350 c correlated with the decomposition of the chemisorbed catechol, releasing the surface sites for more adsorbates. in the present study, the most interesting is the change in the yields and distribution of pcdd / f with varying dichlorobenzene to chlorophenol ratios, as presented in figure 2. a clear shift in the maximum formation yield of 4,6-dichlorodibenzofuran (4,6-dcdf) from 350 to 250 c can be observed with increasing 1,2-dichlorobenzene content. concurrently, dibenzofuran yields almost double and become the most prominent pcdd / f product. it is important to note that in this particular study, we consider nonchlorinated dibenzo - p - dioxins (dd) and dibenzofuran (df) as a representation of polychlorinated species as they represent condensation products with loss of either 1 or 2 chlorine atoms. this is a consequence of using a simple laboratory model with monochlorophenol or dichlorobenzene. the changing formation profile with ratio r is associated with the different formation mechanism of pcdds and pcdfs, and different reactivity of precursors within this mechanism. adsorbed bidentate catechol formed from 1,2-dcbz adsorption has been found to be a precursor of gas phase o - benzoquinone molecules and adsorbed phenoxyl radical and its keto- structure. surface mediated recombination of two keto - phenoxyl radicals leads to the formation of dibenzofuran (df). from the previous studies of surface assisted 1,2-dcbz decomposition, it is known that on cuo surface, df is formed exclusively from decomposition of 1,2-dcbz. for the mixtures of 1,2-dcbz and 2-mcp, 4,6-dichlorodibenzofuran is a product of the keto - structure of monochlorophenoxyl recombination, from both 1,2-dcbz and 2-mcp. however, in the presence of gas phase chlorophenols, two competitive reactions can occur : surface recombination of two radicals to form pcdfs (l - h mechanism) or reaction of a surface radical with a gas - phase 2-mcp to form dichlorohydroxy biphenyl ethers (e r mechanism) (cf. the formation of dichlorohydroxy biphenyl ethers was not observed for 1,2-dcbz, which implies the e figure 3). assuming the fast adsorption of 2-mcp, the rate expression for the l h mechanism is zero order in the gas phase concentration of 2-mcp and first order for the e - r mechanism. as a result, with decreasing relative concentration of 2-mcp in the gas phase, the l in fact, the observed 4,6-dcdf formation from 2-mcp is of 0.6 order with respect to 2-mcp. schematic representation of the two competing mechanisms of pre - dcdf species in the excess of gas phase 2-mcp (top) and 1,2-dcbz (bottom). r reaction with gas phase precursor. decreasing the 1,2-dcbz/2-mcp ratio, r, had also an impact on the yields of pcdds. pcdd formation is exclusively a result of 2-mcp reaction with an experimental reaction order of 0.60.7, with respect to 2-mcp. observed reduction in both 1-mcdd and dd yield with increasing r is a direct result of the decreasing relative concentration of 2-mcp. a closer look at the total pcdd and pcdf yields as a function of chlorinated benzenes to phenols ratio r indicates interesting differences in reactivity. figure 4 presents average total yields of the particular group of products over entire temperature range (where average is defined as a sum of all product in a category divided by the number of measurement points). for pcdd sum include dd, 1-mcdd, for pcdf sum include df and 4,6-dcdf. a maximum of the pcdf formation can be observed for the reaction feed composed of equal amounts of both 1,2-dichlorobenzene and 2-monochlorophenol. in the case of pcdd, a decreasing trend of pcdd yields with increasing r is prominent, with the exception of pure 2-mcp feed. the sharp drop in the average pcdd yield for pure chlorophenol feed is however an averaging artifact, as in this case a very narrow pcdd formation window was observed, however, with significant yield (at 300320 c, 0.3% yield), average yield of pcdds and pcdfs depending on the feed composition at oxygen rich conditions. the pcdd / pcdf ratio is one of the most important indicators of the performance of predictive models. until now, the laboratory studies using a precursor model of pcdd / f formation were not able to mimic the test fields in the incinerators, where pcdfs are dominant product. as a result it is a common interpretation that in the incinerator systems where pcdf are a major dioxin product, carbonaceous deposits are responsible through a de novo process. the presented in here results indicate the ratio of chlorinated benzenes to chlorinated phenols in the reaction feed of precursor model as a key factor determining the pcdd to pcdf ratio. since pcdf formation is observed over a broad temperature range and the maximum formation windows for pcdfs and pcdds do not always overlap, it is necessary to compare the yields integrated for the entire temperature range of surface mediated reactions. the advantage of such an approach is accommodation of the yields from the entire postcombustion, cool - zone. again, in our calculations, we included the nonchlorinated dds and dfs in their chlorinated group. in full - scale combustor exhausts, where higher chlorinated precursors will be present, the respective products will be (x-1)-chlorodibenzo - p - dioxins for x - chlorophenol precursors and (y-2)-chlorodibenzofuran for y - chlorobenzene precursors, where x and y represent number of chlorine atoms per precursor molecule. table 3 presents the integrated pcdd to pcdf ratios for different feed composition ratios, r, for both oxygen rich and pyrolytic conditions. the pcdd to pcdf ratio is decreasing diagonally from top - left to bottom - right, i.e. with decreasing precursor ratio r and oxygen content. one can claim that the oxygen effect on pcdd / pcdf ratio is anticipated and known due to more oxidized form of the product (2 oxygen atoms included in the structure of pcdd vs 1 for pcdf), however studies indicate that gas phase oxygen is not directly involved in the dioxin formation. the observed pcdd / pcdf ratio depends on the relative concentrations of chlorinated phenols to chlorinated benzenes. the provided matrix can be used as a guide in developing predictive models pcdd / f emissions for copper rich fly ashes. much of the research has been focused on the correlation between the particular component of the exhaust stream, however this studies indicate that rather a benzenes to phenols (btp) ratio should be evaluated in field studies. most importantly, this study shows that the precursor model is absolutely valid in cases where a large excess of pcdfs is formed over pcdds. it has to be mentioned that other factors such as the presence of different than copper metals can also contribute to the varying pcdd / f ratio. in particular we have recently reported the iron oxides are more prone to the formation of pcdf than pcdds. in fact the effects of chlorobenzene / chlorophenol ratio on pcdd to pcdf ratio over iron oxide surfaces is currently under investigation. to verify the chlorobenzenes effect theory, it is necessary to compare the emissions of pcdd / f, chlorinated benzenes and chlorinated phenols from actual field studies. in here we would like to focus on a manuscript reporting the emissions from municipal solid waste incinerators (mswi). general trends in the pcdd / f emissions from different installations indicate that ocdd is the most dominant congener of polychlorinated dibenzo - p - dioxins. as for the polychlorinated dibenzofurans, the dominant congener group is sensitive to the type of facility and combusted material. this is in agreement with the proposed surface mechanism that indicates surface retention of pcdd molecules during ring closure reaction and their chlorination, while pcdfs are liberated to the gas phase while formed. takaoka reported gas phase concentrations of chlorinated benzenes and chlorinated phenols for 2 different mswi and in both cases btp ratio was close to 1 before scrubbers and 2 after scrubbers. the observed pcdd / f concentrations corresponded to the pcdd to pcdf ratio of 0.20.25 for before scrubbers and 0.370.5 behind scrubbers. comparing it with the data in table 3 it is interesting to notice that before scrubber conditions fit better to pyrolytic, while after scrubber conditions fit better oxidation condition predictions. the proposed surface model can also predict the dominant pcdd / f congener group, based on the prevalent cbz concentrations. according to the surface model, pcdfs result from the surface condensation of predioxin radicals. such radicals are formed due to the chemisorption of precursors either as monodentate or bidentate species. in the case of 1,2-dichlorobenzene (model compound used in here) this leads to the formation of dcdf and df. the same model can be adapted to higher chlorinated benzenes as depicted in figure 5 for 1,2,3,4-tetrachlorobenzene. the presence of tetrachlorobenzene will yield the formation of tetra to hexa cdf. unlike chlorophenols, chlorobenzenes, have a propensity to undergo a bidentate adsorption, which results in bottom pathway in figure 5 (through bidentate species). as a result pecdf and tecdf table 4 presents selected cbz and their surface radicals as well as anticipated pcdf congeners as their condensation products. this table can serve as a guide for the estimation of pcdf congener group distribution based on the pcbz concentrations. in the studies reported by takaoka chlorobenzenes concentration in the gas phase in one of tested mswi was heavily skewed toward the lower chlorinated benzenes (up to tetrachlorobenzene)the ratio of tri+tetra cbz to pecbz is 9. according to our prediction table this would correspond to higher concentrations of tecdf and pecdf, as authors reported only the tecdf - ocdf. in fact their measurement indicate tcdf to be the highest concentration, with pecdf following and the remaining congener groups to be at much lower concentrations. the other studied mswi in have shown similar trends in the inlet to the scrubber. however, with the changing tri+tetra / penta cbz to 5.4, as in the case of the outlet from the scrubber, the contribution of higher chlorinated pcdf is increasing with hexa and hepta congeners becoming doiminant. | the discrepancies between polychlorinated dibenzo - p - dioxin to polychlorinated dibenzofuran (pcdd to pcdf) ratios in laboratory and field studies in the exhaust of combustion sources are not fully explained by available formation models. in this paper we present the results of experimental studies of the surface mediated formation of pcdd / f at the conditions mimicking the combustion cool zone from a mixture of 1,2-dichlorobenzene (1,2-dcbz) and 2-monochlorophenol (2-mcp) over a model surface consisting of 5% cuo / silica. the pcdd to pcdf ratio was found to be strongly dependent on the ratio of chlorinated benzenes to chlorinated phenols and oxygen content. the higher the 1,2-dcbz to 2-mcp ratio, the lower the pcdd to pcdf ratio. pcdfs are formed predominantly from chlorinated benzenes, while chlorinated phenols are responsible for majority of pcdds. these laboratory results are in general agreement with full - scale measurement and can be used to improve predictive models of pcdd / f formation. |
backpacks are indispensable, as people regularly use them for a long time to carry objects that are used daily1. therefore, avoidance of postural aberration, by practicing appropriate backpack usage, is vital. recently, it has been suggested that the prolonged use of backpacks by school - aged children aggravates musculoskeletal conditions2, 3. in particular, the increased prevalence of spinal deformities, such as scoliosis and kyphosis, and/or pain and discomfort including low back pain and shoulder pain, may be associated with backpack - carrying habits and the backpack weight4,5,6. occasionally, poor distribution of backpack weight and ineffective absorption of this load leads to postural changes, leading to musculoskeletal impairments3. hence, school - aged children should be educated and made aware of these concerns to protect their spinal health7. to our knowledge, although the use of backpacks has a major influence on the spinal condition of school - aged children, the majority of studies have focused on the effects of backpack carrying on posture and movement during standing and walking of adults8, 9. due to this lack of research on juvenile subjects, it is challenging to establish a resource for educating children on the manner in which their backpacks should be correctly used. hence, we sought to determine the effects of backpack use on children rather than review the outcomes of existing adult studies. thus, we aimed to assess the effects of backpack position on the foot weight distribution of standing school - aged children. thirty healthy children (14 males, 16 females) volunteered to participate in this study. the mean age of the children was 8.43 0.50 years, their mean height was 128.57 6.06 cm, and their mean weight was 30.53 4.17 kg. only the subjects who fulfilled the following criteria were included in this study : no orthopedic, neurological, or cognitive impairments that could have influenced the study procedure or results ; no discrepancy in leg length ; and no regular daily exercise. all subjects signed a written consent form before study participation, and this study was approved by the institutional review board of cheongju university. foot weight distribution during standing was recorded using a force plate (fdm - s system, zebris, germany). this consists of a square plate (1.5 m 1.5 m) containing 17,000 micro - sensors which detect foot pressure. a cable connects the force plate to the main unit, and a computer system with custom computer software for analyzing the measured data. the equipment evaluates the weight pressure distribution of the foot, and analyzes the load distribution of the anterior and posterior foot regions. subjects were asked to stand still on the plate, with their feet shoulder length apart and their arms at sides, while looking at a round target attached to wall located 2 m in front of them. after zero - point calibration, measurement was performed for 1 min and data were averaged over three trials separated by 1 min rest intervals. based on the study of oh and choi10, four types of backpack (height : 34 cm, width : 25 cm, and depth : 13 cm) (bp - x031, prospecs, republic of korea) carrying conditions were studied. the backpack position was adjusted using the shoulder strap of the backpack : condition-1, no backpack ; condition-2, carrying a backpack at c7 ; condition-3, carrying a backpack at 10 cm below c7 ; and condition-4, carrying a backpack at 20 cm below c7. the sequence of the four conditions was randomly determined to avoid an interaction effect of each condition. the randomization process involved blindly drawing a card from an envelope, containing 4 cards marked 1, 2, 3, or 4. we adjusted the weight of each backpack to 15% of the subject s bodyweight (average backpack weight of the subjects : 4.580.63), based on a suggestion made by al - khabbaz.2, who investigated the effects of backpack weight load. when statistical significance was identified, the differences in the pairwise comparison were examined using the post - hoc bonferroni adjustment. table 1table 1.comparison of anterior and posterior pressure values and anterior - to - posterior ratios of the four backpack positionscondition-1condition-2condition-3condition-4anterior pressure values39.4510.9642.0412.1640.6413.5137.5011.18posterior pressure values60.5610.9657.9612.1659.3613.5162.5111.18anterior - to - posterior ratio0.720.390.820.480.800.540.650.31condition-1 : no backpack ; condition-2 : carrying the backpack at c7 ; condition-3 : carrying the backpack at 10 cm below c7 ; and condition-4 : carrying the backpack at 20 cm below c7. statistically significant differences were noted in the anterior and posterior pressure values, as well as the anterior - to - posterior ratio among the four conditions (p < 0.05). post - hoc analysis indicated that the pressure values of condition-4 were significantly lower in the anterior foot region and higher in the posterior foot region than in condition-2 and condition-3. in addition, the anterior - to - posterior ratio was lower in condition-4 than in condition-2 and condition-3. condition-1 : no backpack ; condition-2 : carrying the backpack at c7 ; condition-3 : carrying the backpack at 10 cm below c7 ; and condition-4 : carrying the backpack at 20 cm below c7. significant difference from condition-2. the excess weight load of a backpack causes asymmetry in the anterior and posterior load distribution, eliciting forward leaning of the upper trunk to adapt postural agitation and maintain postural balance8. although the anterior - to - posterior pressure ratios did not significantly differ among the conditions, the ratios of condition-2 and condition-3 were greater than those of condition-1, implying that the weight load was greater in the anterior foot region and less in the posterior region. in condition-3, weight load appeared to be concentrated to a greater extent on the posterior, rather than the anterior foot region. a lower backpack position results in the increase of the moment arm generated by the upper trunk movement axis. therefore, it may be difficult to maintain the appropriate pressure ratio on the anterior and posterior foot regions. similar effects have been observed with increased backpack weight, indicating that carrying a backpack at a higher position, with fastening of the shoulder strap, may be better for normalizing foot weight distribution than other conditions. previous studies have reported that forward leaning of the upper trunk increases to a greater extent, when subjects carry a backpack in a lower position, with a longer shoulder strap10, 11. these studies indicated that the weight load is concentrated on the anterior foot region, which is inconsistent with our present study s findings. however, those studies examined walking of subjects, not standing of subjects, as in the present study. given that additional load from a backpack can impede forward movement of the body during walking, forward trunk leaning may be an effective strategy for reducing energy consumption10, 11. therefore, caution should be exercised when comparing our results with those of these previous studies, as their results require the understanding of the dynamic walking mechanism rather than that of static control that is behind the present study s findings. we believe that carrying a backpack in the upper position may lead to safer use of the backpack, and may help prevent musculoskeletal discomfort in those who regularly use a backpack. our findings suggest that adjusting the backpack position, by fastening the shoulder strap, is a useful strategy for the prevention of musculoskeletal problems in school - aged children. first, as the subjects of the present study included only school - aged children, the findings can not be generalized to other age groups. second, we did not record long - term follow - up data and the findings may not reflect long - term backpack use. finally, the lack of other measures such as electromyography or kinematic analysis may be factors limiting the establishment of a more definite conclusion on the effects of backpack position. | [purpose ] in the present study, we aimed to determine the effects of backpack position on foot weight distribution of standing school - aged children. [subjects ] thirty school - aged children volunteered to participate in this study. [methods ] the subjects randomly performed four types of carrying a backpack : no backpack (condition-1), carrying a backpack at c7 (condition-2), carrying a backpack at 10 cm below c7 (condition-3), and carrying a backpack at 20 cm below c7 (condition-4). [results ] statistically significant differences were noted in the anterior and posterior pressure values, and in the anterior - to - posterior ratio, among the four conditions (p < 0.05). post - hoc analysis indicated that the pressure value of condition-4 was significantly lower in the anterior foot region and higher in the posterior foot region than in condition-2 and condition-3. in addition, the anterior - to - posterior ratio was lower in condition-4 than in condition-2 and condition-3. [conclusion ] these findings suggest that carrying a backpack in a higher position, with fastening of the shoulder strap, may be more favorable for normalizing the foot weight distribution. |
idiopathic inflammatory bowel disease (ibd) is a multifactorial disorder characterized by chronic and relapsing inflammation specific to the gastrointestinal tract, thus resulting in intestinal malabsorption, mucosal immune system abnormalities, and exaggerated inflammatory responses [15 ]. ibd has two main subtypes, namely ulcerative colitis (uc) and crohn s disease (cd). although the precise etiology remains unknown, several environmental factors, such as commensal bacteria, food antigens, and smoking, as well as multiple genetic factors may contribute to the occurrence and development of ibd [15 ]. genome - wide linkage - based family studies, candidate gene - based association studies, and large - scale genome - wide association (gwa) studies using single - nucleotide polymorphisms (snps) have shown possible ibd susceptibility genes and chromosomal loci [610 ]. ibd is involved in a complex interplay of innate and adaptive immune cells, including lymphocytes, macrophages, and dendritic cells. in such a setting, alterations in cytokine synthesis and cytokine signaling pathways are attributed to the pathogenesis of ibd [15 ]. gwa studies have recently indicated that the multiple genes implicated in the interleukin 23 (il-23) and its receptor (il23r) signaling pathway are associated with susceptibility to cd as well as uc [612 ]. for example, il-23, il23r, interleukin 12 precursor (il12b), interleukin 12 receptor (il12r), the janus kinase (jak) families, and the signal transducers and activators of transcription (stat) families belong to a gene network in the il-23/il23r signaling pathway, implying that a subset of these genes can play a central role in the pathogenesis of ibd and may function as a key conductor of innate and adaptive inflammatory responses at multiple levels in the intestinal mucosa of ibd patients. il-23 as well as jak2 [9, 14 ] and stat3 [9, 12, 14 ] jak2 has been recently identified as a cd susceptibility gene in a meta - analysis of gwa data [9, 14 ] but not by any single - marker association studies on cd. likewise, tyrosine kinase 2 (tyk2), which is a member of the jak families located in 1 subunit of il12 receptor (il12rb1) [4, 1517 ] and in gp130 of interleukin 6 receptor (il6r), is also identified as a cd susceptibility gene by the meta - analysis but did not reach significance by single - marker association studies. furthermore, tyk2 is activated via signaling from a broader range of cytokine receptors and induces phosphorylation, homodimerization, and nuclear translocation of stat3 [1519 ], thus resulting in several gene transcriptions and leading to il-23-induced production of il-17, a pro - inflammatory cytokine in natural killer cells, natural killer t cells, cd4 t cells, and cd8 t cells [2022 ]. this signaling cascade also plays a role in the differentiation of cd4 (naive) t cells into th17 cells [17, 2022 ], which is involved in the first line of host defense by controlling immune responses. therefore, we performed a candidate gene - based association study by selecting tyk2 and stat3 as candidate genes. the purpose of this study was to investigate whether snps and their combination polymorphisms, which are referred to as haplotypes, in tyk2 and stat3 are also associated with susceptibility to ibd in a japanese population and whether such polymorphisms can be used as new genetic biomarkers for predicting the onset of ibd. the subjects included 112 patients with uc, 83 patients with cd, and 200 gender - matched, healthy volunteers as control subjects. ibd patients were enrolled from eight general hospitals in nagasaki, japan from october 2003 to october 2008. the clinical characteristics of the subjects at the end point of this study are shown in table i. the study protocol was approved by the committee for ethical issue dealing with the human genome and gene analysis at nagasaki university, and written informed consent was obtained from each subject. table ithe clinical characteristics of study subjectscharacteristicspatients withcontrol subjectsuccdnumber11483200age, mean sd (years)44.2 16.7 34.3 12.532.5 11.2age range (years)148317752060male / female (%) 59/55 (51.7/48.3)50/33 (60.2/39.8)126/74 (62.5/37.5)age at onset 40 years3911 40 years7572extent of uc proctitis14 left - sided colitis43 pancolitis57location of cd ileal16 ileocolonic55 colonic11 isolated upper1disease severity mild5117 moderate3845 severe219 unknown412disease activity active6354 inactive4817 unknown312behavior of cd stricturing44 penetrating40 perianal diseases36sd standard deviationp < 0.01 in comparison to control subjectsnumber of the affected patients with cd the clinical characteristics of study subjects sd standard deviation p < 0.01 in comparison to control subjects number of the affected patients with cd the diagnosis of ibd was made based on the endoscopic, radiological, histological, and clinical criteria established by both the world health organization council for international organizations of medical sciences and the international organization for the study of inflammatory bowel disease [2325 ]. patients with indeterminate colitis, multiple sclerosis, systemic lupus erythematosus, or any other diagnosed autoimmune diseases were excluded from this study. patients with uc were classified into subgroups according to age at onset (40 or 40 years), extension of disease (proctitis, left - sided colitis, or pancolitis), disease severity (mild, moderate, or severe), and disease activity (active or inactive) (table i). likewise, patients with cd were classified into subgroups according to age at onset (40 or 40 years), the location of lesions (ileal, ileocolonic, colonic, or isolated upper), disease severity (mild, moderate, or severe), disease activity (active or inactive), and the behavior of disease (stricturing, penetrating, or perianal ; table i). the location and extension of uc and cd, disease severity of uc and cd, and behavior of cd were stratified in accordance with the montreal classification with slight modification. a high clinical activity index (cai 5) for uc and a high crohn s disease activity index (cdai 150) were regarded as active - phase patients. genomic dna was extracted from a whole blood sample from each subject using a dna extractor wb - rapid kit (wako, osaka, japan) according to the manufacturer s protocol. all of snps in stat3 (genbank accession number, ay572796 ; mim 102582) located on chromosome 17q21 and tyk2 (genbank accession number, ay549314 ; mim 176941) located on chromosome 19p13.2 were obtained using data available on the international hapmap web site. candidate tag snps were selected with priority in a minor allele frequency of more than 5%. subsequently, linkage disequilibrium blocks and genotyped tag snps among the candidate tag snps were determined using the ihap software program. the gene structure and positions of the genotyped tag snp sites in stat3 and tyk2 1locations of the genotyped tag snp sites in stat3 in the international hapmap (upper) and ihap (lower) data. yellow rectangles represent the positions of linkage disequilibrium blocks. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted trianglefig. 2locations of the genotyped tag snp sites in tyk2 in the international hapmap (upper) and ihap (lower) data. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted triangle locations of the genotyped tag snp sites in stat3 in the international hapmap (upper) and ihap (lower) data. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted triangle locations of the genotyped tag snp sites in tyk2 in the international hapmap (upper) and ihap (lower) data. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted triangle three snps, rs8074524 in intron 3, rs2293152 in intron 11, and rs957970 in intron 23, were selected as genotyped tag snps (fig. 1) and were subsequently analyzed by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp). the polymorphic region was amplified by pcr with a geneamp pcr system 9700 thermal cycler (applied biosystems, foster city, ca, usa) using 25 ng of genomic dna in a 25-l reaction mixture containing 0.8 gotaq green master mix (promega, madison, wi, usa) and 15 pmol each of the following primers : forward primer 5-gtctggaaagctccatctgc-3 and reverse primer 5-agaggccagattagtgctgg-3 for rs8074524 ; forward primer 5-tcccctgtgattcagatccc-3 and reverse primer 5-cattcccacatctctgctcc-3 for rs2293152 ; and forward primer 5-ctgggctcaagtgatcttcc-3 and reverse primer 5-gtactcatcgccctccattg-3 for rs957970. the amplification protocol comprised initial denaturation at 95c for 2 min, followed by 30 cycles of denaturation at 95c for 30 s, annealing at 62c for 30 s, and extension at 72c for 30 s and final extension at 72c for 5 min. the pcr products were digested with restriction enzyme, hpa ii (takara bio inc., kyoto, japan) for rs8074524 and rs2293152 ; xsp i (takara bio inc.) for rs957970. the digests were separated by electrophoresis on a 2% agarose gel (nacalai tesque, kyoto, japan) and visualized with an ultraviolet transilluminator (alpha innotech co., san leandro, ca, usa) after ethidium bromide (nacalai tesque) staining. four genotyped tag snps in tyk2, rs280496 in intron 3, rs280519 in intron 14, rs2304256 in intron 18, and rs280523 in intron 20 (fig. 2), were analyzed by pcr - rflp using 15 pmol each of the following primers : forward primer 5-cggggtgatatgctcattgg-3 and reverse primer 5-caacgtgctgctggacaacg-3 for rs280496 ; forward primer 5-ccgccatggtgaaagttagc-3 and reverse primer 5-atttgtgcaggccaagctgc-3 for rs280519 ; forward primer 5-tcaccaggcacttgttgtcc-3 and reverse primer 5-cggcttccagcatgtgtatg-3 for rs2304256 ; and forward primer 5-acatttccccctgcctacac-3 and reverse primer 5-ttacagacatgcgccaccac-3 for rs280523. the amplification protocol comprised initial denaturation at 95c for 2 min, followed by 30 cycles of denaturation at 95c for 30 s, annealing at 64c (rs280496, rs280519, and rs280523) or 62c (rs2304256) for 30 s, and extension at 72c for 30 s and final extension at 72c for 5 min., beverly, ma, usa) for rs280496, hpy99 i (new england biolabs inc.) for rs280519, bsm i (new england biolabs inc.) for rs2304256, and bsie i two tag snps in tyk2, which showed a close association of susceptibility to cd and were located within the same linkage disequilibrium block (fig. 2), were utilized to infer the haplotype structure as well as to analyze the haplotype frequency using the snp alyze 7.0 standard software package (dynacom inc., yokohama, japan) to emphasize the variability and to enhance the power of detecting allelic association of rare variants [33, 34 ]. differences in age and gender between uc or cd patients and control subjects were evaluated by an unpaired student s t test and a chi - square test, respectively, using the spss 17 (spss japan inc., tokyo, japan) and prism 5 (graphpad software inc., san diego, ca, usa) statistical software packages. the frequencies of the expected alleles were calculated from those of the observed genotypes according to the hardy weinberg equilibrium. the frequencies of the observed and expected alleles were compared by the chi - square test with yates correction using the snp alyze 7.0 standard software package. the frequencies and distributions of alleles, genotypes, haplotypes, and diplotypes were statistically compared between uc or cd patients and control subjects by the chi - square test and logistic regression analysis using prism 5 and spss 17. subsequently, a comparison of the genetic risk factors between the statistically significant genotype of stat3 and diplotype of tyk2 for susceptibility to cd was carried out by a multivariate logistic regression analysis using spss 17. the odds ratio (or) with 95% confidence interval (ci) was calculated using spss 17. the subjects included 112 patients with uc, 83 patients with cd, and 200 gender - matched, healthy volunteers as control subjects. ibd patients were enrolled from eight general hospitals in nagasaki, japan from october 2003 to october 2008. the clinical characteristics of the subjects at the end point of this study are shown in table i. the study protocol was approved by the committee for ethical issue dealing with the human genome and gene analysis at nagasaki university, and written informed consent was obtained from each subject. table ithe clinical characteristics of study subjectscharacteristicspatients withcontrol subjectsuccdnumber11483200age, mean sd (years)44.2 16.7 34.3 12.532.5 11.2age range (years)148317752060male / female (%) 59/55 (51.7/48.3)50/33 (60.2/39.8)126/74 (62.5/37.5)age at onset 40 years3911 40 years7572extent of uc proctitis14 left - sided colitis43 pancolitis57location of cd ileal16 ileocolonic55 colonic11 isolated upper1disease severity mild5117 moderate3845 severe219 unknown412disease activity active6354 inactive4817 unknown312behavior of cd stricturing44 penetrating40 perianal diseases36sd standard deviationp < 0.01 in comparison to control subjectsnumber of the affected patients with cd the clinical characteristics of study subjects sd standard deviation p < 0.01 in comparison to control subjects number of the affected patients with cd the diagnosis of ibd was made based on the endoscopic, radiological, histological, and clinical criteria established by both the world health organization council for international organizations of medical sciences and the international organization for the study of inflammatory bowel disease [2325 ]. patients with indeterminate colitis, multiple sclerosis, systemic lupus erythematosus, or any other diagnosed autoimmune diseases were excluded from this study. patients with uc were classified into subgroups according to age at onset (40 or 40 years), extension of disease (proctitis, left - sided colitis, or pancolitis), disease severity (mild, moderate, or severe), and disease activity (active or inactive) (table i). likewise, patients with cd were classified into subgroups according to age at onset (40 or 40 years), the location of lesions (ileal, ileocolonic, colonic, or isolated upper), disease severity (mild, moderate, or severe), disease activity (active or inactive), and the behavior of disease (stricturing, penetrating, or perianal ; table i). the location and extension of uc and cd, disease severity of uc and cd, and behavior of cd were stratified in accordance with the montreal classification with slight modification. a high clinical activity index (cai 5) for uc and a high crohn s disease activity index (cdai 150) were regarded as active - phase patients. genomic dna was extracted from a whole blood sample from each subject using a dna extractor wb - rapid kit (wako, osaka, japan) according to the manufacturer s protocol. all of snps in stat3 (genbank accession number, ay572796 ; mim 102582) located on chromosome 17q21 and tyk2 (genbank accession number, ay549314 ; mim 176941) located on chromosome 19p13.2 were obtained using data available on the international hapmap web site. candidate tag snps were selected with priority in a minor allele frequency of more than 5%. subsequently, linkage disequilibrium blocks and genotyped tag snps among the candidate tag snps were determined using the ihap software program. the gene structure and positions of the genotyped tag snp sites in stat3 and tyk2 are shown in figs. 1 and 2, respectively. 1locations of the genotyped tag snp sites in stat3 in the international hapmap (upper) and ihap (lower) data. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted trianglefig. 2locations of the genotyped tag snp sites in tyk2 in the international hapmap (upper) and ihap (lower) data. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted triangle locations of the genotyped tag snp sites in stat3 in the international hapmap (upper) and ihap (lower) data. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted triangle locations of the genotyped tag snp sites in tyk2 in the international hapmap (upper) and ihap (lower) data. a list and the locations of candidate tag snps are shown in the lower right position using haploview 4.0 software. red inverted triangles indicate the genotyped tag snps sites in this study, and their names are presented above each inverted triangle three snps, rs8074524 in intron 3, rs2293152 in intron 11, and rs957970 in intron 23, were selected as genotyped tag snps (fig. 1) and were subsequently analyzed by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp). the polymorphic region was amplified by pcr with a geneamp pcr system 9700 thermal cycler (applied biosystems, foster city, ca, usa) using 25 ng of genomic dna in a 25-l reaction mixture containing 0.8 gotaq green master mix (promega, madison, wi, usa) and 15 pmol each of the following primers : forward primer 5-gtctggaaagctccatctgc-3 and reverse primer 5-agaggccagattagtgctgg-3 for rs8074524 ; forward primer 5-tcccctgtgattcagatccc-3 and reverse primer 5-cattcccacatctctgctcc-3 for rs2293152 ; and forward primer 5-ctgggctcaagtgatcttcc-3 and reverse primer 5-gtactcatcgccctccattg-3 for rs957970. the amplification protocol comprised initial denaturation at 95c for 2 min, followed by 30 cycles of denaturation at 95c for 30 s, annealing at 62c for 30 s, and extension at 72c for 30 s and final extension at 72c for 5 min. the pcr products were digested with restriction enzyme, hpa ii (takara bio inc., kyoto, japan) for rs8074524 and rs2293152 ; xsp i (takara bio inc.) for rs957970. the digests were separated by electrophoresis on a 2% agarose gel (nacalai tesque, kyoto, japan) and visualized with an ultraviolet transilluminator (alpha innotech co., san leandro, ca, usa) after ethidium bromide (nacalai tesque) staining. four genotyped tag snps in tyk2, rs280496 in intron 3, rs280519 in intron 14, rs2304256 in intron 18, and rs280523 in intron 20 (fig. 2), were analyzed by pcr - rflp using 15 pmol each of the following primers : forward primer 5-cggggtgatatgctcattgg-3 and reverse primer 5-caacgtgctgctggacaacg-3 for rs280496 ; forward primer 5-ccgccatggtgaaagttagc-3 and reverse primer 5-atttgtgcaggccaagctgc-3 for rs280519 ; forward primer 5-tcaccaggcacttgttgtcc-3 and reverse primer 5-cggcttccagcatgtgtatg-3 for rs2304256 ; and forward primer 5-acatttccccctgcctacac-3 and reverse primer 5-ttacagacatgcgccaccac-3 for rs280523. the amplification protocol comprised initial denaturation at 95c for 2 min, followed by 30 cycles of denaturation at 95c for 30 s, annealing at 64c (rs280496, rs280519, and rs280523) or 62c (rs2304256) for 30 s, and extension at 72c for 30 s and final extension at 72c for 5 min., beverly, ma, usa) for rs280496, hpy99 i (new england biolabs inc.) for rs280519, bsm i (new england biolabs inc.) for rs2304256, and bsie i (new england biolabs inc.) for rs280523. two tag snps in tyk2, which showed a close association of susceptibility to cd and were located within the same linkage disequilibrium block (fig. 2), were utilized to infer the haplotype structure as well as to analyze the haplotype frequency using the snp alyze 7.0 standard software package (dynacom inc., yokohama, japan) to emphasize the variability and to enhance the power of detecting allelic association of rare variants [33, 34 ]. differences in age and gender between uc or cd patients and control subjects were evaluated by an unpaired student s t test and a chi - square test, respectively, using the spss 17 (spss japan inc., the frequencies of the expected alleles were calculated from those of the observed genotypes according to the hardy weinberg equilibrium. the frequencies of the observed and expected alleles were compared by the chi - square test with yates correction using the snp alyze 7.0 standard software package. the frequencies and distributions of alleles, genotypes, haplotypes, and diplotypes were statistically compared between uc or cd patients and control subjects by the chi - square test and logistic regression analysis using prism 5 and spss 17. subsequently, a comparison of the genetic risk factors between the statistically significant genotype of stat3 and diplotype of tyk2 for susceptibility to cd was carried out by a multivariate logistic regression analysis using spss 17. the odds ratio (or) with 95% confidence interval (ci) was calculated using spss 17. the frequencies and distributions of alleles and genotypes at the three tag snps in stat3 were identified and compared between uc or cd patients and control subjects (tables ii and iii, respectively). the c allele at rs8074524 snp, g allele at rs2293152 snp, and a allele at rs957970 snp are major alleles, whereas the other alleles are minor alleles (table ii). the distributions of these tag snps in stat3 among ibd patients and control subjects corresponded well to the hardy weinberg equilibrium, thus implying that the subject base has a homogeneous genetic background. table iidistributions of polymorphic alleles at the genotyped tag snp sites in stat3 and tyk2 among study subjectsgenesnpallelenumber (%) of alleles inallele comparisonnumber (%) of alleles inallele comparisonuccontrolp valuecdcontrolp valuestat3rs8074524c144 (64.3)253 (63.3)0.759106 (63.9)253 (63.3)0.892t80 (35.7)147 (36.7)60 (36.1)147 (36.7)rs2293152g151 (66.2)267 (66.7)0.89491 (54.8)267 (66.7)0.007c77 (33.8)133 (33.3)75 (45.2)133 (33.3)rs957970a116 (51.8)218 (54.5)0.65284 (50.6)218 (54.5)0.397g108 (48.2)182 (45.5)82 (49.4)182 (45.5)tyk2rs280496c201 (89.7)362 (90.5)0.784144 (86.7)362 (90.5)0.187g23 (10.3)38 (9.5)22 (13.3)38 (9.5)rs280519a136 (60.7)224 (56.0)0.261109 (65.7)224 (56.0)0.034g88 (39.3)176 (44.0)57 (34.3)176 (44.0)rs2304256c156 (69.6)262 (65.5)0.311129 (77.7)262 (65.5)0.004a68 (30.4)138 (34.5)37 (23.3)138 (34.5)rs280523g207 (92.4)370 (92.5)0.968154 (92.8)370 (92.5)0.911a17 (7.6)30 (7.5)12 (7.2)30 (7.5)total number of alleles228400166400each allele was compared to another allele using a chi - square test.table iiidistribution of genotypes at the tag snp sites in stat3 and tyk2 between cd patients and control subjectsgenesnpgenotypenumber (%) of genotypes ingenotype comparisoncdcontrolor (95% ci)p valuestat3rs8074524c / c34 (41.0)75 (37.5)1.156 (0.6861.951)0.586c / t38 (45.8)103 (51.5)0.795 (0.4761.329)0.382t / t11 (13.3)22 (11.0)1.236 (0.5702.680)0.591rs2293152g / g27 (32.5)84 (42.0)0.666 (0.3891.141)0.139g / c37 (44.6)99 (49.5)0.821 (0.4911.372)0.451c / c19 (22.9)17 (8.5)3.196 (1.5666.523)0.001rs957970a / a19 (22.9)55 (27.5)0.783 (0.4301.424)0.423g / a46 (55.4)108 (54.0)1.059 (0.6331.772)0.827g / g18 (21.7)37 (18.5)1.220 (0.6482.296)0.538tyk2rs280496c / c61 (73.5)162 (81.0)0.650 (0.3561.187)0.161c / g22 (26.5)38 (19.0)1.538 (0.8422.807)0.161g / g00rs280519a / a37 (44.6)63 (31.5)1.749 (1.0342.959)0.037a / g35 (42.2)98 (49.0)0.759 (0.4531.272)0.295g / g11 (13.2)39 (19.5)0.631 (0.3061.302)0.212rs2304256c / c52 (62.7)86 (43.0)2.224 (1.3153.716)0.003c / a25 (30.1)90 (45.0)0.527 (0.3050.909)0.021a / a6 (7.2)24 (12.0)0.571 (0.2251.454)0.240rs280523g / g71 (85.5)170 (85.0)1.044 (0.5062.155)0.907g / a12 (14.5)30 (15.0)0.958 (0.4641.976)0.907a / a00total number of subjects83200or odds ratio, ci confidence intervaleach genotype was compared to other genotypes combined using a logistic regression analysis distributions of polymorphic alleles at the genotyped tag snp sites in stat3 and tyk2 among study subjects each allele was compared to another allele using a chi - square test. distribution of genotypes at the tag snp sites in stat3 and tyk2 between cd patients and control subjects or odds ratio, ci confidence interval each genotype was compared to other genotypes combined using a logistic regression analysis the frequencies of the c allele and its homozygous c / c genotype at rs2293152 in cd patients were significantly higher than those in control subjects (45.2% vs. 33.3%, p = 0.007 and 22.9% vs. 8.5%, p = 0.001, respectively). no significant differences were observed in the frequency of other alleles and genotypes between patients and control subjects. the frequencies and distributions of alleles and genotypes at the four tag snps in tyk2 were identified and compared between uc or cd patients and control subjects (tables ii and iii, respectively). the c allele at rs280496 snp, a allele at rs280519 snp, c allele at rs2304256 snp, and g allele at rs280523 snp are major alleles, whereas other alleles are minor alleles (table ii). the distributions of these tag snps in tyk2 among ibd patients and control subjects corresponded well to the hardy weinberg equilibrium. the frequencies of the a allele and its homozygous a / a genotype at rs280519 in cd patients were significantly higher than those in control subjects (65.7% vs. 56.0%, p = 0.034 and 44.6% vs. 31.5%, p = 0.037, respectively). likewise, the frequencies of the c allele and its homozygous c / c genotype at rs2304256 in cd patients were also significantly higher than those in control subjects (77.7% vs. 65.5%, p = 0.004 and 62.7% vs. 43.0%, p = 0.003, respectively). in contrast, the frequency of the c / a heterozygous genotype at rs2304256 was significantly lower in cd patients in comparison to that in control subjects (30.1% vs. 45.0%, p = 0.021). subsequently, four haplotypes composed of these two tag snps (rs280519 and rs2304256), which displayed a significant association with cd susceptibility and were located within the same linkage disequilibrium block, were constructed and identified using the snp alyze 7.0 standard software package (table iv). a logistic regression analysis revealed the frequency of a hap 1 haplotype (a allele at rs280519 snp and c allele at rs2304256 snp) to significantly increase in cd patients in comparison to that in control subjects (65.7% vs. 55.3%, p = 0.023, or = 1.549). in contrast, the frequency of a hap 2 haplotype (g allele at rs280519 snp and a at rs2304256 snp) was significantly decreased in cd patients in comparison to that in control subjects (22.3% vs. 33.7%, p = 0.007, or = 0.563). table ivdistributions of haplotypes of tyk2 between cd patients and control subjectshaplotypesnpnumber (%) of haplotypes inhaplotype comparisonrs280519rs2304256cdcontrolor (95% ci)p valuehap 1ac109 (65.7)221 (55.3)1.549 (1.0632.256)0.023hap 2ga37 (22.3)135 (33.7)0.563 (0.3700.857)0.007hap 3gc20 (12.0)41 (10.3)1.199 (0.6802.117)0.530hap 4aa03 (0.7)total number of haplotypes166400or odds ratio, ci confidence intervaleach haplotype was compared to other haplotypes combined using a logistic regression analysis distributions of haplotypes of tyk2 between cd patients and control subjects or odds ratio, ci confidence interval each haplotype was compared to other haplotypes combined using a logistic regression analysis furthermore, eight diplotypes composed of four haplotypes were identified (table v). a logistic regression analysis showed that the frequency of the cd patients possessing a hap 1/hap 1 diplotype was significantly higher than that of the control subjects (44.6% vs. 30.5%, p = 0.024, or = 1.833). in contrast, the frequency of the cd patients having a hap 1/hap 2 diplotype was significantly lower than that of the control subjects (24.1% vs. 36.5%, p = 0.045, or = 0.552). the results of the diplotype analysis regarding the hap 1 haplotype of tyk2 coincided well with those of the haplotype analysis between cd patients and control subjects ; however, a hap 2/hap 2 diplotype showed no statistically significant lack of susceptibility to cd (table v). table vdistributions of diplotypes of tyk2 between cd patients and control subjectsdiplotypenumber (%) of diplotypes indiplotype comparisoncdcontrolor (95% ci)p valuehap 1/hap 137 (44.6)61 (30.5)1.833 (1.0823.105)0.024hap 1/hap 220 (24.1)73 (36.5)0.552 (0.3090.986)0.045hap 1/hap 315 (18.1)24 (12.0)1.618 (0.8013.268)0.180hap 1/hap 402 (1.0)hap 2/hap 26 (7.2)23 (11.5)0.600 (0.2351.531)0.285hap 2/hap 35 (6.0)15 (7.5)0.791 (0.2782.251)0.660hap 2/hap 401 (0.5)hap 3/hap 301 (0.5)total number83200or odds ratio, ci confidence intervaleach diplotype was compared to other diplotypes combined using a logistic regression analysis distributions of diplotypes of tyk2 between cd patients and control subjects or odds ratio, ci confidence interval each diplotype was compared to other diplotypes combined using a logistic regression analysis the gene a multivariate logistic regression analysis indicated that two variable genetic factors, the c / c genotype at rs2293152 snp in stat3 and the hap 1/hap 1 diplotype of tyk2, independently contributed to susceptibility to cd (p = 0.002, or = 3.113, 95% ci = 1.5156.399 and p = 0.030, or = 1.783, 95% ci = 1.0423.053, respectively ; table vi). gene interaction between stat3 genotype and tyk2 diplotype for susceptibility to cdfactorfactor comparisonor (95% ci)p valuec / c genotype at rs2293152 in stat33.113 (1.5156.399)0.002hap 1/hap 1 diplotype of tyk21.783 (1.0423.053)0.030or odds ratio, ci confidence intervalfactors were statistically analyzed by a multivariate logistic regression analysis gene gene interaction between stat3 genotype and tyk2 diplotype for susceptibility to cd or odds ratio, ci confidence interval factors were statistically analyzed by a multivariate logistic regression analysis furthermore, with regard to the gene gene combination effect of stat3 genotype and tyk2 diplotype for susceptibility to cd, a multivariate logistic regression analysis showed the or to significantly increase (7.486, p = 0.0008, 95% ci = 2.31024.261) in the individuals possessing both the c / c genotype at rs2293152 snp in stat3 and the hap 1/hap 1 diplotype of tyk2 in comparison to that in the individuals possessing the other genotypes (table vii). gene combination effect of stat3 genotype and tyk2 diplotype for susceptibility to cdfactornumber (%) offactor comparisoncdcontrolor (95% ci)p valuec / c genotype at rs2293152 in stat3 and hap 1/hap 1 of tyk211 (13.3)4 (2.0)7.486 (2.31024.261)0.0008other genotypes72 (86.7)196 (98.0)or odds ratio, ci confidence intervalfactors were statistically analyzed by a multivariate logistic regression analysis the gene gene combination effect of stat3 genotype and tyk2 diplotype for susceptibility to cd or odds ratio, ci confidence interval factors were statistically analyzed by a multivariate logistic regression analysis the frequencies and distributions of alleles and genotypes at the three tag snps in stat3 were identified and compared between uc or cd patients and control subjects (tables ii and iii, respectively). the c allele at rs8074524 snp, g allele at rs2293152 snp, and a allele at rs957970 snp are major alleles, whereas the other alleles are minor alleles (table ii). the distributions of these tag snps in stat3 among ibd patients and control subjects corresponded well to the hardy weinberg equilibrium, thus implying that the subject base has a homogeneous genetic background. table iidistributions of polymorphic alleles at the genotyped tag snp sites in stat3 and tyk2 among study subjectsgenesnpallelenumber (%) of alleles inallele comparisonnumber (%) of alleles inallele comparisonuccontrolp valuecdcontrolp valuestat3rs8074524c144 (64.3)253 (63.3)0.759106 (63.9)253 (63.3)0.892t80 (35.7)147 (36.7)60 (36.1)147 (36.7)rs2293152g151 (66.2)267 (66.7)0.89491 (54.8)267 (66.7)0.007c77 (33.8)133 (33.3)75 (45.2)133 (33.3)rs957970a116 (51.8)218 (54.5)0.65284 (50.6)218 (54.5)0.397g108 (48.2)182 (45.5)82 (49.4)182 (45.5)tyk2rs280496c201 (89.7)362 (90.5)0.784144 (86.7)362 (90.5)0.187g23 (10.3)38 (9.5)22 (13.3)38 (9.5)rs280519a136 (60.7)224 (56.0)0.261109 (65.7)224 (56.0)0.034g88 (39.3)176 (44.0)57 (34.3)176 (44.0)rs2304256c156 (69.6)262 (65.5)0.311129 (77.7)262 (65.5)0.004a68 (30.4)138 (34.5)37 (23.3)138 (34.5)rs280523g207 (92.4)370 (92.5)0.968154 (92.8)370 (92.5)0.911a17 (7.6)30 (7.5)12 (7.2)30 (7.5)total number of alleles228400166400each allele was compared to another allele using a chi - square test.table iiidistribution of genotypes at the tag snp sites in stat3 and tyk2 between cd patients and control subjectsgenesnpgenotypenumber (%) of genotypes ingenotype comparisoncdcontrolor (95% ci)p valuestat3rs8074524c / c34 (41.0)75 (37.5)1.156 (0.6861.951)0.586c / t38 (45.8)103 (51.5)0.795 (0.4761.329)0.382t / t11 (13.3)22 (11.0)1.236 (0.5702.680)0.591rs2293152g / g27 (32.5)84 (42.0)0.666 (0.3891.141)0.139g / c37 (44.6)99 (49.5)0.821 (0.4911.372)0.451c / c19 (22.9)17 (8.5)3.196 (1.5666.523)0.001rs957970a / a19 (22.9)55 (27.5)0.783 (0.4301.424)0.423g / a46 (55.4)108 (54.0)1.059 (0.6331.772)0.827g / g18 (21.7)37 (18.5)1.220 (0.6482.296)0.538tyk2rs280496c / c61 (73.5)162 (81.0)0.650 (0.3561.187)0.161c / g22 (26.5)38 (19.0)1.538 (0.8422.807)0.161g / g00rs280519a / a37 (44.6)63 (31.5)1.749 (1.0342.959)0.037a / g35 (42.2)98 (49.0)0.759 (0.4531.272)0.295g / g11 (13.2)39 (19.5)0.631 (0.3061.302)0.212rs2304256c / c52 (62.7)86 (43.0)2.224 (1.3153.716)0.003c / a25 (30.1)90 (45.0)0.527 (0.3050.909)0.021a / a6 (7.2)24 (12.0)0.571 (0.2251.454)0.240rs280523g / g71 (85.5)170 (85.0)1.044 (0.5062.155)0.907g / a12 (14.5)30 (15.0)0.958 (0.4641.976)0.907a / a00total number of subjects83200or odds ratio, ci confidence intervaleach genotype was compared to other genotypes combined using a logistic regression analysis distributions of polymorphic alleles at the genotyped tag snp sites in stat3 and tyk2 among study subjects each allele was compared to another allele using a chi - square test. distribution of genotypes at the tag snp sites in stat3 and tyk2 between cd patients and control subjects or odds ratio, ci confidence interval each genotype was compared to other genotypes combined using a logistic regression analysis the frequencies of the c allele and its homozygous c / c genotype at rs2293152 in cd patients were significantly higher than those in control subjects (45.2% vs. 33.3%, p = 0.007 and 22.9% vs. 8.5%, p = 0.001, respectively). no significant differences were observed in the frequency of other alleles and genotypes between patients and control subjects. the frequencies and distributions of alleles and genotypes at the four tag snps in tyk2 were identified and compared between uc or cd patients and control subjects (tables ii and iii, respectively). the c allele at rs280496 snp, a allele at rs280519 snp, c allele at rs2304256 snp, and g allele at rs280523 snp are major alleles, whereas other alleles are minor alleles (table ii). the distributions of these tag snps in tyk2 among ibd patients and control subjects corresponded well to the hardy weinberg equilibrium. the frequencies of the a allele and its homozygous a / a genotype at rs280519 in cd patients were significantly higher than those in control subjects (65.7% vs. 56.0%, p = 0.034 and 44.6% vs. 31.5%, p = 0.037, respectively). likewise, the frequencies of the c allele and its homozygous c / c genotype at rs2304256 in cd patients were also significantly higher than those in control subjects (77.7% vs. 65.5%, p = 0.004 and 62.7% vs. 43.0%, p = 0.003, respectively). in contrast, the frequency of the c / a heterozygous genotype at rs2304256 was significantly lower in cd patients in comparison to that in control subjects (30.1% vs. 45.0%, p = 0.021). subsequently, four haplotypes composed of these two tag snps (rs280519 and rs2304256), which displayed a significant association with cd susceptibility and were located within the same linkage disequilibrium block, were constructed and identified using the snp alyze 7.0 standard software package (table iv). a logistic regression analysis revealed the frequency of a hap 1 haplotype (a allele at rs280519 snp and c allele at rs2304256 snp) to significantly increase in cd patients in comparison to that in control subjects (65.7% vs. 55.3%, p = 0.023, or = 1.549). in contrast, the frequency of a hap 2 haplotype (g allele at rs280519 snp and a at rs2304256 snp) was significantly decreased in cd patients in comparison to that in control subjects (22.3% vs. 33.7%, p = 0.007, or = 0.563). table ivdistributions of haplotypes of tyk2 between cd patients and control subjectshaplotypesnpnumber (%) of haplotypes inhaplotype comparisonrs280519rs2304256cdcontrolor (95% ci)p valuehap 1ac109 (65.7)221 (55.3)1.549 (1.0632.256)0.023hap 2ga37 (22.3)135 (33.7)0.563 (0.3700.857)0.007hap 3gc20 (12.0)41 (10.3)1.199 (0.6802.117)0.530hap 4aa03 (0.7)total number of haplotypes166400or odds ratio, ci confidence intervaleach haplotype was compared to other haplotypes combined using a logistic regression analysis distributions of haplotypes of tyk2 between cd patients and control subjects or odds ratio, ci confidence interval each haplotype was compared to other haplotypes combined using a logistic regression analysis furthermore, eight diplotypes composed of four haplotypes were identified (table v). a logistic regression analysis showed that the frequency of the cd patients possessing a hap 1/hap 1 diplotype was significantly higher than that of the control subjects (44.6% vs. 30.5%, p = 0.024, or = 1.833). in contrast, the frequency of the cd patients having a hap 1/hap 2 diplotype was significantly lower than that of the control subjects (24.1% vs. 36.5%, p = 0.045, or = 0.552). the results of the diplotype analysis regarding the hap 1 haplotype of tyk2 coincided well with those of the haplotype analysis between cd patients and control subjects ; however, a hap 2/hap 2 diplotype showed no statistically significant lack of susceptibility to cd (table v). table vdistributions of diplotypes of tyk2 between cd patients and control subjectsdiplotypenumber (%) of diplotypes indiplotype comparisoncdcontrolor (95% ci)p valuehap 1/hap 137 (44.6)61 (30.5)1.833 (1.0823.105)0.024hap 1/hap 220 (24.1)73 (36.5)0.552 (0.3090.986)0.045hap 1/hap 315 (18.1)24 (12.0)1.618 (0.8013.268)0.180hap 1/hap 402 (1.0)hap 2/hap 26 (7.2)23 (11.5)0.600 (0.2351.531)0.285hap 2/hap 35 (6.0)15 (7.5)0.791 (0.2782.251)0.660hap 2/hap 401 (0.5)hap 3/hap 301 (0.5)total number83200or odds ratio, ci confidence intervaleach diplotype was compared to other diplotypes combined using a logistic regression analysis distributions of diplotypes of tyk2 between cd patients and control subjects or odds ratio, ci confidence interval each diplotype was compared to other diplotypes combined using a logistic regression analysis the gene gene interaction between stat3 and tyk2 was analyzed between cd patients and control subjects. a multivariate logistic regression analysis indicated that two variable genetic factors, the c / c genotype at rs2293152 snp in stat3 and the hap 1/hap 1 diplotype of tyk2, independently contributed to susceptibility to cd (p = 0.002, or = 3.113, 95% ci = 1.5156.399 and p = 0.030, or = 1.783, 95% ci = 1.0423.053, respectively ; table vi). gene interaction between stat3 genotype and tyk2 diplotype for susceptibility to cdfactorfactor comparisonor (95% ci)p valuec / c genotype at rs2293152 in stat33.113 (1.5156.399)0.002hap 1/hap 1 diplotype of tyk21.783 (1.0423.053)0.030or odds ratio, ci confidence intervalfactors were statistically analyzed by a multivariate logistic regression analysis gene gene interaction between stat3 genotype and tyk2 diplotype for susceptibility to cd or odds ratio, ci confidence interval factors were statistically analyzed by a multivariate logistic regression analysis furthermore, with regard to the gene gene combination effect of stat3 genotype and tyk2 diplotype for susceptibility to cd, a multivariate logistic regression analysis showed the or to significantly increase (7.486, p = 0.0008, 95% ci = 2.31024.261) in the individuals possessing both the c / c genotype at rs2293152 snp in stat3 and the hap 1/hap 1 diplotype of tyk2 in comparison to that in the individuals possessing the other genotypes (table vii). gene combination effect of stat3 genotype and tyk2 diplotype for susceptibility to cdfactornumber (%) offactor comparisoncdcontrolor (95% ci)p valuec / c genotype at rs2293152 in stat3 and hap 1/hap 1 of tyk211 (13.3)4 (2.0)7.486 (2.31024.261)0.0008other genotypes72 (86.7)196 (98.0)or odds ratio, ci confidence intervalfactors were statistically analyzed by a multivariate logistic regression analysis the gene gene combination effect of stat3 genotype and tyk2 diplotype for susceptibility to cd or odds ratio, ci confidence interval factors were statistically analyzed by a multivariate logistic regression analysis this study is the first demonstration of the single - marker association of stat3 and tyk2 polymorphisms with cd susceptibility in the japanese population, although meta - analyses using gwa data previously indicated that stat3 and tyk2 appear to be the genetic determinants of cd in the european and north american populations [9, 10, 14 ]. furthermore, stat3 is associated with only cd, but not uc, in the japanese population by a candidate gene - based association study, thereby supporting the meta - analysis of the gwa data in populations of european and north american ancestry. the presence of the c allele and its homozygous c / c genotype at rs2293152 snp in stat3 conferred susceptibility to cd. cd- and uc - susceptible rs744166 snp, which was identified by gwa studies [9, 12, 14 ], was not analyzed in this study because this snp was not selected as a genotyped tag snp by the ihap software program. because rs744166 and rs957970 snps are located within the same linkage disequilibrium block (fig. 1) and rs957970 snp was not associated with susceptibility to cd in this study, rs744166 snp may not be associated with cd in the japanese population. the difference in susceptibility to cd at the snp site between caucasian and japanese subjects can be attributed to genetic background, although stat3 may contribute to the same mechanisms of the immunopathogenesis of cd in both caucasian and japanese patients. the presence of the a allele and its homozygous a / a genotype at rs280519 snp in tyk2, the c allele and its homozygous c / c genotype at rs2304256 snp in tyk2, the hap 1 haplotype (a allele at rs280519 snp and c allele at rs2304256 snp) of tyk2, and its homozygous hap 1/hap 1 diplotype of tyk2 showed susceptibility to cd. cd - susceptible rs12720356 snp, which was identified by gwa studies, was not analyzed in this study because this snp was not selected as a genotyped tag snp by the ihap software program. although rs280519 and rs2304256 snps, which were examined in this study, are located within the same linkage disequilibrium block, rs12720356 snp does not belong to any linkage disequilibrium blocks (fig. 2). furthermore, because rs280496 snp, nearby rs12720356 snp, was not associated with susceptibility to cd in this study, rs12720356 snp may thus not be associated with cd in the japanese population. this disparity can be also attributed to genetic differences between caucasian and japanese individuals, although tyk2 may contribute to the same immunopathogenesis in both caucasian and japanese cd patients. in addition, the presence of both the c / c genotype at rs2293152 in stat3 and the hap 1/hap 1 diplotype of tyk2 independently contributed to the pathogenesis of cd and remarkably increased the odds ratio for cd, thus indicating an approximately 7.5-fold increase in susceptibility to cd in this study, although such cd patients account for only approximately 13% (11 of 83 = 13.3% in table vii) of the genetic variance observed in cd. these findings imply that stat3 and tyk2 are genetic determinants for the predisposition to the onset and/or development of cd in japanese individuals. however, this study population was relatively small, and further studies on a larger number of japanese subjects and on other ethnicities are necessary to confirm the association between the stat3 and tyk2 polymorphisms and cd. additional studies are needed because different populations will often have different allele frequencies and haplotype structures. recent gwa studies on the il-23/il23r signaling pathway have shifted the focus to the il-23 cytokine [612 ]. after il-23 binds to the receptor, which comprises il23r and il12rb1, il-23 signaling may induce the activation of jak2 in il23r as well as tyk2 in il12rb1 because the il-12rb1 and il-23r require tyk2, thus resulting in the phosphorylation of stat3 as well as stat1, stat4, and stat5 in activated macrophages and dendritic cells. the signaling cascade eventually leads to the differentiation of cd4 (naive) t cells into th17 cells [17, 2022 ]. th17 cells produce il-17a, il-17f, and il-22, which are involved in the first line of the host defense by controlling the immune responses. indeed, the expression of il-12, il-23, stat3, il-17, and il-22 has been reported to increase in the lamina propria of the intestinal mucosa in cd patients [3641 ]. taken together, the il-23/il23r signaling pathway is central to the inflammation leading to cd and modifies an individual s risk of developing cd. for these reasons, it may be speculated that the polymorphisms of stat3 and tyk2, especially the c / c genotype at rs2293152 in stat3 and the hap 1/hap 1 diplotype of tyk2, may affect the gain - of - function of both stat3 and tyk2, thus altering the efficiency of the il-23/il23r signaling pathway. these changes can lead to the perpetuation of the chronic intestinal inflammatory process, thereby resulting in the onset and/or development of cd. as tyk2 and stat3 appear to be the genetic determinants of cd in the japanese population, the combination polymorphism of tyk2 and stat3 may be useful as a new dna - based diagnostic biomarker for identifying high - risk individuals susceptible to cd. finally, stat3 and tyk2 may be good target molecules for the development of novel drugs in the future. | objectivean association between susceptibility to inflammatory bowel disease (ibd) and polymorphisms of both the tyrosine kinase 2 gene (tyk2) and the signal transducer and activator of transcription 3 gene (stat3) was examined in a japanese population in order to identify the genetic determinants of ibd.methodsthe study subjects comprised 112 patients with ulcerative colitis, 83 patients with crohn s disease (cd), and 200 healthy control subjects. seven tag single - nucleotide polymorphisms (snps) in tyk2 and stat3 were detected by pcr - restriction fragment length polymorphism.resultsthe frequencies of a c allele and its homozygous c / c genotype at rs2293152 snp in stat3 in cd patients were significantly higher than those in control subjects (p = 0.007 and p = 0.001, respectively). furthermore, out of four haplotypes composed of the two tag snps (rs280519 and rs2304256) in tyk2, the frequencies of a hap 1 haplotype and its homozygous hap 1/hap1 diplotype were significantly higher in cd patients in comparison to those in control subjects (p = 0.023 and p = 0.024, respectively). in addition, the presence of both the c / c genotype at rs2293152 snp in stat3 and the hap 1/hap 1 diplotype of tyk2 independently contributes to the pathogenesis of cd and significantly increases the odds ratio to 7.486 for cd (p = 0.0008).conclusiontyk2 and stat3 are genetic determinants of cd in the japanese population. this combination polymorphism may be useful as a new genetic biomarker for the identification of high - risk individuals susceptible to cd. |
for the past 50 years, social and epidemiologic surveys have been employed to estimate and track the substance use patterns of representative samples of both adolescents and adults in the united states and other countries. although many of these surveys are of exceptional quality and rigor (e.g., the monitoring, the future survey, the national survey of drug use and health, and the youth risk behavior survey), for almost as long, there have been methodological criticisms and skepticism regarding their ability to accurately portray the behaviors they seek to measure [17 ]. addressing these questions is important given the lack of alternative methodologies for efficiently monitoring substance use behavior within large national and subnational populations. the goal of this paper is to review and summarize the available empirical evidence addressing these questions, to identify gaps in our knowledge base regarding this issue, and to make some recommendations for needed future research to address those knowledge gaps. a useful framework for conceptualizing error in substance use surveys is the total survey error (tse) model. the tse model first delineated by groves focused on sampling, coverage, nonresponse, and measurement errors in surveys. an expanded elaboration of the tse model has been more recently presented by lavrakas, in which he identifies two general classes of errors, measurement and representation, and then explores multiple subclasses of errors within each. briefly, errors of representation are those concerned with technical problems that may impede a survey 's ability to accurately mirror the population that the survey seeks to represent. these include failure to use sample frames that provide adequate coverage of the population being studied (coverage errors), imprecision in the sample(s) drawn from a sample frame (sampling error), errors associated with failure to contact or complete interviews with all sampled respondents, and failure to obtain answers to all questions included in a survey instrument (nonresponse errors), as well as failure to make adequate adjustments for complex sample designs and survey nonresponse (adjustment errors). in contrast, errors of measurement involve failures to adequately assess the variables of interest in a survey. these include specification errors, which involve failures to correctly conceptualize survey constructs, and measurement errors, which include factors external to the construct being measured that nonetheless influence measurement quality. processing errors are defects in the construction of survey data sets and/or final analytic variables and inferential errors which involve difficulties or failures when making adequate sense of the final survey data. the following two sections organize the empirical literature concerned with errors in substance use and misuse surveys within this tse framework. each of these error sources, of course, is broadly relevant to health survey research in general. errors in coverage are generally a consequence of employing a survey sampling frame that does not include all individuals in the population being studied, or, alternatively, by employing methods that do not provide all members of the population of interest some probability of being sampled. as with all other elements of the tse framework, because likelihood of falling into a potential sample frame may in some cases be associated with substance use behaviors, substance use research may be uniquely vulnerable to coverage error. in most community epidemiological surveys, there are many social groups that may be systematically excluded from commonly available sample frames. some of these groups include homeless persons, individuals currently hospitalized, college students living in dormitories, persons incarcerated in the criminal justice system, and members of the military living on military bases. substance use may be particularly high within some of these nonresidential populations [10, 11 ]. weisner and colleagues investigated this problem by comparing prevalence estimates from a general population community survey with data obtained from interviews with nonhousehold populations found in several inpatient and outpatient settings, such as alcohol, drug or mental health treatment, criminal justice, and/or welfare services. for example, 11.3% of the household sample was defined as problem drinkers, compared to 43.1% of those found in nonhousehold agency settings. the disparities were even greater for indicators of weekly drug use (5.5% in the household sample versus 36.5% in the agency sample) and both problem drinking and weekly drug use combined (2.2% in household sample versus 18.7% in agency sample). other research provides similar evidence of increased substance use and misuse among persons less likely to be sampled within single family households as part of community - based epidemiologic surveys. there is also some evidence in the us that failure to incorporate cell - phone - only households into random digit dialed (rdd) telephone samples can lead to underrepresentation of young adults who are at higher risk for substance use behaviors. found significantly decreased measures of binge drinking and heavy alcohol consumption between 20012003 and 20032005 in the national behavioral risk factor surveillance system (brfss) telephone surveys. other research, employing the us national health interview survey, which relies on face - to - face interviews, has demonstrated that adults in cell - phone - only households are more likely to report past year binge drinking behavior (37.6%), compared to those residing in households with landlines (18.0%), and to those in households with no telephone service (23.0% ; blumberg.). the effects of excluding cell phone - only households from survey estimates of binge drinking are particularly serious for young adults (aged 1829 years) and low income persons. similar relationships between type of phone subscribership and substance use reports have been identified in australia and in other us studies. as rates of cell - phone - only residences continue to grow, the coverage error associated with excluding them from telephone samples will only increase, and it will become increasingly difficult to produce credible prevalence estimates using traditional landline - only sample frames. school - based surveys are also subject to coverage errors, as substance use rates have been shown to be higher among adolescents who drop out of school [19, 20 ]. hence, surveys of adolescents that are school based often underestimate substance use within this population, although it is important to acknowledge that many school - based surveys attempt to make no generalizations to nonschool populations. a recent analysis by gfroerer and colleagues using pooled data from the 20022008 nsduh (national survey of drug use and health, previously known as the national household survey of drug abuse or nhsda) surveys reported that substance use estimates were higher for most substances among school dropouts, compared to same - aged students. the effects of dropouts on overall estimates increased from the 8th to the 12th grades, as the numbers of dropouts increased. at the 12th grade level, they found that failure to account for dropouts would miss more than half of past year cocaine users, more than half of all lifetime ecstasy users, 30% of current binge alcohol users, and 25% of current alcohol users. because school absenteeism is also known to be associated with increased substance use [2225 ], gfroerer and colleagues additionally investigated the effects of school absenteeism on substance use prevalence estimates in the nsduh. they reported that those students who missed more days of school were also more likely to be current alcohol users, binge drinkers, and marijuana users. in recognition of this problem, some surveys, such as the yrbs, conduct make - up sessions to maximize student opportunities to participate and minimize coverage errors. both probability and nonprobability sampling methods are commonly applied in substance use surveys. when probability sampling strategies are employed, all elements within the sample frame have a known, the precision of survey statistics derived from such samples can be calculated with a good degree of confidence and used to estimate the sampling error associated with those statistics. all other things being equal, the size of a random survey sample is inversely associated with the degree of potential sampling error associated with it. the precision of survey estimates also decreases as probability samples deviate from simple random sampling designs, a commonplace occurrence designed to reduce survey costs. of all the sources of total survey error, the sampling errors related to probability - based sample designs are probably the most well understood, and definable, in practice. nonprobability samples are commonly used when research questions focus on special populations believed to be at increased risk for substance use and misuse. there are a variety of well - known nonprobability, or convenience, sample designs commonly used in practice. one of the more popular approaches currently is known as respondent driven sampling (rds), which was developed by heckathorn [26, 27 ] and which has been used in numerous substance use studies [2830 ]. other popular nonprobability strategies in substance use research include venue and facility - based sampling [3134 ], snowball sampling [35, 36 ], time - space sampling [3739 ], and advertising for volunteers [4042 ]. an important advantage of these designs is their cost effectiveness when researching rare or hidden populations, such as illicit drug users. because probabilities of selection are unknown, however, there are no definable sampling errors associated with these designs. rather, nonprobability based sample designs typically suffer from large coverage errors and sampling errors are completely unknown. it is common knowledge that unit response rates in general population surveys have been declining for some time [4345 ]. survey response rates have been historically employed as a proxy indicator of survey quality in general and nonresponse error in particular. recent research, though, has demonstrated that response rates per se are not necessarily associated with nonresponse bias [4749 ]. rather, it is the degree to which survey respondents and nonrespondents differ from one another in terms of variables of interest to the survey, combined with the survey 's response rate that defines nonresponse bias. a british study reported by plant., for example, compared two sets of survey data, with 25% and 79% response rates, respectively.. when considering substance use behaviors, there are reasons to be concerned about differences between survey respondents and nonrespondents. pernanen many years ago suggested that persons who drank heavily might be more difficult to contact as part of survey efforts and would be less likely to cooperate when contacted. in a canadian survey, de lint reported that more in - person contact attempts were required to interview those respondents who reported greater numbers of purchases of alcoholic beverages. cottler. additionally reported that those respondents diagnosed with alcohol abuse and dependence required greater numbers of contact attempts in order to complete interviews. crawford also reported more alcohol consumption among those respondents most difficult to contact. using a population register in sweden, tibblin found higher rates of survey nonparticipation among middle aged men who were known to have experienced alcohol related problems. there is also some general evidence that survey nonresponse is greater among persons with poor health [53, 54 ]. a swedish study has reported that survey respondents were less likely to have been hospitalized with alcohol diagnoses, compared to nonrespondents. these findings are generally interpreted as evidence that heavy drinking may be a barrier to participation in social surveys due to difficulty in making contact and also in convincing those individuals who are contacted to agree to participate. other investigations, though, have reported no differences in alcohol use between those who do and do not participate in epidemiologic surveys [5760 ], and alcohol abstainers have also been found to be underrepresented. it should also be noted that standard field procedures in many surveys actually exclude active substance users from participation. much research explicitly requires interviewers not to conduct interviews with individuals who are visibly intoxicated or appear high on other substances. kish commented on this problem nearly 50 years ago, referencing a case in which a respondent was drunk by the time they came home after work every day throughout a survey 's field period. while such protocols are necessary for orderly data collection and are invoked only infrequently in practice, the potential effects of such protocols on nonresponse bias must nonetheless be considered. in addition, despite some claims to the contrary, knowledge that a survey is concerned with substance use appears to have no effect on respondent willingness to participate [55, 63 ]. other relevant information comes from studies of attrition in panel surveys, in which the same respondents are interviewed at multiple time points. a number of such investigations have documented higher levels of attrition among high alcohol and drug users [6473 ]. in contrast, some other research has found higher attrition among nonusers, and thygesen and colleagues have found both high alcohol intake and abstinence to be associated with increased likelihood of panel attrition. in their study, attrition was also found to be predictive of increased mortality from alcoholic liver cirrhosis and alcoholic liver diseases. still other research has found no differences between those who do and do not drop out of panel studies. one type is known as follow - up surveys, which typically involve attempting to obtain survey data from nonrespondents to the primary survey. caspar, for example, conducted follow - up face - to - face interviews with a sample of nonrespondents to the 1990 nhsda, concluding that initial nonrespondents were more likely to report lifetime drug use. provide an example of a nonresponse follow - up survey with individuals who initially did not respond to a mail survey and who were subsequently visited by interviewers to complete a face - to - face interview.. conducted a telephone follow - up survey of nonrespondents to a face - to - face survey, concluding that there were only small effects of nonresponse on self - reporting of alcohol consumption. an important potential limitation when interpreting findings from follow - up surveys such as these is the use of different modes of data collection between the primary survey and the follow - up effort. given what is known about mode differences in reporting of substance use behaviors (see section 4.2), it would not be surprising that a telephone follow - up to a self - administered survey might suggest that the initial survey overestimated substance use, whereas a self - administered nonresponse follow - up survey to an initial interviewer - assisted effort might suggest that it had underestimated substance use. in each case, the effects being attributed to nonresponse bias may actually be a consequence of mode differences rather than systematic nonresponse. indeed, there are several examples in the literature of surveys that relied on interviewer - assisted follow - up interviews (cf., hill. ; lahaut.) that produced data suggesting that primary survey respondents overreport substance use behaviors. examples of other types of nonresponse bias analyses that focus on respondent substance use patterns include studies that compare early versus late respondents [56, 7981 ]. an example is a study reported by zhao., who compared the answers of persons responding early and late to the canadian addictions survey. respondents were more likely to have higher incomes and to be educated, males, young adults, and substance users. such studies employ a continuum of resistance framework that assumes that respondents who require greater effort to contact and interview are more similar to nonrespondents than are those who initially agree to survey requests. other strategies compare estimates from multiple surveys, compare frame data for respondents, nonrespondents, and the full sample, or compare estimates from surveys that have high versus low response rates. another useful strategy for assessing nonresponse bias is to supplement survey data with information obtained from other sources, such as administrative records. for example, gfroerer. examined response patterns in the 1990 national household survey on drug abuse by merging survey findings with records from the 1990 decennial census. of course, this required special authorization from the government, given the strict data protections associated with the census. they found that persons with some characteristics known to be associated with substance use (i.e., living in urban areas, being male) had lower response rates and that persons with other characteristics believed to be associated with nonsubstance use (older age and higher income levels) also had lower response rates and concluded that these various nonresponse correlates would likely cancel out much of the bias either set might have introduced into the survey estimates. finally, it is also important to recognize that high nonresponse rates to individual survey questions (a.k.a., item nonresponse) may also be an indicator of data quality problems in substance use surveys.. found that african americans and persons who were separated or divorced were less likely, and females and persons aged 55 and older were more likely, to answer questions concerned with their use illicit drugs. increased item nonresponse rates to substance use questions among minority groups have also been reported by witt., although aquilino reported no differences. an item nonresponse study of adolescents additionally found higher nonresponse rates to questions concerned with alcohol and marijuana use among male, compared to female respondents. errors of adjustment involve failures to account for the potential effects that a survey 's sample design and execution may have upon empirical findings. these may include instances in which sample weights fail to incorporate all sample design and/or nonresponse factors, when variances are unadjusted for the clustering of respondents within sampled geographic areas, or when the available sample weights are not correctly used. an unfortunate example of the failure to properly employ sample weights occurred about a decade ago when a report concerned with illegal sales of alcohol to underage minors in the us seriously overestimated the proportion of all alcohol sales that were reportedly being made to underage youth. the researchers were conducting a secondary analysis of a public release version of the 1998 nhsda and failed to weigh their data for the survey 's stratified sample design, in which young persons aged 1220 were significantly oversampled. because only persons under the age of 21 purchase alcohol illegally in the us, their overrepresentation in the unweighted nhsda data file led to an overrepresentation of illegal sales in those data. this was an error that could have easily been avoided through the use of the preexisting sample weights. failure to employ nonresponse weights when survey response rates across different demographic subgroups vary considerably and those same variables which are correlated with substance use patterns can also result in biased substance use estimates. in addition, adjustment errors associated with clustered sample designs (when clustering is not taken into account) can lead to survey estimates with artificially small standard errors that can be misinterpreted as being overly precise. in general, avoidance of adjustment errors would seem to require analysts who possess both substantive knowledge of the addiction processes being examined and methodological knowledge and expertise regarding complex sample design and analysis procedures. when survey questionnaires do not correctly conceptualize and/or operationalize constructs of interest, they are understood to have specification errors. for example, the street terminology used by drug users is often unique, constantly changing, and varies across locations. not surprisingly, research demonstrates that the drug names employed in survey questionnaires are not always consistent with the names employed by users in the community [90, 91 ]. the continued introduction of new substances of course also contributes to specification errors. in order to adequately assess substance use, it is necessary to ask respondents about all the forms of alcohol and/or drug they may have consumed. hence, survey questions intended to measure any alcohol or drug use must be able to capture experience with each form of these substances. global questions that ask about use of the substances in general can be expected to miss some experiences with less common varieties of each. although these points may seem obvious, they can lead to specification errors more often than most researchers would prefer to admit. avoiding specification errors requires careful attention during the instrument design process to the specific goals for which the survey is intended to be used. measurement error occurs when survey questions fail to measure what they were designed to measure. there are several potential sources of measurement error which must be considered when constructing a survey instrument or analyzing survey data. broadly speaking, virtually every element of a survey that is exposed to respondents is likely to provide them with cues regarding the information being sought. although many if not most of these cues are unintentional from the researcher 's perspective, they can nonetheless be expected to influence self - reports in ways that can not always be anticipated or controlled. some important design issues discussed below include methods for asking about substance use, mode effects, use of skip patterns, and reference periods. other design factors that may influence measurement quality include how clearly a survey is introduced as being concerned with substance use, the survey 's sponsor, the procedures employed to obtain respondent informed consent, the use of incentives, and the survey 's focus as either primarily concerned with substance use or concerned with a more broad set of topics. regarding this later point, it has been suggested that survey respondents are more willing to discuss negative personal behaviors when they are also asked to report about positive personal behaviors and characteristics. of course, the wording and structure of survey questions can be expected to have a strong influence on the answers obtained, and experimental comparisons have revealed differences in the magnitude of substance use reports obtained using various question measurement strategies. have documented the fact that open - ended questions seriously underestimate drug use prevalence rates. other research has compared methods for measuring alcohol consumption. rehm. has reported findings from a within - subjects experiment that documents consistently higher prevalence rates for several indicators of harmful drinking when graduated - frequency measures are used, in comparison to the more commonly employed quantity - frequency question response format [97, 98 ], and weekly drinking recall questions. other studies have also found graduated - frequency measures to produce higher estimates of alcohol use in comparison to quantity - frequency measures [99, 100 ]. the superior performance of the graduated - frequency format appears to be based on its ability to more precisely measure irregularly high levels of consumption, although there is some evidence suggesting that the graduated - frequency approach may actually overestimate consumption [100, 101 ]. other less commonly used measurement strategies, such as the yesterday (or recent recall) method of reporting in which respondents are asked to report on their alcohol use during the previous day only, have been found to produce higher estimates than either the quantity - frequency or graduated - frequency measures. the use of a daily diary protocol for collection of alcohol consumption is frequently considered to be a gold standard measurement approach [100, 103 ], but not very practical for most survey applications. the design of response categories for use in quantity and frequency questions can also influence respondent self - reports. for example, schwarz has shown how simple changes in the sets of response options presented to respondents, such as emphasizing low versus high frequency events or behaviors, can have effects on overall response patterns. indeed, poikolainen and krkkinen have reported obtaining higher alcohol consumption reports when employing quantity and frequency questions that include more heavier intake response options. it is somewhat ironic that quantity - frequency measures remain commonly utilized in practice, despite the fact that it is conventional wisdom among most substance use researchers that alcohol and drug consumption behaviors are far more variable across even brief time intervals than are assumed by these questions [92, 106 ]. by their very nature, quantity - frequency items ask for average amounts of use, essentially insuring that they will not capture episodes of heavy or binge drinking. hasin and carpenter have documented in a community sample that as many as 30 percent of all respondents report having difficulty when answering typical survey questions concerned with usual drinking patterns due to changes in their drinking behavior during the time period in question and that this problem was particularly acute for persons with symptoms of alcohol dependence. the key advantages of the quantity - frequency measures that make them continue to be popular are their simplicity, ease of answering, and the relatively small amount of space they require in survey instruments. provide comprehensive overviews of the strengths and limitations of various approaches to measuring alcohol consumption in survey questionnaires. various reference periods are used to restrict and specify the time intervals for which respondents are asked to retrospectively report their substance use activities. most often used in practice are 30-day and 12-month reference periods, although there are many variations. it is common knowledge that recall accuracy decays with increasing length of these time intervals, as research suggests that greater alcohol prevalence is obtained when shorter reference periods are employed in survey questions [109, 110 ]. although more susceptible to recall concerns, a 12-month recall period would have the advantage of being less affected by seasonal variations in substance use [92, 111 ]. a 30-day reference period, in contrast, hence, some surveys may ask questions about multiple reference periods in order to address the limitations of each. also problematic are questions concerned with age of initiation of alcohol and other drug use. age of first substance use, of course, is considered an important risk factor for subsequent substance abuse, and accurate measurement is hence important. unfortunately, the length of recall necessary to correctly answer this question can be problematic for many respondents. forward telescoping, in particular, when respondents underestimate the length of time since an event took place, is an important threat to the quality of self - reports of age of first use. numerous studies have documented problems with accurate recall of this information [64, 114121 ]. a common issue when designing substance use questionnaires is the question of whether it is best to employ skip patterns, which allow respondents to avoid answering follow - up questions that are clearly not applicable to them or to instead require all respondents to provide all answers to all items. first, there may be privacy concerns associated with the use of skip patterns, as those who report substance use will require more time to complete all follow - up questions, presumably allowing interviewers and/or other observers to conclude that they are in fact substance users. second, although it is somewhat burdensome for respondents, it is likely that the presence of skip patterns will be quickly detected by many respondents and possibly motivate some to provide negative answers to filter questions in order to skip out longer blocks of questions that request details regarding substance use experiences. as an example of a skip pattern, a question that asks respondents if they have ever used marijuana might be employed as a filter item. those respondents indicating that they had used marijuana would then be eligible to answer a series of follow - up questions that queried about frequency of use, age of initiation, and so forth. in contrast, avoidance of skip patterns would require respondents to answer all follow - up questions, typically by selecting a have never used marijuana response option, which would be available for use with each follow - up question. such an approach can considerably increase the burden and amount of time necessary to complete a questionnaire for nonusers of the substances being examined. investigated the effects of using skip patterns as part of the nhsda. in their random experiment, they found significantly lower prevalence rates for the five illicit drugs examined when skip patterns were employed. because no differences were found in alcohol use estimates, it was concluded that privacy concerns associated with answering the most sensitive questions was a more likely explanation for the findings. survey data can be collected using a variety of modalities, including self - administered paper - and - pencil or electronic questionnaires, and telephone or in - person interviews. the presence of mode effects in surveys is well recognized, and there is now a considerable body of evidence documenting the effects of mode on the quality of self - reports of substance use behaviors. in general, survey modes that rely on respondent self - administration are found to obtain greater reports of alcohol and drug use than do those modes that require interviewers to directly ask about use of these substances [55, 58, 122130 ]. there is additionally some evidence that these mode effects are greater for more sensitive illicit substances, such as cocaine and marijuana, compared to alcohol use. among self - administered modes, audio - computer - assisted - self - interviews (acasi) appear to generate higher reporting of substance use behaviors than do paper - and - pencil (papi) self - administered answer sheets [132, 133 ]. computer - assisted questionnaires produce data that is more internally consistent and more complete, helping to reduce the need for editing, imputation, and other processing activities that may lead to processing errors (see section 4.3). research has also begun to explore the reliability and validity of substance use surveys conducted via the internet. eaton and colleagues randomly assigned classes of high school students to respond to papi or web questionnaires, concluding that there were few differences in prevalence estimates obtained across the two modes. ramo and colleagues examined the quality of self - reported marijuana use in a convenience sample of young adults who completed a web - based questionnaire, concluding that such data can be reliably collected. have reported on the use of respondent driven sampling to more systematically sample young adults to participate in a substance use survey. other investigators have compared internet reporting of alcohol use with reports obtained from self - administered mail questionnaires and both face - to - face and telephone interviews, concluding that online reports have similar levels of measurement quality [136138 ]. among interviewer assisted modes, some evidence suggests that face - to - face interviews appear to produce greater reports than do telephone interviews [86, 123, 139, 140 ], other evidence suggests no differences in substance use estimates between these two interviewer assisted modes [141, 142 ], and one study suggests that higher rates of some alcohol - related measures can be obtained by telephone. some research has also investigated the use of interactive voice recording (ivr) systems (a.k.a., t - acasitelephone audio computer - assisted self - interviewing) to improve the quality of substance use data collected by phone [144, 145 ]. survey respondents vary considerably in their abilities and willingness to provide accurate answers to questions regarding substance use behaviors. respondent behaviors can be understood within the framework of the generally accepted cognitive model of survey response, which recognizes four basic tasks required from respondents when they are answering each survey question. these include (a) question interpretation, (b) memory retrieval, (c) judgment formation, and (d) response editing. this is a useful model for understanding how variability across respondents may influence the quality of self - reported substance use information. evidence regarding how three of these information processing tasks may influence the quality of substance use behavior reporting is reviewed below. question interpretation. because respondents sometimes employ substance use terminology that differs from that employed in research questionnaires [91, 147 ], the risk of miscommunication may be greater in substance use surveys, compared to other topics. this may be of particular concern in surveys of adolescents, who may not always have sufficient knowledge to correctly respond to questions regarding the use of various drugs [147149 ]. johnston and o'malley have presented evidence suggesting that respondents sometimes are more likely to deny, or recant, ever having used certain substances that they had previously reported having used (see also additional discussion of recanting in section below on response editing). of particular relevance here is their finding that recanting varies by type of drug being asked about, with the recanting of tranquilizers and barbiturates found to be greater than that for marijuana and cocaine, a finding that they suggest to be related to the complexity of the definitions of these two substances, relative to marijuana and cocaine definitions, which of course also have some complexity. in alcohol research, recent reviews have found that respondents commonly misinterpret standard drink sizes, suggesting that alcohol intake may be systematically underestimated in survey research [151, 152 ]. a related concern is the degree to which respondent cultural background may influence the interpretation and/or comprehension of survey questions. substance use patterns and practices are known to vary cross - culturally [153155 ], and those varied experiences and beliefs regarding substance use can also be expected to influence respondent knowledge and familiarity with the topic in general and related terminology in particular. experienced researchers, of course, recognize the importance of investigating and addressing these potential problems by employing focus groups, cognitive interviews, and ethnographic methods during survey development (c.f., gardner and tang ; midanik and hines ; ridolfo ; and thrasher.). memory retrieval. the accuracy of respondent recall has been the focus of much attention among methodologists [160, 161 ] and has been historically considered one of the more common explanations for inaccurate reporting of substance use behaviors [4, 120, 121 ]. indeed, when answering survey questions concerned with substance use, the retrieval of the memories necessary to report accurately can be particularly difficult for several reasons. poorly worded survey questions may present respondents with difficult cognitive challenges in terms of the effort necessary to retrospectively retrieve specific and/or detailed information that may not be readily accessible in memory. there is also evidence that heavy drinking [4, 162 ], cocaine [163, 164 ], and mdma use [165167 ] may be associated with impaired memory. mensch and kandel have found inconsistent reporting of marijuana use to be associated with degree of drug use frequency, with the more involved users providing less consistent survey responses, a finding they associate with faulty memory. although considerable research has been invested in experimenting with strategies for aiding respondents with memory retrieval in general [169, 170 ], few efforts have focused on aiding recall of substance use information. hubbard, however, has reported a series of experiments that used anchoring manipulations to improve respondent recall, although these were not found to be very effective. once respondents have successfully interpreted a survey question and retrieved the relevant information necessary to form an answer, they must decide whether that answer is to be accurately shared with the researcher. given the illicit and sometimes stigmatizing nature of substance use behaviors, conventional wisdom often suggests that some respondents will make conscious decisions to underreport, or deny altogether, any such behavior. that survey respondents will sometimes attempt to present themselves in a favorable, albeit not completely accurate, light during survey interviews concerns about the potential effects of social desirability bias have been the subject of considerable research in the survey methodology literature [172175 ]. in general, respondents are known to overreport socially desirable behaviors, such as voting and exercise, and underreport socially undesirable behaviors, including drug and alcohol use. bradburn and sudman have explored and documented the sensitive nature of substance use questions by asking a national sample of respondents in the us how uneasy discussing various potentially sensitive topics would make them feel. they found that 42.0 percent reported that they believed most respondents would be very uneasy discussing their use of marijuana and that 31.3 and 29.0 percent, respectively, would also be uneasy discussing stimulant and depressant use, and intoxication. only 10.3 percent indicated that they believed most people would be uneasy discussing drinking in general. this survey, though, was conducted more than 30 years ago and it is unclear to what degree these topics would elicit similar feelings of discomfort today. respondents may be uneasy discussing their substance use for several reasons, including the need to avoid the social threat and feelings of shame and embarrassment associated with violating social norms [179, 180 ]. reporting illicit substance use may also be viewed by some respondents as a sign of weakness and, hence, something not to disclose. these points are consistent with research findings that indicate that substance use underreporting increases with the perceived stigma of the substance being discussed [182184 ]. respondents may also elect not to admit to substance use behaviors in order to avoid potential legal sanctions, out of fear that a breach of confidentiality might risk their employment or reputation, and/or because they believe that such information is highly personal and not to be shared. some research suggests that questions about current use of illicit substances are more likely to produce underestimates when confidentiality is less certain, compared to questions concerned with past use. experimental studies that have compared substance use reporting patterns when provided with assurance of anonymity versus confidentiality have generally found few differences across conditions [186188 ]. some measures of the propensity to provide socially desirable answers have been found to be associated with substance use reporting such that likelihood of providing socially desirable responses in general is associated with less likelihood of reporting alcohol and/or drug use behavior [172, 189191 ]. these findings have been interpreted alternatively as (a) evidence that underreporting of substance use is a consequence of respondent attempts to conceal illicit behavior or as (b) evidence that persons who engage in socially desirable behaviors in general also report, accurately, that they do not engage in substance use behaviors. although this question remains unresolved, we note that other research has demonstrated the absence of an association between one measure of social desirability, the crowne - marlowe scale, and a measure of cocaine use underreporting that was based on comparisons of self - reports with biological assays. the accuracy of self - reports of substance use behaviors may also vary by the race / ethnicity of the respondent. a literature review of 36 published studies conducted in the us found consistent evidence of lower reliability and validity rates of substance use reporting among racial and ethnic minority populations. models that have been proposed suggest that greater reporting errors among minority groups may be a consequence of differential group educational achievement and question comprehension, greater minority concerns with privacy, discrimination and risk of prosecution, and/or stronger effects of social desirability pressures on minority groups to report behaviors that conform to majority cultural values. internationally, cultural differences in normative patterns of alcohol consumption and other substance use may also influence degree of response editing. in nations where wine is considered part of a meal, rather than mood - altering substance, underreporting might be expected to be much less of a concern. one limitation in much of the research reviewed here is the assumption that greater self - reports of substance use behaviors are more valid [196, 197 ]. indeed, overreporting is another measurement concern [197, 198 ]. there have been cases of respondents providing daily alcohol use reports that are physically impossible. in surveys of adolescents, there is also a widespread belief that some respondents overreport their alcohol and other drug use, possibly to impress peers and improve one 's social status or as part of a general desire for attention [3, 149, 199202 ]. gfroerer and colleagues have speculated that such overreporting of substance use might be more likely to happen during school - based surveys, usually conducted in classroom settings, where peers may be more likely to be aware of respondent answers. it has also been suggested that respondents may in some situations elect to present themselves in a highly negative manner, perhaps for personal amusement or to obtain treatment services [11, 148, 203, 204 ]. in an effort to identify such overreporters, it is notable that these studies have found very low self - reported rates of use of these fictitious substances. for example, found that 4% of his sample of high school students reported the use of the nonexisting drug bindro. they also found that those who reported the use of a nonexistent drug also reported more use of all other drugs included in their survey, compared to those who indicated, correctly, that they did not use bindro. others have reported similar findings when asking survey respondents about the use of nonexistent substances [202, 207209 ]. of course, it may be that heavy drug users just assume, incorrectly, that they have used all available substances at one time or another in their past. others have questioned whether or not it is correct to assume that all substance users will be hesitant to accurately report on their patterns of use. wish., for example, have suggested that heavy substance users may be less concerned about social and other consequences of reporting such information. interviews with persons receiving treatment, though, have found little interest in publicly discussing their patterns of use. concern with the accuracy of substance use reporting has led to a variety of attempts to validate or corroborate survey responses. for example, several panel surveys have demonstrated considerable stability in respondent reporting of substance use over time [22, 212, 213 ]. research, however, has also investigated the recanting of drug and alcohol use, which is the tendency of some panel survey respondents to claim no lifetime experience with a given substance, when they have previously reported having used it. recanting has been identified in responses to both alcohol and drug use questions [119121, 150, 201, 215220 ]. depending on the age group being surveyed (adults versus adolescents), recanting may represent deliberate efforts to deny previously reported activity, exaggerations regarding behaviors that never actually took place, poor comprehension of survey questions during at least one wave of interviews, poor recall of information, or simple carelessness when answering [200, 217 ]. research by martino. suggests that recanting is a consequence of both deliberate misreporting and errors in understanding of survey questions. in surveys of adolescents, one possible explanation for recanting is that younger and less mature respondents may be more likely to exaggerate substance use during surveys conducted in classroom settings in which peers might be aware of one another 's answers and that they may then provide more accurate answers during subsequent survey waves as they subsequently become more mature. interestingly, longitudinal follow - ups with monitoring and the future survey respondents have found that recanting is greater among adults with occupations that might be expected to strongly sanction the use of illicit substances, such as those associated with the military and law enforcement. have also documented increased recanting among adolescents who had received drug education during the study period, suggesting a potentially biasing effect of education on self - reports. higher recanting among low level substance users has also been reported [201, 216 ]. other research has sought to validate self - reported substance use behavior by comparing those reports to toxicological findings from biospecimens collected at the time that interviews are conducted. one of the first studies conducted with a community sample (in chicago) by fendrich. indicated that recent cocaine and heroin use estimates obtained from hair testing were considerably higher than were self - reports obtained from the same respondents. a follow - up survey found that higher rates of cocaine and heroin were obtained from drug assays of hair, saliva, and urine samples, compared to self - reports from respondents to a community survey. a higher estimate of marijuana use, though, was derived from self - reports, compared to drug test assays, a finding that was interpreted as evidence of the limitations of hair testing for the detection of marijuana use. similar findings of underreporting of cocaine and heroin have also been obtained from general population surveys conducted in puerto rico by coln and colleagues [223, 224 ] and of men who have sex with men in chicago. another study conducted as part of the nsduh investigated agreement between self - reported use of marijuana and cocaine and urine tests concluded that most youths aged 12 to 17 and young adults aged 18 to 25 reported their recent drug use accurately examined the association between hair testing and self - reported illicit drug use, concluding that agreement between tests and self - reports to be substantial for marijuana and cocaine, moderate for opiates, and fair for methamphetamines. other research has employed urinalysis and hair assays to document drug use frequency underreporting among drug users receiving treatment. while providing valuable insights, it is important to acknowledge that each of these sources of confirmatory biological information is also imperfect measure of substance use, suffering from a variety of limitations, including imprecise and variable detection windows, vulnerability to contamination, and individual and race / ethnic group variability in rates of chemical absorption and retention [229, 230 ]. another approach to validating self - reports of substance use is to compare information obtained from respondents with those of significant others, a strategy that has found good but far from perfect levels of corroboration [202, 208, 231233 ]. parents and children have also been asked to corroborate one another 's reports of alcohol use. in a dutch study, engels. found that both children and parents underestimate one another 's alcohol consumption to some extent and that underestimation of adolescent alcohol consumption by parents was related to lack of knowledge and control of their children 's activities. an important caveat when employing this approach is that proxy and self - reports generally suffer from the same sources of error. interestingly, perceptions of untrustworthiness by others have also been found to be associated with drug use recanting among adolescents in a study reported by weinfurt and bush. an aggregate level strategy for evaluating self - reports of alcohol use is through comparisons of alcohol sales and tax information. a number of studies have taken this approach and have consistently found evidence suggestive that survey self - reports in some cases vastly underestimate total alcohol consumption [237240 ]. state - level estimates from self - reports, though, do correlate fairly strongly with the estimates from sales / tax data, suggesting sensitivity to variations in substance use behavior. one recent study that compared self - reports of alcohol purchases, rather than self - reported alcohol consumption, found much closer agreement between total estimates developed from those self - reports in comparison to total retail alcohol sales in sweden. interestingly, this study also found considerable variability by type of alcohol, with sales of wine far more accurately reported than beer and spirits, suggesting the possibility that social desirability concerns may be at least partially responsible, given that wine is likely viewed as a more socially desirable alcoholic beverage, at least in the swedish context. reporting of wine consumption was also found to be more complete in a canadian study. one strategy designed to provide respondents with greater privacy when speaking with interviewers about highly sensitive questions such as substance use behavior is the randomized response technique, first proposed by warner. goodstadt and grusin found higher drug use reporting for five of six substances among high school students in ontario. weissman. compared substance use self - reports obtained with and without the use of the randomized response technique during telephone interviews conducted as part of a general household survey in new york city and also found increased reporting for three of four substances when using the randomized response technique. an important drawback noted, though, was that only 52% of those randomly assigned to respond using this technique actually agreed to do so. in contrast, mcauliffe. reported no differences in reports of illicit drug use among those responding via the randomized response technique, compared to those answering direct questions. some limitations of this technique include the challenge of correctly administering it in practice and its ability to provide aggregate estimates only. the bogus pipeline is another approach that has been employed in attempts to induce more accurate reporting of substance use behavior. this involves the ethically questionable practice of leading respondents to believe that their questionnaire responses will be validated using some alternative means, when in fact the investigator has no intention of doing so. rather, the implied threat of validating respondent answers is used to exert pressure on respondents to answer more truthfully. in general, however, the use of the bogus pipeline procedure has failed to obtain higher estimates of substance use behavior, at least among adolescents [247249 ]. a meta - analysis has confirmed the nonefficacy of the bogus pipeline procedure for improved reporting of alcohol consumption and marijuana use., did however demonstrate the effectiveness of the bogus pipeline technique for increasing respondent reporting of sensitive behaviors, including alcohol and illicit drug use. in addition, a special population study has suggested that the bogus pipeline procedure may be successful in improving self - reports under certain conditions. lowe. found that, among pregnant women, those randomly assigned to a bogus pipeline condition were nearly twice as likely to report alcohol consumption when completing a self - administered questionnaire. finally, when considering respondent related reporting errors, it is highly likely that multiple sources of respondent related reporting errors are operating simultaneously. for example, johnson and fendrich demonstrated, using latent measures of cognitive processing difficulties constructed using debriefing probes, that social desirability concerns were predictive of discordant drug use reporting and drug use underreporting, while memory difficulties were predictive of drug use overreporting. interviewers can introduce errors by misreading questions, failing to probe answers correctly, not following other elements of standardized survey protocols, and by deliberate falsification of survey interviews [254, 255 ]. interviewer affiliation with governmental agencies may also influence respondent willingness to report substance use behaviors. interestingly and somewhat counterintuitively, interviewers with no prior project - related experience have been found to generate higher levels of marijuana and cocaine reporting in a national substance use survey [130, 257 ]. research by chromy. also finds that more experienced interviewers achieve higher response rates, in addition to eliciting fewer reports of substance use behaviors, suggesting they may be more successful in gaining cooperation from nonsubstance users who might find a survey on this topic to be less personally salient or interesting, although they do not believe that this fully accounts for the observed differences, which remain unaccounted for. it is possible that the social distance between respondents and interviewers may influence respondent willingness to report sensitive behaviors such as substance use. johnson and colleagues found that adult respondents in a telephone survey regarding substance use treatment needs in illinois were more likely to report recent and lifetime drug use when respondent - interviewer dyads were characterized as having relatively little social distance. in that study, social distance was measured using a simple count of the number of shared demographic identities (i.e., same gender, same race / ethnicity, similar age, and similar educational attainment). johnson and colleagues also explored the effects of social distance between race / ethnic groups in a study in which they probed respondents regarding how comfortable or uncomfortable they would feel when interviewed about their alcohol consumption patterns by interviewers from the same and from other cultural groups. when asked how they would feel if interviewed by an interviewer with the same background, large majorities of african american (88.8%), mexican american (74.7%), puerto rican (85.9%), and non - hispanic white (92.9%) respondents indicated they would feel comfortable. however, when asked how they would feel if the interviewer asking about their alcohol use was from another cultural group, the proportions indicating they would continue to feel comfortable decreased to 60.0% of african americans and mexican americans and 69.4% of puerto ricans. among non - hispanic whites, though, the proportion indicating they would continue to be comfortable remained very high (89.3%), suggesting group differences in reactions to interviewers of similar versus different race / ethnic backgrounds. other research has also examined the effects on substance use reporting of similarities and differences in various demographic characteristics between interviewers and respondents. in studies conducted in iowa many years ago, female respondents were more likely to report alcohol consumption to male interviewers, and conversely, male respondents were more likely to report alcohol use to female interviewers. johnson and parsons found that homeless respondents were more likely to report drug use to male interviewers, a finding that they linked to a likely user hypothesis that suggests that male interviewers were more likely to elicit positive substance use reports because their gender is perceived as being more likely to be substance users themselves and more tolerant of substance use by others. in contrast, a study conducted by darrow and colleagues reported that gay males were more likely to report drug use to female interviewers, who were viewed as having greater empathy and sympathy for deviant behavior than would male interviewers. in a survey conducted in the netherlands, higher rates of alcohol use were reported by turkish and moroccan respondents to dutch interviewers, compared to interviewers who were ethnically matched. these researchers also hypothesized that minority respondents may have either (a) exaggerated their alcohol consumption to comply with the perceived norms of the person interviewing them or (b) underreported, or denied altogether, the use of alcohol when interviewed by interviewers from an islamic background who would have been perceived as having a far less permissive opinion of alcohol use. this limited evidence does not suggest a clear pattern of effects of any interviewer characteristics on respondent self - reports of substance use behaviors, although it does seem likely that interviewer characteristics do matter in many situations. interviewer - respondent familiarity with one another may also influence the quality of self - reported substance use behaviors. for example, mensch and kandel found that, in the panel survey of national longitudinal survey of youth, marijuana use reporting was lower among respondents who had been interviewed more times previously by the same interviewer, suggesting that interviewer familiarity cued respondents regarding social desirability expectations, which depressed their drug use reporting. ironically, again, the use of experienced survey interviewers, something that would typically be considered an important strength of any study, would appear in some circumstances to be a factor contributing to lower quality data, at least when interviewers are serially assigned to the same subsets of respondents. various aspects of the social and physical environment within which survey data are collected may also influence the quality of the information collected. one aspect of the social environment that has received attention is the absence or presence of other individuals during the interview, as this is believed to influence the social desirability demands or pressures that respondents may perceive. in general, the presence of others during survey interviews is known to be associated with lower reporting of sensitive behaviors, including substance use. in an early study, wilson noted that, when interviews were conducted in the presence of another person, average weekly alcohol consumption was lower, compared to interviews conducted in private. several studies of adolescent reporting of alcohol and drug use also found that the presence of a parent during a household interview reduces respondent willingness to report such behaviors [127, 265268 ]. in contrast, hoyt and chaloupka also reported that the presence of friends during an interview increased substance use reporting, and aquilino. reported that the presence of a spouse or significant other had no effect on reports of alcohol and drug use. it is important to recognize, though, some potential confounding, as those most likely to have another person present during an interview are those who are married, and those who have children, and these variables are also commonly associated with less substance use behavior. the physical context within which interviews take place may also influence social desirability pressures and self - report quality. much of this evidence comes from comparisons of adolescent survey responses when the surveys are completed at home versus in a school setting. in school settings, parental monitoring is likely to be perceived as less of a concern and confidentiality assurance likely to be more credible. others [21, 269271 ] have reported that adolescents will underreport substance use during household surveys, relative to school - based surveys. needle and colleagues and zanes and matsoukas, though, did not find differences in the reports obtained from students in school- versus home - based settings. once data collection is complete, the construction of a final survey data set requires the implementation of numerous coding and editing rules. the integrity of these rules is particularly critical in substance use surveys, as they typically involve assumptions about the reporting intentions and substance use behaviors of respondents. fendrich and johnson have documented important differences in the editing assumptions made across national surveys of substance use in the us that can substantially influence the prevalence estimates generated by each. investigators also use a variety of techniques to screen completed substance use questionnaires for inclusion in final data files. farrell and colleagues examined the effects of excluding respondents (1) who provided a large number of inconsistent answers and (2) who reported use of a fictitious substance. the effects of excluding these responses on prevalence estimates were considered to be minimal, although they cautioned that exclusionary criteria should be used carefully in order to avoid producing nonrepresentative results. also, a past report by the us general accounting office identified imputation problems in the national household survey on drug abuse in which the estimated number of past year heroin users in the us ranged from 232,000 to 701,000, as a consequence of whether missing data imputation procedures were or were not used. the same report also indicated that sample weights used to construct subgroup estimates of the total number of illicit drug users were in some instances based on extremely small numbers of individuals in some weighting cells who reported current drug use. in one case from the 1991 nhsda, a single 79-year - old woman was projected to represent approximately 142,000 persons believed to have used heroin during the previous year. in such instances, a single erroneous data entry could be expected to have dramatic effects on overall survey estimates. inferential errors can be avoided by insuring that the survey questions being employed and the respondents being sampled are representative of the constructs and populations to which the researcher plans to make inferences. to the degree that either the measures or sample fail to represent their intended objects, experimental findings are considered the strongest evidence for internal validity, and representative samples provide the strongest evidence for external validity. when research designs deviate from these ideals or measures do not adequately assess the constructs of interest, there is a risk of inferential errors that will limit the generalizability of empirical findings. in substance use research some are a consequence of erroneously concluding that associations between constructs do not exist, due to poor measures and/or research designs. others involve falsely concluding that associations do exist between constructs when they in fact do not, also as a consequence of inadequate designs and/or measures. the failure to properly adjust a high quality substance use survey for its stratified sample design, discussed earlier in section 3.4, is an example of an adjustment error that led to a serious inferential error when investigators erroneously concluded that a large fraction of all alcohol sales in the us were being made to underage minors. over several decades, considerable knowledge has been accumulated regarding sources of error in the survey assessment of substance use behaviors. below, i highlight some of the most important questions that i see relevant to each source of survey errors that have been considered in this paper. regarding coverage errors, the challenge of constructing representative sample frames for both adolescents and adults continues to increase as electronic communications platforms further diversify. this is a general problem that afflicts all survey research efforts but one that can be particularly problematic for substance use research given the associations between these behaviors and likelihood of being covered by many of the potential sources of sample frames. identification of supplemental frames that might provide better coverage of heavy substance users and which could be employed, with appropriate weights, as supplements to more traditional sample frames when conducting population surveys should be considered. when survey estimates are reported, sampling errors, in the form of standard errors or confidence intervals, are commonly included. although reporting these errors is important to survey transparency, it is important to recognize that sampling errors make strong assumptions that can seldom be met in practice. most importantly, they assume the absence of all other sources of survey error. given the unlikeliness of this assumption, merely reporting sampling errors can leave survey consumers with a false sense of the precision of survey estimates, as any sampling errors could be completely overwhelmed by any measurement and/or nonresponse errors, for example, in practice. understanding how sampling errors in substance use surveys may be influenced by other sources of survey error thus seems to be an important research question to be addressed in the future. nonresponse errors seem to be another permanent concern that substance use surveys will need to continually address. of course, the degree to which nonresponse may bias survey findings will vary from topic to topic and question to question. given the strong associations detected between substance use and nonresponse patterns, it appears that this error source is also particularly relevant for surveys on this topic. an important issue for additional research is the relative usefulness for substance use surveys of the various nonresponse bias analytic strategies reviewed earlier in this paper. similarly, research into the relative efficacy of various types of adjustments for nonresponse and other forms of error in substance use surveys would seem to be an important future research topic. in general, there has been little research into specification errors in substance use surveys. this is an oversight, given general acknowledgment that researchers and potential respondents do not always have a shared understanding of the behaviors being examined. development of strategies for identifying and investigating potential errors of specification is another research topic in need of attention. it is my personal opinion that the multiple sources of measurement errors reviewed earlier in this paper pose the greatest threat to the accurate assessment of substance use behaviors. there are several practical questions that remain unresolved, such as the predictive power of social desirability measures, the reasons why experienced interviewers appear to obtain fewer reports of substance use behaviors, and the degree to which adolescents might actually overreport their use of alcohol and/or other drugs. perhaps even more important, how these widely diverse sets of measurement errors interact with one another is poorly understood and remains largely unexamined. evaluation of how various sources of measurement errors in substance use surveys interact together to influence survey estimates should be a priority for future research. in terms of processing errors, surveys concerned with substance use would appear on the surface to be no more vulnerable than other types of survey research. yet, the complexity of most substance use questionnaires, combined with greater item nonresponse rates in many instances, likely provide greater risks for processing errors that can be linked to complex editing rules and assumptions. a general rule of thumb is that the likelihood of experiencing processing errors is inversely associated with the amount of documentation provided with a survey, as careful documentation is an important indicator of quality research. continued research into the veracity of data editing decision rules, particularly when handling missing data and/or inconsistent self - reports in substance use surveys, would certainly be welcomed. as with all other sources of survey related error, inferential errors are not unique to substance use surveys. they are in general a product of poor study design and execution that can seriously limit the value of otherwise commendable efforts. study findings take on additional credibility and are accorded stronger inference to the degree that they can be replicated in subsequent investigations. substance use researchers should seek opportunities to replicate findings from other researchers when conducting their own original studies. and journal editors can provide additional service to science by finding ways to make space available for publishing replication studies that are essential to addressing problems of inferential errors that may otherwise go undetected. it is important to note that the review presented in this paper was not based on a systematic database search. rather, it is based on the author 's personal familiarity with and experience working with this literature over the past several decades. finally, it is strongly recommended that substance use researchers who plan to employ survey research methods recognize and report on their efforts to address each of the potential sources of survey related error discussed in this paper. developing strategies to systematically and rigorously confront each source of errors and transparently sharing one 's successes and failures remains the best approach to minimizing the effects of each when using survey methods to investigate substance use patterns and behaviors. | population - based estimates of substance use patterns have been regularly reported now for several decades. concerns with the quality of the survey methodologies employed to produce those estimates date back almost as far. those concerns have led to a considerable body of research specifically focused on understanding the nature and consequences of survey - based errors in substance use epidemiology. this paper reviews and summarizes that empirical research by organizing it within a total survey error model framework that considers multiple types of representation and measurement errors. gaps in our knowledge of error sources in substance use surveys and areas needing future research are also identified. |
one category of amyotrophic lateral sclerosis (als) occurring in 7.59% of the total incidence of als is associated with biological abnormalities including monoclonal gammopathy of unknown significance. monoclonal protein igm (immunoglobulin m) with anti - mag (myelin - associated glycoprotein) antibodies may be associated with a sensory polyneuropathy. the onset is insidious with numbness and paresthesia predominant in the lower limbs with possible symmetrical weakness and wasting in later stages. an electroneuromyogram (emg) in anti - mag neuropathy demonstrates a demyelinating or mixed axonal demyelinating pattern. peripheral sensory neuropathy is not usually recognized as a feature of als [5, 6, 7 ]. the present exceptional case involving als, waldenstrm macroglobulinemia and anti - mag gammopathy presents an opportunity to discuss the potential relations between als and anti - mag antibodies. a 71-year - old woman with no relevant past medical history presented with insidious onset of dysarthria and slurred speech. she developed weakness in the left hand and after 12 months neurological examination demonstrated diffuse asymmetrical wasting and weakness of the upper limbs, with a slight predominance on the right side. these signs were present to a lesser degree in the lower limbs, associated with fasciculations only in the forearms and in the tongue. emg was in accordance with als criteria but there was reduced amplitude of distal sensory nerve action potentials in the upper and lower limbs (table 1). serum creatine kinase level was increased : 1,237 u / l (normal : 50145). g / l (normal : 0.552.10), with normal iga and igg proteins. igm protein demonstrated high anti - mag activity at 76,200 btu (normal : < 1,000). anti - gm2 activity was at a 1/50 level (normal : < 1/100), without anti - gm1 activity. bone marrow biopsy examination revealed an infiltration by a mixture of small lymphocytes, plasma cells and plasmacytoid lymphocytes of igm type, suggesting the diagnosis of waldenstrm macroglobulinemia. the evolution was marked by asymmetrical global weakness, mainly distal and right - sided, aphagia, and sialorrhea. according to recommendations, 4 weeks treatment with rituximab (375 mg / m / week for a body surface of 1.3 m) and chlorambucil (6 mg / m for 42 days) was administered without significant effect. after 17 months, physical examination revealed symmetrical upper and lower limb weakness, with no additional sensory complaints. in spite of no sensory clinical outcome, 5 months after the first emg there was aggravation of sensory signs with severe reduction of amplitude in the upper and lower limbs for all sensory responses. the patient died 3 months later from aspiration pneumonia, 20 months after symptom onset. the lesions consisted of severe loss of anterior horn neuronal cells, with atrophic corticospinal tracts from cervical to lumbar levels and preservation of the posterior columns. neuronal loss was observed in the motor nuclei of the brainstem, particularly in the hypoglossal nucleus which contained chromatolytic cells, and to a lesser degree in the vestibular nucleus. rare ubiquitin - positive round or skein - like inclusions were observed in the spinal cord (fig. 1b, c) and brainstem, whereas the supratentorial structures were negative, except for the dentate gyrus where rare neuronal intracytoplasmic inclusions could be observed (fig. neither neuronophagia nor lymphocytic cuffing were observed in the spinal cord, and no lesion suggestive of lymphoma was found. immunodetection of inflammatory cells using anti - cd3, cd5, cd20, cd68 and cd138 antibodies was entirely negative. we did observe neither significant decrease in the myelinated fiber density nor widenings of the myelinated lamellae as previously described in anti - mag neuropathy. ubiquitin inclusions in the dentate gyrus are the marker of als with dementia, but in our case these inclusions were rare in accordance with the absence of objective cognitive disorder. the emg examination for sensory nerve conduction can be abnormal, suggesting a coexisting disease, as in our patient. paraproteinemia was reported in 7.59% of als cases compared with 1% in the general population. in this category of als with paraproteinemia and monoclonal gammopathies, anti - gm1 antibodies are found in 90% of cases and anti - mag antibodies are exceptionally reported [5, 11, 12 ]. among 5 patients with sporadic als and sensory neuropathy of unknown cause, 4 had clinical and 5 electrophysiological sensory signs, and only 1 case had a serum igm paraprotein, but anti - mag antibodies were not reported. high titer of anti - mag antibodies, as in our case, has been reported in some waldenstrm macroglobulinemia cases. the association of anti - mag antibodies with waldenstrm macroglobulinemia and motor neuron disease was only reported in 1 case but with borderline titer. the argument suggesting a neuropathy in the present case is the severe reduction of sensory features on the second emg, 5 months after the previous one (table 1). then, in the present case, we raise questions about the possible associations between neuropathy, als and anti - mag antibodies. sensory involvement (electrophysiological or polyneuropathy) has been found in 1020% of als cases. among 88 als patients, another study reported 3 cases associating als with clinical and emg chronic inflammatory demyelination polyradiculoneuropathy., there is only 1 case fulfilling both criteria for als and peripheral sensory anti - mag neuropathy, but neuropathological examination was not performed. initial symptoms were distal paresthesias (unlike in our patient) and rapidly progressive weakness in all four limbs. clinical examination revealed mildly decreased sensation of vibration and reduced sensation of warmth in the legs and hands and only patellar reflexes were found (unlike in our patient). the possible discussion about the relations between als and anti - mag antibodies relies on the immune mechanisms reported in some als cases. several pathogenic mechanisms which may contribute to motor neuron injury and lead to death have been identified in als. oxidative stress, genetic factors, glutamatergic toxicity and mitochondrial dysfunction are well known [14, 15 ]. there are also arguments suggesting that cells of the immune system are implicated in the pathogenesis or propagation of als. microglia, which play a major role in inflammatory cascades, have been proven to be activated in spinal ventral horn neurons in als. otherwise, inflammatory markers such as activated macrophages, mast cells and t cells have been reported in spinal cord and brain in als. anti - mag neuropathy is the consequence of antibodies that react with myelin - associated glycoprotein via a complement - mediated mechanism. the very high titer of anti - mag antibodies, as in the present case, could have been considered an argument for a causative link, but absence of correlation between anti - mag titer and severity of neuropathy has been reported. in fact, several arguments suggest there was no relation between neuropathy and the anti - mag antibodies, because (i) there were no demyelinating neuropathy symptoms, (ii) there was no significant decrease of conduction velocities and (iii) no suggestive neuropathological features. moreover, 6675% of anti - mag neuropathies respond to rituximab therapy after 1 year of treatment but no responses were observed in patients with als and sensory involvement [5, 6, 11 ]. our case is original because of the association between als, waldenstrm macroglobulinemia and high titer of anti - mag antibodies. our investigations including neuropathological examination demonstrate that there was no argument for a causative link between anti - mag antibodies and als. | we report the case of a 71-year - old woman with typical signs of bulbar amyotrophic lateral sclerosis (als) associated with immunoglobulin m (igm) monoclonal gammopathy and anti - mag (myelin - associated glycoprotein) antibodies. this unusual association between als and anti - mag antibodies has previously been reported in a single case. our present case, at neuropathological examination, demonstrated no causative link between anti - mag antibodies and als. |
as all of these events occur during early postnatal life, often a causal relationship is assumed, resulting in widespread use of anti - reflux medications to reduce the occurrence of cardiorespiratory events. recent documentation has shown that 25% of all extremely low birth weight infants are discharged on promotility and/or antacid therapy 1 with one of the most common indicators being apnea, bradycardia and oxygen desaturation 2. pharmacologic treatment for acid suppression and gastric motility have been associated with a wide range of adverse consequences 3,4, including late - onset sepsis 5, necrotizing entercolitis 6,7, respiratory infections 8, dystonia and irritability 4. hence, characterization of a relationship between cardiorespiratory events and ger is imperative in order to address the risk benefit ratio of this treatment modality. early studies finding no relationship between apnea and ger have been limited by the use of a ph probe, allowing only for the detection of acid ger. with frequent feeds and resultant non - acid refluxate, use of a ph probe more recent studies have employed multiple intraluminal impedance (mii)for detection of both acid and non - acid ger, again with conflicting results 1113,15,16. guidelines for detection of ger by mii 17 alone have shown that mii underestimates the number of acid ger events 18. as limitations of using only ph or mii in detecting ger may account for discrepancies between studies, the aim of this study was to characterize cardiorespiratory events in preterm infants following both acid and non - acid ger as detected by ph and mii. twelve hour overnight studies were performed in preterm infants who were referred for cardiorespiratory and ger overnight monitoring by the attending physician. inclusion criteria included infants 34 wks gestational age at birth and a post menstrual age of less than 44 weeks at the time of study. exclusion criteria included clinically significant congenital malformations and need for ventilator support (mechanical ventilation or cpap) at the time of study. each overnight study consisted of monitoring of respiration via respiratory inductance plethymsography (somnostar pt, cardinal health, yorba linda, ca) which provides an estimate of flow and volume, heart rate and oxygen saturation (masimo radical set, masimo, irvine, ca). sao2 data were acquired with pulse oximeter monitor settings of 2 second averaging and 2 second sample rate. the ph probe measures changes in esophageal acidity, whereas the mii probe detects the presence of liquid in the esophagus. a 6.4 french catheter was placed in the esophagus between t7 and t9 and verified by chest radiograph. the impedance catheter contained 7 electrodes placed 1.2 cm apart corresponding to a total of 6 channels. a ph electrode was located 1 cm from the tip of the catheter corresponding to impedance channel six. ger events were defined by mii as a fall in impedance to 50% of the baseline on 2 or more sequential channels followed by stratification as acid with a ph 12% of all cardiorespiratory events preceded by ger. the first infant had 1 of 7 cardiorespiratory events preceded by ger which included an apnea of 11 seconds in duration. all were oxygen desaturation events ranging from 7884% and were accompanied by respiratory pauses of 12% of all cardiorespiratory events preceded by ger. the first infant had 1 of 7 cardiorespiratory events preceded by ger which included an apnea of 11 seconds in duration. all were oxygen desaturation events ranging from 7884% and were accompanied by respiratory pauses of 12%of cardiorespiratory events preceded by ger, all of short duration and relatively low severity, casts considerable doubt on the effectiveness of anti - reflux medication to treat apnea of prematurity. criticism of studies that have found no association between ger and cardiorespiratory events has been the limitation in detection of ger in infants. although mii technology has improved the detection of non - acid ger, it is also has limitations in its ability to detect acid ger 18. detection of both acid and non - acid events has been maximized in this study by documentation of ger by both mii and ph technologies. by combining these modes of ger detection, 9% of all ger events this rate may occur by chance due to the high incidence of both ger and cardiorespiratory events detected in this study. however, as apnea has been associated with a reduction in lower esophageal sphincter tone 21, it is possible that cardiorespiratory events trigger some ger 23. although 656 mixed apnea and 43 obstructive apnea were detected, a limitation of this study is the possibility that the use of respiratory inductance plethysmography may have missed short intermittent obstructive events. rip has been found to have good agreement with the gold standard of nasal mask pneumotachograph 24 and discrepancies in apnea detection have been shown between all additional modes of respiratory monitoring 25. although it is possible that a limited number of short intermittent obstructive apnea may have gone undetected, laryngeal stimulation, the hypothesized mechanism by which ger initiates apnea, has been predominantly associated with central apnea in piglets 26. in addition, the majority of apnea associated with ger in infants have also been found to be central in nature 11. therefore, it is unlikely that clinically meaningful obstructive events were missed in this study and, if a respiratory event was undetected, any associated changes in heart rate or saturation would have been recorded under the scoring criteria for bradycardia and/or desaturation. this was not a random sample of infants as only infants referred for cardiorespiratory or ger monitoring by the attending physician were included in this study. this infant cohort may not represent the general population and may be at higher risk for persistent apnea, a high incidence of ger and corresponding increased chance of ger being associated with a cardiorespiratory event. even in this at risk cohort the association of these events was rare. the mean post menstrual age of 37 weeks may have limited the ability of this study to apply to younger convalescing preterm infants. however, previous findings in infants of 33 weeks post menstrual age were unable to show a temporal relationship between cardiorespiratory events and ger15. in conclusion, less than 3% of all cardiorespiratory events were preceded by ger with a median of 1.4% per infant. the findings of this study have important implications pertaining to the administration of anti - reflux medications for the treatment of apnea, bradycardia and desaturation. if every cardiorespiratory event preceded by ger in this study could be resolved by anti - reflux medications, this would only decrease the incidence of cardiorespiratory events by 3%. given the modest impact in this best case scenario, and the potential side effects of pharmacologic therapy, careful consideration should be given to the risk - benefit ratio of anti - reflux medications before administration of these treatments. | objectiveto characterize cardiorespiratory events in preterm infants following both acid and non - acid ger as detected by ph and multiple intraluminal impedance (mii).study designtwelve hour overnight studies were performed in 71 preterm infants (gestational age 29.43.0 wks, birth weight 1319496 gm). apnea 10 seconds in duration, bradycardia 80 bpm and oxygen desaturation 85% that occurred within 30 seconds after the initiation of ger were classified as associated with ger.result12,957 cardiorespiratory events and 4164 ger episodes were documented. less than 3% of all cardiorespiratory events were preceded by ger constituting 3.4% of apnea, 2.8% of oxygen desaturation and 2.9% of bradycardia events. ger did not prolong cardiorespiratory event duration or increase severity. in contrast, ger was associated with a shorter duration of oxygen desaturation events (7.84.6 vs 6.35.6 sec, p<.05).conclusionger is rarely associated with cardiorespiratory events, and has no detrimental effect on cardiorespiratory event duration or severity. |
the loss of tooth enamel and dentin may occur due to various pathologies such as caries, tooth wear, and trauma. in addition to dental caries, acidic beverages also cause demineralization of tooth enamel leading to tooth wear in the form of erosion. dental erosion is the loss of dental hard tissues by chemical process without the involvement of cariogenic bacteria. several investigations have been conducted to evaluate the effects of aerated drinks and citrus juices on dental hard tissues. dental enamel is a crystalline lattice of various minerals the principle component of which is hydroxyapatite. saliva contains supersaturated solution of calcium and phosphate which maintains the ph of the oral environment. the shift of saliva ph to a critically low value (5 or low) either due to citreous drinks or due to cariogenic results in the demineralization of tooth enamel and leaching of mineral ions. the important factors responsible for the balance of remineralization and demineralization are salivary ph, buffering capacity, and salivary flow rate, with ph and buffering capacity increases as flow rate increases. saliva plays an important role by neutralizing the acids and providing calcium and phosphate ions to aid in remineralization. aerated drinks such as colas (ph = 2.6) and noncolas (ph = 3.5) lower the ph of oral environment thus resulting in leaching out of calcium and phosphate ions from the enamel because of the dissolution of hydroxyapatite crystals. since the introduction of adhesive resin composites in 1955 although the performance of resin composites has improved by incorporation of nanomaterials, the long - term success depends on the bond strength of these materials to tooth tissues. the acidic treatment (from carbonated beverages or any other source) results in loss of minerals from enamel and alters the surface properties compromising the bond strength. it has been hypothesized that teeth exposed to carbonated drinks will often yield lower bond strength corresponding to demineralization of enamel. this study is aimed to establish the microtensile bond strength of enamel following exposure to an aerated drink at various time intervals with / without application of remineralization agent. in addition, degree of remineralization and demineralization of tooth enamel has been assessed using polarized light microscopy. the current study was conducted using seventy permanent first premolars freshly extracted during orthodontic treatment. all teeth were cleared of debris ultrasonically and polished for 30 s using nonfluoridated and oil - free pumice slurry. each tooth was sectioned into two identical halves longitudinally in buccolingually direction using a microtome. each tooth section was treated with sand paper (600 grit ; a-797455 ; foredom 's blackstone industries bethel, ct 06801) to get homogeneously uniform experimental surfaces. all specimens were treated with chilled (57c) carbonated soft drink (coca - cola ; atlanta, ga 30301, usa) for 5 min followed by storage in deionized distilled water (ph, 6.5;37c). prepared samples were randomly divided into following study groups [figure 1 ] : remineralizing group (n = 70) : specimens treated for remineralization using casein phosphopeptides and amorphous calcium phosphate (cpp - acp) remineralization agent (recaldent ; gc europe)control group (n = 70) : no remineralization treatment ; specimens were kept in artificial saliva. remineralizing group (n = 70) : specimens treated for remineralization using casein phosphopeptides and amorphous calcium phosphate (cpp - acp) remineralization agent (recaldent ; gc europe) control group (n = 70) : no remineralization treatment ; specimens were kept in artificial saliva. study groups and sample distribution for surface characterization of dental hard tissues for the remineralization group, cpp - acp was applied to all specimens (3 min every day) following manufacturer 's instructions. all specimens were tested for microtensile bond strength at regular intervals (1 h, 1 day, 2 days, 1 week, and 2 weeks) using the protocol described below. briefly, the labial surface of all specimens was etched using 37% orthophosphoric acid (total etch, ivoclar vivadent, schaan) for 15 s followed by washing and air drying. the adhesive bonding agent (prime and bond nt, dentsply, usa) was applied and cured for 20 s using a qth lamp (astralis, ivoclar vivadent, schaan). an attachment was built (4 mm diameter) on etched enamel surface using a resin composite (filtek z350 ; 3 m espe) and cured for 20 s. the prepared specimens were used to measure the microtensile bonding strength. universal mechanical tester (controls groups ; milan, italy) was used to test the tensile bonding strength. specimens were trimmed using a hard tissue microtome and a custom made jig with specific slot dimensions (1 mm 2 mm 5 mm). the tensile testing was performed by moving the crosshead at an acceleration of 0.5 mm / min until the failure of specimen. the extent of demineralization and remineralization was observed using a polarized light microscope (omano om349p, omano, uk). the data was collected and analyzed statistically using spss computer software (version 22, ibm corporation, new york, usa). the results were statistically evaluated using a dependent variable (tensile bond strength) and two - way anova test ; p 0.05). in contrast, the bond strength of cpp - acp group was significantly increased at day 2, week 1, and week 2 compared to control group (p < 0.05). these findings suggested that remineralizing tooth tissues using cpp - acp may improve the bonding strength of restorative dental materials. polaroid microscopic images showing effects of various surface treatments (a) buccolingual section of premolar surface enamel, inner enamel and dentin (b) treatment of enamel specimen with carbonated drink for 5 min (c) tooth specimen from casein phosphopeptides and amorphous calcium phosphate group assessed 2 days after remineralization treatment ; penetration of casein phosphopeptides and amorphous calcium phosphate in enamel and corresponding changes (arrows) microtensile bond strength (mpa) with / without application of remineralizing agents (meanse) we have determined the effect of carbonated beverage on microtensile bond strength of enamel as function of time. the tensile bond strength was improved in response to both saliva and cpp - acp ; however, cpp - acp treatment showed better results compared to saliva regardless of time interval. the structural integrity of the dental hard tissues (enamel / dentin) depends on the hierarchy and dynamic balance between demineralization and remineralization. saliva is a dynamic medium that is supersaturated with minerals. under physiological conditions, saliva has the ability to remineralize tooth using bio - available calcium and phosphates. the action of salivary phosphor - proteins (statherin) causes precipitation of calcium and phosphorus salt. aerated drinks contain acids (carbonic acid, citric / phosphoric acid) that are known to cause dental erosion. these acids are next stronger to battery acids with a ph of 2.52 and strong enough to demineralize enamel. in the previous investigations, the influence of time interval in relation to microtensile bond strength after exposure to an aerated drink reynolds showed that exposure of inset enamel plaque to solution containing tryptic peptides of casein remarkably reduced the enamel subsurface demineralization using a caries model in situ. the cpp - acp is a sticky protein that binds calcium / phosphorus and maintains the amorphous state. cpp stabilizes the nanoclusters of amorphous calcium / phosphate in the meta - stable solution. it contains the sequence : ser (p)-ser (p)-ser (p)-glu - glu ; pse is phosphoryl residue that stabilizes calcium / phosphorus ions in aqueous solution, hence confirming their bioavailability. the cpp - acp is a well proven remineralizing agent. the alkaline cpp - acp when added to sports drinks decreases the acidity and erosive effects. the role of fluoride in inhibiting tooth demineralization and prevention of caries is well known. the fluoride incorporated cpp - acp complex are also available where acp has been replaced by amorphous calcium - fluoride - phosphate. this fluoride incorporated cpp - acp may have additional benefits of releasing fluoride in the oral cavity and anticarcinogenic properties. microtensile bond strength testing gives more accurate values than conventional tensile strength because the small size of the specimens allows better stress distribution during the test loading. the lower variance of data results could be attributed to the reduction in flow density at the interface. in the present study, it was found that samples treated with cpp - acp have shown better bond strength at all - time intervals. the decreased bond strength of samples not treated by cpp - acp can be linked to the increased demineralization due to acidic drinks as well as etching agent. it is well - known that increasing the etching time leads to reduction in bond strength. the bond strength of cpp - acp applied teeth after 2 days following exposure to an aerated drink was equal to 7 days without application of remineralizing agent. the 1-week bond strength of teeth that were not exposed to remineralizing agent was statistically similar to that of 2 days bond strength achieved with cpp - acp treated teeth. there are a few limitations ; this study was conducted in in vitro and the experimental conditions did not reflect the dynamic conditions of oral environment. a number of factors (temperature, ph, saliva chemistry, food interaction, presence of microorganism) may interfere the remineralization process and bond strength. this study has provided the baseline results hinting that demineralization treatment before adhesive restorations has the potentials to improve the bond strength. however, in vivo and clinical studies are required to validate these claims. the mineralization ability of saliva is not enough to cope with the dimerization caused by aerated drinks. the rapid remineralization of enamel in response to remineralizing agent increased the bond strength significantly at a shorter interval compared to saliva. in clinical prospects, the remineralizing agents can be applied on adherent tooth surface to improve the bond strength. | objective : this study is aimed to establish the microtensile bond strength of enamel following exposure to an aerated drink at various time intervals with / without application of remineralization agent. in addition, degree of remineralization and demineralization of tooth enamel has been assessed using polarized light microscopy.materials and methods : seventy extracted human incisors split into two halves were immersed in aerated beverage (cola drink) for 5 min and stored in saliva until the time of microtensile bond testing. prepared specimens were divided randomly into two study groups ; remineralizing group (n = 70) : specimens were treated for remineralization using casein phosphopeptides and amorphous calcium phosphate (cpp - acp) remineralization agent (recaldent ; gc europe) and control group (n = 70) : no remineralization treatment ; specimens were kept in artificial saliva. all specimens were tested for microtensile bond strength at regular intervals (1 h, 1 days, 2 days, 1 week, and 2 weeks) using a universal testing machine. the results statistically analyzed (p = 0.05) using two - way anova test.results:results showed statistically significant increase in bond strength in cpp - acp tested group (p < 0.05) at all - time intervals. the bond strength of remineralizing group samples at 2 days (~13.64 megapascals [mpa ]) is comparable to that of control group after 1 week (~12.44 mpa).conclusions : cpp - acp treatment of teeth exposed to an aerated drink provided significant increase in bond strength at a shorter interval compared to teeth exposed to saliva alone. |
new composites or hybrid materials were recently developed, based on the association of imidazolium moieties with an inorganic part, like metallic silver, tin oxide, or mainly silica. these materials were designed in order to take advantage of the highly interesting features of imidazolium moieties and, thus, were developed for numerous applications like catalysis, anion exchange, selective gas trapping,(12) drug delivery,(6) or electrochemistry.(5) self - organization was observed in some genuine imidazolium moieties previously : this organization was first evidenced for imidazolium compounds possessing long alkyl chains, which are ionic liquid crystals. self - organization of short - chain imidazolium units by means of stacking of the imidazolium aromatic rings was also observed to a much lower extent. in numerous of the developed composite materials, or in chemical - bonded imidazolium units, the confinement of the imidazolium moieties within the pores of a matrix forced, and thus enhanced strongly, the stacking of the imidazolium aromatic rings. recently we published the synthesis of new hybrid materials, ionic silica nanoparticles networks, which will hereafter be referred as isnn. in those hybrid materials silica nanoparticles the imidazolium unit formation was performed through nucleophilic substitution between the nanoparticle grafted ligands in a the covalent grafting of the ligands onto the nanoparticle surface, the formation of the imidazolium unit to link the nanoparticles, was evidenced by combining solid - state nmr, dynamic light scattering, and thermogravimetric analysis.(21) in a second stage, to get a better picture of the network extension, small - angle x - ray scattering (saxs) experiments were carried out. the main result was the observation of short - range order in the hybrid materials. in light of this observation and of the above cited literature, we put forward the hypothesis that the imidazolium aromatic rings bridging the silica nanoparticles were the driving force for this short - range order, as probably organized by stacking of the imidazolium aromatic rings.(33) in this paper, this hypothesis is further confirmed with the aid of first photoluminescence results obtained on various isnn. 1,4-bis(chloromethyl)benzene, sodium tetrafluoroborate (nabf4), potassium hexafluoroborate (kpf6), and lithium bis(n - trifluoromethylsulfonyl)imide (lin(so2cf3)2) were obtained from aldrich. saxs was performed using a rotating anode generator equipped with a pinhole camera (nanostar from bruker axs with cu k radiation from crossed gbel mirrors). the x - ray patterns were recorded with an area detector (vantec 2000) and radially averaged to obtain the scattering intensity in dependence on the scattering vector q = (4/) sin, with 2 being the scattering angle and = 0.1542 nm the x - ray wavelength. fluorescence spectra were recorded at 25 c with a horiba jobin yvon fluoromax-4 spectrofluorometer with a xenon arc lamp as excitation source and a photomultiplier tube as detector. samples were measured, after grinding, in a solid state sample holder in an angle of 60 to the excitation beam to minimize stray light influence. quantum yield measurements were carried out on an edinburgh instruments spectrometer (fsp920) with a xenon arc lamp, double grating monochromators, and a photomultiplier tube equipped with a barium sulfate coated integrating sphere (150 mm internal diameter). all spectra were recorded at room temperature with excitation and emission slit width of 3.0 nm. the dwell time was of 0.5 s, and the recording steps were 0.2 nm. the fluorescence yield was calculated by dividing the number of emitted photons by the number of absorbed excitation photons. the number of absorbed photons was determined from the decrease of the scattered excitation light intensity compared to the intensity measured with an empty suprasil glass cuvette. the spectra were corrected for system - specific effects such as the detector sensitivity, monochromator efficiency, and baso4 coating. the spectral distribution of the lamp intensity was corrected using a si photodiode reference detector. digital photos were made with a 5.0 megapixels canon digital ixus 50, on the mode photo macro with the highest resolution. in previous works we described the following : the syntheses of silica nanoparticles, the surface functionalization of the silica nanoparticles with chloropropyltrimethoxysilane or n-(3-propyltrimethoxysilane)imidazole, and their reaction leading to the compound im / cl.(21) ; the anion exchange reaction on im / cl to obtain im / pf6, im / bf4, or im / n(so2cf3)2;(34) the reaction of n-(3-propyltrimethoxysilane)imidazole modified silica nanoparticles with 1,6-dichlorohexane which led to the product im / cl / hex / im / cl.(33) the synthesis is carried out under argon atmosphere. in a 100 ml round - bottom flask, 1.88 g (10.7 mmol) of 1,4-bis(chloromethyl)benzene is added at once to 48 ml of a methanol suspension of silica nanoparticles modified by means of 4.95 ml (21.5 mmol) of n-(3-propyltrimethoxysilane)imidazole. the solvent is then removed under reduced pressure (3 mbar), and the crude product is washed twice with 20 ml of deionized water and ethanol, respectively, and dried in a desiccator over p2o5. a white powder is obtained, which will be labeled as im / cl / benz / im / cl. the starting compound im / cl / benz / im / cl was dispersed in 20 ml of acetone. the salt for the exchange, nabf4, kpf6, or lin(so2cf3)2, was added in a mass ratio of 1:1. then the products are collected by centrifugation and washed with acetone, deionized water, and ethanol, 40 ml each. white powders are obtained, which will be labeled as im / bf4/benz / im / bf4, im / pf6/benz / im / pf6, and im / n(so2cf3)2/benz / im / n(so2cf3)2. recently published saxs investigations concerning ionic silica nanoparticle networks have highlighted a short - range order in the issn due to a peak in the scattering intensity in the range of the scattering vector q of about 4 nm.(33) in figure 1 the saxs intensities for seven different silica nanoparticle - based hybrid materials are shown, in the region from q = 2 to 7 nm. the compound im / cl corresponds to bridging ligand containing one imidazolium chloride unit, im / cl / hex / im / cl and im / cl / benz / im / cl are corresponding to bridging ligands containing two imidazolium chloride units with a hexyl chain (hex) or a benzene (benz) group in between (table 1). in the case of im / cl the peak maximum was observed at 5 nm, whereas the peak maximum for im / cl / benz / im / cl is located at 3.3 nm (figure 1, table 1), which corresponds to a longer distance of the objects in real space in comparison with im / cl. the maximum of the scattering peak characteristic for short - range order in the isnn hybrid material, observed in figure 1 and reported in table 1, can be related to the length of the ligand introduced to connect the silica nanoparticles. the peak maximum in reciprocal space from the saxs curves decreases ; i.e., the distance in real space increases in accordance with the increasing length of the ligands. this directly confirms the successful bridging of the ligands in the synthesis of the material. given that the scattering peak is directly related to the length of the nanoparticles bridging ligands, the maximum of the scattering peak did not shift when the chloride anion was exchanged by metathesis reaction toward hexafluorophosphate, tetrafluoroborate, or bis(trifluoromethylsulfonyl)imide (figure 1, table 1).(34) scattering intensities show a short - range order peak for various isnn hybrid materials. the short - range order of the hybrid material was shown to be dependent on the rigidity of the bridging ligand.(33) the results obtained for the considered isnn hybrid materials indicate a stronger short - range order for the im / cl / benz / im / cl samples, with rigid bridging units, in comparison to the im / cl materials, whereas the ordering in im / cl / hex / im / cl, with a highly flexible bridging unit, was much weaker as in im / cl (figure 1). the saxs experiments clearly indicated that the exchange of the chloride anion did not influence the short - range order of the hybrid materials (figure 1 and table 1). the diffraction peak intensities are a signature for the extent of short - range order, in other words, the higher the diffraction peak intensity, the stronger the short - range order in the material. the excitation spectra of the hybrid materials im / cl and im / cl / benz / im / cl are characterized by a broad band in the region 320360 nm with maxima at 340 and 350 nm, respectively (figure 2b). in the case of the hybrid materials im / cl / benz / im / cl, a second maximum can be distinguished in the excitation spectrum at 300 nm ; this second maximum seems also to be present in the case of im / cl, but to a lower extent. the origin of the second maximum in the excitation spectra can not be assigned at this stage ; however the presence of different absorption and fluorescence maxima due to various associated species was already observed in a previous work devoted to fluorescence phenomena in imidazolium ionic liquids.(35) blue emission is observed for the samples when excited at the wavelength of maximum adsorption. at an excitation wavelength of 340 nm, the hybrid material im / cl emits with a maximum at 390 nm, while im / cl / benz / im / cl presents an emission maximum at 410 nm after excitation at 350 mn (figure 2a). it can be noted that the emission spectra of the isnn hybrid materials remain unchanged when excited at 300 nm, while the intensities are much lower (figure 2c). (b) excitation spectra of im / cl and im / cl / benz / im / cl. (c) emission spectra of im / cl / benz / im / cl for various excitation wavelengths. in aromatic - containing compounds, fluorescence phenomena can often be observed as a consequence of the transition of the excited state to the energy level. conjugation length refers to the imidazolium ring stacking, which occurred when networking the silica nanoparticles.(33) this interpretation is supported by previous work concerning photoluminescence investigations of imidazolium based ionic liquids and hybrid materials containing those.(7) the self - organization of the aromatic heterocycle under constraint conditions, like confinement or in the present case due to covalent linking to nanoparticles surface, was already reported and characterized by means of raman, saxs or infrared spectroscopy investigations. this self - arrangement tendency was also highlighted by luminescence experiments in the case of silica - confined imidazolium ionic liquids. the authors pointed out two main features of the emission spectra of confined imidazolium, which were also observed in our isnn hybrid materials based on covalently bonded imidazolium units. the first observation concerns the excitation wavelength for genuine ionic liquids. for those imidazolium compounds, which are not submitted to any constraint, the wavelength corresponding to the maximal emission is strongly dependent on the excitation wavelength. however in the case of the isnn the emission maximum is constant, at 410 nm (figure 2c). the second highly interesting feature, when comparing genuine imidazolium salts and their corresponding hybrid materials, is an enhanced fluorescence. the first measurements indicated, in conformity with the observations reported in the literature, weak luminescence for the genuine imidazolium salts ; whereas once confined or in the present case covalently bridging nanoparticles the fluorescence is strongly enhanced (figure 2a ; table 2). both observations were explained by the fact that in pure imidazolium salts numerous different lengths for the imidazolium stacking are simultaneously present, while in isnn, as well as in confined in silica matrixes, this stacking length is much more homogeneous over the whole studied sample.(7) aggregation - induced emission was also already reported for materials derived from triphenylethylene,(47) as well as in zn based metal oxide frameworks containing bipyridinedicarboxylate,(48) where the aggregation - induced stacking between the aromatic rings originated in strongly enhanced emission. considering the emission spectra of the two isnn hybrid materials im / cl and im / cl / benz / im / cl, a clear shift of the emissions toward longer wavelengths can be observed ; the maxima are at 390 and 410 nm, respectively (figure 2a). a red - shifted luminescence is typical for the presence of extensive stacking between adjacent aromatic rings. it has to be noted that the bridging unit in im / cl / benz / im / cl is not fully conjugated ; the presence of the ch2 groups between the imidazolium and benzene units hinders the conjugation between the aromatic groups (table 1). as a consequence, increased stacking can only originate if the ligands in the isnn hybrid materials exhibit a stronger short - range order. the observation of almost no emission for the sample im / cl / hex / im / cl (figure 2a) speaks also for a minimum short - range order of the ligands as criterion for an increased fluorescence. indeed, the saxs measurements on im / cl / hex / im / cl revealed a very weak ordering (figure 1, dots). we interpret this weak ordering, and as a consequence the very weak fluorescence, by the flexibility of the dichlorohexane which can backbite on the same nanoparticle instead of bridging two different silica nanoparticles.(33) this backbite phenomenon hinders the stacking between adjacent ligands. im / cl / benz / im / cl im / cl / hex / im / cl. the very low value obtained for im / cl / hex / im / cl, can be explained by the fact that the material is so poorly organized, due to the flexible hexane linker, that only very few stacking is possible. the difference in the quantum yield values measured for im / cl and im / cl / benz / im / cl, 0.13 and 0.26, respectively, are in accordance with the results published by carlos. indicating first that the morphology of the hybrid materials influences the quantum yield values and second that a too high degree of organization induced a decrease in the emission quantum yield. the difference in the degree of organization of those two hybrid materials was given by the extent of short - range order from saxs measurements (the peak intensities in figure 1), i.e., im / cl / benz / im / cl > im / cl. the observation of simultaneously : the necessity of a high degree of molecular order from molecular self - assembly for becoming an efficient energy transfer and a decrease of the quantum yield value when self - aggregation is too pronounced were already reported in the case of organic nanoparticles formed upon self - assembly of an anthracene and a fluorophore.(56) in addition to the above - discussed results, measurements of isnn hybrid materials with various anions, obtained by metathesis reactions, revealed that the excitation and emission wavelengths of the hybrid materials after anion exchange are not modified (figure 3) ; also it did not influence the short - range order of the ligands as was visible from saxs experiments (figure 1). this leads to the conclusion that the imidazolium bridging units are responsible for both the short - range order and the photoluminescence of the hybrid material independently of the anion. however, by exchanging the anion, the quantum yield values of some materials varied (table 2). given that for those the saxs curves remained unchanged, thus the short - range order within the material, it seems that some anions could interfere as luminescence quenchers. this observation will constitute one of the starting points for future investigations of this class of hybrid materials. emission and excitation (insert) spectra of (top) im / x and (bottom) im / x / benz / im / x hybrid materials. the excitation wavelength for the emission spectra was the wavelength of maximal absorption in the excitation spectra. comparison of the quantum yield values of the hybrid materials with those of genuine ionic liquids highlights the strong luminescence enhancement obtained owing to the self - organization force induced by the presence and the linking to silica nanoparticles. the isnn hybrid materials present high quantum yields for metal - free solid - state hybrid materials. an exception has to be made for the hybrid material im / cl / hex / im / cl where the coordination to the silica nanoparticles is not sufficient to induce self - organization of the material, the hexane ligand being too flexible. in summary, the photoluminescence investigations of ionic silica nanoparticle networks indicated that the imidazolium bridging units present similar features as imidazolium moieties confined in an inorganic matrix. this results from a self - organization of the aromatic imidazolium rings by stacking. due to this self - organization, a strongly enhanced fluorescence can be observed in comparison with neat imidazolium salts. for the hybrid materials quantum yields of 0.26 can be reached while the quantum yield of neat 1-butyl-3-methylimidazolium ionic liquids is 0.050.07. additionally by varying the imidazolium containing bridging ligand, through introduction of additional aromatic rings, while maintaining a not - full conjugation in the bridging units, the photoluminescence emission maximum wavelength was shifted toward longer wavelengths, i.e., lower energy, which is in accordance with a stronger stacking. the stability of the system is confirmed by saxs experiments, where the short - range order peak was correlated to the length of the bridging chain and remains unchanged even after anion exchange. these results, in combination with saxs measurements, confirmed our previous results suggesting a short - range order in the materials originating from stacking interactions. some open questions have remained, e.g., the influence of the anion exchange on the quantum yield of the hybrid material, and will be investigated further. | recently we published the synthesis of new hybrid materials, ionic silica nanoparticles networks (isnn), made of silica nanoparticles covalently connected by organic bridging ligands containing imidazolium units owing to a click - chemistry - like reaction. among other techniques small - angle x - ray scattering (saxs) experiments were carried out to get a better picture of the network extension. it turned out that the short - range order in isnn materials was strongly influenced by the rigidity of the bridging ligand, while the position of the short - range order peaks confirmed the successful linking of the bridging ligands. the photoluminescence experiments reported in this communication revealed strongly enhanced emission in the hybrid material in comparison with neat imidazolium salts. moreover the shift of the emission maximum toward longer wavelengths, obtained when varying the aromatic ring content of the bridging ligand, suggested the existence of strong stacking in the hybrid material. experiments revealed a stronger luminescence in those samples exhibiting the higher extent of short - range order in saxs. |
anterior palatal fistula or oronasal fistula is the most common complication of cleft palate repair, the incidence ranging from 4% to 35%. palatal fistulae can be classified according to their size as small (5 mm). the common symptoms include hypernasality of speech due to nasal escape of air and leakage of fluid and food into the nasal cavity leading to poor oral hygiene and foul smell. the various treatment options available are - local tissue flaps, revision palatoplasty, regional flaps - such as buccal mucosal flaps, pharyngeal flaps, tongue flaps (anteriorly or posteriorly based), microvascular free tissue transfer (radial forearm flap) and lastly prosthetic rehabilitation. the aim and objective of this study were to analyse the utility of tongue flap in anterior palatal fistula repair. the analysis of 41 previously operated cases of cleft lip and palate with residual anterior palatal fistula, which reported to our centre between the period of 2006 - 2012, were done. out of which outside operated were 32 and 9 were from those operated at our hospital. the following parameters were taken into the consideration taste sensation, swallowing reflex, functional deformity of the tongue, cessation of regurgitation of foods / fluids from nose. the nasal lining was repaired with turn - over flaps from the fistula edges [figures 1 and 2 ] and the tongue flaps provided oral lining [figure 3 ]. length of flap was adjusted long enough to fill anteroposterior dimensions of fistula as measured with the help of a pattern and an additional 1 cm to allow smooth turning of flap, but not exceeding beyond the circumvallate papillae [figure 4 ]. the width of the flap varied according to the fistula size but did not exceed more than 2/3 the tongue width [figure 5 ]. flaps were raised in a fashion that up to 5 - 7 mm thickness of the muscle depth was taken in all cases to protect the underlying submucosal plexus. donor site closure was done primarily with chromic catgut ; care was taken not to close it too tight near the pedicle so as not to hamper the vascularity of the flap [figure 6 ]. proper edge to edge approximation of the flap margins to the mucoperiosteal margins the tongue flaps were routinely divided after 21 days postoperatively [figure 8 ], except in one patient wherein the detachment and insetting had to be done on the 9 postoperative day because of bleeding from one of the edges. preoperative intraoral view of the anterior palatal fistula and after paring of the cleft edges after the repair of the nasal lining by local turn down flaps tongue flap in situ providing the oral lining base of flap lies beneath the posterior border of fistula when tongue is in neutral position facilitated by the extra 1 cm. length of flap than the ap dimension of the fistula tongue flap design, not exceeding beyond circumvallate papillae primary closure of donor site maintaining the vascularity of the flap pedicle, width of flap up to 2/3 width of tongue, depth of flap incorporating 5 - 7 mm thickness of the muscle good edge to edge approximation of tongue flap to the mucoperiosteal margins using mattress sutures tongue flap after detachment and inset after the necessary debulking the patients were followed - up for 3 months postoperatively, and all the findings were recorded and analysed [figure 9 ]. the males comprised 66% and females 34% of the total cases [pie chart 1 ]. the distribution among the unilateral and bilateral cleft lip and palate cases is as depicted [pie chart 2 ]. the size of the fistula varied from 2 cm 1.5 cm to 5.5 cm 3 cm with majority being in the range, 2 - 2.5 cm [graph 2 ]. age range of the patients in whom tongue flap was done sex distribution of the fistulae distribution of anterior palatal fistulae among unilateral and bilateral cleft cases anterior palatal fistula size range the complications noted were bleeding in one case and flap dehiscence in one case, both the flaps survived with no residual fistula. none of the cases had interference with functioning of the tongue [figure 10 ]. cessation of regurgitation of food / fluids from nose was 100% and improvement in the nasality of speech was observed in 75% cases. tongue flap has been a work horse for difficult palatal fistulae with shortage of tissue. the use of the lingual flap for repair of hard palate fistulae was first reported by guerrero - santos and altamirano. the rich vascular supply from the lingual artery and its four branches and the extensive anastomotic network with the contralateral side contributes to the versatility of the tongue flap. good amount of tissue available from the tongue can be used for effectively closing even large palatal fistulae. success depends upon proper flap elevation, tension free nasal layer closure, edge to edge approximation of the flap with palatal tissues and not too tight closure of the donor area near the base of the flap. while raising the flap one should not take more than 5 - 7 mm. thickness of the muscle in order to avoid a bulky flap which may cause difficulty in swallowing and may also cause articulation problems later. all our patients had the flap flush with the adjacent palatal tissues, and none of the patients complained of any difficulty due to bulk. tension free closure of the nasal layer is possible only if nasal layer is widely undermined and raised. in order to have proper apposition of margins mattress sutures are useful. all the patients had cessation of nasal regurgitation and improvement in the nasality of speech was observed in 75% of the cases. the tongue flap is safe and well - tolerated by children, and there is no need to put naso - gastric tube for feeding. in our study, 17% population were children below 5 years of age. in order to restrict tongue movement this was not done in any of our patients and we feel this is not required. too much of the tongue movement is automatically restricted because of the pain associated with it. flap division has been done by various authors varying from 10 to 21 days. in our series, we chose to do flap division after a period of 3-week except in one case where we had to do division and inset per force on the 9 postoperative day because of bleeding. the patients, usually from far off areas, are generally discharged on the 5 postoperative day and it is convenient for them to come back after a gap of 3-week after surgery. in the case of any marginal necrosis wounds will usually heal well by that time and division can be carried out safely. since, while doing insetting, one has to do some flap thinning and may have to raise part of the already healed flap, 3-week period makes it safer. while child is being anaesthetized, it is always a good idea for the surgeon to be ready to divide the flap immediately under local anaesthesia in case there is any difficulty in intubation. though we have always kept ourselves ready, there never has been an occasion when our anaesthesiologist was unable to intubate. this is probably because of the experienced anaesthesiologist being always available for such a case. there is no impairment of speech or movement, and there may only be temporary loss of tongue sensation and taste. the tongue flap in addition can also be used to close the alveolar fistula along with anterior palatal fistulae [figure 11 ]. large tongue flap used to close the cleft alveolus in addition to the anterior palatal fistula tongue flap remains the flap of choice for managing anterior palatal fistulae, leaving apart its only drawback of two - staged procedure and transient patient discomfort. anteriorly based tongue flap is a safe and dependable procedure and gives consistently good results in closure of anterior palatal fistulae. | introduction : despite the improved techniques of repair of cleft palate, fistula occurrence is still a possibility either due to an error in the surgical technique or due to the poor tissue quality of the patient. though commonly the fistula closure is established by use of local flaps but at times the site and the size of the fistula make use of local flaps for its repair a remote possibility. the use of tongue flaps because of the central position in the floor of the mouth, mobility and the diversity of positioning the flaps make it a method of choice for closure of anterior palatal fistulae than any other tissues. the aim of this study was to analyse the utility of tongue flap in anterior palatal fistula repair.materials and methods : we had 41 patients admitted to our hospital during the period 2006 - 2012 for repair of palatal fistula and were enrolled into the study. in the entire 41 cases, fistula was placed anteriorly. the size of the fistulae varied from 2 cm 1.5 cm to 5.5 cm 3 cm. the flaps were divided after 3-week and final inset of the flap was done.observation and result : none of the patients developed flap necrosis, in one case there was the dehiscence of the flap, which was reinset and in one patient there was bleeding. none of our patients developed functional deformity of the tongue. speech was improved in 75% cases.conclusion:leaving apart its only drawback of two - staged procedure and transient patient discomfort, tongue flap remains the flap of choice for managing very difficult and challenging anterior palatal fistulae. |
an i. javanica isolate, which was isolated from a fungus - infected aleyrodidae adult in korea and showed high pathogenicity against the b. tabaci biotype q, was kept in 10% (v / v) glycerol at -20. the isolate was initially cultivated on potato dextrose agar (pda) at 25 1 for 14 days. for the mass production of this i. javanica isolate, brown rice and barley were purchased and stored in a refrigerator at 4 until use. to use grains as a substrate for conidia production, the 2 grains were soaked in tap water for 2 hr and placed into a plastic basket for 1 hr to remove water. one hundred grams of grain was put into an autoclavable plastic bag and autoclaved at 121 for 60 min. to understand the effects of different additives on conidia production by solid - state cultivation using grains as a substrate, a carbon or nitrogen source or trace minerals were added into 15ml conidia suspension (1 10 conidia / ml) and dissolved. the additives investigated were as follows : 2% (w / v) soluble starch (daejung co., busan, korea), 2% (w / v) mannose (sigma - aldrich co., st. louis, mo, usa), 2% (w / v) caco3 and 2% (w / v) caso4 (sigma - aldrich co.), 1.32% and 3% (w / v) of hydrolyzed casein (sigma - aldrich co.), 1.32% and 3% (w / v) of difco yeast extract (becton dickinson co., franklin lakes, nj, usa), 0.6% and 1% (w / v) of kno3 (sigma - aldrich co.), and 1.32% and 3% (w / v) of hydrolyzed wheat gluten protein (sigma - aldrich co.). the conidia suspension was then inoculated onto barley or brown rice in a plastic bag, mixed well, and incubated at 25 1 for 15 days. the grain was added to 27 ml 0.05% sterilized tween 80, vortexed for 1 min, and then the suspension was filtered through 4 layers of sterilized cheesecloth. the number of conidia was counted using an improved neubauer haemocytometer (sigma - aldrich co.). in each trial, each cultivation condition was replicated in 3 different bags, and the trial was repeated 4 times. to test the virulence of conidia produced using solid substrates and different additives, each conidia suspension was sprayed onto second instar nymphs of tobacco whitefly, as previously described. briefly, to prepare synchronized nymphs, fifteen pairs of adult whiteflies were infested overnight on a potted eggplant seedling with 1 leaf, which was prepared by removing the other leaves from a 30-day - old pruned whole eggplant seedling, and then the adult whiteflies were removed. the seedlings were grown in a plexiglas cage (35 40 40 cm) at a constant temperature of 25 1 with a 16l : 8d lighting schedule for 11 days until the whiteflies reached their second instar. for virulence tests, conidia were harvested as described above from the barley or brown rice cultures amended with different additives after 15 days cultivation. the eggplant leaf discs infested with second instar nymphs of b. tabaci were sprayed with each conidia suspension at 3 different concentrations (10, 10, and 10 conidia / ml). one milliliter of each suspension was applied to leaf discs using a spray box (90 90 90 cm) fitted with a polyvinyl acetal cone nozzle (1.5 mm). the spray tower was a plexiglas cylinder and the spray nozzle was installed through the top layer and connected to a vacuum pump, which was fixed at the pressure setting of 100 kpa. the number of conidia applied to each leaf disc was estimated by counting the spores that landed on a petri dish (3.5 cm) containing 2 ml of 0.05% tween 80 that was placed beside the eggplant seedlings during the spore application. the total number of conidia sprayed within the petri dish area was determined using a hemocytometer. the average conidia deposition on each leaf disc was calculated to be 2.8~3.2 conidia / mm for the application of 10 conidia / ml and 10.7~13.6 conidia / mm for the application of 10 conidia / ml. spore viability was determined by germination tests of the conidia suspensions after 24 hr incubation at 25 1 on pda medium. one - way analysis of variance was used to compare conidia production and mortality among the different additives used for cultivation on each grain substrate. median lethal time (lt50) was estimated using the lifereg procedure and data were fitted to a weibull distribution. the differences in conidia yield, mortality and lt50 at 2 different time points (4 and 6 days after treatment) among different additives in each grain were compared using the least significant difference test (< 0.05). the production of i. javanica conidia differed among the substrate and additive combinations investigated after 15 days cultivation (brown rice, f = 8.48 ; df = 11, 47 ; p < 0.0001 and barley, f = 3.78 ; df = 11, 47 ; p = 0.0012). barley (3.43 10 conidia / g) was a higher - yielding solid substrate for conidia production than brown rice (3.05 10 conidia / g). on barley, the addition of yeast extract, casein, or gluten to barley improved conidia production compared with cultivation on barley without additives. when starch or mannose was mixed with barley as the solid substrate, the number of conidia produced was lower (2.33 10 and 2.15 10 conidia / g, respectively) than the number produced by cultivation without additives (3.43 10 conidia / g). the addition of gluten (3% and 1.32%) to brown rice improved the conidia production yield by 14 and 6 times, respectively, compared with that of brown rice without additives (3.05 10 conidia / g). the addition of potassium nitrate (kno3), yeast extract, 2% caco3 and 2% caso4, or mannose to brown rice did not increase conidia production compared with cultivation on brown rice without additives. the addition of gluten stimulated conidia production on both solid substrates, but the addition of mannose and kno3 suppressed the production of conidia of the i. javanica isolate following cultivation on barley and brown rice. the mortality of sweet potato whitefly treated with the conidia suspension produced on barley was generally higher (13% at 10 conidia / ml, 36% at 10 conidia / ml, and 62% at 10 conidia / ml) than that of whitefly treated with the conidia suspension produced on brown rice (14%, 23%, and 53%, respectively) at 4 days after treatment (pooled t tests : 10 conidia / ml, t = 0.16, p = 0.8880 ; 10 conidia / ml, t = -0.55, p = 0.6374 ; 10 conidia / ml, t = -0.84, p = 0.4889). however, these changes were not significant, and conidia virulence showed non - significant trends toward differences between the solid substrates. differences in mortality at the sixth day after application showed trends similar to those on the fourth day, with mortality rates of 57%, 81%, and 100% for barley - cultivated conidia and 46%, 74%, and 100% for brown rice at the 3 concentrations of conidia applied, respectively. the inclusion of additives in the solid - state cultivation procedure changed conidia virulence (fig. conidia produced on barley with different additives did not statistically increase whitefly mortality at 10, 10, and 10 conidia / ml treatment (anova : 10 conidia / ml, f = 0.41, df = 11, 23, p = 0.9236 ; 10 conidia / ml, f = 1.04, df = 11, 23, p = 0.4681 ; 10 conidia / ml, f = 4.19, df = 11, 23, p = 0.0103). the median lethal time (lt50) was shorter for conidia produced with the addition of mannose and starch at 10 (f = 0.58 ; df = 11, 23 ; p = 0.8096), 3% gluten and 1% kno3 at 10 (f = 0.41 ; df = 11, 23 ; p = 0.9228), and 1.32% and 3% yeast extract, 0.6% and 1% kno3, and 1.32% and 3% gluten at 107 conidia / ml treatment (f = 30.80 ; df = 11, 23 ; p < 0.0001). mortality of whitefly treated with conidia that were cultivated on brown rice with additives was lower than that of the conidia produced on brown rice without additives although this difference was not significant (table 2). the lt50 differed among additives ; at 10 conidia / ml, the addition of 1.32% gluten and 1.32% casein resulted in lower lt50 values (6.2 days for both additives, compared with 6.3 days for substrate without additives) (f = 1.00 ; df = 11, 23 ; p = 0.4962), while at 10 conidia / ml, the addition of 0.6% and 1% kno3, 1.32% gluten, and 1.32% yeast extract resulted in lt50 values of 4.6 days, 4.4 days, 4.5 days, and 4.6 days, respectively, compared with 4.7 days for conidia produced by cultivation without additives (f = 0.50 ; df = 11, 23 ; p = 0.8716), and at 10 conidia / ml, the addition of 1.32% and 3% gluten, 0.6% and 1% kno3 and 3% yeast extract led to lt50 values of 3.1 days, 3.3 days, 3.5 days, 3.4 days, and 3.5 days, which were significantly decreased compared with the lt50 of 3.8 days for conidia produced by cultivation without additives (f = 11.73 ; df = 11, 23 ; p < 0.0001). the production yield and virulence of i. javanica conidia varied among different combinations of additives and solid substrates used for cultivation. gluten and kno3 addition commonly increased the conidia production and virulence of the korean isolate of i. javanica under the different conditions investigated. a p. fumosoroseus isolate produced more blastospores in liquid medium that was supplemented with casein or yeast extract than in unsupplemented medium. similarly, tobacco whitefly pathogenic p. fumosoroseus produced more blastospores in liquid culture media supplemented with glucose and casamino acid. the addition of casein, yeast extract, or corn gluten as nitrogen sources, and of glucose or casein as carbon sources into liquid culture media increased the production yield of p. fumosoroseus spores, and the spores also showed enhanced tolerance to dessication. casein and yeast extract supplementation commonly increased the production of p. fumosoroseus blastospores in different liquid media. while many studies have previously investigated the effects of different liquid media and additives on the production and virulence of entomopathogenic fungal spores, few studies p. fumosoroseus, which was isolated from the cadavers of lepidopteran caterpillars in india, showed higher conidia production when cultivated on sorghum (10.4 10 spores/100 g) than when cultivated on corn, rice, pearl millet, or wheat. sporulation of m. anisopliae was stimulated on agar medium by the addition of starch, mannose, or kno3. soluble starch and mannose were the most effective stimulators of m. anisopliae sporulation. in this study, when the i. javanica isolate was cultivated on brown rice or barley with the addition of starch or mannose, conidia production was decreased. conidia of m. anisopliae cultured in sabouraud dextrose agar with added kcl showed a decreased germination rate and the strength of spore attachment onto the host cuticle differed from that of conidia produced in liquid culture media. nomuraea rileyi produced more conidia when cultivated on wheat with added yeast extract than when cultivated on wheat with added brewer 's yeast or whole milk powder. rice was the best solid substrate for the production of v. lecanii spores, compared with sorghum, corn, ragi, and wheat. the sporulation of p. lilacinus, which was isolated from nematodes, was stimulated when it was cultivated on agar medium containing added mannose, sucrose, or starch. the addition of 2% caco3 and 2% caso4 into grain cultivation bags prevented grains from sticking together and thus provided an increased surface area for fungal growth. in our study, the i. javanica isolate showed good sporulation on barley. when the isolate was cultivated on barley with added caco3 plus caso4 or gluten, conidia production was significantly increased, but mannose, starch, or kno3 addition inhibited conidia production. thus, the use of additives for the solid phase cultivation of entomopathogenic fungi led to differences in the yields of conidia production. for the mass production of fungal conidia, each isolate needs to be studied to determine the optimal solid substrate and additive ingredient(s). spore attachment, germination, and the activity of enzymes, including proteases such as pr1, are important factors for fungal virulence. these characteristics may be influenced by spore production conditions such as nutritional ingredients in the cultivation media. the influence of additives such as casamino acid on the spore production and virulence of various fungal isolates has been reported to vary among different media. for example, a m. anisopliae isolate cultured in medium with added casamino acids showed increased 40% mortality against a. aegypti compared with the same isolate cultured in medium without added casamino acids. according to shah., m. anisopliae produced highly virulent conidia when cultivated in osmotic stress medium, and the conidia showed higher pr1 enzyme activity. the addition of yeast extract and peptone to cultivation media also increased the pr1 activity of m. anisopliae conidia, but the relationship between virulence and pr1 activity was not clear in that study. the addition of lecithin, collagen, or lactic acid into culture broth increased the production of m. anisopliae blastospores, but the virulence of the spores was similar or lower than that of spores produced in media without additives. when conidia of lecanicillium sp. were produced by cultivation in media with the addition of 2% glucose and sprayed at low concentration (10 conidia / ml), the infection rate of whitefly was higher than that of conidia produced by cultivation in media with the addition of maltose and peptone. however, at a higher dosage (10 conidia / ml), there was no significant difference between the infection rates of the conidia produced by cultivation with different additives. the addition of trace minerals such as kj, znso4, or kbr to agar medium increased the pathogenicity of b. bassiana conidia. for i. javanica, which is a sweet potato whitefly pathogen, the virulence of conidia was increased when the isolate was cultured with the addition of gluten on both brown rice and barley solid substrates. conidia cultivated on barley with added kno3 or yeast extract also showed improved mortality against tobacco whitefly, compared with conidia cultivated on barley without additives. we expected that when the isolate was cultured with additives, highly virulent conidia would be produced, allowing us to spray a low concentration of spores onto crops but still achieve a high control efficacy against whitefly. however, against our expectations, the virulence of the conidia was only improved at a high spraying concentration (10 conidia / ml). at spraying concentrations of 10 and 10 conidia / ml, the lt50 did not significantly differ among the conidia produced by cultivation on solid substrates with different additives., whose study found that the addition of glucose to cultivation media led to the production of v. lecanii conidia that produced a higher mortality rate in their host (t. vaporariorum). we will conduct future studies to investigate why virulence was increased by the addition of gluten, kno3, or yeast extract at the highest spraying concentration of conidia. in conclusion, the production of sweet potato whitefly pathogenic i. javanica conidia was increased on barley by the addition of caco3+caso4 and on brown rice by the addition of gluten. gluten addition also improved the virulence of conidia cultivated on both substrates. compared with conidia produced on barley without additives, conidia produced on barley with added kno3 or yeast extract also showed improved virulence against tobacco whitefly. | recently, the q biotype of tobacco whitefly has been recognized as the most hazardous strain of bemisia tabaci worldwide, because of its increased resistance to some insecticide groups. as an alternative control agent, we selected an isaria javanica isolate as a candidate for the development of a mycopesticide against the q biotype of sweet potato whitefly. to select optimal mass production media for solid - state fermentation, we compared the production yield and virulence of conidia between 2 substrates (barley and brown rice), and we also compared the effects of various additives on conidia production and virulence. barley was a better substrate for conidia production, producing 3.43 1010 conidia / g, compared with 3.05 1010 conidia / g for brown rice. the addition of 2% caco3 + 2% caso4 to barley significantly increased conidia production. addition of yeast extract, casein, or gluten also improved conidia production on barley. gluten addition (3% and 1.32%) to brown rice improved conidia production by 14 and 6 times, respectively, relative to brown rice without additives. conidia cultivated on barley produced a mortality rate of 62% in the sweet potato whitefly after 4-day treatment, compared with 53% for conidia cultivated on brown rice. the amendment of solid substrate cultivation with additives changed the virulence of the conidia produced ; the median lethal time (lt50) was shorter for conidia produced on barley and brown rice with added yeast extract (1.32% and 3%, respectively), kno3 (0.6% and 1%), or gluten (1.32% and 3%) compared with conidia produced on substrates without additives. |
ras signaling begins with the stimulation of a vast array of upstream receptors including receptor tyrosine kinases (rtks) of which the epidermal growth factor receptor (egfr) is perhaps most relevant to lung cancer. adaptor proteins (e.g., grb2) interact with the intracellular domain of activated egfr and in turn recruit guanine nucleotide exchange factors (gefs) such as son of sevenless (sos) to the cellular membrane where they can associate with ras to promote the exchange of gdp for gtp (figure 2). ras signaling is terminated upon the hydrolysis of gtp to gdp by the intrinsic gtpase activity of ras through the interaction with gtpase - activating proteins (gaps). cancer - causing mutations in ras drastically impair the gtpase activity, resulting in ras proteins that are locked in the active gtp - bound conformation, regardless of the upstream signal. in their active, gtp - bound conformations, the four ras proteins engage and activate a large number of downstream signaling pathways to varying degrees. signaling through these downstream pathways regulates diverse cellular responses, including proliferation, survival, and differentiation (figure 2). the canonical ras / raf / mek / erk pathway controls cellular proliferation by modulating the levels of many cell cycle regulators and is frequently hyperactivated in cancers. / pdk1/akt signaling, a pathway that is also commonly deregulated in many cancer types. ralgds and ralgds - like proteins and tumor invasion and metastasis - inducing protein 1 (tiam1) can also be activated by ras to control vesicle trafficking and cytoskeletal organization, respectively,. both ralgds and tiam1 have been shown to be required for ras - dependent tumor formation in a mouse skin cancer model,. many of these downstream signaling pathways are involved in feedback regulation and crosstalk that together further contribute to the complexity of the ras signaling network. the first 165 amino acids of ras proteins are nearly identical and include motifs responsible for the binding and hydrolysis of gtp, binding of downstream effectors, and interactions with gap and gef. the hypervariable domain at the carboxy - terminus, including the caax motif, contains sequences important for the post - translational modification and membrane targeting of ras proteins. the cysteine residue in the caax motif is a target for prenylation (i.e., farnesylation or geranylgeranylation). the cysteine residues (orange boxes) near the carboxy - termini of hras, nras, and kras4a are targets for palmitoylation. the poly - lysine track (green boxes) helps kras4b to associate with the membrane. lung cancer is a common form of cancer in men and women, and it is responsible for the highest number of cancer - related deaths globally. because of the high rate (> 50%) of late diagnosis, the 5-year overall survival rate of patients with lung cancer has improved very little over the past 3 decades, hovering around the 13%16% range. tobacco use is the most important risk factor for lung cancer, with 80% of cases arising in smokers. kras is the most commonly mutated member of the ras family in lung cancer, although hras and nras mutations have also been reported in a few cases. kras mutations predominantly occur in lung adenocarcinomas, the most common histological subtype of non - small cell lung cancer (nsclc), with frequencies ranging from 16% to 40% of the samples analyzed. kras mutations have also been observed at a low frequency in squamous cell carcinoma (another subtype of nsclc), but never in small cell lung cancer (sclc),. mutations predominantly occur at codon 12, occasionally at codon 13, and rarely at codon 61 of kras. lung adenocarcinomas are usually associated with tobacco smoking, and kras mutations have been found to occur at a higher frequency in tumors in smokers compared to those in non - smokers. in addition, mutations involving nucleotide transversions (i.e., g / c or g / t), which are known to be associated with exposure to tobacco smoke, are more common among smokers than non - smokers. indeed, the two most common kras mutations in nsclc, g12c (40%) and g12v (22%), arise from g / t transversions,. many studies have suggested that the presence of kras mutations in nsclc is associated with shortened survival and time to relapse, although some studies have reported a lack of association. the specific type of kras mutation may also provide information on disease aggressiveness or drug sensitivity. for example, the g12d mutation in nsclc has been associated with better prognosis compared to the g12v or g12r mutations. in contrast, the g12d and g13d mutations in human colorectal cancer (crc) have been associated with a decreased response to chemotherapy compared to other mutations at these codons. further research is needed to conclusively establish the relationship between kras mutation specificity and prognosis in nsclc. in contrast, there is compelling data demonstrating the usefulness of kras status as a marker for predicting therapeutic response. adjuvant treatment with cisplatin and vinorelbine has been found to increase the survival of nsclc patients with wild - type (wt) kras but not those with kras mutations in their lung tumors,. the presence of kras mutations is also associated with a lack of response to egfr inhibitors, not only for nsclc but also for human crc where kras is also frequently mutated. given that kras is downstream of egfr, it seems intuitive that egfr inhibition would have no impact on the activity of mutant kras. however, it is surprising that concurrent treatment of kras - mutant nsclcs with erlotinib and chemotherapy resulted in shortened overall survival and progression - free survival compared with chemotherapy alone. these observations underscore the complexity of kras biology and further emphasize the advantage of having molecular information available (e.g., kras mutational status) when deciding the appropriate course of treatment. activation of rtks by growth factors (gfs) creates intracellular docking sites for adaptor proteins (e.g., grb2 and shp2) that recruit gef to the membrane to interact with ras and promote the exchange of gdp for gtp. in the active gtp - bound conformation, ras engages and activates an array of downstream effector pathways to regulate many cellular responses. the ras signaling is terminated upon hydrolysis of the bound gtp by the intrinsic gtpase activity of ras with the help of gap. oncogenic mutations in ras lock the proteins in a constitutively active state, resulting in the deregulation of many cellular functions that together contribute to the cancer phenotype. the mouse is an invaluable in vivo model system that has been widely used to study the importance of genes and pathways in different physiologic and pathologic contexts. the extensive sequence homology together with the broad overlapping pattern of expression in mice and humans suggests a high degree of functional redundancy among the ras family members. however, studies using mice have shown that knockout of the kras locus results in embryonic lethality, whereas knockout of either hras or nras has no effect on embryonic development and welfare of adult mice. furthermore, genetic disruption involving exon 4a of kras results in viable and healthy mice that express only kras4b. these observations indicate that kras, namely kras4b, is developmentally essential with unique functions that can not be compensated by other ras family members. surprisingly, mice in which hras is inserted into the kras locus are viable despite completely lacking kras proteins. these data suggest that the role of kras in development is not related to unique kras protein functions, but rather involves regulatory properties that are specific to the kras locus. several mouse models of mutant kras - driven lung carcinogenesis have been widely used to study the role of kras in nsclc in vivo. one model involves chemical carcinogen exposure, resulting in lung tumors that almost invariably harbor activating mutations at codon 12 or 61 of kras. a second involves a transgenic strategy in which the mutant kras allele is incorporated into the genome at random locations and expression is induced by treating mice with doxycycline. the third employs a knock - in strategy that involves targeting the endogenous kras locus to generate a mutationally activated kras allele that is maintained in a latent state by inclusion of a stop cassette flanked by loxp elements,. removal of the stop cassette, either by spontaneous recombination in the kras model or by intranasal administration of an adenovirus containing cre recombinase in the kras model, results in mutant kras expression and the development of lung tumors. these mouse models have demonstrated that mutational activation of kras alone is sufficient for lung tumor development, suggesting that it is an early event during lung carcinogenesis. the role of kras during the early stages of lung carcinogenesis is further supported by studies in human nsclc showing that kras mutations can be detected in precancerous lesions. in addition, lung tumors induced by both carcinogen and genetic approaches stain positively for sp - c and negatively for cca / cc10,,, suggesting that they derive from the same cell lineage as human lung adenocarcinomas. however, the majority of mouse lung tumors are adenomas,,, which are thought to be adenocarcinoma precursors. progression from adenomas to adenocarcinomas in these mutant kras - driven mouse models of nsclc can be accelerated by loss of the p53 tumor suppressor gene. likewise, kras and p53 are mutated at similar frequencies in human lung adenocarcinomas and occur concurrently in many cases. while mutant kras is potently oncogenic, studies in the mouse have elegantly demonstrated that wt kras functions as a suppressor of this activity. mice with only one copy of kras were found to be more susceptible to carcinogen - induced lung carcinogenesis than mice with two copies of kras. these findings suggest that the remaining wt allele of kras in mice of the latter genotype inhibits lung tumor development. this was further confirmed by in vitro studies in which ectopic expression of wt kras attenuated the growth of cancer cells containing mutant kras. analyses of lung tumors from mutant kras - driven nsclc models have shown that chromosomal duplication involving the kras locus is the most common genetic event in these tumors that harbor kras mutations,. studies of human lung adenocarcinomas have also shown frequent kras copy number gains and corresponding increases in rna levels of kras. in addition, loss of heterozygosity spanning the kras locus has been observed in human lung tumors that relate with kras mutation and preferentially target the wt kras allele. the imbalance in favor of mutant kras in human and mouse lung tumors is consistent with the requirement of tumor cells to overcome the tumor suppressor effect of wt kras. inbred strains of mice exhibit differential susceptibility to lung carcinogenesis, and genetic crosses between different mouse strains have led to the identification of pulmonary adenoma susceptibility 1 (pas1) as a major locus regulating predisposition to mutant kras - driven lung cancer. of the genes located within pas1, kras emerged as the most likely candidate ; however, a lack of coding sequence variants in kras among the different strains of mice called into question the mechanisms through which kras would regulate lung cancer susceptibility. however, mice that are susceptible to lung carcinogenesis were found to express higher levels of kras compared with mice classified as resistant. we proposed that lung cancer susceptibility is regulated by the balance between the levels of mutant and wt kras, taking into consideration the respective oncogenic and suppressor functions of these alleles. consistent with this notion, kras mutations occur preferentially on the more highly expressed kras allele inherited from the more susceptible parent. in humans, a polymorphism in the 3-untranslated region of kras, which results in increased kras expression via interference of binding by let-7 microrna, is associated with an increased risk of developing nsclc. a number of additional genetic variants in the human kras gene have also been associated with the risk of developing lung adenocarcinomas,, and in some cases the susceptible allele is found to be expressed at relatively higher levels in normal lung tissues. however, many of these associations were not reproducible in later studies,. one possible explanation for the lack of consensus among human case - control studies may be the variations in frequency (16%40%) of kras mutations in different cohorts of patients with nsclc. therefore, it may be necessary to consider the somatic events that occur in tumors (i.e., kras mutational status) in association studies to identify lung cancer susceptibility genes. as a result of alternative splicing, the human and mouse kras loci encode two highly similar proteins, kras4a and kras4b, that are jointly affected by activating mutations commonly found in cancer (figure 1). while kras4b is ubiquitously expressed, albeit at varying levels across tissues, kras4a expression is tissue - specific and not essential for embryonic development, suggesting that kras4a has a minor role in kras biology. however, we have shown that mice lacking kras4a are highly resistant to carcinogen - induced lung tumor development. the requirement for kras4a in carcinogenesis is compatible with the observation that kras4a is expressed in the lung and a number of other tissues from which arising tumors frequently harbor kras mutations, namely the colon and the pancreas,. in the lung, kras4a is highly expressed in a subset of epithelial cells, which could potentially be the originating cells of nsclc. studies of the cellular origin of nsclc in the mouse have identified a number of candidates,, but their relationship to kras4a remains to be determined. nevertheless, the identification of kras4a as an essential component of mutant kras - driven lung tumors may have important implications for the design and development of kras - targeted therapeutics. the development of kras - targeting cancer therapy has proven to be a challenging endeavor. because cancer - causing mutations render kras oncogenic by impairing its gtpase activity, in contrast, a number of drugs have been designed to inhibit the post - translational modification of the ras proteins to prevent proper localization and function. however, farnesyl transferase inhibitors (ftis) failed to inhibit kras due to alternative prenylation by geranylgeranyl transferase (ggtase), and combined treatment with ftis and the more recently developed ggtase inhibitors have displayed unacceptable levels of off - target effects and toxicity,. an alternative strategy involving the use of rna interference (rnai) to deplete kras in cells has been shown to be effective in some human kras - mutant nsclc cell lines, demonstrating that some tumors harboring kras mutations remain highly dependent on oncogenic kras for survival. while sirna knockdown of gene expression is a promising strategy to treat such kras addicted tumors in the clinic, the lack of effective methods to deliver sirna to tumors has precluded development of such therapeutics. however, recent advances in the use of nanoparticles for systemic sirna delivery in humans may potentially help overcome this limitation. a great deal of effort has been placed on developing inhibitors of effector pathways downstream of ras with the understanding that inhibition of these pathways could counteract the activity of oncogenic ras. the raf / mek / erk pathway was the first ras effector pathway identified and the best characterized. activating mutations in braf have been identified in different human cancers, including lung cancer, but generally never together with ras mutations. braf - selective inhibitors that effectively block proliferation of braf - mutant cell lines have been developed but are surprisingly ineffective against ras - mutant cells. in fact, these inhibitors paradoxically potentiate raf / mek / erk signaling in ras - mutant cells, and may cause severe adverse effects when given to patients with ras - mutant cancers. indeed, a subset of patients with melanoma treated with braf inhibitors developed cutaneous squamous cell carcinomas that contained ras mutations. the mechanistic explanation for the paradoxical activation of raf / mek / erk in ras - mutant cells treated with braf inhibitors involves craf activation, suggesting that craf - selective inhibitors could potentially be effective against cancers driven by mutant ras. in support of this notion, studies in the mouse have shown that craf, rather than braf, is essential for the development of mutant kras - driven nsclc. recently, there has been growing interest in exploiting the concept of synthetic lethality to target kras - mutant cancer cells. this approach involves the selective killing of kras - mutant cancer cells through inhibition of a second protein. studies in the mouse identified a synthetic lethal interaction between mutant kras and cdk4, where cdk4 ablation caused lung cells expressing mutant kras to undergo senescence and prevented tumor growth. a number of rnai - based synthetic lethal screens in cell lines have identified many potential synthetic lethal therapeutic targets that preferentially cause death of kras - mutant cells. some of these targets, including cdk4, stk33, tbk1, and plk1, encode protein kinases and thus may be tractable to inhibition by selective small molecular inhibitors, such as those that have demonstrated success against mutant egfr and braf. much progress has been made over the years in our understanding of ras genes and the critical role they play in tumorigenesis, yet we have been unable to exploit this knowledge to significantly improve the outcome of cancers driven by mutant kras. while recognizing that nsclc and crc with kras mutations are not likely to be responsive to egfr inhibitors is an important step forward in improving patient treatment, there remains a pressing need for the development of effective kras - directed cancer therapies. although efforts to develop kras - targeting therapies have so far been met with disappointment, we have gained important insights into the complex biochemistry of kras proteins and kras signaling networks. at the same time, in vivo studies in mice have and will continue to make important contributions to our understanding of the underlying biology of kras proteins and their roles in cancer. going forward, it will be critical to continue interrogating the role of kras in cancer through mouse and molecular studies and to bridge this knowledge with clinical studies to facilitate the development of truly effective therapies against mutant kras - driven cancers. | mutational activation of kras is a common oncogenic event in lung cancer and other epithelial cancer types. efforts to develop therapies that counteract the oncogenic effects of mutant kras have been largely unsuccessful, and cancers driven by mutant kras remain among the most refractory to available treatments. studies undertaken over the past decades have produced a wealth of information regarding the clinical relevance of kras mutations in lung cancer. mutant kras - driven mouse models of cancer, together with cellular and molecular studies, have provided a deeper appreciation for the complex functions of kras in tumorigenesis. however, a much more thorough understanding of these complexities is needed before clinically effective therapies targeting mutant kras - driven cancers can be achieved. |
developmental processes occurring from the neural plate stages and leading to the identification of all main brain regions entail dramatic changes, including a pronounced cephalic flexure. as the anterior neural tube closes, the embryonic brain subdivides into three primary vesicles : the rhombencephalon, the mesencephalon, and the prosencephalon, from caudal to rostral. subsequently, the primary prosencephalon subdivides into two major components, the caudal diencephalon and the rostral secondary prosencephalon. the secondary prosencephalon constitutes the entire prechordal (rostralmost) portion of the neural tube, which gives rise to the hypothalamus ventrally, the eye vesicles dorsolaterally, the telencephalic vesicles dorsally, and a ventral telencephalic preoptic area (poa ; reviewed in garca - lpez., 2009 ; like the caudal vesicles, the secondary prosencephalon is formed by alar and basal derivatives. thus, the hypothalamus includes alar and basal plate components, whereas the eye field and the telencephalon are completely derived from the alar region (figure 1). the topological arrangement of these forebrain subdivisions is a consequence of the location of their primordia in the neural plate (couly and le douarin, 1987 ; eagleson and harris, 1990 ; rubenstein., 1998 ; inoue., 2000 the mechanisms leading to the formation of the prosencephalon and its subsequent patterning are highly conserved in evolution, especially the genetic codes that result in the production of forebrain subdivisions recognized along the rostrocaudal and dorsoventral axes in different vertebrates. schematic drawing of a lateral view of the brain showing the main subdivisions of the prosencephalon, as currently accepted. ma, mammillary area ; oc, optic chiasm ; p13, prosomeres 13 ; pa, pallidum ; po, preoptic region ; poc, commissural preoptic area ; sc, suprachiasmatic area ; spv, supraopticparaventricular area ; stn, subthalamic nucleus ; str, striatum ; tu, tuberal hypothalamus. apart from the evaginated telencephalic and eye vesicles, the rest of the secondary prosencephalon (here referred to as the non - evaginated secondary prosencephalon, regardless the diverse evaginations that may occur in the hypothalamus) has been traditionally considered as the ventral portion of the diencephalon. in particular, the term hypothalamus (hypo from the old greek p : under) literally describes its topographical position beneath the thalamus. thus, in the literature, frequent definitions describe the preoptic region (po) and the hypothalamus proper as special regions of the ventral diencephalon that are involved in regulation of the endocrine system, the autonomic nervous system, and related brain systems (bruce, 2008 ; hodos, 2008). these definitions, older or current, treat the brain as a column, so that a transverse section of an adult brain shows the hypothalamus beneath the thalamus, and molecular and developmental data are not taken into account. however, detailed comparison of diverse developmental gene expression patterns gathered in the last decades led puelles and rubenstein to postulate in 1993 the prosomeric model, modified 10 years later (puelles and rubenstein, 1993, 2003) as a morphological framework (paradigm), which divides the forebrain into transverse segments (prosomeres) and longitudinal zones. in this prosomeric context, we refer in the present analysis to the non - evaginated portion of the secondary prosencephalon, only with the intention to use a short name that includes the hypothalamus and the po of the telencephalon. classically, the po has been considered part of the hypothalamus (for review butler and hodos, 2005), but in recent years this view has changed and the po been considered part of the telencephalon, due to its topological position in the neural plate and its genetic specification (flames., 2007 ; garca - lpez., 2008 ; snchez - arrones., 2009). therefore, in the present analysis of the po and hypothalamus we pay special attention to the boundaries between them in diverse vertebrate species in order to gain a better understanding of the evolution of these forebrain regions. the regions under analysis are very complex in terms of genetic specification, morphological, hodological and neurochemical organization, and, especially, function (for review see ten donkelaar, 1998 ; butler and hodos, 2005 ; medina, 2008). however, it is interesting to note that in the last years, numerous data have shown conserved patterns in the evolution of vertebrates related to the embryological origin, cell types, neurochemical organization, hodology, and functional implications of the subregions in the secondary prosencephalon (reviewed in medina, 2008 ; moreno., 2009). in addition, it has been increasingly reported that in these regions many differences can also be observed among vertebrates, which may reflect not only the existence of divergent evolution in the different lineages but also secondary adaptations of each group. moreover, to this complex evolutionary equation has to be added a high divergence of sizes, shapes, and complexity of centers related to sophisticated behaviors like human consciousness, songbird vocalization, electrical responses in some fishes, etc. in this evolutionary context, when one accomplishes a comparative analysis the main aim is to establish homologous relationships. to this end, however, many different characteristics can be considered, which sometimes make this comparison extremely difficult and dense. at least in the last years, most comparative studies of the forebrain in different vertebrates have been framed within the prosomeric model as a paradigm to interpret the results (puelles and rubenstein, 2003). those studies suggest that some characteristics are more important than others in this analysis, as they determine the topology of the neural plate, the genetic specification of a topographic domain in the neural tube, the histogenetic pattern, the neuronal differentiation, hodology and, finally, neuronal organization. with these ideas in mind, the most convenient approach to establishing homologies is to assess the number of shared features, keeping in mind that homologous structures with common morphogenetic origin may have varied enormously in the course of evolution. substantial data reported in the last years have demonstrated that the combined expression of regulatory genes is very conserved in the evolution (puelles and rubenstein, 1993, 2003 ; bachy., 2001, 2002 ; gonzlez., 2008 ; abelln and medina, 2009 ; gonzlez and northcutt, 2009 ; domnguez., 2010), suggesting that the molecular specifications unravel the morphogenetic field formation. when comparing across species, two main concepts are to be kept in mind, developmental regulatory genes and basic histogenetic domains. both concepts allow and help explain forebrain organization in the comparative approach. in mammals, hypothalamic neurogenesis occurs in individual nuclei between embryonic day 10 (e10) and e16 in mice (shimada and nakamura, 1973 ; shimogori., numerous studies have identified many transcription factors that regulate the development of specific hypothalamic nuclei and neuronal subtypes (schonemann., 1995 ;, 1997 ; wehr., 1997 ; michaud., 1998 ;, seeking terminological continuity, in the prosomeric model the hypothalamus constitutes the rostral diencephalon (puelles and rubenstein, 2003). it includes alar and basal components, while the eye field and the telencephalon are exclusively derived from the alar region. the alar portion, includes the suprachiasmatic (sc) and the supraopticparaventricular (spv) regions, which will give rise their respective hypothalamic nuclei. the basal hypothalamus includes the tuberal hypothalamus, which contains among other structures, the ventromedial nucleus, the arcuate nucleus, and the mammillary hypothalamic region, which includes the subthalamic nucleus (reviewed in medina, 2008). in molecular terms, shh is essential for the specification of the hypothalamus (chiang., 1996 ; szbo., 2009) through the action, among others, of the homeobox gene nkx2.1, whose expression is triggered by prechordal shh signals (kimura., 1996). in addition, in mammalian forebrains, nkx2.1 is expressed in the basal telencephalon poa and the basal hypothalamus (price., 1992), which is severely malformed and reduced in size in knockout mice (kimura., 1996 ; sussel., 1999 specially, the neural shh has a very important and specific role in the development of the lateral hypothalamus, possibly mediated by regulation of dlx2, dbx1, and foxd. the lack of shh expression in the hypothalamic neuroepithelium results in a very reduced lateral hypothalamus, in which some of the most functionally important and characteristic neuronal subpopulations are either very reduced or completely missing, whereas the poa and the sc nucleus show normal development (szbo., 2009). the transcription factor orthopedia (otp) is expressed in the spv of the alar hypothalamus, the arcuate nucleus, and the oblique perimammillary band (simeone., 1994 ; puelles and rubenstein, 2003 ; del giacco., 2006 ; bardet., 2008), where it operates in the proper differentiation of several neurohormone - secreting nuclei (acampora., 1999 ; wang and lufkin, 2000 ; michaud, 2001 ; eaton and glasgow, 2007 ; blechman., 2007 ; otp also contributes to progenitor cell proliferation, survival, and migration (goshu. the transcription factor sim1, in parallel to otp, acts to differentiate a large population of neurohormone- containing cells (michaud., 1998 ; acampora., 1999 ; wang and lufkin, 2000 ; sim1 mutants, the prospective spv cells are born and specified, but the cells do not terminally differentiate to express neuroendocrine hormone genes. in terms of anatomical subdivisions the poa, which was previously believed to be derived from the ventral diencephalon, is currently regarded as derived from the foxg1-positive telencephalic neuroepithelium (zhao., 2008 ; roth., 2010), whereas the hypothalamus can be subdivided into the sim1-positive anterodorsal hypothalamic neuroepithelium, which contain the primordium of the paraventricular nucleus, and the nkx2.1-positive posteroventral hypothalamic neuroepithelium, which contains the primordia of the arcuate nucleus and the ventromedial hypothalamic region, the premammillary neuroepithelium, and the mammillary neuroepithelium (reviewed in medina, 2008 ; zhao., the rostral hypothalamus correlates topographically with the early expression of six3, which extends from the septum to the neurohypophysis. the boundary between the caudal hypothalamus and p3 is well defined by the expression of several genes. in the alar plate, it coincides with the sharp caudal limit of the sim1, otp, and brn2 genes, which are expressed in the spv, and with the sharp rostral limit of arx, dlx, and pax6 transcripts in p3. finally, a territorial boundary between the telencephalon and the hypothalamus has been recently defined and called the preoptohypothalamic boundary (poh;bardet. it is delimited between the po in the subpallium and the spv of the hypothalamus and occupies the region just outside the nkx2.1- and shh - expressing poa and it also lies within the contiguous thin corridor, expressing only dlx5 among the studied subpallial markers (bardet., 2006). to summarize, in mammals, shh and nkx2.1 are expressed in the entire non - evaginated prosencephalon, with the exception of the alar hypothalamus (reviewed in medina, 2008). the absence of nkx2.1 expression in the alar hypothalamus in mice correlates well with the absence of lhx6 or lhx7 expression in this area (rtaux., 1999). in the alar hypothalamus, the spv also contains an important population of otp - expressing cells (bardet., 2008). in modern classification schemes, living vertebrates consist of two main groups, the jawless agnatha (represented by hagfishes and lampreys) and the jawed gnathostomata. jawed vertebrates are divided again into two groups, the cartilaginous chondrichthyes (represented by sharks, rays, and chimeras) and the bony osteichthyes. the two main groups of bony vertebrates are the ray - finned fishes (actinopterygii) and the lobe - limbed vertebrates (sarcopterygii). the latter comprises three groups : coelacanths (actinistia), lungfishes (dipnoi), and limbed vertebrates, or tetrapoda, encompassing amphibians, reptiles, birds, and mammals (clack, 2002). in recent years, evolutionary / developmental studies have revealed a segmentary pattern in the developing forebrain of all the vertebrates analyzed. in this kind of comparative analysis it is essential to consider the highest possible number of groups, especially those with crucial evolutionary positions. in particular, the phylogenetic position of reptiles, especially turtles, makes their study interesting because they are purported to be the most closely related to the extinct therapsids from which mammals arose (northcutt, 1970) but, alternatively, they have been considered the sister group to crocodiles and birds (zardoya and meyer, 2001a, b). it is also essential to consider amphibians in analyzing ancestral brain organization, because they constitute the only group of tetrapod anamniotes and represent a key model in the anamniote / amniote transition, as they show shared features with other tetrapods (amniotes) and also shared features with fishes (anamniotes). in the context of this transition, the colonization of land by tetrapod ancestors is presumably one of the evolutionary events that could entail more neural changes. molecular phylogenetic research addressing this transition and the most recent paleontological evidence, suggest that lungfishes are closest living relatives of tetrapods and that several of the features defining this group were highly conserved throughout the entire evolutionary history of land vertebrates (meyer and wilson, 1990 ; meyer and dolven, 1992 ; hedges., 1993 ; zardoya., 1998 ;, 2004a, b ; takezaki., 2004 ; hallstrom and janke, 2009), making this group of fishes of particular interest in comparative studies. and finally, lampreys are jawless vertebrates, belonging to the agnatha, the sister group of gnathostome vertebrates. their phylogenetic position makes them an invaluable model in the study of evolutionary developmental biology, as they allow us to gain new insights into ancestral characters in vertebrates, and they also help to reveal, but also to understand the emergence of novelties at important evolutionary transitions, such as the agnathan / gnathostome transition (kuratani., 2002). according to the rationale outlined above, the evolutionary analysis of transcription factors involved in the patterning of the non - evaginated secondary prosencephalon in key vertebrate groups would be of special interest for understanding its evolution (figures 2 and 3). in recent years, cell groups and regions that were characterized according to gene expression patterns in the prosencephalon of mammals have been similarly analyzed in the brains of multiple vertebrates (figures 2 and 3 ; puelles and rubenstein, 1993, 2003 ; bachy. b ; brox., 2003 ; moreno., 2004, 2005, 2008a, b, 2010 ; osorio., 2008 ; abelln and medina, 2009 ; domnguez., 2010). these cell groups and regions in the secondary prosencephalon, and many of their neurochemical and connectional features, appear to be highly conserved in the evolution of tetrapods, since these features show similarities from amphibians through mammals (for review see reiner., 1998 ; marn., 1998 ; ten donkelar, 1998 ; moreno., 2009). nevertheless, although many characteristics are shared, the degree of complexity increases during evolution from the common ancestor of tetrapods to birds and mammals photomicrographs of transverse sections through non - evaginated portion of the secondary prosencephalon of a turtle [pseudemys scripta ; (a, d) ], a frog [xenopus laevis ; (b, e) ], and a lungfish [neoceratodus forsteri ; (c, f) ] illustrating by immunohistochemistry the developing expression of different transcription factors indicated in each figure. mea, medial amygdala ; poa, preoptic area ; poc, commissural preoptic area ; pthe, prethalamic eminence ; th, thalamus. photomicrographs of transverse sections through non - evaginated portion of the secondary prosencephalon of a mouse (a), a turtle [pseudemys scripta ; (d, g) ], a frog [xenopus laevis ; (b, e, h) ], and two lungfishes [neoceratodus forsteri ; (c, f) and protopterus dolloi ; (i) ] illustrating by immunohistochemistry the developing expression of different transcription factors indicated in each figure. mea, medial amygdala ; p3, prosomere 3 ; pv, paraventricular nuclei ; so, supraoptic nuclei ; sc, suprachiasmatic area ; spv, supraopticparaventricular area. current theories regarding the organization of the forebrain in mammals consider that the subpallium encompasses the non - evaginated telencephalon, anterior (topologically dorsal) to the optic chiasm (see figure 1 ; flames. it is composed of the po, which contains the commissural preoptic division (poc) and the poa proper, which is located at the base of (topologically rostral to) the former (for review see moreno., 2009) ; both express nkx2.1 and shh in the ventricular layer during development. in chicks, the po has been similarly recognized as a part of the subpallium and is characterized by the expression of both nkx2.1 and shh (abelln and medina, 2009). in addition to the distinct expression of shh, the po is also distinguished from the subpallial pallidal division (derived from the medial ganglionic eminence, mge) by its expression of islet1 (abelln and medina, 2009). the po in birds is also said to contain the two major subdivisions : the commissural poa, comparable to the same - named subdivision in mice (ppo1 of flames., 2007 ; poc of garca - lpez., 2008) and characterized by strong expression of shh and lhx7 and by its relation to the anterior commissure ; and a basal poa, comparable to the ventral preoptic subdivision (ppo2 of flames. 2008). in the vertebrates analyzed in our laboratory (figures 2a f), the situation in mice and chicks largely agrees with the results obtained in the po of pseudemy scripta (moreno., 2010), xenopus laevis (moreno., 2008a ; domnguez., 2010), and the lungfishes protopterus dolloi and neoceratodus forsteri (unpublished observations). we identified the po (figures 2a c) and the poc (figures 2d e) by the expression of shh, nkx2.1, lhx7, and isl1 and the absence of tbr1 and pax6 (moreno. in teleost fishes (including zebrafish), shh is expressed in a small subdivision of the nkx2.1-expressing domain of the basal telencephalon (rohr., 2001 ; scholpp., 2006 ; menuet., 2007), suggesting that the subpallium of early jawed vertebrates likely included striatal, pallidal, and preoptic subdivisions. the existence of pallidal and preoptic domains in the subpallium of teleost fishes expressing nkx2.1 is correlated with expression of lhx6 and/or lhx7/8 in the same domains, and the presence of pallidal - like cell groups as well as cholinergic cells resembling those of the corticopetal system (prez., 2000 ; mueller., 2004 ; wullimann and mueller, 2004 ; menuet., 2007). however, more data are needed to determine whether the preoptic part of the subpallium in other jawed fishes (such as zebrafish) includes subdivisions similar to those of tetrapods. in a strikingly different situation, the homolog of nkx2.1 in lampreys (ljnkx2.1, and a putative homolog of sonic hedgehog (lfhh) in gnathostomes have no expression domain in the ventral telencephalon in lampreys(ogasawara., 2001 ; the forebrain expression pattern of lfhh comprises two separate domains, a small hypothalamic domain and a large and robust coma - shaped domain in the diencephalon, showing continuity with floor plate expression all along the neural tube. the alar part of the hypothalamus (figures 3a f) has been said to contain the spv area and the sc region. the spv is a component of the central neural circuitry that regulates several homeostatic variables through numerous connections among others with the posterior pituitary. the homeodomain transcription factor otp is particularly expressed in this region in mice, chicks, amphibians, and lungfishes (figures 3a c), allowing it to be distinguished from the shh- nkx2.1- dlx - expressing adjacent domains (bardet.. other shared features of this region, at least shared among amniotes, include pax6 ventricular expression (flames., 2007 ; the spv region in xenopus could differ a little from that described in amniotes because, among other peculiarities, it does not include pax6 expressing cells (moreno. the zebrafish genome contains two otp orthologs, expressed in almost identical forebrain domains (del giacco., 2006), largely comparable to the otp - expressing zones in mice (simeone., 1994). in lampreys there are no detailed data on otp expression, however it has been reported that its hypothalamic distribution resembles that of the otp - positive neurons in zebrafish (joly., 2007). however, pax6 expression has not been described in the hypothalamic region (murakami., 2001). in the case of the sc region (figures 3d f), it has been shown to be dlx - positive territory in all the vertebrates examined (bachy., 2002 ; brox. the expression of nkx2.1, however, has been detected only in non - mammalian vertebrates (bachy., 2002 ; moreno., 2008a ; van den akker., 2008 ; abelln and medina, 2009 ; domnguez., 2010 ; moreno., this region of nkx2.1 expression runs parallel to the hypothalamic zone of shh expression, in a zone containing arx- and gad67-positive cells, and it is also defined by the expression of lhx6, lhx8, and lhx1. in xenopus, isl1, xlhx1, and xlhx5 expression has been described as restricted to the sc area (moreno., 2004, 2008a). some distinction was noted in distribution, as the cells located just above and rostral to the optic chiasm only expressed xlhx1, whereas more caudally in the sc nucleus two regions could be distinguished : a dorsal portion that only expressed xlhx5 and a ventral portion that expressed both xlhx1 and xlhx5 (moreno., 2004). in regard to the basal hypothalamus, the ventral tubero - mammillary cells in chick embryogenesis arise from a set of floor plate - like precursors that initially express shh (manning., the transcription factor tbx2 is expressed only transiently in the mammillary region, for a slightly longer period in the tuberal region, and it is retained only at the junction of the retrochiasmic / tuberal hypothalamus (manning., 2006). in the tuberal hypothalamus of pseudemy scripta (moreno., 2010), x. laevis (moreno., 2008a ; domnguez., 2010), and the two lungfishes (unpublished observations) it was observed that the nkx2.1 expression is strong in the vz of the tuberal area, whereas isl1 is restricted to the svz ; and the expression of otp is restricted to the arcuate nucleus (figure 3i). in xenopus, xshh expression is strong in the basal hypothalamus, specifically in the mammillary band and the tuberal area. this expression is mainly restricted to the anterior levels, and xshh expression was not detected in the most posterior basal hypothalamic levels (domnguez., 2010). the basal hypothalamus contains some populations of xgad67- and xdll-4-expressing cells (brox., 2003). the relatively few cells that express xlhx7 in the mammillary region in adults could be distinguished in the caudal hypothalamus from early premetamorphic stages (moreno., 2004). the dorsomedial hypothalamic area and the superficial mammillary and mammillary nuclei contained a small to moderate number of xgad67-expressing perikarya. the main conclusion of this comparative study is that major histogenetic processes thought to be a hallmark of so - called higher vertebrates actually existed early in vertebrate phylogeny (summarized in figure 4). the expressions of different gene families during development in different vertebrates correspond to the conserved boundaries of the proliferative domains. (2010) ; : abelln and medina (2009) ; bardet. the developmental stages showed are not comparable. at anterior levels in the po, lampreys (jawless fish) 2001), and this is correlated with the absence of shh expression (osorio., 2005). this suggests that the subpallium of the first vertebrates not only lacked a pallidal domain (as noted above), but also appear to have lacked a subdivision comparable to the preoptic subpallium (for review see osorio and rtaux, 2008). this would correlate with the apparent lack of both pallidal - like cells (like those of the pallidal part of the basal ganglia) and basal forebrain cholinergic cells (pombal., the detection of both shh and dlx expression shows that shh expression can be localized in the basal telencephalon / poa at late developmental stages (scholpp., 2006 ; a pallidum expressing nkx2.1 and a poc region comparable to that in amphibians have been demonstrated in the telencephalon of lungfishes (gonzlez and northcutt, 2009), although the presence of shh expression in the telencephalon needs to be investigated. therefore, based on those lack of nkx2.1 and shh expression in lampreys (osorio., 2005) and the organization of the poa demonstrated in amphibians (moreno., 2008a ; domnguez., 2010) it can be postulated that the de novo expression of shh and nkx2.1 in the basal telencephalon of gnathostomes may be responsible for the emergence of new brain structures in the subpallium, the pallidum, and the po, which probably emerged in the transition from agnathans to gnathostomes (see also murakami., 2005 ; osorio and rtaux, 2008). in the alar hypothalamus, the first thing to consider is the presence of the spv throughout vertebrates. in all groups examined, this area expresses otp and lacks nkx2.1/shh / dlx, in a very conserved pattern (reviewed in medina, 2008). in addition, the maintenance of otp expression throughout development seems to be a conserved feature in vertebrates, as inferred from the cases in which late developmental stages or adult specimens were studied. it is noteworthy that otp - expressing cells were consistently noted in some subdivisions of the amygdala (bardet., 2008 ; moreno., 2010), subdivisions whose cells most likely have a hypothalamic origin (soma., 2009). at present, there are no anatomical data regarding possible otp expression in the amygdala in teleosts or lampreys, but given the otp expression found in the amygdala in adult lungfish (gonzlez and northcutt, 2009) it will be an interesting aspect to investigate, in the context of evolution of the amygdaloid complex. it is possible that the double nkx2.1/pax6 expression is an exclusive feature of the forebrain of anamniotes, not only present in the pallium but also in the hypothalamus. consistent with this idea is the finding that, in mice, the absence of nkx2.1 expression in the alar hypothalamus correlates with expression of pax6 in the whole alar diencephalon, including the alar hypothalamus (puelles., 2000 ; thus, the mutually exclusive balance between nkx2.1 and pax6 in the telencephalon and diencephalon may be important for the relative size of pallial versus subpallial and thalamic versus hypothalamic territories. any alteration of this balance, as occurs in nkx2.1 knockout mice and nkx2.1 knockdown xenopus sussel., 1999 ;, 2002 ; van den akker., 2008) or in pax6 knockout mice (stoykova., 2000 ; yun., 2001), modifies the relative size of these subdivisions. in the case of the sc region, the phylogenetic diagram in figure 4 shows the expression of the orthologous homeobox gene nkx2.1 in several vertebrate species, including a jawless fish (a lamprey), a jawed fish (a zebrafish), an amphibian (a frog), a reptile (a turtle), a bird (a chicken), and a mammal (a mouse). this comparative representation highlights the peculiar lack of expression in this region only in the case of the forebrain of mammals. considering the expression patterns observed in chicks and mice (shimamura., 1995 ; puelles., 2000), it appears that the alar hypothalamic expression of nkx2.1 was either dramatically reduced or disappeared during the evolution to birds and mammals (medina, 2008 ; van den akker., analysis of the xnkx2.1 knockdown xenopus embryos indicates that xnkx2.1 controls the relative size of major regions in both the telencephalon (subpallium versus pallium) and the diencephalon (thalamus versus hypothalamus) of the forebrain (van den akker., changes in the regulation of nkx2.1 expression have played an important role in the evolution of forebrain development, and they emphasize the potential of combined analysis of expression and function of master control genes in different vertebrates for revealing the origins of brain complexity and diversity. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | the secondary prosencephalon (telencephalon plus hypothalamus) is probably the most complex area of the brain, with complicated patterning specifications. as yet, no prosomeric subdivisions have been reported and only distinct histogenetic territories have been recognized. in the present comparative study we analyzed cross - correlated expression maps in the non - evaginated territories of the secondary prosencephalon in different vertebrates throughout development, to assess the existence of comparable divisions and subdivisions in the different groups. each division is characterized by expression of a unique combination of developmental regulatory genes, and each appears to represent a self - regulated and topologically constant histogenetic brain compartment that gives rise to a specific cell group. the non - evaginated area of the telencephalon corresponds to the preoptic region, whereas the hypothalamus, topologically rostral to the diencephalic prethalamus, includes basal (mammillary and tuberal) and alar (paraventricular and suprachiasmatic) parts. this complex area is specified by a cascade of transcription factors, among which the dlx family members and nkx2.1 are essential for the correct development. the only exception is found in the subdivision named termed the supraoptoparaventricular area, in which the transcription factor orthopedia is essential in restricting the fate of multiple categories of neuroendocrine neurons, in the absence of the dlx / nkx2.1 combination. our analysis, based on own data and published results by other researchers, suggests that common features are shared at least by all tetrapods and, therefore, they most likely were present in the stem tetrapods. the available data for agnathans (lampreys) and other fish groups indicate that not all subdivisions of the secondary prosencephalon were present at the origin of vertebrates, raising important questions about their evolution. |
phyllodes tumor is a rare fibroepithelial tumor of the breast that accounts for less than 1% of all breast tumors. few cases have been published involving a synchronous invasive carcinoma component within a phyllodes tumor [2 - 7 ]. this is the first documented case of a borderline malignant phyllodes tumor with both tubular carcinoma and lobular carcinoma in situ components within the same tumor mass. a 53-year - old female presented with a several year history of a right painless breast mass in the right upper outer quadrant that had grown in size significantly during the last two years until referral for treatment. the patient had no increased risk factors and family history was negative for both breast and ovarian cancers. the patient had been started on progesterone 10 mg daily one year prior to presentation for oligomenorrhea. the patient was then followed biennially by the ontario breast screening program until the significant growth increase when a second core biopsy was recommended and was also consistent with fibroadenoma. given the sudden increase in size, with the lesion now measuring 5.51.8 cm by mammogram (figures 1, 2), the radiological / pathological correlation rounds recommended a surgical assessment and excisional biopsy for definitive diagnosis. grossly, the well - circumscribed pale - tan mass measured 6.53.62.4 cm, which in areas had a whorled appearance. histologically, focal areas of fibroadenomatous changes were noted (figure 3a) while areas of leaf like architecture with variable cellularity of stromal component were present as well. cytologic atypia was present throughout the stromal component ranging from minimal to moderate atypia and a variable number of mitoses were present (up to 7 to 8 per 10 high power fields), consistent with a diagnosis of phyllodes tumor with borderline malignant potential (figure 3b). at the lateral portion of the excised mass there was a tubular carcinoma area with a greatest dimension of 2.4 cm (figure 3c). the carcinoma cells were immunohistochemically positive for epithelial membrane antigen, estrogen receptor, progesterone receptor and e - cadherin, but negative for s100 protein. absence of basal myoepithelial lining in tubules was confirmed by negativity for smooth muscle myosin heavy chain and p63 (figure 3d). lobular carcinoma in situ was also noted in this area and the diagnosis was confirmed by negativity of immunohistochemical stain for e - cadherin (figure 3e, 3f). the surgical margins were not involved by the tumor, but the tubular carcinoma was very close (0.1 mm) to the inked surface at the lateral and posterior edges. following the pathology review the patient had complete staging workup, including bone scan, liver ultrasound, and chest x - ray. these were all negative for metastases. a dedicated breast magnetic resonance image (mri) was completed prior to reexcision that demonstrated a few, scattered enhancing masses of unknown significance. subsequently the patient underwent a re - excision for margin safety and sentinel lymph node biopsy. pathology review of the re - excision found scerlosing adenosis and atypical ductal hyperplasia with no evidence of residual invasive or in situ carcinoma or phyllodes tumor components. the previously close margins of tubular carcinoma at the lateral and posterior aspect are now 4.0 cm and 2.8 cm from those margins, respectively. this patient 's unique presentation was reviewed at breast tumor rounds at the juravinski cancer centre with the decision for the patient to next be evaluated by the radiation oncology team for adjuvant therapy. they are usually classified as benign, borderline, or malignant, depending upon stromal cellularity and overgrowth, cellular atypia, mitotic activity, and margin status. the local recurrence for these tumors range from 4.3 - 36% with wide local excision (> 1 cm margin), the standard primary treatment modality. an invasive ductal carcinoma that is incidentally found within a borderline phyllodes tumor has been reported only once before in literature. our case showed a similar presentation to the previous one, and a lobular carcinoma in situ component was also accompanied with the phyllodes tumor and invasive tubular carcinoma. this patient was followed routinely for several years with a stable lesion, originally biopsied as fibroadenoma. during the interim the mass followed what is expected as a normal course for a fibroadenoma : overall stable size, sometimes minimal size alteration with menstrual cycles, painless, and well - circumscribed on radiographs. given that both fibroadenomas and phyllodes tumors have similar clinical presentation of a painless breast mass, physicians rely on accurate diagnostic imaging, biopsy, and routine clinical follow - up for surveillance. a retrospective review of thirty - one histologically proven masses (19 fibroadenomas and 12 phyllodes tumors) examined the mammographic and sonographic differences between fibroepithelial lesions. from that review they suggested that assured accurate diagnosis can only be made by core or excisional biopsy. as a less invasive diagnostic modality to excisional biopsy, core biopsy of fibroepithelial lesions is a strong alternative for differentiating these tumors. in a study of sixty - seven finnish females examining the efficacy of core biopsy of diagnosing between fibroadenoma and phyllodes tumor, they found a sensitivity, specificity, and positive and negative predictive values of 83%, 92%, 71%, and 96%, respectively, for phyllodes tumors. in this patient, even though the most recent repeat needle core biopsy confirmed again a fibroadenoma it was the significant change in size of the tumor that raised concern of a more aggressive mass, leading to excision and incidentally finding the invasive carcinoma component unfortunately, review of this patient 's mammograms did not suggest an invasive carcinoma component, given that it was within the well - circumscribed borderline phyllodes component. in conclusion, a phyllodes tumor with a synchronous invasive carcinoma component is rarely presented and routine follow - up that involves clinical and radiologic assessment of fibroadenomas is important to curtail progression of a masked malignant component. | phyllodes tumors are an infrequent breast tumor presentation. a phyllodes tumor with a synchronous invasive ductal carcinoma is rarely described and has never been reported with lobular carcinoma in situ component. a 53-year - old female presented with a nine - year history of twice core biopsy proven fibroadenoma. after an increase in the tumor 's growth velocity it was decided upon to undergo an excisional biopsy. microscopic examination of the well - circumscribed pale - tan mass found focal areas of leaf like architecture with variable number of mitoses present, representing a phyllodes tumor of borderline malignant potential. incidentally, at one edge of the mass was found a tubular carcinoma and lobular carcinoma in situ components. thorough, routine follow - up of patients with biopsy proven benign breast masses is important to finding a masked malignant component. |
thyroids were excised from c57bl/6 male mice that were sacrificed at 18 weeks (n=2), 6 months (n=2), 15 months (n=2), or 30 months (n=2) of age. the mice had been fed a normal chow diet since birth and were maintained in accordance with the principles of laboratory animal care. for analysis, they were grouped as follows : control mice (18 weeks old), adult mice (six months old), and aged mice (15 and 30 months old). blood was collected from the retro - orbital sinus in anesthetized 11-week - old (n=7) or 20-month - old (n=7) c57bl/6 male mice in order to measure serum thyroid hormone levels. each thyroid was fixed in 10% neutral buffered formalin and paraffin - embedded in the transverse plane using standard procedures. paraffin - embedded tissue sections (4-m - thick) were stained with hematoxylin and eosin (h&e) for histological analysis, which included follicle size and shape, follicular cell height, and characteristics of the cytoplasm and intrafollicular colloid. tissue sections were stained with periodic - acid - schiff (pas), which stains the glycoprotein thyroglobulin (tg) in the colloid purple - red. the pas stain intensity of the intrafollicular colloid was then compared between samples. in hypoactive follicles all mouse experiments were approved by the university committee for animal experiments and were performed in accordance with the national research council guide for the care and use of laboratory animals and in accordance with the guidelines for the care and use of laboratory animals prepared by the institute for laboratory animal research, national academy of sciences. retro - orbitally collected, clotted mouse blood was centrifuged at 3,000 g for 10 minutes. total t3 and t4 levels were measured using an enzyme - linked immunosorbent assay kit (merck millipore, darmstadt, germany) according to the manufacturer s instructions. serum thyroid - stimulating hormone (tsh) was measured using a specific mouse tsh radioimmunoassay provided by dr. (center for cancer research, national cancer institute, bethesda, md, usa). sections of the whole thyroid gland (approximately 20 serial sections) at 100 magnification were used to assess the area of the follicles. the inner area of the thyroid gland at half the maximum length in the longitudinal planes was considered the central zone, and the area surrounding the central zone was considered the peripheral zone (fig. the same approach was taken in the transverse planes of the thyroid gland (fig. the size of the follicles in the central zone was compared with the size of the follicles in the peripheral zone. follicles that were neither ovoid / round nor smooth in outline were considered to be of irregular shape. irregularly - shaped follicles were assessed in 20 serial sections using 100 magnification images of the whole thyroid gland section. to assess mitochondrial content, tissue sections were immunostained with an antibody against the translocase of the outer mitochondrial membrane (tom20). paraffin - embedded tissue sections (4-m - thick) were placed in an oven and incubated at 56c for three hours before immunohistochemistry. specimens were stained using the ventana hx automatic system from benchmark (ventana medical systems, sa, illkirch cedex, france) with an anti - tom20 rabbit polyclonal antibody (santa cruz biotechnology, santa cruz, ca, usa). all procedures, including antigen retrieval and blocking of endogenous peroxidase activity, were performed automatically by the benchmark system. immunoperoxidase staining was performed using the lsab neuvision system, according to the manufacturer s instructions (ventana medical systems), and sections were counterstained with hematoxylin. tissue slides were analyzed using an olympus bx51 microscope (olympus, tokyo, japan). the intensity of tom20 immunoexpression was scored as follows : 0, negative ; 1, weakly positive ; 2, moderately positive ; and 3, strongly positive. h&e - stained, paraffin - embedded thyroid tissue and thyroid function test results (serum concentrations of tsh, t3, and t4) from 10 women over the age of 70 and 10 women between 30 and 50 years of age who underwent total thyroidectomy between january 2002 and december 2005 at daejeon st. the study protocol was reviewed and approved by the institutional review board of daejeon st. the baseline characteristics and thyroid function test results of each human participant are summarized in table 1. group comparisons of categorical variables were evaluated using a linear - by - linear association. thyroids were excised from c57bl/6 male mice that were sacrificed at 18 weeks (n=2), 6 months (n=2), 15 months (n=2), or 30 months (n=2) of age. the mice had been fed a normal chow diet since birth and were maintained in accordance with the principles of laboratory animal care. for analysis, they were grouped as follows : control mice (18 weeks old), adult mice (six months old), and aged mice (15 and 30 months old). blood was collected from the retro - orbital sinus in anesthetized 11-week - old (n=7) or 20-month - old (n=7) c57bl/6 male mice in order to measure serum thyroid hormone levels. each thyroid was fixed in 10% neutral buffered formalin and paraffin - embedded in the transverse plane using standard procedures. paraffin - embedded tissue sections (4-m - thick) were stained with hematoxylin and eosin (h&e) for histological analysis, which included follicle size and shape, follicular cell height, and characteristics of the cytoplasm and intrafollicular colloid. tissue sections were stained with periodic - acid - schiff (pas), which stains the glycoprotein thyroglobulin (tg) in the colloid purple - red. the pas stain intensity of the intrafollicular colloid was then compared between samples. in hypoactive follicles all mouse experiments were approved by the university committee for animal experiments and were performed in accordance with the national research council guide for the care and use of laboratory animals and in accordance with the guidelines for the care and use of laboratory animals prepared by the institute for laboratory animal research, national academy of sciences. retro - orbitally collected, clotted mouse blood was centrifuged at 3,000 g for 10 minutes. total t3 and t4 levels were measured using an enzyme - linked immunosorbent assay kit (merck millipore, darmstadt, germany) according to the manufacturer s instructions. serum thyroid - stimulating hormone (tsh) was measured using a specific mouse tsh radioimmunoassay provided by dr. (center for cancer research, national cancer institute, bethesda, md, usa). sections of the whole thyroid gland (approximately 20 serial sections) at 100 magnification were used to assess the area of the follicles. the inner area of the thyroid gland at half the maximum length in the longitudinal planes was considered the central zone, and the area surrounding the central zone was considered the peripheral zone (fig. the same approach was taken in the transverse planes of the thyroid gland (fig. the size of the follicles in the central zone was compared with the size of the follicles in the peripheral zone. follicles that were neither ovoid / round nor smooth in outline were considered to be of irregular shape. irregularly - shaped follicles were assessed in 20 serial sections using 100 magnification images of the whole thyroid gland section. to assess mitochondrial content, tissue sections were immunostained with an antibody against the translocase of the outer mitochondrial membrane (tom20). paraffin - embedded tissue sections (4-m - thick) specimens were stained using the ventana hx automatic system from benchmark (ventana medical systems, sa, illkirch cedex, france) with an anti - tom20 rabbit polyclonal antibody (santa cruz biotechnology, santa cruz, ca, usa). all procedures, including antigen retrieval and blocking of endogenous peroxidase activity, were performed automatically by the benchmark system. immunoperoxidase staining was performed using the lsab neuvision system, according to the manufacturer s instructions (ventana medical systems), and sections were counterstained with hematoxylin. tissue slides were analyzed using an olympus bx51 microscope (olympus, tokyo, japan). the intensity of tom20 immunoexpression was scored as follows : 0, negative ; 1, weakly positive ; 2, moderately positive ; and 3, strongly positive. h&e - stained, paraffin - embedded thyroid tissue and thyroid function test results (serum concentrations of tsh, t3, and t4) from 10 women over the age of 70 and 10 women between 30 and 50 years of age who underwent total thyroidectomy between january 2002 and december 2005 at daejeon st. the study protocol was reviewed and approved by the institutional review board of daejeon st. the baseline characteristics and thyroid function test results of each human participant are summarized in table 1. group comparisons of categorical variables were evaluated using a linear - by - linear association. we measured serum levels of tsh, t3, and t4 in 20-month - old mice (aged group ; n=7) and compared them with levels of the same hormones in 11-week - old mice (control group ; n=7) (table 2). serum t3 concentrations were lower in the aged group than in the control group, but there was no difference in serum t4 and serum tsh levels between the two groups. the thyroid activation index, expressed as the ratio of follicular cell volume to colloid volume, reflects changes in thyroid function caused by alterations in tsh level. thyroid tissue from 18-week - old control mice was composed of a relatively homogeneous population of small- to medium - sized round follicles lined with cuboidal epithelium (fig. in contrast, the 30-month - old mouse thyroid was composed of dilated or irregularly - shaped follicles (fig. the intrafollicular colloid volume was larger in 30-month - old mice than in the controls. consequently, the younger thyroid, which was composed of cuboidal or columnar epithelium and a small amount of colloid, had a higher thyroid activation index than the older thyroid, which was composed of a flat epithelium and significantly more colloid. next, we examined whether the histological changes in the thyroid follicles of aged mice correlated with thyroid function. the 18-week - old control thyroid glands were composed of small - to medium - sized round follicles. thyroid follicles of the central zone were smaller than those of the peripheral zone, with a ratio of the area of central zone to peripheral zone follicles of 1:2 (fig. the adult mouse thyroid gland (6 months old) was composed of variable - sized follicles with a relatively round shape. the zonal variation in the size of thyroid follicles was well preserved ; however, follicles in the peripheral zone were three times larger than those in the central zone (fig. smaller follicles found in the central zone were lined with a cuboidal epithelium, and larger follicles located in the peripheral zone were lined with a cuboidal or low cuboidal epithelium (fig. thyroid glands from aged mice (15 months old and 30 months old) were composed of irregularly dilated follicles, which were considered to be inactive (fig. there was a greater incidence of inactive large follicles in the aged mice compared to the thyroid of adult mice. dilated follicles were not only present in the periphery of the gland, but were also seen in the central zone. the aged thyroids were extremely heterogeneous in appearance, exhibiting variability in the height of the follicular epithelium and in the amount of intrafollicular colloid. this was more frequently observed in the 30-month than in the 15-month - old mice (fig. most irregularly - shaped follicles were surrounded by a cuboidal or high cuboidal epithelium. in the thyroid glands of aged mice, some of the large dilated follicles had cellular areas composed of small follicles lined by a crowded epithelium with scant colloid in a small lumen ; some of the large dilated follicles also had asymmetric pseudopapillary projections of crowded columnar cells (fig., many endocytic vacuoles were seen near the margin of the colloid - filled lumen (fig. these vacuoles emerge with dissolution of colloid by acid phosphatase that is secreted from lysosomes in conjunction with hormonal release during follicular cell activity. thus, colloid endocytosis correlates with follicular cell activity. in 6-month - old adult thyroids, several follicles in the peripheral zone were empty and were lined with a high cuboidal epithelium. the follicular cells of the empty follicles had clear cytoplasm and a centrally located, shrunken nucleus (fig. these clear cell changes were suggestive of a distended endoplasmic reticulum, which was interpreted as a sign of follicular degeneration. colloid vacuolation was frequently observed in the adult thyroid. in the aged thyroid (15 months old and 30 months old), colloid - filled follicles lined with a flat epithelium stained homogeneously pink with h&e, while colloid within the follicles lined with a high cuboidal epithelium was pale and coarsely granular (fig. there were numerous small, fragmented, and clumped tg globules within the intrafollicular colloid that were surrounded by a high cuboidal or oncocytic epithelium (figs. the formation of tg multimers allows for storage of tg at excessively high concentrations, and they are more frequently observed in hypofunctioning follicles. some of the thyroid follicles were empty, and others contained only small amounts of colloid in their larger lumen (fig. empty follicles are inactive follicles that may have lost accumulated colloid over time due to stasis of colloid circulation. in 30-month - old mouse thyroids, follicles lined with a low columnar epithelium showed oncocytic changes that were characterized by an abundant oxyphilic, granular cytoplasm and a large hyperchromatic nucleus (fig. an oncocytic appearance is the result of mitochondrial proliferation, which is a mechanism to compensate for mitochondrial defects. compared with nononcocytic follicular cells, the tom20 score was 1 and 3 in non - oncocytic follicular cells and oncocytic follicular cells, respectively (fig. 3d). after analyzing the intensity and multimerization of pas - stained intrafollicular colloid, we compared intrafollicular colloid concentrations and follicular activity for each age group. regular thyroid follicles of the adult group (6 months old) were filled with homogeneously pas - positive colloid. pas - positive colloid in the peripheral zone was more darkly stained than the pas - positive colloid in the central zone. the markedly enlarged follicles of the aged group (15 months old and 30 months old) had an increased pas - positive density (dark blue - purple pas stain) (fig. the colloid in irregularly - shaped follicles and in small follicles was stained purple - red by pas. these aggregated, small follicles are composed of a high cuboidal epithelium and lead to depletion of luminal colloid. abundant colloid in large follicles was stained purple - red or dark blue - purple by pas, while cellular areas composed of small follicles were pale - red or negative (fig. thus, the markedly enlarged follicles seen in aged mice were considered to be hypoactive compared with small follicles, irregularly - shaped follicles, and sanderson s polsters. we also analyzed age - related histological changes in the thyroid glands of 10 women over 70 years of age and compared them with thyroids from women between 30 and 50 years of age. we observed findings similar to those seen in aged mice, including (1) variable size and enlargement of follicles (fig. 5b) ; (3) sanderson s polsters in the walls of large follicles (fig. 5b) ; (4) a large tg globule or numerous, small, fragmented tg globules in the follicular lumen (fig. however, unlike the thyroids of aged mice, fibrosis or fatty infiltration within the extracellular matrix was observed in elderly human thyroids (fig. 5f, g). among the 10 elderly women, thyroid hormone levels were unremarkable when compared with controls (t3, p=.551 ; t4, p=.138 ; tsh, p=.085) (table 1). age - related histological changes and functional activities in the human thyroid follicles were not associated with changes in serum thyroid hormone levels. we measured serum levels of tsh, t3, and t4 in 20-month - old mice (aged group ; n=7) and compared them with levels of the same hormones in 11-week - old mice (control group ; n=7) (table 2). serum t3 concentrations were lower in the aged group than in the control group, but there was no difference in serum t4 and serum tsh levels between the two groups. the thyroid activation index, expressed as the ratio of follicular cell volume to colloid volume, reflects changes in thyroid function caused by alterations in tsh level. thyroid tissue from 18-week - old control mice was composed of a relatively homogeneous population of small- to medium - sized round follicles lined with cuboidal epithelium (fig. in contrast, the 30-month - old mouse thyroid was composed of dilated or irregularly - shaped follicles (fig. the intrafollicular colloid volume was larger in 30-month - old mice than in the controls. consequently, the younger thyroid, which was composed of cuboidal or columnar epithelium and a small amount of colloid, had a higher thyroid activation index than the older thyroid, which was composed of a flat epithelium and significantly more colloid. next, we examined whether the histological changes in the thyroid follicles of aged mice correlated with thyroid function. the 18-week - old control thyroid glands were composed of small - to medium - sized round follicles. thyroid follicles of the central zone were smaller than those of the peripheral zone, with a ratio of the area of central zone to peripheral zone follicles of 1:2 (fig. the zonal variation in the size of thyroid follicles was well preserved ; however, follicles in the peripheral zone were three times larger than those in the central zone (fig. smaller follicles found in the central zone were lined with a cuboidal epithelium, and larger follicles located in the peripheral zone were lined with a cuboidal or low cuboidal epithelium (fig. thyroid glands from aged mice (15 months old and 30 months old) were composed of irregularly dilated follicles, which were considered to be inactive (fig. there was a greater incidence of inactive large follicles in the aged mice compared to the thyroid of adult mice. dilated follicles were not only present in the periphery of the gland, but were also seen in the central zone. the aged thyroids were extremely heterogeneous in appearance, exhibiting variability in the height of the follicular epithelium and in the amount of intrafollicular colloid. this was more frequently observed in the 30-month than in the 15-month - old mice (fig. most irregularly - shaped follicles were surrounded by a cuboidal or high cuboidal epithelium. in the thyroid glands of aged mice, some of the large dilated follicles had cellular areas composed of small follicles lined by a crowded epithelium with scant colloid in a small lumen ; some of the large dilated follicles also had asymmetric pseudopapillary projections of crowded columnar cells (fig. intrafollicular colloid of control and adult thyroids stained homogeneously pink by h&e (figs. 1b, 2a). in the control group, many endocytic vacuoles were seen near the margin of the colloid - filled lumen (fig. these vacuoles emerge with dissolution of colloid by acid phosphatase that is secreted from lysosomes in conjunction with hormonal release during follicular cell activity. thus, colloid endocytosis correlates with follicular cell activity. in 6-month - old adult thyroids, several follicles in the peripheral zone were empty and were lined with a high cuboidal epithelium. the follicular cells of the empty follicles had clear cytoplasm and a centrally located, shrunken nucleus (fig. 2a, arrow). these clear cell changes were suggestive of a distended endoplasmic reticulum, which was interpreted as a sign of follicular degeneration. colloid vacuolation was frequently observed in the adult thyroid. in the aged thyroid (15 months old and 30 months old), colloid - filled follicles lined with a flat epithelium stained homogeneously pink with h&e, while colloid within the follicles lined with a high cuboidal epithelium was pale and coarsely granular (fig. there were numerous small, fragmented, and clumped tg globules within the intrafollicular colloid that were surrounded by a high cuboidal or oncocytic epithelium (figs. the formation of tg multimers allows for storage of tg at excessively high concentrations, and they are more frequently observed in hypofunctioning follicles. some of the thyroid follicles were empty, and others contained only small amounts of colloid in their larger lumen (fig. empty follicles are inactive follicles that may have lost accumulated colloid over time due to stasis of colloid circulation. in 30-month - old mouse thyroids, follicles lined with a low columnar epithelium showed oncocytic changes that were characterized by an abundant oxyphilic, granular cytoplasm and a large hyperchromatic nucleus (fig. an oncocytic appearance is the result of mitochondrial proliferation, which is a mechanism to compensate for mitochondrial defects. compared with nononcocytic follicular cells, oncocytic follicular cells were intensely stained by the tom20 antibody. the tom20 score was 1 and 3 in non - oncocytic follicular cells and oncocytic follicular cells, respectively (fig. after analyzing the intensity and multimerization of pas - stained intrafollicular colloid, we compared intrafollicular colloid concentrations and follicular activity for each age group. fig. regular thyroid follicles of the adult group (6 months old) were filled with homogeneously pas - positive colloid. pas - positive colloid in the peripheral zone was more darkly stained than the pas - positive colloid in the central zone. 4a). the markedly enlarged follicles of the aged group (15 months old and 30 months old) had an increased pas - positive density (dark blue - purple pas stain) (fig. the colloid in irregularly - shaped follicles and in small follicles was stained purple - red by pas. these aggregated, small follicles are composed of a high cuboidal epithelium and lead to depletion of luminal colloid. abundant colloid in large follicles was stained purple - red or dark blue - purple by pas, while cellular areas composed of small follicles were pale - red or negative (fig. thus, the markedly enlarged follicles seen in aged mice were considered to be hypoactive compared with small follicles, irregularly - shaped follicles, and sanderson s polsters. we also analyzed age - related histological changes in the thyroid glands of 10 women over 70 years of age and compared them with thyroids from women between 30 and 50 years of age. we observed findings similar to those seen in aged mice, including (1) variable size and enlargement of follicles (fig. 5b) ; (3) sanderson s polsters in the walls of large follicles (fig. 5b) ; (4) a large tg globule or numerous, small, fragmented tg globules in the follicular lumen (fig. however, unlike the thyroids of aged mice, fibrosis or fatty infiltration within the extracellular matrix was observed in elderly human thyroids (fig. 5f, g). among the 10 elderly women, thyroid hormone levels were unremarkable when compared with controls (t3, p=.551 ; t4, p=.138 ; tsh, p=.085) (table 1). age - related histological changes and functional activities in the human thyroid follicles were not associated with changes in serum thyroid hormone levels. in this study, we identified age - related histological changes in the thyroid glands of aged mice. these changes included a decrease in the entire thyroid size, formation of markedly dilated follicles with a flat epithelium, irregularly - shaped follicles, aggregations of small follicles with oncocytic epithelia, the presence of colloid - depleted follicles, a large tg globule or multimeric tg globules within the colloid, and loss of zonal variation. fibrosis, inflammation, and fatty infiltration were common in elderly human thyroids but rarely observed in aged mouse thyroids. of these changes, the height of the follicular epithelium, cytoplasmic features of follicular cells, pas staining properties of the colloid, and characteristic tg globules are representative of functional activity of the thyroid follicles. a commonly encountered pattern in the aged mice thyroid was prominent cystically dilated follicles with flimsy walls composed of scant fibrous stroma. cystic atrophy is not infrequently found in the postmenopausal endometrium. in the absence of ovarian function, the endometrium experiences cystic atrophy, having a thin uterine mucosa, cystically dilated endometrial glands, and a flattened inactive epithelium. the pathogenesis of cystic atrophy of the endometrium has not yet been established. in cystic atrophy of the aged thyroid, low cuboidal or flattened and inactive follicular cells line the distended follicles. larger or dilated follicles lined with a low cuboidal epithelium contain colloid that stains purple - red with pas, while larger or dilated follicles with a flat epithelium contain thick colloid that stains dark blue - purple with pas. as organisms get older, the function of thyroid follicular cells tends to diminish, thus decreasing endocytosis of luminal colloid into follicular cells. eventually, the accumulation of luminal colloid increases intrafollicular pressure and increases tension in the follicular wall, which may contribute to the flattening of follicular cells. oncocytic change in cells is increasingly observed with advancing age in thyroid glands and in other organs. in the involuted thyroids of old mice, a follicular epithelium with oncocytic change the characteristic oncocytic appearance, which consists of an abundant oxyphilic, granular cytoplasm and a large hyperchromatic nucleus, is the result of mitochondrial proliferation that compensates for mitochondrial defects. mitochondrial function is very important for maintaining functional activity in most endocrine organs ; therefore, age - related changes in the mitochondria are likely to impair endocrine organ function. intrafollicular colloid took on the appearance of a large tg globule or of numerous, small, fragmented tg globules within oncocytic follicles. a large globule or multimeric tg globules are more frequently present in hypofunctioning follicles than in active follicles. in aged mice, we observed oncocytic follicular epithelia lining lumens lacking colloid, which is a characteristic sign of an inactive follicle. we found that follicles composed of oncocytic epithelia are more likely to be irregular in shape, lack colloid, or have highly insoluble colloid. these results demonstrate the important role of mitochondrial function in maintaining proper activity of the thyroid follicular cell. with advancing age, conversion of functional tissue to fatty or fibrous tissue occurs in most organs, and fundamental tissue loss in a variety of organs is associated with a decrease in proper function. in the aged mouse, however, there is a paucity of conversion to fatty or fibrous tissue. instead, cystic atrophy is common. although conversion of thyroid follicles to fatty or fibrous tissue is occasionally observed in elderly women, the proportion of fatty or fibrous tissue within thyroid is not so pronounced. in conclusion, follicular cells and the follicles of aged thyroids show characteristic morphological changes, which include cystic atrophy, empty colloid, tg globules, and oncocytic follicular cells. | backgroundalthough both thyroid histology and serum concentrations of hormones are known to change with age, only a few reports exist on the relationship between the age - related structural and functional changes of the thyroid follicles in both mice and humans. our objectives were to investigate age - related histological changes of the thyroid follicles and to determine whether these morphological changes were associated with the functional activity of the follicles.methodsthe thyroid glands of mice at 18 weeks and at 6, 15, and 30 months of age were histologically examined, and the serum levels of thyroid hormones were measured in 11-week - old and 20-month - old mice. samples of human thyroid tissue from 10 women over 70 years old and 10 women between 30 and 50 years of age were analyzed in conjunction with serum thyroid hormone level.resultsthe histological and functional changes observed in the thyroid follicles of aged mice and women were as follows : variable sizing and enlargement of the follicles ; increased irregularity of follicles ; sanderson s polsters in the wall of large follicles ; a large thyroglobulin (tg) globule or numerous small fragmented tg globules in follicular lumens ; oncocytic change in follicular cells ; and markedly dilated follicles empty of colloid. serum t3 levels in 20-month - old mice and humans were unremarkable.conclusionsthyroid follicles of aged mice and women show characteristic morphological changes, such as cystic atrophy, empty colloid, and tg globules. |
the hippocampus has long been known as a brain structure fundamental for memory formation and retrieval. recent technological advances of cellular tracing techniques and optogenetic manipulation strategies have allowed to unravel important aspects of the cellular origin of memory, and have started to shed new light on the neuronal networks involved in encoding, consolidation and retrieval of memory in the hippocampus. in particular, memory traces, or engrams, that are formed during encoding in the dentate gyrus and ca3 region are crucial for memory retrieval and amenable to modulation by neuroplastic mechanisms, including adult hippocampal neurogenesis. here, we will discuss how memory traces are being encoded at the cellular level, how they may contribute to pattern separation and pattern completion in the hippocampus, and how they can be associated with different experiences to express memories of opposite valence. we propose a mechanism by which adult hippocampal neurogenesis may contribute to the formation of engrams, which may be relevant not only for the encoding of contextual information, but also for mood abnormalities, such as anxiety and depression. |
|
patients who underwent an agv implantation with fibrin sealant for part of the procedure during june 2009 all surgeries were performed by 1 of the 2 authors (nsc, an) using an identical technique. the conjunctival incision was made 4 - 5 mm behind and parallel to the corneal limbus for approximately 100 in the supero - temporal quadrant. the agv (model fp7 or fp8, new world medical, rancho cucamonga, la) was primed by injecting 1 - 2 ml balanced salt solution. the plate of an agv was placed at 8 mm behind the corneal limbus and secured to the sclera with 8 - 0 nylon suture material (m / s gn corporation ltd. this was followed by placement of the silicone tube into the anterior chamber or pars plana region through a 23-gauge needle track. the anterior part of the tube was covered with previously prepared human donor scleral patch graft. the fibrin sealant (tisseel kit, baxter ag, vienna, austria) was used for gluing the patch graft. an overlying conjunctiva was sutured with 8 - 0 polyglactin suture material (ethicon inc., the eye was inspected for any leaks as the anterior chamber was inflated to a proper pressure using balanced salt solution. postoperatively, all cases were prescribed ciprofloxacin eye drops (cipla ltd, mumbai, india) 4 times a day for a week and 6 weeks tapering regimen of prednisolone acetate eye drops (allergan india private limited, bangalore, india). the donor scleral tissue preserved in absolute alcohol the tissue was cleaned of all the uveal tissue attachments, washed thoroughly with balanced salt solution and cut into the desired size (4 - 5 4 - 5 mm). tissel kit (baxter ag, vienna, austria), a biodegradable, 2 component fibrin sealant, was used in every case. before use the fibrinolysis inhibitor, aprotinin, was added to the sealer protein concentrate vial followed by warming in a patented fibrotherm device. the second component was prepared by injecting calcium chloride solution into the thrombin 4 vial, which was then warmed. we preferred thrombin 4 over thrombin 500 as it allows sufficient time (60 seconds versus 10 seconds, respectively) for approximation of the patch graft to the underlying sclera. the required dose of the fibrin sealant was 0.1 to 0.2 ml of each of thrombin and fibrinogen solutions. after application, the donor tissue was pressed gently over the sealant for 3 minutes for firm adhesion. the data collection included information on patient demography, diagnosis of glaucoma, prior ocular surgeries, measurements of visual acuity ; intraocular pressure (iop) ; number of anti - glaucoma medications at the pre - operative and every post - operative follow - up visit and complications, if any. goldmann tonometer (haag - streit, switzerland), a hand - held perkin 's tonometer (haag - streit, essex, uk) or by tonopen xl (reichert ophthalmic instruments, walden ave. the cause(s) for low vision and post - operative reduction in visual acuity, if any, were also recorded. surgical success was defined as a final iop between 5 and 22 mm hg without (complete success) or with topical anti - glaucoma medication(s) (qualified success) and without any vision threatening complication. paired t test was used to compare the measurements of iop and the number of anti - glaucoma medications at the pre - operative and the final visits. patients who underwent an agv implantation with fibrin sealant for part of the procedure during june 2009 all surgeries were performed by 1 of the 2 authors (nsc, an) using an identical technique. the conjunctival incision was made 4 - 5 mm behind and parallel to the corneal limbus for approximately 100 in the supero - temporal quadrant. the agv (model fp7 or fp8, new world medical, rancho cucamonga, la) was primed by injecting 1 - 2 ml balanced salt solution. the plate of an agv was placed at 8 mm behind the corneal limbus and secured to the sclera with 8 - 0 nylon suture material (m / s gn corporation ltd. this was followed by placement of the silicone tube into the anterior chamber or pars plana region through a 23-gauge needle track. the anterior part of the tube was covered with previously prepared human donor scleral patch graft. the fibrin sealant (tisseel kit, baxter ag, vienna, austria) was used for gluing the patch graft. an overlying conjunctiva was sutured with 8 - 0 polyglactin suture material (ethicon inc., the eye was inspected for any leaks as the anterior chamber was inflated to a proper pressure using balanced salt solution. postoperatively, all cases were prescribed ciprofloxacin eye drops (cipla ltd, mumbai, india) 4 times a day for a week and 6 weeks tapering regimen of prednisolone acetate eye drops (allergan india private limited, bangalore, india). the tissue was cleaned of all the uveal tissue attachments, washed thoroughly with balanced salt solution and cut into the desired size (4 - 5 4 - 5 mm). tissel kit (baxter ag, vienna, austria), a biodegradable, 2 component fibrin sealant, was used in every case. before use the fibrinolysis inhibitor, aprotinin, was added to the sealer protein concentrate vial followed by warming in a patented fibrotherm device. the second component was prepared by injecting calcium chloride solution into the thrombin 4 vial, which was then warmed. we preferred thrombin 4 over thrombin 500 as it allows sufficient time (60 seconds versus 10 seconds, respectively) for approximation of the patch graft to the underlying sclera. the required dose of the fibrin sealant was 0.1 to 0.2 ml of each of thrombin and fibrinogen solutions. after application, the donor tissue was pressed gently over the sealant for 3 minutes for firm adhesion. the data collection included information on patient demography, diagnosis of glaucoma, prior ocular surgeries, measurements of visual acuity ; intraocular pressure (iop) ; number of anti - glaucoma medications at the pre - operative and every post - operative follow - up visit and complications, if any. goldmann tonometer (haag - streit, switzerland), a hand - held perkin 's tonometer (haag - streit, essex, uk) or by tonopen xl (reichert ophthalmic instruments, walden ave. the cause(s) for low vision and post - operative reduction in visual acuity, if any, were also recorded. surgical success was defined as a final iop between 5 and 22 mm hg without (complete success) or with topical anti - glaucoma medication(s) (qualified success) and without any vision threatening complication. paired t test was used to compare the measurements of iop and the number of anti - glaucoma medications at the pre - operative and the final visits. 13 patients underwent implantation of agv with fibrin sealant for part of the procedure during the study period. the mean number of intra - ocular surgeries prior to an implantation of agv was 1.8. the mean pre - operative iop in 9 eyes, in which it could be measured, was 34.4 mm hg. its mean value in these eyes was 9.5 mm hg at the final post - operative visit. the mean number of anti - glaucoma medications decreased from 2.5 at the pre - operative visit to 0.6 at the final post - operative visit. the postoperative reductions in iop and in number of anti - glaucoma medications were statistically significant (p < 0.01, paired t test). the scleral patch graft was posteriorly retracted in case 1 at the last follow - up [fig. 2 ]. conjunctival retraction occurred in case 10 in the second post - operative week, the scleral patch graft was partly exposed, and it epithelized over the next 3 weeks. there was no case of agv tube exposure, tube - cornea touch, or conjunctival erosion. the iop was 38 mm hg, and the bleb was shallow in case 12 at 5 weeks post - surgery. the agv tube was suspected to be blocked by vitreous strands, requiring anterior vitrectomy and intra - cameral flushing of the tube with balanced salt solution. two patients (cases 2 and 11) experienced a decrease in visual acuity due to late post - operative rhegmatogenous retinal detachment. photograph of case 7 taken under surgical microscope at 26 weeks post - surgery. the scleral patch graft (outlined by arrows) was secured with the fibrin sealant slit lamp photograph of case 1. in this series, we used a fibrin sealant to secure the human scleral patch graft over the extra - ocular portion of the agv tube. although other fibrin adhesives are available, we preferred tisseel since it is more extensively researched as a suture substitute in ophthalmic surgery. we did not encounter any case of agv tube exposure, tube - cornea touch, or conjunctival erosion. during an implantation of a gdd as soon as the silicone tube is inserted into the anterior chamber, aqueous drains and hypotony follows. thus, the globe is hypotonic unless an anterior chamber maintainer is used while the scleral patch graft is being sutured in place. while superficial suture bites can make the graft unstable, deep scleral bites carry a considerable risk of globe perforation. use of the fibrin sealant to stick the patch graft over the implant tube can avoid suturing the graft to the underlying sclera and thereby offers a safety advantage to the gdd implantation surgery. kahook and noecker used a 6 6 mm pericardial patch graft of 0.4 mm thickness. availability and cost are barriers to the routine use of pericardial patch graft to cover the gdd tube in our scenario. potential for pericardial graft thinning and possible tube erosion are additional concerns. in a series, 5 (11.3%) out of 44 eyes developed thinning of the pericardial patch graft over a mean follow - up of 10.2 months. the thickness of human sclera obtained from formalin - fixed eyes, varied from 0.39 0.17 mm near the equator to 0.9 to 1.0 mm near the optic nerve. there is a concern about fibrin sealant not providing enough tensile strength to keep the patch graft in place. shigemitsu and majima compared tensile strength of the cataract surgery wounds sutured with various methods and glued with bio - tissue adhesives in rabbit eyes. the strength of the wound treated with fibrin sealant was much less (43 gf / mm) compared to the wound sutured with a single 10 - 0 nylon suture (131 gf / mm) at 4 days after surgery although the respective strengths were comparable at 28 days after surgery. prospectively studied 15 eyes for the safety and efficacy of a fibrin sealant to secure the human scleral patch graft during an agv implantation. the scleral patch graft was found in place at each check during the follow - up period. we did find scleral patch graft retraction in 1 eye [fig. 2 ]. use of fibrin sealant to stick the patch graft during gdd implantation can cut down the surgical time. our retrospective and non - comparative study design did not allow commenting on this aspect. earlier kahook and noecker did report reduced mean surgical time by 10 minutes in the tisseel - assisted group. they retrospectively compared 28 cases of gdd implantation using traditional suture material with 14 cases of gdd implantation using fibrin sealant for portions of the procedure. kahook and noecker used the fibrin sealant to close the conjunctiva besides sticking the scleral patch graft. most of our patients had undergone more than 1 ocular surgery with an inevitable conjunctival manipulation. freely mobile conjunctiva is also necessary to limit postoperative conjunctival retraction, which can overcome the tensile strength of the glue. for these reasons, we did not use the fibrin sealant to close the conjunctival wound. in our study, 2 patients experienced retinal detachment after implantation of agv. case 2 was a high myope, suffered from post - traumatic retinal detachment, and developed secondary glaucoma following retinal detachment repair. retinal detachment as a complication of gdd has been reported in as many as 16% cases. suggested causes of retinal detachment in the literature include previous intraocular operations other than gdd implantation, posterior vitreous detachment in patients with an underlying retinal pathologic condition, chorioretinal scar, trauma, uveitis, retinal apposition from suprachoroidal hemorrhage, vitreous incarceration, inadvertent scleral perforation, and retinal dialysis from the pars plana - positioned tube. nevertheless, the retinal detachment was unlikely to be related to the use of fibrin sealant to stick the scleral patch graft. the duploject application system, a double - barrel syringe, is supplied with the tisseel kit. the required dose of the sealant in ophthalmic surgery is much less than the dose in general and cardiovascular surgery. therefore, we prefer 2 separate 1 ml syringes over the double - barrel syringe to load the reconstituted components. moreover, the quantity of the sealant lost in the joining piece of the duploject system is utilized. the preparation of the components of the smallest available tisseel kit gives 1.0 ml each of thrombin and fibrinogen solutions. this amount is sufficient for the gluing of surfaces for an area of at least 10 cm. we elect cases requiring the fibrin sealant for various conditions besides gdd implantation e.g. pterygium surgery, forniceal reconstruction, amniotic membrane transplantation etc. and operate them simultaneously in adjacent operation theatres. the required amount of the reconstituted components of the fibrin sealant is taken into separate 1 ml syringes and used. we are able to use the components of 1 kit for an average of 4 patients. this approach allows significant reduction in per - patient - price of the fibrin sealant. the retrospective and non - comparative nature of the study did not allow us to compare the safety and efficacy of the newer technique to the current practice of suturing the scleral patch graft during implantation of agv. also, a higher number of patients are necessary to understand the possible complications or difficulties with this technique. our study at least presents an alternative to the suture - assisted scleral patch grafting during implantation of agv. | aim : to report our experience with the fibrin sealant as a suture substitute for securing the human scleral patch graft during implantation of ahmed glaucoma valve (agv).materials and methods : a retrospective, non - comparative study of 12 eyes of 12 patients who underwent an agv implantation with fibrin sealant for part of the procedure during june 2009 to september 2010.results:the mean patient age was 21.5 20.6 years. male : female ratio was 2 : 1. seven (58.3%) patients were monocular. the indications for agv were varied. the mean number of intra - ocular surgeries prior to an implantation of agv was 1.8. the mean follow - up duration was 24.5 17.9 weeks. there was a statistically significant reduction in the mean iop and in the mean number of anti - glaucoma medications at the final visit compared to the pre - operative values (p < 0.01, paired t test). conjunctival retraction was seen in 1 (8.3%) case. the scleral patch graft was retracted posteriorly in another (8.3%) case. there was no case of agv tube exposure, tube - cornea touch, or conjunctival erosion. vision threatening complication viz. late post - operative rhegmatogenous retinal detachment, unlikely to be related to the use of the fibrin sealant, occurred in 2 (16.6%) eyes.conclusion:the fibrin sealant offers the advantages of safety and convenience to the placement of a scleral patch graft during an agv implantation. |
ionizing radiation (ir) produces reactive oxygen species (ros) such as oh, h2o2, oh, o. these toxic substances are highly chemically reactive and can react with cellular biomolecules including proteins, lipids, and dna, resulting in varieties of oxidative lesions. elevated ros level if dna lesions ca nt be effectively repaired by endogenous defense systems, it leads to genome instability and chromosome abnormalities. radiation - induced genome aberration has a crucial role in the mechanisms underlying radiation - induced carcinogenesis. with respect to radiation damage to humans, it is important to protect biological systems from radiation - induced genotoxicity. amifostine is a powerful radioprotective agent with a thiol group in its structure, but this drug has limited usage in clinical practice due to its side effects and toxicity. the search for less toxic radioprotectors has spurred interest in the development of natural products. achillea millefolium l (compositae ; acm) is a well - known medicinal plant. in iranian folk medicine, several species of achillea called bumadaran in persian have been used to treat bleeding, wounds, inflammation, pain, and spasmodic diseases. phytochemical studies on acm have shown that this plant is rich in flavonoids and caffeic acid derivatives. the presence of other secondary metabolites including essential oil, sesquiterpenes, alkaloid, steroids, and triterpenes has also been reported in this plant. previous studies on the extracts of acm have reported antioxidant, antiviral, antispasmodic, hepatoprotective, gastroprotective, estrogenic, immunological, and anti - inflammatory activities. in addition, the aqueous extract of this plant showed protective effects against cyclophosphamide - induced testicular toxicity. based on the above findings and the anti - inflammatory and antioxidant potential of acm extract, the present study aimed to evaluate the radioprotective activity of the plant by using in vitro radiation - induced genetic damage in volunteers human blood lymphocytes. the aerial parts of acm were collected from kalat naderi, khorasan - e - razavi province, iran, at 1586 m above sea level, during the flowering stage. a voucher specimen (44246) was deposited at the herbarium of the research center for plant sciences, ferdowsi university of mashhad, mashhad, iran. dried aerial parts of the plant were cut into small pieces and then extracted with methanol by maceration at room temperature. the extract was concentrated by using a rotary evaporator (heidolph, germany) and dried by a freeze dryer (zirbus, germany). healthy, nonsmoking human male volunteers aged between 20 and 25 years were enrolled in this study. twelve milliliter whole blood were collected in heparinized tubes and divided in centrifuge tube at 0.9 ml. blood samples were treated with 100 l solution of herbal extract at the concentrations 10, 50, 100, or 200 g / ml (final concentrations). ethanol concentration was same in control and extract solutions (0.5%). at each concentration and for each volunteer, tubes were irradiated at 37c with 6 mv x - ray beam produced by a radiotherapy machine (linear accelerator, siemens, primus, germany) at a dose of 2.5 gy with a dose rate of 190 cgy / min. subsequently, 0.5 ml of each sample (control and irradiated samples in duplicate) was added to 4.4 ml of roswell park memorial institute (rpmi) 1640 culture medium (gibco, paisley, uk), which contained 10% fetal calf serum. then phytohemagglutinin (100 l/5 ml, gibco) was added to the cultures as lymphocyte stimulator. all cultures were incubated at 37c in a humidified atmosphere of 5% co2 and 95% air. cytochalasin b (sigma, usa ; final concentration : 6 l / ml) was added after 44 hours of culture. following 72 hours of incubation, the cells were collected by centrifugation for 7 minutes at 3500 rpm and resuspended in cold potassium chloride. cells were immediately fixed in a fixative solution as methanol acetic acid (6:1) 2 times. the fixed cells were dropped onto clean microscopic slides, air - dried, and stained with giemsa solution (10%). all slides were evaluated at 100 magnification in order to determine the frequency of micronuclei in the cytokinesis - blocked binucleated cells with a well - preserved cytoplasm. at each concentration and for each volunteer, 1000 binucleated lymphocyte cells were examined from the irradiated and control cultures in duplicate to record the frequency of micronuclei. the free radical - scavenging capacity of the methanolic extract of acm was determined as bleaching of the stable 1,1-diphenyl-2-picryl hydrazyl radical (dpph). different concentrations of the extract (0.05 - 1 mg / ml) were added, at 2 ml, to 2 ml methanol solution of dpph (10 mg/250 ml). after 15 minutes at room temperature, the absorbance was recorded at 517 nm. the experiment was performed in triplicate, and butylated hydroxytoluene (bht) was used as a standard antioxidant agent. percentage of scavenging was calculated using the formula : [(control test)/control ] 100. for each volunteer, at each concentration, the incidence of radiation - induced micronuclei was recorded. the aerial parts of acm were collected from kalat naderi, khorasan - e - razavi province, iran, at 1586 m above sea level, during the flowering stage. a voucher specimen (44246) was deposited at the herbarium of the research center for plant sciences, ferdowsi university of mashhad, mashhad, iran. dried aerial parts of the plant were cut into small pieces and then extracted with methanol by maceration at room temperature. the extract was concentrated by using a rotary evaporator (heidolph, germany) and dried by a freeze dryer (zirbus, germany). healthy, nonsmoking human male volunteers aged between 20 and 25 years were enrolled in this study. twelve milliliter whole blood were collected in heparinized tubes and divided in centrifuge tube at 0.9 ml. blood samples were treated with 100 l solution of herbal extract at the concentrations 10, 50, 100, or 200 g / ml (final concentrations). ethanol concentration was same in control and extract solutions (0.5%). at each concentration and for each volunteer, tubes were irradiated at 37c with 6 mv x - ray beam produced by a radiotherapy machine (linear accelerator, siemens, primus, germany) at a dose of 2.5 gy with a dose rate of 190 cgy / min. subsequently, 0.5 ml of each sample (control and irradiated samples in duplicate) was added to 4.4 ml of roswell park memorial institute (rpmi) 1640 culture medium (gibco, paisley, uk), which contained 10% fetal calf serum. then phytohemagglutinin (100 l/5 ml, gibco) was added to the cultures as lymphocyte stimulator. all cultures were incubated at 37c in a humidified atmosphere of 5% co2 and 95% air. cytochalasin b (sigma, usa ; final concentration : 6 l / ml) was added after 44 hours of culture. following 72 hours of incubation, the cells were collected by centrifugation for 7 minutes at 3500 rpm and resuspended in cold potassium chloride. cells were immediately fixed in a fixative solution as methanol acetic acid (6:1) 2 times. the fixed cells were dropped onto clean microscopic slides, air - dried, and stained with giemsa solution (10%). all slides were evaluated at 100 magnification in order to determine the frequency of micronuclei in the cytokinesis - blocked binucleated cells with a well - preserved cytoplasm. at each concentration and for each volunteer, 1000 binucleated lymphocyte cells were examined from the irradiated and control cultures in duplicate to record the frequency of micronuclei. the free radical - scavenging capacity of the methanolic extract of acm was determined as bleaching of the stable 1,1-diphenyl-2-picryl hydrazyl radical (dpph). different concentrations of the extract (0.05 - 1 mg / ml) were added, at 2 ml, to 2 ml methanol solution of dpph (10 mg/250 ml). after 15 minutes at room temperature, the absorbance was recorded at 517 nm. the experiment was performed in triplicate, and butylated hydroxytoluene (bht) was used as a standard antioxidant agent. percentage of scavenging was calculated using the formula : [(control test)/control ] 100. for each volunteer, at each concentration, the incidence of radiation - induced micronuclei was recorded. the percentage of micronuclei in binucleated lymphocytes in 3 donors treated with 2.5 gy x - ray was 5.41 1.25, while the percentage in nontreated control lymphocytes was 0.69 0.17. it showed a significant increase of 8-fold in frequency of micronuclei in lymphocytes exposed to 2.5 gy of x - ray (p <.01 ; table 1). the frequency of micronuclei after pretreatment with acm at doses of 10, 50, 100, or 200 g / ml was 2.76 0.23, 1.85 0.05, 1.13 0.06, and 0.73 0.06, respectively (figure 1). the data demonstrate that human blood incubated with acm, and then exposed in vitro to x - ray radiation, exhibited a significant reduction in micronuclei frequency compared to blood samples incubated with x - ray alone. the extract at all concentrations exhibited significantly lower micronuclei frequency than irradiation control sample (p <.01). the values of total micronucleated binucleated cells were 49%, 66%, 79%, and 86% fold less in the 10, 50,100 and 200 g / ml concentrations of acm extract (table 1). a concentration - dependent effect for acm was observed in the reduction of chromosome damage in lymphocytes exposed to ionizing radiation (p <.01). the acm did not exhibit any genotoxicity in cultured lymphocytes at concentration of 200 g / ml without exposure to radiation. it is interesting to observe that the frequency of micronuclei in acm at a concentration of 200 g / ml was lower than control group (p <.01). the frequency of micronuclei induced in vitro by 250 cgy x - ray radiation (ir) in cultured blood lymphocytes from human volunteers examined at different doses of achillea millefolium (acm). one thousand bn cells were examined in each sample. in vitro protection by achillea millefolium (acm) at different concentrations (10, 50, 100, and 200 g / ml) against genetic damage induced by x - ray (ir ; 2.5 gy) in cultured whole blood lymphocyte. p <.01 : ir sample compared to 10 acm + ir, 50 acm + ir, 100 acm + ir, and 200 acm + ir samples. p <.01 : 10 acm + ir and 50 acm + ir ; 50 acm + ir and 100 acm + ir ; and 100 acm + ir and 200 acm + ir samples. scavenging effects of the methanolic extract of acm on dpph radicals increased with the increase in concentrations, it was 90% at 1 mg / ml which was similar to bht (figure 2). scavenging effect of different concentrations of achillea millefolium (extract) and butylated hydroxytoluene (bht) on the 1,1-diphenyl-2-picrylhydrazyl (dpph) free radical at 517 nm. in this study, we demonstrated that the methanolic extract of acm has potent radioprotective effect against genotoxicity induced by x - ray in human lymphocytes. achillea millefolium reduced the frequency of micronuclei in binucleated lymphocytes that increased by ir and exhibited protective effect at concentrations of 10, 50, 100, and 200 g / ml by factors 1.9, 2.9, 4.8, and 7.4, respectively. furthermore, considerable antioxidant activity with free radical - scavenging property was observed from the extract. with respect to side effects induced by ir in patients undergoing radiotherapy, the radioprotective agents have an important role for reduction of side effects in patients. increase in the intracellular level of ros produced by ir is a potentially toxic insult. natural compounds may play a role in scavenging free radicals such as hydroxyl radicals generated by ionizing radiation. in this study, treatment of whole blood with acm for 2 hours prior irradiation reduced the frequency of micronuclei. oral administration of the hydroalcoholic extract of acm reduced gastric ulcers induced by acetic acid. in other study, the aqueous extract of this plant with antioxidant and anti - inflammatory potential showed protective effect against cyclophosphamide - induced testicular toxicity. in the present study, acm extract showed a potent radical - scavenging effect against free radical dpph. the ros may attack dna, proteins, and lipid that can initiate degenerative diseases. antioxidant compounds such as phenolic acids, poly phenols, and flavanoids can scavenge free radicals and protect normal tissues against disease related to oxidative stress. chemical analysis of the methanol extract of the aerial parts of acm showed the presence of several phenolic compounds such as flavonoids (eg, rutin, luteolin glucosides, apigenin glucosides) and quinic acid derivatives such as chlorogenic acid. chlorogenic acid and other caffeoylquinic acid derivatives exhibit antioxidant activities and dna damage protective effects. in a research, chlorogenic acid and quinic acid showed radioprotective effects and decreased the dna damage induced by x - ray irradiation in human blood lymphocytes in vitro. some flavonoids such as luteolin 7-o - glucoside isolated from the whole plant of pilea microphylla exhibited protective effect against radiation - induced cytotoxicity. these compounds showed significant antioxidant activity and reduced lipid peroxidation, formation of intracellular ros, and dna strand breaks. apigenin, as a dietary antioxidant, protected human peripheral blood lymphocytes from radiation - induced oxidative damages. previously, we showed that extracts of zataria multiflora and hawthorn as medicinal plants protected human lymphocytes against genotoxicity induced by gamma irradiation. hawthorn and zataria extracts that have strong antioxidant activities may affect scavenging free radicals such as hydroxyl radicals generated by -rays in cells. it is interesting, acm at high concentration, 200 g / ml, completely normalized dna damage induced by ir on human lymphocyte. this result is promising that a natural product showed an excellent radioprotective effect. in this study, it was found that acm with antioxidant properties can contribute to reduce genotoxicity induced by ir in human lymphocytes as well as can be used as herbal medicine in several diseases, and it can help to protect body against side effects induced by irradiation during radiotherapy. | the radioprotective effect of achillea millefolium l (acm) extract was investigated against genotoxicity induced by ionizing radiation (ir) in human lymphocytes. peripheral blood samples were collected from human volunteers and incubated with the methanolic extract of acm at different concentrations (10, 50, 100, and 200 g / ml) for 2 hours. at each dose point, the whole blood was exposed in vitro to 2.5 gy of x - ray and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis - blocked binucleated cell. antioxidant capacity of the extract was determined using free radical - scavenging method. the treatment of lymphocytes with the extract showed a significant decrease in the incidence of micronuclei binucleated cells, as compared with similarly irradiated lymphocytes without any extract treatment. the maximum protection and decrease in frequency of micronuclei were observed at 200 g / ml of acm extract which completely protected genotoxicity induced by ir in human lymphocytes. achillea millefolium extract exhibited concentration - dependent radical - scavenging activity on 1,1-diphenyl-2-picryl hydrazyl free radicals. these data suggest that the methanolic extract of acm may play an important role in the protection of normal tissues against genetic damage induced by ir. |
although unicompartmental knee arthroplasty (uka) has been accepted as a reliable procedure for the management of patients with arthritis limited to one compartment of the knee1,2), it is still considered as a technically demanding procedure3). while a good size match between the component and the resected surface of the knee is an important factor in reducing the occurrence of complications and in achieving successful outcome of knee replacement4,5), morphological dimensions of a single compartment of the knee undergoing uka are small. it has been reported that implant overhang may cause pain after uka6,7) or total knee arthroplasty (tka)8,9). the design of uka prosthesis continues to evolve in terms of geometry, materials, fixation techniques, and bearing surfaces10). bearing components can be either fixed or mobile. whereas the mobile bearing components, such as the oxford prosthesis (biomet, warsaw, in, usa), are fully congruent to minimize point contact forces, fixed bearing designs, such as the miller - galante ii (m - g ii) prosthesis (zimmer inc., warsaw, in, usa), incorporate round - on - flat geometries to lessen constraints10). although several studies have reported long - term success of both oxford mobile and m - g ii fixed bearing ukas1,2,11,12), to the best of our knowledge, the prevalence of implant overhang after uka has not been well known. the purpose of this study was to compare the prevalence of implant overhang between the oxford and m - g ii uka prostheses and to determine whether overhang was associated with postoperative clinical results. we hypothesized that both prostheses would show similarly low prevalence of implant overhang with successful clinical outcomes. approval for this retrospective study was obtained from the institutional review board of our hospital. between january 2002 and december 2009, 122 ukas were performed in 109 patients by a single surgeon for the treatment of medial unicompartmental osteoarthritis of the knee. forty - six knees were treated with the oxford phase 3 uka prosthesis that had a single or twin peg femoral component, and 76 with the m - g ii uka prosthesis. the indications for uka were based on the kozinn and scott13) criteria, i.e., medial osteoarthritis or medial avascular necrosis, intact anterior cruciate ligament, range of motion larger than 90, flexion contracture less than 5, varus deformity less than 10, and valgus deformity less than 15. the lateral compartment and patellofemoral joint were well preserved on standing anteroposterior (ap), lateral, and skyline knee radiographs. four knees (three knees treated with the oxford uka prosthesis and one knee treated with the m - g ii uka prosthesis) were revised during the follow - up period and were excluded. the reasons for failure in the oxford uka group were one dislocation of the polyethylene, one femoral component loosening, and one tibial component loosening. the cause of revision in the m - g ii uka group was tibial component loosening. one hundred and seven knees (37 knees treated with the oxford uka prosthesis and 70 knees treated with the m - g ii uka prosthesis) in 95 patients were included in the final radiographic and clinical outcome analysis. among the 37 oxford ukas, 30 knees had a single peg femoral component and 7 had a twin peg femoral component. the mean age at operation was 60 years (range, 45 to 75 years) and the mean follow - up period was 48 months (range, 24 to 111 months). operation was performed according to the oxford unicompartmental knee placement operating manual and the m / g unicompartmental knee minimally invasive surgical technique manual. the size of the tibial component was determined by laying metal tibial templates on the excised tibial plateau for both the oxford and m - g ii prostheses. the tibial component should cover the tibial cut surface as much as possible so as to maximize the load distribution. when the tibial component did n't fit perfectly the resected proximal tibia, we made the maximum amount of effort to fit the anteromedial edge between the implant and the resected proximal tibia. while the size of the femoral component of the oxford prosthesis was determined using preoperative x - ray templates, the size of the femoral component of the m - g ii prosthesis was determined intraoperatively using a femoral cutting guide. the foot of the femoral cutting guide was inserted into the flat surface against the cut distal femoral condyle. a femoral cutting guide having 2 - 3 mm of the exposed bone above the anterior edge of the guide was considered to be the proper size for m - g ii uka. we inserted the tibial and femoral trial components and finally ensured the size of implant in both the oxford and m - g ii prostheses. the concept and technique for gap balancing were different between the two prostheses. in the oxford uka group, gap balancing was achieved by milling the distal femoral bone to obtain an equal extension gap with the flexion gap. however, in the m - g ii uka group, gap balancing was evaluated after bone cutting. a tension gauge with 2 mm thickness was used to ensure that flexion and extension gaps were not too tight in the m - g ii uka group. overhang of the implant was measured in the posterior femoral, anterior tibial, medial tibial, and posterior tibial zones on the ap and lateral knee radiographs. no reference on criteria for implant overhang after m - g ii uka could be found in the literature. considering one millimeter of measured error, we defined overhang as a measured distance of larger than 3 mm between the implant and the resected bone edge in any zone6,7,14). the average of these measurements was calculated and the prevalence of implant overhang was determined. clinical results were evaluated using the range of knee motion, the knee society scores15), and the western ontario and mcmaster universities osteoarthritis index (womac)16). the knees were divided into two groups according to the type of prosthesis ; those treated with the oxford uka prosthesis and those with the m - g ii uka prosthesis. the prevalence of implant overhang in each location was compared according to the prosthesis type. clinical results were compared not only between the oxford uka and m - g ii uka groups, but also between the overhang group and the non - overhang group at the final follow - up. the fisher 's exact test was used to examine the prevalence of overhang of each implant component. the chi - square test was used to examine categorical demographic data in both groups. the independent t - test any test was considered statistically significant if the p - value was equal to or less than 0.05. spss ver. 13.0 (spss inc., chicago, il, usa) was used to analyze the data. of 107 knees, 33 had overhang in at least one location of the femoral or tibial component. the prevalence of posterior overhang of the femoral component was significantly higher in the oxford uka group than that in the m - g ii uka group (p<0.001) (table 1). the prevalence of tibial component overhang ranged from 2.7% to 16.2% according to the type of prosthesis and location. although the oxford uka group showed a higher prevalence of posterior overhang of the tibial component than did the m - g ii uka group, there were no significant differences in the prevalence of tibial component overhang at each location between the groups (table 1). function as measured by the knee society function and knee score and womac score was compared between the oxford uka group and the m - g ii uka group at a minimum follow - up of 24 months (table 2). even though the oxford uka group showed a significantly higher prevalence of the posterior overhang of the femoral component than did the m - g ii uka group, there were no significant differences in clinical results between the two groups. we compared patients with overhang in at least one location of the femoral or tibial component with patients without overhang. table 3 shows the pre- and postoperative clinical data of the overhang group and non - overhang group regardless of the prosthesis type. at a mean follow - up of 48 months, both overhang group and non - overhang group showed improvements in knee alignment and clinical outcomes. there were no significant differences in clinical results between the overhang group and non - overhang group. the majority of implant overhang occurred in the posterior part of the femoral component of the oxford uka prosthesis. even with the high prevalence of posterior overhang of the femoral component in our uka study population, we did not find any significant correlation between posterior overhang of the femoral component and clinical results regardless of the presence of overhang or implant type. mahoney and kinsey8) reported that overhang of the femoral component was highly prevalent in tka, and overhang of 3 mm in at least one zone was associated with a 90% increase in the odds of clinically important knee pain at 2 years after surgery. clarius.7) analyzed implant position of the oxford uka prosthesis according to the oxford x - ray rating system. they reported that 17 of 56 knees (30.4%) were overhanging 3 mm or more in the femoral component and it was not correlated with the postoperative oxford knee score and pain. after uka, there was no mediolateral overhang of the femoral component because of its narrow shape. we believe that the posterior compartment of the medial side of the knee was forgiving of soft tissue irritation and pain. among cases of femoral or tibial overhang, chau.6) reported that surgeons must avoid medial tibial overhang of 3 mm or more, as this severely compromised the outcome and might cause irritation of soft tissue and pain. in an in vitro study by gudena.17), they demonstrated that medial collateral ligament load almost doubled from overhang of 2 to 4 mm. when compared with the results of a study by chau.6) which demonstrated a 9% prevalence of medial overhang of the tibial component, the prevalence of medial overhang of the tibial component in our study population was not that high (2.7% in the oxford uka group and 5.7% in the m - g ii uka group). with low prevalence of medial overhang of the tibial component, the effect of femoral or tibial component overhang on clinical outcomes was negligible in both the oxford and m - g ii uka groups. it is believed that the tibial sizing method using the metal template during surgery was reliable and the shapes and sizes of the tibial components of both the oxford and m - g ii implants were appropriate. we speculated the reasons for the high prevalence of posterior overhang of the femoral component of the oxford uka prosthesis as follows. bothra.18) reported that the use of x - ray template systems in the oxford uka lacked reliability. they indicated that variability in prosthetic sizing might be attributable to the difficulty in distinguishing the medial from the lateral femoral condyle on lateral radiographs. second, the medial femoral condyle of a small asian female patient was smaller than the x - small femoral component of the oxford uka prosthesis. among 19 knees which showed posterior overhang of the femoral component of the oxford uka system, 6 knees were implanted with the x - small femoral component (fig. the m - g ii uka prosthesis offers seven different femoral ap sizing options, and the oxford uka prosthesis has five different radii of curvature. these differences could allow for a limited number of options in choosing the appropriate size of the femoral component for the oxford uka. third, subsequent milling of the femoral condyle during the oxford uka in order to balance the flexion - extension gap might have affected posterior overhang of the femoral component (fig. 2). subsequent milling to increase the extension gap resulted in proximal positioning of the femoral component. during the surgical procedure, it was difficult to identify whether the femoral component was overhanging posteriorly or not. as a technical tip, we recommend that adequate amount of proximal tibial cut should be performed to decrease subsequent distal femoral milling. appropriate implant size matching could be one of the important factors for a successful outcome of uka19). to the best of our knowledge, no study has compared the prevalence of implant overhang between oxford and m - g ii uka. in our study, implant overhang had no relation with postoperative clinical results at a mean of 48-month follow - up. in this sense, long - term follow - up studies are needed and we suggest some recommendations to prevent implant overhang. the femoral and tibial components should be flush with all edges of the resected bone as much as possible5,10). if the tibial cut is done as a single piece, this can be helpful for tibial sizing. in cases where the tibial component does not fit perfectly the resected bone, the anteromedial edge should be observed directly between the tibial component and the resected proximal tibia to prevent medial overhang of the tibial component. sometimes, the patient 's height and gender can be helpful to estimate the size of the component20). also, it may be possible to determine the size of the component from templating using pre - operative radiographs. during uka procedure, perioperative fluoroscopy may be helpful to determine the size of implant and to prevent implant overhang. first, it was a retrospective study and the number of cases of overhang in each location was too small to provide enough statistical power to the study. if we had a larger number of patients in the cohort, patients could have been divided into 3 groups ; proper fit group, overhang group, and underhang group. despite these limitations, our study population consisted of patients who were operated on by a single surgeon with a mean of 48 months follow - up. it could have influenced the clinical results, but it was not clear whether the component overhang caused the pain or tenderness of knee joint7,11). in conclusion, posterior overhang of the femoral component was highly prevalent in the oxford uka patients. however, the posterior overhang of the femoral component had no significant relationship with postoperative clinical results in both oxford iii and m - g ii ukas at a mean of 48 months follow - up. | purposethe purpose of the present study is to compare the prevalence of implant overhang between the oxford and the miller - galante ii (m - g ii) unicompartmental knee arthroplasty (uka) prostheses and determine whether overhang is associated with postoperative clinical results.materials and methodswe retrospectively reviewed one hundred and seven ukas which consisted of 37 oxford ukas and 70 m - g ii. overhang was considered present if 3 mm overhang was observed in any zone. the range of motion, the knee society scores and the western ontario and mcmaster scores were compared after a mean follow - up duration of 48 months.resultsthirty three of 107 knees (30.8%) had overhang in at least one zone of the femoral or tibial component. in the tibial side, there were no significant differences between the groups in component overhang in each zone. in the femoral side, the oxford uka group showed a significantly higher prevalence of the posterior overhang of the femoral component (19/37, 51.4%) than did the m - g ii uka group (3/70, 4.3% ; p<0.001). however, no significant differences in clinical results were observed between the two groups. there were also no significant differences in clinical results between the overhang and the non - overhang groups.conclusionsposterior overhang of the femoral component was highly prevalent in oxford uka patients. however, posterior overhang of the femoral component had no significant relationship with postoperative clinical results in both oxford and m - g ii ukas at a mean of 48 months follow - up. |
in singapore in 2005, as part of an integrated vector - control program, laboratory - based dengue virus surveillance was established for close monitoring and investigation of the circulating dengue virus serotypes. samples were sent to the environmental health institute from tan tock seng hospital, which cares for 40% of all reported dengue patients in singapore, and from a network of participating general practitioners throughout the country. pcr to detect dengue virus rna and serotyping were performed at the environmental health institute according to its in - house real - time pcr protocol (7). the numbers of dengue - positive samples serotyped were 186 in 2006, 889 in 2007, and 918 in 2008, and represent 10% of the total dengue cases reported each year by the ministry of health. the envelope protein gene of denv (1,480 nt) was amplified by reverse transcription pcr and directly sequenced by using an automated dna sequencer (abi 3100 ; applied biosystems, foster city, ca, usa). phylogenetic analysis of denv sequences was conducted by using the maximum - likelihood method as implemented in paup software, version 4.0b10 (8), and compared with sequence data obtained from genbank. during 20062008, denv-1 (21.7%) and denv-2 (69.3%) were the predominant serotypes throughout the study period ; denv-3 (7.8%) and denv-4 (1.2%) were less prevalent. in 2006, the number of denv cases was relatively low, and denv-1 remained the predominant serotype after the major 20042005 outbreak. during january september 2006, 75%100% of samples collected each month contained denv-1. in early january 2007, the predominant circulating serotype switched from denv-1 to denv-2. early detection of this switch warned of a possible upcoming dengue outbreak. in response, the proportion of denv-2positive samples detected by pcr rose from 57.9% in january 2007 to a peak of 91.0% in july 2007. this increase was accompanied by an increase in the total number of dengue cases reported by the ministry of health ; cases peaked at 432 in the first week of july 2007. by late august, the number of dengue cases fell to below the warning level (warning level = 256 cases / epidemiologic week) as reported by the ministry of health (9). during the switch in predominant serotype, fatality rates (0.32% in 2006 and 0.27% in 2007) and dengue hemorrhagic fever rates (2.4% in 2006 and 2.1% in 2007) did not differ substantially among the reported cases. during this same period of extensive surveillance, 5.2% of the samples in 2007 and 10.8% in 2008 our spatial analysis indicated localized emergence of denv-3 in the eastern region of the country in 2007 and in the central region in 2008. enhanced control was also attempted in these areas to prevent the spread of the serotype that had been uncommon in singapore. trends of monthly dengue cases in singapore, 20052008, showing a switch in predominant serotype from dengue virus serotype 1 (denv-1) to denv-2 in january 2007 and cocirculation of all 4 serotypes with general dominance of denv-1 and denv-2 and lesser circulation of denv-3 and denv-4. from 10% of all dengue cases. phylogenetic analysis of denv-2 envelope gene sequences showed that the switch in predominant serotype in early 2007 coincided with a clade replacement within denv-2. during 20002008, 2 distinct subclades, with strong temporal topology, specifically, denv-2 isolates obtained before 2007 formed the subclade herein referred to as the old clade, whereas isolates obtained in 2007 and later formed the new clade with strong bootstrap support. because 1 of the denv-2 isolates sampled in 2005 clustered with the new clade but fell closer to the root of that clade, in situ evolution giving rise to denv-2 viruses that subsequently replaced the old clade viruses is highly likely. a genbank sequence that belonged to denv-2, sampled in 2007 in vietnam, grouped within the new clade, indicating that this virus strain was not restricted to singapore and may have been circulating in this region. maximum - likelihood tree showing the phylogenetic relationship of a) dengue virus serotype 2 (denv-2) and b) denv-3 from singapore and global isolates based on the envelope protein gene. ehi, sequence data generated at environmental health institute ; new clade, isolates obtained in 2007 and later ; old clade, isolates obtained before 2007. our dengue surveillance also indicated sporadic emergence of denv-3 from localized areas throughout the country (6,10). phylogenetic analysis of isolates from singapore from 2006 through 2008 identified 3 genotypes of denv-3. these isolates were closely related to those found in indonesia, malaysia, philippines, thailand, saudi arabia, and cte divoire (figure 2), which suggests multiple importations of denv-3 viruses into singapore. analysis of denv-1 sequences showed that all except 3 belonged to genotype i and were similar to those responsible for the 2005 outbreak (data not shown). our dengue surveillance provided early warning of the outbreak in 2007 and contributed to early activation of enhanced vector control. although we were unable to assess the effectiveness of the control measures, considering the regional situation in 2007 (11,12), we believe that without these measures the dengue situation in singapore in 2007 would have been worse than or comparable to that in 20042005. after a lull year in 2006, dengue cases were expected to rise for a few years. the integrated vector control program has interrupted the dengue trend, with 7,032 cases reported in 2008 and 4,498 in 2009. as a travel hub, although some become established at various levels, some develop into outbreaks and subsequently get replaced. it also highlights the complexity of the disease and the challenges faced by affected states that seek to understand the epidemiology for purposes of disease control. to shed further light on the complex interplay among the various factors that affect dengue transmission, studies are being conducted on complete genome sequences of dengue viruses, vectorial capacity of local aedes spp. | in singapore, after a major outbreak of dengue in 2005, another outbreak occurred in 2007. laboratory - based surveillance detected a switch from dengue virus serotype 1 (denv-1) to denv-2. phylogenetic analysis showed a clade replacement within denv-2 cosmopolitan genotype, which accompanied the predominant serotype switch, and cocirculation of multiple genotypes of denv-3. |
the authors report a unique case of malignant mesothelioma with distant metastasis to the brain. pleural mesothelioma was thought to spread directly to local structures with distant metastasis found only on autopsy. the authors present this case to demonstrate that mesothelioma can have distant metastasis and that surgery can improve both survival and quality of life. we report a case of a 62-year - old gentleman who presented with shortness of breath and right - sided chest pain over the period of 12 months. a chest x - ray showed a right pleural plaque that subsequently caused a right pleural effusion (fig. 1). he declined chemotherapy or radiotherapy, although he was having regular follow - up by the oncologist. he confirmed being exposed to asbestos, having worked for 20 years in aluminum and margarine factories. eighteen months after his initial presentation, he experienced progressive left - sided weakness and left - sided homonymous hemianopia. an mri scan of his brain showed extra - axial right occipitoparietal enhancing mass (43 43 47 mm) with surrounding vasogenic oedema. it was isointense to grey matter on t2 and abutting the falx cerebri with midline shift and effacement of the right lateral ventricle (fig. 2). the lesion was approached through a right occipital craniotomy and entirely removed in a piecemeal fashion. at surgery, it appeared as a greyish - white firm vascular mass with a clear cleavage plane with the surrounding brain. the patient had good recovery and was discharged home. on 6-week follow - up, the patient continued to improve with significant improvement in his quality of life. figure 2:mri image of brain with gadolinium contrast showing enhancing mass in the right occipital lobe. mri image of brain with gadolinium contrast showing enhancing mass in the right occipital lobe. section from the intraoperative specimen showed a large cell - epithelioid malignancy composed of markedly pleomorphic epithelial cells with large hyperchromatic nuclei. immunoperioxidase stains demonstrated in fact that the cells expressed vimentin, calretinine, ae1/ae3 and cam 5.2 as in the previously biopsied pleural lesion. histologically mesotheliomas may be purely epithelioid (50%) or of mixed histology (30%). more than 80% of tumors arise from the pleura ; however, primary mesothelioma of the peritoneum, pericardium and tunica vaginalis have also been reported. the risk of developing mesothelioma after asbestos exposure is 8%, depending on the duration and intensity of exposure. malignant pleural mesothelioma usually presents between the fifth and seventh decade of life and can be difficult to diagnose due to the vagueness of the presenting symptoms including dyspnea and nonpleuritic chest pain. the median survival is only 9months from diagnosis with death usually due to thoracic disease. this rapid progression may have contributed to the belief that mesothelioma rarely has distant metastasis. reviewed 171 cases of malignant mesothelioma at autopsy and discovered that over 54% of patients had distant metastases. the sites most commonly affected were the liver, adrenal glands and kidneys (56, 31 and 30%, respectively). wronski and burt also reviewed the post - mortem findings and they concluded that the incidence of distant metastasis from pleural mesothelioma is around 50% and the incidence of cerebral metastasis around 510%. although there are numerous reported cases of cerebral metastasis from malignant pleural mesothelioma, the majority of cases were post - mortem findings. furthermore, only eight patients had neurological manifestation of cerebral disease at the time of diagnosis [3, 6, 8 ]. the case we report is interesting for the presentation of the intracranial pathology 18months after the initial presentation, well beyond the reported median survival for this lesion. the good postoperative neurological recovery also emphasizes the role of surgery to extend survival in the presence of locally controlled primary disease. to the best of our knowledge, this case represents the third report of symptomatic cerebral metastases from malignant mesothelioma that was treated by surgical excision via craniotomy, where ante - mortem histological diagnosis was obtained. with an increasing incidence of mesothelioma, the treatments available are also expanding. in the past, single - modality therapy alone (surgery, radiotherapy and chemotherapy) has failed to improve survival significantly. furthermore, several new therapeutic techniques, such as photodynamic therapy, immunotherapy and intracavitary chemotherapy, are increasing survival and retarding disease progression. we present this case to demonstrate that metastatic mesothelioma can have distant metastasis to the brain. as a direct result of improvements in treatment, the survival of patients with malignant mesothelioma will continue to improve, and more patients are likely to present with metastatic disease. as we have demonstrated in this case, surgical excision of cerebral metastases can provide symptomatic relief, and surgical treatment may therefore be justified in enhancing the patient 's quality of life. | malignant mesothelioma is an uncommon, highly invasive tumor derived from the mesothelial cells of pleura or peritoneum characterized by poor outcome. mesothelioma was thought to metastasize locally only via direct invasion and not have distant spread. distant metastases were discovered mostly on post - mortem examination. the authors present a case of 62-year - old man with pleural mesothelioma and brain metastasis. |
neglect frequently appears due to brain damage and impedes everyday life as well as rehabilitation1. found that stroke patients with neglect showed postural instability more clearly compared to those without neglect2. the syndrome frequently occurs in left hemiplegic patients because while the right hemisphere of the brain recognizes left and right spaces, the left hemisphere recognizes only right spaces. damage to the left hemisphere can be compensated by the right hemisphere, while damage to the right hemisphere can not be compensated by the left hemisphere but the converse is not true3. stroke patients with neglect show diverse kinds of damage to their eye movements, such as decreased saccadic movements and difficulties in space observation4. hornak showed that patients with neglect showed difficulties in perceiving and drawing pictures placed in the left visual field and that stimulating these affected visual fields was important5, kerkhoff. reported that optokinetic stimulation activates many regions involved in vision and hearing such as the temporo - parietal cortex, basal ganglia, brain stem, and cerebellum they emphasized the importance of optokinetic stimulation for patients with damage to the nervous system6. the roles of vision, the vestibular system, and the somatosensory system are very important in postural control. visual inputs provide important information about one s environment, postures, and head movements7, 8. in the present study, proprioceptive neuromuscular facilitation (pnf) training was implemented in combination with eye movements in order to induce head movements. pnf can activate the neuromuscular system by stimulating the proprioceptors in the muscles and tendons thereby activating or suppressing certain muscle groups9. the main characteristic of pnf patterns is the occurrence of spiral and diagonal movements. kim and oh reported that neck proprioceptive training has a good chance of improving the balance function of stroke patients with hepiparesis12. although the importance of eye and head movements for postural control has been emphasized, most previous studies have been conducted on healthy adults8, 13. the present study was conducted to examine the effects of eye movement and pnf training in neglect patients. the present study was conducted on 20 patients in two rehabilitation hospitals located in gyeonggi - do. patients who were diagnosed at least six months previously with hemiplegia due to stroke, had at least moderate neglect symptoms, and had a total score of at least 11 points on the catherine bergego scale (cbs) were selected14. the study was limited to those who had scored not lower than 24 points on the korean version of the mini - mental state examination (mmse - k), and who could carry out instructions15, had no visual or hearing impairment, and could stand independently for at least one minute. the subjects agreed to participate in the study after receiving explanations regarding its purpose and procedures. the protocol for this study was approved by the local ethics committee of yongin university (2 - 1040966-ab - n-01 - 201503-hsr-025 - 1). the general characteristics of the subjects and their cbs values are summarized in table 1table 1.general characteristics of subjectsem grouppem groupgendermale4 5 female65age (years)60.27.8561.18.17time since stroke (months)22.210.2123.37.00stroke typeinfarction53hemorrhage57affected sideleft1010right00cbs (score)13.11.6613.11.60. balance ability, static balance, and dynamic balance were measured using a biorescue apparatus (sycomore, france). subjects stood on the footplate with their feet spread at an angle of approximately 30 while looking straight ahead. static balance was measured by measuring the sway length and sway area while standing for one minute with eyes open and eyes closed. dynamic balance was measured as the limit of stability (forward / backward and left / right). lower values for static balance and higher values for dynamic balance denoted better balance ability. head alignment was measured as the craniovertebral angle (cva) and cranial rotation angle (cra) using the global postural system (gps) (fig. smaller values for cva indicated increased bending of the lower cervical vertebrae and larger values for cra indicated increased extension of the upper cervical vertebrae leading to upward turning of the head. measurement of cva and cra the eye movements program used cards and comprised four steps : intermittent saccadic eye movements, pursuit eye movements, adapting movements 1, and adapting movements 2 (table 2table 2.eye movements exercise programstagecontentsaccadic eye exercisewith the subject s face stationary, the therapist held up a card in each hand at a distance of 30 cm from the subject s eyes and the subject looked at the card in the two hands alternately. pursuit eye exercisewith the subject s face stationary, the therapist repeatedly moved a hand - held card from the left to the right, and from the right to the left, at a distance of 30 cm from the subject s eyes and the subject kept their eyes on the card, moving their eyeballs to follow the card. adaptation exercise 1the therapist held a card in one hand at a fixed distance of 30 cm from the subject s eyes and the subject repeatedly turned their head from the right to the left, and from the left to the right, while keeping their eyes on the card. adaptation exercise 2the therapist held a card in one hand at a fixed distance of 30 cm from the subject s eyes. the subject repeatedly turned their head from the right to the left, and from the left to the right, while keeping their eyes on the card and the therapist moved the card in the direction opposite to the direction of the movement of the subject s head while the subject s head was turning. 2.eye movements exercises using a card1 : saccadic eye exercise, 2 : pursuit exercise, 3 : adaptation exercise 1, 4 : adaptation exercise 2)13. the movements in each step were performed 10 times, constituting one set, and two sets were performed in total. eye movements exercises using a card 1 : saccadic eye exercise, 2 : pursuit exercise, 3 : adaptation exercise 1, 4 : adaptation exercise 2 with the subject s face stationary, the therapist held one card in each hand at a distance of 30 cm from the subject s eyes. next, the therapist held a card stationary at the same distance from the subject s eyes. the subject repeatedly turned their head from right - to - left and from left - to - right while maintaining their gaze on the card. next, the therapist held a card at a fixed distance of 30 cm from the subject s eyes. the subject repeatedly turned their head from right - to - left and from left - to - right while keeping their eyes on the card, with the therapist moving the card in the direction opposite to the direction of the head movement. the pnf training was implemented using the neck extensor pattern while the subject was in a sitting position. the movements began with the contraction of the neck extensor group by neck flexion, right rotation, and left lateral flexion, and ended with neck extension involving left rotation and right lateral flexion. the therapist verbally instructed the subject to push their head in the opposite direction, with another instruction to the subject to move their eyes upward along with the movement of the head. the contract - relax training was implemented by performing the movements 10 times as a set and repeating the set three times with a rest period of two minutes after each set. the pnf training was implemented by one researcher who had completed pnf level i and level ii courses. the mean and standard deviation of the general characteristics were calculated using descriptive statistics. anova was used to evaluate the changes in balance and head alignment. in all analyses, measurements of balance and head alignment were performed before and after testing. in measurements of static balance, the em group showed significant differences in sway length and sway area in the eyes - open condition (p0.05). the pem group, however, showed significant differences in both these conditions (p0.05). however, the pem group showed significant differences in both measurements (p0.05). in measurements of dynamic balance, both the em and pem groups showed significant differences after intervention (p0.05). we conclude therefore that eye movements alone have no positive effect on head alignment. however, the pem group showed significant differences in both the craniovertebral angle and the cranial rotation angle (p<0.05). sarig - behat reported that proprioceptive training is effective for mechanical problems in the neck region21. this indicates that pnf training has stimulated the proprioceptors, leading to positive effects on head alignment. hindle. reported that the contract - relax method in pnf is effective for the improvement of the range of motion22 and they concluded that pnf training has a positive effect on the range of motion of cervical vertebrae. in the present study, the eye movements had positive effects on static balance with the eyes open, and on dynamic balance, but they had no positive effects on static balance with the eyes closed, or with head alignment. however, pnf training with eye movements had positive effects on both static and dynamic balance as well as on head alignment. therefore, we conclude that for neglect patients, pnf training with eye movements is a more effective intervention than eye movements alone. since the present study was conducted with only 20 neglect patients, the results can not be generalized to all neglect patients. nevertheless, our study demonstrates the importance of eye and head movements for postural control. more extensive studies should now be done to test the effectiveness of this treatment for neglect patients. | [purpose ] the purpose of this study was to assess the effect of eye movements and proprioceptive neuromuscular facilitation (pnf) on patients with neglect syndrome. [subjects and methods ] the subjects were randomly allocated to 2 groups : the eye movements (em) group ; and the pnf with eye movements (pem) group. the program was conducted five times each week for 6 weeks. balance (both static and dynamic) and head alignment (craniovertebral angle and cranial rotation angle) were measured before and after testing. [results ] in measurements of static balance, the em group showed significant improvement in sway length and sway area when examined in the eyes - open condition, but not when examined in the eyes - closed condition. the pem group showed significant improvement when examined under both conditions. in the assessment of dynamic balance, both groups showed significant improvement in measurements of sway areas. with respect to head alignment, there were no significant differences pre- and post - testing in either the craniovertebral angle or the cranial rotation angle in the em group, but the pem group showed significant differences in both measurements. [conclusion ] these results suggest that in stroke patients with neglect syndrome, pnf with eye movements, rather than eye movements alone, has a greater positive effect on balance and head alignment. |
the increase in the incidence of solid organ transplantation has led to the need for highly potent immunosuppressive drugs for the prevention of allograft rejection and autoimmune conditions. mycophenolate mofetil (mmf or cellcept) is commonly used for maintenance of immunosuppression and the treatment of ongoing rejection in organ transplant patients. its effectiveness is comparable to other immunosuppressive agents such as tacrolimus, cyclosporine and azathioprine, yet it holds a lower toxicity profile, making it an attractive alternative agent in allograft rejection. gastrointestinal mucosal injuries related to mmf use are common and include those related to suppression of immune function (infectious) and effects due to drug interaction and metabolism (suppression of de novo purine synthesis). afebrile diarrhea is the most frequently reported primary manifestation of toxicity related to mmf, with an incidence in renal transplant patients ranging from 12 to 40%. while diffuse colitis has been described as a typical endoscopic feature of mmf use, its presentation as segmental colitis is unknown. we report the rare case of segmental colitis in a patient with autoimmune autonomic dysfunction treated with mmf. our patient presented unusually with abdominal pain and diarrhea after a recent increase of his mmf dose. a 64-year - old gentleman with a history of autoimmune autonomic dysfunction and gastroparesis on a background of type 2 insulin - dependent diabetes mellitus presented to our hospital with abdominal pain and diarrhea. the patient was initially started on mmf 1,000 mg twice daily 2 years prior for autoimmune autonomic dysfunction. this was subsequently raised to 1,500 mg twice daily 4 months preceding the patient 's presentation. the diarrhea had started 2 months prior to presentation, with up to eight episodes of non - bloody diarrhea per day. abdominal pain was of recent onset with a 4-day history of lower abdominal stabbing pain most severe in the left lower quadrant and reported as constant and progressive in nature. he also had decreased appetite, weight loss, sensation of bloating and intermittent nausea. on examination, his vital signs were normal, and he had diffuse abdominal tenderness with increased intensity in the left lower quadrant area. initial investigations revealed an elevated white blood cell count of 17.9 10 cells / l (normal 3.510.5 an elevated creatinine of 1.8 mg / dl (normal 0.61.2 mg / dl) was also noted. further work - up with ct of the abdomen and pelvis with contrast revealed segmental wall thickening and pericolic inflammation at the splenic flexure and proximal descending colon without evidence of diverticulitis. flexible sigmoidoscopy revealed a segmental erythematous mucosa and multiple ulcers in the sigmoid colon, descending colon, splenic flexure and proximal transverse colon, suggesting a mucosal injury pattern consistent with ischemic colitis (fig. 1). however, biopsies showed dilated damaged crypts, eosinophilic epithelial changes and crypt abscesses with apoptotic bodies, a pattern of injury highly suggestive of mmf - related colitis (fig. mmf therapy was subsequently discontinued and the patient was discharged following improvement of symptoms with follow - up in an outpatient clinic 5 weeks later. during this visit, his abdominal pain and diarrhea had improved rapidly and significantly. he is scheduled for a repeat colonoscopy in 4 months time to assess the extent of mucosa recovery. while the diffuse pattern of mucosal injury with mmf use has been described previously, segmental mucosal injury similar to ischemic colitis has not been reported to date. our case is interesting due to the fact that despite endoscopic appearance of ischemic colitis in a segmental fashion, histology did not show any changes suggestive of ischemia, but rather suggested mmf - related injury, which was confirmed by resolution of symptoms following discontinuation of the offending agent. the hallmark for the diagnosis of mmf - related colitis is increased epithelial cell apoptosis, which can be accompanied by an inflammatory bowel disease - like histological pattern. complicating the diagnosis is the similarity of this pattern to acute intestinal graft - versus - host disease and inflammatory bowel disease, and diagnostic distinction is critical in these patients, as mmf - related colitis is managed with reduction in dosage, while graft - versus - host disease and inflammatory bowel disease should be managed with immunosuppression, such as mmf. several histological features suggestive of mmf injury include crypt architectural disarray, lamina propria edema, increased lamina propria inflammation, dilated damaged crypts and increased crypt epithelial apoptosis. in addition to preventing allograft rejection, mmf is also used to treat autoimmune conditions, including psoriasis, rheumatoid arthritis and autoimmune uveoretinitis. mmf is converted to mycophenolic acid, which non - competitively inhibits the inosine monophosphate dehydrogenase enzymes required for purine synthesis in b and t lymphocytes. this subsequently causes a reduction in humoral and cytotoxic t cell response to immunogenic stimuli. although the exact pathogenesis is unknown, gastrointestinal mucosal injury is thought to occur due to insult of enterocytes and the formation of toxic immunogenic reactions in the bowel. enterocyte damage arises as 50% of these cells use the inosine monophosphate dehydrogenase pathway for de novo purine metabolism. increased incidence of duodenal villous atrophy has been reported with chronic mmf use, and injury via infective etiologies due to immunosuppression may also occur in addition to mmf - related colitis. however, the mechanism for segmental mucosal injury, as seen in our case, is not clear. we speculate that the disease process begins as a segmental colitis, as seen in our case, and then progresses to diffuse colitis, as reported in previous studies. clinically, mmf - related colitis commonly manifests as chronic diarrhea unresponsive to antibiotics or steroid therapy, with improvement of symptoms following reduction or cessation of mmf. other presenting symptoms may include nausea, vomiting, abdominal colic, gastritis, gastric ulcers and intestinal perforation. patients with poor responses 6 months following discontinuation of therapy may require other alternative strategies, including surgical intervention for curative treatment. in summary, mmf - induced segmental colitis is an uncommon but important clinical condition in patients presenting with chronic diarrhea. early recognition of this entity along with discontinuation of mmf may result in improved clinical outcome. | mycophenolate mofetil (mmf) is a commonly used drug in the prevention of allograft rejection in patients with solid organ transplants. although diffuse colitis has been described in mmf - related colitis, segmental colitis has not been reported. we report the case of a 64-year - old male on mmf therapy who presented for evaluation of afebrile diarrhea and abdominal pain. flexible sigmoidoscopy revealed a segmental erythematous mucosa with ulceration in the sigmoid colon, descending colon, splenic flexure and proximal transverse colon. biopsies of these areas showed dilated damaged crypts, eosinophilic epithelial changes and crypt abscesses with apoptotic bodies consistent with mmf - induced injury. mmf was discontinued, leading to a significant improvement of his symptoms. |
postprandial hypotension (pph) was defined in the past as a fall in systolic blood pressure of 20 mmhg within 2 hours of eating a meal, comparable with the definition of postural hypotension. a recent study using sphygmomanometer readings has shown that in subjects with hypertension the cutoff should be a fall of 30 mmhg and not 20 mmhg in systolic pressure. potential symptoms of a fall in blood pressure include dizziness, syncope, and falls. it has been described frequently in elderly individuals, with a higher incidence in certain risk groups, namely, in 24%33% of elderly residents of nursing homes, in almost 50% of elderly patients with unexplained syncope and in 67% of hospitalized geriatric patients. risk groups are patients with autonomic dysfunction in diabetes mellitus, hypertension [7, 8 ], alzheimer 's disease and parkinson 's disease [10, 11 ] although pph has been reported to occur in 33% of healthy individuals. in the long term, pph is associated with an increased incidence of falls, syncope, new coronary events, new stroke, and higher total mortality. the pathophysiology of pph is probably multifactorial, possibly involving an attenuated baroreflex, an attenuated reflex increase in sympathetic activity by activation of stretch receptors in the stomach (gastrovascular reflex), sympathetic dysfunction (e.g., autonomic neuropathy in diabetes mellitus, parkinson 's disease), and patients with an incapability to increase cardiac output due to heart failure or any combination of these factors. there is no consensus on the test conditions that should be used to diagnose pph. the characteristics of the actual decrease in blood pressure in pph are not clearly defined : when does blood pressure start to decrease after a meal, how long does the decrease last, and what is the magnitude of the decrease in blood pressure ? it is not clear what time interval and which method of blood pressure measurement should be used. in most studies, blood pressure was measured with a sphygmomanometer at intervals varying from 3 up to 60 minutes (table 1) [3, 5, 1621 ]. if the frequency of measurements is too high, participants are likely to experience discomfort. some authors used continuous blood pressure measurements (finger arterial blood pressure) [11, 22, 23 ], which might be uncomfortable during prolonged measurements. in most studies the aims of this study were to establish the prevalence, duration, and association with symptoms of pph in patients admitted to a geriatric ward, compared with healthy elderly individuals. we measured beat - to - beat blood pressure and tested different intervals between blood pressure measurements to diagnose postprandial hypotension adequately. consecutive patients admitted to the geriatric ward of the university medical center utrecht over 6 months were eligible for inclusion. this ward serves elderly patients with a wide range of acute and chronic diseases and referrals for diagnostic work - up or therapy. inclusion criteria were being able to give informed consent and to walk (with or without walking aid). exclusion criteria were myocardial infarction less than 3 months earlier, any acute illness, uncontrolled metabolic disease, resting systolic blood pressure more than 200 mmhg, cardiovascular disease (aortic stenosis, intermittent claudication, and angina pectoris), dysphagia, life expectancy less than 3 months, and use of medication that affects blood pressure and which could not be discontinued for 24 hours. healthy elderly individuals, recruited by advertisement in a weekly newspaper, were screened by telephone and underwent a physical examination before being matched for age with the geriatric patients. all subjects were healthy according to modified exclusion criteria defining medically stable elderly subjects for exercise studies. subjects who were taking medications that affect blood pressure that could not be withdrawn for at least 24 hours were excluded as well. blood pressure was recorded using portapres (tno - tpd biomedical instrumentation, netherlands organization for applied scientific research). portapres is a non - invasive method to record beat - to - beat blood pressure alternately from two adjacent fingers and is accurate for measuring changes over time, but not for measuring absolute values. the method is based on the volume - clamp method of pez and the physiocal criteria of wesseling [26, 27 ]. it also measures heart rate and hydrostatic height of the hand (to correct for pressure changes due to vertical heart - hand distance changes). beatscope software was used to analyze the measurements (beatscope 1.0 ; tno - tpd biomedical instrumentation netherlands organization for applied scientific research). twenty - four hours before the measurements, all medication that affects blood pressure was withdrawn. after an overnight fast, the subjects were allowed to take their regular medication (with the above exception) with some water 2 hours before breakfast. they could select the ingredients of their continental breakfast, to mimic normal conditions as closely as possible, but were offered weak tea, because caffeine in coffee might affect blood pressure. after breakfast, subjects were not allowed to eat or drink until measurements were completed. blood pressure was measured after a supine period of 15 minutes and from 15 minutes before until 135 minutes after the start of breakfast, with subjects in sitting position. to avoid vasoconstriction - related inconsistency in measurements, the hand connected to the portapres device was placed on a warm cherry stone pillow. symptoms or complaints associated with pph (light - headedness, dizziness, tiredness, hazy vision) were scored every 15 minutes on a 3-point scale (absent, moderate, or severe). the study protocol was approved by the medical, ethical testing committee of the university medical center utrecht. differences between the groups at baseline and maximum changes from baseline were determined with a mann - whitney u test. the chi - square and fisher exact tests were used for comparison of the presence of pph with pph - related symptoms. in this study, pph was defined as a decline in systolic blood pressure of at least 20 mmhg, determined by calculating the difference between the minimum systolic blood pressure values before and within 120 minutes after the start of breakfast. according to the results of a recent study, we also analyzed the presence of pph defined as a fall in systolic blood pressure of 30 mmhg or more. because we were not interested in beat - to - beat blood pressure variations the start of pph and the time of maximal fall in blood pressure are expressed in minutes after the completion of breakfast. to determine the minimum interval between blood pressure measurements for diagnosis of pph, the maximum decrease in blood pressure was calculated for different intervals between measurements, which was done by omitting 1 or more of the 2-minute blood pressure measurements from calculations. during the study period, 101 patients were admitted to the geriatric ward, 68 of whom were excluded because they met one or more of the exclusion criteria (in most cases because they suffered form an acute illness). five patients did not give consent, and five others could not participate because of technical difficulties. they were admitted because of metabolic disorders (4 patients), mobility disorders (4), anemia (3), parkinsonism (3), delirium (2), syncope (2), or miscellaneous (4). resting blood pressure was 140/65 mm hg (sd 27/15, range 104200/3190) and heart rate 67/min (sd 10, range 4080). of these patients, 45% had a history of cardiovascular disease (other than hypertension), 32% had a history of hypertension (partially overlapping the group 45% of subjects with cardiovascular disease), 27% had parkinson 's disease (all were hoehn and yahr stage 2 to 3), 18% had a history of cerebrovascular disease, and 14% had diabetes mellitus. of the 53 healthy elderly subjects who responded to the advertisement, 20 were excluded because they met one or more of the exclusion criteria, 2 did not give consent after reading the patient information, and 11 were excluded because of age mismatching. there were no differences in baseline characteristics between the two groups (table 2). of the healthy elderly individuals, 23% had with hypertension ; other diseases were not present. figure 1 shows mean systolic and diastolic blood pressure, and heart rate during and after breakfast of individuals. there were no significant differences between the two groups, and blood pressure and heart rate increased in both groups during the meal. the increase in diastolic blood pressure and heart rate tended to be higher in the healthy elderly group (p =.05 and p =.13, resp.). after the meal, systolic and diastolic blood pressure decreased below baseline values in both groups, but the fall in systolic blood pressure was significantly greater in the patient group (mean values over period 4060 minutes after the start of breakfast : 24.1 19.6 versus 9.6 15.8 mmhg ; p =.01). thus pph, defined as a systolic fall of 20 mmhg or more, occurred in 20 (91%) of 22 patients and in 8 of 20 (40%) healthy elderly individuals (p =.001, mwu test). pph defined as a fall of 30 mmhg or more was present in 10 (45%) patients and three (15%) healthy persons (p 30 mmhg. to calculate the sensitivity and specificity of pph - related symptoms for the diagnosis of pph, we considered patients to be symptomatic if at least one of the symptoms was scored as moderate during the test. the sensitivity of pph - related symptoms for a significant decrease in blood pressure was 17/28 = 0.61, the specificity was 13/14 = 0.93, the positive predictive value was 17/18 = 0.94, and the negative predictive value was 13/24 = 0.54. the presence of pph - related symptoms was strongly associated with a decrease in blood pressure greater than 20 mmhg (p 30 mmhg, an association could not be found. the sensitivity of pph - related symptoms for a significant decrease in blood pressure was 7/13 = 0.53, the specificity was 18/29 = 0.62, the positive predictive value was 7/18 = 0.39, and the negative predictive value was 18/24 = 0.75. also an association between supine systolic hypertension > 160 mmhg in rest and pph could not be found. only four patients and one healthy subject had a supine blood pressure > 160 mmhg after 15 minutes rest ; three of them had pph. we found pph to be present in a high number of frail elderly patients, but also in a considerable number of healthy elderly. the prevalence of pph we found in elderly patients (91%) is higher than previously reported [3, 7, 30 ]. recently a study suggested that the cutoff of orthostatic fall in the elderly should be 30 mmhg in systolic pressure or more and not 20 mmhg. using this cutoff value, the prevalence of pph in our study is 45% in the patient group and 15% in the healthy group. the higher prevalence in the patient group is probably inherent on the comorbidity of this group. there was considerable variability in the time of onset, duration, and maximum decrease in blood pressure in the subjects with pph, which supports the hypothesis that pph is not caused by the failure of a single mechanism, but rather by the failure of several, and that there is considerable variation between subjects. despite this variability, pph started within an hour of eating breakfast in 18 of 20 patients (90%). assuming that changes in systolic blood pressure measured with a sphygmomanometric device are similar to the 2-minute mean portapres values that were used in this study, blood pressure measured at 6-minute intervals would have identified at least 18 patients with pph (90%) ; measurement with 10-minute intervals would have identified at least 17 patients (85%) with pph (figure 2). however with definition of pph as > 30 mmhg systolic fall, measuring blood pressure every 6 minutes will miss pph in one of four and with 10 minutes interval in one of three patients. beat - to - beat measurements will increase the number of short - lasting falls in postprandial blood pressure detected relative to the number detected when blood pressure is measured with a sphygmomanometer. however, in general practice, this method is not attractive because continuous blood pressure measurements are comprehensive, require skill to get reliable results, and require relatively expensive equipment. for this reason, automatic sphygmomanometer measurements, with devices such as spacelab, are preferred (table 1). in this study, the subjects ate a continental breakfast, in which the release of glucose is quite constant whereas other studies used standardized liquid meals, in which the release of glucose is rapid. moreover, we hypothesize that differences in the severity of pph observed with high and low carbohydrate - containing meals can be explained by differences in the rate of glucose uptake rather than by differences in the total number of calories consumed, a hypothesis that is supported by the findings of o'donovan.. they found that the fall in systolic blood pressure was greater with a higher rate of glucose delivery into the duodenum. despite the probably relatively low rate of glucose delivery with our continental breakfast, a large proportion of our subjects developed pph. we chose to withdraw medication that affects blood pressure for 24 hours to eliminate transient effects of medication that could occur directly after ingestion. however, we can not rule out the possibility that these medications affect the mechanism of pph. we calculated mean values of blood pressure over 2-minute periods and may thus have missed a short - lasting fall (less than 2 minutes) in blood pressure. however, such a short - lasting fall in blood pressure may not be of clinical relevance. it was our goal to mimic the normal physiological condition by using a continental breakfast instead of standardized meal and to allow subjects to choose their breakfast. because subjects were not active after they finished their breakfast, we can not rule out that the fall in blood pressure was the effect of drowsiness or even sleep as might occur during the period of inactivity after the meal, adding to the occurring hypotension, although a researcher was constantly in the room and a symptom - questionnaire was evaluated every 15 minutes, limiting the presence of drowsiness and its potential influence. there is currently no standardized clinically meaningful definition of pph. in the current study, it is surprising that blood pressure fall up to more than 30 mm hg is still well tolerated in six subjects (3 patients and 3 healthy subjects) without any symptoms. the mechanism(s) mediating the effects of meal on the hypotensive response remain uncertain, and there are a number of possibilities which warrant further exploration. symptoms due to reduced cerebral perfusion can be manifested as dizziness, light - headedness, weakness, dyspnea, sweating, or syncope. cerebral autoregulation is ineffective when the blood pressure falls to < 70 mmhg, and here the cerebral perfusion might be severely compromised. however, patients with chronic orthostatic hypotension may have an expansion of their autoregulated range to lower bp. such patients and perhaps elderly patients need much greater fall in blood pressure than 30 mmhg. as neurogenic orthostatic hypotension, severely afflicted patients are unable to stand without experiencing symptoms. we can not, however, exclude the possibility that there is some kind of autonomic dysfunction in this very elderly group. aging, leading to abnormalities of parasympathetic and sympathetic control of heart rate and blood pressure, may be associated with concomitant autonomic afferent sensory fiber neuropathy. degeneration or dysfunction of these afferent fibers may result in interruption of pain or other autonomic disorders sensations. reduced appreciation for ischemic events such as pain or dizziness can impair timely recognition of ischemia or infarction, thereby delaying appropriate therapy. hence, patients who have no symptoms of this hypotension could be jeopardized because the longer threshold permits them to continue the event (meal) despite increasing ischemia. in this study, we did not search for signs of cardiovascular pathology, and we did not follow our patients and the healthy subjects up. therefore we do not know the prognostic significance of pph in this study groups. from the clinical perspective, it is important to measure blood pressure following a meal in patients who have unexplained syncope and whose orthostatic stress test result is normal. reduced fluid and food intake and common medical conditions in the elderly, such as gastrointestinal disease (malabsorption syndromes), can cause dehydration. dehydration and the use of diuretics or other agents may reduce the cardiac output in the elderly. in subjects with pph, these factors might induce severe hypotension, and this is a potential trigger for ischemic stroke or acute myocardial infarction. in conclusion, pph occurs in a high number of frail elderly patients admitted to a geriatric ward. measuring blood pressure every 10 minutes, starting from 15 minutes before until 60 minutes after the end of a regular breakfast, using a sphygmomanometer, will detect about 70% of geriatric patients with pph. this seems a patient friendly, practical, and adequate way to evaluate pph in clinical practice. the presence of postprandial complaints during the measurement is not a good indicator of the existence of pph. | the aims of this study were to find out whether postprandial hypotension (pph) occurs more frequently in patients admitted to a geriatric ward than in healthy elderly individuals, what the optimal interval between blood pressure measurements is in order to diagnose pph and how often it is associated with symptoms.the result of this study indicates that pph is present in a high number of frail elderly, but also in a few healthy older persons. measuring blood pressure at least every 10 minutes for 60 minutes after breakfast will adequately diagnose pph, defined as > 20 mmhg systolic fall, in most patients. however with definition of pph as > 30 mmhg systolic fall, measuring blood pressure every 10 minutes will miss pph in one of three patients. with the latter definition of pph the presence of postprandial complaints is not associated with the existence of pph. |
the disparity between in vitro silica cytotoxicity toward macrophages and their in vivo resistance to injury following inhalation of silica at physiologic concentrations is unresolved. it is probable that inhaled silica particles absorb a variety of biological substances including proteins and alveolar lining material (alm) thus altering the in vivo response of the macrophage to these particles. silica (si) particles coated with rat alm and uncoated si particles were studied for their ability to injure rat alveolar macrophages (am) in vitro. suspensions of particles were tested at concentrations from 0 to 400 micrograms per 2 x 10(6) cells. cytotoxicity was assessed by the percent of total cellular lactate dehydrogenase (ldh) released by am into the culture medium during incubation. comparable physical association by alm - coated and uncoated si particles with am was shown by scanning electron microscopy combined with x - ray energy spectrometry. these data show that si coated with alm is effectively phagocytosed by am in vitro but is much less cytotoxic than uncoated si. the surfactant lipids which presumably coat inhaled si particles in the lung may reduce or delay their toxicity for am.imagesfigure 1.figure 2. |
|
papillary thyroid carcinoma (ptc) is the most common form of malignant thyroid neoplasm. its diagnosis is based on nuclear features such as nuclear clearing, overlapping, grooves and pseudoinclusions. however, identification of these features remains, at times, difficult because of its focal presence and thus the distinction of ptc from other thyroid lesions may not be possible., follicular variant of papillary thyroid carcinoma (fvptc) is the most common one, which is characterized by an almost exclusive follicular growth pattern and a set of nuclear features identical to those of the usual type of papillary thyroid carcinoma (utptc). diagnostic dilemma may arise when an encapsulated nodule with a follicular pattern of growth exhibits clear nuclei with grooves or darkly staining colloid and distinguishing follicular adenoma (fa) from encapsulated fvptc becomes difficult. there are several other thyroid lesions that may contain papillary processes with nuclear features in a focal manner, which pose diagnostic difficulties with ptc. multinodular goiter (mng) with delicate papillary budding and focal nuclear clearing may be confused with ptc. several immunohistochemical stains have been investigated for their possible role as diagnostic markers for ptc. they are cytokeratin19 (ck19), hbme1, galectin-3 and ret and thyroid transcription factor 1.[49 ] although galectin-3 was initially shown to have utility in the differential diagnosis between benign and malignant thyroid lesions, recent studies suggest that it is not reliable.[1214 ] several studies have shown conflicting results regarding the usefulness of ck19 as a diagnostic marker in ptc.[1320 ] this study was carried out to investigate the role of ck19 as a possible diagnostic marker of ptc and its utility in differentiating ptc from the above - mentioned thyroid lesions. forty - two cases were collected for a period of 2 years and 6 months. immunohistochemical stains were performed on 5-m - thick paraffin sections taken on poly - l - lysine - coated slides. enzyme pretreatment was done for ck19 staining by incubating the sections with pepsin at 37c for 5 min for antigen retrieval. semi quantitative scoring of ck19 expression according to the percentage of positively stained cells all the statistical analyses were performed with stastistica version 6.0 (statsoft inc. ; 2001, tulsa, ok, usa) and windows software. twenty - two cases were ptc, of which eight cases were follicular variant ptc, a single case was diffuse sclerosing variant and the remaining 13 were utptc. regarding age distribution, on applying the non - parametric kruskal - wallis test in the three groups (ptc, mng and fa), there was no difference in median age distribution. median age was taken because there was variation in the number of samples. on analyzing the sex distribution of ptc, it was observed that 19 cases (86.36%) were females and three cases (13.64%) were males, with a male : female ratio of 1:6.3. in mng and fa, ck19 expression was seen in all 22 ptc (100%) in a diffuse manner, whereas only four cases of mng and six cases of fa showed focal expression. this observation was highly significant (chi - square test 2-tailed p value=0.003) [table 2 ]. presence / absence of cytokeratin 19 expression in papillary thyroid carcinoma, follicular adenoma and multinodular goiter when ck 19 semiquantitative score was analyzed, 17 out of 22 (77.27%) ptc showed 4 + positivity and the remaining five (22.73%) expressed 3 + positivity. only four cases out of eight mng (50%) focally expressed ck19, two with 2 +, one with1 + positivity and only one showed 3 + positivity. six cases out of eight fa (75%) were also focally positive for ck19, five (62.50%) with 1 + positivity and a single case showed 2 + positivity. taking ck19, 4 + as a cut - off value for positivity for detecting ptc versus mng and fa, there was 77% sensitivity with a very high specificity and positive predictive value each of 100% [table 3 ]. semiquantitative expression of ck19 in papillary thyroid carcinoma, follicular adenoma and multinodular goiter comparing usual type ptc and follicular variant ptc, it was observed that in utptc, 4 + positivity was seen in 11 of 13 cases (84.62%) and the remaining showed 3 + positivity [figure 1 ]. in cases of fvptc, it was found that five of eight (62.50%) showed 4 + positivity and the remaining three showed 3 + positivity (37.50%) [figure 2 ]. applying the fisher exact test seventy - five percent of fa showed ck19 focal expression of weak intensity [figure 3 ]. 3 + or 4 + expression in fa and all the cases of fvptc showed either 3 + or 4 + ck19 expression, using the non - parametric chi - square test, the 2-sided p value was found to be 0.001, which was highly significant. all the cases of utptc showed either 4 + or 3 + expression, whereas fa showed only 1 + or 2 + positivity. thus, semiquantitative ck19 score was found to be useful to differentiate fa from both utptc and fvptc [table 4 ]. (a) a usual type papillary thyroid carcinoma (hematoxylineosin, lp), (b) a usual type papillary thyroid carcinoma, immunostaining for cytokeratin 19-diffuse and strong (4 +) positive (ihc, lp, inset hp) (a) a follicular variant papillary thyroid carcinoma with typical nuclear features (hematoxylin - eosin, hp) (b) a follicular variant papillary thyroid carcinoma, immunostaining for cytokeratin 19 (ihc, hp) (a) photomicrograph of a follicular adenoma (hematoxylineosin, lp) (b) a follicular adenoma, immunostaining for cytokeratin 19, focal positive (1 +) (ihc, lp) semiquantitative expression of ck19 in usual type papillary thyroid carcinoma, follicular variant papillary thyroid carcinoma, follicular adenoma and multinodular goiter in mng, only 50% (4/8) cases showed ck19 expression. only one case of mng showed 3 + positivity and all the remaining showed either 1 + or 2 + positivity (three cases) or negative staining (four cases) [figure 4 ]. however, 84.62% (11/13) of utptc expressed 4 + positivity and the remaining 15.38% showed 3 + positivity. applying chi - square test, the 2-sided p value was found to be < 0.001, which was highly significant. similar to fa, the semiquantitative score of ck19 of mng was useful to differentiate it from both utptc and fvptc [table 4 ]. (a) photomicrograph of multinodular goiter (hematoxylineosin, lp) (b) a multinodular goiter, negative immunostaining for cytokeratin 19 (ihc, lp) with an age range of 1553 years, the median age of ptc was 34.50 years in this study, which was similar to the study done by mazzaferri. and preston - martin. they found that most of the ptc were diagnosed in the third to fifth decades. sahoo., showed a higher mean age of 46 years, with an age range of 33 - 78 years in their five cases of utptc, which may be explained by the small number of cases compared with our study. in this study, the male : female ratio, in case of ptc, was 1:6.3, which was slightly more than that already published in the literature. according to negri., ptc was about four - times more common in females than in males, and this was also seen in the study done by mazzaferri. this excessive female predominance may be due to the small number of cases in our study. however, sahoo., have shown a male : female ratio of 1:9 in their 10 cases of fvptc, which is slightly higher than our findings of 1:6. the role of cytokeratin 19 in the diagnosis of ptc is not yet fully established. different studies have shown varying percentage distribution of ck19 positivity in ptc and other lesions of the thyroid. in this study, fifty percent (4/8) of mng and 75% (6/8) of fa also showed immunoreactivity to ck19, except one case of mng. in all of these cases, expression was focal and weak in intensity. only one case of mng out of eight was strongly positive, but only in focal areas. the study by nasr. also noted a high rate of ck19 positivity in benign lesions in as much as 68% (39/57), but staining intensity was weak in these benign lesions. in their study, three out of four papillary hyperplastic nodules were negative for ck19, whereas focal staining was present in one. they suggested that combination of hbme1 and ck19 attained a high sensitivity and specificity for the diagnosis of ptc. showed that ckl9 stained strongly and diffusely in all the cases of 10 utptc and all 26 fvptc. fonseca., in a similar study on paraffin - embedded material, found that ck19 was strongly expressed in papillary carcinoma and focally in follicular carcinoma. miettinen., in their study of keratin subsets on paraffin - embedded papillary and follicular thyroid lesions, confirmed that ck19 is a useful marker for differentiating between papillary carcinoma and papillary hyperplasia. however, they also identified expression of ck19 in follicular neoplasms and, hence, this particular cytokeratin was alone not useful in differentiating it from follicular tumors. if ck19 4 + positivity was taken as a cut - off value to differentiate ptc from other mimicking lesions, it showed 77% sensitivity and 100% specificity and positive predictive value in this study. they showed that 93% of ptc had 4 + positivity. in a large study, cheung., reported diffuse and moderate to strong ck19 staining in 80% (43/54) of ptcs and 57% (48/84) of fvptc. they also observed that 17% (6/35) of fa showed focal staining with ck19, and a single case (1/35) showed diffuse cki9 staining. in addition, they showed that immunoreactivity for hbme-1 and ret represents a reliable indicator of malignancy in nodules of follicular epithelial derivation. however, unlike other studies, they did not estimate the percentage of positively stained cells. according to kragsterman., all 35 patients (100%) with thyroid papillary carcinoma were positive for ck19 and in 80%, more than half of the tumor cells were immunostained, and the intensity of the immunoreactivity was strong. guyetant. noted similar findings with 16 usual - type ptcs and 43 fvptc. ck19 was diffusely expressed in all their utptc and fvptc. according to them, sensitivity and specificity of ck19 as a marker of papillary carcinomas was 100% and 82.5%, respectively. beesley. got similar results with diffuse and strong expression of ck19 in all four fvptc (100%) and 20 out of 22 utptc (91%). in this study, most of the utptc (84.62%) were diffuse 4 + positive. in case of fvptc, 4 +, 93% of fvptc and all cases of utptc showed strong 4 + positivity for ck19. the study done by cheung. observed that 48 cases of fvptc out of 84 (57%) were positive for ck19, and that this was diffuse and moderate to strong in intensity, resembling our 3 + and 4 + positivity. observed 26 cases of fvptc with multifocal distribution of papillary cancer nuclei and 10 cases of usual type ptc ; ckl9 stained strongly and diffusely in all cases of papillary carcinoma, and fvptc cases showed strong staining of the areas with papillary cancer nuclei in all cases and moderate to strong staining in areas of tumor without obvious nuclear features of papillary cancer. similar to this study, beesley. and nasr. 50% of mng were positive for ck19 expression, but only one case showed 3 + positivity. beesley. found that ck19 expression was seen in two out of eight mng with only focal staining. nasr. noted that only one out of four papillary hyperplastic nodules was positive, and only 15% of the cells stained, with the staining intensity being weak in most of these benign lesions. cheung. demonstrated that 20% (8/40) of the nodular goiters were focally ck19 positive, and immunoreactivity was seen mainly in areas of degeneration, indicating the reactive nature of ck19 positivity. it was evident from this study that 4 + immunoreactivity was helpful in distinguishing ptc from mng. when studied separately, both fvptc and utptc maintained a similar status of significance. in our study, 75% of fa showed ck19 positivity. sahoo. also found ck19 positivity in 100% (20/20) of fa ; of these, 75% showed 1 + positivity. however, in 25% of the fa, ck19 immunoreactivity was more extensive (2 + in one and 3 + in four), although none was 4 +. in all these cases, cki9 staining was patchy and often restricted to follicular cells located at the periphery of the nodule or those lining the cystic follicles. nasr. also demonstrated the ck19 status in five out of six fas, and the staining intensity was weak. beesley. observed that only five out of 20 fas were positive for ck19, and this was in a focal distribution. guyetant. showed that 90% of the fas (26/29) and four out of 11 atypical adenomas were focally positive for ck19. low - ck19 expression was also observed by rorive. in the fas as compared with ptc. further studies are necessary to show whether these tumors have a different clinical behavior or molecular profile. in our study, we found that diffuse and strong expression of ck19 is characteristic of ptc, both usual type and follicular variant. although weak ck19 staining is common in mng and fas, a negative stain is a good evidence against ptc. | context : the diagnosis of papillary thyroid carcinoma (ptc) is based on nuclear features. these features may be present in focal areas in benign thyroid diseases and follicular adenoma (fa), leading to diagnostic difficulty.aims:to evaluate the expression and pattern of the distribution of cytokeratin 19 (ck19) in ptc and compare its reactivity with other neoplastic and non - neoplastic conditions to assess its potential as a useful marker for ptc.materials and methods : twenty two cases of papillary carcinoma (usual type, follicular and diffuse sclerosing variant), eight follicular adenomas, eight multinodular goiters (mng) were collected for a period of two years and six months. sections were taken from thyroidectomy specimens fixed in 10% buffered neutral formalin. hematoxylin and eosin staining and immunohistochemical staining for ck19 were done using standard protocol. results were semiquantitatively scored as follows : 1 + (75%), and then analyzed.statistical analysis and results : all 22 (100%) papillary carcinomas showed diffuse and strong (3 + and 4 +) ck19 expression. six out of eight (75%) fas and four out of eight (50%) mng were positive for ck19, but it was of weaker intensity (1 + and 2 +) and focal in distribution.conclusion:focal ck19 staining may be found in benign disease, but diffuse and strong positivity is characteristic of ptc, which can be used in the diagnosis of ptc in lesions of equivocal morphological appearances. |
in the united states, 1520 individuals for every 100 000 annually receive a diagnosis of brain cancer. the primary brain tumors that constitute this cancer category continue to gain the attraction of biomedical investigators and clinicians for a multitude of reasons. much of the current interest in primary brain tumors stems from past endeavors that focused on identifying overall incidence and survival rates [27 ] as well as race - associated incidence and survival [812 ]. although ambiguity exists among past reports as to the precise incidence trends, evidence regarding the stability of a dismal prognosis for brain cancer is both overwhelming and unsettling. the overall most common histological subtypes of primary brain cancer have been identified as follows, in order of decreasing magnitude : glioblastoma, lymphoma, non - specific astrocytoma, glioma, anaplastic astrocytoma, and meningioma. not only are the prognoses of the aforementioned subtypes alarmingly poor, but their elusive etiologies and risk factors are cause for concern. only recently have genetic studies begun to surface that attempt to explain the molecular characteristics of certain histological subtypes of brain cancer [1420 ]. prior to discovering specific molecular differences between subtypes, knowledge regarding genetic associations of other forms of cancer guided several studies to retrospectively examine relationships between the incidence of primary brain tumors and different races and ethnicities. the results of racially - based incidence and survival data are contrary to the normal health disparity profile of the united states. strong evidence shows that populations of white individuals experience higher incidence and death due to brain cancer in relation to hispanic and black individuals. despite the compelling evidence of a racial disparity in primary brain tumors, incidence studies often preclude minority groups from sub - analysis and many prospective studies fail to recruit multiple racial demographics or plan for sub - analysis of data within and between populations of different races. furthermore, past investigations of the effect of socioeconomic, genetic, or other factors on the overall prognosis of primary brain tumors have been rather limited in resolution of analysis. specifically, the incidence and survival trends within the black population with respect to factors such as sex, age, and tumor histology, are sparsely described in the literature. the purpose of this study is, therefore, to determine the 6 most common primary brain tumors specific to the u.s. black population and to analyze the incidence and survival trends within this population from 1973 to 2008, with respect to sex and age of diagnosis. the surveillance, epidemiology, and end results (seer) program of the national cancer institute was utilized for this retrospective study. seer is a program that collects data regarding cancer incidence and survival from 17 cancer registries, encompassing roughly 26% of the u.s. overall incidence rates were obtained using parameters for year of diagnosis, age, sex, and major histological subtype. inclusion criteria included black racial demographic, adults older than 20 years of age, and a confirmed diagnosis received between 1973 and 2008. individuals meeting these specified parameters were then grouped into 15-year age groups ; 2034, 3549, 5064, 6579, and 80 +. racial demographic was specifically defined by seer as code race 02 (black), which included a stated - race of african - american, black, or negro. exclusion criteria were specified as diagnosis of anything other than first primary brain tumor and diagnosis at autopsy. based on these parameters, 4704 cases met the inclusion criteria ; of these cases, 3272 remained after screening for exclusion criteria. incidence and survival analyses were performed for the 6 most common histological subtypes of primary brain cancer (n=3272). all of the tumor subtypes compared in our study have been previously defined and published as standards by the central brain tumor registry of the united states (cbtrus). joinpoint 3.5.2 software was used to analyze trends in the incidence of diagnosis during the period 19732008 using a weighted mean squares methodology with random permutations to fit a model of a 03 joinpoints via a grid search method. the year of diagnosis was plotted against the natural log - transformation of age - adjusted incidence rate for specified adult brain tumors to obtain average annual increase in incidence. kaplan - meier (km) survival analysis was performed with an end - point defined as death, for a time frame limited to 60 months post - diagnosis, as this was found to be the convention for brain cancer statistics (figure 1). log - rank analysis was performed and a cox proportional hazards model was generated to calculate hazard ratios (hr). survival data was calculated regardless of treatment decisions, to reflect the reality of variable treatment regimens and variations in patient compliance. all statistical analyses were performed with 95% confidence intervals (ci) ; statistical significance was determined at p<0.05. cox proportional hazards model was generated via graphpad prism version 5.04 (graphpad software inc. the 6 most common tumor subtypes for the overall black population were in agreement with previous publications. however, upon sub - analysis within the black population, differences were identified that would have been masked if we not investigated further into this population. joinpoint analysis revealed that the annual rate of change for incidence of brain cancer in the u.s. black population increased by a factor of 0.11 each year between 1973 and 1989. following this period of increasing incidence rates, a moderate decrease by a factor of 0.06 per annum was observed from 1989 to 2008 (figure 1). sub - analysis of tumor histology incidence uncovered unique distributions of the 6 most common brain cancer subtypes by age (figure 2). lymphoma was found to be the most common primary tumor subtype for black individuals ages 20 to 34 years (37% of cases). in contrast, glioblastoma was identified as the most common tumor subtype for black individuals in the age groups of 3549, 5064, 6579, and 80 +, accounting for 34%, 58%, 60%, and 54% of cases, respectively. further differences in the distribution of the 6 most common brain cancer subtypes by age were identified (figure 2). sub - analysis of incidence by sex revealed that black men had higher incidence of each brain cancer subtype except for malignant meningioma (0.19 vs. 0.34 ; men vs. women, respectively). sizable differences in incidence between black men and women are those for glioblastoma (2.61 vs. 1.65), lymphoma (0.81 vs. 0.47), and non - specific astrocytoma (0.31 vs. 0.18) (table 1). a marked increase in primary lymphomas occurred among men in the late 1980s through the 1990s when compared to women (figure 3a, 3b ; table 1). data also revealed a decrease in the incidence of astrocytoma during the same time period for both black men and women (figure 3a, 3b ; table 1). the kaplan - meier curve reveals differences in median time to death between the brain cancer subtypes. our analysis showed that more than half of the patients diagnosed with lymphoma and glioblastoma died prior to 1 year after diagnosis. half of the patients diagnosed with anaplastic astrocytoma, non - specific astrocytoma, and non - specific glioma had died by 18 months. interestingly, a narrow majority of the patients diagnosed with malignant meningioma survived longer than 60 months after diagnosis (figure 4a). survival at 60 months after diagnosis differed from the trends identified for median time to death. our analysis shows that glioblastoma patients reached far greater mortality during the 60 months after diagnosis than any other subtype analyzed ; the population of patients diagnosed with lymphoma and anaplastic astrocytoma had a similar and higher proportion of survivors at 60 months relative to glioblastoma ; the population of patients diagnosed with non - specific astrocytoma and non - specific glioma attained a similar and higher proportion of survivors than the aforementioned ; and the favorable survival profile of malignant meningioma patients remained at 60 months after diagnosis (figure 4a). meningioma cases demonstrated the most favorable long - term survival when compared to all other brain cancer subtypes ; conversely, glioblastoma cases demonstrated the most dismal long - term prognosis (figure 4a, 4b). hazard ratios in relation to malignant meningioma were calculated to be 1.65, 2.01, 2.06, 2.79, and 5.81 for glioma, lymphoma, astrocytoma, anapestic astrocytoma, and glioblastoma, respectively (p<0.0001) (table 2). joinpoint analysis revealed that the annual rate of change for incidence of brain cancer in the u.s. black population increased by a factor of 0.11 each year between 1973 and 1989. following this period of increasing incidence rates, a moderate decrease by a factor of 0.06 per annum was observed from 1989 to 2008 (figure 1). sub - analysis of tumor histology incidence uncovered unique distributions of the 6 most common brain cancer subtypes by age (figure 2). lymphoma was found to be the most common primary tumor subtype for black individuals ages 20 to 34 years (37% of cases). in contrast, glioblastoma was identified as the most common tumor subtype for black individuals in the age groups of 3549, 5064, 6579, and 80 +, accounting for 34%, 58%, 60%, and 54% of cases, respectively. further differences in the distribution of the 6 most common brain cancer subtypes by age were identified (figure 2). sub - analysis of incidence by sex revealed that black men had higher incidence of each brain cancer subtype except for malignant meningioma (0.19 vs. 0.34 ; men vs. women, respectively). sizable differences in incidence between black men and women are those for glioblastoma (2.61 vs. 1.65), lymphoma (0.81 vs. 0.47), and non - specific astrocytoma (0.31 vs. 0.18) (table 1). a marked increase in primary lymphomas occurred among men in the late 1980s through the 1990s when compared to women (figure 3a, 3b ; table 1). data also revealed a decrease in the incidence of astrocytoma during the same time period for both black men and women (figure 3a, 3b ; table 1). the kaplan - meier curve reveals differences in median time to death between the brain cancer subtypes. our analysis showed that more than half of the patients diagnosed with lymphoma and glioblastoma died prior to 1 year after diagnosis. half of the patients diagnosed with anaplastic astrocytoma, non - specific astrocytoma, and non - specific glioma had died by 18 months. interestingly, a narrow majority of the patients diagnosed with malignant meningioma survived longer than 60 months after diagnosis (figure 4a). survival at 60 months after diagnosis differed from the trends identified for median time to death. our analysis shows that glioblastoma patients reached far greater mortality during the 60 months after diagnosis than any other subtype analyzed ; the population of patients diagnosed with lymphoma and anaplastic astrocytoma had a similar and higher proportion of survivors at 60 months relative to glioblastoma ; the population of patients diagnosed with non - specific astrocytoma and non - specific glioma attained a similar and higher proportion of survivors than the aforementioned ; and the favorable survival profile of malignant meningioma patients remained at 60 months after diagnosis (figure 4a). meningioma cases demonstrated the most favorable long - term survival when compared to all other brain cancer subtypes ; conversely, glioblastoma cases demonstrated the most dismal long - term prognosis (figure 4a, 4b). hazard ratios in relation to malignant meningioma were calculated to be 1.65, 2.01, 2.06, 2.79, and 5.81 for glioma, lymphoma, astrocytoma, anapestic astrocytoma, and glioblastoma, respectively (p<0.0001) (table 2). our review of 3272 cases of primary malignant brain tumors in black individuals in the united states reveals interesting results that would have been masked had we not conducted sub - analysis within this population. additionally, the steady increase in incidence of brain cancer that we observed in the overall u.s. black population from 1973 to 1989, followed by a moderate decrease to present day (figure 2), is congruent with previous studies that had differing aims. past attempts to explain this peak in incidence have pointed to various associations, including : changes in histological classification of brain tumors, an interaction between hiv / aids and brain lymphoma [2831 ], a transient spike in the magnitude of the elderly population, as well as increased use and quality of advanced imaging modalities. changes in histological classification and subsequent deviations in data recording is a likely contributing factor to the drop in non - specific astrocytoma diagnoses observed in our results (figure 4a). the coincidence of the aids epidemic with the rise in lymphoma diagnosis that we report (figures 1 and 4a, 4b) has been previously described. nevertheless, we do not find satisfaction in attempting to fully explain these phenomena with covariates that were not specified in our analysis. furthermore, we do not find these associations to provide adequate explanations to the current disparities within and between racially - based sub - groups. the contribution of increased quality of care in the united states is unlikely to be a major influence on the decline in incidence of brain cancer for the black population. rather, it is well - documented that black patients are still at a great disadvantage in terms of access to care, which has been identified as strong evidence of the higher incidence and poorer prognoses in this population for almost all other types of cancer. although access to care is certainly a problem that warrants great resources and attention, the reversed disparity profile for brain cancer may suggest that the mechanisms behind the different primary brain tumors depend more strongly on other factors. alternatively, the reversal of the typical disparity profile may reflect a negative, unintended, consequence of the overuse of imaging by those with high access to healthcare [3436 ], which results in an increase in the only well - known risk factor for brain cancer ionizing radiation. recent research has identified several molecular alterations that are consistent with incidence risk [1416,19,20,38 ], as well as treatment response and survival [18,3841 ] of certain brain cancers. none of these recent genetic studies regarding brain cancer implemented sub - analysis of the black population. the 2 studies that did compare racial differences limited their analysis to the caucasian and asian populations and were not conducted with patients from the united states. performing sub - analysis and molecular level comparisons between populations with lower incidence and higher survival (i.e., black brain cancer patients) and those with particularly high incidence and low survival (i.e., white brain cancer patients) may increase the speed at which important etiological discoveries are made. for instance, a recent study found an interesting occurrence of a 42% reduction of risk for glioma in patients with a history of diabetes. not only do blacks have lower incidence and higher survival in brain cancer, it has also been well established that the higher trend of diabetes in blacks in the u.s. continues. therefore, it would be beneficial to identify and compare polymorphisms between blacks and whites in an attempt to uncover possible genetic alterations and variations in signaling pathways that may guide future research and treatments. furthermore, recently identified germ - line and gene - gene interactions in brain cancers may explain the racial clustering of incidence. we believe that it would be wise to begin exploring the genetic mechanisms of the 6 most common brain cancer subtypes in black adults in the united states, by age and sex, using results from our study as a guide for comparisons. variations between these subtypes according to age and sex may help to identify characteristics that influence etiology, lower risk, and improved responses to treatments, as the black population has lower incidence and higher survival of brain cancer. from our data it is apparent that differences in brain cancer incidence and survival exist beyond the level of race alone. important differences have been identified within the black population that should be used to direct future research regarding brain cancer. it is likely that comparisons between the black and white populations in the united states at various levels (e.g., population, molecular) will yield interesting results that will enhance our understanding and treatment of brain cancer. the present study has several limitations, including limited data available from the seer database and the limitations inherent in all retrospective and exploratory investigations. also, despite the high regard given to the seer database due to its quality and breadth of representation, it is not without limitation. those limitations that may have influenced this study include the coding for risk factors associated with primary central nervous system lymphoma (pcnsl), including congenital disorders, iatrogenic immunosuppression, hiv status, incomplete data, and the lack of central pathogenic review. national and state population estimates are provided by the census bureau to estimate the years between actual censuses. inter - census population estimates are based on numbers from updated death and birth data and are subject to increased inaccuracies compared to estimates of actual counts. although these population estimates are thought to be the most precise way of generating these data, errors in estimates have the potential to amplify over time. nonetheless, seer provides the largest national cancer database for obtaining the large sample sizes required to supply population - based estimates of the end results of research, clinical trials, incidence, and survival information. cbtrus reported the incidence of meningioma as 7.5 per 100 000 in the adult black population, whereas our analysis calculated an incidence of 0.2. this discrepancy can be explained by the requirement for seer mandatory reporting to only include malignant tumors, whereas the higher rate published by cbtrus also takes into account benign meningiomas. malignant meningiomas have been estimated as accounting for only 13% of the total cases of meningiomas elsewhere. in response to our results, as well as new molecular - level research regarding brain cancer, we believe it would be wise to conduct prospective studies that incorporate racial sub - analysis and comparisons. this population - based retrospective study of brain cancer in black adults in the united states revealed sex and age differences in the incidence of the 6 most common brain tumor subtypes from 1973 to 2008. we feel as though the paradoxical relationship between health care access and brain cancer incidence, on the population - level, warrants quantitative investigation into imaging exposures and the incidence of brain cancer sub - types. furthermore, we find the recent data regarding molecular alterations in certain brain tumor subtypes to be an exciting new avenue for brain cancer research one that may benefit from our data regarding differences found within the black population for future study design. ultimately, we believe that identifying the differences among cancer subtypes within the black population will help to elucidate etiologies for certain brain cancers through further comparison studies, which will influence the development of more targeted treatments, identification of better predictors of prognosis, and discovery of preventable risks that will be of benefit to all individuals, regardless of race. | backgrounddespite much epidemiological research on brain cancer in the united states, the etiology for the various subtypes remains elusive. the black population in the united states currently experiences lower incidence but higher survival rates when compared to other races. thus, the aim of this study is to analyze the trends in incidence and survival for the 6 most common primary brain tumors in the black population of the united states.material/methodsthe surveillance, epidemiology, and end results (seer) database was utilized in this study to analyze the incidence and survival rates for the 6 most common brain tumor subtypes. joinpoint 3.5.2 software was used to analyze trends in the incidence of diagnosis from 1973 to 2008. a kaplan - meier curve was generated to analyze mean time to death and survival at 60 months.resultsjoinpoint analysis revealed that per year the incidence of brain cancer in the u.s. black population increased by 0.11 between 1973 and 1989. after this period, a moderate decrease by 0.06 per annum was observed from 1989 to 2008. lymphoma was the most common primary tumor subtype for black individuals ages 2034, and glioblastoma was identified as the most common tumor subtype for black individuals in the age groups of 3549, 5064, 6579, and 80+.conclusionsthis population - based retrospective study of brain cancer in black adults in the united states revealed significant sex and age differences in the incidence of the 6 most common brain tumor subtypes from 1973 to 2008. |
during rehabilitation, maxillectomy patients are treated with obturators, which are essential for oral functions such as speech, swallowing, mastication, and esthetics.123 loss of or damage to an obturator results in both functional disability and cosmetic disfigurement. conventional fabrication of an obturator is complex and needs multiple scheduled visits, and an alternative process is needed for rapid fabrication in emergency cases, such as disaster - related damage or loss. digital technology is creating exciting opportunities for improving the delivery of maxillofacial prostheses.4 digitized data for any object can be obtained from various resources, such as computerized tomography (ct) imaging, three - dimensional (3d) photography, and 3d scanning. intraoral scanners are among the 3d scanning tools that can be used to directly obtain 3d models of the mouth. these scanners record the geometry of the object by measuring the distance between the tip of scanner 's sensor and the target object by utilizing different technologies to convert the optical data to a 3d model. lava cos is a 3d scanner and uses a 3d video system comprising active wavefront sampling that captures 20 3d frames per second, resulting in a high - resolution final model.5 construction of a 3d model can be performed using surface - based recording, as it is commonly used in various types of 3d modeling software. this model does not contain any volumetric data but instead uses a triangle - based mesh representation of the 3d surface geometry. standard tessellation language (stl) is an open - source surface - based format, which is easily accessible through most commercial software applications. surface models using such a method have been widely used for rapid prototyping and computer - aided manufacturing. stl data can be saved in two different formats, american standard code for information interchange (ascii) and binary.6 the ascii format is less commonly used due to the large size of the resultant file, but it has an advantage in that it can be easily modified for debugging purposes. the binary format is more complicated in its syntax, but it generates smaller file sizes and is therefore used more often. the development of 3d printing technologies has allowed for the accurate printing of the complex shapes of maxillofacial prosthetics with details including the precise simulation of the undercut areas.7 these technologies utilize an additive process of building an object geometry in layers from a virtually sectioned 3d model. fused deposition modeling (fdm) is one such technology, in which a thermoplastic material is extruded from a nozzle layer by layer, controlled by temperature difference.8 this technique has been successfully used for the fabrication of maxillofacial prosthetics for over the past two decades.9 assessing the accuracy of 3d models can be done by linear or 3d measurements. in the case of 3d assessment, the scan of an object 's geometric form is compared to the original and the difference between the two forms is taken as the scanning accuracy. the working concept of intraoral scanners involves merging multiple 3d images scanned with different angles to build the final 3d model. this process may possibly lead to recording errors of varying degrees, which depend on the scanning technology and the recording algorithms used.10 the accuracy of intraoral scanners has been tested and verified in many published studies.11121314 using 3d models of fabricated obturators that have been saved in a digitized database will enable the rapid restoration of the basic oral function and appearance provided by an obturator while avoiding time - consuming conventional fabrication methods. this study aimed for and successfully provided a proof - of - concept of a digitized database of fabricated obturators at the maxillofacial prosthetics clinic of tokyo medical and dental university, to be kept for patients ' potential emergency needs. the creation of the digitized database was initiated by creating a 3d model of a patient 's and using this model as a basis for 3d printing from an appropriate material for a hypothetical emergency case. an acrylic resin obturator for edentulous maxillectomy patients with a open hollowed bulb was sprayed with titanium oxide powder (lava powder ; 3d espe, st. paul, mn, usa), recording the 3d surface sections during scanning, according to the manufacturer 's instructions for accurate 3d scanning (fig. the scanning was performed using a chairside intraoral scanner (lava cos ; 3d espe, st. the lava scanning protocol was done, consisting of calibration using a small calibration block followed by scanning of the obturator surfaces according to an arbitrary scan path. four scans were performed with a 10-min interval between the scans, with two scans of the obturator 's polished surfaces and the other two of the fitting surfaces. the scanning time was 7 min (the maximum time limit) for each scan. the scanned data was checked for surface scanning quality on the lava cos monitor (fig., the scan files were sent to the scanner provider, as a post - processing cycle was needed to recalculate the recorded surfaces and compensate for potential errors. the scanned data was saved as binary stl files, which were imported into 3d modeling software (artec 3d studio ; artec, palo alto, ca, usa). the files were compiled into a single stl file representing surface shape to provide the data for the combined surfaces of the obturator. small artifacts such as holes and/or triangulation errors were repaired according to the modeling software 's built - in algorithm. the final stl file was evaluated for 3d printing suitability using a 3d printing software (netfabb studio basic ; netfabb gmbh, lupburg, germany). three - dimensional printing, comprising an automatic production technique guided by a digital 3d model, is a suitable technology for manufacturing complex geometric shapes. obturators have complex geometry that necessitates a layer - by - layer printing technique ; therefore, fused deposition modeling was selected for 3d printing. a digitized 3d obturator model was manufactured using acrylonitrile butadiene styrene (portabee go ; romscraj, singapore) on a 3d printer (portabee go) (fig. the obturator was manufactured on the building platform of the 3d printer layer - by - layer ; once a layer was light - cured, a new layer of resin was applied until the obturator was completely constructed. the print nozzle was 0.4 mm and the layer thickness was set to 0.1 mm. the working dimensions on the platform were 120 cm for x, 168 mm for y, and 135 mm for z. the obturator was cleaned and any excess supporting structures were removed from the surface as they were deposited in thin and brittle walls., the original obturator was scanned using a cone - beam ct scanner (3dx multi - image micro ct fpd, morita, japan), and the scanned obturator was saved as a digital imaging and communication in medicine (dicom) file format. dicom data was imported into 3d modeling software mimics 11.11 (materialise nv, belgium) to produce a virtual 3d reference model and saved as a binary stl file. the original ct scanned obturator (reference scan model), the original lava scanned obturator (scan1 model), and the manufactured ct scanned obturator (scan2 model) were geometrically evaluated using a computer aided testing (cat) software (spgauge, armonicos co., ltd., japan) with an error scale of 1.0 mm. the geometry of the scan1 model was compared with the geometry of the reference scan model. the geometry of the scan2 model was also compared with the geometry of the reference scan model. the entire surface of the obturator was successfully scanned regardless of its structural complexity, modeled as 3d data, and stored in the maxillofacial prosthetics clinic digital system in a relatively small file size (19.6 mb). the model was used to successfully manufacture an obturator that had accurate dimensions compared to the original. 5 compares the geometries of the original ct scanned obturator and the original lava scanned obturator. 6 compares the geometries of the original ct scanned obturator and the manufactured ct scanned obturator. obturators have become especially important in complicated emergency cases, such as disaster - related maxillofacial damage or loss of obturators. in such situations, conventional fabrication of obturators therefore, we aimed to show the feasibility of an alternative method to produce these prostheses more rapidly from a digitized database for rehabilitation of patients in an emergency situation. conventionally, fabrication of an obturator is a complex task that requires a highly skilled maxillofacial prosthodontist and a dental laboratory technician. therefore, there is an approximately five - week long lead time before the patient is able to use the obturator. modern digital technology, including 3d scanning and printing, opens up the possibility of manufacturing maxillofacial prostheses more efficiently and with shorter lead time.15 the advantage of using a chairside intraoral scanner is apparent in that the obturator can be scanned while the patient is sitting in the dental chair and the scanner allows a shorter scanning time ; furthermore, the patient can walk out the clinic with the obturator. however, multiple scans were needed for the surfaces of the obturator in order to combine the images and achieve a higher accuracy. the scanning time was also limited to 7 min, which made it impossible to scan the entire obturator surface in a single scanning session. as a consequence, 4 scanning sessions over a total of 38 min were necessary. three - dimensional printing technology allows for the production of objects with complex 3d geometry. in this study, an obturator, one such complex object, could be accurately printed out from a scanned model using fused deposition modeling technique. evidence of the clinical applicability of the re - fabricated obturators is limited to our current experimental results, and further studies are needed. however, 3d printing of a virtual model and its individual testing usually lead to further developmental treatment phases before further clinical experience can be obtained. in addition, as the obturator in this study was a proof - of - concept, the manufacturing material used was not a standard dental material. consequently, more studies using 3d printable dental materials are needed. furthermore, the material for supporting structures in this study was non - soluble, but it may be better to use a soluble material that can make the removal of material more efficient and hands - free. like other 3d printing technologies, fused deposition modeling has some disadvantages that include : 1) laying out multiple layers often leads to seams which can be seen as lines between each layer, andan extra procedure of finishing and polishing can be added to remove these lines 2) support structures are required for complex geometrical objects, which can lead to difficulty in removing them, and a soluble material can be used to solve it. in the future, further studies will be needed to validate the feasibility of the presented method in a case series. this study provides a proof - of - concept for the use of digital technology to create a digitized database of obturators for edentulous maxillectomy patients. such a database could provide an advanced technological solution for the rapid rehabilitation of maxillectomy patients in disaster - related emergency cases. | purposethis study aimed to create a digitized database of fabricated obturators to be kept for patients ' potential emergency needs.materials and methodsa chairside intraoral scanner was used to scan the surfaces of an acrylic resin obturator. the scanned data was recorded and saved as a single standard tessellation language file using a three - dimensional modeling software. a simulated obturator model was manufactured using fused deposition modeling technique in a three - dimensional printer.resultsthe entire obturator was successfully scanned regardless of its structural complexity, modeled as three - dimensional data, and stored in the digital system of our clinic at a relatively small size (19.6 mb). a simulated obturator model was then accurately manufactured from these data.conclusionthis study provides a proof - of - concept for the use of digital technology to create a digitized database of obturators for edentulous maxillectomy patients. |
lack of specific hallmark of cancer is reason for using of whole tumor cells (tumor apoptotic bodies, tumor cell lysates, or tumor cell - derived rna), which represent full characteristics of tumor identity, as common source of tumor antigens in clinical trials of dendritic cell (dc) based cancer vaccines [1, 2 ]. among these antigen preparation procedures, ultraviolet b (uvb) irradiation is a safe, inexpensive, and easy method of inducing a mixed population of viable, early apoptotic, and late apoptotic / necrotic cells with various proportions during tumor antigen preparation [3, 4 ]. however, the immunogenic properties of prepared tumor antigens depend on the cell death stage. engulfment of the early apoptotic body leads to silent phagocytosis with anti - inflammatory activity, whereas phagocytes are activated when encountering late apoptotic / necrotic cells ; as a result, the latter gives rise to an inflammatory response [5, 6 ]. in our previous studies, apoptotic cells or dying tumor cells, used as a tumor antigen source, showed high antitumor induction efficacy of dcs to t cells [7, 8 ]. to develop novel techniques for tumor antigen preparation, we induced immunogenic cell death using jsi124 combined with bortezomib in multiple myeloma (mm). recently, superparamagnetic iron oxide nanoparticles (spions) have been reported to enhance reactive oxygen species (ros) production. based on our previous studies on dcs, we suppose that spions accelerate tumor cell death to an immunogenic induction stage ; hence, the antigen can be more highly immunogenic than uvb irradiated tumor antigens. spions are an interesting tool for cell labeling, cell therapy, and diagnostic imaging. however, uncoated spions can cause toxicity to living cells, and coating materials have been developed to stabilize aqueous spion suspensions and reduce toxicity. branched polyethylenimine- (bpei-) spions, iron oxide nanoparticles coated with bpei, are less toxic than spions and readily bind to the cell membrane to enhance their uptake. here, we investigated the immunogenicity of tumor antigen sources prepared from uvb irradiated tumor cells in the presence of bpei - spions during t cell responses elicited by dcs loaded with these tumor antigens. we showed that bpei - spions accelerated uvb irradiated cell death to the late apoptotic / necrotic stage after 2 h incubation. furthermore, prepared antigen with bpei - spions induced the highest production of il-12p70 of dcs, and these dcs favored th1 polarization during the t cell response. bpei - spion was synthesized by conjugation of low molecular weight bpei (mw 1,800 da, aldrich) onto thermally cross - linked spion (tcl - spion) via amide linkage. the physical - chemical properties of bpei - spion were further characterized by using zetasizer nano z (malven instruments, malvern, uk), the transmission electron microscopy (tem) (jeol jem-2000 fxii, japan), and tga analysis (mettler - toledo, sdt851, columbus, usa) in order to confirm its successful synthesis. bpei - spion uptake by the u266 mm cell line was evaluated using a quantitative spectrophotometric method. briefly, 5 10 u266 cells were put in contact with different amount of bpei - spions with shaking for 1 h at room temperature. cells were collected and washed three times in 1x phosphate - buffered saline (pbs) (sigma aldrich, st. the pellet was resuspended in 30% hcl (sigma aldrich) for 2 h at 60c. next, 0.08% potassium persulfate, 8% potassium thiocyanate, and 3.6% hcl (sigma aldrich) were added to form the iron - thiocyanate complex. the absorbance at 490 nm was measured using a microplate reader (tecan infinite m200 pro, tecan, mnnedorf, switzerland) after 10-min incubation. aqueous fecl36h2o (sigma aldrich) solution was treated in the same manner to create the standard curve. u266 cells were put in contact with bpei - spion conjugated with fnr-675 dye (bioacts, namdong - gu, incheon, korea), which appears as a red color under confocal microscopy (carl zeiss, jena, germany). cells were fixed on a glass slide and the nuclei were stained with dapi (thermo scientific pierce, rockford, usa), which appears as a blue color. 2,7-dichlorofluorescein - diacetate (dcfh - da) (sigma aldrich) and n - acetylcysteine (nac) (sigma aldrich), which blocks ros production, were used to determine intracellular ros levels based on fluorescence measurements. briefly, cells were incubated in warm rpmi-1640 medium (invitrogen life technologies, carlsbad, ca, usa) containing 10% fetal bovine serum (fbs) (paa, murarrie, australia) and 1% penicillin / streptomycin (p / s) (lonza, walkersville, md, usa) with 6 m dcfh - da at 37c for 1 h. the probe was then removed and cells were used for preparation of several types of antigen. ros levels for each type of antigen were then measured using flow cytometry with a facs calibur instrument (becton dickinson, mountain view, ca, usa) at 488 nm. the data were analyzed using winmdi, version 2.9 (bio - soft net). monocytes were isolated from peripheral blood mononuclear cells (pbmcs) obtained from healthy donors using a cd14 magnetic activating cell sorting (macs) system (miltenyi biotec inc., auburn, ca, usa) and were cultured at 2 10 cells / well in six - well plates (bd falcon, san jose, ca, usa) in imdm with l - glutamine and 25 mm hepes buffer (gibco - brl, grand island, ny, usa) supplemented with 10% fbs and 1% p / s in the presence of recombinant human il-4 (50 ng / ml, peprotech, rocky hill, nj, usa) and gm - csf (20 ng / ml, peprotech). cell culture medium was replenished every 2 days. at day 6, 2 10 immature dcs / well were matured with 50 ng / ml tnf- (peprotech) in 24-well plates (bd falcon). two hours after tnf- stimulation, dcs were loaded with several types of tumor antigen. three types of tumor antigen were prepared, as follows : (1) 2 h postirradiated antigen - containing u266 cells, which were induced by high - dose uvb irradiation (120 mj / cm) (international light, newburyport, ma, usa) followed by 2 h culture in rpmi-1640 with l - glutamine (gibco - brl) ; (2) bpei - spion 2 h postirradiated antigens, which were u266 cells pretreated with 16 g / ml of bpei - spions for 1 h, followed by high - dose uvb irradiation (120 mj / cm) and 2 h culture in rpmi-1640 ; (3) 16 h postirradiated antigen composed of u266 cells, which were irradiated in the same manner as 2 h postirradiated antigen followed by overnight culture in rpmi-1640. the apoptotic cells were analyzed using the annexin - v - fitc apoptosis detection kit (bd bioscience, san jose, ca, usa) and pulsed with immature dcs at 2 h after maturation at a dc : apoptotic cell ratio of 2 : 1. fitc - hsp70 antibody (ab) (cell signaling technology, danvers, ma, usa) and pe - hsp90 ab (abcam, cambridge, ma, usa) were used to measure surface expression of hsp70 and hsp90, respectively, on tumor antigens by flow cytometry. results represent the percentage of cells positive for expression of the total number of cells analyzed. cell death mechanism of prepared antigens was evaluated by apoptotic markers such as caspase 3, cleaved caspase 3, caspase 8, cleaved caspase 8, and bcl - xl (cell signaling technology, danvers, ma, usa) at 1 : 1000. -actin (santa cruz biotechnology, dallas, texas, usa) was used as the control marker at 1 : 500. three types of antigen were incubated in rpmi-1640 medium with 1% p / s (10 cells/10 l) for 24 h. the supernatants were collected for detection of immunogenic molecule release (hsp70, hsp90, and hmgb1). a total of 10 l of supernatant of each type of antigen was denatured with sodium dodecyl sulfate (sds) (sigma aldrich) and loaded into one well. primary antibodies specific to human hsp70 (biolegend, san diego, usa) at 0.25 g / ml, hsp90 (enzo, life sciences, pa, usa) at 1 : 10,000, and hmgb1 (abcam) at 1 g / ml were used. to measure dc tumor antigen uptake capacity, after maturation, tumor - loaded dcs were marked with cd11c - pe (bd bioscience). monoclonal antibodies against human cd80-pe, cd83-fitc, cd86-fitc, cd40-fitc, cd38-pe, cd74-fitc (bd biosciences), and ccr7-fitc (r&d systems, minneapolis, mn, usa) were used to detect surface markers on dcs using flow cytometry. the data were analyzed with winmdi, version 2.9 (bio - soft net). dc migration capacity was measured based on the percentage of mature dcs migrating toward ccl21 (r&d system) using a 24-well transwell plate with polycarbonate filters of 5-m pore size (costar, corning incorporated, ny, usa). a total of 5 10 dcs in 100 l of imdm with 10% fbs and 1% p / s were added to the upper chamber, and the lower chamber contained 600 l of imdm with 10% fbs and 1% p / s supplemented with 250 ng / ml of ccl21. the plate was incubated at 37c for 3 h. a total of 500 l of medium in the lower chamber was collected and cells were counted for 1 minute using flow cytometry. mature dcs were cultured in 96-well plates at 2 10 cells / well and stimulated with cd40l - transfected j558 cells (5 10 cells / well), which mimics the interaction between dcs and t cells. human il-12p70 and il-10 cytokine levels after maturation with cd40l stimulation were determined using a bd opteia elisa set (bd biosciences). allogeneic human t cells (2 10 cells) isolated from pbmcs using the macs system (miltenyi biotec) were cocultured with 2 h postirradiated dcs, bpei - spion 2 h postirradiated dcs, and 16 h postirradiated dcs (2 10 cells) at a ratio of 5 : 1 in rpmi-1640 medium containing 10% fbs and 1% p / s. on day 5, rhil-2 (10 u / ml, r&d systems) was added to the t cell culture. the medium was replenished with cytokines every 2 days for 6 days. on day 11, t cells were harvested for intracellular staining. then, 1 10 harvested t cells were stimulated with dynabead human t - activator cd3/cd28 (gibco - brl) for 24 h. the supernatants were collected for ifn- and il-4 dosage by elisa kit (bd biosciences). cells were activated with 100 ng / ml of phorbol myristate acetate (pma) and 1 g / ml of ionomycin (sigma aldrich) for 4 h. after 2 h of treatment with pma and ionomycin, brefeldin a (ebioscience, san diego, usa) was added to inhibit protein transport from the endoplasmic reticulum to the golgi apparatus. cells were superficially stained by cd8-apc and intracellularly marked using ifn--fitc and il-4-pe (bd biosciences). cells were analyzed by flow cytometry and data analysis was performed using win mdi, version 2.9 (bio - soft net). to optimize the concentration of bpei - spions for uptake by u266 cells, 5 10 u266 cells were put in contact with different amount of bpei - spion from 0 to 32 g with shaking for 1 h at room temperature. the ferric mass was significantly increased at bpei - spion amount greater than 16 g / ml (figure 1(a)). in addition, the presence of nanoparticles inside the cells was confirmed based on confocal microscopy (figure 1(b)). therefore, treatment of 16 g of bpei - spions per 5 10 cells was considered optimal for further experiments based on its cost effectiveness. after uvb irradiation, more than half of the total bpei - spion - pretreated cell population was in a death state, whereas cells without bpei - spions exhibited a low percentage of cell death (figure 2(a)). in addition, 2 h after uvb irradiation, only the pretreated bpei - spion u266 cells showed a homogenous population in the late apoptotic / necrosis stage (figure 2(b)). the cell viability was also confirmed with trypan blue exclusion method (figure 2(c)). for this reason, tumor antigen preparation was stopped 2 h after uvb irradiation. tumor antigen ros analysis showed that bpei - spions enhanced intracellular ros production by up to threefold compared to nonpretreated cells, and ros production in pretreated cells was inhibited by the antioxidant agent, nac (figure 2(d)). a recent report showed that spions induce intracellular ros production correlated to increase of cell death. by western blot, we showed that 2 h postirradiated cells, bpei - spion 2 h postirradiated cells, and 16 h postirradiated cells expressed the cleaved caspase 3 and the cleaved caspase 8 similar to cell treated with apoptosis inducer, 5-fluorouracil (figure 2(e)). hence, pretreated bpei - spion to u266 cells accelerated cell death after uvb irradiation to the late apoptotic / necrotic stage. as expected, more than 90% of bpei - spion 2 h postirradiated cells expressed surface hsp70 and hsp90 (figure 3(a)). although 2 h postirradiated cells also showed a high percentage of hsp70 and hsp90 expression (figure 3(a)), more than 60% of cells were viable (figure 2(a)). similarly, 16 h postirradiated cells presented high percentage of cells which were positive for these two markers. for this reason, the release of danger signals such as hsp70, hsp90, and hmgb1 was investigated by western blotting. the 2 h postirradiated cells and bpei - spion 2 h postirradiated cells released higher levels of three danger signals compared to 16 h postirradiated cells (figure 3(b)). although danger signals were released in antigen (2 h postirradiated cells and bpei - spion 2 h postirradiated cells) contact milieu, surface maturation - marker expression by 2 h postirradiated dcs and bpei - spion 2 h postirradiated dcs was similar to that by 16 h postirradiated dcs (figure 4(a)). in addition, there were no differences in antigen uptake and migration toward ccl21 among the groups (figures 4(b) and 4(c)). interestingly, 2 h postirradiated dcs and bpei - spion 2 h postirradiated dcs showed a significant increase in il-12p70 production but unchanged il-10 levels, compared with 16 h postirradiated dcs (figure 4(d)). il-23 production by the three types of dcs was similar (data not shown). based on evaluation of t cell polarization by intracellular cytokine staining, bpei - spion 2 h postirradiated dcs showed the strongest expression of the th1 polarizing cytokine, ifn-, reaching 18% of total analyzed t cells (figure 5(a)). although the il-12p70 level produced by 2 h postirradiated dcs was higher than that by 16 h postirradiated dcs, th1 polarization capacity was slightly increased in 2 h postirradiated dcs compared to 16 h postirradiated dcs. besides, amount of released ifn- and il-4 was measured by elisa and we observed a significant increase of ifn- in t cells polarized by bpei - spion 2 h postirradiated dcs (figure 5(b)). recent advances have suggested that apoptosis can be immunogenic in particular death states. upon the onset of the death stage, early apoptotic cells are characterized by an intact plasma membrane and superficial exposure of phosphatidylserine (ps) as an eating - me signal. early apoptotic cells can progress to the late apoptotic state, also known as secondary necrotic cells, when the plasma membrane becomes permeabilized. importantly, depending on the death state (e.g., early versus late apoptotic), recognition and internalization by phagocytes can result in an anti- or proinflammatory response [5, 16 ]. we showed here that bpei - spions accelerated cell death through apoptotic pathway after uvb irradiation with increasing intracellular ros production and induced extracellular release of hsp70, hsp90, and hmgb1. increases in intracellular ros give rise to high level expression of several hsps to protect cells from damage. in this study, considerable quantities of hsp70, hsp90, and hmgb1 were released from 2 h postirradiated cells and bpei - spion 2 h postirradiated cells. we hypothesized that uvb irradiation causes only mild damage to cells ; hence, part of the cell population entered the death stage, resulting in gradual damage signal accumulation. on the other hand, excessive cell damage occurred in the bpei - spion - pretreated cell population, which caused simultaneous death ; hence the damage signals were released immediately. therefore, bpei - spion 2 h postirradiated cells were the most immunogenic tumor antigens. in literature, even so, these stimuli were not sufficient to change dc surface marker expression. however, hsp and hmbg1 effectively promoted t cell polarization to the th1 subset. this result was in agreement with previous findings regarding the effect of hsp and hmgb1 on th1 polarization [19, 20 ]. in conclusion, bpei - spions, which are nontoxic elements, accelerate tumor cell death to the immunogenic late apoptotic / necrosis stage after a short incubation after uvb irradiation and can serve as an antigen for loading onto dcs. dcs loaded with the antigen exhibited basic properties of dcs loaded with 16 h postirradiated cells with high th1 polarization characteristics. these results suggest that bpei - spion 2 h postirradiated cells are useful and promising tumor antigens in dcs for induction of antigen - specific t cell immune responses against mm. | nanoparticles in the field of dendritic cell (dc) research are emerging as a promising method of enhancing the efficacy of cancer immunotherapy. we investigated the effect of branched polyethylenimine - superparamagnetic iron oxide nanoparticles (bpei - spions) on tumor cells loaded onto dcs. the tumor antigens were prepared as follows : (1) apoptotic u266 cells with ultraviolet b (uvb) irradiation followed by a 2 h incubation in the absence (2 h postirradiated cells) or (2) presence of bpei - spions (bpei - spion 2 h postirradiated cells) and (3) apoptotic u266 cells with uvb irradiation followed by an overnight 16 h incubation (16 h postirradiated cells). bpei - spions render u266 cells sensitive to uvb irradiation through reactive oxygen species production to accelerate apoptotic death. the 2 h postirradiated cells and bpei - spion 2 h postirradiated cells released immunogenic proteins, including hsp70, hsp90, and hmgb1. the dcs loaded with bpei - spion 2 h postirradiated cells showed the highest il-12p70 production and th1 polarization compared with other dcs. these results suggest that bpei - spions are a promising method of enhancing the immunogenicity of tumor cells and promoting th1 polarization of dcs loaded with these tumor cells. |
colonic intussusceptions are less common than small bowel intussusceptions and are mostly caused by malignant neoplasms. in children, intussusceptions however, there are pathologic leading points in 90% of all adult intussusceptions and surgical resection is required for adult cases. laparoscopic surgery requires smaller incisions than open surgery and postoperative pain is less severe than in open surgery. in addition, laparoscopic colectomy for curable cancers is not inferior to open surgery in terms of long - term oncologic endpoints. here we describe laparoscopic anterior resection in 2 patients with sigmoidorectal intussusceptions and 1 patient with a rectorectal intussusceptions. a 49-year - old woman was admitted to hallym university sacred heart hospital with a chief complaint of intermittent rectal bleeding and abdominal discomfort for 2 days. digital rectal examination revealed a mass in the rectum about 7 cm proximal to the anal verge. simple abdominal x - rays showed no abnormal bowel gas pattern. in laboratory tests, her neutrophil percentage was mildly elevated to 80.4% and her carcinoembryonic antigen level was slightly increased to 5.34 ng / ml. flexible sigmoidoscopy revealed an irregular mass at the prolapsed mucosal head of the rectum (fig. 1). abdominal computed tomography (ct) demonstrated a sigmoidorectal intussusception, probably due to a sigmoid colon cancer (fig. the patient took 2 l of polyethylene glycol (peg) solution for bowel preparation the day before operation. although the simple abdomen x - rays did not show any bowel obstruction, the patient could not defecate anything after taking peg. on laparoscopic exploration the sigmoid colon telescoped into the lumen of the rectum and the mesentery of the sigmoid colon was trapped between the involved bowels. it was difficult to establish a dissection plane of the mesentery. in order to continue laparoscopic surgery, reduction of the sigmoid the operator pulled out the proximal bowel using a laparoscopic bowel grasper and the assistant push up the invaginated distal end from the anus with a lubricated sponge - on - the - stick. after the resection of the proximal segment, we found out stool impaction within the end of the proximal segment. it is thought that the stool impaction caused bowel obstruction and failure of bowel preparation with peg solution. the pathologic finding was a 6 4 4-cm mucinous adenocarcinoma with moderate differentiation. six years have elapsed after the first operation, and the patient shows neither regional recurrence nor distant metastasis. ct scans demonstrated that a sigmoidorectal intussusception and a intraluminal enhancing polypoid mass at the distal portion of the sigmoid colon (fig. sigmoidoscopy showed a polypoid mass with nodular mucosa in the rectum 10 cm from the anal verge, as well as a partial downward displacement of the proximal bowel (fig. 4). sigmoidoscopic forceps biopsy was carried out from the mass. during air inflation of sigmoidoscope, the intussusception was partially restored., we could not find the intussusception site or the tumor lesion because there were no serosa deformities. we were thus forced to carry out intraoperative colonoscopy in order to find the tumor location. intraoperative colonoscopy revealed that a 5-cm polypoid mass located 17 cm from the anal verge as well as an approximately 5-cm segmented mucosal edema and congestion (fig. we marked distal resection margin using laparoscopic clipping with the help of the colonoscopic light (fig. although the sigmoid colon was distended immediately after intraoperative colonoscopy, decompression was completed using a rectal tube. histopathological examination revealed a 6 4.5-cm villotubular adenoma with focal high - grade dysplasia. the patient was referred to our hospital for further evaluation and treatment. at our institute, the ct scans showed rectorectal intussusceptions with irregular enhancing wall thickening at the upper rectum and we could not evaluate perirectal fat invasion due to the intussuscepted state (fig. 7). bowel preparation was carefully performed with 4 l of peg solution the day after ct because the patient had no abdominal symptoms. laparoscopy did not show an intussusception lesion but a distorted serosa in the rectum which was suggestive of cancer. we performed intraoperative colonoscopy to in order to indentify mucosal abnormalities, such as edema or congestion. we thought that the intussuseception had been restored by itself and that the lesion might be a transient intussusception. a 49-year - old woman was admitted to hallym university sacred heart hospital with a chief complaint of intermittent rectal bleeding and abdominal discomfort for 2 days. digital rectal examination revealed a mass in the rectum about 7 cm proximal to the anal verge. simple abdominal x - rays showed no abnormal bowel gas pattern. in laboratory tests, her neutrophil percentage was mildly elevated to 80.4% and her carcinoembryonic antigen level was slightly increased to 5.34 ng / ml. flexible sigmoidoscopy revealed an irregular mass at the prolapsed mucosal head of the rectum (fig. 1). abdominal computed tomography (ct) demonstrated a sigmoidorectal intussusception, probably due to a sigmoid colon cancer (fig. the patient took 2 l of polyethylene glycol (peg) solution for bowel preparation the day before operation. although the simple abdomen x - rays did not show any bowel obstruction, the patient could not defecate anything after taking peg. on laparoscopic exploration the sigmoid colon telescoped into the lumen of the rectum and the mesentery of the sigmoid colon was trapped between the involved bowels. it was difficult to establish a dissection plane of the mesentery. in order to continue laparoscopic surgery, reduction of the sigmoid the operator pulled out the proximal bowel using a laparoscopic bowel grasper and the assistant push up the invaginated distal end from the anus with a lubricated sponge - on - the - stick. after the resection of the proximal segment, we found out stool impaction within the end of the proximal segment. it is thought that the stool impaction caused bowel obstruction and failure of bowel preparation with peg solution. the pathologic finding was a 6 4 4-cm mucinous adenocarcinoma with moderate differentiation. six years have elapsed after the first operation, and the patient shows neither regional recurrence nor distant metastasis. simple abdominal x - rays showed a nonspecific bowel gas pattern. on physical examination, the patient showed periumbilical tenderness. for the evaluation of tenderness, we first performed abdomino - pelvic ct. ct scans demonstrated that a sigmoidorectal intussusception and a intraluminal enhancing polypoid mass at the distal portion of the sigmoid colon (fig. sigmoidoscopy showed a polypoid mass with nodular mucosa in the rectum 10 cm from the anal verge, as well as a partial downward displacement of the proximal bowel (fig. 4). sigmoidoscopic forceps biopsy was carried out from the mass. during air inflation of sigmoidoscope, laparoscopic anterior resection was then performed. during the laparoscopy, we could not find the intussusception site or the tumor lesion because there were no serosa deformities. we were thus forced to carry out intraoperative colonoscopy in order to find the tumor location. intraoperative colonoscopy revealed that a 5-cm polypoid mass located 17 cm from the anal verge as well as an approximately 5-cm segmented mucosal edema and congestion (fig. we marked distal resection margin using laparoscopic clipping with the help of the colonoscopic light (fig. although the sigmoid colon was distended immediately after intraoperative colonoscopy, decompression was completed using a rectal tube. histopathological examination revealed a 6 4.5-cm villotubular adenoma with focal high - grade dysplasia. the patient was referred to our hospital for further evaluation and treatment. at our institute, the ct scans showed rectorectal intussusceptions with irregular enhancing wall thickening at the upper rectum and we could not evaluate perirectal fat invasion due to the intussuscepted state (fig. 7). bowel preparation was carefully performed with 4 l of peg solution the day after ct because the patient had no abdominal symptoms. laparoscopy did not show an intussusception lesion but a distorted serosa in the rectum which was suggestive of cancer. we performed intraoperative colonoscopy to in order to indentify mucosal abnormalities, such as edema or congestion. we thought that the intussuseception had been restored by itself and that the lesion might be a transient intussusception. intussusceptions in adults are different from those in children in that pathologic leading points are found in 70 to 90% of all adult intussusceptions. en block resection without reduction is recommended for adult intussusceptions because of the possibility of malignancy. if reduction is attempted during operation in patients with malignant tumors, it can lead to intraluminal seeding or venous embolization of malignant cells. from an oncologic viewpoint, minimal of manipulation of the tumor cells azar and berger have proposed that resection with primary anastomosis can be achieved on right - side intussusceptions in unprepared bowels but the construction of colostomies should be performed on left - side or rectosigmoid lesions. when intussusception involves the lower rectum, operation without reduction requires extensive surgeries, such as abodminoperitoneal excision or very low anterior resection. in contrast, when a sigmoidorectal intussusception is reduced, unnecessary extensive operations sacrificing the anus, such as abdominoperineal excisions, can be avoided. permanent colostomy affects the quality of life. though previous studies reported the possibility of intraluminal seeding and venous emboli after reduction of colonic intussusceptions, there is not yet definite evidence to support this possibility because of the scarcity of clinical cases. reduction of sigmoidorectal intussuscpetions that can avoid unnecessary resections and permanent colostomies has clinical implications for the quality of life. it would be easier to reduce sigmoidorectal or rectorectal intussusceptions than ileo - colic or colo - colic intussusceptions. we found in case 1 that the abdominal approach only allows for pulling the intussusception. the laparoscopic intraabdominal approach carries a high risk of tearing of the colon wall by traction. the sigmoidoscopic approach to the intussusceptions site is able to be useful as in case 2. in our case, partial reduction was achieved during air inflation of a sigmoidoscope, and thus patient discomfort was improved. if a patient shows a complete obstruction or ischemic signs, such as fever, rigid abdomen, leukocytosis and nonenhanced bowel wall on contrast ct, sigmoidoscopy should be avoided to prevent bowel perforation. however, when there are no complete obstruction or ischemic signs, flexible sigmoidoscopy may help identify the lesion. another problem with reduction is the possibility that the bowel is vital externally but mucosa is necrotized internally. the distal colon of intussusceptions (intussuscipiens) would be too edematous to safely permit primary anastomosis. we performed intraoperative colonoscopy in order to detect the location of the tumor in case 2. during colonoscopy, we marked the resection margin, excluding the congested mucosa, with the help of colonoscopic light. in case 3 it is thought that intra - operative colonoscopy is a useful means of estimating mucosal state and of determining the resection margin. complete resection along with lymphatic drainage should be performed due to the possibility of malignancy in all colonic intussusceptions. the validity of laparoscopic oncologic colon surgery has been supported by the results of previous studies. anatomical knowledge of the mesentery is essential for complete resection along with lymphatic drainage during laparoscopic surgery. in intussusceptions, the proximal bowel is driven distally into the bowel and the mesentery of the involved bowel is trapped. when of intussusceptions reduction is achieved, the folded mesentery becomes unfolded as in case 1. it is thought that intussusceptions reduction clearly visualizes the mesentery and allows for successful laparoscopic surgery. previous reports have shown that most transient intussusceptions occur in the small bowel in adults. however, our case was occurred directly related to a cancer. most intussusceptions in the small bowel are incidentally discovered and do not cause specific symptoms. in our case, if an asymptomatic colonic intussusception is incidentally found, the possibility of transient intussusceptions should be considered. we conclude that sigmoidrectal or rectorectal intussusceptions can be managed by laparoscopic colectomy combined with perineal reduction. | adult intussusception is a rare entity. most adult intussusceptions require surgical intervention because they have a high rate of pathologic leading point. mandatory laparotomy and en bloc resection is recommended in colonic intussusceptions due to the possibility of malignancy. we report herein 3 cases of adult colonic intussusceptions. the intussusceptions were located in the sigmoid and rectum, which were managed by laparoscopic colectomy. case 1 was managed by laparoscopic anterior resection and diverting ileostomy combined with perineal reduction. perineal approach facilitated laparoscopic reduction. in case 2, intraoperative colonoscopy was performed to determine the distal resection margin. intraoperative colonoscopy showed edematous bowel mucosa as well as leading point after reduction of intussusceptions. case 3 showed asymptomatic transient rectorectal colonic intussusceptions. |
with the introduction of dental adhesive technology, tooth - colored composite restorations have gained wide popularity in recent decades. despite innovative improvements over the years, and the long - term stability of composite restorations, failures continue to occur. in cases of failure or fracture of an adhesive restoration, repair is an alternative to complete removal that preserves the tooth, as it is often difficult to remove an adhesive restoration without removing an integral part of the tooth. for all of these reasons, a qualitative, optimal method of repairing composite restorations has become a desirable alternative. the evidence as it currently stands seems to favor repair over replacement, but it is not conclusive. the success of such repairs depends not only on the type of the material used for the initial restoration and the repair procedure utilized, but also on the surface treatment applied to the surface to be repaired. treating the surface is mandatory, owing to the lack of reactive methacrylate (mc) groups in the old resin, and because the sorption of water by the old restoration restricts the addition of new material. however, the problem for the clinician is that the type and brand of the composite used in the previous restoration is often unknown. the problem is aggravated when different composites with organic matrices other than dimethacrylates have been introduced. furthermore, various protocols of intraoral repair systems are exploring on the repair potential of indirect aesthetic restorations, ceramic, feldspathic porcelain, and zirconia. the repair of mc - based composite restorations is widely considered to be a reliable procedure in modern dentistry. with regard to the repair of silorane moreover, few studies investigating interactions between mc - based composite and sil - based restorations have been published. in addition, studies comparing the bond quality facilitated by different combinations of surface pretreatments (grinding and conditioning) have not been published. the purpose of this in vitro study was to investigate the influence of different pretreatment procedures - grinding (by diamond bur [db ] or air abrasion [aa ]) and conditioning (orthophosphoric acid [oa ] or sodium hypochlorite [hy])-and different organic matrix compositions (mc- and sil - based composite), on the shear bond strength of repairs to artificial aged composite. the null hypotheses tested were that (a) composites with different organic matrix compositions demonstrate the same reparability, and (b) different surface pretreatment procedures have no influence on the shear bond strength of repaired composites. the following composite systems were tested : the filtek ultimate (3 m espe, st. paul, mn, usa) (mc), a nanofilled mc - based composite with the corresponding bonding agent single bond adhesive, and the filtek silorane (3 m espe, st. paul, mn, usa) (sil), a sil - based composite with the corresponding bonding agent silorane adhesive system. materials, batch numbers, and composition standardized disc - shaped composite substrates of 4 mm diameter and 2 mm thickness were prepared for the test specimens, using cylindrical shaped teflon molds with a hole in their center, of those same dimensions. prior to filling the holes with the resin material, the molds were set on a glass microscope plate in order to achieve a flat surface of the specimen after light - curing. resin composite was condensate into the hole with a hand instrument, to a thickness of 2 mm, and a second microscope slide was firmly pressed onto the top of the mold to remove excess resin composite, and to create a flat surface. polymerization of the composite specimens was achieved through the application of a light - emitting diode (led) polymerisation device for 20 s (heraeus kulzer translux power blue germany 50/60 hz 15 va), at a light intensity of 1000 mw / cm. light intensity was periodically verified by a radiometer after the processing of every five specimens (bluephase meter ivoclar, vivadent, schaan, liechtenstein). the specimens were then left to age in artificial saliva for 7 days at room temperature. artificial saliva was prepared according to donmez. and was comprised of the following components (mmol / l) : cacl2.2h2o (0.7), mgcl2.6h2o (0.2), kh2po4 (4.0), kcl (30.0), hepes buffer (20.0), nan3 (3.0). the specimens were then removed from the artificial saliva, air - dried and randomly assigned to the test groups. the chosen combination of surface treatments (grinding procedure and conditioning procedure) for each group was performed. the two types of surface treatment tested were a grinding procedure with the use of a db (komet 107 m - grit, for 10 s at high speed with water cooling) (db) and aa (ems air - flow prep k1 max device, 50-m aluminum oxide for 10 s, with the device set with the nozzle at a distance of 5 mm) (aa). in addition, two different conditioning procedures prior to the application of the corresponding bonding agent and the repair composite were compared ; application of 37% oa, and application of 2.5% sodium hy. for the conditioning procedure, 37% oa was placed on the specimens, left for 30 s, washed away with water, and the specimens were then air dried for 10 s. for the 2.5% naocl conditioning procedure, the specimens were rinsed with 5 ml naocl and air dried for 10 s. thereafter, bonding procedures were performed on the aged treated composite surfaces using the corresponding bonding agent of the repair composite resin, strictly in accordance with the manufacturer 's instructions. following the curing of the adhesive, a teflon matrix which manufactured to fit exactly on the aged surface and to specify the exact dimensions of the repair material (3 mm in diameter and 4 mm in height) was placed over the aged specimen substrates and filled in two increments with the repair composite. each increment was light - cured for 20 s using the same led polymerization device and the bonded specimen was removed from the matrix with slight pressure on the top of the repair composite cylinder. this procedure resulted in cylinders of repair composite measuring 3 mm in diameter and 4 mm in height being bonded to the aged resin [figure 1a ]. (a) the teflon matrix that used to form the specimens along with a prepared specimen. (b) the shear device and the specimen positioned in it prior loading in the manner described above, 160 cylinders of repair composite resin specimens comprised of 16 groups of 10 specimens were prepared. the grey cells represent the pretest failure groups the bonded specimens were subjected to an aging procedure, undergoing storage for 7 days in artificial saliva followed by thermo - cycling (5000 cycles between 5c and 55c, dwell time 30 s). shear testing was conducted using a universal testing machine (testometric ax, m 350 - 10kn, rochdale, england) at a crosshead speed of 0.5 mm / min until fracture. the specimens were fixed on a modified shear fixture, and the load was applied with the use of a chisel [figure 1b ]. the load at fracture, expressed in mpa, was calculated by dividing the peak load by the bonding area, and this value was used in statistical analyses. levene 's test for equality of error variances was used to test the assumption of homogeneity. according to the results of the above tests, the nonparametric mann - whitney u - test was used for comparisons of the interaction between base material and surface treatment. adjustment for type i error resulting from multiple tests was achieved via the bonferroni method. armonk, ny : ibm corp., and statistical significance was set at p 0.05), ignoring all other factors. collectively, the above results indicate that the combination of composite based materials (mc - based composite and sil - based composite) used is the most significant factor influencing the bond strength in this experimental context, as none of the other factors had a significant influence on the bond quality of the repaired composite resins. no statistically significant differences were observed between any combination of surface treatments when mc was used as the base resin (mann whitney u - test with bonferroni adjustment, p > 0.05). when the base resin was sil, statistically significant differences (mann - whitney u - test with bonferroni adjustment) were observed between db + oa and db + hy (p = 0.012), and db + oa and aa + oa (p = 0.012), while no other comparisons yielded statistically significant differences (p > 0.05). table 3 shows the failure mode results of the specimens that were analyzed under a stereomicroscope after shear bond tests. in almost all groups, most fracture modes were mixed, but in the sil - sil - db - oa group, most were adhesive. the first hypothesis that composites with different organic matrix compositions demonstrate the same reparability has to be rejected since at this study composites with different matrix compositions demonstrated repair bond strength variations. this study shows that mc - based composite can be repaired with a mc - based composite and the corresponding bonding agent, and adequate bonding could be achieved in every combination of surface treatment that was investigated in this study [figure 3a and b ]. conversely, mc - based composite could not be effectively repaired with sil - based composite and the corresponding bonding agent, since no combination of surface treatment investigated in this study established an adequate bond. scanning electron microscopy images of a specimen where aged methacrylate (mc) has been repaired with mc after the aged surface was treated with air abrasion and conditioned with orthophosphoric acid. inorganic fillers originating on the repair resin are detectable on the aged resin surface (500) artificial aging of mc - based composites creates degradation processes which cause increased water sorption. owing to their hydrophobic siloxane component, sil - based composites are more hydrophobic than mc - based composites. this may influence the reparability of mc - based composites with sil - based composite and the corresponding bonding agent. in contrast, sil - based composite could be effectively repaired with sil- and mc - based composite and the corresponding bonding agent [figure 4a and b ]. sil - based composite is more resistant to water sorption and disintegration, and this leads to smaller changes in material properties resulting from aging. sil - based composite could only be used effectively as a repair composite for aged sil with the surface treatment combination of db and oa. although, the poor shear bond strength results along with the high rate of adhesive failures that the failure mode analysis showed for this group are evidence of the weakness of bonding in this context. this result is concordant with a study reported by ivanovas., who concluded that sil - based composite can be effectively repaired with mc - based composite. the results of this study show that the mc resins that were repaired with the same composite reached the highest possible bond strength. the failure pattern analysis affirms the above results, as a large number of mixed and cohesive fractures were observed. scanning electron microscopy images of a specimen where aged silorane was repaired with methacrylate after the aged surface was treated with diamond bur and conditioned with hypochlorite. distinguishable are the repair composite (r), the adhesive failure area (a) and the base composite (b). (b) magnification of the failure line in the base composite area (500) the reparability of mc - based composite and sil - based composite differs depending on whether the material is used as a base material or as a repair material. mc - based composite yielded good results when used as a base material, as well as a repair material. in contrast, sil - based composite yielded good results when used as a base material, but was evidently a poor repair material. these results are concordant with those of a study reported by baur and ilie who concluded that the properties of materials can differ strongly depending on whether they are used as a base material, or a repair material. some sources propose the additional use of silane as an intermediate agent, in order to effectively repair a sil - based composite. on the contrary a new research on microtensile bond strength of sil - based composites repaired with mc - based composites showed that silane did not contributed to an increase in the tensile strength. in this study, a silane solution was not employed prior to the adhesive, in order to focus on the influence of grinding and conditioning of the repair surface on the bond. implementation of silane enhances the wetting of the surface for the bonding agent, which then infiltrates easily into the irregularities created by the surface roughening. and the silane - coated composite surface becomes more reactive to the mc groups of the repair resin. with regard to surface treatment, it should be noted that both db and aa procedures are used to increase the micromechanical retention of the new material onto the aged composite. in this study, there were no statistically significant differences between the two grinding procedures tested. these results are concordant with the results of rathke. and yesilyurt., who investigated the influence of different methods of mechanical roughening on bond quality between new and aged composite. a treated composite either with db or with aa infiltrates on its surface organic and inorganic particles originated on the environment. the conditioning procedure using oa does not aim to decalcification of the resin surface similar with what happens in the hard dental tissues surfaces. it is used is an order to obtain a cleansing effect by removing inorganic particles. on the contrary, sodium hy is a known organic solvent, which is used in many dental applications in order to remove the organic particles from surfaces. statistical analysis in this study has shown that there were no statistically significant differences between the two types of conditioning procedures. the statistical analysis of the results of this study showed that the best two combinations of surface treatment were db with oa, and aa with oa. therefore, the second hypothesis should be rejected up to a point, and it is concluded that within the limitations of this study the surface treatment has a minor influence on the shear bond strength of repaired composites. this is concordant with loomans., who attested that none of the common surface treatments can be recommended as a universally applicable repair technique, since their effectiveness is material - dependent. the authors are in agreement with the conclusions of a recent systematic review that repair of restorations is a valuable method of improving their quality, and can yield acceptable results. however, methodologically sound, randomized, controlled, long - term clinical trials are required, in order to facilitate evidence - based recommendations. within the limitations of this study, it can be concluded that : material is the major contributory factor for the successful repair of a composite resin restoration.knowing the composition of the aged composite should allow the operator to achieve reliable bond strength with the new composite resin.mc-based composites showed better reparability than sil - based composites.regarding the surface treatment, no statistical differences in repair bond strength were evident. material is the major contributory factor for the successful repair of a composite resin restoration. knowing the composition of the aged composite should allow the operator to achieve reliable bond strength with the new composite resin. mc - based composites showed better reparability than sil - based composites. regarding the surface treatment, | objective : the objective was to investigate the way that various surface treatments could influence the bond strength of the repair of methacrylate (mc) and silorane (sil) composites.materials and methods : a total of 160 mc and sil cylindrical specimens were polymerized and aged in artificial saliva solution for 7 days. depending on the following surface treatment (diamond bur or air abrasion), and the conditioning procedure (orthophosphoric acid or sodium hypochlorite), 16 groups were formed and repaired either with mc, either with sil composite. repaired specimens were subjected to an additional aging procedure in artificial saliva for 7 days, followed by thermo - cycling and then stressed in shear at a rate of 0.5 mm / min until failure. failure patterns were analyzed using stereomicroscope and scanning electron microscopy.results:mc composite showed statistically significant higher bond strength both as a base or repair material than sil (p < 0.001). statistically significant differences were not observed, when grinding and conditioning procedures was compared. pretest failures were observed when aged mc - based composite was repaired with sil-based.conclusions:type of composite seems to be the main factor influencing the bond strength of the repair. mc - based composite showed better repairability than sil composite. optimum repair conditions should include knowledge of the composite 's composition. |
this work was supported in part by grants - in - aid for scientific research from the ministry of education, science, sports and culture of japan and the kawano masanori memorial foundation for the promotion of pediatrics, japan. | asymmetric cell division (acd) is a characteristic of cancer stem cells, which exhibit high malignant potential. however, the cellular mechanisms that regulate symmetric (self - renewal) and asymmetric cell divisions are mostly unknown. using human neuroblastoma cells, we found that the oncosuppressor protein tripartite motif containing 32 (trim32) positively regulates acd. |
a retrospective review was performed of biopsied and/or surgically resected cases diagnosed as nsclc and tested for egfr, kras, and alk mutations in the department of pathology and translational genomics at samsung medical center, seoul, korea from 2006 to 2014. of 6,637 nsclc patients, 6,595 egfr, 5,177 kras, and 4,869 alk mutation tests were performed. among them, 2,387 patients (36.2%) had egfr, 398 (7.7%) had kras, and 281 (5.8%) had alk mutations. based on these tests, we selected 12 patients with concomitant mutations in the same tumor. clinical data, including gender, age, smoking history, and previous concurrent chemoradiation therapy history were extracted from electronic medical records. all hematoxylin and eosin stained slides of selected cases were reviewed by two pathologists (t.l. and b.l.). histologic classification was made according to the iaslc / american thoracic society (ats)/european respiratory society (ers) international multidisciplinary classification of lung adenocarcinoma, and typical pathologic features were re - evaluated, including cell type, nuclear and cytoplasmic characteristics. we used two methods to evaluate egfr mutations in the 18th, 19th, 20th, and 21st exon : sanger sequencing and real time polymerase chain reaction after peptide nucleic acid (pna)clamping using the pna clamping egfr mutation detection kit (panagene, inc., daejeon, korea). extracted genomic dna isolated from formalin - fixed paraffin - embedded (ffpe) tissue was used for egfr and kras analyses. alk mutation tests were performed using immunohistochemical (ihc) (1:40, ncl - alk, clone 5a4, novocastra, newcastle upon tyne, uk) and fluorescence in situ hybridization (fish) analysis (lsi alk dual color break - apart probe, dako, glostrup, denmark) with ffpe tissue. diffuse strong positivity in the cytoplasm was regarded as positive in alk ihc. in the fish analysis, 50 non - overlapping nuclei were counted and 15% of break - apart probes were used as a cutoff value for positivity. diffuse strong positive alk ihc results were interpreted as a surrogate marker of alk rearrangement. clinical data and pathologic features of the 12 patients with concomitant mutations are summarized in table 1. the age at diagnosis of patients ranged from 48 to 78 years old (median, 62 years old) and 75% of patients were female. all three male patients had a history of smoking (1557 pack - years) in contrast to none of female patients. five surgically resectable patients underwent lobectomy and seven clinical stage iv patients had a needle aspiration or biopsy from the lung, bronchus, lymph node, or adrenal gland. 7 had a history of lobectomy at an outside hospital without any other treatment 9 years prior to the lymph node biopsy. 12 previously underwent lobectomy and was treated with gefitinib for 1 month and crizotinib for 4 months in china. she had no egfr or kras mutation at the time of diagnosis at china. aside from this patient, all other patients had no history of prior targeted therapy before mutation testing. egfr mutations of three patients were only detected with the pna clamping method, while nine were confirmed by sanger sequencing. five (41.7%) had the missense l858r mutation in exon 21, four (33.3%) had the missense g719x mutation in exon 18, and three (25%) had a deletion in exon 19. 12 had a missense r803w mutation at exon 20 that was not identified before targeted therapy at the outside hospital. six patients had additional kras mutations and the other six had an alk mutation (referred to as egfr - kras and egfr - alk hereafter). three among the six patients who showed positivity at alk ihc were confirmed with fish analysis. six egfr - kras patients had papillary, acinar, solid, and micropapillary patterns (fig. 1). among them, five patients had typical hobnail cell features with apical snouts ; the remaining patient (patient no. 4) had a focal columnar cell component and intraand extracytoplasmic mucin, which are similar morphologic features of nsclc with a kras mutation. in contrast, six egfr - alk patients showed solid, cribriform and micropapillary patterns. three of six patients had signet ring cells and intracytoplasmic mucin with or without extracytoplasmic mucin production, which are typical features of alk - positive nsclc. seven patients had surgical resection and two were alive without targeted therapy and any evidence of relapse or progression, including one patient who received neoadjuvant concurrent chemoradiation therapy. one patient who had surgery and decided not to receive targeted therapy due to old age and underlying diabetes mellitus died of disease after 53 months from surgery (patient no. egfr tki included gefitinib (250 mg, orally, every day) and erlotinib (150 mg, orally, every day) and the alk inhibitor was crizotinib (250 mg, orally, every day or twice daily). all three patients showed partial responses, but two also showed disease progression after 7 and 20 months. in the egfr - alk patients, two were treated with egfr tki, two with alk inhibitor and one received both egfr tki and alk inhibitor therapy. most patients treated with alk inhibitor or egfr tki showed partial response or stable disease, but patient no. 12 with clinical data and pathologic features of the 12 patients with concomitant mutations are summarized in table 1. the age at diagnosis of patients ranged from 48 to 78 years old (median, 62 years old) and 75% of patients were female. all three male patients had a history of smoking (1557 pack - years) in contrast to none of female patients. five surgically resectable patients underwent lobectomy and seven clinical stage iv patients had a needle aspiration or biopsy from the lung, bronchus, lymph node, or adrenal gland. 7 had a history of lobectomy at an outside hospital without any other treatment 9 years prior to the lymph node biopsy. 12 previously underwent lobectomy and was treated with gefitinib for 1 month and crizotinib for 4 months in china. she had no egfr or kras mutation at the time of diagnosis at china. aside from this patient, egfr mutations of three patients were only detected with the pna clamping method, while nine were confirmed by sanger sequencing. five (41.7%) had the missense l858r mutation in exon 21, four (33.3%) had the missense g719x mutation in exon 18, and three (25%) had a deletion in exon 19. 12 had a missense r803w mutation at exon 20 that was not identified before targeted therapy at the outside hospital. six patients had additional kras mutations and the other six had an alk mutation (referred to as egfr - kras and egfr - alk hereafter). three among the six patients who showed positivity at alk ihc were confirmed with fish analysis. six egfr - kras patients had papillary, acinar, solid, and micropapillary patterns (fig. 1). among them, five patients had typical hobnail cell features with apical snouts ; the remaining patient (patient no. 4) had a focal columnar cell component and intraand extracytoplasmic mucin, which are similar morphologic features of nsclc with a kras mutation. in contrast, six egfr - alk patients showed solid, cribriform and micropapillary patterns. three of six patients had signet ring cells and intracytoplasmic mucin with or without extracytoplasmic mucin production, which are typical features of alk - positive nsclc. seven patients had surgical resection and two were alive without targeted therapy and any evidence of relapse or progression, including one patient who received neoadjuvant concurrent chemoradiation therapy. one patient who had surgery and decided not to receive targeted therapy due to old age and underlying diabetes mellitus died of disease after 53 months from surgery (patient no. egfr tki included gefitinib (250 mg, orally, every day) and erlotinib (150 mg, orally, every day) and the alk inhibitor was crizotinib (250 mg, orally, every day or twice daily). all three patients showed partial responses, but two also showed disease progression after 7 and 20 months. in the egfr - alk patients, two were treated with egfr tki, two with alk inhibitor and one received both egfr tki and alk inhibitor therapy. most patients treated with alk inhibitor or egfr tki showed partial response or stable disease, but patient no. concomitant genetic alteration of nsclc is unusual because egfr, kras, and alk mutations are widely known as mutually exclusive. most are associated with acquired mutation after targeted therapy and are related to drug resistance. in previous studies, underlying intratumoral heterogeneity of egfr mutations, different tumor cells may have different oncogenic driver mutations. some authors have also suggested coexistence of mutations in the same tumor cells using ihc expression and mutation - specific antibodies. egfr, as a growth factor membrane - bound receptor tyrosine kinase, controls cell proliferation and survival. approximately 36% of patients with nsclc in east asia have egfr mutations and 90% of mutations are exon 21 l858r or exon 19 deletion. these mutations increase tyrosine kinase activity, resulting in hyperactivation of the ras - raf - mek - erk pathway, which regulates cell proliferation, and the phosphoinositide 3-kinase akt mammalian target of rapamycin pathway, which is associated with cell survival. ras proteins are central mediators in egfr tyrosine kinase signaling and also associated with cell proliferation and differentiation. in particular, kras mutations are common in lung cancer but less common in east asia than in western countries. alk fusion with various n - terminal fusion partners stimulates kinase activity, and signaling of these mutations are involved in cell growth, proliferation and apoptosis. among nsclcs, 3%7% harbor alk fusion and the majority of these fusions are associated with the echinoderm microtubule - associated proteinlike 4 (eml4) gene. typical histologic features of lung cancer with egfr, kras, and alk mutations have been described [20 - 24 ]. all of these mutations are frequent in adenocarcinoma and egfr mutations are more common in tumors with papillary, micropapillary, acinar, and lepidic patterns than in others. hobnail cell type and intranuclear inclusion are typical nuclear features of egfr mutation - harboring tumors. extracellular mucin production, cribriform patterns, and signet ring cell features are more common in alk mutation tumors. in this study, we analyzed six egfr - kras (1.5%) and six egfr - alk patients (2.1%) among kras and alk mutation - positive nsclc patients. the majority of egfr - kras tumors showed typical histologic features of egfr mutation, except one case which had focal morphologic features similar to kras mutation. in a previous study, nsclc harboring a kras mutation showed resistance to egfr tki and adjuvant chemotherapy. in our study, however, three patients showed partial response to gefitinib and erlotinib for 729 months. intratumoral heterogeneity of the mutation may explain this discordance between coexistent kras mutation and response to egfr tki therapy. 2 had short progression - free survival (pfs) and disease progressed rapidly, while the other two patients had 2029 months of pfs. based on the evidence of morphologic phenotypes and responsiveness to targeted therapy, we expected these two patients to have coexistent mutations in the same tumor cells, and hypothesized that the egfr mutation was the dominant driver oncogene in lung cancer, even if the tumor cells harbored an additional kras mutation. in egfr - alk patients, some previous studies have shown acquired egfr mutations as a mechanism of resistance to alk inhibitor therapy. in our case, patient no. 12 had no egfr or kras mutation and was treated with crizotinib in an outside hospital. after 4 months of alk - targeted therapy, she acquired an additional egfr mutation and showed poor response to erlotinib, which is consistent with previous studies. in the other four patients, however, conflicting results of responsiveness to egfr tki and alk inhibitor in egfr - alk patients in nsclc have been reported in a few limited studies. investigated relationships between receptor phosphorylation and response to targeted therapy, and suggested that relative phospho - egfr and alk levels can be used to predict response. although the g719x mutation accounts for about 7% of egfr mutated nsclc in east asia, three of five egfr - alk patients had the egfr exon 18 g719x missense mutation in our study. in addition, three of five egfr - alk patients showed typical histologic features of alk - expressing adenocarcinoma. from these findings, we suspect that the alk mutation determines morphological phenotypes and acts as a driver oncogene, and the egfr mutation may also play an important role in oncogenesis. accordingly, based on evaluation of the signaling pathway, including phosphorylation of receptor kinase, the modification or combination of egfr tki and alk inhibitor offer promising treatments. revealed the possibility of more frequent concomitant mutations than expected and won. reported an increased proportion of nsclc harboring concomitant egfr and alk mutations from 4.4% to 15.4% using more sensitive methods such as targeted next - generation sequencing (ngs) and mutant - enriched ngs. greater numbers of previously undisclosed concomitantly mutated tumors can be identified using more sensitive and advanced techniques. some authors also suggest that tumor burden of each mutation affects responses to targeted therapy. there may be a limitation to evaluating mutations from biopsy specimens, but the majority of patients who need to receive targeted therapy might be clinically unresectable and may only be diagnosed with biopsy. thus, the expectation of tumor burden in a limited biopsy specimen is unfavorable. to the best of our knowledge, morphologic findings might help predict underlying driver mutations and further studies are warranted. in conclusion, we investigated genetic driver mutations in 12 double mutated nsclcs with analysis of clinical and pathologic features. the majority of egfr - kras tumors showed typical histologic patterns and cell features of egfr - mutated tumors and partially responded to egfr tki. in egfr - alk tumors, however, some showed alk - mutated features and partially responded to alk inhibitor or egfr tki. egfr and alk play an important role in the oncogenesis of nsclc and tumor morphology can provide important clues to predict treatment response. we suggest that patients with histologic features of egfr mutations can be treated with egfr tki, even with coexistence of a kras mutation. we also suggest that egfr - alk patients should have underlying mutational pathways evaluated and be treated with a selection and/or combination of alk inhibitor and egfr tki. | background : although epidermal growth factor receptor (egfr), v - ki - ras2 kirsten rat sarcoma viral oncogene (kras), and anaplastic lymphoma kinase (alk) mutations in non - small cell lung cancer (nsclc) were thought to be mutually exclusive, some tumors harbor concomitant mutations. discovering a driver mutation on the basis of morphologic features and therapeutic responses with mutation analysis can be used to understand pathogenesis and predict resistance in targeted therapy. methods : in 6,637 patients with nsclc, 12 patients who had concomitant mutations were selected and clinicopathologic features were reviewed. clinical characteristics included sex, age, smoking history, previous treatment, and targeted therapy with response and disease - free survival. histologic features included dominant patterns, nuclear and cytoplasmic features. results : all patients were diagnosed with adenocarcinoma and had an egfr mutation. six patients had concomitant kras mutations and the other six had kras mutations. five of six egfr - kras mutation patients showed papillary and acinar histologic patterns with hobnail cells. three of six received egfr tyrosine kinase inhibitor (tki) and showed partial response for 729 months. all six egfr - alk mutation patients showed solid or cribriform patterns and three had signet ring cells. five of six egfr - alk mutation patients received egfr tki and/or alk inhibitor and four showed partial response or stable disease, except for one patient who had acquired an egfr mutation. conclusions : egfr and alk mutations play an important role as driver mutations in double mutated nsclc, and morphologic analysis can be used to predict treatment response. |
subepithelial tumors (sets) may arise from deep mucosa to serosa depending on the histological type. sets are mostly asymptomatic lesions with normal overlying mucosa, which are often incidentally found during endoscopic or radiologic examinations (overall frequency 0.3%).1 endoscopic ultrasonography (eus) is a method which has led to a demonstrable improvement of the differential diagnosis of sets, but a definite diagnosis can not be done on eus features alone in some cases, especially for hypoechoic lesions.2 - 4 and, although most sets used to be considered as benign, they do have malignant potential, especially when they originate from the muscularis propria layer such as gastrointestinal stromal tumor (gist). in general, a tissue diagnosis may not be necessary for large (> 3 cm in diameter) and/or symptomatic lesions that require surgery regardless of the histology.4,5 currently, there are no firm guidelines for the management of small (<3 cm in diameter, < 2 cm if gist is suspected), asymptomatic sets that originate from the muscularis propria layer. periodic follow - up examination by endoscopy and eus remains the recommended management strategy. however, this approach involves issues related to patient compliance, cost - effectiveness, and the risk associated with repeated endoscopic procedures and delayed diagnosis of malignancy. there is a growing body of evidence that a pathological diagnosis is necessary for small (<3 cm in diameter), asymptomatic, hypoechoic sets originating from the muscularis propria on eus. however, the diagnostic efficacy of current tissue sampling techniques such as eus - guided find needle aspiration or eus - guided trucut biopsy appears to be limited.6,7 the use of endoscopic submucosal resection (esmr) or endoscopic submucosal dissection to resect submucosal lesions is another technique to obtain tissue specimens for accurate histologic diagnosis. endoscopic resection of lesions arising in the muscularis propria using an insulated - tip electrosurgical knife has also been reported in several small case series. in the present review, a various endoscopic techniques ranging from simple snare resection to endoscopic submucosal tunnel dissection for the management of sets will be introduced. sets which are pedunculated or sessile with less than 1 to 2 cm of base can be resected by using a snare with or without saline injection with diluted epinephrine (1:100,000) and indigo carmine dye into the submucosal layer using an injection needle (fig. one series included 45 patients with sets in various parts of the gastrointestinal tract which were resected with a polypectomy snare with or without assistance of a grasping forceps using a double - channel endoscope.8 kojima.9 reported their experience with polypectomy of 31 sets with snare electrocautery and a grasping forceps via double channel endoscope with no perforations and 9% of bleeding rate. a lifted lesion is aspirated into the ligation device, followed by deployment of the elastic band. snare resection is performed using blended electrosurgical current, and the resected specimen is then removed by aspiration into the cap or retrieved with a net (fig. the maximum size of a resectable set is limited because the diameter of ligation devices is usually between 9 and 11 mm. wehrmann.10 demonstrated a successful resection of esophageal sets 13 mm and less in size using esmr - l. in another series, esmr - l was performed in all 25 esophageal sets in less than 10 minutes of procedure time, and the en bloc resection rate was 100%. there were no serious complications such as perforation or massive bleeding.11 a lifted lesion is sucked into the cap thus creating a pseudo - polyp which is immediately captured by closing the prepositioned emr - snare. finally the lesion is removed using electrocoagulation. kajiyama.12 proved that esmr - c was a safe, effective and less invasive procedure for small esophageal leiomyomas (< 2 cm in diameter) derived from the muscularis mucosa. lee.13 evaluated the diagnostic yield and safety of endoscopic partial resection using the unroofing technique in 16 patients with hypoechoic sets of <3 cm in diameter, originating from the muscularis propria on eus (14 gastric and 2 esophageal lesions). the overlying mucosa of the set was removed using an electrosurgical snare with electrical current under minimal aeration to expose the tumor sufficiently (unroofing). next, the snare grasped the half upper portion of the exposed target lesion and cut the lesion. they showed a high diagnostic yield (93.7%) and no major complications using unroofing technique. there are a few small case series on endoscopic resection of sets originating from the muscularis propria by using an insulated - tip knife (table 1).14 - 19 it must be emphasized that characterization of the layer of origin with eus is necessary before attempting endoscopic resection because the risk of the procedure, especially perforation, is directly related to the depth of the tumor within the esophageal or gastric wall. it is possible to achieve complete tumor dissection using the insulated - tip knife if the endoluminal set is loosely adherent to the muscularis propria regardless of their size and shape (fig. endoscopic submucosal dissection preserves the integrity of the stomach, and shortens patient recovery time. inoue.20 reported a new technique of submucosal endoscopic tumor resection using submucosal tunnel created by the technique of peroral endoscopic myotomy, which was introduced for esophageal achalasia. following initial mucosal incision approximately 5 cm proximal to the edge of the set, saline and indigo carmine are injected to create a mucosal bleb. all muscle bundles that connect to the set are cut with the triangle - tip knife. because the overlying mucosal layer is kept intact, this technique may be effective in preventing mediastinitis and peritonitis. in recent three reports about this technique, the success rate was high and the complication rate was acceptable (table 2).20 - 22 the endoscopic resection of sets has been reported in many series and variety of techniques has been introduced. but the efficacy and safety of endoscopic resection for sets are not established yet. in all series, follow - up was limited and studies have not included examination of surgical or autopsy specimens to confirm complete resection. therefore, regular follow - up is mandatory after endoscopic resection, and wide resection should be considered if an endoscopically resected set proves to have been incompletely removed and where the histology report shows the tumor to be a gist with a high risk of malignancy. | subepithelial tumors (sets) are often incidentally found during endoscopic examinations. endoscopic ultrasonography (eus) is a good method for differential diagnosis of sets, but a definite diagnosis can not be made based on eus features alone in some cases. periodic follow - up examinations by endoscopy and eus remains the recommended management strategy, which involves issues related to patient compliance, cost - effectiveness, and the risk associated with repeated endoscopic procedures and delayed diagnosis of malignancy. endoscopic resection of the sets is another technique to treat them as well as to obtain tissue specimens for accurate histologic diagnosis. herein, a various endoscopic techniques ranging from simple snare resection to endoscopic submucosal tunnel dissection for the management of sets will be reviewed. |
breast cancer is one of the most common cancers affecting women, accounting for approximately 30% of all cancers among women worldwide1, 2. breast cancer - related lymphedema (bcrl) is chronic swelling that can occur in the arms and/or hands, trunk, or breasts of patients who have received treatment for breast cancer. lymphedema is characterized by the accumulation of interstitial fluid and tissue alterations due to insufficient lymph drainage1, 3. removal of axillary lymph nodes is the primary risk factor, and the risk increases significantly when this is followed by postoperative radiation therapy4, 5. bcrl usually develops gradually, and the swelling can range from mild to severe. lymphedema may reduce the range of movement of the affected limb, and impair functional ability as well as cause physical disfigurement, pain, skin problems, anxiety, depression, and overall poorer quality of life1, 3, 4. the clinical diagnosis of lymphedema is typically based on measurements of arm circumference or volume. measuring circumference with a tape measure is the most frequently used technique in clinical practice9, 10. an alternative for measuring hand swelling is the figure - of - eight method11,12,13,14, which involves a simple measuring tape passed in a figure - of - eight configuration around the hand and wrist at specified points, particularly areas of the hand where swelling is known to accumulate, such as the dorsum11. the obvious advantage of this method is that only a simple measuring tape is required. the figure - of - eight method may also provide a clinically useful measure of hand size. concordantly, borthwick.15 report that the figure - of - eight method is a valid and reliable method of measuring hand swelling related to bcrl. hand edema that negatively affects daily activities and functional mobility is also present in 6070% of upper - extremity lymphedema cases16,17,18,19. however, there is insufficient research related to the evaluation of hand function in upper - extremity lymphedema. therefore, this study evaluated the functional mobility and kinesthetic sense of the hands as well as daily living skills in patients with upper - extremity lymphedema. all patients had completed breast cancer therapy (surgery, radiotherapy, or chemotherapy) at least 3 months before the beginning of the study. the inclusion criteria were as follows : (a) age over 18 years, (b) able to understand and speak the turkish language, (c) unilateral lymphedema ranging from mild to severe, and (d) willingness and ability to participate in the study. meanwhile, the exclusion criteria were as follows : previous contralateral breast disease, cancer recurrence, disorders related to muscles or joints, severe axillary pain, and conditions that would make participation difficult (e.g., dementia). all patients were informed of the purpose and procedures of the study, and signed an informed consent form in accordance with the guidelines approved by the university hospital ethical committee and the declaration of helsinki. the patients were divided into two groups according to the presence of hand edema : 29 patients with hand edema (he+ group) and 28 patients without hand edema (he group). the lymphedema severity of the arms was assessed, and functional mobility, kinesthetic sense, daily living skills, and hand size were evaluated for both hands. before testing, a complete medical history was obtained from each patient, including demographic information (i.e., age, sex, height, weight, body mass index [bmi ], profession, dominant hand, and affected hand) and disease characteristics (i.e., type and side of the operation, number of excised axillary lymph nodes, number of tumors and positive lymph nodes, radiotherapy technique used, adjuvant systemic treatment, lymphedema duration, and previous infection episodes). edema of the arm was assessed by circumference measurements taken with a standard 1-inch retractable fiberglass tape measure. for the arm circumference measurements, patients were in a supine position with their arms relaxed by their sides and elbows straight. arm circumference was measured at 5-cm intervals beginning at the third phalanx nail fold and continuing 45 cm proximally. the severity of lymphedema was classified according to a modified version of the criteria described by the american physical therapy association : circumference differences between arms 5 cm were defined as mild, moderate, and severe lymphedema, respectively9, 20. a standard 0.25-inch - wide retractable fiberglass tape measure was used to perform figure - of - eight measurements of hand size14. the testing position required the patient to sit with their arms abducted and externally rotated 90, elbow flexed 90, wrist neutral, fingers adducted and extended, and thumb abducted in the plane of the hand. hand functional mobility was assessed by modified kapandji index (mki)21. the assessment was conducted twice by the physiotherapist with a 1-hour interval between trials. the mki score was obtained by summing the scores of the following three tests. the first test assessed the opposition of the thumb, which was scored from 0 (impossible to do) to 10 (completely accomplished). the second test evaluated the flexion of each long finger, which was scored from 0 (impossible to do) to 5 (completely accomplished). the third test assessed finger extension, which was scored from 0 (impossible to do) to 5 (completely accomplished). total scores for the scale of finger flexion (maximum 20 points), scale of finger extension (20 points), and total opposition test (10 points) were recorded. the total score of the three tests (maximum 50 points) was calculated and used for analysis. the kinesthetic sense of the hand was measured by examining the patients ability to copy hand positions22, 23. the test was conducted in a quiet well - lit room, with the patient comfortably seated using a table and chair of appropriate height. a masking box was placed on the table to occlude the patient s vision while allowing the physiotherapist to have a full view of the patient s hand. the box had an internal shelf that supported the forearm along predetermined lines drawn at an angle of 20 to the table edge ; the hands hung freely over the edge of the self. 2.hand positions), and the patient was instructed to produce mirror images of these gestures with the tested hand, which was hidden inside the masking box. the time taken to copy each test pattern was recorded in seconds rounded to two decimal places, and the accuracy of replication was graded using the criteria described by lynch : 0, failure to move the hand from the resting position ; 1, no resemblance to the test position ; 2, incomplete replication, which may include use of the wrong fingers in the correct relationship, one finger out of place, inappropriate opposition, or a reversal of the gesture ; 3, complete and accurate replication. any position that could not be held by the non - test hand during kinesthetic cuing was also noted. the final score was reported as the mean replication accuracy in the tested hand for all eight test positions or the percent of kinesthetic sense lost with respect to the total possible score achievable. daily living skills were assessed by the 62-item hand function sort (hfs)24. this instrument is a self - rating of ability to perform the task by reviewing the item and judging one s ability on a five - point scale (4, able ; 3, slightly restricted ; 2, moderately restricted ; 1, very restricted ; 0, unable). a total score for the items in each physical demand characteristic level was obtained along with an overall rating of perceived capacity score ranging 0248. the items represent tasks with a broad range of physical demand, including activities of daily living. the significance of differences between two groups was determined by the mann - whitney u - test. pearson s product - moment correlation coefficients were calculated to determine the relationships between measurement techniques. however, one patient withdrew because of infection, and two patients declined to participate, resulting in a final study group of 57 patients. the mean ages of the he+ and he groups were 55.3 11.1 years (range : 3373 years) and 56.7 10.6 years (range : 4070 years), respectively ; mean bmi was 26.9 4.2 and 27.7 3.8 kg / m, respectively. the demographic characteristics and evaluation results of the 57 women are presented in table 1table 1.patients characteristicslymphedema severityhe+ group (n = 29)he group (n = 28)mild (n = 6)moderate (n = 15)severe (n = 8)mild (n = 5)moderate (n = 13)severe (n = 10)age (years, mean sd (range)52.3 10.0354.7 11.260.2 9.754.8 7.856.3 11.858.2 11.04(3368)(3570)(5173)(4666)(3370)(4275)bmi (kg / m, mean sd)24.1 2.926.03 3.531.0 3.526.9 1.726.4 3.929.7 3.9dominant armright51265117left132 - 23affected arm (%) dominant51053119non - dominant153221duration of lymphedema (years, mean sd)2.7 1.34.5 3.45.7 9.62.3 0.55.9 6.66.7 4.8lymph nodes removed (mean sd)5.8 2.212 8.922.7 6.89.2 3.515.6 10.127.1 11.2treatmentset+rt242132ct+rt13 - -22et+ct+rt396486history of recurrent cellulitis (%) -4087.5 - 53.889.2hand size (mean sd)36.1 5.941.7 7.252.9 9.635.5 7.736.8 9.538.4 10.8kinesthetic sense (% lost)16.653.387.5 - 30.860functional mobility score (mean sd)46.8 13.539.6 4.330.3 4.949.1 9.741.9 8.2 34.9 2.5hfs score (mean sd)214.0 11.8199.4 16.6178.4 44.5235.2 1.09217.5 15.1199.1 51.3he : hand edema, et : endocrine therapy, rt : radiotherapy, ct : chemotherapy, hfs : hand function sort. he : hand edema, et : endocrine therapy, rt : radiotherapy, ct : chemotherapy, hfs : hand function sort according to arm circumference, 18 (31.58%), 28 (49.14%), and 11 (19.28%) patients had severe, moderate, and mild lymphedema, respectively. using the figure - of - eight method of hand size measurement, 10 (34.5%), 12 (41.4%), and 7 (24.1%) women in the he+ group had severe, moderate, and mild hand lymphedema, respectively. hand size differed significantly between the affected and unaffected hands in the he+ group (p 0.05, table 2table 2.hand size, mki scores, kinesthetic sense, and hfs scorehe+ groupn = 29he groupn = 28hand size (cm)43.5 7.236.9 9.3mki score38.9 11.541.3 9.2kinesthetic sense score1.8 2.512.6 1.78hfs score199 10.2217.7 12.4p 0.05, table 2). there was no significant difference regarding the functional mobility of the hand in patients whose dominant or non - dominant arm was affected (p > 0.05). meanwhile, the functional mobility of the affected hand was significantly lower than that of the unaffected hand in the he+ group (p 0.05). hfs scores revealed that increasing edema severity was significantly correlated with diminished ability to perform activities of daily living (p < 0.05, table 3). the hfs score was significantly higher in the he group than the he+ group (p < 0.05, table 2). within each group, the score of the affected hand was significantly lower than that of the unaffected hand (p < 0.05). in both groups, daily living skills were significantly better in women with lymphedema of the dominant arm than the non - dominant arm (p < 0.05). the present study assessed the functionality and kinesthetic sense of the hand in women who developed bcrl. in both groups, hand function decreased with increasing arm edema. furthermore, the kinesthetic sense of the hand was significantly better in patients without hand edema than those with hand edema. this result may indicate stronger interactions between edema severity and reductions in hand function and kinesthetic sense. lymphedema results in pain, loss of sensation, muscular weakness, lack of movement in articulations, and impairment of upper extremity functional skills25,26,27. most studies used standard circumference measurement and volumetric measuring to assess edema severity9, 28, 29. borthwick.15 demonstrated that the figure - of - eight method, the primary assessment used in the present study, is a valid and reliable technique for measuring hand swelling related to bcrl. the present results also show the results of the figure - of - eight method are strongly correlated with those of the circumference measurement method. it was reported in previous studies that the hand area was affected in 6070% of cases of upper - extremity lymphedema. however, no studies have evaluated how functional mobility of the hand is affected by lymphedema occurring after breast cancer treatment. lefevre - calau.21 report that the mki is a valid and reliable assessment method for evaluating the functional mobility of the hand in patients with rheumatoid arthritis. meanwhile, we examined the effect of upper - extremity lymphedema on the functional mobility of the hand using the mki. the results show the functional mobility of the hand decreased with increasing edema severity in the affected arm of both groups. the functional mobility of the hands in the he group tended to be better than in the he+ group, although the difference was not significant. furthermore, there was no difference in the functional mobility of the hand between cases in which the dominant and non - dominant arm was affected. this could be attributable to the small number of cases in which the non - dominant arm was affected in the present study. loss of sensation in the upper extremity due to surgery is another post - treatment problem that is exacerbated by edema31,32,33. bosompra.32 and maunsell.33 report that 63% and 61% of patients with lymphedema experience loss of sensation, respectively. hwang.22 hypothesize that a loss of kinesthetic sense of the hand results in inability to perform many functional skills of the hand, consequently causing difficulty performing activities of daily living. the present findings indicate that a loss of kinesthetic sense is correlated with hand edema severity. loss of kinesthetic sense was observed in 65.3% of patients, who also had impaired daily activity skills. these findings patients who develop lymphedema during the post - treatment period of breast cancer experience a sense of heaviness in the arm, muscle weakness, restricted shoulder mobility, difficulty performing activities of daily living, and overall diminished quality of life5, 17,18,19. gosselink.34 evaluated postoperative upper - extremity function in breast cancer patients and found that restriction of the movement of the upper and lower extremities negatively affects patients ability to perform activities of daily living, necessitating immediate physiotherapy. voogd.19 evaluated the effect of upper - extremity lymphedema on quality of life and found it caused a loss of daily functional skills, energy and motivation ; they conclude that such factors lead to diminished quality of life. as mentioned in the above examples, most previous studies focused on the functional impact of lymphedema of the shoulder and upper extremities. however, the hand plays a significant role in upper - extremity function32, 33. thus, it is surprising how few studies have employed activities of daily living questionnaires to assess the functional skill of the hands in lymphedema patients. the present study examined the effect of upper - extremity lymphedema on hand function by using the hfs questionnaire. the results indicate that daily life activity skills decreased with increasing edema severity in the affected hand ; this relationship was even stronger when the dominant arm was affected. this study is valuable in that few studies have evaluated hand function in upper - extremity lymphedema. however, the relatively small number of patients may limit the study s power. further studies on larger samples are needed to support the findings. in conclusion, the functional mobility, kinesthetic sense, and daily living skills of the hand can be negatively affected in upper - extremity lymphedema. therefore, greater emphasis should be placed on addressing the long - term post - treatment complications of lymphedema in patients with breast cancer, such as sensation loss and functional impairment of the hand, which significantly impact quality of life. | [purpose ] this study evaluated the functional ability and kinesthetic sense of the hands of women with breast cancer - related lymphedema. [subjects and methods ] fifty - seven women experiencing lymphedema after breast surgery and adjuvant radiotherapy were included. the patients were divided into two groups : women with hand edema (he+, n = 29) and without hand edema (he, n = 28) after breast cancer treatment. arm edema severity, hand size, functional mobility and kinesthetic sense of the hand, and daily living skills were evaluated. [results ] the mean age of the patients was 55.8 years. in both groups, functional mobility, kinesthetic sense, and daily living skills decreased significantly with increasing edema severity. however, there was no significant difference between groups with respect to functional mobility or daily living skills. the kinesthetic sense of the hand was better in the he group than the he+ group. there was a significant negative relationship between the severity of edema and hand function. [conclusion ] breast cancer - related lymphedema can negatively impact women s functional mobility and kinesthetic sense of the hands as well as daily living skills. |
metabolic syndrome (mets) is a constellation of risk factors associated with increased risk of diabetes and cardiovascular disease (1). in 2001, the national cholesterol education program (ncep) adult treatment panel iii (atp iii) provided a new definition for mets, focusing on easily measurable clinical parameters such as abdominal obesity, increased fasting blood sugar, increased serum concentration of triglyceride, low serum high - density lipoprotein cholesterol (hdl - c) and/or elevated blood pressure. in 2004, the threshold for fasting blood sugar was reduced to 100 mg / dl (5.6 mmol / l) in concordance with the american diabetes association criteria for impaired fasting glucose (2). the new international diabetes federation (idf) definition in 2006 included a lower waist circumference (3). metabolic syndrome is a growing public health problem worldwide in both developed and developing countries (4). there is a general agreement that high prevalence of mets (33.2% and 10.1% among iranian adults and adolescents, respectively) basically deals with increasing incidence of obesity (5 - 7). a strong relationship between mets and dietary pattern, tobacco use and physical inactivity has been reported in the literature (8). the most important therapeutic intervention that has been proven to be effective in metabolic syndrome is lifestyle modification with focus on dietary change and physical activity (2, 9). metformin has been proven to improve insulin sensitivity and shown to be effective on weight loss, although it has minor side effects (10). recent evidence suggests that lifestyle modifications can be a decent way to reduce metabolic and cardiovascular risk factors. the study of giugliano. reported that lifestyle modification (25%) has more benefits in resolution of mets compared to drug therapy (19%) (9). in 1981, jenkins classified carbohydrate - containing foods, based on the glycemic index (gi). the incremental area under the blood glucose response curve to a test food, relative to a standard control food (glucose or white bread) with the same amount of carbohydrate. glycemic index differs according to the rate of digestion and absorption, which depends on the type of carbohydrate and protein, fat and fiber content of the food (11). high gi carbohydrates have been shown to be positively associated with insulin resistance and metabolic syndrome (12). a low gi diet will improve postprandial glycemia and as a result reduce insulin resistance, b - cell dysfunction and hyperinsulinemia (11). the results of a recent pilot study showed that a short - duration low glycemic index fitness program could improve anthropometric and physiological measures in mets subjects (13). also, a systemic review in 2013 provided evidence that administrating a low gi diet was helpful in prevention of obesity - associated diseases (14). the present study was designed to examine the effect of low gi diet versus metformin on adults with mets. sixty adults with mets, aged 25 to 65 years, participated in this randomized clinical controlled trial. patients were included in the study according the following inclusion criteria : having metabolic syndrome characteristics according to idf, waist circumference 90 for males or 80 cm for females (3), blood pressure 135 (systole) or 85 (diastole) mmhg, fasting blood sugar (fbs) 100 mg / dl (newly diagnosed pre - diabetic patients), triglycerides 150 mg / dl, and high - density lipoproteins (hdl) 30) subjects significantly reduced body weight and waist circumference (28). regarding the beneficial effect of metformin on lipid profiles, it diminished rho (small gtp - binding protein, which is generated in the process of cholesterol biosynthesis) kinase activity in hyperlipidemic rats (29). reported that metformin administration can ameliorate tg, total cholesterol and ldl - c levels in six weeks ; this is in agreement with the results obtained in the current study (30). low gi diet may also have the ability to decrease blood lipids by reducing hyperinsulinemia. the investigation of bouch. showed that eight - week consumption of low gi diet improved lipid profiles significantly in overweight and non - diabetic subjects (31). moreover, heilbronn. observed that low gi diet leads to a noticeable decrease in tg, total cholesterol and ldl in type 2 diabetic patients (32). also, the outcome of a systemic review suggested that low gi diet could significantly reduce ldl and total cholesterol (33). the present study also showed that low gi diet diminished lipid profile and lipoprotein ratio (ldl / hdl) in patients with metabolic syndrome. low gi has positive effects on blood pressure through its reducing effects on obesity, hyperglycemia and hyperinsulinemia (11). moreover, low insulin concentration can lead to a reduction in sympathetic nervous system activities, which decrease heart rate, cardiac output and sodium retention and thus blood pressure (34). in the present study, low gi diet reduced systolic and diastolic blood pressure. these results are consistent with those of sloth. who found that low gi diet decreased systolic blood pressure markedly in overweight females during a ten - week trial (35). moreover, in the study of melanson., low gi diet decreased blood pressure, although not significantly (27). the current study showed that metformin intake was also associated with significant alterations in blood pressure (bp). the mechanism underlying the bp - lowering effect of metformin is obscure, yet a decrease in peripheral hyperinsulinemia may be implicated (36). moreover, a reduction in sympathetic nervous system activity, as suggested by a 25% decrease in plasma norepinephrine after metformin consumption could also contribute to the condition (37). giugliano obtained similar results for bp in obese - hypertensive subjects ; a considerable reduction was detected after three months (21). the inconsistent results of studies on the effects of low gi diet or metformin on metabolic parameters in obese or diabetic individuals can be attributed to different features of studies. these include differences in design, sample size, dosage and length of metformin administration. in conclusion, eight weeks of low gi diet and metformin therapy in mets could significantly decrease body weight, waist circumference, blood pressure, fbs, hba1c, lipid profiles and lipoprotein ratio (ldl / hdl), although the difference between groups was not noticeable except for fbs, for which the reduction was slightly larger (3 mg / dl) in the metformin group, yet this was not clinically considerable. since metformin may cause side effects such as lactic acidosis, vitamin b12 deficiency, gastrointestinal disturbances and hepatotoxicity (38), low gi diet can be safer in managing mets. to the best of our knowledge, the present study was the first trial on mets that compared low gi diet and metformin drug therapy although with a small sample size. in addition, the short duration of the intervention was the other limitation of this clinical trial. further investigations with larger populations and longer periods are required to confirm whether metformin therapy can be replaced by low gi diet. | background : metabolic syndrome (mets) continues to be highly prevalent and contributes to a rapidly growing problem worldwide. the most important therapeutic intervention for metabolic syndrome is diet modification, an intervention whose efficacy has been proven for metabolic syndrome.objectives:the aim of the present study was to compare the effects of low glycemic index diet versus metformin on mets components in adults with mets.patients and methods : fifty - one adults with mets participated in this randomized controlled clinical trial. patients were randomly allocated to two groups of metformin and low glycemic index diet. the intervention period was eight weeks. the studied participants were compared at baseline and the end of the trial, regarding the following factors : weight, blood pressure, waist circumference, fasting blood sugar, hemoglobin a1c and lipid profiles (triglyceride (tg), total cholesterol (tc), low - density lipoprotein (ldl) cholesterol, and high - density lipoprotein (hdl) cholesterol).results : the anthropometric measurements, fasting blood sugar (fbs), hemoglobin a1c, serum lipid profiles (tg, tc, ldl - c, hdl - c) and lipoprotein ratio (ldl / hdl) showed a significant decrease after the intervention in both groups (p < 0.05). comparison of the difference between the two groups was not significant, except for the mean reduction in fbs, which was more in the metformin group although this was not clinically significant.conclusions:this study supports the assumption that low glycemic index diet as well as metformin can positively affect metabolic syndrome components. |
sexually transmitted diseases (stds) are markers of high risk sexual behavior. herpes simplex virus (hsv) types 1 and 2 cause genital herpes infections and are the most common cause of genital ulcer disease worldwide. considering that herpes is a life long infection, not cured by antimicrobial treatment, hsv-2 antibodies are a much more reliable indicator of risky behavior than treponema palladium antibodies. as large number of genital infections the present study was thus undertaken for finding igm antibodies against hsv-1 and 2. while most herpetic infections are asymptomatic or mild, some can be transmitted to neonates and are associated with other stds and cervical neoplasia. genital herpes may contribute more to human immunodeficiency virus (hiv) infection because of its higher frequency than other stds, the recurrence of genital herpes and large number of herpes infected persons who continue their sexual activities despite being infectious. serology is the only practical way to diagnose hsv infection in individuals without any relevant clinical history or presentation with lesions. immunoglobulin m (igm) antibodies to hsv are increased to four times the normal value 24 weeks after the infection and the enzyme linked immuno sorbent assay (elisa) is a specific, sensitive, and simple test which confirms the infection by hsv. one hundred fifty (150) blood samples were collected from patients attending the std clinic attached to a tertiary care hospital of ahmedabad. these patients clinical diagnosis was made by department of skin and venereal diseases, b. j. medical college, ahmedabad. the consecutive patients only whose clinical history suggested that clinical manifestations of stds were established by sexual route were included in this study. along with blood sample, all clinical history especially regarding the recurrence of genital herpes was also taken. the sera were tested for hsv-1 and 2 igm type specific antibodies by elisa ca, usa. as we wanted to study genital herpes infection as a whole, irrespective type of causative agent, we used elisa assay that detects hsv-1 and 2 igm antibodies simultaneously. results are analyzed here. in our study, out of 150 patients we found 23 (15.66%) for serum hsv-1 and 2 igm from all the std patients. in disease wise analysis, numbers of genital herpes were the highest among all diseases and their proportional positivity for serum hsv-1 and 2 igm was 18% only. all other stds and their proportional positivity of serum hsv-1 and 2 igm antibodies are as described in table 1. disease pattern in std clinic patients 98 male and 52 female out of 150 std patients, 10 male and 13 female were positive for serum hsv-1 and 2 igm. female has statistically significant higher positive proportion than male (p 97%) of all stds fall into 1549 age group. overall 2130 age group has more number of hsv positive patients (52%) than any other age group and especially in female 76% hsv positive in this age group [table 2 ]. age - sex wise distribution of std patients out of 150 patients, 65 were of clinical symptoms of genital herpes. from these 65 patients, 30 and 35 serum hsv-1 and 2 igm were found positive in 16.66% (6 out o 30) in patients with recurrent history of genital herpes and also in 20% (7 out of 35) in patients without any clinical history of herpes in the past [table 3 ]. this study applies the clinical presentation of std and serological herpetic infection correlation in a std clinic of a tertiary care hospital in india. that will help in interpreting the test result of serum hsv-1 and 2 igm in any std patients as many times serological test reports are given unnecessary higher importance without knowing the surrounding factors. the herpes simplex virus persists life - long in neuronal cells (especially in trigeminal and sacral ganglia) and is frequently reactivated with or without clinical manifestations. this study effectively shows that when the disease (e.g., genital herpes) whose prevalence is more in population concerned and have latent period, its serological test especially detecting igm only helps in screening to detect the burden of the disease, but has less correlation with the clinical symptoms. all 23 serum hsv-1 and 2 igm positive are also distributed in other type of stds (48% 11 out of 23), apart form genital herpes cases (52% 12 out of 23). now a days, serum hsv-1 and 2 igm found positive in patients of non - herpetic clinical manifestations in std patients is quite common and also reported by many authors in india as well as outside india too. this shows that these patients may have either simultaneous co - infection with hsv-1 and or 2, though the virus is not clinically manifesting the herpetic symptoms and patients shows asymptomatic sero conversion or hsv-1 and /or 2 igm reactivation. despite the relatively high number of genital herpes positive among all stds (12 out of 23), proportional positivity rate of serum hsv-1 and 2 igm is highest in cervico - vaginal discharge (27%). female as compare to male has tendency more number and early sero conversion. in our study, a high proportion (25%) of female patient was hsv positive and even from this positive female, 84% were up to age of 30 year. detection of sub clinical hsv co - infection in this group, by serology facilitates counseling regarding advisability of acyclovir therapy when needed (in addition to treating the other coexisting std). transmission of infection from hsv positive males to their sexual partners may further cascade the situation. in genital herpetic patients, those having no history of clinical herpetic manifestation in past, only 20% of them show positivity by serum hsv-1 and 2 igm while others remain negative. this is not due to false clinical diagnosis but rather due to earlier primary hsv infection that did not converted in to clinical herpetic presentation and remained unrecognizable at that time. in earlier studies of comparing the relationship between a history of herpes, symptoms suggestive of herpes, and hsv antibody prevalence, overall, only one - third of those with antibodies to hsv had a clinical diagnosis of herpes. while the patients having recurrent genital herpes in genital herpes group, 16.66% of them show positivity by serum hsv-1 and 2 igm. it suggests that along with igg some people with recurrent herpetic infection get reactivated igm. so igm can not be taken as diagnostic for primary episode of herpetic infection only. this study has shown that among stds patients, overall the clinical genital herpes are increased than other std of earlier time like gonorrhea, syphilis. serological analyses have also shown less prevalence of syphilis than herpes in general as well as std group as shown in last some studies too.[1619 ] a higher seroprevalence of hsv-1 and or 2 among females as compared to males has been recorded in the studies in india and outside india. higher seroprevalence among younger women as compared to men of a similar age group was observed.[2022 ] in our study, the prevalence of hsv-1 and 2 among males was 10% (10 out of 98) which was less compared to females 25% (13 out of 52), particularly the 21 - 30 age group, the male to female difference is more 8 to 35%, respectively. since most genital hsv infections are unrecognized and undiagnosed, serum hsv-1 and 2 igm helps to see the iceberg part of the infection among the population concerned. but when serum hsv-1 and 2 igm come positive in any of the patients having std, it may be sero - conversion of primary infection or reactivation. in the community, when hsv transmissibility increased by sexual route, case of primary infection and reactivation of herpes virus also increased. this phenomenon directly leads the level of serum hsv-1 and 2 igm in std patients both herpetic and non - herpetic groups are also going to be increased. so, serum hsv-1 and 2 igm can be used for periodically screening in std patient to know the trend, transmissibility and load of hsv. | background : herpes simplex virus type 2 (hsv-2) is the cause of most genital herpes. now, hsv-1 has become an important cause and represents even about 30% of genital herpes in some countries. so, study related to genital herpes should consider both hsv-1 and hsv-2.aim:to examine trends in hsv-1 and 2 seroprevalence by serum hsv-1 and 2 igm in all type of sexually transmitted disease (std) patients and also to evaluate correlation of serum hsv-1 and 2 igm in std.materials and methods:150 patients attending the std clinic attached to a tertiary care hospital of ahmedabad were included in the study. serum hsv-1 and 2 igm correlations with clinical manifestations of recurrent and non - recurrent type of genital herpes patients and other non - herpetic std patients were studied.results:the overall serum hsv-1 and 2 igm in std seroprevalence were 15.66%. female has significant higher prevalence (p < 0.05). std cases and hsv seroprevalence were specially concentrated in persons aged 21 to 30 years. among those positive with hsv, the distribution of std are wide spread and found in non - herpetic group at high frequency. out of total 23 serum hsv-1 and 2 igm positive, 12 and 11 are distributed in herpetic and non - herpetic stds, respectively.discussion and conclusion : though serum hsv-1 and 2 igm in stds are less diagnostic, they help to see the iceberg part of the infection among the population concerned in recent scenario or in another words, it provides recent infective burden. |
the information on chloroplast genome sequences is essential in different fields of plant biology : plant physiology, population genetics, phylogenetics, and evolution. as a consequence, the number of published chloroplast genome sequences greatly increased in past three years allowing an assessment of general trends of their evolution (reviewed in wicke. in contrast, little is known about the structure of the plastid genome in nonphotosynthetic species. in higher plants, complete plastid genome sequences are available now for only three completely nonphotosynthetic species : two angiosperms the parasitic plant epifagus virginiana from orobanchaceae (wolfe. 1992) and mycoheterotrophic orchid, rhizanthella gardneri (delannoy. 2011), and a mycoheterotrophic liverwort aneura mirabilis (wickett, zhang,. 2008). while there is about 3,000 nonphotosynthetic plants representing more than ten families, this is obviously insufficient to infer general patterns of the evolution of plastome in the absence of photosynthetic activity. we have increased this set by sequencing the entire plastome of a mycoheterotrophic orchid, neottia nidus - avis. neottia belongs to the same family as rhizanthella whose plastid genome was recently characterized (delannoy. 2011) but the shift to heterotrophy occurred independently in these two lineages (molvray. thus, this information is useful for revealing both general features of nonphotosynthetic plant plastomes and for comparing the plastid genome structure under the parallel loss of photosynthetic activity in a specific plant group, the family orchidaceae. the sequence of neottia plastome was assembled from the partial genomic dna sequence produced using high - throughput pyrosequencing technology (454 sequencing) complemented with sanger sequencing. as expected, the plastid genome of neottia is highly reduced in length (92 kb compared with 146149 in photosynthetic orchids) and in gene content (fig. all genes encoding photosystem i and ii components are lost or pseudogenized. the same is true for the genes of the cytochrome b6f components and photosystem i assembly proteins ; ccsa (involved in c - type cytochrome synthesis) and cema (chloroplast envelope membrane protein) genes are also lost. the latter does not however seem to be related to heterotrophic way of life since it is characteristic for several photosynthetic angiosperms, including orchids (chang. 2006 ; wu. 2010 ; blazier. 2011) and for gymnosperms (braukmann. it contains a region with high similarity to matk but its comparison with orthologous sequences from photosynthetic orchids reveals strong divergence of its 5end (including the substitution in the start codon) ; this suggests that in neottia, matk is a pseudogene. despite this, the genes that are supposed to require matk protein activity for the splicing of their mrnas (rpl2, rps12, clpp, trna - ugc, trng - ucc) have retained their introns. this is in contrast with the genus cuscuta where matk loss observed in the species of subgenus grammica is correlated with the loss of group iia introns (mcneal. first, we can imagine that matk - like region retained in neottia is still functional this is possible under the condition that alternative start codon is adopted or that multiple rna editing events occur in the 5end of this region. second, the involvement of matk in the splicing of group iia introns could be not as essential in orchids as it is cuscuta (and, apparently, in most plants). this hypothesis is supported by the fact that rhizanthella, another nonphotosynthetic orchid, totally lacks any matk - like regions but also retains group iia introns and that matk is pseudogenized in corallorhiza trifida (freudenstein and senyo 2008), a mycoheterotrophic species that partially retains photosynthetic activity. genes shown inside the circle are transcribed clockwise, those outside the circle are transcribed counterclockwise. asterisks indicate intron - containing genes, dark gray bars inside the inner circle indicate guanine - cytosine content. the only class of genes that is unaffected by the reduction is the ribosomal rna genes. all four rrna genes characteristic for typical plant plastomes are present in neottia and share very high similarity (9699%) with their orthologs from other orchids. genes encoding another essential component of the ribosome, the ribosomal proteins, are also mostly retained : neottia plastome encodes a complete set of large subunit proteins genes (including rpl23 and rpl22 that are turned to pseudogenes in many photosynthetic species) and most small subunit proteins genes. though they are highly similar to the orthologous genes from photosynthetic orchids in silico translation reveals internal stop codons in both sequences. these stop codons are however in frame and thus are potential targets of rna editing so additional experiments are required to confirm the nonfunctionality of rps16 and rps18. in anthoceros formosae, conversion of nonsense codons into sense is found in 52 genes, including rps18 (kugita. rna editing system was shown to be active in rhizanthella so it is presumably active in neottia plastids too. another possible target of rna editing is the rpl2 gene that has atypical start codon acg. as for transfer rna, we were able to find the sequences with high similarity for all trnas characteristic for plant plastomes with the exception of trni - gau. most of them seem to be functional genes because they share high similarity with their orthologs from photosynthetic orchids (overall similarity of trna sequences between neottia and phalaenopsis is 0.95) and have conserved secondary structure typical for trnas. trnv - uac and trnp - ugg are putative pseudogenes since they differ from their orthologs by multiple substitutions and indels. in contrast to translation apparatus that seems to be almost unaffected, genes of the transcription machinery those encoding plastid rna polymerase subunits are lost (rpoa, rpoc1) or pseudogenized (rpob, rpoc2). two genes involved in plastid metabolism accd and clpp are retained, as well as two large orf encoding proteins of the unknown function ycf1 and ycf2. the retention of ycf1 is the most enigmatic since it is presumably pseudogenized in photosynthetic orchids oncidium and phalaenopsis. the regions with strong similarity to ycf1 are present in their plastomes but their in silico translation reveals multiple stop codons due to nontriplet indels. pseudogenization of ycf1, as well as another large plastid orf, ycf2 occurs in grasses where a region similar to ycf1 is present but highly reduced due to multiple deletions (hiratsuka. the structure of ycf1 in phalaenopsis and oncidium may represent a first stage of gene degradation. another explanation of the frameshifts might be related to sequencing errors because ycf1 is rich in homopolymer regions, which are prone to errors. broader survey of ycf1 integrity in orchids is required to see if this gene is indeed nonfunctional in either lineage of orchids. despite about one - third reduction in length, the overall structure of neottia plastid genome is conserved and collinear with that of photosynthetic orchids. the only alteration of the gene order is the position of inverted repeat - large single copy border that resides in the rps3-rpl16/rps3-trnk spacer in neottia. the expansions of the ir are well documented in different lineages of angiosperms ; in particular in orchids ir was found to include complete sequence of rps19, trnh, and a part of rpl22 (wang. 2008). in neottia, further expansion of the ir is observed it includes rpl22 and rps3. such structure of jir - lsc is by now unique however it is possible that increased taxon sampling will reveal similar structures in other orchids, either photosynthetic or not. to assess the selection constraint on plastid genes in neottia, we performed the analysis of synonymous and nonsynonymous substitutions in protein - coding genes. the examination of dn / ds ratio for individual genes that are shared between neottia, rhizanthella, and photosynthetic orchids demonstrates that most of them are evolving under purifying selection in both nonphotosynthetic species (fig. the sequence of this gene is highly conserved between phalaenopsis and oncidium, differing by single substitution (nonsynonymous). rpl23 is a pseudogene in several photosynthetic plants, for example, caryophylalles (logacheva. 2008). high dn / ds in this gene may indicate on the relaxation of selection constraint as initial stage of pseudogenization. however, in rps14 and rpl33, dn / ds is found to be increased in photosynthetic species. such increase may be caused by the unreliability of dn / ds estimates for short genes (haddrill. 2007) and thus not be biologically relevant. to get a generalized view on substitution rate in neottia and rhizanthella, we calculated substitution values for all genes combined in one sequence. this demonstrated that dn / ds ratios are very similar between two photosynthetic species and between photosynthetic and nonphotosynthetic ones. in contrast, the number of both synonymous and nonsynonymous substitutions differs greatly being about 2.5 times much in neottia and 5 times in rhizanthella (fig. the apparent effect of higher substitution rate is often observed in annual plants when compared with perennials (e.g., yue. 2010) ; however, both neottia and rhizanthella are perennials as well as phalaenopsis and oncidium. this suggests that this effect is related to their heterotrophic way of life and that plastid genes in nonphotosynthetic orchids have higher mutation rate but retain their functionality. pairwise dn / ds (the ratio of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site) for all genes shared between photosynthetic orchids and neottia and/or rhizanthella. n / a indicates the cases where it was not possible to estimate the ratio due to the absence of synonymous substitutions or the absence of gene in the plastid genome. dn / ds ratio and dn and ds values for all genes shared between neottia, rhizanthella, oncidium, and phalaenopsis, combined in one sequence. the broader comparison that includes all nonphotosynthetic higher plant plastomes two orchids, parasitic dicot epifagus and liverwort aneura reveals both parallelisms and dissimilarities in their structure. the plastome sizes differ almost twice from about 108 kb in aneura to 59,190 bp in rhizanthella. the degree of gene loss and pseudogenization also differs in aneura, the plastome is much less affected than in angiosperms. it retains all transfer rna, rna polymerase, and ribosomal protein genes and also many photosynthesis - related genes (wickett, zhang,. 2008) being similar in this respect to the species of cuscuta that retain photosynthetic activity (funk. 2007 ; not a single photosynthesis - related gene is retained as intact reading frame, even in neottia that is characterized by the least reduction in both size and gene content and is only 16 kb less than in aneura. in general, the structure and gene content of plastome are more similar within nonphotosynthetic angiosperms than in any of them compared with aneura. it is well known that the activity of the plastid is not provided by plastid genome only but also is highly dependent on and coordinated with that of nuclear genome (reviewed in taylor 1989 and woodson and chory 2008). the results from an ongoing genomic project of a liverwort, marchantia polymorpha, indicate that the organization of the nuclear genomes in angiosperms and in liverworts differs greatly ; in particular, the diversity of gene families is lower in marchantia (yamato. this and other differences in nuclear genome organization can account for the difference in the structure of plastid genomes in nonphotosynthetic angiosperms and nonphotosynthetic liverworts. other possible explanation is the time past from the moment of switch to heterotrophy. though there are no direct (paleontological) observations on this subject, we suggest that heterotrophy is the most recent in a. mirabilis since another species of the genus aneura are photosynthetic (wickett, fan,. 2008) and the most ancient in epifagus which shares the holoparasitic way of life with related genera conopholis and orobanche (depamphilis. neottia and rhizanthella represent genera that include only nonphotosynthetic species but their most closely related genera are completely or partially photosynthetic. moreover, in some systematic treatments, neottia is merged with photosynthetic genus listera (chase. 2003) that suggests recent switch to heterotrophy, similar to aneura. in terms of gene content, neottia is more similar to epifagus than to rhizanthella (fig. phylogenetically neottia is close to rhizanthella thus the dissimilarity of their plastid genomes is unlikely to be related to major dissimilarities in the organization of their nuclear genomes but rather to lineage - specific variations in rate and pattern of plastid genome evolution. this phenomenon is well known with regard to single nucleotide substitutions (reviewed in muse 2000), and it is likely to be applicable to length mutations as well. gene and pseudogene content in the plastomes of nonphotosynthetic angiosperms, numbers indicate different groups of genes / pseudogenes according to their presence in the plastomes : (1) genes that are present in all plastid genomes of nonphotosynthetic angiosperms, (2) pseudogenes that are present in all plastid genomes of nonphotosynthetic angiosperms, (3) genes that are unique for neottia, (4) pseudogenes that are unique to neottia, (5) genes present only in neottia and rhizanthella, (6) pseudogenes present only in neottia and rhizanthella (no), (7) genes unique to rhizanthella (no), (8) pseudogenes unique to rhizanthella, (9) genes present only in rhizanthella and epifagus (no), (10) pseudogenes present only in rhizanthella and epifagus, (11) pseudogenes unique to epifagus, (12) genes unique to epifagus, (13) pseudogenes present only in epifagus and neottia, and (14) genes present only in epifagus and neottia. among about 100 of genes encoded in typical plastid genome, only 29 are shared in all the plastid genomes of nonphotosynthetic plants. this core set of genes constitutes of ribosomal and transfer rna and ribosomal protein genes, translation initiation factor (infa), protease subunit (clpp), acetyl - coa carboxylase subunit (accd) genes, and two large orf of unknown function ycf1 and ycf2 (fig. this is in contrast with the situation observed in experimental systems of transition to heterotrophy. calli cultured for a long time on sucrose - rich media also undergo multiple deletions in plastid dna and extensive gene losses. however, their size and location drastically differ in different calli samples and do not seem to converge to a gene set shared by all samples. moreover, the loss of circular structure typical for plastid dnas, is reported (harada. 1992 ; abe. 2002). the stability of plastid genomes of nonphotosynthetic plants the conservation of typical quadripartite structure, the existence of shared gene set, and the evidence of purifying selection acting on these genes suggests that the evolution of plastid genomes in nonphotosynthetic plants is dictated by the constraints that are obviously different from those acting on photosynthetic plant plastomes but are not less strong. orchidaceae is a perfect model system for deeper study of the evolution of nonphotosynthetic plants plastomes because it contains many cases of independent transition to nonphotosynthetic lifestyle (molvray. 2000), from rather ancient occurring at the level of tribe to extremely recent occurring in certain individuals within the population (tranchida - lombardo. moreover, many photosynthetic orchids are able to use fungi as secondary carbon source being mixotrophic rather than fully autotrophic (bidartondo. 2004 ; abadie. mixotrophy is thought to be the preadaptation that mediates the transition to completely heterotrophic lifestyle (selosse and roy 2009). it is interesting to infer if any of the changes in the plastome characteristic for strictly nonphotosynthetic plants (e.g., gene loss, increase of substitution rate) are observed in mixotrophic species. we expect that further sampling of the plastid genome sequences from the species of orchidaceae representing different stages and different times of transition to mycoheterotrophy will provide valuable information about the evolution of plastomes in nonphotosynthetic plants. total dna was extracted from the above - ground part of three neottia plants using nucleospin plant ii kit (macherey - nagel, germany). dna (10 g) was used for sequencing with roche genome sequencer flx system using the titanium kit (454 life sciences). then a blast database was made from the resulting fasta file and a plastome of oncidium, the photosynthetic orchid was queried against it. all reads with e - value lower then 10 were used for de novo assembly using mira assembler ver. two largest contigs (11,141 and 49,963 nucleotides) were used as a basis for the generation of draft version of the neottia plastome. they were aligned with oncidium plastome using the whole vista genome alignment tool (frazer. then sanger sequencing was used to fill the gaps. to check the accuracy of the assembly and to correct possible 454 sequencing errors associated with homopolymer runs, several regions were sequenced by sanger sequencing using the primers designed on the base of the assembly (supplementary table s1, supplementary material online). average coverage for regions derived from 454 assembly is assessed as 29.8 ; for regions sequenced by sanger sequencing it is 2. polymerase chain reaction (pcr) amplification was performed on biometra t300 thermal cycler using encyclo pcr kit (evrogen, russia). pcr conditions were as follows : initial denaturation 3 min at 94 c, then 35 cycles of 15 s at 94 c, 25 s at 59 c, and 15 min (depending on the expected length of the product) at 72 c. sanger sequencing was performed in the interinstitutional sequencing center at engelhardt institute of molecular biology (moscow, russia) using abi prism bigdye terminator kit v. 3.1 with following analysis on abi prism 3730 genetic analyzer (applied biosystems). 2004). then manual correction and adjustment, that included alignment of every oncidium and phalaenopsis gene with neottia plastome sequence was performed. the regions with similarity to known protein - coding genes but lacking intact orf were classified as pseudogenes. to detect trnas pseudogenes, each trna - like sequence was analyzed using trnascan - se (lowe and eddy 1997) and by comparison with its putative orthologs from oncidium and phalaenopsis. those sequences that lack typical trna folding and/or differ from their orthologs by multiple indels or substitutions were considered to be pseudogenes. the map of neottia plastome was visualized using ogdraw online tool (lohse. 2007) with further manual correction. assembled and corrected sequence of neottia plastome was deposited in the genbank under accession number jf325876. for dn / ds calculation, all protein - coding plastid genes that are shared between neottia, rhizanthella, oncidium, and phalaenopsis were included in the analysis. 1994) ; dn / ds ratios were calculated using codeml program from the package paml 4.3 (yang 2007). in the alignments of several genes, nontriplet indels or in - frame only the part of sequence before presumable indel or stop codon was used for dn / ds calculation. | plastids are the semiautonomous organelles that possess their own genome inherited from the cyanobacterial ancestor. the primary function of plastids is photosynthesis so the structure and evolution of plastid genomes are extensively studied in photosynthetic plants. in contrast, little is known about the plastomes of nonphotosynthetic species. in higher plants, plastid genome sequences are available for only three strictly nonphotosynthetic species, the liverwort aneura mirabilis and two flowering plants, epifagus virginiana and rhizanthella gardneri. we report here the complete sequence of a plastid genome of nonphotosynthetic mycoheterotrophic orchid neottia nidus - avis, determined using 454 pyrosequencing technology. it was found to be reduced in both genome size and gene content ; this reduction is however not as drastic as in the other nonphotosynthetic orchid, r. gardneri. neottia plastome lacks all genes encoding photosynthetic proteins, rna polymerase subunits but retains most genes of translational apparatus. those genes that are retained have an increased rate of both synonymous and nonsynonymous substitutions but do not exhibit relaxation of purifying selection either in neottia or in rhizanthella. |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.