A rapid diagnosis method for organ specific disease state is based on obtaining an infrared absorbance spectra of a patient""s blood at a frequency range from about 400 cmxe2x88x921 to about 2000 cmxe2x88x921 and further from about 3000 cmxe2x88x921 to about 3100 cmxe2x88x921. Comparing it with the predetermined normal infrared absorbance spectra of known healthy subjects for the presence or absence of predetermined features such as increase or decrease of infrared absorbance, peaks at particular frequencies allows for accurate diagnosis of a disease of an organ, including most major organs such as a heart, lungs, stomach, liver, kidneys, brain, etc.

1. Field of the Invention
The present invention related generally to an infrared (IR) spectroscopy method for the diagnosis of a disease state of a mammal such as a human being. More particularly, the method of the invention allows for specific and quick diagnosis of a variety of pathological conditions by analyzing the IR spectra of the patient""s blood.
2. Description of the Prior Art
Diagnosis of a disease state of a human being is done using a variety of laboratory techniques. That, in conjunction with the general medical assessment of the patient""s condition and symptoms allows a modern medical practitioner to determine in most cases the accurate diagnosis of a patient. It is well known that the ease of treatment depends greatly on the speed of such diagnosis. Thus, the sensitivity and precise discriminatory nature of the early diagnostic methods is very important in the effective treatment of patients.
There is a variety of sources of biological information about the general medical state of a patient that is used in the laboratory analysis as various tissue and cell samples as well as biological fluids that may be taken from a human being. An example of a biological fluid is blood, saliva, sweat, urine, semen, various gland secretions, joint lubricant fluids, lymphatic fluids, and so on. Typically, these fluid or tissue samples come from a specific organ so that a further laboratory analysis can reveal a certain pathologic condition of that particular organ. Blood, however, represents a universal fluid circulating throughout the human body and as such provides an almost unique cumulative source of biological information about the body as a whole as well as about individual organs.
A number of laboratory techniques have been developed in the recent years to study blood and other biological fluids and tissues. Many of them are aimed specifically to identify a certain pathological condition. However, no universal technique exists today that would allow for a quick, minimally invasive broad diagnosis of various organ specific conditions.
Usually, a simple elevation of a certain particle count in blood (such as leukocytes) is indicative of some inflammation process. Also, in recent years physicians have used such serum enzymes as creatine phosphokinase, lactic dehydrogenase, and aminotransferases, which are released in large quantities into the blood from necrotic tissue, for diagnostic purposes (for example for diagnosis of myocardial infarction or hepatitis). However, elevation of the serum enzymes level, as well as an increased level of leukocytes, is not organospecific and can be used only as an additional method of diagnostics. In that case, it is up to a physician to determine the exact organ or a group of organs responsible for a pathologic condition based on the symptom analysis, which may be not present, misleading, or confusing at times.
Among methods other than IR spectroscopy the method of microparticle enzyme immunoassay technology is the closest one to the suggested by us method by its aim and problem solving. This technique is used primarily for diagnostics of acute myocardial infarction and based on determination of a cardiac form of Troponin-1, which is the regulatory submit of the troponin complex associated with the actin thin filament within muscle cells. This method also has numerous limitations because any conditions resulting in myocardial cell damage can potentially increase cardiac Troponin-1 levels. These conditions include, but are not limited to: angina, unstable angina, congestive heart failure, myocarditis, cardiac surgery or invasive testing. Thus, this method provides an opportunity to diagnose the pathology of the specific organ, in this particular case the heart, but does not allow for making the specific diagnosis, that is for determining the particular heart disease. This leads to an incorrect diagnosis or a complete misdiagnosis of a patient with obvious consequences.
IR spectroscopy is a very sensitive chemical analysis method, which is routinely used by organic chemists and biochemists as a molecular probe. When infrared light is passed through a sample of an organic compound, some of the frequencies are absorbed and some are transmitted through the sample without being absorbed. As a result, this selective light absorbance is recorded as a chart by the machine and can be further used to determine the exact chemical composition of the sample under investigation. The term xe2x80x9cIR spectroscopyxe2x80x9d is used here to include laser-Raman spectroscopy, Raman con-focal laser spectroscopy, Fourier Transform infrared spectroscopy, or any other infrared spectroscopy technique. Organic applications of IR spectroscopy are almost entirely concerned with frequencies in the range of 650-4000 cmxe2x88x921. Frequencies lower than 650 cmxe2x88x921 are called far infrared and those greater than 4000 cmxe2x88x921 are called near infrared.
There are several important advantages in using this technique: results are obtained relatively quickly with less labor input than many other diagnostic techniques; the use of IR spectroscopy may provide a more precise information on the exact nature of a disease based on sampling of blood or other biological fluid; the method also allows to monitor the dynamics of the characteristic change, which is important in determining the exact stage of the disease.
It has been established in the prior art that certain diseases such as cancer can substantially change the IR characteristic of blood and some other biological fluids. Therefore, IR can be used to determine the presence or absence of cancer cells and even to determine whether the tumor is malignant or not.
Several US and international patents contain a description of utilizing IR spectroscopy for cancer diagnosis. U.S. Pat. No. 5,261,410 by Alfano discloses a method of determining the cancerous state of a human tissue, particularly in breast tissue after exposing a sample of that tissue to IR light. This method has limitations due to the need for tissue biopsy and also since it does not allow for diagnosis of other organs.
U.S. Pat. No. 5,186,162 by Oong illustrates a method of using the IR spectroscopy of a tissue sample or a culture for differentiation between the presence of normal and cancerous cells. The specimen under investigation may be a Papanicolu smear, a cervical specimen, an endocervical specimen, or a vaginal or uterus specimen. This method allows only for cancer diagnosis and is limited to the particular organ under investigation, which has to be suspected to contain cancerous cells even before the test is done. Thus, early diagnosis, especially other than cancer, is not feasible with this technique.
PCT Patent Application No. WO 97/14961 by Antipov describes the use of IR spectroscopy means for cancer diagnosis of various organs by analyzing the IR spectra of blood. This method allows for diagnosis of malignant disorders using the method of multiple irregular total internal reflection in the IR range. It utilizes blood as a source of biological information and allows for limited determination of a specific organ that contains cancerous cells. At the same time, in addition to requiring some special IR tools and exotic evaluation technique (which are not routinely available or known in the medical laboratory community), this method is limited to the diagnosis of other cancer only and does not allow for diagnosis of other important diseases and conditions such as inflammation of a certain internal organ.
A multivariate classification techniques are applied to spectra from cell and tissue samples irradiated with IR light according to the U.S. Pat. No. 5,596,992 by Haaland in order to determine if the samples are normal or cancerous. Classification can be made using infrared spectroscopy and analysis tools such as partial least square technique (PLS), principal component regression (PCR), and linear discriminant analysis. These classifications can be used to distinguish normal, hyperplastic, and neoplastic cells. Lymphatic fluid and tissue samples are used as the object of evaluation. The important limitation of this invention is that it requires a complex apparatus and a special microscope to be used and, as described before, allows only for cancer diagnosis.
U.S. Pat. No. 5,733,739 by Zakim discloses yet another IR spectroscopy machine-based cancer diagnosis method that allows differentiating between normal and abnormal (cancerous) cells. It has all the same limitations as the patents described above such as the need for a difficult organ tissue sample collection procedure (biopsy), which can not be done routinely but rather is performed very rarely due to its traumatic nature. In addition, this method can not include other than cancer diseases of internal organs.
Other than cancer diagnostic methods have also been suggested in the prior art. PCT Patent Application No. WO 97/32194 by Ashdown discloses the use of IR spectroscopy of blood or its components for determination of cellular immunity. Fourier Transform infrared spectroscopy is a preferred method of analysis according to this invention. This invention can be used for the determination of cellular immunity in patients with immunodeficiency, autoimmunity, and contact with infectious diseases, allergies, hypersensitivity, and cancer. It is also claimed to be used for determination of tissue transplant compatibility. Although a useful technique, this method by itself does not allow for differentiation between various internal organs as being the source of the disease let alone diagnosing other inflammatory conditions.
Chemical composition of a biological fluid such as blood can be determined using a near IR spectroscopy method described in U.S. Pat. No. 4,975,581 by Robinson. This method can be used effectively to determine the glucose level in blood and thus is of interest to diabetic patients. However, a simple determination of glucose level cannot be used effectively in diabetic diagnosis and screening since no determination can be made as to whether the source of higher glucose levels is physiologically acceptable or pathologic. This method is also claimed as capable of determining other blood parameters such as alcohol presence, fatty acid content and others and as such can be used as a replacement method to the currently employed techniques used for such determinations. However, it cannot produce clinically important information about the presence of inflammatory processes in various internal organs and therefore cannot be used as a broad diagnostic tool. Hemoglobin and hematocrit concentration in blood can be determined by using a near IR spectroscopy technique suggested by Osten in U.S. Pat. No. 5,830,133. Although fast and reliable, this method is limited to just the claimed purpose of hemoglobin evaluation and does not allow for internal organ diagnosis.
Eysel in the U.S. Pat. No. 5,473,160 suggests another specific application of IR spectroscopy of synovial fluid for diagnosing arthritis disorders. Osteoarthritis is diagnosed by comparing the synovial fluid near IR spectra retrieved from the patient with that of either a known healthy or diseased spectra. The method described in the patent is limited to only arthritis diagnosis.
Finally, my Russian Patent # SU 1,698,775 describes a method of determining the level of septic state in children using IR spectra. The presence of a xe2x80x9cwavedxe2x80x9d-shape peak in the range of about 1095 cmxe2x88x921 to about 1105 cmxe2x88x921 of the same elevation as the liver portion of the chart is used as an indicator of a presepsis condition. Same at about 1080 cmxe2x88x921 points out to the initial stage of sepsis. A peak at about 1040 cmxe2x88x921 points to a medium level of sepsis, and finally a peak at about 1010 cmxe2x88x921 is associated with extended sepsis. This method is very useful but applicable only in small children and cannot differentiate between specific internal organs.
All IR spectroscopy techniques suggested in the prior art have similar limitations: they allow for selective diagnosis of some global disorders, mostly cancer, and sometimes capable of pointing to a certain internal organ. However, no universal technique exists for a broad diagnosis of organ specific disorders and the stage of their development in various internal organs, for example, a variety of inflammation conditions. Therefore, the need exists for such a broad diagnosis method.
Accordingly, it is an object of the present invention to overcome these and other drawbacks of the prior art by providing a novel machine-based method for a broad and comprehensive diagnosis of various pathological conditions of the human being by using IR spectroscopy of blood.
It is another object of the invention to provide a method for diagnosis for a variety of organ specific disorders using the IR spectra of patient""s blood.
It is another object of the present invention to provide a method for early stage diagnosis of such conditions in patients of any age when conventional chemical analysis techniques are not capable of determining the diagnosis and no or little clinical symptoms are present.
It is a further object of the present invention to provide a method of monitoring the progression of the disease; the progression and results of the treatment of that disease; the effectiveness of the treatment therapy including the effect of medication; and the determination of the final cure from the disease, such method based on IR spectroscopy of blood.
It is yet a further object of the present invention to provide a method of fast and comprehensive diagnosis of a variety of pathological conditions of a human being soon after the patient is first presented to the medical professional.
It is another object of the invention to provide a method for selecting especially healthy people for certain special needs such as fighter pilots, astronauts, and other professions requiring physical endurance.
The method of the invention is based on the discovery that various major organs and their disease state can affect the IR spectra of human blood in a very specific individual way. In other words, certain ranges of frequencies in the IR spectra are indicative of certain specific organs and their pathologies.
The physiological importance of blood flow is that it transports all necessary ingredients to the internal organs and then removes all the components that have to be discarded by these organs. Whole blood contains over 1000 chemical components. Although IR spectroscopy is a very sensitive analysis tool, it cannot pick up reliably the presence of components having less than about 5% of the total mass of the sample. In a dried blood sample, the distribution of mass is such that most of it is represented by hemoglobin ranging from about 64 to 87%. The next highest number is lipids occupying about 8.8% of the total mass. All other components are less than 5% and should not affect significantly the IR spectra of the whole blood. Of course, special techniques are available to separate certain components from the whole blood. Themselves may in turn, use their selective IR spectra for diagnostic purposes.
Hemoglobin has a very complex chemical protein structure capable of reacting to the presence in blood of a variety of compounds. Such compounds entering the blood stream may be the result of metabolic processes, sub-components of certain cells that have to be carried away from diseased organs, or some other products of pathological processes in the body. All of these complex compounds have the ability to attach to hemoglobin proteins or in some other way effect its chemical structure. Since most of pathological reactions are organ specific, it is suggested that the unique compounds released in the blood stream can be used as an organ specific diagnostic tool provided that the information can be properly extracted and analyzed. The similar process takes place by the interaction of the above-mentioned pathologic products with phospholipids of erythrocytes.
Thus, the basis of the suggested by us method of diagnostics is not the identification of products of pathological processes by their spectra. The basis of this method is the change of blood spectra of a healthy human being (frequency shift, changes in the intensity and shape of the absorbance band) under the influence of intermolecular forces, which are brought about by the interaction of the bonds found in the whole blood of a healthy human being with the bonds of the corresponding metabolites and/or the products of pathological processes, entering the blood, if a specific disorder is taking place in the body.
Whereat, the concentration of the above-mentioned metabolites and/or the products of the decay of the specific organs and tissues, not found in the normal blood, is, as a rule, significantly lower than the threshold concentration which is necessary to diagnose a disorder of a specific organ using routine biochemical methods. The method of IR spectroscopy suggested by us detects organ specific molecules that precede the appearance of a whole complex of organ specific antigens in the blood.
It is possible that the mentioned above decay products of the specific organs can be determined by using immunology or other spectroscopy methods, based on the diagnosis of specific antigens or substances. However, in variety of illnesses, some of which are appendicitis, cholecystitis, pancreatitis e.t.c. it is impossible to make a diagnosis using the antigens or substances because in most cases the nature of this antigens or substances is unknown.
Besides, it should be noted that in overwhelming majority of cases (if not in all of them), the specific substance characteristic to a certain disease enters the blood in concentrations that are significantly less than 5% of the sample specimen and can not be seen by themselves. We can see this substance only if it is involved in some kind of intermolecular interaction with the hemoglobin bonds or that of phospholipids, which make up the basis for the blood spectra of a healthy human being.
Apparently, the said changes can be observed in those parts of spectra where chemical bonds absorb, which are mostly typical of (mostly absorbing) the substance entering the blood. If in the particular region of spectra the vibrations of hemoglobin bonds and/or the bond of phospholipids of erythrocytes are not found or faintly pronounced, we will observe the appearance of new ranges of absorbance absent in the blood spectra of a healthy human being, as for example, in the case of kidney disorder. However if in the specific region of the spectra the vibrations of hemoglobin bonds and/or the bonds of erythrocyte phospholipids are well pronounced (peaks, bands), then the result of intermolecular interaction will depend on the chemical nature and the number of bonds of the substance entering the blood.
As a rule, when a small amount of the substance enters the blood, the intensity of the corresponding absorbance range will increase, as for example in the case of acute myocarditis. By contrast when a large amount of the substance enters the blood, the intensity of the absorbance range will reduce, as for example in case of myocardial infarction. If the band of absorbance in norm is well pronounced, as for example in the region in which nervous system diseases are diagnosed, then the appearance of the products of the organ decay with the similar chemical bonds will result in the reduction of the absorbance band. The larger the amount of the decay products that enter the blood, the more significant the reduction of the absorbance band will be. For example, the brain injury of different degree of the severity would correspond to the different degree of the intensity reduction.
If the absorbance of the bonds of pathological products entering the blood occurs beyond the region where the bonds of substances found in the normal blood absorb, namely, next to the said region, then we would observe the absorbance range shift, as it happens in case of allergy or hepatitis.
At the same time, it is possible that the complex molecule of hemoglobin can be a molecular system of correspondence of the body, on which the condition of internal organs and the small changes of their metabolism are represented. These changes can be detected by us due to conformational changes of hemoglobin structure. It is similar to such known correspondence systems as reflexogenic areas of skin or the iris of an eye.
According to the method of the invention, a sample of patient""s blood is taken and processed in a known way to allow for recording of IR spectra. One common way to prepare the blood sample is to dry the blood on glass plate and then remove the dried particles from the glass, mix with a filler and compress into a tablet or, in case of a liquid filler, place the suspension in a shape allowing the use of an IR recording apparatus. That example of a preparation technique is described in greater detail in my Russian Patent No. SU 1,698,775 and allows for easy transportation of the sample to the IR laboratory as well as for subsequent easy handling and storage. Once in the lab, the IR light is passed through the sample, the IR spectra of the patient""s blood is recorded and compared with the general norm from known healthy subjects as well as with the IR charts from known disease conditions. The presence or absence of certain graphical elements on the chart in comparison to the normal or known disease chart is used for a variety of organ specific diagnostic purposes described in greater detail below.
The importance of the method in the present invention is in utilization of a newly discovered fact that specific organs and their respective disease states have particular and distinct ranges of frequency in which the IR absorbance spectra changes (specific pictures). Therefore, once a change or deviation from an established norm is detected, the range of frequency where the change occurs points out to a specific organ in question. Most of the times, based on a particular frequency with a pronounced change, clinician would be able to diagnose a specific pathological condition for a particular organ. The regions responsible for individual organs and organ systems are described below progressing from lower frequencies towards higher frequencies (FIGS. 1 and 2).
REGION 1, with frequency ranging from about 600 cmxe2x88x921 to about 700 cmxe2x88x921 is indicative of various hypoxic conditions associated with reduction in oxygen availability in organism. Hypothetically, in this region out-of-plane deformation vibrations of NH-bonds of hemoglobin and OCN-turns of the peptide groups of hemoglobin are observed, which increase by lack of oxygen in blood as a result of activation of vibrations of the iron-containing group of hemoglobin""s hem.
REGION 2, with frequency ranging from about 700 cmxe2x88x921 to about 800 cmxe2x88x921, is indicative of liver failure. Hypothetically, in this region we observe deformation rocking vibrations of CH2-groups in lipids, when they enter the blood with concentration significantly exceeding the norm, which occurs only in case of liver failure.
REGION 3, with frequency ranging from about 800 cmxe2x88x921 to about 1000 cmxe2x88x921 is indicative of disorders of gastrointestinal tract, kidneys, endocrine glands, as well as some types of cancer. This region, depending on the structure of chemical compounds shown in the spectra, is divided into Subregion 3A with frequency ranging from about 800 cmxe2x88x921 to about 900 cmxe2x88x921 and Subregion 3B with frequency ranging from about 900 cmxe2x88x921 to about 1000 cmxe2x88x921.
Subregion 3A is indicative of the disorders of colon, kidneys and endocrine glands. Hypothetically, if a colon disorders exists, in the range of about 850 cmxe2x88x921 to about 900 cmxe2x88x921 we observe deformation vibrations of NH, CH and CHxe2x95x90CH bonds of some toxic products, developed in large intestine as a result of proteins putrefaction (indole, skatole, phenylpropeonic, phenylacetic, parahydroxylphenyllactic acids, etc.). These products are normally not found, but can enter blood in very low amounts as the result of colon disorders.
Hypothetically, in the case of kidney disorders, in the range of about 850 cmxe2x88x921 to about 860 cmxe2x88x921 we observe deformation vibrations of NH, CH and CHxe2x95x90CH bonds of the uric acid, urea and decay products of protein-lipids antigens from damaged kidneys cells. It happens, when the concentration of the specified above metabolites in the blood deviate from the conventional standard, determined by the IR spectra.
Hypothetically, when an endocrine disorders take place in the body, in the range of about 880 cmxe2x88x921 to about 900 cmxe2x88x921 we observe deformation vibrations of CH and CHxe2x95x90CH bonds of a number of hypophysiotropic and steroid hormones. The spectra of a healthy human being do not show these hormones.
Subregion 3B, is indicative of the disorders of stomach, pancreas, small intestine and some types of cancer. Hypothetically, if a patient suffers from stomach disorders, in the range of about 930 cmxe2x88x921 to 950 cmxe2x88x921 we observe deformation vibrations of NH and CH bonds of protein products, which are a result of stomach mucous membrane inflammation.
Hypothetically, in the area of about 940 cmxe2x88x921 we can observe deformation vibrations of NH and CH bonds of protein products, which form as a result of pancreas tissue inflammation.
Hypothetically, in case of duodenum, jejunum and ileum disorders, in the frequencies ranging from about 950 cmxe2x88x921 to about 1000 cmxe2x88x921 we observe deformation vibrations of CH and CHxe2x95x90CH bonds of phospholipid products, as well as NH and CH bonds of protein products formed by inflammation of these organs.
Hypothetically, if a patient suffers from certain types of cancer which correlate with immunodeficiency conditions, in the area of about 908 cmxe2x88x921 we observe deformation vibrations of CH and NH bonds of protein-lipid antigens and RNA from the cancerous tissue.
REGION 4, with frequency ranging from about 1000 cmxe2x88x921 to 1140 cmxe2x88x921, is indicative of the disorders of liver and immune system (different kinds of immunodeficiency and allergy). This region, depending on the functional characteristics, can be subdivided into Subregion 4A with frequency ranging from about 1100 cmxe2x88x921 to 1140 cmxe2x88x921 or more exactly the peak at a frequency of about 1130 cmxe2x88x921, and Subregion 4B with frequency ranging from about 1000 cmxe2x88x921 to 1100 cmxe2x88x921.
Subregion 4A, especially the peak at a frequency of about 1130 cmxe2x88x921, to be exact, is indicative of normal liver function. This subregion represents a spectra of SH-groups of hemoglobin, consisting of sulfur containing amino acids (cysteine, cystine and methionine) and responsible for respiratory function of hemoglobin. Also, sulfur-groups of hemoglobin determine its reactivity, that is its ability to react not only with oxygen but also with other compounds. In connection with this, when toxic metabolites, products of decay of organs and tissues enter the blood and/or the liver detoxification function is upset, the peak at a frequency of about 1130 cmxe2x88x921 disappears or modifies. This is a result of interaction between SH-groups of hemoglobin and the above-mentioned toxins, as well as of the formation of other compounds with a new conformation structure. We cannot also rule out that sulfa lipids of erythrocytes and possibly ether-sulfonic acids, which are associated with liver detoxification function, form the peak at about 1130 cmxe2x88x921. In view of this, the normal position of the peak in the range of about 1130 cmxe2x88x921 defines the normal liver condition, while its disappearance or changes in its conformation correlate with liver function disorders.
Subregion 4B is indicative of the disorders of the liver (hepatitis) and such immune disorders as allergy; T- and B-cells immunodeficiency; and nonspecific phagocytic immunodeficiency (sepsis: SU Patent No. 1,698,775).
Hypothetically, when hepatitis takes place in the frequency range of about 1030 cmxe2x88x921 to about 1000 cmxe2x88x921 we observe vibration modes of glycogen. In case of allergy in the range of about 1080 cmxe2x88x921 to about 1090 cmxe2x88x921 we observe the vibrations of Cxe2x80x94Oxe2x80x94P bonds of phospholipids from decayed cells, and Pxe2x80x94O bonds of ADP and AMP. If a patient suffers from T- and B-cells immunodeficiency associated with a tumor, we observe vibrations of Cxe2x80x94Oxe2x80x94P bonds of DNA and RNA at frequencies of about 1030 cm31 1 and 1060 cmxe2x88x921.
Hypothetically, if nonspecific phagocyte immunodeficiency (sepsis) exists, we observe vibrations of Cxe2x80x94Oxe2x80x94P and Pxe2x80x94OH bonds of all above-mentioned components, namely ADP, AMP, DNA, RNA, phospholipids, as well as vibrations of metal containing groups of cytochromes from decayed mitochondria of cells.
REGION 5, with frequency ranging from about 1140 cmxe2x88x921 to about 1350 cmxe2x88x921, is indicative of the disorders of the heart. Depending on the chemical compounds, revealed in this region of the spectrum, we subdivide this region into three subregions:
Subregion 5A, with frequency ranging from about 1140 cmxe2x88x921 to about 1200 cmxe2x88x921, primarily the peak at a frequency of about 1160 cmxe2x88x921, reflects the condition of a myocardial muscle in norm (FIG. 1);
Subregion 5B: the straight descendent under acute angle line in the range of about 1180 cmxe2x88x921 to about 1200 cmxe2x88x921, reflects the condition of the heart valves in norm and/or absence of heart insufficiency (FIG. 1), and
Subregion 5C: the area ranging from about 1200 cmxe2x88x921 through about 1350 cmxe2x88x921, including a big peak at a frequency of about 1250 cmxe2x88x921, reflects the cardiac rhythm in norm (FIG. 1).
Changes of IR absorbance in these areas reflect the appropriate pathologies of myocardial muscle, disorders of heart valves and/or presence of heart insufficiency of different degree, and cardiac arrhythmia.
Hypothetically, the peak at about 1160 cmxe2x88x921 (Subregion 5A) normally is the spectra of Cxe2x80x94Oxe2x80x94C and Pxe2x80x94Oxe2x80x94C bonds of phospholipids of erythrocytes. When Cxe2x80x94Oxe2x80x94C and Pxe2x80x94Oxe2x80x94C bonds of phospholipids from the damaged heart cells, especially cardiolipin (serinephosphatide) appears in the blood and in the spectra, it allows for diagnosis of myocard diseases and heart valves disorders, including heart failure.
Hypothetically, the peak at about 1250 cmxe2x88x921 (Subregion 5C), normally and primarily represents amid III of the hemoglobin peptid chain (NH turn in-plane of peptide chain of hemoglobin), and to the lower extend, Pxe2x95x90O and Cxe2x80x94Oxe2x80x94C bonds of phospholipids of erythrocytes. The appearance in the blood and in the spectra of Pxe2x95x90O, Pxe2x80x94OH and Cxe2x80x94Oxe2x80x94C bonds of phospholipids, and, probably, NH bonds of protein from the damaged myocard cells, in the range of about 1200 cmxe2x88x921 to about 1350 cmxe2x88x921 strictly correlates with the heart rhythm disorder, that is evident of the damage of the heart conductive system, and allows for diagnosis of various types of arrhythmia.
REGION 6 (MIXED), with frequency ranging from about 1100 cmxe2x88x921 to about 1200 cmxe2x88x921 includes confluence of Subregion 4A (which is indicative of the normal liver function) and Subregion 5A (which is indicative of the normal myocard function of the heart). Simultaneous increase of IR absorbance in this region, especially of the peaks at about 1130 cmxe2x88x921 and 1160 cmxe2x88x921, is indicative of the inflammation disorders of the upper respiratory tract and lungs. Hypothetically, that is because by the above-mentioned diseases one can see in this region Pxe2x95x90O and Pxe2x80x94OH vibrations of ATP; SS and SH vibrations of sulfolipids and also Cxe2x80x94Oxe2x80x94C and Pxe2x80x94Oxe2x80x94C vibrations of phospholipids, which appear in the blood as a result of lung cells damage and throw of ATP into the blood, which is necessary to provide energy for a general inflammation process.
REGION 7, with frequency ranging from about 1400 cmxe2x88x921 to about 1700 cmxe2x88x921, is indicative of the disorders of brain and central nervous system. Depending on the degree of severity of brain disorders, shown in the spectra, we subdivided this region into Subregion 7A, with frequency ranging from about 1400 cmxe2x88x921 to about 1500 cmxe2x88x921, or more definitely, the peaks at frequencies of about 1410 cmxe2x88x921 and 1460 cmxe2x88x921. And Subregion 7B, with frequency ranging from about 1500 cmxe2x88x921 to about 1700 cmxe2x88x921, or more definitely, the peaks at frequencies of about 1550 cmxe2x88x921 and 1650 cmxe2x88x921.
Hypothetically, the peak at a frequency of about 1410 cmxe2x88x921 (Subregion 7A) represents symmetric CO stretching vibrations and symmetric CH3 bending vibrations of hemoglobin amino acids. The peak at a frequency of about 1460 cmxe2x88x921 (Subregion 7A) is probably indicative of CH2 bending vibrations and asymmetric CH3 bending vibrations of all components of the whole dry blood.
If a brain damage take place, the appearance in the blood of the protein products of the nerve tissue decay, namely, of neuroglobulin and neurostromin, containing CO bonds of amino acids, results in the changes of the peak conformation at a frequency of about 1410 cmxe2x88x921. And the appearance of even small amounts of products of nerve cell membranes decay, containing CH2 and CH3 bonds, results in the changes of peak conformation at a frequency of about 1460 cmxe2x88x921.
On the whole, the changes in spectra in the Subregion 7A are indicative of less severe, typical for early stage of central nervous system diseases and/or functional brain disorders, such as a minor degree of intracranial hypertension, neurosis or early stage of convulsion syndrome.
Hypothetically, in Subregion 7B the peak at a frequency of about 1550 cmxe2x88x921 represents primarily asymmetric CO stretching vibrations and, secondly, NH bending and CN bending vibrations of hemoglobin amino acids. The peak at a frequency of about 1650 cmxe2x88x921 represents primarily CO bonds of a-spiral of the hemoglobin peptide chain and, secondly, CO bonds of acetalphosphatides (plasmalogens) of erythrocyte lipids and Nxe2x89xa1CH3)3 bonds of cholinephosphatides (lecithins), which are part of erythrocyte lipids.
When brain is severely damaged, the blood shows not only the products of protein decay of brain tissues, containing asymmetric CO bonds with vibrations at a frequency of about 1590 cmxe2x88x921 and NH bending vibrations at a frequency of about 1570 cmxe2x88x921. Also one can see the products of a more severe brain damages, such as a sphingomyelin with asymmetric CO and CN bonds vibrations in the frequency range of about 1550 cmxe2x88x921-1655 cmxe2x88x921. These products consist of sphingomyelin, phosphatidylserine, phosphatidylcholine and other phosphoric acids with CC-cis bonds, vibration of which one can see at frequencies of about 1660 cmxe2x88x921 and 1670 cmxe2x88x921. Since phosphatidylcholine also contain with Nxe2x89xa1(CH3)3 bonds, one can identify vibrations at a frequency of about 1630 cmxe2x88x921. On the whole, the changes in spectra in the Subregion 7B are indicative of more severe damages of brain and central nervous system, such as head trauma, hypoxic encephalopathy with hemorrhage, etc.
REGION 8, with frequency ranging from about 3000 cmxe2x88x921 to about 3100 cmxe2x88x921, especially the peak at a frequency of about 3060 cmxe2x88x921, is indicative of severe chronic infections, such as pulmonary tuberculosis. Hypothetically, the peak at a frequency of about 3060 cmxe2x88x921 primarily represents xe2x80x94NH3+ bond of associated molecules of secondary amids, constituting side chains and terminal groups of the hemoglobin protein molecules. Also, in the frequency range of about 3010 cmxe2x88x921 to about 3095 cmxe2x88x921 one can see xe2x95x90Cxe2x80x94H stretching vibration of erythrocyte phospholipids.
In the case of severe chronic infections, including pulmonary tuberculosis, the intoxication of the body is strongly pronounced, which results in products of protein decay entering the blood. These products contain xe2x80x94NH3+ bond of associated molecules of secondary amids, constituting side chains and terminal groups of protein molecules. Besides, we cannot rule out the appearance in blood of great amounts of decay products of the tuberculosis bacillus itself, particularly, of the tuberculostearic acid, containing xe2x95x90Cxe2x80x94H bonds, which absorb in the range of about 3010 cmxe2x88x921 to about 3095 cmxe2x88x921.
On the base of outlined above yet another application of the method is that it allows for monitoring the progression of the disease or the success of the treatment, by comparing subsequent blood samples spectra to the initial one.
Also, the use of the method is possible when selecting the healthiest men for the professions where extreme physical endurance is needed.
In addition the method of the invention allows for rapid and accurate early diagnosis, frequently pointing out to the initial stage of the disorder when no clinical manifestations are yet observed.
The method is also particularly useful when a physician cannot count on the patient""s response, as for example, in case of a patient being unconscious after an accident or with a pediatric patient.
Another advantage is that the method of the present invention allows for a rapid differentiation between a number of likely medical conditions, all of them presenting with similar clinical symptoms. This method has real advantages because it gives us a chance to simultaneously diagnose different organs pathology in contrast to some well known diagnostic methods such as ECG or EEG, which can be used for diagnostic diseases of only one organ (heart or brain).
The method can be used in insurance medicine, military and space medicine, forensic and veterinary medicine, pharmacology and parasitology. It can also be helpful for diagnostics of some oncology (retroperitoneal lymphoma) and infectious diseases (HIV, hepatitis B, etc.) on the earliest stage, when no clinical manifestations and antibodies are present. Moreover, the method can be used in different branches of biology (zoology, entomology, ichthyology, ornithology) and botany.

Method for blood infrared spectroscopy diagnosing of inner organs pathology


A lung-governed breathing valve used in a breathing mask for excess pressure operation. The valve has a casing with a spring-loaded control diaphragm which divides the casing into an outer chamber connected to the outside environment and a breathing chamber for conveying the gas for breathing. The control diaphragm controls a breathing gas inlet valve via a lever system and a manually released actuating button. The control diaphragm has a resilient raised portion formed integrally thereon with a recess therein. A push-rod integrally molded on the actuating button is positively engageable in the recess of the raised portion to prevent the lung-governed valve from being accidentally released or switched from the stand-by to the operative position.

A breathing mask is a device used to enable breathing in environments where such would be difficult or impossible without mechanical aid. Typically, a breathing mask incorporates an inlet valve which controls the flow of gas for breathing between a supply of breathing gas and the user of the breathing mask. A lung-governed valve controls the flow of gas through the inlet valve via the respiration of the user.
A diaphragm divides the lung-governed valve into an inner chamber at a pressure corresponding to the pressure within the breathing mask and an outer chamber at a pressure corresponding to the pressure in the environment. The diaphragm is coupled to a mechanism which opens and closes the inlet valve. The user's respiration creates a pressure differential between the inner and outer chambers of the valve which, in turn, causes displacement of the diaphragm thereby controlling the inlet valve closure mechanism.
A lung-governed valve of this type is described in German Patent No. DE 35 39 669 A1. In that device, the diaphragm controlling the inlet valve lever has at its central point a cam projecting outwardly into the outer chamber. A tilt lever is pivoted to the outer chamber casing and is clamped by transversely tensioned spring elements to the outer chamber so that the tilt lever is movable in a tilting joint. The tilt lever is movable out of an idle position into a first pressure position, exerting force on the diaphragm, and into a second stand-by position, lifting the diaphragm off the valve lever by engaging the cam and moving it and the diaphragm through a predetermined stroke. Because of the transversely tensioned spring elements, which are pivoted to the shorter end of the tilt lever, the end position of the tilt lever may be unstable. Subjection of the valve to external stress may cause the tilt lever to tilt from one position to the other. This instability is particularly disadvantageous when the control diaphragm is in the stand-by position. In this position, external vibration could tilt the lever, entraining the control diaphragm and opening the inlet valve via the valve lever. The limited amount of breathing gas available to the person using the apparatus can thus be inadvertently lost.
It would be desirable, therefore, to develop a lung-governed valve with an improved structural design wherein the valve is protected from accidental release or switching on when in the stand-by position.


Embodiments disclosed herein relate to an apparatus (1) for manufacture of tobacco industry products (21). The apparatus (1) comprising a rolling unit (9) comprising: • a rotatable drum (14) having a plurality of recesses formed in an outer surface of the drum (14) to receive tobacco industry product components (5/6); • a roll hand (15) spaced from the outer surface of the drum (14) to define a space between the outer surface of the drum (14) and the roll hand (15), within which space tobacco industry product components (5/6) are rollable upon rotation of the drum (14); and • a kicker bar (16) disposed adjacent to the roll hand (15). • The kicker bar (16) is moveable relative to the roll hand (15) and in a direction towards and away from the drum (14) during operation of the apparatus (1), and the kicker bar (16) is biased towards the drum (14).

It is known to provide apparatuses for receiving, arranging and assembling components for the manufacture of tobacco industry products, such as smoking articles. Such apparatuses can include a series of drums that convey components along a manufacturing path as the drums rotate. Components are transferred from one drum to the next at a point where the circumferential surfaces of the drums are closest to each other. As the components are conveyed along the path they are subject to several processes, for example cutting, sliding, combining with other components, gluing, and rolling or wrapping with a paper patch. Cigarette components typically include filters, filter components and wrapped tobacco rods.

This patent is subject to a terminal disclaimer.

Apparatus and method for manufacturing tobacco industry products

BRITISH AMERICAN TOBACCO (INVESTMENTS) LIMITED


In a screw machine, to extend the material to be prepared with the latter, at least one processing element is configured as an extension-kneading element, the crest region of which has a reduced thickness compared to a flank region and, with adjacent processing elements, forms an extension channel for the material in the region between the triangles, which tapers in cross section in the flow direction of the material. Because of the tapering of the extension channel, the material is accelerated in the flow direction thereof and extended, so that high-molecular particles in the material are divided and the latter is therefore homogenized.

Screw machines of this type are used to prepare plastics materials and are, for example, known from EP 0 522 390 B2. The plastics material to be prepared is subjected to shear flows in these screw machines and thus melted, mixed and homogenized. To avoid thermally damaging shear peaks, the kneading discs of EP 0 522 390 B2 have an enlarged passage cross section produced by material removal in the axial direction. The drawback is that plastics materials with high-molecular particles can only be inadequately homogenized with the forced shear flows, so so-called spots—also called "white spots"—remain in the materials.

Screw machine with at least one extension-kneading element in a flow direction


An electric vehicle comprising a battery charges the battery through an electric vehicle power supply device, and transmits to a user terminal device a first charging information notification message, which includes information on a current charging status of the battery. The electric vehicle detects an interruption to the charging of the battery on the basis of the current charging status of the battery, and checks the cause for the interruption to the charging of the battery. When the interruption to the charging of the battery is detected, the electric vehicle transmits to the user terminal device a second charging information notification message, comprising information on battery charging status that indicates the interruption to the charging of the battery and charging error information that indicates the cause of the interruption to the charging of the battery.

The present disclosure relates to an electric vehicle and an operating method thereof, and particularly, to a method of charging a battery of an electric vehicle.
In order to control global warming through the G20 and G8 countries, a smart grid solution and real-time use of an electric vehicle are suggested to reduce CO2.
An electronic vehicle includes at least one electric motor using an electrical energy of a rechargeable battery. This rechargeable battery may be connected to an electric outlet in order for recharging. In general, it takes a few hours to charge a battery of an electric vehicle. During this process, a user may want to obtain several information.
Moreover, electric grids may have a time interval at which an electrical demand exceeds a limitation. In order to prevent this, constructing a power plant additionally may rather disturb the reduction of CO2. Accordingly, adaptively coping with the demand of electricity by using a large capacity battery of an electric vehicle is required.
For this, users, electric vehicles, and electric power supply facilities may need to exchange information. To this end, various communication techniques may be used.
A wireless home area network (W-HAN), a wireless personal area network (W-PAN), and a wireless frequency transceiver perform communication through a wireless frequency in a short distance area between devices. A Zigbee transceiver may be used as an example. Zigbee is a standard protocol designed for wireless frequency communication in a short distance area. The Zigbee transceiver establishes a mesh network on the basis of IEEE 802.15.4.
A wireless local area network (WLAN) transceiver performs communication through a wireless frequency between devices away from each other by more than 10 m. A Wi-Fi device may be used as an example. The Wi-Fi device follows the IEEE 802.11 standards.
A power line communication (PLC) technique may be used to establish a network between devices through a power line. This technique is limited to an area of up to 100 m.
A wide area network (WAN) is a communication network covering a wide area. The Transmission Control Protocol/Internet Protocol (TCP/IP), that is, a standard protocol, may be used as an example. Additionally, a CDMA or GSM network may be used as an example of a wireless broadband network.
In order to adaptively cope with the demand of electricity by using a high capacity battery of an electric vehicle, demands for exchanging information between users, electric vehicles, and electric power supply facilities are emerging.

Method for charging an electric vehicle including a battery


A system and method for creating a consistent view of previously backed up data. In one embodiment, a point in time copy of a set of data is requested. The point in time copy includes a base image of the set of data and additional data corresponding to transactions targeted to the set of data which are received during creation of the point in time copy. A subset of the additional data usable to generate a consistent view of the set of data from the base image is determined. A file that includes a set of changes to the base image represented by the subset is created and stored in association with the base image. Responsive to a request for access to the copy, the previously stored subset is utilized to create a consistent virtual view of the copy from the base image.

1. Field of the Invention
This invention is related to performing backup and restore operations and, more particularly, to performing granular restorations of data.
2. Description of the Relevant Art
Backup systems are used to protect data against loss. Typically, a backup system includes software that copies the content of a set of data from a first storage device, or location, to a backup storage device. Frequently, the data is backed up and stored as a backup image, which may be a single file representing the content and structures of the backed up data. Examples of sets of data include data structures such as files, directories, databases, and volumes. If data is lost in the original set of data, the backed-up content of the set of data can be retrieved from the backup storage device and restored. Once the backed-up content is restored, it is available for use.
Conventional backup systems have traditionally created full-data structure images for full sets of data. These full-data structure images have enabled users to restore an entire data structure, such as a database, to its status at a particular time. Recently, backup systems have developed that allow selective restoration, commonly called granular restoration, of parts of a set of data, such as individual entries in a database, to the state of those entries at a particular time.
Unfortunately, existing systems for granular restoration of a set of data require the use of cumbersome processes that require the evaluation of overbroad change histories to reconstruct the status of the data structure at a particular time. These cumbersome and inefficient processes must be executed prior to allowing a user select items for restoration. The value of granular restoration technology is diminished by the delays inherent in current conventional methods for reconstructing the status of a set of data.

Generating data set views for backup restoration


A clock signal capable of changing the frequency in a wide range and with high resolution is generated.

The present invention relates to techniques effective in semiconductor devices with a clock oscillation circuit generating a clock signal used for circuit operation.
Semiconductor devices, such as general-purpose microcomputers, need to reduce the number of external components for miniaturization and cost reduction of equipment. Some semiconductor devices have an on-chip oscillator, which uses no external components, such as a crystal oscillator, in a clock oscillation circuit which generates a clock signal supplied to internal modules, such as a CPU (Central Processing Unit) and a peripheral functional block.
Frequency accuracy is an important characteristic of the on-chip oscillator. This is a frequency variation relative to a power supply variation or a temperature variation. The low frequency accuracy of the on-chip oscillator limits the application of the on-chip oscillator as the alternative component of an external oscillator.
Widening the application range needs to increase the frequency accuracy of the on-chip oscillator. The range can be further widened by heightening the frequency accuracy and covering a wide oscillation frequency range. General-purpose microcomputers usually allows users to change the settings according to an intended application.
For example, the on-chip oscillator, requiring high frequency accuracy, is performed temperature trimming in order to improve the frequency accuracy. Temperature trimming sets a control signal to cancel the temperature dependence of a circuit and that of a device that is being used.
Trimming at two temperature points can cancel the first-order temperature coefficient. Trimming at three temperature points can cancel the second-order temperature coefficient, too.
An example of this type of clock oscillation circuits is the one which stabilizes the oscillation frequency and heightens the oscillation accuracy by connecting a current-controlled oscillator, a frequency divider, a period comparison circuit, an integration circuit, and a voltage-current conversion circuit in series, returning the output current of the voltage-current conversion circuit at the final stage to the input side of the current-controlled oscillator at the first stage, and using the output of the current-controlled oscillator as the oscillation output (see Japanese Patent Laid-Open No. 2002-300027 (Patent Document 1)).
Moreover, a voltage generation circuits generating a plurality of voltages with high accuracy is the one including a reference voltage generation circuit, a differential amplifier, a P-channel MOS (Metal Oxide Semiconductor) transistor, a switching transistor, an output node, and a first to a third resistor strings. The third resistor string is between other input terminal of the differential amplifier and a ground. Even when the resistance of the first resistor string between the drain of the P-channel MOS transistor and an output node and that of the second resistor string between the output node and the other input terminal of the differential amplifier, which differs from that of the first resistor string, are selected and controlled, these resistances are selected and controlled so that a sum of the resistance of the first resistor string and that of the second resistor string is constant (see Japanese Patent Laid-Open No. 2007-293545 (Patent Document 2)).

Semiconductor device, with high-resolution and wide-range adjustable clock

Abstract: An operational amplifier AMP1 is subject to feedback control so that the voltage of a positive input part equals that of a negative input part. The voltage of a circuit node fbck equals a reference voltage VREFI. A decoder DEC decodes control signals CNT7 and CNT6 and turns on one of transistors T2 to T5. This configuration provides feedback control so that the voltage of the circuit node fbck equals the reference voltage VREFI. This significantly reduces the on-resistances of the transistors T2 to T5 and prevents the degradation of the frequency accuracy.


A delayed locked loop (DLL) system and method for determining a forward clock path delay are disclosed. One embodiment of the DLL system includes a delay line having a plurality of delay stages. The DLL system also includes a measure shot device configured to determine a forward clock path delay of the DLL system. The measure shot device is configured to provide a calibration sequence into the DLL loop and to detect the calibration sequence after the calibration sequence has passed through the DLL loop. The measure shot device is further configured to count the number of clocks for a period of time between providing and detecting the calibration sequence. The number of clocks can be used to calibrate components of the DLL system.

1. Field of the Invention
Embodiments of the invention relate to electronic devices, and more particularly, to electronic devices including a delay-locked loop (DLL).
2. Description of the Related Art
High speed electronic devices such as DRAM are often provided with external or reference clock signals for timing the operations of their internal components. When an external clock signal is propagated to internal components, a time delay of the clock signal occurs. The internal components may not be well synchronized with the external clock signal, which can affect the performance of the internal components at relatively high speeds.
In addition, various components in an electronic device can have different manufacturing parameters. Thus, the components in an electronic device may not be relatively well synchronized even though they are clocked by the same external or reference clock signal. In addition, variations in operating conditions such as operating temperature and external power supply may also vary propagation delay, resulting in changes to clock delay.
FIG. 1 illustrates a conventional delay-locked loop (DLL) system 100. The DLL system 100 includes an input buffer 105, a delay line 115, a clock tree 117, an output buffer 120, a replica model 125, a phase detector 140, a shift register controller 142, a shift register 145, and internal circuits 170. The input buffer 105 receives an external clock signal CLK and the output buffer 120 outputs an output clock signal CLK_DLL.
The delay line 115, the replica model 125, the phase detector 140, the shift register controller 142, and the shift register 145 together form a delay-locked loop (DLL). The DLL is configured to synchronize the external clock signal CLK with the output clock signal CLK_DLL.
The input buffer 105, the delay line 115, the clock tree 117, and the output buffer 120 together form a forward or internal clock path. In the context of this document, the term "forward clock path delay" refers to a clock delay occurring along the forward clock path from the input buffer 105 to the output buffer 120. Other systems can have additional components along their forward clock path. In such cases, part of the forward clock path delay can be associated with the additional components.
The input buffer 105 receives the external clock signal CLK. The input buffer 105 generates a reference clock signal clk with a delay from the external clock signal CLK. The input buffer 105 provides the delay line 115 and the phase detector 140 with the reference clock signal clk.
The delay line 115 receives the reference clock signal clk from the input buffer 105 and delays the signal. The delay line 115 includes a plurality of delay stages, for selection of a desired delay from the reference clock signal clk. The delay line 115 can include a daisy chain of logic gates.
The clock tree 117 receives the delayed signal from the delay line 115. The clock tree 117 is configured to distribute clocks for timing the internal circuits 170. The clock tree 117 also provides an output signal to the output buffer 120.
The output buffer 120 receives the output signal from the clock tree 117. The output buffer 120 provides an output signal CLK_DLL which is delayed from the clock tree output signal.
The replica model 125 receives a delayed clock signal from the delay line 115 and further delays the delayed signal. The replica model 125 is configured to emulate delays along the forward clock path of the system 100 except for a delay associated with the delay line 115. In the illustrated system 100, the replica model 125 can include a dummy output buffer, a dummy clock tree, a dummy input buffer, and additional gates (not shown) to form a replica of the forward clock path delay. The replica model 125 provides a replica model output signal to the phase detector 140.
The phase detector 140 compares the reference clock signal clk with the replica model output signal from the replica model 125. The phase detector 140 generates a comparison signal corresponding to a phase difference between the reference clock signal clk and the replica model output signal. The phase detector 140 provides the comparison signal to the shift register controller 142.
The shift register controller 142 receives the comparison signal, and controls the shift register 145 in response to the comparison signal. The shift register 145 is configured to select the delay amount of the delay line 115.

Delay-locked loop (DLL) system for determining forward clock path delay


Embodiments for lighted see-through displays include a plurality of light elements positioned thereon at select points arranged to convey information. The light elements may be selectively lit to provide fixed or moving information, or may even be programmed to change or even display like a marquee or even a television. Particular embodiments of the invention employ a lightweight, flexible substrate to support the light elements. Other embodiments employ one or more comparatively rigid substrates supporting rows of light elements. Particular applications for the lighted sign embodiments include display in vehicle windows such as police vehicle windows, construction and utility vehicle windows, and school bus and public transportation windows. Global Positioning System and radio signal receiver components allow for regional display of pertinent information in particular embodiments of the invention.

1. Technical Field
This invention generally relates to lighted displays for vehicles, and more particularly to see-through outwardly facing, lighted displays for use in vehicle windows.
2. State of the Art
Lighted displays generally are well known in the art. Businesses use lighted "OPEN" and "CLOSED" signs and shaped neon bulbs for signs, companies regularly display their trademarks in lights to draw more attention, and lights displayed behind colored translucent signs are found in nearly every strip mall today. These examples of lighted displays typically include fixed information. In other words, the sign displays one message (or possibly two messages in the case of "OPEN" and "CLOSED" signs), and the lights can only display their given message or light the fixed image printed on the translucent sign board. Relating to lighted vehicle displays see, for example, U.S. Pat. Nos. 5,604,480 to Thompson and 5,648,756 to Zadok. Many of these signs are also contained within a housing and given a black or dark background to enhance the visibility of the message in the display.
Another example of a lighted information display is that of a marquee. Conventional marquees include an array of light emitting diodes (LEDs) housed in a box that contains all of the electronics for the array. The box and array of LEDs are coupled to a microcontroller that controls which LEDs are "on" and which are "off" at any given time to display particular information on the display, and in some cases the color and intensity of the lights emitted. These types of LED marquees housed in a box are known to be displayed on vehicles (see, for example, U.S. Pat. Nos. 6,590,502 B1 to Pederson (showing an LED display on a police light bar) and 4,868,542 to Thompson (showing an LED display at the bottom of a back car window)) and in store windows. If in a window, such marquees are typically displayed near an edge of the window so the marquee display box does not disrupt the view through the window. The marquee display box is given a dark background to enhance the visibility of the message in the display and hide the electronics contained within the marquee.


Immunity against protozoan is conferred on an animal by a method comprising orally administering to an animal a transformed plant cell comprising a polynucleotide encoding a protective antigen against protozoiasis development, a transformed plant or its progeny or clone comprising the transformed cell, a propagation material of the plant or its progeny or clone, a processed material or extract of the above cell, plant, or its progeny or clone, or propagation material, or a protective antigen against protozoiasis development isolated from the transformed plant cell or the transformed plant or its progeny or clone.

Chicken leucocytozoonosis is a disease resulting from infection by Leucocytozoon caulleryi belonging to the suborder Haemosporina. L. caulleryi was recognized for the first time in 1909 in Vietnam in the blood of chicken as round gametocytes that do not carry malaria particles, and was named as a protozoan belonging to the genus Leucocytozoon.
In Japan, chicken leucocytozoonosis was confirmed for the first time in 1954 in Hyogo prefecture. The pathogen was identified as L. caulleryi, and thereafter, experiments demonstrated that this protozoan is biologically transmitted by a type of blood-sucking insect, Culicoides arakawae (hereinafter abbreviated as C. arakawae) belonging to the order Diptera and family Ceratopogonidae. The life cycle of this pathogen has been almost fully elucidated. Leucocytozoon caulleryi has three stages of development: schizogony (multiplicative reproduction) and gametogony (gamete formation) in the body of the chicken host, and sporogony (spore formation) in the body of the C. arakawae transmitter.
When C. arakawae infected with this protozoan sucks blood, sporozoites that enter the body of chicken with the saliva become parasitic on vascular endothelial cells, mainly in the lung, liver, spleen, kidney and such, and form first-generation schizonts. On the fifth to seventh day after infection, first-generation merozoites are released into the blood. These first-generation merozites reenter the vascular endothelial cells distributed throughout the body, grow into second-generation schizonts, leave the host cells and grow in the intercellular space in the late stage. On the 14th day, second-generation merozoites are released into the blood by these schizonts. The merozoites enter erythroid cells and grow, and on the 18th to 19th day, they separate from the host cells and mature as macrogametocytes (female gametocytes) and microgametocytes (male gametocytes).
When C. arakawae sucks blood, both gametocytes are taken up into the midgut together with the blood of the chicken. These gametocytes form macrogametes (female gametes) and microgametes (male gametes), which then conjugate (fertilization) to form zygotes that subsequently form ookinetes which enter the intercellular space in the midgut wall and develop into oocysts after migrating to beneath the outer membrane of the midgut wall. Several tens of sporozoites are formed inside the oocysts, and as the wall of the oocysts breaks down, sporozoites are released into the celom of C. arakawae. They then migrate to the salivary gland to complete the developmental cycle.
Symptoms and lesions in chicken caused by this protozoan infection are observed mainly in the late stages of schizogony when second-generation schizonts grow, and in the gametogony stage when second-generation merozoites become parasitic on erythroid cells and develop into gametocytes. Anemic symptoms and bleeding lesions in various organs and tissues, which are most characteristic of this disease, are thought to be triggered by vascular emboli-accompanying bleeding caused by second-generation schizonts, and disruption of red blood cells due to parasitization by protozoans in the gametogonic stage.
Since C. arakawae, the mediator of the protozoan, inhabits wetlands, this infection has been observed to be prevalent in Southeast Asia, China, North Korea, and South Korea where paddy fields are found. In Japan, since the first observation of leucocytozoonosis, the epidemic has recurred every summer and inflicted a significant loss on poultry productivity. However, the occurrence sharply decreased after feeds started to be supplemented with pyrimethamine (since 1964) and a mixture of pyrimethamine and sulfa drugs (since 1968) as preventive agents. Since then however, acquired resistance of protozoans and residuality issues have restricted the duration of use of effective drugs, and the use of some of these drugs have been banned for safety reasons. Such restrictions are making the prevention of the disease difficult and the damages are also becoming constant issues.
Conventional experimentally produced vaccines against chicken leucocytozoonosis include live vaccines that use sporozoites, inactivated vaccines in which the antigen is a parasite-derived substance, and inactivated vaccines produced out of organ emulsions of protozoan-infected chicken. In the case of live vaccines using sporozoites, chickens are immunized with a small number of sporozoites separated from the salivary glands of C. arakawae [see Non-Patent Document 1]. Examples of inactivated vaccines in which the antigen is a parasite-derived substance are as follows.
Examples of parasite-derived antigens include first-generation schizonts, first-generation merozoites, second-generation schizonts, second-generation merozoites, and soluble antigens derived from the body of chicken and from developing chick embryos. Of these, soluble antigens detected in the serum of infected chicken on the tenth to fifteenth day after sporozoite inoculation have been observed to have nearly the same antigenic characteristics as second-generation schizonts, and a relatively strong immunogenicity similar to that of the second-generation schizonts. Utility has been confirmed for experimental vaccines produced by inactivating such serum-derived antigenic material with formalin [see Non-Patent Document 2].
Furthermore, inactivated vaccines made from organ emulsions were developed to improve the difficulties of obtaining large amounts of the above-described protozoan antigens. The vaccines were experimentally produced out of organ emulsions collected from the thymus, lung, liver, spleen, kidney, and F sac from infected chicken on the thirteenth day after sporozoite inoculation, and then inactivated with formalin. The effects of such vaccines have been confirmed [see Non-Patent Document 3].
To enable a stable and abundant supply of vaccines, the present inventors have provided vaccines that use genetically modified microorganisms made to express immunogenic proteins derived from second-generation schizonts of Leucocytozoon caulleryi [see Patent Document 1].
However, with vaccines developed so far, there is the premise that they will be administered by injection, and is problematic in that their use is limited due to cost and inconvenience.    [Patent Document 1] Granted/Registered Japanese Patent No.3582663    [Non-Patent Document 1] Shiihara et al., "Shizuokaken Yokei Shikenjo Kenkyu Hokoku (Bulletin of Shizuoka Poultry Experiment Station)" 13:25-27 (1978)    [Non-Patent Document 2] Isobe and Suzuki, Jpn, J. Parasitol., 37:214-219 (1988)    [Non-Patent Document 3] Morii, T. et al., J. Parasitol. Res., 76:630-632 (1990)

Methods for conferring immunity against protozoans in an animal

Kitasato Daiichi Sankyo Vaccine Co., Ltd.

National Institute of Advanced Industrial Science and Technology


The present invention is a method of identifying ligands that interact will cellular processes involved in the life-cycle of HIV. In particular, an oligonucleotide, corresponding to specific RNA sequence within an infected cell, is modified by the substitution of 2-aminopurine. As a result, interactions between the oligonucleotide and the ligand can be measured via fluorescence. This technique can be use to find inhibitors of binding between rev and its response element (RRE), dimerization initiation sequences, and topoisomerases and DNA.

This invention pertains to a method for selective substitution of highly fluorescent nucleotide base analogs within a sequence of nucleic acid drug targets. The substitution is particularly selected such that the base can be used as a probe to monitor and screen for interactions of ligands with a nucleic acid target.
The development of specific inhibitors of protein-RNA interactions, that are unique and required for retroviral viability, is of significant interest to pharmaceutical industries since these complexes provide new and potentially powerful targets to inhibit retroviral infection.
As described in U.S. Pat. No. 6,153,382, herein incorporated by reference, the HIV genome is tightly compressed. At least 30 different RNA transcripts are produced by splicing using the six splice acceptors and two splice donor sequences. The structural proteins encoded by HIV are chemically similar to those of the C-type retroviruses and, like them, are encoded as polyproteins by the gag (group antigen), pol (polymerase) and env (envelope) genes. Cleavage of the polyproteins by the viral protease or cellular enzymes generates eight functional virion proteins. In addition to these structural genes, HIV-1 also carries genes for three regulatory proteins, rev (regulator factor); and two proteins involved in virus maturation, vif (virion infectivity factor) and vpu (viral protein U). The vpr (viral protein R) gene encodes a low copy number virion component. In the closely related viruses HIV-2 and simian immunodeficiency virus (SIV), vpr is replaced by vpx (viral protein X), a unique virion protein.
Transcription of the HIV genome during virus replication shows distinct kinetic phases. The initial products of HIV gene expression are short, multiply spliced mRNAs approximately 1.8 to 2.0 kb in length, which encode the trans-acting regulatory proteins including rev. As infection by the virus develops, and the levels of the rev protein rise in the infected cells, mRNA production shifts progressively towards synthesis of a family of singly-spliced 4.3 kb mRNAs encoding env and other HIV gene products such as vif and vpr. Finally, late in the infection process, production switches to full-length, unspliced, transcripts that act both as the virion genomic RNA and the mRNA for the gag-pol polyprotein.
To achieve this control of gene expression, HIV relies on the interaction of cellular and virus-encoded trans-acting factors with cis-acting viral regulatory sequences. Initiation of transcription relies largely on the presence of binding sites for cellular transcription factors in the viral long terminal repeat (LTR). In contrast, the virally encoded regulatory proteins tat and rev exert their activity via cis-acting sequences encoded within HIV messenger RNAs. After a systematic search, a cis-acting sequence required for rev activity, was mapped to a complicated RNA stem-loop structure located within the env reading frame. This sequence has been named the rev-responsive element (RRE) and has been localized to a 234-nucleotide long sequence within the env gene. Similar regulatory proteins and target sequences are used by HIV-2 and SIV. The human T-cell leukemia (HTLV-1) virus rex gene product appears to function analogously to rev, and can functionally substitute for rev to promote viral gene expression.
The distinct kinetic phases of HIV transcription are now believed to reflect the intracellular levels of the regulatory proteins tat and rev. Initially, binding of host transcription factors to the LTR induced basal level transcription of the early mRNAs including tat. As tat levels rise, increased transcription from the LTR is stimulated by the trans-activation mechanism. This leads to further increases in tat levels, and also stimulates production of rev. Production of the viral structural proteins begins once rev levels have risen to sufficiently high levels to promote export of messenger RNAs carrying the RRE sequence. The HIV growth cycle may also include a latent stage where viral gene expression is silent because transcription from the viral LTR produces insufficient amounts of regulatory proteins to initiate the lytic growth cycle.
Rev recognition of the RRE RNA element, like tat recognition of HIV-1 trans activation region (TAR) RNA, is due to direct binding. Binding is tight (KD=1-3 nM) and highly specific for the RRE. However, the binding behavior of rev to RRE is much more complex than the binding of tat to TAR RNA. As the concentration of rev increases, progressively larger complexes with RRE RNA are formed, whereas tat only forms one-to-one complexes with TAR RNA.
The simplest explanation for the RNA binding behavior of rev is that the protein binds initially to a high affinity site and that subsequently additional rev molecules occupy lower affinity sites.
Many methods for nucleic acid-protein binding assays, to measure interactions of, for example, RRE with rev, have been developed, such as gel electrophoresis, filter binding and chromatography. However, these procedures lack sufficient speed, sensitivity and accuracy to be useful as high-throughput binding assays. Additionally, fluorescence methods, such as fluorescence resonance energy transfer (FRET), provide many advantages, however do not detect both direct and indirect interactions of rev peptide or ligand binding that cause subtle conformational changes in the nucleic acid structure of RRE.
The present invention pertains to a generally useful approach for detecting and quantifying the interaction of nucleic acids with macromolecules and/or small molecule ligands that can be adapted into high-throughput screens (HTS). A fluorescence-based binding assay, i.e. fluorescence emission perturbation (FREP) has been developed that provides such a method for screening and optimizing inhibitors of nucleic-acid protein interactions that are potential and so far unexploited targets for anti-viral and anti-cancer drugs. The FREP method described and claimed herein, provides a general, rapid and sensitive fluorescence assay that allows the direct detection and quantification of the interaction of ligand with nucleic acid targets. Using the FREP assay, multiple binding classes of small molecules to nucleic acid targets can be directly detected, as well as specific inhibition of macromolecular interactions with nucleic acid targets by small molecule antagonists. The ability to detect both macromolecular (mostly protein) and small molecule binding using the same fluorescently labeled nucleic acid constructs allows the application of dual screening; where analysis for both direct binding of small molecules, as well as the potential competitive inhibition of the nucleic acid-protein interaction by these molecules may be assayed simultaneously.
In particular, the FREP binding assay, in one form, includes an RRE oligonucleotide labeled with the fluorescence adenosine analog, 2-aminopurine (2-AP) in specific nucleotide. Modified RRE oligonucleotides show measurable changes in fluorescence emission that can be directly correlated with rev and small molecule binding to RRE; thereby providing a general method for monitoring these binding interactions. Rev monomers (or small peptides derived from rev) act by first binding to a high affinity site within the response element (RRE), a 34-nucleotide stem-loop structure (stem-loop IIB) found in nuclear HIV-1 mRNA transcripts (FIG. 1).
The FREP method is applicable in the context of many nucleic acid systems. Specifically, however, the FREP method is particularly advantageous for use in measuring interactions with (1) the above-described HIV-1 RRE-rev system; (2) HIV-1 dimerization initiation sequences (DIS); and (3) topoisomerase DNA systems.
Previous studies have shown the 2-AP can be used as a valuable probe of the structure and dynamics of specific sites in DNA, as a monitor for enzyme-DNA interaction, and to study Mg2+ dependent conformational changes in certain ribozymes. The fluorescence of 2-AP is usually highly quenched when it is stacked with other bases, but increases as much as 100 fold when fully exposed to solvent. Thus, the quantum yield of 2-AP is highly sensitive to changes in its microenvironment, which allows the detection of subtle conformational changes in the nucleic acid upon interaction with ligands. In addition, 2-AP is a generally non-perturbing substitution because it is similar in structure to adenine (6-aminopurine) and will form a thermodynamically equivalent base pair with uridine.
The FREP method of the invention has proven to be a more general and more easily applied approach for detecting and quantifying interactions with nucleic acids because the fluorescence base reporter groups do not need to directly interact with either the macromolecule or the small molecules to show a measurable change in fluorescence. Instead, subtle conformational changes in the nucleic acid structure caused by ligand binding could either directly or indirectly result in significant changes in the fluorescence intensity of the fluorescent base reported that could be used as a monitor for binding. This feature makes the FREP assay a more general reporting technique for binding interactions, than, for example, a specific FRET pair, allowing both for screening of direct small molecule RNA interactions, as well as for binding events that result in an inhibition of macromolecular complex formation. Moreover, the use of fluorescent nucleotide base labeling of the nucleic acid target allows screening of molecular libraries without any need for further labeling of macromolecules (proteins or peptides) or small molecules. This is a large advantage in terms of versatility and generality of this screening method. A summary comparison of the 2-AP FREP method of the invention with other conventional methods is shown in Table 1.

Rapid fluorescence detection of binding to nucleic acid drug targets labelled with highly fluorescent nucleotide base analogs

The United States of America as represented by the Secretary of the Commerce


The quality that is good or bad of an injection molded product is judged. The temperature T of a metal mold, of a resin in the mold cavity, barrel or nozzle of an injection molding machine is measured at a time t elapsed after comencement of a pressure holding step of the injection molding machine. The present P of resin in the mold cavity or barrel or nozzle is measured at the time t, and a time function A=f(t) is calculated using the temperature T and the pressure P, where A represents a calculating parameter T/P, P/T, T.times.P, T+P or T.+-.P. This is compared with a reference time function Ao=f(t) of a shot in which satisfactory products can be obtained, with a time function An=f(t) of another shot.

This invention relates to a method of judging quality (good or bad) of an injection molded product, and more particularly a method of judging the accuracy of the configuration and dimensions of the injection molded product.
According to a prior art method of judging the configuration of a product of an injection molding machine, a resin pressure or an injection pressure in a mold cavity is measured at different times during a pressure holding time to obtain a time function P=f(t), then this function is compared with a function P.sub.1 =f(t) which holds when a satisfactory product is obtained. Alternatively, a function T=f(t) representing a mold temperature is compared with a function T.sub.1 =f(t) representing the mold temperature at which a satisfactory product is obtained, thereby judging the quality of the molded product.
The state of the resin in the mold cavity changes in accordance with a state equation PV=RT (where P: pressure, V: volume, T: temperature and R: constant). Accordingly, where the quality of the product is judged by measuring only presssure P or temperature T as in the prior art, it has been impossible to grasp volume V.

Method and apparatus of judging quality of injection molded products


The present disclosure describes a handle system for use with finishing tools utilized to finish wallboard joints. A finishing tool may be connected to the handle system and moved relative to a wallboard joint to appropriately apply mastic over the joint. In one embodiment, the handle system includes an elongate handle body having a tear drop cross-sectional shape. The shape enables the handle body to fit ergonomically within a user's hand and improves the user's ability to resist torque tending to cause the handle body to twist and a connected finishing tool to come out of contact with wallboard during use. The handle system further includes a pivoting connector assembly that permits a connected finishing tool to rotate relative to the handle body and that has a unique pivot pin that allows mastic to flow through the handle body, through the pivoting connector assembly, and into the connected finishing tool.

Today, many construction materials come in the form of substantially planar panels or rolls which are unrollable into, essentially, planar panels. The construction materials are, in both cases, secured with appropriate fasteners and/or adhesives to the framing members, sheathing and/or decking of building walls, floors, ceilings and roofs. Such construction materials comprise, without limitation: (a) drywall, gypsum board, plasterboard, cement board, greenboard, blueboard, wood, and foam board for use in forming interior, and in some cases exterior, building walls and/or ceilings; (b) felt paper, underlayments, membranes, wraps, and other similar materials that are generally applied to sheathing and/or decking to protect underlying building materials from the effects of weather, to reduce the amount of air and/or moisture infiltration through a building's structure, and/or to provide insulation against heat and sound transmission; and (c) other similar construction materials. The term "wallboard" is used herein to mean and refer, collectively, to such construction materials.
When individual panels or unrolled rolls of such wallboard are positioned side-by-side in/on a building structure as is often the case, the panels or unrolled rolls define gaps, seams or joints therebetween and are joined together with tape, sealant, filler and/or adhesive substances to form a monolithic structure. The tape may be manufactured from paper, fiberglass, or other material, be woven or non-woven, and be or not be backed with an adhesive substance. The tape is, generally, applied over the gaps, seams or joints either by hand or through use of a taping tool. The sealant, filler and/or adhesive substances may include, but not be limited to, drywall compound, glue, caulk, resin, epoxy, and other similar substances that are collectively referred to herein by the term "mastic". The tape and mastic may be used alone or in combination to seal, fill and/or hide the gaps, seams or joints and secure the individual panels or unrolled rolls together. When the tape and mastic are used in combination to hide a joint between pieces of wallboard, the mastic aids in securing the tape to the wallboard while being featherable away from the joint to cause the joint and tape to blend in and provide a smooth, substantially level surface with the wallboard to either side of the joint.
To apply, screed, smooth and/or feather the mastic relative to a gap, seam, joint, and/or tape, an appropriate finishing tool is selected and used based, at least, on the particular wallboard product, tape, and/or mastic being employed, and the location and orientation of the gap, seam or joint. In situations where the wallboard on both sides of a gap, seam or joint therebetween substantially defines a single plane, a finishing tool known as a "finisher" is often used to quickly apply a thin, uniform layer of mastic over tape covering the gap, seam or joint. A finisher comprises a partial enclosure having a slotted opening in the base thereof and that is filled with mastic. When the base of the finisher is placed in contact with two pieces of wallboard and moved over, for example a joint therebetween taped previously by a user, mastic exits the partial enclosure via the slotted opening, is applied in a layer of desired thickness over the taped joint, and is feathered to cause the tape to blend in with the wallboard on both sides of the taped joint. Typically, a round handle is coupled to the finisher to extend the reach of the user with the finisher and to allow the user to move the finisher over taped joints.
Unfortunately, the finisher may become somewhat heavy when filled with mastic and the finisher's shape/geometry can cause the finisher to become difficult to manipulate. For vertical taped joints, the finisher's weight and shape/geometry are not particularly problematic. However, for horizontal and other non-vertical taped joints, the user must hold the round handle in a non-vertical plane while moving the finisher over the taped joints. Due to the weight and shape/geometry of the finisher, the finisher sometimes becomes difficult to support and keep in appropriate contact with the wallboard while being moved over the horizontal and other non-vertical taped joints. In such circumstances, the finisher exerts a torque on the round handle, tending to twist the handle in the user's hands. In addition, the user may desire to maintain a constant angle between the finisher and handle while moving the finisher over the taped joints in order to apply a uniform layer of mastic and a constant angle may be difficult for the user to maintain. Consequentially, the mastic may not be applied over taped joints in a uniform, consistent manner and the user may be fatigued while attempting to keep the finisher in appropriate contact with the wallboard and at a desired angle relative to the handle.
Therefore, there is a need in the industry for systems, apparatuses, and methods that improve the control and/or usability of finishing tools or other similar devices, that minimize fatigue encountered by users during use of finishing tools or other similar devices, and that may address other problems, difficulties, and/or shortcomings of current technology that may or may not be described herein.


Chlorine dioxide is continuously produced at high efficiency in a single vessel process by reduction of chlorate ions with hydrogen peroxide in an aqueous acidic medium. The hydrogen peroxide is pre-mixed with at least a portion of the aqueous chlorate solution feed or the sulfuric acid feed or a mixture thereof. By pre-mixing the hydrogen peroxide feed, the occasional decomposition of hydrogen peroxide and the resulting foaming of the generator liquor is eliminated.

It is known to produce chlorine dioxide by reduction of an acid aqueous chlorate solution using various reducing agents such as methanol, chloride ions, hydrogen peroxide, etc. In U.S. Pat. No. 2,833,624 (Sprauer), a process is described whereby chlorine dioxide is produced by reacting chlorate ions, a strong acid and hydrogen peroxide. The process can be carried out in the presence of chloride ions, which are said to catalyze the reaction. U.S. Pat. No. 2,833,624, however, does not teach carrying out the process in a single vessel under subatmospheric pressure.
U.S. Pat. No. 4,421,730 (Isa et al) also discloses a process to produce chlorine dioxide using hydrogen peroxide. This process, however, requires the reduction to be effected using chloride ions in the presence of a complex palladium (II) catalyst and the hydrogen peroxide in order to achieve commercially attractive production rates.
Recently, there issued U.S. Pat. Nos. 5,091,166 (Engstrom et al) and 5,091,167 (Engstrom et al) wherein chlorine dioxide is described as being produced in a single-vessel generator-evaporator-crystallizer process using hydrogen peroxide as the reducing agent. Hydrogen peroxide for use in the process generally is employed in its commercially-available form, generally an aqueous solution containing about 30 wt % hydrogen peroxide or higher. Alkali metal chlorate, hydrogen peroxide and sulfuric acid are continuously fed to the generator. Commercially acceptable production rates and efficiencies are said to be obtained. The main distinction between both processes is that U.S. Pat. No. 5,091,166 covers the acidity range 2 to 5N whereas U.S. Pat. No. 5,091,167 covers the acidity range 5 to 11N.
The operation of a hydrogen peroxide-based chlorine dioxide generator using one of the processes described in U.S. Pat. Nos. 5,091,166 and 5,091,167 generally leads to satisfactory results in terms of the chemical yield of chlorine dioxide on chlorate. These processes, however, have certain deficiencies and disadvantages which can seriously affect the operation of the generator. For example, there can occasionally be an excessive consumption of hydrogen peroxide, leading to inefficient operation. Also there can be excessive foaming of the liquor in the generator, potentially leading to unsafe operation and uneven production rates of chlorine dioxide. Additionally, the process can have high sensitivity to metallic impurities (such as iron) inadvertently but often present in variable and uncontrollable amounts in the liquor, there can be occasional variations in the purity of the product, there is enhanced corrosion of the equipment, etc. In other words, continuous operation of the processes in U.S. Pat. Nos. 5,091,166 and 5,091,167 can, on occasion, become highly inefficient and unpredictably unstable, leading to potentially unsafe operation.

Hydrogen peroxide-based chlorine dioxide process


The disclosure provides an antigenic peptide targeting the extracellular, but not the intracellular, form of CETP to reduce the level of LDL (including oxLDL and other derivatives of LDL) and increase the level of HDL. Accordingly, the disclosure provides a CETP vaccine to overcome the low immunogenicity of CETP and for long-term inhibition of CETP activity to prevent or treat oxLDL or other LDL derivative-related diseases/symptoms.

HDL continues to attract interest because its level is inversely associated with the risk of cardiovascular disease. This may be attributed to its various potentially anti-atherogenic properties, such as reverse cholesterol transport, anti-inflammatory, anti-oxidative, and anti-thrombotic effects. Clinical studies have shown that low HDL-C is also found in non-alcoholic steatohepatitis (NASH). NASH shares several characteristics with atherosclerosis, including lipid accumulation, inflammation, and macrophage infiltration. The pathogenesis of NASH involves scavenger receptor-mediated uptake of ox-LDL by macrophages in the liver.
Cholesteryl ester transfer protein (CETP) is considered a therapeutic target for increasing HDL-C. Interest in CETP as a therapeutic target began as a result of the high HDL-C and low LDL-C observed in Japanese people carrying the homozygous, defective CETP gene who showed no evidence of premature atherosclerosis, even though they had hypercholesterolemia. This CETP is bound mainly to HDL particles and transfers cholesterol ester from HDL to triglyceride-rich lipoproteins. CETP action results in a CE enrichment of non-HDL lipoproteins, which could contribute to atherosclerosis. Small molecule CETP inhibitors, including dalcetrapib, evacetrapib, and anacetrapib that are at various phases of clinical development inhibit CETP activity and significantly increase HDL-C. However, torcetrapib failed in phase 3 clinical trials due to compound specific off-target effects, and dalcetrapib failed in phase 3 clinical trials due to less meaningful outcomes. Two other CETP inhibitors, evacetrapib and anacetrapib, are still under clinical trials. Inducing an immune response against specific self-peptides is potentially beneficial for the treatment of certain diseases. The drawbacks of peptide-based immunization include low immunogenicity of self-peptides, a low efficiency of chemical conjugation, and the heterogeneous nature of antigen preparations.
U.S. Pat. No. 7,074,407 B1 provides a method for increasing HDL cholesterol in a mammal by stimulating an immune response that inhibits the function of CETP. U.S. Pat. No. 6,284,533 B1 provides a plasmid-based vaccine based on the combination of DNA segments coding for one or more B cell epitopes of cholesteryl ester transfer protein (CETP) and one or more broad range helper T cell epitopes. US20090104211 provides a CETP mimotope which, in their amino acid sequence are completely different from the amino acid sequence of CETP or of fragments of CETP. U.S. Pat. No. 6,410,022 B1 relates to peptides comprising a helper T cell epitope portion and a B cell epitope portion for eliciting an immune response against endogenous cholesteryl ester transfer protein (CETP) activity to prevent or treat cardiovascular disease, such as atherosclerosis. WO2014003531 provides a novel vaccine composition of micellar nanoparticles consisting of carboxyl terminal of CETP peptide immunogen for intranasal administration for the treatment and/or prevention of atherosclerosis disease or non-alcoholic fatty liver resulting from an abnormal metabolism of circulating lipids. Although a number of antigens are developed, their immunogenicity and efficiency are not sufficient.
Therefore, there is still a need to develop a CETP peptide immunogen having advantageous immunogenicity.

CETP antigenic peptide and fusion protein and their composition and applications