Extensive colorectal lymphomatous polyposis complicated by acute intestinal obstruction: a case report

Jaques Waisberg
Amanda Do
Val Anderi
Pedro Augusto
Soffner Cardoso
José Henrique
Miranda Borducchi
Demetrius Eduardo Germini
Maria Isete
Fares Franco
Cidia Vasconcellos
Extensive colorectal lymphomatous polyposis complicated by acute intestinal obstruction: a case report
10.1186/s13256-017-1340-1C A S E R E P O R T Open Access
Background: Multiple lymphomatous polyposis is a rare type of gastrointestinal lymphoma that extensively infiltrates the intestine. Multiple lymphomatous polyposis originates from the mantle zone of the lymphoma follicle and is considered to be a mantle cell lymphoma, which is a relatively aggressive type of B-cell non-Hodgkin's lymphoma. We report an unusual case of a patient with multiple lymphomatous polyposis with extensive colorectal involvement and acute intestinal obstruction, an atypical complication of this rare disease. On the basis of this case study, the pitfalls in gastrointestinal tract lymphomatous polyposis diagnosis and prognosis, as well as the treatment options, are discussed. Case presentation: Our patient was a 76-year-old white woman with asthenia, cramps, and swelling in the lower left quadrant of the abdomen, as well as weight loss within the previous 5 months. A colonoscopy revealed polyps in the rectum, sigmoid colon, descending colon, and right and left colic flexures. A biopsy revealed lymphomatous infiltration of the intestinal wall. Because of the large size of the polypoid masses, which narrowed the colonic lumen in multiple locations, the patient developed acute intestinal obstruction and was referred for laparotomy. She underwent a total proctocolectomy with a permanent ileostomy and a left salpingo-oophorectomy. Microscopic examination showed the presence of a multicentric, low-grade, small lymphocytic lymphoma. Immunohistochemical analysis revealed positive immunostaining for CD79a, CD20, and CD45. These results were consistent with the diagnosis of mantle cell lymphoma. Two weeks after surgery and prior to discharge, but before the beginning of chemotherapy, the patient's general condition worsened as she experienced a severe and progressive respiratory tract infection, advanced respiratory insufficiency, and septic shock, and she ultimately died. Conclusions: Mantle cell lymphoma develops as a progressive and aggressive disease with widespread polyposis of the gastrointestinal tract. The intensive chemotherapeutic regimens usually result in the regression of macroscopic and microscopic lesions; however, remissions are short in duration, and the median length of patient survival is 3-4 years. Mantle cell lymphoma is a rare disease that should be part of the differential diagnosis of polypoid diseases of the large intestine.

Background

Although the intestinal diffuse mucosa-associated lymphoid tissue (MALT) found in the mucosa of the digestive system, including the tonsils, Peyer's patches, and appendix, contains the most lymphocytes of any organ in the immune system following the compartment that comprises the peripheral lymph nodes and spleen, only 10% of all lymphomas occur in the gastrointestinal (GI) tract [1][2][3]. Non-Hodgkin's lymphoma (NHL) of the GI tract is the most frequent extranodal lymphoma. Approximately 15-30% of primary extranodal lymphomas occur in the GI tract, and primary GI tract lymphomas account for only 1-10% of all GI malignancies [1][2][3].

Mantle cell lymphoma, a distinct clinicopathological entity of NHL, is an aggressive B-cell neoplasm that is characterized first by a monotonous proliferation of small-to medium-sized lymphocytes coexpressing CD5, CD20, and cyclin D1 epitopes and second by frequent t(11;14)(q13;q32) chromosomal translocation [4][5][6][7]. Mantle cell lymphoma comprises 2.5-7.0% of all NHLs, and the GI tract is involved in approximately 20% of cases [8,9]. Mantle cell lymphoma is often diagnosed at an advanced stage. The response rate to multiagent chemotherapy is nearly 90%, and the expected 5-year survival rate in selected cases is close to 60% [2,[8][9][10]. Importantly, mantle cell lymphoma is clinically aggressive and has a shorter median survival period than other types of small B-cell neoplasms, such as the extranodal marginal zone of MALT lymphoma and follicular lymphoma [10][11][12].

Lymphomatous polyposis of the GI tract is a rare, heterogeneous group of small B-cell lymphomas including mantle cell lymphoma, follicular lymphoma, and MALT lymphoma [3]. Multiple lymphomatous polyposis (MLP) is characterized by the presence of multiple lymphomatous polyps along one or more segments of the digestive tract [2,9,10]. MLP is characterized by multiple white, superficially infiltrated nodular or polypoid lesions involving long segments of the GI tract [12]. In most MLP cases, lymphoma cells originate from the mantle zone of the lymphoid follicle, so this disease is actually considered a subtype of mantle cell lymphoma [8,9], which accounts for 2-9% of primary GI lymphomas [2,9,10].

Lymphoma of the colorectal region is mostly of B-cell lineage, as it is in other sites in the GI tract. Cornes [13] coined the disease's name, multiple lymphomatous polyposis, in 1961 to designate numerous polypoid lesions in the GI tract comprising lymphoma with mucosal involvement. Since this first description, only a few cases have been reported [2,9,10]. We describe and discuss the clinical and pathological features of a rare case of a patient with mantle cell lymphoma with diffuse involvement of the colon and rectum and complicated by an intestinal obstruction.

Case presentation

A 76-year-old white woman presented to our hospital with asthenia, cramps, and swelling in the lower left quadrant of the abdomen, as well as weight loss within the previous 5 months. A physical examination revealed pallid mucosa and no palpable superficial lymph nodes. Her Eastern Cooperative Oncology Group/Karnofsky Performance Status score was 1/80. Blood test examinations upon admission revealed anemia (hemoglobin 8.2 g/dl), a hematocrit level of 24.6%, and a white blood cell count of 9300/μl (neutrophils 72%, lymphocytes 20%), as well as proteinemia, albuminemia, carcinoembryonic antigen, blood urea nitrogen, creatinine, lactate dehydrogenase, and human immunodeficiency virus (HIV) serology that were in the normal ranges. Her myelogram result was normal. A colonoscopy was performed because of her complaints of weight loss, fatigue, and anemia and the fact that she was elderly. This procedure revealed polyps in the rectum, sigmoid colon, descending colon, and right and left colic flexures. A rectal biopsy revealed lymphomatous infiltration of the intestinal wall. The pathological diagnosis was of primary colorectal lymphoma. A computed tomography (CT) scan of the patient's abdomen and pelvis did not reveal extraintestinal involvement, and her liver and spleen were apparently normal. A thoracic CT scan did not show alterations related to lymphoma. A gastroduodenoscopic examination revealed no important findings.

Owing to the large size of the polypoid masses, which narrowed the colonic lumen in multiple locations, the patient developed acute intestinal obstruction and was referred for laparotomy. During surgery, a large polypoid lesion was found to be causing an intestinal obstruction in the descending colon, and periaortic lymph nodes with increased dimensions were exposed. A frozen section biopsy of these lymph nodes revealed a welldifferentiated small lymphocytic lymphoma. The surgical team decided to remove the colorectal segments involved in lymphomatosis because new episodes of intestinal obstruction could occur and because, in the presence of large intestinal tumors such as these, chemotherapy can lead to intestinal perforation. Thus, the patient was referred for a total proctocolectomy with a permanent ileostomy and a left salpingo-oophorectomy. Macroscopic examination of the surgical specimen revealed colorectal mucosa with polypoid nodules of variable size (1-5 cm in diameter) situated from the ascending colon to the rectum. Microscopic examination revealed the presence of a multicentric, low-grade, small lymphocytic lymphoma originating from the mantle zone of the lymphoid follicle in both the tumor-infiltrated colorectal mucosa and the pericolic/perirectal fat (Fig. 1). Moreover, the lymphoma had also infiltrated the left ovary and fallopian tube as well as all of the 43 dissected mesenteric lymph nodes. Immunohistochemical analysis revealed positive immunostaining for CD79a and CD20 (Fig. 1), as well as CD45, but there was no immunostaining for CD10 or CD23. These results were consistent with a diagnosis of mantle cell lymphoma. The final diagnosis was made 5 months after the onset of symptoms. In this case, the stage of the disease at presentation was assumed as stage II (locally advanced disease). In the postoperative period, the patient received intravenous fluid replacement and antibiotics. Two weeks after surgery and before discharge and the initial chemotherapy, the patient presented with a worsening of her general condition, a respiratory infection caused by pneumonia with fulminant evolution, and septic shock. This status was accompanied by progressive respiratory insufficiency leading to death.

Discussion

Any part of the GI tract may be involved in mantle cell lymphoma, but diffuse GI involvement is rare [2,4,12,14,15]. The colon and rectum are usually the most affected segments (approximately 90% of mantle cell lymphoma cases, including this one), followed by the small intestine, stomach, and duodenum [2,12]. The ileocecal region is frequently the original focus of the mantle cell lymphoma, and it generally remains the primary site of the disease [2,9]. The macroscopic appearance of mantle cell lymphoma in the GI tract is variable, and it can look like tumor masses, ulcers, mucosal thicknesses, or multiple polypoid lesions, with the latter being characteristic of MLP [2,9]. The main extradigestive sites affected are the bone marrow, peripheral lymph nodes, Waldeyer's ring, and liver [2,9].

Mantle cell lymphoma expresses B-lymphocyte markers (CD20 and CD79a), T-lymphocyte markers (CD5), and cyclin D1. These markers are used in routine immunohistochemistry to characterize the mantle cell lymphoma. However, CD10 is a marker of diffuse, large B-cell lymphoma, and CD23 is a marker for follicular lymphoma; both are usually negative in mantle cell lymphoma. In our patient, immunohistochemical analysis for lymphoma revealed positive immunostaining for CD79a, CD20, and cyclin D1, but there was no immunostaining for CD10 or CD23. A conclusive diagnosis of MLP requires the histological examination of a specimen in a histomorphological, immunohistochemical, and/or immunophenotypic study [6,7,16].

Most MLP cases occur in male patients over 50 years of age (median age 60 years) [2,9,17]. In general, patients with MLP have no specific clinical manifestations, but the most frequent symptoms are abdominal pain, diarrhea, palpable masses, melena, hematochezia, weight loss, and fatigue [2,9,18]. The patient in this study complained of fatigue, diarrhea, abdominal pain, and weight loss, but not of palpable masses or GI bleeding. Less frequently, intestinal obstruction, protein-losing enteropathy, intestinal malabsorption, chylous ascites, and acute abdomen consequent to perforation have also been reported [2,9,10,14,17]. In the acute intestinal obstruction in cases of MLP, the most frequent manifestation consists of multiple intussusceptions that usually occur in the ileocecal region [14,18]. The mean interval between the onset of symptoms and the diagnosis is 3 months (range 1-9 months), which was the interval for our patient [2,9,10,17]. By the time of diagnosis, more than half of the patients have bone marrow infiltration and involvement of the liver, spleen, and peripheral and mesenteric lymph nodes [2,9,12]. Our patient showed a significant infiltration of the mesenteric lymph nodes but had clean bone marrow, liver, spleen, and peripheral lymph nodes.

In general, small or nodular polypoid tumors and sessile tumors of different sizes are found throughout the GI tract during upper GI endoscopy, enteroscopy, or colonoscopy examinations that are performed to confirm the locations of the polyps and to collect tissue biopsies [2,9,10,12]. Our patient presented with many large polyps, some with a diameter up to 5 cm, which are capable of occluding the intestinal lumen.

Most MLPs originate from mantle cell lymphoma, but others originate from follicular lymphoma or MALT lymphoma. Other entities need to be included in the differential diagnosis, particularly regarding chronic lymphoid leukemia; peripheral T-cell lymphoma; subtypes of lymphocytic lymphoma such as diffuse large B-cell lymphoma; and immune proliferative small intestinal disease, a subtype of MALT lymphoma [9,10,17,19]. It is also judicious to differentiate MLP from colonic polyposis-like adenomatous polyps, familial adenomatous polyposis, and variant syndromes such as Turcot, Gardner, Peutz-Jeghers, and Cronkhite-Canada syndromes [9,15].

Current multiagent chemotherapy and arrangements of monoclonal antibodies have led to important progress in mantle cell lymphoma response rates. The overall response rates fluctuate from 80% to 95%, and complete response rates of 30-50% are often reached [8,9,[20][21][22][23][24][25][26][27].

For patients in good condition who are younger than 65 years of age, the treatment of choice is an intensive immunochemotherapy combining rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) [20][21][22]. A therapy with single-agent rituximab, a chimeric monoclonal antibody that binds specifically to the CD20 antigen, produced response rates of approximately 30%; when combined with an anthracycline-containing regimen, the response rate can increase to more than 90% [22][23][24]. Standard immunochemotherapy with R-CHOP reduces lesions, and the initial response to treatment reaches 94% [20][21][22]. Nevertheless, the resulting remissions are of short duration, and the thorough response rate is only 31% [22,23].

Other alternative treatments include a regimen of dexamethasone, high-dose cytarabine, and cisplatin or one of hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, and highdose cytarabine (hyper-CVAD). After disease control, therapy can be followed by autologous peripheral blood stem cell transplant. The initial therapeutic response rate of the hyper-CVAD regimen is over 90% [24].

However, the general poor outcome of MLP has led to the use of more aggressive therapies. Myeloablative therapies with stem cell support have significantly improved outcomes. If the patient is in good physical condition and is younger than 65 years old, intensive frontline immunochemotherapy with a multidrug regimen, followed by an autologous stem cell transplant, should be considered [2,24,28]. However, nonintensive strategies are reserved for older patients over 65 years of age or those who have important comorbidities. Intensive immunochemotherapy, both with and without stem cell support, has been used effectively to extend progressionfree survival to 5 years or more [2,22,[28][29][30]. In relapsed or refractory cases, hyper-CVAD plus rituximab can be an alternative therapeutic scheme [22][23][24].

For older patients, those who cannot receive a peripheral blood stem cell autograft, or those with an anthracycline contraindication, purine nucleoside analogues associated with rituximab are a plausible option [8,23,25]. Surgery may be appropriate and/or necessary for patients with complications such as recurrent GI bleeding and perforation or obstruction of the intestine [14,18], as in our patient.

Mantle cell lymphoma has one of the poorest prognoses of all NHL subtypes, with a median survival time of less than 3 years [2,9,10,25,26] and frequent relapses [3,8,27]. The poor prognostic factors include unsatisfactory general clinical condition, involvement of multiple extranodal sites, advanced age (older than 70 years), elevated lactate dehydrogenase levels, and bone marrow infiltration [2,9,10,17]. Several morphological variants can be recognized in peculiar aggressive variants, either blastoid or pleomorphic and adverse prognosis [2]. To identify subsets and classify patients with mantle cell lymphoma according to their prognoses, several researchers have attempted to establish a prognostic index. Ki-67 positivity, which represents cell proliferation, has been reported as a predictor. The number of Ki-67-positive cells lies between 5% and 50% of the cells [2]. In cases of high positivity for the Ki-67 cell proliferation index, high mitotic rates are associated with adverse prognoses [23,26,27]. The expected 5-year survival rate in selected cases is close to 60%, which is lower than that for other GI B-cell NHLs [2,9,10,20,25].

Conclusions

Mantle cell lymphoma develops as a progressive disease with a relatively aggressive clinical evolution and a high response rate to multiagent chemotherapy, but the outcomes may be poor in some cases, particularly in older patients. In the GI tract, so-called multiple lymphomatous polyposis is the counterpart of peripheral mantle cell lymphoma. Mantle cell lymphoma must be considered in patients with widespread polyposis of the GI tract. Prognosis has been significantly improved since, in younger patients, intensive frontline immunochemotherapy with autologous stem cell transplantation was proposed.

Availability of data and materials

The datasets analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions JW designed and wrote the manuscript, revised the manuscript, and edited the manuscript language and style. DEG corevised the manuscript. AdVA coprepared the manuscript. CV revised the manuscript language and style. JHMB and PASC coprepared the manuscript. MIFF codesigned the manuscript, performed the technical analysis, and corevised the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Ethics approval and consent to participate

The patient involved in this study (or her legal guardian) provided written consent prior to study enrollment. The data presented are anonymized, and there is negligible risk of subject identification or of any harm to the patient or her family.

Fig. 1
1Pathological findings of mantle cell lymphoma. a Neoplastic cells involving the small intestine (hematoxylin and eosin counterstain, original magnification ×100). The cell proliferation can be nodular or diffuse, with a mixed nodular pattern. b The CD20 antigen was positive in neoplastic cells (immunoperoxidase with hematoxylin counterstain, original magnification ×40). Tumor cells are small-to medium-sized and are composed of a dense, monotonous infiltration of small to intermediate cleaved cells with small, irregularly shaped nuclei and scanty cytoplasm. c The CD79a antigen was positive in neoplastic cells (immunoperoxidase with hematoxylin counterstain, original magnification ×200). d The CD20 antigen was positive in neoplastic cells (immunoperoxidase with hematoxylin counterstain, original magnification ×400)

Abbreviations CT: Computed tomography; GI: Gastrointestinal; Hyper-CVAD: Hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, and high-dose cytarabine; MALT: Mucosaassociated lymphoid tissue; MLP: Multiple lymphomatous polyposis; NHL: Non-Hodgkin's lymphoma; R-CHOP: Combination of rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone Funding None declared.
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Heterologous expression of human costimulatory molecule B7-2 and construction of B7-2 immobilized polyhydroxyalkanoate nanoparticles for use as an immune activation agent

Ming-Chuan Li
†
Qian-Qian Liu
Xiao-Yun Lu
Ya-Li Zhang
Lei-Lei Wang
Heterologous expression of human costimulatory molecule B7-2 and construction of B7-2 immobilized polyhydroxyalkanoate nanoparticles for use as an immune activation agent
R E S E A R C H A R T I C L E Open Access
Background: Costimulation of T cells via costimulatory molecules such as B7 is important for eliciting cell-mediated antitumor immunity. Presenting costimulation molecules by immobilizing recombinant B7 on the surface of nanovectors is a novel strategy for complementary therapy. Polyhydroxyalkanoates (PHAs) are a family of biodegradable, non-toxic, biocompatible polyesters, which can be used as a nonspecific immobilizing matrix for protein presentation. Recombinant protein fusion with PHA granule binding protein phasin (PhaP) can be easily immobilized on the surface of PHA nanoparticles through hydrophobic interactions between PhaP and PHA, and therefore provides a low-cost protein presenting strategy. Results: In this study, the extracellular domain of the B7-2 molecule (also named as CD86) was fused with PhaP at its N-terminal and heterogeneously expressed in recombinant Escherichia coli strain BL21 (DE3). The purified B7-2-PhaP protein was immobilized on the surface of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)-based nanoparticles. Loading of 240 μg (3.2 pMol) of B7-2-PhaP protein per mg nanoparticles was achieved. Immobilized B7-2-PhaP on PHBHHx nanoparticles induced T cell activation and proliferation in vitro.Conclusions: A PHA nanoparticle-based B7-2 costimulation molecule-presenting system was constructed. The PHA-based B7 presenting nanosystem provided costimulation signals to induce T cell activation and expansion in vitro. The B7-2-PhaP immobilized PHA nanosystem is a novel strategy for costimulation molecule presentation and may be used for costimulatory molecule complementary therapy.

Background

It is generally accepted that the activation of naïve T cells requires two signals. One is provided by association of the T-cell receptor with the major histocompatibility complex-peptide complex. The other, known as costimulation, is necessary for optimal T-cell activation, which leads to clonal T-cell expansion, cytokine secretion and development of effector functions. Two main costimulatory molecules, B7-1 and B7-2, accomplish the coactivation function through binding with their ligand CD28 on the surface of T cells [1]. Lack of costimulation however, results in the induction of T-cell tolerance, named anergy. This is important for tumor immunity because a variety of tumors are potentially immunogenic but do not stimulate an effective anti-tumor immune response in vivo. That is partly because some tumors can deliver antigen-specific signals to T cells, but do not deliver costimulatory signals necessary for full activation of T cells [2].

Low levels of B7-1 and B7-2 have been detected on certain human cancers [3,4] and in a series of cell lines derived from human carcinomas [2,5]. Absence of costimulatory B7 molecule expression renders tumors "invisible" to the immune system. Accordingly, expression of B7 on tumor cells induced tumor rejection in a murine model, suggesting that providing extra costimulation molecules might render tumor cells capable of effective antigen presentation, leading to their eradication in vivo. Based on the safety considerations of expressing B7 using a virus vector, we propose a novel strategy to present costimulatory molecules by immobilizing recombinant B7 on the surface of polyhydroxyalkanoate (PHAs)-based nanoparticles.

PHAs are a family of biodegradable, non-toxic, biocompatibility polyesters, which are synthesized by a wide range of microorganisms [6,7], have promise as biomedical materials and have been investigated for many transplant applications [8]. Moreover, PHAs could also be used as a nonspecific immobilizing matrix for protein purification and presentation, mediated by several kinds of PHA granule associated proteins, which contain a hydrophobic granule-binding domain [9][10][11][12][13]. Among these proteins, PHA granule binding protein phasin (PhaP) has a high affinity for hydrophobic materials and has been utilized to develop a low-cost protein production system [11,12], as well as a fluorescence activated cell sorting-based diagnostic technique [13].

In this study, the extracellular domain of the B7-2 molecule was fused with PhaP at its N-terminal and the recombinant B7-2-PhaP protein was immobilized on the surface of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)-based nanoparticles. The immunocompetence of the resulting fusion protein and functionality of the PHBHHx nanoparticles were analyzed.

Results

Microbial production and purification of GST-B7-2-PhaP fusion proteins by Escherichia coli

The PhaP gene was fused with the coding sequence for the extracellular domain of the B7-2 molecule and was expressed in Escherichia coli BL21 (DE3), with a glutathione S-transferase (GST) tag at the N-terminal of the resulting fusion protein. A flexible peptide linker (GGGGSGGGSGGGS) was inserted between B7-2 and PhaP to ensure correct folding. The GST-B7-2-PhaP fusion protein was efficiently produced by recombinant E. coli BL21 (DE3) (Figure 1). However, most of the expressed intracellular fusion protein formed inclusion bodies, probably owing to the hydrophobic nature of PhaP. In order to increase the soluble proportion of the fusion protein to facilitate purification, 5% (v/v) ethanol was added to the broth during cultivation. At the same time, the process of inducing expression was undertaken at a low temperature of 20°C. The soluble crude proteins obtained from lysates of E. coli were collected by sonication and centrifugation. The GST-B7-2-PhaP fusion protein was purified by affinity chromatography using a glutathione sepharose 4B (GS4B) column. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis showed that soluble GST-B7-2-PhaP fusion protein with a molecular mass of 73 kDa was successfully produced in the recombinant E. coli BL21 (DE3) and a single band of the purified GST-B7-2-PhaP fusion protein was detected from the eluted sample ( Figure 2).

Preparation and characterization of nanoparticles

PHBHHx nanoparticles were prepared using the emulsification-solvent evaporation technique described previously [9,14]. The average diameter of prepared nanoparticles was 198 ± 5 nm with a relative low polydispersity index of 0.009. Figure 3 shows the size distribution of resulting PHBHHx nanoparticles, which formed an incipient stable colloidal suspension in aqueous medium that could be stored for at least 1 month without aggregation. The concentration of the PHBHHx nanoparticle suspension was adjusted to 5 mg ml -1 for further application.

Characterization of GST-B7-2-PhaP fusion protein immobilized nanoparticles

To characterize the nanoparticles, 250 μl of 5 mg ml -1 PHBHHx nanoparticles were incubated with an equal volume of purified GST-B7-2-PhaP fusion proteins (1-2.5 mg ml -1 ) for 16 h and then collected by centrifugation. After the immobilization process and removal of the nanoparticle proportion, no proteins were detected in the supernatant of samples with the initiation GST-B7-2-PhaP concentration of 1, 1.5 and 2 mg ml -1 . In the sample to which 2.5 mg ml -1 GST-B7-2-PhaP protein was added, 0.236 mg ml -1 protein remained in the supernatant after removing the coated nanoparticles. This indicated that approximately 400 μg of GST-B7-2-PhaP fusion protein could be immobilized on the surface of 1 mg PHBHHx nanoparticles. Western blot analysis also demonstrated that GST-B7-2-PhaP fusion proteins could be detected on the samples eluted from the nanoparticles ( Figure 4, Lane 3, 4). A slight amount of protein could still be detected in the supernatant when the initial protein concentration was 2.5 mg ml -1 (Figure 4, Lane 6). However, when the protein concentration of the initial sample was 2 mg ml -1 , all GST-B7-2-PhaP fusion proteins could be immobilized on the surface of PHBHHx nanoparticles and no protein was detected in the supernatant after incubation with PHBHHx nanoparticles (Figure 4, Lane 5). This demonstrated that the highest achievable amount of immobilized GST-B7-2-PhaP fusion protein on the PHBHHx nanoparticles could be as high as 400 μg per 1 mg nanoparticles.

Proliferation of human T cells stimulated with B7-2-PhaP-PHBHHx nanoparticles

Human lymphocytes were isolated from peripheral blood donated by a healthy volunteer. The T cell population was expanded by phytohemagglutinin stimulation. The assembled GST-B7-2-PhaP-PHBHHx nanoparticles were digested with thrombin to remove the GST tag. The immunocompetence of B7-2-PhaP-PHBHHx nanoparticles was evaluated by T cell proliferation assay. Figure 5 shows the relative proliferation of human T cells under different treatments.

Following in vitro phytohemagglutinin (5 μg ml -1 ) stimulation, activated T cells proliferated. T cells treated with anti-CD3 antibody (5 ng ml -1 ) alone received only the primary signal for T cell activation, and thus did not become activated or proliferate. However, addition of either immobilized B7-2-PhaP (1 μg ml -1 ) or free B7-2-PhaP protein led to significant cell expansion. The B7-2-PhaP loaded PHBHHx nanoparticles showed a higher activity for stimulated cell expansion than nonimmobilized free B7-2-PhaP protein. PHBHHx nanoparticle treatment also induced the stimulation of cell proliferation. Immobilized B7-2-PhaP on PHBHHx   nanoparticles together with anti-CD3 antibody demonstrated the highest activity for the stimulation of cell proliferation. These results indicated that PHBHHxbased B7-2-PhaP presenting nanoparticles can be used to activate T cells.

Discussion

PHAs are linear biopolyesters produced as energy-and carbon-storage materials by many bacteria. The medical applications of PHA have been extensively explored in recent years for implant biomedical applications including sutures, nerve conduits, patches, slings, cardiovascular patches, stents, guided tissue repair/regeneration devices, bone marrow scaffolds and so on. PHA nanoparticles have also been developed as controlled drug-release vectors owing to their biocompatibility and biodegradability.

The surface of PHA-based nanoparticles can be modified by many different types of signaling molecules and thus can be used for purposes other than targeted delivery. This can also be achieved by engineering proteins other than PhaP such as other PHA surface associated proteins including PhaC, PhaR, and PhaZ. These proteins provide targets for displaying various molecules on polymer nanoparticle surfaces for protein immobilization and display. The phasin PhaP is a small amphiphilic protein that can associate with most hydrophobic polymer granule surfaces via strong hydrophobic interactions. PhaP is abundant on the surface of natural PHA granules, and can reach approximately 5% (wt/wt) of the total cell protein in PHA-accumulating bacteria. Thus, multifunctional polymer nanoparticles can be obtained through protein engineering of PhaP and applied to a variety of versatile situations. The functional fusion proteins were able to produce easily by recombinant bacteria cultivation. This method provides a simple and convenient way to immobilize and present proteins on the surface of hydrophobic biomaterials without complicated procedures to modify both proteins and materials.

Human B7-2 is a 329-amino-acid (aa) protein containing a putative 23-aa signal peptide, a 224-aa extracellular domain, a 21-aa transmembrane domain, and a 61-aa cytoplasmic domain. The absence of the signal peptide, the transmembrane domain, and the cytoplasmic domain did not affect the costimulatory activity of B7 molecules. In this study, the 224-aa extracellular domain was subcloned and fused with PhaP and immobilized on the surface of PHBHHx nanoparticles through the hydrophobic interactions between PhaP and polymer. As measured by western blot, 400 μg of GST-B7-2-PhaP fusion proteins could be immobilized on the surface of 1 mg PHBHHx nanoparticles, which by extrapolation implies 240 μg (3.2 pMol) B7-2-PhaP fusion protein was present on the surface of 1 mg of nanoparticles. When 1 μg ml -1 immobilized B7-2-PhaP proteins was applied to cultured cells, the amount of nanoparticles used was approximately 4.2 μg ml -1 . Previously we Figure 5 Relative proliferation ratio of human peripheral blood lymphocytes by B7-2-PhaP immobilized PHBHHx nanoparticle treatment. Lymphocytes were seeded in triplicate at 1 × 10 4 cells per well in 96-well culture plates, followed by incubation with the indicated treatments for 72 h. 1: control cells; 2: cells treated with 5 μg ml -1 phytohemagglutinin; 3: cells treated with 500 ng ml -1 anti-CD3 antibody; 4: cells treated with 5 μg ml -1 PHBHHx nanoparticles; 5: cells treated with 1 μg ml -1 B7-2-PhaP fusion protein loaded PHBHHx nanoparticles; 6: cells treated with 1 μg ml -1 B7-2-PhaP fusion protein loaded PHBHHx nanoparticles plus 500 ng ml -1 anti-CD3 antibody; 7: cells treated with 1 μg ml -1 non-immobilized B7-2-PhaP fusion protein; 8: cells treated with 1 μg ml -1 non-immobilized B7-2-PhaP fusion protein plus 500 ng ml -1 anti-CD3 antibody. The cell proliferation was analyzed by MTT assay and the relative proliferation ratio was calculated relative to the cell proliferation level of the control group, which was defined as 100%.

demonstrated that 5 μg ml -1 PHBHHx nanoparticles could enhance the cell viability of rodent lymphocytes from spleen. Interestingly, the main degradation product of the PHBHHx polymer, 3-hydroxybutyrate, can promote interleukin-2 and interferon-γ secretion by rodent T lymphocytes (data not published). This may explain the slight T cell proliferation resulting from the noncoated PHBHHx nanoparticle treatment in this study.

Phytohemagglutinin is a lectin found in plants, and is used as a mitogen to trigger T lymphocyte cell division. Isolated lymphocytes were treated with 5 μg ml -1 phytohemagglutinin to increase cell numbers prior to the proliferation assay. Thus, the expanded cells were mainly T cells, although small numbers of B cells and other monocytes remained in the culture. This may explain why non-immobilized B7-2-PhaP fusion proteins also resulted in cell proliferation. PhaP is a bacterial protein that might be recognized by B cells, thus triggering an immune response resulting in lymphocyte proliferation. However, whether the B7-2-PhaP fusion protein was immobilized or not, the combined treatment of B7-2-PhaP protein and anti-CD3 antibody achieved the highest proliferation ratio and number of activated T cells. Compared with the non-immobilized free B7-2-PhaP protein, the B7-2-PhaP protein loaded nanoparticles showed a higher activity for cell proliferation stimulation. This can be explained by the advantage of the immobilization system, which can be used to provide several signal molecules that interact concurrently with ligands on cell surfaces.

Conclusions

In this study, a novel PHA nanoparticle-based B7 costimulation molecule-presenting system was developed. Both the vehicle and the presented functional protein were produced by microorganism fermentation and purified from the culture broth. The extracellular domain of the B7-2 molecule was fused with PhaP, and the B7-2-PhaP protein was hetero-expressed by recombinant E. coli BL21 (DE3). The purified B7-2-PhaP protein was immobilized on the surface of PHBHHx nanoparticles through the hydrophobic interaction between PhaP and polymer. The immunocompetence study showed that the PHA-based B7 presenting nanosystem could provide costimulatory signals for T cell activation, leading to T cell expansion in vitro. The B7-2-PhaP immobilized PHA nanosystem provides a novel strategy for the presentation of costimulatory molecules and may be a useful tool for costimulation molecule complementary therapy.

Methods

Plasmid construction

The DNA sequence coding for the extracellular domain of human B7-2 molecule (52-672bp) was subcloned from the plasmid B7-2(IgV + C)/TEasy, which contains the cDNA sequence of B7-2. The gene encoding PhaP of Ralstonia eutropha H16 was subcloned from the plasmid pTwin2-EPhaP (donated by the Laboratory of Microbiology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing, China). The primers B1 (5'-GCGGATCCATGGCTCCTCTGAAGATTC-3') (the double line marks the BamHI restriction site) and B2 (5'-ACCAGAGCCACCTCCTGAACCGCCTCCACCTG-TAATCCAAGGAATG-3') were used to amplify extracellular domain of B7-2 coding sequence. The primers P1 (5'-GGTTCAGGAGGTGGCTCTGGTGGATCGCTC ACCCCGGAACAAGTT-3') and P2 (5'-CGCTCGAGA GCCGTCGTCTTCTTTGCCGT-3') (the double line marks the XhoI restriction site) were used to amplify the PhaP gene. The PCR products of B7-2 and PhaP were mixed at the ratio of 1:1 and fusion with each other by overlap extension PCR. Then BamHI and XhoI digested B7-2-phaP segment was inserted into the corresponding sites downstream of the GST tag coding sequence of pGEX-4T-3 expression vector to generate the final plasmid pGEX-B72P ( Figure 6).

Cultivation of strains and expression of recombinant protein

E. coli strain BL21 (DE3) was used to express GST-B7-2-PhaP fusion proteins. E. coli BL21 (DE3) transformants carrying the pGEX-B72P plasmid was cultured at 37°C in 500 ml shake flasks containing 200 ml Luria Bertani medium (1% w/v Bacto tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with 100 mg ml -1 ampicillin and 3% (v/v) ethanol in a shaking incubator at 200 rpm. At an OD 600 of 0.4-0.6, 0.1 mM isopropyl β-Dthiogalactopyranoside was added to induce the expression of the fusion gene and the recombinant E. coli BL21 (DE3) carrying pGEX-B72P was continued in culture at 20°C for another 6 hours.

Recombinant protein purification and characterization

Cells were harvested by centrifugation at 6000 × g for 10 min and re-suspended in 20 ml phosphate-buffered saline (PBS) buffer followed by sonication (Ultrasonic crasher, Scientz-II D, Ningbo, China) for 10 min at 40% output. After centrifugation at 12000 × g for 30 min at 4°C, the clarified soluble protein fraction was collected. The target recombinant GST-B7-2-PhaP fusion protein was purified by GS4B affinity chromatography (GE Healthcare, USA) according to the manufacturer's protocol.

The eluted protein was purified and condensed by the dialysis bag method. Sample collected after purification was analyzed by 12% SDS-PAGE. The purified recombinant protein was stored at −20°C after lyophilization for further usage.

Preparation of PHBHHx nanoparticles

The PHBHHx material was produced, isolated and characterized as described previously [15,16]. The PHBHHx nanoparticles were fabricated by a modified emulsification/solvent diffusion method [17,18]. Briefly, 20 mg of PHBHHx polymer was added into 1 ml chloroform. Five milliliters of 1% poly(vinyl alcohol) (PVA) (w/v) was sonicated for 1 min and was slowly added to 1 ml of organic solution. The double emulsion was then sonicated using a probe sonicator (Sonics & Materials, Newtown, CT, USA) for 5 min and the mixed solution was moderately stirred with a magnetic mixer for 6 h to solidify the nanodroplets. Chloroform was removed by volatilization at room temperature. The nanoparticles were collected by centrifugation at 38000 rpm for 60 min, followed by washing twice with deionized water. The particle size was analyzed by a laser light scattering machine (Zetasizer Nano ZS, Malvern, UK).

Immobilization and characterization of fusion proteins on nanoparticles

Fusion protein GST-B7-2-PhaP was dissolved in PBS at a final concentration of 1-2.5 mg ml -1 . A concentration of 5 mg ml -1 of PHBHHx nanoparticles was mixed with various amounts of purified GST-B7-2-PhaP fusion proteins, followed by stirring and incubation at 4°C for 12-16 h. Subsequently, the colloidal system was collected by centrifugation at 12,000 × g for 30 min and washed twice with PBS. The immobilized GST-B7-2-PhaP fusion protein was calculated according to the difference between the amounts of protein in the initial sample and that in the supernatant after incubation, and was quantified using the bicinchoninic acid method using a protein assay kit Beyotime (#P0010, Beyotime Institute of Biotechnology, China) according to the manufacturer's protocol.

The assembled GST-B7-2-PhaP-PHBHHx nanoparticles were resuspended in protein loading buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 1% β-mercaptoethanol) and boiled for 2 min to allow detachment of denatured GST-B7-2-PhaP fusion protein from PHBHHx nanoparticles. After 12,000 × g centrifugation for 10 min, the supernatant was analyzed by SDS-PAGE and western blot to verify the immobilization of GST-B7-2-PhaP on the surface of PHBHHx nanoparticles. Briefly, proteins were transferred to poly (vinylidene fluoride) (PVDF) membranes after electrophoresis, and were then blocked for 1 h at room temperature with 5% bovine serum albumin in TBST buffer. PVDF membranes were incubated overnight with an anti-GST antibody (CW0084, Beijing CWBio Co., Ltd., China). The PVDF membrane was subsequently hybridized with secondary horseradish peroxidase-conjugated goat anti-mouse IgG (CW0084, Beijing CWBio Co., Ltd., China) and the recombinant protein was detected by incubating with a chemiluminescence Figure 6 Plasmid map of pGEX-B72-P. pGEX-B72-P was derived from plasmid pGEX-4T-3 which contains the pBR322 origin. B7-2: coding sequence for 224-aa extracellular domain of human B7-2 costimulatory molecule; phaP: coding sequence of PHA associated protein phasin (PhaP); Amp: ampicillin resistance gene; Lac I: coding sequence of Lac operon DNA-binding transcriptional repressor. solution purchased from Thermo Scientific (#34079, Thermo Scientific Inc., Bremen, Germany) according to the manufacturer's instructions.

Lymphocyte separation, cell culture and proliferation assay Five milliliters of whole blood was donated by a healthy volunteer. Lymphocytes were separated from noncoagulated whole blood using a human lymphocyte separation medium according to the manufacturer's instructions (PAN Biotech, Aidenbach, Germany). The isolated cells were suspended in a culture bottle with high-glucose RPMI1640 medium (Gibco, UK) supplemented with 10% fetal bovine serum (Gibco, UK) and maintained at 37°C in a humidified 5% CO 2 atmosphere overnight. After disposal of adherent cells, the suspended cells were recovered and treated with 5 μg ml -1 phytohemagglutinin for 72 h to expand the T cell population. For the T cell proliferation assay, cells were passaged and seeded in triplicate at 1 × 10 4 cells per well in 96-well culture plates, followed by incubation with the corresponding treatment for another 72 h. The cell proliferation was analyzed by MTT assay. Cells treated with 5 μg ml -1 phytohemagglutinin served as the positive control. T-cell surface glycoprotein CD3 antibody (500 ng ml -1 ) was used to provide the first signal for T cell activation.

Statistical analysis

All results were expressed as mean ± standard deviation of experiments performed in triplicate. Statistical differences were evaluated using a one-way analysis of variance with a Student's t-test. Differences were considered to be statistically significant when P < 0.05.

Figure 1
1Coomassie blue-stained SDS-PAGE of heteroexpressed GST-B7-2-PhaP fusion protein by recombinant E. coli BL21 (DE3) (pGEX-B72P). Lane 1: whole crude lysate of E. coli BL21 (DE3) transformed with pGEX-4T-3 plasmid. Lane 2: whole crude lysate of E. coli BL21 (DE3) transformed with pGEX-B72P plasmid. Protein weight marker (Institute of Biochemistry and Cell Biology, SIBS, CAS, China) was loaded in lane M.

Figure 2
2Coomassie blue-stained SDS-PAGE of soluble GST-B7-2-PhaP fusion protein. Lane 1: the soluble fraction was recovered after centrifugation of whole crude lysate of E. coli BL21 (DE3) transformed with pGEX-B72P plasmid. Lane 2: purified soluble GST-B7-2-PhaP fusion protein. Protein weight marker (Institute of Biochemistry and Cell Biology, SIBS, CAS, China) was loaded in lane M.

Figure 3
3Size distribution of PHBHHx nanoparticles determined by dynamic light scattering.

Figure 4
4Western blot analysis of GST-B7-2-PhaP fusion protein immobilized on the surface of PHBHHx nanoparticles. Lanes 1-2: the original GST-B7-2-PhaP protein samples with an initial concentration of 2 and 2.5 mg ml -1 , respectively. Lanes 3-4: GST-B7-2-PhaP protein eluted from the surface of PHBHHx nanoparticles after the immobilization procedure. Lanes 5-6: the remnant GST-B7-2-PhaP proteins remaining in the supernatant after the immobilization procedure. The concentration of the initial protein sample of Lanes 3 and 5 was 2 mg ml -1 , and that of Lanes 4 and 6 was 2.5 mg ml -1 . Protein weight marker (Institute of Biochemistry and Cell Biology, SIBS, CAS, China) was loaded in lane M, which was not detected by western blot analysis.
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AcknowledgmentsThis work has been supported by grants from the National Natural Science Foundation of China (30801059, 81172170) and the Fundamental Research Funds for the Central Universities of Xi'an Jiaotong University.Competing interestsThe authors declare that they have no competing interests.
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