Response In Murine Model Research Articles

Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4(+) and CD8(+) immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207(+), CD11c(+), CD4(+), CD8(+), iNOS(+) cellular densities. Cytokine (IFN-γ, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207(+) and CD11c(+) were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4(+) and CD8(+) were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS(+) was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-γ was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

Myeloid suppressor cell depletion augments antitumor activity in lung cancer.

BackgroundMyeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer.Principal FindingsIndividual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell IL-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had 20 fold reductions in tumor burden compared to controls.SignificanceOur data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.

Abstract PL03-03: The rational combination of BRAF inhibition with immunotherapy for the treatment of metastatic melanoma

Abstract Patients with metastatic melanoma have a median survival of less than one year and new treatments are clearly needed. Targeted therapy with BRAF inhibitors (BRAFi) in patients whose tumors contain mutated BRAF has been promising with response rates of over 50%. However, the responses are transient with the median duration of response under 7 months. In contrast, immunotherapies such as interleukin-2 or anti-CTLA-4 can induce long term survival in some patients, but the overall response rates are low. The combination of targeted therapies with immunotherapies has the potential to result in higher, more durable clinical responses and improved survival in patients with metastatic cancer. Besides inducing the release of antigens following tumor destruction, targeted therapies may also reverse the immunosuppressive microenvironment found at the tumor site. Rationally combining immunotherapy with targeted therapy requires in-depth knowledge of the effects of the targeted therapies on immune effector cells and the immune tumor microenvironment. We have taken 3 appoaches in defining the interaction of mutated BRAF and BRAF inhibitors with the immune system by studying: 1) the direct effects of mutated BRAF on the tumor immune microenvironment 2) the in vivo consequences of BRAF inhibition on anti-tumor T-cell responses in murine models and 3) T-cell function in patients receiving BRAF inhibitors. To determine the role of mutated BRAF on the tumor microenvironment, we performed RNA analysis of melanocytes expressing control, wild type BRAF or mutated BRAF genes. This revealed the preferential upregulation of several immune molecules in cells expressing mutated BRAF, including IL-1. In turn, we found that IL-1 could induce the expression of immune inhibitory molecules on tumor-associated fibroblasts and myeloid cells such as PDL1 (program death ligand-1). Furthermore, fibroblasts exposed to IL-1 were capable of inhibiting T-cell function in vitro. Inhibition of the MAPK pathway using a BRAF-specific inhibitor resulted in the reversal of these immune inhibitory effects. We next evaluated the effects of BRAF inhibition on T-cell migration to the tumor site. Mice bearing a BRAF mutated tumor were treated with antigen-specific T-cells, a BRAF inhibitor or a combination of the two agents. Tumor regression was highest in mice receiving both T-cells and the BRAF inhibitor. T-cells were monitored in vivo using a sensitive modified luciferase gene, and were found to migrate more efficiently to the tumor following addition of the BRAF inhibitor. This was partially due to the downregulation of VEGF expression by BRAF inhibition, since tumors which constitutively expressed VEGF driven by a viral promoter failed to demonstrate enhanced T-cell migration following addition of the BRAF inhibitor in vivo. In addition, anti-VEGF therapy also resulted in enhanced migration of T-cells to the tumor site. We are currently investigating the mechanisms by which VEGF inhibits T-cell migration to tumor. Finally, we have investigated the effects of BRAF inhibitors on T-cell function in patients. Peripheral blood mononuclear cells were isolated from patients before and after initiation of therapy with a BRAF inhibitor. Functional T-cell assays were performed to assess the amount of interferon gamma produced following stimulation with class I and class II restricted pools of peptides from recall antigens, in order to assess the function of CD8+ and CD4+ T-cells, respectively. We found that therapy with the BRAF inhibitor did not adversely affect T-cell function in these patients with metastatic melanoma. Luminex assays were performed from sera following therapy with the BRAF inhibitor to evaluate the expression of multiple cytokines. No changes in circulating cytokines were observed following therapy with BRAFi, except for a slight elevation in circulating TNF-alpha levels. In summary, we have found that: 1) mutated BRAF induces an immunosuppressive tumor microenvironment which can be reversed by BRAFi; 2) BRAFi enhances the migration of T-cells to tumor; and 3) therapy with BRAFi does not inhibit immune function in patients with metastatic melanoma. This work further underscores the potential synergy in combining BRAFi with immunotherapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr PL03-03. doi:1538-7445.AM2012-PL03-03

Human TH2 cells respond to cysteinyl leukotrienes through selective expression of cysteinyl leukotriene receptor 1

Allergic asthma is characterized by reversible airway obstruction and bronchial hyperresponsiveness associated with T(H)2 cell-mediated inflammation. Cysteinyl leukotrienes (CysLTs) are potent lipid mediators involved in bronchoconstriction, mucus secretion, and cell trafficking in asthmatic patients. Recent data have implicated CysLTs in the establishment and amplification of T(H)2 responses in murine models, although the precise mechanisms are unresolved. Preliminary microarray studies suggested that human T(H)2 cells might selectively express cysteinyl leukotriene receptor 1 (CYSLTR1) mRNA. We sought to establish whether human T(H)2 cells are indeed a CysLT target cell type. We examined the expression of CYSLTR1 using real-time PCR in human T(H)1 and T(H)2 cells. We functionally assessed cysteinyl leukotriene receptor 1 protein (CysLT(1)) expression using calcium flux, cyclic AMP, and chemotaxis assays. We show that human T(H)2 cells selectively express CYSLTR1 mRNA at high levels compared with T(H)1 cells after invitro differentiation from naive precursors. Human T(H)2 cells are selectively responsive to CysLTs in a calcium flux assay when compared with T(H)1 cells with a rank order of potency similar to that described for CysLT(1) (leukotriene [LT] D(4) > LTC(4) > LTE(4)). We also show that LTD(4)-induced signaling in T(H)2 cells is mediated through CysLT(1) coupled to G(α)q and G(α)i proteins, and both pathways can be completely inhibited by selective CysLT(1) antagonists. LTD(4) is also found to possess potent chemotactic activity for T(H)2 cells at low nanomolar concentrations. These findings suggest a novel mechanism of action for CysLTs in the pathogenesis of asthma and provide a potential explanation for the anti-inflammatory effects of CysLT(1) antagonists.

A Novel Synbiotic Concept Derived From Cow's Milk-free Source Materials Beneficially Affects In Vivo Immune Responses In Murine Models For Influenza Vaccination And Cow'S Milk Allergy

A Novel Synbiotic Concept Derived From Cow's Milk-free Source Materials Beneficially Affects In Vivo Immune Responses In Murine Models For Influenza Vaccination And Cow'S Milk Allergy

Peripheral blood gene expression profiles in metabolic syndrome, coronary artery disease and type 2 diabetes

To determine if individuals with metabolic disorders possess unique gene expression profiles, we compared transcript levels in peripheral blood from patients with coronary artery disease, type 2 diabetes and their precursor state, metabolic syndrome to those of control subjects and subjects with rheumatoid arthritis. The gene expression profile of each metabolic state was distinguishable from controls and correlated with other metabolic states more than with rheumatoid arthritis. Of note, subjects in the metabolic cohorts over-expressed gene sets that participate in the innate immune response. Genes involved in activation of the pro-inflammatory transcription factor, NF-κB, were over-expressed in coronary artery disease while genes differentially expressed in type 2 diabetes play key roles in T cell activation and signaling. RT-PCR validation confirmed microarray results. Furthermore, several genes differentially expressed in human metabolic disorders have been previously shown to participate in inflammatory responses in murine models of obesity and Type 2 diabetes. Taken together, these data demonstrate that peripheral blood from individuals with metabolic disorders display overlapping and non-overlapping patterns of gene expression indicative of unique, underlying immune processes.

Honokiol rescues sepsis-associated acute lung injury and lethality via the inhibition of oxidative stress and inflammation

Sepsis has a high mortality rate despite the recent advances in intensive care medicine and antibiotics. Honokiol, a low molecular weight natural product, is known to possess anti-inflammatory activity. Here, we investigate whether honokiol can ameliorate acute lung injury and lethal response in murine models of sepsis. Mice were intraperitoneally given vehicle or honokiol 30 min after the induction of sepsis by cecal ligation and puncture (CLP) and endotoxemia by administration of E. coli lipopolysaccharide (LPS). The productions of serum tumor necrosis factor-α (TNF-α), nitric oxide (NO), and high mobility group box 1 (HMGB 1) were increased in mice during sepsis, which could be reversed by honokiol. Honokiol could also effectively reduce the increased blood lipid peroxidation and nitrotyrosine in septic mice. Honokiol significantly reversed the inductions of inducible NO synthase and nuclear factor-κB (NF-κB) activation in the lungs of mice during sepsis. Honokiol also effectively rescued the lung edema, lung pathological changes, and lethality in septic mice. These findings suggest that honokiol is capable of suppressing the lethal response and acute lung injury associated with sepsis, and support the potential use of honokiol as a therapeutic agent for the conditions associated with septic shock.

Airway epithelium mediates the anti-inflammatory effects of exercise on asthma

Airway epithelium plays an important role in the asthma physiopathology. Aerobic exercise decreases Th2 response in murine models of allergic asthma, but its effects on the structure and activation of airway epithelium in asthma are unknown. BALB/c mice were divided into control, aerobic exercise, ovalbumin-sensitized and ovalbumin-sensitized plus aerobic exercise groups. Ovalbumin sensitization occurred on days 0, 14, 28, 42, and aerosol challenge from day 21 to day 50. Aerobic exercise started on day 22 and ended on day 50. Total cells and eosinophils were reduced in ovalbumin-sensitized group submitted to aerobic exercise. Aerobic exercise also reduced the oxidative and nitrosative stress and the epithelial expression of Th2 cytokines, chemokines, adhesion molecules, growth factors and NF-kB and P2X7 receptor. Additionally, aerobic exercise increased the epithelial expression of IL-10 in non-sensitized and sensitized animals. These findings contribute to the understanding of the beneficial effects of aerobic exercise for chronic allergic airway inflammation, suggesting an immune-regulatory role of exercise on airway epithelium.

GMCSF-Interleukin fusion cytokines induce novel immune effectors that can serve as biopharmaceuticals for treatment of autoimmunity and cancer

We created the GIFTs, fusions of granulocyte-colony macrophage-stimulating-factor with IL-2, or IL-15 or IL-21, in order to stimulate distinct, but complimentary elements of the immune response. We found that the physical coupling of two functionally distinct cytokines as a bifunctional hybrid allowed for synergistic bioactivity not seen by the simple combined use of parent components. Indeed, despite how these interleukins are pro-inflammatory cytokines that serve essential roles in the maturation of CD8(+) T cells and NK cells, the GIFTs were remarkably different from one another, with GIFT-2 and GIFT-21 promoting and GIFT-15 downregulating inflammation. The common denominator to the biochemistry of these fusokines was their ability to hijack the signalling machinery associated with common to their respective γ-chain interleukin receptors, radically altering the activation status of responding lymphomyeloid cells. By studying the GIFTs, we found that both secreted and cell surface factors presented by GIFT-activated lymphomyeloid cells were required to modulate the immune responses in murine models of multiple sclerosis and cancer. The ability of GIFTs to co-opt the normal signalling machinery of interleukin receptors leads to the acquisition of functional responder cell phenotypes unparalleled in nature. These novel properties provide opportunities to alter maladapted immune responses in health and disease.

Identification of immunodominant linear B-cell epitopes within the major outer membrane protein of <italic>Chlamydia trachomatis</italic>

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. Chlamydial major outer membrane protein (MOMP) can induce strong cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of MOMP was analyzed using computer-assisted techniques to scan B-cell epitopes, and three possible linear B-cell epitopes peptides (VLKTDVNKE, TKDASIDYHE, TRLIDERAAH) with high predicted antigenicity and high conservation were investigated. The DNA coding region for each potential epitope was cloned into pET32a(+) and expressed as Trx-His-tag fusion proteins in Escherichia coli. The fusion proteins were purified by Ni-NTA agarose beads and followed by SDS-PAGE and western blot analysis. We immunized mice with these three fusion proteins. The sera containing anti-epitope antibodies from the immunized mice could recognize C. trachomatis serovars D and E in ELISA. Antisera of these fusion proteins displayed an inhibitory effect on invasion of serovar E by in vitro neutralization assays. In addition, serum samples from convalescent C. trachomatis-infected patients were reactive with the epitope fusion proteins by western blot assay. Our results showed that the epitope sequences selected by bioinformatic analysis are highly conserved C. trachomatis MOMP B-cell epitopes, and could be good candidates for the development of subunit vaccines, which can be used in clinical diagnosis.

Polymorphisms in genes of interleukin 12 and its receptors and their association with protection against severe malarial anaemia in children in western Kenya

BackgroundMalarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined.MethodsFifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya.ResultsAn increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21).ConclusionsThis study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients.

818 HUMAN GENE POLYMORPHISM AND RESPONSE TO BACILLUS CALMETTE-GUERIN IMMUNOTHERAPY FOR SUPERFICIAL BLADDER CANCER

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore C61 Objectives: Superficial bladder cancers (SBC) have a 60-70% recurrence rate after transurethral resection, and 10-20% of recurrences progress to invasive cancer. Post-transurethral resection intravesical Bacillus Calmette-Guerin (BCG) instillation has been the ‘gold-standard’ treatment for high risk SBC, for which 20-40% of patients do not respond. There are also frequent, potentially serious side effects to BCG therapy. Although clinical and pathologic variables to predict BCG response and cancer recurrence have been extensively studied, little is known of the role of underlying host genetic susceptibility factors, such as genetic polymorphisms. The NRAMP1 gene has been implicated in susceptibility to tuberculosis and to BCG response in murine models. The Human glutathione peroxidase 1 (hGPX1) is a selenium-dependent enzyme that participates in detoxification of hydrogen peroxide, organic peroxides, and possibly cigarette smoke-derived oxidative radicals. An association between hGPX1 and NRAMP1, and bladder cancer recurrence has been suggested. We aim to determine the predictive value of NRAMP1 and hGPX1 gene polymorphisms in superficial bladder cancer recurrence and response to BCG therapy. Methods: Peripheral blood DNA was prospectively obtained from 99 high risk superficial bladder cancer patients, who underwent post-resection intravesical regimes of BCG (81mg, n=50 or 27mg, n=19) or BCG (27mg) with interferon alpha (IFNa) (n=30), and followed-up for a mean of 4.5 years. The (GT)n and D534N polymorphisms in the NRAMP1 gene, and the Pro198Leu polymorphism in the hGPX1 gene were tested with restriction fragment length polymorphisms and DNA sequencing following PCR amplification. Data was analyzed using Chi-square analysis, multiple logistic regression and Kaplan-Meier curves. Results: The D534N G:A genotype was found to be protective towards cancer recurrence (p=0.016) and cancer-specific death (p=0.036). The presence of allele 3 in the (GT)n gene polymorphism was found to correlate with higher recurrence in BCG treated patients (p=0.003), and had shorter recurrence-free survival (p=0.024) and progression-free survival (p=0.005) in patients treated with a combination of BCG and IFNa. The hGPX1 CC genotype was shown to be protective (p=0.014), while the variant CT genotype (Pro/Leu) was associated with increased bladder cancer recurrence and decreased time to recurrence (p=0.03) after BCG therapy. Conclusion: Our findings suggest that polymorpisms in the NRAMP1 and hGPX1 genes correlate with response to BCG therapy in bladder cancer patients. They may serve as molecular markers to predict BCG failure and cancer recurrence.

MP-13.01: Human Gene Polymorphism and Response to Bacillus Calmette-Guerin Immunotherapy for Superficial Bladder Cancer

The NRAMP1 gene has been implicated in susceptibility to tuberculosis and to BCG response in murine models. The Human glutathione peroxidase 1 (hGPX1) is a selenium-dependent enzyme that participates in detoxification of hydrogen peroxide, organic peroxides, and possibly cigarette smoke-derived oxidative radicals. An association between hGPX1 and NRAMP1, and bladder cancer recurrence has been suggested. We aim to determine the predictive value of NRAMP1 and hGPX1 gene polymorphisms in superficial bladder cancer recurrence and response to BCG therapy.

CD137, implications in immunity and potential for therapy

CD137 is a member of the TNF receptor family and a potent T cell costimulatory molecule. Crosslinking of CD137 on activated T cells has shown promise in enhancing anti-tumor immune responses in murine models, and agonistic anti-CD137 antibodies are currently being tested in phase I clinical trials. Surprisingly, these very same agonistic anti-CD137 antibodies have also been found to ameliorate autoimmune disease under certain circumstances. At the current state of knowledge these circumstances cannot be clearly defined. Therefore, anti-CD137 antibodies in man will need to be used with caution. CD137 ligand is expressed by antigen presenting cells. Antagonistic anti-CD137 ligand antibodies have shown efficacy in dampening disease in murine autoimmune models. A similar effect would be expected from antagonistic anti-CD137 antibodies, soluble CD137, or any other compound interfering with CD137 / CD137 ligand interaction. CD137 ligand is expressed as a transmembrane protein on the cell surface and it too can transmit signals into antigen presenting cells. Agonistic anti-CD137 ligand antibodies or a recombinant CD137 protein could stimulate the activity of antigen presenting cells.

Helminth Products as a Potential Therapeutic Strategy for Inflammatory Diseases

Helminths secrete several molecules that can modulate the immune responses, favoring their evasion and perpetuate their survival in the host. These molecules interfere with antigen presentation, cell proliferation and activation, antibody production, cause cell death, and stimulate regulatory responses. Here, we focus on some helminth products and address their immunomodulatory effects in the host immune system and, also, we describe some anti-inflammatory properties of an Ascaris suum-derived immunomodulatory molecule, named PAS-1. This protein is a 200-kDa molecule isolated by affinity chromatography using MAIP-1 (monoclonal antibody which recognizes PAS-1), coupled to Sepharose 4B. It suppresses the inflammatory responses in murine models of delayed-type hypersensitivity, lung allergic inflammation and LPS-induced inflammation into air pouches. PAS-1 also stimulates the secretion of regulatory cytokines such as IL-10 and TGF-beta and primes IFN-gamma-secreting CD8+ and IL-10/ TGF-beta-secreting CD4+CD25+ cell clones that avoid the lung inflammation. Thus, this protein is a potent immunomodulatory component that may be used for therapeutic interventions in inflammatory diseases.

HUMAN GENE POLYMORPHISM AND RESPONSE TO BACILLUS CALMETTE-GUERIN IMMUNOTHERAPY FOR SUPERFICIAL BLADDER CANCER

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore C61 Objectives: Superficial bladder cancers (SBC) have a 60-70% recurrence rate after transurethral resection, and 10-20% of recurrences progress to invasive cancer. Post-transurethral resection intravesical Bacillus Calmette-Guerin (BCG) instillation has been the ‘gold-standard’ treatment for high risk SBC, for which 20-40% of patients do not respond. There are also frequent, potentially serious side effects to BCG therapy. Although clinical and pathologic variables to predict BCG response and cancer recurrence have been extensively studied, little is known of the role of underlying host genetic susceptibility factors, such as genetic polymorphisms. The NRAMP1 gene has been implicated in susceptibility to tuberculosis and to BCG response in murine models. The Human glutathione peroxidase 1 (hGPX1) is a selenium-dependent enzyme that participates in detoxification of hydrogen peroxide, organic peroxides, and possibly cigarette smoke-derived oxidative radicals. An association between hGPX1 and NRAMP1, and bladder cancer recurrence has been suggested. We aim to determine the predictive value of NRAMP1 and hGPX1 gene polymorphisms in superficial bladder cancer recurrence and response to BCG therapy. Methods: Peripheral blood DNA was prospectively obtained from 99 high risk superficial bladder cancer patients, who underwent post-resection intravesical regimes of BCG (81mg, n=50 or 27mg, n=19) or BCG (27mg) with interferon alpha (IFNa) (n=30), and followed-up for a mean of 4.5 years. The (GT)n and D534N polymorphisms in the NRAMP1 gene, and the Pro198Leu polymorphism in the hGPX1 gene were tested with restriction fragment length polymorphisms and DNA sequencing following PCR amplification. Data was analyzed using Chi-square analysis, multiple logistic regression and Kaplan-Meier curves. Results: The D534N G:A genotype was found to be protective towards cancer recurrence (p=0.016) and cancer-specific death (p=0.036). The presence of allele 3 in the (GT)n gene polymorphism was found to correlate with higher recurrence in BCG treated patients (p=0.003), and had shorter recurrence-free survival (p=0.024) and progression-free survival (p=0.005) in patients treated with a combination of BCG and IFNa. The hGPX1 CC genotype was shown to be protective (p=0.014), while the variant CT genotype (Pro/Leu) was associated with increased bladder cancer recurrence and decreased time to recurrence (p=0.03) after BCG therapy. Conclusion: Our findings suggest that polymorpisms in the NRAMP1 and hGPX1 genes correlate with response to BCG therapy in bladder cancer patients. They may serve as molecular markers to predict BCG failure and cancer recurrence.

Suppression of colon inflammation by CD80 blockade: Evaluation in two murine models of inflammatory bowel disease

Human inflammatory bowel disease (IBD) is a chronic condition mediated by aberrant immune responses to the luminal antigens by activated CD4+ T cells. The CD80/CD86:CD28/CD152 costimulatory pathways transmit signals critical for T cell activation and suppression. Macrophages and epithelial cells are the chief antigen-presenting cells in the gut. Macrophages from the IBD colon express significantly elevated levels of CD80 and CD86 costimulatory molecules. The CD28-CD80 interaction primarily participates in breaking the tolerance and inducing the immune response in murine models of colitis. Blockade of CD80-costimulatory axis is an attractive strategy in the treatment of IBD. Incorporating the structural information of the CD80:CD152 complex together with the preferences of interface residues to form polyproline type II helix, we designed novel peptide agents that selectively blocked CD80 receptor interactions. Administration of CD80 blocking agent at the time of adoptive transfer prevented the SCID mice from CD4+CD45Rb(high) T-cell mediated colitis. Significantly, CD80-CAP (competitive antagonist peptide) treatment suppressed established inflammation in TNBS-induced colitis, a model for Th1-mediated Crohn's disease. The colons of the mice receiving the CD80 blocking agent appeared unaffected macroscopically and exhibited negligible microscopic inflammation. The CD80-CAP treatment was associated with significantly reduced Th1 cytokines in the colon. The CD80 blocking peptide appeared to mediate protection against colitis by inducing Th2 skewing of the cytokine response.

The Relevance of the Inhibitory Receptor FcγIIB for the Regulation of Anti-Factor VIII Antibody Responses in Murine Models.

The prevention of neutralizing anti-factor VIII (FVIII) antibodies remains the major challenge in the treatment of hemophilia A patients with FVIII products. Therefore, it is important to understand how B cells differentiate into antibody-producing plasma cells, how this process is regulated and how it can be inhibited. A number of inhibitory receptors have been identified that are expressed on B cells and might provide targets for the prevention of antibody development against FVIII. The inhibitory receptor FcγIIB has emerged as a key regulator of B cell responses in the later phase of antibody responses. It has been described as a potent inhibitor of B-cell-receptor (BCR) signaling when co-ligated to the BCR by engagement of antigen-containing immune complexes. Based on these findings we asked whether the inhibitory receptor FcγIIB is involved in the natural regulation of anti-FVIII antibody responses in murine models. If this receptor were an important negative regulator of anti-FVIII antibody responses, mice that do not express this receptor should develop higher titers of anti-FVIII antibodies than mice that express the receptor. We treated wildtype mice and FcγRIIB knockout mice, both on a C57BL/6J background, with four intravenous doses of 200ng of human FVIII (80U/kg) and analyzed titers of anti-FVIII antibodies after the second, the third and the fourth dose. Surprisingly, we did not see any significant difference in antibody titers between both strains of mice. We extended our study and asked under which conditions FcγRIIB knockout mice would develop higher titers of anti-FVIII antibodies than wildtype mice. We tested different application routes (i.v. and i.p.) of FVIII, compared low doses (200ng) and high doses (80μg) and combined the i.p. application with or without Freund's adjuvant. Furthermore, we included KLH-TNP (i.p. application of 100μg with Freund's adjuvant) as a positive control that was previously shown to induce different levels of antibodies in wildtype mice compared with FcγRIIB knockout mice. We found significant differences in the development of specific antibodies between both strains of mice when we used the positive control (i.p. KLH-TNP with adjuvants) and when we applied high dose FVIII (80μg) with Freund's adjuvant i.p. We did not see any differences in antibody titers between both strains of mice with any of the other treatment schedules. Our results indicate that the inhibitory receptor FcγIIB is only active as a negative regulator for antibody responses against foreign proteins in vivo when a certain threshold level of antibodies is reached in the circulation. This critical threshold level is obviously not reached when mice are treated with clinically relevant doses of FVIII. Based on our results we conclude that the inhibitory receptor FcγIIB is not involved in the regulation of antibody responses against therapeutically relevant doses of human FVIII in murine models. Whether the same situation is true for patients with hemophilia A remains to be shown. Furthermore, the relevance of the inhibitory receptor FcγIIB might differ between patients who react to the FVIII product as a foreign protein and patients who react to FVIII as an altered self protein or self protein.

The Public Health Need and Present Status of a Vaccine for the Prevention of Coccidioidomycosis

Although the epidemiology of coccidioidomycosis has been well described, there is a paucity of recent data on the public health burden associated with this disease. Accordingly, California's Inpatient Hospital Discharge Data Set from 1997 to 2002 was used to calculate the incidence of hospitalization for coccidioidomycosis by county, year, age, race, ethnicity, and gender. The overall finding that coccidioidomycosis has a significant impact in endemic areas supports the conclusion that the need for a preventive vaccine is great. Investigators of the Valley Fever Vaccine Project (VFVP) have successfully identified a number of recombinant coccidioidal protein antigens and two attenuated mutant strains that have been evaluated as vaccines, demonstrating protective responses in murine models. Efforts to select and develop a vaccine for human clinical trials are in progress.

Assessment of a chemically induced model of lung squamous cell carcinoma in mice by 18F-FDG small-animal PET

Small-animal imaging has become a relevant research field in pre-clinical oncology. In particular, metabolic information provided by small-animal positron emission tomography (PET) is very useful to closely monitor tumour growth and assess therapy response in murine models of human disease. There are various murine models for human lung adenocarcinoma, but those for squamous cell lung carcinoma, the most common form of human cancer, are lacking. To assess the feasibility of 18F-FDG small-animal PET to monitor tumour growth in a chemically induced model of squamous cell carcinoma of the lung. Nineteen NIH Swiss mice were skin painted by N-nitroso-tris-chloroethylurea (NTCU) twice a week, with a 3 day interval, for 8 months and 10 NIH Swiss mice skin painted with NTCU solvent (acetone) were used as controls. 18F-FDG PET was performed under sevofluorane anaesthesia and oxygen supplementation at 2, 4, 6 and 8 months from initial treatment. Images were assessed by visual analysis and semi-quantitatively. When a diffuse distribution of tumour was noted, the mean of the counts/pixel measured at three lung levels, corrected for the effective dose injected and for decay, was used for comparison between mutagen-painted and control mice. Pathological evaluation was carried out from the time of the first positive PET results in a subgroup of the whole population to assess correlation with PET findings. Small animal CT was performed at 8 months in another subgroup. In both terms of visual analysis and measurement of total lung activity, 18F-FDG PET at 2 and 4 months from initial treatment were comparable in mutagen-painted and controls. At 6 months, PET images showed a faint and diffuse uptake over both lung fields in mutagen-painted mice with multiple focal areas of increased tracer uptake that merged into confluent masses at 8 months and seriously subverting lung architecture on computed tomography. Total lung activity was significantly higher in mutagen-painted versus control mice at 6 (P=0.00000668) and 8 months (P=0.00000043) from initial treatment and paralleled the progressive lung involvement and histological severity. 18F-FDG PET may be useful in the assessment of this chemically induced murine model of lung squamous cells carcinoma. The total lung activity may be used as a measure of tumour metabolic activity of the tumour-bearing animals and may be useful in new drug testing studies.