Langerhans cells (LCs) serve as epidermal sentinels of the adaptive immune system. Conventional wisdom suggests that LC encounter antigen in the skin, and then migrate to the draining lymph nodes, ...where the antigen is presented to T cells thus initiating an immune response. Platelet-activation factor (PAF) is a phospholipid mediator with potent biological effects. During inflammation, PAF mediates recruitment of leukocytes to inflammatory sites. Here we tested a hypothesis that PAF induces LC migration. Applying 2,4-dinitrofluorobenzene (DNFB) to wild-type (WT) mice activated LC migration. In contrast, applying DNFB to PAF receptor (PAFR)-deficient mice or mice injected with PAF receptor antagonists failed to induce LC migration. Moreover, after FITC application the appearance of hapten-laden LCs (FITC
+
, CD11c
+
, Langerin
+
) in the lymph nodes of PAF receptor deficient mice was significantly depressed compared with that found in WT mice. LC chimerism indicates that PAF receptor on keratinocytes but not LCs is responsible for LC migration. Contrary to the diminution of LC migration in PAF receptor-deficient mice, we did not observe any difference in the migration of hapten-laden dDCs (FITC
+
, CD11c
+
, Langerin
−
) into the lymph nodes of PAF receptor-deficient mice. In addition, the contact hypersensitivity response generated in WT or PAF receptor-deficient mice was identical. Finally, dDCs, but not LCs isolated from the draining lymph nodes after hapten application activated T cell proliferation. These findings suggest that LC migration may not be responsible for the generation of CHS, and dDCs may play a more important role.
Abstract 3735
Poster Board III-671
Recent clinical trials demonstrated a promising clinical activity of the HDAC inhibitor MGCD0103 in heavily pretreated patients with relapsed HL. However, the ...mechanisms underlying MGCD0103 activity in HL remains unclear. Here we show that MGCD0103 preferentially inhibited HDACs1, 2 with no effect on HDAC6. Using three HL-derived cell lines, the IC50 for MGCD0103 after 72 hour of incubation was in the submicromolar concentrations, and was more effective than vorinostat in inducing cell death. Using gene expression profiling (GEP) studies, pathway PCR array, and western blot analysis, we identified several pathways that are modulated by MGCD0103. MGCD0103 downregulated the X-linked inhibitor of apoptosis (XIAP) protein, activated the intrinsic caspase pathway, and synergized with TRAIL agonistic antibodies. MGCD0103 downregulated CD30 mRNA and surface protein expression in a dose dependent manner, but had no significant effect on B cell-associated proteins, including CD19 and CD20, or on the costimulatory receptors CD40 and CD80. MGCD0103 induced TNF-alfa expression and secretion, which was associated with NF-kB activation, upregulation of the silencer of cytokine signaling (SOCS)-3, downregulated STAT6, and upregulation of STAT1 and 2. Selective inhibition of TNF-alfa expression by short interfering mRNA enhanced MGCD0103-induced cell death. Similarly, inhibition of NF-kB by proteasome inhibitors also potentiated the effect of MGCD0103, thus demonstrating synergy independent of HDAC6 inhibition. These findings demonstrate that MGCD0103 has a potent anti lymphoma activity by modulating the expression of a variety of survival proteins, and provides mechanistic rationale for combining class-I HDAC inhibitors with proteasome inhibitors and TRAIL.
Mamidipudi:Celgene: Employment. Heise:Celgene Corporation: Employment, Equity Ownership. Besterman:Methylgene: Employment. Martell:Methylgene: Employment. MacBeth:Celgene Corporation: Employment, Equity Ownership. Younes:MethylGene: Honoraria, Research Funding.
Abstract
HIV infection is characterized by profound changes in the B cell compartment, which includes, impaired antigen specific response and a significant loss of IgM memory B cells, amongst other ...defects. Despite HAART’s success in reducing the incidence of opportunistic infections, invasive pneumococcal disease in the HIV infected population continues to be high. PPV-23 vaccine is recommended as a prophylactic measure in the HAART treated population; however, its serological benefit is currently unclear. Although, HAART has been highly successful in reconstituting T cell function, the effect of HAART on antigen specific B cell reconstitution remains to be elucidated. We developed a novel method to identify pneumococcal polysaccharide responding B cells in circulation by conjugating vaccine serotype 14 and 23 to fluorescent molecules. Using this method, we investigated the polysaccharide responding B cells in HIV cohorts on long term HAART. Preliminary data suggests severe depletion of polysaccharide responding IgM memory B cells in HAART treated adults comparable to HAART naïve group; a striking contrast to HIV uninfected adults. This is in alignment with the diminished anti pneumococcal antibody response observed in the HAART treated group. HAART is thus unable to fully reconstitute the functional B cell subset required for protection against S.pneumoniae.
The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide ...(PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.
Pneumococcal polysaccharide vaccines have been used to elicit a protective anti-pneumococcal polysaccharide antibody response against Streptococcus pneumoniae in healthy individuals. Identifying ...human B cells which respond to T-cell independent type-2 antigens, such as pneumococcal polysaccharides, has been challenging. We employed pneumococcal polysaccharides directly conjugated to fluorophores in conjunction with flow cytometry to identify the phenotype of B cells that respond to pneumococcal polysaccharide vaccination. We have previously identified that the majority of pneumococcal polysaccharide-selected cells responding to vaccination are CD27+IgM+ (IgM+ memory) cells. In this study, we further characterized pneumococcal polysaccharide-selected cells in the peripheral blood to better identify how the various B cell phenotypes responded 7 and 30 days post-immunization. We show that 7 days post-immunization the majority of pneumococcal polysaccharide-selected IgM+ memory cells (PPS14+ 56.5%, PPS23F+ 63.8%) were CD19+CD20+CD27+IgM+CD43+CD5+/aCD70a, which was significantly increased compared to pre-immunization levels. This phenotype is in alignment with recent publications describing human B-1 cells. PPS-responsive B cells receded to pre-immunization levels by day-30. These findings suggest that this B-1 like cell population plays an important role in early responses to S. pneumoniae infection and possibly other T-cell independent type-2 antigens in humans
Objective Based on promising in vitro and in vivo activity of several histone deacetylase inhibitors in Hodgkin lymphoma (HL), we investigated SNDX-275, an oral class 1 isoform–selective histone ...deacetylase inhibitors in HL-derived cell lines. Materials and Methods Proliferation and cell death were examined by MTS assay, Annexin V/propidium iodide, and fluorescence-activated cell sorting analysis. Gene and protein expression were measured by reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemical analysis. A multiplex assay was used to determine cytokines and chemokines. Results SNDX-275 induced cell death in a dose- and time-dependent manner with an IC50 at the sub- and lower micromolar range at 72 hours. At the molecular level, SNDX-275 increased histone H3 acetylation, upregulated p21 expression, and activated the intrinsic apoptosis pathway by downregulating the X-linked inhibitor of apoptosis protein. SNDX-275 downregulated expression of antiapoptotic Bcl-2 and Bcl-xL proteins without altering Mcl-1 or Bax levels. Combination studies demonstrated that two Bcl-2 inhibitors (ABT-737 and obatoclax) significantly enhanced the effect of SNDX-275. SNDX-275 modulated the level of several cytokines and chemokines, including interleukin-12 p40-70, interferon-inducible protein-10, RANTES (regulated on activation, normal T expressed and secreted), interleukin-13, interleukin-4, and thymus and activation-regulated chemokine and variably induced the cancer/testis antigen expression of MAGE-A4 and survivin in HL cell lines. Conclusions SNDX-275 has antiproliferative activity in HL cell lines, involving several mechanisms: induction of apoptosis, regulation of cytokines and chemokines, and alteration of cancer/testis antigens. Clinical investigation of SNDX-275 alone or in combination with Bcl-2 inhibitors is warranted in patients with HL. Phase 2 studies with SNDX-275 in HL are ongoing, and future clinical studies should investigate combinations with SNDX-275.
Background Cutaneous tuberculosis is widespread in Pakistan but has not been fully documented. This study was conducted to determine the clinical pattern, nature and existence of the disease in ...Larkana, Sindh province, Pakistan.
Methods We are reporting 153 cases of patients with cutaneous tuberculosis who visited our department from 1996 to 1999. All cases were diagnosed at the clinic, and the biopsies were examined for histopathological evidence. The patients received three antituberculous treatments during a 9 month course.
Results Clinically, 63 (41.2%) cases of lupus vulgaris, 54 (35.3%) of scrofuloderma, 29 (19.59%) of lupus verrucosa cutis, six (3.92%) of tuberculosis cutis orificialis and one (0.64%) case of disseminated cutaneous tuberculosis were observed in our department from 1996 to 1999. All patients were aged between 3 and 50 years and had experienced the present complaints for 1 to 12 years. Sixty‐nine (45.1%) cases were children aged under 10 years, 50 cases (37.25%) were aged between 10 and 20 years, and 27 cases (17.65%) were aged over 20 years. There was no considerable ratio difference of the disease between male and female patients. Histopathologically, all the specimens showed chronic granulomatous changes; the majority was infiltrated with epitheloid cells, langhans giant cells, plasma cells and other inflammatory cells, such as lymphocytes, eosinophils and neutrophils in ulcerated lesions. Increased numbers of mast cells were seen in upper and lower dermis in two‐thirds of the specimens. Caseating necrosis was visible in half of the specimens while Ziehl‐Neelsen stain was negative in all the sections.
Conclusions The observed number of patients was moderately large, thus indicating a high incidence of cutaneous tuberculosis in Larkana. Lupus vulgaris, a form of cutaneous tuberculosis, was widespread in this area and prevalent in adults, while scrofuloderma was prevalent in children. Moreover, the existing rate of the disease was higher in children aged under 10 years and lower in adults. This indicates that children are more prone to this disease than adults.
Abstract 2850
Based on recent favorable in vitro and in vivo activity of several HDACi (histone deacetylase inhibitors) in HL (Hodgkin lymphoma), we investigated the in vitro activity of SNDX-275, an ...oral, class 1 isoform of selective HDACi in HL-derived cell lines.
Proliferation and cell death were examined by MTS assay, Annexin-V/PI and FACS analysis. For combination studies, cells were incubated with SNDX-275 (0.1-2 μM) and either ABT-737 (0.01-0.2 μM), Obatoclax (0.1-2 μM), Gemcitabine (1-20 nM) or Bortezomib (1-20 nM) for 72 hours. Gene and protein expression were measured by RT-PCR, Western blot, and immunohistochemistry. A multiplex assay was used to determine 30 cytokines and chemokines.
SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased histone-3 acetylation, up-regulated p21 expression, and activated the intrinsic apoptosis pathway by down-regulating the XIAP (X-linked inhibitor of apoptosis protein), which was associated with activation of Caspase 9 and 3. Similarly to other HDACis, SNDX-275 decreased the expression of anti-apoptotic Bcl-2 and Bcl-xL, while level of Mcl-1 and pro-apoptotic Bax remained the same level. Combination studies demonstrated that SNDX-275 had more synergistic effect when combined with Bcl-2 inhibitors ABT-737 or Obatoclax and less when combined with Gemcitabine or Bortezomib. Dysregulated cytokine/chemokine production has been shown previously to contribute to HL pathology, including immune tolerance of the cancer cells. Hence, we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Increased IL12 p40-70, IP10, and RANTES, and decreased IL13, IL4 and TARC levels were found, thus favoring Th1-type cytokines/chemokines. Recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of CTAs (cancer testis antigens), leading to a favorable immune response. SNDX-275 was able to induce CTA expression of SSX2 and NY-ESO only in one cell line whereas MAGE-A4 was induced in both HL cell lines.
Our studies demonstrate that SNDX-275 has a dual effect on apoptotic and immunomodulatory pathways in HL, which can be enhanced by the addition of agents targeting cell survival pathways. Phase II studies with SNDX-275 in HL are ongoing, future clinical studies should investigate combinations with SNDX-275.
No relevant conflicts of interest to declare.
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