RESUMO
Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.
Assuntos
Quimiocinas CC , Interleucina-23 , Humanos , Animais , Camundongos , Quimiocinas CC/genética , Receptores CCR7 , Ligantes , Linfócitos T , Inflamação , Receptores CCR6RESUMO
PURPOSE: Chimeric antigen receptor-modified T cells (CAR-T) have demonstrated encouraging results in early-phase clinical trials. Successful adaptation of CAR-T technology for CEA-expressing adenocarcinoma liver metastases, a major cause of death in patients with gastrointestinal cancers, has yet to be achieved. We sought to test intrahepatic delivery of anti-CEA CAR-T through percutaneous hepatic artery infusions (HAIs). EXPERIMENTAL DESIGN: We conducted a phase I trial to test HAI of CAR-T in patients with CEA(+) liver metastases. Six patients completed the protocol, and 3 received anti-CEA CAR-T HAIs alone in dose-escalation fashion (10(8), 10(9), and 10(10) cells). We treated an additional 3 patients with the maximum planned CAR-T HAI dose (10(10) cells × 3) along with systemic IL2 support. RESULTS: Four patients had more than 10 liver metastases, and patients received a mean of 2.5 lines of conventional systemic therapy before enrollment. No patient suffered a grade 3 or 4 adverse event related to the CAR-T HAIs. One patient remains alive with stable disease at 23 months following CAR-T HAI, and 5 patients died of progressive disease. Among the patients in the cohort that received systemic IL2 support, CEA levels decreased 37% (range, 19%-48%) from baseline. Biopsies demonstrated an increase in liver metastasis necrosis or fibrosis in 4 of 6 patients. Elevated serum IFNγ levels correlated with IL2 administration and CEA decreases. CONCLUSIONS: We have demonstrated the safety of anti-CEA CAR-T HAIs with encouraging signals of clinical activity in a heavily pretreated population with large tumor burdens. Further clinical testing of CAR-T HAIs for liver metastases is warranted.
Assuntos
Adenocarcinoma/terapia , Quimioterapia do Câncer por Perfusão Regional/métodos , Imunoterapia/métodos , Neoplasias Hepáticas/terapia , Receptores de Antígenos de Linfócitos T/administração & dosagem , Linfócitos T/transplante , Adenocarcinoma/secundário , Idoso , Feminino , Neoplasias Gastrointestinais/patologia , Neoplasias Gastrointestinais/terapia , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-IdadeRESUMO
Chimeric antigen receptor-modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded threefold in response to LM, and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias Hepáticas/patologia , Células Mieloides/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fígado/citologia , Fígado/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição STAT3/metabolismo , Carga TumoralRESUMO
Delayed-type hypersensitivity (DTH) is a T-cell-mediated immune response that may be used for immunotoxicity testing in non-clinical species. However, in some cases DTH assays using T-dependent antigens may be confounded by the production of antibodies to the antigen. The authors have previously modified a DTH assay, initially validated in the mouse, for use in juvenile rats to assess the effect of immunosuppressive drugs on the developing rat immune system. The assay measures footpad swelling induced by subcutaneous footpad injection of Candida albicans (C. albicans) derived-chitosan in rats previously sensitized with C. albicans. Antibodies to chitosan are not produced in this model. However, considerable inter-animal variability inherent in the footpad swelling assay can make it difficult to precisely quantify the magnitude of the immune response and inhibition by immunosuppressants, particularly if complete suppression is not observed. This report describes the development of an ex vivo assay to assess DTH in rats using interferon (IFN)-γ production by splenocytes, obtained from rats sensitized with C. albicans, as the quantifiable measure of the DTH response. Adult and neonatal rats administered dexamethasone (DEX), a known immunosuppressant, exhibited immunosuppression as evidenced by a reduction in ex vivo IFNγ production from splenocytes challenged with C. albicans-derived chitosan. Current data indicate that the ex vivo based DTH assay is more sensitive than the conventional footpad swelling assay due to a lower background response and the ability to detect a response as early as post-natal day (PND) 12. The ex vivo based rat DTH assay offers a highly sensitive and quantitative alternative to the footpad swelling assay for the assessment of the immunotoxic potential of drugs. The increased sensitivity of the ex vivo DTH assay may be useful for identifying smaller changes in response to immunotoxic drugs, as well as detecting responses earlier in animal development.
Assuntos
Candida albicans/imunologia , Candidíase/diagnóstico , Candidíase/imunologia , Quitosana/imunologia , Hipersensibilidade Tardia/imunologia , Animais , Quitosana/química , Feminino , Hipersensibilidade Tardia/induzido quimicamente , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
LM escape immune surveillance, in part, as a result of the expansion of CD11b+MC, which alter the intrahepatic microenvironment to promote tumor tolerance. HBC make up a significant proportion of liver lymphocytes and appear to delay tumor progression; however, their significance in the setting of LM is poorly defined. Therefore, we characterized HBC and HBC/CD11b+MC interactions using a murine model of LM. Tumor-bearing livers showed a trend toward elevated absolute numbers of CD19+ HBC. A significant increase in the frequency of IgM(lo)IgD(hi) mature HBC was observed in mice with LM compared with normal mice. HBC derived from tumor-bearing mice demonstrated increased proliferation in response to TLR and BCR stimulation ex vivo compared with HBC from normal livers. HBC from tumor-bearing livers exhibited significant down-regulation of CD80 and were impaired in inducing CD4(+) T cell proliferation ex vivo. We implicated hepatic CD11b+MC as mediators of CD80 down-modulation on HBC ex vivo via a CD11b-dependent mechanism that required cell-to-cell contact and STAT3 activity. Therefore, CD11b+MC may compromise the ability of HBC to promote T cell activation in the setting of LM as a result of diminished expression of CD80. Cross-talk between CD11b+MC and HBC may be an important component of LM-induced immunosuppression.
Assuntos
Subpopulações de Linfócitos B/imunologia , Antígeno CD11b/metabolismo , Neoplasias Hepáticas/imunologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Receptores de Superfície Celular/metabolismo , Transferência Adotiva , Animais , Subpopulações de Linfócitos B/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Medições Luminescentes/métodos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Imagem Molecular/métodos , Metástase Neoplásica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Carga TumoralRESUMO
Biliary obstruction is a common clinical problem that is associated with intrahepatic inflammation and impaired immunity. PD-1 is well known to mediate T cell dysfunction but has been reported to promote and attenuate acute inflammation in various injury models. With the use of a well-established murine model of BDL, we studied the effects of intrahepatic PD-1 expression on LTC function, inflammation, and cholestasis. Following BDL, PD-1 expression increased significantly among LTCs. Increased PD-1 expression following BDL was associated with decreased LTC proliferation and less IFN-γ production. Elimination of PD-1 expression resulted in significantly improved proliferative capacity among LTC following BDL, in addition to a more immunostimulatory cytokine profile. Not only was LTC function rescued in PD-1(-/-) mice, but also, the degrees of biliary cell injury, cholestasis, and inflammation were diminished significantly compared with WT animals following BDL. PD-1-mediated acute inflammation following BDL was associated with expansions of intrahepatic neutrophil and Th17 cell populations, with the latter dependent on IL-6. PD-1 blockade represents an attractive strategy for reversing intrahepatic immunosuppression while limiting inflammatory liver damage.
Assuntos
Colestase/imunologia , Colestase/fisiopatologia , Inflamação/imunologia , Fígado/patologia , Neutrófilos/patologia , Receptor de Morte Celular Programada 1/metabolismo , Células Th17/patologia , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Proliferação de Células , Colestase/complicações , Colestase/patologia , Inflamação/complicações , Inflamação/patologia , Interleucina-6/metabolismo , Icterícia/complicações , Icterícia/imunologia , Icterícia/patologia , Icterícia/fisiopatologia , Ligadura , Fígado/imunologia , Fígado/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Regulação para CimaRESUMO
BACKGROUND: Imatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options. Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells. METHODS: Human anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions. RESULTS: We successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates. CONCLUSIONS: We have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc.
Assuntos
Tumores do Estroma Gastrointestinal/terapia , Proteínas Proto-Oncogênicas c-kit/imunologia , Linfócitos T/citologia , Animais , Sequência de Bases , Proliferação de Células , Primers do DNA , Tumores do Estroma Gastrointestinal/patologia , Masculino , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase , Linfócitos T/imunologiaRESUMO
Establishing an in vivo cell-mediated immunity (CMI) assay, such as the delayed-type hypersensitivity (DTH) assay, has been identified as an important gap and recommended to receive highest priority for new model development in several workshops on developmental immunotoxicity. A Candida albicans DTH model has recently been developed that has the advantage over other DTH models, which use alternative sensitizing antigens, in that antigen-specific antibodies, which may interfere with the assay, are not produced. In addition, the in vivo C. albicans DTH model was demonstrated to be more sensitive in detecting immunosuppression than DTH models using keyhole limpet hemocyanin (KLH) or sheep red blood cells as antigens, as well as some ex vivo CMI assays. While KLH and sheep red blood cells are non-physiological immunogens, C. albicans is an important human pathogen. The present studies were conducted in order to optimize and validate the C. albicans DTH model for use in developmental immunotoxicity studies using juvenile rats. Three known immunosuppressive compounds with different mechanisms of action were tested in this model, cyclosprorin A (CsA), cyclophosphamide (CPS), and dexamethasone (DEX). Animals were sensitized with formalin-fixed C. albicans on postnatal day (PND) 28 and challenged with chitosan on PND 38. Drug was administered beginning on PND 23 and continued until PND 37. Exposure to each of the three immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans-derived chitosan. Decreases in footpad swelling were observed at ≥10 mg CsA/kg/day, ≥5 mg CPS/kg/day, and ≥0.03 mg DEX/kg/day. These results demonstrate that the C. albicans DTH model, optimized for use in juvenile rats, can be used to identify immunotoxic compounds, and fills the need for a sensitive in vivo CMI model for assessments of developmental immunotoxicity. Abbreviations Ab, antibody APC, antigen presenting cell BSA, bovine serum albumin C. albicans, Candida albicans CI, challenge interval CMI, cell-mediated immunity CO, challenge only CPS, cyclophosphamide CsA, cyclosporin A CTL, cytotoxic T lymphocyte DEX, dexamethasone DIT, developmental immunotoxicity DTH, delayed-type hypersensitivity ip, intraperitoneal KLH, keyhole limpet hemocyanin MLR, mixed lymphocyte reaction OVA, ovalbumin PBS, phosphate-buffered saline PND, postnatal day sc, subcutaneous SEM, standard error of the mean SRBC, sheep red blood cells.
Assuntos
Candida albicans/imunologia , Modelos Animais de Doenças , Hipersensibilidade Tardia/tratamento farmacológico , Animais , Antígenos de Fungos/imunologia , Bovinos , Quitosana/imunologia , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Ciclosporinas/administração & dosagem , Ciclosporinas/efeitos adversos , Dexametasona/administração & dosagem , Dexametasona/efeitos adversos , Feminino , Humanos , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/microbiologia , Imunidade Celular/efeitos dos fármacos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , OvinosRESUMO
BACKGROUND: There is concern about embryo-fetal exposure to antibody-based biopharmaceuticals based on the increase of such therapies being prescribed to women of childbearing potential. Therefore, there is a desire to better characterize embryo-fetal exposure of these molecules. The pregnant rat is a standard model for evaluating the potential consequences of exposure but placental transfer of antibody-based biopharmaceuticals is not well understood in this model. METHODS: The relative embryo-fetal distribution of an antibody-based biopharmaceutical was evaluated in the rat. Bevacizumab (Avastin) was chosen as a tool antibody since it does not have significant target binding in the rat that might influence embryo-fetal biodistribution. Avastin was labeled with a fluorescent dye, characterized, and injected into pregnant rats at different gestation ages. Labeled Avastin in fetal tissues was visualized ex vivo using an IVIS 200 (Caliper, A PerkinElmer Company, Alameda, CA). RESULTS: Avastin localized to the fetus as early as 24-hr post intravenous injection of the dam, and was taken up by the fetus in a dose-dependent manner. Avastin was detectable in the developing embryo as early as gestation day 13 and continued to be transferred until the end of gestation. Fetal transfer of Avastins mutated in the portion of the antibody that binds the neonatal Fc receptor (FcRn) was tested in late gestation and was found to correlate with affinities of the mutant Avastin antibody to FcRn. CONCLUSIONS: The novel application of this imaging technology was used to characterize the onset and duration of Avastin maternal-fetal transfer in rats and the importance of FcRn binding.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Embrião de Mamíferos/metabolismo , Feto/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Troca Materno-Fetal , Receptores Fc/metabolismo , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Bevacizumab , Relação Dose-Resposta a Droga , Membranas Extraembrionárias/metabolismo , Feminino , Gravidez , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: It has been postulated that brain inflammatory processes associated with autoimmune diseases may be causative factors in emotional disorders. Accordingly, we examined emotional behaviors in autoimmune-prone cytokine B-cell-activating factor (BAFF) transgenic mice, a model of systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. METHODS: Male BAFF transgenic mice were examined on a series of standard laboratory assays of emotionality. Mice were also tested for brain inflammation, stress-induced c-Fos expression, hippocampal progenitor cell proliferation, and hippocampal neurogenesis-dependent and neurogenesis-independent long-term potentiation (LTP). RESULTS: Our study revealed that older BAFF transgenic mice exhibit an anxiety-like phenotype associated with brain inflammation. Furthermore, anxious mice display an abnormal neuronal activation within the limbic system in response to mild anxiogenic stimuli. Proliferation of newly formed neurons in the subgranular zone of adult hippocampus was significantly decreased in anxious BAFF transgenic mice that also showed impaired neurogenesis-dependent and neurogenesis-independent dentate gyrus LTP. CONCLUSIONS: Our results suggest that anxiety associated with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome can be linked to brain inflammation, impaired neurogenesis, and hippocampal plasticity. BAFF transgenic mice can be used in future studies to test compounds of therapeutic value for the treatment of mood disorders associated with autoimmune diseases.
Assuntos
Ansiedade , Fator Ativador de Células B/genética , Emoções/fisiologia , Encefalite , Regulação da Expressão Gênica/genética , Neurogênese/genética , Fatores Etários , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Ansiedade/genética , Ansiedade/patologia , Ansiedade/fisiopatologia , Comportamento Animal/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Estimulação Elétrica/métodos , Encefalite/genética , Encefalite/patologia , Encefalite/fisiopatologia , Ensaio de Imunoadsorção Enzimática/métodos , Comportamento Exploratório/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Elevação dos Membros Posteriores/métodos , Hipocampo/citologia , Imunoglobulina G/sangue , Técnicas In Vitro , Potenciação de Longa Duração/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células-Tronco/fisiologia , Natação/psicologiaRESUMO
Overexpression of BAFF is believed to play an important role in systemic lupus erythematosus and elevated levels of serum BAFF have been found in lupus patients. Excess BAFF also leads to overproduction of anti-dsDNA antibodies and a lupus-like syndrome in mice. In the present study, we use mice transgenic for the R4A-Cmu (IgM) heavy chain of an anti-dsDNA antibody, to study the effects of BAFF overexpression on anti-dsDNA B-cell regulation. We observe that overexpression of BAFF promotes anti-dsDNA B-cell maturation and secretion of antibody and enriches for transgenic anti-dsDNA B cells in the marginal zone and follicular splenic compartments. In addition, our data suggests that BAFF rescues a subset of anti-dsDNA B cells from a regulatory checkpoint in the transitional stage of development.
Assuntos
Anticorpos Antinucleares/imunologia , Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Diferenciação Celular , DNA/imunologia , Animais , Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
OBJECTIVE: The objective of this article is to study the effect of PC-815, a novel combination microbicide containing carrageenan and the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150, in blocking HIV-1 and HIV-2 infections in vitro as compared with Carraguard alone. GOAL: The goal of this study was to develop a combination microbicide that is more efficacious than Carraguard against HIV-1 and HIV-2. STUDY DESIGN: The microtiter syncytial assay was used to evaluate: 1) the antiviral and virucidal activity of MIV-150 against HIV-1MN; 2) the additive effect of MIV-150 when combined with carrageenan; and 3) a possible interference of seminal fluid in the antiviral activity of these compounds. RESULTS: MIV-150 effectively inactivated free virus. Combination of MIV-150 and Carraguard demonstrated an additive antiviral effect. Seminal fluid had no effect on the antiviral activity of MIV-150 or Carraguard. The average concentration that blocks 50% of infection (EC50) for PC-815 was approximately 10 times stronger than Carraguard for the different clinical isolates used in the study. CONCLUSION: Theoretically, PC-815 is likely to be a more efficacious microbicide than Carraguard.
Assuntos
Anti-Infecciosos/farmacologia , Carragenina/farmacologia , Chondrus , Fitoterapia , Piridinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Ureia/análogos & derivados , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/uso terapêutico , Carragenina/administração & dosagem , Carragenina/uso terapêutico , Quimioterapia Combinada , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Masculino , Testes de Sensibilidade Microbiana , Piridinas/administração & dosagem , Piridinas/uso terapêutico , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/uso terapêutico , Sêmen/virologia , Ureia/administração & dosagem , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
Mice transgenic for the R4A-Cmu heavy chain of an anti-dsDNA antibody, maintain tolerance by anergy and deletion. In C57BL/6 mice overexpressing CD19, a molecule, which lowers the threshold for B cell activation, elevated levels of serum autoantibodies have been observed. In the present study, we wished to determine whether CD19 overexpression could alter the induction of tolerance in R4A-Cmu mice and lead to the secretion of transgenic anti-dsDNA antibodies. We, therefore, bred R4A-Cmu transgenic mice-to-mice transgenic for human CD19 (hCD19) and generated R4A-Cmu mice heterozygous and homozygous for hCD19. We, now report the spontaneous secretion of transgenic IgM anti-dsDNA antibody in the sera of R4A-Cmu mice overexpressing CD19, indicative of a loss of B cell tolerance. We observe that transgenic B cells secreting anti-dsDNA antibody in these mice are T independent and display a marginal zone like phenotype althought they do not reside in the MZ. In addition, they appear to be derived from the conventional B2 subset rather than the B1 subset. Interestingly, a subset of the anti-dsDNA B cells in these mice still display the phenotype and functional characteristics of anergic B cells. These B cells cannot be activated to secrete antibody following BCR crosslinking, however, they are hyper-responsive to activation by innate signaling mechanisms. This suggests that CD19 overexpression may promote anergic B cells to escape tolerance by converging with BCR independent pathways, thereby rendering these B cells hyper-responsive to innate signaling.
Assuntos
Antígenos CD19/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , DNA/imunologia , Tolerância Imunológica/imunologia , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos CD19/genética , Antígenos CD19/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Anergia Clonal/imunologia , Expressão Gênica , Humanos , Imunidade Inata/imunologia , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peritônio/citologia , Transdução de Sinais , Baço/citologiaRESUMO
OBJECTIVES: To develop an accurate, rapid, and inexpensive method for verifying vaginal applicator use. GOAL: To develop a method for assessing compliance in microbicide clinical trials. STUDY DESIGN: Single use Microlax applicators containing a placebo formulation either were or were not exposed to the vagina. Three assays were developed to determine whether the applicators had been used vaginally. RESULTS: Blinded examiners were able to discern 63% of the time whether or not applicator tips had been exposed to the vagina. Optical density (to measure lactobacilli), increased in media exposed to used applicators but not in media exposed to unused applicators. When tips of applicators were stained with trypan blue, used applicators could be distinguished easily from unused applicators. CONCLUSION: Staining of applicator is accurate, simple, rapid, and inexpensive. This method could be be used in clinical settings in the developing world. Dying applicator tips could prove useful in excluding non-compliant subjects, analyzing data, or developing social intervention strategies to improve compliance.