RESUMO
In the dose-escalation phase of a Phase I clinical trial in which six subjects each were vaccinated with PepCan at the 50, 100, 250, and 500 µg per peptide dose, the 50 µg dose showed the best histological regression rate. Ten additional subjects were vaccinated at this dose in the final dose phase. As with the dose-escalation phase, no dose-limiting toxicities were observed. Overall, the histological regression rates were 50% at the 50 µg dose (7 of 14) and 100 µg dose (3 of 6), and 45 % overall (14 of 31). Of subjects in whom HPV type 16 (HPV 16) was detected at entry, it became undetectable in three subjects after vaccination, and the viral loads significantly decreased in nine subjects in whom HPV 16 infection was detected at entry and exit (p = 0.008). Immune profiling revealed increased T-helper type 1 cells after vaccinations (p = 0.02 and 0.0004 after 2 and 4 vaccinations, respectively). T-helper type 2 cells initially increased after two vaccinations (p = 0.01), but decreased below the baseline level after four vaccinations although not significantly. Pre-vaccination regulatory T cell levels were significantly lower in histological responders compared to non-responders (p = 0.03). Feasibility of testing plasma for multiplex cytokine/chemokine analysis and of performing proteomic analysis of PBMCs was examined for potentially identifying biomarkers in the future. While these analyses are feasible to perform, attention needs to be given to how soon the blood samples would be processed after phlebotomy. As sufficient safety of PepCan has been demonstrated, enrollment for the Phase II clinical trial has been opened.
Assuntos
Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/imunologia , Carga Viral/imunologia , Adulto , Cromatografia Líquida , Citocinas/sangue , Citocinas/imunologia , Relação Dose-Resposta a Droga , Feminino , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/fisiologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Infecções por Papillomavirus/tratamento farmacológico , Infecções por Papillomavirus/virologia , Proteoma/imunologia , Proteoma/metabolismo , Proteômica/métodos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Espectrometria de Massas em Tandem , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/virologia , Vacinação/métodos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Carga Viral/efeitos dos fármacos , Adulto JovemRESUMO
OBJECTIVE: Our group and others have shown that serial intra-lesional injections of common warts with skin testing reagents such as Candida, mumps and Trichophyton are effective in regressing injected and non-injected warts. Anti-HPV T-cell responses appear to be induced. The goal of this study was to understand the mechanisms of how Candida skin testing reagent enhances immune responses. METHODS: The following immunological features were studied to understand how Candida induces immune responses in healthy subjects: (1) proliferative capacity of T-cells upon exposure to Candida through monocyte-derived human Langerhans cells (LCs) measured using alamarBlue, (2) cytokine (IL-1ß, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ, and TNF- expression upon Candida stimulation of LCs by quantitative reverse transcription (qRT)-PCR and cytokine secretion by ELISA, (3) expression of pattern recognition receptors (PRRs) known to associate with Candida albicans (DC-SIGN, dectin-1, dectin-2, galectin-3, mincle, mannose receptor, Toll-like receptors 1, 2, 4, 6, and 9) on LCs by qRT-PCR, (4) role of dectin-1 in IL-12 production by antibody blocking, and (5) induction of Th1, Th2, and/or Th17 responses by intracellular cytokine staining of CD4 cells exposed to Candida pulsed LCs for IFN-γ, IL-4, and IL-17A. RESULTS: T-cell proliferation upon stimulation with Candida-pulsed LCs was significantly higher compared to proliferation in the absence of Candida (p=0.004). The most frequently expressed cytokine in stimulated LCs was IL-12p40 mRNA, and IL-12p40 and IL-12p70 were also detected at protein levels. All other cytokine mRNAs examined were detected in the following order of decreasing frequency: IL23Ap19, IFN-γ, IL-1ß, IL-6, IL-8, and IL-10. LCs expressed all PRRs examined. Anti-dectin-1 inhibited IL-12p40 mRNA production upon Candida stimulation of LCs from some healthy subjects. IFN-γ secretion was increased and IL-4 secretion was decreased in CD4 cells of a few healthy subjects, but IL-17A was essentially unchanged upon Candida treatment. CONCLUSIONS: Proliferation of T-cells in a substantial majority of healthy subjects can be demonstrated with Candida stimulation. We show Th1 promotion and dectin-1 stimulation of LCs as potential mechanisms in some healthy subjects.
Assuntos
Candida/química , Saúde , Interleucina-12/metabolismo , Células de Langerhans/metabolismo , Lectinas Tipo C/metabolismo , Antígenos CD/metabolismo , Antígenos CD1/metabolismo , Caderinas/metabolismo , Candida/imunologia , Proliferação de Células/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Indicadores e Reagentes/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/imunologia , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testes Cutâneos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
The distribution of human papillomavirus (HPV) types and T-cell immune responses were compared among European American, African American, and Hispanic American populations being followed for abnormal Papanicolaou smear results who were attending the same university medical center clinic in Central Arkansas. Statistically significant differences were found for HPV types 55, 58, and 83 among the 37 HPV types tested. However, there were no differences in T-cell immune responses among these racial/ethnic groups. These results are unlikely to have an impact on therapeutic HPV vaccine development since the most prevalent HPV type among all racial/ethnic groups was HPV type 16.
Assuntos
Papillomavirus Humano 16/isolamento & purificação , Infecções por Papillomavirus/etnologia , Infecções por Papillomavirus/imunologia , Grupos Raciais/estatística & dados numéricos , Linfócitos T/virologia , Adulto , Negro ou Afro-Americano/estatística & dados numéricos , Arkansas/epidemiologia , Feminino , Hispânico ou Latino/estatística & dados numéricos , Papillomavirus Humano 16/classificação , Humanos , Fatores de Risco , Linfócitos T/imunologia , População Branca/estatística & dados numéricosRESUMO
The association between the CD8+ T-cell responses to human papillomavirus type 16 (HPV-16) E6 protein and a favorable clinical trend has been demonstrated previously. The roles of human papillomavirus (HPV)-specific CD4+ T-cell responses and of regulatory T-cells (Tregs) were examined. Subjects with a recent history of abnormal Papanicolaou smear were eligible, and colposcopy-guided biopsy was performed at enrollment. Interferon-γ enzyme-linked immunospot assay and fluorescent-activated cell sorter analysis to measure the frequencies of Tregs were performed. Subjects with histological diagnoses of cervical intraepithelial neoplasia 1, 2, or 3 were considered to have short-term persistence of cervical abnormality and were called "persistors" (n = 51) while those of normal histology were designated to be "regressors" (n = 33). A significantly higher percentage CD4+ T-cell response was detected in the regressors (15/33 or 45.5%) compared with the persistors (10/51 or 19.6%) (P = .015) for the E6 peptides but not for the E7 peptides. The CD4+ responses to certain E6 regions [E6(16-40), E6(91-115), E6(106-130), and E6(136-158)] were also significantly higher in the regressors. Although there was no difference in the frequencies of Tregs between the two groups, low frequencies of Tregs were significantly associated with positive CD4+ T-cell responses within certain E6 regions [E6(16-40), E6(31-55), E6(76-100), E6(91-115), and E6(106-130)]. The CD4+ and CD8+ T-cell responses to the HPV-16 E6 protein are associated with a favorable clinical trend. The HPV-16 E6 protein should be incorporated in the design of an HPV therapeutic vaccine.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Papillomavirus Humano 16/imunologia , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Proteínas Repressoras/imunologia , Linfócitos T Reguladores/imunologia , Displasia do Colo do Útero/imunologia , Neoplasias do Colo do Útero/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/patologia , Linhagem Celular Tumoral , DNA Viral/análise , DNA Viral/genética , Feminino , Papillomavirus Humano 16/genética , Humanos , Pessoa de Meia-Idade , Teste de Papanicolaou , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal , Adulto Jovem , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologiaRESUMO
Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 ± 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.
Assuntos
Colo do Útero/microbiologia , Colo do Útero/virologia , DNA/genética , Microbiota/genética , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Adulto , Bancos de Espécimes Biológicos , Citodiagnóstico/métodos , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Lactobacillus/genética , Neoplasias do Colo do Útero/microbiologia , Neoplasias do Colo do Útero/virologiaRESUMO
OBJECTIVE: The goal of this study was to examine the role of CD8 T-cell responses to human papillomavirus type 16 (HPV-16) in a favorable clinical trend in women being studied for abnormal Pap smear results. MATERIALS AND METHODS: Human papillomavirus-deoxyribonucleic acid testing and enzyme-linked immunospot assay using the HPV-16 E6 and E7 antigens were performed. The subjects with subsequent normal histologic diagnoses were considered to be "regressors" (n = 28), whereas those with histologic diagnoses of cervical intraepithelial neoplasia 1, 2, or 3 were considered to have short-term persistence of cervical abnormality and were designated to be "persistors" (n = 37). RESULTS: There was a higher percentage of CD8 T-cell responses to the E6 antigen in the regressors (15/28 or 53.6%) when compared with the persistors (10/37 or 27.0%; p = .04), but there was no recorded response difference for the E7 antigen. Results were the same when the analyses for E6 included only subjects who were high-risk HPV-positive (p = .01). CONCLUSIONS: The CD8 T-cell immune responses to the HPV-16 E6 antigens but not to E7 antigens are associated with a favorable clinical trend regardless of HPV types currently detected.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/patologia , Proteínas Repressoras/imunologia , Adulto , Colo do Útero/patologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/análise , Teste de Papanicolaou , Papillomaviridae/isolamento & purificação , Proteínas E7 de Papillomavirus/análise , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/epidemiologia , Reação em Cadeia da Polimerase , Proteínas Repressoras/análise , Esfregaço Vaginal , Adulto JovemRESUMO
Gut metagenome profiling using the Oxford Nanopore Technologies (ONT) sequencer was assessed in a pilot-sized study of 10 subjects. The taxonomic abundance of gut microbiota derived from ONT was comparable with Illumina Technology (IT) for the high-abundance species. IT better detected low-abundance species through amplification, when material was limited.
Assuntos
Bactérias/classificação , Microbioma Gastrointestinal/genética , Neoplasias de Cabeça e Pescoço/epidemiologia , Metagenoma/genética , Sequenciamento por Nanoporos/métodos , Idoso , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Análise de Sequência de DNA/métodosRESUMO
Human papillomavirus (HPV) infection is associated with the vast majority of cervical cancer cases as well as with other anogenital cancers. PepCan is an investigational HPV therapeutic vaccine for treating cervical high-grade squamous intraepithelial lesions. The present study was performed to test whether the cervical microbiome influences vaccine responses and to explore host factors as determinants of the cervical microbiome composition in women with biopsy-proven high-grade squamous intraepithelial lesions. In a recently completed Phase I clinical trial of PepCan, histological response rate of 45% (14 of 31 patients), a significant increase in circulating T-helper type 1 cells, and a significant decrease in HPV 16 viral load were reported. DNA, extracted from liquid cytology specimens collected before and after vaccinations, were amplified and then hybridized to a G4 PhyloChip assay to characterize the microbiome. We describe trends that certain bacterial taxa in the cervix may be enriched in non-responders in comparison to responders (Padj = .052 for phylum Caldithrix and Padj = .059 for phylum Nitrospirae). There was no difference in bacterial diversity between the 2 groups. A permutational analysis of variance performed for various demographic and immune parameters showed significant clustering with microbiome beta diversity for race, HPV 16 status, peripheral T-helper type 1 cells, and HLA-B40 (P = .001, .014, .037, and .024, respectively). Further analyses showed significant differences at the empirical Operational Taxonomic Unit level for race and HPV 16 status. As these results are from a small Phase I study, further studies are needed to examine the role of cervical microbiome in response to HPV therapeutic vaccines.
Assuntos
Colo do Útero/microbiologia , Microbiota/imunologia , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Lesões Intraepiteliais Escamosas/imunologia , Neoplasias do Colo do Útero/imunologia , Adulto , Colo do Útero/imunologia , Feminino , Papillomavirus Humano 16/imunologia , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/microbiologia , Lesões Intraepiteliais Escamosas/microbiologia , Neoplasias do Colo do Útero/microbiologia , Carga Viral/imunologia , Adulto JovemRESUMO
Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Diabetes Mellitus Tipo 1/fisiopatologia , Osteogênese/fisiologia , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Regulação para Baixo , Feminino , Insulina/farmacologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Osteogênese por Distração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genéticaRESUMO
Numerous versions of human papillomavirus (HPV) therapeutic vaccines designed to treat individuals with established HPV infection, including those with cervical intraepithelial neoplasia (CIN), are in development because approved prophylactic vaccines are not effective once HPV infection is established. As human papillomavirus 16 (HPV-16) is the most commonly detected type worldwide, all versions of HPV therapeutic vaccines contain HPV-16, and some also contain HPV-18. While these two HPV types are responsible for approximately 70% of cervical cancer cases, there are other high-risk HPV types known to cause malignancy. Therefore, it would be of interest to assess whether these HPV therapeutic vaccines may confer cross-protection against other high-risk HPV types. Data available from a few clinical trials that enrolled subjects with CINs regardless of the HPV type(s) present demonstrated clinical responses, as measured by CIN regression, in subjects with both vaccine-matched and nonvaccine HPV types. The currently available evidence demonstrating cross-reactivity, epitope spreading, and de novo immune stimulation as possible mechanisms of cross-protection conferred by investigational HPV therapeutic vaccines is discussed.
Assuntos
Proteção Cruzada , Epitopos/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/terapia , Vacinas contra Papillomavirus/imunologia , Displasia do Colo do Útero/terapia , Humanos , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/administração & dosagem , Resultado do Tratamento , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologiaRESUMO
PURPOSE: Non-surgical treatments for cervical intraepithelial neoplasia 2/3 (CIN2/3) are needed as surgical treatments have been shown to double preterm delivery rate. The goal of this study was to demonstrate safety of a human papillomavirus (HPV) therapeutic vaccine called PepCan, which consists of four current good-manufacturing production-grade peptides covering the HPV type 16 E6 protein and Candida skin test reagent as a novel adjuvant. PATIENTS AND METHODS: The study was a single-arm, single-institution, dose-escalation phase I clinical trial, and the patients (n = 24) were women with biopsy-proven CIN2/3. Four injections were administered intradermally every 3 weeks in limbs. Loop electrical excision procedure (LEEP) was performed 12 weeks after the last injection for treatment and histological analysis. Six subjects each were enrolled (50, 100, 250, and 500 µg per peptide). RESULTS: The most common adverse events (AEs) were injection site reactions, and none of the patients experienced dose-limiting toxicities. The best histological response was seen at the 50 µg dose level with a regression rate of 83% (n = 6), and the overall rate was 52% (n = 23). Vaccine-induced immune responses to E6 were detected in 65% of recipients (significantly in 43%). Systemic T-helper type 1 (Th1) cells were significantly increased after four vaccinations (P = 0.02). CONCLUSION: This study demonstrated that PepCan is safe. A significantly increased systemic level of Th1 cells suggests that Candida, which induces interleukin-12 (IL-12) in vitro, may have a Th1 promoting effect. A phase II clinical trial to assess the full effect of this vaccine is warranted.
RESUMO
Haemophilus influenzae is a human-adapted commensal and pathogen that can cause mucosal infections such as sinusitis, otitis media and bronchitis. Certain strains also cause bacteraemia and meningitis. Clinical isolates are genetically heterogeneous and are often recalcitrant to standard genetic manipulation. H. influenzae strain Rd KW20 has traditionally been considered avirulent, since it does not survive in the bloodstream of animals, is readily killed by normal adult human sera and cannot colonize the nasopharynx of infant rats. The purpose of this study was to determine whether Rd KW20 could be used in certain infection models. It is shown here that strain Rd KW20 can invade certain human epithelial cell lines grown either as monolayers or as differentiated epithelium at the air-liquid interface. In addition, Rd KW20 can invade a monolayer of immortalized human brain microvascular endothelial cells. Finally, this strain can replicate and survive in human bronchial xenografts for up to 3 weeks. The complete genomic sequence of Rd KW20 is available and it is readily amenable to genetic manipulation. These properties and the results reported here indicate that this strain is a viable alternative to the use of clinical isolates for the investigation of H. influenzae virulence.
Assuntos
Haemophilus influenzae/patogenicidade , Animais , Brônquios/citologia , Brônquios/microbiologia , Brônquios/transplante , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/microbiologia , Células Epiteliais/microbiologia , Feminino , Humanos , Camundongos , Ratos , Transplante Heterólogo , VirulênciaRESUMO
Cell-mediated immune responses to the human papillomavirus type 16 (HPV 16) E6 protein have been shown to be important in viral clearance and in regression of cervical lesions. Here, detailed analyses of a novel HPV 16 E6 CD4 T-cell epitope from a subject with cervical intraepithelial neoplasia 1 are described. This subject had demonstrated HPV 16 CD4 T-cell responses to multiple regions within the E6 protein. Isolation and cloning of CD4 T-cells were performed by magnetic selection of interferon-γ secreting cells and limiting dilution. A single HPV 16-specific T-cell clone isolated was shown to have a specificity to HPV 16 E6 52-62 restricted by the HLA-DR11 molecule. Homologous sequences (≥70% amino acid homology) were identified for HPV types 31, 33, 45, 58, 73, but cross-recognition was demonstrated only for HPV 45. Based on work performed by our group and others, it is known that this short peptide contains multiple CD4 and CD8 T-cell HPV epitopes and would be an ideal region to incorporate into a design of vaccines and immunotherapies against HPV-associated malignancies.
RESUMO
A vaccine adjuvant that can effectively promote cell-mediated immunity is currently not available. Because of the ability of a Candida skin test reagent injection to induce common wart regression, our group is using it as a novel adjuvant in a clinical trial of a peptide-based human papillomavirus therapeutic vaccine. The goal of this current study was to investigate the mechanisms of how Candida enhances the vaccine immune responses. Maturation effects on Langerhans cells, capacity to proliferate T-cells, expression of cytokines and pattern recognition receptors by Langerhans cells, and ability to induce Th1, Th2, and Th17 responses were investigated in healthy subjects. The vaccine, human papillomavirus peptides with Candida, demonstrated partial maturation effects on Langerhans cells indicated by significantly up-regulated CD40 (p=0.00007) and CD80 (p<0.00001) levels, and showed T-cell proliferative capacity (p<0.00001) when presented by Langerhans cells in vitro. Interestingly, the maturation effects were due to the peptides while Candida was responsible for the T-cell proliferation. The cytokine profile (IL-1ß, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ and TNF-α) of Langerhans cells treated with the vaccine or Candida alone showed that IL-12p40 mRNA was most frequently induced, and IL-12p70 protein was detected in the supernatants. The presence of pattern recognition receptors known to associate with Candida albicans (DC-SIGN, dectin-1, dectin-2, galectin-3, mincle, mannose receptor, Toll-like receptors-1, 2, 4, 6 and 9) were demonstrated in all subjects. On the other hand, the induction of Th1 response demonstrated by IFN-γ secretion by CD4 cells stimulated with the vaccine or Candida pulsed Langerhans cells was demonstrated only in one subject. In summary, the Langerhans cell maturation effects of the vaccine were due to the peptides while the T-cell proliferative capacity was derived from Candida, and the most frequently induced cytokine was IL-12.
Assuntos
Adjuvantes Imunológicos/farmacologia , Candida/imunologia , Imunidade Celular , Vacinas contra Papillomavirus/imunologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Citocinas/imunologia , Humanos , Células de Langerhans/imunologiaRESUMO
A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected(1). A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)(2) in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 10(5) cells per well typically used in ELISPOT assays(1,3), 1,000 T-cell clone cells in the presence of 1 x 10(5) autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes(4).
Assuntos
ELISPOT/métodos , Epitopos de Linfócito T/análise , Papillomavirus Humano 16/imunologia , Interferon gama/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Epitopos de Linfócito T/imunologia , HumanosRESUMO
CD8 T-cell responses were examined in subjects with incident (new following negative visits) or prevalent (lasting ≥ 4 months) human papillomavirus type 16 (HPV16) or human papillomavirus (HPV18) infection. The groups were chosen from a cohort of women being followed every 4 months with cervical cytology and HPV-DNA testing. Enzyme-linked immunospot (ELISPOT) assay was performed at enrollment (time zero) and one year later. At time zero, 1 (6%) of 17 subjects with incident HPV 16/18 infections had positive ELISPOT results which increased to 6 (35%) at one year. For the subjects with prevalent HPV 16/18 infections, the ELISPOT results were similar at time zero (2 (15%) of 15 subjects positive) and at one year (3 (20%)). While all of the 11 women with prevalent HPV16 infection showed clearance one year later, unexpectedly only 1 (25%) of 4 women with prevalent HPV18 infection demonstrated clearance one year later (P = .009).
RESUMO
OBJECTIVE: Dysregulation of matrix metalloproteinase (MMP)-2 may contribute pathologically to the development of diabetes complications, including diabetic retinopathy and coronary and peripheral arterial disease. Our objective was to explore whether systemic MMP-2 dysregulation could be demonstrated in type 1 diabetes and to determine how MMP-2 concentration relates to disease status. RESEARCH DESIGN AND METHODS: In this cross-sectional study, MMP-2 concentrations and MMP-2 activity were measured in plasma and timed urine samples from 93 type 1 diabetic and 50 healthy control subjects, aged 14-40 years. Relationships between MMP-2 concentrations in these biological fluids and subject characteristics (sex, age, and duration of type 1 diabetes), indexes of glycemic control (A1C, fasting plasma glucose, and continuous glucose monitoring system average daily glucose), and measurements of renal function (urinary albumin excretion and glomerular filtration rate) were examined. RESULTS: Urine and plasma MMP-2 concentrations and plasma MMP-2 activity were all significantly elevated in type 1 diabetic subjects compared with those in control subjects. Urine MMP-2 concentrations, in particular, were correlated with several clinical parameters that infer increased risk for diabetic comorbidity and specifically for diabetic nephropathy, including higher A1C, longer duration of disease, evidence of renal hyperfiltration, and the presence of microalbuminuria. CONCLUSIONS: Urine and plasma MMP-2 concentrations are dysregulated in type 1 diabetes; urinary excretion of MMP-2, in particular, might provide a unique biomarker of diabetes-induced intrarenal pathologic processes.
Assuntos
Complicações do Diabetes/etiologia , Diabetes Mellitus Tipo 1/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Adolescente , Adulto , Estudos Transversais , Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Nefropatias Diabéticas/etiologia , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/urinaRESUMO
A chemically defined medium that supports the growth of both encapsulated and nontypeable Haemophilus influenzae strains in broth to densities that are >/= 10(9) CFU/ml or on agar plates is described. The mean generation time of a panel of clinical isolates was comparable to that in rich, chemically undefined media (brain-heart infusion broth supplemented with heme and beta-NAD).