RESUMO
The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIVmne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1.
Assuntos
Farmacorresistência Viral , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , DNA Viral/sangue , Feminino , Trato Gastrointestinal/virologia , HIV-1/genética , Humanos , Leucócitos Mononucleares , Linfonodos/virologia , Macaca mulatta , Especificidade de Órgãos , RNA Viral/sangue , Inibidores da Transcriptase Reversa/uso terapêutico , Vírus da Imunodeficiência Símia/genética , Vagina/virologia , ViremiaRESUMO
INTRODUCTION: Oral squamous cell carcinoma is a major cause of death throughout the developed world. It is associated with smoking and alcohol consumption. Human papillomavirus (HPV) type 16 has also been suggested to play a role in etiology of head and neck squamous cell carcinoma (HNSCC). p16 expression is now being used as a surrogate marker of HPV infection in squamous cell carcinoma and provides important prognostic information and future therapy planning. MATERIALS AND METHODS: In this prospective study, total of 75 cases of HNSCC were taken. Tumor grade was determined according to World Health Organization (WHO) criteria. p16 expression was determined by immunohistochemical staining. The obtained results were analyzed and evaluated using Chi-square test (Statistical Package for Social Sciences (SPSS) version 20), value of P <0.05 was taken significant. RESULTS: Out of 75 cases, 78.7% cases were positive for p16 (inclusive of all grades), while 21.3% cases were negative. Expression of p16 was higher in nonsmokers and nonalcohol consumers and significantly associated with paan chewing habit. No significant correlation was seen with history of abnormal sexual habits, but p16 expression was significantly correlated in cases with multiple sexual partners (P = 0.003), with increasing histological grade (P = 0.045) and in cases with lymph node metastasis (P = 0.03). CONCLUSION: As HPV integration with transcription of viral oncoprotein induces overexpression of p16, immunohistochemical expression of p16 can be used as a surrogate marker of HPV. This approach can be implemented in diagnostic laboratories and can provide support for vaccination program in high risk group.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Proteínas de Neoplasias/biossíntese , Infecções por Papillomavirus/genética , Adulto , Idoso , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Inibidor p16 de Quinase Dependente de Ciclina , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Humanos , Linfonodos/patologia , Linfonodos/virologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
BACKGROUND: Lymph nodes and gut-associated lymphatic tissue are important reservoirs of the human immunodeficiency virus (HIV). Little is known about these reservoirs in different geographic populations. We report the surgical outcomes of excisional lymph node and anorectal mucosal biopsies performed internationally and describe the lessons learned. METHODS: Patients were recruited through the Joint Clinical Research Center (JCRC) in Kampala, Uganda, where procedures were performed. Studies were approved by the Institutional Review Boards of the JCRC and the University of Minnesota. Instruments and supplies were shipped to Uganda and prepared onsite. Drugs and skin preparations were purchased locally. Lymph nodes were removed through 1-3 cm incisions with ligatures on lymphovascular pedicles. Incisions were closed with subcuticular sutures and epidermal tape. Two to four pieces of anorectal mucosa were obtained through anoscopes using biopsy forceps. RESULTS: One hundred thirty-eight lymph node biopsies and 98 anorectal mucosal biopsies were performed on 71 patients. Forty-one patients were HIV-positive. Many patients had multiple procedures. Two minor complications resulted: One hematoma and one lymphocele. Despite the cost of travel and lodging, cost per biopsy was lower in Uganda compared with the United States. CONCLUSION: Invasive clinical research can be performed with minimal morbidity in emerging nations with outcomes similar to those found in the United States, but with lower cost.
Assuntos
Pesquisa Biomédica/métodos , Biópsia/métodos , Infecções por HIV/diagnóstico , Infecções por HIV/virologia , HIV/isolamento & purificação , Mucosa Intestinal/virologia , Linfonodos/virologia , Adolescente , Adulto , Pesquisa Biomédica/economia , Biópsia/economia , Países em Desenvolvimento , Feminino , Custos de Cuidados de Saúde , Humanos , Masculino , Resultado do Tratamento , Uganda , Adulto JovemRESUMO
Combination Antiretroviral Therapy (cART) can suppress plasma HIV below the limit of detection in normal assays. Recently reported results suggest that viral replication may continue in some patients, despite undetectable levels in the blood. It has been suggested that the appearance of the circularized episomal HIV DNA artifact 2-LTR following treatment intensification with the integrase inhibitor raltegravir is a marker of ongoing viral replication. Other work has suggested that lymphoid organs may be a site of reduced antiviral penetration and increased viral production. In this study we model the hypothesis that this ongoing replication occurs in lymphoid follicle sanctuary sites and investigate the patterns of 2-LTR formation expected after raltegravir application. Experimental data is used to estimate the reaction and diffusion parameters in the model, and Monte-Carlo simulations are used to explore model behavior subject to variation in these rates. The results suggest that conditions for the formation of an observed transient peak in 2-LTR formation following raltegravir intensification include a sanctuary site diameter larger than 0.2mm, a viral basic reproductive ratio within the site larger than 1, and a total volume of active sanctuary sites above 20mL. Significant levels of uncontrolled replication can occur in the sanctuary sites without measurable changes in the plasma viral load. By contrast, subcritical replication (where the basic reproductive ratio of the virus is less than 1 in all sites) always results in monotonic increases of measured 2-LTR following raltegravir intensification, occurring at levels below the limit of detection.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Modelos Biológicos , Pirrolidinonas/farmacologia , Viremia/virologia , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Linfonodos/virologia , Método de Monte Carlo , Pirrolidinonas/administração & dosagem , Pirrolidinonas/uso terapêutico , Raltegravir Potássico , Carga Viral , Viremia/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Monocyte-derived dendritic cells (DCs) loaded with heat-inactivated HIV are used in therapeutic immunizations. It is not known whether they migrate in vivo to lymph nodes. We used an (111)In-oxine-labeled DC (ILDC) method to visualize the migration of DCs. The activity, time and incubation medium were investigated to obtain the highest cellular viability and radiolabeling yield. A trypan-blue exclusion test was used to determine the cellular viability. In five patients, 2 x 10(6) ILDCs were injected subcutaneously in the arm. An initial dynamic study was performed during the first 5 min after injection. This was followed by static acquisitions at several time points, using a high-resolution (general electric) gamma-camera and quantifying the activity at regions of interest drawn on the injection point. The sensitivity of the gamma-camera was evaluated. The highest number of viable DCs (>83%) and the best radiolabeling yield (>70%) were obtained with 1.11 MBq (111)In-oxine, after 10 min of incubation at 37 degrees C in sodium chloride solution 0.9%. We did not observe migration of ILDCs to local lymph nodes in any patient. However, focal uptake at the place of injection continued during the study period. We observed a higher than expected loss of activity from the injection point (median A(t)/A(0) = 0.60 at day 2), which correlated with an increase in total cytotoxic T lymphocytes (CD8(+) and granzyme B(+) cells) in the lypmphoid tissue observed after immunization (R(2) = 0.92, p = 0.03). If more than 20,000 ILDCs had migrated, they could have been detected. In future trials, a higher number of DCs or alternative methods should be used to assess the migration of DCs to lymph nodes.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/metabolismo , Infecções por HIV/terapia , HIV-1/imunologia , Linfócitos T Citotóxicos/metabolismo , Contagem de Células , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Infecções por HIV/imunologia , HIV-1/patogenicidade , Humanos , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/virologia , Ativação Linfocitária , Monócitos/patologia , Compostos Organometálicos/metabolismo , Oxiquinolina/análogos & derivados , Oxiquinolina/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , VacinasRESUMO
Endotheliotropic herpesvirus causes a fatal disease in young Asian elephants, but there are no methods for identifying latent carriers of the virus. During the postmortem study of one female African elephant and three male and two female Asian elephants, a lymph node located bilaterally caudoventral to the parotid gland, approximately 1.5 to 5 cm below the skin, was identified as suitable for transcutaneous ultrasound-guided biopsy. An ultrasonographic assessment and two biopsies were performed on 39 Asian elephants, and these lymph nodes were classified ultrasonographically as active, inactive or chronically active. The calculated mean (se) volume of 10 active lymph nodes was 17.4 (6.9) cm(3), and that of three chronically active lymph nodes was 10.6 (1.0) cm(3), whereas the mean volume of 17 inactive lymph nodes was 3.1 (0.6) cm(3). The presence of lymph node tissue in samples obtained by ultrasound-guided biopsy from three animals that were maintained under conditions that allowed for additional sampling was confirmed histologically. The dna extracted from the lymphoid tissue and the whole blood of all the elephants was negative for endotheliotropic herpesvirus by PCR.
Assuntos
DNA Viral/isolamento & purificação , Elefantes , Infecções por Herpesviridae/veterinária , Herpesviridae/isolamento & purificação , Linfonodos/patologia , Animais , Animais de Zoológico , Biópsia por Agulha Fina/métodos , Biópsia por Agulha Fina/veterinária , Reservatórios de Doenças/veterinária , Reservatórios de Doenças/virologia , Feminino , Herpesviridae/patogenicidade , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/patologia , Linfonodos/diagnóstico por imagem , Linfonodos/virologia , Masculino , Reação em Cadeia da Polimerase/veterinária , Prevalência , Ultrassonografia , Latência ViralRESUMO
AIDS: Studies on early HIV infection funded by the National Institute of Allergy and Infectious Diseases (NIAID) are reported. Four-year awards were made to researchers at six different institutions to pursue studies on immune functions of cytotoxic T-lymphocytes in HIV infection, distribution of virus between blood and tissues, and whether treatment during acute HIV infection allows the immune system to recover its function.^ieng
Assuntos
Infecções por HIV , Apoio à Pesquisa como Assunto , Progressão da Doença , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Linfonodos/virologia , National Institutes of Health (U.S.) , Linfócitos T Citotóxicos/imunologia , Estados Unidos , ViremiaRESUMO
OBJECTIVE: To assess the antiretroviral effect of a combination of zidovudine, didanosine, lamivudine and saquinavir in plasma, peripheral blood mononuclear cells (PBMC) and lymph-node mononuclear cells (LNMC) after 8 weeks. METHODS: Ten HIV-1 antiretroviral therapy-naive patients were given a combination of oral zidovudine (200 mg three times daily), oral didanosine (200 twice a day), oral lamivudine (150 mg twice a day) and oral saquinavir (600 mg three times daily). HIV-1 plasma RNA was measured by quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR). Infectious HIV-1 in PBMC and LNMC was measured by a coculture technique. HIV-1 RNA in PBMC and LNMC was quantified by RT-PCR. Proviral DNA titres in PBMC and LNMC were measured by endpoint dilution PCR. CD4 T-cells were analysed by flow cytometry. RESULTS: CD4 cell counts rose in all patients (mean increase of 125 +/- 71 CD4 cells x 10(6)/l) and the benefit was greater for patients with fewer than 350 CD4 cells x 10(6)/l (mean increase of 159 +/- 74 CD4 cells x 10(6)/l). Plasma HIV-1 RNA decreased exponentially in all patients (mean decrease of 3.1 log10 after 8 weeks with a mean half-life of 2.2 +/- 0.6 days). HIV-1 RNA showed a decrease of 3.07 log10 in PBMC and of 2.1 log10 in LNMC. The decrease in plasma HIV-1 RNA was consistently associated with the decrease in LNMC. These data were supported by a concomitant drop of HIV-1 infectious titres in PBMC (mean decrease of 1.41 log10) and in LNMC (mean decrease of 2.54 log). CONCLUSIONS: These data show a significant antiretroviral effect of this four-drug combination in blood and lymphoid tissues. However, a greater decrease in HIV-1 RNA was observed in PBMC and in plasma than in lymph node cells.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Leucócitos Mononucleares/virologia , Linfonodos/virologia , Carga Viral , Adulto , Fármacos Anti-HIV/administração & dosagem , Contagem de Linfócito CD4 , DNA Viral , Didanosina/administração & dosagem , Quimioterapia Combinada , Feminino , Infecções por HIV/sangue , HIV-1/genética , Humanos , Lamivudina/administração & dosagem , Linfonodos/patologia , Masculino , Reação em Cadeia da Polimerase , RNA Viral/sangue , Saquinavir/administração & dosagem , Viremia/virologia , Zidovudina/administração & dosagemRESUMO
In vitro amplification of DNA by PCR is a powerful tool to detect small amounts of DNA. It is now widely used for detection of pathogenic agents from extracellular fluids and organs. The use of anion exchange HPLC to quantify the PCR product resulting from the specific amplification of the DNA from the replicative-defective viral DNA responsible for MAIDS is described. This technique allows precise quantification of MAIDS virus DNA in different organs and circumvents the use of radioactivity and gel electrophoresis.