RESUMEN
Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-21-4. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection5,6. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors5-8. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.
Asunto(s)
Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/virología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Vacunas Sintéticas/inmunología , Vacuna nCoV-2019 mRNA-1273 , Adulto , Anciano , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Vacuna BNT162 , Vacunas contra la COVID-19/genética , Microscopía por Crioelectrón , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/ultraestructura , Femenino , Humanos , Inmunización Secundaria , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Memoria Inmunológica/inmunología , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación , Pruebas de Neutralización , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/genética , Vacunas de ARNmRESUMEN
To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines 1,2 . These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known 3-6 . Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers 3,5,6 . Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection 7,8 . However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.
RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.
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Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Inmunidad Humoral/inmunología , SARS-CoV-2/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/genética , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Biopsia , COVID-19/sangre , Estudios de Cohortes , Técnica del Anticuerpo Fluorescente , Humanos , Inmunidad Humoral/genética , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Memoria Inmunológica/inmunología , Intestinos/inmunología , Persona de Mediana Edad , Mutación , Hipermutación Somática de Inmunoglobulina , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Factores de Tiempo , Adulto JovenRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 4 months after coronavirus disease-2019 (COVID-19) onset, using immunofluorescence, or polymerase chain reaction, revealed persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.
RESUMEN
A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs.
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Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Fragmentos de Péptidos/inmunología , Animales , Femenino , Macaca mulatta , Polisacáridos/inmunologíaRESUMEN
During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/análisis , Anticuerpos Antivirales/análisis , Especificidad de Anticuerpos , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/prevención & control , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Pandemias , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Virales/inmunología , Adulto JovenRESUMEN
During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
RESUMEN
Antibody cloning from single B cells is an essential tool for characterizing humoral immune responses and obtaining valuable therapeutic and analytical reagents. Antibody cloning from individuals with high serologic titers to HIV-1, Influenza, Malaria and ZIKV has led to new insights that inform vaccine design efforts. In contrast to humans and mice, less is known about antibody cloning from single B cells in macaques. Here, we describe a protocol to identify and purify single antigen-specific macaque B cells, and subsequently clone and produce macaque monoclonal antibodies. The sorting strategy requires the use of a combination of fluorochrome labeled antigens and omission of anti-IgG antibodies that can interfere with antigen binding and vice versa. Optimized methods for macaque antibody gene amplification, DNA preparation for antibody production and antibody screening by ELISA are also presented.
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Anticuerpos Monoclonales/aislamiento & purificación , Linfocitos B/inmunología , Separación Celular/métodos , Clonación Molecular/métodos , Anticuerpos Anti-VIH/aislamiento & purificación , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/aislamiento & purificación , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/metabolismo , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , Antígenos VIH/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Inmunidad Humoral/inmunología , Macaca mulatta/sangre , Macaca mulatta/inmunología , Macaca mulatta/virología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
BACKGROUND: Commercial sex venues (CSV), bathhouses and sex clubs, have a long history of serving a high-risk population. In those facilities, patrons engage in multiple sexual encounters and often in high-risk sexual behaviors. Designing prevention interventions specifically for CSVs could be an effective way to increase testing and control HIV transmission. METHODS: In collaboration with the AIDS Healthcare Foundation (AHF), our team distributed free HIV self-test kits using vending machines located at two CSVs in Los Angeles, California. Test kit dispensing rate was monitored remotely. Patrons receiving a test kit were surveyed regarding their testing experience, test result and follow up. Linkage to care was offered to participants. RESULTS: During 18 months, 1,398 kits were dispensed. The survey was completed by 110 patrons (response rate =7.9%). Among those who reported that they used the test kit (n=96), 17 (17.7%) participants reported a first-time reactive HIV result. At the time of the survey, six participants with reactive results reported seeking confirmatory testing and linkage to care and four had initiated treatment. Two participants requested linkage-to-care assistance. Participants reported valuing the privacy and convenience of the vending machine but were skeptical on the accuracy of their result. The startup cost, including the purchase of two vending machines, was $10,000 and the recurring cost (monitoring, test kits, personnel) was $33.81 per kit vended. CONCLUSIONS: While survey response was low, our results demonstrate that an intervention using vending machines and HIV self-test kits in CSVs was acceptable, feasible, used by the CSV patrons and can help identify new HIV cases.
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Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections are frequently asymptomatic, requiring highly accurate diagnostic tests and proper management to prevent further transmission. We compared two nucleic acid tests, Xpert® CT/NG (Cepheid, Sunnyvale, CA) point-of-care platform and at an offsite clinical laboratory with Aptima Combo 2® (Hologic, Inc., San Diego, CA) assay, for the detection of extragenital infection in patients at an STI clinic in Hollywood, CA.We calculated concordance between the two assays and used the exact binomial method to calculate 95% confidence intervals (CIs) for each specimen type and pathogen.The concordance between the two assays was 97.7% (95% CI: 95.7%,99.0%) for 393 paired CT rectal results, 98.2% (95% CI: 96.4%,99.3%) for 391 paired NG rectal results and 98.4% (95% CI: 96.8%,99.4%) for 448 paired NG pharyngeal results.The performance of Xpert® CT/NG assay in point-of-care testing in extragenital specimens was highly similar to the laboratory-based platform.
Asunto(s)
Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/aislamiento & purificación , Gonorrea/microbiología , Neisseria gonorrhoeae/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Faringe/microbiología , Recto/microbiología , Adulto , California/epidemiología , Chlamydia trachomatis/genética , Intervalos de Confianza , Investigación sobre Servicios de Salud , Homosexualidad Masculina , Humanos , Masculino , Salud del Hombre , Neisseria gonorrhoeae/genética , Sistemas de Atención de PuntoRESUMEN
BACKGROUND: Patients with chronic kidney disease (CKD) experience high rates of atherosclerotic cardiovascular disease and death that are not fully explained by traditional risk factors. In animal studies, defective cellular cholesterol efflux pathways which are mediated by the ATP binding cassette transporters ABCA1 and ABCG1 are associated with accelerated atherosclerosis. We hypothesized that cholesterol efflux in humans would vary in terms of cellular components, with potential implications for cardiovascular disease. METHODS: We recruited 120 CKD patients (eGFR<30mL/min/1.73m2) and 120 control subjects (eGFR ≥60mL/min/1.73m2) in order to measure cholesterol efflux using either patients' HDL and THP-1 macrophages or patients' monocytes and a flow cytometry based cholesterol efflux assay. We also measured cell-surface levels of the common ß subunit of the IL-3/GM-CSF receptor (IL-3Rß) which has been linked to defective cholesterol homeostasis and may promote monocytosis. In addition, we measured plasma inflammatory cytokines and plasma metabolite profiles. RESULTS: There was a strong positive correlation between cell-surface IL-3Rß levels and monocyte counts in CKD (P<0.001). ABCA1 mRNA was reduced in CKD vs. control monocytes (P<0.05), across various etiologies of CKD. Cholesterol efflux to apolipoprotein A1 was impaired in monocytes from CKD patients with diabetic nephropathy (P<0.05), but we found no evidence for a circulating HDL-mediated defect in cholesterol efflux in CKD. Profiling of plasma metabolites showed that medium-chain acylcarnitines were both independently associated with lower levels of cholesterol transporter mRNA in CKD monocytes at baseline (P<0.05), and with cardiovascular events in CKD patients after median 2.6years of follow-up. CONCLUSIONS: Cholesterol efflux in humans varies in terms of cellular components. We report a cellular defect in ABCA1-mediated cholesterol efflux in monocytes from CKD patients with diabetic nephropathy. Unlike several traditional risk factors for atherosclerotic cardiovascular disease, plasma metabolites inversely associated with endogenous cholesterol transporters predicted cardiovascular events in CKD patients. (Funded by the National Institute of Diabetes and Digestive and Kidney DiseasesK23DK097288 and others.).
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Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/epidemiología , Colesterol/metabolismo , Metaboloma , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/complicaciones , Transportador 1 de Casete de Unión a ATP/metabolismo , Anciano , Transporte Biológico , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Carnitina/análogos & derivados , Carnitina/metabolismo , Línea Celular , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/complicaciones , Nefropatías Diabéticas/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Factores de RiesgoRESUMEN
Alcohol use is correlated with unprotected sex, which may place young men who have sex with men (YMSM) who use alcohol with sex at increased risk for contracting HIV. However, little is known about how this link develops. This study used qualitative interviews to explore how alcohol became associated with sex and sexual risk among YMSM. We purposively sampled 20 Black and 20 Latino YMSM (N = 40), ages 21 to 24, who used substances (alcohol, marijuana, and crystal methamphetamine) with sex. Interviews focused on participants' personal histories to trace how these associations developed for each individual. Drawing on sexual script, emotion regulation, and alcohol expectancy theories, analyses followed a modified grounded theory approach. Participants stated that alcohol enabled them to engage in sexual behaviors with men that they wanted to try, allowing them to be more "bold," overcome stigma about homosexuality, and feel increased comfort with their sexual desires and identities. The use of alcohol during sex was helpful to some of the participants but could also lead to sexual risk behaviors. Intervention programs seeking to reduce alcohol misuse and sexual risk should take into account how YMSM conceptualize associations between alcohol and sex. These programs may be more effective if they provide support for sexual identity exploration.
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Consumo de Bebidas Alcohólicas/etnología , Negro o Afroamericano/etnología , Hispánicos o Latinos/etnología , Homosexualidad Masculina/etnología , Asunción de Riesgos , Conducta Sexual/psicología , Adulto , Consumo de Bebidas Alcohólicas/efectos adversos , Humanos , Masculino , Conducta Sexual/efectos de los fármacos , Adulto JovenRESUMEN
OBJECTIVES: We examined the effectiveness of peer ethnography to gain insider views on substance use and sex among a diverse range of high-risk substance-using Black and Latino young men who have sex with men. METHODS: We recruited 9 peer ethnographers aged 21 to 24 years from youth programs for the lesbian, gay, bisexual, and transgender community in Los Angeles, California, and trained them in ethnography, study protocol, and human participant protection. Peer ethnographers collected 137 single-spaced pages of field notes in 2009 and 2010 derived from observation of 150 members of the target population. RESULTS: Peer ethnography revealed local language and phrasing and provided a window into new and different social contexts. Peers provided valuable information on current trends in substance use, revealing themes that needed to be addressed in further research, such as the use of substances during sex to "clock coin" (exchange sex for money and substances). These data enabled us to refine our recruitment strategies and ask more culturally relevant questions in a later phase of the study. CONCLUSIONS: The peer ethnography method can provide a sound basis for further research phases in multistage studies on numerous other social issues and with other hard-to-reach populations.
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Antropología Cultural/métodos , Infecciones por VIH/etnología , Homosexualidad Masculina/psicología , Grupo Paritario , Trastornos Relacionados con Sustancias/psicología , Negro o Afroamericano , Comorbilidad , Infecciones por VIH/epidemiología , Investigación sobre Servicios de Salud/métodos , Disparidades en el Estado de Salud , Hispánicos o Latinos , Homosexualidad Masculina/etnología , Humanos , Los Angeles/epidemiología , Masculino , Factores de Riesgo , Trastornos Relacionados con Sustancias/etnología , Adulto JovenRESUMEN
Texas-Red-asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles.
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Proteoma/análisis , Vesículas Transportadoras/química , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Animales , Transporte Biológico , Línea Celular , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/ultraestructura , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Hígado/química , Hígado/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Factores de Intercambio de Guanina Nucleótido Rho , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteínas de Unión al GTP rab1/genéticaRESUMEN
Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.
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Dineínas/fisiología , Cinesinas/fisiología , Microtúbulos/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Anticuerpos/inmunología , Transporte Biológico , Complejo Dinactina , Dineínas/análisis , Cinesinas/análisis , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/inmunología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Motoras Moleculares/inmunología , Ratas , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab4/análisis , Proteínas de Unión al GTP rab4/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Upon thymus entry, thymic-homing progenitors undergo distinct phases of differentiation as they migrate through the cortex to the capsule, suggesting that the signals that induce these differentiation steps may be stratified in corresponding cortical regions. To better define these regions, we transplanted purified stem cells into nonirradiated congenic recipients and followed their differentiation with respect to both tissue location and time. The earliest progenitors (DN1) remained confined to a very narrow region of the cortex for about the first 10 d of intrathymic residence; this region virtually overlaps the sites of thymic entry, suggesting that DN1 cells move very little during this lengthy period of proliferation and lineage commitment. Movement out of this region into the deeper cortex is asynchronous, and corresponds to the appearance of DN2 cells. Differentiation to the DN3 stage correlates with movement across the midpoint of the cortex, indicating that stromal signals that induce functions such as TCR gene rearrangement reside mainly in the outer half of the cortex. The minimum time to reach the capsule, and thus transit to the DP stage, is approximately 13 d, with the average time a few days longer. These findings reveal for the first time the kinetics of steady-state progenitor differentiation in the thymus, as well as defining the boundaries of cortical regions that support different phases of the differentiation process. We also show that the first lineage-positive progeny of transplanted stem cells to appear in the thymus are dendritic cells in the medulla, suggesting that each new wave of new T cell production is preceded by a wave of regulatory cells that home to the medulla and ensure efficient tolerance and selection.
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Diferenciación Celular/fisiología , Células Madre Hematopoyéticas/fisiología , Transducción de Señal/fisiología , Trasplante de Células Madre , Timo/metabolismo , Animales , Movimiento Celular , Células Dendríticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Linfocitos T/metabolismo , Timo/citología , Quimera por TrasplanteRESUMEN
In this manuscript, we describe modifications to a commercial three-laser benchtop flow cytometer, as well as relevant biological methods, that allow analysis of up to five immunofluorescent parameters together with an ultraviolet (UV)-excitable DNA stain. This method allows expanded capacity for multiparameter immunophenotyping of complex mixed cell populations, together with accurate measurements of DNA content (cell cycle) or cell viability, on a stable, end-user operated platform.
Asunto(s)
ADN/análisis , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente/métodos , Ciclo Celular , Supervivencia Celular , Citometría de Flujo/instrumentación , Técnica del Anticuerpo Fluorescente/instrumentación , Colorantes Fluorescentes , Indoles , Rayos Láser , Propidio , Coloración y Etiquetado/métodosRESUMEN
During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus.