HIV-1 Infection of DC: Evidence for the Acquisition of Virus Particles from Infected T Cells by Antigen Uptake Mechanism

Dendritic cells (DC) play a pivotal role in transmission and dissemination of HIV-1. Earlier studies reported that DC present at the site of infection trap virus particles via DC-SIGN and transfer the virus to the interacting naïve T cells. This prompted us to ask the question whether DC could acquire virus from infected T cells during DC-T cell interaction. To address this, we investigated the likely transfer of virus from HIV-1 infected T cells to DC and the underlying mechanisms involved. Results indicate that DC acquire virus from infected T cells via antigen uptake mechanism and this results in infection of DC with expression of proteins directed by viral DNA. Further studies with HIV-1 lacking the Env protein also resulted in infection of DC. The use of antibodies against DC-SIGN and DC-SIGN-R ruled out a role for receptor in the infection of DC. Additional data show that DC infection is directly correlated with the ability of DC to take up antigen from infected T cells. Overall, these studies provide evidence to suggest that HIV-1, besides infecting immune cells, also utilizes immunological mechanism(s) to acquire and disseminate virus

Temporal transcriptional response to latency reversing agents identifies specific factors regulating HIV-1 viral transcriptional switch

Background: Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4+ T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. Results and discussion: In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-aα, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. Conclusion: These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4+ T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency

The co-receptor signaling model of HIV-1 pathogenesis in peripheral CD4 T cells

HIV-mediated CD4 depletion is the hallmark of AIDS and is the most reliable predictor of disease progression. While HIV replication is associated with CD4 depletion in general, plasma viremia by itself predicts the rate of CD4 loss only minimally in untreated patients. To resolve this paradox, I hypothesize the existence of a subpopulation of R5X4-signaling viruses. I also suggest that the gradual evolution and emergence of this subpopulation are largely responsible for the slow depletion of peripheral CD4 T cells

Comparative Expression Profile of miRNA and mRNA in Primary Peripheral Blood Mononuclear Cells Infected with Human Immunodeficiency Virus (HIV-1)

Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA) and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1

Chemokine Coreceptor Signaling in HIV-1 Infection and Pathogenesis

Binding of the HIV-1 envelope to its chemokine coreceptors mediates two major biological events: membrane fusion and signaling transduction. The fusion process has been well studied, yet the role of chemokine coreceptor signaling in viral infection has remained elusive through the past decade. With the recent demonstration of the signaling requirement for HIV latent infection of resting CD4 T cells, the issue of coreceptor signaling needs to be thoroughly revisited. It is likely that virus-mediated signaling events may facilitate infection in various immunologic settings in vivo where cellular conditions need to be primed; in other words, HIV may exploit the chemokine signaling network shared among immune cells to gain access to downstream cellular components, which can then serve as effective tools to break cellular barriers. This virus-hijacked aberrant signaling process may in turn facilitate pathogenesis. In this review, we summarize past and present studies on HIV coreceptor signaling. We also discuss possible roles of coreceptor signaling in facilitating viral infection and pathogenesis

Manipulation of Costimulatory Molecules by Intracellular Pathogens: Veni, Vidi, Vici!!

Some of the most successful pathogens of human, such as Mycobacterium tuberculosis (Mtb), HIV, and Leishmania donovani not only establish chronic infections but also remain a grave global threat. These pathogens have developed innovative strategies to evade immune responses such as antigenic shift and drift, interference with antigen processing/presentation, subversion of phagocytosis, induction of immune regulatory pathways, and manipulation of the costimulatory molecules. Costimulatory molecules expressed on the surface of various cells play a decisive role in the initiation and sustenance of immunity. Exploitation of the “code of conduct” of costimulation pathways provides evolutionary incentive to the pathogens and thereby abates the functioning of the immune system. Here we review how Mtb, HIV, Leishmania sp., and other pathogens manipulate costimulatory molecules to establish chronic infection. Impairment by pathogens in the signaling events delivered by costimulatory molecules may be responsible for defective T-cell responses; consequently organisms grow unhindered in the host cells. This review summarizes the convergent devices that pathogens employ to tune and tame the immune system using costimulatory molecules. Studying host-pathogen interaction in context with costimulatory signals may unveil the molecular mechanism that will help in understanding the survival/death of the pathogens. We emphasize that the very same pathways can potentially be exploited to develop immunotherapeutic strategies to eliminate intracellular pathogens

Novel assay reveals a large, inducible, replication-competent HIV-1 reservoir in resting CD4+ T cells.

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication-competent virus persist in resting CD4+ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that can accurately and rapidly quantify inducible, replication-competent latent HIV-1 from resting CD4+ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible, replication-competent latent HIV-1. This assay has several advantages over existing technology in that it (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive; and (iv) can be readily adapted into a high-throughput format. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates

High T-cell immune activation and immune exhaustion among individuals with suboptimal CD4 recovery after 4 years of antiretroviral therapy in an African cohort

<p>Abstract</p> <p>Background</p> <p>Antiretroviral therapy (ART) partially corrects immune dysfunction associated with HIV infection. The levels of T-cell immune activation and exhaustion after long-term, suppressive ART and their correlation with CD4 T-cell count reconstitution among ART-treated patients in African cohorts have not been extensively evaluated.</p> <p>Methods</p> <p>T-cell activation (CD38+HLA-DR+) and immune exhaustion (PD-1+) were measured in a prospective cohort of patients initiated on ART; 128 patient samples were evaluated and subcategorized by CD4 reconstitution after long-term suppressive treatment: Suboptimal [median CD4 count increase 129 (-43-199) cells/μl], N = 34 ], optimal [282 (200-415) cells/μl, N = 64] and super-optimal [528 (416-878) cells/μl, N = 30].</p> <p>Results</p> <p>Both CD4+ and CD8 T-cell activation was significantly higher among suboptimal CD4 T-cell responders compared to super-optimal responders. In a multivariate model, CD4+CD38+HLADR+ T-cells were associated with suboptimal CD4 reconstitution [AOR, 5.7 (95% CI, 1.4-23, <it>P </it>= 0.014)]. T-cell exhaustion (CD4+PD1+ and CD8+PD1+) was higher among suboptimal relative to optimal (<it>P </it>< 0.001) and super-optimal responders (P < 0.001). T-cell exhaustion was significantly associated with suboptimal responders [AOR, 1.5 (95%CI, 1.1-2.1), <it>P </it>= 0.022].</p> <p>Conclusion</p> <p>T-cell activation and exhaustion persist among HIV-infected patients despite long-term, sustained HIV-RNA viral suppression. These immune abnormalities were associated with suboptimal CD4 reconstitution and their regulation may modify immune recovery among suboptimal responders to ART.</p

Análisis de la endotoxemia en el postoperatorio de cirugía cardiaca

Desde el inicio de la cirugía cardiaca, el desarrollo de nuevas técnicas quirúrgicas, los avances en los métodos de circulación extracorpórea (CEC) y los tratamientos en cuidados intensivos hacen que la mortalidad de estos pacientes haya disminuido de casi el 100% hasta un 5-6% en la actualidad. No obstante, estas cifras se mantienen estables durante los últimos años. La respuesta inflamatoria asociada a la CEC es un proceso que puede llegar a desarrollar complicaciones mayores incluyendo insuficiencia respiratoria, shock, fracaso renal e incluso fracaso multiorgánico. La cirugía con CEC provoca cambios sistémicos importantes que inician el proceso de SIRS. Entre ellos se encuentra la hipoperfusión esplénica, que podría producir daños en la mucosa intestinal alterando la permeabilidad de la barrera favoreciendo así la traslocación bacteriana y la endotoxemia. La endotoxina es un lipopolisacárido de la pared celular de las BGN y es reconocido como un importante estímulo para el desarrollo del SIRS. Su presencia en pacientes sometidos a CEC ha sido ampliamente reconocida, pero la magnitud de la endotoxemia así como su correlación con la evolución clínica y la aparición de complicaciones varía ampliamente entre los estudios. Según algunos estudios, la concentración sistémica de endotoxinas se correlaciona estrechamente con el grado de disfunción cardiovascular, duración de la cirugía, tiempo de CEC, tiempo de isquemia y necesidad de aminas vasoactivas, lo que se podría resumir en todas aquellas situaciones que potencialmente podrían favorecer una situación de hipopefusión esplácnica. Existen diferentes técnicas para detectar la presencia de la endotoxemia. Tradicionalmente, se había cuantificado la cantidad de endotoxina mediante un análisis in vitro denominado lisado de Amebocitos del Limulus Polyphemus (LAL), pero este test nunca ha sido aprobado por la FDA pasa su uso en sangre. Esto ha motivado el descubrimiento de un nuevo test llamado ensayo de actividad de endotoxina (EAA) (Spectral Diagnostics, Toronto, ON, Canadá), que consta de un kit de prueba rápida de quimioluminiscencia inmunodiagnóstica que se puede realizar en menos de 1 hora, aprobado por la FDA para su realización en líquidos biológicos como es la sangre. Los objetivos marcados del estudio fueron detectar la presencia de endotoxemia en el postoperatorio de cirugía cardiaca utilizando un nuevo método diagnóstico así como establecer los factores de riesgo de presentar endotoxemia y establecer la implicación pronóstica de esta. El estudio se ha realizado en la Unidad de Cuidados Intensivos del Hospital Germans Trías i Pujol, Badalona. Incluimos un total de 107 pacientes, la mayoría varones (69%), con una edad media de 66 años (36-87). El 38% tenían DM, 71% HTA y un 12% vasculopatía periférica. La mediana del EuroSCORE I fue de 7 (0-16). Solo 24 pacientes presentaron endotoxemia alta (≥0,4EA). La duración mediana de la CEC fue de 95 min (24-300) con un tiempo de isquemia (ClAo) de 72 min (17-175). El 37% de los pacientes requirieron transfusión de concentrados de hematíes. Los resultados de nuestro estudio indican que en el postoperatorio de cirugía cardiaca existe endotoxemia al menos en grado moderado y que esta puede tener utilidad en la detección de aquellos pacientes que pueden presentar infección postoperatoria precoz. Como factor de riesgo de endotoxemia, hemos observado que aquellos pacientes con vasculopatía periférica y los que requieren trasfusión de mayor cantidad de concentrados de hematíes durante la intervención son los que presentan mayor riesgo de presentar endotoxemia en el postoperatorio inmediato.Since the beginning of cardiac surgery, development of new surgical techniques, advances in methods of cardiopulmonary bypass (CPB) and intensive care treatments, mortality has decreased from almost 100% to 5- 6% today. However, these figures have remained stable in recent years. The inflammatory response to CPB has been implicated in many of the postoperative clinical problems that often occur in these patients including respiratory failure, postoperative shock states, renal failure and multiple organ dysfunction syndrome. These systemic changes may be due to many different mechanisms. One mechanism, is attributed to splanchnic hypoperfusion that might cause harm to the intestinal mucosa by altering the barrier's permeability, thus favouring bacterial translocation and endotoxemia. Endotoxin is a lipopolysaccharide in the membrane of GNB and is known to be one of the most potent activators known of innate immunity and the inflammatory response in humans. In patients subjected to cardiac surgery, transient endotoxemia has been shown in many occasions, which seems to be closely related to extracorporeal circulation, but the magnitude of endotoxemia and their correlation with clinical evolution and the development of complications vary widely between studies. According to some studies, systemic endotoxin concentration is closely correlated with the degree of cardiovascular dysfunction, duration of CPB, ischemic time and need for vasoactive amines, which can be summarized in all situations that could potentially favor a situation of splanchnic hypoperfusion. There are different techniques to detect the presence of endotoxemia. The amount of endotoxin has traditionally been quantified by the analysis known as "Limulus amebocyte lysate" (LAL), but this test has never been approved by the FDA for clinical use in humans. This has led to the discovery of a new test called endotoxin activity assay (EAA) (Spectral Diagnostics, Toronto, ON, Canada), comprising a rapid chemiluminiscent immunodiagnostic test kit that can be performed in less than 1 hour, approved by the FDA for its realization in biological fluids such as it is blood. The purpose of the study was to assess the prevalence of endotoxemia related to CPB in a cohort of patients undergoing cardiac surgery, using the EAA test. There was also investigated the criteria for high risk of endotoxemia and the association between endotoxemia and the development of adverse clinical events including length of stay and mortality. The study was performed in the Intensive Care Unit of Germans Trias i Pujol Hospital, in Badalona. A total of 107 patients were enrolled. Of these 107 patients, the median age was 66 years (36-87), most were males (69%), 38% had DM, 71% HTA and 12% peripheral vascular disease. Median EuroSCORE I was 7 (0-6). Only 23 patients had EAA ≥0,4 EA. Median CBP time was 95 (24-300) and isquemic time 68 (17-175) minutes. 37% required blood transfusion. The results of the study indicate that in postoperative cardiac surgery there is endotoxemia at least in moderate degree, and that the presence of endotoxemia is significantly related to early postoperative infection. As a risk factor, we found that patients with peripheral vascular disease and transfused more than 2 during surgery are those with increased risk of endotoxemia