Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19.

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B(0). We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G --> A and 0.001 for 718C --> T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

Preserved central memory and activated effector memory CD4+ T-cell subsets in human immunodeficiency virus controllers: an ANRS EP36 study.

Human immunodeficiency virus (HIV) controllers are rare individuals who spontaneously control HIV type 1 replication for 10 years or more in the absence of antiretroviral treatment. In the present study, HIV controllers (n = 11) maintained potent HIV-specific CD4 responses in spite of very low antigenic loads. Their CD4+ central memory T (T(CM)) cells were characterized by near-normal numbers and preserved interleukin-2 (IL-2) secretion in response to HIV antigens and uniformly high expression of the survival receptor IL-7 receptor alpha (IL-7Ralpha). Controllers expressed CCR7 at higher levels than uninfected controls, suggesting differences in T(CM)-cell homing patterns. CD4+ effector memory T (T(EM))-cell responses were polyfunctional in HIV controllers, while IL-2 secretion was lost in viremic patients. Cytokine production was three times higher in controllers than in treated patients with undetectable viral loads, suggesting an intrinsically more efficient response in the former group. The total CD4+ T(EM)-cell pool underwent immune activation in controllers, as indicated by increased HLA-DR expression, decreased IL-7Ralpha expression, a bias towards gamma interferon production upon polyclonal stimulation, and increased macrophage inflammatory protein 1beta secretion associated with chronic CCR5 down-regulation. Thus, HIV controllers showed a preserved CD4+ T(CM)-cell compartment and signs of potent functional activation in the CD4+ T(EM)-cell compartment. While controllers did not show the generalized immune activation pattern associated with disease progression, they had signs of immune activation restricted to the effector compartment. These findings suggest the induction of an efficient, nondetrimental type of immune activation in patients who spontaneously control HIV.

Assessment of drug resistance mutations in plasma and peripheral blood mononuclear cells at different plasma viral loads in patients receiving HAART.

BACKGROUND: HIV drug resistance mutations both in peripheral blood mononuclear cells (PBMCs) and plasma have the ability to influence the outcome of highly active antiretroviral therapy for HIV patients. PBMCs harbor archival proviral DNA, are a major source of HIV and also underdo latent infection during suppressive HAART. OBJECTIVES: The main objectives of this study were to assess whether specific viral load groups are better predictors of drug resistance and to examine the utility of PBMCs for drug resistance testing during HAART. STUDY DESIGN: Patients were grouped into a plasma panel comprising of 100 patients and a PBMC/plasma panel of 45 patients. These two groups were further divided according to plasma viral load (low, medium and high). Therapy naive patients were also included. Resistance to protease and reverse transcriptase inhibitors was assessed in each group over different viral load categories. RESULTS: Our data indicated that in addition to plasma, PBMCs also are a reliable predictor of drug resistance. Drug resistance mutations analyzed from each panel demonstrated that intermediate and high viral loads were strong indicators of drug resistance in both the plasma and PBMC compartments. Despite this, a significant portion of patients with high viral loads showed reduced levels of drug resistance indicating that factors including poor compliance, drug pharmacokinetics and host genetic factors are also likely to contribute to therapy failure. A significant degree of resistance to NRTI and PI resistance was found in treatment-naive individuals, demonstrating the transmission of circulating drug resistant HIV-1 variants. CONCLUSIONS: Our data emphasize the need for stronger pharmacokinetic evaluation during HAART, especially for patients with intermediate or high plasma viremia. The utility of PBMCs as an alternative source of resistance profiling was also demonstrated, and this approach may benefit the assessment of future drug regimens for HIV-infected patients.

HIV-1 compartmentalization in diverse leukocyte populations during antiretroviral therapy.

CD4+ T lymphocytes are the primary target of human immunodeficiency virus type 1 (HIV-1), but there is increasing evidence that other immune cells in the blood, including CD8+ T lymphocytes and monocytes, are also productively infected. The extent to which these additional cellular reservoirs contribute to ongoing immunodeficiency and viral persistence during therapy remains unclear. In this study, we conducted a detailed investigation of HIV-1 diversity and genetic structure in CD4+ T cells, CD8+ T cells, and monocytes of 13 patients receiving highly active antiretroviral therapy (HAART). Analysis of molecular variance and nonparametric tests performed on HIV-1 envelope sequences provided statistically significant evidence of viral compartmentalization in different leukocyte populations. Signature pattern analysis and predictions of coreceptor use provided no evidence that selection arising from viral tropism was responsible for the genetic structure observed. Analysis of viral genetic variation in different leukocyte populations demonstrated the action of founder effects as well as significant variation in the extent of genetic differentiation between subpopulations among patients. In the absence of evidence for leukocyte-specific selection, these features were supportive of a metapopulation model of HIV-1 replication as described previously among HIV-1 populations in the spleen. Compartmentalization of the virus in different leukocytes may have significant implications for current models of HIV-1 population genetics and contribute to the highly variable way in which drug resistance evolves in different individuals during HAART.

Reservoirs of HIV-1 in vivo: implications for antiretroviral therapy.

The eradication of HIV-1 from infected individuals remains the ultimate goal of all anti-HIV therapeutics. Although highly active antiretroviral therapy (HAART) has led to a profound decrease in morbidity and mortality in infected people by suppressing HIV replication, the virus continues to evolve slowly during therapy even when patients achieve below detectable levels of HIV in plasma. HIV-1 persists in latently infected memory CD4+ T cells and there is minimal decay of HIV in this compartment despite prolonged HAART. Various other reservoirs and sanctuary sites harboring HIV are also established in vivo during antiretroviral therapy. Collectively these sites represent a major impediment to the eradication of HIV-1. This review presents a detailed overview of various reservoir sites in vivo, and discusses their impact on the success and failure of HAART for HIV patients. In addition, it addresses the effect of sub-optimal drug concentrations on reservoir establishment and outlines future therapeutic strategies to counteract these reservoirs and sanctuaries.

Obstacles to successful antiretroviral treatment of HIV-1 infection: problems & perspectives.

Mutations in human immunodeficiency virus type 1 (HIV-1) are a major impediment to successful highly active antiretroviral therapy (HAART) and the design of anti-HIV vaccines. Although HAART has made long-term suppression of HIV a reality, drug resistance, drug toxicity, drug penetration, adherence to therapy, low levels of continued viral replication in cellular reservoirs and augmentation of host immune responses are some of the most important challenges that remain to be sorted out. Continuing viral replication in the face of HAART leads to the accumulation of drug resistance mutations, increase in viral loads and eventual disease progression. Patients who fail therapy have minimal options for their clinical management. Therefore, a clear understanding of the pathogenesis of drug-resistant HIV-1, and all of the issues that influence the success of HAART is urgently needed. In the present article, we discuss various obstacles to HIV therapy, and provide perspectives relating to these issues that are critical in determining the success or failure of HAART.

Human immunodeficiency virus interactions with CD8+ T lymphocytes.

Human immunodeficiency virus (HIV)-specific CD8+ T cells can mediate anti-HIV activity by both cytolytic (cytotoxic T lymphocyte or CTL) and non-cytolytic mechanisms (antiviral) and play a crucial role in HIV pathogenesis. Both mechanisms actively contribute to the control of HIV in vivo. The non-cytolytic CD8+T cells from individuals infected with HIV suppress virus replication in CD4+ T cells in vitro by a non-cytolytic mechanism that involves interplay of several chemokines and an unidentified secreted soluble CD8 (+)-cell antiviral factor (CAF). There is immense value of these two distinct CD8 activities in anti-HIV responses and their necessity to be maintained during highly active antiretroviral therapy (HAART). The aim of this review is to provide an overview of some of the novel aspects of CD8+ T cell interactions with HIV, their role in HIV pathogenesis, HAART therapy, HIV disease progression, gene expression and interactions with other cell types during HIV infection.

Genetic analyses reveal structured HIV-1 populations in serially sampled T lymphocytes of patients receiving HAART.

HIV-1 infection and compartmentalization in diverse leukocyte targets significantly contribute to viral persistence during suppressive highly active antiretroviral therapy (HAART). Longitudinal analyses were performed on envelope sequences of HIV-1 populations from plasma, CD4+ and CD8+ T lymphocytes in 14 patients receiving HAART and 1 therapy-naive individual. Phylogenetic reconstructions and analysis of molecular variance revealed that HIV-1 populations in CD4+ and CD8+ T cells remained compartmentalized over time in most individuals. Analyses of viral genetic variation demonstrated that, despite compartmentalization remaining over time, viral subpopulations tended not to persist and evolve but instead broke down and became reconstituted by new founder viruses. Due to the profound impact of HAART on viral evolution, it was difficult to discern whether these dynamics were ongoing during treatment or predominantly established prior to the commencement of therapy. The genetic structure and viral founder effects observed in serially sampled T lymphocyte populations supported a scenario of metapopulation dynamics in the tissue(s) where different leukocytes become infected, a factor likely to contribute to the highly variable way that drug resistance evolves in different individuals during HAART.

Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification.

The severe acute respiratory syndrome (SARS) epidemic of 2003 was responsible for 774 deaths and caused significant economic damage worldwide. Since July 2003, a number of SARS cases have occurred in China, raising the possibility of future epidemics. We describe here a rapid, sensitive, and highly efficient assay for the detection of SARS coronavirus (SARS-CoV) in cultured material and a small number (n = 7) of clinical samples. Using rolling circle amplification (RCA), we were able to achieve sensitive detection levels of SARS-CoV RNA in both solid and liquid phases. The main advantage of RCA is that it can be performed under isothermal conditions with minimal reagents and avoids the generation of false-positive results, a problem that is frequently encountered in PCR-based assays. Furthermore, the RCA technology provides a faster, more sensitive, and economical option to currently available PCR-based methods.

Differential cellular distribution of HIV-1 drug resistance in vivo: evidence for infection of CD8+ T cells during HAART.

This study presents a detailed analysis of HIV-1 populations isolated from total PBMC, plasma, CD4+ T cells, CD8+ T cells, and monocytes/macrophages in 13 patients receiving HAART. Sequence analysis of the reverse transcriptase and protease genes indicated that viral strains isolated from different blood leukocytes were genetically distinct in each subject. Notably, HIV variants isolated from CD8+ T cells were distantly related to strains derived from other blood cell types, providing evidence for the strain-specific infection of CD8+ T cells in vivo. Compartmentalization of drug resistance mutations in specific blood cell types was observed in approximately 50% of patients. The prevalence of resistance mutations was higher in either CD4+ T cells or monocytes/macrophages in these subjects. However, CD8+ T cells showed markedly lower levels of viral drug resistance in these patients, indicating a lack of viral replication in this compartment. This study is the first to demonstrate the differential distribution of HIV drug resistance in different blood cell types during HAART and provides new insights into the infection of CD8+ T cells in vivo.