Measurement of absolute T cell receptor rearrangement diversity.

T cell receptor (TCR) diversity is critical for adaptive immunity. Existing methods for measuring such diversity are qualitative, expensive, and/or of uncertain accuracy. Here, we describe a method and associated reagents for estimating the absolute number of unique TCR Vβ rearrangements present in a given number of cells or volume of blood.

A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers.

HIV-infected individuals maintaining undetectable viremia in the absence of therapy (HIV controllers) often maintain high HIV-specific T cell responses, which has spurred the development of vaccines eliciting HIV-specific T cell responses. However, controllers also often have abnormally high T cell activation levels, potentially contributing to T cell dysfunction, CD4+ T cell depletion, and non-AIDS morbidity. We hypothesized that a weak T regulatory cell (Treg) response might contribute to the control of viral replication in HIV controllers, but might also contribute to generalized immune activation, contributing to CD4+ T cell loss.

The human fetal immune response to hepatitis C virus exposure in utero.

Background: Although the rate of mother-to-child transmission of hepatitis C virus (HCV) is low, the effect of HCV exposure in utero on the fetal immune system is unknown.

Methods: Umbilical cord blood was obtained from 7 neonates born to HCV-seropositive, HCV RNA-positive women and 8 neonates born to HCV-seronegative women. Cord blood mononuclear cells were analyzed by immunophenotyping and by intracellular cytokine staining after HCV-specific and polyclonal stimulation.

HIV-specific CD4+ T cells may contribute to viral persistence in HIV controllers.

Background: Human immunodeficiency virus (HIV)--infected individuals maintaining plasma HIV RNA levels <75 copies/mL in the absence of therapy ("HIV controllers") often maintain high HIV-specific T cell responses, which likely contribute to the control of viral replication. Despite robust immune responses, these individuals never eradicate HIV infection. We hypothesized that HIV-specific CD4(+) T cells might serve as target cells for HIV, contributing to viral persistence in this setting.

HIV disease progression correlates with the generation of dysfunctional naive CD8(low) T cells.

HIV infection can result in depletion of total CD4(+) T cells and naive CD8(+) T cells, and in the generation of dysfunctional effector CD8(+) T cells. In this study, we show that naive CD8(+) T cells in subjects with progressive HIV disease express low levels of CD8α and CD8β chains. Such naive CD8(low) T cells display broad signaling defects across the T-cell receptor complex, and their appearance correlates with generalized up-regulation of major histocompatibility complex class I (MHC-I) antigens on peripheral blood mononuclear cells (PBMCs).

Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans.

Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development.

Pharmacogenetics of intravenous and oral busulfan in hematopoietic cell transplant recipients.

Kinetics-based dose targeting is often conducted in hematopoietic cell transplant (HCT) patients conditioned with intravenous (IV) or oral busulfan to lower rates of rejection, nonrelapse mortality, and relapse. Using the candidate gene approach, the authors evaluated whether busulfan clearance was associated with polymorphisms in the genes regulating the predominant metabolizing enzymes involved in busulfan conjugation, specifically glutathione S-transferase (GST) isoenzymes A1 (GSTA1) and M1 (GSTM1). Busulfan clearance was estimated after the morning dose on days 1, 2, and 3; each patient's average clearance was used for analyses.

IL-22+ CD4+ T cells are associated with therapeutic trichuris trichiura infection in an ulcerative colitis patient.

Ulcerative colitis, a type of inflammatory bowel disease, is less common in countries endemic for helminth infections, suggesting that helminth colonization may have the potential to regulate intestinal inflammation in inflammatory bowel diseases. Indeed, therapeutic effects of experimental helminth infection have been reported in both animal models and clinical trials. Here, we provide a comprehensive cellular and molecular portrait of dynamic changes in the intestinal mucosa of an individual who infected himself with Trichuris trichiura to treat his symptoms of ulcerative colitis.

Design, construction, and validation of a modular library of sequence diversity standards for polymerase chain reaction.

Methods to measure the sequence diversity of polymerase chain reaction (PCR)-amplified DNA lack standards for use as assay calibrators and controls. Here we present a general and economical method for developing customizable DNA standards of known sequence diversity. Standards ranging from 1 to 25,000 sequences were generated by directional ligation of oligonucleotide "words" of standard length and GC content and then amplified by PCR.

Effects of Garlic on Cytochromes P450 2C9- and 3A4-Mediated Drug Metabolism in Human Hepatocytes.

Several reports suggest garlic supplements may inhibit the metabolism of cytochrome P450 (CYP) 2C9 and CYP3A4 substrates, such as warfarin and saquinavir. To characterize the effects of garlic extract on CYP2C9 and CYP3A4 enzyme activity immortalized human hepatocytes (Fa2N-4 cells) were exposed to garlic extract (0-200 μg/mL). CYP2C9 and CYP3A4 enzyme activities were evaluated in parallel with enzymatic activities, expression of respective RNA transcripts was also assessed.

Naive human T cells are activated and proliferate in response to the heme oxygenase-1 inhibitor tin mesoporphyrin.

Heme oxygenase-1 (HO-1) and its catabolic by-products have potent anti-inflammatory activity in many models of disease. It is not known, however, if HO-1 also plays a role in the homeostatic control of T cell activation and proliferation. We demonstrate here that the HO-1 inhibitor tin mesoporphyrin (SnMP) induces activation, proliferation, and maturation of naive CD4(+) and CD8(+) T cells via interactions with CD14(+) monocytes in vitro.

Colony-stimulating factor prescribing patterns in patients receiving chemotherapy for cancer.

Objective: To examine variables influencing colony-stimulating factor (CSF) prescription as primary prophylaxis versus other use during patients' initial chemotherapy course among a large sample of health insurance records.

Study Design: Retrospective cohort study.

Methods: Adults 25 years or older with a diagnosis of breast, colorectal, or non-small cell lung cancer (NSCLC) between January 1, 2002, and December 31, 2005, were identified from the western Washington State Surveillance, Epidemiology, and End Results Seattle Puget Sound registry.

The role of transplacental microtransfusions of maternal lymphocytes in in utero HIV transmission.

Background: The mechanisms of HIV transmission from mothers to infants are poorly understood. A possible mechanism of in utero transmission is transplacental transfer of HIV-infected maternal leukocytes into the fetal circulation during pregnancy.

Objective: To determine if the frequency of in utero HIV infection correlates with presence or levels of maternal cells (MCs) in placenta-derived cord blood.

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates.

HSC transplantation using genetically modified autologous cells is a promising therapeutic strategy for various genetic diseases, cancer, and HIV. However, for many of these conditions, the current efficiency of gene transfer to HSCs is not sufficient for clinical use. The ability to increase the percentage of gene-modified cells following transplantation is critical to overcoming this obstacle.

Th17 and regulatory T cells: implications for AIDS pathogenesis.

Purpose Of Review: The present review discusses recent reports showing that reciprocal changes in T helper interleukin-17-secreting CD4 Th17 cells and CD4CD25FoxP3 regulatory T cells (Tregs) may play a role in the progressive disease caused by the HIV and by simian immunodeficiency virus.

Recent Findings: Studies in nonhuman primate models of lentiviral infection and in HIV-infected human individuals have shown that pathogenic infection is associated with loss of Th17 cells and an increase in the frequency of Tregs. Because interleukin-17 serves to maintain the integrity of the mucosal barrier, loss of Th17 cells may permit the increase in microbial translocation across the gastrointestinal mucosa that is observed in pathogenic lentiviral disease.

Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease.

The pathogenesis of human and simian immunodeficiency viruses is characterized by CD4(+) T cell depletion and chronic T cell activation, leading ultimately to AIDS. CD4(+) T helper (T(H)) cells provide protective immunity and immune regulation through different immune cell functional subsets, including T(H)1, T(H)2, T regulatory (T(reg)), and interleukin-17 (IL-17)-secreting T(H)17 cells. Because IL-17 can enhance host defenses against microbial agents, thus maintaining the integrity of the mucosal barrier, loss of T(H)17 cells may foster microbial translocation and sustained inflammation.

Expression of the autoimmune susceptibility gene FcRL3 on human regulatory T cells is associated with dysfunction and high levels of programmed cell death-1.

CD4(+)FoxP3(+) regulatory T cells (T(reg)) play a critical role in maintaining self-tolerance and inhibiting autoimmune disease. Despite being a major focus of modern immunological investigation, many aspects of T(reg) biology remain unknown. In a screen for novel candidate genes involved in human T(reg) function, we detected the expression of an autoimmune susceptibility gene, FcRL3, in T(reg) but not in conventional CD4(+) T cells.

IFN-alpha-induced upregulation of CCR5 leads to expanded HIV tropism in vivo.

Chronic immune activation and inflammation (e.g., as manifest by production of type I interferons) are major determinants of disease progression in primate lentivirus infections.

Correlating cellular and molecular signatures of mucosal immunity that distinguish HIV controllers from noncontrollers.

HIV "controllers" are persons infected with human immunodeficiency virus, type I (HIV) who maintain long-term control of viremia without antiviral therapy and who usually do not develop the acquired immune deficiency syndrome (AIDS). In this study, we have correlated results from polychromatic flow cytometry and oligonucleotide expression arrays to characterize the mucosal immune responses of these subjects in relation to untreated HIV(+) persons with high viral loads and progressive disease ("noncontrollers"). Paired peripheral blood and rectosigmoid biopsies were analyzed from 9 controllers and 11 noncontrollers.

Cytomegalovirus-specific T cells persist at very high levels during long-term antiretroviral treatment of HIV disease.

Background: In healthy, HIV seronegative, CMV seropositive adults, a large proportion of T cells are CMV-specific. High-level CMV-specific T cell responses are associated with accelerated immunologic aging ("immunosenesence") in the elderly population. The impact of untreated and treated HIV infection on the frequency of these cells remains undefined.

Development of a population pharmacokinetics-based sampling schedule to target daily intravenous busulfan for outpatient clinic administration.

Therapeutic drug monitoring of daily intravenous (IV) busulfan currently requires hospital admission. Population pharmacokinetic modeling and determination of an optimal pharmacokinetic sampling schedule over 6 hours could allow for personalizing these busulfan doses in the outpatient clinic. A retrospective evaluation of daily IV busulfan pharmacokinetics was conducted in 37 adults.

A limited sampling schedule to estimate individual pharmacokinetic parameters of fludarabine in hematopoietic cell transplant patients.

Purpose: Fludarabine monophosphate (fludarabine) is frequently administered to patients receiving a reduced-intensity conditioning regimen for allogeneic hematopoietic cell transplant (HCT) in an ambulatory care setting. These patients experience significant interpatient variability in clinical outcomes, potentially due to pharmacokinetic variability in 2-fluoroadenine (F-ara-A) plasma concentrations. To test such hypotheses, patient compliance with the blood sampling should be optimized by the development of a minimally intrusive limited sampling schedule (LSS) to characterize F-ara-A pharmacokinetics.

Establishment of long-term tolerance to SRBC in dogs by recombinant canine CTLA4-Ig.

Background: Blockade of the CD28 costimulatory molecule by recombinant human cytotoxic T lymphocyte (CTL)-associated antigen (CTLA4)-Ig or CD40-CD154 interaction with the monoclonal antibody 5C8 together with donor-specific transfusion led to enhanced engraftment in the canine model of dog leukocyte antigen (DLA)-identical marrow transplantation after 1 Gy total body irradiation. To reduce or eliminate total body irradiation conditioning regimens, we have sought to develop canine specific reagents.

Methods: We have created a fusion protein of the extracellular domain of canine (c) CTLA-4 linked to the hinge-CH2-CH3 domains of canine IgG1 in a pcDNA3.

Busulfan in hematopoietic stem cell transplant setting.

This paper focuses primarily on the data published in the last decade about the pharmacokinetics and pharmacodynamics of oral and intravenous (i.v.) busulfan, therapeutic drug monitoring and clinical outcome in hematopoietic stem cell transplant (HCT) patients.