Transient renewal of thymopoiesis in HIV-infected human thymic implants following antiviral therapy.

Stem cell gene therapy strategies for AIDS require that differentiation-inducing stromal elements of HIV-infected individuals remain functionally intact to support the maturation of exogenous progenitor cells into mature CD4+ cells. To investigate the feasibility of stem cell reconstitution strategies in AIDS, we used the SCID-hu mouse to examine the ability of HIV-infected CD4+ cell-depleted human thymic implants to support renewed thymopoiesis. Here we report that following treatment of these implants with antiretroviral drugs, new thymopoiesis is initiated. This suggests that antiviral therapies might allow de novo production of T lymphocytes and provides support for the concept of therapeutic strategies aimed at reconstitution of the peripheral CD4+ T-cell compartment.

T cell memory. Long-term persistence of virus-specific cytotoxic T cells.

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.

[Delleman syndrome: anesthetic considerations]. / Syndrome de Delleman: prise en charge anesthésique.

We herein report a case of a 5-year-old patient with Delleman Syndrome, a rare congenital disorder affecting the eyes, skin, and central nervous system, who underwent general anesthesia for conjuctivoplasty. This is only the second report of the anesthetic management of a patient with this condition. We attempt to summarize some of the anesthetic implications of this syndrome.

Differential tropism of HIV-1 isolates for distinct thymocyte subsets in vitro.

OBJECTIVE: Understanding the interaction between HIV and developing thymocytes is crucial in determining how HIV infection perturbs the immune system. We determined which thymocyte subsets can harbor and express HIV. DESIGN: HIV expression in mature and immature thymocytes obtained from surgical specimens from non-infected children was determined after in vitro infection with the syncytium-inducing, cytopathic NL4-3 and the non-syncytium-inducing, relatively noncytopathic JR-CSF isolates. METHODS: Intracellular staining for the HIV p24gag antigen was combined with cell surface phenotyping to determine thymocyte subsets expressing HIV. Infection was quantitated by polymerase chain reaction on sorted subsets. RESULTS: NL4-3 replicated faster and to higher titers and caused a more severe decrease of all CD4-bearing thymocytes than did JR-CSF. In addition, both immature CD1+ and mature CD1-thymocytes expressed NL4-3, whereas only mature CD1-cells expressed JR-CSF. The tropism of NL4-3 for these immature cells suggests a mechanism for a more profound impact on T-cell maturation than that seen with JR-CSF. We also found that thymocytes lacking cell surface CD4 (CD4-CD8- and CD4-CD8+ subsets) expressed virus with either isolate late in infection, when viral levels were high. The CD4-CD8- cells expressing HIV were mature CD3bright T-cell receptor (TCR) alpha/beta bright cells. CONCLUSIONS: These results show that NL4-3 can be expressed by thymocytes at immature and mature stages of differentiation and cause severe loss of CD4+ cells. Thus, tropism of a virus for immature cells can affect the capability of the thymus to produce new T lymphocytes leading to a greater impact on development and functions of the immune system. It is proposed that this in vitro model can be used to study pathogenic mechanisms in the thymus.

Effects of megakaryocyte growth and development factor on survival and retroviral transduction of T lymphoid progenitor cells.

Murine retroviral vectors have the potential to mediate stable gene transfer into hematopoietic progenitor cells. A known drawback to the use of these vectors is that transduction can only take place in cells actively progressing through the cell cycle. Thrombopoietin, the c-mpl ligand, is known to support division of hematopoietic precursors of primitive origin. Polyethylene glycol (PEG)-conjugated recombinant human megakaryocyte growth and development factor (MGDF) is a polypeptide related to thrombopoietin that stimulates megakaryocyte production. To investigate whether MGDF would also induce stem cell division and support retroviral transduction of CD34+ cells, we compared the effects of MGDF, stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, alone or in combination, using amphotropic and vesicular stomatitis virus (VSV-G) pseudotyped murine retroviral vectors. Similar transduction efficiency was observed when CD34+ cells were transduced in the presence of SCF and MGDF as compared to SCF, IL-3, and IL-6. Using the SCID-hu mouse model of thymopoiesis, we investigated whether CD34+ cells transduced in the presence of these cytokines could reconstitute irradiated thymic implants, and whether vector sequences were present in mature thymocytes. At early timepoints, no significant differences were observed on engraftment of donor progenitors incubated with each cytokine combination. However, a significant difference in the percentage of donor derived CD4+/CD8+ immature thymocytes was observed 9 weeks after implantation of CD34+ cells exposed to the combination of SCF and MGDF as compared to SCF, IL-3, and IL-6 (p = 0.04), indicating that MGDF/SCF better supported the survival of thymocyte precursor cells. Approximately 4% of thymocytes in both cytokine groups harbored vector sequences. These studies provide evidence that MGDF and SCF in combination can mediate transduction of hematopoietic progenitors capable of contributing to long-term thymopoiesis. These results may have important applications for the implementation of gene therapy strategies in disorders affecting the T lymphoid system.

Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation.

Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 1x PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.

Loss of T cell receptor Vbeta repertoires in HIV type 1-infected SCID-hu mice.

Late-stage HIV-1 disease in humans has been associated with perturbations of the T cell receptor (TCR) Vbeta repertoire. It is not known if the observed loss of certain Vbeta families is attributable directly to HIV-1 infection or whether this is a consequence of multiple opportunistic infections. Putative HIV-1-associated superantigens have been postulated to be the cause of the perturbed TCR Vbeta repertoire and the subsequent CD4+ T cell depletion in HIV-1-infected humans. In this study, we examined the human TCR Vbeta repertoire in SCID-hu mice, housed in a pathogen-free environment and infected with a molecularly cloned virus strain, to ascertain directly the effect of HIV-1 on the human TCR Vbeta repertoire in the absence of other infectious agents. We demonstrate that mock-infected human thymus/liver (Thy/Liv) implants in SCID-hu mice have complete TCR Vbeta repertoires, reflective of a normal human thymus. However, HIV-1-infected implants in SCID-hu mice had depleted TCR Vbeta repertoires, corresponding with thymocyte depletion. These results indicate that HIV-1-specific mechanisms are the cause of the TCR Vbeta repertoire depletion in infected implants. However, these thymocyte depletions were not restricted to specific TCR Vbeta subsets. These results are not consistent with the hypothesis that HIV-1 acts as a superantigen in vivo. The disruption of the TCR Vbeta repertoire in the human Thy/Liv implants of the SCID-hu mice suggests that HIV-1 infection may be influencing T cell development in the thymus, contributing to both the overall CD4+ T cell depletion in AIDS and limited TCR repertoire diversity.

Driving offenders and the defensive driving course--an archival study.

Two hundred and seventy-five drivers who had been required by court order to attend a Defensive Driving Course (DDC) were compared on six posttreatment driving measures obtained from archival data with 275 drivers who also had had a court appearance and standard treatment. The DDC group showed greater reductions in serious and accident-promoting convictions but no greater reduction in accidents when compared with the standard treatment comparison group.

Preferential depletion of gut CD4-expressing iNKT cells contributes to systemic immune activation in HIV-1 infection.

Chronic inappropriate immune activation is the central defect-driving loss of CD4(+) T helper cells and progression to AIDS in persons with HIV-1 infection, but the mechanisms remain controversial. We examined key regulatory invariant receptor natural killer T (iNKT) cells in the gut, the largest reservoir of lymphocytes and a key arena of HIV-1 pathogenesis. In healthy control persons, the anti-inflammatory CD4(+) iNKT-cell subset predominated over the pro-inflammatory CD4(-) iNKT-cell subset in the gut, but not in the blood, compartment. HIV-1 infection resulted in a preferential loss of this anti-inflammatory CD4(+) iNKT-cell subset within the gut. The degree of loss of the CD4(+) iNKT-cell subset in the gut, but not in the blood, correlated to the systemic immune activation and exhaustion that have been linked to disease progression. These results suggest a potentially important contribution of gut iNKT-cell imbalance in determining the systemic immune activation that is the hallmark of HIV-1 pathogenesis.

T-cell tolerance: exposure to virus in utero does not cause a permanent deletion of specific T cells.

This study documents the curing of a congenitally acquired chronic viral infection and the acquisition of T-cell competence by a previously tolerant host. Infection of mice with lymphocytic choriomeningitis virus (LCMV) is a classic model of viral persistence and antigen-specific T-cell unresponsiveness. Mice infected at birth or in utero become lifelong carriers with no detectable virus-specific cytotoxic T lymphocyte (CTL) responses. This chronic infection can be eliminated by adoptive transfer of Lyt-2+ T cells from LCMV-immune mice. To determine whether these cured carriers were capable of generating their own LCMV-specific CTL response, mice congenic at the Thy-1 locus (Thy-1.1 and Thy-1.2) were used in the adoptive transfer experiments. Host-derived T-cell responses were checked after treating the cured carriers with a monoclonal antibody to deplete the immune donor T cells. Such cured carrier mice were able to generate a host-derived virus-specific CTL response and resisted a second LCMV challenge in the absence of any donor T cells. In addition, bone marrow cells from these cured carriers could functionally reconstitute irradiated mice. Thus this report demonstrates the acquisition of LCMV-specific T-cell competence by previously unresponsive carrier mice infected in utero. These results show that exposure to a virus even during embryonic life does not cause a permanent deletion of specific T cells. These findings are of significance to the understanding of tolerance mechanisms and have implications for the treatment of chronic viral infections.

In vivo pathogenesis of a human immunodeficiency virus type 1 reporter virus.

Our understanding of human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis is hampered by the inability to detect HIV-1 gene expression in infected viable cells. In this report, we describe two HIV-1 reporter constructs that are replication competent and cytopathic in vivo. These constructs contain DNA regions of two different lengths that bear the cDNA for the murine heat-stable antigen in the vpr region of a CXCR4-tropic virus. We used the SCID-hu mouse model and these reporter viruses to perform detailed kinetic studies of HIV-1 infection of human thymocytes in vivo. We document that the CD4(+)/CD8(+) thymocytes are the first to express virus and that this subset demonstrates the most rapid and extensive HIV-1-induced cell depletion. Following depletion of this subset, subsequent virus expression occurs predominantly in phenotypically CD4(-) cells, suggesting that CD4 down-regulation occurs in HIV-1-infected thymocytes in vivo. These results demonstrate the utility of these HIV-1 reporter constructs to monitor HIV pathogenesis in vitro and in vivo.

Effective clearance of a persistent viral infection requires cooperation between virus-specific Lyt2+ T cells and nonspecific bone marrow-derived cells.

The lifelong chronic lymphocytic choriomeningitis virus (LCMV) infection established in neonatally or congenitally infected mice can be eliminated by adoptive transfer of lymphoid cells from LCMV-immune mice. In this study, we have identified the effector cells mediating the clearance of persistent and disseminated LCMV infection. Using mice that are recombinant in the H-2 region and by selective depletion of lymphocyte subpopulations, we show that viral clearance was mediated by LCMV-specific Lyt2+ L3T4- T cells that are restricted to the class I genes of the major histocompatibility complex. In addition, our results show a requirement for host-derived bone marrow cells for the effective elimination of virus from the liver. These studies emphasize the importance of virus-specific T cells and an intact bone marrow function in viral clearance.

Immune therapy of a persistent and disseminated viral infection.

The mechanism of viral clearance was studied by using the mouse model of chronic infection with lymphocytic choriomeningitis virus. Distinct patterns of viral clearance and histopathology were observed in different organs after adoptive immune therapy of persistently infected (carrier) mice. Clearance from the liver occurred within 30 days and was accompanied by extensive mononuclear cell infiltrates and necrosis of hepatocytes. Infectious virus and viral antigen were eliminated concurrently. This pattern of viral clearance was also seen in most other tissues (i.e., lung, spleen, lymph nodes, pancreas, etc.). In contrast, a different pattern of clearance was observed in the brain. Infectious virus was eliminated within 30 days, but viral antigen persisted in the central nervous systems of treated carrier mice for up to 90 days. The urinary system was the most resistant to immune therapy. Elimination of infectious virus and viral antigen from the kidney took greater than 200 days and even then was not complete; trace levels of infectious virus were still present in the kidneys of some treated carrier mice. After immune therapy, viral antigen in the kidney was located within renal tubules that costained for intracellular mouse immunoglobulin G. This unusual staining pattern, coupled with the observation of large numbers of plasma cells within the kidney, suggests that virus-immunoglobulin G complexes found in the tubules may represent in situ immune complex formation as opposed to deposition of circulating immune complexes. In conclusion, these results suggest that the site (organ) of viral persistence is an important consideration in developing treatment strategies for controlling chronic viral infections.

Abrogation of tolerance to a chronic viral infection.

This study documents failure of peripheral tolerance mechanisms in a chronic viral infection and shows that T cell tolerance to a viral Ag seen as self from fetal life can be broken despite the presence of this Ag in extrathymic tissues. Congenital infection of mice with lymphocytic choriomeningitis virus (LCMV) results in T cell tolerance to the virus. Such mice become carriers for life harboring virus in many tissues including the thymus and exhibit no LCMV-specific CTL responses. Our previous studies have documented the curing of this congenitally acquired chronic infection after adoptive transfer of CD8+ T cells from LCMV-immune mice and the presence of host-derived, LCMV-specific CTL in these "cured" carriers. In this study we have examined the mechanism by which these carriers acquired T cell competence and show that these CTL differentiated from the bone marrow after elimination of viral Ag from the thymus. These results demonstrate that even when a chronic infection has been established in utero, the adult thymus retains the ability to restore immunocompetence to the host and to provide protection against reinfection. Surprisingly, these LCMV specific CTL were acquired at a time when infectious virus and intracellular viral Ag, although cleared from the thymus, were readily detectable in organs such as the kidney, testes, and brain. In fact, active viral replication in peripheral tissues was ongoing when these mice acquired new virus-specific T cells. These results show that clearance of virus form the thymus was sufficient to abrogate tolerance to a congenitally acquired chronic infection and that Ag in peripheral tissues did not tolerize newly developing T cells. These findings suggest that mechanisms that operate on immature cells within the thymus to silence self-reactive T cells are effective in induction of tolerance to viruses, but mechanisms of tolerizing mature T cells are likely to breakdown. This has implications for virus-induced autoimmunity and for treatment of chronic infections.

The SCID-hu mouse: an in-vivo model for HIV-1 pathogenesis and stem cell gene therapy for AIDS.

Animal models are critical to the investigation of human immunodeficiency virus type 1 (HIV-1) pathogenesis. However, normal animal models are either uninfectable with HIV-1, or if infected, do not display HIV-1 induced pathology. Here, we describe how the severe combined immunodeficient mouse (SCID), implanted with human fetal thymus and liver, has been used to model HIV-1 pathogenesis and anti-retroviral gene therapy. Unable to reject the human tissue, these chimeric mice provide the investigator with a human hematolymphoid organ which, following infection by HIV-1, may more closely mimic the situation seen in humans than standard in-vitro culture systems.

cAMP up-regulates cell surface expression of lymphocyte CXCR4: implications for chemotaxis and HIV-1 infection.

The chemokine receptor CXCR4 mediates lymphocyte chemotaxis in response to stromal cell-derived factor-1 (SDF-1) and functions as a coreceptor for T cell-tropic strains of HIV-1. We examined the role of the cAMP-protein kinase A (PKA) signaling pathway in regulating expression of CXCR4. In response to exogenous dibutyryl cAMP or cAMP-inducing ligands, cell surface expression of CXCR4 was increased by up to 10-fold on CD3/CD28-stimulated PBMC and by up to sixfold on unstimulated PBMC. cAMP did not alter receptor mRNA levels or affect the size of the total CXCR4 pool. However, cAMP did significantly reduce CXCR4 internalization rates and thereby increased the fraction of the total CXCR4 pool expressed on the cell surface. cAMP-induced increases in CXCR4 expression counteracted SDF-1-induced receptor internalization and enhanced both chemotactic response to SDF-1 and cellular vulnerability to HIV-1 infection. Thus, altered chemokine receptor expression may provide one mechanism by which cAMP-inducing ligands influence lymphocyte localization and HIV pathogenesis.