Christel H. Uittenbogaart

A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry

A sensitive method for quantification of cells undergoing apoptosis that permits the simultaneous measurement of dual-color cell surface immunofluorescence is presented. Unfixed cells are stained with 7-amino-actinomycin D (7-AAD) for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). Owing to its spectral characteristics 7-AAD can be combined with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) cell surface staining. After staining, the samples can be treated with paraformaldehyde (PF) solution to eliminate the risk for exposure of laboratory personnel to biohazardous agents and to preserve the cells through fixation for later analysis on the flow cytometer. The value of the method is shown on the measurement of apoptosis in human thymocytes and in human peripheral blood mononuclear cells (PBMC) exposed to various inducers of active cell death. The method is validated by fluorescent activated cell sorting in combination with morphologic examination of the sorted cells. The technique we are presenting is particularly valuable in a clinical setting because it allows rapid multiparameter analysis of apoptosis in combination with cell surface phenotype on biohazardous samples with single laser instrumentation.

Nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes

We have investigated the mechanism of nitric oxide-induced damage in glial cells. Genomic DNA isolated from astrocytes and microglia, treated for 18 h with varying concentrations of a nitric oxide donor, was analysed by electrophoresis. No DNA damage was evident. Oligodendrocytes, treated with 2 mM nitric oxide for 3-48 h, showed single stranded breaks at 48 h but no laddering of nucleosomic fragments of DNA. When analysed by electron microscopy, ultrastructural changes in oligodendrocytes treated with 1 mM nitric oxide for 24 h showed intact nuclei but alterations in membranes and organelles characteristic of necrosis, including disrupted mitochondria with dissolution of their christae. Astrocytes, a glial cell type that we have previously shown to be much less sensitive to nitric oxide-induced damage, did not show ultrastructural changes. DNA analysis by flow cytometry of glial cells treated with nitric oxide supported the apparent necrotic-type death in oligodendrocytes. Double staining of oligodendrocytes, using Hoechst 33342 and propidium iodide for the simultaneous assessment of both apoptotic and necrotic cells, demonstrated that, while the proportion of dead cells increased with time and increasing concentrations of nitric oxide, the death was due to necrosis and not apoptosis. In this study, we demonstrate that direct exposure to soluble nitric oxide, produced in vitro from a nitric oxide donor chemical, ultimately kills oligodendrocytes by necrosis. Microglia and astrocytes maintain DNA and organelle integrity when exposed to exogenous nitric oxide.

Endogenous IFN-α Production by Plasmacytoid Dendritic Cells Exerts an Antiviral Effect on Thymic HIV-1 Infection

Plasmacytoid dendritic cells (pDC) are the principal producers of IFN-α in response to viral infection. Because pDC are present in the thymus, we investigated the consequences of HIV-1-induced IFN-α production by thymic pDC. We observed that thymic pDC as well as thymocytes express intracellular IFN-α upon infection with HIV-1. However, only the pDC could suppress HIV-1 replication, because depletion of pDC resulted in enhancement of HIV-1 replication in thymocytes. Thymic pDC could also produce IFN-α in response to CpG oligonucleotides, consistent with the observations of others that peripheral pDC produce IFN-α upon engagement of TLR-9. Importantly, CpG considerably increased IFN-α production induced by HIV-1, and addition of CpG during HIV-1 infection enhanced expression of the IFN response protein MxA in thymocytes and strongly reduced HIV-replication. Our data indicate that thymic pDC modulate HIV-1 replication through secretion of IFN-α. The degree of inhibition depends on the level of IFN-α produced by the thymic pDC.

Use of a novel chimeric mouse model with a functionally active human immune system to study human immunodeficiency virus type 1 infection

ABSTRACT The goal of this study was to develop a small-animal model to study human immunodeficiency virus type 1 (HIV-1) pathogenesis in blood and primary and secondary lymphoid organs. Rag2−/−γc−/− mice that are neonatally injected with human CD34+ cells develop a functional human immune system (HIS), with human hematopoietic cells being found in the thymuses, peripheral blood, spleens, and bone marrow of the animals (hereafter these animals are referred to as HIS-Rag2−/−γc−/− mice). HIS-Rag2−/−γc−/− mice were infected with small amounts of CCR5-tropic HIV-1. Viral replication and immunophenotypic changes in the human cells in peripheral blood and lymphoid organs were examined. The productive infection of human cells in peripheral blood, thymus and spleen tissue, and bone marrow was detected. Ratios of CD4+ T cells to CD8+ T cells in the infected animals declined. Although no specific anti-HIV-1 immune responses were detected, immunoglobulin M (IgM) and IgG antibodies to an unidentified fetal calf serum protein present in the virus preparation were found in the inoculated animals. Thus, we have shown that the HIS-Rag2−/−γc−/− mouse model can be used for infection with low doses of CCR5-tropic HIV-1, which is most commonly transmitted during primary infections. HIS-Rag2−/−γc−/− mice can serve as a small-animal model for investigating HIV-1 pathogenesis and testing potential HIV-1 therapies, and studies with this model may replace some long and costly studies with nonhuman primates.

Progression to end-stage renal disease in children with obstructive uropathy.

The course of 54 patients (35 boys and 19 girls) with end-stage renal disease resulting from obstructive uropathy was reviewed. The mean age at the initial sign of obstructive uropathy was 3.5 years. Twenty-two patients (41%) manifested evidence of obstructive uropathy during the first year of life. The mean age at the time of onset of ESRD (dialysis) was 12.2 years and was similar in boys and girls. The mean time interval between the first sign of obstructive uropathy and the initiation of dialysis was nine years. Fourteen patients operated upon at less than one year of age developed ESRD one to 20 years (mean ten years) following their initial surgery. Progression to ESRD occurred despite appropriate surgical management, including corrective as well as diversionary urologic procedures. However, because the patients were selectively referred for care of ESRD, no assessment of the incidence of ESRD caused by obstructive uropathy was possible. The data indicate that prolonged follow-up periods are necessary to assess the ultimate outcome of renal function in young patients with obstructive uropathy. Despite early intervention and intact renal function for many years during childhood, progression to ESRD may occur.

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

ObjectiveUnderstanding 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. DesignHIV 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. MethodsIntracellular 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. ResultsNL4–3 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)α/βbright cells. ConclusionsThese 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.

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.

Focal glomerulosclerosis and renal transplantation

Eighteen patients with corticosteroid-resistant nephrotic syndrome developed end-stage renal disease and received one or more renal allografts. The lesion of focal segmental glomerulosclerosis and/or of focal glomerular obsolescence was demonstrable in the native kidneys of each patient. Following transplantation, nephrosis developed in three recipients. Two recipients developed nephrosis at two weeks and nine months posttransplant in association with rejection; the lesion of FGS was present in association with chronic rejection. Only one recipient developed recurrence of nephrosis and FGS unrelated to rejection. This was manifested by immediate onset of nephrosis in two successive allografts and histologic evidence of the lesion of FGS. The immediate recurrence in successive allografts suggests a circulating factor responsible for the renal lesion in this patient and indicates a separate etiology for a small number of patients with corticosteroid-resistant nephrosis and FGS.

Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo.

ABSTRACT Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses. For the noninvasive and continuous monitoring of the interaction between gammaherpesvirus and the host, a recombinant murine gammaherpesvirus 68 (MHV-68, a gammaherpesvirus 68) was constructed to express a firefly luciferase gene driven by the viral M3 promoter (M3FL). Real-time monitoring of M3FL infection revealed novel sites of viral replication, such as salivary glands, as well as acute replication in the nose and the lung and progression to the spleen. Continuous monitoring of M3FL infection in individual mice demonstrated the various kinetics of transition to different organs and local clearance, rather than systemically synchronized clearance. Moreover, in vivo spontaneous reactivation of M3FL from latency was detected after the initial clearance of acute infection and can be induced upon treatment with either a proteasome inhibitor Velcade or an immunosuppressant cyclosporine A. Taken together, our results demonstrate that the in vivo replication and reactivation of gammaherpesvirus are dynamically controlled by the locally defined interaction between the virus and the host immune system and that bioluminescence imaging can be successfully used for the real-time monitoring of this dynamic interaction of MHV-68 with its host in vivo.

The role of the thymus in HIV infection: a 10 year perspective.

Despite substantial progress over the last 10 years the exact role of the thymus in HIV-1 infection and HIV-1 pathogenesis is still under investigation. Much has been learned of the types of cells in the thymus that are targets for CXCR4 and CCR5 HIV-1 isolates. In addition, it has become clear that even the adult thymus continues to function, although at a much lower level in uninfected patients, and is able to export naive T cells to the periphery. Changes in thymus function can be evaluated by several methods, including determination of naive T-cell subsets using multicolor flow cytometry and T-cell receptor excision circles (TREC), as well as thymus size and metabolic labeling assays [1–5]. Although each of these measures has its advantages and drawbacks [6], combinations of these parameters provide a picture of the contribution of thymic output and homeostatic proliferative expansion (HPE) to peripheral blood T-cell homeostasis. The importance of the thymus in regenerating a functional immune system has been clearly shown after chemotherapy, bone marrow transplantation and highly active retroviral therapy (HAART) in HIV infection [5,7–10]. The data published during the last 10 years also show that a possible increase in thymic output has an instrumental role in the immunopathogenesis that takes place during the clinically asymptomatic phase of HIV-1 infection.