Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model.

AIMS/HYPOTHESIS: We sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells. METHODS: Wild-type, gp130(-/-) and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp(+) chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood. RESULTS: BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b(+)/CD45(hi)/ CCR2(+)/Ly6C(hi) inflammatory monocytes. Diabetic gp130(-/-) mice were protected from development of diabetes-induced changes in their HSCs. CONCLUSIONS/INTERPRETATION: The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.

Impaired replication of protease inhibitor-resistant HIV-1 in human thymus.

Many HIV-1-infected patients treated with protease inhibitors (PI) develop PI-resistant HIV-1 variants and rebounds in viremia, but their CD4+ T-cell counts often do not fall. We hypothesized that in these patients, T-cell counts remain elevated because PI-resistant virus spares intrathymic T-cell production. To test this, we studied recombinant HIV-1 clones containing wild-type or PI-resistant protease domains, as well as uncloned isolates from patients, in activated peripheral blood mononuclear cells, human thymic organ cultures and human thymus implants in SCID-hu Thy/Liv mice. In most cases, wild-type and PI-resistant HIV-1 isolates replicated to similar degrees in peripheral blood mononuclear cells. However, the replication of PI-resistant but not wild-type HIV-1 isolates was highly impaired in thymocytes. In addition, patients who had PI-resistant HIV-1 had abundant thymus tissue as assessed by computed tomography. We propose that the inability of PI-resistant HIV-1 to replicate efficiently in thymus contributes to the preservation of CD4+ T-cell counts in patients showing virologic rebound on PI therapy.

Experimental transmission of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) to SCID-hu Thy/Liv mice.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a novel human lymphotropic herpesvirus linked to several human neoplasms. To date, no animal model for infection by this virus has been described. We have examined the susceptibility of C.B-17 scid/scid mice implanted with human fetal thymus and liver grafts (SCID-hu Thy/Liv mice) to KSHV infection. KSHV virions were inoculated directly into the implants, and viral DNA and mRNA production was assayed using real-time quantitative polymerase chain reaction. This revealed a biphasic infection, with an early phase of lytic replication accompanied and followed by sustained latency. Ultraviolet irradiation of the inoculum abolished all DNA- and mRNA-derived signals, and infection was inhibited by ganciclovir. Viral gene expression was most abundant in CD19(+) B lymphocytes, suggesting that this model faithfully mimics the natural tropism of this virus. Short-term coinfection with HIV-1 did not alter the course of KSHV replication, nor did KSHV alter levels of HIV-1 p24 during the acute phase of the infection. Although no disease was evident in infected animals, SCID-hu Thy/Liv mice should allow the detailed study of KSHV tropism, latency, and drug susceptibility.

Human herpesvirus 6 (HHV-6) causes severe thymocyte depletion in SCID-hu Thy/Liv mice.

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that may act as a cofactor in the progression of AIDS. Here, we describe the first small animal model of HHV-6 infection. HHV-6 subgroup A, strain GS, efficiently infected the human thymic tissue implanted in SCID-hu Thy/Liv mice, leading to the destruction of the graft. Viral DNA was detected in Thy/Liv implants by quantitative polymerase chain reaction (PCR) as early as 4 d after inoculation and peaked at day 14. The productive nature of the infection was confirmed by electron microscopy and immunohistochemical staining. Atypical thymocytes with prominent nuclear inclusions were detected by histopathology. HHV-6 replication was associated with severe, progressive thymocyte depletion involving all major cellular subsets. However, intrathymic T progenitor cells (ITTPs) appeared to be more severely depleted than the other subpopulations, and a preferred tropism of HHV-6 for ITTPs was demonstrated by quantitative PCR on purified thymocyte subsets. These findings suggest that thymocyte depletion by HHV-6 may be due to infection and destruction of these immature T cell precursors. Similar results were obtained with strain PL-1, a primary isolate belonging to subgroup B. The severity of the lesions observed in this animal model underscores the possibility that HHV-6 may indeed be immunosuppressive in humans.

CCR5- and CXCR4-utilizing strains of human immunodeficiency virus type 1 exhibit differential tropism and pathogenesis in vivo.

CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.

Coinfection of SCID-hu Thy/Liv mice with human herpesvirus 6 and human immunodeficiency virus type 1.

Human herpesvirus 6 (HHV-6) has been proposed as a potential cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) disease. We used the SCID-hu Thy/Liv mouse model to evaluate the in vivo interactions between HHV-6 and HIV-1. Our results demonstrate that HHV-6 and HIV-1 can simultaneously replicate in the human thymus in vivo. In this model, however, the presence of one virus appears not to modify the replication or cytopathicity of the other.

Inhibition of human immunodeficiency virus type 1 infection in SCID-hu Thy/Liv mice by the G-quartet-forming oligonucleotide, ISIS 5320.

Viral replication was inhibited in a dose-dependent manner after administration of the phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) to human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu Thy/Liv mice. Potent in vivo antiviral activity was observed against the T-cell-tropic molecular clone NL4-3; the agent was found to have weak activity against one primary HIV-1 isolate, and the agent was inactive against a second primary isolate.

R5 strains of human immunodeficiency virus type 1 from rapid progressors lacking X4 strains do not possess X4-type pathogenicity in human thymus.

Some individuals infected with only R5 strains of human immunodeficiency virus type 1 progress to AIDS as quickly as individuals harboring X4 strains. We determined that three R5 viruses were much less pathogenic than an X4 virus in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid disease progression is associated with host, not viral, factors.

Antiviral activity of 2'-deoxy-3'-oxa-4'-thiocytidine (BCH-10652) against lamivudine-resistant human immunodeficiency virus type 1 in SCID-hu Thy/Liv mice.

Oral administration of 2'-deoxy-3'-oxa-4'-thiocytidine (BCH-10652), a nucleoside analog structurally similar to lamivudine (3TC), caused dose-dependent inhibition of viral replication in SCID-hu Thy/Liv mice infected with human immunodeficiency virus type 1 NL4-3 and with an NL4-3 clone containing the M184V mutation in reverse transcriptase that confers resistance to 3TC. These experiments demonstrate the utility of this mouse model for evaluating drug resistance and for performing direct comparisons between antiviral compounds in vivo.