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In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir., (© 2022. The Author(s).)

Viral rebound upon stopping combined antiretroviral therapy poses a major barrier toward an HIV cure. Cellular and anatomical sources responsible for reinitiating viral replication remain a subject of ardent debate, despite extensive research efforts. To unravel the source of rebounding viruses, we conducted a large-scale HIV-STAR (HIV-1 sequencing before analytical treatment interruption to identify the anatomically relevant HIV reservoir) clinical trial. We collected samples from 11 participants and compared the genetic composition of (pro)viruses collected under treatment from different cellular and anatomical compartments with that of plasma viruses sampled during analytical treatment interruption. We found a remarkably heterogeneous source of viral rebound. In addition, irrespective of the compartment or cell subset, genetically identical viral expansions played a significant role in viral rebound. Our study suggests that although there does not seem to be a primary source for rebound HIV, cellular proliferation is an important driver of HIV persistence and should therefore be considered in future curative strategies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human immunodeficiency virus (HIV) is associated with an increased risk of developing Hodgkin lymphoma (HL). South Africa (SA) has the highest HIV prevalence rate in the world. There is currently no outcome-based data for HIV-associated HL from SA. A bone marrow database was compiled of all bone marrow biopsies (BMB) reported at National Health Laboratory Service (NHLS) Groote Schuur Hospital (GSH) between January 2005 and December 2012. Patients who had a BMB performed for staging of HL or where HL was diagnosed on the BMB were included for further analysis. Clinical and laboratory data was extracted from medical and laboratory records. Primary outcome measures included histological subtype, bone marrow infiltration (BMI) by HL, CD4 count, HIV-viral load (HIV-VL), tuberculosis (TB) data, treatment with chemotherapy and 5-year overall survival (OS). The database included 6569 BMB and 219 patients of these had HL and were included for analysis. The median age at presentation (32 years) was similar in the HIV+ and HIV- populations. While males predominated in the HIV- group, females predominated in the HIV+ group (male:female ratio of 1.5:1 vs 0.7:1, respectively). The majority of patients (71%) were HIV negative (HIV-) and 29% were HIV positive (HIV+). The diagnosis of HL was made on BMB in 17% of cases. BMI was seen in 37% (82/219) overall, and was found in more HIV+ patients (61%; 39/64) than HIV- patients (28%; 43/155; p = 0.03). The histological subtype varied according to HIV status with nodular sclerosis classical Hodgkin lymphoma (NSCHL) being most frequent in the HIV- group and classical Hodgkin lymphoma (CHL)-unclassifiable the most frequent in the HIV+ group. HIV+ patients had a median CD4 count of 149 × 10 6 /L and 39% were anti-retroviral therapy (cART) naive at HL diagnosis. HIV+ patients had received anti-TB therapy more frequently than HIV- patients (72% vs 17%; p = 0.007). More HIV+ patients did not receive chemotherapy than HIV- patients (31% vs 3%; p = 0.001). The 5-year OS was 56%. HIV+ patients with BMI had a 5-year OS of 18%. BMI, HIV status, low CD4 count, histological subtype and TB therapy had a statistical significant impact on 5-year OS (p < 0.01). The 5-year OS was 56%, with both BMI and HIV+ status being associated with poor survival. BMB provided the diagnosis of HL in 17% of cases, confirming its diagnostic utility in our setting. Our cohort showed similar survival outcomes to other countries in Africa, Asia and Central America with comparable socio-economic constraints to SA.

Background: The nucleoside reverse transcriptase inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) in preclinical development exhibits improved safety and antiviral activity profiles with minimal drug resistance compared to approved NRTIs. However, the systemic antiviral efficacy of EFdA has not been fully evaluated. In this study, we utilized bone marrow/liver/thymus (BLT) humanized mice to investigate the systemic effect of EFdA treatment on HIV replication and CD4+ T cell depletion in the peripheral blood (PB) and tissues. In particular, we performed a comprehensive analysis of the female reproductive tract (FRT) and gastrointestinal (GI) tract, major sites of transmission, viral replication, and CD4+ T cell depletion and where some current antiretroviral drugs have a sub-optimal effect., Results: EFdA treatment resulted in reduction of HIV-RNA in PB to undetectable levels in the majority of treated mice by 3 weeks post-treatment. HIV-RNA levels in cervicovaginal lavage of EFdA-treated BLT mice also declined to undetectable levels demonstrating strong penetration of EFdA into the FRT. Our results also demonstrate a strong systemic suppression of HIV replication in all tissues analyzed. In particular, we observed more than a 2-log difference in HIV-RNA levels in the GI tract and FRT of EFdA-treated BLT mice compared to untreated HIV-infected control mice. In addition, HIV-RNA was also significantly lower in the lymph nodes, liver, lung, spleen of EFdA-treated BLT mice compared to untreated HIV-infected control mice. Furthermore, EFdA treatment prevented the depletion of CD4+ T cells in the PB, mucosal tissues and lymphoid tissues., Conclusion: Our findings indicate that EFdA is highly effective in controlling viral replication and preserving CD4+ T cells in particular with high efficiency in the GI and FRT tract. Thus, EFdA represents a strong potential candidate for further development as a part of antiretroviral therapy regimens.

Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human.

Several potent and broadly neutralizing Abs to HIV-1 have been isolated recently from peripheral blood B cells of infected individuals, based on prescreening of Ab activity in the serum. However, little is known regarding the cells that make the Abs that circulate in the blood. Accordingly, we investigated the most likely source, the bone marrow, of chronically HIV-1-infected individuals who were not receiving antiretroviral therapy. Increased frequencies of plasma cells, as well as B cell precursors, namely preB-I and preB-II, and decreased frequencies of mature B cells were observed in bone marrow aspirates of these individuals compared with HIV-negative counterparts. Increased frequencies of bone marrow plasma cells are consistent with known hallmarks of HIV-1 infection, namely hypergammaglobulinemia and increased frequencies of peripheral blood plasmablasts. Levels of HIV-1 envelope (Env)-binding and HIV-1-neutralizing Abs were measured in serum, and corresponding frequencies of Ab-secreting or Env-binding cells were measured in the blood (plasmablasts and memory B cells) and in the bone marrow (plasma cells). A strong correlation was observed between serum HIV-1-specific Abs and Env-specific bone marrow-derived plasma cells, but not circulating plasmablasts or memory B cells. These findings demonstrate that, despite HIV-1-induced phenotypic and functional B cell dysregulation in the peripheral blood and secondary lymphoid tissues, bone marrow plasma cells remain a primary source for circulating HIV-1-specific Abs in HIV-1-infected individuals.

Background: Hematopoietic progenitor cells (HPCs) in the bone marrow of human immunodeficiency virus (HIV)-infected individuals have been proposed as a persistent reservoir of virus. However, some studies have suggested that HIV genomes detected in HPCs arise from T-cell contamination., Methods: CD133-sorted HPCs and CD133-depleted bone marrow cells were purified from bone marrow specimens obtained from 11 antiretroviral-treated donors in whom the HIV load had been <48 copies/mL for at least 6 months. CD133 and CD3 expression on the cells was assessed by flow cytometry. HIV DNA was quantified by real-time polymerase chain reaction analysis., Results: HIV genomes were detected in CD133-sorted samples from 6 donors, including 2 in whom viral loads were undetectable for >8 years. CD3(+) T cells represented <1% of cells in all CD133-sorted samples. For 5 of 6 CD133-sorted samples with detectable HIV DNA, the HIV genomes could not be explained by contaminating CD3(+) T cells. Donors with detectable HIV DNA in HPCs received their diagnosis significantly more recently than the remaining donors but had had undetectable viral loads for similar periods., Conclusions: HIV genomes can be detected in CD133-sorted cells from a subset of donors with long-term viral suppression and, in most cases, cannot be explained by contamination with CD3(+) T cells.

During the course of human immunodeficiency virus type 1 (HIV-1) disease, the virus has been shown to effectively escape the immune response with the subsequent establishment of latent viral reservoirs in specific cell populations within the peripheral blood (PB) and associated lymphoid tissues, bone marrow (BM), brain, and potentially other end organs. HIV-1, along with hepatitis B and C viruses (HBV and HCV), are known to share similar routes of transmission, including intravenous drug use, blood transfusions, sexual intercourse, and perinatal exposure. Substance abuse, including the use of opioids and cocaine, is a significant risk factor for exposure to HIV-1 and the development of acquired immune deficiency syndrome, as well as HBV and HCV exposure, infection, and disease. Thus, coinfection with HIV-1 and HBV or HCV is common and may be impacted by chronic substance abuse during the course of disease. HIV- 1 impacts the natural course of HBV and HCV infection by accelerating the progression of HBV/HCV-associated liver disease toward end-stage cirrhosis and quantitative depletion of the CD4+ T-cell compartment. HBV or HCV coinfection with HIV-1 is also associated with increased mortality when compared to either infection alone. This review focuses on the impact of substance abuse and coinfection with HBV and HCV in the PB, BM, and brain on the HIV-1 pathogenic process as it relates to viral pathogenesis, disease progression, and the associated immune response during the course of this complex interplay. The impact of HIV-1 and substance abuse on hepatitis virus-induced disease is also a focal point.

Background: We address the key emerging question of whether Lin(-)/CD34(+) hematopoietic precursor cells (HPCs) represent an important latent reservoir of human immunodeficiency virus type 1 (HIV-1) during long-term suppressive therapy., Methods: To estimate the frequency of HIV-1 infection in bone marrow, we sorted Lin(-)/CD34(+) HPCs and 3 other cell types (Lin(-)/CD34(-), Lin(-)/CD4(+), and Lin(+)/CD4(+)) from 8 patients who had undetectable viral loads for 3-12 years. Using a single-proviral sequencing method, we extracted, amplified, and sequenced multiple single HIV-1 DNA molecules from these cells and memory CD4(+) T cells from contemporaneous peripheral blood samples., Results: We analyzed 100,000-870,000 bone marrow Lin(-)/CD34(+) HPCs from the 8 patients and found no HIV-1 DNA. We did isolate HIV-1 DNA from their bone marrow Lin(+)/CD4(+) cells that was genetically similar to HIV-1 DNA from lymphoid cells located in the peripheral blood, indicating an exchange of infected cells between these compartments., Conclusions: The absence of infected HPCs provides strong evidence that the HIV-1 infection frequency of Lin(-)/CD34(+) HPCs from bone marrow, if it occurred, was <.003% (highest upper 95% confidence interval) in all 8 patients. These results strongly suggest that Lin(-)/CD34(+) HPCs in bone marrow are not a source of persistent HIV-1 in patients on long-term suppressive therapy.

Anemia is common in HIV infection, but the pathophysiology is poorly understood. Bone marrow analysis in 329 severely anemic (hemoglobin <5 g/dl) Malawian children with (n = 40) and without (n = 289) HIV infection showed that HIV-infected children had fewer CD34(+) hematopoietic progenitors (median 10 vs. 15‰, P = 0.04) and erythroid progenitors (2.2 vs. 3.4‰, P = 0.05), but there were no differences in erythrocyte viability and maturation in later stages of erythropoiesis. Despite an HIV-associated reduction in early red cell precursors, subsequent erythropoiesis appears to proceed similarly in HIV-infected and HIV-uninfected children with severe anemia.

Background: Anaemia is the most common haematological complication of HIV and associated with a high morbidity and a poor prognosis. The pathogenesis of HIV-associated anaemia is poorly understood and may include a direct effect of HIV on erythropoiesis. In vitro studies have suggested that specific HIV strains, like X4 that uses the CXCR4 co-receptor present on erythroid precursors, are associated with diminished erythropoiesis. This co-receptor affinity is determined by changes in the hypervariable loop of the HIV-1 envelope genome. In a previous case-control study we observed an association between HIV and severe anaemia in Malawian children that could not be fully explained by secondary infections and micronutrient deficiencies alone. We therefore explored the possibility that alterations in the V1-V2-V3 fragment of HIV-1 were associated with severe anaemia., Methods: Using peripheral blood nucleic acid isolates of HIV-infected children identified in the previous studied we assessed if variability of the V1-V2-V3 region of HIV and the occurrence of X4 strains were more common in HIV-infected children with (cases, n = 29) and without severe anaemia (controls, n = 30). For 15 cases bone marrow isolates were available to compare against peripheral blood. All children were followed for 18 months after recruitment., Results: Phylogenetic analysis showed that HIV-1 subtype C was present in all but one child. All V1-V2-V3 characteristics tested: V3 charge, V1-V2 length and potential glycosylation sites, were not found to be different between cases and controls. Using a computer model (C-PSSM) four children (7.8%) were identified to have an X4 strain. This prevalence was not different between study groups (p = 1.00). The V3 loop characteristics for bone marrow and peripheral blood isolates in the case group were identical. None of the children identified as having an X4 strain developed a (new) episode of severe anaemia during follow up., Conclusion: The prevalence of X4 strains in these young HIV-1-subtype-C-infected children that were most likely vertically infected and naïve to anti-retroviral therapy can be considered high compared to previous results from Malawi. It is unlikely that V1-V2-V3 fragment characteristics and HIV co-receptor affinity is an important feature in the development of severe anaemia in Malawian children.

Human parvovirus 4 (PARV4) is a recently discovered member of the Parvoviridae. We investigated the presence of this virus in bone-marrow aspirates of 35 Italian patients with AIDS. Viral DNA was detected by polymerase chain reaction in over 40% of patients (16/35). The infection was most prevalent in injection drug users (IDU; 12/18; 66.7%) as opposed to non-IDU (4/17; 23.5%). PARV4 infection is widespread in Italian patients with AIDS.

Murine primary cells are poorly permissive to human immunodeficiency virus type 1 (HIV-1) vector infection. Retroviral infectivity is influenced by dominant inhibitors such as TRIM5alpha. Sensitivity to TRIM5alpha is altered by interactions between cyclophilin A and the HIV-1 capsid. Here we demonstrate that competitive inhibitors of cyclophilins, cyclosporine or the related Debio-025, stimulate HIV-1 vector transduction of primary murine cells, including bone marrow and macrophages, up to 20-fold. Unexpectedly, the infectivity of an HIV-1 mutant or a simian lentivirus that does not recruit cyclophilin A is also stimulated by these drugs. We propose that cyclosporine and related compounds will be useful tools for experimental infection of murine primary cells. It is possible that HIV-1 infection of murine cells is inhibited by dominant factors related to immunophilins.

Efficient gene transfer to hematopoietic stem cells by Moloney murine leukemia virus-derived retroviral vectors benefits from ex vivo culture and cytokine support. Both also increase the risks of apoptosis and differentiation among cells targeted for transduction. In an effort to maximize the retention of stem cell properties in target cells, we developed a transduction protocol with a focus on minimizing graft manipulation, cytokine stimulation, and ex vivo exposure duration. Based on their wide host range and ability to transduce quiescent cells, human immunodeficiency virus (HIV)-derived lentivirus vectors are ideally suited for this purpose. Our present studies in a murine model show that whole bone marrow cells are readily transduced after a 1-hour vector exposure in the presence of stem cell factor and CH296 fibronectin fragment. Using this rapid transduction protocol, we achieved long-term, multilineage reconstitution of murine recipients with up to 25% GFP-expressing cells in primary and secondary recipients. Our results demonstrate the unique ability of HIV-derived vectors to transduce hematopoietic stem cells in the absence of enrichment, under minimal cytokine stimulation, and following brief exposures.

Health-care workers are at risk to acquire HIV through occupational exposure to blood of HIV-infected patients. The mean risk after a percutaneous exposure is approximately 0.3%. A large inoculum and a source patient with a high plasma viral load increases the transmission risk. To ensure the safety of the operating team, we try to reduce HIV viral load in plasma prior to high-risk interventions (cardiothoracic and orthopaedic surgery). However, in 15.7% of the exposures occurring in the operating room, the possible source material is bone marrow. To make more accurate exposure risk assessments, we measured HIV-1 RNA in both plasma and bone marrow of five HIV-infected patients undergoing surgery. We found that the plasma viral load was not different from the viral load in bone marrow.

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.

The human immunodeficiency virus (HIV-1) causes various hematopoietic abnormalities, with thrombocytopenia (TP) occurring in 30% of infected individuals. In the present study, we aimed to determine whether HIV-1 in the bone marrow of TP patients can infect primary megakaryocytes in vitro, which may contribute to the development of thrombocytopenia. We amplified the V3 loop of HIV-1 envelope from the bone marrow of TP and non-TP patients and constructed recombinant viruses. We demonstrate that the bone marrow of TP and non-TP patients contained R5 strains, whereas X4 strains were present only in the bone marrow of TP patients. Furthermore, HIV-1 from the bone marrow of TP and non-TP patients infected megakaryocytes to similar levels, suggesting that the V3 loop of HIV-1 may not contain the viral determinants of HIV-associated TP. Chemokine receptor analysis determined that CD34+-cell-derived megakaryocytes express CD4, CXCR4, and CCR5 and are productively infected by both X4 and R5 HIV-1 isolates. Finally, we showed that CD34+-cell-derived megakaryocytes express the chemokine receptor CCR3., (Copyright 2000 Academic Press.)

We analyzed bone marrow and blood from human immunodeficiency virus type 1 (HIV-1)-infected individuals and described the HIV-1 quasispecies in these cellular compartments. HIV-1 isolates from the bone marrow of thrombocytopenic patients contained distinct amino acids in the V3 loop and infected T-cell lines, implicating this virus in the development of thrombocytopenia.

Patients with human immunodeficiency virus-1 (HIV-1) infection often present with bone marrow (BM) failure that may affect all hematopoietic lineages. It is presently unclear whether this failure reflects a direct viral impairment of the CD34+ hematopoietic progenitor cells or whether the virus affects the BM microenvironment. To study the effects of HIV-1 on the BM microenvironment, we examined the stromal cell monolayers in long-term BM culture (LTBMC), which are the in vitro equivalent of the hematopoietic microenvironment. We assessed the hematopoietic support function (HSF) of human stromal layers by determining the cellular proliferation and colony-forming ability of hematopoietic progenitors from BM cells grown on the stromal layers. We show that the HSF is reduced by in vitro infection of the human stromal cell layer by a monocytotropic isolate of HIV-1 (JR-FL). There is no loss of HSF when the stromal cell layer is resistant to HIV-1 replication, either using murine stromal cell layers that are innately resistant to HIV-1 infection or using human stromal cells genetically modified to express a gene that inhibits HIV-1 replication (an RRE decoy). Decreased HSF was seen using either human or murine hematopoietic cells, if the stromal cells were human cells that were susceptible to HIV-1 infection. These in vitro studies implicate HIV-1 replication in the stroma as the essential component causing decreased hematopoietic cell production in HIV-1 infection.

Bone marrow of 61 HIV-1-infected patients and 23 control patients was examined to determine the incidence of B19 infection and its clinical impact in HIV-1-infected persons. Of the 61 HIV-infected patients studied, ages ranged from 22-47 years with a mean of 33.2 years. There was a man:woman ratio of 3.8:1. With regard to staging of HIV disease at the time of bone marrow sampling, 52 patients were CDC group 4, 5 patients were CDC group 3, and 4 patients were CDC group 2. Control patients, were not known to be HIV-1-infected, and had one of the following conditions: lymphoma, leukaemia, thrombocytopenia, thrombocytosis, anaemia, multiple myeloma, raised serum IgM. Thirteen of 61 HIV-infected patients and 0 of 23 control patients were positive for B19 DNA in bone marrow (two-tailed P value = 0.016). Within the HIV-infected group, the average haemoglobin among persons testing B19 DNA positive (n = 13) was 11.1 g/dl, compared with 11.5 g/dl among persons testing B19 DNA negative (n = 48). In conclusion, B19 persistence may be common and frequently subclinical in AIDS patients.

Similar to other lymphoid organs, the bone marrow (BM) is a potential reservoir for HIV-1. Although hematologic abnormalities are common in AIDS patients, the mechanisms by which HIV-1 contributes to these abnormalities are poorly understood. Hemapoietic suppression can be mediated by HIV-1 and some of its associated proteins. We have previously reported that AIDS peripheral blood sera inhibit normal CFU-granulocyte macrophage (GM). In this study, we have found that sera obtained from AIDS BM patients are more inhibitory to CFU-GM than those from peripheral blood (p < 0.05). To determine a candidate suppressor factor, we studied the role of p24 and found that it exerts varying degrees of suppression to CFU-GM, but minimal inhibition on erythroid colonies. We have found that the suppressive levels of p24 in in vitro assays are similar to the average circulating levels in AIDS patients. We have also observed that most of the inhibition caused by p24 is mediated by soluble factor(s) produced by the BM stroma. Cross-linking studies with membrane proteins from normal BM stroma and CD34+ cells indicated a putative 26-kDa p24 receptor, indicating that p24 binds to a receptor present on normal BM cells. These results implicate another HIV-1 protein, p24, in hemapoietic suppression and add another plausible mechanism by which HIV-1 contributes to BM failure in AIDS. Furthermore, these findings suggest a potential risk for similar in vivo suppression in the early phase of HIV-1 infection when viremia is elevated, and also in long-term survivors who have low, although persistent, HIV-1 replication.

By animal-to-animal passage of simian/human immunodeficiency virus (SHIV) in pig-tailed macaques, we have developed a macaque model of human immunodeficiency virus type 1 (HIV-1) disease in humans. Passaging was begun with a chimeric virus containing the env gene of HIV-1 HXBc2 and the gag and pol genes of simian immunodeficiency virus SIVmac239. SHIV was passaged serially in cohorts of two macaques each, using bone marrow-to-bone marrow transfers at 5, 5, and 16 weeks for passages 2, 3, and 4, respectively. The fifth passage was done by using cell-free virus isolated from cerebrospinal fluid of a passage 4 macaque. The virus became more virulent with each passage. Virus replication was restricted in all three animals in passages 1 and 2 but not in five of the six animals in passages 3, 4, and 5. In these animals, intense virus replication in the lymphoid tissues resulted in almost total elimination of CD4+ T cells within weeks of inoculation, and three of these animals developed AIDS in less than 1 year. The more uniform virus-host interaction initiated by the cell-free virus in the passage 5 animals contrasted with a more variable pattern of disease initiated by infectious bone marrow cells during earlier passages. The virulent cell-free SHIV can now be used to screen the efficacy of vaccines directed against the envelope of HIV-1.

A small number of patients seropositive for the human immunodeficiency virus (HIV) have been reported as developing acute non-lymphoblastic leukemia (ANLL). In the cases previously published, the authors never reported a study of the link joining HIV infection and leukemia. We describe here the case of a 41-year-old HIV positive patient who developed ANLL (FAB classification M5). Using molecular techniques, we looked for a direct link between these two co-existing diseases. We showed the absence of HIV expression in the malignant clone, suggesting that the association of ANLL and Acquired Immune Deficiency Syndrome is not a direct consequence of the myeloid precursors infection. Nevertheless a relationship may exist through a disorganization of the bone marrow micro-environment.

Haematological abnormalities are often seen in patients infected with HIV. A number of mechanisms are thought to contribute to this bone marrow suppression, including impaired stromal function and direct infection of progenitor cells. Evidence suggests that both bone marrow progenitor cells and perhaps stromal cells are open to infection by HIV, which raises the possibility that bone marrow stromal cells may serve as a reservoir for HIV. This study investigated the cellular targets and kinetics of in vitro infection of stroma in long-term bone marrow culture (LTBMC) using both mono- and lymphocytotropic strains of HIV-1. p24 ELISA and reverse transcriptase (RT) assay demonstrated that stroma could be infected with HIV and release infectious virions. The target cells for infection were shown to be macrophages by immunohistochemistry (APAAP), dual immunofluorescence staining (using CD68 and p24) and electron microscopy. The data show that it was possible to infect stroma in LTBMC with HIV and that such infection was productive. The main target for infection was bone marrow macrophages. In contrast to peripheral blood derived macrophages, these cells were susceptible to both lymphocytotropic and monocytotropic strains of HIV-1. The data suggests that these bone marrow macrophages may act as a reservoir for HIV, Infection of bone marrow macrophages may affect haemopoiesis either by transmission of HIV infection to developing progenitor cells through direct cell-to-cell contact or by altering the ability of the stroma to support normal development.

The majority of human immunodeficiency virus (HIV)-seropositive patients develop bone marrow abnormalities associated with hematopoietic malfunction during the progression of disease. One important manifestation of HIV-associated hematopoietic dysfunction is that after myelosuppression, bone marrow recovery, a process known to be mediated in part by the production of stromal cell-derived hematopoietic growth factors, is impaired. We sought to test the hypothesis that bone marrow stromal cells are infected by HIV-1 in vivo and that production of certain stromal cell-derived hematopoietic growth factors is deficient as a consequence. In this report, we demonstrate that bone marrow microvascular endothelial cells (MVEC), a key element of the stroma, are the predominant cells infected by HIV (5% to 20%) in bone marrow stromal cultures obtained from 11 consecutive HIV-seropositive patients. Although HIV-infected stromal cultures enriched for MVEC constitutively express normal levels of interleukin (IL)-4, IL-6, granulocyte (G)-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and Steel factor, IL-1 alpha-induced release of IL-6 and G-CSF is significantly reduced in these cultures. These observations suggest that HIV infection of bone marrow MVEC reduces the capacity of hematopoietic stroma to respond to regulatory signals that normally augment blood cell production during periods of increased demand.

In order to investigate the potential susceptibility of bone marrow stromal myoid cells to human immunodeficiency virus (HIV), a myoid cell population devoid of all other cellular components of marrow environment was isolated from 3 normal adult bone marrow samples. The bone marrow myoid cells were infected with 3 different strains of HIV-1, 2 strains isolated from patients with acquired immune deficiency syndrome (AIDS) and HTLV-IIIB strain. To demonstrate successful infection and HIV production, culture supernatants, harvested weekly until 2 months post-infection, were tested for the presence of p24 antigen and infectious virus. Myoid cell monolayers obtained from the 3 different bone marrow samples were shown to be susceptible to infection. In particular, infection led to the presence of p24 antigen and of infectious virus in culture supernatants up to day 49 post-infection. After day 49, it was not possible to demonstrate the presence of infectious virus in culture supernatants and HIV-DNA polymerase chain reaction (PCR) failed to show viral genome in any of the cultures assayed. Our results demonstrate the susceptibility of myoid stromal cells to HIV infection and may provide an in vitro model for studying the effects of HIV infection in disregulation of the haemopoietic function of bone marrow environment.

As an attempt to elucidate the pathogenesis of human immunodeficiency virus type 1 (HIV-1)-related cytopenia, the effects of infection of long-term primary bone marrow culture (LTBMC)-derived adherent cells on hematopoiesis were investigated. Productive infection could then be established only when using monocytotropic strains HIV-1Ba-L, HIV-1Ada, and HIV-1JR-FL but not with lymphocytotropic strain HIV-1LAI. Culture supernatants were tested for major cytokines involved in the regulation of hematopoiesis: neither IL-3 nor GM-CSF were detectable in the infected or noninfected cultures; in contrast, TGF-beta, TNF-alpha, MIP-1 alpha, Steel Factor, and IL-6 were detected at all times in established LTBMCs, but their levels were not consistently altered by virus replication. In vitro functional analysis by colony and long-term culture assays showed that HIV-1 infection failed to alter either the kinetics or the number of hematopoietic progenitors produced by the stromal layers; it did not interfere with the clonogenicity of exogeneous CD34+ cells in semisolid assays, and no difference was observed relative to the controls when HIV-1-infected stromal layers were tested for their ability to sustain long-term hematopoiesis. These results show that productive and sustained virus replication in the macrophage component of LTBMCs does not significantly alter the profile of major cytokines involved in regulating hematopoiesis, nor is it sufficient by itself for altering in vitro hematopoiesis under the baseline conditions used.

We investigated whether cells belonging to the megakaryocytic lineage could be infected in vitro with human immunodeficiency virus type-1 (HIV-1). Primary GPIIb/IIIa+ bone marrow (BM) cells and HEL continuous cell line were first phenotypically characterized for the presence of megakaryocytic markers and CD4 antigen, then challenged in vitro with the laboratory strain IIIB of HIV-1. Both GPIIb/IIIa+ BM and HEL cells expressed significant levels of CD4 receptor (> 50%) and were efficiently infected with HIV-1, as judged by the presence of proviral DNA after polymerase chain reaction analysis and by quantitative evaluation of gag p24 antigen in the culture supernatants. Of note, infection with HIV-1 in both primary BM megakaryocytes and HEL cells was specifically blocked by soluble recombinant CD4. To ascertain whether the CD4 receptor was essential for infection of megakaryocytic cells, HEL were subcloned into CD4+ and CD4- cells. Although unfractionated and CD4+ HEL cells were productively infected with HIV-1, CD4- HEL cells could not be infected. Infection of HEL cells did not induce gross cytotoxic effects or a significant increase of apoptosis. On the other hand, treatment of unfractionated or CD4+ HEL cells with cross-linked recombinant env gp120 or Leu3a anti-CD4 monoclonal antibody markedly (P < 0.01) increased the degree of apoptosis with respect to HEL cells infected with HIV-1 or treated with cross-linked gag p24 or anti-GPIIb/IIIa antibody. Taken together, these data indicate that the CD4 receptor represents the main route of infection in cells belonging to the megakaryocytic lineage. Moreover, an inappropriate engagement of CD4 by either free env gp120 or anti-CD4 monoclonal antibody could be more relevant than a direct infection with HIV-1 in the induction of the frequent BM megakaryocyte abnormalities found in HIV-1 seropositive thrombocytopenic patients.

This report presents results concerning the potential role of negative regulators in hematopoietic suppression observed in human immunodeficiency virus (HIV)-infected long-term cultures (LTC) of human bone marrow cells. Confluent stromal cell layers established from human bone marrow cells were exposed to HIV-1ADA, a monocytotropic strain of HIV-1. A progressive increase in the concentration of HIV-1 p24 antigen in cultures exposed to HIV-1ADA demonstrated that there was a productive infection. Cells from both noninfected and HIV-infected stromal cell layers produced factors that stimulated the proliferation of colony-forming units for granulocytes and macrophages (CFU-GM) from non-infected CD34+ cells. In contrast, when noninfected CD34+ cells were directly cocultured on intact stromal cell layers fewer CFU-GM and burst-forming units for erythroid cells (BFU-E) were detected in HIV-infected LTC than in noninfected LTC. One week after the addition of CD34+ cells, the number of CFU-GM in HIV-infected LTC in six of nine experiments was reduced compared to noninfected control LTC. In those six experiments, the number of CFU-GM was only 53 +/- 5% (SEM) of the number in noninfected LTC. The number of BFU-E in HIV-1-infected LTC was only 46 +/- 5% of the number in noninfected LTC (n = 5). There were fewer BFU-E in HIV-1-infected LTC, whether or not there was a reduced number of CFU-GM. Neutralizing antibody to tumor necrosis factor alpha (TNF-alpha) had no effect on the number of BFU-E in HIV-infected LTC. The number of BFU-E, however, was 2.1 +/- 0.2-fold greater (n = 3) in HIV-infected LTC incubated with neutralizing antibody to interferon-alpha. In HIV-infected LTC with decreased numbers of CFU-GM, the number of CFU-GM was approximately 2-fold greater after incubation of HIV-infected LTC with anti-interleukin-4 (IL-4). The effect of anti-TNF-alpha was variable, and anti-transforming growth factor-beta had no effect on the number of CFU-GM in HIV-infected LTC. After 2 weeks, the number of CFU-GM in HIV-infected LTC incubated with anti-IL-4 and anti-TNF-alpha was 2- to 4-fold greater than in untreated HIV-infected LTC. Antibody treatment did not promote an increase in the number of CFU-GM in noninfected LTC or in LTC in which CFU-GM numbers were not reduced after HIV infection.(ABSTRACT TRUNCATED AT 400 WORDS)

It is not known whether impaired hematopoiesis noted during human immunodeficiency virus (HIV) infection results from infection of stem/progenitor cells or of cells of the bone marrow microenvironment. Normal adherent primary stromal layers were exposed to HIV to determine which of this mixture of endothelial cells, fibroblasts, and macrophages are susceptible to the virus. Viral p24 in supernatants was noted with monocytotropic HIV-1Ada, HIV-1Ba-L, and HIV-1JR-FL but not with lymphotropic HIV-1LAI nor HIV-1MN strain, and only stromal macrophages expressed the viral antigens. Coculture of the layers with PHA-activated normal lymphocytes failed to rescue lymphotropic virus. No p24 was produced when macrophage-depleted stromal cells were exposed to either HIV-1Ba-L or HIV-1LAI; proviral DNA was then amplified by PCR in cells exposed to either virus, though coculture with lymphocytes rescued only HIV-1Ba-L. Altogether, these data indicate that macrophages are the major targets of HIV in cultured stromal layers. As virus replication in macrophages did not affect the profile of major cytokines involved in regulating hematopoiesis, HIV infection could alter hematopoiesis by other as yet unspecified mechanisms.

The pathogenesis of peripheral blood cytopenias in AIDS patients is clearly multifactorial. Among the various contributing mechanisms, those involving a direct role of HIV-1 have been actively investigated in the past few years. It has now been convincingly demonstrated that HIV can impair the survival/proliferative capacity of purified haematopoietic progenitor cells. Although a subset of haematopoietic progenitor cells are perhaps susceptible to HIV-1 infection, both in vitro and in vivo, the suppressive effect does not require either active or latent infection and is probably mediated by the interaction of viral or virus-associated proteins with the cell membrane of haematopoietic progenitor cells. Both the viral load and the biological characteristics of the virus play an important role in suppression, since different isolates displayed different inhibitory activity. Haematosuppression is not a specific property of monocytotropic versus lymphocytotropic or low-replicating versus high-replicating isolates, and it will be important to exactly establish which viral component is crucial to suppression of haematopoietic progenitor cells. Since the haematopoietic stem cell is the common progenitor to both the myeloid and lymphoid lineages, the capacity of HIV to impair the growth of early haematopoietic progenitor cells could contribute not only to the frequent occurrence of anaemia, granulocytopenia and thrombocytopenia in AIDS patients, but also to the inability of the bone marrow to reconstitute a functional pool of mature CD4+ T-cells. It is also possible that haematopoietic progenitor cells committed to the T-lymphoid lineage are impaired by HIV in their differential pathway within the thymus (Bonyhadi et al, 1993). Infection of megakaryocytes could result in underproduction of platelets and possibly represents a major pathogenetic mechanism of HIV-related thrombocytopenia. Infection of monocytes and T-lymphocytes leads in vitro and probably also in vivo to deranged cytokine production. In the first stages of the disease, increased cytokine production, consequent to a chronic immune activation, is probably responsible for the myelodysplastic/hyperplastic alterations observed at the bone marrow level. In more advanced stages of the disease, the general decline in immune function, the consequent imbalance in cytokine production, and the increase in viral burden, may contribute to dysregulated haematopoiesis and peripheral blood cytopenias.