Search

Persons living with HIV (PLWH) receiving tenofovir disoproxil fumarate (TDF)-based antiretroviral therapy (ART) risk suffering TDF-associated nephrotoxicity (TDFAN). TDFAN can result in short- and long-term morbidity, including permanent loss of kidney function, chronic kidney disease (CKD), and end-stage kidney disease (ESKD) requiring dialysis. Currently, there is no model to predict this risk or discern which patients to initiate TDF-based therapy. Consequently, some patients suffer TDFAN within the first few months of initiating therapy before switching to another suitable antiretroviral or a lower dose of TDF. In a prospective observational cohort study of adult Zambian PLWH, we modelled the risk for TDFAN before initiating therapy to identify individuals at high risk for experiencing AKI after initiating TDF-based therapy. We enrolled 205 HIV-positive, ART-naïve adults initiating TDF-based therapy followed for a median of 3.4 months for TDFAN at the Adult Infectious Disease Research Centre (AIDC) in Lusaka, Zambia. We defined TDFAN as meeting any of these acute kidney disease (AKD) criteria: 1) An episode of estimated glomerular filtration rate (eGFR)< 60ml/ min/1.73m2 within 3 months, 2) reduced eGFR by> 35% within 3 months or 3) increased serum creatinine by> 50% within 3 months. A total of 45 participants (22%) developed acute kidney disease (AKD) after TDF-based therapy. The development of AKD within the first 3 months of commencing TDF-based therapy was associated with an increase in baseline serum creatinine, age, baseline eGFR and female sex. We concluded that baseline characteristics and baseline renal function biomarkers predicted the risk for AKD within the first 3-months of TDF-based therapy., Competing Interests: NO authors have competing interests. EDS reports consulting for Akebia, Inc. 4/19, honorarium for an invited educational talk at the Annual Da Vita Physician Leadership Conference 2/19, royalties as an author for UpToDate, and serving on the editorial board for the Clinical Journal of the American Society of Nephrology. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Increased antimicrobial resistance among Human Immunodeficiency Virus (HIV)-infected individuals to commonly used antibiotics in the treatment of gastroenteritis is a public health concern, especially in resource-limited settings. We set out to compare the antimicrobial susceptibility pattern of Escherichia coli (E. coli) isolates from HIV-infected and HIV-uninfected individuals at a tertiary hospital in Lusaka, Zambia. An analytical cross-sectional study was conducted at the University Teaching Hospital from May 2019 to August 2019. Stool samples were screened, and 79 HIV-infected individuals matched by age and sex with 84 HIV-uninfected individuals that presented with E. coli associated gastroenteritis were studied. Demographics were collected from the Laboratory Information System (LIS) and stool samples were collected in a sterile leak-proof container. Samples were cultured and only those where E. coli was isolated were included in the study and tested for antimicrobial susceptibility by the Kirby-Bauer disk diffusion technique. HIV-positive individuals were 3 times (adjusted odds ratio (AOR) = 3.17; 95% CI (1.51, 6.66); p < 0.001) more likely to be resistant to quinolones compared with their HIV-negative counterparts. Similarly, HIV-positive individuals were almost 4 times (AOR = 3.97, 95% CI (1.37, 11.46); p = 0.011) more likely to have multidrug-resistant E. coli compared with those who were HIV-negative. HIV infection was associated with reduced E. coli susceptibility to commonly used antibiotics, and most cases showed resistance.

Effective suppression of HIV-1 replication requires inhibition of critical viral target molecules. Tat and Rev are indispensable regulatory factors for HIV-1 replication, whereas Env mediates virus entry by direct interaction with surface receptor CD4 and coreceptor CCR5 or CXCR4. Anti-HIV-1 tat-rev and env ribozymes and Rev aptamers were previously demonstrated to provide relatively long-term protection against HIV-1 infection in vitro. However, further improvements in these constructs for clinical application in a stem-cell-based gene therapy setting requires in vivo characterization. Toward this end, we introduced these constructs into CD34(+) hematopoietic progenitor cells by retrovirus-mediated gene transduction. Ribozyme- and aptamer-transduced CD34(+) cells differentiated normally into multiple lineages of erythroid and myeloid progenies in a colony-forming unit assay. Macrophages that differentiated from the transduced CD34(+) cells expressed anti-tat-rev and -env ribozymes and Rev aptamers and displayed their normal characteristic surface markers CD14, CD4, and CCR5. Using the SCID-hu mouse in vivo human thymopoiesis model, we demonstrated that ribozyme- and aptamer-transduced CD34(+) cells retained their normal capacity to reconstitute human fetal thymus and liver tissue (thy/liv) grafts. Reconstitution by ribozyme- and aptamer-transduced CD34(+) cells reached levels of up to 87% based on HLA surface marker staining. Differentiated thymocytes derived from reconstituted grafts expressed anti-tat-rev and -env ribozymes and Rev aptamers and showed significant resistance to HIV-1 infection upon challenge. Analysis of reconstituted thymocytes by hybridization revealed an average of 0.4 to 2 copies of vector sequences per cell. Southern analysis of proviral integration junctions in progeny thymocytes demonstrated that the human thy/liv grafts were reconstituted by a few primitive hematopoietic stem cells. These results highlight the utility of RNA-based anti-HIV-1 gene therapeutic approaches and their preclinical testing in a surrogate animal model harboring human tissue.

The cellular entry of HIV is mediated by the specific interaction of viral envelope glycoproteins with the cell-surface marker CD4 and a chemokine receptor (CCR5 or CXCR4). Individuals with a 32-base-pair (bp) deletion in the CCR5 coding region, which results in a truncated peptide, show resistance to HIV-1 infection. This suggests that the downregulation of CCR5 expression on target cells may prevent HIV infection. Therefore, ribozymes that inhibit the CCR5 expression offer a novel approach for anti-HIV gene therapy. To assess the effect of an anti-CCR5 ribozyme (R5Rbz) on macrophage differentiation, CD34+ hematopoietic progenitor cells were transduced with a retroviral vector carrying RSRbz and allowed to differentiate in the presence of appropriate cytokines. R5Rbz-transduced CD34+ cells differentiated normally into mature macrophages that carried CD14 and CD4 surface markers, expressed the anti-CCR5 ribozyme, and showed significant resistance to viral infection upon challenge with the HIV-1 BaL strain. Using an in vivo thymopoiesis model, the effect of RSRbz on stem cell differentiation into thymocytes was evaluated by reconstituting SCID-hu mice thymic grafts with ribozyme-transduced CD34+ cells. FACS analysis of cell biopsies at 4 and 6 weeks postengraftment for HLA, CD4, and CD8 markers showed comparable levels of reconstitution and similar percentages of subpopulations of thymocytes between grafts receiving R5Rbz-transduced and control CD34+ cells. RT-PCR assays demonstrated the expression of the anti-CCR5 ribozyme in CD4+, CD8+, and CD4+/CD8+ thymocyte subsets derived from RSRbz-transduced CD34+ cells. These results indicate that anti-CCR5 ribozyme can be introduced into hematopoietic stem cells without adverse effects on their subsequent lineage-specific differentiation and maturation. The expression of anti-CCR5 ribozymes in HIV-1 target cells offers a novel gene therapy strategy to control HIV infection.

Pancytopenia as a consequence of bone marrow abnormalities is commonly seen in HIV-infected individuals. To examine the effect that HIV-1 has on hematopoietic cells, we compared hematopoietic properties of bone marrow samples from HTV+ patients at various stages of disease with bone marrow samples from uninfected donors. While the absolute number of recovered CD34+ cells and the cloning efficiency of these cells did not differ significantly in HIV+ donors, the percentage of CD34+ CD4+ cells was significantly depleted in late-stage HIV+ patients. We observed a direct correlation between the numbers of CD34+ CD4+ cells in the bone marrow and the peripheral CD4 count. Further characterization of the CD34+ CD4+ subpopulation demonstrated that these cells expressed lower levels of HLA-DR on their surface compared with CD34+ CD4- cells, suggesting an immature phenotype. We also found evidence for expression of HIV-1 coreceptors CXCR-4 and CKR-5 message and protein in CD34+ bone marrow cells. While this finding suggested that hematopoietic cells might be susceptible to HIV infection at an early stage of maturation, thus affecting different cell lineages as they matured, we did not find any evidence for infection of HIV in these cells. These data suggest that HIV affects early hematopoietic progenitor cells either directly or indirectly, and in particular CD34+ CD4+ cells. This finding has important implications for disease pathogenesis and for application of gene therapy approaches that use CD34+ hematopoietic cells.

The reconstitutive potential of CD34+-derived cord blood (CB) cells, transduced with a regulated diphtheria toxin A (DT-A) chain gene, was examined in SCID-hu mice harboring a conjoint organ composed of human thymus and liver (thy/liv). The DT-A-transduced cells, injected directly into the thy/liv organ, showed the same engraftment potential as control CB cells transduced with the non-DT-A parental vector. CB cells, distinguishable from the thy/liv cells by the HLA marker B7, were preferentially maintained in ex vivo culture. In the thy/liv organ, the engrafted CB cells represented >80% of the total cells. A majority of cells (>70%) in the thy/liv organ were also CD4+CD8+, as would be expected of maturing thymocytes. The incidence of double-positive cells was highest at 44 days (compared with 30 days and 80 days) after injection of CB cells. This suggested that a minimum time was required to achieve optimal proliferation of cells in the thy/liv organ but that, at later times, all of the early cells had matured. Thus, the population used for engraftment contained early cells but not self-renewing cells. The double-positive cells matured rapidly into single-positive cells (either CD4+ or CD8+) when placed in ex vivo culture. Marked cells (neo+) could readily be detected in the thy/liv-derived cells. The cells transduced with DT-A showed long-term protection in ex vivo culture against HIV T lymphotropic isolate NL4-3. This study shows that DT-A-transduced cells had no apparent disadvantage in engraftment of the thy/liv organ and did not have any toxic effects in vivo. Such cells were protected against HIV infection even when challenged more than 2 months after transduction and after a 44-day engraftment period in the thy/liv mice. These data support the feasibility of toxin gene therapy as a strategy for HIV infection.

Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Although twelve years have passed since the identification of HIV as the cause of AIDS, we do not yet know how HIV kills its target, the CD4+ T cell, nor how this killing cripples the immune system. Prominent theories include direct killing of infected CD4+ T cells by the action or accumulation of cytopathic viral DNA, transcripts or proteins, or by virus-specific cytotoxic T lymphocytes, and indirect killing of uninfected CD4+ T cells (and other immune cells) by autoimmune mechanisms, cytokines, superantigens, or apoptosis. In the past year, studies have provided tantalizing clues as to why infected cells may not die and how these infected cells kill innocent bystander cells.