Late-stage AIDS Patients Research Articles

Novel heparan sulfate-binding peptides for blocking herpesvirus entry.

Human cytomegalovirus (HCMV) infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV) infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs), serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.

Long-term highly suppressed HIV-infected children and adolescents with negative rapid HIV tests due to significant antibody loss

BackgroundRapid HIV test devices are widely used throughout the world and are important as diagnostic tools with relatively high sensitivity and specificity. Loss of HIV specific antibodies in late-stage AIDS patients has previously been reported in patients with advanced disease (i.e., AIDS). ObjectiveTo study rate of antibody loss that may lead to false negative HIV-antibodies results in children and adolescents who received long term antiretroviral (ARV) treatment with persistently undetectable viral loads. Study designFive FDA approved rapid HIV test kits including Trinity Uni-Gold Recombigen HIV-1, OraQuick Advance HIV-1/2, Reveal G3 HIV-1, Clearview STAT-PAK HIV-1/2, and Clearview COMPLETE HIV-1/2 were used to test 98 stored samples from 27 patients. Samples were tested at baseline and at least twice in 6–14 years post initiation of ARV treatment and full viral load suppression. ResultsOf the 403 tests, 43 (10.7%) were found to be false-negative using rapid HIV kits. Loss of positivity was correlated with decrease of HIV antibody titer. ConclusionsThere is a slow but persistent loss of HIV specific antibodies in highly suppressed HIV infected children and adolescents that may lead to false-negative results in rapid HIV antibody tests. The temporal loss of signal is dependent on the baseline level of antibodies and the type of HIV rapid test kit used.

Circulating Dendritic Cells Isolated from Healthy Seropositive Donors Are Sites of Human Cytomegalovirus Reactivation In Vivo

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34(+) progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.

Cardiac effects in perinatally HIV‐infected and HIV‐exposed but uninfected children and adolescents: a view from the United States of America

IntroductionHuman immunodeficiency virus (HIV) infection is a primary cause of acquired heart disease, particularly of accelerated atherosclerosis, symptomatic heart failure, and pulmonary arterial hypertension. Cardiac complications often occur in late-stage HIV infections as prolonged viral infection is becoming more relevant as longevity improves. Thus, multi-agent HIV therapies that help sustain life may also increase the risk of cardiovascular events and accelerated atherosclerosis.DiscussionBefore highly active antiretroviral therapy (HAART), the two-to-five-year incidence of symptomatic heart failure ranged from 4 to 28% in HIV patients. Patients both before and after HAART also frequently have asymptomatic abnormalities in cardiovascular structure. Echocardiographic measurements indicate left ventricular (LV) systolic dysfunction in 18%, LV hypertrophy in 6.5%, and left atrial dilation in 40% of patients followed on HAART therapy. Diastolic dysfunction is also common in long-term survivors of HIV infection. Accelerated atherosclerosis has been found in HIV-infected young adults and children without traditional coronary risk factors. Infective endocarditis, although rare in children, has high mortality in late-stage AIDS patients with poor nutritional status and severely compromised immune systems. Although lymphomas have been found in HIV-infected children, the incidence is low and cardiac malignancy is rare. Rates of congenital cardiovascular malformations range from 5.6 to 8.9% in cohorts of HIV-uninfected and HIV-infected children with HIV-infected mothers. In non-HIV-infected infants born to HIV-infected mothers, foetal exposure to ART is associated with reduced LV dimension, LV mass, and septal wall thickness and with higher LV fractional shortening and contractility during the first two years of life.ConclusionsRoutine, systematic, and comprehensive cardiac evaluation, including a thorough history and directed laboratory assays, is essential for the care of HIV-infected adults and children as cardiovascular illness has become a part of care for long-term survivors of HIV infection. The history should include traditional risk factors for atherosclerosis, prior opportunistic infections, environmental exposures, and therapeutic and illicit drug use. Laboratory tests should include a lipid profile, fasting glucose, and HIV viral load. Asymptomatic cardiac disease related to HIV can be fatal, and secondary effects of HIV infection often disguise cardiac symptoms, so systematic echocardiographic monitoring is warranted.

Current antibody-based immunoassay algorithm failed to confirm three late-stage AIDS cases in China: case report

BackgroundImmunoassays composed of screening and confirmation are the established algorithm to confirm HIV infection in China, with a Western blot result as the final diagnosis.Case presentationIn this report, three late-stage AIDS patients were initially tested HIV antibody positive using multiple screening kits, but tested indeterminate using Western blot. HIV infection diagnosis was confirmed based on nucleic acid assays, clinic manifestations and epidemiological history. Case A was identified positive at 30 months, using Western blot, Case B at 8 months, and case C remained indeterminate until he died of Kaposi's sarcoma 4 months after HAART.ConclusionThe report indicates that current antibody-based testing algorithms may miss late-stage AIDS patients and therefore miss the opportunity for preventing these cases from further transmission. The report also implies that viral load assays is not easy to be universely applicated in developing country like China although it is helpful in diagnosing complicated cases of HIV infection, so the counselling before and after testing is imperative to the diagnosis of HIV infection and risk behavior survey on the examinee should be as detailed as possible.

CD4 and MHC-I downregulation are conserved in primary HIV-1 Nef alleles from brain and lymphoid tissues, but Pak2 activation is highly variable

HIV-1 compartmentalization in the CNS has been demonstrated for gag, pol, and env genes. However, little is known about tissue compartmentalization of nef genes and their functional characteristics in brain. We have cloned 97 nef genes and characterized 10 Nef proteins from autopsy brain and lymphoid tissues from 2 patients with AIDS and HIV-1-associated dementia. Distinct compartmentalization of brain versus lymphoid nef genes was demonstrated within each patient. CD4 and MHC-I downregulation were conserved in all tissue-derived Nefs. However, MHC-I downregulation by brain-derived Nefs was weaker than downregulation by lymphoid-derived Nefs. The motifs KEEE- or EKEE- at the PACS-1 binding site represented brain-specific signature patterns in these 2 patients and contributed to the reduced MHC-I downregulation activity of brain-derived Nefs from these patients. Pak2 association was highly variable in Nefs from both patients. Three of 10 tissue-derived Nefs coimmunoprecipitated activated Pak2, with strong association demonstrated for only 2 Nefs. The ability of Nef to associate with activated Pak2 did not correlate with brain or lymphoid tissue origin. Nef genes from viruses isolated from brain by coculture with PBMC were not closely related to sequences amplified directly from brain tissue, suggesting that viral selection or adaptation occurred during coculture. This study of tissue-derived HIV-1 Nefs demonstrates that CD4 and MHC-I downregulation are highly conserved Nef functions, while Pak2 association is variable in late stage AIDS patients.

Characterization of HIV-related periodontitis in AIDS patients: HIV-infected macrophage exudate in gingival crevicular fluid as a hallmark of distinctive etiology.

In an attempt to clarify the immunobiological events featuring periodontitis lesions of AIDS patients in the late stage of the disease, peripheral blood (PB) and gingival crevicular fluid (GCF) leucocytes from periodontitis lesions of 23 late-stage AIDS patients were analysed by three-colour flow cytometry for detection and identification of intracytoplasmic p24+ cell fractions. The cells were reacted with CD14 and CD68 for mononuclear phagocytes or with CD4 and CD14 for Th cells, then with anti-p24 MoAb. To detect HIV proviral sequences and intracellular p24 RNA sequences, genomic DNA and cellular RNA from leucocytes were extracted for semi-nested polymerase chain reaction (PCR) amplification. CD68+/p24+ and CD14+/CD68+/p24+ fractions were larger in GCF than in PB (P<0.0001; P < 0.003). CD14+/p24+ fraction was lower in GCF than in PB (P < 0.05). The fluorescence intensities (FI) for intracellular p24 in CD68+ and CD14+/CD68+ cells were higher in GCF than in PB (P < 0.003; P < 0.02), whereas those of CD14+ macrophages did not differ. The p24 FI of CD68+ macrophages in GCF correlated with CD4+ lymphocyte counts in PB (P < 0.005). p24 FI levels of CD14+ monocytes in GCF and PB significantly correlated (P < 0.02), whereas that of CD68+ macrophages did not. PCR and reverse transcriptase (RT)-PCR of cellular DNA and RNA yielded positive signals, demonstrating viral integration and production in GCF leucocytes. These results show that periodontitis lesions in AIDS patients can be characterized by a rapid macrophage turnover, and these HIV-infected macrophage exudates in GCF may be considered as a within-mouth source of virus.

Postoperative follow-up of anal condylomata acuminata in HIV-positive patients.

The purpose of this study was to analyze the postoperative follow-up of HIV-positive patients with anal condylomata acuminata, associating recurrence to the AIDS status. Ninety-seven male, homosexual patients with anal condylomata were submitted to surgical treatment from August 1992 to December 1997. Specimens were obtained for histologic investigation of Ki-67 cell proliferation marker and for polymerase chain reaction to define the human papillomavirus type. The patients were advised to return weekly during the first month, and monthly up to the sixth month, to evaluate recurrence. Patients with high-grade anal intraepithelial neoplasia remain in follow-up. Histology revealed low-grade anal intraepithelial neoplasia in 81.44 percent of patients and high-grade lesions in 18.56 percent. The results showed that high-grade lesions were not more frequent in late-stage AIDS patients. Ki-67 expression, a cell proliferation marker, was greater in high-grade than in low-grade anal intraepithelial neoplasia, but had no association with AIDS status. Nononcogenic human papillomavirus 6 and 11 were the commonest types. Comparing papillomavirus types and anal intraepithelial grade, we noticed that both oncogenic and nononcogenic types were responsible for high-grade lesions. All patients healed and 51 (52.6 percent) had recurrence up to the sixth month. AIDS status and papillomavirus type were not associated with recurrence. However, high-grade anal intraepithelial neoplasia patients had more recurrence than those with low-grade lesions. Topical treatment failed in 20 patients (41.6 percent), and these were submitted to new surgical treatment. All of them were in the late stage of AIDS. Three who had high-grade lesions in the first operation had low-grade lesions in specimens from the second surgery. The same histologic pattern was observed in 17 patients who had low-grade lesions in warts removed in the first operation. Other patients with high-grade lesions had no recurrence or evolution to invasive carcinoma from five to ten years of follow-up. High-grade anal intraepithelial neoplasia and late-stage AIDS are risk factors for recurrence of anal condylomata.

Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS.

The evolution of human immunodeficiency virus type 1 (HIV-1) quasispecies at the envelope gene was studied from the time of infection in 11 men who experienced different rates of CD4+ cell count decline and 6 men with unknown dates of infection by using DNA heteroduplex mobility assays. Quasispecies were genetically homogeneous near the time of seroconversion. Subsequently, slower proviral genetic diversification and higher plasma viremia correlated with rapid CD4+ cell count decline. Except for the fastest progressors to AIDS, highly diverse quasispecies developed in all subjects within 3 to 4 years. High quasispecies diversity was then maintained for years until again becoming more homogeneous in a subset of late-stage AIDS patients. Individuals who maintained high CD4+ cell counts showed continuous genetic turnover of their complex proviral quasispecies, while more closely related sets of variants were found in longitudinal samples of severely immunocompromised patients. The limited number of variants that grew out in short-term PBMC cocultures were rare in the uncultured proviral quasispecies of healthy, long-term infected individuals but more common in vivo in patients with low CD4+ cell counts. The slower evolution of HIV-1 observed during rapid progression to AIDS and in advanced patients may reflect ineffective host-mediated selection pressures on replicating quasispecies.

ANTISENSE GENE THERAPY DIRECTED AGAINST EBNA-1 IN EBVASSOCIATED B CELL LYMPHOMAS RESULTS IN POTENTIATION OF CHEMOTHERAPY IN VITRO AND DECREASED TUMOR VIRULENCE IN SCID MICE

Background: With prolonged survival of late-stage AIDS patients, AIDS-related lymphomas (ARL) are occurring with an increased frequency, resulting in approximately 3000-9000 new cases per year. Overall prognosis remains exceedingly poor. EBV is highly associated with ARL (30-70% of systemic lymphomas and up to 100% of CNS lymphomas) and likely plays a significant role in pathogenesis. We investigated the effects of ablating EBNA-1, an EBV transforming gene, using antisense oligodeoxynucleotides (oligos). Methods: Antisense (AS) oligos corresponded to codons 16-30 of theEBNA-I open reading frame. Scrambled AS (SAS) control was created using the same nucleotides in random order. Chemotherapeutic agents included adriamycin, etoposide (VP-16), and taxol. Cell lines included human B cells transformed with EBV strain B95-8, EBV negative B cell lymphomas, and EBV negative, non-B cell tumors. Cellular proliferation was determined by tritiated thymidine uptake as a measure of cytotoxicity. Apoptosis was measured by JAM assay or fluorescent staining. In vivo studies utilized SCID mice inoculated by intraperitoneal injection with B cell tumors. Results: The addition of as little as 3 µM AS resulted in a significant reduction of the LD50 of chemotherapeutic agents (equivalent to 0.007 µg/ml of VP-16 in the presence of the LD50 of adriamycin, for example). Although both AS and SAS potentiated the effect of chemotherapy on all tumor cell lines tested, AS potentiated the cytotoxicity of chemotherapy significantly more than SAS in EBV positive cell lines, but not in EBV negative cell lines (p<0.005,t-test). Apoptosis due to chemotherapy also appeared to be enhanced by prior AS treatment in EBV positive cell lines but not in EBV negative cell lines. In SCID mice inoculated with EBV positive human B cells, prior in vitro AS treatment of tumors (without chemo) enhanced survival by two-fold (median 102 days vs 56 days for saline and 42 days for SAS). Experiments using chemo plus oligos in SCID mice are underway. Conclusions: Ablation of EBNA-1 by AS potentiates the cytotoxicity of chemotherapy in an in vitro model of ARL. Non-specific cytotoxicity due to SAS was also seen. Enhanced cytotoxicity may be related to increased apoptosis as a result of EBNA-1 ablation and/or other downstream effects. Ex vivo EBNA-1 ablation by AS also increased survival in vivo in SCID mice inoculated with these tumors. These studies provide evidence for a significant role played by EBV in ARL and may lead to therapies for ARL based on EBV gene ablation.

Isolation and characteristics of Mycobacterium avium complex from water and soil samples in Uganda

Setting: Mycobacterium avium complex organisms have not been isolated from late stage AIDS patients in Uganda. This could possibly be due to the absence of M. avium complex in the Uganda environment. Objective and Design: Determine whether M. avium complex organisms could be isolated from water and soils collected in the living environment of Ugandan AIDS patients. Results: Representatives of the M. avium complex were isolated from 3 of 7 (43%) water and 3 of 7 (43%) soil samples collected in Kampala, Uganda. The average number of colony-forming units per ml water was 3.3 and average colony-forming units per gram of soil was 7825. In terms of growth characteristics, antimicrobial susceptibility patterns, and the presence or absence of plasmids and IS 901, Ugandan M. avium complex isolates were similar to those isolated from the US and European AIDS patients and their environment. Conclusion: M. avium complex organisms sharing genetic and physiological characteristics of M. avium complex isolates recovered from patients with AIDS can be isolated from water and soil samples in Uganda.