Amebiasis in HIV-1-infected Japanese men: clinical features and response to therapy

Invasive amebic diseases caused by Entamoeba histolytica are increasing among men who have sex with men and co-infection of ameba and HIV-1 is an emerging problem in developed East Asian countries. To characterize the clinical and epidemiological features of invasive amebiasis in HIV-1 patients, the medical records of 170 co-infected cases were analyzed retrospectively, and E. histolytica genotype was assayed in 14 cases. In this series of HIV-1-infected patients, clinical presentation of invasive amebiasis was similar to that described in the normal host. High fever, leukocytosis and high CRP were associated with extraluminal amebic diseases. Two cases died from amebic colitis (resulting in intestinal perforation in one and gastrointestinal bleeding in one), and three cases died from causes unrelated to amebiasis. Treatment with metronidazole or tinidazole was successful in the other 165 cases. Luminal treatment was provided to 83 patients following metronidazole or tinidazole treatment. However, amebiasis recurred in 6 of these, a frequency similar to that seen in patients who did not receive luminal treatment. Recurrence was more frequent in HCV-antibody positive individuals and those who acquired syphilis during the follow-up period. Various genotypes of E. histolytica were identified in 14 patients but there was no correlation between genotype and clinical features. The outcome of metronidazole and tinidazole treatment of uncomplicated amebiasis was excellent even in HIV-1-infected individuals. Luminal treatment following metronidazole or tinidazole treatment does not reduce recurrence of amebiasis in high risk populations probably due to amebic re-infection.     

Monocyte activation by circulating fibronectin fragments in HIV-1-infected patients

To identify signals that can alter leukocyte function in patients receiving highly active antiretroviral therapy (HAART), we analyzed single blood samples from 74 HIV-1-infected patients and additional blood was collected at 90-day intervals from 51 HIV-1-infected patients over a 516 +/- 172 (mean +/- SD) day interval. Despite the absence of circulating immune complexes and normalization of phagocytic function, compared with controls, the fraction of patients' monocytes expressing CD49e and CD62L was decreased and expression of CD11b and CD86 increased. Plasma from 63% of patients but none from normal controls contained 110-120 kDa fibronectin fragments (FNf). Presence of FNf did not reflect poor adherence to therapy. Addition of FNf to normal donor blood in vitro replicated changes in monocyte CD49e, CD62L, CD11b, and CD86 seen in vivo. FNf also induced monocytes to release a serine proteinase, nominally identified as proteinase-3, that hydrolyzed cell surface CD49e. alpha(1)-Antitrypsin blocked FNf-induced shedding of CD49e in a dose-dependent manner. Plasma with a normal frequency of CD49e(+) monocytes contained antiproteases that partially blocked FNf-induced monocyte CD49e shedding, whereas plasma from patients with a low frequency of CD49e(+) monocytes did not block this effect of FNf. Electrophoretic analyses of plasma from the latter group of patients suggested that a significant fraction of their alpha(1)-antitrypsin was tied up in high molecular mass complexes. These results suggest that monocyte behavior in HIV-1-infected patients may be influenced by FNf and the ratio of protease and antiproteases in the cells' microenvironment.     

Uridine metabolism in HIV-1-infected patients: effect of infection, of antiretroviral therapy and of HIV-1/ART-associated lipodystrophy syndrome

Background

Uridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS), although its metabolism in HIV-1-infected patients is poorly understood.

Methods

Plasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR.

Results

Median plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60), and for controls 4.60 µmol/l (IQR: 1.8) (P = 0.0009). In fat, they were of 6.0 (3.67), and 2.8 (4.65) nmol/mg of protein, respectively (P = 0.0118). Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40–4.80) whereas in those with isolated lipoatrophy it was 3.25 (2.55–4.15) µmol/l/l (P = 0.0066). The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls.

Conclusions

HIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations.     

Cellular immunity function in HIV-1-infected persons

The data on the state of cell-mediated immunity in patients with AIDS-related complex are presented. The synthetic peptide of membrane protein gp120 of HIV-1 was shown to inhibit leukocyte adhesion in persons under examination, as well as to have the tendency towards inhibiting the chemotaxis of migratory cells. The maximum effect was achieved at a peptide concentration of 10(-6) M. The data obtained in this investigation suggest the presence of specific cell-mediated sensitization to the fragment of protein gp120, detected by the adhesion inhibition test with the use of spectrophotometric techniques and the capillary evaluation of the chemotaxis of migrating cells, in patients with AIDS-related complex.     

Cerebrospinal fluid proteomic profiling of HIV-1-infected patients with cognitive impairment

Advanced HIV-1 infection is commonly associated with progressive immune suppression and the development of cognitive, motor, and behavior disturbances. In its most severe form, it is diagnosed as HIV-1 associated dementia (HAD) and can progress to profound functional disability and death. Despite prodigious efforts to uncover biomarkers of HAD, none can adequately reflect disease onset or progression. Thus, we developed a proteomics platform for HAD biomarker discovery and used it to perform a pilot study on cerebrospinal fluid (CSF) from HIV-1-infected people with or without HAD. A 2-dimensional electrophoresis (2-DE) map of a HAD CSF proteome was focused on differentially expressed proteins. 2-DE difference gel electrophoresis (2-D DIGE) analysis showed >90 differences in protein spots of which 20 proteins were identified. Differential expression of 6 proteins was validated by Western blot tests and included vitamin D binding protein, clusterin, gelsolin, complement C3, procollagen C-endopeptidase enhancer 1, and cystatin C. We posit that these proteins, alone or together, are potential HAD biomarkers.     

Mitochondrial glutaminase enhances extracellular glutamate production in HIV-1-infected macrophages: linkage to HIV-1 associated dementia

Dysfunction in mononuclear phagocyte (MP, macrophages and microglia) immunity is thought to play a significant role in the pathogenesis of HIV-1 associated dementia (HAD). In particular, elevated extracellular concentrations of the excitatory neurotransmitter glutamate, produced by MP as a consequence of viral infection and immune activation, can induce neuronal injury. To determine the mechanism by which MP-mediated neuronal injury occurs, the concentration and rates of production of extracellular glutamate were measured in human monocyte-derived macrophage (MDM) supernatants by reverse phase high-performance liquid chromatography (RP-HPLC). Measurements were taken of supernatants from MDM infected with multiple HIV-1 strains including ADA and DJV (macrophage tropic, M-tropic), and 89.6 (dual tropic). High levels of glutamate were produced by MDM infected with M-tropic viruses. AZT, an inhibitor of HIV-1 replication, inhibited glutamate generation, demonstrating a linkage between HIV-1 infection and enhanced glutamate production. In our culture system, glutamate production was dependent upon the presence of glutamine and was inhibited by 6-diazo-5-oxo-L-norleucine, a glutaminase inhibitor. Supernatants collected from HIV-1-infected MP generated more glutamate following glutamine addition than supernatants isolated from uninfected MP. These findings implicate the involvement of a glutamate-generating enzyme, such as phosphate-activated mitochondrial glutaminase (PMG) in MP-mediated glutamate production.     

Antiviral activity of fattiviracin FV-8 against human immunodeficiency virus type 1 (HIV-1)

A novel antiviral agent, fattiviracin FV-8, purified from the culture broth of Streptomyces microflavus strain No. 2445, showed potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV), and influenza A and B viruses. The action mechanism of fattiviracin FV-8 against HIV-1 was examined. As a result, the agent was thought to act on HIV-1 particles directly without lysis of the particles, and it affords the inhibition of viral entry into the host cells.     

Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E_GSH; Grx1-roGFP2) and measured subcellular changes in E_GSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E_GSH (approximately −310 mV), active viral replication induces substantial oxidative stress (E_GSH more than −240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H₂O₂), induces significant deviations in subcellular E_GSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E_GSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E_GSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.     

Induction of apoptosis by HIV-1-infected monocytic cells

We have previously described a soluble 6000-Da peptide produced by an HIV-1-infected human macrophage cell line, clone 43(HIV), which induces apoptosis in T and B cells. We have identified this factor as the novel cDNA clone FL14676485 that encodes for the human hypothetical protein, FLJ21908. The FL14676485 cDNA clone was isolated from a 43(HIV) lambda ZAP Escherichia coli expression library and screened with a panel of rabbit and mouse anti-apoptotic Abs. We transfected the FL14676485 clone into Bosc cells and non-HIV-1-infected 43 cells. Western blot analysis of lysates from the FL14676485-transfected 43 cells and Bosc cells using anti-proapoptotic factor Abs revealed a protein with a molecular mass of 66 kDa corresponding to the size of the full-length gene product of the FL14676485 clone, while Western blot of the supernatant demonstrated a doublet of 46-kDa and 6000-Da peptide that corresponds to our previously described proapoptotic factor. Primary HIV-1(BaL)-infected monocytes also produce the FLJ21908 protein. Supernatants from these transfected cells induced apoptosis in PBMC, CD4(+), and CD8(+) T and B cells similar to the activity of our previously described proapoptotic factor. PCR analysis of 43 cells and 43(HIV) cells revealed a base pair fragment of 420 bp corresponding to the FL14676485 gene product in 43(HIV) cells, but not in 43 cells. The FLJ21908 protein induces apoptosis through activation of caspase-9 and caspase-3. We have further demonstrated that the FLJ21908 protein has apoptotic activity in the SH-SY5Y neuronal cell line and can be detected in brain and lymph tissue from HIV-1-infected patients who have AIDS dementia. The FLJ21908 protein may contribute to the apoptosis and dementia observed in AIDS patients.     

Antiviral activity of steric-block oligonucleotides targeting the HIV-1 trans-activation response and packaging signal stem-loop RNAs

Mixmer oligonucleotides consisting of residues of both 2'-O-methylnucleosides (OMe) and locked nucleic acids (LNA) were designed targeting two stem-loops in the 5'-UTR of HIV-1 RNA, the transactivation response region (TAR), which is the site of binding of the Tat protein, and the SL3 loop, which is the primary packaging element that binds the Gag polyprotein. These oligonucleotides were found to inhibit syncitia formation dose- and sequence-dependently when delivered to HeLa T4 LTR beta-Gal cells and subsequently infected with HIV-1.     

Plasma interleukin-18 is associated with viral load and disease progression in HIV-1-infected patients

Recent studies demonstrate persistent elevation of interleukin-18 (IL-18) concentration in human immunodeficiency virus type 1 (HIV-1)-infected patients. Due to pleiotropic action of IL-18 on the immune system, dysregulation of its synthesis may lead to inappropriate immune activation. The aim of this study was to determine possible correlation between IL-18 levels and the natural stages of HIV-1 infection. IL-18 plasma concentrations were determined in 42 patients in different stages of an HIV-1 infection and in 15 healthy controls. HIV infection resulted in a more than fourfold increase of plasma IL-18 concentration compared to healthy individuals (865 +/- 87 vs. 206 +/- 32 pg/ml, P < 0.001). Moreover, a positive correlation between plasma IL-18 concentration and HIV viral load was found (r = 0.44, P < 0.01). Further analysis showed marked elevation of IL-18 levels in late-stage symptomatic patients. Plasma IL-18 concentrations in patients receiving high-activity antiretroviral treatment (HAART) were significantly lower than in those not undergoing antiretroviral treatment. Individuals who did not reach viral suppression showed higher IL-18 plasma concentration than the group with achieved viral suppression. Excessive production of IL-18 observed in our study may promote viral replication and disease progression in advanced, especially late-stage HIV-infected patients. Furthermore, reduction of IL-18 concentration can be an important step in HAART-related immune restoration.     

Comparison of heterologous neutralizing antibody responses of human immunodeficiency virus type 1 (HIV-1)- and HIV-2-infected Senegalese patients: distinct patterns of breadth and magnitude distinguish HIV-1 and HIV-2 infections

Neutralizing antibody responses against heterologous isolates in human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections were compared, and their relationships with established clinical markers of progression were examined. Neutralizing responses against 7 heterologous primary isolates and 1 laboratory strain were compared between 32 untreated HIV-1-infected subjects and 35 untreated HIV-2-infected subjects using a pseudotyped reporter virus assay. The breadth of the neutralizing response, defined as the proportion of panel viruses positively neutralized by patient plasma, was significantly greater among HIV-2-infected subjects than among HIV-1-infected subjects. Notably, for fully one-third of HIV-2 subjects, all viruses were effectively neutralized in our panel. Magnitudes of responses, defined as reciprocal 50% inhibitory concentration (IC(50)) titers for positive reactions, were significantly greater among HIV-1-infected subjects than among HIV-2-infected subjects. When plasma samples from HIV-1 patients were tested for cross-neutralization of HIV-2 and vice versa, we found that these intertype responses are very rare and their prevalences comparable in both HIV-1 and HIV-2 infection. The significantly higher magnitude of heterologous responses for HIV-1 compared to HIV-2 prompted us to examine associations with viremia, which is known to be significantly higher in HIV-1 infection. Importantly, there was a significant positive correlation between the IC(50) titer and viral load within both the HIV-1 and HIV-2 groups, suggesting heterologous antibodies may be driven by viral replication. We conclude that HIV-2 infection is characterized by a broad, low-magnitude intratype neutralization response, while HIV-1 is characterized by a narrower but higher-magnitude intratype response and that a significant positive association between the IC(50) titer and viremia is common to both HIV-1 and HIV-2 infections.     

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

Background

Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.

Results

A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank^GAG1D4 (16.5 kDa) was isolated. Ank^GAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K _d ~ 1 ??M, and the Ank^GAG1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank^GAG1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank^GAG1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank^GAG1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank^GAG1D4-CA with the Gag assembly and budding pathway.

Conclusions

The resistance of Ank^GAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank^GAG1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.     

Cytoskeletal protein transformation in HIV-1-infected macrophage giant cells

The mechanisms linking HIV-1 replication, macrophage biology, and multinucleated giant cell formation are incompletely understood. With the advent of functional proteomics, the characterization, regulation, and transformation of HIV-1-infected macrophage-secreted proteins can be ascertained. To these ends, we performed proteomic analyses of culture fluids derived from HIV-1 infected monocyte-derived macrophages. Robust reorganization, phosphorylation, and exosomal secretion of the cytoskeletal proteins profilin 1 and actin were observed in conjunction with productive viral replication and giant cell formation. Actin and profilin 1 recruitment to the macrophage plasma membrane paralleled virus-induced cytopathicity, podosome formation, and cellular fusion. Poly-l-proline, an inhibitor of profilin 1-mediated actin polymerization, inhibited cytoskeletal transformations and suppressed, in part, progeny virion production. These data support the idea that actin and profilin 1 rearrangement along with exosomal secretion affect viral replication and cytopathicity. Such events favor the virus over the host cell and provide insights into macrophage defense mechanisms used to contain viral growth and how they may be affected during progressive HIV-1 infection.