Increased rates of CD4+ and CD8+ T lymphocyte turnover in simian immunodeficiency virus-infected macaques

Defining the rate at which T cells turn over has important implications for our understanding of T lymphocyte homeostasis and AIDS pathogenesis, yet little information on T cell turnover is available. We used the nucleoside analogue bromodeoxyuridine (BrdUrd) in combination with five-color flow cytometric analysis to evaluate T lymphocyte turnover rates in normal and simian immunodeficiency virus (SIV)-infected rhesus macaques. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labeling and elimination rates of both CD4+ and CD8+ BrdUrd-labeled cells were increased by 2- to 3-fold as compared with normal controls. In normal and SIV-infected animals, the rates of CD4+ T cell BrdUrd-labeling and decay were closely correlated with those of CD8+ T cells. The elimination rate of BrdUrd-labeled cells was accelerated in both naive and memory T lymphocytes in SIV-infected animals. Our results provide direct evidence for increased rates of both CD4+ and CD8+ T cell turnover in AIDS virus infection and have important implications for our understanding of T cell homeostasis and the mechanisms responsible for CD4+ T cell depletion in AIDS.

Identification of inflections in T-cell counts among HIV-1-infected individuals and relationship with progression to clinical AIDS

Studies of circulating T (CD3+) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5–2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log10 CD3+ cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3+ lymphocyte counts before the inflection point was close to zero for HIV− and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3+ cell counts relative to HIV− men and HIV+ men not developing AIDS. A CD3+ cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.

Effect of preventive treatment for tuberculosis in adults infected with HIV: systematic review of randomised placebo controlled trials

Objective: To determine whether preventive treatment for tuberculosis in adults infected with HIV reduces the frequency of tuberculosis and overall mortality.

The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals

The HIV-1 regulatory proteins Rev and Tat are expressed early in the virus life cycle and thus may be important targets for the immune control of HIV-1-infection and for effective vaccines. However, the extent to which these proteins are targeted in natural HIV-1 infection as well as precise epitopes targeted by human cytotoxic T lymphocytes (CTL) remain to be defined. In the present study, 57 HIV-1-infected individuals were screened for responses against Tat and Rev by using overlapping peptides spanning the entire Tat and Rev proteins. CD8+ T cell responses against Tat and Rev were found in up to 19 and 37% of HIV-1-infected individuals, respectively, indicating that these regulatory proteins are important targets for HIV-1-specific CTL. Despite the small size of these proteins, multiple CTL epitopes were identified in each. These data indicate that Tat and Rev are frequently targeted by CTL in natural HIV-1 infection and may be important targets for HIV vaccines.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria

Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.

A unique phospholipid organization in bovine erythrocyte membranes

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A2, an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A2 to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.

The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (i) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (ii) In an in vitro substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes.

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.

Salmonella typhimurium invasion induces apoptosis in infected macrophages.

Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.

Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

Whole body positron emission tomography imaging of simian immunodeficiency virus-infected rhesus macaques.

Pathogenesis of simian immunodeficiency virus (SIV) infection in rhesus macaques begins with acute viremia and then progresses to a distributed infection in the solid lymphoid tissues, which is followed by a process of cellular destruction leading to terminal disease and death. Blood and tissue specimens show the progress of infection at the cellular level but do not reveal the pattern of infection and host responses occurring throughout the body. The purpose of this investigation was to determine whether positron emission tomography (PET) imaging with intravenous 2-18F-2-deoxyglucose (FDG) could identify activated lymphoid tissues in a living animal and whether this pattern would reflect the extent of SIV infection. PET images from SIV-infected animals were distinguishable from uninfected controls and revealed a pattern consistent with widespread lymphoid tissue activation. Significant FDG accumulation in colon along with mesenteric and ileocaecal lymph nodes was found in SIV infection, especially during terminal disease stages. Areas of elevated FDG uptake in the PET images were correlated with productive SIV infection using in situ hybridization as a test for virus replication. PET-FDG images of SIV-infected animals correlated sites of virus replication with high FDG accumulation. These data show that the method can be used to evaluate the distribution and activity of infected tissues in a living animal without biopsy. Fewer tissues had high FDG uptake in terminal animals than midstage animals, and both were clearly distinguishable from uninfected animal scans.

Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen.

Plasmodium chabaudi adami causes a nonlethal infection in mice. We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing. The factors that mediate spleen-dependent immunity are not known. An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis [Wyler, D. J., Quinn, T. C. & Chen, L.-T. (1982) J. Clin. Invest. 67, 1400-1404]. To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P. chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis. After intravenous injection, bacteria were localized predominantly in the marginal zone. In contrast, parasitized erythrocytes were found in the red pulp. We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased. These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis). Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P. chabaudi adami infection in mice.