Comparison of CD8(+) T-cell subsets in HIV-infected rapid progressor children versus non--rapid progressor children.

BACKGROUND: CD8(+) T-cell subsets have not been adequately described in HIV-infected (HIV(+)) children classified with respect to disease progression as rapid-progressors (RPs) and non-rapid progressors (non-RPs). OBJECTIVE: The purpose of this investigation was to determine the distribution of CD8(+) T-cell subsets in HIV(+) children and correlate the findings with degree of immunosuppression and HIV viral burden. METHODS: By means of 3-color flow cytometry, percentages of CD38(+)DR(+), CD28(+), and CD57(+) CD8(+) T-cell subsets were examined in RP (n = 15) and non-RP (n = 36) HIV(+) children and in HIV-exposed but uninfected (n = 11) and HIVunexposed (n = 8) children. The CD8(+) T-cell subsets were correlated with mean CD4(+) T-cell percentages and HIV RNA levels. Analysis of covariance was used for group comparisons for the control of the covariate of age. RESULTS: The HIV-exposed and HIV-unexposed controls were not different from each other in CD8(+) T-cell subset percentages, except that the DR(-)CD38(+)CD8(+) T-cell percentages were higher in the exposed controls than in the unexposed controls. RPs had a higher mean percentage of DR(+)CD38(+)CD8(+) T cells than non-RPs and both control groups, and RPs had higher viremia than non-RPs. CD38(+)CD8(+) T-cell percentages did not correlate with viral burden as it has been seen to do in HIV(+) adults. Percentages of CD28(+)CD8(+) T cells were lower in HIV-infected children than in controls. There was a positive correlation of percentage of CD28(+)CD57(-)CD8(+) T cells with CD4(+) T-cell percentages in each HIV-infected group. CONCLUSION: CD8(+) T cells become activated (dual expression of DR and CD38) and lose CD28, some acquiring CD57, in relation to rapidity of disease progression in pediatric HIV infection.

Expression of IL-15 and IL-4 in IFN-gamma-independent control of experimental human Cryptosporidium parvum infection.

We have previously demonstrated interferon gamma (IFN-gamma) in intestinal mucosa after experimental human Cryptosporidium parvum infection, but expression was limited to sensitized volunteers. To characterize IFN-gamma-independent mechanisms in control of infection, jejunal biopsies from immunocompetent volunteers experimentally challenged with C. parvum were examined by in situ hybridization for interleukin (IL-)15 and IL-4 mRNA with confirmation by immunohistochemistry. Cytokine expression was correlated with prechallenge anti- C. parvum IgG, symptoms, oocyst shedding, and prior IFN-gamma expression data. IL-15 expression was noted only in those without prior sensitization, who did not express IFN-gamma. By contrast, expression of IL-4 was associated with prior sensitization. IL-15 was only detected in those with symptoms (6/14 symptomatic vs 0/3 asymptomatic, P<0.05). Among 14 volunteers who did not express IFN-gamma, oocyst shedding was lower in those expressing IL-15. Overall, 14/15 volunteers who did not shed oocysts expressed either IFN-gamma or IL-15. There was no correlation between expression of IL-4 and symptoms or oocyst shedding. In conclusion, IL-15 expression was associated with control of oocyst shedding in those not expressing IFN-gamma. These data suggest that IL-15 is involved in IFN-gamma independent mechanisms of control of human cryptosporidiosis, perhaps via activation of the innate immune response.

CD86 expression correlates with amounts of HIV produced by macrophages in vitro.

Primary macrophages from different donors produce variable levels of HIV; however, the mechanisms are unclear. We tested whether variations in cell-surface or cell-cycle characteristics influenced HIV production. We found that greater basal proliferation of the macrophages prior to infection resulted in more arrested in G2M 3 days post-infection (r2=0.7, P<0.04). Likewise, the number of G2M-arrested macrophages correlated with p24 production (r2=0.78, P<0.02) and apoptosis (r2=0.67, P<0.05) later in the infection. Serum-starvation or reduction, which limit HIV spread, reduced G2M arrest and HIV amounts. Surprisingly, the amount of HIV produced correlated with expression levels of the costimulating ligand, CD86, but not with other important molecules, including class II, CD40, or CD54 (r2=0.96, P<0.0005). These data establish donor characteristics related to variable HIV production in vitro and suggest that altered expression of costimulatory ligands may influence HIV production in vivo.

Expression of tumor necrosis factor alpha and interleukin 1 beta in jejuna of volunteers after experimental challenge with Cryptosporidium parvum correlates with exposure but not with symptoms.

Jejunal biopsies from volunteers challenged with Cryptosporidium parvum were examined for tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1 beta mRNA. Postchallenge biopsies from 15 of 28 (54%) volunteers expressed TNF-alpha; 14% expressed IL-1 beta. Cytokine expression did not correlate with enteric symptoms, suggesting that TNF-alpha and IL-1 beta are not key mediators of diarrhea in human cryptosporidiosis.

Antiapoptotic function of Cdk9 (TAK/P-TEFb) in U937 promonocytic cells.

Cdk9 is the catalytic subunit of TAK (cyclinT1/P-TEFb), a cellular protein kinase that mediates human immunodeficiency virus type 1 (HIV-1) Tat transcriptional activation function. To examine Cdk9 function in cells relevant to HIV-1 infection, we used a murine leukemia virus retrovirus vector to transduce and overexpress the cDNA of a dominant negative mutant Cdk9 protein (Cdk9-dn) in Jurkat T cells and U937 promonocytic cells. In Jurkat cells, overexpression of Cdk9-dn specifically inhibited Tat transactivation and HIV-1 replication but had no inhibitory effect on induction of CD69, CD25, and interleukin-2 following T-cell activation. In U937 cells, overexpression of Cdk9-dn sensitized cells to apoptosis, especially after phorbol myristate acetate (PMA) treatment to induce differentiation to macrophage-like cells. Because Cdk9 function is induced in PMA-treated U937 cells, Cdk9 may play an antiapoptotic role during monocyte differentiation.

Activation of HIV in human skin by ultraviolet B radiation and its inhibition by NFkappaB blocking agents.

To determine whether ultraviolet B (UVB) irradiation leads to activation of HIV in human skin, we conducted prospective and controlled studies in two academic medical centers in Texas from July 1995 to April 1999. HIV-positive patients with UV-treatable skin diseases were enrolled at each center, 18 subjects at one and 16 at the other. In one center, specimens from lesional and nonlesional skin biopsies were taken before and after sham- or UVB-irradiation administered in vivo or in vitro. In the other center, UVB phototherapy was administered three times weekly and specimens from skin biopsies were taken before and after 2 weeks (six treatments). Cutaneous HIV load was assessed using reverse transcriptase-polymerase chain reaction and reverse transcriptase-polymerase chain reaction in situ hybridization. UVB irradiation led to a 6-10-fold increase in the number of HIV in skin. To ascertain a role for nuclear factor kappa B (NFkappaB) in UVB-inducible HIV activation, two types of blockers, NFkappaB oligonucleotide decoy and sodium salicylate, were tested; each inhibited UVB-inducible HIV activation in skin partially. We conclude that UVB irradiation leads to increased numbers of HIV in human skin via processes that include release of cytoplasmic NFkappaB.

In vivo expression of the novel CXC chemokine BRAK in normal and cancerous human tissue.

Using differential display, we cloned a gene with reduced expression in short-term explants of head and neck squamous cell carcinoma (HNSCC) tumors compared to cultured normal oral epithelial cells. The differentially expressed gene was identical to the recently cloned CXC chemokine BRAK, which is ubiquitously expressed in normal tissue extracts but is absent from many tumor cell lines in vitro. To define the cell populations expressing BRAK in vivo, in situ mRNA hybridization was performed on normal and cancerous tissues from six different histological sites. The predominant normal cell type constitutively expressing BRAK in vivo was squamous epithelium. Expression in tumors was heterogeneous, with the majority of HNSCCs and some cervical squamous cell carcinomas (SCCs) showing loss of BRAK mRNA. Although absent in unstimulated peripheral blood mononuclear cells, high levels of BRAK were consistently found in infiltrating inflammatory cells (with lymphocyte morphology) in nearly all cancers examined. Furthermore, BRAK expression was demonstrated in B cells and monocytes, after stimulation of peripheral blood mononuclear cells with lipopolysaccharide. This study demonstrates for the first time up-regulation of BRAK mRNA by inflammatory cells in the tumor microenvironment and lost expression from certain cancers in vivo. The data suggest that BRAK may have a role in host-tumor interactions.

Interferon-gamma expression in jejunal biopsies in experimental human cryptosporidiosis correlates with prior sensitization and control of oocyst excretion.

To investigate the role of interferon (IFN)-gamma in human cryptosporidiosis, jejunal biopsies from experimentally infected volunteers and chronically infected AIDS patients were examined for IFN-gamma expression by in situ hybridization. IFN-gamma expression was compared with oocyst excretion, baseline serum anti-Cryptosporidium antibody, and symptoms. IFN-gamma mRNA was detected in biopsies from 13 of 26 volunteers after experimental infection but not in biopsies taken before C. parvum exposure or in biopsies from patients with AIDS-associated cryptosporidiosis. After challenge, 9 of 10 volunteers with baseline C. parvum antibody produced IFN-gamma, compared with 4 of 16 volunteers without baseline antibody (P<.01). Furthermore, IFN-gamma mRNA was detected in 9 of 13 volunteers who did not excrete oocysts, compared with 4 of 13 with organisms (P<.05). Thus, expression of IFN-gamma in the jejunum was associated with prior sensitization and absence of oocyst shedding. IFN-gamma production may explain the resistance to infection noted in sensitized persons but may not be involved in control of human primary infection.

Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

Costimulatory pathways mediate monocyte-dependent lymphocyte apoptosis in HIV.

Examination of annexin V binding, an indicator of early apoptosis, on lymphocytes from HIV+ people immediately after isolation showed that both CD4(+) and CD8(+) T cells were apoptotic, whereas B cell apoptosis was induced mainly after incubation. CD8(+) T cell apoptosis correlated with fewer CD4(+) T cells, but not the level of viremia. To determine potential mechanisms for apoptosis, we examined FasL expression, which was dramatically elevated on CD14(+) monocytes; however, antibody to FasL did not reproducibly inhibit apoptosis. Rather, CD8(+) T cell apoptosis was caused by antigen-presenting cells because removal of monocytes or addition of antibodies to CD80 and CD86 reduced apoptosis. B cell apoptosis also involved costimulatory signals delivered by T cells but not monocytes. A unique CD8(bright)CD28(dim) T cell population died after costimulation by monocytes. Because this population was increased in patients with undetectable viremia, abnormal antigen-presenting cells may contribute to continued CD8(+) T cell exhaustion by inducing apoptosis.

Alterations in HIV expression in AIDS patients with psoriasis or pruritus treated with phototherapy.

BACKGROUND: Ultraviolet light (UVL) upregulates HIV transcription in vitro and in transgenic mice. AIDS-associated psoriasis and pruritus respond to phototherapy. OBJECTIVE: Our goal was to determine the effect of phototherapy on viral load and immunologic parameters in HIV-positive patients. METHODS: T cell subsets, p24, plasma cytokines, serum or plasma HIV-RNA, dosage, and antivirals were assessed in HIV-positive patients and negative controls receiving 6 weeks of phototherapy with UVB and in untreated controls. RESULTS: Phototherapy improved skin conditions without significantly affecting T cell numbers. Plasma p24 increased 2-fold (P = .055) and HIV-RNA levels 4-fold (P = .022) 6 weeks from baseline in patients who entered the trial before March 1995. Later patients who were mostly receiving combination antiviral therapy showed a 4-fold reduction in serum HIV-RNA (P = .012) at 2 weeks. The effect of UVB on viral load at 6 weeks was dependent on the baseline level (P = .006). IL-10 increased and was inversely related to HIV-RNA levels (P = .0267). CONCLUSION: Phototherapy is associated with HIV load alterations, depending on patients' initial HIV-RNA, antiviral therapy, skin type, and UVL dosage.

Presence of HLA-C-restricted cytotoxic T-lymphocyte responses in long-term nonprogressors infected with human immunodeficiency virus.

Approximately 5% of people with human immunodeficiency virus type 1 (HIV-1) infection remain free of disease for 10 or more years. These long-term nonprogressors (LTNPs) exhibit lower viral loads and stable CD4+ lymphocyte counts. The immunologic basis for this disease-free condition is not known. Because cytotoxic T lymphocytes (CTLs) constitute a major immune defense mechanism for sustained recovery from viral infections, we analyzed HIV-specific CTL responses in three asymptomatic LTNPs. We observed the presence of HIV-1 envelope-specific CTL responses mediated by HLA class I C-restricted CD8+ cells in these individuals. Using autologous target cells and a panel of HLA-matching and -mismatching B-cell lines as targets, we determined that HLA-Cw7 is the restricting element for the observed CTL activity. Additionally, we identified three peptides, one previously not reported, from conserved regions in the envelope protein as CTL epitopes. We previously reported these peptides to be efficient in inducing HIV-specific cellular immune responses in murine and nonhuman primate models. Our results support the role of the HLA-C locus in generating CTL responses and constitute the first report of an HLA-Cw7-restricted HIV-1 envelope-specific CTL response in HIV+ LTNPs, which may be important in the control of HIV replication in vivo.

Distinct serum cytokines in AIDS-related skin diseases.

To determine whether common skin diseases associated with human immunodeficiency virus (HIV) were distinguishable based on the pattern of serum cytokine expression, we studied patients with psoriasis, pruritus, and Kaposi's sarcoma (KS) for levels of tumor necrosis factor (TNF)-alpha, interferon-gamma (IFN-y), interleukin (IL)-10, and IL-4. Thirty-two HIV-positive (HIV+) patients including 8 with KS, 11 with psoriasis, and 13 with pruritus along with 16 HIV-negative subjects with psoriasis were studied. IFN-gamma levels were highest in sera of HIV+ patients with psoriasis (p = 0.040). By contrast, TNF-alpha and IL-10 levels were highest in sera of HIV+ patients with pruritus (p = 0.012). Detectable levels of all cytokines in these patients were remarkably higher than for healthy adults. These results suggest that common skin diseases associated with HIV infection and AIDS can be distinguished by the production of unique cytokines.

Smoking policy in long-term care: a survey of administrators in San Francisco.

Despite the fact that smoking is a ubiquitous yet potentially hazardous behavior in long-term care settings, little previous investigation has been made into the construction or implementation of policy to manage smoking by elderly residents. This survey of administrators in long-term care facilities in San Francisco city and county reveals that fire and safety issues were the prime forces motivating smoking policy, which operated through control of behavior rather than by other available means, such as the use of fire retardant aprons. Although all facilities permitted smoking, a hierarchy of limit setting strategies was adopted, strategies which successively and evermore intrusively overrode the resident's autonomy and turned smoking policy from beneficent philosophy into a coercive moral statement. Arranged in order of frequency of occurrence and increasing degree of restriction, these strategies were: (a) designating appropriate locations; (b) controlling smoking opportunities and materials; (c) requiring staff supervision or help; (d) limiting the availability of staff help; and (e) writing Doctor's orders. By acknowledging the tensions between ambivalent goals inherent in smoking in long-term care, administrators could devise policies and procedures that are both supportive of collective rights and less corrosive of individual ones.

Granuloma cytokines in murine cysticercosis.

Neurocysticercosis, caused by Taenia solium, is one of the most common causes of seizures worldwide. The symptoms result from granulomatous inflammation associated with dying cyst forms of the parasite. Although the invasive larvae can be killed by immune serum plus complement, immunity to the cyst stage depends on a cellular response. This dichotomous immune response is reminiscent of the extremes of the immune response associated with T helper 1 (Th1) and Th2 cytokine profiles. To characterize the cytokine response in cysticercosis, granulomas were removed from the peritoneal cavity of mice infected with Taenia crassiceps cysts and examined for cytokine message by in situ hybridization using 35S-labeled RNA probes. The granulomas were staged based on histologic appearance of the degenerating parasite. Message for gamma interferon (IFN-gamma) was identified by light microscopy in 11 of the 12 granulomas, and interleukin-2 (IL-2) message was identified in 9 of the 12. By laser scanning confocal microscopy, significantly increased IFN-gamma and IL-2 pixel intensity was identified in nearly all of the granulomas from early histologic stages. Message for IL-4 was seen in 6 of the 12 granulomas. Only granulomas with complete destruction of the parasite architecture displayed more than minimal amounts of IL-4 message by light microscopy, and only 2 of 12 granulomas had IL-4 pixel intensity significantly above background. Only minimal amounts of IL-10 message were detected in 4 of 11 granulomas. Thus, early granulomas in cysticercosis are predominantly associated with a Th1 response, whereas later granulomas, in which parasite destruction is complete, have a mixture of Th1 and IL-4. The Th1 response appears to play an important role both in the pathogenesis of disease as well as in the clearing of the parasites, with IL-4 involved in downregulation of the initial response.

Role of placental cytokines and inflammation in vertical transmission of HIV infection.

In light of new evidence suggesting that maternal human immunodeficiency virus (HIV) infection produces at least a three-fold increase in the number of early spontaneous abortions, it is important to search for factors that may predispose to fetal wastage. Immunological factors are thought to play an important role in permitting the HLA-disparate fetus to continue to term, despite powerful maternal immune forces capable of rejection. In the context of a heightened incidence of spontaneous abortion in HIV infection, evidence is now accumulating that implicates an imbalance in immune factors in contributing to this fetal loss. Soluble immune factors, such as cytokines, have been suggested as contributing agents to recurrent spontaneous abortions. Inflammatory cytokines-interleukin 1beta, interleukin 6 and tumor necrosis factor alpha-have been measured in isolated placental trophoblastic cells in HIV-infected and non-infected pregnant women in an attempt to explore this hypothesis. These inflammatory cytokines and their messenger RNAs were significantly elevated before and after stimulation in HIV-infected women, supporting the belief that HIV-infected women present their fetuses a milieu of imbalanced immune factors capable of contributing to immunological rejection. In addition, these elevated inflammatory cytokine levels may contribute to HIV disease progression in fetuses by virtue of activation of HIV gene transcription factors similar to what has been demonstrated in in vitro systems. We therefore propose that HIV infection in pregnant women produces an altered state of certain soluble immune factors, which in concert with other immune factor abnormalities, such as loss of immune selection in the fetal thymus, predisposes the fetus to advanced HIV infection and possible spontaneous abortion.

Protection of primary human T cells from HIV infection by Trev: a transdominant fusion gene.

Gene therapy is one of several approaches that are being tested in the search for an effective anti-human immunodeficiency virus (HIV) treatment. In this strategy, a "protective" gene would be introduced into target cells, rendering them relatively resistance to the virus-induced cytopathicity. Tat and Rev are viral proteins essential for HIV gene expression. Tat increases viral gene transcription and Rev is responsible for the nuclear export of mRNA encoding structural viral proteins. A fusion protein (Trev) was constructed, joining Tat and Rev transdominant mutant gene sequences. Previously, we showed that Trev inhibits both Tat and Rev activities in Jurkat T cells. To determine whether Trev could inhibit HIV replication in primary cells, we transferred the trev gene to peripheral blood lymphocytes and challenged them with different HIV strains. Levels of HIV p24 antigen (Ag) were reduced 4- to 15-fold in cultures of Trev-CD4+ T cells infected with two HIV primary clinical isolates and were not detectable in cultures infected with HIV strains NL4-3 and SF2. In contrast, cultures of nontransduced CD4+ T cells infected with the same viruses had levels of HIV p24 Ag up to 10 ng/ml. Trev-transduced CD4+ T cells demonstrated increased survival following HIV challenge for the length of the experiments (30 days). We did not observe rapid emergence of Trev-resistant HIV in our cultures. Following HIV challenge, cell-associated Trev protein was increased, supporting the hypothesis that cells surviving Trev expression provided a cell survival advantage. This work showed that Trev was able to inhibit HIV replication in primary CD4+ T cells, and, therefore the trev gene could be a candidate for gene therapy against HIV.

Effect of hepatocyte proliferation and cellular DNA synthesis on hepatitis B virus replication.

BACKGROUND & AIMS: Interrelationship between hepatitis B virus (HBV) replication and the stage of hepatocyte proliferation and differentiation may play an important role in the pathogenesis of HBV infection. The aim of this study was to assess the effect of hepatocyte proliferation and/or cell arrest on HBV replication. METHODS: Hepatoblastoma cells transfected with HBV were subjected to serum deprivation or treatment with aphidicolin or camptothecin. Cell cycle analysis was performed using flow cytometry, and cellular DNA synthesis was analysed by assessing 5-bromo-2'-deoxyuridine incorporation. Distribution of episomal HBV DNA and proliferating cell nuclear antigen in liver specimens was assessed by simultaneous in situ hybridization and immunohistochemistry. RESULTS: Serum deprivation inhibited cellular DNA synthesis and increased levels of HBV messenger RNA (mRNA). Aphidicolin treatment resulted in cell arrest in C1, with concomitant increases in levels of HBV mRNA and viral DNA. Cell entry into S phase inhibited expression of HBV mRNA. Camptothecin induced G2 cell arrest and inhibited cellular DNA synthesis with increased amounts of viral replication and levels of HBV mRNA. In vivo studies showed an inverse correlation between expression of proliferating cell nuclear antigen and presence of episomal HBV DNA in individual hepatocytes. CONCLUSIONS: HBV replication is cell cycle dependent, supporting the concept of enhanced viral replication in quiescent hepatocytes. The results may explain the mechanism of viral elimination during cell regeneration.

Detection of low-grade mosaicism in fetal cells isolated from maternal blood.

Recovering and analysing fetal erythrocytes from maternal blood is being pursued for non-invasive prenatal genetic diagnosis. We report the observation of 46,XY/47,XXY mosaicism in fetal cells from a woman whose first-trimester chorionic villus sampling (CVS) initially showed only 46,XY. Only after exhaustive (500 cells) analysis were four XXY cells found in cultured villi.

Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells.

Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.