Complement and c4 null alleles in severe chronic adult periodontitis.

Severe forms of chronic periodontitis affect up to 10% of adults. Tumour necrosis factor and lymphotoxin-alpha genes in the major histocompatibility complex are associated with severe periodontitis. Complement factor C4 is a nearby, polymorphic, functionally relevant gene region. Although associated with chronic mucosal infections, C4 deficiencies have not been assessed in adult periodontitis patients. We tested whether complement levels are systemically altered and C4 deficiencies associated with severe chronic periodontitis. In a case-control study, we analysed levels of plasma C3, and C4, serum classical pathway haemolytic activity, C4 allotypes and C4 gene numbers in 37 patients with severe chronic periodontitis and in 150 voluntary controls. Plasma levels of C3 were higher, and classical pathway haemolytic activity was lower in patients than in controls. Partial C4 gene deficiencies were more frequent in patients than in controls (odds ratio 2.4, 95% confidence interval 1.1-5.5, P = 0.032). Changes in complement levels may reflect chronic, recurring inflammation. C4 gene deficiencies are associated with predisposition to chronic periodontitis.

Open-label phase II trial of amprenavir, abacavir, and fixed-dose zidovudine/lamivudine in newly and chronically HIV-1--infected patients.

A Phase II clinical trial was designed to evaluate the efficacy and tolerability of twice-daily abacavir, amprenavir, and zidovudine (ZDV)/lamivudine (3TC) in HIV-1-infected study subjects naive to protease inhibitors and 3TC. Plasma and cerebrospinal fluid (CSF) HIV-1 RNA levels and T-cell subsets were measured. In all, 27 newly diagnosed and 12 chronically HIV-1-infected study subjects are included in the analysis. Week 48 plasma HIV-1 RNA levels were <500 copies/ml in 100% of study subjects, and <50 copies/ml in 80% of chronically infected and 100% of newly infected study subjects. The mean change in CD4 was (+)150 cells/microl (newly infected, p <.001), and (+)155 cells/microl (chronically infected, p <.001). At Week 48, evidence of cellular activation persisted in both cohorts. A twice-daily regimen of amprenavir, abacavir, and ZDV/3TC affords potent viral suppression and significant increases in total CD4(+) cells in HIV-1--infected study subjects. Patient intolerance may limit the efficacy of this combination.

Virologic and immunologic effect of antiretroviral therapy on HIV-1 in gut-associated lymphoid tissue.

OBJECTIVES: We evaluated virologic and immunologic responses to antiretroviral therapy in gut-associated lymphoid tissue (GALT) compared with those found in peripheral blood. METHODS: Eight HIV-1-infected individuals were treated with three reverse transcriptase inhibitors and one protease inhibitor. Endoscopic biopsies were performed at baseline, and at months 1, 2, and 6. We measured the level of cell-associated multiply spliced and unspliced HIV-1 mRNA in GALT and in peripheral blood mononuclear cells. Immunologic responses were assessed by flow cytometry. RESULTS: Levels of multiply spliced HIV-1 mRNA declined in parallel fashion both in peripheral blood and GALT. After 6 months of therapy, unspliced HIV-1 mRNA in the GALT was below assay detection although it persisted in peripheral blood mononuclear cells in 4 study subjects. Although the percentage of CD4+ lymphocytes increased significantly in peripheral blood, only modest increases occurred in GALT. The percentage of activated CD8+ T cells decreased significantly in peripheral blood whereas only modest reductions occurred in GALT. CONCLUSIONS: Antiretroviral therapy effectively suppressed HIV-1 replication in GALT. The percentage of CD4+ T cells in peripheral blood uniformly increased in all study subjects, whereas it was more variable in the GALT.

An antigenic threshold for maintaining human immunodeficiency virus type 1-specific cytotoxic T lymphocytes.

BACKGROUND: Using the lymphocytic choriomeningitis virus (LCMV) model in mice, a number of studies show that memory cytotoxic T-lymphocyte (CTL) responses are maintained in the presence of continuous antigenic stimulation. Yet, other groups found that memory CTL specific for LCMV could last for a lifetime in mice without viral antigens. Thus, the extent to which an antigen is required for the maintenance of virus-specific CTL remains controversial. In humans, very few studies have been conducted to investigate the relationship between the quantity of antigen and the magnitude of CTL responses. MATERIALS AND METHODS: We quantified CTL precursors (CTLp) using a limiting-dilution analysis (LDA) and CTL effectors (CTLe) using a new Major Histocompatibility Complex (MHC) class I tetramer technology in six long-term nonprogressors (LTNPs) with human immunodeficiency virus type-1 (HIV-1) infection, as well as in eight patients whose viral loads were well suppressed by antiretroviral therapy. The viremia levels in these patients were measured using an reverse transcription polymerase chain reaction (RT-PCR) assay. The proviral DNA load in peripheral blood mononuclear cell (PBMC) was also measured by PCR in four LTNPs. RESULTS: The LTNPs had high levels of HIV-1-specific memory CTLp and CTLe, while maintaining a low plasma viral load. Despite also having low viral loads, patients whose plasma viremia was well-suppressed by effective therapy had low levels of CTLe. CONCLUSIONS: Our findings suggest that a complex, rather than a monotonic, relationship exists between CTL levels and HIV-1 viremia, including what appears to be an antigenic threshold for the maintenance of CTL at a measurable level. Under conditions of "antigen excess,", CTLe levels correlate inversely with viral load. On the other hand, under conditions that are "antigen limited," the correlation appears to be direct.

Passive infusion of immune serum into simian immunodeficiency virus-infected rhesus macaques undergoing a rapid disease course has minimal effect on plasma viremia.

Antibody responses are often considered to play only a limited role in controlling viremia during chronic infections with human or simian immunodeficiency virus (SIV). We investigated this by determining the effect of passively infused antibody on plasma viremia in infected rhesus macaques. The emphasis of the study was to understand the mechanism(s) underlying any observed effects. We infused serum immunoglobulins (SIVIG) purified from SIV(mac)251-infected macaques into other SIV(mac)251-infected macaques. The rapid progressor recipients had high viral loads but negligible titers of antibodies to SIV. Thus, we could significantly increase antibody titers with exogenous SIVIG. Despite restoring anti-SIV titers to levels typical of macaques with a normal disease course, SIVIG had only a modest effect on plasma SIV RNA and cell-associated viral load; the maximum, transient, reduction was threefold. The decrease in plasma RNA commenced within 1-2 h of SIVIG infusion, the nadir was at 12 h, and then a rebound occurred. A two- to threefold drop in cell-associated viral RNA was simultaneous with the decrease in plasma RNA. The kinetics of the viremia changes are inconsistent with neutralization of new cycles of infection. More likely, perhaps unexpectedly, is that infused antibodies killed SIV-infected cells, via an effector mechanism such as antibody-dependent cellular cytotoxicity.

Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy.

The role of the thymus in HIV-1 pathogenesis remains unclear. We developed an assay to quantify the number of recent thymic emigrants in blood based on the detection of a major excisional DNA byproduct (termed alpha1 circle) of T cell receptor rearrangement. By studying 532 normal individuals, we found that alpha1 circle numbers in blood remain high for the first 10-15 yr of life, a sharp drop is seen in the late teen years, and a gradual decline occurs thereafter. Compared with age-matched uninfected control individuals, alpha1 circle numbers in HIV-1-infected adults were significantly reduced; however, there were many individuals with normal alpha1 circle numbers. In 74 individuals receiving highly active antiretroviral therapy, we found no appreciable effect on alpha1 circle numbers in those whose baseline values were already within the normal range, but significant increases were observed in those with a preexisting impairment. The increases in alpha1 circle numbers were, however, numerically insufficient to account for the rise in levels of naive T lymphocytes. Overall, it is difficult to invoke thymic regenerative failure as a generalized mechanism for CD4 lymphocyte depletion in HIV-1 infection, as alpha1 circle numbers are normal in a substantial subset of HIV-1-infected individuals.

Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy.

We have designed a novel, precise, and sensitive assay to measure unspliced (US) human immunodeficiency virus type 1 (HIV-1) mRNA in peripheral blood mononuclear cells of HIV-1-infected individuals by using real-time PCR and molecular beacons. Individuals were classified as either well suppressed (WS) or partially suppressed, based on longitudinal measurements of plasma HIV-1 RNA. The proportion of individuals with US mRNA undetectable over time was significantly higher among WS individuals; however, 30% of WS subjects still had detectable US mRNA after 24 months of effective antiviral therapy.

Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy.

BACKGROUND: In patients infected with human immunodeficiency virus type 1 (HIV-1), combination antiretroviral therapy can result in sustained suppression of plasma levels of the virus. However, replication-competent virus can still be recovered from latently infected resting memory CD4 lymphocytes; this finding raises serious doubts about whether antiviral treatment can eradicate HIV-1. METHODS: We looked for evidence of residual HIV-1 replication in eight patients who began treatment soon after infection and in whom plasma levels of HIV-1 RNA were undetectable after two to three years of antiretroviral therapy. We examined whether there had been changes over time in HIV-1 proviral sequences in peripheral-blood mononuclear cells, which would indicate residual viral replication. We also performed in situ hybridization studies on tissues from one patient to identify cells actively expressing HIV-1 RNA. We estimated the rate of decrease of latent, replication-competent HIV-1 in resting CD4 lymphocytes on the basis of the decrease in the numbers of proviral sequences identified during primary infection and direct sequential measurements of the size of the latent reservoir. RESULTS: Six of the eight patients had no significant variations in proviral sequences during treatment. However, in two patients there was sequence evolution but no evidence of drug-resistant viral genotypes. In one patient, extensive in situ studies provided additional evidence of persistent viral replication in lymphoid tissues. Using two independent approaches, we estimated that the half-life of the latent, replication-competent virus in resting CD4 lymphocytes was approximately six months. CONCLUSIONS: These findings suggest that combination antiretroviral regimens suppress HIV-1 replication in some but not all patients. Given the half-life of latently infected CD4 lymphocytes of about six months, it may require many years of effective antiretroviral treatment to eliminate this reservoir of HIV-1.

Molecular tracking of an Human Immunodeficiency Virus nef specific cytotoxic T-cell clone shows persistence of clone-specific T-cell receptor DNA but not mRNA following early combination antiretroviral therapy.

The mechanisms that lead to maintenance of an active effector cytotoxic T-cell (CTL) response in Human Immunodeficiency Virus type-1 (HIV-1) infection are not well understood. We have investigated the role of antigen in maintenance of an HIV-1 specific CTL response by studying a patient (313-7) whose antigenic stimulus was reduced using antiretroviral drug therapy started within 90 days of HIV-1 infection. This patient made a primary monospecific CTL response to an HLA-C*0802 restricted epitope in nef (KAAVDLSHFL) prior to treatment. Within 7 days of starting treatment the nef specific CTL precursor frequency (CTLp) had decreased from 60/10(6) to 4/10(6) peripheral blood mononuclear cells (PBMC), coincident with a decline in viremia from 18 470 to 615 copies/ml. Both plasma viremia and nef specific CTLp remained at low levels for 180 days. The nef-specific CTL clone T-cell receptor (TCR) mRNA transcripts also decreased after treatment, but clone specific TCR DNA persisted. It appears that removal of antigen alters the state of HIV specific CTL from an activated effector population (detected in the CTLp assay and by measurement of clone specific RNA) to a non-activated quiescent population (detected by measurement of clone-specific DNA). This latter population may represent persisting HIV specific memory CTL.

The effect of commencing combination antiretroviral therapy soon after human immunodeficiency virus type 1 infection on viral replication and antiviral immune responses.

Twelve subjects were treated with zidovudine, lamivudine, and ritonavir within 90 days of onset of symptoms of acute infection to determine whether human immunodeficiency virus type 1 (HIV-1) infection could be eradicated from an infected host. In adherent subjects, with or without modifications due to intolerance, viral replication was suppressed during the 24-month treatment period. Durable suppression reduced levels of HIV-1-specific antibodies and cytotoxic T lymphocyte responses in selected subjects. Proviral DNA in mononuclear cells uniformly persisted. The persistence of HIV-1 RNA expression in lymphoid tissues and peripheral blood mononuclear cells suggests that elimination of this residual pool of virus should be achieved before considering adjustments in antiretroviral therapeutic regimens. In addition, given the reduction in levels of virus-specific immune responses, it would seem prudent to consider enhancing these responses using vaccine strategies prior to the withdrawal of antiviral therapy.

Human immunodeficiency virus type-1 mRNA splicing pattern in infected persons is determined by the proportion of newly infected cells.

Plasma viremia during HIV-1 infection is regulated by a dynamic balance between viral replication and removal of infected cells and cell-free virus. Administration of novel potent antiretroviral drugs provides an opportunity to study the consequences of perturbing this equilibrium by blocking de novo infections. In this study, we examined the expression of differentially spliced forms of HIV-1 mRNA, unspliced (US) and multiply spliced (MS), in peripheral blood mononuclear cells (PBMCs) of patients treated with HIV protease inhibitors or combination therapy. In all nine patients studied, a significant reduction in the MS/US mRNA ratio was observed after 1 week of treatment, suggesting that the majority of HIV MS mRNA in the steady-state situation prior to therapy was expressed by cells which had been infected during the previous couple of days. This idea was supported by a detailed analysis of serial PBMC specimens collected from two of the patients during the first hours and days after initiation of therapy. In both cases, a substantial decrease in MS mRNA expression was evident already after 48 hr, whereas the expression of US mRNA at this time was virtually unaffected. These data indicate that the HIV mRNA splicing pattern in vivo is mainly determined by the relative proportion of newly infected cells and suggest that examination of this pattern could be useful in evaluating the potency of antiretroviral therapies and in studying dynamics of HIV-1 infection.

Decay characteristics of HIV-1-infected compartments during combination therapy.

Analysis of changes in viral load after initiation of treatment with potent antiretroviral agents has provided substantial insight into the dynamics of human immunodeficiency virus type 1 (HIV-1). The concentration of HIV-1 in plasma drops by approximately 99% in the first two weeks of treatment owing to the rapid elimination of free virus with a half-life (t1/2) of < or =6 hours and loss of productively infected cells with a t1/2 of 1.6 days. Here we show that with combination therapy this initial decrease is followed by a slower second-phase decay of plasma viraemia. Detailed mathematical analysis shows that the loss of long-lived infected cells (t1/2 of 1-4 weeks) is a major contributor to the second phase, whereas the activation of latently infected lymphocytes (t1/2 of 0.5-2 weeks) is only a minor source. Based on these decay characteristics, we estimate that 2.3-3.1 years of a completely inhibitory treatment would be required to eliminate HIV-1 from these compartments. To eradicate HIV-1 completely, even longer treatment may be needed because of the possible existence of undetected viral compartments or sanctuary sites.

Stability in controlling viral replication identifies long-term nonprogressors as a distinct subgroup among human immunodeficiency virus type 1-infected persons.

Long-term nonprogressors (LTNPs) of human immunodeficiency virus type 1 (HIV-1) infection are characterized by low levels of HIV-1 replication and viral load. However, it has not been established whether they differ in this regard from progressors from the very early stage of infection. By studying peripheral blood mononuclear cell (PBMC) specimens from a longitudinally monitored cohort of HIV-1-infected men, we found that HIV-1 proviral copy numbers and HIV-1 mRNA expression levels as low or lower than those seen in seven carefully selected LTNPs were commonly observed in specimens collected soon after seroconversion from 28 subjects who became infected while under observation. However, only the LTNPs were able to stably maintain such an efficient viral control over time. Because of the instability of the early control of HIV-1 replication, the predictive value of HIV-1 mRNA expression in PBMCs at postseroconversion was found to be limited but significantly increased during the first year of infection. Besides their diagnostic implications, these data support the idea that LTNPs may be a pathophysiologically distinct subgroup among persons infected with HIV-1.

Detection of herpes simplex virus DNA in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization.

As conventional polymerase chain reaction (PCR) procedures are time-consuming and laborious, we developed and evaluated a rapid semi-automatic microplate method to detect the amplified PCR products. The use of PCR, with subsequent hybridization in microplates, is described for the detection of herpes simplex virus (HSV) DNA in cerebrospinal fluid samples. The principle of the method is based on two phases. Firstly, the amplification of the viral DNA in the sample is undertaken using a pair of primers of which one is biotinylated. Secondly, the amplified viral genomic sequences are bound to the wells of streptavidin-coated microplates and hybridized with digoxigenin-labeled oligonucleotide probes which are then detected using anti-digoxigenin antibody enzyme conjugates and either a photometric, fluorometric or luminometric substrate and microplate reader. The method is highly sensitive allowing the detection of as few as five purified DNA molecules. Compared to conventional gel electrophoresis followed by Southern blotting the established microplate hybridization is also much less time-consuming and involves less manual work. The applicability of the method is described for use as a routine diagnostic procedure for detection of early central nervous system infections caused by HSV-1 and HSV-2.

Morphological differentiation of human SH-SY5Y neuroblastoma cells inhibits human immunodeficiency virus type 1 infection.

We have studied human immunodeficiency virus type 1 (HIV-1) infection in human SH-SY5Y neuroblastoma cells at various stages of morphological differentiation. Two days' treatment of the cells with retinoic acid (RA) or dibutyryl cAMP (db-cAMP) resulted in the appearance of elongated neurites and enhanced production of 160K to 200K neurofilament proteins as shown by indirect immunofluorescence. DNA synthesis was reduced only in RA-treated cells as detected by 5-bromo-2'-deoxyuridine incorporation. The cells were infected with two T-lymphotropic virus strains (IIIB and NDK) and two fresh isolates (39001 and 46001) from bronchoalveolar lavage samples of AIDS patients. The latter two isolates were unable to form syncytia in infected CD4-positive T-lymphoblastoid C8166 cells which was in contrast to our T-lymphotropic virus strains. Interphase in situ hybridization showed that 14 to 16% of SH-SY5Y cells become positive for HIV-1 DNA. Regardless of the virus strain, morphological differentiation of the cells with RA or db-cAMP inhibited infection by 50% at a single cell in situ resolution. Nested PCR confirmed the presence of proviral DNA in the infected cells. These results show that human neuroblastoma cells, tumour cells of neuroectodermal origin, can be infected by different HIV-1 isolates and that the infection is inhibited by neurotypic cell differentiation.

Bovine uterine, cervical and ovarian cytosol estrogen and progesterone receptor concentrations in cystic ovarian disease.

Bovine cytosol estrogen (ERC) and progesterone receptor (PRC) concentrations were measured simultaneously in various regions of the uterus and in ovarian stromal tissue in cows with cystic ovarian disease (follicular cysts), and the concentrations compared with those in animals with normal cycles. In cystic ovarian disease, ERC concentrations in endometrium (550 fmol/mg cytosol protein (c.p.)) and in myometrium (405) were significantly higher than in control animals. Very high PRC contents were measured in the endometrium (3115) and myometrium (2761) of cows with cystic ovarian disease. In control animals, PRC concentrations in the endometrium and myometrium were significantly lower than in diseased animals. No statistical differences were observed in ERC or PRC contents between the endometrium and the myometrium in cows with cystic ovarian disease. ERC and PRC concentrations in the uterine cervix and ovaries were low compared to those detected in the uterus. Bovine serum estradiol-17 beta concentrations were higher (p < 0.001) in cows with cystic ovarian disease than in control animals in postpartum anestrus or during the normal estrous cycle. Serum sex hormone-binding globulin (SHBG) concentrations were of the same magnitude as in control cows during their estrous cycles. These findings show that long standing low endogenous progesterone and elevated estradiol concentrations in serum are associated with elevated ERC and PRC concentrations in bovine uterus.

Human spumaretrovirus in the etiology of sudden hearing loss.

The etiology of sudden sensorineural hearing loss, so called sudden deafness, has for long puzzled researchers. Recently we have studied the possibility that a hitherto relatively unknown retrovirus group consisting of human spumaretroviridae (HSRV) might be the causative agent of sudden deafness. During the last 3 months we have screened about 30 cases of sudden deafness. In 4 of them antibodies against HSRV were detected. Three of them had suffered from a flu-like condition about 2 weeks before the onset of hearing loss. In 2 cases the hearing of both ears was involved, in 1 case a relapsing hearing loss was observed, and 1 case developed a Meniere-like symptomatology with a fluctuant hearing loss. Vertigo was present in 3 patients and all suffered from tinnitus. Full recovery of hearing was observed in 4 of 6 affected ears whereas 2 ears became practically deaf with poor speech discrimination. At present it seems likely that a significant part of sudden deafness is caused by HSRV infection. The course of infection follows the spontaneous course of sudden deafness described by many authors. We encourage otologic units to screen for HSRV when assessing the etiology of sudden deafness.

Identification of human type D retrovirus as a contaminant in a neuroblastoma cell line.

In this report we describe a type D virus isolated from a human neuroblastoma cell line (Paju). The viral RNA was isolated, partially molecularly cloned and sequenced. Our clones were shown to be identical to a human D-type retrovirus previously isolated from a human lymphoblastoid cell line. However, we obtained no evidence for the virus in earlier passage of the Paju cell line and therefore we must consider this isolate a laboratory contamination. contamination.

Activation of integrated human immunodeficiency virus type 1 in human neuroblastoma cells by the cytokines tumour necrosis factor alpha and interleukin-6.

Human immunodeficiency virus type 1 (HIV-1) infection was studied in two different human neuroblastoma cell lines, SK-N-MC and SH-SY5Y. Results from immunofluorescence analysis indicate that SK-N-MC cells express a 68K neurofilament, and SH-SY5Y cells express additionally a 160K to 200K neurofilament complex and thus represent a more differentiated state. HIV-1 infection in these cell lines was demonstrated by nested polymerase chain reaction and further characterized by in situ hybridization, which showed that about 50% of SK-N-MC cells and 20% of SH-SY5Y cells were infected by HIV-1 and contained integrated proviral HIV-1 DNA. Among the cytokines and growth factors studied, tumour necrosis factor alpha (TNF-alpha) enhanced virus production in both cell lines, but to a differing extent, according to our mRNA and p24 antigen capture assay. In SK-N-MC cells the enhancement of HIV-1 mRNA was detected after 24 h of stimulation, and declined to the control level by 48 h. In SH-SY5Y cells a clear-cut stimulation was seen at both time points. By contrast, interleukin-6 (IL-6) enhanced the virus replication only in SK-N-MC cells, as shown at the mRNA level. Immunochemical staining showed no differences in the proportion of HIV-1-positive cells after 48 h of stimulation by TNF-alpha or IL-6 when compared to the control cells. In addition, based on a thymidine incorporation assay, TNF-alpha inhibited, but IL-6 strongly increased, the DNA synthesis in SK-N-MC cells, whereas in the SH-SY5Y cell line no such differences were seen. We discuss the possibility that developing, less-differentiated neurons may be more readily infected by HIV-1 than fully differentiated neurons, and that cytokines such as TNF-alpha and IL-6, which are elevated in HIV-1-infected individuals, may enhance HIV production.