Evidence of recombination producing allelic diversity in MHC class I Mafa-B and -A alleles in cynomolgus macaques.

The MHC class I-A and -B genes of cynomolgus macaques are highly polymorphic. These genes encode proteins presenting peptides to CD8+ T cells to initiate adaptive immune response. Recombination events are one way the diversity of these alleles can be increased. Such events have been well characterized in humans, but have not been as well characterized in macaques. In order to identify and examine recombinations that create new alleles, it is important to analyze intron sequences. Intron sequences have been shown to be important to understand the evolutionary mechanisms involved in the generation of major histocompatibility complex (MHC) alleles and loci. Thus far, there have been relatively few intron sequences reported for MHC class I alleles in macaques, and this has hampered the understanding of MHC organization and evolution in macaques. In this study, we present evidence of a gene conversion event generating the Mafa-B*099 allele lineage by the combination of Mafa-B*054 and Mafa-B*095 allele lineages. A potential recombination between the Mafa-A3*13 and Mafa-A4:14 lineages was also observed, but it is less clear due to lack of intron 2 sequence. This report stresses the role that recombination can play in MHC class I diversity in cynomologus macaques, and the importance of introns in identifying and analyzing such events.

Mucosal correlates of isolated HIV semen shedding during effective antiretroviral therapy.

Effective antiretroviral therapy (ART) suppresses the blood HIV RNA viral load (VL) below the level of detection. However, some individuals intermittently shed HIV RNA in semen despite suppression of viremia, a phenomenon termed "isolated HIV semen shedding (IHS)". In a previously reported clinical study, we collected blood and semen samples from HIV-infected men for 6 months after ART initiation, and documented IHS at ≥1 visit in almost half of the participants, independent of ART regimen or semen drug levels. We now report the mucosal immune associations of IHS in these men. Blood and semen plasma cytokine levels were assayed by multiplex enzyme-linked immunosorbent assay, T-cell populations were evaluated by flow cytometry in freshly isolated blood and semen mononuclear cells, and semen cytomegalovirus (CMV) DNA levels were measured by PCR. Although IHS was not associated with altered blood or semen cytokine levels, the phenomenon was associated with a transient, dramatic increase in CD4+ and CD8+ T-cell activation that was restricted to the semen compartment. All participants were CMV infected, and although semen CMV reactivation was common despite ART, this was not associated with T-cell activation or IHS. Further elucidation of the causes of compartmentalized mucosal T-cell activation and IHS may have important public health implications.

Identification of 23 novel MHC class I alleles in cynomolgus macaques of Philippine and Philippine/Mauritius origins.

We report here novel Mafa-A, -AG and -B alleles identified in two groups of cynomolgus macaques.

Discordance in HIV-1 viral loads and antiretroviral drug concentrations comparing semen and blood plasma.

OBJECTIVES: For individuals not on antiretroviral therapy, the risk of heterosexual transmission of HIV appears negligible when blood plasma (BP) viral loads are <1500 HIV-1 RNA copies/mL. It is not clear whether this observation can be extrapolated to individuals on highly active antiretroviral therapy (HAART). Because of differential tissue penetration, antiretroviral drug concentrations may be sufficient to maintain an undetectable viral load in the BP yet not achieve adequate levels to suppress HIV in the genital tract. Therefore, we wanted to correlate HIV viral loads and drug concentrations in semen plasma (SP) and BP. METHODS: Thirty-three men were included. All were on combination antiretroviral therapy with an undetectable BP viral load for at least 1 year. Blood and semen samples were collected within 2 h of each other and tested for HIV RNA by the NucliSens QT (bioMerieux, St Laurent, QC, Canada) method; drug concentrations were determined by liquid chromatography tandem mass spectrometry. RESULTS: Two of the 33 patients (6.1%) with BP viral loads below detection had time-matched HIV viral loads in SP > or =700 copies/mL. Both patients were on efavirenz, the SP concentrations of which were < or =10% of the levels in BP and well below the minimal therapeutic drug monitoring target concentration required to suppress HIV. CONCLUSIONS: Because, at least in part, of poor drug penetration into the genital tract, an undetectable HIV viral load in the BP does not guarantee an undetectable viral load in semen. In view of this, caution should be taken in concluding that patients on HAART with suppressed viraemia are sexually non-infectious.

Affordable CD4 T-cell enumeration for resource-limited regions: a status report for 2008.

BACKGROUND: The global struggle with human immunodeficiency virus (HIV) and the battle to develop affordable CD4 T-cell counting technology are both unfulfilled goals in 2008. The need for such instrumentation is more critical now as implementation of antiretroviral therapy (ART) is in progress in many resource limited regions. Major scaling-up efforts in rural situations are difficult to implement without laboratory infrastructure. CD4 T-cell counting is especially critical when trying to reach individuals with HIV to have them enrolled in ART as soon as they qualify for treatment based on CD4 count. METHOD: This review covers both the chronological evolution and the scientific milestones of technological development of affordable immunophenotyping. It is more focused on flow cytometry but does consider the potential contribution by digital image cytometry. RESULTS: Thus far flow cytometry offered only modest progress toward affordable immunophenotyping. A list with desirable features is offered for side by side comparison. Digital image cytometry has yet to show its enormous affordable market potential. CONCLUSIONS: It is possible to develop truly affordable, portable flow cytometry but it is not here yet. There are some hopeful signs as there are innovative and practical technical components appearing at regular intervals. However, so far the technical breakthroughs have been fragmented efforts without any attempts to consider intercorporate collaboration to optimize critical mass and synergy. The smaller players in the industry have made some progress toward meeting the monumental needs in Africa and Asia. Digital image cytometry may well be the ultimate winner in the affordable technology race.

A randomized control trial of an Ultra-Short zidovudine regimen in the prevention of perinatal HIV transmission in rural Zimbabwe.

OBJECTIVE: To assess the practicality and effectiveness of an Ultra-Short zidovudine regimen for prevention of perinatal HIV transmission in rural Zimbabwe. DESIGN: Double-blinded placebo-controlled randomized clinical trial. SETTING: The Salvation Army Howard Hospital, a district hospital in rural Zimbabwe. SUBJECTS: 222 HIV positive pregnant women presenting for antenatal care prior to 36 weeks were randomized. Twenty nine women were lost to follow up. INTERVENTION: In the Thai regimen, mothers received zidovudine (300 mg po bid) from 36 weeks gestation until labour, and zidovudine (300 mg po q3h) during labour, and the neonates received a placebo. In the Ultra-Short regimen, the mothers received a placebo from 36 weeks to labour, then zidovudine (300 mg po q3h) in labour. The neonates received zidovudine (2 mg/kg po qid) for the first three days of life. MAIN OUTCOME MEASURE: Infant HIV RNA status at six weeks of life. RESULTS: Results were available for 90 infants from the Thai group and 89 infants from the Ultra-Short group. Infant HIV seroconversion rates at six weeks of life were 18.9% (95%CI 10.8 to 27.0) with the Thai regimen, and 15.7% [95% Confidence Interval (CI) 8.1 to 23.4] with the Ultra-Short regimen. The upper bound of seroconversion in the Ultra-Short group was lower than the 25% seroconversion boundary that was specified to show equivalence. CONCLUSIONS: Although the Ultra-Short regimen has equivalent efficacy to the Thai regimen, it also has many practical advantages. Ultra-Short is thus a preferable protocol.

Promotion of morphological transformation by Di-n-butyltin dichloride in C3H/10T1/2 cells: prediction by prior expression of tumour promoter-responsive genes.

Previous studies in our laboratory have shown that chemical treatments may induce increases in proliferin gene family mRNA accumulation in cultured murine embryonic cells. Proliferin inductions are highly correlated with subsequent promotional outcomes during two-stage focus-formation assays in C3H/10T1/2 cell cultures. In work reported here, the strong affiliation between these two responses was further validated after treating cells with di-n-butyltin dichloride which is a polyvinyl chloride (PVC) plastic additive that often contaminates food and water. Increased proliferin expression and promotion of morphological transformation occurred at similar concentrations. Promotion of transformation was detected at di-n-butyltin dichloride concentrations of 80 nM (24 ng/ml) and above, if added to initiated cultures before confluent monolayers had formed. Proliferin induction and morphological transformation were both reduced in confluent cultures treated with di-n-butyltin dichloride, as compared to subconfluent cultures. Proliferin expression measured in near-confluent cultures was induced up to 10-fold during the 36-hr period following di-n-butyltin dichloride exposure and was accompanied by increased accumulation of transcripts from many genes regulated by oxidative stresses, growth-inducing agents, and/or other promoting agents (asbestos, superoxide radicals ). Di-n-butyltin dichloride-induced mRNA species included members of the fos and jun proto-oncogene families, c-myc, egr1, ribonucleotide reductase (R2 subunit), odc, macrophage chemotactic protein/je, hsp70, metallothionine IIA, c-sod and mn-sod. The observed patterns of RNA accumulation suggested that a small subset of mRNA species, including proliferin, exhibit regulatory behaviour as a response to dissimilar agents or conditions that promote focus-formation in C3H/10T1/2 cultures. Plausible predictions of promotional effects in two-stage morphological transformation assays can be made from gene-expression responses to test agents.

Virus levels in untreated African infants infected with human immunodeficiency virus type 1.

In developed areas, human immunodeficiency virus (HIV)-infected infants have high virus levels and rapidly progress to death. HIV levels were assessed in 1994-1997 in untreated infants in Malawi by analysis of dried blood spots tested by nucleic acid silica-bound amplification. Of 24 umbilical cord blood (CB)-positive samples, 83% had >10,000 copies/mL. The median virus level was 78,000 copies/mL. First positive sample median levels were 355,000 copies/mL among 52 perinatally infected infants and 130,000 copies/mL among 43 infants infected by breast-feeding. Virus levels were stable, and initial levels predicted levels 1 year after infection (P=.005), at which time levels did not significantly differ among in utero, perinatally, or postnatally infected infants. Thus, neither age at infection nor route of infection significantly influenced HIV levels measured 1 year after infection. Most (87%) CB-positive infants were infected before labor onset, since virus levels greatly exceeded those expected in their mothers.

Diagnosis and Direct Automated Sequencing of HIV-1 From Dried Blood Spots (DBS) Collected on Filter Paper.

Since its discovery in 1981, human immunodeficiency virus type 1 (HIV-1) has rapidly emerged as one of the most devastating infectious pathogens of this century (1-3). The World Health Organization (WHO) estimates that, as of 1995, there were at least 15 million HIV- infected men, women, and children worldwide, with the vast majority of infections occurring in developing countries and isolated rural regions where specimen collection, preparation and shipment are difficult (4). Simple and improved sampling methods that can be widely applied under difficult field conditions are needed to effectively monitor the changing dynamics of the HIV-1/AIDS pandemic, track the spread of HIV-1 variants among different population groups, and ensure that research and interventive activities are directed against biologically important variants of the virus. To date, at least eight major HIV-1 subtypes, designated A through H, have been identified (5,6). More recently, a ninth subtype, I, has been detected (7), as well as several highly divergent, or "outlying" variants of HIV-1 that have been tentatively classified as group O (8,9). This subtyping is based on a relatively small number of specimens collected from a few geographic areas and the full range and distribution of HIV-1 variants remains to be established. The collection of whole blood on filter paper provides an innovative and powerful approach for the systematic and unbiased collection of large numbers of field specimens for diagnostic and surveillance purposes (10-19).

Quantification of HIV-1 RNA in Dried Plasma Spots (DPS) : A Field Approach to Therapeutic Monitoring.

The ability to accurately measure viral RNA in the plasma (1-3) and intracellular (4-7) compartments of HIV-1-infected persons has led to a dramatic improvement in the understanding of the natural history of HIV-1 and AIDS. A number of recent studies have convincingly demonstrated that high levels of viral replication occur at all stages of disease (8-10), and that changes in viral RNA load are predictive of disease outcome (11,12), and response to therapy (13,14). These findings, combined with the introduction of potent new antivirals (15,16), have stimulated a growing interest in viral load monitoring, both as a function of disease status, and as a predictor of disease progression and therapeutic efficacy.

Amplicor HIV monitor, NASBA HIV-1 RNA QT and quantiplex HIV RNA version 2.0 viral load assays: a Canadian evaluation.

BACKGROUND: HIV-1 viral load quantitation is now recognized as a useful tool to monitor the efficiency of antiviral treatment and a powerful predictor of disease outcome. Three HIV-1 viral load quantitation methods have been currently available as commercial kits in Canada since 1996. OBJECTIVE: To evaluate the ability to quantify HIV-1 RNA in plasma of the Amplicor HIV Monitor Test, the NASBA HIV-1 RNA QT Assay and the Quantiplex HIV RNA Assay, version 2.0, at comparable lower detection limits. STUDY DESIGN: Blood was collected from 50 HIV-1-infected patients at various stages of infection and therapy. CD4+ cell count were estimated by flow cytometry. Plasma was isolated and tested in duplicate on four occasions using viral load kits from a single lot. HIV RNA data, performance, sensitivity and intra- and inter-assay variability were compared. RESULTS: RNA could be quantified in 33 patients by each technique. An inverse correlation was observed between viral load level and CD4+ cell counts in patients with counts below 200. Monitor could detect RNA in 94% of patients, but it showed the greatest variability and failure rate. Quantiplex 2.0 could detect HIV-1 RNA in 78%, and NASBA in 88% of the patients at theoretically equivalent lower detection limits, suggesting that the detection limit of Quantiplex 2.0 may be higher than 500 HIV-1 RNA copies per ml. NASBA had the fewest invalid tests and good reproducibility, comparable to that of Quantiplex 2.0. The mean values from NASBA and Monitor were the most similar but the best correlation was observed between Monitor and Quantiplex 2.0 results. CONCLUSIONS: Monitor, NASBA and Quantiplex results were comparable, although those obtained by Quantiplex were significantly lower. Performing this study at comparable detection limits showed that the detection limit of Quantiplex 2.0 may be higher than stated by the manufacturer.

Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper.

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.

Asbestos promotes morphological transformation and elevates expression of a gene family invariably induced by tumor promoters in C3H/10T1/2 cells.

The murine proliferin gene family, which has been shown to respond consistently to tumor promoters and other cellular pro-oxidant agents in C3H/10T1/2 cells, was used to monitor responses after treatment of these cell cultures with toxic, pro-oxidant asbestos fibres. Proliferin mRNA levels were increased by amosite, crocidolite or chrysotile asbestos fibres, especially in the presence of fresh serum and at low cell densities. Promotion of morphological transformation was confirmed in two-stage focus formation assays using crocidolite at a fibre density that induced proliferin expression. Asbestos-induced gene expression was inhibited by millimolar levels of N-acetylcysteine (NAC), supporting a linkage between: (i) induced oxidant stress that was sufficient to promote morphological transformation; (ii) induction of proliferin expression. Other anti-oxidant compounds (dithiothreitol and pyrrolidine dithiocarbamate) or enzymes (superoxide dismutase and catalase) did not inhibit induced expression. Non-fibrous powders (titanium dioxide, quartz or silica gel) were also effective inducers of proliferin mRNA accumulation. Latex beads and activated charcoal were effective at higher particle densities, implying that ubiquitous particle-induced surface membrane effects can lead to an NAC-reversible step necessary for proliferin induction. The results showed that asbestos resembled all other promoters of morphological transformation in C3H/10T1/2 cells in that an antioxidant-sensitive induction of the proliferin gene family occurred following treatment.

Oxidative stress-regulated gene expression and promotion of morphological transformation induced in C3H/10T1/2 cells by ammonium metavanadate.

Promoters of C3H/10T1/2 cell morphological transformation that elevate intracellular oxidant levels can be distinguished by a spectrum of induced gene expression, which includes the oxidant-responsive murine proliferin gene family. Proliferin transcripts were induced 40- to 100-fold by 20 microM ammonium metavanadate, 20-fold by 5 microM vanadium pentoxide but only three-fold by vanadium oxide sulfate. Consistent with its response to other oxidant chemicals, induction of proliferin by ammonium metavanadate was inhibited almost completely by the antioxidant N-acetylcysteine (8 mM). Ammonium metavanadate (5 microM), added as promoter in two-stage morphological transformation assays, amplified yields of Type II and Type III foci in monolayers of 20-methylcholanthrene-initiated C3H/10T1/2 cells. Ammonium metavanadate also induced formation of Type II foci in single-step transformation assays. The results suggest that pentavalent vanadium compounds could promote morphological transformation in C3H/10T1/2 cells by creating a cellular state of oxidative stress, sufficient to induce elevated expression of the proliferin gene family.

Somatothymia in chronic pain patients.

Somatothymia is the use of somatic language to communicate affective distress. A total of 152 chronic pain patients completed a systems review checklist and the Minnesota Multiphasic Personality Inventory. Associated features of somatic symptoms and the meaningfulness of somatic symptoms as a communication, common physical areas of somatic focus, patterns of affective distress in high and low somatothymics, and the utility of select variables classifying high and low somatothymics were evaluated. The results indicate that a systems review checklist can be used as a quick, useful, and initial screen for somatothymia and that somatic symptoms can in fact communicate affective distress.

Tri-n-butyltin chloride promotes morphological transformation and induces proliferin expression in C3H10T1/2 cells.

Transcripts from the murine gene family proliferin, which are increased by a wide assortment of chemical promoters of C3H10T1/2 cell morphological transformation, were shown to be induced by tri-n-butyltin chloride at concentrations above 50 nM. Two-stage transformation assays, with 3-methylcholanthrene as inducer and tri-n-butyltin chloride as promoter, were performed to determine if promotion of morphological transformation and proliferin induction were properties shared by this compound. Tri-n-butyltin chloride synergistically enhanced focus formation at concentrations ranging from 20 to 75 nM. Di-n-butyltin dichloride, n-butyltin trichloride and tin (II) chloride, but not tin (IV) chloride, were also effective inducers of proliferin. Changes in patterns of TPA-inducible, secreted proteins, including those likely to be proliferin, were detected following organotin treatment of confluent monolayers. Tri-n-butyltin chloride resembles other agents active as promoters in C3H10T1/2 two-stage transformation assays by possessing an ability to induce proliferin expression.

Identification and partial characterization of a candidate gene for X-linked retinopathies using a lateral approach.

Using library to library cross-screening we have identified a number of genomic clones that harbor X-linked sequences expressed in the human choroid/retina. We describe the characterization of one of these, designated XEH.8 (DXS542), which is localized to Xp11.3-q12. Isolation, partial sequencing, and Northern analysis of the cognate cDNA (XEH.8c), has shown that the cDNA has some homology to the dystrophin gene and hybridizes to a 10-kb mRNA present in the choroid and retina but not in fibroblasts. This expressed sequence maps to the same region of the X chromosome as several known X-linked ophthalmic diseases, including Norrie disease, retinitis pigmentosa 2, congenital night blindness and Aland Island eye disease.

Oxidative stress mediates tumor promoter-induced proliferin gene-expression in c3h10t1/2 cells.

Increased expression of the proliferin gene family occurs upon exposure of C3H10T1/2 cells to a broad range of chemical agents known to promote morphological transformation and likely to increase cellular reactive oxygen species. To determine if proliferin gene expression is influenced by reactive oxygen species, superoxide radicals were generated in culture by the xanthine/xanthine oxidase couple. The 1 kb cytoplasmic proliferin transcript accumulated up to ten-fold following extracellular superoxide production. Within certain concentration ranges of catalase and superoxide dismutase activity, xanthine oxidase-induced proliferin expression was reduced to control levels, while expression was increased at other concentrations. Induction of proliferin by the tumour promoters butylated hydroxytoluene or TPA was efficiently inhibited at certain concentrations of catalase and superoxide dismutase, but retinoic acid had no effect. Proliferin induction by a recently identified promoter of transformation, tri-n-butyltin chloride, was stimulated by catalase, superoxide dismutase and retinoic acid, but inhibited at higher concentrations of N-acetyl cysteine. c-fos preceded proliferin induction by butylated hydroxytoluene, but several other oncogenes and growth-regulated genes were unaffected. The results support a mechanism for tumour promoter-induced proliferin gene expression that involves a response to superoxide anions, hydrogen peroxide or other shifts in the cellular equilibrium between pro-oxidant and anti-oxidant moieties.

An identification of low back pain groups using biobehavioral variables.

A multivariate predictive model of low back pain (LBP) was developed. Following a semi-structured interview, 73 participants were assigned to dysfunctional chronic low back pain (DCLBP), functional chronic low back pain (FCLBP), acute low back pain (ALBP), and healthy control (HC) groups. All participants underwent a comprehensive physical, psychophysiological, and psychological evaluation. Multivariate analyses indicated no psychophysiological, few physical, and many psychological differences among the groups. The DCLBP group was found to be most impaired in flexion (p<.001), and the HC group performed the most total work (ft-lb) in extension (p<.001). Psychologically, the DCLBP group displayed greater levels of emotional distress and characterological disturbances and were more functionally impaired (p<.001). Few differences between FCLBP and HC were found. A classification analysis using physical and psychological variables correctly classified 83.3% of DCLBP patients, and it was found that the ALBP group was heterogeneous with some patients having a dysfunctional profile and other patients having a functional profile. The psychological variables were more potent predictors of group membership than were the physical variables. These findings indicate that potential DCLBP and FCLBP patients can be identified shortly following an injury, suggesting important implications for assessment and treatment of low back pain in general, and more specifically, for reducing health care costs and human suffering.

Protein Import into and Sorting inside the Chloroplast Are Independent Processes.

Plastocyanin is a nuclear-encoded chloroplast thylakoid lumen protein that is synthesized in the cytoplasm with a large N-terminal extension (66 amino acids). Transport of plastocyanin involves two steps: import across the chloroplast envelope into the stroma, followed by transfer across the thylakoid membrane into the lumen. During transport the N-terminal extension is removed in two parts by two different processing proteases. In this study we examined the functions of the two cleaved parts, C1 and C2, in the transport pathway of plastocyanin. The results show that C1 mediates import into the chloroplast. C1 is sufficient to direct chloroplast import of mutant proteins that lack C2. It is also sufficient to direct import of a nonplastid protein and can be replaced functionally by the transit peptide of an imported stromal protein. C2 is a prerequisite for intraorganellar routing but is not required for chloroplast import. Deletions in C2 result in accumulation of intermediates in the stroma or on the outside of the thylakoids. The fact that C1 is functionally equivalent to a stromal-targeting transit peptide shows that plastocyanin is imported into the chloroplast by way of the same mechanism as stromal proteins, and that import into and routing inside the chloroplasts are independent processes.