An improved method for HLA-B and -C supratyping.

A growing body of evidence links the analysis of the KIR genotype and the presence of their HLA-B and -C ligands to a wide repertoire of human diseases. We noticed that, using a panel of 184 Caucasoid donors, a limited number of HLA alleles were incorrectly supratyped by previously described pyrosequence-based assays. Here we describe a simple implementation of the reported methods that corrects all the discrepancies found with HLA-B and -C molecular typing and allows establishing a quick and high-throughput method for the determination of HLA-Bw4 I(80), Bw4T(80), Bw6 and HLA-C1 or -C2 supratype.

Dynamics of adaptive and innate immunity in patients treated during primary human immunodeficiency virus infection: results from Maraviroc in HIV Acute Infection (MAIN) randomized clinical trial.

We evaluated the dynamics of innate and adaptive immunity in patients treated with combined antiretroviral therapy (cART) during primary human immunodeficiency virus infection (PHI), enrolled in a prospective randomized trial (MAIN, EUDRACT 2008-007004-29). After 48 weeks of cART, we documented a reduction in activated B cells and CD8(+) T cells. Natural killer cell and dendritic cell frequencies were measured and a decrease in CD16(+) CD56(dim) with a reciprocal rise in CD56(high) natural killer cells and an increase in myeloid and plasmacytoid dendritic cells were recorded. In conclusion, 48 weeks of cART during PHI showed significant benefits for both innate and adaptive immunity.

Maraviroc 150 mg daily plus lopinavir/ritonavir, a nucleoside/nucleotide reverse transcriptase inhibitor-sparing regimen for HIV-infected naive patients: 48-week final results of VEMAN study.

Non-conventional strategies with nucleoside/nucleotide reverse transcriptase inhibitor-sparing regimens in antiretroviral naive human immunodeficiency virus (HIV) -infected patients have been explored in clinical trials. A prospective, open-label, randomized (1:1), multicentre, proof-of-concept trial (VEMAN study, EUDRACT number 2008-006287-11) was conducted assigning HIV-infected naive patients to once-daily maraviroc plus lopinavir/ritonavir (MVC group) or to tenofovir/emtricitabine plus lopinavir/ritonavir (TDF/FTC group). Clinical and laboratory data were collected at baseline, and after 4, 12, 24, 36 and 48 weeks with the objective to evaluate the 48-week virological and immunological efficacy. HIV-1 DNA load and CD4(+) T-cell subsets were analysed on frozen peripheral blood mononuclear cells collected at baseline, 4 and 48 weeks to explore the trend in HIV reservoirs. Fifty patients were randomized and included in the analysis. During follow up, HIV-1 RNA decreased similarly in both groups and, at week 48, all patients in the MVC group and 22/24 (96%) in the TDF/FTC group had < 50 copies/ml of HIV-1 RNA. CD4(+) trend during follow up was higher in maraviroc-treated patients (MVC group: 286 (183-343) versus TDF/FTC group: 199 (125-285); Mann-Whitney U-test: p 0.033). A significant 48-week increase of CCR5(+) CD4(+) T cells and CD4(+) effector memory cells was observed among maraviroc-treated patients (Wilcoxon signed rank test: p 0.016 and p 0.007, respectively). No significant variations were found in naive and central memory CD4(+) T cells. Among naive patients with an R5 virus, treatment with maraviroc and lopinavir/ritonavir was shown to provide a virological response compared to a triple therapy and a greater immunological benefit.

In vitro antifungal activity of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens.

The aim of this study was to examine the effect of five naturally occurring compounds from essential oils on 10 different species of mycotoxigenic fungi involved in several plant diseases. The antifungal activities of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol were observed in vitro on Fusarium subglutinans, Fusarium cerealis, Fusarium verticillioides, Fusarium proliferatum, Fusarium oxysporum, Fusarium sporotrichioides, Aspergillus tubingensis, Aspergillus carbonarius, Alternaria alternata and Penicillium sp. The naturally occurring compounds tested showed toxic effects on in vitro mycelium growth of all fungal species but with different level of potency. The results are encouraging for further investigations of in planta antifungal activities of these essential oils components.

Clock drawing interpretation scale (CDIS) and neuro-psychological functions in older adults with mild and moderate cognitive impairments.

The clock drawing test (CDT) is an easy to apply, well accepted and reliable test that is widely used to screen for visuo-constructional difficulties in the aged people. Yet, besides visual agnosia and constructional apraxia, executive control influences performances in this task. Execution modalities and rating schemes vary widely, as for the way stimuli are proposed, the time to which the clock is set, and the elements that are considered for scoring. The scoring system we have selected is called clock drawing interpretation scale (CDIS) contains 20 items in 3 areas: visuo-perceptual, attentional and numerical factors. Our aim is to know the meanings of the CDIS total and sub-areas score, investigating the relationships with specific neuropsychological tests, in elderly persons with moderate cognitive impairment. CDIS has been administered to 90 people, aged about 75 years, attending our rehabilitative day hospital or our memory clinic. A neuropsychological battery has been administered to a sample of 47 outpatients, selected by contingence. Respective mean MMSE for the 2 samples are 24 and 25.1 (adjusted for age and education). The score method shows good internal consistency, with Cronbach's a about 0.75, either for total score or for the 3 sub-scores. Both total score and all sub-scores share correlations with mini mental state examination (MMSE), geriatric depression scale (GDS 5-item form), digit-symbol test, phonetic fluency and constructional apraxia tests. Total score and Group A (visuo-perceptive items) correlate also with cancellation attentional matrices, trail making test A and B, Corsi's cubes and Raven's colored matrices. Group B (attention items) and Group C (numerical factors) lack some of these correlations (Pearson correlation coefficients between 0.264 and 0.629). Neither CDIS total score nor sub-scores (except for numerical factors) correlate with verbal learning and memory. CDIS total cores correlates also with the level of education (r=0.418; p=0.001), but not with age. In conclusion, the clock drawing test, scored by the 20-item CDIS, looks as a homogeneous and analytic test, which is focused on visuo-perceptive and executive skills, while it disregards verbal learning and memory, in elderly people with moderate cognitive impairment. Its 3-item groups show good internal consistency; they also plausibly correlate to specific neuropsychological tests. The almost overlapping results of total and visuo-perceptual sub-score come from the way the test is performed and evaluated. The correlation between CDIS and GDS confirms the relevance of mood towards executive functions.

Impaired facial emotion recognition and preserved reactivity to facial expressions in people with severe dementia.

The ability of decoding the emotional facial expressions may be early damaged in frontotemporal dementia, but relatively well preserved in the Alzheimer's disease (AD). Nevertheless, the data about the relationship of the dementia severity with the ability of recognizing the face emotions are conflicting and insufficient, mainly for the moderate-severe stage of the disease. The present study extends to the existing literature by: (1) assessing people in the moderate and severe stage of dementia, compared with people without cognitive impairment; (2) assessing not only recognition but also reactivity to the facial expression of emotion. The capability of understanding the facial emotions has been evaluated in 79 patients with dementia compared to 64 healthy elderly people. The test consisted in showing them 14 photographic representations of 7 emotions both from male and from female faces, representing happiness, sadness, fear, disgust, boredom, anger and surprise. Patients were asked to observe the face and to recognize the emotion either with a denomination or a description. Then the spontaneous reactivity to the face expressions was videotaped and classified as a congruous or incongruous reaction by two independent observers who showed a good inter-rater reliability. Of the patients, 53% with dementia recognized up to 5 emotions out of 14, while in the healthy controls this number of mean recognition raised to 8.4, a value reached by the patients who scored 16 at MMSE. The most identified emotion is happiness both for the patients and for the controls. In general, positive emotions are better recognized than the negative ones, confirming the literary data. About the reactions to face emotion stimuli, there is no significant difference for any of the face emotion between the control group and the people with dementia. These data show that patients with dementia can recognize and react to facial emotions also in the severe stage of the disease, suggesting the usefulness of a non-verbal, emotional communication and supporting the need for more emotional education for care givers, both relatives and professionals.

In vitro antifungal activity of the tea tree (Melaleuca alternifolia) essential oil and its major components against plant pathogens.

AIMS: The aim of this study was to examine the effect of Melaleuca alternifolia essential oil (TTO) and its principal components on four cereal-pathogenic fungi. METHODS AND RESULTS: The antimycotic properties of TTO and of terpinen-4-ol, gamma-terpinen and 1,8-cineole (eucalyptol) were evaluated in vitro on Fusarium graminearum, Fusarium culmorum and Pyrenophora graminea. Moreover, barley leaves infected with Blumeria graminis were treated with whole TTO. All the tested fungi were susceptible to TTO and its components. CONCLUSIONS: TTO exerted a wide spectrum of antimycotic activity. Single TTO purified components were more active than the whole oil in reducing in vitro growth of fungal mycelium and, among the tested compounds, terpinen-4-ol was the most effective. SIGNIFICANCE AND IMPACT OF THE STUDY: TTO and its components can be considered potential alternative natural fungicides.

Quadriceps and hamstring isokinetic strength and electromyographic activity measured at different ranges of motion: a reproducibility study.

Isokinetic strength measurements of the quadriceps and hamstring that are commonly conducted using a 90 degrees range of motion (RoM) may involve some risk to specific knee patient groups. Testing these muscles at a much shorter RoM may reduce the risk but in order to render this method clinically acceptable the reproducibility of the derived test findings has to be established. Therefore the main objective of this study was to assess the reproducibility of isokinetic peak torque and normalized EMG scores of these muscles based on 90 degrees (0-90 degrees flexion, LR) and three successive short RoMs: 0-30 degrees (SR1), 30-60 degrees (SR2) and 60-90 degrees (SR3). Eight healthy subjects were tested three times with a 2 week between-session interval. All tests were performed on the dominant limb and consisted of maximal concentric and eccentric exertions. The velocities applied were 90 degrees /s for LR and 30 degrees /s for each of the SRs. Findings indicated no between-session improvement in strength. Based on the coefficient of variation the measurement error for all isokinetic strength scores remained stable throughout the testing sessions ranging 0.6-13.9% with the absolute majority of instances less than 10%. The reproducibility of the EMG scores was poorer ranging 1.5-25% and 0.5-19% for the quadriceps and hamstring, respectively. It is concluded that testing of knee muscles at short (30 degrees ) RoMs does not compromise the reproducibility of the strength or EMG scores derived from the commonly used RoM of 90 degrees . However, whereas strength was reproducible to within the accepted clinical standards, the corresponding EMG scores were characterized by a wider error band.

Productive infection of HUVEC by HHV-8 is associated with changes compatible with angiogenic transformations.

Kaposi's Sarcoma (KS) is an angioproliferative disease associated with human herpesvirus 8 (HHV-8) infection. We have characterized the morphologic and phenotypic modifications of HUVEC in a model of productive HHV-8 infection. HHV-8 replication was associated with ultra-structural changes, flattened soma and a loss of marginal folds and intercellular contacts, and morphologic features, spindle cell conversion and cordon-like structures formation. Phenotypic changes observed on cordon-like structures included partial loss and redistribution of CD31/PECAM-1 and VE-cadherin, uPAR up-regulation and de novo expression of CD13/APN. Such changes demonstrate the induction, in HUVEC, of an angiogenic profile. Most of these findings are directly linked to HHV-8-encoded proteins expression, suggesting that HHV-8 itself may participate to the initial steps of the angiogenic transformation in KS.

Natural killer-cell cytotoxicity in HIV-positive and HIV-negative patients with and without severe course of hepatitis B virus infection.

Natural killer (NK) cells represent the first line of defence against viral infections but, in the case of hepatitis B virus (HBV), may also be involved in liver injury. We here compared NK-cell activity of 11 patients with acute HBV infection, either HIV-positive or HIV-negative, with that of 11 healthy subjects. One of the HIV-positive patients, characterized by a severe immunodeficiency, died 3 weeks after hospitalization for HBV-related fulminant hepatitis (FH). He displayed a remarkable NK-cell cytotoxicity against both cell lines and autologous dendritic cells, whereas the NK-cell activity of the remaining patients was significantly reduced as compared with healthy individuals. Our findings suggest that NK-cell-mediated cytotoxicity could contribute to the development of HBV-related acute liver failure in HIV-positive patients with severe immunodeficiency. An immunopathological model of FH in immunocompromised patients was proposed.

Testing knee extension and flexion strength at different ranges of motion: an isokinetic and electromyographic study.

The main objective of this study was to explore the mechanical and electrical output of thigh muscles derived from a range of motion (RoM) of 90 degrees (0-90 degrees flexion, LR) and the three successive RoMs: 0-30 degrees (SR1), 30-60 degrees (SR2) and 60-90 degrees (SR3). Thirteen men took part in the study. In view of the torque-velocity relationship and in order to render the test conditions as equivalent as possible, LR was tested at 90 degrees /s while the corresponding velocity for all SRs was 30 degrees /s. The findings indicated very good agreement between LR and SR2 in terms of absolute strength (particularly the concentric), within muscle eccentric to concentric strength ratios, between muscles (agonist to antagonist) strength ratios and the normalized IEMG (expressed in muV/Nm). An agreement was also noted between the mean eccentric peak torque of the knee flexors at LR and SR1 and between the mean eccentric peak torque of the knee extensors at LR and SR3. However, in general there was a lesser agreement between LR and SR1 or SR3. It is suggested that testing thigh muscles in the middle sector of knee motion (SR2) yields strength and EMG data that are close and well correlated with those derived from testing the knee along the commonly used (0-90 degrees ) RoM.

Detection of minimal residual disease by real-time PCR can be used as a surrogate marker to evaluate the graft-versus-myeloma effect after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) is an effective and potentially curative treatment for some cases of multiple myeloma (MM). The curative efficacy of allo-SCT may be largely attributed to its immunological activity, the graft-versus-myeloma (GVM) effect. To evaluate the kinetics of residual myeloma cells, we analyzed the follow-up bone marrow samples of three MM patients by means of a real-time molecular assay. We identified a consistent correlation between onset of graft-versus-host disease and disease response. These data suggest that real-time molecular follow-up can be used to monitor the GVM effect and that it can be employed in the clinical setting to tailor post transplant immunomodulation.

Suppression of CCR5- but not CXCR4-tropic HIV-1 in lymphoid tissue by human herpesvirus 6.

HIV-1 infects target cells via a receptor complex formed by CD4 and a chemokine receptor, primarily CCR5 or CXCR4 (ref. 1). Commonly, HIV-1 transmission is mediated by CCR5-tropic variants, also designated slow/low, non-syncytia-inducer or macrophage-tropic, which dominate the early stages of HIV-1 infection and frequently persist during the entire course of the disease. In contrast, HIV-1 variants that use CXCR4 are typically detected at the later stages, and are associated with a rapid decline in CD4+ T cells and progression to AIDS (refs. 2,7-11). Disease progression is also associated with the emergence of concurrent infections that may affect the course of HIV disease by unknown mechanisms. A lymphotropic agent frequently reactivated in HIV-infected patients is human herpesvirus 6 (HHV-6), which has been proposed as a cofactor in AIDS progression. Here we show that in human lymphoid tissue ex vivo, HHV-6 affects HIV-1 infection in a coreceptor-dependent manner, suppressing CCR5-tropic but not CXCR4-tropic HIV-1 replication, as shown with both uncloned viral isolates and isogenic molecular chimeras. Furthermore, we demonstrate that HHV-6 increases the production of the CCR5 ligand RANTES ('regulated upon activation, normal T-cell expressed and secreted'), the most potent HIV-inhibitory CC chemokine, and that exogenous RANTES mimics the effects of HHV-6 on HIV-1, providing a mechanism for the selective blockade of CCR5-tropic HIV-1. Our data suggest that HHV-6 may profoundly influence the course of HIV-1 infection.

Real-time quantitative PCR for human herpesvirus 6 DNA.

The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5' nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 10(6) viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.

Coinfection of SCID-hu Thy/Liv mice with human herpesvirus 6 and human immunodeficiency virus type 1.

Human herpesvirus 6 (HHV-6) has been proposed as a potential cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) disease. We used the SCID-hu Thy/Liv mouse model to evaluate the in vivo interactions between HHV-6 and HIV-1. Our results demonstrate that HHV-6 and HIV-1 can simultaneously replicate in the human thymus in vivo. In this model, however, the presence of one virus appears not to modify the replication or cytopathicity of the other.

Analysis of serum and plasma beta chemokines in primary HIV infection (PHI).

The levels of certain beta-chemokines in biological fluids do not necessarily reflect their circulating concentrations as they may be dramatically influenced by ex vivo release during sample manipulation. In the present study beta-chemochine levels were evaluated in sequential paired plasma and serum samples collected from a cohort of 18 patients with primary HIV infection (PHI), as well as from 17 HIV-seronegative individuals. In plasma of PHI patients, a significant increase of RANTES (mean 119.1 vs 15.85 ng/ml; p=0.0001) and MIP-1beta (mean 53.4 pg/ml vs 33.6 pg/ml; p=0.0001) was documented. Intra-patient covariance analysis demonstrated no significant association between the variations of RANTES in plasma and serum or between RANTES levels and platelet counts. Reproducibility tests of RANTES measurements in plasma from PHI patients showed a mean coefficient of variation of 1.8%. These data demonstrate that the plasma levels of RANTES and, to a lesser extent, MIP-1beta are persistently perturbed during the early phase of HIV infection. Furthermore they indicate that plasma and serum levels are not directly correlated, being influenced by different physiological phenomena that occur during the ex vivo preparation procedures of the two biological fluids.

Human herpesvirus 6 (HHV-6) causes severe thymocyte depletion in SCID-hu Thy/Liv mice.

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that may act as a cofactor in the progression of AIDS. Here, we describe the first small animal model of HHV-6 infection. HHV-6 subgroup A, strain GS, efficiently infected the human thymic tissue implanted in SCID-hu Thy/Liv mice, leading to the destruction of the graft. Viral DNA was detected in Thy/Liv implants by quantitative polymerase chain reaction (PCR) as early as 4 d after inoculation and peaked at day 14. The productive nature of the infection was confirmed by electron microscopy and immunohistochemical staining. Atypical thymocytes with prominent nuclear inclusions were detected by histopathology. HHV-6 replication was associated with severe, progressive thymocyte depletion involving all major cellular subsets. However, intrathymic T progenitor cells (ITTPs) appeared to be more severely depleted than the other subpopulations, and a preferred tropism of HHV-6 for ITTPs was demonstrated by quantitative PCR on purified thymocyte subsets. These findings suggest that thymocyte depletion by HHV-6 may be due to infection and destruction of these immature T cell precursors. Similar results were obtained with strain PL-1, a primary isolate belonging to subgroup B. The severity of the lesions observed in this animal model underscores the possibility that HHV-6 may indeed be immunosuppressive in humans.

Longitudinal analysis of serum chemokine levels in the course of HIV-1 infection.

OBJECTIVES: To investigate the correlation between the serum levels of the CC-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, and the progression of HIV-1 disease. DESIGN: Retrospective analysis of serial serum samples from HIV-1 seroconverters selected according to clinical outcome. METHODS: Twenty-one patients, derived from a cohort recruited between 1985 and 1996 for a prospective study of the natural history of HIV infection, were analysed. All patients had at least one HIV-1-seronegative sample within 1 year prior to the first seropositive test and were followed longitudinally throughout the course of HIV-1 infection (mean follow-up, 73.5 months). Nine were rapid progressors (RP; patients who developed AIDS within 60 months of antibody seroconversion), seven were slow progressors (SP; patients who developed AIDS after 60 months), and five were long-term asymptomatic (LTA; patients with circulating CD4+ cells higher than 400 x 10(6)/l, no signs of HIV disease, no antiretroviral therapy for more than 96 months). A total of 339 serum samples was studied (mean, 16.1 per patient). The levels of RANTES, MIP-1alpha and MIP-1beta were measured by enzyme-linked immunosorbent assay and correlated with different immunological and clinical parameters. RESULTS: Over the entire follow-up period, the geometric mean of serum RANTES was significantly higher in RP [68.6 ng/ml; 95% confidence interval (CI), 56.9-82.7] than in SP (23.7 ng/ml; 95% CI, 20.0-28.2; P < 0.001) and LTA (19.5 ng/ml; 95% CI, 15.5-24.5; P < 0.001). This difference was already significant during the early clinical stages, when patients had peripheral blood CD4+ cell counts still greater than 400 x 10(6)/l (P < 0.001). By contrast, the mean serum levels of MIP-1alpha and MIP-1beta did not differ significantly between the three study groups. Multivariate analysis using the Cox proportional hazard model demonstrated that the mean serum concentration of RANTES before the development of AIDS was independently associated with the time to AIDS (relative risk, 4.5; 95% CI, 1.1-18.2; P = 0.035). In patients with low versus high mean serum RANTES before the fall of CD4+ cells below 400 x 10(6)/l, the median AIDS-free time was 117.5 and 42.7 months, respectively (P = 0.037). CONCLUSION: These data suggest that an elevation of serum RANTES predicts a rapid progression of the disease since the early stages of HIV-1 infection.

CD46 is a cellular receptor for human herpesvirus 6.

Human herpesvirus 6 (HHV-6) is the etiologic agent of exanthema subitum, causes opportunistic infections in immunocompromised patients, and has been implicated in multiple sclerosis and in the progression of AIDS. Here, we show that the two major HHV-6 subgroups (A and B) use human CD46 as a cellular receptor. Downregulation of surface CD46 was documented during the course of HHV-6 infection. Both acute infection and cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Nonhuman cells that were resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. The use of a ubiquitous immunoregulatory receptor opens novel perspectives for understanding the tropism and pathogenicity of HHV-6.