Diagnostic performances of clinical laboratory tests using Triton X-100 to reduce the biohazard associated with routine testing of Ebola virus-infected patients.

BACKGROUND: Ebola virus, an enveloped virus, is the cause of the largest and most complex Ebola virus disease (EVD) outbreak in West Africa. Blood or body fluids of an infected person may represent a biohazard to laboratory workers. Laboratory tests of virus containing specimens should be conducted in referral centres at biosafety level 4, but based on the severity of clinical symptoms, basic laboratories might be required to execute urgent tests for patients suspected of EVD. The aim of this work was to compare the analytical performances of laboratory tests when Triton X-100, a chemical agent able to inactivate other enveloped viruses, was added to specimens. METHODS: Results of clinical chemistry, coagulation and haematology parameters on samples before and after the addition of 0.1% (final concentration) of Triton X-100 and 1 h of incubation at room temperature were compared. RESULTS: Overall, results showed very good agreement by all statistical analyses. Triton X-100 at 0.1% did not significantly affect the results for the majority of the analytes tested. CONCLUSIONS: Triton X-100 at 0.1% can be used to reduce the biohazard in performing laboratory tests on samples from patients with EVD without affecting clinical decisions.

Spike-in SILAC proteomic approach reveals the vitronectin as an early molecular signature of liver fibrosis in hepatitis C infections with hepatic iron overload.

Hepatitis C virus (HCV)-induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal-restricted histological distribution of pathological iron deposits has hampered the attempt to perform large-scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload-induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike-in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron-associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV-infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV-infected patients without iron overload.

O(2)-dependent efficacy of novel piperidine- and piperazine-based chalcones against the human parasite Giardia intestinalis.

Giardia intestinalis is the most frequent protozoan agent of intestinal diseases worldwide. Though commonly regarded as an anaerobic pathogen, it preferentially colonizes the fairly oxygen-rich mucosa of the proximal small intestine. Therefore, when testing new potential antigiardial drugs, O2 should be taken into account, since it also reduces the efficacy of metronidazole, the gold standard drug against giardiasis. In this study, 46 novel chalcones were synthesized by microwave-assisted Claisen-Schmidt condensation, purified, characterized by high-resolution mass spectrometry, (1)H and (13)C nuclear magnetic resonance, and infrared spectroscopy, and tested for their toxicity against G. intestinalis under standard anaerobic conditions. As a novel approach, compounds showing antigiardial activity under anaerobiosis were also assayed under microaerobic conditions, and their selectivity against parasitic cells was assessed in a counterscreen on human epithelial colorectal adenocarcinoma cells. Among the tested compounds, three [30(a), 31(e), and 33] were more effective in the presence of O2 than under anaerobic conditions and killed the parasite 2 to 4 times more efficiently than metronidazole under anaerobiosis. Two of them [30(a) and 31(e)] proved to be selective against parasitic cells, thus representing potential candidates for the design of novel antigiardial drugs. This study highlights the importance of testing new potential antigiardial agents not only under anaerobic conditions but also at low, more physiological O2 concentrations.

Analysis of single-nucleotide polymorphisms (SNPs) in human CYP3A4 and CYP3A5 genes: potential implications for the metabolism of HIV drugs.

BACKGROUND: Drug metabolism via the cytochrome P450 (CYP450) system has emerged as an important determinant in the occurrence of several drug interactions (adverse drug reactions, reduced pharmacological effect, drug toxicities). In particular, CYP3A4 and CYP3A5 (interacting with more than 60% of licensed drugs) exhibit the most individual variations of gene expression, mostly caused by single nucleotide polymorphisms (SNPs) within the regulatory region of the CYP3A4 and CYP3A5 genes which might affect the level of enzyme production.In this study, we sought to improve the performance of sensitive screening for CYP3A polymorphism detection in twenty HIV-1 infected patients undergoing lopinavir/ritonavir (LPV/r) monotherapy. METHODS: The study was performed by an effective, easy and inexpensive home-made Polymerase Chain Reaction Direct Sequencing approach for analyzing CYP3A4 and CYP3A5 genes which can detect both reported and unreported genetic variants potentially associated with altered or decreased functions of CYP3A4 and CYP3A5 proteins. Proportions and tests of association were used. RESULTS: Among the genetic variants considered, CYP3A4*1B (expression of altered function) was only found in 3 patients (15%) and CYP3A5*3 (expression of splicing defect) in 3 other patients (15%). CYP3A5*3 did not appear to be associated with decreased efficacy of LPV/r in any patient, since none of the patients carrying this variant showed virological rebound during LPV/r treatment or low levels of TDM. In contrast, low-level virological rebound was observed in one patient and a low TDM level was found in another; both were carrying CYP3A4*1B. CONCLUSIONS: Our method exhibited an overall efficiency of 100% (DNA amplification and sequencing in our group of patients). This may contribute to producing innovative results for better understanding the inter-genotypic variability in gene coding for CYP3A, and investigating SNPs as biological markers of individual response to drugs requiring metabolism via the cytochrome P450 system.

Low plasma concentrations of albumin influence the affinity column-mediated immunoassay method for the measurement of tacrolimus in blood during the early period after liver transplantation.

BACKGROUND: Monitoring of tacrolimus (TAC) concentrations in transplanted patients is necessary to ensure effective immunosuppression and to avoid adverse side effects. The fully automated analysis of TAC by the affinity column-mediated immunoassay (ACMIA), which does not require a precipitation step, may represent an efficient alternative to liquid chromatography-tandem mass spectrometry (LC-MS/MS), including in the clinically urgent situation. The aim of this work was to compare the analytical performances of ACMIA with those of LC-MS/MS and to evaluate the influence of hematological parameters, time posttransplant, and type of transplant on the results obtained from routine blood samples. METHODS: Performance characteristics of ACMIA were evaluated using quality control materials and samples spiked with TAC from the International Proficiency Testing Scheme. One hundred and fifty-eight whole-blood samples from patients who received a liver (n = 55) or kidney (n = 14) transplant were assayed by ACMIA and LC-MS/MS, and hematologic, biochemical, and demographic data were collected. Univariate and multivariate statistical analyses were also performed to assess associations between the interassay differences with clinical and laboratory parameters. RESULTS: For artificially spiked samples, the average difference between results obtained by ACMIA and LC-MS/MS was 0.24 ± 0.51 ng/mL (2.91 ± 7.03%). Crosschecking of calibrators and controls by both methods was in accordance with the nominal concentrations of TAC. The lower limit of quantification of ACMIA was found to be 3.0 ng/mL. The results with the 2 methods using routine samples from the transplant recipients correlated well (Spearman's r = 0.90). However, the ACMIA method demonstrated a positive mean bias of 1.78 ng/mL in comparison with LC-MS/MS. Multivariate analysis showed that liver transplant and albumin plasma concentrations significantly and independently affected ACMIA results (P = 0.033 and P = 0.001, respectively). Samples from liver transplant recipients early postsurgery were associated with a larger method bias than those from renal transplant recipients. CONCLUSIONS: Results obtained by ACMIA must be interpreted cautiously, particularly at lower TAC concentrations. Patients with low plasma concentrations of albumin are likely to display higher concentrations of TAC compared with LC-MS/MS in the early postsurgery period.

Ferritin heavy chain is the host factor responsible for HCV-induced inhibition of apoB-100 production and is required for efficient viral infection.

Hepatic fat export occurs by apolipoprotein B-100-containing lipoprotein production, whereas impaired production leads to liver steatosis. Hepatitis C virus (HCV) infection is associated to dysregulation of apoB-100 secretion and steatosis; however, the molecular mechanism by which HCV affects the apoB-100 secretion is not understood. Here, combining quantitative proteomics and computational biology, we propose ferritin heavy chain (Fth) as being the cellular determinant of apoB-100 production inhibition. By means of molecular analyses, we found that HCV nonstructural proteins and NS5A appear to be sufficient for inducing Fth up-regulation. Fth in turn was found to inhibit apoB-100 secretion leading to increased intracellular degradation via proteasome. Notably, intracellular Fth down-regulation by siRNA restores apoB-100 secretion. The inverse correlation between ferritin and plasma apoB-100 concentrations was also found in JFH-1 HCV cell culture systems (HCVcc) and HCV-infected patients. Finally, Fth expression was found to be required for robust HCV infection. These observations provide a further molecular explanation for the onset of liver steatosis and allow for hypothesizing on new therapeutic and antiviral strategies.

Effects of myosin heavy chain (MHC) plasticity induced by HMGCoA-reductase inhibition on skeletal muscle functions.

The rate-limiting step of cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme reductase (HGMR), whose inhibitors, the statins, widely used in clinical practice to treat hypercholesterolemia, often cause myopathy, and rarely rhabdomyolysis. All studies to date are limited to the definition of statin-induced myotoxicity omitting to investigate whether and how HMGR inhibition influences muscle functions. To this end, 3-mo-old male rats (Rattus norvegicus) were treated for 3 wk with a daily intraperitoneal injection of simvastatin (1.5 mg/kg/d), and biochemical, morphological, mechanical, and functional analysis were performed on extensor digitorum longus (EDL) muscle. Our results show that EDL muscles from simvastatin-treated rats exhibited reduced HMGR activity; a 15% shift from the fastest myosin heavy-chain (MHC) isoform IIb to the slower IIa/x; and reduced power output and unloaded shortening velocity, by 41 and 23%, respectively, without any change in isometric force and endurance. Moreover, simvastatin-treated rats showed a decrease of maximum speed reached and the latency to fall off the rotaroad (∼-30%). These results indicate that the molecular mechanism of the impaired muscle function following statin treatment could be related to the plasticity of fast MHC isoform expression.

Hepatitis C virus production requires apolipoprotein A-I and affects its association with nascent low-density lipoproteins.

BACKGROUND/AIMS: The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. METHODS: A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naïve HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. RESULTS: A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. CONCLUSIONS: This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy.

Giardia intestinalis escapes oxidative stress by colonizing the small intestine: A molecular hypothesis.

Giardia intestinalis is the microaerophilic protozoon causing giardiasis, a common infectious intestinal disease. Giardia possesses an O(2) -scavenging activity likely essential for survival in the host. We report that Giardia trophozoites express the O(2) -detoxifying flavodiiron protein (FDP), detected by immunoblotting, and are able to reduce O(2) to H(2) O rapidly (∼3 µM O(2) × min × 10(6) cells at 37 °C) and with high affinity (C(50) = 3.4 ± 0.7 µM O(2)). Following a short-term (minutes) exposure to H(2) O(2) ≥ 100 µM, the O(2) consumption by the parasites is irreversibly impaired, and the FDP undergoes a degradation, prevented by the proteasome-inhibitor MG132. Instead, H(2) O(2) does not cause degradation or inactivation of the isolated FDP. On the basis of the elevated susceptibility of Giardia to oxidative stress, we hypothesize that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress.

Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis.

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN=116+/-10s(-1) at 1microM NO, T=37 degrees C). The activity is [O(2)]-dependent and characterized by an apparent K(M,O2)=22+/-7microM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with 5mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.

Determination of antituberculosis drug concentration in human plasma by MALDI-TOF/TOF.

Therapeutic drug monitoring allows to determine the best dosage regimen adapted to each patient optimizing the therapeutic benefits, while minimizing the risk for side effects. Here, the first methodological approach based on matrix-assisted laser desorption/ionization source equipped with tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry for the determination of the antituberculosis (anti-TB) drugs ethambutol, pyrazinamide, rifampicin, and streptomycin concentration in the plasma of tuberculosis-infected patients is reported. The volume of the plasma sample was 200 microL. Plasma samples were cleaned-up by protein precipitation and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with 200 microL of 50% methanol-0.03% formic acid solution (v/v), spiked with known amounts of anti-TB drugs, mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid solution; v/v), and spotted onto the MALDI-TOF/TOF sample target plate. The anti-TB drug concentration was determined by standard additions analysis. Regression of standard additions was linear over the whole anti-TB drug concentration range explored (the final anti-TB drug concentration ranged from 0.20 to 200 pmol/microL). The absolute recovery of the anti-TB drugs ranged between 87 and 110%. The minimal ethambutol, pyrazinamide, rifampicin, and streptomycin concentration detectable by MALDI-TOF/TOF is 0.08, 0.20, 0.12, and 0.15 pmol/microL, respectively.

Simultaneous determination of maraviroc and raltegravir in human plasma by HPLC-UV.

Therapeutic drug monitoring is pivotal to improve the management of HIV infection. Here, a new HPLC-UV method to quantify simultaneously maraviroc and raltegravir levels in human plasma is reported. Remarkably, this is the first method for maraviroc determination in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (30 mg divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 200 microL 50/50 of mobile-phase solution (0.01 M KH(2)PO(4) and acetonitrile). Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical dC18 Atlantis column (150 mm x 4.6 mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with isocratic elution. The total run time for a single analysis was 10 min; maraviroc and raltegravir were detected by UV at 197 and 300 nm. The calibration curves were linear up to 2,500 ng/mL. The absolute recovery ranged between 93 and 100%. The HPLC-UV method reported here has been validated and is currently applied to monitor plasma levels of maraviroc and raltegravir in HIV-infected patients.

Therapeutic drug monitoring in the management of HIV-infected patients.

The rate of HIV-positive patients that fails to reach or to maintain a durable virological suppression under anti-retroviral (ARV) therapy might be as high as 50%, therefore new tools to improve ARV drug efficacy are urgently needed. Among others, therapeutic drug monitoring (TDM) is a strategy by which the dosing regimen for a patient is guided by measurement of plasma drug levels, enabling physicians to optimize ARV drug efficacy and to avoid drug-related toxicity. The most used analytical methods to determine plasma levels of ARV drugs are HPLC-UV and HPLC-MS(/MS), recently MALDI-based methods and enzyme immunoassay (EIA) technologies have been also employed. The wide inter-patient variability in ARV drug pharmacokinetic supports the application of TDM to the clinical management of HIV-infected patients. Drug-drug and drug-food interactions, drug binding to plasma proteins, drug sequestering by erythrocytes, hepatic impairment, sex, age, pregnancy, and host genetic factors are sources of inter-patient variability affecting ARV drug pharmacokinetics. Combining the information of TDM and resistance tests in genotypic inhibitory quotient (GIQ) is likely to be of great clinical utility. Indeed, only two clinical trials on GIQ, both conducted using ARV drugs not more commonly in use, have shown clinical benefits. The design of new trials with long follow-up and sample size representative of the current HIV prevalence is urgently needed to give indications for GIQ as an early predictor of virological response. Here, the basic principles and the available methods for TDM in the management of HIV-infected patients are reviewed.

Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography.

Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC-UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 microL methanol. Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical C(18) Symmetry column (250 mm x 4.6mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 microg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005-10 microg/mL) in plasma has been studied at 24.0 degrees C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC-UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.

First external quality assurance program of the Italian HLA-B*57:01 Network assessing the performance of clinical virology laboratories in HLA-B*57:01 testing.

BACKGROUND: Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA-B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. OBJECTIVES: The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new methods, develop and maintain external quality assurance (EQA) programs. STUDY DESIGN: A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01-negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. RESULTS: DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicα Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. CONCLUSIONS: The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine.

Dendritic cells derived from BCG-infected precursors induce Th2-like immune response.

Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a- DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli. In addition, BCG-DCs produced proinflammatory cytokines (tumor necrosis factor alpha, IL-1beta, IL-6) and IL-10 but not IL-12. BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells. The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.

Comparison between the traditional and a rapid screening test for cryoimmunoglobulins detection.

OBJECTIVES: A new rapid, automatic, and sensitive screening test useful to detect cryoglobulins in serum samples is proposed. DESIGN AND METHODS: The increase of turbidity during the cryoglobulin aggregation was monitored spectrophotometrically in sera from 400 patients with clinical evidence of cryoglobulinemia related disorders and 100 controls. Results were correlated to those obtained by the traditional method. RESULTS: Kinetics of the aggregation curves were described by their maximum turbidity increase, lag time, and slope. Despite a partial correspondence between the traditional and the rapid test, patients with symptomatic cryoglobulinemia showed turbidity values significantly higher than the determined cutoff. Moreover, a functional classification of cryoglobulins is proposed. CONCLUSIONS: Due to its high reproducibility, operator independence, low cost, and results obtained within 2 hours, the rapid test can be used as a "real time" monitoring of cryoglobulinemia related diseases and for the evaluation of plasmapheresis efficacy.

Expansion of pre-terminally differentiated CD8 T cells in chronic HIV-positive patients presenting a rapid viral rebound during structured treatment interruption.

OBJECTIVE: The influence of structured treatment interruption on effector/memory CD8 T cell dynamics was analysed in chronic HIV-infected patients showing a rapid or delayed viral rebound. DESIGN: Structured treatment interruption consisted of at least one month of discontinuation, followed by highly active antiretroviral therapy (HAART) resumption. Two groups of HIV structured treatment interruption patients were selected on the basis of plasma viral HIV-RNA value (> 30 000 copies/ml, branched DNA): group A (n = 14), patients with a rapid viral rebound (within one month) and group B (n = 6), patients with a delayed or no viral rebound (after a minimum of 4 months). METHODS: A clinical and immunological follow-up was performed at HAART suspension (t 0), one month from suspension (t 1), at HAART resumption (t 2), and 30 days from resumption (t 3). RESULTS: A sustained viral rebound was observed in group A patients, showing a rapid expansion of circulating CD8 T lymphocytes. In this group, the frequencies of CD8 T cells releasing IFN-gamma after mitogen-induced or Gag-specific stimulation were highly increased after HAART discontinuation. Nevertheless, these CD8 T lymphocytes were mainly composed of pre-terminally differentiated cytotoxic T lymphocytes (CTL) expressing a CCR7 CD27 CD45RA phenotype and a reduced amount of perforin. In contrast, group B patients showed no significant changes in immunological parameters during a prolonged drug-free period. CONCLUSION: These data indicate that monitoring CD8 T cell dynamics during structured treatment interruption could be clinically relevant, and new therapeutic strategies should aim qualitatively to restore CTL effector functions.

Short Communication: Yersinia pseudotuberculosis septicemia in an HIV-infected patient failed HAART.

The first case of septicemia due to Yersinia pseudotuberculosis in an HIV-infected person was reported. The 42-year-old woman was severely immunosuppressed despite a prolonged exposure to HAART. Specific amplicons for inv, yadA, and lcrF genes showed the pathogenetic potential of the Y. pseudotuberculosis serotype O1 isolate. A favorable clinical response to ceftriaxone and levofloxacin was observed.

Lymphocyte distribution and intrahepatic compartmentalization during HCV infection: a main role for MHC-unrestricted T cells.

Hepatitis C virus (HCV) infection induces an acute and chronic liver inflammation through an immune-mediated pathway that may lead to cirrhosis and liver failure. Indeed, HCV-related hepatitis is characterized by a dramatic lymphocyte infiltrate into the liver which is mainly composed by HCV non-specific cells. Several data indicated that interferon (IFN)-gamma secretion by intrahepatic lymphocytes (IHL) may drive non-specific cell homing to the liver, inducing interferon inducible protein-10 (IP-10) production. An interesting hallmark of these IHL is the recruitment of lymphocytes associated with mechanisms of innate immunity, such as natural killer (NK), natural killer T (NKT) and gamma delta T lymphocytes. CD81 triggering on NK cell surface by the HCV envelope glycoprotein E2 was recently shown to inhibit NK cell function in the liver of HCV-infected persons, resulting in a possible mechanism contributing to the lack of virus clearance and to the establishment of chronic infection. In contrast, intrahepatic NKT cells restricted to CD1d molecules expressed on the hepatocyte surface may contribute to a large extent to liver damage. Finally, an increased frequency of T cells expressing the gamma delta T cell receptor (TCR) was observed in HCV-infected liver and recent observations indicate that intrahepatic gamma delta T cell activation could be directly induced by the HCV/E2 particle through CD81 triggering. These cells are not HCV specific, are able to kill target cells including primary hepatocytes and their ability to produce T helper (Th)1 cytokines is associated with a higher degree of liver disease. Together, CD1d/NKT and/or E2/CD81 interactions may play a major role in the establishment of HCV immunopathogenesis. In the absence of virus clearance, the chemokine-driven recruitment of lymphocytes with an innate cytotoxic behavior in the liver of HCV-infected patients may boost itself, leading to necroinflammatory and fibrotic liver disease.