dLLME-µSPE extraction coupled to HPLC-ESI-MS/MS for the determination of F2α-IsoPs in human urine.

Oxidative stress is a pathological condition characterized by an imbalance between body's antioxidant defenses and oxidizing agents, resulting in damage of endogenous molecules. These products can be used as markers of oxidative conditions; in particular, isoprostanes (IsoPs) come from the reaction of arachidonic acid with reactive oxygen species (ROS) and are currently defined as gold markers of oxidative stress in urine. Our main goal was the development of a reliable analytical method for the determination and quantification of the IsoPs in human urine by dispersive Liquid-Liquid Micro Extraction (dLLME) coupled with micro Solid Phase Extraction (µSPE) clean-up and HPLC-MS/MS analysis. The selected compounds are present in very small concentration in urine, furthermore, due to relevant matrix effect, they are challenging for ESI-MS/MS analysis. This approach provided selectivity and sensitivity for 8-isoprotaglandine F2α (8-iso-PGF2α), the "gold" OS marker, together with the main isomers. dLLME extraction allowed a significant enrichment factor and µSPE clean-up provided the removal of ion-suppressing compounds from the sample resulting in low matrix effect. The chromatographic separation was also challenging as the target compounds possess very similar chemical characteristics, so experimental conditions were carefully tuned. The reported method represents a useful tool for the detection of IsoPs in urine taking advantage of the combination of dLLME extraction and µSPE clean-up; overall recoveries were above 50 % and matrix effects were ≤15 %, with LOQs ranging between 0.020 and 0.060 ng mL-1. The procedure is easy to use and rapid allowing the removal of interfering compounds and matrix effect maintaining a highly sensitive determination.

Mobile health clinic for the medical management of clinical sequelae experienced by survivors of the 2013-2016 Ebola virus disease outbreak in Sierra Leone, West Africa.

An Ebola survivor Mobile Health Clinic (MHC) was established to implement lasting changes in communities it operates by providing effective and efficient mobile healthcare. After months of development, the MHC solution was operationalised in February 2015, aiming to provide integrated primary healthcare services to address the medical and psychosocial needs of Ebola virus (EBOV) survivors living in areas with low medical coverage. A total of 910 medical consultations for 246 EBOV survivors were performed between 7 February 2015 and 10 June 2016. Females constituted 148 (60.2%) whereas 6 (2.44%) were children under 5 years of age. The most common complication was arthralgia 185 (75.2%), headache 98 (39.8%), abdominal pain 167 (68%), myalgia 182 (73.6%), and skin disease 25 (10%). Moreover, ocular problems were diagnosed in 84 survivors (34.1%), and 60 (24.4%) suffered from psycho-trauma. Some 16 female survivors (10.8%) had miscarriages, whereas 9 (6.1%) had complaints of oligomenorrhea, 7 (4.7%) loss of sexual desire and 4 (2.7%) premature menopause. Five male survivors (5.1%) reported erectile dysfunction and 10 (10.2%) loss of sexual desire. At least 221 (89.8%) reported more than one complication. Other infectious diseases were common and no clinically relevant differences were established from haematology and clinical biochemistry laboratory results. Ibuprofen, paracetamol, anti-malaria drugs and antibiotics were the most common medicines prescribed. Community participation is critical for implantation of MHC among EBOV survivors. Future strategies for the mobile clinics should include enrolment of survivors at discharge from treatment centres with close monitoring follow-up activities, to address sequelae as they arise, to reduce the potential for development of long-term disabilities.

Neonatal protection and preterm birth reduction following maternal group B streptococcus vaccination in a mouse model.

PURPOSE: Evaluate effects of maternal immunization in a mouse model of Group B Streptococcus (GBS) vaginal colonization using clinical isolates. MATERIALS AND METHODS: Female pregnant mice were immunized with heat-killed GBS 21 days before pregnancy and were inoculated intravaginally with GBS cultures (5 × 107 CFU twice a day for three days) from the 16th day of pregnancy. Gestation period and mice survival were monitored. Maternal anti-GBS IgG levels have been determined by ELISA analysis in vaccinated, unvaccinated mothers and newborns. RESULTS: Maternal immunization before pregnancy provided protection to newborns for three of the four GBS strains used. Evaluation of the immunogenicity showed that this vaccination induced higher levels of IgG in vaccinated compared to unvaccinated dams and the presence of antibodies in the offspring at embryonic and postnatal age, and a Th1 response and high levels of IgG2a subclass antibody and IFN-γ were detected. A significant reduction of preterm births was observed in vaccinated mothers (p< 0.05). CONCLUSIONS: Our finding suggest that vaccinated mothers could protect their progeny from GBS infection and preterm birth through passive immunization. The proposed mouse model may represent a noninvasive and effective tool to investigate pathogenetic mechanisms of GBS ascending infection and for vaccine protection studies.

Neutral loss and precursor ion scan tandem mass spectrometry for study of activated benzopyrene-DNA adducts.

Methodology for detection of activated benzo[a]pyrene (B[a]P)-nucleoside adducts by liquid chromatography-tandem mass spectrometry is reported. Adducts of B[a]P-dihydrodiol epoxide (B[a]PDE) with guanosine and adenosine have been detected for the first time by use of precursor ion scan and neutral loss scan. B[a]P was then activated by use of UV irradiation and some of the products obtained have been identified by taking advantage of the information obtained for B[a]PDE. Photoactivation has also been carried out in the presence of hydrogen peroxide; this resulted in a higher yield of products with increased production of BaP diones. The reactivity of these compounds toward nucleosides has been tested. The proposed method was successfully used for detection of one stable guanosine-B[a]P dione adduct.

Innate T-cell immunity in HIV infection: the role of Vgamma9Vdelta2 T lymphocytes.

There is growing interest in the use of innate immune reactions in the therapy and prophylaxis of various diseases. Natural T (NT) lymphocytes that recognize infected cells or microbial compounds without the classical genetic restriction by polymorphic MHC molecules are crucial components of innate immunity. NT cells bearing the Vgamma9Vdelta2 T-cell receptor (TCR) are broadly reactive against intracellular pathogens, can lyse human immunodeficiency virus (HIV) infected cells, and release cytokines capable of regulating HIV replication. The potent antiviral activities of Vgamma9Vdelta2 T cells may help to contain viral spread during acute HIV infection and/or to prevent the establishment of viral persistence. Substantial changes in the composition and function of circulating gammadelta T-cell pools occur in HIV-infected patients. These changes a) may contribute to the etiopathogenesis of opportunistic infections and neoplasms, and b) are partly reversed by highly active anti-retroviral therapy (HAART). In addition to direct antiviral activities, activated gammadelta T cells influence dendritic cell maturation and the adaptive alphabeta T-cell response. Vgamma9Vdelta2 T cells can be stimulated in vivo and in vitro by various nonpeptidic antigens (NpAgs) and recent animal experimental data suggest that activated Vgamma9Vdelta2 T cells may help to control SIV replication. Currently, NpAgs are being assessed as potential therapeutic agents in AIDS, tuberculosis and certain cancers susceptible to Vgamma9Vdelta2 T-cell effector mechanisms.

Antiviral activity and anergy of gammadeltaT lymphocytes in cord blood and immuno-compromised host.

Gammadelta T lymphocytes recognize nonpeptidic microbial antigens without MHC restriction and display both lytic and proliferative responses to human immunodeficiency virus (HIV)-infected cells. This innate recognition involves both T Cell Receptor (TCR) and NK-receptor mediated signalling through non-peptidic metabolites and HLA class I down-regulation. We observed that HLA-masking and nonpeptidic phosphoantigens induce the expression of CD25 and CD69 activation markers on the surface of gammadelta T cells. Interestingly, CD94+ cell depletion by magnetic beads showed that the expression of this antigen is essential for Vdelta2 T cell activation by HLA-masking. Moreover, both phosphoantigen-stimulation and in vitro HIV infection resulted in marked Vgamma9Vdelta2 T cell expansion, whereas HLA-masking was unable to induce proliferative responses. Finally, we observed a relevant hyporesponsiveness to non-peptidic antigens in HIV-infected persons and in cord blood cells from healthy donors when compared to adult PBMC from uninfected donors. Altogether, the reduced ability to naturally recognize the infected cells may contribute to HIV-disease progression and may facilitate maternal transmission of HIV infections.

Discrepancy between polymerase chain reaction assay and Western blot analysis in the assessment of CagA status in dyspeptic patients.

BACKGROUND: Infection with CagA-positive Helicobacter pylori may be diagnosed by detecting cagA gene by polymerase chain reaction assay (PCR) or serum antibodies against CagA by Western blot analysis. The aim of this study is to evaluate whether results of PCR and Western blot analysis are in agreement in CagA status assessment. PATIENTS AND METHODS: Thirty-six dyspeptic patients with unknown H. pylori status underwent upper gastrointestinal endoscopy to assess the presence of mucosal lesions and to collect six gastric biopsies (three from the antrum and three from the body) for evaluation of H. pylori infection (rapid urease test, histology and PCR for ureA gene) and gastritis. CagA status was assessed by PCR (cagA gene) on two biopsy specimens and by Western blot analysis of serum (CagA-antibodies) in each patient. RESULTS: At endoscopy, nine patients showed normal mucosa, 15 a duodenal ulcer and 12 antral erosions. Twenty-eight patients were found to be H. pylori-positive and eight H. pylori-negative. Of the 28 H. pylori-positive patients, 17 were CagA-positive and five were CagA-negative by both methods, five were CagA-positive by Western blot analysis but not by PCR and one was CagA-positive by PCR but not by Western blot analysis. Of the eight H. pylori-negative patients, none was CagA-positive by PCR, while six were CagA-positive by Western blot analysis. Therefore, the two tests agreed in only 24 patients (67%). In those patients in whom the PCR and Western blot analysis were not in agreement, the histological features appear to suggest that the results of the Western blot analysis should be considered false positives or false negatives. CONCLUSIONS: PCR and Western blot analysis failed to provide comparable data in many cases. Western blot analysis seems to be more likely to give misleading results than PCR. Thus, PCR seems to be the method of choice to assess CagA status.

Mycobacterium tuberculosis-induced apoptosis in monocytes/macrophages: early membrane modifications and intracellular mycobacterial viability.

Apoptosis has been observed in monocytes/macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis (MTB) infection. In order to define the early events of MTB-induced apoptosis, membrane CD14 expression and the exposure of Annexin V-binding sites in MTB-infected monocytes/macrophages have been monitored. Moreover, the role of MTB-induced apoptosis was further analyzed in vitro in terms of mycobacterial viability. Results show that monocyte/macrophage apoptosis is a very early event that is strictly dependent on the MTB amount, and this apoptosis is associated with a selective down-regulation of surface CD14 expression. Furthermore, no statistically significant decrease in mycobacterial viability was observed, which indicates that the apoptotic pathway triggered by high doses of MTB is associated with parasite survival rather than with killing of the parasite.

The role of HIV gp120 in the disruption of the immune system.

The existence of HIV positive individuals who do not appear to progress to disease, or do so only very slowly (LTNPs), strongly suggest that factors other than virus pathogenicity determine disease. The occurence of HIV infected chimpanzees that remain disease free and other African SIV infected primates where disease is apparently species specific underscores the importance of host factors [1,2]. We have examined the immune response of LTNP patients using a variety of techniques including intracellular cytokine FACscan, anchor PCR analysis of the T cell receptor and HLA typing of class II genes by DNA sequencing. Our results to date confirm that the development of disease is consistent with activation of a susceptible immune system, and that this could be due to the fact that HLA-like sequences of HIV may 'allo' activate the host immune response. In order to test this hypothesis further we have examined whether gp120 itself can bind and present specific peptides which may be capable of eliciting 'allo' activation responses in particular hosts.

The presence of antibodies against HIV peptides in the sera of alloimmune mice and thalassemic patients is due to a polyclonal activation mechanism.

This paper analyses the HIV-1 gp120 epitope specificity and activation mechanisms (i.e., polyclonal versus oligoclonal) of antibodies present in the sera of alloimmune mice and humans. Sera from CBA mice engrafted with C57BL/6 lymphoid cells significantly reacted against the gp120-derived peptide as 261-270, which shares high homology with the membrane-proximal domain of HLA class II beta-chains (HLA/ gp120) and against the HIV gp120 V3 loop-derived peptides DP32 (HIV-1 MN-derived as 302-334) and C53 (HIV-1 IIIB-derived as 304-318). The same sera also reacted against the HIV-unrelated peptide necdin. Moreover, sera from BALB/c mice injected with LPS presented antibodies reacting against both HIV-related and -unrelated peptides, suggesting that similar mechanisms are shared in alloimmune and LPS-treated mice. A similar analysis was then performed on the sera of patients affected with beta-thalassemia major, receiving at least 10 blood transfusions/year. In particular, 15 of 58 (26%) sera from HIV-uninfected thalassemic patients showed a significantly reactivity against the HLA/gp 120-derived peptides. Moreover, 22 of 58 (38%) sera from the same cohort showed a significant reactivity against DP32 peptide. This reactivity was related to a polyclonal activation mechanism since the DP32-reactive sera significantly bound a panel of HIV-unrelated peptides, as observed by testing 22 sera against necdin, 21 against HSP65 kDa, 21 against amyloid-1, and 17 against MAGE-1 peptides. Moreover, a significant increase of IgG concentration was also observed in all thalassemic sera, when compared to healthy controls, without regard to the anti-gp120 antibody reactivity. Taken together, these results indicate that (i) allogeneic stimuli may induce anti-gp120 antibodies in CBA and in 38% of polytransfused patients and (ii) this reactivity is related to a polyclonal activation mechanism but not to a heightened concentration of IgG.

Infection of human monocytes with Mycobacterium tuberculosis enhances human immunodeficiency virus type 1 replication and transmission to T cells.

Mycobacterium tuberculosis and human immunodeficiency virus type 1 (HIV-1) are virulent intracellular pathogens that invade and multiply within macrophages. The effect of M. tuberculosis on HIV-1 infection and replication was analyzed in vitro using human monocyte-derived macrophages (MDM) isolated from peripheral blood mononuclear cells by countercurrent centrifugal elutriation. Preinfection of MDM with M. tuberculosis followed by HIV-1 infection resulted in an increase in p24 release, reverse transcriptase activity, and infective virus production. In contrast, no increase in HIV-1 production was observed when MDM were infected with Mycobacterium avium complex or heat-killed M. tuberculosis. Coinfected MDM were potent stimulators of T cell proliferation, while HIV-1-infected MDM failed to present exogenous tuberculin to T cells. Furthermore, coinfected MDM showed an increased capacity to transmit HIV-1 to activated T cells. These results suggest that M. tuberculosis infection can both up-regulate HIV-1 infection and replication within MDM and increase the efficiency of virus transmission from infected MDM to T cells.

Epitope specificity of anti-HIV antibodies in human and murine autoimmune diseases.

This article reports the HIV epitope specificity of antibodies present in the sera of HIV-negative patients with autoimmune diseases. Recombinant gp120 and a panel of synthetic peptides derived from the amino acid consensus sequences of either related (gp120, gp41, and p24) or unrelated (Mage-1, necdin, heat shock protein [65 kDa], and amyloid) HIV proteins were tested by a specific ELISA. The first set of experiments performed on four patients with Sjögren's syndrome (SjS) and four patients with systemic lupus erythematosus (SLE) revealed a significant anti-gp120 antibody reactivity in autoimmune patients when compared to healthy HIV-negative controls. Moreover, such binding could be almost completely inhibited by preincubation with free gp120. A significant anti-p24 reactivity was observed in 18 of 29 sera from SjS patients and in 13 of 25 sera from SLE patients, while anti-gp41 was observed only in 3 of 14 SjS and in 2 of 20 SLE-affected patients. Similar analyses were performed in the murine model of autoimmunity, showing that sera from MRL/lpr mice were able to bind all HIV-related peptides in an age-dependent manner. The analysis of a panel of HIV-unrelated peptides showed that SLE as well as MRL/lpr sera bind both HIV-related and unrelated peptides, while SjS sera failed to do so, revealing the polyclonal nature of the SLE and MRL/lpr repertoire and the oligoclonal reactivity of SjS sera. This is also supported by inhibition experiments, which showed that SLE, but not SjS, sera competitively inhibited the binding to HIV gp120 peptide of sera from autoimmune MRL/lpr mice. These results indicate that an overlapping polyclonal repertoire is present in both SLE and MRL/lpr sera, while the oligoclonal specificity of SjS antibodies may be related to a specific, nonpolyclonal, activation against putative retroviral antigens.

Induction of graft versus host disease in SCID mice by MRL/lpr cell transfer.

We have investigated the graft versus host (GvH) disease induced in immunodeficient SCID (H-2d) mice by intravenous (iv) or intraperitoneal (ip) transfer of either spleen or lymph node cells from autoimmune (MRL/lpr and MRL/++ mice, H-2k) and normal (CBA, H-2k) mice. Rapid and lethal GvH disease was observed when cells from MRL/lpr or MRL/++ were iv transferred into SCID mice, while spleen cells from nonautoimmune CBA donors were partially tolerized into SCID recipients and induced only lower levels of GvH reaction. No GvH reaction (complete tolerance) was observed when CBA lymph node cells were iv transferred into SCID recipients. In contrast, the ip injection of MRL/lpr or CBA spleen cells induces similar levels of GvH. The development of GvH disease in SCID recipients was due to the expansion of alloreactive CD8+ cells displaying significant cytotoxic activity against H-2d, but not against autologous targets. Also, a significant decrease of CD4/CD8 ratio was observed in the course of GvH caused by the iv transfer of cells from MRL/lpr mice. Altogether, these data support the hypothesis that lymphocytes from the MRL/lpr mice may escape tolerance in the GvH reaction.

Analysis of the adoptive transfer of delayed hypersensitivity and T cell repertoire in severe combined immunodeficiency mice reconstituted with human lymphocytes.

The functional capacity of human T cells to passively transfer delayed hypersensitivity (DH) was analysed in severe combined immunodeficiency (SCID) mice. The tissue distribution of human peripheral blood lymphocytes (PBL) was analysed by 51chromium labelling 1 and 24 h after intravenous cell injection. Labelled PBL from purified protein derivative (PPD)-positive healthy individuals mainly localize in the spleen, liver and lungs, with no arrival in the peripheral lymphoid organs and at the site of antigen challenge (footpad). According to such defective distribution, human PPD-immune cells failed to passively transfer PPD-specific DH to SCID recipients when cells were injected either intravenously or intraperitoneally (systemic transfer). On the contrary, PPD-immune cells were able to transfer DH to PPD when injected directly into the footpad (local transfer). Both memory (CD45RA-) and naive (CD45RA+) enriched subsets were equally able to transfer local DH. The long-term reconstitution of the human immune system in SCID mice was analysed after intraperitoneal PBL transfer (hu-PBL-Scid) by phenotypic analysis, immunoglobulin level, and human DNA detection. Moreover, the reconstitution of the V beta T cell receptor (TCR) repertoire in SCID mice was analysed by anchored polymerase chain reaction (PCR) showing that all the 22 V beta families were expressed in the spleen of hu-PBL-SCID mice. Moreover, scanner analysis of Southern blotting revealed the selective expansion of distinct V beta families (V beta 3, V beta 6, V beta 8, V beta 13.1, V beta 14, V beta 17), suggesting that human lymphocytes could recognize specific antigens or superantigens in the SCID environment.