A universal UHPLC-CAD platform for the quantification of polysaccharide antigens.

Several glycoconjugate-based vaccines against bacterial infections have been developed and licensed for human use. Polysaccharide (PS) analysis and characterization is therefore critical to profile the composition of polysaccharide-based vaccines. For PS content quantification, the majority of Ultra High Performance Liquid Chromatography (UHPLC) methods rely on the detection of selective monosaccharides constituting the PS repeating unit, therefore requiring chemical cleavage and tailored development: only a few methods directly quantify the intact PS. The introduction of charged aerosol detector (CAD) technology has improved the response of polysaccharide analytes, offering greater sensitivity than other detector sources (e.g., ELSD). Herein, we report the development of a universal UHPLC-CAD method (UniQS) for the quantification and quality evaluation of polysaccharide antigens (e.g., Streptococcus Pneumoniae, Neisseria meningitidis and Staphylococcus aureus). This work laid the foundation for a universal UHPLC-CAD format that could play an important role in future vaccine research and development helping to reduce time, efforts, and costs.

Transglutaminase Type 2 is Involved in the Hematopoietic Stem Cells Homeostasis.

Type 2 transglutaminase (TG2) is a multifunctional protein involved in various biological processes playing a key regulatory role in cell homeostasis such as cell death and autophagy. New evidence is emerging that support an important role of autophagy in regulating normal hematopoiesis. Prompted by these findings, in this study we investigated in vivo involvement of TG2 in mouse hematopoiesis under normal or nutrient deprivation conditions. We found that the number and rate of differentiation of bone marrow hematopoietic stem cell was decreased in the TG2 knockout mice. We present evidence showing that these effects on hematopoietic system are very likely due to the TG2-dependent impairment of autophagy. In fact, stimulation of autophagy by starvation is able to rescue the block of the differentiation of stem cells progenitors in the TG2 KO mice. It was also shown that the RhoA/ERK½ pathway, known to be essential for regulation of the bone marrow progenitor cells homeostasis, was significantly impaired in the absence of TG2. Hence, this study expanded our knowledge about TG2 discovering a role of this enzyme in regulation of hematopoiesis.

N-terminal pro B-Type natriuretic peptide is inversely correlated with low density lipoprotein cholesterol in the very elderly.

BACKGROUND AND AIMS: Laboratory studies on human adipose tissue and differentiated adipocytes indicate that natriuretic peptides (NPs) affect lipid metabolism and plasma cholesterol. Few previous clinical studies in non-elderly populations found associations between NPs in the physiological range and cholesterol. AIM: evaluate the association between NT-proBNP and lipid profile in very elderly hospitalized patients characterized by a wide range of NT-proBNP levels. METHODS AND RESULTS: Cross-sectional study on 288 very elderly patients hospitalized for medical conditions, in which increased NT-proBNP levels are very common. NT-proBNP, total cholesterol (TC), HDL cholesterol (HDLc) and triglycerides were collected just few days before discharge. Patients taking lipid-lowering drugs and patients with an admission diagnosis of acute heart failure were excluded. Calculated LDL-cholesterol (LDLc) was used for the analyses. Mean age: 87.7 ± 6.2 years; female prevalence (57.3%). Median NT-proBNP: 2949 (1005-7335) pg/ml; mean TC: 145.1 ± 40.3 mg/dl; mean HDLc: 38.4 ± 18.6 mg/dl; median triglycerides: 100 (75-129) mg/dl; mean LDLc: 84.0 ± 29.5 mg/dl. We found negative correlations between NT-proBNP and both TC and LDLc (Rho = -0.157; p = 0.008 and Rho = -0.166; p = 0.005, respectively), while no correlations emerged between NT-proBNP and HDLc (Rho = -0.065; p = 0.275) or triglycerides (Rho = -0.009; p = 0.874). These associations were confirmed considering NT-proBNP tertiles. The inverse association between NT-proBNP and LDLc was maintained even after adjusting for confounding factors. CONCLUSION: Our real-life clinical study supports the hypothesis that NPs play a role on cholesterol metabolism, given the association found between LDLc and NT-proBNP even in very elderly patients where NT-proBNP values are often in the pathological range.

Intrahepatic Vγ9Vδ2 T-cells from HCV-infected patients show an exhausted phenotype but can inhibit HCV replication.

Hepatitis C virus (HCV) persistence results from inefficiencies of both innate and adaptive immune responses to eradicate the infection. A functional impairment of circulating Vγ9Vδ2 T-cells was described but few data are available on Vγ9Vδ2 T-cells in the liver that, however, represents the battlefield in the HCV/host interaction. Aim of this work was to compare circulating and intrahepatic Vγ9Vδ2 T-cells in chronic HCV-infected patients (HCVpos) and in HCV-negative (HCVneg) subjects. Phenotypic and functional analysis was performed by flow cytometry. Anti-HCV activity was analyzed by using an in vitro autologous liver culture system. Independently from HCV infection, the liver was enriched of Vγ9Vδ2 T-cells expressing an effector/activated phenotype. In contrast, an enrichment of PD-1 expressing Vγ9Vδ2 T-cells was observed both in the peripheral blood and in the liver of HCVpos patients, probably due to a persistent antigenic stimulation. Moreover, a lower frequency of IFN-γ producing Vγ9Vδ2 T-cells was observed in the liver of HCVpos patients, suggesting a functional impairment in the cytokine production in HCVpos liver. Despite this hypo-responsiveness, intrahepatic Vγ9Vδ2 T-cells are able to exert an anti-HCV activity after specific stimulation. Altogether, our data show that HCV infection induced a dysregulation of intrahepatic Vγ9Vδ2 T cells that maintain their anti-HCV activity after specific stimulation. A study aimed to evaluate the mechanisms of the antiviral activity may be useful to identify new pathways able to improve Vγ9Vδ2 T-cells intrahepatic function during HCV infection.

Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection.

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

Bone marrow CD34+ progenitor cells may harbour HIV-DNA even in successfully treated patients.

The issue about bone marrow hematopoietic progenitor cells harbouring HIV-DNA in infected patients is still under scrutiny. We studied nine HIV-infected individuals undergoing bone marrow aspiration for diagnostic purposes. In all patients, even in those receiving successful antiretroviral therapy for several years, HIV-DNA was detected in purified CD34+ lineage-bone marrow progenitor cells. This finding, although not conclusive due to the low number of patients examined, adds further evidence that current treatment strategies may be insufficient to resolve latent infection in bone marrow CD34+ hematopoietic progenitor cells.

Modulation of polyfunctional HIV-specific CD8 T cells in patients responding differently to antiretroviral therapy.

Antiretroviral therapy allows a restoration of immune cell homeostasis associated with a normal immune competence. Our goal was to analyze the modulation of polyfunctional HIV-specific CD8+ T-cell responses during antiretroviral therapy. HIV-infected individuals were divided into four groups according to CD4+ cell count and viral load at the moment of recruitment. Whole blood was stimulated with a pool of CD8-specific HIV-antigens to assess cytokine/chemokine production and cytotoxicity activity by using flow cytometry. The groups show different modulation in HIV-specific CD8+ T-cell responses. In particular, immunological failure showed different distributions of polyfunctional HIVspecific CD8+ responses, mainly due to an increase of cells producing CD107alpha/IFNgamma/IL-2/MIP-1beta. Our results indicate that this particular 4+ functional subset is a possible correlate of immunological failure. Considering the complexity of interactions among HAART, immune system and HIV, work is in progress to find correlates of therapy efficacy.

HIV impairs CD34+-derived monocytic precursor differentiation into functional dendritic cells.

Dendritic cells (DCs) perform a basic role in the immune system by allowing the initiation of the primary T-cell-dependent immune response. Given previous indirect evidence that DC maturation and function are impaired by HIV, we have developed an in vitro culture system in order to verify the effect of HIV infection on DC function during the development from hematopoietic progenitors. Considering that monocytic (Mo) differentiating cells efficiently replicate monocytotropic HIV, we examined whether HIV-infected monocytic precursors (MoP) were able to generate functional DCs. CD34+ hematopoietic progenitor cells (HPCs) were induced along Mo differentiative pathway in liquid cultures and at an early stage of culture, MoP were infected with M-tropic BaL HIV strain, and after 2 days they were switched to DC differentiation with GM-CSF and IL-4. Derived DCs were actively infected, as detected by HIV-p24 production. HIV did not significantly affect cell viability, but induced a reduction in cell proliferation and an inefficient functional activity in terms of uptake capability and stimulation of allogenic T cells. These results indicate that HIV-infected MoP lost the capacity to generate functional DCs, and this may represent one of the many mechanisms of immunosuppression exploited by HIV.

A novel 8-color flow cytometry panel to study activation, maturation and senescence of CD4 and CD8 T lymphocytes in HIV-infected individuals at different stages of disease.

Multicolor flow cytometry allows to study the markers differentially expressed during maturation, activation, function and senescence on immune cells. Despite the availability of reagents and technology, scarce agreement has been gained regarding phenotypic markers of HIV disease progression other than CD4 T-cell count. In this work, we present a novel high-throughput global analysis of CD4 and CD8 T-lymphocyte profiles by standardized 8-color combinations of antibodies aimed at analyzing HIV disease course progression. For this purpose, two tubes with lyophilized reagent cocktails (CD4- and CD8-specific tubes) were designed to compare the immunological characteristics of HIV-infected persons (37 "high CD4" HAART-treated and 32 "low CD4" naïve or failed-treatment patients) with healthy donors (HD). In particular, T-cell activation (CD25, CD38, CD69), differentiation (CD45RA, CCR7), apoptosis (CD95) and immune suppression profiles (CD25(high)CD127-) in HIV+ patients were compared with HD. Statistical analysis was performed by identifying the parameters associated with disease progression, namely markers that were found to be significantly different between groups with high CD4 counts (including HD) and low CD4 counts (restricted to HIV patients) but not between the HD and the "high CD4" group. This set of markers, including those identifying different maturation and senescence subtypes of CD4 and CD8 T cells, was found to be associated with therapy failure, and it is in fact evaluated in an ongoing study aimed to verify its prognostic value. This robust assay was found feasible on a semi-routine scale for HIV-infected persons, and allows for broader clinical studies aimed at defining markers associated with treatment outcome, possibly having a high impact on the clinical management of HIV disease.

Isolation and characterization of a murine resident liver stem cell.

Increasing evidence provides support that mammalian liver contains stem/progenitor cells, but their molecular phenotype, embryological derivation, biology and their role in liver cell turnover and regeneration remain to be further clarified. In this study, we report the isolation, characterization and reproducible establishment in line of a resident liver stem cell (RLSC) with immunophenotype and differentiative potentiality distinct from other previously described liver precursor/stem cells. RLSCs, derived from fetal and neonatal murine livers as well as from immortalized hepatocytic MMH lines and established in lines, are Sca+, CD34-, CD45-, alpha-fetoprotein+ and albumin-. This molecular phenotype suggests a non-hematopoietic origin. RLSC transcriptional profile, defined by microArray technology, highlighted the expression of a broad spectrum of 'plasticity-related genes' and 'developmental genes' suggesting a multi-differentiative potentiality. Indeed, RLSCs spontaneously differentiate into hepatocytes and cholangiocytes and, when cultured in appropriate conditions, into mesenchymal and neuro-ectodermal cell lineages such as osteoblasts/osteocytes, chondrocytes, astrocytes and neural cells. RLSC capability to spontaneously differentiate into hepatocytes, the lack of albumin expression and the broad differentiative potentiality locate them in a pre-hepatoblast/liver precursor cells hierarchical position. In conclusion, RLSCs may provide a useful tool to improve liver stem cell knowledge and to assess new therapeutic approaches for liver diseases.

Effect of cyclosporine, mycophenolate mofetil, and their combination with steroids on apoptosis in a human cultured monocytic U937 cell line.

Transplant patient plasma produces an increased rate of mononuclear cell apoptosis despite a normal serum creatinine value. Immunosuppressive medications may be one factor that causes an altered apoptotic pattern. We evaluated the in vitro effects of various doses of cyclosporine, mycophenolate mofetil, and steroids on apoptosis of a cultured human monocytic U937 cell line, using estimates by fluorescence microscopy and annexin V assays. Increasing cyclosporine concentrations (100 to 800 ng/mL) progressively increased apoptosis rates (16% to 32%). The combination of steroid (0.01 microg/mL) and cyclosporine increased the apoptosis rate to 45%. Mycophenolate mofetil alone (0.3 microg/mL) led to an apoptosis rate of 34%. Therapeutic levels of mycophenolate mofetil from 3 to 7 microg/mL led to apoptosis rates from 56% to 67%. The combination of cyclosporine, steroid, and mycophenolate mofetil increased the rate of apoptosis to 95%. Immunosuppressive therapy may contribute to the high rate of apoptosis observed among mononuclear cells of transplanted patients. This effect may alter patient susceptibility to infections and contribute to a unique mechanism of immunosuppression.

[Rationale for the use of extracorporeal treatments for sepsis].

Sepsis is the leading cause of disability and mortality among critical patients; moreover, it causes high economic expenditures. Although very much is known about the pathophysiology of this condition and its mediators despite great investments directed to its control, mortality rates remain high. Recent treatment manuals emphasize the value of early goal-oriented therapy and also point to the high efficacy of activated protein C. Extracorporeal blood clearance may potentially become a new approach to treating this condition. There are reports on its positive clinical results that are likely associated with the effective removal of septic mediators. Human and animal studies, few and rather alike as they are, have yielded promising results. It is evident that the use of these procedures is justified; however, their efficiency in sepsis requires large-scale, correctly conducted studies.

Pulse high volume hemofiltration.

The sepsis syndrome is the most common cause of acute renal failure and multiple organ dysfunction in critically ill subjects and continues to have an alarmingly high mortality. Normal immune homeostasis is interrupted by a complex storm of inflammatory mediators responsible for the deleterious effects. Extracorporeal blood purification techniques can confer benefits in sepsis by proven non-specific removal of these mediators (pro- or anti-inflammatory), and provide a logical and adequate approach to treat this syndrome. High volume hemofiltration (HVHF) has had the most dramatic effect conferring benefits in hemodynamics, reduction in vasopressor doses and improvement in survival. "Pulse HVHF" is the latest approach which may offer the most efficient results: a daily schedule of 6-8 hours followed by standard CVVH. This paper describes the rationale and potential of this technique. Reliability and tolerance of this technique and biological effects are described.

Extracorporeal treatments in sepsis: are there new perspectives?

Sepsis continues to provide a major challenge to clinicians. Despite vast advancements achieved in the understanding of its pathways and mechanisms, the incidence of sepsis is increasing and the mortality and morbidity rates remain high, generating a considerable burden to health budgets worldwide. Unfortunately, no definitive therapy yet exists that can successfully treat sepsis and its complications. At variance with targeting single mediators, therapeutic intervention aimed at the non-selective removal of pro- and anti-inflammatory mediators seems a rational concept and a possible key to successful extra-corporeal therapies. A further advantage may lie in the continuous nature of such therapy. With such continuous therapy, sequentially appearing peaks of systemic mediator overflow may be attenuated and persistently high plasma levels reduced. This theoretical framework is proposed as the underlying biological rationale for a series of innovative modalities in sepsis. In this editorial, we will review recent animal and human trials which lend support to this concept. We will also review the importance of treatment dose during continuous renal replacement therapy as a major factor affecting survival in critically ill patients with acute renal failure. We will also review novel information related to other blood purification techniques using largo pore membranes or plasma filtration with adsorbent perfusion. Although these approaches are still in the early stages of clinical testing, they are conceptually promising and might represent an important advance.

Caspase-3 and -8 activation and cytokine removal with a novel cellulose triacetate super-permeable membrane in an in vitro sepsis model.

Pro-apoptotic molecules are generated during sepsis which may be responsible for alteration of organ function in sepsis. Removal of systemic apoptotic activity may affect recovery from sepsis. Current high flux membranes might not be sufficiently permeable to eliminate pro-apoptotic factors. We evaluated the elimination of pro-apoptotic factors induced by LPS in human whole blood by a super-permeable cellulose triacetate membrane (SUREFLUX FH 150, Nipro, Osaka, Japan) in comparison to a standard high flux cellulose triacetate membrane (UT 700, Nipro, Osaka, Japan) and a polyethersulfone plasmafilter (Bellco, Mirandola Italy) in an in vitro blood circulation. We spiked human whole blood with lipopolysaccharide from Escherichia coli (Serotype 026-86, 10 mg/ml), incubated it for 3 hours to allow cytokine generation and recirculated it at 300 ml/min for 3 hours. The UF line was first returned to the blood module at 10 min. After this, the UF was drained from 10 to 60 min at a rate of 1000 ml/h. Zero balance was obtained by re-infusion of bicarbonate buffered hemofiltration fluid. Apoptosis was assessed on U937 monocytes (incubated with plasma or ultrafiltrate) by fluorescence microscopy dyes (Hoechst 33342, propidium iodide) and annexin V flow cytometry. Caspase-3 and Caspase-8 activity was assessed on the recirculated blood monocytes by spectrophotometric methods. IL-2, IL-10 and TNFalpha were determined by commercially available ELISAs. Sieving coefficients and clearances were determined for the different cytokines. Caspase-3 and Caspase-8 were activated by LPS and remained either stable or increased during in vitro circulation. Apoptosis activity of U937 cells, when incubated with the ultrafiltrate, increased in parallel with arterial plasma values (for Uf: UT700 = 23.1%; Sureflux FH150 = 42.5%). However, by 60 min the apoptotic activity recorded with the ultrafiltrate was reduced to the levels of arterial plasma (for Uf: UT700 = 19.8%; Sureflux FH150 = 11.2%). Sieving coefficients in the super-permeable membrane were significantly higher for all measured cytokines in comparison to the standard high flux membrane (e.g. TNFalpha 0.72 vs 0.03 p < 0.001) and close to the values observed for the plasmafiltration membrane. Nevertheless protein losses measured by albumin leakage were much lower with the Sureflux filter in comparison to the plasmafilter. In conclusion, pro-apoptotic factors can be eliminated by dialytic membranes with the removal rate maximized by using super high flux dialysers which may represent a compromise between hemofiltration and plasmafiltration membranes.

Iron management in hemodialysis patients: optimizing outcomes in Vicenza, Italy.

The management of anemia in uremic patients undergoing hemodialysis requires the appropriate combination of erythropoietin treatment, iron supplementation, and on occasion androgen therapy. Identifying and correcting functional iron deficiency is crucial to optimizing erythropoietin efficiency. Recently, however, the trend to administer maintenance iron with resultant high serum ferritin and high transferrin saturation has led to an increase in reports of iron overload. Oral iron supplementation is inexpensive and safe, but poor patient compliance and reduced intestinal absorption may limit its efficacy. Intravenous iron, on the other hand, is effective, and its safety is related to the iron salt used. Currently available data suggest that iron saccharate may be the safest iron salt available for intravenous administration, although iron gluconate is safer than the dextran forms of intravenous iron. It should be kept in mind, however, that all forms of intravenous iron may have the potential of inducing iron overload. At this time, the levels of ferritin that define iron overload are not clearly established. The side effects of iron overload are well recognized (infections, malignancies, vascular diseases); however, no guidelines exist for safe practice. There are many markers of iron deficiency, with serum ferritin and hypochromic red cell percentage currently the best markers available in clinical practice.

Fibroblast growth factor receptor mutational screening in newborns affected by metopic synostosis.

A number of craniosynostotic disorders have recently been ascribed to mutations in genes coding for the fibroblast growth factor receptors(FGFRs). The common feature of these FGFR-associated conditions is the unilateral or bilateral premature ossification of the coronal suture. One distinct craniosynostotic condition is trigonocephaly, which results from the premature fusion of the metopic suture. Trigonocephaly mostly occurs as isolated cranial defect; however, the premature closure of the metopic suture may represent a feature of more complex craniosynostotic conditions in which a progressive involvement of other cranial sutures with age is observed. The possible involvement of mutated FGFRs in trigonocephaly was investigated in nine newborns affected by isolated premature synostosis of the metopic suture. All except one of these cases carried no mutations in the FGFR1-3 domains indicated as hot spots for craniosynostosis-associated mutations. A T(978)C transition in the FGFR2 exon IIIa was found in a patient who had a phenotype that apparently fitted the trigonocephalic condition at birth, but showed additional facial anomalies, which worsened progressively with age towards a Crouzon-like profile. The present finding points out the importance, from both diagnostic and prognostic points of view, of early FGFR mutational screening in craniosynostotic conditions, even in forms that apparently do not involve closure of the coronal suture at birth.