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In an attempt to study size and shape variations induced in red cells by fixation of relatively large amounts of antibody protein in the absence of haemagglutination, microscopic analysis has been performed on human O red cells incubated with rabbit univalent anti-human red cell antibodies. These were obtained by recombining half-molecules of anti-red cell γG-immunoglobulin with half-molecules of non-specific γG-immunoglobulin. Changes in cell morphology accounted essentially for a diameter increase during incubation at 37° in the presence of the recombined fraction. These changes were a progressive variation towards the sphere and then the disk shape. The amount of univalent material effective in determining such variation was estimated to be near to 45,000 molecules/cell, i.e. about twice the amount of bivalent molecules per cell giving 50 per cent agglutination. The possibility that the mechanism of action of either the bivalent γG-immunoglobulin antibody or the recombined univalent molecule might be the same in respect to cytotoxicity is discussed. [ABSTRACT FROM AUTHOR]

Background: We previously showed that the 10-year cardiovascular disease (CVD) risk threshold to initiate statins for primary prevention depends on the baseline CVD risk, age, sex, and the incidence of statin-related harm outcome and competing risk for non-CVD death. As these factors appear to vary across countries, we aimed in this study to determine country-specific thresholds and provide guidelines a quantitative benefit-harm assessment method for local adaptation., Methods: For each of the 186 countries included, we replicated the benefit-harm balance analysis using an exponential model to determine the thresholds to initiate statin use for populations aged 40 to 75 years, with no history of CVD. The analyses took data inputs from a priori studies, including statin effect estimates (network meta-analysis), patient preferences (survey), and baseline incidence of harm outcomes and competing risk for non-CVD (global burden of disease study). We estimated the risk thresholds above which the benefits of statins were more likely to outweigh the harms using a stochastic approach to account for statistical uncertainty of the input parameters., Results: The 5 th and 95 th percentiles of the 10-year risk thresholds above which the benefits of statins outweigh the harms across 186 countries ranged between 14 and 20% in men and 19-24% in women, depending on age (i.e., 90% of the country-specific thresholds were in the ranges stated). The median risk thresholds varied from 14 to 18.5% in men and 19 to 22% in women. The between-country variability of the thresholds was slightly attenuated when further adjusted for age resulting, for example, in a 5 th and 95 th percentiles of 14-16% for ages 40-44 years and 17-21% for ages 70-74 years in men. Some countries, especially the islands of the Western Pacific Region, had higher thresholds to achieve net benefit of statins at 25-36% 10-year CVD risks., Conclusions: This extensive benefit-harm analysis modeling shows that a single CVD risk threshold, irrespective of age, sex and country, is not appropriate to initiate statin use globally. Instead, countries need to carefully determine thresholds, considering the national or subnational contexts, to optimize benefits of statins while minimizing related harms and economic burden.

Learning in biologically relevant neural-network models usually relies on Hebb learning rules. The typical implementations of these rules change the synaptic strength on the basis of the co-occurrence of the neural events taking place at a certain time in the pre- and post-synaptic neurons. Differential Hebbian learning (DHL) rules, instead, are able to update the synapse by taking into account the temporal relation, captured with derivatives, between the neural events happening in the recent past. The few DHL rules proposed so far can update the synaptic weights only in few ways: this is a limitation for the study of dynamical neurons and neural-network models. Moreover, empirical evidence on brain spike-timing-dependent plasticity (STDP) shows that different neurons express a surprisingly rich repertoire of different learning processes going far beyond existing DHL rules. This opens up a second problem of how capturing such processes with DHL rules. Here we propose a general DHL (G-DHL) rule generating the existing rules and many others. The rule has a high expressiveness as it combines in different ways the pre- and post-synaptic neuron signals and derivatives. The rule flexibility is shown by applying it to various signals of artificial neurons and by fitting several different STDP experimental data sets. To these purposes, we propose techniques to pre-process the neural signals and capture the temporal relations between the neural events of interest. We also propose a procedure to automatically identify the rule components and parameters that best fit different STDP data sets, and show how the identified components might be used to heuristically guide the search of the biophysical mechanisms underlying STDP. Overall, the results show that the G-DHL rule represents a useful means to study time-sensitive learning processes in both artificial neural networks and brain., Competing Interests: The authors have declared that no competing interests exist.

Recent evidence has indicated that leptin, an adipocyte-secreted hormone belonging to the helical cytokine family, significantly influences immune and autoimmune responses. We investigate here the mechanisms by which in vivo abrogation of leptin effects protects SJL/J mice from proteolipid protein peptide PLP(139-151)-induced EAE, an animal model of MS. Blockade of leptin with anti-leptin Abs or with a soluble mouse leptin receptor chimera (ObR:Fc), either before or after onset of EAE, improved clinical score, slowed disease progression, reduced disease relapses, inhibited PLP(139-151)-specific T cell proliferation, and switched cytokine secretion toward a Th2/regulatory profile. This was also confirmed by induction of forkhead box p3 (Foxp3) expression in CD4 T cells in leptin-neutralized mice. Importantly, anti-leptin treatment induced a failure to downmodulate the cyclin-dependent kinase inhibitor p27 (p27) in autoreactive CD4 T cells. These effects were associated with increased tyrosine phosphorylation of both ERK1/2 and STAT6. Taken together, our data provide what we believe is a new molecular basis for leptin antagonism in EAE and envision novel strategies of leptin-based molecular targeting in the disease.

We analyzed the serum and cerebrospinal fluid (CSF) leptin secretion and the interaction between serum leptin and CD4(+)CD25+ regulatory T cells (T(Regs)) in naive-to-therapy relapsing-remitting multiple sclerosis (RRMS) patients. Leptin production was significantly increased in both serum and CSF of RRMS patients and correlated with IFN-gamma secretion in the CSF. T cell lines against human myelin basic protein (hMBP) produced immunoreactive leptin and up-regulated the expression of the leptin receptor (ObR) after activation with hMBP. Treatment with either anti-leptin or anti-leptin-receptor neutralizing antibodies inhibited in vitro proliferation in response to hMBP. Interestingly, in the RRMS patients, an inverse correlation between serum leptin and percentage of circulating T(Regs) was also observed. To better analyze the finding, we enumerated T(Regs) in leptin-deficient (ob/ob) and leptin-receptor-deficient (db/db) mice and observed the significant increase in T(Regs). Moreover, treatment of WT mice with soluble ObR fusion protein (ObR:Fc) increased the percentage of T(Regs) and ameliorated the clinical course and progression of disease in proteolipid protein peptide (PLP(139-151))-induced relapsing-experimental autoimmune encephalomyelitis (R-EAE), an animal model of RRMS. These findings show an inverse relationship between leptin secretion and the frequency of T(Regs) in RRMS and may have implications for the pathogenesis of and therapy for multiple sclerosis.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal, acquired hematopoietic disorder characterized by a phosphatidylinositol (PI) glycan-A gene mutation, which impairs the synthesis of the glycosyl-PI (GPI) anchor, thus causing the absence of all GPI-linked proteins on the membrane of the clonal-defective cells. The presence of a consistent GPI-defective monocyte compartment is a common feature in PNH patients. To investigate the functional behavior of this population, we analyzed its in vitro differentiation ability toward functional dendritic cells (DCs). Our data indicate that GPI-defective monocytes from PNH patients are unable to undergo full DC differentiation in vitro after granulocyte macrophage-colony stimulating factor and recombinant interleukin (IL)-4 treatment. In this context, the GPI-defective DC population shows mannose receptor expression, high levels of the CD86 molecule, and impaired CD1a up-regulation. The analysis of lipopolysaccharide and CD40-dependent, functional pathways in these DCs revealed a strong decrease in tumor necrosis factor alpha and IL-12 production. Finally, GPI-defective DCs showed a severe impairment in delivering accessory signals for T cell receptor-dependent T cell proliferation.

Monocytes are circulating precursors of the dendritic cell subset, professional antigen-presenting cells with a unique ability to initiate the innate and adaptive immune response. In this study, we have investigated the effects of wild-type Helicobacter pylori strains and their isogenic mutants with mutations in known bacterial virulence factors on monocytes and monocyte-derived dendritic cells. We show that H. pylori strains induce apoptosis of human monocytes by a mechanism that is dependent on the expression of a functional cag pathogenicity island. This effect requires an intact injection organelle for direct contact between monocytes and the bacteria but also requires a still-unidentified effector that is different from VacA or CagA. The exposure of in vitro-generated monocyte-derived dendritic cells to H. pylori stimulates the release of inflammatory cytokines by a similar mechanism. Of note is that dendritic cells are resistant to H. pylori-induced apoptosis. These phenomena may play a critical role in the evasion of the immune response by H. pylori, contributing to the persistence of the infection.

Immature dendritic cells (iDCs) can be instructed to polarize the immune response toward a noninflammatory pathway by mediators that increase the intracellular concentration of cAMP. This phenomenon is associated with the ability of the cyclic nucleoside to inhibit the release of pro-inflammatory cytokines without affecting the differentiation process of the dendritic cells (DCs). Here we investigated the ability of cAMP to modulate the endotoxin signaling by exposing DCs to exogenous 8-bromium-cyclic AMP in the presence or absence of H89, a selective inhibitor of the protein kinase A, one of the major molecular targets of the cyclic nucleoside. cAMP affects the early lipopolysaccharide-induced signaling cascade dissociating the activation of NF-kappa B, p38, and ERK pathways from the stimulation of c-Src and Lyn kinases. This phenomenon was prevented by H89. The pharmacological block of Src-like tyrosine kinases induces comparable results confirming the involvement of this family of enzymes in the mechanism controlling the release of cytokines in human monocyte-derived iDCs. We propose that the cAMP-protein kinase A-dependent pathway regulates the functional plasticity of iDCs by gating the Toll-like receptor signaling at the level of Src-like kinases.

This study addresses the analysis of the human leucocyte antigen (HLA) allele distribution in 54 historical and in 68 recently diagnosed tuberculosis (TB) patients. The historical cohort was characterized by the presence of large fibrocavernous lesions effectively treated with therapeutic pneumothorax during the period 1950-55. Patients and healthy controls enrolled in the study were from the Campania region of southern Italy. No significant association between HLA alleles and TB in the population of recently diagnosed TB patients was observed. On the contrary, among the historical TB patients there was a strong association with an increased frequency of the HLA-DR4 allele alone and/or in the presence of the HLA-B14 allele (P = 0.000004; Pc = 0.0008), as well as with a decreased frequency of the HLA-A2+,-B14-,DR4- allele association (P = 0.00005; Pc = 0.01). In order to exclude any interference from age-related factors, these results were confirmed by comparing the historical cohort of TB patients with an age-matched healthy control population of the same ethnic origin (P = 0.00004; Pc = 0.008; and P = 0.0001; and Pc = 0.02, respectively).

The ability of the envelope glycoprotein gp120 [human immunodeficiency virus (HIV) env] to induce intracellular signals is thought to contribute to HIV-1 pathogenesis. In the present study, we found that the exposure of CD4+ CD45RA+ naive T cells to HIVenv results in a long-lasting hyporesponsiveness to antigen stimulation. This phenomenon is not dependent on CD4-mediated signals and also can be generated by the exposure of naive T cell to soluble CD4-HIVenv complexes. The analysis of the proximal signaling reveals that HIVenv does not activate Lck as well as the mitogen-activated protein kinase intermediate cascade. Conversely, the envelope glycoprotein stimulates the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity and induces the progressive accumulation of the phosphorylated form of the cAMP-responsive element binding. Of note, the ligation of CXCR4 by stromal cell-derived factor-1alpha but not the engagement of CD4 by monoclonal antibody stimulates the PKA activity and induces a long-lasting hyporesponsivity state in naive CD4+ lymphocytes. The pretreatment of lymphocytes with H89, a cell-permeable PKA inhibitor, prevents the induction of anergy. These findings reveal a novel mechanism by which HIVenv may modulate the processes of clonal expansion, homeostatic proliferation, and terminal differentiation of the naive T lymphocyte subset.

In the work presented here, we explored the influence of leptin on the kinetics of experimental autoimmune encephalomyelitis (EAE) onset, in the EAE-associated inflammatory anorexia, and in the development of pathogenic T cell responses. We found that the expression of serum leptin increased before the clinical onset of EAE in disease-susceptible C57BL/6J (H-2(b)) and SJL/J (H-2(s)) strains of mice, which are models of chronic-progressive and relapsing-remitting EAE, respectively. This increase in serum leptin correlated with disease susceptibility, reduction in food intake, and decrease in body weight. Indeed, acute starvation, which is able to prevent the increase in serum leptin, delayed disease onset and attenuated clinical symptoms by inducing a T helper 2 cytokine switch. Furthermore, immunohistochemical analysis revealed a parallel in situ production of leptin in inflammatory infiltrates and in neurons only during the acute/active phase of both chronic-progressive and relapsing-remitting EAE. We also found that leptin secretion by activated T cells sustained their proliferation in an autocrine loop, since antileptin receptor antibodies were able to inhibit the proliferative response of autoreactive T cells in vitro. Given that leptin appears to regulate EAE susceptibility, inflammatory anorexia, and pathogenic T-cell immune function, we postulate that it may offer a potential target in the treatment of multiple sclerosis.

Natural killer (NK) cells are physiologically involved in the immune response against viruses, intracellular bacteria, and parasites as well as against malignant diseases. In addition to the cytotoxic activity, NK lymphocytes mediate a variety of homeostatic effects by producing cytokines. This study focused on the differential role of CD40 and CD80 costimulatory molecules and major histocompatibility complex class I (MHC-I) antigens in the regulation of cytotoxicity and of interferon (IFN)-gamma secretion of resting and interleukin (IL)-2-activated human NK cells. CD40 and CD80 molecules were observed to play a specific role in the induction of cytotoxic function but not in IFN-gamma production of IL-2-activated NK effectors. In addition, a critical role of CD94-dependent MHC-I recognition for the regulation of IFN-gamma production and target lysis was demonstrated. These data provide a possible mechanism underlying functional interactions between NK lymphocytes and CD40/CD80-expressing cell targets, as represented by dendritic cells.

We have recently shown that leptin, the product of the obese gene, can directly influence T-cell function. In the work presented here, we explored the role of leptin in the development of spontaneous autoimmunity in the nonobese diabetic (NOD) mouse, an animal model for the study of human insulin-dependent diabetes mellitus (type 1 diabetes). We found that expression of serum leptin increased soon before the onset of hyperglycemia and diabetes in susceptible females. A pathogenetic role of leptin was assessed by administering recombinant leptin to young female and male NOD mice. Intraperitoneal injections of leptin accelerated autoimmune destruction of insulin-producing beta-cells and significantly increased interferon-gamma production in peripheral T-cells. These findings indicate that leptin can favor proinflammatory cell responses and directly influence development of autoimmune disease mediated by Th1 responses.

The expression of major histocompatibility complex (MHC) Class I antigens has been studied, by means of monoclonal antibodies directed against nonpolymorphic determinants of MHC Class I molecules, in two epithelial differentiated cell lines (FRTL-5 clone 2 and PC clone 3) and in one fibroblast cell line (FRT Fibro) of Fischer rat thyroid origin, before and after infection with various acute retroviruses carrying the v-ras-Ha, v-mos, v-src, polyoma middle T, and c-myc oncogenes. The results obtained indicate that a single virus does not produce identical changes in MHC Class I molecule expression in all tested lines, but a general increase occurs in lines derived from FRTL-5 clone 2 and a decrease occurs in lines derived from PC clone 3 and from FRT Fibro. Thus the modulation of expression seems to proceed always in the same direction in each cell line regardless of the infecting retrovirus and appears to involve posttranscriptional mechanisms, since no modification of expression of mRNA levels has been observed between normal and transformed cells. Only one line of PC clone 3 origin, transformed by the cooperation of two oncogenes (human c-myc and middle T), almost completely lost MHC Class I antigens on the cell surface and presented a significantly reduced synthesis of Class I mRNA.

Recent evidence suggests that the expression of abnormally high amounts of major histocompatibility complex (MHC) class I molecules may be a feature of at least some kinds of transformed cells. To investigate this aspect of neoplastic transformation we studied the expression of MHC class I antigens in an experimental model of normal, tumor-derived, and virus-transformed thyroid epithelial cell lines. The expression of MHC class I antigens has been studied by means of several monoclonal antibodies directed against either monomorphic or polymorphic epitopes and quantified by flow cytometry. Class I specific mRNA transcripts have been also analyzed by Northern blot hybridization, using a mouse genomic H-2 DNA probe. Our results indicate a modulation of MHC class I expression associated with loss of the differentiated phenotype and with transformation of thyroid epithelial cell lines. Undifferentiated cells in fact show a small quantity of these antigens, because acquisition of a fully differentiated phenotype is associated with an increase in their expression. Cell lines derived from thyroid tumors show reduced expression of MHC class I antigens, as compared to differentiated cells. Conversely, cells transformed in vitro by a retrovirus carrying the v-raski oncogene exhibit an increase in these antigens in comparison to their normal differentiated counterparts. Cells infected with a mutant virus able to transform cells only at the permissive temperature of 33 degrees C show a similar increased expression. After a shift to the nonpermissive temperature of 39 degrees C, infected cells, even those losing the transformed phenotype retain the same increased amount of MHC class I antigens. Our data suggest that the modulation of MHC class I antigen expression is strongly associated with transformation in thyroid epithelial cells.