Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis.

Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated-soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.     

A p65/p95 Neural Surface Receptor is Expressed at the S-G2 Phase of the Cell Cycle and Defines Distinct Populations

A surface receptor complex of M _r˜65 000 (p65) and ˜95 000 (p95) is expressed in cells of the central nervous system of mice. This receptor is recognized by monoclonal antibody 87.92.6 or by reovirus type 3 haemagglutinin as unnatural ligands. The p65/p95 receptor is expressed mostly in neural embryonic precursors undergoing proliferation, especially those in the S-G₂ phase of the cell cycle. Receptor expression decreases progressively throughout embryogenesis to low but detectable levels in the adult brain. Biochemical characterization revealed that the neural p65/p95 receptor complex is indistinguishable from the p65/p95 receptor expressed in T cells, where receptor ligation leads to a mitogenic block. In neural and lymphoid tissues the p65/p95 receptor (or an associated protein) possesses a tyrosine kinase enzymatic activity. Receptor ligation in neural cells resulted in the rapid tyrosine phosphorylation of cellular proteins which are different from substrates phosphorylated in T cells. Differential substrate coupling to the receptor may account for differences in signal transduction and biology between neural cells and T cells. Further study of this receptor complex may help define important features of neural proliferation, differentiation and survival.     

Myosin isozymes in avian skeletal muscles. I. Sequential expression of myosin isozymes in developing chicken pectoralis muscles

Myosin has been purified from chicken pectoralis muscle at various stages of development, from 10 days' incubation to approximately 10 months after hatching. Embryonic myosin from the earliest stage showed a high level of ATPase activity, similar to that obtained for adult pectoralis myosin. Two-dimensional peptide mapping of partial chymotryptic digests showed, however, that is heavy chain is quite different from that of adult fast myosin. The immunological crossreactivity observed between embryonic myosin and adult fast (pectoralis) myosin is therefore due to shared antigenic determinants rather than the presence of any adult isoforms. In an accompanying paper we will show that embryonic myosin at 10 days' incubation is not a single species, but consists of at least two heavy chain isozymes. The minor fraction binds slow light chains preferentially, and appears to be largely responsible for the observed crossreactivity with slow (ALD) myosin. None of the embryonic myosins is equivalent to the adult forms. Prior to hatching, LC3f is present only in very small amounts (less than 5%), and the adult light chain pattern, containing LC1f and LC3f in equimolar amounts, is not generated until after one week post-hatching. At about that time a new heavy chain population is detected, different from either the embryonic heavy chain or the adult heavy chain. The adult heavy chain peptide pattern appears from about three weeks' post-hatching, but a map indistinguishable from that of adult myosin is not observed until about 26 weeks. None of the observed differences in peptide maps can be related to different strains of chicken; pectoralis myosin from adult White Rock gave an identical map to that from White Leghorn. Unexpectedly, posterior latissimus dorsi (PLD) myosin from White Leghorn appears to be different from pectoralis myosin from the same strain, despite the histochemical and immunocytochemical similarity of the two muscles. We conclude that myosin polymorphism is widespread in muscle tissue, and that the expression of myosin isozymes and their subunits is under developmental regulation.     

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB-/-) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.     

Bruton tyrosine kinase gene mutations in Argentina

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.     

The Role of Complement in Pregnancy and Fetal Loss

In the United States, between 1 and 3% of women suffer recurrent miscarriages; 50-70% of all conceptions fail. [1,2] Although in the majority of affected women the cause of recurrent miscarriages is unknown, an immune mechanism involving the inappropriate and subsequently injurious recognition of the conceptus by the mother's immune system has been proposed. Murine models have recently been developed that are relevant to this issue. We and others have identified a novel role for complement as an early effector in the pathway leading to pregnancy loss associated with placental inflammation. Indeed, it appears that inhibition of complement activation is an absolute requirement for normal pregnancy, and that in the antiphosphospholid syndrome overwhelming activation of complement triggered by antibodies (Ab) deposited in placenta leads to fetal injury. Identification of complement activation as a mediator of pregnancy loss and definition of the complement components necessary to trigger such injury is likely to lead to a better understanding of its pathogenesis and to new and improved treatments.     

Boosting can explain patterns of fluctuations of ratios of inapparent to symptomatic dengue virus infections

Dengue is the most prevalent arboviral disease worldwide, and the four dengue virus (DENV) serotypes circulate endemically in many tropical and subtropical regions. Numerous studies have shown that the majority of DENV infections are inapparent, and that the ratio of inapparent to symptomatic infections (I/S) fluctuates substantially year-to-year. For example, in the ongoing Pediatric Dengue Cohort Study (PDCS) in Nicaragua, which was established in 2004, the I/S ratio has varied from 16.5:1 in 2006–2007 to 1.2:1 in 2009–2010. However, the mechanisms explaining these large fluctuations are not well understood. We hypothesized that in dengue-endemic areas, frequent boosting (i.e., exposures to DENV that do not lead to extensive viremia and result in a less than fourfold rise in antibody titers) of the immune response can be protective against symptomatic disease, and this can explain fluctuating I/S ratios. We formulate mechanistic epidemiologic models to examine the epidemiologic effects of protective homologous and heterologous boosting of the antibody response in preventing subsequent symptomatic DENV infection. We show that models that include frequent boosts that protect against symptomatic disease can recover the fluctuations in the I/S ratio that we observe, whereas a classic model without boosting cannot. Furthermore, we show that a boosting model can recover the inverse relationship between the number of symptomatic cases and the I/S ratio observed in the PDCS. These results highlight the importance of robust dengue control efforts, as intermediate dengue control may have the potential to decrease the protective effects of boosting.

Dengue virus (DENV) is the most prevalent vector-borne viral disease of humans, with recent estimates of around 105 million individuals infected annually (1). It comprises four antigenically distinct serotypes, DENV-1 to -4 (2), and is transmitted to humans by Aedes aegypti and, less frequently, Aedes albopictus mosquitoes (3–5). While most studies have focused on symptomatic infections, epidemiologic studies have shown that for dengue, the majority of infections are inapparent (3, 5), that is, infections that do not cause detected disease but result in a fourfold or greater rise in antibody titers. However, large fluctuations in annual dengue inapparent:symptomatic (I/S) ratios have been documented worldwide (5). For example, cohort studies able to detect inapparent DENV infections in Nicaragua (6–9), Peru (10), and Thailand (11) have shown that the I/S ratio of DENV infections ranges widely year to year. In the Pediatric Dengue Cohort Study (PDCS) in Nicaragua, the longest running dengue cohort study, the I/S ratio has varied widely, from 16.5:1 in 2006–2007 (7) to 1.2:1 in 2009–2010 (9). We currently do not understand the drivers of these fluctuations; however, we do know that potential extrinsic drivers, such as differences in replication rates of the predominating serotype, cannot explain them (5). Gaining a mechanistic understanding of these fluctuations in the I/S ratio is likely to be critical for understanding potential drivers of epidemic potential and severe dengue disease and for enacting effective control policies.Extensive research has been conducted into the causes of DENV infection and disease, and there is now some evidence to suggest that immune interactions among viruses and strains may be responsible for fluctuating patterns (12–14). In particular, this extensive body of work has shown that severe disease occurs due to immunopathology (4, 15, 16). The most important risk factor for severe dengue disease is secondary heterologous infections (4), due in part to a phenomenon called antibody-dependent enhancement (ADE), in which antibodies from a first infection cross-react with virus from a secondary infection, leading to incomplete neutralization. The resulting partially neutralized immune complexes enhance infection into Fc receptor-bearing cells (17). Low to intermediate titers of cross-reactive anti-DENV antibodies have been shown to enhance subsequent dengue disease severity in human populations (15, 18, 19). However, neutralizing antibody titers are thought to be protective against dengue disease, and a recent study showed that higher preinfection neutralizing antibody titers correlated with lower probability of symptomatic infection in children in the PDCS (20). Importantly, individuals with inapparent heterologous secondary infections had significantly higher preinfection titers than individuals with symptomatic heterologous secondary infections (20–22), providing direct evidence that preinfection neutralizing antibody titer is an important determinant of disease outcome. Therefore, it is plausible that the variability in preinfection antibody titer could explain fluctuations in I/S ratios.Recent work has suggested that frequent exposure to DENV may boost the immune response and result in modest increases in neutralizing antibody titer (20), which in turn may protect individuals against symptomatic infection. Evidence for boosting comes from analysis of neutralizing antibodies following primary infection. Here we have defined boosting as exposures to DENV that do not lead to extensive viremia and that result in a less than fourfold rise in antibody titers. Traditionally, the temporary period of cross-protection against heterotypic serotypes following a primary infection is explained by waning cross-reactive antibodies, resulting in a decrease in neutralizing antibody titers (23). However, an analysis of neutralizing antibody titers from the PDCS showed that neutralizing antibody titers did not decrease in the time between primary and secondary DENV infection, but in fact increased marginally (20). A comparable trend was seen in Thailand (24) and in a long-term hospital-based study in Nicaragua (25, 26). The increase in neutralizing antibody titer may be due to immune boosts (20), suggesting that children may be regularly exposed to DENV without experiencing symptoms or meeting the criteria for inapparent infection. There is also evidence of a phenomenon similar to boosting in a human vaccine study (27) and in a study in nonhuman primates (28), where in both cases there was initial exposure that resulted in viremia and seroconversion and a second challenge that did not result in viremia but did result in increased antibody titers. Clearly, in years with a high incidence of dengue, we would expect boosting to occur more frequently, and thus in the years immediately following high dengue incidence, we would expect fewer symptomatic infections, as individuals would be protected against symptomatic infection due to boosts (5).Here we used mathematical models to determine which mechanisms can recover the fluctuations in the I/S ratio in DENV infections. Since our aim was to gain a conceptual qualitative understanding of the role of the impact of a range of mechanisms, we took the classic simplifying approach of not explicitly modeling the mosquito population dynamics. All models are adapted from existing dengue epidemiologic models (12, 29) and include immunity against homologous reinfection, a period of cross-protection following infection, and seasonality. For simplicity, we model the whole population but also present results from a model of the pediatric cohort from which our data are taken. With only these factors, a year-to-year variation in case number is seen, but not a variation in I/S ratio. This model was first modified to include the basic assumption that antibody titer decreases with time since infection and is predictive of infection outcome (20), to evaluate whether I/S fluctuations can be recovered by shorter periods of cross-protection between primary infections and secondary heterotypic infections for inapparent secondary infections than for symptomatic secondary infections, as previously suggested (6, 23).We then explored whether I/S ratio differences can be explained by protection against symptomatic disease due to boosting of the immune response. We define boosts as exposures to homotypic or heterotypic DENV serotypes that “boost” the immune response and result in a modest rise in antibody titers (less than fourfold rise, below the threshold of classification as an inapparent infection), possibly due to limited viremia. It is important to note that with boosting, the antibody titer that we measure might not fall. Although it was previously thought that homologous DENV infection confers lifelong immunity against the infecting serotype (30), recent work has shown that homologous DENV reinfections do occur (31). We hypothesize that a boost in antibody titer can protect an individual during subsequent infections, resulting in the development of inapparent infection instead of symptomatic infection. We show that a boosting model can recover the fluctuations in the I/S ratio, recover the inverse relationship between the number of symptomatic cases and the I/S ratio in the PDCS, and recover a positive relationship between the I/S ratio in a given year and the number of cases in the previous year, as has been previously noted (5, 11). These models suggest that boosts may be occurring frequently in endemic areas and need to be considered when constructing effective dengue control policies.     

Genetic Variation of Echinococcus canadensis (G7) in Mexico

We evaluated the genetic variation of Echinococcus G7 strain in larval and adult stages using a fragment of the mitochondrial cox1 gen. Viscera of pigs, bovines, and sheep and fecal samples of dogs were inspected for cystic and canine echinococcosis, respectively; only pigs had hydatid cysts. Bayesian inferences grouped the sequences in an E. canadensis G7 cluster, suggesting that, in Mexico, this strain might be mainly present. Additionally, the population genetic and network analysis showed that E. canadensis in Mexico is very diverse and has probably been introduced several times from different sources. Finally, a scarce genetic differentiation between G6 (camel strain) and G7 (pig strain) populations was identified.Echinococcus granulosus sensu lato (s.l.) includes species that cause cystic echinococcosis (CE), one of the most important and widespread parasitic zoonoses. Recent phylogenetic studies based on both mitochondrial and nuclear DNA genes show that E. granulosus s.l. consists of at least four valid species: E. granulosus sensu stricto (s.s.; genotypes G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). Genotypes G6/G7 are closely related and referred to as camel and pig strains, respectively.^1–3 The pig–dog cycle is mainly present in Mexico and maintains the G7 strain.^4,5 Although there are isolated reports of E. oligarthrus in a wild cat,⁶ E. ortleppi (E. granulosus s.l.; G5) in a patient,⁷ and E. granulosus s.s. (G1) in a rural pig, there is no evidence that these species are maintained in Mexico.⁸ No data of CE caused by G7 have been documented in Mexican patients, although there is a high number of E. canadensis G7-infected patients in central Europe, pointing to the importance of this strain as a cause of human CE.^9,10 There are only two genetic studies performed in samples from Mexico. Cruz-Reyes and others⁵ documented that G7 parasites of Mexican and Polish pig isolates showed similar patterns by restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) and random amplified polymorphic DNA (RAPD) techniques, and although polymerase chain reaction (PCR) -sequencing analysis of mitochondrial cox1 gen fragment was performed, no polymorphism data were reported. Sharma and others¹¹ identified two variants (A and B) inside of the G6/G7 group consisting of samples from Mexico and Argentina using five nuclear markers (elongation factor 1α, transforming growth factor-β receptor kinase, thioredoxin peroxidase, calreticulin, and ezrin-radixin-moesin-like protein). Because some local slaughter records from northern Mexico indicate the presence of Echinococcus spp. in livestock animals,⁵ the objective of this study was to investigate if parasites in pigs and dogs correspond to G7 and if so, describe its genetic variation.Infected animals were identified in the municipal slaughterhouse of Calera, Zacatecas (north central Mexico), where farm and backyard livestock animals coming from the whole state and other surrounding states were included. For this purpose, viscera from 387 pigs, 243 bovines, and 32 sheep were inspected for the larval stage of Echinococcus. Nine pigs (six pigs from Zacatecas, two pigs from Aguascalientes, and one pig from Morelos) were found infected, and hydatid cysts were obtained under aseptic conditions. After cyst contents were aspirated and centrifuged, aliquots were examined under microscopy to confirm the presence of protoscolices, and pellets were kept in 70% ethanol at −20°C until DNA extraction. Each cyst from each animal was considered as an isolate.Based on the presence of the parasites previously identified in Calera''s slaughterhouse, a rural community located in the central area of Zacatecas at 22°55′ N, 102°48′ W was selected to look for the adult stage of this parasite. For this search, all dogs (60) present in the community were sampled one time for feces after obtaining verbal consent from the owner; samples were used to identify taeniid eggs by the Faust technique, antigens in stool samples (copro-antigens) by enzyme-linked immunosorbent assay (ELISA; CpAg ELISA), and DNA by Copro-PCR. The CpAg ELISA was performed as described by Allan and others¹² and Moro and others.¹³ For Copro-PCR, only positive samples by CpAg ELISA were analyzed using JB3 and JB4 primers to amplify a cox1 gen fragment.¹⁴ Coprological analysis of dogs showed that 11 samples were positive by CpAg ELISA (18.3%); only 2 of these samples had taeniid tapeworms (3.4%), and 3 of 11 samples yielded products of approximately 450 bp. All amplicons obtained of hydatid cysts and fecal samples were purified, sequenced on both strands, submitted to GenBank (accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"KF734649-KF734660","start_term":"KF734649","end_term":"KF734660","start_term_id":"570963772","end_term_id":"570963794"}}KF734649-KF734660), and compared with several mitochondrial DNA sequences of cox1. Dogs positive for taeniid eggs or antigens were purged and treated with praziquantel at 30 mg/kg and arecoline bromide at 2 mg/kg. The protocol was previously approved by the Ethics and Research Committees of the General Hospital “Dr. Manuel Gea Gonzalez”; government and health authorities of the municipality and community also authorized our study.All sequences were subjected to the Basic Local Alignment Search Tool (BLAST) search in the GenBank database; multiple alignments were performed with the CLUSTAL W and MUSCLE programs,^15,16 with manual adjusted in MEGA program v5¹⁷ to determine the appropriate model of molecular evolution in the Modeltest 3.7 program.¹⁸ The phylogenetic reconstruction using Bayesian inference was performed with Mr Bayes 3.2.1 program.¹⁹ Unrooted haplotype networks were created using NETWORK 4.611 software and nested according to the rules in median-joining networks.²⁰ An analysis of genetic diversity within and between populations was performed using DnaSPv4²¹ and included nucleotide diversity (π), haplotype polymorphism (θ), genetic differentiation index (F_ST), and Tajima''s D test. Analysis of molecular variance (AMOVA) was used to examine the population genetic structure between populations by Φ_ST as the genetic fixation index (analogous to F_ST) obtained by ARLEQUIN software.²²After multiple alignments, all sequences of larval and adult stages showed 98% or higher identity with E. canadensis, whereas the Bayesian phylogenetic tree and the haplotype network inference grouped these sequences in the E. canadensis G7 cluster. Sequences for cox1 of E. canadensis from Africa, Asia, Europe, Latin America, and North America deposited in the GenBank databases (N = 58) as well as our sequences (accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"KF734649-KF734660","start_term":"KF734649","end_term":"KF734660","start_term_id":"570963772","end_term_id":"570963794"}}KF734649-KF734660) were analyzed. The results for π and θ were 0.0118 and 0.718, and the result of Tajima''s D test was −2.1885 (P < 0.01). Genetic differentiation indexes between different paired sequences of E. canadensis genotypes are shown in

High levels of the p53 inhibitor MDM4 in head and neck squamous carcinomas

The p53 tumor suppressor is mutated in most human tumors. MDM2, a well-known inhibitor of p53, is overexpressed in a large number of tumors, suggesting that increased levels of MDM2 also contribute to tumorigenesis. A novel p53 inhibitor, MDM4, was more recently identified. The role of MDM4 in cancer development is not well understood. We set out to examine the levels of MDM4 by immunohistochemistry in head and neck squamous carcinomas (HNSC) to ask whether high MDM4 levels could contribute to its development and progression. In addition, MDM2 and p53 levels were examined to identify overlapping expression patterns. MDM4 is present at high levels in 50% of HNSC. In addition, overexpression of MDM2 was detected in 80% of tumors, many of which were also positive for MDM4. A subset of tumors displayed high levels of all 3 proteins. Sequencing of the p53 gene revealed that tumors with positive immunoreactivity for MDM2 or MDM4, some of which also had high levels of p53, did not carry mutations in this gene. Thus, the detection of p53 by immunohistochemistry was not synonymous with the presence of p53 mutations. Expression of both MDM2 and MDM4 in tumors without p53 mutations strongly suggests that MDM2 and MDM4 inhibit the activity of this tumor suppressor in HNSC.     

Effect of hemodialysis schedules and membranes on hepatocyte growth factor and hepatitis C virus RNA levels

Hemodialysis induces production of the hepatocyte growth factor (HGF) and decrease of serum hepatitis C virus (HCV) RNA in patients with HCV infection, but it is not known if the hemodialysis schedule or type of membrane affect both the HGF production and HCV viremia. The effects on both parameters of alternate‐day intermittent hemodialysis and short‐daily hemodialysis and high and low flux membranes were investigated in 41 patients treated by hemodialysis. Sixteen (39%) patients were anti‐HCV positive and 11 (69%) had HCV RNA. Twenty‐six patients were on alternate‐day intermittent and 15 on short‐daily hemodialysis. High flux membranes were used for 29 patients and low flux membranes for 12 patients. A decrease in HCV RNA was observed at the end of hemodialysis (8.6 × 10⁵ ± 1.1 × 10⁶ IU/ml vs. 4.4 × 10⁵ ± 7.3 × 10⁵ IU/ml, P = 0.003). The proportion of HCV RNA decrease was similar in patients dialyzed with both schedules and with both types of membranes. The HGF levels increased from 2,605.9 ± 1,428.7 to >8,000 pg/ml at 15 min. At the end of the session, the HGF levels decreased to 5,106.7 ± 2,533.9 pg/ml. The HGF levels at the start of the next session were similar to those at baseline (2,680.0 ± 1,209.3 pg/ml). The increase and dynamics of the HGF levels were similar in patient's hemodialyzed with both schedules and with both types of membranes. These results suggest that changes in HCV RNA and HGF levels during hemodialysis are not influenced by the schedule or type of membrane used. J. Med. Virol. 82: 763–767, 2010. © 2010 Wiley‐Liss, Inc.