Inhibition of Hippocampal Regeneration by Adjuvant Dexamethasone in Experimental Infant Rat Pneumococcal Meningitis

Pneumococcal meningitis (PM) causes neurological sequelae in up to half of surviving patients. Neuronal damage associated with poor outcome is largely mediated by the inflammatory host response. Dexamethasone (DXM) is used as an adjuvant therapy in adult PM, but its efficacy in the treatment of pneumococcal meningitis in children is controversially discussed. While DXM has previously been shown to enhance hippocampal apoptosis in experimental PM, its impact on hippocampal cell proliferation is not known. This study investigated the impact of DXM on hippocampal proliferation in infant rat PM. Eleven-day-old nursing Wistar rats (n = 90) were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis. Treatment with DXM or vehicle was started 18 h after infection, concomitantly with antibiotics (ceftriaxone 100 mg/kg of body weight twice a day [b.i.d.]). Clinical parameters were monitored, and the amount of cells with proliferating activity was assessed using in vivo incorporation of bromodeoxyuridine (BrdU) and an in vitro neurosphere culture system at 3 and 4 d postinfection. DXM significantly worsened weight loss and survival. Density of BrdU-positive cells, as an index of cells with proliferating activity, was significantly lower in DXM-treated animals compared to vehicle controls (P < 0.0001). In parallel, DXM reduced neurosphere formation as an index for stem/progenitor cell density compared to vehicle treatment (P = 0.01). Our findings provide clear evidence that DXM exerts an antiproliferative effect on the hippocampus in infant rat PM. We conclude that an impairment of regenerative hippocampal capacity should be taken into account when considering adjuvant DXM in the therapeutic regimen for PM in children.

Proteins encoded in genomic regions associated with immune-mediated disease physically interact and suggest underlying biology

Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein-protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in line with observations in Mendelian disease.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

Molecular characterization of HOXA13 polyalanine expansion proteins in hand-foot-genital syndrome

We report on a father and daughter with hand-foot-genital syndrome (HFGS) with typical skeletal and genitourinary anomalies due to a 14-residue polyalanine expansion in HOXA13. This is the largest (32 residues) polyalanine tract so far described for any polyalanine mutant protein. Polyalanine expansion results in protein misfolding, cytoplasmic aggregation and degradation; however, HOXA13 polyalanine expansions appear to act as loss of function mutations in contrast to gain of function for HOXD13 polyalanine expansions. To address this paradox we examined the cellular consequences of polyalanine expansions on HOXA13 protein using COS cell transfection and immunocytochemistry. HOXA13 polyalanine expansion proteins form cytoplasmic aggregates, and distribution between cytoplasmic aggregates or the nucleus is polyalanine tract size-dependent. Geldanamycin, an Hsp90 inhibitor, reduces the steady-state abundance of all polyalanine-expanded proteins in transfected cells. We also found that wild-type HOXA13 or HOXD13 proteins are sequestered in HOXA13 polyalanine expansion cytoplasmic aggregates. Thus, the difference between HOXA13 polyalanine expansion loss-of-function and HOXD13 polyalanine expansion dominant-negative effect is not the ability to aggregate wild-type group 13 paralogs but perhaps to variation in activities associated with refolding, aggregation or degradation of the proteins.

Molecular cloning and characterization of NcROP2Fam-1, a member of the ROP2 family of rhoptry proteins in Neospora caninum that is targeted by antibodies neutralizing host cell invasion in vitro.

Recent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

C1 esterase inhibitor reduces lower extremity ischemia/reperfusion injury and associated lung damage

BACKGROUND Ischemia/reperfusion injury of lower extremities and associated lung damage may result from thrombotic occlusion, embolism, trauma, or surgical intervention with prolonged ischemia and subsequent restoration of blood flow. This clinical entity is characterized by high morbidity and mortality. Deprivation of blood supply leads to molecular and structural changes in the affected tissue. Upon reperfusion inflammatory cascades are activated causing tissue injury. We therefore tested preoperative treatment for prevention of reperfusion injury by using C1 esterase inhibitor (C1 INH). METHODS AND FINDINGS Wistar rats systemically pretreated with C1 INH (n = 6), APT070 (a membrane-targeted myristoylated peptidyl construct derived from human complement receptor 1, n = 4), vehicle (n = 7), or NaCl (n = 8) were subjected to 3h hind limb ischemia and 24h reperfusion. The femoral artery was clamped and a tourniquet placed under maintenance of a venous return. C1 INH treated rats showed significantly less edema in muscle (P<0.001) and lung and improved muscle viability (P<0.001) compared to controls and APT070. C1 INH prevented up-regulation of bradykinin receptor b1 (P<0.05) and VE-cadherin (P<0.01), reduced apoptosis (P<0.001) and fibrin deposition (P<0.01) and decreased plasma levels of pro-inflammatory cytokines, whereas deposition of complement components was not significantly reduced in the reperfused muscle. CONCLUSIONS C1 INH reduced edema formation locally in reperfused muscle as well as in lung, and improved muscle viability. C1 INH did not primarily act via inhibition of the complement system, but via the kinin and coagulation cascade. APT070 did not show beneficial effects in this model, despite potent inhibition of complement activation. Taken together, C1 INH might be a promising therapy to reduce peripheral ischemia/reperfusion injury and distant lung damage in complex and prolonged surgical interventions requiring tourniquet application.

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K(d)=68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.

The role of tissue damage in whiplash-associated disorders: discussion paper 1

Nonsystematic review of cervical spine lesions in whiplash-associated disorders (WAD).

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

Background DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. Methods mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. Results We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. Conclusions mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

SUMMARY: In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of approximately 67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

Cardiac channelopathies: genetic and molecular mechanisms

Channelopathies are diseases caused by dysfunctional ion channels, due to either genetic or acquired pathological factors. Inherited cardiac arrhythmic syndromes are among the most studied human disorders involving ion channels. Since seminal observations made in 1995, thousands of mutations have been found in many of the different genes that code for cardiac ion channel subunits and proteins that regulate the cardiac ion channels. The main phenotypes observed in patients carrying these mutations are congenital long QT syndrome (LQTS), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), short QT syndrome (SQTS) and variable types of conduction defects (CD). The goal of this review is to present an update of the main genetic and molecular mechanisms, as well as the associated phenotypes of cardiac channelopathies as of 2012.

In bacterial meningitis cortical brain damage is associated with changes in parenchymal MMP-9/TIMP-1 ratio and increased collagen type IV degradation

Adverse outcome in bacterial meningitis is associated with the breakdown of the blood-brain barrier (BBB). Matrix-metalloproteinases (MMPs) facilitate this process by degradation of components of the BBB. This in turn results in acute complications of bacterial meningitis including edema formation, increased intracranial pressure and subsequent ischemia. We determined the parenchymal balance of MMP-9 and TIMP-1 (tissue inhibitor of MMP) and the structural integrity of the BBB in relation to cortical damage in an infant rat model of pneumococcal meningitis. The data demonstrate that the extent of cortical damage is significantly associated with parenchymal gelatinolytic activity and collagen type IV degradation. The increased gelatinolysis was found to be associated with a brain parenchymal imbalance of MMP-9/TIMP-1. These findings provide support to the concept that MMPs mediated disruption of the BBB contributes to the pathogenesis of bacterial meningitis and that protection of the vascular unit may have neuroprotective potential.