Combined VEGF/PDGF improves olfactory regeneration after unilateral bulbectomy in mice.

The olfactory receptor neurons lining the nasal cavity have a remarkable capacity to regenerate throughout life. They are replenished continuously and their axons make new connections within the olfactory bulb. However, some factors such as head trauma and skull base surgery damage the olfactory nerve which lead to olfactory dysfunction. Losing the sense of smell has considerable effects on quality of life and life-expectancy. Therefore, there is a clear need to find a treatment for olfactory dysfunction. One such potential treatment is growth factor therapy which showed promising results in the spinal cord and brain injuries. The aim of the present study was to investigate whether combined delivery of two growth factors, vascular endothelial growth factor and platelet-derived growth factor treatment can improve the olfactory neurons regeneration in mice. The degeneration of the olfactory neurons was induced by unilateral bulbectomy. The treatment group received 1.5 µg of the combined growth factors intranasally, while the control injured group received saline. Growth factor treatment significantly increased the number of immature neurons at 5 and 7 days post injury and also the number of mature olfactory neurons at 10 and 14 days post bulbectomy. Regenerating axons extended over a larger volume in the operated cavity in the treatment group compared to control group at 14 days post bulbectomy. The growth factor treatment also significantly reduced astrocytic glia scar in the operated cavity. The results indicate that the combined delivery of the growth factors has the potential to improve olfactory dysfunction.

Predicted Coverage and Immuno-Safety of a Recombinant C-Repeat Region Based Streptococcus pyogenes Vaccine Candidate.

The C-terminal region of the M-protein of Streptococcus pyogenes is a major target for vaccine development. The major feature is the C-repeat region, consisting of 35-42 amino acid repeat units that display high but not perfect identity. SV1 is a S. pyogenes vaccine candidate that incorporates five 14mer amino acid sequences (called J14i variants) from differing C-repeat units in a single recombinant construct. Here we show that the J14i variants chosen for inclusion in SV1 are the most common variants in a dataset of 176 unique M-proteins. Murine antibodies raised against SV1 were shown to bind to each of the J14i variants present in SV1, as well as variants not present in the vaccine. Antibodies raised to the individual J14i variants were also shown to bind to multiple but different combinations of J14i variants, supporting the underlying rationale for the design of SV1. A Lewis Rat Model of valvulitis was then used to assess the capacity of SV1 to induce deleterious immune response associated with rheumatic heart disease. In this model, both SV1 and the M5 positive control protein were immunogenic. Neither of these antibodies were cross-reactive with cardiac myosin or collagen. Splenic T cells from SV1/CFA and SV1/alum immunized rats did not proliferate in response to cardiac myosin or collagen. Subsequent histological examination of heart tissue showed that 4 of 5 mice from the M5/CFA group had valvulitis and inflammatory cell infiltration into valvular tissue, whereas mice immunised with SV1/CFA, SV1/alum showed no sign of valvulitis. These results suggest that SV1 is a safe vaccine candidate that will elicit antibodies that recognise the vast majority of circulating GAS M-types.

Combined Immune Therapy for the Treatment of Visceral Leishmaniasis.

Chronic disease caused by infections, cancer or autoimmunity can result in profound immune suppression. Immunoregulatory networks are established to prevent tissue damage caused by inflammation. Although these immune checkpoints preserve tissue function, they allow pathogens and tumors to persist, and even expand. Immune checkpoint blockade has recently been successfully employed to treat cancer. This strategy modulates immunoregulatory mechanisms to allow host immune cells to kill or control tumors. However, the utility of this approach for controlling established infections has not been extensively investigated. Here, we examined the potential of modulating glucocorticoid-induced TNF receptor-related protein (GITR) on T cells to improve anti-parasitic immunity in blood and spleen tissue from visceral leishmaniasis (VL) patients infected with Leishmania donovani. We found little effect on parasite growth or parasite-specific IFNγ production. However, this treatment reversed the improved anti-parasitic immunity achieved by IL-10 signaling blockade. Further investigations using an experimental VL model caused by infection of C57BL/6 mice with L. donovani revealed that this negative effect was prominent in the liver, dependent on parasite burden and associated with an accumulation of Th1 cells expressing high levels of KLRG-1. Nevertheless, combined anti-IL-10 and anti-GITR mAb treatment could improve anti-parasitic immunity when used with sub-optimal doses of anti-parasitic drug. However, additional studies with VL patient samples indicated that targeting GITR had no overall benefit over IL-10 signaling blockade alone at improving anti-parasitic immune responses, even with drug treatment cover. These findings identify several important factors that influence the effectiveness of immune modulation, including parasite burden, target tissue and the use of anti-parasitic drug. Critically, these results also highlight potential negative effects of combining different immune modulation strategies.

Pathogenesis of fallopian tube damage caused by Chlamydia trachomatis infections.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease worldwide resulting in 4-5 million new cases of Chlamydia annually and an estimated 100 million cases per annum. Infections of the lower female genital tract (FGT) frequently are asymptomatic; thus, they often remain undiagnosed or untreated. If infections are either not resolved or left untreated, chlamydia can ascend to the upper FGT and infect the fallopian tubes (FTs) causing salpingitis that may lead to functional damage of the FTs and tubal factor infertility (TFI). Clinical observations and experimental data have indicated a role for antibodies against C. trachomatis proteins such as the 60-kDa heat shock protein 60 (cHSP60) in the immunopathogenesis of TFI. When released from infected cells, cHSP60 can induce proinflammatory immune responses that may functionally impair the FTs leading to fibrosis and luminal occlusion. Chlamydial pathogenesis of irreversible and permanent tubal damage is a consequence of innate and adaptive host immune responses to ongoing or repeated infections. The extracellular matrix that is regulated by metalloproteinases may also be modified by chlamydial infections of the FGT. This review will highlight protective and pathogenic immune responses to ongoing and repeated chlamydial infections of the FGT. It will also present two recent hypotheses to explain mechanisms that may contribute to FT damage during a C. trachomatis infection. If Chlamydia immunopathology can be controlled, it might yield a method of inducing fibrosis and thus provide a means of nonsurgical permanent contraception for women.

In silico identification and in vivo analysis of a novel T-cell antigen from Chlamydia, NrdB.

Chlamydial infections are a serious economic burden and health threat to developed and developing countries. Development of an efficacious vaccine is thought to be the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Currently there are very few efficacious vaccine candidates that have been identified and characterized. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. The chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. 'promiscuous' epitopes). A selection of target proteins were cloned, expressed, and purified. Recombinant proteins were screened against sera samples from patients with Chlamydia trachomatis genital tract infections. Two proteins, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. Using a mouse model, we found that intranasal immunization with NrdB conferred a CD4+ T-cell driven degree of protection similar to that seen with CD4+ T-cells primed from a whole organism, live challenge. In addition, serum from immunized mice was found to neutralize chlamydial infection of a cell monolayer in vitro. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could be a useful component of a multi-subunit vaccine against chlamydial genital tract infections.

Expression library immunization confers partial protection against Chlamydia muridarum genital infection.

Protective sequences of Chlamydia muridarum were identified as potential vaccine candidates by screening a genomic DNA expression library and assessing the immune responses of mice immunized with individual library clones following vaginal challenge with live Chlamydia. Groups of female BALB/c mice were immunized intra-abdominally by gene gun delivery of DNA three times at three-weekly intervals with individual library clones expressing chlamydial protein fragments and humoral and cell-mediated immune responses were evaluated. Chlamydia-specific cytokines including tumour necrosis factor-alpha (TNF-alpha) interleukin-10 (IL-10), interleukin-4 (IL-4), interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) were detected in mice immunized either with selected DNA clones in spleen cells (0.2-135.2 pg/mL) or lymph nodes (0.15-84.9 pg/mL). The most protective antigen identified was TC0512, a putative outer membrane protein (OMP). Immunization of mice with this clone elicited T-helper type-1 (Th-1) and T-helper type-2 (Th-2) cytokines as well as and IgG1 and IgG2a in sera of these animals. Ten days after the last immunization, animals were challenged intra-vaginally with 5 x 10(4) inclusion-forming units (IFUs) of C. muridarum. At 9 days following challenge TC0512 showed a 73% reduction in the number of recoverable Chlamydia compared with vector only immunized controls. Six additional clones were identified that also conferred varying degrees of protection against live chlamydial challenge. Significant protection against the initial stages of infection was shown by two DNA clones (encoding hypothetical proteins) and five clones showed enhanced clearance of chlamydial infection following DNA immunization and live chlamydial challenge. These results demonstrate that the C. muridarum genome can be screened for individual vaccine candidates by genetic immunization and that the screen produces novel and partially protective vaccine candidates.

Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus.

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.

Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11.

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His(6)). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His(6) and CFTR peptides or His(6) peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.