Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.     

Genome-wide comparative analysis of Mg transporter gene family between Triticum turgidum and Camelina sativa

Magnesium (Mg) as a bimetal plays critical roles in biochemical processes, membrane stability, and enzyme activity. Mg transporters (MGTs) are involving in maintaining Mg homeostasis in cells. Although the MGT family members have been identified in different plant species, there is no comprehensive analysis of the other plants' MGT genes. In the current study, 62 and 41 non-redundant putative MGT proteins were recognized into the genome of Camelina sativa, and Triticum turgidum and they were compared based on physicochemical properties, protein structure, expression, and interaction. All identified MGTs were classified into three subgroups, NIPA, CorA, and MRS2/MGT, based on conserved-motifs distribution. The results showed that the secondary structure pattern in NIPA and MRS2 subfamily members in both studied plant species were highly similar. Furthermore, MGTs encompass the conserved structures and the critical sites mainly in the metal ion and Mg²⁺ binding centers as well as the catalytic sites were observed. The highest numbers of protein channels were predicted in CorA proteins in both C. sativa and T. turgidum with 24 and 17 channel numbers, respectively. The Ser, Pro, Gly, Lys, Tyr, and Arg amino acids were predicted as the binding residues in MGTs channel regions. The expression pattern of identified genes demonstrated that MGT genes have diverse tissue-specific expression and stress response expression patterns. Besides, 147 co-expressed genes with MGTs were clustered into the eight co-expression nodes involved in N-glycan biosynthesis, protein processing in the endoplasmic reticulum, carbon metabolism, biosynthesis of amino acids, and endocytosis. In the present study, all interpretations are based on in silico predictions, which can be used in further studies related to functional genomics of MGT genes.

     

Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB^-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB^-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB^-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.     

Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.     

Dynamic Changes in Mucus Thickness and Ion Secretion during Citrobacter rodentium Infection and Clearance

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle.     

Effects of Pro-Tex® on the expression of Hsp70 gene and immune response parameters in the Persian sturgeon fingerlings,Acipenser persicus,infected with Aeromonas hydrophila

The induction of Hsps (heat shock protein) recognized as a promising approach to limiting disease and improving health in aquaculture. This investigation aimed to study the impacts of Pro-Tex®, an extract from the prickly pear cactus (Opuntia ficus indica), on the expression of Hsp70 gene and induction of immune response parameters in Acipenser persicus infected with Aeromonas hydrophila ATCC^®7966^TM. Fish were pretreated with 25, 50 and 100 mg/L of Pro-Tex and then injected in the intra-peritoneal cavity with A. hydrophila. The expression level of Hsp70 gene, lysozyme activity (LYZ) and complement C3 (C3), and immunoglobulin M (IgM) levels were assessed in liver, gill, and intestine on the days 3 and 7 post-infection. Tex-OE® increased expression of Hsp70 in a dose-dependent way in A. persicus, but this expression significantly reduced on the 7-days post-injection. The Hsp70 expression pattern was variable in each tissue, also, LYZ activity, C3, and IgM increased, depending on the concentration, and showed a decreasing trend in a time-dependent way. In conclusion, our data indicated that Pro-Tex as an Hsp70 inducer increases the resistance of sturgeon fry against fish pathogens by induction of different immunity factors.     

Cytokine gene polymorphism and graft-versus-host disease: a survey in Iranian bone marrow transplanted patients

Graft versus host disease (GVHD) is a major complication of bone marrow transplantation (BMT). Numerous studies have shown the potential role of cytokine genotypes in the occurrence of GVHD. In this retrospective, case–control study we aimed to investigate the association between 13 cytokine genes and acute GVHD (aGVHD) after HLA-identical sibling BMT in 91 Iranian subjects. Negative association was found between aGVHD and donor IL-10/GCC haplotype or donor IL-4Ra-A allele in the population study. When compared within the leukemia subgroup, we observed positive association between recipient IL-1α ?889/C allele and aGVHD. Also there were negative association between recipient IL-10/CAA haplotype and donor IL-4Ra/A allele and development of aGVHD. Among the different genotypes only donor IL-4Ra and donor IL-12 showed significant association. We conclude that several cytokine polymorphisms are positively and negatively associated with aGVHD in Iranian HLA matched siblings, of which IL-4Ra and IL-12 may play important roles.     

Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.