Interplay between Inflammation and Pathological Bone Resorption: Insights into Recent Mechanisms and Pathways in Related Diseases for Future Perspectives.

Bone is a mineralized and elastic connective tissue that provides fundamental functions in the human body, including mechanical support to the muscles and joints, protection of vital organs and storage of minerals. Bone is a metabolically active organ that undergoes continuous remodeling processes to maintain its architecture, shape, and function throughout life. One of the most important medical discoveries of recent decades has been that the immune system is involved in bone remodeling. Indeed, chronic inflammation has been recognized as the most significant factor influencing bone homeostasis, causing a shift in the bone remodeling process toward pathological bone resorption. Bone osteolytic diseases typified by excessive bone resorption account for one of the greatest causes of disability worldwide, with significant economic and public health burdens. From this perspective, we discuss the recent findings and discoveries highlighting the cellular and molecular mechanisms that regulate this process in the bone microenvironment, in addition to the current therapeutic strategies for the treatment of osteolytic bone diseases.

Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor.

DNAX-associated protein 12 kD size (DAP12) is a dominant immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis. Although several DAP12-associated receptors (DARs) have been identified in osteoclasts, including triggering receptor expressed on myeloid cells 2 (TREM-2), C-type lectin member 5 A (CLEC5A), and sialic acid-binding Ig-like lectin (Siglec)-15, their precise role in the development of osteoclasts and bone remodeling remain poorly understood. In this study, mice deficient in Trem-2, Clec5a, Siglec-15 were generated. In addition, mice double deficient in these DAR genes and FcεRI gamma chain (FcR)γ, an alternative ITAM adaptor to DAP12, were generated. Bone mass analysis was conducted on all mice. Notably, Siglec-15 deficient mice and Siglec-15/FcRγ double deficient mice exhibited mild and severe osteopetrosis respectively. In contrast, other DAR deficient mice showed normal bone phenotype. Likewise, osteoclasts from Siglec-15 deficient mice failed to form an actin ring, suggesting that Siglec-15 promotes bone resorption principally by modulating the cytoskeletal organization of osteoclasts. Furthermore, biochemical analysis revealed that Sigelc-15 activates macrophage colony-stimulating factor (M-CSF)-induced Ras-associated protein-1 (RAP1)/Ras-related C3 botulinum toxin substrate 1 (Rac1) pathway through formation of a complex with p130CAS and CrkII, leading to cytoskeletal remodeling of osteoclasts. Our data provide genetic and biochemical evidence that Siglec-15 facilitates M-CSF-induced cytoskeletal remodeling of the osteoclasts.

Molecular and Cellular Mechanisms of Bone and Cartilage Diseases.

Bones and cartilage, the two most important parts of the musculoskeletal system, provide mobility and maintain the body's posture [...].

Low-Grade Inflammation in the Pathogenesis of Osteoarthritis: Cellular and Molecular Mechanisms and Strategies for Future Therapeutic Intervention.

Osteoarthritis (OA) is a musculoskeletal disease characterized by cartilage degeneration and stiffness, with chronic pain in the affected joint. It has been proposed that OA progression is associated with the development of low-grade inflammation (LGI) in the joint. In support of this principle, LGI is now recognized as the major contributor to the pathogenesis of obesity, aging, and metabolic syndromes, which have been documented as among the most significant risk factors for developing OA. These discoveries have led to a new definition of the disease, and OA has recently been recognized as a low-grade inflammatory disease of the joint. Damage-associated molecular patterns (DAMPs)/alarmin molecules, the major cellular components that facilitate the interplay between cells in the cartilage and synovium, activate various molecular pathways involved in the initiation and maintenance of LGI in the joint, which, in turn, drives OA progression. A better understanding of the pathological mechanisms initiated by LGI in the joint represents a decisive step toward discovering therapeutic strategies for the treatment of OA. Recent findings and discoveries regarding the involvement of LGI mediated by DAMPs in OA pathogenesis are discussed. Modulating communication between cells in the joint to decrease inflammation represents an attractive approach for the treatment of OA.

Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein-70.

Serological immunoscreening was used to identify a gene encoding heat shock protein-70 from Babesia gibsoni (BgHSP-70) that showed high homology with HSP-70s from other apicomplexan parasites. This gene corresponded to a full-length cDNA containing an open reading frame of 1968 bp predicted to result in a 70-kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP-70 indicated elevated transcription from cultured parasites incubated at 40 degrees C for 1 h, but not at 30 degrees C. Interestingly, antiserum raised against recombinant BgHSP-70 protein reacted specifically not only with a 70-kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP-70), indicating the high degree of conservation of this protein. The BmHSP-70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP-70 (rBgHSP-70) and rBmHSP-70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN-gamma production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP-70 and rBmHSP-70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP-70s as a molecular adjuvant vaccine.

Characterization of the Babesia gibsoni 12-kDa protein as a potential antigen for the serodiagnosis.

A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced an anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 days post-infection, even when the dog became chronically infected and showed a low level of parasitemia. Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be an immunodominant antigen for B. gibsoni infection that could be used as a diagnostic antigen for B. gibsoni infection with high specificity and sensitivity.

Genetic diversity of two selected antigen loci in Babesia gibsoni Asian genotype obtained from Japan and Jeju island of South Korea.

Previous reports have shown that the secreted antigen 1 of Babesia gibsoni (BgSA1) and the thrombospondin-related adhesive protein of B. gibsoni (BgTRAP) are promising diagnostic reagents and vaccine candidates. Therefore, we determined the extent of nucleotide sequence variation in the BgSA1 and BgTRAP genes, obtained from eight isolates of B. gibsoni got from clinically infected dogs in geographically distinct areas of Japan and one isolate from Jeju island of South Korea. Sequence analyses have revealed that nucleotide diversity is lower in BgSA1 than that in BgTRAP. The mean number of non-synonymous (dn) nucleotide substitutions was significantly greater than that of synonymous (ds) ones per site in region II of BgTRAP. Overall, the results predict more allele-specific immunity to BgTRAP than that to BgSA1, which could be useful in designing and testing efficacy of diagnostic reagents as well as vaccine candidates for the B. gibsoni isolates from Japan and Jeju island of South Korea.

A shared antigen among Babesia species: ribosomal phosphoprotein P0 as a universal babesial vaccine candidate.

Babesia gibsoni ribosomal phosphoprotein P0 (BgP0) was previously identified as a cross-protective antigen against Babesia microti infection in mice. Interestingly, the same protein showed considerable antigenicity when tested with serum samples collected from Babesia-infected animals. Moreover, the polyclonal antibody raised against the recombinant BgP0 (rBgP0) recognized the P0 homologues from other Babesia species either by immunoblotting or by immunoscreening. The P0 genes from Babesia caballi, Babesia equi, and Babesia bigemina were then cloned and sequenced. The phylogenic analyses based on the amino acid sequences indicated that BgP0 has high identities with B. caballi P0 (88.1%), B. bigemina P0 (85.6%), Babesia bovis P0 (81.4%), and B. equi P0 (64.9%). Western blot analyses revealed that the corresponding native proteins ranged between 31 and 34 kDa, consistent with predicated molecular weight of Babesia P0. Furthermore, the immunogenic property of anti-rBgP0 IgG was evaluated against a B. bovis in vitro culture. The growth of B. bovis parasites was restricted by anti-rBgP0 IgG in a concentration-dependent manner, and significant reductions in parasitemia were observed only at 1 mg/ml in the culture. Taken together, these data suggest that P0 is a conserved protective antigen among Babesia species and might be a potentially universal vaccine candidate for babesiosis.

Four promising antigens, BgP32, BgP45, BgP47, and BgP50, for serodiagnosis of Babesia gibsoni infection were classified as B. gibsoni merozoite surface protein family.

We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results.

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test.

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.

Artesunate, a potential drug for treatment of Babesia infection.

The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260microM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6microM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10mg/kg of body weight on days 8-10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.

Identification of secreted antigen 3 from Babesia gibsoni.

A cDNA expression library of Babesia gibsoni was screened with the serum collected from a dog experimentally infected with B. gibsoni. A novel antigen sharing homology with secreted antigen 1 of B. gibsoni was isolated. The genomic analysis indicated that the BgSA3 gene exists as multicopies in the genome of B. gibsoni. The putative peptide encoded by the BgSA3 gene showed some characteristics of secreted proteins. The serum raised in mice immunized with the recombinant BgSA3 expressed in Escherichia coli could recognize a native parasite protein with a molecular mass of 70 kDa. Moreover, a sandwich enzyme-linked immunosorbent assay with anti-BgSA3 antibodies could detect the circulating BgSA3 in the blood plasma of dogs experimentally infected with B. gibsoni. The identification of BgSA3 provided a useful target for the development of a diagnostic test for detecting specific antibodies and circulating antigens.

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP.

The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.

Babesia gibsoni: identification of an immunodominant, interspersed repeat antigen.

In this report, an immunodominant antigen called BgIRA from Babesia gibsoni is identified and described. A highly repetitive antigen was screened from a cDNA library. The genomic BgIRA gene exists as single cope gene and contains 10 introns. BgIRA plays a dominant role in the immune response in dogs infected with B. gibsoni. The specificity and sensitivity of the rBgIRA in an ELISA indicated that this antigen might be useful in a diagnostic test.

Babesia gibsoni ribosomal phosphoprotein P0 induces cross-protective immunity against B. microti infection in mice.

Babesia gibsoni ribosomal phosphoprotein P0 (BgP0) was identified as an immunodominant cross-reactive antigen with B. microti. The BgP0 gene is a single copy with a predicted open reading frame of 942 bp and 314 amino acids. The BgP0 was expressed as a glutathione S-transferase fusion protein in Escherichia coli. The serum raised in mice with the recombinant BgP0 showed a specific band with a 34-kDa molecular mass in the extracts of B. gibsoni and B. microti merozoites. Furthermore, the intraperitoneal (i.p.) immunization of rBgP0 and Freund's adjuvant induced strong humoral response consisting of mixed immunoglobulins IgG1 and IgG2a in BALB/c mice. Following the challenge with B. microti, these mice delayed the onset of parasites and significantly reduced the peripheral parasitemia. On the other hand, passive-transfer of purified anti-BgP0 IgG into SCID mice showed partial protection against B. microti challenge infection. It was only effective in restricting the initial parasitemia but not later during its progress. Taken together, the immunological response elicited by rBgP0 protected the mice against B. microti challenge infection. These data suggest that BgP0 is a potentially universal vaccine candidate for both B. gibsoni and B. microti infections.