RANKL expression in chondrocytes and its promotion by lymphotoxin-alpha in the course of cartilage destruction during rheumatoid arthritis

We investigated the expression and localization of the receptor activator nuclear factor kappa B ligand (RANKL) in cartilage from patients with rheumatoid arthritis (RA) of relevance to cartilage degeneration. We also examined the role of exogenous lymphotoxin (LT)-alpha on RANKL expression in human chondrocytes and its effect on in vitro osteoclast differentiation. Cartilage and synovial fluid samples were obtained from 45 patients undergoing total joint replacement surgery or joint puncture, including 24 patients with osteoarthritis (OA) and 21 patients with RA. RANKL expression in articular cartilage was examined by immunohistochemistry. LT-alpha concentrations in synovial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). Normal human chondrocytes were stimulated with LT-alpha, and the relative mRNA levels of RANKL, osteoprotegerin (OPG), matrix metalloproteinase-9, and vascular endothelial growth factor were examined by real-time polymerase chain reaction. Soluble RANKL protein in culture media was measured using ELISA, and membrane-bound RANKL protein in cells was examined by western blotting. Co-cultures of human chondrocytes with peripheral blood mononuclear cells (PBMCs) were stimulated with macrophage-colony stimulating factor and LT-alpha, and osteoclast differentiation was evaluated by staining for tartrate-resistant acid phosphatase. LT-alpha concentrations were higher in RA synovial fluid than in OA samples. The population of RANKL-positive chondrocytes of RA cartilage was higher than that of OA cartilage, and correlated with cartilage degeneration. Stimulation of cultured human chondrocytes by LT-alpha increased RANKL expression, the RANKL/OPG ratio, and angiogenic factors. Membrane-bound RANKL in chondrocytes was up-regulated after stimulation of LT-alpha, whereas soluble RANKL in culture medium did not increase. Co-cultures of human chondrocytes and PBMCs demonstrated that LT-alpha stimulated human chondrocytes to produce RANKL and induced osteoclastic differentiation of PBMCs. RANKL produced by chondrocytes may contribute to cartilage destruction during RA and LT-alpha could promote the expression of RANKL in human chondrocytes

Adipose-Derived Extract Suppresses IL-1 beta-Induced Inflammatory Signaling Pathways in Human Chondrocytes and Ameliorates the Cartilage Destruction of Experimental Osteoarthritis in Rats

We investigated the effects of adipose-derived extract (AE) on cultured chondrocytes and in vivo cartilage destruction. AE was prepared from human adipose tissues using a nonenzymatic approach. Cultured human chondrocytes were stimulated with interleukin-1 beta (IL-1 beta) with or without different concentrations of AE. The effects of co-treatment with AE on intracellular signaling pathways and their downstream gene and protein expressions were examined using real-time PCR, Western blotting, and immunofluorescence staining. Rat AE prepared from inguinal adipose tissues was intra-articularly delivered to the knee joints of rats with experimental osteoarthritis (OA), and the effect of AE on cartilage destruction was evaluated histologically. In vitro, co-treatment with IL-1 beta combined with AE reduced activation of the p38 and ERK mitogen-activated protein kinase (MAPK) pathway and nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-kappa B), and subsequently downregulated the expressions of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, IL-6, and IL-8, whereas it markedly upregulated the expression of IL-1 receptor type 2 (IL-1R2) in chondrocytes. Intra-articular injection of homologous AE significantly ameliorated cartilage destruction six weeks postoperatively in the rat OA model. These results suggested that AE may exert a chondroprotective effect, at least in part, through modulation of the IL-1 beta-induced inflammatory signaling pathway by upregulation of IL-1R2 expression

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

AbstractMammal–fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements

High interleukin-6 levels induced by COVID-19 pneumonia correlate with increased circulating follicular helper T cell frequency and strong neutralization antibody response in the acute phase of Omicron breakthrough infection

BackgroundAcute immune responses to coronavirus disease 2019 (COVID-19) are influenced by variants, vaccination, and clinical severity. Thus, the outcome of these responses may differ between vaccinated and unvaccinated patients and those with and without COVID-19-related pneumonia. In this study, these differences during infection with the Omicron variant were investigated.MethodsA total of 67 patients (including 47 vaccinated and 20 unvaccinated patients) who were hospitalized within 5 days after COVID-19 symptom onset were enrolled in this prospective observational study. Serum neutralizing activity was evaluated using a pseudotyped virus assay and serum cytokines and chemokines were measured. Circulating follicular helper T cell (cTfh) frequencies were evaluated using flow cytometry.ResultsTwenty-five patients developed COVID-19 pneumonia on hospitalization. Although the neutralizing activities against wild-type and Delta variants were higher in the vaccinated group, those against the Omicron variant as well as the frequency of developing pneumonia were comparable between the vaccinated and unvaccinated groups. IL-6 and CXCL10 levels were higher in patients with pneumonia than in those without it, regardless of their vaccination status. Neutralizing activity against the Omicron variant were higher in vaccinated patients with pneumonia than in those without it. Moreover, a distinctive correlation between neutralizing activity against Omicron, IL-6 levels, and cTfh proportions was observed only in vaccinated patients.ConclusionsThe present study demonstrates the existence of a characteristic relationship between neutralizing activity against Omicron, IL-6 levels, and cTfh proportions in Omicron breakthrough infection

Regulation of Transgene Expression in Tumor Cells by Exploiting Endogenous Intracellular Signals

Recently, we have proposed a novel strategy for a cell-specific gene therapy system based on responses to intracellular signals. In this system, an intracellular signal that is specifically and abnormally activated in the diseased cells is used for the activation of transgene expression. In this study, we used protein kinase C (PKC)α as a trigger to activate transgene expression. We prepared a PKCα-responsive polymer conjugate [PPC(S)] and a negative control conjugate [PPC(A)], in which the phosphorylation site serine (Ser) was replaced with alanine (Ala). The phosphorylation for polymer/DNA complexes was determined with a radiolabel assay using [γ-32P]ATP. PPC(S)/DNA complexes were phosphorylated by the addition of PKCα, but no phosphorylation of the PPC(A)/DNA complex was observed. Moreover, after microinjection of polymer/GFP-encoding DNA complexes into HepG2 cells at cation/anion (C/A) ratios of 0.5 to 2.0, significant expression of GFP was observed in all cases using PPC(S)/DNA complexes, but no GFP expression was observed in the negative control PPC(A)/DNA complex-microinjected cells at C/A ratios of 1.0 and 2.0. On the other hand, GFP expression from PPC(S)/DNA complexes was completely suppressed in cells pretreated with PKCα inhibitor (Ro31-7549). These results suggest that our gene regulation system can be used for tumor cell-specific expression of a transgene in response to PKCα activity

SDOP-DB: a comparative standardized-protocol database for mouse phenotypic analyses

Summary: This article reports the development of SDOP-DB, which can provide definite, detailed and easy comparison of experimental protocols used in mouse phenotypic analyses among institutes or laboratories. Because SDOP-DB is fully compliant with international standards, it can act as a practical foundation for international sharing and integration of mouse phenotypic information

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

Validation of the FIB4 index in a Japanese nonalcoholic fatty liver disease population

<p>Abstract</p> <p>Background</p> <p>A reliable and inexpensive noninvasive marker of hepatic fibrosis is required in patients with nonalcoholic fatty liver disease (NAFLD). FIB4 index (based on age, aspartate aminotransferase [AST] and alanine aminotransferase [ALT] levels, and platelet counts) is expected to be useful for evaluating hepatic fibrosis. We validated the performance of FIB4 index in a Japanese cohort with NAFLD.</p> <p>Methods</p> <p>The areas under the receiver operating characteristic curves (AUROC) for FIB4 and six other markers were compared, based on data from 576 biopsy-proven NAFLD patients. Advanced fibrosis was defined as stage 3-4 fibrosis. FIB4 index was assessed as: age (yr) × AST (IU/L)/(platelet count (10⁹/L) × √ALT (IU/L))</p> <p>Results</p> <p>Advanced fibrosis was found in 64 (11%) patients. The AUROC for FIB4 index was superior to those for the other scoring systems for differentiating between advanced and mild fibrosis. Only 6 of 308 patients with a FIB4 index below the proposed low cut-off point (< 1.45) were under-staged, giving a high negative predictive value of 98%. Twenty-eight of 59 patients with a FIB4 index above the high cut-off point (> 3.25) were over-staged, giving a low positive predictive value of 53%. Using these cutoffs, 91% of the 395 patients with FIB-4 values outside 1.45-3.25 would be correctly classified. Implementation of the FIB4 index in the Japanese population would avoid 58% of liver biopsies.</p> <p>Conclusion</p> <p>The FIB4 index was superior to other tested noninvasive markers of fibrosis in Japanese patients with NAFLD, with a high negative predictive value for excluding advanced fibrosis. The small number of cases of advanced fibrosis in this cohort meant that this study had limited power for validating the high cut-off point.</p

Identification of mutations through dominant screening for obesity using C57BL/6 substrains

The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon, and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work