Group A Streptococcus Subcutaneous Infection-Induced Central Nervous System Inflammation Is Attenuated by Blocking Peripheral TNF.

Group A streptococcus (GAS) infection causes a strong inflammatory response associated with cytokine storms, leading to multiorgan failure, which is characterized as streptococcal toxic shock syndrome. However, little is known about GAS subcutaneous infection-mediated brain inflammation. Therefore, we used a bioluminescent GAS strain and reporter mice carrying firefly luciferase under transcriptional control of the nuclear factor-kappa B (NF-κB) promoter to concurrently monitor the host immune response and bacterial burden in a single mouse. Notably, in addition to the subcutaneous inoculation locus at the back of mice, we detected strong luminescence signals from NF-κB activation and increased inflammatory cytokine production in the brain, implying the existence of central nervous system inflammation after GAS subcutaneous infection. The inflamed brain exhibited an increased expression of glial fibrillary acidic protein and nicotinamide adenine dinucleotide phosphate oxidase components and greater microglial activation and blood-brain barrier (BBB) disruption. Furthermore, Fluoro-Jade C positive cells increased in the brain, indicating that neurons underwent degeneration. Peripheral tumor necrosis factor (TNF), which contributes to pathology in brain injury, was elevated in the circulation, and the expression of its receptor was also increased in the inflamed brain. Blockage of peripheral TNF effectively reduced brain inflammation and injury, thereby preventing BBB disruption and improving survival. Our study provides new insights into GAS-induced central nervous system inflammation, such as encephalopathy, which can be attenuated by circulating TNF blockage.

Sciellin mediates mesenchymal-to-epithelial transition in colorectal cancer hepatic metastasis.

Hepatic metastasis is the major cause of mortality in colorectal cancer (CRC) patients. Using proteomic analysis, we found sciellin (SCEL) to be specifically expressed in hepatic metastatic CRC cell lines. SCEL knockdown increased CRC cell migration and invasion, while overexpression had the opposite effect. SCEL knockdown also caused cancer cells to form more invasive structures within 3D cultures, increased the mesenchymal marker vimentin, and attenuated the epithelial marker E-cadherin. SCEL increased WNT signaling by activating ß-catenin and its downstream target c-myc, and activated mesenchymal-to-epithelial transition (MET) through a SCEL-ß-catenin-E-cadherin axis. SCEL showed higher expression in late stage primary CRC than in its hepatic metastatic counterpart. SCEL expression is dynamically modulated by TGF-ß1 and hypoxia, revealing a plastic MET mechanism for tumor colonization. Intrahepatic injection in immunodeficient mice revealed that SCEL is necessary for metastatic CRC tumor growth in the liver. These results demonstrate that SCEL is a MET inducer dynamically regulated through the metastasis process. They suggest SCEL may be a useful therapeutic target for preventing or eliminating CRC hepatic metastasis.

Albumin stimulates renal tubular inflammation through an HSP70-TLR4 axis in mice with early diabetic nephropathy.

Increased urinary albumin excretion is not simply an aftermath of glomerular injury, but is also involved in the progression of diabetic nephropathy (DN). Whereas Toll-like receptors (TLRs) are incriminated in the renal inflammation of DN, whether and how albumin is involved in the TLR-related renal inflammatory response remains to be clarified. Here, we showed that both TLR2 and TLR4, one of their putative endogenous ligands [heat shock protein 70 (HSP70)] and nuclear factor-κB promoter activity were markedly elevated in the kidneys of diabetic mice. A deficiency of TLR4 but not of TLR2 alleviated albuminuria, tubulointerstitial fibrosis and inflammation induced by diabetes. The protection against renal injury in diabetic Tlr4(-/-) mice was associated with reduced tubular injuries and preserved cubilin levels, rather than amelioration of glomerular lesions. In vitro studies revealed that albumin, a stronger inducer than high glucose (HG), induced the release of HSP70 from proximal tubular cells. HSP70 blockade ameliorated albumin-induced inflammatory mediators. HSP70 triggered the production of inflammatory mediators in a TLR4-dependent manner. Moreover, HSP70 inhibition in vivo ameliorated diabetes-induced albuminuria, inflammatory response and tubular injury. Finally, we found that individuals with DN had higher levels of TLR4 and HSP70 in the dilated tubules than non-diabetic controls. Thus, activation of the HSP70-TLR4 axis, stimulated at least in part by albumin, in the tubular cell is a newly identified mechanism associated with induction of tubulointerstitial inflammation and aggravation of pre-existing microalbuminuria in the progression of DN.

Bone marrow transplantation rescues intestinal mucosa after whole body radiation via paracrine mechanisms.

PURPOSE: Our previous study reveals bone marrow transplantation (BMT) recruits host marrow-derived myelomonocytic cells to radiation-injured intestine, enhancing stromal proliferation, leading secondarily to epithelial regeneration. In this study, we propose BMT ameliorates intestinal damage via paracrine mechanisms. MATERIALS AND METHODS: Angiogenic cytokines within the intestinal mucosa of mice after whole body irradiation (WBI) with or without BMT were measured by cytokine array and ELISA. BM conditioned medium (BMCM) with or without treatment with neutralizing antibodies to angiogenic cytokines were continuously infused into mice for three days after radiation. Carrageenan was used to deplete myelomonocytic cells of mice. RESULTS: BMT increased VEGF, bFGF and other angiogenic and chemotactic cytokines in the intestinal mucosa within 24h after WBI. Infusion of BMCM ameliorated radiation-induced intestinal damage with improved stromal activity and prolonged survival of mice. Neutralization of bFGF, PDGF and other angiogenic cytokines within BMCM abolished the mitigating effect to the intestine. Pretreatment of carrageenan to recipient mice reversed some of the cytokine levels, including VEGF, bFGF and IGF within the intestinal mucosa after BMT. CONCLUSIONS: Our result suggests BMT recruits host myelomonocytic cells and enhances intestinal stroma proliferation after radiation by secreting cytokines enhancing angiogenesis and chemotaxis. Host myelomonocytic cells further uplift the paracrine effect to enhance intestinal mucosal recovery.

Bone marrow transplantation enhances trafficking of host-derived myelomonocytic cells that rescue intestinal mucosa after whole body radiation.

BACKGROUND: Bone marrow (BM)-derived cells were demonstrated within intestines after radiation damage and were reported to be responsible for intestine repair. However, there was a discrepancy between intestine epithelial clonogenic regeneration, and mouse survival after BM transplantation (BMT) and radiation. The contribution of BM to acute intestine repair after radiation needed further investigation. METHODS: Mouse survival, intestine microcolony assay, immunohistochemical studies of both intestine and BM were evaluated in mice after whole body irradiation (WBI) and BMT. Immunoblotting, flowcytometry, and double immunostaining were used to evaluate the amount and the character of stroma cells within intestines of recipient mice after receiving gender-mismatched BMT or BMT from green fluorescence donors. RESULTS: Stromal cell proliferation within the lamina propria correlated with the beneficial effect of BMT to intestine recovery and day-8 survival of mice. Few donor-derived cells were found before the completion of intestine repair. The number of host but not donor-derived myelomonocytic and stromal cells increased dramatically within one week after radiation and BMT. Depletion of myelomonocytic cells of recipient mice abolished the mitigating effect of BMT. CONCLUSIONS: Besides rescuing injured BM from aplasia, BMT triggers trafficking of host CD11b(+) myelomonocytic cells from the host marrow to the radiation-injured intestinal mucosa, enhancing the proliferation of intestinal stroma cells, leading secondarily to epithelial regeneration.

SEPT12 deficiency causes sperm nucleus damage and developmental arrest of preimplantation embryos.

Oocytes fertilized with spermatozoa obtained from Septin 12+/- chimeric mice failed to develop beyond the morula stage after IVF and intracytoplasmic sperm injection because of significant DNA defects in the spermatozoa. Given that SEPT12 is expressed at the edge of the sperm nucleus in both humans and mice, we hypothesized the vital roles of Septin 12 in sperm head shaping, nuclear DNA condensation, and early embryonic development.

ENU mutagenesis identifies mice with morbid obesity and severe hyperinsulinemia caused by a novel mutation in leptin.

BACKGROUND: Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals. METHODOLOGY/PRINCIPAL FINDINGS: To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin(145E/145E) mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin(145E/145E) mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin(145E/145E) mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region. CONCLUSIONS/SIGNIFICANCE: Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome.

Comparative proteomic analysis of proteins involved in the tumorigenic process of seminal vesicle carcinoma in transgenic mice.

We studied the seminal vesicle secretion (SVS) of transgenic mice by using one-dimensional gel electrophoresis combined with LTQ-FT ICR MS analysis to explore protein expression profiles. Using unique peptide numbers as a cut-off criterion, 79 proteins were identified with high confidence in the SVS proteome. Label-free quantitative analysis was performed by using the IDEAL_Q software program. Furthermore, western blot assays were performed to validate the expression of seminal vesicle proteins. Sulfhydryl oxidase 1, glia-derived nexin, SVS1, SVS3, and SVS6 showed overexpression in SVS during cancer development. With high sequence similarity to human semenogelin, SVS2 is the most abundance protein in SVS and is dramatically decreased during the tumorigenic process. Our results indicate that these protein candidates could serve as potential targets for monitoring seminal vesicle carcinoma. Moreover, this information can provide clues for investigating seminal vesicle secretion-containing seminal plasma for related human diseases.

Involvement of p29 in DNA damage responses and Fanconi anemia pathway.

Human p29 is a chromatin-associated protein and the silencing of p29 expression increases cell population in G(1) phase and decreases phosphorylation levels of Chk1 and Chk2 in response to UV treatment. To further characterize the function of p29, U2OS and Fanconi anemia complementation group G (FA-G) cells with constitutive p29 expression have been established. Analyses of these cells identified increased phosphorylation levels of Chk1 and Chk2, which were accompanied by elevated amounts of chromatin-associated Mre11-Rad50-Nbs1 complex and ATR-IP. Monoubiquitination of the FA ID complex was restored in p29 stably expressing FA-G cells. Moreover, lower tumor incidence was observed in mp29 transgenic mice after UV irradiation. These results suggest the involvement of p29 in the DNA damage responses and Fanconi anemia pathway.

Pluripotency of mouse spermatogonial stem cells maintained by IGF-1- dependent pathway.

Recent studies indicate that neonatal spermatogonial stem cells (SSCs) possess pluripotency. However, the mechanisms that regulate the pluripotent differentiation capacity of SSCs remain unclear. Here, we describe a new method to clonally derive pluripotent SSCs from neonatal mouse testis. By coculturing with testicular stromal cells, SSCs can be maintained and expanded in serum-free conditions. Unlike endogenous SSCs, these in vitro expanded SSCs showed strong alkaline phosphatase (AP) activity and displayed characteristics of embryonic stem cells and primordial germ cells, which were therefore designated as AP(+) germline stem cells (AP(+)GSCs). The pluripotency of AP(+)GSCs was confirmed by in vitro differentiation toward hepatic and neuronal lineages and formation of embryonic chimeras after injection into blastocysts. Further investigation revealed that insulin-like growth factor-1 (IGF-1) secreted from Leydig cells was a key factor involved in maintaining the pluripotency of AP(+)GSCs. The blockage of IGF-1 receptor phosphorylation and its downstream PI3K pathway by PPP or LY294002 dramatically reduced their AP activity and expression of pluripotent genes, such as Oct-4, Blimp1, and Nanog. In conclusion, the present study demonstrated that IGF-1 secreted by testicular Leydig cells plays an important role in maintaining the pluripotency of SSCs in culture, which provides an insight into the molecular mechanism underlying germ cell pluripotency.