Expression profiles of MMP-1 and TIMP-1 in lumbar intervertebral disc degeneration.

Lumbar intervertebral disc degeneration (IDD) is a common clinical pathology and has become a focus for research in recent years. Matrix metalloproteinases (MMPs) are enzymes responsible for the degradation of almost all extracellular matrix proteins (ECM). The over-expression of MMPs or tissue inhibitors of metalloproteinases (TIMPs) may disrupt the dynamic balance of the ECM. Therefore, in the current study, the expression levels of MMP-1 and TIMP-1 in lumbar IDD patients were evaluated in an attempt to elucidate their role in IDD pathogenesis and progression. In total, 60 IDD patients were recruited as the experimental group, along with 20 cases of lumbar vertebral injury without disc degeneration as the control group. Preoperative venous blood samples were collected, and intervertebral disc tissues were collected from the lesion during surgery. Serum and tissue levels of MMP-1 and TIMP-1 were quantified by enzyme-linked immunosorbent assay and immunohistochemical staining, respectively. Serum and tissue MMP-1 levels in IDD patients were significantly higher than those in the control group (P < 0.05). Additionally, sub-group analysis revealed that severe IDD patients had higher MMP-1 levels compared with mild or moderate IDD patients (P < 0.05). However, there were no significant differences in TIMP- 1 levels in either the serum or tissues of IDD patients compared to patients in the control group (P > 0.05). These results demonstrate that MMP-1 expression is increased in IDD, with higher expression observed in more severe cases, whereas TIMP-1 expression was similarly expressed in both normal and degenerated discs.

Ncor1 Deficiency Promotes Osteoclastogenesis and Exacerbates Periodontitis.

Nuclear receptor corepressor 1 (Ncor1) has been reported to regulate different transcription factors in different biological processes, including metabolism, inflammation, and circadian rhythms. However, the role of Ncor1 in periodontitis has not been elucidated. The aims of the present study were to investigate the role of Ncor1 in experimental periodontitis and to explore the underlying mechanisms through an experimental periodontitis model in myeloid cell-specific Ncor1-deficient mice. Myeloid cell-specific Ncor1 knockout (MNKO) mice were generated, and experimental periodontitis induced by ligation using 5-0 silk sutures was established. Ncor1 flox/flox mice were used as littermate controls (LC). Histological staining and micro-computed tomography scanning were used to evaluate osteoclastogenesis and alveolar bone resorption. Flow cytometry was conducted to observe the effect of Ncor1 on myeloid cells. RNA sequencing was used to explore the differentially targeted genes in osteoclastogenesis in the absence of Ncor1. Coimmunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) experiments, and dual luciferase assays were performed to explore the relationship between NCoR1 and the targeted gene. Alveolar bone resorption in the MNKO mice was significantly greater than that in the LC mice after periodontitis induction and osteoclastogenesis in vitro. The percentage of CD11b+ cells, particularly CD11b+ Ly6G+ neutrophils, was substantially higher in gingival tissues in the MNKO mice than in the LC mice. Results of RNA sequencing demonstrated that CCAAT enhancer binding protein α (Cebpα) was one of the most differentially expressed genes between the MNKO and LC groups. Mechanistically, Co-IP assays, ChIP experiments, and dual luciferase assays revealed that NCOR1 interacted with peroxisome proliferator-activated receptor gamma (PPARγ) and cooperated with HDAC3 to control the transcription of Cebpα. In conclusion, Ncor1 deficiency promoted osteoclast and neutrophil formation in mice with experimental periodontitis. It regulated the transcription of Cebpα via PPARγ to promote osteoclast differentiation.

Oral Pathobionts Promote MS-like Symptoms in Mice.

Dysbiotic oral microbiota has been associated with multiple sclerosis. However, the role and mechanism of oral microbiota in the development of multiple sclerosis are still elusive. Here, we demonstrated that ligature-induced periodontitis (LIP) aggravated experimental autoimmune encephalomyelitis (EAE) in mice, and this was likely dependent on the expansion of T helper 17 (Th17) cells. LIP increased the splenic richness of Enterobacter sp., which was able to induce the expansion of splenic Th17 cells and aggravate EAE in mice. LIP also led to enrichment of Erysipelotrichaceae sp. in the gut and increased Th17 cells in the large intestinal lamina propria of EAE mice. Fecal microbiota transplantation from EAE mice with LIP also promoted EAE symptoms. In conclusion, periodontitis exacerbates EAE, likely through ectopic colonization of oral pathobionts and expansion of Th17 cells.

Prevotella intermedia Aggravates Subclinical Hypothyroidism.

Subclinical hypothyroidism (SCH) has been shown to be associated with microbiota. However, the association between SCH and oral microbiota has not yet been elucidated. The results of our previous clinical studies showed that Prevotella intermedia was abundant in the oral microbiota of SCH patients. This study aimed to investigate the relationship between SCH and oral microbiota, verify the pathogenicity of P. intermedia in SCH, and preliminarily explore the possible mechanism. The SCH mouse model with oral application of P. intermedia was established, and the variance in the mouse oral microbiota and changes in thyroid function and metabolism were detected in mice. Student's t test and analysis of variance were used for statistical analysis. Oral application of P. intermedia changed the composition of the oral microbiota of SCH mice, which enhanced the damage to the thyroid and decreased the expression of functional genes of the thyroid. Moreover, P. intermedia decreased oxygen consumption and aggravated glucose and lipid metabolism disorders in SCH mice. Glucose tolerance and insulin tolerance decreased, and the triglyceride content of the liver and inflammatory infiltration in adipose tissue increased in SCH mice after P. intermedia stimulation. Mechanistically, P. intermedia increased the proportion of CD4+ T cells in cervical lymph nodes and thyroids in SCH mice. Th1 cells were suggested to play an important role in the pathogenesis of SCH involving P. intermedia. In conclusion, P. intermedia aggravated SCH manifestations, including thyroid dysfunction and glucose and lipid metabolism disorders, by causing immune imbalance in mice. This study sheds new light on the pathogenesis of SCH from the perspective of oral microbiota.

Role of reactive oxygen species in mediating hepatic ischemia-reperfusion injury and its therapeutic applications in liver transplantation.

Increasing evidence has shown that reactive oxygen species (ROS) are important mediators in liver ischemia/reperfusion injury(IRI). ROS include hydrogen peroxide (H(2)O(2)), superoxide anion (O(-2)), and hydroxyl radical (HO(-)), which may be generated by activated Kupffer cells in the liver, contributing to reperfusion injury. Hepatic IRI is a multistep process that damages liver graft function. To establish a series of therapeutic strategies to improve the outcome of liver transplantation, a good understanding of the mechanisms of IRI is essential. However, the detail mechanisms of how ROS lead to hepatocyte damage in IRI remains unclear. The aim of this review was to describe recent developments in the field of oxidative stress research. The first part of this review focused on the key roles and possible mechanisms of ROS in hepatic IRI. The second part of this review summarizes some findings including novel and classic antioxidant methods to ameliorate the hepatocyte damage during IRI.

PCD1, a novel gene containing PDZ and LIM domains, is overexpressed in several human cancers.

In an effort to discover novel genes differentially expressed in human pancreatic cancer, we have identified a gene named PCD1 (pancreatic cancer derived) that is up-regulated in pancreatic dysplasia and cancer relative to normal pancreatic ductal epithelium. We cloned the full length (4572 bp) of this gene, which encodes a novel protein of 1064 amino acids containing a PDZ domain and a LIM domain. An alternatively spliced form with a deletion of 30 bp in the coding region was also found. In situ hybridization results showed that PCD1 is highly expressed in a significant percentage of colon, breast, liver, lung, pancreas, stomach, and prostate tumor tissues but is expressed in very few normal tissues. Northern blot hybridization confirmed the overexpression of PCD1 in colon and breast tumor tissues and also showed strong expression of PCD1 in the heart as well as in HeLa cells. Real-time quantitative reverse transcription-PCR verified the overexpression of PCD1 in primary colon tumors or in liver metastases relative to normal colon tissues in five of eight patients. The PCD1 gene maps to human chromosome 13q21.33. Because of its high levels of expression in neoplastic tissues and the presence of both PDZ and LIM domains, we suggest that PCD1 may play an important role in cytoskeletal reorganization during carcinogenesis.

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Production of monoclonal antibody to Clostridium difficile toxin A which neutralizes enterotoxicity but not haemagglutination activity.

Nine monoclonal antibodies (mAb) to Clostridium difficile toxin A were produced. The isotype of one mAb (37B5) was IgG2b, kappa, and that of the other eight mAbs was IgM, kappa. Immunoblot analysis after non-denatured PAGE showed that with the exception of one mAb (112G6) all mAbs gave a positive reaction with the 540 kDa band of toxin A. Immunoblot analysis showed that four mAbs (2E15, 3B4, 37B5 and 49C4) gave a positive reaction with the 240 kDa major band of toxin A. In neutralisation tests with these mAbs for enterotoxicity, mouse lethality, haemagglutination activity and cytotoxicity, 37B5 neutralised enterotoxicity in a rabbit ileal loop response test but did not neutralise any other biological activities. None of the other eight mAbs showed any neutralising activities at all.

Haemagglutination activity of toxigenic and non-toxigenic strains of Clostridium difficile.

Cell extract of Clostridium difficile strains was fractionated by ammonium sulfate precipitation and sulfated cellulofine column chromatography to detect haemagglutination (HA) activity. HA activity without cytotoxicity was detected in fractions eluted at 0.79-0.91 M NaCl in sulfated cellulofine column chromatography of the cell extract in both toxigenic strain VPI 10463 and non-toxigenic strain KZ 1678, while toxin A was detected in fractions eluted at 0.27-0.29 M NaCl. Antisera were prepared with HA substance-containing fractions of the chromatography. Antiserum to the HA substance(s) of strain VPI 10463 neutralised the HA activity of the fractions of strains VPI 10463 and KZ 1678 at nearly the same titres. Antiserum to the HA substance(s) of strain KZ 1678 also neutralised the HA activity of both strains at nearly the same titres as above. These findings suggest that haemagglutinin(s) is commonly produced by C. difficile strains irrespective of toxin A-producing ability.

Correlation between cytotoxin production and sporulation in Clostridium difficile.

Correlation between cytotoxin production and sporulation was demonstrated when a Clostridium difficile culture was inoculated into fresh broth to give an initial count of less than 10 vegetative cells/ml with no spores. Under these conditions, cytotoxin was produced and released during sporulation. Addition of a sporulation inhibitor (acridine orange, 30 mg/L), resulted in a marked decrease in both sporulation and cytotoxin production, despite there being no change in the number of vegetative cells in the culture. These results indicate that sporulation might be closely related to cytotoxin production.

Toxin production by Clostridium difficile in a defined medium with limited amino acids.

Basal defined medium (BDM) containing vitamins, minerals and seven amino acids--(/L) tryptophan 0.1 g, methionine 0.2 g, valine 0.3 g, isoleucine 0.3 g, proline 0.3 g, leucine 0.4 g and cysteine 0.5 g--which appeared to be essential for good growth of Clostridium difficile was prepared. Addition of glycine 0.2 g/L and threonine 0.4 g/L to BDM produced better growth of strain VPI 10463, and this defined medium was designated minimum amino acid-defined medium (MADM). Production of toxins A and B by strain VPI 10463 in 6 x MADM containing (/L) tryptophan 0.6 g, methionine 1.2 g, valine 1.8 g, isoleucine 1.8 g, proline 1.8 g, leucine 2.4 g, cysteine 0.5 g, glycine 0.2 g and threonine 0.4 g, was much greater than in MADM. Toxin production by 20 C. difficile strains was examined in two defined media--6 x MADM and complete amino acid-defined medium (CADM) containing 18 amino acids--and one complex medium, modified brain heart infusion medium (m-BHI). Simultaneous production of toxins A and B by all test strains was demonstrated in m-BHI and the two defined media. It was also shown that 6 x MADM was generally better than CADM and as effective as m-BHI for stimulating toxin production by 13 strains. This defined medium would be useful for studies on the physiology, metabolism and pathogenicity of C. difficile.

Purification and characterisation of intracellular toxin A of Clostridium difficile.

After sonic disintegration of Clostridium difficile cells, intracellular toxin A was purified to homogeneity by thyroglobulin affinity chromatography (TGAC) followed by anion-exchange (Mono Q) by fast protein liquid chromatography (FPLC). High haemagglutinating (HA) activity was detected in TGAC-unbound fractions (2(9)/50 microliters), but not in TGAC thermal eluates (2(0)/50 microliters). The low HA titre of the thermal eluates was markedly increased to 2(5)/50 microliters after dialysis against 0.02 M Tris-HCl (pH 7.5). A disparity in the position of the peaks containing cytotoxic and HA activity was observed in the first Mono Q-FPLC step. Intracellular toxin A without HA activity was obtained by a second Mono Q-FPLC step. The M(r) of the intracellular toxin A was estimated by polyacrylamide gel electrophoresis (PAGE) to be 580 kDa under non-denaturing conditions. The minimum doses of the toxin causing cytotoxicity, mouse lethality and enterotoxicity were 0.83 ng, 8.7 ng and 5 micrograms, respectively.

Sperm penetration of immature and maturing oocytes does not affect phosphorylation of mitogen-activated protein kinase in pigs.

Pig oocytes cultured in vitro for 0, 25, 33 and 44 h were inseminated by frozen-thawed ejaculated sperm. At specified times after insemination, sperm penetration, cell cycle progression and mitogen-activated protein kinase (MAPK) phosphorylation were evaluated. It was shown that: (1) oocytes at various maturational stages could be penetrated by sperm; (2) sperm penetration did not affect meiotic cell cycle progression; (3) sperm penetration of germinal vesicle (GV) oocytes and maturing oocytes did not alter MAPK phosphorylation; and (4) when premetaphase I (pre-MI) and metaphase I (MI) oocytes, in which MAPK was activated, were fertilised, no evident MAPK dephosphorylation was detected as in metaphase II oocytes. The data suggest that sperm penetration before oocyte maturation does not affect MAPK phosphorylation and that the machinery inactivating MAPK upon fertilisation is not developed in maturing (pre-MI to MI) oocytes.

Development of PCR-Based Assays for Detecting Xanthomonas campestris pv. carotae, the Carrot Bacterial Leaf Blight Pathogen, from Different Substrates.

Detection of the carrot bacterial leaf blight pathogen, Xanthomonas campestris pv. carotae, was achieved using polymerase chain reaction (PCR) along with primer pairs developed from sequences of cloned random amplified polymorphic DNA (RAPD) fragments. Primer pairs 3S and 9B directed the amplification of ∼350-bp and ∼900-bp (or ∼2 kb) DNA fragments, respectively, from genomic DNA of all known X. campestris pv. carotae strains tested, but not from that of 13 other X. campestris pathovars or other bacterial species, including yellow non-xanthomonad bacteria isolated from carrot tissues and seeds. In tests conducted with an extensive collection of X. campestris pv. carotae-like strains isolated from different substrates from California, Idaho, Oregon, Washington, and Canada, the 3S primer pair directed the amplification of the ∼350-bp target fragment from all strains. These results indicated that the 3S primer pair is highly specific for X. campestris pv. carotae detection. Using the 3S primer pair, PCR assays were developed for detection of X. campestris pv. carotae from colonies on agar media, carrot leaf and stem tissues, and seeds. These tests could be performed in a single day. The PCR-based seed assay detected X. campestris pv. carotae from lots with contamination rates ranging from 2 × 102 to 2.3 × 108 CFU per gram of seed. This assay gave results similar to a seed-wash dilution plating assay and proved more sensitive than an enzyme-linked immunosorbent assay (ELISA)-based assay.

[Freeze-etch study on the brush border structure of Cysticercus cellulosae].

The images of C. cellulosae detected by freeze-itch technique were exactly similar to those observed in the thin section. The scolex microtriches might be divided into two parts, a thick proximal portion and a thinner spinal distal end, the former measuring about 0.2-0.4 micron in length, the latter measuring about 1-2 microns in length. On the extracellular surface of the proximal portion of microthrix, 1 or 2 particles could occasionally be seen, while numerous particles appeared on the protoplasmic fracture face (PF face) of the proximal portion. The PF face were closely packed and irregularly distributed with membranous particles, which were spherical or subspherical in shape, measured 0.01-0.02 micron in diameter. The extracellular fracture face (EF face), of proximal portion and all four membranous faces of spinal end were smooth. The tegumentary plasmic membrane was 0.02 micron in thickness, with smooth ES face. The PF face of this membrane packed closely with numerous particles, which were similar with those on the PF face of the microthrix. Numerous globular or discoidal vesicles scattered at random in the tegumentary matrix. Most mitochondria were near the basement membrane of the matrix. In the parenchymal layer, the parenchymal cells, calcareous corpuscles and flame-cells could easily be seen. Most of the calcareous corpuscles and flame-cells were located closely beneath the muscle layer. The flame-cell of C. cellulosae mainly consisted of three portions, cell body, a bundle of cilia and a filtered cell lumen. The flame-cell body had a large nucleus and relatively few cytoplasm with several mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)