Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.

AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS: Bovine nasal cartilage explants were cultured with and without recombinant human interleukin 1 alpha (IL-1 alpha), recombinant human tumour necrosis factor alpha, or retinoic acid. Proteoglycan and type II collagen release were determined by colorimetric assay and immunoassay, respectively, in the absence and presence of matrixin inhibitors. Potential toxic effects of the inhibitors were assessed by measuring rates of glycolysis. RESULTS: Loss of proteoglycan and type II collagen from nasal cartilage was inhibited by batimastat, a broad spectrum matrixin inhibitor. BB-3437, a selective inhibitor of stromelysin, neutrophil collagenase, and collagenase 3, at the concentrations used in this study, showed a weak but dose dependent inhibitory effect on the IL-1 stimulated degradation of type II collagen, but had virtually no effect on proteoglycan breakdown. Neither inhibitor affected rates of glycolysis. CONCLUSIONS: Stromelysin 1, neutrophil collagenase, and collagenase 3 are unlikely to contribute to chondrocyte mediated proteoglycan degradation in our model system. The modest effect of a selective inhibitor of these enzymes on IL-1 stimulated collagen breakdown suggests a minor role for one or more of these proteinases; potent inhibition by an inhibitor of interstitial collagenase and the gelatinases suggests that these enzymes play a major role in IL-1 stimulated, chondrocyte mediated type II collagen breakdown from nasal cartilage.     

Pulmonary C-fibers do not play a role in ammonia-induced brief tachypnea in the rabbit.

To determine the role of pulmonary C-fibers in evoking a brief tachypnea induced by ammonia vapor, we examined the responses of diaphragm electromyogram (DIAP EMG) to ammonia inhalation before and after procaine treatment to the contralateral vagus nerve in urethane-anesthetized, spontaneously breathing rabbits with unilateral vagotomy. Procaine treatment that blocked the conduction of vagal afferent C wave did not significantly alter the response of brief tachypnea to ammonia. Furthermore, the stimulation of pulmonary C-fibers by ammonia inhalation did not coincide with the induction of brief tachypnea. In addition, we also examined the responses of rapidly adapting receptors (RARs) and slowly adapting receptors (SARs) to ammonia inhalation in rabbits, particularly in which inhalation of this chemical gas produced a brief tachypnea. The burst activity of RARs evoked by ammonia inhalation coincided with the phase of rapid shallow breathing for a few breaths. The discharge rates of SARs during both inspiration and expiration increased when ammonia inhalation caused a brief tachypnea. From these results, it can be suggested that the ammonia-induced brief tachypnea is probably mediated by transient stimulation of both SAR and RAR activities but does not occur as a result of the pulmonary C-fiber stimulation.     

Gene delivery GAD 500 autoantigen by AAV serotype 1 prevented diabetes in NOD mice: transduction efficiency do not play important roles

We previously found that adeno-associated viral vector serotype 2 (AAV-2) muscle gene delivery of GAD 500-585 autoantigen efficiently prevented autoimmune diabetes in NOD mice. Recent reports suggest that AAV vectors based on serotype 1 (AAV-1) transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2 to 1000-fold. To determine whether this increased efficacy of AAV-1 could result in increased therapeutic effects in mice, we constructed rAAV1/GAD 500-585 vectors and compared their effects in preventing autoimmune diabetes in NOD mice with those of rAAV2/GAD 500-585 after muscle injection. rAAV(1)/GAD(500-585) gene therapy prevented diabetes in NOD mice. However, although much higher level of GAD 500-585 expression was found in mice using AAV-1 as gene delivery vector than those using AAV-2, no increased efficiency of AAV-1 vectors were found in their capability to prevent autoimmune diabetes, as higher titers of rAAV1/GAD 500-585 virus (3x10(11)v.g./mouse) were needed to obtain therapeutic effects in NOD mice, a titer not different from that of AAV-2. Protection resulted from rAAV1/GAD 500-585 gene therapy were marked by enhanced Th2 immune response and up-regulated CD4+ Foxp3+T regulatory cells, which might actively suppress effector T cells in NOD mice. As here we found that the therapeutic effects of AAV1 were not positively correlated to it's transduction efficiency, our data suggested that the safety and other factors besides efficiency should be considered when use different AAV serotype to treat autoimmune disease.     

The major risk alleles of age-related macular degeneration (AMD) in CFH do not play a major role in rheumatoid arthritis (RA)

Because activation of the alternative pathway (AP) of the complement system is an important aspect of both age-related macular degeneration (AMD) and rheumatoid arthritis (RA), we wished to address the question whether genetic risk factors of the AP inhibitor complement factor H (CFH) for AMD would also be risk factors for RA. For this purpose we genotyped single nucleotide polymorphisms (SNPs) in a Dutch set of RA patients and controls. Similarly, a meta-analysis using a Spanish cohort of RA as well as six large genome-wide association studies (GWAS) studies was performed. For these SNPs we analysed more than 6000 patients and 20,000 controls. The CFH variants, I62V, Y402H, IVS1 and IVS10, known to associate strongly with AMD, did not show a significant association with the risk of developing RA despite a strong statistical power to detect such differences. In conclusion, the major risk alleles of AMD in CFH do not have a similar effect on developing RA.     

Endogenous tachykinins play a role in IL-1-induced neutrophil accumulation: involvement of NK-1 receptors.

Pretreatment of mice with capsaicin resulted in approximately 40% inhibition of the polymorphonuclear leucocyte (PMN) influx elicited by interleukin-1 (IL-1) injected into a murine air-pouch. This inhibition was mimicked by two selective antagonists of neurokinin-1 (NK-1) tachykinin (TK) receptors, i.e. CP-96,345 and RP-67,580, but not by the inactive enantiomer RP-68,651. A selective NK-2 antagonist, SR-48,968, was inactive. The natural peptide, substance P (SP), and a selective NK-1 agonist, (Sar9)SP, induced PMN infiltration into the murine air-pouch, whereas a selective NK-2 agonist, (beta Ala8)NK-A(4-10), was ineffective. Moreover, SP-induced PMN accumulation was prevented by co-administration of RP-67,580 and CP-96,345, but not by RP-68,651. These findings suggest that the release of an endogenous TK, possibly SP, may occur following IL-1 injection in vivo, indicating a contributory role for neuropeptides in the PMN migration elicited by this cytokine. The action of selective agonists and antagonists suggests the involvement of NK-1 receptors.     

"Aggrecanase" activity is implicated in tumour necrosis factor alpha mediated cartilage aggrecan breakdown but is not detected by an in vitro assay.

AIMS: To develop an in vitro assay for the putative glutamyl endopeptidase, "aggrecanase", which is thought to degrade cartilage aggrecan, and to examine the role of the enzyme in tumour necrosis factor stimulated aggrecan cleavage. METHODS: Aggrecan fragments released by bovine nasal cartilage explants, with and without exposure to tumour necrosis factor alpha, were purified and analysed by western blotting and N-terminal sequencing. Intact bovine aggrecan was incubated with extracts of cartilage, lysed chondrocytes, or cartilage explant conditioned culture medium under a variety of conditions. Deglycosylated aggrecan was incubated with nasal cartilage explants. Proteoglycan breakdown was assessed by metachromatic assay of fragments in culture media, and cleavage of the substrate at the aggrecanase cleavage site was detected and measured using the antibody BC3, which recognises a neoepitope produced by aggrecanase cleavage of aggrecan. RESULTS: Aggrecan fragments generated from explants treated with tumour necrosis factor had N-terminal sequences consistent with cleavage of aggrecan at a restricted number of glutamyl bonds. Aggrecanase generated fragments were found in cartilage explant culture medium and chondrocyte monolayers. However, no aggrecanase activity could be detected in extracts of cartilage, or chondrocytes from which endogenous aggrecan fragments had been removed, under a variety of assay conditions. Deglycosylated aggrecan, added to explant cultures, efficiently inhibited endogenous aggrecan breakdown. CONCLUSIONS: Aggrecanase is active in cartilage and in chondrocyte monolayers, and its action is stimulated by tumour necrosis factor alpha. However, activity due to this enzyme could not be detected in vitro under our assay conditions, although a deglycosylated version of the substrate inhibited aggrecan breakdown in explant cultures.     

GABA-A receptor genes do not play a role in genetics of Lesch's typology in Caucasian subjects

Introduction

Lesch''s typology differentiates alcoholics into different treatment response subgroups. The effects of ethanol are mediated, to an important extent, via the GABA-ergic system.

Material and methods

We have evaluated the linkage disequilibrium patterns and haplotype frequencies of GABRG1 and GABRA2 genes in 133 alcoholics divided according to Lesch''s typology and in 145 matched controls.

Results

Besides several relationships at a threshold of statistical significance, we found no significant differences in the haplotype distribution of these genes between alcoholics and controls.

Conclusions

Lesch''s typology may not be related with the genotype of alcoholics – at least in terms of genes with an established role in the development of dependency.     

TIM-3 and CEACAM1 do not interact in cis and in trans

TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells.     

Natural killer cells do not play a dominant role in CD4+ subset differentiation in Candida albicans-infected mice.

The effects of in vivo administration of monoclonal antibodies against NK-1.1-bearing cells on the early production of gamma interferon (IFN-gamma) in vitro and development of Th1-associated immunity were studied in mice infected with a live vaccine strain of Candida albicans. At 1 and 4 days postinfection, natural killer (NK) cell-enriched fractions from the spleens of antibody-treated mice displayed a dramatic reduction in 5E6+ lymphocytes and negligible anti-YAC-1 cytotoxic activity in vitro. Nevertheless, the frequency of IFN-gamma-producing cells in those fractions was reduced by less than half, on average, by anti-NK-1.1 treatment in vivo. In addition, the antibody-treated and infected mice demonstrated unchanged T helper cell responses, as measured by yeast-specific footpad reactions, resistance to reinfection, occurrence of antibodies of different isotypes, and production in vitro of interleukin-2 (IL-2), IFN-gamma, IL-4, and IL-10 by CD4+ cells. Therefore, although NK cells may contribute to early IFN-gamma production in Candida-vaccinated mice, these cells apparently do not play a dominant role in the qualitative development of yeast-specific T helper responses.     

Decreased chondrocyte proliferation and dysregulated apoptosis in the cartilage growth plate are key features of a murine model of epiphyseal dysplasia caused by a matn3 mutation

Disruption to endochondral ossification leads to delayed and irregular bone formation and can result in a heterogeneous group of genetic disorders known as the chondrodysplasias. One such disorder, multiple epiphyseal dysplasia (MED), is characterized by mild dwarfism and early-onset osteoarthritis and can result from mutations in the gene encoding matrilin-3 (MATN3). To determine the disease mechanisms that underpin the pathophysiology of MED we generated a murine model of epiphyseal dysplasia by knocking-in a matn3 mutation. Mice that are homozygous for the mutation develop a progressive dysplasia and have short-limbed dwarfism that is consistent in severity with the relevant human phenotype. Mutant matrilin-3 is retained within the rough endoplasmic reticulum of chondrocytes and is associated with an unfolded protein response. Eventually, there is reduced proliferation and spatially dysregulated apoptosis of chondrocytes in the cartilage growth plate, which is likely to be the cause of disrupted linear bone growth and the resulting short-limbed dwarfism in the mutant mice.     

Expression of hypoxia-inducible factors in human endometrium and suppression of matrix metalloproteinases under hypoxic conditions do not support a major role for hypoxia in regulating tissue breakdown at menstruation

BACKGROUND: Classical studies in monkeys suggested that menstruation results from vasoconstriction, hypoxia and necrosis. The heterodimeric hypoxia-inducible factor (HIF) complex is critical in oxygen homeostasis via increased stability of HIF-1alpha/2alpha monomers, and these act as markers of hypoxia. We hypothesized that focal hypoxia in perimenstrual endometrium results in locally increased matrix metalloproteinases (MMP), leading to tissue destruction. METHODS: HIF-1alpha, HIF-2alpha and HIF-1beta were immunolocalized in cycling endometrium. Endometrial stromal cells were cultured under hypoxic and normoxic conditions and MMP measured by zymography and Western blots. RESULTS: HIF-1alpha and HIF-2alpha were detected in only some endometrial samples, and not confined to the perimenstrual tissue. Where present, they were primarily cytoplasmic, not nuclear. HIF-1beta was localized in epithelium, leukocytes and some decidual cells. Cultured endometrial stromal cells responded to hypoxia with increased cellular HIF-1alpha and secreted vascular endothelial growth factor. ProMMP-1 and proMMP-3 production was reduced in response to hypoxia regardless of the steroidal milieu (no added steroids, estrogen or estrogen plus progesterone). Active MMP-2 and membrane type 1 MMP but not proMMP-2 or proMMP-9 production were also inhibited by hypoxia. CONCLUSIONS: These results do not support a role for hypoxia in the focally increased production and activation of MMP observed prior to and during menstruation.     

ICAM-1 can play a major role in mediating P. falciparum adhesion to endothelium under flow

We have investigated the importance of adhesion molecule co-operation in mediating Plasmodium falciparum adhesion to endothelial cells under flow conditions. Using three laboratory parasite lines and a patient isolate which differ in their ICAM-1 and CD36-binding avidity, we found that blockade of ICAM-1 and CD36 separately reduced IRBC adhesion by up to 95 and 50%, respectively. These results confirm previous data showing that ICAM-1 and CD36 synergize to mediate adhesion, but differ in demonstrating that without ICAM-1, binding under flow conditions is severely impaired. Thus, in this system, ICAM-1 is critical for P. falciparum adhesion to activated endothelium and once bound, synergy with CD36 mediates the majority (> or =98%) of adhesion.     

CD27 and CD70 do not play a critical role in the development of experimental allergic conjunctivitis in mice

CD27, which belongs to the TNF receptor family, is a costimulatory molecule that participates in T-cell activation. Unlike costimulatory molecules such as OX40 and 4-1BB, little is known about the role CD27 plays a role in the development of experimental diseases. We asked whether CD27 and its ligand CD70 participate in the development of experimental allergic conjunctivitis (EC) in BALB/c mice, which is generated by immunization with ragweed (RW) in alum and challenged 10 days later with RW in eye drops. The roles of CD27 and CD70 were tested by intraperitoneally injecting the mice with anti-CD27, anti-CD70 or a control Ab during the induction or effector phase. Twenty-four hours after challenge, the conjunctivas, blood and spleens were harvested for histological analysis, measuring Ig levels and cytokine analysis, respectively. Regardless of when the mice were treated, anti-CD27 or anti-CD70 Ab treatment did not significantly affect the severity of EC as evaluated by conjunctival eosinophil numbers. However, anti-CD27 or anti-CD70 Ab treatment during the induction phase did significantly modulate systemic humoral and cellular immune responses. In vitro treatment of RW-primed splenocytes with anti-CD27 or anti-CD70 Ab did not affect the EC-inducing capability of the splenocytes. Taken together, CD27 and CD70 do not play a critical role in the development of EC.     

中性粒细胞蛋白-1、-2、-3在膀胱癌患者组织中的表达及与预后的相关性

目的:研究中性粒细胞蛋白(HNP)-1、-2、-3在膀胱癌患者组织中的表达及与预后的关系.方法:采用免疫组织化学法检测膀胱癌组织中HNP-1、HNP-2、HNP-3的表达情况,分析HNP-1、HNP-2、HNP-3在膀胱癌组织中的表达与膀胱癌临床病理特征的相关性及预后相关因素的关系.结果:HNP-1、HNP-2、HNP...     

Critical role for caspases 3 and 8 in neutrophil but not eosinophil apoptosis.

BACKGROUND: Apoptosis is a necessary process to control cell numbers in multicellular organisms. In many chronic inflammatory diseases, reduced cell death of different types of granulocytes is one important mechanism for cell accumulation. Here, we studied the role of caspases in neutrophil and eosinophil apoptosis in the presence or absence of granulocyte-macrophage stimulating factor and anti-CD95 monoclonal antibodies, respectively. METHODS: Granulocytes were isolated from human blood using standard protocols. Immunoblot and functional studies with cell-permeable specific peptide inhibitors were performed to analyze caspase involvement. Fas receptor and Fas ligand expression was analyzed by RT-PCR, flow cytometry, and immunoblotting. Cell death was analyzed by ethidium bromide exclusion test. RESULTS: Caspases 3 and 8 are critically involved in the regulation of neutrophil apoptosis in vitro. In contrast, these two caspases did not appear to play a major role in the regulation of eosinophil apoptosis. However, the broad-range caspase inhibitor VAD prevented eosinophil death, indicating that caspases are also involved within the apoptotic machinery of eosinophils. Functional inhibitor studies suggested that caspase 9 is crucial for both caspase 3 and 8 activation, at least in neutrophils. In contrast, spontaneous apoptosis of neutrophils or eosinophils is unlikely to be the consequence of Fas ligand/Fas receptor molecular interactions. CONCLUSION: The data of this study indicate differences in the usage of caspases between neutrophils and eosinophils.     

Stromelysin, gelatinase A and TIMP-1 in prosthetic interface tissue: a role for macrophages in tissue remodelling

Aseptic loosening of prosthetic components is the most important long-term complication of total joint replacement. To investigate the underlying destructive mechanisms, periprosthetic tissues from both well-fixed and loosened sites from six patients, undergoing surgery for aseptic loosening of knee or hip prostheses, were analysed in detail by immunohistochemical methods for the presence, of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 (TIMP-1). The tissues contained small numbers of cells positive for either collagenase, stromelysin, gelatinase A or TIMP-1; these were randomly distributed, neither specifically next to the bone interface nor to wear particles, and the number of positive cells did not correlate with macroscopic observations at operation. Gelatinase A was co-localized in cells with prolyl-4-hydroxylase, an enzyme involved in collagen synthesis. The predominant cell type in these tissues was shown to be the macrophage by the use of cell marker antibodies. Dual localization was not technically possible but the results strongly suggest that monocyte/macrophages were the primary source of gelatinase A and TIMP-1. Stromelysin was immunolocalized on connective tissue matrix in four patients, and gelatinase A in one patient, and were also observed in tissues in which there was no evidence of cellular synthesis of these enzymes. This suggests that secretion had taken place previously, resulting in enzyme bound to matrix for some time. Taken together, these data indicate that localized focal connective tissue remodelling occurs in periprosthetic tissues from both well fixed and loosened sites.     

Human neutrophil alloantigen-1a, -1b, -2, -3a and -4a frequencies in Brazilians

Human neutrophil reactive antibodies may cause clinical disorders such as transfusion-related acute lung injury, febrile transfusion reactions, alloimmune neonatal neutropenia, immune neutropenia after stem cell transplantation, refractoriness to granulocyte transfusion, drug-induced neutropenia and autoimmune neutropenia. Using the granulocyte immunofluorescence test by flow cytometry, the phenotypic frequencies of the human neutrophil alloantigens (HNA)-1a, -1b, -2, -3a and -4a were determined in 100 healthy Brazilian persons. Neutrophils were separated from blood samples by sedimentation, centrifugated and incubated with HNA-specific alloantibody plus fluorescein isothiocyanate-labeled F(ab')₂ fragments of anti-human IgG. The results showed that the phenotype frequencies of HNA-1a, -1b, -2a, -3a and -4a were 65%, 83%, 97%, 95% and 94%, respectively. We detected that neutrophils from 17% of Brazilians typed positive only with anti-HNA-1a (HNA-1a/a), 35% only with anti-HNA-1b (HNA-1b/b) and 48% reacted with both antibodies (HNA-1a/b). The frequencies found for HNA-1a and -1b were quite similar to that reported among Africans and American-Africans, but different from those found in Japanese and Chinese. In addition, our data showed that the frequencies of HNA-2, -3a and -4a in Brazilians were comparable with those observed in Caucasians. The determination of HNAs frequencies among populations with distinct racial backgrounds is important not only for anthropological reasons, but also for neonatal typing in suspected cases of alloimmune neutropenia or when patients are severely neutropenic.     

Enhanced expression of matrilysin, collagenase, and stromelysin-1 in gastrointestinal ulcers.

Programmed expression of several matrix metalloproteinases is an important feature of cutaneous wound healing. To study whether this also applies to gastrointestinal ulcer healing, we used in situ hybridization with 35S-labeled probes to localize sites of collagenase, stromelysin-1, and matrilysin expression in 26 samples representing peptic ulcers, Crohn's disease, and ulcerative colitis. In contrast to skin wounds, collagenase mRNA was not detected in the surface epithelium bordering gastrointestinal ulcer areas. However, together with stromelysin-1 mRNA, it was abundantly expressed by the granulation tissue in all types of ulcers. Signal for matrilysin mRNA and protein was detected in the mucosal epithelium bordering the ulcerations but never in the ulcer stroma. The gut basement membrane was disrupted under the matrilysin-producing epithelial cells as assessed by immunostaining for laminin. Tissue inhibitor of metalloproteinases (TIMP-1) mRNA never co-localized with matrilysin-positive mucosal epithelial cells. These data indicate that matrilysin plays a significant role in epithelial remodelling occurring in gastrointestinal ulcerations whereas collagenase and stromelysin-1 are involved in the reparative processes in the ulcer bed.