Sonja Blöcher

Presence of intestinal Mycobacterium avium subspecies paratuberculosis(MAP) DNA is not associated with altered MMP expression in ulcerative colitis

BackgroundMycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in human Crohns disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohns disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.MethodsColonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.ResultsMAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.ConclusionsThe presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

Different CREM-isoform gene expression between equine and human normal and impaired spermatogenesis

Histone-to-protamine exchange causes chromatin condensation ceasing gene expression in elongating spermatids. Gene expression of protamines is regulated by the transcription factor cAMP-responsive element modulator (CREM). Altered CREM expression results in male infertility, as shown by CREM-knock-out mice being sterile due to round spermatid maturation arrest and patients exhibiting round spermatid maturation arrest revealing a lack or substantial reduction of both CREM-mRNA and CREM-protein. Similar defects in histone-to-protamine exchange have been suggested in infertile stallions exhibiting enlarged sperm heads. The CREM-gene consists of 14 exons. Alternative exon splicing results in the production of both activator and repressor proteins. To further clarify the role of different CREM-isoforms for male infertility, the expression pattern of various CREM-isoforms during equine and human normal and impaired spermatogenesis was investigated by RT-PCR. Stallions with normal spermatogenesis expressed six activators and three repressors. In men three activators and seven different repressors were detected. In one stallion and patients with impaired spermatogenesis, only repressors were found. It is concluded that (i). stallion and man reveal a different CREM expression pattern, (ii). the expression of CREM activators is a prerequisite for normal spermatogenesis, and (iii). the lack of CREM activator expression results in male infertility.

Elements of the kallikrein–kinin system are present in rat seminiferous epithelium

Peptide hormones are involved in the paracrine regulation of several physiological processes. A possible function of the kallikrein-kinin system (KKS) in mammalian reproduction has been discussed. To evaluate its putative role in spermatogenesis, we searched for components of the KKS (kallikrein, kininases, kinin receptor) in the rat testis. Specific immunostaining demonstrated that the kininogenase tissue kallikrein was present in round and elongated spermatids. Leydig cells, Sertoli cells, peritubular cells, spermatogonia and spermatocytes were not stained. Bradykinin in the supernatant of Sertoli cell cultures was effectively degraded. The resulting metabolites were analysed by high-performance liquid chromatography (HPLC). Specific protease inhibition in the degrading experiments confirmed the occurrence of several metalloproteases on Sertoli cell membranes, including neutral metalloendopeptidases (NEP 24.11 and NEP 24.15), kininase type II (angiotensin converting enzyme, ACE), and kininase type I (metallocarboxypeptidase). Northern blots hybridized with a bradykinin B2 receptor probe showed the presence of B2 receptor mRNA in testis homogenate and Sertoli cell extract. All components of the kallikrein-kinin system are present within the seminiferous epithelium of the rat. Therefore, this paracrine peptide system may play a role in the regulation of Sertoli cell function or in the Sertoli cell-germ cell crosstalk.

Bradykinin increases intracellular calcium levels in rat testis peritubular cells via the B2 receptor subtype

RT–PCR and Western blots were used to detect bradykinin B2 receptors in testis and isolated peritubular cells of pre‐pubertal rats. RT–PCR demonstrated expression of a single transcript, whereas Western blots showed up to three specific bands that were in accordance with the described native, glycosylated and dimeric form of B2 receptor proteins, respectively. Fura‐2‐loaded peritubular cells responded with an instantaneous, linear and transient rise in [Ca2+]i after adding bradykinin. Stimulation of cells with bradykinin concentrations between 1 μM and 1 pM showed a dose dependent increase of [Ca2+]i. The calcium response to bradykinin was diminished after stimulation of peritubular cells in calcium‐free buffer. After blocking the SERCA‐pumps by thapsigargin and subsequent stimulation with bradykinin, no rise of [Ca2+]i was appreciated. Multiple stimulation of a single peritubular cell by local perfusion with a brief addition of BK (10 nM) resulted in a fast and immediate response. However, the second and third stimuli had slower rise rates and diminished [Ca2+]i peaks, showing desensitization of the kinin receptor. Addition of the bradykinin B1 receptor agonist [des‐Arg9]‐bradykinin (100 nM) to Fura‐2‐loaded peritubular cells did not change the [Ca2+]i. However, the B2 receptor antagonist HOE 140 (100 nM) strongly inhibited the bradykinin‐induced calcium response. We conclude that the bradykinin‐induced increase in [Ca2+]i, in testicular peritubular cells is mediated by the stimulation of kinin receptors of the B2 subtype.

Expression and Location of the Bradykinin B2 Receptor in Rat Testis

Abstract To investigate the possible role of the local tissue kallikrein-kinin system in spermatogenesis, we analyzed gene expression and cellular distribution of the bradykinin subtype-2 receptor (B2 receptor) in the rat testis. Reverse transcription-polymerase chain reaction revealed B2 receptor expression in testis and primary cultures of Sertoli cells and peritubular cells isolated from immature and mature rats. In situ hybridization of the B2-receptor mRNA showed intense labeling of cells on the base of the seminiferous tubule, whereas the autoradiographic signals gradually decreased toward the lumen. Immune histochemistry using testicular sections of pubertal and adult rats showed specific staining for the B2-receptor protein in cells of the adluminal compartment of the seminiferous tubules, especially on pachytene spermatocytes and spermatids. This immunostaining varied with the stages of the seminiferous cycle. The receptor protein was also observed on peritubular cells of pubertal rats. In conclusion, we demonstrated a stage-specific expression of the bradykinin B2 receptor in different cells of the seminiferous tubules of the rat testis. The results point to a possible function of the tissue kallikrein-kinin system in the local regulation of spermatogenesis.

Induction of matrix metalloproteinases and TLR2 and 6 in murine colon after oral exposure to Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohns disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1β, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP.

Leukocyte accumulation in graft blood vessels during self-limiting acute rejection of rat kidneys.

During self-limiting acute rejection preceding chronic vasculopathy, large amounts of leukocytes, predominantly monocytes, interact with the endothelium of renal allografts. We aim to characterize them and to identify targets for functional and interventional studies. Leukocytes were harvested by vascular perfusion from Fischer 344 to Lewis renal allografts or Lewis isografts, followed by flow cytometry, quantitative RT-PCR and genome-wide transcriptional profiling. Leukocyte accumulation peaked in allografts on day 9. The percentage of monocytes expressing MHC class II and CD161 was increased whereas CD4, CD11a, CD43, and CD71 expression remained unchanged. IFN-γ, IL-1β, IL-2, IL-10, TNF-α, and iNOS mRNA increased in allograft leukocytes but IL-4, IL-6, IL-12, TGF-β, and tissue factor did not. During acute rejection, 1783 genes were differentially expressed. In conclusion, graft blood leukocytes display a unique state of partial activation during self-limiting rejection. Numerous differentially expressed genes deserve further investigation as potential factors in deciding the fate of the allograft.

Tissue kallikrein and bradykinin B2 receptors in the reproductive tract of the male rat

The setting of a local tissue kallikrein kinin system (tKKS) within the reproductive organs of the male rat was investigated by analysing bradykinin subtype 2 receptor (B2R) gene expression and cellular distribution of B2R protein and the kinin‐liberating protease tissue kallikrein (tK). Reverse transcription–polymerase chain reaction showed B2R expression in testis, epididymis and prostate from prepubertal and sexually mature rats. In mature testis, in situ hybridization and immunohistochemistry localized B2R mRNA and protein besides endothelial cells of blood vessels exclusively on pachytene spermatocytes and round and elongated spermatids. B2R expression within the seminiferous tubules was found to be dependent on the stage of the spermatogenic cycle. In pre‐pubertal rat testis, B2R mRNA and protein were additionally located in peritubular cells. In the testis, specific staining for tK occurred in addition to endothelial cells of blood vessels on the acrosomal cap of round and elongated spermatids. This immunostaining was also stage‐dependent. In the epididymis, tK was detected on epithelial cells near the apical surface. The stage‐dependent specific expression of tK and bradykinin B2Rs in developing germ cells and peritubular cells suggests a potential role of the tKKS in the local regulation of spermatogenesis and seminiferous tubule function.

Expression of CREM isoforms in human and equine testis with normal and impaired spermatogenesis

Introduction. Calcium regulation and protein tyrosine phosphorylation play an important role during sperm capacitation and acrosome reaction. Genistein, a protein tyrosine kinase (PTK) inhibitor, reduces tyrosine phosphorylation of sperm proteins and inhibits progesteroneand zona pellucida-induced acrosome reaction in mammals. Previously, we demonstrated that pre-incubation of cryopreserved bovine spermatozoa with genistein inhibits progesteroneand ZP3-6 peptideinduced acrosome reaction. Our preliminary data showed no major changes in protein tyrosine phosphorylation during capacitation. However, protein tyrosine phosphorylation was more intense in cryopreserved spermatozoa than in fresh spermatozoa. This could be an indication for precapacitation due to cryopreservation. Because the assessment of changes of protein tyrosine phophorylation might not be the adequate indicator for capacitation in cryopreserved spermatozoa, we applied the chlortetracycline (CTC) fluorescence assay. Materials and methods. Cryopreserved bovine spermatozoa were incubated for 4 h with dimethyl sulphoxide (DMSO) (0.2 l1 ml) or genistein (2 lg ml). Two independent experiments yielded an increased proportion of capacitated spermatozoa (B pattern) after 4-h incubation. Results. The percentage of capacitated spermatozoa at t0 was 8 ± 0.82% and 5.25 ± 0.85% for control spermatozoa and genistein treated semen, respectively. After a 4-h capacitation period we found 35.5 ± 6.44% capacitated spermatozoa in the control sample and 33 ± 4.08% in the probe pre-incubated with genistein. Conclusion. Our preliminary results suggest that genistein has no effect upon capacitation in cryopreserved bovine spermatozoa. Effect of anti-porin type 2 antibodies upon bovine sperm motility and acrosomal status