Inhibition of Matriptase Activity Results in Decreased Intestinal Epithelial Monolayer Integrity In Vitro.

Barrier dysfunction in inflammatory bowel diseases implies enhanced paracellular flux and lowered transepithelial electrical resistance (TER) causing effective invasion of enteropathogens or altered intestinal absorption of toxins and drug compounds. To elucidate the role of matriptase-driven cell surface proteolysis in the maintenance of intestinal barrier function, the 3-amidinophenylalanine-derived matriptase inhibitor, MI-432 was used on porcine IPEC-J2 cell monolayer. Studies with two fluorescent probes revealed that short (2 h) treatment with MI-432 caused an altered distribution of oxidative species between intracellular and extracellular spaces in IPEC-J2 cells. This perturbation was partially compensated when administration of inhibitor continued for up to 48 h. Significant decrease in TER between apical and basolateral compartments of MI-432-treated IPEC-J2 cell monolayers proved that matriptase is one of the key effectors in the maintenance of barrier integrity. Changes in staining pattern of matriptase and in localization of the junctional protein occludin were observed suggesting that inhibition of matriptase by MI-432 can also exert an effect on paracellular gate opening via modulation of tight junctional protein assembly. This study confirms that non-tumorigenic IPEC-J2 cells can be used as an appropriate small intestinal model for the in vitro characterization of matriptase-related effects on intestinal epithelium. These findings demonstrate indirectly that matriptase plays a pivotal role in the development of barrier integrity; thus matriptase dysfunction can facilitate the occurence of leaky gut syndrome observed in intestinal inflammatory diseases.

Effects of Lactobacillus plantarum 2142 and sodium n-butyrate in lipopolysaccharide-triggered inflammation: comparison of a porcine intestinal epithelial cell line and primary hepatocyte monocultures with a porcine enterohepatic co-culture system.

This study was based on our previously developed double-layered enterohepatic co-culture system, composed of nontumorigenic porcine intestinal epithelial cell line (IPEC-J2) and primary culture of porcine hepatocytes. The anti-inflammatory effect of spent culture supernatant of Lactobacillus plantarum 2142 (Lp2142; 13.3%) and sodium n-butyrate (2 mM) was tested on IPEC-J2 and hepatocyte monocultures as well as on the gut-liver co-culture. To mimic inflammation, lipopolysaccharide (LPS; 1 and 10 µg/mL) was applied. Production of IL-8 and IL-6 was measured as a marker of inflammatory responses. The paracellular permeability of the intestinal epithelium was also monitored by fluoresceinisothiocyanate-labeled dextran 4 assay. Significant increase of IL-8 concentration was observed in the IPEC-J2 monoculture (P < 0.01) while the level of IL-6 was not changed following LPS treatment. Concentration of IL-8 and IL-6 was grown significantly in hepatocyte monocultures (P < 0.05 and P < 0.001) as well as in the co-culture after 10 µg/mL LPS treatment (P < 0.001 and P < 0.001). One microgram per milliliter LPS caused elevated IL-8 level in the co-culture (P < 0.001) and in the hepatocyte monoculture (P < 0.01), while it caused increased IL-6 level only in the hepatocytes (P < 0.001). Production of IL-8 was significantly decreased by butyrate in case of 1 µg/mL as well as 10 µg/mL LPS exposure in the co-culture (P < 0.001). Application of butyrate also reduced IL-6 level in the co-culture after 10 µg/mL LPS treatment (P < 0.01). Lactobacillus plantarum 2142 decreased IL-8 level after incubation with 1 µg/mL LPS (P < 0.001), while in case of 10 µg/mL LPS treatment only a marginal lowering in IL-8 (P = 0.064) release was measured. The IL-6 concentration was significantly reduced (P < 0.01 in case of 1 µg/mL LPS treatment) by Lp2142 in the co-culture. Contrarily, the elevated IL-8 and IL-6 level of hepatocytes has not been reduced in case of either butyrate or Lp2142 addition. The enterohepatic co-culture model offers a possibility for fast and reliable screening of new candidates against enteric inflammation, which are of special interest in porcine medicine and health management. According to our results, Lp2142 and butyrate both seem to be effective as anti-inflammatory agents in LPS-triggered inflammatory response, tested in the gut-liver co-culture model.

Effects of dietary sodium butyrate on hepatic biotransformation and pharmacokinetics of erythromycin in chickens.

Butyrate, a commonly applied feed additive in poultry nutrition, can modify the expression of certain genes, including those encoding cytochrome P450 (CYP) enzymes. In comparative in vitro and in vivo experiments, the effect of butyrate on hepatic CYP genes was examined in primary cultures of chicken hepatocytes and in liver samples of chickens collected from animals that had been given butyrate as a feed additive. Moreover, the effect of butyrate on the biotransformation of erythromycin, a marker substance for the activity of enzymes of the CYP3A family, was investigated in vitro and in vivo. Butyrate increased the expression of the avian-specific CYP2H1 both in vitro and in vivo. In contrast, the avian CYP3A37 expression was decreased in hepatocytes following butyrate exposure, but not in the in vivo model. CYP1A was suppressed by butyrate in the in vitro experiments, and overexpressed in vivo in butyrate-fed animals. The concomitant incubation of hepatocytes with butyrate and erythromycin led to an increased CYP2H1 expression and a less pronounced inhibition of CYP3A37. In in vivo pharmacokinetic experiments, butyrate-fed animals given a single i.m. injection of erythromycin, a slower absorption phase (longer T(half-abs) and delayed T(max)) but a rapid elimination phase of this marker substrate was observed. Although these measurable differences were detected in the pharmacokinetics of erythromycin, it is unlikely that a concomitant application of sodium butyrate with erythromycin or other CYP substrates will cause clinically significant feed-drug interaction in chickens.

Lactobacillus plantarum 2142 prevents intestinal oxidative stress in optimized in vitro systems.

Recently, there has been a growing interest to replace antibiotics' administration with the application of probiotics. The aim of our investigations was to reveal the influence of spent culture supernatant of Lactobacillus plantarum 2142 on the response of enterocytes to oxidative stress, and the spent culture supernatant's ability to protect them from oxidative injury. The experiments were performed on non-carcinogenic porcine epithelial cell line, IPEC-J2 isolated from a neonatal piglet and on human colon adenocarcinoma cell line, Caco-2. The cells cultured on membrane inserts were treated with millimolar hydrogen peroxide solution to provoke oxidative stress. The peroxide-triggered cell response profile was evaluated via determination of change in transepithelial electrical resistance, quantification of extent of cell death by 4',6-diamidino-2 phenylindole (DAPI) staining and via estimation of proinflammatory cytokine, IL-8 production using ELISA technique. Non-starter lactobacilli supernatant-mediated inhibition of peroxide-triggered upregulation of IL-8 production confirmed the antiinflammatory properties of active metabolites produced by Lactobacillus plantarum 2142 in acute oxidative stress.

Expression of claudin-1 in canine peripheral nerve sheath tumours and perivascular wall tumours. Immunohistochemical study.

AIMS: A peripheral nerve sheath tumour consists of neoplastic Schwann cells or perineurial cells, or a mixture of Schwann cells, perineurial cells and fibroblasts. The first aim of the present study was to characterise the expression of the claudin-1 tight junction protein in canine intact peripheral nerves, canine benign peripheral nerve sheath tumours (cBPNSTs), such as schwannomas, neurofibromas, perineuriomas and canine malignant peripheral nerve sheath tumours (cMPNSTs), and in different other benign and malignant canine spindle cell tumours. The second aim of the present study was to examine whether claudin-1 can help to distinguish the subgroups of canine perivascular wall tumours. METHODS AND RESULTS: The biopsy and necropsy samples (n=203) included 10 intact peripheral nerves, 20 cBPNSTs (4 schwannomas, 8 neurofibromas, 8 perineuriomas), 16 cMPNSTs, 6 psammomatous meningiomas, 6 dermatofibromas, 6 leiomyomas, 6 myxomas, 4 spindle cell hemangiomas, 2 spindle cell lipomas, 6 fibrohistiocytic nodules, 8 fibrosarcomas, 8 leiomyosarcomas, 6 myxosarcomas, 8 hemangiosarcomas, 8 anaplastic sarcomas, 8 amelanotic spindle cell melanomas, 8 histiocytic sarcomas, 8 spindle cell carcinomas, 8 myoepitheliomas, 8 complex carcinomas, 5 cardiac rhabdomyosarcomas, 4 synovial sarcomas, 5 osteosarcomas, 4 chondrosarcomas and 4 liposarcomas; 31 canine perivascular wall tumours: 10 hemangiopericytomas, 8 myopericytomas, 6 angioleiomyomas, 4 angioleiomyosarcomas, 3 angiofibromas. The immunohistochemical panel consisted of humanized antibodies: anti-claudin-1, anti-neuron specific enolase, anti-S-100 protein, anti-α-smooth muscle actin, anti-vimentin, anti-cytokeratin AE1-AE3, anti-claudin-5, anti-Melan-A and anti-heavy caldesmon, anti-calponin and anti-desmin. The intact perineurial cells, all perineuriomas, neurofibromas, cMPNSTs, spindle cell carcinomas and epithelial components of the complex carcinomas, all hemangiopericytomas and myo-pericytomas showed claudin-1 positivity. The schwannomas and other spindle shape cell tumours were negative for claudin-1. CONCLUSION: Our findings suggest that an antibody against claudin-1, in combination with other antibodies, can be used as a novel diagnostic tool to differentiate canine peripheral nerve sheath tumours from other fusocellular tumours, and anti-claudin-1, together with other antibodies, can also be used to subclassify cBPNSTs. Furthermore, analysis of claudin-1 expression can help to differentiate between subgroups of canine perivascular wall tumours.

Efficacy of protected sodium butyrate, a protected blend of essential oils, their combination, and Bacillus amyloliquefaciens spore suspension against artificially induced necrotic enteritis in broilers.

Necrotic enteritis caused by Clostridium perfringens leads to serious economical losses to the poultry industry. There is a growing need to find effective, nontoxic, antibiotic alternatives to prevent and cure the disease. In our study, the efficacy of protected sodium butyrate at 1.5 g/kg (BP70), a Bacillus amyloliquefaciens spore suspension with 10(9) cfu/g (BAL; Ecobiol), a protected blend of essential oils (1%) at 1.5 g/kg (EO), and a combination of sodium butyrate with essential oils (1%) protected with vegetable fat at 1.5 g/kg (BP70+EO; Natesse) was investigated in an artifical C. perfringens-infection model. Body weight gain, gross pathological and histopathological lesion scores, villus lengths, and villus length:crypt depth ratio was determined and compared with the control group. Broilers infected with C. perfringens and treated with essential oils or the combination of sodium butyrate and essential oils showed significantly better BW gain (P < 0.05), increased villus length and villus length:crypt depth ratio (P < 0.001), and decreased gross pathological and histopathological lesion scores (P < 0.05) compared with the control. Sodium butyrate alone and B. amyloliquefaciens spore suspension had no beneficial effects on the course of the disease in this study. According to our results, the protected combination of sodium butyrate and essential oils, as well as the protected essential oils, can be potential candidates for the prevention and treatment of necrotic enteritis in broiler chickens.

Expression of claudin-4 molecule in canine exocrine pancreatic acinar cell carcinomas.

AIMS: Claudins, integral membrane proteins are components of the tight junction structures between epithelial and endothelial cells. These transmembrane proteins create a primary barrier to prevent paracellular transport of solutes, and also restrict the lateral diffusion of membrane lipids and proteins to maintain the cellular polarity. The aim of the present study was to characterise the expression pattern of claudin-4 tight junction molecule in canine normal pancreatic tissues and in the well-differentiated and poorly-differentiated pancreatic acinar cell carcinomas in canines. METHODS AND RESULTS: The necropsy samples included canine intact pancreatic tissues, and canine well-differentiated and poorly-differentiated pancreatic acinar cell carcinomas samples. Claudin-4 was detected as an intense lateral membrane labelling of acinar cells in all intact pancreatic tissues. The intact epithelial cells of the different ducts were negative for the claudin-4 molecule. All primary and secondary canine well-differentiated exocrine pancreatic acinar cell carcinoma tissues showed intense apical lateral positivity for the claudin-4 molecule. All primary and secondary poorly-differentiated pancreatic acinar cell carcinoma tissues showed diffusely the loss of claudin-4 expression. CONCLUSION: Consequently, we hypothesize that the loss of expression of claudin-4 plays a role in the progression of canine pancreatic acinar cell carcinoma and may lead to cellular detachment, disorientation and invasion of these pancreatic cancers. Furthermore, claudin-4 can be used as an immunohistochemical marker to distinguish canine well-differentiated and undifferentiated exocrine pancreatic acinar cell carcinomas.

Claudin-7 protein differentiates canine cholangiocarcinoma from hepatocellular carcinoma.

UNLABELLED: The aim of the present study was to characterise the expression pattern of claudin-7 tight junction protein in canine normal liver, hyperplastic and primary neoplastic lesions of the canine liver and whether this tight junction protein can help differentiate canine cholangiocarcinomas from canine hepatocellular carcinomas. METHODS AND RESULTS: Necropsy samples included 15 canine normal liver tissue samples, 10 hepatocellular nodular hyperplasias, 6 hepatocellular adenomas, 15 well-differentiated and 6 poorly differentiated hepatocellular carcinomas, 6 cholangiocellular hyperplasias, 10 cholangiocellular adenomas, 15 well-differentiated and 6 poorly differentiated cholangiocarcinomas, 6 normal extrahepatic bile ducts, 8 normal gall bladder tissue samples, and 5 cystic mucinous hyperplasias of the gall bladder. In all canine normal liver tissue samples the hepatocytes were negative for claudin-7 and the normal biliary epithelial cells showed intense basolateral membrane claudin-7 positivity. In all cholangiocellular hyperplasia samples and in all cholangiocellular adenoma samples the benign cholangiocytes showed intense basolateral membrane positivity for claudin-7. In all samples of the well-differentiated and poorly differentiated cholangiocarcinomas, the malignant neoplastic biliary epithelial cells showed intense basolateral membrane positivity for claudin-7. Neither the hyperplastic nodules of the liver cells nor the hepatocellular adenomas reacted with claudin-7. The well-differentiated and poorly differentiated hepatocellular cancers were negative for claudin-7. The epithelial cells of canine normal extrahepatic bile ducts, gall bladder and cystic mucinous hyperplasias of the gall bladder showed intense basolateral membrane positivity for claudin-7. Differences in the intensity of claudin-7 reaction were not apparent among different types of proliferative lesions of cholangiocytes or degrees of cellular differentiation of neoplastic biliary epithelial cells. CONCLUSION: Consequently, we hypothesize that claudin-7 is an excellent immunohistochemical marker of the cholangiocellular differentiation in canines and can be used to detect benign and malignant proliferative lesions of the canine biliary tract. It can also help to differentiate canine cholangiocarcinomas from hepatocellular carcinomas.

Expression of claudin-1, -3, -4, -5 and -7 proteins in low grade colorectal carcinoma of canines.

UNLABELLED: The aim of the present study was to characterise the expression pattern of claudin-1, -3, -4, -5 and -7 tight junction proteins in canine normal colorectum and in the low-grade, tubulopapillary colorectal carcinoma in canines. METHODS AND RESULTS: The biopsy samples included 10 canine normal colorectal tissues and 20 canine low grade colorectal carcinomas (CLGCCs). The canine normal colorectal mucosa was negative for claudin-1. Claudin-1 was detected as a non-diffuse intense membrane labelling of neoplastic epithelial cells in low grade colorectal cancer in canines. Fifty five per cent of all tumours showed a weak cytoplasmic pattern of staining for claudin-1 protein. The normal colorectal mucosa showed diffuse punctate positivity for claudin-3. Claudin-3 was detected as an intense lateral membrane labelling of tumour cells in CLGCCs. Claudin-4 expression in surface and crypt epithelial cells of the intact colorectal mucosa in canines was punctate. Claudin-4 molecule was detected as a lateral membrane labelling of neoplastic cells in CLGCCs. The epithelium of the CLGCCs and the low grade colorectal carcinoma were negative for claudin-5. The surface and crypt epithelial cells of the canine normal colorectal mucosa showed a diffuse lateral membranous pattern of staining for claudin-7. Claudin-7 molecule was detected as an intense membrane labelling of neoplastic cells in CLGCCs. Seventy per cent of all tumours showed weak cytoplasmic positivity for claudin-7. CONCLUSION: Consequently, we hypothesize that claudin-1 plays a role in the progression of CLGCCs. Further functional studies are needed to clarify the biological role of the mislocalization of the claudin-1 molecule from cell membrane to the cytoplasm in CLGCCs. Lower claudin-4 expression suggests that reduced expression of claudin-4 molecule may lead to cellular disorientation, detachment and invasion of CLGCCs. Further functional studies are needed to clarify the biological role of overexpression and mislocalisation of claudin-7 in CLGCCs.

Claudin-5 protein is a new differential marker for histopathological differential diagnosis of canine hemangiosarcoma.

AIMS: Claudin-5 protein is an endothel-specific claudin, present in tight junctions. To evaluate its usefulness as a differential diagnostic marker of canine hemangiosarcomas, the expression of claudin-5 molecule was studied in different canine tumours of vascular origin. METHODS AND RESULTS: Ninety two canine neoplastic tissue samples obtained from necropsies and biopsy specimens were routinely processed and stained immunhistochemically for claudin-5. The neoplastic endothelial cells of canine hemangiosarcomas, hemangiomas, and lymphangiomas showed a strong membrane immunoreactivity for claudin-5, but the other investigated canine malignant and benign tumours, including fibrosarcomas, myxo-, leiomyo-, cardiac rhabdomyo-, neurofibro-, synovial-, osteo-, and chondrosarcomas, spindle cell melanomas, hemangio-pericytomas, benign fibroblast proliferations, and leiomyomas were negative for this endothelial marker. In these non-vascular canine tumours intense immunostaining was detected in the endothelial cells of the incorporated intratumoural vessels and neovasculature. The canine splenic hematomas induced by hemangiosarcomas were distinguished from splenic hematomas induced by non-neoplastic lesions by the means of claudin-5 protein. In hemangiosarcomas the percentage of positive neoplastic endothelial cells was higher, and stronger when using the claudin-5 molecule compared to CD31 and vWf. CONCLUSION: The results show that claudin-5 molecule can be used as a new differential marker, and could also be of a diagnostic value in the differential diagnosis of canine hemangiosarcomas from sarcomas of other origin with hemorrhages or increased vascularization. Claudin-5 could help to reveal neoplastic proliferation of endothelial cells causing splenic hematomas and differentiate these tumours from non-vascular neoplastic splenic lesion. The immunohistochemical detection of the claudin-5 protein had a higher sensitivity than CD31, and vWf antigen in case of canine hemangiosarcomas.

Expression of claudin-1, -2, -3, -4, -5 and -7 proteins in benign and malignant canine mammary gland epithelial tumours.

Claudins are tight junction proteins expressed by epithelial and endothelial cells. The present study has evaluated the expression of claudin-1, -2, -3, -4, -5 and -7 in 115 hyperplastic and neoplastic lesions of the canine mammary gland and compared this expression with that of normal mammary epithelium. The lesions studied included lobular hyperplasia (n=15), simple adenoma (n=20), non-infiltrating carcinoma in situ (n=20) and infiltrating carcinomas of histological grades I, II and III (n=20 of each). There was strong expression of claudin-1, -3, -4, -5 and -7 by epithelia within examples of lobular hyperplasia and simple adenoma, and strong expression of claudin-3 and -4 by non-infiltrating carcinomas and all three grades of infiltrating carcinoma. By contrast, there was reduced expression of claudin-5 and -7 by non-infiltrating and infiltrating carcinomas and the expression of these two molecules was inversely correlated with histological grade. Claudin-1 was expressed focally within carcinoma in situ, but this molecule was not detected in any invasive carcinoma. Claudin-2 was weakly expressed within areas of lobular hyperplasia and by simple adenomas, but was not expressed by any carcinoma cells. These results suggest that loss or reduction of expression of claudin-1, -2, -5 and -7 may lead to cellular disorientation, detachment and invasion in canine mammary neoplasia.

Na+/H+ exchange in primary, secondary and n-butyrate-treated cultures of ruminal epithelial cells: short communication.

Rate of amiloride-sensitive Na+ uptake into cultured rumen epithelial cells was studied in order to clarify the influence of culture conditions on Na+/H+ exchange (NHE). Cell cultures were exposed to Na-n-butyrate or not for seven days or subcultured. On the 14th day of culturing, primary cell cultures without butyrate exposure showed both non-stratified and stratified growth. Na-n-butyrate treated 14-day-old cultures and 3-day-old subcultures contained mostly non-stratified, i.e. non-keratinised cells. Both n-butyrate treatment and subculturing increased total and amiloride-sensitive Na+ uptake. Our results indicate that Na+ uptake via NHE is determined by the amount and the ratio of non-stratified (non-keratinised) cells.

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157.

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.

Inverse hierarchy of vimentin epitope expression in primary cultures of chicken and rat astrocytes: a double-immunofluorescence study.

Vimentin contributes to the cytoskeleton of different cell-types, among them glial cells. We report here that different forms of this protein, distinguishable by the monoclonal antibodies Vim3B4 and V9, are species-specifically expressed in cultures of glial fibrillary acidic protein (GFAP) positive, primary astrocytes of the chicken and rat. Most cells in the cultures co-expressed GFAP and one of the two vimentin epitopes. The Vim3B4 positive epitope was present in chicken astrocytes, while the V9 positive was not. Inverse situation was found in the astrocytes of rat. In vitro age of the cells did not influence the hierarchy of vimentin epitope expression with respect to species-specificity. Our result shows that the different vimentin expression program of cultured astrocytes of the chicken and rat is preserved under in vitro conditions. The presented data support the concept of the species-specific regulation of vimentin forms in glial cells of the central nervous system.

Attachment of different Escherichia coli strains to cultured rumen epithelial cells.

The attachment to fully characterized primary rumen epithelial cell cultures of Escherichia coli strains isolated from different animal species and expressing F1-F4 or F17 fimbriae was examined. As the cell cultures contained stratified (keratinized) and non-stratified (non-keratinized) cells which grew either confluently or non-confluently, the strength of attachment of the different bacterial strains was assessed in relation to the differentiation state of the cells. Thus, strains having F1 fimbriae attached to all types of cultured cells, while strains with F2 and F3 fimbriae did not bind at all. E. coli strains having F4 or F17 fimbriae attached only to non-keratinized cells, particularly to confluent areas. As membrane glycosylation is known to change with differentiation (keratinization), our results suggest that the attachment of fimbriated E. coli strains which were capable of binding to rumen cells was more likely to be dependent on differentiation than the host specificity of the bacteria.

Characterization of Na+/H+ exchange in sheep rumen epithelial cells kept in primary culture.

The study aimed at testing whether Na+/H+ exchange activity is maintained during isolation and cultivation of rumen epithelial cells (REC) and whether Na+/H+ exchange is altered during proliferation and differentiation. REC were isolated from sheep rumen epithelium by fractional trypsinization and further cultivated for up to 21 days. Characteristics of Na+ uptake into cultured REC were the same as previously described for the Na+/H+ exchange in the intact rumen epithelium. Na+ uptake was increased by intracellular acidification and was inhibited by high doses of amiloride. Amiloride inhibited 3H-thymidine incorporation into REC at concentrations similar to those found for the inhibition of Na+ uptake. Fetal calf serum, a growth factor, stimulated amiloride-sensitive Na+ uptake. We therefore conclude that cultured rumen epithelial cells express Na+/H+ exchange and that the activity of the exchange is at least correlated to the intensity of proliferation.

Species-specificity of glial vimentin as revealed by immunocytochemical studies with the Vim 3B4 and V9 monoclonal antibodies.

Two monoclonal antibodies directed against vimentin, Vim 3B4 and V9 could distinguish between vimentins originating from certain species, when tested on cell lines (Bohn et al, 1992). Our comparative immunohistochemical studies in the rat and chicken brain with the same antibodies suggest the coexistence of two vimentin forms in the glial cells of both species. One of these forms bearing the epitope present in the respective non-glial cell lines is present in astrocytes and Bergmann glia independently of the ontogenic state of the animal. The other epitope appeared also mutually in both species, albeit its expression was more restricted. These patterns suggest that in these two species, the expression of the different vimentin forms might be differently regulated.

Lectins as markers of rumen epithelial cell differentiation.

Lectins of different carbohydrate specificities (GNA (Galanthus nivalis), con A (Canavalia ensiformis), VFL (Vicia faba), PSL (Pisum sativum), LCA (Lens culinaris), PNA (Arachis hypogaea; with or without prior neuraminidase treatment), WGA (Triticum vulgare), SBA (Glycine max), UEA-I (Ulex europaeus), LPA (Limulus polyphemus), BS-I B4 (Bandeiraea simplicifolia, isolectin B4)) were explored for use as differentiation markers of rumen epithelial cells in vivo and in vitro. Lectins specific for mannose (GNA), mannose/glucose (con A, VFL, PSL and LCA), N-acetylglucosamine (WGA) or for N-acetylneuraminic acid (LPA) reacted generally with all types of rumen epithelial cell from both rumen tissue and cell culture. They were, therefore, not suitable markers of epithelial differentiation. SBA was unsuitable because, although it reacted with both tissue and cultured rumen epithelial cells, it was also bound to non-stratified areas of primary rumen epithelial cell cultures. Both BS-I B4 and PNA (after neuraminidase treatment) had to be ruled out because they did not react with differentiated rumen tissue epithelial cells, although they did bind to both stratified and non-stratified cultured cells. In contrast, UEA-I reacted strongly with differentiated rumen epithelial cells both from rumen tissue and cell cultures and therefore appears to be a good general marker for rumen epithelial cell differentiation.

The adherence of three Streptococcus bovis strains to cells of rumen epithelium primoculture under various conditions.

Three Streptococcus bovis strains were tested in biotype assay and examined for the adherence to cells of rumen epithelium primoculture. The adherence pattern of ruminal streptococci in phosphate buffered saline at pH values ranging from 4.1 to 8.5 was determined. Our isolates of Streptococcus bovis strains adhered best at pH 7.0-7.3. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. Protease treatment dramatically decreased the adherence of all Streptococcus bovis strains, thus suggesting that the determinants responsible for the adherence are largely proteinaceous. Carbohydrates could be also significantly involved in the active sites of bacterial surface because metaperiodate-treated cells adhered much more poorly than control, sodium iodate-treated cells. Addition of carbohydrates (lactose, maltose and saccharose) had no significant effect on the adherence of Streptococcus bovis strains although a slight decrease in the adhesion was detected.

Acute metabolic and hormonal effects of intravenously administered sodium n-butyrate in untreated and alloxan-diabetic sheep.

Experimental diabetes was induced in 4 wethers of the Mutton Merino breed by intravenous injection of alloxan (75 mg.kg-1) in order to determine its impact on plasma glucose, immunoreactive insulin, free fatty acids (FFA), cholesterol, phospholipids, triglycerides, D-(-)-3-hydroxybutyrate (D-3-HB), bilirubin and aspartate aminotransferase (ASAT) as well as on the changes of these parameters brought about by an intravenous infusion of sodium n-butyrate (1 mmol.kg-1). Alloxan administration caused a significant elevation of plasma glucose, FFA, triglycerides, cholesterol, phospholipids, D-3-HB and bilirubin and a decrease of the level of immunoreactive insulin. The increase in glucose level brought about by a bolus injection of sodium n-butyrate in untreated sheep did not appear in alloxanized animals. Thus, it is suggested that the lack of hyperglycaemic response in diabetic sheep was due to the absence of liver glycogen stores. Unexpectedly in alloxan-diabetic sheep, a decrease in the plasma level of FFA occurred after the administration of sodium n-butyrate. Therefore, it may be assumed that beside insulin other factors may contribute to the decrease of FFA under these conditions.