Short communication: progression of Johne's disease curtailed by a probiotic.

The naturally occurring inflammatory bowel disease Johne's, caused by Mycobacterium avium ssp. paratuberculosis (MAP), has many clinical manifestations in common with the human inflammatory bowel disease Crohn's disease. In addition, both lack preventive and curative therapies. Because a high percentage of Crohn's patients harbor MAP, it is not surprising that MAP is at the center of controversy as to its contribution. Special concern is being raised as to what role, if any, food animals play in transmission of MAP to humans. Because management practices, presently considered the best way to control the spread of MAP, have not and most likely will not eliminate MAP from food animals, other preventive or curative measures are needed. The results presented herein show that a unique bacterium, Dietzia ssp. C79793-74, used as a probiotic, was therapeutic for adult paratuberculosis animals, and resulted in a cure rate of 37.5%.

Proinflammatory properties of IL-4 in the intestinal microenvironment.

IL-4 is involved in type 2 T helper cell (Th)2-type immune responses and, in some cases, can promote Th1 responses. However, the proinflammatory potential of IL-4 alone is unclear. In this study, we examined the ability of IL-4 to induce colitis after its overexpression in the colon using an adenoviral vector (Ad5) and compared results with those obtained after overexpression of IL-12, a cytokine implicated in several models of colitis. Overexpression of IL-4 or IL-12 caused a fatal colitis within 24 h in 60% of animals and was dose and strain dependent. IL-12-induced colitis was accompanied by the local expression of IFN-gamma and TNF-alpha but not IL-4 mRNA and protein. Conversely, IL-4-induced colitis was accompanied by the local expression of IL-4 and TNF-alpha but not IFN-gamma mRNA and protein. The Ad5-IL4-induced colitis did not persist beyond 3 days and was present in recombinase activation gene-2 (RAG-2)-/- mice but not in STAT6-/- mice. Acute lethal colitis induced by Ad5IL12 was T cell mediated and IFN-gamma receptor (IFN-gamma R) dependent. Furthermore, TNF-alpha was found to be important in the pathogenesis of Ad5IL-4 and Ad5IL-12-induced colitis. Results of this study indicate that IL-4 alone can act as a proinflammatory cytokine in the gut of normal mice, inducing a rapid onset and short-lived colonic injury while maintaining a Th2-type cytokine profile that functions via a local T cell-independent mechanism involving TNF-alpha.

Regulation of bovine E-selectin expression by recombinant tumor necrosis factor alpha and lipopolysaccharide.

Induction of adhesion molecules by cytokines and LPS is an important mechanism of regulating leukocyte migration into tissue. Expression and regulation of E-selectin may be differentially influenced by the stimuli involved with effects on mRNA or surface protein kinetics. Surface protein and mRNA expression kinetics of bovine E-selectin were measured and compared in primary cultures of bovine aortic endothelial cells (BAEC) stimulated for various periods of time with recombinant bovine tumor necrosis factor alpha (rbTNF-alpha) or Escherichia coli lipopolysaccharide (LPS). E-selectin mRNA expression was measured via quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) using a construct that contained multiple synthetic oligonucleotides for several bovine adhesion molecules and cytokines. Surface expression of E-selectin was measured by flow cytometry. Unstimulated BAECs expressed minimum or no E-selectin on the surface. A low number of endothelial cells expressed surface E-selectin as early as 1h post-stimulation and surface expression was sustained after both stimuli for 24-72h. Mean fluorescence intensity (MFI) indicated peak surface concentration of E-selectin at 6 h post-stimulation after LPS followed by a gradual decrease to 72h without returning to baseline values. Mean fluorescence intensity following stimulation with TNF-alpha increased slightly between 0 and 72h. The pattern of mRNA expression differed between stimuli. LPS-stimulated BAECs expressed peak amounts of E-selectin mRNA at 6 h, followed by a decline to baseline by 24 h. Conversely, BAECs stimulated with rbTNF-alpha expressed significantly (p pound 0.05) higher amounts of mRNA at 1h than compared to unstimulated controls (0 h), but this decreased to below baseline levels by 6h; followed by a gradual increase and eventually a sharp increase between 18 and 72 h. To account for the lack of correlation between mRNA and protein expression, it was hypothesized that shedding of surface E-selectin accounted at least in part, for the large increase in mRNA expression seen at 18-72h. Culture supernatants from rbTNF-alpha-treated BAECs were harvested, and tested for the presence of shed E-selectin using ELISA. Unstimulated culture supernatants contained little or no E-selectin. Between 6 and 48 h, the concentration of E-selectin in culture supernatants from rbTNF-alpha-stimulated BAECs increased approximately two-fold, suggesting that the sharp increase in E-selectin mRNA expression around 18 h may be related to significant loss of surface E-selectin during this period.

Rejection of a kidney transplant does not always lead to priming of cytotoxic T cells against mismatched donor HLA class I antigens.

BACKGROUND: Previous studies showed that graft rejection is often associated with the presence of primed cytotoxic T cells (CTLs) with a high avidity for donor cells. Similar high avidity CTLs have been found in individuals who have formed IgG anti-HLA antibodies. The presence of such CTLs to a specific HLA mismatch is therefore considered to be a reflection of an activated immune system, and a contraindication for retransplantation with a donor sharing this particular HLA class I mismatch. METHODS: In our study we investigated whether patients have always primed CTLs against all individual HLA class I mismatches present on a rejected graft. Therefore, 14 patients who had undergone transplantectomy after irreversible kidney graft rejection were analyzed with respect to donor-specific CTLp frequencies and the presence or absence of high avidity CTLs directed against HLA class I mismatches present on the rejected graft. RESULTS: Patients, who have not formed anti-HLA antibodies against the donor have mainly naive CTLs. Most of the patients, that have developed IgG anti-HLA antibodies against a donor mismatch, have primed CTLs directed against that particular mismatch. However, patients with IgM anti-HLA antibodies only, and patients with IgG anti-HLA antibodies in historical sera but no IgG anti-HLA antibodies in current sera, have mainly naive CTLs against the donor HLA mismatch. CONCLUSION: Our results suggest that it is not always necessary to exclude repeated HLA class I mismatches for a subsequent transplantation. In addition to good anti-HLA antibody screening, the CTLp-assay may be a useful tool for donor-selection in retransplant candidates.

Regulation of bovine intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on cultured aortic endothelial cells.

Endothelial cell adhesion molecules (AM) intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) are important mediators of cell migration from blood into tissue. The kinetics of ICAM-1 mRNA and VCAM-1 protein expression in bovine aortic endothelial cells (BAEC) were determined using quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) and flow cytometric analysis, respectively. Stimulation of BAEC with recombinant bovine tumor necrosis factor alpha (rbTNF-alpha) resulted in protein expression of VCAM-1 on less than 5% of all cultured BAECs at 1h post-stimulation, followed by a significant increase at 3h that was maintained until 48h when the proportion of VCAM-1 positive (+) cells decreased significantly, but not to baseline proportions. The expression kinetics for VCAM-1 were similar on cells stimulated with lipopolysaccharide (LPS) except at 24h, when there was a significantly higher proportion of BAEC expressing VCAM-1 than at any other time. The expression of ICAM-1 mRNA differed significantly between stimuli. Expression of ICAM-1 mRNA peaked at 12-18h and then diminished but remained at amounts above baseline up to 72h after stimulation. Stimulation with LPS induced a significant increase in ICAM-1 mRNA expression between 1 and 12h after which the amounts rapidly decreased to baseline. In summary, different stimuli produced similar expression kinetics of VCAM-1 surface protein but different kinetics of ICAM-1 mRNA expression.

Pregnancy can induce long-persisting primed CTLs specific for inherited paternal HLA antigens.

Previous studies showed that pregnancy can prime the maternal cellular immune response directed against paternal HLA antigens. Primed CTLs specific for inherited paternal HLA antigens (IPA) were found in women who had formed HLA allo antibodies, whereas naive CTLs were present in women who did not form antibodies against the paternal HLA antigens. As HLA allo antibodies may disappear in time, it is not clear which women on the waiting list for transplantation have been sensitized to paternal HLA antigens and are at risk for graft rejection if paternal HLA antigens are shared by the donor organ. The presence of primed CTLs specific for a particular antigen is considered to be a reflection of sensitization.In the present study we investigated whether these primed CTLs persist in women who had been pregnant and had formed antibodies against the inherited paternal HLA class I antigens. For this purpose 14 women who had their last pregnancy 10 years ago were analyzed with respect to IPA-specific CTLp frequencies and the presence of high avidity CTLs directed against inherited paternal HLA class I antigens. Although primed CTLs specific for IPA's were found more frequently in women with persisting alloantibodies, they still can be detected when the antibodies have disappeared. The current data show that primed CTLs directed against inherited paternal HLA antigens towards which antibodies have been formed in the past can persist for more than 10 years after pregnancy. The cellular test used in our study can be useful to detect presensitization in women with a history of pregnancy, who enter the waiting list for transplantation.

Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows.

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4+, CD8+, and WC1+ (Workshop Cluster 1; marker for gammadelta T cells) lymphocyte phenotypes and expression of LFA-1 and L-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4+ T cells were higher (p < or = 10.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8+ cells. Expression of L-selectin was lower (p < or = 0.05) on CD4+ cells and higher on CD8+ cells from milk. The WC1+ subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of L-selectin+ WC1+ cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in L-selectin expression may indicate a role for this molecule in regulating the movement of CD8+ and WC1+ T cells into the bovine mammary gland.

Effects of peripartum stress and health on circulating bovine lymphocyte subsets.

Alterations in the proportions of peripheral blood lymphocyte subsets was monitored using flow cytometry, before, during and after the peripartum period (week-8 to week (wk) 16) of dairy cows, when pregnancy and lactational stresses prevail and disease incidence is highest. A health code was assigned to each animal at each sampling time, and a subset of non-pregnant, non-lactating (NPNL) cows were sampled for comparisons, and to examine the effect of pregnancy and lactation only, on lymphocyte subset proportions. Results were expressed as the percentage of positive stained cells expressing CD2, CD4, CD8, WC1(gamma delta), IgM and class II major histocompatibility complex (MHC-II) molecules, as determined by flow cytometry. Comparisons of subset proportions were made across time and between health status categories. Mean fluorescence intensity (MFI) values were also examined across the periparturient period for CD2, CD4, CD8 and gamma delta T cell subsets. All T cell subsets varied significantly during the perparturient period in healthy cows, especially around parturition. B cell and MHC-II+ populations remained relatively constant until after calving and then significantly (P < or = 0.06) decreased. Compared to NPNL cows, all T cell subsets were significantly lower in healthy cows at most time points, whereas B cell and MHC-II+ cells were comparable to the NPNL values. CD2, CD4, and CD8 subsets decreased significantly at wk-3 and returned to initial subset proportions by wk 16. Conversely, the WC1 population increased at wk-3 relative to calving but declined to approximate initial proportions at calving. Health status had no significant overall effect on any subset unless it was separated by weeks in which case there were interactions between health and week for CD2 and CD8. Results indicate that variations in T lymphocyte subsets and in the concentrations of surface marker molecules (MFI) occur more as a result of pregnancy and lactation than health status in the dairy cow and that these factors have the least influence upon B cell and MHC-II+ populations during the periparturient period.

Biomechanics of synthetic augmentation of ligament reconstructions.

Synthetic augmentation of an anterior cruciate ligament reconstruction is intended to provide stress protection to the biological graft during the early healing period when the graft tissue is weak. It is hypothesized that the ligament augmentation device carries the majority of the load during the early post-operative period, and as the graft tissue heals the load is gradually transferred from the ligament augmentation device to the biological graft. This transfer of load to the biological graft occurs as the stiffness of the remodeling graft tissue increases. Cadaver studies of biological grafts augmented with the Kennedy LAD¿ demonstrated that the LAD carried 28% of the load with a high-stiffness bone-patellar tendon-bone graft and 45% of the load with a lower-stiffness semitendinosus/gracilis graft, which supports the hypothesis that load sharing is dependent on the stiffness of the graft tissue. Cadaver studies have also demonstrated high fixation strength with the Kennedy LAD¿. A new bioabsorbable LAD with long-term strength retention has been developed to avoid potential complications caused by the implant material.

Use of a phosphophoryn-Ca(+2)-collagen composition that mimics a mineralization front in unicortical defects in long bones.

The present study was designed to ascertain if dynamic ionic matrices that mimic the mineralization front could be used as active scaffolds for bone repair. Dentinal phosphophoryn calcium salts were extracted from unerupted bovine dentine using chatopic buffers and EDTA. The phosphophoryns were subsequently isolated following precipitation with CaCl2. The phosphophoryn-Ca+2 salts were then mixed with pepsin solubilized bovine skin collagen and lyophilized into hardened sponges. Three groups of 4 beagle dogs were employed such that one leg served as an experimental test site for a mechanical wound, while the contralateral leg served as a control. Animals were sacrificed at 1,3, and 6 month intervals. The test specimens were harvested, fixed, and processed for routine histology, examined with image histomorphometric analysis, and scored. Tabulation of these data indicated that phosphophoryn-Ca(+2)-collagen enhances the repair of mechanically formed osseous defects in the distal femur of beagle dogs. This enhanced rate of bone repair was manifest by earlier filling of bony voids with osteoid and trabecular bone. Eventually, this process was followed by recortification of the surface defects. These data suggest that components derived from a mineralization front may influence bone formation in unicortical defects within long bones.

Expression of constitutive and inducible HSP70 and HSP47 is enhanced in cells persistently spread on OPN1 or collagen.

Cells persistently spread on OPN or collagen survive heat shock better than cells transiently spread on fibronectin or tissue culture plates. Thus, a central question is whether constitutively or inducible stress proteins are enhanced in cells grown on adhesive proteins that maintain a persistent spread cell shape. Levels of Hsp 72,73, and colligin/Hsp47 were determined by Western blot analyses. The inducible Hsp 72 was prominently expressed following heat shock in cells grown on OPN or collagen, but not in cells plated on fibronectin coated substratum or on tissue culture plates. Colligin/Hsp 47 and Hsp 73 manifested a similar pattern of expression indicating that these adhesive attachment proteins accommodate cell function through organization of cell architecture.

Persistent spreading of ligament cells on osteopontin/bone sialoprotein-I or collagen enhances tolerance to heat shock.

Fibronectin (FN), bone sialoprotein-I (BSP-I), Type I collagen, and a number of synthetic peptides containing the integrin attachment sequence (RDG) were evaluated for their ability to affect stress tolerance in osteo-ligament cells (OL). The attachment and spreading of OL cells was determined by the method of Klebe (1974) and Akiyama et al. (1986). Survival from heat shock was evaluated after the methods of Gerner et al. (1976). These studies showed that FN, BSP-I, and synthetic RGD peptides enhance attachment of OL cells. Increased survival from heat was limited to cells spread on fibronectin, BSP-I, and Type I collagen. OL cells that persistently spread on BSP-I and Type I collagen had more survivors than cells demonstrating transient spreading on FN. These studies indicate that (a) cell spreading is a prerequisite for stress tolerance and (b) enhanced stress tolerance is mediated by protein sequences other than those immediately surrounding the RGD sites in native proteins.

[Nedocromil sodium therapy in asthma patients. Therapeutic effect in addition to treatment with oral theophylline and inhaled bronchodilator agents]. / Nedocromil-Natrium-Therapie bei Asthmapatienten. Therapeutische Wirkung zusätzlich zu einer Behandlung mit oralem Theophyllin und inhalativen Bronchodilatatoren.

131 asthmatics aged 12-65 years, who still had symptoms despite inhaled or oral bronchodilators, were included in this double-blind group comparative study involving nedocromil sodium (2 puffs of 2 mg each twice daily) and placebo (2 puffs twice daily). The study was carried out at 8 centers over six weeks. Under nedocromil sodium, cough, dyspnea and severity of attacks were reduced significantly. Overall patient assessment also clearly favoured the active substance. Nedocromil sodium was also superior to placebo in terms of the improvement of lung function (FEV1, FVC and PEFR). 26 patients complained of unusual symptoms (12 under nedocromil sodium, 14 under placebo). Nedocromil sodium proved to be an effective, safe and well-tolerated drug in the antiinflammatory long-term treatment of reversible obstructive airways disease.

Biomechanics of fascia lata ligament replacements: early postoperative changes in the goat.

Mechanical properties of fascia lata autografts used to replace the anterior cruciate ligament (ACL) in the goat were measured at 0, 2, 4, and 8 weeks after surgery. The ACL was replaced in the right knee of 50 animals divided equally into two groups according to graft fixation technique: (a) two smooth staples at each end, with the tissue pulled back toward the joint over the first staple and (b) reinforced fixation with a spiked bushing placed through the tissue and a 3-cm-long flat polypropylene braid sutured to each end of the graft. Eleven unoperated contralateral knees were tested as controls. All statistically significant effects of the reinforced versus staple fixation were observed at 0 weeks, with the reinforced group showing less anteroposterior (AP) translation of the joint and greater maximum force and stiffness of the femur-graft-tibia units. The reinforced group had increased AP translation and decreased strength and stiffness by 2 weeks after surgery. Increased AP translation resulted primarily from increases in the low-stiffness region of the force-displacement curve (primary AP translation) and to a lesser extent from increased translation in the high-stiffness region (secondary anterior translation). Failures at 0 weeks with the reinforced fixation occurred at the bushing or end of the reinforcing braid, while all but one of the later failures occurred in the tissue mid-substance. In the staple group, maximum force was greater at 8 weeks than at 0 weeks, as the failure locations changed from the fixation to the tissue mid-substance.(ABSTRACT TRUNCATED AT 250 WORDS)

Noncollagenous phosphoprotein derived from teleostean fish-scales.

A noncollagenous phosphoprotein derived from teleostean fish-scales is extracted by the use of a dissociative extraction scheme, utilizing 4 M guanidine-HCl, pH 7.4, followed by a 4 M guanidine-HCl, 0.5 M EDTA, pH 7.4. DEAE-cellulose chromatography and repeated Sepharose CL-6B chromatography are used to purify this protein. The isolated protein contains extensive quantities of aspartic and glutamic acids in addition to O-phosphoserine and O-phosphothreonine. The apparent molecular weight of the phosphoprotein as determined by Sepharose CL-6B chromatography is 13 000.

Electron-microscopical studies of conformational changes in dentinal phosphophoryn.

p67tinal phosphophoryn was isolated and purified from unerupted calves molars by the methods of Butler et al. (1981). The resulting proteins were concurrently analyzed by circular dichroism and electron microscopy after low-angle rotary shadowing. Electron microscopy of these proteins in aqueous solutions revealed extended bead-like chains that possessed intramolecular variations in morphology. The addition of calcium ion or methanol to solutions of these proteins produced circular dichroism spectra indicative of more ordered structures. Electron microscopy of these preparations revealed aggregates of 25-30 nm disc-like structures. Although correlations of domain sequences and structure were not possible, the resulting structures did possess molecular morphologies that are compatible with some of the functional roles advocated for these proteins as calcium hydroxyapatite nucleating sites in the mineralization of dentin (Lechner et al., 1981).

Effect of implant surface chemistry upon arterial thrombosis.

A series of poly(alpha-amino acid)s with controlled chemical variations were investigated in order to assess the effect of different chemical moieties upon arterial thrombosis. The gross implant surface properties ranged from hydrophobic to hydrophilic ionic and nonionic. The materials were tested by implantation within canine femoral and carotid arteries. Results were compared with the response to the polyurethane Biomer. The changes in implant surface chemistry elicited a range of response that varied from intense thrombosis and rapid vessel occlusion to minimal thrombosis and endothelialization. The results showed that no simple relationship exists between a gross surface property, such as hydrophobicity, and the degree of thrombosis resistance. Some hydrophobic and hydrophilic materials were found to have good thrombosis resistance, while others were found to have poor thrombosis resistance. Leukocytes were shown to play an important role in both initial thrombosis and endothelialization. The major difference between materials that progressed to rapid vessel occlusion and materials that remained patent was the degree of direct leukocyte adherence and spreading on the implant surface prior to extensive platelet aggregation (less than 30 min). It was consistent for both hydrophobic and hydrophilic materials that the lack of direct leukocyte adherence to the implant surface was associated with intense thrombosis and rapid vessel occlusion. Conversely, the presence of numerous leukocytes directly adherent to either hydrophobic or hydrophilic surfaces appeared to have a moderating effect upon thrombosis and vessels with these implants remained patent. In instances when thrombosis was nonocclusive, the surfaces of the thrombi became endothelialized, primarily through the transformation of mononuclear leukocytes into endothelial cells. This article includes a hypothetical model representing the sequence of events and alternative pathways occurring at the blood-material interface, with special attention given to the involvement of leukocytes in arterial thrombosis.

An in vivo method to evaluate the effect of materials upon arterial thrombosis.

An in vivo method to evaluate the effect of materials upon arterial thrombosis was developed that minimized the effect of surgical artifacts and provided a test which was critically sensitive to the surface properties of the materials. The procedure has general applicability to elastomers, either solvent cast or mold polymerized, and involves the implantation of segments of the test materials within canine femoral and carotid arteries. The sensitivity of this technique was demonstrated by comparing two surface preparations of a segmented polyurethane: as-cast and ion sputtered. There was a striking difference in the rate of thrombus development on as-cast polyurethane implants compared with sputtered polyurethane implants. After 1 hr. of implantation the surface of the as-cast polyurethane was covered with a monolayer of platelets and leukocytes, whereas thrombus development progressed more rapidly on the sputtered polyurethane surface and at 1 hr. it was covered with pillars of platelets and leukocytes with fibrin accumulation between pillars. The method was also useful for long-term studies, which showed that a thin layer of thrombus developed on both polyurethane surfaces and by 1 wk. after implantation the surfaces of the thrombi became endothelialized.