A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp.

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury.

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

The modulation of corneal keratocyte and epithelial cell responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases collagenase production in vitro.

We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.

Synthesis, physical characterization, and biological performance of sequential homointerpenetrating polymer network sponges based on poly(2-hydroxyethyl methacrylate).

A limitation in the use of hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA) sponges as implantable devices is their inherently poor mechanical strength. This precludes proper surgical manipulation, especially in the eye where the size of the implant is usually small. In this study a new method was developed to produce mechanically stronger PHEMA sponges. Sequential homointerpenetrating polymer network (homo-IPN) sponges were made by using HEMA as the precursor for generating both the first network and the successive interpenetrated networks. Following the formation of network I, the sponge was squeezed to remove the interstitial water, soaked in the second monomer (also HEMA), and squeezed again to remove the excess monomer from the pores before being subjected to the second polymerization leading to the formation of network II. Two two-component IPN sponges (K2 and K4) with increasing HEMA content in the network II and a three-component IPN sponge (K3) were produced, and their properties were compared to those of a homopolymer PHEMA sponge (control). Apart from elongation, the tensile properties were all significantly enhanced in the IPN sponges; the water content was the same as in the control sponge, except for sponge K4, which was lower. Light microscopy revealed similar pore morphologies of the control and IPN sponges K2 and K3, and the majority of the pores were around 25 microm. Sponge K4 displayed smaller pores of around 10 microm. Cellular invasion into the sponges was examined in vitro (incubation with 3T3 fibroblasts) and in vivo (implantation in rabbit corneas). Although the in vitro assay detected a change in the cell behavior in the early stage of invasion, which was probably due to the formation of IPNs, such changes were not reflected in the longer term in vivo experiment. There was a proper integration of sponges K2 and K3 with the corneal stroma, but much less cellular invasion and no neovascularization in sponge K4. We concluded that IPN formation is a valid method to enhance the strength of PHEMA sponges, provided that the content of HEMA in the successive networks is not too high.

In vitro assessment of the biological activity of basic fibroblast growth factor released from various polymers and biomatrices.

The kinetics of controlled release of basic fibroblast growth factor (bFGF) from polymers (sutures, polycarbonate, Hydron, and Elvax), biopolymers (alginate), and biomatrices (lens capsules), and conditions for storage of bFGF (temperature, plastic type, heparin) were evaluated in vitro. Tissue culture proliferation bioassays with 3T3 fibroblasts, showed that only lens capsules with bFGF had a sustained release of bFGF for up to three weeks. The other materials released all of the 'bound' bFGF with two hours or produced an inflammatory response in vivo. Therefore, the lens tissue had the most potential for controlled long-term delivery of bFGF in vivo. These studies emphasise the importance of in vitro analysis of release kinetics of growth factors from a range of materials as a basis for potential in vivo applications.

Extracellular matrix, growth factors, genetics: their influence on cell proliferation and myotube formation in primary cultures of adult mouse skeletal muscle.

Cell proliferation and myotube formation in response to growth factors on various extracellular matrices (ECM) were investigated in primary skeletal muscle cultures from adult SJL/J and BALB/c mice. There was no difference between the rates of proliferation from primary cultures of SJL/J and Balb/c mice measured at 48 h in response to a range of concentrations of PDGF-AA, -AB, -BB, TGF beta 1, or LIF (added at 24 h). SJL/J primary cultures were more responsive to bFGF (which was the most potent mitogen) than were BALB/c cultures. Comparison of dose response curves to bFGF and TGF beta 1 grown on gelatin or Matrigel showed that the nature of the ECM did not have a significant affect. More myotubes formed at 4 days in SJL/J than in parallel BALB/c cultures on gelatin or Matrigel (P < 0.05). On gelatin more myotubes with 4 or more nuclei were formed in cultures from SJL/J than BALB/c muscles (P < 0.05); however, on Matrigel these myotubes occurred with similar frequency. Myotube formation examined in BALB/c muscle cultures grown on collagen i.v., entactin-free laminin, and fibronectin showed that none of these ECM components alone supported large myotube formation (4 or more nuclei) as well as did Matrigel, although fibronectin was as effective as Matrigel with respect to the total number of myotubes formed. Parallel experiments carried out using the myogenic H-2Kb(27) cell line showed similar effects with the exception of laminin which enhanced large myotube formation and desmin expression in the H-2Kb(27) but not in the primary muscle cultures. The greater sensitivity in mitogenic response to bFGF and the more extensive myotube formation seen in SJL/J compared with BALB/c cultures in vitro reflects the superior capacity for muscle regeneration of SJL/J mice in vivo.

Intrinsic differences in MyoD and myogenin expression between primary cultures of SJL/J and BALB/C skeletal muscle.

The time course of expression of the skeletal muscle-specific regulatory genes MyoD and myogenin was studied in primary cultures of skeletal muscle from adult SJL/J and BALB/c mice. In situ detection of expression with MyoD and myogenin riboprobes and myogenin antibody showed that the onset of expression of these genes occurred earlier in cells from SJL/J mice. Probe-positive cells and myotubes were also more frequent in cultures from SJL/J mice than in BALB/c. The onset of expression of MyoD and myogenin was delayed in cultured cells relative to the time course seen following injury in vivo. Myogenin protein was demonstrated in replicating cells and all myogenin-positive cells expressed desmin. The observed strain-specific difference infers a greater intrinsic myogenicity of cells in SJL/J muscle in vitro and reflects the superior capacity for new muscle formation previously reported in SJL/J mice in vivo.

The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis.

The results from this investigation suggest that chemotactic factor(s) from damaged myofibers attract polymorphonuclear leukocytes (PML) and macrophages to the site of injury, while exudate macrophages but not PML induce a strong positive chemotactic response in myogenic cells. The AB and BB isoforms of platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) (all of these are secreted by macrophages) were also shown to be chemoattractants for muscle precursor cells (MPC). The AA isoform of PDGF did not appear to have any such chemotactic effect on MPC. Macrophages were also shown, under tissue culture conditions, to stimulate the proliferation of MPC. This mitogenic effect was similarly demonstrated for the BB isoform of PDGF, bFGF, and TGF-beta but not for the AA or AB isoforms of PDGF nor for LIF. The results indicate that macrophages play a pivotal role in the orchestration of muscle fiber reconstitution.

Phenotype alteration in colon carcinoma cells: effect of in vivo passage?

A panel of rat colon adenocarcinoma cell lines (the Per series) were used to investigate the phenotype and karyotype changes induced by in vivo passage in the subcutis of athymic nude mice. One poorly and one well-differentiated tumor cell line were serially passaged through the athymic nude mouse and then back to the syngeneic rat host. Each of the primary and xenograft cell lines expressed fetal crypt cell ("CaCo") antigens. The well differentiated primary and xenograft lines (Per305, Per305N1, and Per305N2a) were different in each of their growth factor responsiveness in vitro [i.e. epidermal growth factor (EGF), bombesin, vasoactive intestinal peptide], their EGF receptor expression, their secretion of transforming growth factor-alpha, and their exhibition of anchorage independent (A-I) growth capabilities. The poorly differentiated primary and xenograft cell lines were also different but were all capable of A-I growth; their responsiveness to exogenous growth factor stimulation decreased with progressive in vivo passage, as did their basal unstimulated proliferation rate. Cytogenetic alterations detected were those associated with clinical specimens from various stages of malignancy, i. e. aneuploidy, structural aberrations, and marker chromosomes. Genetic and mitogenic individuality of each line demonstrated the diversity of the growth control mechanisms in neoplasms at different stages of progression.

Three new rat colon tumour cell lines from 1,2-dimethylhydrazine induced adenocarcinomas: morphology, karyotype and clonogenicity.

Three different tumour cell lines (Per113, Per192, Per237) were established from colon adenocarcinomas induced by 1,2-dimethylhydrazine (DMH) inbred male Wistar/Wag rats. Each cell line had epithelial morphology and an abnormal karyotype. The three cell lines were clonogenic in semi-solid medium, but differed in growth factor response, culture characteristics and karyotype. Each cell line was stimulated differentially by epidermal growth factor (EGF) and bombesin in culture. Only Per192 would passage in vivo in athymic nude mice. Per 113 had modal chromosome number of 70 with multiple structural abnormalities; Per237 had 43 chromosomes with consistent trisomy of chromosome I; 20-25% of the cells in line Per192 were in the tetraploid range with multiple copies of chromosome I.

Single chromosome defect, partial trisomy 1q, in a colon cancer cell line.

The characterization of a single chromosome defect previously reported in a case of inherited nonpolyposis colorectal cancer is the essence of this communication. The defect, originally described as 13p+, is now being defined and the karyotype designated as 46,XY,-13,+der(13)t(1;13)(q32.1;p11).

Growth factor-induced proliferative responses of human and DMH-induced rat colorectal tumour cell lines.

The proliferative response of three human and three 1,2-dimethylhydrazine (DMH)-induced rat colon tumour cell lines to mouse epidermal growth factor (mEGF) and human urogastrone was examined in two in vitro assays (3H-thymidine uptake in liquid microculture and clonogenicity in semi-solid medium). In the 3H-thymidine uptake assay, one of three rat and one of three human colon tumour cell lines, i.e., r237 and LIM1215, were stimulated by mEGF. In the clonogenic assay, mEGF stimulated the one clonogenic rat tumour cell line r113, which did not respond in the 3H-thymidine uptake assay, and the human tumour cell line LIM1215. Urogastrone stimulated LIM1215 in both assay systems. The significance of these in vitro patterns of response to growth stimulatory factors is discussed.

Colorectal carcinoma in a rat model: suppression of tumour development and altered host immune status following treatment with anti B-lymphocyte serum.

The rate of colonic tumour development and immune capability in rats whose B-lymphocyte function was suppressed by injections of rabbit anti-rat IgM and also given the carcinogen dimethylhydrazine (DMH) were studied. Four rat groups were arranged to receive either DMH + anti-IgM, DMH + normal rabbit serum (NRS), saline + anti-IgM, or saline + NRS. Tumour weight, blood and mesenteric lymph node B-lymphocyte numbers, in vivo allograft response, in vitro lymphocytotoxicity, and leucocyte migration inhibition response (LMI) were recorded fortnightly. Tumour induction was delayed in the DMH + anti-IgM (treated tumour) group, which developed less tumour than the DMH + NRS (untreated tumour) group (p less than 0.001). Spleen cell lymphocytotoxicities were depressed in treated rats when compared to either the saline + anti-IgM (treated control) rats or the untreated rats (P less than 0.02), whereas anti-IgM treatment suppressed lymphocytotoxicity responses in control rats (p less than 0.05). The untreated tumour rats were tumour immune by LMI; however, the treated tumour rats did not express this in vitro tumour immunity. The B-lymphocyte levels in the mesenteric lymph nodes of untreated tumour rats increased with tumour induction (p less than 0.05), whereas in the treated tumour rats B-lymphocyte levels were not similarly stimulated.

Sequential study of gamma-glutamyltransferase during complete and two-stage mouse skin carcinogenesis.

The histochemical pattern of gamma-glutamyltransferase (GGT) was studied in benign and malignant tumors produced by two different experimental protocols, two-stage carcinogenesis and complete carcinogenesis. Six percent of all papillomas produced by two-stage carcinogenesis were GGT positive, whereas 14% of benign tumors produced by complete carcinogenesis exhibited GGT-positive areas. The incidence of GGT-positive papillomas in the two-step carcinogenesis protocol increased up to wk 28 of treatment. After 32 wk, the incidence decreased abruptly, coinciding with an abrupt increase in the incidence of squamous cell carcinomas. On the other hand, the incidence of GGT-positive benign tumors produced during the course of complete carcinogenesis increased gradually up to wk 32 of treatment, coinciding with the increased incidence of squamous cell carcinomas. The incidence of GGT-positive keratoacanthomas and GGT-positive papillomas produced with the complete carcinogenesis protocol exhibited different patterns, suggesting different histogenesis and biological behavior of these two types of tumors. In addition, the labeling index of GGT-positive areas was lower (17 +/- 3%) than that of the GGT-negative areas (41 +/- 0.18%) of the same papillomas, indicating that the presence of GGT may be related to abnormal keratocyte differentiation rather than to proliferative changes.

Immune response to storage-induced injury in non-ischaemic renal autografts.

Graft sensitization was measured in dogs receiving autotransplants of perfused, flushed or hypothermically preserved kidneys. Five kidney treatments were studied: (1) continuous pulsatile perfusion with Ross solution for 24 h; (2) flush with Ross solution followed by 24h cold storage; (3) removal and immediate reimplantation; (4) flush with Ross solution and immediate reimplantation; (5) cool and immediate reimplantation. Continuous perfusion resulted in a lower mean creatinine (measured over the first five days after transplant) than did flushing followed by cold storage (P less than 0.001). Creatinines were lower in the groups in which kidneys were not stored (regardless of treatment) than in the stored groups (P less than 0.001). Dogs were said to be immune if their leucocytes were inhibited by nephrectomy kidney antigen in the leucocyte migration inhibition assay [LMI]. Immune dogs had a higher mean creatinine than non-immune animals (P less than 0.05). All autografts were examined for deposition of IgG, IgM and C3 at post-mortem by tissue immunofluorescence [IF]. Positive immune responses (LMI or IF) were seen more often in dogs receiving long preserved grafts than in those receiving immediate graft implantation (P less than 0.05).

Immune capability of rats with colorectal carcinoma treated by resection with or without 5-fluorouracil or levamisole.

The immune status of rats with chemically induced colorectal tumors was investigated for 8 weeks following treatment by resection, resection plus 5-fluorouracil (5-FU), and resection plus levamisole. Normal rats without tumors were given identical treatment and acted as controls. Tumor rats regained their ability to respond to an allograft of allogeneic lymphocytes after surgical excision of the tumor for a short period. Adjuvant Levamisole treatment enhanced this responsiveness, but adjuvant 5-FU depressed it. The spleen and lymph node cells of tumor-bearing rats showed greater spontaneous and PHA-induced cytotoxicity for chicken red blood cells than control rats (P less than 0.001), and adjuvant therapy did not alter this response. However in control animals, levamisole treatment did augment the antibody-dependent cytotoxicity of their spleen and lymph node cells. Rats receiving tumor resection alone were immune by leukocyte migration inhibition at 8 weeks only, while those receiving either adjuvant exhibited an immune response to the same homogenate for 4 and 8 weeks.

Clinical and in vivo response following surgery or surgery plus adjuvant chemotherapy or immunotherapy for colorectal carcinoma in a rat model.

Two cohorts of rats, 240 with colon cancer and 150 controls, were assessed clinically and immunologically for their response to tumour and its management which was either by surgical excision alone or by surgical excision combined with either adjuvant chemotherapy or immunotherapy. The histology and invasion characteristics were observed for similarity with those of human lesions. Metastases were found in liver, lymph nodes, the peritoneum or lungs in 27% of animals during follow up. Significantly fewer adjuvant-treated rats had metastases than those receiving surgery alone (P less than 0.05), and less total tumour weight was found in the adjuvant-treated rats at four (P less than 0.03) and six (P less than 0.001) weeks postoperatively. Animals in the adjuvant immunotherapy group survived longer than in either other group (P less than 0.001). The crude parameters of host response to tumour, body, spleen and mesenteric lymph node weight were recorded and the latter two indexed to body weight. The body weight of tumour and control rats increased significantly with time (P less than 0.04). The spleen and mesenteric node indices were significantly (P less than 0.04) greater in tumour than control rats and were varied by recurrent tumour growth and by the adjuvant treatment administered postoperatively.

The immune status of the rat with carcinoma of the bowel.

Studies were made to determine whether rats receiving 1,2-dimethylhydrazine (DMH) to induce bowel cancer were immunologically compromised during tumour development. General cellular immunodepression was observed in DMH rats in nonspecific assays of T-cell and killer-cell (K-cell) function and later in allograft response and in vitro tumour immunity tests. B-cell levels in local lymph nodes increased significantly very late in tumour growth. It is suggested that DMH itself was exerting immunosuppressive as well as carcinogenic effects while it was administered. After withdrawal of DMH some immune faculties (tumour immunity, B-cell response) reappeared, so it is unlikely that the immune suppression observed was a result of factors produced by the developing neoplasm. There are indications that DMH administration in the rat is immunosuppressive and that tumour growth may be facilitated by this action and not by tumour reactive lymphocytic depletion or paralysis associated with blocking factors or suppressor cell production.