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Using a polymerase chain reaction (PCR) test for sequences of ovine herpesvirus 2 (OHV2), this virus was shown to be significantly associated with sheep-associated malignant catarrhal fever (SA-MCF) in terminal cases of disease in 34 cattle and 53 bison. Ovine herpesvirus 2 was not detected in cattle (38) and bison (10) that succumbed to other diseases. Other persistent herpesviruses, retroviruses, and pestivirus, some of which have been previously isolated from cases of SA-MCF, were not associated with the disease. These included bovine herpesvirus 4 (BHV4), bovine lymphotrophic herpesvirus (BLHV), bovine syncytial virus (BSV, also known as bovine spumavirus), bovine immunodeficiency virus (BIV), and bovine viral diarrhea virus (BVDV). A PCR survey for OHV2 in DNA from individual cow's peripheral blood lymphocytes in 4 dairies showed that the 1 dairy that was in close contact to sheep had a prevalence of OHV2 of 21.3%, whereas the 3 other dairies had no OHV2. Prevalence of the other herpesviruses and retroviruses in the dairy cows was variable, ranging from 2% to 51% for BHV4, 52% to 78.7% for BLHV, and 10% to 34% for BSV. Bovine lymphotrophic herpesvirus and BSV were also found in a few (1-4 of 21 tested) cases of terminal SA-MCF, but BIV and BVDV were not found in either the dairy cows sampled, or in the cases of SA-MCE No significant correlation was found between the presence of any 2 viruses (OHV2, BHV4, BLHV, BSV) in the dairy cows or terminal cases of SA-MCE

Accurate staging of cancer is important, as the presence or absence of systemic spread determines treatment. The sensitivity of current imaging and biochemical techniques is suboptimal for the detection of minimal residual disease and latent metastases. This results in understaging and potential undertreatment. To improve detection of disseminated epithelial malignancy, immunohistochemical and molecular methods have been employed that search for epithelial cell-specific proteins in nonepithelial tissue. Bone marrow is mesenchymal tissue (that does not normally express epithelial cell components) and represents an accessible window for detection of micrometastatic carcinoma cells. Detection methods for epithelial cell components (cytokeratins, epithelial membrane antigen, carcinoembryonic antigen) include immunohistochemistry, flow cytometry, reverse transcriptase polymerase chain reaction (rt-PCR), and enzyme linked immunoassay (ELISA). Micrometastatic cells in bone marrow are viable, capable of proliferation, resistant to immune attack, and insensitive to s-phase chemotherapeutic agents. Patients with carcinomas of the lung, breast, prostate, or gastrointestinal tract and in whom bone marrow micrometastases are detected have a foreshortened interval to recurrence and impaired survival. Detection of micrometastases deserves serious consideration in treatment protocols, and standardization of methods is now required.

Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59 degrees C. An air outlet temperature of 80 to 85 degrees C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 x 10(9) CFU/g for NFBC 338 and 5.2 x 10(7) CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at approximately 1 x 10(9) CFU/g during 2 months of powder storage at 4 degrees C, while a decline in the level of survival of approximately 1 log (from 7.2 x 10(7) to 9.5 x 10(6) CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.

Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.

Cheddar cheese has previously been shown to be an effective vehicle for delivery of viable cells of a probiotic Enterococcus faecium strain to the gastrointestinal tract. The particular strain, E. faecium PR88, has proven efficacy in the treatment of irritable bowel syndrome, and in this study it was evaluated for suitability as a starter adjunct for Cheddar cheese manufacture. When added to cheesemilk at an inoculum of 2 x 10(7) cfu/mL, the enterococcal adjunct maintained viability in Cheddar cheese at levels of up to 3 x 10(8) cfu/g during 9 months of ripening. Increased proteolysis and higher levels of some odor-active volatile compounds were observed in Cheddar cheeses containing the PR88 adjunct compared with the control throughout the ripening period. In addition, the enterococcal adjunct strain did not affect cheese composition. Although sensory evaluation showed no significant difference in flavor/aroma and body/texture scores between control and experimental cheeses, repeated comments by the commercial grader consistently described the cheeses containing PR88 as 'more advanced than the control' and as having 'better flavor'. These findings indicate that the presence of the PR88 adjunct strain in Cheddar cheese at levels of >/=10(8) cfu/g may positively influence Cheddar flavor.

A polymerase chain reaction (PCR) test was compared with culture for the detection and diagnosis of bovine Mycoplasma intramammary infection. The PCR test was applied to 24-hour Mycoplasma enrichment cultures of milk from cows with suspected mastitis and from bulk tank milk. In comparison to culture, the sensitivity and specificity of the PCR method were 96.2% and 99.1% for individual cow milk and 100% and 99.8% for the bulk tank milk, respectively. However, in discrepant cases where PCR was positive and culture was negative, the PCR test was correct; subsequent PCR tests and culturing of the individual cow's milk yielded positive results. The PCR test simultaneously detected and differentiated among 11 bovine Mycoplasma species.

Breast cancers have been shown to elicit tumor-specific immune responses. As in other types of cancer, the antitumor immune response fails to contain breast tumor growth, and a reduction in both the quantity and cytotoxic effectiveness of tumor-infiltrating lymphocytes (TILs) is associated with a poorer prognosis. Fas ligand (FasL) induces apoptotic death of activated lymphocytes that express its cell surface receptor, FasR (CD95/APO-1). FasL-mediated apoptosis of activated lymphocytes contributes to normal immune downregulation through its roles in tolerance acquisition, immune response termination, and maintenance of immune privilege in the eye, testis, and fetus. In this report, we demonstrate that breast carcinomas express FasL. Using in situ hybridization and immunohistochemistry, we show that breast tumors constitutively express FasL at both the mRNA and protein levels, respectively. FasL expression is prevalent in breast cancer: 100% of breast tumors (17 of 17) were found to express FasL, and expression occurred over more than 50% of the tumor area in all cases. By immunohistochemistry, FasR was found to be coexpressed with FasL throughout large areas of all the breast tumors. This suggests that the tumor cells had acquired intracellular defects in FasL-mediated apoptotic signaling. FasL and FasR expression were independent of tumor type or infiltrative capacity. FasL expressed by tumor cells has previously been shown to kill Fas-sensitive lymphoid cells in vitro and has been associated with apoptosis of TILs in vivo. We conclude that mammary carcinomas express FasL in vivo as a potential inhibitor of the antitumor immune response.

Cheddar cheese was evaluated as a food carrier for the delivery of viable microorganisms of Enterococcus faecium (Fargo 688; Quest Int., Naarden, The Netherlands) to the gastrointestinal tract. This strain had previously been shown to possess properties required of a probiotic microorganism including the ability to relieve irritable bowel syndrome. The strain was found to survive to high numbers in Cheddar cheese during ripening at 8 degrees C for 15 mo (4 x 10(8) cfu/g) and in yogurt during storage at 4 degrees C for 21 d (4 x 10(7) cfu/g). In an in vitro model system, Cheddar cheese was found to have a greater protective effect than yogurt upon exposure of the probiotic culture to porcine gastric juice at pH 2. Subsequently, a feeding trial involving 8 pigs per group was performed in which a rifampicin-resistant variant of the probiotic strain was fed for 21 d at a mean daily intake of 1.3 x 10(10) cfu/d from Cheddar cheese or 3.7 x 10(9) cfu/d from yogurt. During the feeding period, Cheddar cheese yielded a significantly higher mean fecal probiotic count (2 x 10(6) cfu/g of feces) than did yogurt (5.2 x 10(5) cfu/g of feces). These data indicate that mature Cheddar cheese compares very favorably with fresh yogurt as a delivery system for viable probiotic microorganisms to the gastrointestinal tract.

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two members of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probiotic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.

The efficacy and safety of a gene-deleted bovine herpesvirus-1 (BHV-1) vaccine was determined in a bovine herpesvirus challenge trial in calves. Three different doses of the vaccine were administered intramuscularly at 10(5), 10(6) and 10(7) PFU/ml and compared to a commercial vaccine and non vaccinated control calves. Challenge was performed by intranasal aerosolization with the Cooper strain of BHV-1 (3 x 10(4) PFU/ml). The non-vaccinated calves shed significantly (P < 0.05) more virus than all other groups on days 4, 8 and 10 post challenge. By day 14 post challenge, antibody titers for BHV-1 of calves vaccinated with 10(7) PFU/ml were significantly (P < 0.05) higher than the commercial or non-vaccinated calves. Clinical scores of non-vaccinated calves were significantly (P < 0.05) higher than all other groups on days 4-14 post challenge. With both radioimmunoprecipitation and competitive enzyme-linked immunosorbent assays (C-ELISA), calves in the gene-deleted vaccine groups mounted comparable specific responses against gB, gC and gD post vaccination as calves in the commercial vaccine group, but in a dose dependent manner. These data suggest that the gene-deleted BHV-1 vaccine tested may be used as an effective vaccine in controlling BHV-1 infections.

Monitoring the progression of hepatitis C virus (HCV) includes clinical, biochemical, and histological parameters. Quantitation of viral load by reverse-transcription polymerase chain reaction (RT-PCR) may offer a more reliable marker of disease status. Conflicting reports on viral titers may reflect heterogeneity of patient population, mode of infection, and viral type/subtype. The aim of this study was to correlate quantitative serum viral load with alanine transaminase (ALT) and histological status in a homogenous population. The study population consisted of 77 Rhesus-negative women with chronic hepatitis C type 1b. Homogenous features of this study population included: same defined source of infection (contaminated anti-D immunoglobulin); same duration of disease (17 years at the time of study); same viral type/subtype; same ethnic origin; all healthy child-bearing females at the time of infection; and an absence of competing risk factors for infectious and other liver diseases. None of the patients had received antiviral treatment at the time of study. Liver biopsy was performed on all patients. All biopsies were scored by a single histopathologist who was blinded to the clinical and viral status of each patient. A weak, but statistically significant, correlation (rs =.26; P <.05) between serum viral load and the degree of inflammation (mean value: 3.87 +/- 2.17 [SD]) was found. There was no significant correlation between serum viral load and the degree of fibrosis (mean value: 0.84 +/- 0.8 [SD]; P =.06). There was no significant correlation between serum viral load and ALT, although there was a correlation between ALT and the degree of inflammation (rs =.241; P =.035).

Background & Aims: Micrometastases within bone marrow indicate a poor prognosis. We prospectively examined micrometastases in patients undergoing resection of esophagogastric cancers for (1) prevalence in rib marrow; (2) comparative detection rates in rib and iliac crest marrow; (3) responsiveness to neoadjuvant therapy; and (4) viability and tumorigenicity., Methods: In 50 consecutive patients, marrow was obtained before manipulation of the primary tumor. Micrometastatic cells were detected by staining contaminant cytokeratin-18-positive cells. Viability and tumorigenicity were determined by culture and xenograft., Results: Micrometastases were detected in rib marrow from 88% of patients (44 of 50). When bilateral iliac crest marrow was also obtained, micrometastases were found in 15% (4 of 27) compared with 89% (24 of 27) for ribs (P < 0.001). Detection rates were independent of histological type or nodal status and were similar in patients with and without neoadjuvant therapy. Metastatic cells were cultured from rib marrow of 5 of 7 patients and were tumorigenic in nude mice., Conclusions: Most patients undergoing resection of esophagogastric malignancies have micrometastases in rib marrow. Detection rates based on iliac crest marrow are underestimates. Hematogenous spread of metastatic cells is independent of histological type or nodal status. The metastatic cells are viable, tumorigenic, and resistant to neoadjuvant therapy.

Cats with clinical signs suggestive of ocular infection with feline herpesvirus type 1 (FHV 1) and cats without such signs were assayed by 3 methods to detect FHV. Comparison of polymerase chain reaction (PCR), virus isolation, and indirect fluorescent antibody staining techniques for the detection of FHV demonstrated higher sensitivity of PCR in detecting this common infectious agent of cats. Compared with PCR, sensitivity and specificity for virus isolation was 49% and 100%, respectively, and those of indirect immunofluorescence were 29% and 96%, respectively. FHV was detected in 13.7% of client-owned cats with conjunctivitis and in 31% of shelter cats with no ocular signs. The use of FHV PCR as a diagnostic test for FHV-associated disease is limited because of the occurrence of healthy carriers.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.

Objective: To determine the value of virus isolation (VI), immunofluorescent antibody (IFA) assay, serum neutralization (SN), and ELISA for the diagnosis of clinical feline herpesvirus-1 (FHV-1) infection in cats., Animals: 46 clinically normal cats, 17 cats with signs of acute respiratory tract disease, and 38 cats with signs of chronic ocular disease., Procedure: Conjunctival swabs for VI, conjunctival scrapings for IFA testing, and venous blood samples for SN or ELISA testing were obtained from all cats., Results: FHV-1 was detected in 10.9 and 28.3% of clinically normal cats and in 18.2 and 33.3% of cats with FHV-1-associated disease by VI and the IFA assay, respectively. There were no significant differences in the viral detection rate between cats with acute respiratory tract disease and cats with chronic ocular disease or between diseased cats and clinically normal cats; however, FHV-1 was never detected by both methods in clinically normal cats. Overall FHV-1 seroprevalence was 97% when tested by ELISA and 66% when tested by SN. Seroprevalence did not vary significantly among the 3 groups for either serologic test. Magnitude of SN and ELISA titers varied greatly but independently of presence or absence of clinical signs of FHV-1-associated disease. Sensitivity, specificity, and positive and negative predictive values were assessed for VI and the IFA assay--jointly and individually--and for each SN and ELISA titer magnitude. Values never all exceeded 50%., Clinical Implications: Because FHV-1 can be detected commonly in clinically normal cats by the IFA assay or VI, neither test appears to aid in the clinical diagnosis of FHV-1 infection. Seroprevalence does not appear to vary between affected and clinically normal cats. SN, ELISA, VI, and the IFA assay appear to be of limited value in the diagnosis of FHV-1-associated disease in cats. Concurrent assessment of the IFA assay and VI results may permit exclusion of FHV-1 as an etiologic agent if results of both tests are negative.

Background: Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape., Aim: To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer., Specimens: Thirty paraffin wax embedded human gastric adenocarcinomas., Methods: FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL)., Results: Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour., Conclusions: Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.

Early metastasis contributes to the very poor prognosis of esophageal carcinoma. The recent immunohistochemical finding that invasive esophageal carcinomas express elevated levels of urokinase (uPA) and urokinase receptor (uPA-R) in vivo suggest that the plasminogen activation system may contribute to metastasis in esophageal cancer. The aim of our study was to functionally investigate, at the molecular level, the relative contribution of uPA and uPA-R to the invasiveness of esophageal cancer cells in vitro. The three esophageal cancer cell lines, OC1-3, generated in our laboratory, were analyzed for uPA and uPA-R expression by RT-PCR, immunoenzymatic staining, and quantitative ELISA. Invasiveness of all cell lines was quantified as percentage cellular invasiveness in a standardized Matrigel in vitro assay. OC1 and OC3, which were found to coexpress both uPA and uPA-R, displayed stronger invasiveness (44% and 32.5% respectively) relative to OC2 (19%) which expressed uPA-R but was negative for uPA. Transfection of OC2 cells with the uPA cDNA resulted in two variants, OC2.uPA1 and OC2.uPA2, stably expressing functional uPA. Both transfectants exhibited enhanced invasiveness (60% and 50% respectively) relative to the parent uPA-negative OC2 cells (19%). Antisense oligonucleotide inhibition of either uPA or uPA-R expression resulted in a similar, marked reduction in invasiveness of esophageal tumor cells which normally coexpress both molecules (OC1, OC3 and the uPA-expressing OC2-transfectant clones). Neither antisense treatment altered the basal invasiveness of OC2, which expresses uPA-R but not uPA. In conclusion, coexpression of uPA with its receptor, uPA-R, is required for functional involvement of the urokinase system in invasion by esophageal carcinoma cells. Our results suggest that these synergistic mediators of invasiveness are quantitatively major contributors to the invasiveness of esophageal carcinoma.

Fas (CD95/Apo-1) receptor (FasR) is a cell-surface receptor that mediates apoptotic cell death upon triggering by Fas ligand (FasL). We sought to determine whether normal human esophageal epithelial cells express FasL and/or FasR and whether their localization is consistent with a role in the turnover of normal esophageal epithelium. Normal esophageal epithelium was immunohistochemically positive for FasL in upper prickle cell layers and in mature squamous cells, but the proliferative basal layer was negative. FasL mRNA was detected in the same epithelial cell layers by in situ hybridization. Co-localization of FasL mRNA and protein therefore confirmed that FasL expression is induced in esophageal epithelial cells as they reach terminal differentiation. FasR was immunohistochemically detected throughout the esophageal epithelium. Positive TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling) staining confirmed cell death of the FasL and FasR coexpressing mature epithelial cells. CD45-positive immunocytes were notably absent from FasL-expressing upper epithelial layers. The findings are consistent with a contributory role for Fas-mediated autocrine suicide or paracrine fratricide in the apoptotic turnover of normal esophageal epithelium.

The mechanisms by which esophageal tumors escape immunologic recognition and clearance are only partly understood at the molecular level. Esophageal cancers have been shown to evade host recognition by down-regulation of antigen presentation and production of immunosuppressive factors. Recently, two independent reports have shown that esophageal tumor cells abundantly express Fas ligand (FasL) in vivo. As the triggering agonist for Fas receptor (Fas or APO-1/CD95)-mediated apoptosis of lymphocytes, FasL normally plays immune down-regulatory roles, including activation-induced cell death of T and B cells, as well as maintaining immune privilege in certain organs. Fas ligand expressed by esophageal cell lines has been shown to induce apoptosis of cocultured Fas-sensitive lymphoid cells in vitro. FasL expression by esophageal carcinomas in vivo has been associated with significantly reduced tumor-infiltrating lymphocytes (TILs) in FasL-positive tumor nests, concomitant with significantly increased TIL apoptosis in these nests. These studies support a 'Fas counterattack' mechanism of immune escape in esophageal cancer. By expressing functional Fas ligand, esophageal cancer cells can deplete antitumor lymphocytes by inducing apoptosis. To express functional FasL, esophageal carcinomas also acquire molecular mechanisms to resist autocrine Fas-mediated apoptosis of tumor cells.

Objective: To determine the extent to which prehospital patient care protocols incorporate the findings of the peer-reviewed scientific EMS literature., Methods: Using a computerized literature search, articles published from eight institutions known to be active in prehospital care research were identified and obtained from the local health sciences library. Animal or bench research, analysis of administrative practices, evaluation of educational or quality assurance techniques, collective reviews, and air medical articles were excluded. We compared the findings of each article with the guidelines contained in 12 sets of prehospital care protocols, ranking them as: 1) consistent; 2) partially consistent; 3) not discussed; or 4) not consistent. The rankings for the article-protocol comparisons for each EMS system were compared using the Kruskal-Wallis test., Results: Forty-nine papers were compared with 12 sets of protocols, resulting in 588 comparisons. More than half (53.1%, n = 312) of the comparisons were ranked as "consistent." Only 28 (4.8%) of the comparisons were found to be "not consistent." There was no significant difference in the rankings assigned to the comparisons for protocols from each individual system, nor in the rankings for protocols from the EMS system associated with the source of the article, from other systems with academic affiliations, and from systems without academic affiliations., Conclusion: Most EMS protocols are consistent with the published peer-reviewed research. There is no difference in the level of consistency when comparing protocols from EMS systems associated with the source of the articles, those associated with other academic institutions, and those without strong academic affiliations.

In recent years, the time-honored reputation of lactobacilli as promoters of gastrointestinal and female urogenital health has been qualified. This has occurred due to a rare association with human infection in the presence of certain predisposing factors and their potential to act as a source of undesirable antibiotic resistance determinants to other members of the indigenous microbiota. This necessitates greater caution in their selection for use in microbial adjunct nutrition and disease management (prophylaxis and therapy). It was against this background that 46 Lactobacillus strains from human and dairy sources were assayed for susceptibility to 44 antibiotics. All strains were resistant to a group of 14 antibiotics, which included inhibitors of cell wall synthesis (cefoxitin [30 microg] and aztreonam [30 microg]), protein synthesis (amikacin [30 microg], gentamicin [10 microg], kanamycin [30 microg], and streptomycin [10 microg]), nucleic acid synthesis (norfloxacin [10 microg], nalidixic acid [30 microg], sulphamethoxazole [100 microg], trimethoprim [5 microg], co-trimoxazole [25 microg], and metronidazole [5 microg]), and cytoplasmic membrane function (polymyxin B [300 microg] and colistin sulphate [10 microg]). All strains were susceptible to tetracycline (30 microg), chloramphenicol (30 microg), and rifampicin (5 microg). Four human strains and one dairy strain exhibited atypical resistance to a penicillin, bacitracin (10 microg), and/or nitrofurantoin (300 microg). One human strain was also resistant to erythromycin (15 microg) and clindamycin (2 microg). These resistances may have been acquired due to antibiotic exposure in vivo, but conclusive evidence is lacking in this regard. Seven microorganism-drug combinations were evaluated for beta-lactamase activity using synergy and nitrocefin tests. The absence of activity suggested that cell wall impermeability appeared responsible for beta-lactam resistance. The occurrence of a minority of lactobacilli with undesirable, atypical resistance to certain antibiotics demonstrates that not all strains are suitable for use as probiotics or bacteriotherapeutic agents. The natural resistance of lactobacilli to a wide range of clinically important antibiotics may enable the development of antibiotic/probiotic combination therapies for such conditions as diarrhea, female urogenital tract infection, and infective endocarditis.

There is widespread interest in capacitance immunosensor systems which directly detect antigen binding to immobilized antibody. Our system comprises an active biolayer of antibodies bound to a silicon--silicon dioxide--silicon nitride (Si-SiO2-Si3N4) surface. As with other groups, our system initially gave poorly reproducible responses on addition of antigen. We mechanically degraded the Si-SiO2-Si3N4 surface, and the responses on addition of transferrin were monitored. The mechanical degradation allowed the affinity reaction to be 'seen' capacitively. Once the system was established, a comparison of capture antibodies was performed to establish the most effective biolayer. Three affinity reactions were examined: (a) 1D2A4, monoclonal antibody (mAb) to human transferrin, as the capture layer; (b) polyclonal goat anti-human transferrin antibody (PcAb) as the capture layer; and (c) 1D2A4 with transferrin (Tf) prebound as the capture layer. There was no response to addition of transferrin where 1D2A4 was the capture layer. Addition of transferrin when the polyclonal antibody was used as the primary layer resulted in a drop in measured capacitance. Addition of goat anti-human transferrin antibody to a device with 1D2A4 plus transferrin as the capture layer also resulted in a measured capacitance decrease. There is a difference in dielectric/blocking effectiveness between the monoclonal and polyclonal antibodies.

Fas ligand (FasL) kills sensitive Fas receptor (FasR)-bearing cells by inducing apoptosis. FasL expressed by non-lymphoid cells within the eye and the testis mediates immune privilege by inducing apoptosis of Fas-sensitive infiltrating pro-inflammatory immune effector cells. It has previously been demonstrated by the present authors that the colon cancer cell SW620 expresses FasL and can kill lymphoid cells by Fas-mediated apoptosis in vitro. This 'Fas counterattack' was subsequently confirmed by others as a potential mechanism of immune privilege in various malignancies. The aim of the present study was to ascertain the prevalence of FasL expression in human colon cancer and to confirm that neoplastic colonic epithelial cells express FasL in vivo. The study of FasL expression by colon cancer cell lines was extended: it was shown that seven of eight colon adenocarcinoma cell lines expressed FasL mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). Prevalent expression of FasL was confirmed in vivo: all the resected colonic tumours examined (31/31) were found to express FasL. In the tumours, FasL were co-localized to neoplastic colonic epithelial cells, using immunohistochemistry and in situ hybridization, respectively. FasL expression was independent of Dukes' stage, suggesting that it may occur throughout colon cancer progression. These results suggest that FasL is a common mediation of immune privilege in colon cancer.

Reciprocal communication between the immune system and the neuroendocrine system is mediated via a common chemical language of shared ligands and receptors. The neuropeptide substance P (SP) has been implicated as a mediator of immunomodulation. The evidence for substance P receptors on human lymphocytes is, however, controversial. The aims of the present study are to investigate substance P receptor (SPR) expression in human peripheral and mucosal mononuclear cells and to identify cellular sites of expression in human colonic mucosa. Using reverse-transcriptase PCR, we demonstrate that PBMC isolations are negative for SPR mRNA expression, whereas lamina propria mononuclear cell (LPMC) isolations express on average eight SPR mRNA transcripts per cell. In situ hybridization performed on surgically resected colonic tissue confirms the expression of SPR mRNA in LPMC in vivo. SPR mRNA signal was detected in LPMC, lymphoid follicles, and epithelium. The complementary technique of immunohistochemistry gave a similar distribution of SPR expression that colocalized with CD45 immunoreactivity. Dual-fluorochrome flow cytometry revealed SPR expression by CD4, CD45RO, CD45RA, CD8, CD19, and CD14 LPMC subsets, but not PBMC. Our findings suggest that SPR expression is distinctive of human colonic mucosal mononuclear cells and support a direct role for SP in mucosal immunomodulation.

Objective: To develop a system to monitor and detect acute infections of the upper respiratory tract (i.e., nares, nasopharynx, and pharynx) in horses and to assess the association among specific viral infections, risk factors, and clinical signs of disease., Design: Prospective study., Animals: 151 horses with clinical signs of acute infectious upper respiratory tract disease (IURD) from 56 premises in Colorado., Procedure: Health management data, blood samples, and nasal or nasopharyngeal swab samples were obtained for 151 horses with clinical signs of acute IURD. Of these horses, 112 had an additional blood sample obtained during convalescence and were considered to have complete sample sets. Samples were tested for evidence of respiratory tract infection by use of ELISA, virus isolation, and serologic testing of paired serum samples., Results: Viral infections were identified in 65 horses with complete sample sets; influenza virus infection was identified in 43 horses, equine herpesvirus (EHV) infection in 18, and mixed influenza virus and EHV infections in 4. On 14 premises, samples were obtained from more than 1 affected horse. Viral infections were identified in horses on 11 of 14 premises. Equine herpesviruses were isolated from 10 horses. A relationship was not found between vaccination history and identification of EHV or influenza virus infections. An infection with EHV was less likely to be identified in horses with initial (acute) antibody titers > 1:16 to EHV., Clinical Implications: Influenza virus (specifically, A/equine/2) was the most common virus associated with acute IURD. Use of multiple diagnostic tests and obtaining samples from more than 1 horse in an outbreak may improve detection of viral infections.

Acute malignant catarrhal fever (MCF) was diagnosed in 10 bison from 6 herds and ranging from 1 to 6 years of age. The pattern of clinical signs and morphologic lesions differed among bison. Combinations of corneal opacity, lacrimation, nasal discharge, depression, excess salivation, anorexia, diarrhea, melena, and hematuria were observed. Vasculitis characterized by lymphoid infiltrates in the adventia with variable extension into media and intima was found in multiple tissues in each animal. Fibrinoid vascular necrosis was rare. Ulceration in the alimentary tract was found in 9/10 bison, and ulceration or hemorrhage in the urinary bladder was found in 8/10 bison. Lymphoid infiltrates were present in 7 of 9 livers and 9 of 9 kidneys examined histologically. Hyperplasia of lymph nodes was observed in 5 bison. Chronic MCF was diagnosed in 1 bison with an 80-day course of illness that began with lacrimation, corneal opacity, mucoid nasal discharge, depression, and anorexia. These signs ceased after 15 days but circling and blindness developed on day 76. Chronic vascular lesions characterized by endothelial cell hypertrophy, intimal thickening, fragmentation of the internal elastic membrane, smooth muscle hypertrophy, and adventitial infiltrates of lymphocytes and plasma cells were found in many organs. The retinal arteries had chronic inflammation and acute transmural fibrinoid necrosis. The retinas were infarcted. Polymerase chain reaction technique for amplification of ovine herpesvirus 2 sequences was performed on formalin-fixed tissues, and viral sequences were detected in 1-7 tissues from each animal. These viral sequences were not found in tissues of 4 bison not affected by MCF.

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10(7) CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.

Various cancer cell lines express Fas ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression has been demonstrated in several human malignancies in vivo. We sought to determine whether human esophageal carcinomas express FasL, and whether FasL expression is associated with increased apoptosis of tumor-infiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Using in situ hybridization and immunohistochemistry, respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n = 6; adenocarcinoma, n = 2). The Extent of FasL expression was variable, with both FasL-positive and FasL-negative neoplastic regions occurring within tumors. TIL were detected by immunohistochemical staining for the leukocyte common Ag, CD45. FasL expression was associated with a mean fourfold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6- to 12-fold, n = 6,p < 0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean twofold increase in detectable cell death among TIL in FasL-positive areas compared with FasL-negative areas (range 1.6- to 2.4-fold, n = 6, p < 0.05). In conclusion, we demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest Fas-mediated apoptotic depletion of TIL in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer.

An in vitro methodology which mimics in vivo human upper gastrointestinal transit was developed. The transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species was determined by exposing washed cell suspensions at 37 degrees C to a simulated gastric juice (pH 2.0), containing pepsin (0.3% w/v) and sodium chloride (0.5% w/v), and a simulated small intestinal juice (pH 8.0), containing pancreatin USP (1 g l-1) and sodium chloride (5 g l-1), and monitoring changes in total viable count periodically. The methodology was also employed to determine the effect of adding milk proteins (1 g l-1), hog gastric mucin (1 g l-1) and soyabean trypsinchymotrypsin inhibitor [SBTCI] (1 g l-1) on transit tolerance. The majority (14 of 15) of isolates lost > 90% viability during simulated gastric transit. Only one isolate, Lactobacillus fermentum KLD, was considered intrinsically resistant. The addition of milk proteins, singly and in combination, generally improved gastric transit tolerance. In this regard, two isolates, Lact. casei 212.3 and Bifidobacterium infantis 25962, exhibited 100% gastric transit tolerance in the presence of milk proteins. In general, the addition of hog gastric mucin did not influence simulated gastric transit tolerance of lactobacilli but tended to increase that of bifidobacteria. However, it increased that of Lact. casei 242 and Lact. salivarius 43338 but diminished that of B. bifidum 2715 and B. animalis Bo. Selected bile salts-resistant isolates were intrinsically tolerant to simulated small intestinal transit. Only Lact. casei F19 and B. adolescentis 15703T showed significant reduction in viability after 240 min. In general, the addition of milk proteins and SBTCI did not affect simulated small intestinal transit tolerance. However, they significantly improved the intrinsic resistance of Lact. casei F19 but diminished that of B. breve 15700T. It is concluded that, whereas the majority of bile salts-resistant lactobacilli and bifidobacteria may be intrinsically sensitive to gastric transit, they are intrinsically resistant to small intestinal transit. In addition, it is postulated that milk proteins and mucin may function as both buffering agents and inhibitors of digestive protease activity in vivo, thereby protecting ingested bacterial strains during upper gastrointestinal transit.

Sixteen Bifidobacterium isolates from the human gastrointestinal tract were assayed for susceptibility to 44 antibiotics by soft agar overlay disc diffusion on TPY agar. Five isolates (3/7 B. bifidum and 2/3 B. breve) exhibited atypical antibiotic susceptibility profiles. Poor growth in the agar overlay accounted for susceptibility of B. bifidum but not B. breve isolates. All other isolates were resistant to cefoxitin (30 micrograms), aztreonam (30 micrograms), vancomycin (30 micrograms), amikacin (30 micrograms), gentamicin (10 micrograms), kanamycin (30 micrograms), streptomycin (10 micrograms), fusidic acid (10 micrograms), trimethoprim (5 micrograms), norfloxacin (10 micrograms), nalidixic acid (30 micrograms), metronidazole (5 micrograms), polymyxin B (300 micrograms) and colistin sulphate (10 micrograms), and they were susceptible to the six penicillins studied, cephalothin (30 micrograms), cefuroxime (30 micrograms), cefaclor (30 micrograms), ceftizoxime (30 micrograms), cefotaxime (30 micrograms), bacitracin (10 micrograms), chloramphenicol (30 micrograms), erythromycin (15 micrograms), clindamycin (2 micrograms), rifampicin (5 micrograms) and nitrofurantoin (300 micrograms). In addition, they varied in their susceptibility to cephradine (30 micrograms), cephazolin (30 micrograms), cefoperazone (75 micrograms), ceftriaxone (30 micrograms), ofloxacin (5 micrograms) and furazolidone (15 micrograms). They were resistant, or only marginally moderately susceptible, to ceftazidime (30 micrograms), netilmicin (10 micrograms), sulphamethoxazole (100 micrograms), cotrimoxazole (25 micrograms) and ciprofloxacin (5 micrograms), and susceptible or marginally moderately susceptible to tetracycline (30 micrograms). All B. bifidum isolates were susceptible to cefixime (5 micrograms). Four microorganism-drug combinations were evaluated for beta-lactamase activity but its absence suggested that cell wall impermeability was responsible for cephalosporin resistance among bifidobacteria. The antibiotic susceptibility of B. animalis 25527T was similar to that of the human isolates.

Background: The bacterial chemotactic peptide N-formyl-methionine-leucine-phenylalanine (FMLP) is produced by enteric flora and is one of the factors implicated in contributing to inflammatory bowel disease. Expression of receptors for FMLP on human phagocytes (polymorphs and monocytes) is well established, but there is conflicting evidence regarding the potential expression of FMLP receptors on other cells within the mucosa, particularly the epithelial cells., Aims: To map FMLP receptors within intestinal mucosa using several different experimental approaches., Methods and Results: Radioligand binding assays with 'H-FMLP' revealed no specific binding to primary cultured colonic enterocytes or to the cell line HT29, whereas neutrophils, as expected, exhibited specific binding with a Kd of 19 nM and approximately 2 x 10(4) receptors per cell. FITC labelled FMLP exhibited specific, displaceable binding on flow cytometry to neutrophils and monocytes but not to 10 gastrointestinal epithelial cell lines. Isolated lamina propria lymphocytes and peripheral blood lymphocytes exhibited no binding. To confirm the absence of receptors on epithelia, reverse transcription polymerase chain reaction for mRNA for the classic FMLP receptor was performed. While the presence of message was detected in activated peripheral blood phagocytes, it was not detected in epithelial cell lines. To exclude the possibility of FMLP binding to other receptors such as tachykinin receptors on epithelia, FITC labelled FMLP binding in tissue sections confirmed that the binding is subepithelial--that is, in the lamina propria., Conclusion: Receptors for FMLP are subepithelial and map to the lamina propria of the gastrointestinal mucosa.

We have previously described the isolation, cloning, and characterization of a tumorigenic murine fibrosarcoma, designated JBS. Growth of JBS tumors in syngeneic mice initiates an anti-tumor immune response that initially manifests as progressive splenic hyperplasia and an increased proliferative ability in cultured splenocytes. In animals with tumors progressing beyond the 2 cm stage there is a reduction in spleen size and a gradual decrease in splenocyte proliferative abilities, leading to anergy at heavy tumor burdens (>3.5 cm). During the phase of immune hyperresponsiveness in tumor-bearing mice clearance of Listeria monocytogenes by components of the innate immune system is increased. This heightened resistance to infection is most likely macrophage-mediated because these mice demonstrate an increased ability to recruit macrophages to the peritoneal cavity during Listeria infection. In addition, these macrophages are highly activated in vivo as evidenced by an elevated capacity to express class II MHC (Ia) molecules. This increase in macrophage activation status is coincident with an increased capacity of splenocytes from tumor-bearing mice to secrete IFN-gamma. In mice with much heavier tumor burdens (>3.5 cm), down-regulation of the immune response leads to a reduction in peritoneal macrophage numbers, decreased macrophage Ia expression, and diminished splenic clearance of L. monocytogenes. Our data demonstrate that activation of macrophages distal to the tumor site occurs as an initial consequence of tumor growth. It is only in mice with very heavy tumor burdens that functionality of macrophages is sufficiently suppressed to allow increased splenic growth of L. monocytogenes.

A sensitive method for simultaneously detecting and discriminating between bovine herpesviruses types 1 and 5 (BHV-1 and BHV-5) was developed using a nested polymerase chain reaction (PCR) technique. Following amplification using type-common primers derived from gC sequences, amplification using type-specific nesting primers produced different-sized bands specific to the corresponding types, as demonstrated by blot hybridization. Less than 0.1 plaque-forming units (PFU) of each virus and 75 fg or less of viral DNA were routinely detected. The PCR technique amplified correct product from 4 BHV-5 isolates and from 48 BHV-1 isolates, all from the United States, and did not amplify heterologous herpesviruses. The PCR technique was more sensitive than virus isolation in detection of BHV-1 or BHV-5 in nasal secretions from experimentally and naturally infected calves, and it detected BHV-1 or BHV-5 in trigeminal ganglia from these calves.

Ninety-one isolates of Pasteurella multocida (Pm)and 124 of Pasteurella haemolytica (Ph) were recovered from the lungs of calves that died of bovine respiratory tract disease (BRTD). Nine Pm enzyme profiles (A through I) and 9 Ph enzyme profiles (J through R) were determined for the Pasteurella isolates. The Pm isolates were relatively evenly divided among the enzyme profiles, with one exception, profile I. The Ph isolates were not evenly distributed among the profiles. Fifty of the 91 Pm isolates were serotyped. Forty-two Pm isolates were positive for capsule type A, and 8 were untypable. Five somatic type antigen profiles (3; 3,4; 3,7; 3,4,7; and 4) were identified among the 50 serotyped Pm isolates; one isolate was untypable. The Ph isolates were further divided through serotyping and grouped as follows: 74 (60%) Pasteurella haemolytica A1 (PhA1), 12 (10%) PhA2, 4 (3%) PhA5, and 34(27%) PhA6. Eighty-one percent of the Ph serotypes were clustered in the M and N enzyme profile. The P enzyme profile was almost unique to PhA2 (8 of 12, 67% of PhA2 isolates). Results of this study indicate a need to collect more data on Ph serotypes at the state veterinary diagnostic laboratories.

Background: Micrometastases within bone marrow have been shown to indicate a poor prognosis in patients with epithelial tumours. However, the degree to which micrometastases represent true residual disease or cell shedding and metastatic potential, is unclear., Aim: To explore whether micrometastases represent residual disease, bone marrow taken from carefully staged patients before and after (> 6 months) "curative" resection of a primary gastrointestinal cancer was studied prospectively., Patients/methods: Seventy two consecutive patients were studied; the only exclusions were patients with known overt metastatic disease at the time of surgery. Micrometastatic cells were quantified per 10(5) marrow cells by flow cytometry after staining for contaminant cytokeratin-18 positive cells., Results: Micrometastases were detected preoperatively in 22% (16/72) of all patients, comprising 11 (23%) of 48 with colorectal cancer, five (33%) of 15 with gastric adenocarcinoma and none (0%) of nine with oesophageal squamous cancer. Although fewer metastatic cells were detected in postoperative bone marrow, and clearance of marrow deposits was evident in most patients, the persistence of micrometastases in five of 16 patients after resection, without evidence of tumour recurrence, indicates a subset with true residual disease. Detection of micrometastases postoperatively (persistent or newly developed) was significantly associated with development of overt metastases during the subsequent 12-18 months of follow up (nine of 19 patients) when compared with patients testing negative for micrometastases (eight of 53; p < 0.01)., Conclusions: Preoperative detection of micrometastases may reflect either transient shedding of cells, metastatic potential or residual disease, but postoperative micrometastases indicate minimal residual disease. Identification of these patients is important because they may benefit from adjuvant therapy.

Lactobacillus and Bifidobacterium species constitute a significant proportion of probiotic cultures used in developed countries in 'microbial adjunct nutrition'. A number of differential plating methodologies have been developed which seek to selectively detect and enumerate these bacterial groups in bioproducts. Differences in oxygen tolerance, nutritional requirements, antibiotic susceptibility, and colony morphology and colour constitute the bases of differentiation in these methods. The choice of methodology depends on the nature of the bioproduct to be examined (wet or dry) and the presence of other bacteria such as starter cultures. In addition, a number of nucleic acid methods have been developed in recent years which enable the specific detection of these bacterial groups at species, subspecies and strain level in mixed populations. The methods use synthetic 16S and 23S rRNA-targeted hybridisation probes, the specificity of which can be adjusted to fit any taxonomic ranking from genus to genotype, for detection, enumeration and identification in situ or after differential plating. The combined use of differential plating and molecular strain typing methodologies provides food and medical microbiologists with a powerful and targeted approach to the detection, enumeration and identification of these bacterial groups and their members in a wide range of food and biological materials. An overview of these methods is presented in this review.

Equine herpesvirus-1 (EHV-1) infection in a few widely scattered neurons and astrocytes plus endothelial cells in brain and spinal cord of two horses with naturally occurring paralytic disease was demonstrated by use of an immunoperoxidase technique. These horses were euthanatized less than 48 hours after the onset of clinical signs. No staining for EHV-1 was demonstrated in brain or spinal cord of three horses that had a longer duration of clinical disease or in two uninfected horses.

Although production of immunosuppressive factor(s) by esophageal squamous cancer has been demonstrated, systemic immunosuppression occurs late. Whether local immunosuppression by tumor-derived factors occurs in vivo as a potential mechanism of escape from immune surveillance is unknown. We found that lymphocytes from nodes draining distal esophageal squamous tumors in 23 consecutive patients had depressed proliferative and cytotoxic responsiveness relative to both lymphocytes from a reference node outside the field of drainage and matched PBL from the same patient. In a subset of patients in which more than one tumor-draining node was examined, a radial or zonal immunosuppression relative to the primary tumor was evident. The findings were unrelated to surgery or anatomic location because all but 2 of 26 control patients with esophagogastric adenocarcinoma had normal or enhanced lymphocyte responsiveness in the tumor-draining node. The absence of overt or even micrometastatic nodal disease, as determined by immunostaining for cytokeratin expression, coupled with the long-term survival of several of the patients, strongly suggests that the immunosuppressive effect is due to mechanisms other than metastases, and may be a premetastatic occurrence. We conclude that regional immunesuppression does exist in patients with esophageal squamous cancer when systemic immunity is still well preserved. The local immune suppression inhibits the generation of lymphokine-activated killer (LAK) cells and may be an impediment to potential immunotherapeutic strategies.

Tumors escape immunological rejection by a diversity of mechanisms. In this report, we demonstrate that the colon cancer cell SW620 expresses functional Fas ligand (FasL), the triggering agent of Fas receptor (FasR)-mediated apoptosis within the immune system. FasL mRNA and cell surface FasL were detected in SW620 cells using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining, respectively. We show that SW620 kills Jurkat T cells in a Fas-mediated manner. FasR-specific antisense oligonucleotide treatment, which transiently inhibited FasR expression, completely protected Jurkat cells from killing by SW620. FasL-specific antisense oligonucleotide treatment of SW620 inhibited its Jurkat-killing activity. FasL has recently been established as a mediator of immune privilege in mouse retina and testis. Our finding that colon cancer cells express functional FasL suggests it may play an analogous role in bestowing immune privilege on human tumors. HT29 and SW620 colon cancer cells were found to express FasR mRNA and cell surface FasR using RT-PCR and immunofluorescence flow cytometry, respectively. However, neither of these cells underwent apoptosis after treatment by the anti-FasR agonistic monoclonal antibody CH11. Our results therefore suggest a Fas counterattack model for immune escape in colon cancer, whereby the cancer cells resist Fas-mediated T cell cytotoxicity but express functional FasL, an apoptotic death signal to which activated T cells are inherently sensitive.

Background & Aims: Immunohistochemical detection of bone marrow micrometastases has been reported as a prognostic marker in colorectal cancer. The aims of this study were to evaluate the potential advantage of flow cytometry as an objective method of identifying and quantifying micrometastatic deposits within bone marrow and to determine the prevalence and quantity of micrometastases in patients undergoing surgery for gastrointestinal cancers., Methods: Flow cytometry was first validated by a controlled "spike" experiment in which varying numbers of neoplastic epithelial cells were added to bone marrow, and cytometry was performed in a blinded fashion. Three neoplastic cell lines (colonic and esophageal) with varying degrees of expression of cytokeratin-18 were used. Epithelial cells were detected by dual staining with fluorescence-labeled, monoclonal anti-cytokeratin, and propidium iodide., Results: Cytometry reproducibly detected the presence of > or = 10 neoplastic cells per 10(5) marrow cells. Micrometastases were found in 20%-30% of patients undergoing potentially curative resection of colorectal and gastroesophageal adenocarcinomas. There was a trend toward increasing positivity for marrow deposits with advanced Dukes' staging of colorectal cancer., Conclusions: Flow cytometric assessment of bone marrow is a reliable, objective, and quantitative method of detecting micrometastatic deposits found in a substantial subset of patients undergoing surgery for gastrointestinal adenocarcinomas.

Immunoglobin G (IgG) quantitation was performed by a commercially available single radial immunodiffusion kit on 528 plasma samples obtained from llamas. Fifty samples encompassing the range of values obtained were selected for further evaluation. The IgG concentration of these samples was measured by a second commercially available single radial immunodiffusion kit and a sodium sulfite precipitation kit. gamma-Globulins also were measured. IgG values predicted by the single radial immunodiffusion kits were not in agreement. Failure to appreciate this lack of agreement could result in misinterpretation of a llama's IgG level derived from one kit when clinical guidelines established using the other kit are followed.

This study determined the efficacy of treatment of chronic Staphylococcus aureus IMI of lactating dairy cows with a biological response modifier consisting of a killed preparation of Propionibacterium acnes. Fifty-one lactating dairy cows with chronic S. aureus IMI on two commercial dairies were enrolled in a controlled, randomized field trial. Twenty-four cows received 1 ml of the immunostimulant twice weekly for 4 wk, and the remainder of the cows served as untreated controls. Quarter milk samples were collected for SCC from all cows at 3 and 6 wk after initiation of treatment and for culture at 6 wk after initiation of treatment. If samples were negative for S. aureus at 6 wk, cows were sampled again for culture 1 wk later for 2 successive d before being considered cured. For the cows treated with immunostimulant, 16.7% (36.7% of infected quarters) were cured of S. aureus IMI; for the untreated group, 11.1% of cows (32.2% of quarters) were cured. The difference was not statistically significant. Immunostimulant treatment had no effect on quarter SCC in infected quarters overall, but, in those quarters that were cured of S. aureus infection, quarter SCC of treated cows tended to be lower than SCC of control cows.

Intraocular production of Toxoplasma gondii-specific antibody in cats has been estimated by comparing the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of total immunoglobulins in serum and aqueous humor (Goldmann-Witmer coefficient; aqueous antibody coefficient; C value). It has been proposed that in human beings, comparison of the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of antibodies against a nonocular pathogen in serum and aqueous humor is more accurate than methods using total immunoglobulin quantification. We developed an ELISA for detection of calicivirus-specific antibodies in the serum and aqueous humor of cats. By evaluating calicivirus-specific antibody concentrations in the aqueous humor of healthy and diseased cats, calicivirus was assessed as a nonintraocular pathogen. The ratio of T gondii-specific antibodies in the aqueous humor and serum and the ratio of calicivirus-specific antibodies in serum and aqueous humor were evaluated as a means of estimating intraocular T gondii-specific antibody production. A field strain of feline calicivirus was isolated, cultured, and purified. A calicivirus-specific IgG ELISA was developed for detection of feline calicivirus-specific IgG in serum and aqueous humor. Calicivirus-specific IgG was measured in the serum and aqueous humor from 3 groups of control cats. Results suggested that calicivirus is a nonintraocular pathogen in cats and that calicivirus IgG detected in aqueous humor is attributable to leakage across a damaged blood-ocular barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Aims: A survey was undertaken to assess the effectiveness of an integrated approach to the provision of low visual aids (LVAs) in south Devon over a 2 year follow up period. This integrated approach includes the assessment of patient needs by low vision therapists, followed by the provision of suitable LVAs, with particular emphasis on training in their use., Methods: A total of 125 patients were selected at random from the 445 patients seen in the low vision clinic at Torbay Hospital in the year 1991. These patients were sent questionnaires relating to the service over a 2 year period. Questionnaires from 111 patients were analysed at 1 year and 75 questionnaires together with 46 clinical reassessments, after 2 years., Results: Using a similar questionnaire to one used in a previous study in the UK from a unit where LVA training was not provided, not only was a higher rate of satisfaction found with the services provided, but also the LVAs dispensed were used more frequently. The majority of the LVAs provided were of the simple, inexpensive variety and wastage was very low., Conclusions: It was concluded that this integrated approach to low vision rehabilitation with emphasis on training in the use of less complex LVAs exceeds the performance of other types of service that rely on the dispensing of more complex LVAs.

Isogenic mutant strains of Listeria monocytogenes demonstrating graded attenuation in mice were used to analyse the correlation between bacterial virulence, ability to induce class II MHC (la) molecules in antigen presenting cells and ability to vaccinate against secondary infection. The mutants used differed only in the amino acid sequence of the thiol-activated hemolysin, Listeriolysin O (LLO). The results indicate that L. monocytogenes mutants of reduced virulence have the potential to act as vaccines only if they are sufficiently persistent to induce la expression in antigen presenting cells. The findings also suggest that specific mutagenesis of virulence factors, including LLO, could provide an approach for creating Listeria monocytogenes strains with potential for use as attenuated live vaccines.

In this study we compared the host response to Listeria monocytogenes in 129 REJ mice with listeria-resistant (C57Bl/6j) and susceptible (Balb/c) mouse strains. In all experiments mice were inoculated by the i.p. route. 129 REJ mice and Balb/c mice were sensitive to listeriosis whilst C57Bl/6j mice were relatively resistant to i.p. infection. Relatively large numbers of viable bacteria could be detected in the spleens of 129 REJ mice as early as 6 h following i.p. inoculation suggesting that dissemination of listeria from the peritoneal cavity is rapid in this mouse strain. This contrasted with Balb/c mice which exhibited an early lag phase during which only low numbers of bacteria could be isolated from the spleens of infected animals. In response to both proteose peptone and live listeria, 129 REJ mice demonstrated a greater capacity to recruit neutrophils to the peritoneal cavity than Balb/c and C57Bl/6j mice. In addition, inflammatory phagocytes from 129 REJ mice were as bactericidal in vitro as phagocytes from the Balb/c and C57Bl/6j strains. However, in vivo, inflammatory neutrophils elicited by proteose peptone prior to i.p. infection with L. monocytogenes were not protective in the three mouse strains tested. Despite the apparent inadequacy of peritoneal neutrophils in controlling early bacterial proliferation, depletion of neutrophils in 129 REJ mice severely exacerbated i.p. infection with L. monocytogenes. The results indicate that neutrophils provide an inefficient but essential means of controlling early outgrowth of listeria in the peritoneal cavity of 129 REJ mice. The excessive inflammatory response seen in 129 REJ mice may facilitate the early dissemination of L. monocytogenes from the peritoneal cavity to peripheral sites.