Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilis by PCR.

A polymerase chain reaction (PCR) assay was developed for discrimination of Bacillus subtilis from other members of B. subtilis group as well as rapid identification from environmental samples. Primers ENIF and EN1R from endoglucanase gene were used to amplify a1311 bp DNA fragment. The specificity of the primers was tested with seven reference strains and 28 locally isolated strains of endoglucanase positive Bacillus species. The PCR product was only produced from B. subtilis. The results demonstrated high specificity of two oligonucleotides for B. subtilis. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of B. subtilis. To our knowledge this is the first report of a B. subtilis specific primer set.

Outbreak of bovine brucellosis in County Clare, Ireland, in 2005.

This paper describes an investigation of an outbreak of bovine brucellosis in County Clare, Ireland, during 2005. It is likely that infection on the index farm was linked to a previous outbreak of brucellosis in County Clare. During March to May 2005, transmission of brucellosis within the herd was rapid; this was facilitated by a range of factors, including close contact between cattle kept in winter housing, and the mixing of animals, both during grazing and at housing, throughout the year. Containment of the disease, including only limited spread to one contiguous herd, was facilitated by the recent construction of a shed for winter housing.

Survival and dispersal of a defined cohort of Irish cattle.

An understanding of livestock movement is critical to effective disease prevention, control and prediction. However, livestock movement in Ireland has not yet been quantified. This study has sought to define the survival and dispersal of a defined cohort of cattle born in Co. Kerry during 2000. The cohort was observed for a maximum of four years, from January 1, 2000 to December 31, 2004. Beef and dairy animals moved an average 1.31 and 0.83 times, respectively. At study end, 18.8% of the beef animals remained alive on Irish farms, including 6.7% at the farm-of-birth, compared with 48.6% and 27.7% for dairy animals respectively. Beef animals werae dispersed to all Irish counties, but mainly to Cork, Limerick, Tipperary and Galway. Dairy animals mainly moved to Cork, Limerick, and Tipperary, with less animals going to Galway, Meath and Kilkenny. The four-year survival probability was 0.07 (male beef animals), 0.25 (male dairy), 0.38 (female beef), and 0.72 (female dairy). Although there was considerable dispersal, the number of moves per animal was less than expected.

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis.

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results.

Hospital outreach service: helping to prevent nursing home placement.

An outreach service from a post-acute metropolitan teaching hospital delivered an intensive, multidisciplinary and coordinated allied health service, and achieved both early hospital discharge and the prevention or delay of nursing home placement. This article reports on three types of cases which illustrate how the service assisted ward teams, families and patients to determine whether nursing home placement was essential. For a group of 20 cases, the total reduction in hospital length of stay was 556 days, and home accommodation as an alternative to nursing home accommodation was achieved for a total of 7505 days. The article outlines a matrix of advantages and disadvantages, both tangible and intangible, of home versus nursing home accommodation. It is suggested that a full costing of this matrix would inform debate on the comparative merits of long-term home and nursing home accommodation.

The home based rehabilitation service: rationale, operation and outcomes.

The Home Based Rehabilitation Service was established as an allied health early discharge and outreach service from a major metropolitan post-acute teaching hospital. Two hundred and eighty-two patients were discharged to the service according to established criteria from the following specialities: neurology, neurosurgery, rheumatology, amputation, orthopaedic and spinal. Inpatient length of stay was reduced by 19 days on average (the range was 3-75 days). Inpatient throughput was increased equivalent to 10 extra beds on an annual basis. The cost of home-based services was 11 per cent of the cost of the inpatient services they replaced. There were low rates of hospital readmissions, and users registered high levels of satisfaction with the service.

B7-1/CD80-transduced tumor cells elicit better systemic immunity than wild-type tumor cells admixed with Corynebacterium parvum.

Tumor cells genetically modified by transduction of B7 (B7-1/CD80), a natural ligand for the T-cell costimulatory molecules CD28 and CTLA-4, can elicit potent tumor immunity, and they can be effective for treatment of established cancers in animal models. In this study, three tumor lines, the EL4 lymphoma, the P815 mastocytoma, and the MCA102 sarcoma were transduced with recombinant retrovirus containing the murine B7 gene, and their potency to induce systemic immunity protective against challenge with wild-type tumor was compared to that of the same tumor cells admixed with the commonly used adjuvant Corynebacterium parvum. While admixture of tumor cells with C. parvum resulted in complete regression of tumors in syngeneic mice, it did not induce protective immunity against a subsequent challenge of wild-type cells from any of the 3 tumors tested. In contrast, B7-transduced EL4 and P815 tumors regressed locally and induced a potent systemic immunity to wild-type tumors and a higher level of cytotoxic T-cell activity than did tumor cells admixed with C. parvum. No systemic immunity was induced by B7-transduced nonimmunogenic MCA102 sarcoma cells. Our results demonstrate that immunogenic tumor cells transduced with the B7 gene are superior to tumor cells mixed with C. parvum for the induction of systemic tumor immunity.

Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity.

A costimulatory signal through B7 to its counter-receptor CD28 on T cells enhances T cell activation. We have generated recombinant retroviruses containing cDNA for murine B7 and transduced a panel of murine tumor lines with varying immunogenicity to study the effect of B7 costimulation on antitumor immunity. In contrast to the progressive outgrowth of all wild-type (B7-) tumors in unimmunized syngeneic mice, four immunogenic tumors, lymphoma RMA, EL4, mastocytoma P815, and melanoma E6B2, regressed completely when transduced with the B7 gene. In contrast, four nonimmunogenic tumors, sarcomas MCA101, MCA102, and Ag104, and melanoma B16, remained tumorigenic after transduction of the B7 gene. Immunization with B7-transduced immunogenic tumors enhanced protective immunity and increased specific cytotoxic T lymphocyte (CTL) activity against the respective wild-type tumors as compared to immunization with nontransduced or mock-transduced tumors. Moreover, cocultivation of CTL with B7-transduced EL4 cells augmented the specificity of tumor-reactive CTL in long-term cultures. Treatment by injection of B7-transduced tumor cells cured 60% of mice with established wild-type EL4 lymphoma. In contrast, immunization with nonimmunogenic tumors transduced with B7 did not provide protective immunity and did not increase specific CTL activity. Our results show that tumor immunogenicity is critical to the outcome of costimulation of T cell-mediated tumor immunity by B7.

Metastatic conversion of cells by expression of human papillomavirus type 16 E6 and E7 genes.

The human papillomavirus type 16 (HPV-16) is a DNA tumor virus highly associated with cervical carcinoma. Viral DNA from HPV-16 is found in primary tumors and their metastatic lesions. To investigate the role of HPV-16 oncoproteins in the development of cancer metastasis, the E6 and E7 genes from HPV-16 were inserted into retrovirus and introduced into nonmetastatic mouse cell lines. Expression of either of the viral genes from HPV-16 made the cells metastatic in nude mice. In contrast, expression of the E6 and E7 genes of HPV type 6 (HPV-6b), which is frequently found in nonmalignant HPV-associated diseases, did not. The metastatic ability of cells transduced with viral genes of HPV-16 did not correlate with their growth rate or sensitivity to destruction by natural killer cells. Our results demonstrate that expression of oncogenic proteins of HPV-16 can cause tumor metastasis and implicate HPV-16 in an important role regarding the progression of HPV-associated human cancers.

Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4.

Interaction of the B7 molecule on antigen-presenting cells with its receptors CD28 and CTLA-4 on T cells provides costimulatory signals for T cell activation. We have studied the effects of B7 on antitumor immunity to a murine melanoma that expresses a rejection antigen associated with the E7 gene product of human papillomavirus 16. While this E7+ tumor grows progressively in immunocompetent hosts, cotransfection of its cells with B7 led to tumor regression by a B7-dependent immune response mediated by CD8+ cytolytic T lymphocytes. The immune response induced by E7+B7+ tumor cells also caused regression of E7+B7- tumors at distant sites and was curative for established E7+B7- micrometastases. Our findings suggest that increasing T cell costimulation through the CD28 and CTLA-4 receptors may have therapeutic usefulness for generating immunity against tumors expressing viral antigens.

Stereospecific effects of ascorbic acid and analogues on D1 and D2 agonist binding.

Ascorbic acid inhibited the specific binding of both the D1 agonist, [3H] SKF 38393, and the D2 agonist, [3H] N-0437 at physiologically relevant concentrations. This inhibition was both stereospecific and receptor selective. Using ligand concentrations approximating their KD's, the IC50's for ascorbate and two structural analogues, isoascorbate and D-glucoascorbate, were determined. The rank order of IC50's at both D1 and D2 were D-glucoascorbate greater than isoascorbate greater than ascorbate. However, the IC50 for each compound was greater at D1 than D2. Evaluation of the relationship between the IC50 for ascorbate and the ligand concentration using both the D1 and the D2 ligand yielded data inconsistent with competitive inhibition models. Preliminary experiments were conducted to evaluate the site and type of inhibition with results consistent with an allostearic effect at the level of the receptor.

Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin C and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates.

Alkaline phosphatase (AP) was covalently linked to the two antitumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), forming conjugates that could bind to antigen-positive tumor cells. The conjugates were able to convert the prodrugs, mitomycin phosphate (MOP) and etoposide phosphate (EP), into an active mitomycin C derivative, mitomycin alcohol, and etoposide, respectively. MOP and EP were less toxic to cultured cells from the H2981 lung adenocarcinoma than their respective hydrolysis products, mitomycin alcohol and etoposide, by a factor greater than 100, and they were also less toxic in mice. Pretreatment of H2981 cells with L6-AP greatly enhanced the cytotoxic effects of MOP and EP, while 1F5-AP caused no such enhancement. A strong antitumor response was observed in H2981-bearing mice that were treated with L6-AP followed 24 h later by either MOP or a combination of MOP and EP. This response was superior to that of MOP or combinations of MOP and EP given alone.