Effect of ingested sperm on fecundity in the rat.

The intragastric administration of homologous strain epididymal sperm to adult virgin NZBW (Rt 1) female rats was shown to induce short-to long-term infertility. Infertility was associated with an early rise of genital secretory fluid IgA antisperm antibody preceding mating.

Australasian Laparoscopic Colon Cancer Study shows that elderly patients may benefit from lower postoperative complication rates following laparoscopic versus open resection.

BACKGROUND: A retrospective analysis of age-related postoperative morbidity in the Australia and New Zealand prospective randomized controlled trial comparing laparoscopic and open resection for right- and left-sided colonic cancer is presented. METHODS: A total of 592 eligible patients were entered and studied from 1998 to 2005. RESULTS: Data from 294 patients who underwent laparoscopic and 298 who had open colonic resection were analysed; 266 patients were aged less than 70 years and 326 were 70 years or older (mean(s.d.) 70.3(11.0) years). Forty-three laparoscopic operations (14.6 per cent) were converted to an open procedure. Fewer complications were reported for intention-to-treat laparoscopic resections compared with open procedures (P = 0.002), owing primarily to a lower rate in patients aged 70 years or more (P = 0.002). Fewer patients in the laparoscopic group experienced any complication (P = 0.035), especially patients aged 70 years or above (P = 0.019). CONCLUSION: Treatment choices for colonic cancer depend principally upon disease-free survival; however, patients aged 70 years or over should have rigorous preoperative investigation to avoid conversion and should be considered for laparoscopic colonic resection. REGISTRATION NUMBER: NCT00202111 (http://www.clinicaltrials.gov).

Classifying algorithms for SIFT-MS technology and medical diagnosis.

Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) is an analytical technique for real-time quantification of trace gases in air or breath samples. SIFT-MS system thus offers unique potential for early, rapid detection of disease states. Identification of volatile organic compound (VOC) masses that contribute strongly towards a successful classification clearly highlights potential new biomarkers. A method utilising kernel density estimates is thus presented for classifying unknown samples. It is validated in a simple known case and a clinical setting before-after dialysis. The simple case with nitrogen in Tedlar bags returned a 100% success rate, as expected. The clinical proof-of-concept with seven tests on one patient had an ROC curve area of 0.89. These results validate the method presented and illustrate the emerging clinical potential of this technology.

The rapid evaluation of bacterial growth in blood cultures by selected ion flow tube-mass spectrometry (SIFT-MS) and comparison with the BacT/ALERT automated blood culture system.

We compared the performance of the BacT/ALERT automated blood culture system with real-time, quantitative volatile organic compound (VOC) detection by selected ion flow tube-mass spectrometry (SIFT-MS). Blood samples from healthy donors were artificially infected with 5 or 100 CFU of organisms commonly causing bacteremia. Positive results by SIFT-MS analysis of headspace gases were recorded for 53/60 (88.3%) bottles at 8h, and 58/60 (96.6%) bottles at 24 h. We conclude that SIFT-MS is a sensitive method for the detection of microbial VOCs. Furthermore, profiles of the VOCs detected may allow simultaneous identification of infecting organisms.

A microculture technique for rat lymphocyte transformation.

We report the development of an economical microculture technique suitable for measuring rat lymphocyte response to mitogens and in mixed lymphocyte reactions. The effects of varying culture conditions, i.e. source of serum, addition and concentration of 2-mercaptoethanol, mitogen concentrations, culture incubation times, absorption of serum, lymphocyte numbers and microtitre plate well shape are described.

Dual silver staining to characterise Helicobacter spp. outer membrane components.

Helicobacter pylori is a bacterial pathogen, estimated to infect half the world's population. The bacterium is the aetiological cause of gastritis, the common precursor for peptic ulcer disease and gastric cancer. Immunisation of at-risk individuals is the most cost-effective means of dealing with such a widespread pathogen. Potential vaccine candidates need to be identified and characterised. Conventional silver staining is commonly used for the sensitive detection of bacterial protein components separated by SDS-PAGE. Modified silver stains employing periodate oxidation have also been developed for the analysis of purified bacterial lipopolysaccharide. By using these methods in parallel, as a dual silver stain, bacterial fractions can be characterised in terms of protein and LPS content. Strain differences can also be readily identified by comparing protein and LPS profiles. When combined with differential immunoblotting, the dual silver stain is a useful analytical tool for characterising potential vaccine candidate antigens.

A micro macrophage migration inhibition test for the detection of cellular immunity in vitro.

A simple, economical, macrophage migration inhibition assay enabling multiple tests to be performed using microculture plates has been developed. The system confers many advantages over the standard in vitro assay of macrophage migration inhibition factor. It requires only 5 X 10(5) macrophages for each assay, small volumes (100 microliter) of test factors are consumed, 96 tests in each culture plate are easily assessed microscopically and the technique is easily and reproducibly executed. An adherent monolayer of activated fresh mouse peritoneal exudate cells (PEC) is established in each microculture well. A 'starting line' is scored across the monolayer by the application of a trimmed razor blade and the cells are dislodged and aspirated from one side of the line. Test factors from cultures of stimulated lymphocytes were added to the 'wounded' monolayer and the migration of macrophages from test and control cultures was measured after staining. The cells across the line were counted after incubation. Miration was shown to be time and temperature dependent, markedly stimulated by colchicine and appeared to associate with tuberculin antigen (PPD) skin testing and lymphocyte transformation in human subjects.

Antibacterial action of the urease inhibitor acetohydroxamic acid on Helicobacter pylori.

The urease inhibitor acetohydroxamic acid (AHA) was assessed for its bacteriostatic and bactericidal effects on Helicobacter pylori. For eight isolates of H pylori, the minimum inhibitory concentration (MIC) was either 200 mg/l or 400 mg/l. Interactions between AHA and antimicrobial drugs used to treat H pylori were also determined. For most isolates AHA reduced the MIC for colloidal bismuth subcitrate (CBS), tetracycline, metronidazole, and amoxicillin. In a few isolates, however, AHA increased the minimum bactericidal concentration (MBC) for these antimicrobial treatments. In vitro AHA is active against H pylori and it interacts with other agents directed against H pylori.

Lack of protection following immunisation with H. pylori outer membrane vesicles highlights antigenic differences between H. felis and H. pylori.

Helicobacter pylori-induced inflammation is associated with the development of gastritis, peptic ulcer disease and gastric cancer in humans. Immunisation against this bacterium would ultimately have a major impact on H. pylori-related disease, notably global gastric cancer rates. To date, several potential H. pylori vaccine candidates have been identified. In this study, the Helicobacter felis/murine model was used to assess the immunogenicity of a previously undescribed H. pylori outer membrane vesicle fraction in immune protection.

Iron influences the expression of Helicobacter pylori outer membrane vesicle-associated virulence factors.

BACKGROUND: Helicobacter pylori shed outer membrane vesicles (OMV) in vitro and in vivo. These OMV, which contain active VacA, provide a potential vehicle for the delivery of H. pylori virulence factors to the gastric mucosa. OBJECTIVE: To assess the influence of environmental iron levels on H. pylori OMV VacA and protease expression in vitro. METHODS: Three well-characterized H. pylori type-strains were grown for 72 h under normal (Brucella broth, 5% fetal calf serum) and iron-limiting (Brucella broth, 5% fetal calf serum, 50 micromol/l deferoxamine) conditions. Following harvesting by differential centrifugation, the ratio of whole cells to OMV was determined. OMV VacA levels in response to iron availability were determined by ELISA and immunolabelling of washed bacteria. Protease activity was detected by zymography of OMV in the presence and absence of enzyme inhibitors and activators. HEp-2 cells were used to assay for OMV-associated cytopathogenic toxins. RESULTS: Decreased iron availability, which limited bacterial growth but not OMV release, also influenced the expression of OMV-associated virulence factors. VacA levels were reduced, whereas two new proteolytic enzymes were expressed on these OMV. When an iron salt was added to counteract the effect of the deferoxamine, VacA levels were restored in the outer membrane and the proteolytic activity disappeared. CONCLUSIONS: These results suggest that OMV release by H. pylori is influenced by environmental iron levels, and that the qualitative changes that occur in outer membrane composition may contribute to the clinical patterns of H. pylori-associated disease.

Polymorphonuclear leukocyte-mediated bone degradation and influence of blood mononuclear cells on the mouse calvarial model.

OBJECTIVE: To investigate the relationships between degradation of bone and activated blood polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (ML) as well as their soluble products in vitro. MATERIALS AND METHODS: A neonatal mouse calvarial bone model was used to assess the activity of degradation (by measuring the amount of 45Ca release) by normal human blood leukocytes, separated PMNL and ML following 24-hour incubation. The effects of conditioned culture medium obtained from Staphylococcus aureus-stimulated ML on PMNL-mediated calvarial bone loss were also studied. RESULTS: It was demonstrated that isolated human PMNL rapidly degraded bone in a dose and time dependent manner. The PMNL-mediated bone degradation was enhanced by conditioned medium obtained from Staphylococcus aureus-stimulated ML. CONCLUSION: These findings implicate PMNL as major contributors to early bone loss in infectious diseases such as acute haematogenous osteomyelitis.

Soluble products of Staphylococcus aureus-stimulated blood mononuclear cells in enhancing polymorphonuclear leukocyte degradation of bone.

Rapid, extensive loss of infected bone implies abnormal localized inflammatory cell activity. We have demonstrated, using a live bone Ca-45 release model, that polymorphonuclear leukocytes degrade bone in a dose dependent manner. Staphylococcus aureus-stimulated blood mononuclear leukocytes release soluble products in vitro that enhance that process. Despite the usually accepted roles of osteoclasts and their blood-borne monocytic precursors in normal bone remodelling, these results indicate that considerable early pathological infected bone loss may be attributable to inflammatory polymorphonuclear leukocytes.

Toxocaral infection in hydatid control officers: diagnosis by enzyme immunoassay.

An enzyme-linked immunosorbent assay for Toxocara canis larva excretory/secretory antigen has been set up in Christchurch. Employing this test, elevated specific antibody levels for toxocara larvae were found in 3.3% (3/90) of urban adult blood donors and 4.4% (4/90) of secondary school students. In comparison, elevated levels were present in 25.6% (23/90) of New Zealand hydatid control officers. It can be concluded from the present study that the higher risk of toxocariasis experienced by hydatid officers is reflected in elevated specific antibody titres in a significant proportion of individuals when compared with controls.

Mechanisms of bone degradation in infection: a review of current hypotheses.

Bone infection in clinical practice results in loss of structural integrity, leading to complications such as fracture and implant loosening. The biology of this process has received relatively little attention. In experimental models it seems clear that both polymorphonuclear leukocytes, whose effect predominates in the early stages, and mononuclear phagocytes are capable of bone degradation. An indirect effect by stimulation of physiologic mechanisms of resorption has not been ruled out, but the evidence points to direct degradation by inflammatory cell enzymes.

Is the port site really at risk? Biology, mechanisms and prevention: a critical view.

Early case reports of port site tumour implants led to debate regarding the appropriateness of laparoscopic techniques for abdominal malignancies. Review of clinical, animal and cell culture studies addresses the relationships between tumour staging and shed cell behaviour that contributes to the peri-operative spread of tumours. In addition, a porcine model was used to test tumour cell distribution after laparoscopic and open colonic resections. Clinical evidence indicate that wound recurrence rates are 0.60 and 0.85% for open and laparoscopic colon cancer operations, respectively. Tumour staging and operative techniques are the most important factors determining wound implantation. Port site and open wounds are at equal risk of tumour implantation.

The in vitro discrimination of rat immune and natural cytotoxicity by serum choice.

Natural in vitro cytotoxicity of non-immune lymphoid cells against a variety of transformed and normal iso- and allogeneic target cells has been described in a number of species (for review see Herberman, Djeu, Kay, Ortaldo, Riccardi, Bonnard, Holden, Fagnani, Santoni & Puccetti, 1979). This background cytotoxicity is a hindrance to the estimation of immune lymphocyte cytolysis (Herberman & Oldham, 1975). Other workers have demonstrated that the choice of serum in which effector (EC) or target cells (TC) are prepared or tested may greatly influence the degree of killing (Irie, Irie & Morton, 1974; Zielske & Golub, 1976; Akira & Takasugi, 1977; Ortaldo, Bonnard & Herberman, 1977). It was found that high levels of rat normal effector cell (NEC) cytotoxicity against normal rat fibroblasts observed in the presence of foetal calf serum (FCS) were markedly reduced by the inclusion of normal rat serum (NRS). This effect on NEC activity was consistently noted in eleven batches of FCS and eight batches of NRS, whereas, normal human serum (NHuS) gave variable results. Decreased TC lysis was proportional to the concentration of NRS, was independent of heat inactivation and was primarily due to an effect on the EC population. The effects of FCS and NRS were not affected by prior absorption on TC or EC nor was NEC activity related to a proliferative response of EC during the 18-48 h microcytotoxicity assay employed in these studies. Since the inclusion of NRS did not markedly diminish the levels of immune effector cell cytotoxicity we have been able consistently to demonstrate that the use of 10% NRS in long-term rat cytotoxicity assays provides a means of dissociating the measurement of immune and normal lymphoid cell cytotoxicity.