Phillip Summers

Induction of Multiple Chemokine and Colony-Stimulating Factor Genes in Experimental Burkholderia pseudomallei Infection

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN‐γ in addition to the chemokines interferon‐γ‐inducible protein 10 (IP‐10) and monocyte interferon‐γ‐inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G‐CSF, M‐CSF, GM‐CSF, IP‐10, Mig, RANTES, MCP‐1, KC and MIP‐2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high‐level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine‐induced neutrophil chemoattractant (KC), macrophage inflammatory protein‐2 (MIP‐2), monocyte chemoattractant protein‐1 (MCP‐1), granulocyte‐macrophage CSF (GM‐CSF) and macrophage CSF (M‐CSF) mRNA was demonstrated in spleen, while MIP‐2, MCP‐1, IP‐10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.

Relevance of postmortem radiology to the diagnosis of fatal cerebral gas embolism from compressed air diving

Aims: To test the hypothesis that artefact caused by postmortem off-gassing is at least partly responsible for the presence of gas within the vascular system and tissues of the cadaver following death associated with compressed air diving. Methods: Controlled experiment sacrificing sheep after a period of simulated diving in a hyperbaric chamber and carrying out sequential postmortem computed tomography (CT) on the cadavers. Results: All the subject sheep developed significant quantities of gas in the vascular system within 24 hours, as demonstrated by CT and necropsy, while the control animals did not. Conclusions: The presence of gas in the vascular system of human cadavers following diving associated fatalities is to be expected, and is not necessarily connected with gas embolism following pulmonary barotrauma, as has previously been claimed.

Leucocyte population changes in the reproductive tract of the ewe in response to insemination

Leucocyte changes after insemination may affect conceptus implantation, but information regarding leucocyte populations in the ruminant reproductive tract is limited. The present study investigated changes in leucocyte populations and distribution in the ovine reproductive tract following oestrus and insemination. Fifteen ewes were mated with a ram for 1 h and their reproductive tracts collected 3, 6, 18, 24 or 48 h later. Another 15 ewes were used as oestrus controls. Tissues were collected from 10 sites in each reproductive tract and stained with haematoxylin and eosin, Toluidine blue and immunohistochemically using a monoclonal CD68 antibody. Luminal mucus smears were collected from seven sites and stained with a modified Wrights stain and immunohistochemically. Neutrophils, eosinophils, mast cells and macrophages were identified and quantified, and temporal changes in their distribution within tissues were examined. Neutrophils and macrophages increased significantly (P < 0.05) in posterior cervical and uterine tissues following insemination. In uterine tissues, neutrophils peaked at 6 h after insemination, whereas macrophages peaked at 18-24 h. Mast cells decreased and eosinophils remained constant. Neutrophils increased significantly (P < 0.05) in the cervical and uterine lumen following insemination. In conclusion, leucocyte population changes after insemination vary between different sites in the ovine reproductive tract and may contribute to pregnancy establishment.

EFFECTS OF BOVINE OVIDUCTAL PROTEINS ON BULL SPERMATOZOAL FUNCTION

The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/-2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa.

Non-invasive in vivo magnetic resonance imaging assessment of acute aflatoxin B1 hepatotoxicity in rats.

Acute aflatoxin B1 (AFB1)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-300 g) using magnetic resonance imaging (MRI). MRI results were compared to serum enzyme levels, histology and electron microscopy. Twenty-four hours following intraperitoneal delivery of AFB1 (3 mg/kg body weight in a saline/dimethyl sulfoxide (DMSO; 0.03 ml/kg body weight) solution), regions of damage, characterised by increased proton signal intensities in T2-weighted images, were observed in the vicinity of the hepatic portal vein (HPV) and in the right medial regions of the liver. Image analysis of regions of apparent damage around the HPV and right medial regions, following 24 h of AFB1 exposure, indicated statistically significant (P<0.05) increases in proton image signal intensities, when compared to saline/DMSO-treated rats. No significant difference in proton image signal intensities were observed 1-2 h following AFB1 exposure. Twenty-four hours following AFB1 exposure, histopathological assessment was characterised by portal/central vein/artery congestion, sinusoid congestion, nuclear pyknosis and karyolysis, and hepatocyte vacuolation; electron microscopy (EM) examination indicated nuclear debris, swollen cytoplasmic compartments, vacuolation, and the disappearance of the smooth endoplasmic reticulum, and elevated levels of serum aspartate aminotransferase and alanine aminotransferase were found to be significantly different (P<0.01) than controls.

Reduction of post-surgical pericardial adhesions using a pig model

BACKGROUND Post-surgical pericardial adhesions pose an increased risk of complications during redo sternotomies. Adhesive tissue formation is a normal response to tissue injury and involves complex patho-physiological processes including the actions of prostaglandins to cause plasma leakage and fibrin formation. The purpose of this study was to assess the ability of two non-steroidal anti-inflammatory agents (Indomethacin and Rofecoxib) and a barrier (Coseal, a polyethylene glycol) to limit adhesion formation following cardiac surgery in a pig model. METHODS Forty-four piglets were allocated equally to four treatment groups: Group 1: Control, Group 2: intramuscular Indomethacin, Group 3: oral Rofecoxib and Group 4: Coseal sprayed on the heart. A full median sternotomy was performed on each animal and the heart exposed. Adhesions were induced by rubbing tissues with gauze, applying sutures and leaving blood in the pericardial sac before chest closure. Plasma inflammatory markers including prostaglandin E(2) and thromboxane B(2) were measured preoperatively and on Days 2, 5 and 10 after surgery. Eight animals from each group were slaughtered after 12 weeks and 3 after 25 weeks. Adhesions were assessed macroscopically and microscopically. RESULTS Compared to the Control group, the extent of adhesions was significantly less in all other groups whilst adhesion density was least in the Indomethacin and Coseal groups. Indomethacin and less so Rofecoxib, inhibited the synthesis of prostaglandin E(2) and thromboxane B(2) but there were no significant changes in other inflammatory markers. CONCLUSIONS We conclude that systemic Indomethacin, and locally applied Coseal are suitable methods to markedly reduce pericardial and retrosternal adhesions.

In vitro culture and interferon-tau secretion by ovine blastocysts.

Embryos were collected surgically from superovulated ewes on days 7, 8, 9 and 10 (oestrus=day 0) to evaluate the long-term culture and interferon-tau (IFN-tau) secretion of ovine blastocysts. Embryos were cultured in 2 ml Dulbeccos modification of Eagles medium (DMEM) supplemented with 15 mg/ml BSA in 5% CO(2) in air or DMEM without BSA in 5% CO(2), 7% O(2), and 88% N(2) at 39 degrees C, examined daily for morphological features and diameter and each day placed into fresh culture medium to enable daily measurement of IFN-tau secretion. Nine day-7 and two day-9 embryos were cultured in DMEM with BSA and nine continued to develop. The day-7 embryos reached a mean maximum diameter of 370.0+/-50.25 microm after 4 days in culture. Nineteen day-7, 12 day-8 and five day-10 embryos were cultured in DMEM without BSA but only six of the day-7 and one day-8 embryos survived for at least 7 days with the former reaching a mean maximum diameter on day 7 of 357+/-43.75 microm whereas all five day-10 embryos survived for at least 7 days reaching a mean maximum diameter on day 6 of 1038+/-155.8 microm. An anti-viral assay and a ELISA for IFN-tau were developed. There was a considerable variation in the time of onset and amount of IFN-tau secreted that did not seem to be related to embryo morphology. Of 28 day-7 embryos cultured, 60.7% were secreting IFN-tau after 1 day of culture whereas 87.5% of day-8 embryos were secreting IFN-tau after 1 day in culture. The mean concentration of IFN-tau secreted by day-8 embryos after 1 day in culture (10.99+/-2.55 ng/ml) was not significantly different to day-7 embryos after 2 days in culture (8.8+/-1.75 ng/ml).

Spermatozoa and seminal plasma induce a greater inflammatory response in the ovine uterus at oestrus than dioestrus.

Leukocyte infiltration and increased synthesis of cytokines in response to insemination is considered to enhance reproductive success. The present study investigated the inflammatory response to whole semen, spermatozoa and seminal plasma, with and without the addition of antibiotics, in the ovine uterus at oestrus and dioestrus. Seminal plasma and spermatozoa both contributed to increased IL-8 secretion (P < 0.01) by endometrial epithelial cells and a concurrent infiltration by neutrophils (P < 0.01). Increased GM-CSF secretion (P < 0.01) occurred in response to whole semen and spermatozoa when antibiotics were not used. Macrophages and eosinophils increased (P < 0.05) in the endometrial stroma when antibiotics were not used, and fewer mast cells were detected in the deep endometrial stroma after treatments containing antibiotics (P < 0.05). Neutrophil and IL-8 responses to insemination were greater at oestrus (P < 0.01) than at dioestrus and the GM-CSF response followed a similar trend. Eosinophil numbers were increased at oestrus (P < 0.01) but minimally affected by insemination. More macrophages were located in the superficial endometrial stroma at oestrus. These results indicate that spermatozoa, seminal plasma and possibly bacteria contribute to the post-insemination inflammatory response, and that leukocytes, GM-CSF and IL-8 secretion in the ovine uterus are influenced by ovarian hormones.

Controlled trial of hyperbaric oxygen treatment for alkali corneal burn in the rabbit

Purpose: To evaluate the efficacy of hyperbaric oxygen therapy in the treatment of alkali‐induced corneal burns in an animal model.

Effects of 17β-oestradiol or oestrous stage-specific cow serum on the ability of bovine oviductal epithelial cell monolayers to prolong the viability of bull spermatozoa

The effect of 17beta-oestradiol and oestrous stage-specific cow serum on bovine oviductal epithelial cell monolayers to extend the viability of co-cultured bull spermatozoa was examined. Monolayers of cells from ampullary and isthmic segments were pre-treated with medium containing either oestrous cow serum, luteal-phase cow serum, 1 microg/ml 17beta-oestradiol + foetal bovine serum or foetal bovine serum alone (control) before the addition of motile frozen/thawed spermatozoa. Motility was visually assessed throughout a 48 h co-incubation period, while fertilising ability of spermatozoa was evaluated by adding in vitro matured bovine oocytes. Pre-treatment with 17beta-oestradiol or oestrous cow serum resulted in a higher percentage of motile spermatozoa after 18 h in isthmic and after 36 h in ampullary cultures compared with the control, but pre-treatment did not affect fertilisation rates. Only at 42 h in ampullary cultures was motility higher in luteal serum pre-treated cultures compared to the control. Motility was also assessed in medium conditioned by pre-treated monolayers. Pre-treatment with 17beta-oestradiol enhanced the ability of conditioned medium to prolong motility and medium conditioned with oestrous cow serum was superior to medium conditioned by luteal-phase serum at maintaining motility. In conclusion, the ability of oviductal epithelium to prolong the motility of spermatozoa is enhanced by 17beta-oestradiol.