Functional advantage of educated KIR2DL1(+) natural killer cells for anti-HIV-1 antibody-dependent activation.

Evidence from the RV144 HIV-1 vaccine trial implicates anti-HIV-1 antibody-dependent cellular cytotoxicity (ADCC) in vaccine-conferred protection from infection. Among effector cells that mediate ADCC are natural killer (NK) cells. The ability of NK cells to be activated in an antibody-dependent manner is reliant upon several factors. In general, NK cell-mediated antibody-dependent activation is most robust in terminally differentiated CD57(+) NK cells, as well as NK cells educated through ontological interactions between inhibitory killer immunoglobulin-like receptors (KIR) and their major histocompatibility complex class I [MHC-I or human leucocyte antigen (HLA-I)] ligands. With regard to anti-HIV-1 antibody-dependent NK cell activation, previous research has demonstrated that the epidemiologically relevant KIR3DL1/HLA-Bw4 receptor/ligand combination confers enhanced activation potential. In the present study we assessed the ability of the KIR2DL1/HLA-C2 receptor/ligand combination to confer enhanced activation upon direct stimulation with HLA-I-devoid target cells or antibody-dependent stimulation with HIV-1 gp140-pulsed CEM.NKr-CCR5 target cells in the presence of an anti-HIV-1 antibody source. Among donors carrying the HLA-C2 ligand for KIR2DL1, higher interferon (IFN)-γ production was observed within KIR2DL1(+) NK cells than in KIR2DL1(-) NK cells upon both direct and antibody-dependent stimulation. No differences in KIR2DL1(+) and KIR2DL1(-) NK cell activation were observed in HLA-C1 homozygous donors. Additionally, higher activation in KIR2DL1(+) than KIR2DL1(-) NK cells from HLA-C2 carrying donors was observed within less differentiated CD57(-) NK cells, demonstrating that the observed differences were due to education and not an overabundance of KIR2DL1(+) NK cells within differentiated CD57(+) NK cells. These observations are relevant for understanding the regulation of anti-HIV-1 antibody-dependent NK cell responses.

Biliary and plasma copper and zinc in pregnant Simmental and Angus cattle.

Three each of 3-year-old Angus and Simmental heifers, surgically modified to collect bile, were used to measure the effects of pregnancy and breed on bile flow, biliary copper and zinc excretion and plasma copper and zinc concentrations. Bile copper excretion was significantly higher at 7-mo of pregnancy when samples from both breeds were pooled. From then onwards it declined to its lowest, one week post-partum. During pregnancy, plasma copper concentration increased slightly, reaching its highest level at 7-mo of pregnancy and then decreased slightly until full term. In pooled samples from both breeds, the correlation between increase in bile copper excretion and plasma copper concentration from 0 to 7-mo of pregnancy was high (r = 0.85) and significant (p < 0.05). Plasma zinc concentration decreased to the lowest level around 6-mo of pregnancy but increased thereafter until full term. In cows that were dried off one week after parturition, major shifts in bile and plasma copper and zinc parameters occurred at one week following and these coincided with a marked decline of bile flow and bile copper and zinc excretion. By 3-mo post-partum, biliary copper and zinc excretion and plasma copper and zinc concentrations had reached levels observed prior to pregnancy. When the data from all samples were pooled, the bile flow and bile copper excretion were significantly (p < 0.05) higher in Simmental, and plasma copper and zinc concentration higher in the Angus.

Excretion of loline alkaloids in urine and faeces of sheep dosed with meadow fescue (Festuca pratensis) seed containing high concentrations of loline alkaloids.

AIM: To determine the effect of oral dosing of sheep with loline alkaloids on their excretion in urine and faeces, and to monitor for any toxic effects. METHODS: In Experiment 1, six 9-month-old ewe lambs were given a single oral dose of loline alkaloids (52 mg/kg bodyweight (BW); acute exposure) as a suspension of ground meadow fescue (Festuca pratensis) seed in water. In Experiment 2, on six consecutive days, six ewe lambs were given three doses of loline (68 mg/kg BW/day; chronic exposure). Blood was collected at variable intervals up to 72 h in Experiment 1, and up to 8 days in Experiment 2, for haematology and measurement of alkaline phosphatase, aspartate aminotransaminase, creatine kinase and γ-glutamyl transferase in plasma. Urine and faecal samples were collected at similar times for measurement of creatinine in urine and loline alkaloid analysis. A post mortem with histopathology was carried out on two animals at the end of each experiment. RESULTS: The loline alkaloids, N-acetyl norloline, N-formyl loline, N-acetyl loline, N-methyl loline and loline base were detected in urine within 15 minutes after the single dosing. N-formyl loline and loline base were the predominant metabolites in urine in both experiments. The total quantity of lolines excreted in both urine and faeces was 10% and 4% of the amount dosed in Experiments 1 and 2, respectively. In both experiments, the clinical chemistry parameters in blood and urine were within normal ranges. Post-mortem and histopathological examination did not show any abnormalities. CONCLUSIONS: This is the first report of loline alkaloid profiles in both urine and faeces of sheep. The appearance of loline alkaloids and the loline base in urine within 15 minutes suggests rapid uptake, metabolism and excretion. Loline alkaloids were non-toxic to sheep at the concentrations they are exposed to under New Zealand grazing conditions. The low recovery of loline alkaloids in urine and faeces in the absence of toxicity signs suggests lolines are extensively metabolised; probably to forms other than N-formyl loline, N-methyl loline, N-acetyl loline, N-acetyl norloline, and loline base in the digestive tract of sheep prior to absorption, and/or in the liver or other tissues following absorption.

Effects of clonidine and idazoxan on tetrathiomolybdate-induced copper and lysosomal enzyme excretion into sheep bile.

This study investigated the effects of intravenous (IV) administration of tetrathiomolybdate (TTM), and α(2)-adrenergic agonist clonidine (CLO) and α(2)-antagonist idazoxan (IDA), alone or in combination with TTM, on sheep fed low (LCu) and high (HCu) copper diets. Effects on bile flow, biliary Cu concentration and excretion, plasma Cu concentration, and lysosomal enzyme ß-glucuronidase (ß-GLU) activity in bile and plasma were determined. Tetrathiomolybdate alone or with CLO or IDA significantly enhanced biliary Cu excretion most likely by removing Cu from hepatocyte lysosomes as evidenced by a significant increase in ß-GLU enzyme activity in bile. A significant increase in plasma ß-GLU concentration occurred only in sheep treated with CLO in combination with TTM. Because of the lytic nature of the lysosomal enzymes, caution is advocated in use of drugs, especially α(2)-adrenergic agonists, to further enhance TTM-induced biliary Cu excretion in the treatment of chronic Cu poisoning in sheep.

Changes in concentrations of lysergol in urine and prolactin in plasma, rectal temperature and respiration rate in sheep selected for resistance or susceptibility to ryegrass staggers and fed ergovaline.

AIM: To determine the effects of feeding ryegrass seed containing ergovaline to sheep selected for resistance or susceptibility to ryegrass staggers on concentration of lysergol (a metabolite of ergovaline) in urine, prolactin in plasma, rectal temperature and respiration rate. METHODS: Two experiments were carried out using 12 Romney crossbred ewe lambs aged 9 months, comprising animals resistant (n=4), susceptible (n=4) or outcross (n=4) to ryegrass staggers. In Experiment 1, sheep were given either a single (Part A) or six (Part B) feed (s) of endophyte-infected seed containing ergovaline at 30 mg/kg dry matter (DM), at 42 µg ergovaline/kg bodyweight (BW), to simulate acute and chronic exposure to ergovaline, respectively. The concentration and excretion of lysergol in urine and concentration of prolactin in plasma were measured over 3 and 12 days, for Parts A and B respectively. In Experiment 2, after a recovery period of 7 days, the same sheep were fed with ergovaline at 67 µg/kg of BW for 7 days. Soon after the seventh feed the sheep were moved to a hothouse at 36.5°C and 60% humidity, and 3 h later their rectal temperatures and respiration rates were measured. RESULTS: The concentration of lysergol and excretion in urine increased to a peak between 6 and 9 h after exposure to ergovaline whereas the concentration of prolactin in plasma was markedly reduced. Differences in concentration and rate of excretion of lysergol in urine between animals resistant, outcross and susceptible to ryegrass staggers were not significant (p>0.1). The animals resistant to ryegrass staggers had a lower rectal temperature (p<0.05) and a faster respiration rate than the outcross and susceptible groups when exposed to high ambient temperature and high humidity. CONCLUSIONS: This study showed that excretion of lysergol in urine increased with each exposure of sheep to ergovaline. Animals genetically resistant to ryegrass staggers exhibited a lower rectal temperature and a faster respiration rate than those susceptible, demonstrating the possible cross resistance of sheep to ergovaline in a population originally selected for resistance to ryegrass staggers. Hence potential exists to select animals resistant to ryegrass staggers that are also resistant to ergovaline.

Effects of dietary Cu, Mo and S on urinary Cu and Zn excretion in Simmental and Angus cattle.

This study determined the effects of dietary copper (Cu), molybdenum (Mo) and sulphur (S) on urinary Cu and zinc (Zn) excretion in cattle. Four Simmental and four Angus heifers were fed low (L) or high (H) levels (mg/kg DM) of Cu (5,40), Mo (1,10) and S (0.2,0.5%). Initially two of each breed was fed either LCu or HCu (2 mo). Then all eight animals were fed sequentially LCuHS (1.5 mo), HCuHS, HCuHMo and HCuHMoHS (2 mo each). Simmental had a higher urine flow, increased concentration and total excretion of urinary Cu and Zn compared to Angus, but only total Zn excretion was significantly higher. Urinary Cu excretion was greatest with the HCuHMoHS diet. Urinary Zn excretion significantly increased with HS but not HS in combination with HMo and/or HCu. This study, together with previously reported biliary excretion, allows a direct comparison of urinary and biliary Cu and Zn excretion responses to dietary Cu, Mo and S.

Acute and long-term effects of exposure to sodium monofluoroacetate (1080) in sheep.

Acute and long-term effects of a single, relatively high oral dose (0.25 and 0.30 mg/kg) of sodium monofluoroacetate (1080) on the survival and productivity of sheep were evaluated to establish a better understanding of 1080 poisoning and identify more specific changes diagnostic of toxicosis. In survivors, clinical signs of acute 1080 toxicosis such as salivation and lethargy were generally very mild. Fasted animals were more prone to 1080 toxicity. In animals that died, more severe signs, including tachypnoea, dyspnoea, and tremors occurred for 15-20 min prior to death. 1080 concentrations were highest in the blood > heart > skeletal muscle > liver. 1080 could not be detected in any of these organs of the animals that survived. Serum citrate concentrations were elevated for 4 days after dosing. No clinical or biochemical abnormalities were found in any animal after 4 days. Histopathological lesions were most marked in the heart and lung with inflammation, necrosis, and scattered foci of fibrous tissue in the myocardium, pulmonary oedema and inflammation of the lung. No adverse long-term effects on general health or reproductive performance were observed in any sheep that survived the first 4 days following exposure to 1080. The most reliable diagnostic indicators of 1080 exposure in sheep were measurement of its residues in blood, skeletal muscle and ruminal contents, increased serum citrate concentration, elevated heart rate, and characteristic electrocardiograph changes (up to 4 days after exposure). Death from 1080 is most likely to occur within 96 h, and animals that survived this period appeared normal.

Anal fin morphology and gonadal histopathology in mosquitofish (Gambusia holbrooki) exposed to treated municipal sewage effluent.

Feral mosquitofish living in undiluted treated municipal sewage in Queensland, Australia, were examined for morphologic abnormalities indicative of reproductive dysfunction. Male and female mosquitofish were captured at two sites receiving undiluted treated sewage and compared with those captured at a reference site. Several morphologic end points were examined, including length of the fourth and sixth anal fin rays and gonadal histology. Both male and female mosquitofish at one site exhibited minor elongation of the fourth anal fin ray consistent with exposure to androgenic stimulation, although the spatial extent of the effect was limited and not significant at other sites further downstream. No incidences of intersex were found. These findings suggest that the level of treatment of domestic sewage at the two sewage treatment plants assessed is adequate to prevent reproductive abnormalities in exposed mosquitofish populations.

Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples.

Chemicals released into the environment by anthropogenic activities have been linked to estrogenic or androgenic effects in exposed wildlife, and there is a need to develop and validate rapid and cost-effective methods to quantify the total estrogenic and androgenic activity of environmental water samples. In this study, estrogen receptors (ER) were isolated from sheep (Ovis aries) uteri and rainbow trout (Oncorhynchus mykiss) livers and androgen receptors (AR) were isolated from rainbow trout brains. The isolated receptors were used in competitive receptor binding assays to test the affinity of known estrogenic and androgenic chemicals for the receptor binding site, and results were compared with literature values for the rat uterine ER binding assay and the E-Screen. The relative binding affinities of the tested compounds to ER from different species were similar, and binding to the ER was a more responsive endpoint than the cellular effect measured in the E-Screen. Using the sheep ER binding assay in combination with solid-phase extraction, the estrogenic activity in a raw sewage sample from a municipal treatment plant in Brisbane (Queensland, Australia) was measured at 51-73 ng/L estradiol equivalents (EEq).

Bioassay-derived androgenic and estrogenic activity in municipal sewage in Australia and New Zealand.

Raw sewage and sewage at various stages of treatment were sampled from 15 municipal sewage treatment plants in south Queensland (Australia) and Canterbury (New Zealand). Estrogenic and androgenic activities were determined with sheep estrogen receptor and rainbow trout androgen receptor binding assays, respectively. Selected estrogenic chemicals were also analyzed by gas chromatography-mass spectrometry. The raw sewage influents contained significant levels of both estrogenic (<4-185 ng/L estradiol equivalents) and androgenic (1920-9330 ng/L testosterone equivalents) activity. Subsequent treatment of raw sewage successfully removed most of the activity so that the estrogenicity and androgenicity associated with the final effluents were very low (<1-4.2 ng/L estradiol equivalents and <6.5-736 ng/L testosterone equivalents, respectively). Secondary treatment was the most effective treatment step to remove estrogenic and androgenic activity from sewage water. Activated sludge treatment in particular removed 92% to >99% of the estrogenic activity and 82% to >99% of the androgenic activity in sewage.

Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR).

A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l(-1) 17beta-oestradiol (E(2)) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l(-1) E(2). Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.

Efficacy of an advanced sewage treatment plant in southeast Queensland, Australia, to remove estrogenic chemicals.

The estrogenicity profile of domestic sewage during treatment at a medium-sized (3800 EP) advanced biological nutrient removal plant in Queensland, Australia, was characterized using a sheep estrogen receptor binding assay (ERBA) and the MCF-7 breast cancer cell proliferation assay (E-Screen). The raw influent was highly estrogenic (20-54 ng/L EEq), and primary treatment resulted in a slight increase in estrogenicity that was detected in one of the assays (6-80 ng/L). Concurrent chemical analysis suggested that most of the estrogenicity in the influent was due to natural hormones (>48%). Secondary activated sludge treatment followed by nitrification/denitrification effectively removed > 95% of the estrogenic activity (to <0.75-2.6 ng/L), and estrogenicity of the final tertiary-treated effluent was below the detection limit of both assays (<0.75 ng/L).

Herbicide spray drift odor: measurement and toxicological significance.

The toxicological significance of exposure of members of the public to spray drift odors of four herbicide formulations (three 2, 4-dichlorophenoxyacetic [2,4-D] acid derivatives and one MCPA [4-methyl-2-chlorophenoxyacetic acid] derivative) has been studied using a combination of novel odor measurement and classic residue analysis techniques. The mean odor concentrations, generated during the spraying of the commercial herbicide formulations under simulated aerial application conditions, were about twofold higher for 2,4-D ethylhexyl ester (22,500 OU(c)/m(3)) and MCPA (30,100 OU(c)/m(3)) than for 2,4-D butyl ester (12,400 OU(c)/m(3)) and 2,4-D amine (11,800 OU(c)/m(3)). Detailed investigations determined that the odors are due to trace manufacturing impurities and additives in the commercial formulations, whereas the herbicide active ingredients are odorless. Measured airborne herbicide active ingredient concentrations under the simulated aerial application conditions were all below their respective occupational safety and health TLV-TWA values, indicating that exposures of toxicological significance as a result of spray drift are unlikely.

Effect of chelating agents on the excretion of copper, zinc and iron in the bile and urine of sheep.

The effect of tetrathiomolybdate (TM), disodium calcium ethylenediamine tetraacetate (EDTA), D-penicillamine (PEN), 2-3 dimercapto-1-propanol (BAL) and dimethyl dithiocarbamate (DDC) administration on biliary and urinary excretion of copper (Cu), zinc (Zn) and iron (Fe) was investigated in sheep on a low Cu diet (Group A) and a high Cu diet (Group B). Only biliary Cu excretion increased significantly (P<0.01) with TM treatment. Urinary Cu excretion increased (P<0.01) following PEN treatment. TM, EDTA, PEN, BAL and DDC adminstration increased Cu excretion via bile and urine by 254, 11, 266, 46 and 16%, respectively in Group A sheep, and by 354, 13, 196, 20 and (-) 31% in Group B sheep. Urinary Zn excretion increased (P<0.01) following EDTA and PEN treatments. Only urinary Fe excretion increased (P<0.01) with EDTA treatment. The results show that TM and PEN are the most efficient decoppering agents, but PEN unlike TM also removes Zn. The major routes of excretion of Cu chelates by TM and PEN are different. TM increases Cu excretion significantly (P<0.05 in Group A and P<0.01 in Group B) in bile with only a slight increase in urinary Cu whereas PEN increases Cu excretion significantly (P<0.01) in urine. Therefore from a therapeutic view, a combination therapy of TM and PEN would be useful to maximize Cu removal from the body.

Prognostic significance of keratinization in nasopharyngeal carcinoma.

INTRODUCTION: Keratinization may be a vulnerable aid in predicting response to therapy for nasopharyngeal carcinoma. METHODS: The presence or absence of keratin in biopsy specimens was correlated with tumor behavior, locoregional control, patterns of failure, and survival of patients irradiated for nasopharyngeal carcinoma. RESULTS: Patients with keratinizing squamous-cell cancers (World Health Organization [WHO] type 1) had a higher incidence (76%) of locally advanced tumors than those with nonkeratinizing (WHO type 2) and undifferentiated (WHO type 3) cancers (55%). The former group of patients had a lower incidence (29%) of lymph node metastases than the later group (70%). Primary tumor was controlled in 62% and neck nodes were controlled in 82% of all patients. Primary tumor control rates were 29% in patients with keratinizing squamous-cell cancers and 79% in those with nonkeratinizing and undifferentiated cancers (P = .001). Nodal control rates were 76% for keratinizing squamous-cell cancer and 85% for nonkeratinizing and undifferentiated cancers (P = .001). The incidence of distant metastases was 6% in patients with keratinizing squamous-cell cancer and 33% in those with nonkeratinizing and undifferentiated cancers (P = .001). Patients with keratinizing squamous-cell cancers, even though they had a lower incidence of lymphatic and distant metastases, had a poorer survival rate because of a higher incidence of deaths from uncontrolled primary tumors and nodal metastases. The 5-year survival rates were 35% for all patients, 6% for those with keratinizing squamous-cell cancers, and 51% for nonkeratinizing and undifferentiated cancers respectively (P = .001). CONCLUSION: Higher doses of external beam radiotherapy with or without brachytherapy boost may be needed to improve local control and survival of patients with keratinizing squamous-cell carcinoma of the nasopharynx, whereas an effective systemic therapy is needed for nonkeratinizing and undifferentiated cancers, which tend to metastasize.

Persistence of sodium monofluoroacetate in rabbits and risk to non-target species.

1. Sodium monofluoroacetate (1080), a vertebrate pesticide used in New Zealand, was administered orally to rabbits at two dose levels (sub-lethal and lethal) to determine how long 1080 would persist in plasma, liver, kidney, and muscle so that the risk of consumption of meat from lethally or sub-lethally poisoned rabbits by non-target species could be assessed. 2. The plasma elimination half-life in rabbits receiving a sub-lethal dose was 1.1 h. Retention of 1080 in tissue was greater in rabbits dosed with a lethal dose than in those that received a sub-lethal dose. Irrespective of the dose level, concentration of 1080 in muscle, kidney, and liver was substantially lower than in the plasma. 3. Poisoning of dogs is possible because of their extreme susceptibility to 1080. Poisoning of birds is less likely. The risk of secondary poisoning is reduced as the concentration of 1080 declines in putrefying carcasses.

Feminizing Sertoli cell tumors in boys with Peutz-Jeghers syndrome.

We report the pathology findings in two cases of multicentric Sertoli cell testicular tumors in two young boys with probable Peutz-Jeghers syndrome. Four cases of such tumors occurring in boys with Peutz-Jeghers syndrome were previously reported. Each of the two boys reported in this paper had prominent gynecomastia, rapid growth, and advanced bone age. Serum levels of estradiol were markedly elevated. Anti-müllerian hormone was measured in the serum of one of the boys and was in the normal range for age. Bilateral orchiectomy was performed in each case because the neoplastic growth would most likely result in sterility, and curtailment of height potential was threatened from continued elevation of estradiol levels. Microscopically, greatly enlarged seminiferous tubules packed with ovoid Sertoli-like cells were present. Prominent eosinophilic basement membrane surrounded the tubules and intersected between the cells, forming hyalinized ovoid globules and microcalcifications. Ultrastructure revealed lamination of basement membranes surrounding adjacent cells, ovoid cells with abundant cytoplasm, and limited smooth endoplasmic reticulum. Studies of testicular tumor tissue from both cases revealed increased transcription of the aromatase cytochrome P450 gene using promoter II, the promoter directing aromatase expression in the normal ovary and testis. The levels of transcripts were comparable to corpus luteum, thus resulting in increased estrogen synthesis. Transcripts specific for placental-type aromatase promoters (I.1 and I.2) were not detected in significant levels in these tumors.

DNA ploidy, proliferation index, grade, and stage as prognostic factors for vulvar squamous cell carcinomas.

DNA analysis by flow cytometry has been reported to be a useful prognostic technique for a variety of malignant tumors. The goal of this study was to examine DNA ploidy status and proliferation index as potential predictors of recurrence and survival for patients with squamous cell vulvar carcinomas. The DNA ploidy, percentage of cells in the S-phase of the cell cycle, stage, and histologic grade were analyzed in 42 patients. Flow cytometry was performed on archival paraffin-embedded tissue. The histologic specimens were reviewed by a single pathologist. Thirty-four tumors (81%) were diploid and eight (19%) demonstrated an aneuploid peak. The percentage of cells in the S-phase (proliferation index) was calculated for all patients. The 5-year survival rate was 68% for diploid tumors and was 75% for aneuploid tumors. The difference between these rates was not statistically significant (P < or = 0.65). S-phase fraction was also not a useful predictor for recurrence or overall survival. FIGO stage and histologic grade were accurate predictors for both recurrence and 5-year survival. Multiple predictor analysis using stage, grade, ploidy status, and proliferation index did not identify any subgroup which would predict recurrence better than FIGO stage and grade. DNA ploidy and S-phase fraction analysis do not appear to be clinically useful prognostic factors for vulvar squamous cell carcinomas.

Segmental absence of small intestinal musculature.

Three instances of segmental absence of small intestinal musculature are described. Based on their clinical and pathological features and on the 12 cases previously described in the English literature, these can be classified into two groups: primary and secondary. In the primary group, the etiology is unknown, the onset of symptoms is acute, and there are no pathologic findings in the remaining layers of the small bowel except for superimposed perforation, or intussusception. In the secondary group, there is a longer history of intestinal symptoms and of multiple surgical procedures. Histologically, there may be ischemic necrosis of remaining layers, fibrosis, calcification, chronic inflammation, and presence of macrophages. These findings indicate secondary destruction of muscle layers due to ischemia and/or infarction, interruption of the blood supply, or trauma.