A mutation in transmembrane domain II of the 5-hydroxytryptamine(3A) receptor stabilizes channel opening and alters alcohol modulatory actions.

Mutant 5-hydroxytryptamine (5-HT)3A receptors, in which changes were made at Ile294, position 16', of the second transmembrane domain, were assessed for alterations in macroscopic response kinetics and modulation by alcohols. Function of heterologously expressed receptors was measured in Xenopus oocytes in the two-electrode voltage clamp configuration and in human embryonic kidney 293 cells using whole cell patch-clamp electrophysiological recordings with rapid drug application. Compared with the wild-type receptor, a decrease in the 5-HT EC50 value in the Ile294Thr mutant was observed, whereas an increase in the 5-HT EC50 value in the Ile294Leu mutant was measured. Ile294Thr receptors showed a marked reduction in the extent of desensitization. Ethanol and 2,2,2-trichloroethanol (TCEt) enhanced 5-HT-mediated currents in wild-type and Ile294Leu receptors, but inhibited or had little stimulatory effect in the Ile294Thr mutant. Kinetic analysis revealed that in the presence of TCEt, the slope of activation was unchanged in the Ile294Thr mutant and increased in the wild-type receptor. Alcohol cutoff was altered with wild-type = heptanol and Ile294Leu = hexanol. Kinetic changes in the Ile294Thr mutant that favor the open channel state, as well as reduction in the rate of channel activation in the presence of TCEt, likely underlie this mutant's altered response to n-chain alcohols.

Mouse strain-dependent chemokine regulation of the genital tract T helper cell type 1 immune response.

Vaginal infection with the mouse pneumonitis agent of Chlamydia trachomatis (MoPn) produces shorter courses of infection in C57BL/6 and BALB/c mice than in C3H/HeN mice, while C57BL/6 mice are more resistant to oviduct pathology. A robust Th1 response is extremely important in host defense against chlamydia. In this study we examined gamma interferon (IFN-gamma), interleukin 10 (IL-10), and the T-cell-regulatory chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1) to determine if differences in these responses were associated with the differential courses of infection seen in these three strains of mice. Increased and prolonged IFN-gamma responses and lower IL-10 responses were observed in the C57BL/6 strain compared to BALB/c and C3H. Examination of genital tract chemokines revealed a marked predominance of MIP-1alpha over MCP-1 only in the C57 strain. Thus, a pattern of high MIP-1alpha and low MCP-1 levels during the first week of infection is associated with an increased Th1 response and a shorter, more benign chlamydial infection. Inhibition of the MCP-1 response in C3H mice increased their later T-cell production of IFN-gamma but decreased their early IFN-gamma response and had no effect on the course or outcome of infection. Inhibition of MCP-1 is not beneficial in chlamydial infection because of its pleiotropic effects.

Adhesion of monocytes to arterial endothelium and initiation of atherosclerosis are critically dependent on vascular cell adhesion molecule-1 gene dosage.

- Vascular cell adhesion molecule-1 (VCAM-1/Vcam1) is a cytokine-inducible member of the immunoglobulin gene superfamily that is expressed by arterial endothelial cells in regions predisposed to atherosclerosis and at borders of atherosclerotic plaques. To determine whether VCAM-1 expression regulates atherosclerotic lesion formation, we crossed Vcam1 domain 4-deficient (D4D) mice, which partially circumvent the embryonic lethality of Vcam1 null mice, with apolipoprotein E null (Apoe(-/-)) mice, which spontaneously develop hypercholesterolemia and atherosclerosis. In the Apoe(-/-) background, mice homozygous for the Vcam1 D4D allele had markedly reduced arterial VCAM-1 expression, monocyte adherence in the aortic root, and fatty streak formation. Heterozygous Vcam1 D4D mice revealed a Vcam1 gene-dosage effect and had intermediate, yet significant, reductions in these parameters. Our data demonstrate that VCAM-1 plays a pivotal role in the initiation of atherosclerosis in Apoe(-/-) mice.

Early local cytokine profiles in strains of mice with different outcomes from chlamydial genital tract infection.

In this study, we expand on the examination of genetically determined differences in host responses that correlate with clearance of Chlamydia trachomatis from the genital tract. We infected C57BL/6, BALB/c, and C3H/HeN mice with the mouse pneumonitis agent of C. trachomatis (MoPn). C57BL/6 mice had the shortest course of infection (22 days) and the lowest incidence of severe hydrosalpinx. BALB/c mice also had a short course of infection (25 days), but all developed hydrosalpinx. C3H/HeN mice had the longest course of infection (38 days), and all developed severe hydrosalpinx. Determination of local cytokine responses by enzyme-linked immunosorbent assay (ELISA) of genital tract secretions revealed that the levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were significantly increased in the C57BL/6 and BALB/c strains compared to those in the C3H/HeN strain whereas the level of IL-6 was not different. The level of the neutrophil chemokine macrophage inflammatory protein 2 (MIP-2) was increased during the first week of infection in all three strains but was significantly higher in the BALB/c strain, the strain with the most rapid influx of neutrophils into the genital tract. Prolonged detection of MIP-2 in C3H/HeN mice was associated with a protracted presence of neutrophils in the genital tract. Early increases in the levels of the proinflammatory cytokines TNF-alpha and IL-1beta are associated with earlier eradication of infection in the C57BL/6 and BALB/c strains than in the C3H/HeN strain. Increased levels of MIP-2 and neutrophils in BALB/c and C3H/HeN mice relative to C57BL/6 mice suggest that these responses may contribute to pathology.

Apolipoprotein E deficiency effects on learning in mice are dependent upon the background strain.

Apolipoprotein E (apoE) deficient mice were bred onto the C57BL/6 and FVB/N strain backgrounds. The cognitive behavior of food-restricted apoE-deficient and wildtype male mice from these strains was assessed in an olfactory cued 8-arm radial maze. At 6 weeks of age, all four types of mice improved in maze performance over the course of 5 days. However, at 6 months of age, only the apoE-deficient mice on the C57BL/6 background failed to improve their maze performance over the 5 day course, as gauged by the number of incorrect choices made before retrieving both food rewards. Thus, an age-dependent and strain-specific effect of apoE deficiency on cognitive behavior was observed in these mice. The background strain affected activity levels in the maze, as well as in an open field assay. Plasma corticosterone levels were assessed in control, fasted, and post-restraint stress states. Fasting and restraint stress led to increases in plasma corticosterone levels. Although there were strain specific effects on fasting corticosterone levels, and the effect of apoE deficiency on post-stress corticosterone levels, there was no association between fasted corticosterone levels and impaired cognitive behavior in the 8-arm radial maze assay.

An in vitro study of the effect of bony buttressing on fixation strength of a fractured atrophic edentulous mandible model.

PURPOSE: The purpose of this study was to measure the resistance to displacement in an adult bovine rib mandible model as the vertical dimension of the bone decreases, and to compare the fixation strength of titanium miniplates and reconstruction plates. MATERIALS AND METHODS: Five groups of 6 ribs each were tested based on the vertical dimension of the rib and method of fixation (group 1, 40 mm, miniplate), (group 2, 30 mm, miniplate), (group 3, 20 mm, miniplate), (group 4, 10 mm, miniplate), (group 5, 10 mm, reconstruction plate). In the 4 groups stabilized with a miniplate, a 3-hole-per-segment, 2.0-mm titanium miniplate and 6.0-mm monocortical titanium screws were used. In group 5, a 3-screw-per-segment, 2.4-mm titanium reconstruction plate and 2.4-mm bicortical titanium screws were used for fixation. A 2-dimensional beam model (Class III lever) was established, and an Instron machine was used initially to develop a load-displacement curve to 100 N for each specimen. An osteotomy was then created, and the segments were reduced without preload. The ribs were then loaded to failure in the Instron machine. The load-displacement curve and failure load were recorded. RESULTS: There was no significant difference (P < or = .05) between any of the groups before the osteotomy. At 75 N, groups 1 and 2 were superior to group 5 in resistance to displacement. Group 4 showed significantly less (P < or = .05) resistance to displacement than the other experimental miniplated subgroups. As loads increased, groups 1 and 2 continued to show increased resistance to displacement when compared with groups 3, 4, and 5. Loads to failure for groups 1 and 2 were comparable to group 5. CONCLUSIONS: Decreased resistance to displacement occurs with decreasing vertical dimension of the bovine rib. In fractures of the edentulous mandible, a miniplate is more likely to provide adequate fixation if the mandible is 30 to 40 mm in height (nonatrophic). At higher loads, groups with greater vertical height (30 mm and 40 mm) provided resistance to displacement equivalent to the 10-mm group repaired with a reconstruction plate. Therefore, fractures of the atrophic mandible may be better treated with more rigid techniques.

Genetic background determines the extent of atherosclerosis in ApoE-deficient mice.

Two strains of ApoE-deficient mice were found to have markedly different plasma lipoprotein profiles and susceptibility to atherosclerosis when fed either a low-fat chow or a high-fat Western-type diet. FVB/NJ ApoE-deficient (FVB E0) mice had higher total cholesterol, HDL cholesterol, ApoA1, and ApoA2 levels when compared with C57BL/6J ApoE-deficient (C57 E0) mice. At 16 weeks of age, mean aortic root atherosclerotic lesion area was 7- to 9-fold higher in chow diet-fed C57 E0 mice and 3.5-fold higher in Western diet-fed C57 E0 mice compared with FVB E0 mice fed similar diets. Lesion area in chow diet-fed first-generation mice from a strain intercross was intermediate in size compared with parental values. The distribution of the lesion area in 150 chow diet-fed second-generation progeny spanned the range of the lesion area in both parental strains. There were no correlations between total cholesterol, non-HDL cholesterol, HDL cholesterol, ApoA1, ApoA2, ApoJ, or anti-cardiolipin antibodies and lesion area in the second-generation progeny. Thus, a genomic approach may succeed in identifying the genes responsible for the variation in atherosclerosis susceptibility in these 2 strains of ApoE-deficient mice, which could not be explained by measured plasma parameters.

The home environment and salmonellosis in children.

OBJECTIVE: To explore the role of foods and the home environment in the development of Salmonella infections in infants and children. METHODS: Home investigations were conducted of patients younger than 4 years of age infected with Salmonella. Cultures were obtained from foods, persons residing in the home, animals/pets/insects, and environmental sources. Like serotypes encountered in the index patients and isolates from the home underwent typing with pulsed-field gel electrophoresis. RESULTS: Home inspections were conducted in approximately 66% of eligible homes on the average of 3.4 days after the confirmation of the Salmonella isolate. A total of 526 cultures from 50 homes were obtained from foods (120), household members (73), refrigerators (52), water (47), countertops (46), soil (42), can-openers (36), vacuum cleaners (34), animals/pets/insects (26), and others (50). Isolates with a serotype identical to those in the index patient were found in 16 homes, 3 of which included an isolate of a second serotype, and an isolate of a different serotype was recovered in 3 homes. The pulsed-field gel electrophoresis patterns of the isolates of identical serotypes from the subjects and from their environment were indistinguishable in all but 2 patients. Among isolates of the same serotype encountered in different homes, all patterns were different. The identical serotype was found in multiple locations (4), dirt surrounding front doors (4), household members (3), vacuum cleaner (1), animals/pets/insects (1), and a refrigerator shelf (1). CONCLUSIONS: These data illustrate the importance of the child's environment in the development of salmonellosis. Clinicians should concentrate on educating the parents about the environmental spread of Salmonella. Contaminated foods in the home play a less significant role in the infection of infants and children.

Comparison of fixation strengths of locking head and conventional screws, in fracture and reconstruction models.

PURPOSE: Claimed clinical advantages of the locking-head mandibular reconstruction plating system include the ability to achieve stability with fewer numbers of screws per bony segment as compared with conventional screws. The purpose of this study was to test the hypothesis that increased resistance to displacement will be obtained when using locking-head as compared with the same number of conventional screws per segment in both fracture and reconstruction models. MATERIALS AND METHODS: Eight groups were tested based on the screw number (two or four), screw type (locking-head or conventional), and fracture (bony apposition) or reconstruction model (1-cm defect). Two-dimensional beam mechanics using adult bovine ribs and the Instron machine were used to develop a load-displacement curve up to 150 N for each specimen. An osteotomy was then created and the segments were reduced, with preload (fracture model) or with a 1-cm defect (reconstruction model), and plated using the Synthes locking-head plate with either two or four bicortical locking-head (4.0-mm) or conventional (2.7-mm) screws per segment. The fixed ribs were loaded to 150 N, and the displacement was recorded. RESULTS: Locking-head screws provided superior resistance when using two screws per segment in the reconstruction model as compared with conventional screws. Minimal difference was seen between other screw types within a model. The fracture model offered significantly greater (3.1 to 3.7X) resistance to displacement than did the reconstruction model. CONCLUSIONS: Locking-head screws provided significantly increased resistance to displacement when only two screws per segment were used in the reconstruction model. When four screws per segment were used, there was no significant difference between locking-head and conventional screw types in either model. The effect of bony buttressing is significant and may explain why miniplates often fail in the atrophic mandible but are successful in the fully dentate patient.

Human apolipoprotein E allele-specific brain expressing transgenic mice.

Transgenic mice were created using human apolipoprotein E2, E3, and E4 gene fragments driven by the human glial acidic fibrillary protein (GFAP) promoter. Founders were obtained and progeny were assayed for transgene expression in the brain by RNase protection, immunohistochemistry, and Western blotting, demonstrating robust apolipoprotein E (apoE) brain expression, with human apoE representing up to approximately 0.2% of total brain protein. Selected lines were bred to apoE-deficient mice yielding mice which expressed the human transgenic apoE isoforms in the absence of endogenous apoE. Immunohistochemistry revealed accumulation of the transgene encoded human apoE throughout the brain. Double immunofluorescence showed co-expression of the apoE transgene with endogenous glial acidic fibrillary protein. Primary astrocyte cultures from the transgenic mice secreted human apoE into the medium. Aged apoE4 transgenic mouse brain failed to demonstrate any evidence of senile plaques. These mice may be useful for elucidation of the mechanism by which apoE4 is associated with Alzheimer's disease.

Lateral mandibulectomy and partial glossectomy with plate application.

The goals of composite resection with reconstruction plate application include removal of the primary tumor, any compromised portions of the mandible, and any involved lymph-bearing tissue. Recent advances in surgical technique and reconstruction have made this treatment a more appealing choice for patients. Although microvascular free flaps may be the treatment of choice in the younger patient with an excellent prognosis, the use of reconstruction plates with a myocutaneous flap remains a viable alternative for many patients with oral cancer. Regardless of the reconstructive technique utilized, both functional and aesthetic parameters must be addressed in treatment planning for patients with head and neck cancer.

Increasing the rate of blastocyst formation and hatching from in vitro-produced bovine zygotes.

This study was designed to identify parameters that would facilitate early selection of superior embryos, as well as to define culture conditions that could increase the proportion of embryos proceeding to the blastocyst stage. In the first experiment, the developmental potential of bovine embryos that had reached different stages of development after 60 h of culture following insemination was assessed. No 2-cell embryos underwent further cleavage. Of the 4-cell embryos (n = 188) only 12.2% progressed to the blastocyst stage, while 62.5% of 8-cell embryos (n = 480) did so (P < 0.01). In a further experiment, the effects of conditioning the culture medium (TCM 199) either with Buffalo rat liver cells (BRLC) or bovine oviductal epithelial cells (BOEC) and the effects of co-culture with either of these 2 cell types were examined. The percentage of 8-cell embryos proceeding to the morula and blastocyst stages was independent of cell type and culture system. However, BOEC-conditioned medium supported significantly lower production of blastocysts than any of the other culture methods. Only 24.1% of the former proceeded to the blastocyst stage after the full 10 d of culture, and only 3% hatched, values that were significantly lower than in the other 3 groups (P < 0.01). Among the latter, 44% progressed to the blastocyst stage in BRLC-conditioned medium while 44 and 46% reached that endpoint after co-culture with BOEC or BRL cells, respectively. The percentages that hatched among these were 28.2, 31 and 28.5%, respectively.

Expression of two transgenes in in vitro matured and fertilized bovine zygotes after DNA microinjection.

Bovine zygotes produced by in vitro maturation-in vitro fertilization (IVM-IVF) were examined for their potential to serve as recipients of transgenes. Pronuclei, which were maximally visible at about 22 h after IVF, were injected with a SV40-LacZ construct (pSVON). Injected zygotes had lower cleavage rates (49.1%, n = 1162, P < 0.01) than did either noninjected controls (87.4%, n = 1420) or noninjected zygotes in which pronuclei were not visible (67.6%, n = 803). Zygotes that were injected into their pronuclei cleaved as well as zygotes injected cytoplasmically. At 48 h after injection, when most embryos had reached the four- and eight-cell stages, more zygotes in the pronuclear group (22.7%, n = 125) stained positively for LacZ than did zygotes in the cytoplasmic group (8.0%, n = 125). A group of zygotes injected into the pronucleus with pSVON was cultured for 9 days. More morulae (10.8%, n = 134) than blastocysts (3.2%, n = 31) expressed the LacZ gene, indicating that silencing of expression occurred as development progressed. Another group of zygotes was injected with a beta-actin-LacZ gene construct (pbActinLacZ) and, of the embryos assayed at 48 h, 10.6% (n = 255) stained positively. At 9 days, 36.3% of morulae (n = 91) and 21% of blastocysts (n = 33) expressed the transgene. Almost all putative transgenic embryos injected with either construct showed a mosaic pattern of LacZ expression, with an average of only 2-3 cells staining at the eight-cell stage and the majority of cells in positive blastocysts showing no evidence of expression.

Using the PDSA cycle to standardize a quality assurance program in a quality improvement-driven environment.

BACKGROUND: At Parkview Episcopal Medical Center (Pueblo, Colorado), QA means not only quality assurance but also quality alarms-statistical monitoring and analysis of key indicators that lead to the discovery of opportunities for continuous improvement. Data are monitored using statistical process control. Continuous improvement supports quality assurance (QA) just as it supports all other functions at Parkview. METHODS: A PDSA (Plan-Do-Study-Act) analysis form was created for use in conjunction with the data collection tool selected by the user. The data collected and analyzed are made meaningful through the standardization of formats and methods. RESULTS: Successes with this form demonstrate that QA and quality improvement (QI) can be integrated, allowing processes and outcomes to be improved. Since standardization occurred successfully in 1993, QA has been integrated into the strategic-planning process for 1994. Expectations are that duplicate reports and data gathering will be eliminated.

Comparison of colony stimulation factors on in vitro rat and human neutrophil function.

The effects of rhCSFs on in vitro polymorphonuclear leukocyte (PMN) function were studied in Sprague-Dawley neonatal and adult rats and adult and umbilical cord derived human PMN to compare species response. Following in vitro exposure to buffer or rhCSFs (50-100 micrograms/ml), PMN oxidative burst, chemotactic activity and adherence protein expression were measured. RhG-CSF increased the oxidative burst of adult rat PMN as measured by chemiluminescence and altered CD11b/CD18 in resting neonatal rat but not adult rat cells. RhGM-CSF had no effect on adult rat PMN function in vitro, but led to modest changes in adult rat PMN diapedesis across rat peritoneum. No responses were noted to rhM-CSF. Human PMN responded best to GM-CSF (particularly in the neonate), intermediately to G-CSF and none to M-CSF. These experiments show that the profile of cytokine effects is not similar in adult and neonatal rat PMN when compared to human cells. The diversity of actions in other species must be considered when using rhCSFs in animal models.

Expression of bovine trophoblast interferons by in vitro-derived blastocysts is correlated with their morphological quality and stage of development.

Bovine embryos, whether produced naturally or by in vitro techniques, exhibit considerable variability in morphological quality and develop at different rates. Our objectives have been to determine whether initial expression of trophoblast interferon (IFN-tau) was a reflection of conceptus stage of development or age and whether there was an effect of embryo quality on the amount of IFN-tau produced. Early blastocysts (N = 187) were selected at the onset of blastocoele formation and cultured individually. Embryo quality (excellent, good, or fair: E, G, or F) and developmental stage (early, expanded and hatched blastocysts: BL, EBL, and HBL, respectively) were used in a 3 x 3 factorial complete randomized block design, each block (n = 4) consisting of batches of embryos produced from oocytes in different collections. Quality and developmental stage of embryos and IFN-tau released into the medium were assessed every 24 h. Production of IFN-tau (units/embryo/24 h) was greater (P < 0.01) among hatched blastocysts (HBL; 0.91 +/- 0.08) than expanded blastocysts (EBL; 0.23 +/- 0.04) and early blastocysts (BL; 0.05 +/- 0.08). Embryos of similar developmental stage but differing by 2 days in age released equal amounts of IFN-tau. Expression of antiviral activity increased (P < 0.05) from 27% to 57% to 100% as development proceeded from BL to EBL and to HBL respectively. More IFN-tau was produced by HBL graded G (1.0 +/- 0.1) or E (1.3 +/- 0.1) than by those of F quality (0.5 +/- 0.1). All blastocysts, whatever their quality and developmental stage, contained IFN-tau mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of bovine trophoblast interferon in conceptuses derived by in vitro techniques.

Expression of the trophoblast interferon, bovine trophoblast protein-1 (bTP-1), has been studied in embryos produced by in vitro maturation-in vitro fertilization (IVM-IVF). No bTP-1 production was noted until after embryos had reached the expanded blastocyst stage and had begun to hatch (Days 8-9 post-fertilization). Single blastocysts comprising 115 +/- 22 cells released 1.0 +/- 0.1 units of interferon activity/24 h. Amplification of conceptus mRNA by reverse transcription-polymerase chain reaction procedure with bTP-1-specific oligonucleotides confirmed that bTP-1 transcripts were present in blastocysts but were not detectable at earlier stages. Although cultured blastocysts produced by IVM-IVF procedures continued to secrete bTP-1 for a few days, they failed to attach to the substratum and form outgrowths, and soon lost structural integrity. However, when Day 8 blastocysts/morulae were transferred to the uteri of synchronized cows, recovered 4 days later, and placed into individual cultures, they attached and formed outgrowths that produced large amounts of bTP-1 (greater than 2000 units/culture/24 h after 14 days). Embryos thus first expressed bTP-1 when a functional trophectoderm was first formed, and induction did not require a period of in vivo development. However, continued viability of the blastocyst and bTP-1 production were not sustained in vitro and may require some exposure to the uterine environment.

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility.

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.