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.

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.

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.

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.

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.

Effect of repeated flushing and a prostaglandin analogue on the estrous cycle of pony mares.

An experiment was conducted to test the effect of repeated transcervical (non-surgical) uterine flushing and a prostaglandin analogue (PG) on the estrous cycle of pony mares. Uteri in group A were trancervically flushed for embryos 7 to 9 days post ovulation. In addition, group B mares were given 5 ml of PG by intramuscular injection on the day of flushing. Group C served as controls and were not flushed or given PG but were allowed to cycle normally. All mares (except controls) were bred A.I. every other day during estrus. There was no effect on embryo recovery rate from repeated flushing or PG administration. The number of days in estrus was greater for groups A and B than for group C (P<0.05). Length of diestrus was longer for group C than for the other two groups. The total estrous cycle length was similar for all three groups (P>0.05).