Equine endometritis: a review of challenges and new approaches.

Endometritis in the mare begins as a normal physiological inflammatory response to breeding that involves both a mechanical and immunological response pathway activated to rid the uterus of semen and bacteria. With successful resolution of this inflammation, the mare's uterus will provide a hospitable environment for the development of the semi-allogenic conceptus. If the mare fails to resolve this inflammatory response within 48 h of breeding, she will become susceptible to persistent breeding-induced endometritis (PBIE) which will have detrimental effects on her fertility. This condition can then predispose the mare to bacterial or fungal endometritis leading to further degeneration of the endometrium. Optimisation of the mare's fertility requires a fine balance between allowing the natural immune response of the endometrium to its exposure to allogenic semen to run its course, and yet preventing its progression to PBIE or the involvement of infectious agents. This review discusses the challenges presented by PBIE, latent infections, biofilms, fungal infections and the need to utilise diagnostic methods available and implement targeted treatments to optimise fertility in the mare.

What is Comprehensive Geriatric Assessment (CGA)? An umbrella review.

Background: Comprehensive Geriatric Assessment (CGA) is now the accepted gold standard for caring for frail older people in hospital. However, there is uncertainty about identifying and targeting suitable recipients and which patients benefit the most. Objectives: our objectives were to describe the key elements, principal measures of outcome and the characteristics of the main beneficiaries of inpatient CGA. Methods: we used the Joanna Briggs Institute umbrella review method. We searched for systematic reviews and meta-analyses describing CGA services for hospital inpatients in the Cochrane Database of Systematic Reviews, Database of Reviews of Effectiveness (DARE), MEDLINE and EMBASE and a range of other sources. Results: we screened 1,010 titles and evaluated 419 abstracts for eligibility, 143 full articles for relevance and included 24 in a final quality and relevance check. Thirteen reviews, reported in 15 papers, were selected for review. The most widely used definition of CGA was: 'a multidimensional, multidisciplinary process which identifies medical, social and functional needs, and the development of an integrated/co-ordinated care plan to meet those needs'. Key clinical outcomes included mortality, activities of daily living and dependency. The main beneficiaries were people ≥55 years in receipt of acute care. Frailty in CGA recipients and patient related outcomes were not usually reported. Conclusions: we confirm a widely used definition of CGA. Key outcomes are death, disability and institutionalisation. The main beneficiaries in hospital are older people with acute illness. The presence of frailty has not been widely examined as a determinant of CGA outcome.

New horizons in comprehensive geriatric assessment.

In this article, we discuss the emergence of new models for delivery of comprehensive geriatric assessment (CGA) in the acute hospital setting. CGA is the core technology of Geriatric Medicine and for hospital inpatients it improves key outcomes such as survival, time spent at home and institutionalisation. Traditionally It is delivered by specialised multidisciplinary teams, often in dedicated wards, but in recent years has begun to be taken up and developed quite early in the admission process (at the 'front door'), across traditional ward boundaries and in specialty settings such as surgical and pre-operative care, and oncology. We have scanned recent literature, including observational studies of service evaluations, and service descriptions presented as abstracts of conference presentations to provide an overview of an emerging landscape of innovation and development in CGA services for hospital inpatients.

The effects of dexamethasone and prednisolone on pituitary and ovarian function in the mare.

REASONS FOR PERFORMING STUDY: Persistent mating induced endometritis is among the most common causes of infertility in the mare. Recently, improved pregnancy rates have been reported when corticosteroids were administered to 'problem mares' specifically, to modulate the post mating inflammatory response; however, the effect of treatment on pituitary and ovarian function requires further study. OBJECTIVES: To evaluate the effects of prolonged treatment with glucocorticoids on pituitary and ovarian function. METHODS: Eighteen cycling Quarter Horse mares in early oestrus were assigned randomly to one of 3 treatment groups: dexamethasone 0.05 mg/kg bwt i.v. twice a day, prednisolone 0.5 mg/kg per os twice a day, or placebo for 5 days. Mares were examined by ultrasound daily to evaluate reproductive function. Blood samples were collected daily to measure luteinising hormone (LH), progesterone and cortisol levels. RESULTS: Dexamethasone treatment caused greater (P<0.05) suppression of endogenous cortisol concentration (9.4 +/- 1.1 ng/ml) compared to prednisolone- (41.9 +/- 4.0 ng/ml) or placebo-treated mares (32.4 +/- 3.8 ng/ml). After 24 h, mares treated with dexamethasone exhibited lower uterine oedema scores than prednisolone- or placebo-treated mares. An ovulation rate of 40% was observed in dexamethasone-treated mares (2/5) compared to 83% for prednisolone (5/6) and 100% for placebo-treated (6/6) mares. An absence of a LH surge was noted in 3 of 5 dexamethasone-treated mares and one of 6 prednisolone-treated mares. CONCLUSIONS: Repeated administration of dexamethasone to mares in oestrus is associated with decreased uterine oedema, suppression of LH and a high rate of ovulation failure. It is recommended that dexamethasone treatment is limited to only 1 or 2 days and that a lower dose is considered in the management of persistent mating induced endometritis to avoid potential adverse affects on reproductive function.

Evaluation of a veterinary glucometer for use in horses.

BACKGROUND: Glucose assessment and regulation are important factors in the treatment of hospitalized horses and foals. HYPOTHESIS/OBJECTIVES: The purpose of this study was to compare glucose measurement by a veterinary glucometer, adjusted by code for use in horses and foals, to a reference chemistry analyzer. It was hypothesized that the veterinary glucometer and reference analyzer would yield similar results and that interpretation of glucose values obtained from a veterinary glucometer would result in clinically appropriate decisions. ANIMALS: Fifty blood samples from adult horses and 50 blood samples from neonatal foals admitted to the Colorado State University Veterinary Hospital or Equine Reproduction Laboratory for evaluation. METHODS: Glucose concentrations from fresh whole blood samples were evaluated in duplicate with a veterinary glucometer and these values were compared with those obtained with a reference plasma chemistry analyzer. The accuracy of glucometer measurement was evaluated with a Clarke error grid. RESULTS: The veterinary glucometer accurately measured whole blood glucose concentrations in both horses and foals when compared with a reference plasma chemistry analyzer. Nearly 97% of the glucometer values obtained in this study would have resulted in appropriate clinical decisions based on the Clarke error grid analysis. CONCLUSIONS AND CLINICAL IMPORTANCE: The veterinary glucometer evaluated has potential utility for point-of-care whole blood glucose evaluation in both horses and foals.

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells.

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.

Effect of dehydration prior to cryopreservation of large equine embryos.

Cryopreservation of equine embryos>300microm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7-8, grade 1, equine embryos 300-1350microm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6M galactose, 10min in 1.5M glycerol, slow freeze (n=21); (B) 10min in 1.5M glycerol, slow freeze (n=15); (C) 2min in 0.6M galactose, 10min in 1.5M glycerol, followed by exposure to thaw solutions, then culture medium (n=5); (D) transferred directly to culture medium (n=5). Frozen embryos were thawed and subjected to a three-step cryoprotectant removal. Five embryos from each treatment were evaluated morphologically after 24 and 48h culture (1=excellent, 5=degenerate/dead). All treatments had at least 4/5 embryos with a quality score >or=3 at these time points except treatment B (2/5 at 24h, 1/5 at 48h). Subsequent embryos from treatment A (n=16) or B (n=10) were matched in sets of two for size and treatment, thawed, and immediately transferred in pairs to 13 recipients. Only two recipient mares were pregnant; one received two 400microm embryos from treatment A, and the other one 400 and one 415microm embryo from treatment B. There was no advantage of incorporating a 2min dehydration step into the cryopreservation protocol for large equine embryos.

Enhancement of seed vigour following insecticide and phenolic elicitor treatment.

Thiamethoxam (CGA 293'343) is a novel broad-spectrum neonicotinoid insecticide. It is commercially used as a seed treatment under the trademark Cruiser (CRZ). Although many reports detail its insecticidal, plant-protecting properties, there are minimal reports concerning the effect on seed germination activities which can be key control points of seedling vigour. In this report, we investigated the effect of CRZ, fish protein hydrolysates (FPH; a known elicitor of pentose-phosphate pathway) and the combination of CRZ and FPH (CF) on seed vigour of pea, soybean and corn. Seed vigour was investigated by estimating germination percentage, shoot height, shoot weight, total soluble phenolic content, antioxidant content, G6PDH (glucose-6-phosphate dehydrogenase) activity, and GPX (guaiacol peroxidase) activity. Addition of FPH to CRZ (CF) seemed to have a slightly positive effect on seed vigour, especially, CF and FPH treatment for corn and FPH treatment for pea, suggesting that pre-sowing treatments may cause positive/negative effects on seed vigour, depending on the concentration of treatments. Further research will be needed to determine their effects and the optimal concentration for seed priming.

Superovulation in mares.

Embryo recovery from single ovulating mares is approximately 50 per cent per estrous cycle. Superovulation could be used to increase embryo recovery and provide extra embryos for embryo freezing. This review addresses some historical approaches to superovulation, as well as examines factors that affect the response of mares to equine FSH. eCG, GnRH and inhibin vaccines have been of limited success in stimulating multiple ovulation. Numerous studies have shown that injection of equine pituitary extract (EPE) will result in three to four ovulations per estrous cycle and two embryos. A purified, standardized EPE preparation (eFSH) also results in a similar response to EPE. Factors affecting the response to EPE and eFSH include day of initial treatment, size of largest follicle at initial treatment and frequency of injection. Embryos from single ovulating, untreated mares and eFSH-treated mares provide similar pregnancy rates upon nonsurgical transfer. Five to 7 days of eFSH treatment also has been shown to hasten the first ovulation of the breeding season. Potential problems after eFSH injections include anovulatory or luteinized follicles and overstimulation. Studies are needed to further evaluate the criteria for initiation of treatment and to determine how to increase ovulation rate without decreasing embryo recovery per ovulation.

Evaluation of three equine FSH superovulation protocols in mares.

Superovulation could potentially increase embryo recovery for immediate transfer or cryopreservation. The objectives were to evaluate the effect of pretreatment with progesterone and estradiol (P+E) on follicular response to eFSH and compare doses of eFSH and ovulatory agents on follicular development and ovulation in mares. In Experiment 1, 40 mares were assigned to one of four treatment groups. Group 1 consisted of untreated controls. Group 2 mares were administered eFSH without pretreatment with P+E. Group 3 mares were administered P+E for 10 days starting in mid-diestrus followed by eFSH therapy. Group 4 mares were administered P+E for 10 days followed by eFSH therapy. All treated mares were administered 12.5mg eFSH twice daily and prostaglandins were given on the second day of eFSH therapy. Mares were bred with fresh semen the day of hCG administration and with cooled semen the following day. The numbers of preovulatory follicles and ovulations were lower for mares treated with P+E prior to eFSH treatment. Pretreatment with P+E in estrus also resulted in a lower embryo recovery rate per ovulation compared to the other two eFSH treatment groups. In Experiment 2, two doses of eFSH (12.5 and 6.25mg) and two ovulation-inducing agents (hCG and deslorelin) were evaluated. The number of preovulatory follicles was greater for mares given 12.5mg of eFSH compared to mares given 6.25mg. Number of ovulations was greatest for mares given 12.5mg of eFSH twice daily followed by administration of hCG. Embryo recovery per flush was similar among treatment groups, but the percent of embryos per ovulation was higher for mares given the low dose of eFSH. In summary, there was no advantage to giving P+E prior to eFSH treatment. In addition, even though the lower dose of eFSH resulted in fewer ovulations, embryo recovery per flush and embryo recovery per ovulation were similar or better for those given the lower dose of eFSH.

eFSH in clinical equine practice.

Equine follicle stimulating hormone (eFSH) has been used to induce follicular development in transitional mares and problem acyclic mares, as well as superovulate cycling mares. The most efficacious protocol is to administer 12.5 mg eFSH, intramuscularly, twice daily beginning 5 to 7 days after ovulation when the diameter of the largest follicle is 20 to 25 mm. Prostaglandins are to be administered on the second day of eFSH therapy. Treatment with eFSH is continued for 3 to 5 days until follicle(s) are >or=35 mm in diameter. The mare is subsequently allowed to 'coast' for 36 h, after which human chorionic gonadotropin is administered to induce ovulation.

Murine bladder carcinoma cells present antigen to BCG-specific CD4+ T-cells.

Intravesical administration of Bacillus Calmette-Guérin (BCG) is the most effective therapy for superficial transitional cell carcinoma of the bladder although its mechanism of action is not known. To determine if bladder tumors are capable of antigen presentation and thus might interact directly with BCG-specific T-cells, we studied the murine bladder tumor MB49. MB49 (MHC Class II negative) (IA-), when induced to express IA with interferon, presented BCG to specific CD4+ T-cells obtained from bladder-draining lymph nodes following intravesical BCG administration. This interaction resulted in antigen- and IA-dependent interleukin 2 and tumor necrosis factor production. Interferon also induced MB49 IA expression in vivo. This first demonstration of antigen presentation by epithelial tumors supports new approaches to immunotherapy of these malignancies.

Retinoid binding protein activities in murine embryonal carcinoma cells and their differentiated derivatives.

It has been proposed that cellular retinoic acid binding protein is essential for retinoid-induced differentiation of embryonal carcinoma line PCC4.aza1R. To assess the generality of this proposal, we have tested for the presence of cellular retinoic acid binding protein activities in several other embryonal carcinoma lines. Cytosolic extracts from all cells were found to possess binding proteins for retinoic acid and also for retinol, although levels varied widely among the different lines. There was no clear quantitative relationship between binding protein activities and the propensity of the cells for differentiation in tumor form or under various in vitro conditions. Our results suggest that other factors might modulate the response of embryonal carcinoma cells to retinoids and/or that alternate pathways for differentiation which do not involve retinoids and retinoid binding proteins exist in these cells. When embryonal carcinoma cells are stimulated to differentiate, the derivatives can possess higher, lower, or similar levels of retinoic acid binding protein activity. These levels appear to reflect the phenotype of the differentiated cells rather than the conversion from a tumorigenic to a nontumorigenic state.

Effect of human natural killer cells on the metastatic growth of human melanoma xenografts in mice with severe combined immunodeficiency.

An in vivo model for human melanoma was established with the growth of CR3 and DE5 human melanoma tumor cells following i.v. injection into C.B.-17 severe combined immunodeficient mice depleted of murine natural killer (NK) cells. The ability of human NK cells to mediate antitumor activity in vivo was investigated by evaluating the number of lung nodules and survival of mice given injections of human NK cells i.v. early after injection of CR3 tumor cells. Under these conditions, human NK cells effectively reduced lung nodule counts and prolonged survival when coinjected with interleukin 2 (IL-2). Multiple injections of IL-2 given during the first 16 h post-NK injection did not further enhance the tumor reduction. Significantly increased antitumor activity against CR3 tumor cells in vivo was observed in mice receiving NK cells coinjected with IL-2 and interleukin 12 (IL-12) in comparison to NK cells and IL-2 only. However, coinjection of IL-12 with human NK cells alone did not reduce the tumor burden. These results demonstrate the antitumor activity of human NK cells against human melanoma in severe combined immunodeficient mice and its augmentation by IL-2, alone or in combination with IL-12, suggesting that this model can be used to further investigate the interaction between human NK cells and human tumors.

Intravesical gene therapy: in vivo gene transfer using recombinant vaccinia virus vectors.

Intratumoral gene transfer may be a significant tool in active immunotherapy. The ability to insert functional genes into a tumor in vitro and in vivo using recombinant vaccinia vectors was examined in the murine bladder tumor model. Vaccinia recombinants expressing the influenza hemagglutinin or nucleoprotein antigens infected/transfected murine (MB-49 and MBT-2) and human (T24) bladder tumor cell lines in vitro. Systemic vaccinia immunity was induced with as few as 10 plaque-forming units of recombinant vaccinia instilled intravesically, and the encoded protein was expressed in vivo in tumor and urothelium. However, preimmunity to vaccinia did not inhibit intravesical tumor transfection. Thus, recombinant vaccinia virus is effective in introducing foreign antigens locally into tumor in vivo, supporting its use in clinical immunotherapy.

Common regions of deletion in chromosome regions 3p12 and 3p14.2 in primary clear cell renal carcinomas.

Nearly all clear cell renal cell carcinomas (RCCs) exhibit loss of alleles on the short arm of chromosome 3. Loss and mutation at the von Hippel-Lindau (VHL) gene at 3p25 probably occurs in most RCCs and, since the VHL gene was recently cloned, data on VHL involvement in RCCs is accumulating. However, the region 3p14-p12, a region that contains the familial RCC-associated t(3;8)(p14.2;q24) chromosome translocation and the small cell lung carcinoma-associated homozygous deletion at 3p13-12, has also been reported to exhibit allele loss in a large fraction of RCCs. In order to focus future studies on potential suppressor genes in the 3p14-p12 region, we have studied allele loss in 30 RCCs with 9 polymorphic simple sequence repeat markers spanning 3p21.1-p12. Partial losses in the 3p21-p12 region were observed, allowing determination of common regions of loss of heterozygosity overlap in 15 RCCs. Results suggested that most RCCs exhibit loss in a region which brackets the t(3;8) familial chromosome translocation at 3p14.2, and some show additional deletions within the U2020 small cell lung carcinoma deletion at 3p12.

Growth and metastasis of fresh human melanoma tissue in mice with severe combined immunodeficiency.

Cryopreserved cell suspensions of freshly excised melanoma metastases from nine patients were injected s.c. into C.B-17 severe combined immunodeficiency (SCID) mice. All 9 tumors grew as s.c. masses and six of nine were successfully transplanted into other SCID mice. Transplant inocula as low as 5 x 10(5) cells resulted in 100% tumor incidence. Moreover, seven of nine tumors metastasized, five from the original s.c. implants and two from transplanted s.c. tumors. Metastases were detected mainly in the lungs but also were found in abdominal viscera (liver, spleen, and pancreas) and thoracic lymph nodes. Flow cytometric analysis showed that expression of a panel of melanoma antigens, melanoma-associated proteoglycan, ganglioside GD3, and ganglioside GD2, was maintained with SCID passage. The original tumor inocula contained a variable percentage of tumor-associated lymphocytes (1-76%). Flow cytometry analysis indicated that these were mainly CD3+ T-cells. However, there was no correlation between the percentage of tumor-associated lymphocytes and the time required for development of a palpable tumor after s.c. injection or the ability to metastasize. These results demonstrate the growth and spontaneous metastasis of fresh human melanoma in SCID mice and suggest that this model could be important for therapeutic and basic biological studies.

Effects of dietary retinoids upon growth and differentiation of tumors derived from several murine embryonal carcinoma cell lines.

We have examined the effects of dietary retinoids upon the growth and differentiation of seven embryonal carcinoma lines in mice. The control diet contained 4000 IU/mg retinyl palmitate; the other diets contained 2 x 10(5) IU/mg retinyl palmitate, 50 mg/kg all-trans-retinoic acid (RA), 100 mg/kg RA, and no retinoid. The RA-containing diets had little influence on tumor latency or incidence but did suppress growth of many of the tumors. Decreased tumor mass was, in most but not all instances, accompanied by an increased proportion of differentiated cells. Increased differentiation was most commonly quantitative rather than qualitative; i.e., there was a larger proportion of the same types of differentiated cells seen in tumors from the control diet group rather than an increase in the spectrum of cell types observed. Notably, tumors from two differentiation-defective embryonal carcinoma lines were refractory to both the differentiation-inducing and growth-suppressing properties of dietary RA. Taken together, our results suggest that dietary RA can reduce teratocarcinoma growth in part by promoting differentiation but that other mechanisms are likely to be involved. The therapeutic benefits that we observed with dietary RA were compromised by adverse effects, including failure of the mice to gain weight as effectively as those on the control diet. The effects of elevated levels of retinyl palmitate, or its omission from the diet, were much less striking than that of RA. Both modifications tended to decrease tumor latency but had little effect, if any, upon ultimate tumor mass. Elimination of retinoid from the diet failed to significantly reduce degree of differentiation in tumors which normally differentiate extensively in animals on retinoid-containing diets. Excess retinyl palmitate led to a marginal increase in differentiation in F9 tumors and a statistically significant increase in differentiation in OC15-S1 tumors. Tumors from other embryonal carcinoma lines did not contain elevated levels of differentiated cells. The interpretation of these results is complicated by our observations that although our dietary alteration in levels of palmitate were dramatic, they resulted in much more modest differences in circulating retinoid levels when compared with mice on the control diet.

Loss of heterozygosity at the familial RCC t(3;8) locus in most clear cell renal carcinomas.

Previously, we had observed that more than 80% of clear cell renal carcinomas (RCCs) exhibited loss of heterozygosity (LOH) between the microsatellite markers D3S1285 (in 3p14.1) and D3S1295 (in 3p21.1), a region which includes the protein tyrosine phosphatase gamma locus (PTPRG locus, PTP gamma gene) and the 3p14.2 break of the familial RCC-associated translocation, t(3;8)(p14.2;q24), which has been hypothesized to affect expression of an RCC suppressor gene or oncogene. Using seven microsatellite markers and four markers derived from a PTPRG YAC contig, we have further delineated the 3p14.2 region of LOH in RCCs. Eighty-nine % of clear cell RCCs (31 of 35) showed a common region of loss between the D3S1481 and D3S1312 loci which flank the 3p14.2 t(3;8) translocation breakpoint and the PTP gamma gene. The PTP gamma gene occupies approximately 780 kilobase pairs between markers D3S1480 and D3S1312, with its currently defined 5' end greater than 200 kilobase pairs centromeric to the 3p14.2 translocation break. Although most of the RCCs with LOH between D3S1481 and D3S1312 loci have lost at least a portion of one PTP gamma allele, we have tested all known exons of the remaining PTP gamma gene in a number of the kidney tumors and have not observed mutations. Thus, there may be another gene in the vicinity of the 3p14.2 break that is important not only in the familial RCCs in the t(3;8) family but in the majority of clear cell RCCs.

Absence or reduction of Fhit expression in most clear cell renal carcinomas.

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.