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Doxorubicin and nogalamycin are antitumor antibiotics that interact with DNA via intercalation and threading mechanisms, respectively. Because the importance of water, particularly its impact on entropy changes, has been established in other biological processes, we investigated the role of water in these two drug-DNA binding events. We used the osmotic stress method to calculate the number of water molecules exchanged (Δnwater), and isothermal titration calorimetry to measure Kbinding, ΔH, and ΔS for two synthetic DNAs, poly(dA·dT) and poly(dG·dC), and calf thymus DNA (CT DNA). For nogalamycin, Δnwater<0 for CT DNA and poly(dG·dC). For doxorubicin, Δnwater>0 for CT DNA and Δnwater<0 for poly(dG·dC). For poly(dA·dT), Δnwater~0 with both drugs. Net enthalpy changes were always negative, but net entropy changes depended on the drug. The effect of water exchange on the overall sign of entropy change appears to be smaller than other contributions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Extended-spectrum β-lactamase (ESBL) enzymes cause resistance to common β-lactam antibiotics and are associated with poor outcomes. Management of ESBL urinary tract infections (UTIs) is challenging given the limited treatment options available outside the hospital setting. In this study, the primary endpoint of UTI-related 30-day hospital re-admission or emergency department/clinic revisit rates was compared for fosfomycin and ertapenem outpatient ESBL UTI treatments. A retrospective cohort study was performed on patients with ESBL UTIs treated with outpatient fosfomycin or ertapenem from January 2010 to February 2015. Inclusion criteria were age ≥18 years, outpatient treatment with fosfomycin or ertapenem for symptomatic ESBL UTI, and positive urine cultures with microbiologically proven ESBL-producing bacteria. A non-inferiority margin of 0.15 was selected to detect a difference in the primary endpoint. Patient and infection characteristics were compared. A sensitivity analysis with propensity score matching was performed. In total, 178 patients were included (89 fosfomycin-treated and 89 ertapenem-treated). Ertapenem-treated patients received longer outpatient antibiotic treatment (10 days vs. 6 days; P <0.001). ESBL isolates identified were 149 Escherichia coli (83.7%), 26 Klebsiella spp. (14.6%) and 3 other (1.7%). Common dosage regimens were oral dose of 3 g fosfomycin every 72 h (62%), oral dose of 3 g fosfomycin every 48 h (23%) and intravenous dose of 1 g ertapenem daily (76%). The thirty-day re-admission/revisit rates for fosfomycin and ertapenem were 14.6% vs. 13.5% (1.1% difference; 97.5% CI, -0.11 to 0.13). Fosfomycin was non-inferior to ertapenem for treating outpatient ESBL UTIs and should be considered as appropriate step-down therapy for these infections., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 (-1). We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures.

Cellular migration is the movement of cells, cultured as a monolayer; cellular invasion is similar to migration, but requires the cells to move through a three-dimensional material such as basement membrane extract or a synthetic hydrogel. Migration assays, such as the transwell assay, are widely used to study cellular movement because they are amenable to high-throughput screens with minimal experimental setup. These assays offer limited information about cellular responses to gradients in vivo because they oversimplify the threedimensional (3D) environment of a tissue. There are a number of invasion assays that support 3D cultures, some of which provide experimental control over the spatial and temporal gradients imparted on the culture. These assays, in their current form, are difficult to setup and maintain, and often require specialized laboratory equipment or engineering expertise. Here we describe a paper-based invasion assay in which cellular movement can be monitored in real-time with fluorescence microscopy. These assays are easily prepared and utilize materials commonly found in any laboratory: a single sheet of paper. These sheets are wax patterned to contain channels in which cells suspended in a hydrogel are seeded and cultured. Cell-containing sheets of paper are placed in a custom-built holder that allows gradients to form along the length of the channels. In this work, we compare the invasion of cells cultured in the presence and absence of an oxygen gradient. Our result support previous findings that oxygen is a chemoattractant, and selectively directs cellular movement in a 3D culture environment.

Vancomycin-resistant urinary tract infections are often challenging to treat. This retrospective cohort study compared outcomes between patients treated for vancomycin-resistant enterococcal urinary tract infection with an aminopenicillin and those treated with a non-β-lactam antibiotic. Inpatients treated with an enterococcus-active agent for their first symptomatic vancomycin-resistant enterococcal urinary tract infection between 1 January 2012 and 31 December 2013 were considered for inclusion. Patients with colonization, on hospice, or receiving comfort care only were excluded. The primary endpoint of clinical cure was defined as resolution of clinical symptoms, or symptom improvement to the extent that no additional antibacterial drug therapy was necessary, and lack of microbiologic persistence. Secondary endpoints of 30-day readmission or retreatment and 30-day all-cause mortality were also compared. A total of 316 urinary isolates were screened, and 61 patients with symptomatic urinary tract infection were included. Twenty (35%) of the 57 isolates tested were ampicillin susceptible. Thirty-one patients received an aminopenicillin, and 30 received a non-β-lactam. Rates of clinical cure for aminopenicillin versus non-β-lactam treatment were 26/31 (83.9%) and 22/30 (73.3%) (P = 0.315), respectively. Rates of 30-day readmission (6/31, or 19.4%, versus 9/30, or 30%, respectively; P = 0.334), 30-day retreatment (4/31, or 12.9%, versus 4/30, 13.3%, respectively; P = 0.960), and 30-day all-cause mortality (2/31, or 6.5%, versus 1/30, or 3.3%, respectively; P = 0.573) were also not significantly different between groups. Aminopenicillins may be a viable option for treating vancomycin-resistant urinary tract infection regardless of the organism's ampicillin susceptibility. Prospective validation with larger cohorts of patients should be considered., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Purpose: Improvements in medication use achieved by pharmacy generalists using a care bundle approach to antimicrobial stewardship are reported., Methods: A six-month prospective, repeated-treatment, quasi-experimental study involving three month-long intervention periods and three month-long control periods was conducted in the setting of an existing antimicrobial stewardship program at a large hospital. The intervention involved prospective audit and feedback conducted by pharmacy generalists who were trained in an antimicrobial stewardship care bundle approach. During control months, a pharmacy generalist who was not trained in antimicrobial stewardship rounded with the multidisciplinary team and provided standard-of-care pharmacy services. The primary endpoint was compliance with a care bundle of four antimicrobial stewardship metrics: documentation of indication for therapy in the medical record, selection of empirical therapy according to institutional guidelines, documented performance of indicated culture testing, and deescalation of therapy when indicated., Results: Two-hundred eighty-six patients were enrolled in the study: 124 in the intervention group and 162 in the control group. The cumulative rate of full compliance with all care bundle components during the six-month study was significantly greater during intervention months than during control months (68.5% versus 45.7%, p < 0.001). After adjusting for infection type, antimicrobial stewardship provided by an intervention-group pharmacist was associated with improved care bundle compliance (adjusted odds ratio, 2.70; p < 0.001). No significant differences in patient outcomes during intervention and control months were detected., Conclusion: Pharmacy generalists trained to comply with a systematic care bundle approach enhanced the quality of antimicrobial management., (Copyright © 2015 by the American Society of Health-System Pharmacists, Inc. All rights reserved.)

Vancomycin has been in clinical use for over 60 years, during which time renal toxicity has been well documented. Multiple risk factors and outcomes are associated with vancomycin-related nephrotoxicity. Risk factors include vancomycin exposure (trough levels 15 mg/L or higher, larger area under the curve, duration of therapy), host susceptibility to vancomycin (increased body weight, preexisting renal dysfunction, critical illness), and concurrent nephrotoxin therapy. Nephrotoxicity is associated with prolonged hospital stays, mortality, and the need for renal replacement therapy. To what degree vancomycin-associated nephrotoxicity exacerbates these adverse clinical outcomes remains unclear. This article reviews the current evidence on vancomycin-associated nephrotoxicity and explores future research directions with potential implications for improved patient safety., (© 2014 Pharmacotherapy Publications, Inc.)

Introduction: Antimicrobial prescribing in the emergency department is predominantly empiric, with final microbiology results either unavailable or reported after most patients are discharged home. Systematic follow-up processes are needed to ensure appropriate antimicrobial therapy at this transition of care. The objective of this study was to assess the impact of a culture follow-up (CFU) program on the frequency of emergency department (ED) revisits within 72 h and hospital admissions within 30 days compared to the historical standard of care (SOC). Additionally, infection characteristics and antimicrobial therapy were compared., Methods: A single group, pre-test post-test quasi-experimental study was conducted comparing a retrospective SOC group to a prospective CFU group. CFU was implemented using computerized decision-support software and a multidisciplinary team of pharmacists and emergency physician staff., Results: Over the four-month intervention period the CFU group evaluated 197 cultures and modified antimicrobial therapy in 25.5%. The rate of combined ED revisits within 72 h and hospital admissions within 30 days was 16.9% in the SOC group and 10.2% in the CFU group (p = 0.079). When evaluating the uninsured population alone, revisits to the ED within 72 h were reduced from 15.3% in the SOC group to 2.4% in the CFU group (p = 0.044)., Conclusion: Implementation of a multidisciplinary CFU program was associated with a reduction in ED revisits within 72 h and hospital admissions within 30 days. One-fourth of patients required post-discharge intervention, representing a large need for antimicrobial stewardship expansion to ED practice models.

Introduction: Little is known regarding age-related risk of nephrotoxicity during vancomycin therapy after the publication of the 2009 vancomycin consensus guidelines for therapeutic drug monitoring. We sought to evaluate incidence and risk factors for acute kidney injury in three age groups., Methods: Matched cohort study of patients receiving vancomycin, grouped by age: young adults (18-64 years), older adults (65-79 years) and very elderly (≥80 years), matched on previously published risk factors for nephrotoxicity. Outcomes included traditional vancomycin nephrotoxicity and Acute Kidney Injury Network-modified definition of nephrotoxicity., Results: The incidence of acute kidney injury was 34.1% vs. 34.1% vs. 31.8% in the young, older adults and very elderly groups, respectively (p = 0.97). In the logistic regression model, after adjusting for baseline risk factors, age was not a significant predictor of acute kidney injury. Lower respiratory tract infection (adjusted odds ratio [aOR] 5.18; 95% confidence interval [CI] 2.15-12.41) and duration of treatment (aOR 1.12; 95% CI 1.03-1.22) were found to be independently associated with outcome., Conclusion: No differences in risk of acute kidney injury were identified between young, older, and very elderly adults when adjusting for other risk factors. Further research is required to identify strategies to optimize the safety of vancomycin in the aging population.

Protamine P1 amino acid sequences were determined from semen samples of the Przewalski horse, donkey, Somali wild ass, Grevy's zebra, and Grant's zebra (odd-toed perissodactyls), and compared with those of the domestic horse. Although the rate of amino acid variation of protamine P1 is known to be among the most rapidly diverging polypeptides, the equid sequences revealed only little variation. The sequence from the Przewalski horse was identical with that from the domestic horse. The other sequences differed from the corresponding sequences of the domestic and Przewalski horses in two positions-Ser29 was replaced by Cys and Gln32 was replaced by Arg. The presence of the Cys residue at position 29 in the protamine P1 from the zebras, the donkey, and the Somali wild ass may allow formation of one extra protamine disulfide bridge during chromosome condensation in these species. Comparison with protamines from various even-toed animals (artiodactyls) indicated amino acid changes specific for those but different from the equid sequences.

Testicular degeneration is a major cause of subfertility in stallions, although an aetiological diagnosis cannot be made in most cases. In the present study, autoimmune testicular degeneration was induced and evaluated in stallions by immunizing stallions with their own spermatozoa mixed with an adjuvant. The factors evaluated included changes in semen quality and testicular histology. A large decrease in sperm number and quality was observed in response to sperm autoantigens. An ELISA test specific for antisperm antibodies was developed which enabled antibody titres in serum, seminal plasma and accessory sex organs to be measured. Serum antibodies were characterized as being specific for spermatozoa by absorption studies and western blotting. The data obtained and the tests developed in the present study provide a better understanding of the disease in subfertile breeding stallions and the ability to diagnose the disease using ELISA. The results of a clinical trial demonstrate that there is a higher incidence of antisperm antibodies in subfertile stallions compared with fertile stallions. The results of the present study indicate that autoimmunity to spermatozoa plays a role in idiopathic subfertility in stallions. A potentially useful method for tentative diagnosis of autoimmune testicular degeneration in subfertile stallions was also developed.

A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm daily thereafter for 60 days. Changes in the SCSA were then correlated with protamine disulfide content and protamine types and levels. Results of the SCSA indicated that the susceptibility of spermatozoal DNA to denaturation was dependent on the spermatogenic cell stage that the ejaculated sperm was in at the time of the heat stress. Spermatozoa with altered DNA had a decrease in the extent of disulfide bonding that was associated with an increase in the susceptibility of DNA to denaturation. However, there were no detectable changes in either the protamine type or level. Thus, in this model, decreased disulfide bonding is associated with an increased susceptibility of spermatozoal DNA to denaturation in the absence of protamine changes.

The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percentage pregnant per first cycle (FCP; SD alpha t, r = -0.30, P < or = 0.01; % COMP alpha t, r = -0.42, P < or = 0.0001); and the percentage pregnant per cycle (PC; mean alpha t, r = -0.31, P < or = 0.01; SD alpha t, r = -0.32, P < or = 0.01; % COMP alpha t, r = -0.41, P < or = 0.0001). This study describes detectable intrinsic variation in sperm chromatin structure among fertile stallions (SPR, mean = 83%; FCP, mean = 58%; PC, mean = 57%) in an active breeding population (number of mares bred/stallion/year, mean = 37), in the absence of overt reproductive abnormalities and apparent diseases such that an increase in the susceptibility of sperm DNA to denaturation is associated with reduced fertility, both in terms of efficiency of reproduction (FCP and PC) and seasonal pregnancy rate (SPR). Both COMP alpha t and mean alpha t were useful indicators of fertility, with COMP alpha t being the only SCSA value able to identify mean differences between fertility groupings for SPR and FCP, and overall it was the most reliable indicator of fertility in this group of stallions. The SCSA is able to evaluate a compartment of the spermatozoa which is different from that of traditional tests for sperm quality such as motility and morphology.

An 8-year-old stallion was evaluated because of recurrent urinary tract infections and chronic intermittent urospermia. After extensive diagnostic testing, it was hypothesized that the stallion had a reflex dyssynergia of the bladder and urethral sphincter. Initial attempts to manage the urospermia included semen fractionation, semen collection after voluntary urination, and use of semen extenders. None of these efforts reliably yielded a quality ejaculate. Administration of imipramine hydrochloride (1.2 mg/kg of body weight, PO, 4 hours prior to semen collection) was initiated in an attempt to enhance bladder neck closure during ejaculation. This treatment, combined with voluntary urination prior to ejaculation, resulted in ejaculates containing little or no urine. Using this protocol, 19 of 20 mares bred during the subsequent 2 years became pregnant. By the third year, the bladder dysfunction had progressed, and the urospermia was no longer manageable. Bladder catheterization, followed by manual expression of the bladder per rectum, were necessary prior to each semen collection to obtain a urine-free ejaculate. Three-and-a-half years after initial examination, transitional cell carcinoma of the bladder with metastasis was identified, and the stallion was euthanatized. It is not known whether the transitional cell carcinoma was related to the dysfunctional bladder. Imipramine hydrochloride did not eliminate, but did reduce, the frequency and degree of urospermia in the affected stallion for approximately 2 years.

This article provides an update on the methods of collection, preservation, storage, and administration of platelet concentrates. The current indications for platelet transfusions are reviewed, and current knowledge concerning alloimmunization and its prevention is summarized. In addition, the ongoing controversies related to prophylactic administration of platelets versus therapeutic administration are reviewed.

In this study, 2 stallions were immunised with their own spermatozoa to ascertain whether an antisperm autoantibody response could be mounted. The results demonstrated that the stallion can recognise and respond to sperm autoantigens by producing circulating antisperm antibodies, primarily of the IgG class. Such autoantibodies appeared 2-4 weeks after inoculation and persisted for 6-20 weeks. Immunochemical characterisation by western blot identified two major sperm autoantigens, with molecular weights of 70 kD and 62 kD. Control pony stallions immunised with adjuvants alone failed to exhibit such antibodies. IgA antisperm antibodies were measurable in seminal plasma of both stallions. We suggest that, as in other species, autoimmunity to spermatozoa may play a role in idiopathic subfertility in stallions.

A Thoroughbred stallion with erectile dysfunction following paraphimosis was managed to allow consistent ejaculation. Ejaculation was elicited in this stallion by applying manual stimulation to the base of the penis. This technique allowed consistent ejaculation, and the stallion was able to return to natural service. Historically, stallions with this degree of penile dysfunction have been considered incapable of ejaculation and unable to continue as breeding animals.

Two mature breeding stallions were evaluated because of specific ejaculatory dysfunction, and each was found to have aortic-iliac thrombosis occluding 60 to 70% of the aortic lumen. In each case, the stallion had strong libido, normal mounting, and vigorous initial thrusting. With continued exertion, thrusting became weak and dismount was awkward. Treatment aimed at maximizing sexual arousal before mounting and reducing hind limb pain and exertion during breeding allowed these stallions to continue breeding. A program of gradually increasing daily exercise also was associated with improved breeding performance during subsequent breeding seasons. In cases of specific ejaculatory dysfunction, aortic-iliac disease should be included among the differential diagnoses.

Equine venereal infections of concern in the United States include EHV-3, T. equigenitalis, P. aeruginosa, and K. pneumoniae. Stallions may also harbor EAV in the genital tract and transmit the virus to mares during coitus. With the exception of EHV-3, the stallion generally remains asymptomatic while transmitting infections to mares during breeding. Methods for diagnosis, treatment, and control of these infections are discussed.

Anomalies of centriolar derivatives were identified in ejaculates and testicular and tracheal biopsies of a sterile stallion, using light, scanning, and transmission electron microscopy. LM revealed that over half the sperm population had only a vestigial or no tail, while the rest had tails of variable length and shape. The vestigial tail was represented by its anlage, which was implanted on the nucleus and differentiated up to capitulum and collum stage. The stunted tail had an axoneme and its derivatives, but was short in all tail segments. Regardless of the tail length or shape, virtually all axonemes were devoid of the central tubular complex ("9 + 0" defect). Abnormal tail segmentation was associated with missing or defective flagellar sheaths and a profusion of extraneous dense fibers, which contributed to the knobby, bulbous, or lobuliform tail configurations. The gradient of flagellar anomalies seems associated with the inability of the distal centriole to implant on the plasmalemma, to produce the axoneme, or maintain its growth, and to induce the normal differentiation of periaxonemal structures. In contrast to sperm, the tracheal epithelium displayed moderate changes, which are manifest in circumscribed rarefaction of cilia, increased incidence of compound cilia, and disturbed orientation of cilia regarding the plane of central tubular complex. The tracheal cilia were free of "9 + 0" defect.

Histological features of the endometrium, as assessed in biopsy samples, were related to Standardbred mare genotypes for transferrin, esterase (as a control) and equine leucocyte antigens (ELA). Pathological changes were found more frequently in each successively older age group of mares. Among mares aged 6-19 years, there were significant pathologic changes on first examination following an infertile breeding season for 46 of 90 (51%) of transferrin homozygotes and 50 of 146 (34%) of transferrin heterozygotes. The difference between the two groups was significant for the total data (chi 1(2) = 6.56, P = 0.010) and when the data were stratified for mare age at biopsy (chi 1(2) = 7.33, P = 0.0068). The effect of transferrin was similar in both trotters and pacers, especially for frequent genotypes commonly found in horses of both gaits. There was no effect of esterase and, in a smaller set of ELA-typed mares, no significant effect of ELA genotype on uterine biopsy category. Transferrin has a well-established microbiostatic and biocidal effect. Conceivably, heterozygotes for some combinations of transferrin variants could have a slower natural rate of endometrial deterioration than homozygotes.

The semen of 26 stallions, 2-20 years of age, was collected once a day for 7 consecutive days to determine daily sperm output (DSO). After the last collection, 17 stallions were castrated. Testicular volume was estimated using two methods. Length, width, height, of each testis were measured by caliper. Length, width, height, cross-sectional area and circumference at the widest point of the testis were measured by ultrasonography. Both caliper and ultrasound measurements were first made in the live animal, and again in vitro. There were no differences in these measurements because of method or condition of the animal, i.e., whether the measures were taken in the live animal or after castration. The volume of an ellipsoid (4/3 pi abc, a = height/2, b = width/2, c = length/2) is introduced as a method of accurately estimating testicular volume (r = 0.99) as well as predicting DSO (r = 0.92) in the stallion. The authors of this report suggest that this estimation can be performed by substituting values derived by ultrasonography or calipers.

A total of 174 stallions were subjected to a standard fertility examination and classified as fertile, subfertile or sterile. All stallions were phenotypical males involved in breeding programmes with no detectable abnormalities in their reproductive organs. Fertile stallions had no history of any breeding problem. Subfertile stallions were referred with a history of a breeding problem that was subsequently determined not to be attributable to the mares or infectious diseases. They were divided into chromosomally normal and abnormal groups on the basis of karyotype. The relative DNA content of lymphocytes (DI) was estimated by flow cytometry to relate this parameter with chromosome status. Of 62 completed karyotypes, 44 were normal diploid stallions and 18 had chromosomal defects, including mosaics with sex or autosomal deletions or duplications. The seasonal pregnancy rates for the subfertile chromosomally defective and subfertile chromosomally normal stallions were similar but were less than half of those of the fertile stallions. With the present limited data, no association was noted between DI and pregnancy rate. The mean DI for the subfertile stallions with chromosome defects was not different from those of the fertile and the subfertile chromosomally normal stallions. However, the DI for a mosaic stallion with sex chromosome deletion was lower than the DI for the fertile stallions. Extremely high and low values of the DI may be useful for flagging potential subjects for detailed chromosome investigation. The DI also was correlated negatively (P less than 0.002) with progressive sperm motility.

The association of equine granulosa-theca cell tumors with atrophied contralateral ovaries and abnormal estrous cycles suggests that these tumors produce hormones that affect pituitary gonadotropin production. Because inhibin, a heterodimer protein secreted by granulosa cells, decreases FSH production, we examined the presence of inhibin alpha- and beta A-subunits and their mRNAs in ovarian tumors obtained from three mares. These tumors contained neoplastic cords and nodules, multiple fluid-filled cysts, and a predominance of neoplastic granulosa cells. Reduced proteins from tumor-conditioned media were analyzed by electrophoresis and immunoblotting using antibodies directed against peptide fragments of the alpha- and beta A-chains of porcine inhibin. Specific bands at 50-kDa and 36-kDa for the inhibin alpha-subunit and at 44 kDa and 13 kDa for the inhibin beta A-subunit were observed in these tumors. Northern blot hybridization of 32P-labeled rat inhibin alpha- and beta A-subunit complementary RNAs to total RNA from each tumor revealed predominant bands of activity in all three tumors at 1.5 and 7 kb for the alpha- and beta A-subunit mRNAs, respectively. These results demonstrate that equine granulosa-theca cell tumors express the mRNAs for inhibin alpha- and beta A-subunits and also secrete inhibin subunits that could potentially affect gonadotropin production in afflicted mares. Furthermore, cells derived from these tumors may provide a useful model for understanding inhibin gene regulation and ovarian tumorigenesis.

A functional corpus luteum was found in the ovary contralateral to the ovary with a granulosa cell tumor in a 24-year-old Standardbred mare. The mare was ovariectomized because she was to be used as a jump mare for collection of semen from stallions. The blood concentration of progesterone was 2.2 ng/ml, and the luteal tissue progesterone concentration was 6.3 micrograms/mg. Atrophy of the contralateral ovary is one of the major signs used in diagnosis of granulosa cell tumor; however, our findings indicate that the ovary contralateral to a granulosa cell tumor is not invariably nonfunctional.

It is possible that the commonly measured serum level of vitamin B12 may miss some cases when used to detect vitamin B12 malabsorption and deficiency in older persons. Serum levels of vitamin B12 and intrinsic factor antibody (IFAB) were determined on 250 consecutive patients over the age of 70 admitted to a rehabilitation hospital. Patients with abnormal results on either test were given the standard Schilling test when possible. Eight patients had documented B12 malabsorption. Of these, five had a low serum B12 level alone and one had a low serum B12 level and a positive IFAB level; however, two patients had positive IFAB and normal serum B12 levels. Serum IFAB level may serve as a useful adjunct to serum B12 level in detecting vitamin B12 malabsorption in older patients.

Two experiments were conducted to investigate methods for aspirating oocytes from immature preovulatory follicles in the mare. In Experiment 1, the ovary was manipulated per rectum and the follicle was punctured by a needle introduced through the flank. Suction was provided by either a syringe or by a vacuum pump connected to the needle via tubing. The preovulatory follicle was aspirated when it reached a diameter of 32 +/- 2 mm (Group A); 37 +/- 2 mm (Group B); or 42 +/- 2 mm (Group C). There was no significant difference in oocyte recovery rates between the two methods (7/24 vs 3/19). Oocyte recovery rates were higher for Groups B and C (5/14 and 4/12, respectively) than for Group A (1/17; P < 0.05). In Experiment 2, the ovary was held against the internal abdominal wall by the hand inserted into the abdomen via a vaginal incision, and the follicle was flushed after aspiration. Recovery rates were 9/13 (69%) for mares treated with human chorionic gonadotrophin (hCG) and 15/21 (71%) for unstimulated mares. This difference was not significant. The oocyte recovery rate for unstimulated follicles (average diameter 39.7 mm) in Experiment 2 was significantly higher than those for Group B and Group C in Experiment 1 (P < 0.05).

Serum concentration of ampicillin, a semisynthetic penicillin, was measured in mares at various time intervals up to 24 hours after intrauterine infusion of 3 g of ampicillin. Blood samples were drawn immediately before infusion and at 1-, 4-, 10- and 24-hour intervals after infusion. At postinfusion hour 24, two endometrial biopsy specimens were obtained to measure endometrial concentrations of ampicillin. Blood was drawn twice as part of the 24-hour postinfusion sample collection, once before removal of the biopsy specimens and again 5 minutes after removal of the biopsy specimens. After drug infusion, more diestrous mares had detectable serum ampicillin concentration than did estrous mares for all samples, except the 24-hour prebiopsy sample. None of the 24-hour prebiopsy serum samples had detectable ampicillin concentration, but ampicillin was detected in the serum of 4 of 5 diestrous mares after endometrial biopsy. Endometrial concentrations of ampicillin were detectable at postinfusion hour 24 in estrous and diestrous mares, but were not different. All 24-hour biopsy specimens had ampicillin concentrations greater than the ampicillin minimal inhibitory concentration.

Cervical hyperplasia with prolapse through the vulvar lips was documented in a mare. Postmortem examination indicated that the mass originated from the cervical wall. The surface of the prolapsed mass had histologic features of normal cervix. Cervical hyperplasia can be considered in a list of differential diagnoses in cases of prolapse of the internal genitalia in mares.

The endometria of 39 mares were cultured simultaneously using a swab guarded with a double cannula and distal, teflon plug and an unguarded swab with a single, open cannula. Sheep blood (5%) agar, Mac-Conkey's agar, and Sabourad's agar media were innoculated with each swab. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. There were significantly more plates that failed to yield bacterial or fungal growth when streaked with swab specimens obtained with the guarded cannula than when streaked with those obtained with the unguarded cannula. It was concluded that while culturing the endometrium of mares for bacteria or fungi, the use of a guarded instrument consisting of a double cannula with a closed end will result in the recovery of fewer contaminants; therefore, it will be more likely to result in a more accurate representation of uterine bacterial and fungal flora.

Two fetal claws were removed from the cervix of a Holstein cow that had a chronic vulvar discharge. The cow had calved 1 year earlier. After removal of the claws, localized peritonitis developed and the cervix remained open and draining.

The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17 beta and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10-15 mg prostaglandin F-2 alpha was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18-34 of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P less than 0.01) but did not differ for the oestrous cycle or pregnancy (P greater than 0.05).

A breeding trial was conducted to determine if a semen extender containing polymixin-B sulfate would improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa . Twenty-three mares were bred to the stallion by one of three methods: artificial insemination with raw semen (Group 1, n = 10), artificial insemination with semen mixed 1:1 with a nonfat dry skim milk/glucose extender containing 1000 units/ml polymixin-B sulfate (Group 2, n = 9), or natural service immediately following infusion of the uterus with 100 ml of the same extender (Group 3, n = 4). Artificial breedings contained a minimum insemination dose of 500 x 10(6) progressively motile spermatozoa. All mares were bred every other day while in estrus. Pregnancy status was determined by transrectal ultrasound examination 15 d after the last breeding. First-cycle pregnancy rate for Group 2 mares (78%) was greater (P < 0.01) than for Group 1 mares (10%). There was a tendency (P = 0.10) for the pregnancy rate of Group 3 mares (50%) to be greater than Group 1 mares. The use of a semen extender containing polymixin-B sulfate improved the fertility of this stallion.

A study was conducted over a 2-mo period to compare estrus detection results obtained using an androgenized teaser mare with those obtained with a stallion, using the same group of 10 normally cyclic mares. The teaser mare was androgenized by administration of boldenone undecylenate (500 mg i.m. every 1 to 2 wk), and allowed to run loose with the mare group. Estrus was determined by observation of the group for a 30-min period daily. In the second month of the experiment, a marking harness was used on the androgenized mare to help detect mares mounted when in estrus. Estrous periods detected by each teasing method were 1) first month: stallion, 18; androgenized mare, 5; 2) second month: stallion, 16; androgenized mare, 9. There were no estrous periods detected by the androgenized mare that were not also detected by the stallion. Under these conditions, the androgenized mare was not an adequate estrus detection aid. Also discussed are the successful results of an independent trial on a breeding farm using an androgenized mare as an estrus detection aid.

Luminal fluid from the mare uterus was used to investigate its relation to antibacterial defenses. Uterine flushings were collected at Day 3 of estrus, Day 8 postovulation and Day 15 postovulation. Uterine proteins were concentrated by ultrafiltration, dialyzed and examined for chemotactic activity to neutrophils and for antibacterial properties. Serum taken at the time of flushing was dialyzed and studied in a similar manner. Neutrophil migration in response to serum from Day 3 estrus and Day 8 postovulation was increased (P less than 0.05) above controls. Uterine protein from Day 8 postovulation and from Day 3 of estrus also stimulated neutrophil migration (P less than 0.05) above values of controls. Antibacterial activity was measured by incubation of S. zooepidemicus with concentrated uterine flushing or serum. Serum from all three estrous cycle intervals diluted 1:10 or used at a protein concentration equal to the protein concentration of uterine fluid did not inhibit growth. After 4 h of incubation, bacterial growth in estrous serum was significantly greater (P less than 0.01) than serum taken at Day 8 and Day 15 postovulation. Uterine flushings from Day 8 postovulation significantly decreased bacterial colony-forming units (P less than 0.01). Heating flushings at 56 degrees C for 30 min did not abolish the antimicrobial activity, while heating flushings for 30 min at 80 degrees C removed this activity. The antibacterial activity does not appear to be due to agglutinating antibody.

Surgery was performed to repair lacerations which prevented cervical closure in thirteen mares. All lacerations involved the portion of the cervix that protrudes into the vagina. There did not appear to be a predisposition for lacerations to occur in any one position about the perimeter of the cervix. Nine mares had one laceration and four mares had two lacerations. Although the lacerations were presumed to have occurred at parturition, they apparently were not associated with dystocia. The method of surgical repair is described. Eight mares were bred after surgery, and five conceived.

A 49-year-old woman ate N-3-pyridylmethyl-N'-p'nitrophenylurea (PNU; Vacor) and was admitted to the hospital 12 hours later with a blood glucose level of 940 mg/dl and an anion-gap metabolic acidosis. Her diabetes was successfully treated, but she contained to manifest severe orthostatic hypotension. A painless ileus developed, followed by cecal perforation and death. Immunoperoxidase staining of paraffin-embedded pancreatic tissue obtained during postmortem examination clearly demonstrated the pancreatic beta-cell destruction. Results of this same staining technique also suggested that fewer alpha cells were present.

The surprisingly rapid rate of uterine involution detected is consistent with a high rate of conception as the first post-partum heat. Furthermore, many of the immediately post-partum features have attained the pregravid state by the end of the first post-partum oestrus and virtually all by the second post-partum oestrus. There was no specific cause detected for the higher rate of early embryonic death associated with conception at the foal heat.

Normal histologic changes that occurred in concert with the ovarian cycle were evaluated. When combined with behavioral and physical findings, this evaluation enabled determination when there was synchrony between ovarian steroids (estrogen and progesterone) and their target tissues of endometrium. A system for classification as well as definitions of patterns types and degrees of severity of inflammatory and fibrotic changes was developed to facilitate a uniform system for description and communication. An attempt was made to relate the pathologic changes to their effect on fertility when fertility was defined as the ability to conceive, maintain, and deliver a live foal. This was done by dividing the types of endometrium into 3 categories, based on increasing degrees of extensiveness and severity of the various changes, and it was possible to show a high correlation of category to foaling rate. Embryonic and fetal loss from endometria with widespread periglandular fibrosis was observed. This was considered to be the major diagnosable cause of this type of reproductive failure.

Cervical measurements were recorded on palpation per rectum for 603 Bos taurus , Santa Gertrudis, and crossbred cattle. Postmortem cervical measurements were recorded for 77 of these cows. Antemortem measurements were positively correlated with those taken at postmortem examination (cervical diameter: r=0.75, P<0.01 and cervical length: r=0.29, P<0.06). Cervical diameters from antemortem and postmortem specimens were greater in Santa Gertrudis cattle than in Bos taurus and crossbred cattle (P<0.05). Cervical shape was different between Santa Gertrudis cattle and Bos taurus or crossbred cattle (P<0.01), with Santa Gertrudis cows having a higher incidence of conical shaped cervices than other breeds. Within Santa Gertrudis cattle, the incidence of conical cervical shape increased with age (P<0.05). Conical cervical shape was associated with a lower pregnancy rate in all breeds (P<0.01). Within Santa Gertrudis cattle, conical cervical shape was associated with a lower pregnancy rate in 1- to 4-year-old cows (P<0.05) but not in cows more than 4 years of age (P>0.05).

Neutrophils isolated from jugular blood of ovariectomized mares were studied for the effect of estradiol and progesterone on bactericidal activity against Staphylococcus aureus. In experiment 1, neutrophils obtained from 4 mares were tested for bactericidal activity by adding estradiol (43 pg/ml) or progesterone (6.4 ng/ml) to the bactericidal assay. In experiment 2, 3 of the 4 ovariectomized mares were given 2 mg of estradiol, IM, daily for 3 days. Eighteen days after the initial estradiol injection, mares were given 300 mg of progesterone, IM, for 6 days. Neutrophils from these mares were tested for bactericidal activity 4 days after the initial estradiol injection, 17 days after the initial estradiol injection (control), and 7 days after the first progesterone injection. Bactericidal activity was measured at 30 and 120 minutes by counting the number of colony-forming units remaining. Neutrophil antistaphylococcal activity was not altered by adding estradiol and progesterone to the assay or by supplementing ovariectomized mares with estradiol and progesterone (P greater than 0.05).

Pony stallions were given sexual behavior trials in experimental "home" and "novel" environments, similar in all major features except location. In the novel environment, erection latency was increased and erection time decreased. Treatment with an anxiolytic benzodiazepine derivative (diazepam) appeared to block these effects.

The interpretation of red blood cell panels and identification of alloantibodies can be a difficult and time-consuming task. PANEL-IZER, a computer program that performs this interpretation, is described. The panel analysis is based on a matrix algebra algorithm that is presented in detail. Conditional probability logic supplements the analysis by searching for unexpected reactive cells or other possibilities such as the presence of autoantibodies or antibodies to low- or high-frequency antigens. The program considers phases of reactivity, dosage effects, and reaction strengths to aid in antibody identification. Probability levels for antibody identification based on Fisher's "exact" method are calculated. The program also can help suggest a "select cell" panel. This program can be a valuable tool in blood banks to aid in the interpretation of panels, to check technologists' interpretations, to generate "select cell" panels, and to help teach students and residents antibody identification.

The endometria of 77 barren mares was swabbed simultaneously using a swab guarded with a single cannula and distal, gelatin capsule (completely guarded swab - CGS) and a partially guarded swab (PGS) with an open cannula. Sheep blood (5%) agar plates were inoculated with each swab, while MacConkey's agar plates were inoculated with the swabs from 44 mares. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. Organisms present were identified, counted, and categorized as saprophytic or pathogenic flora. The endometria of all mares were biopsied immediately following swabbing. Histologic evidence of inflammation in biopsy specimens was classified as (1) none, (2) slight, discrete, focal, and (3) slight or moderate, diffuse, widespread infiltration of inflammatory cells. The number of inflammatory cells migrating through the luminal epithelium was counted and averaged. There were significantly fewer CGS than PGS cultures that yielded growth at 24 and 48 hours of incubation after being streaked on blood agar and MacConkey's agar plates. There were fewer pathogenic bacterial or fungal colonies present at 48 hours of incubation on blood agar plates after being streaked with CGS as compared to PGS. There were no differences in the number of pathogenic bacterial or fungal colonies present at 24 hours of incubation on blood agar or at 24 and 48 hours of incubation on MacConkey's agar plates. There was no correlation between CGS or PGS culture of pathogens and severity of histologic inflammation. There was a positive correlation between culture of pathogens and number of inflammatory cells migrating through the luminal epithelium.

Swab specimens for bacterial culture were obtained from the uterus, vagina, vestibule, and clitoral fossa of 48 mares that had normal reproductive tracts, no history of reproductive problems, and no inflammation on evaluation of endometrial biopsy. The mares were predominantly Thoroughbred and Standardbred. Swab specimens of the vagina were obtained through a sterile speculum; swab specimens of the uterus were obtained by use of a double-guarded, occluded culture instrument. Fifteen (31%) of the uterine swab specimens and 20 (42%) of the vaginal swab specimens yielded growth on aerobic culture; however, only 2 (4%) of the uterine swab specimens and 4 (8%) of the vaginal swab specimens yielded growth of more than 10 colonies. In contrast, 21 (44%) of the vestibular swab specimens and 45 (94%) of the clitoral fossa swab specimens had moderate (greater than 10 colonies in 1 quadrant) to heavy (colonies in 2 or 3 quadrants) growth of organisms on culture. Of organisms considered to be potential pathogens, Streptococcus zooepidemicus and Escherichia coli were found on bacteriologic culture of several clitoral fossa swab specimens and of some vestibular swab specimens. We did not isolate any potential pathogens from uterine or vaginal swab specimens. It appears that 1 to 10 colonies of nonpathogenic organisms could be recovered from the uterus in a substantial number of clinically normal mares even when double-guarded swabbing techniques are used, and we suggest that prebreeding culture requirements be modified to reflect this. Also, our findings indicate that the vulvovaginal fold, rather than the cervix, might be the major barrier to ascending bacterial contamination of the reproductive tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Two groups of 3 mares were inoculated with Haemophilus equigenitalis or Pseudomonas aeruginosa on the 1st day of estrus. Uterine flushing samples were recovered on day 3 of estrus and day 8 after ovulation for each cycle. Mares were killed 22, 25, and 30 days after inoculation with P aeruginosa and 45, 46, and 49 days after inoculation with H equigenitalis. Pseudomonas aeruginosa was recovered from the uterus of 2 mares 48 hours after inoculation. Although the initial flushing sample of 1 of these 2 mares had an increased total protein concentration, there appeared to be little difference between protein concentrations of other uterine flushing samples. Haemophilus equigenitalis was recovered from the uterus of each of the 3 mares at postmortem. One mare had a slight, purulent discharge from the vulva. Total protein values were not increased in flushing samples from this mare after inoculation with H equigenitalis. Total protein values decreased in the last flushing sample of each of the 2 remaining mares. Swabbing the uterus was more effective than was homogenizing the uterine mucosa in isolating H equigenitalis.