Baseline levels of seminal reactive oxygen species predict improvements in sperm function following antioxidant therapy in men with infertility.

BACKGROUND: Poor sperm function is a major cause of infertility. There is no drug therapy to improve sperm function. Semen oxidative stress is a recently identified pathway for sperm damage. Commercial antioxidants such as L-carnitine and acetyl-L-carnitine (LAL) are commonly self-administered by infertile men. However, concerns have been raised whether inappropriate LAL therapy causes reductive stress-mediated sperm damage. It is imperative to investigate whether: (1) LAL improves sperm function by reducing reactive oxidative species (ROS); (2) LAL has differential effects on sperm function between men with normal and elevated ROS. METHODS: A prospective cohort study of routine clinical practice was performed in infertile men with abnormal sperm quality. Changes in sperm function and semen ROS levels following three months of oral LAL therapy were compared between participants with baseline seminal normal ROS (≤10RLU/SEC/106 sperm; n = 29) and High ROS (>10 RLU/SEC/106 sperm; n = 15) levels measured using an established colorimetric-luminol method. RESULTS: In normal ROS group, sperm function did not change following LAL therapy. In high ROS group, LAL therapy reduced semen ROS fivefold, increased sperm count by 50% (mean count in mill/ml: 21.5 + 7.2, baseline; 32.6 + 9.5, post-treatment, P = .0005), and total and progressive sperm motility each by 30% (mean total sperm motility in % 29.8 + 5.0, baseline: 39.4 + 6.2, post-treatment, P = .004; mean progressive sperm motility in % 23.1 + 4.6, baseline: 30.0 + 5.5, post-treatment, P = .014 vs. baseline). CONCLUSIONS: We report for the first time that LAL only improves sperm quality in infertile men who have baseline high-ROS levels prior to treatment. These data have important potential implications for couples with male infertility and their clinicians.

Optimized Analog Multi-Band Carrierless Amplitude and Phase Modulation for Visible Light Communication-Based Internet of Things Systems.

This paper presents a multi-user Visible Light Communication (VLC)-based Internet of Things (IoT) system using multi band-Carrierless Amplitude and Phase (m-CAP) modulation for IoT applications. The proposed system uses a digital m-CAP modulator embedded in a ceiling LED light fixture and analog receivers, aiming at low-cost, low-power, and small-sized IoT devices. The performance was evaluated in terms of the filtering stage design and the usage of guard bands. Different pairs of emitter and receiver filters were considered. While Bessel and Butterworth analog filters were tested in the analog receiver, the digital m-CAP modulator pulse shaping filter considered raised cosine filters, as well as digital matched filters for the analog Bessel and Butterworth filters. Regarding the guard bands, two approaches were considered: either by using the raised cosine roll-off factor (bandwidth compression) or by suppressing the even bands. The Bit Error Rate (BER) performance was obtained by simulation. The usage of the Bessel filter in the receiver, along with a digital matched filter, proved to be the best solution, achieving a BER lower than 10-3 for an Eb/No of 6 dB, using a third-order filter. Furthermore, guard bands should be used in order to mitigate inter-band interference in order to have improved performance when multiple users intend to simultaneously communicate.

Reduced Testicular Steroidogenesis and Increased Semen Oxidative Stress in Male Partners as Novel Markers of Recurrent Miscarriage.

BACKGROUND: Recurrent pregnancy loss, (RPL) affecting 1%-2% of couples, is defined as ≥3 consecutive pregnancy losses before 20-week' gestation. Women with RPL are routinely screened for etiological factors, but routine screening of male partners is not currently recommended. Recently it has been suggested that sperm quality is reduced in male partners of women with RPL, but the reasons underlying this lower quality are unclear. We hypothesized that these men may have underlying impairments of reproductive endocrine and metabolic function that cause reductions in sperm quality. METHODS: After ethical approval, reproductive parameters were compared between healthy controls and male partners of women with RPL. Semen reactive oxygen species (ROS) were measured with a validated inhouse chemiluminescent assay. DNA fragmentation was measured with the validated Halosperm method. RESULTS: Total sperm motility, progressive sperm motility, and normal morphology were all reduced in the RPL group vs controls. Mean ±SE morning serum testosterone (nmol/L) was 15% lower in RPL than in controls (controls, 19.0 ± 1.0; RPL, 16.0 ± 0.8; P < 0.05). Mean ±SE serum estradiol (pmol/L) was 16% lower in RPL than in controls (controls, 103.1 ± 5.7; RPL, 86.5 ± 3.4; P < 0.01). Serum luteinizing hormone and follicle-stimulating hormone were similar between groups. Mean ±SE ROS (RLU/sec/106 sperm) were 4-fold higher in RPL than in controls (controls, 2.0 ± 0.6; RPL, 9.1 ± 4.1; P < 0.01). Mean ±SE sperm DNA fragmentation (%) was 2-fold higher in RPL than in controls (controls, 7.3 ± 1.0; RPL, 16.4 ± 1.5; P < 0.0001). CONCLUSIONS: Our data suggest that male partners of women with RPL have impaired reproductive endocrine function, increased levels of semen ROS, and sperm DNA fragmentation. Routine reproductive assessment of the male partners may be beneficial in RPL.

[MINICHAL and items predictors of worst quality of life in men with arterial hypertension]. / MINICHAL e items predictores de peor calidad de vida en el varón con hipertension arterial.

OBJECTIVE: Systemic arterial hypertension is the most important modificable risk factor for morbidity and mortality and a Public Health problem. The objective was to estímate ítems of worse quality of life (Qol) in both domains of the MINICHAL questionarie and the associated variables. METHODS: An observational study of prevalence in men was carried out. Sociodemographic, comorbidity, clinical, examination, control and serum parameters variables were collected. The following questionnaires were applied: MINICHAL, International Physical Activity Questionnaire, International Score of Prostatic Symptomatology and International Index of Erectile Function. Apart from the usual descriptive ones, a bivariate and a multivariate logistic regression were performed, determining Odds Ratio values with a 95% confidence interval. RESULTS: 262 hypertensive patients were analyzed, of which 42% reported worse quality of life in the mental state dimensión compared to 47.3% in the somatic manifestacions dimensión. The multivariate logistic regression analysis showed as outstanding predictor variables: Metabolic Syndrome in the chest pain item without making any effort; the presence of filling symptoms in the item urinate more often; pathological cardiopulmonary auscultation in the item numbness or tingling in some part of the body; the presence of erectile dysfunction in the item difficulty falling asleep. CONCLUSIONS: All the items of the MINICHAL questionnaire that assess the Somatic Manifestations dimension have a very negative impact on the quality of life of patients, and only the difficulty falling asleep item in the Mental State dimension.

OBJECTIVE: La hipertensión arterial es el factor de riesgo modificable más importante para la morbimortalidad y un problema de Salud Pública. El objetivo de este trabajo fue estimar los ítems predictores de peor calidad de vida (CV) en ambos dominios del cuestionario MINICHAL y las variables asociadas. METHODS: Se realizó un estudio observacional de prevalencia en varones. Se recogieron variables sociodemográficas, de comorbilidad, clínicas, de exploración, de control y parámetros séricos. Se aplicaron los cuestionarios MINICHAL, Cuestionario Internacional de Actividad Física, Puntuación Internacional de Sintomatología Prostática e Índice Internacional de Función Eréctil. Aparte de los descriptivos habituales, se realizó una regresión logística bivariada y otra multivariada, determinando valores de Odds Ratio con intervalo de confianza al 95%. RESULTS: Se analizaron 262 hipertensos, de los cuales el 42% refirió peor calidad de vida en la dimensión estado mental frente al 47,3% en la dimensión manifestaciones somáticas. El análisis de regresión logística multivariada mostró como variables predictoras destacadas: el Síndrome metabólico en el ítem dolor en el pecho sin hacer ningún esfuerzo; la presencia de síntomas de llenado en el ítem orinar más a menudo; la auscultación cardiopulmonar patológica en el ítem entumecimiento u hormigueo en alguna parte del cuerpo; la presencia de disfunción eréctil en el ítem dificultad para conciliar el sueño. CONCLUSIONS: Todos los ítems del cuestionario MINICHAL que valoran la dimensión Manifestaciones somáticas provocan un impacto muy negativo en la calidad de vida de los pacientes, y solo el ítem dificultad para conciliar el sueño en la dimensión Estado mental.

Expression of Toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva.

OBJECTIVE: Human corneal cells have detectable levels of TLRs 1-10. TLRs 2 and 4 are the major corneal receptors, recognizing the PAMPs associated with fungal invasion in humans. The conjunctiva and limbus contain TLRs 2, 4, and 9. Our purpose was to determine the expression of TLRs 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, conjunctiva, and limbus. METHODS: Corneal, limbal, and conjunctival tissues were collected from seven euthanized horses having no evidence of ocular disease. RNA extraction with DNase-1 digestion was performed followed by RT-PCR to determine expression of TLRs 2, 3, 4, 6, 9, and MD-2. Products were resolved by electrophoresis on 1.5% agarose gels and visualized using ethidium bromide staining. RESULTS: Expression of TLRs 2, 3, 4, 6, 9, and MD-2 was present in the cornea, limbus, and conjunctiva of each horse, except one horse, where TLR3 expression was unable to be demonstrated in the dorsal and ventral conjunctiva. CONCLUSIONS: Confirming the expression of TLRs in equine ocular tissues is an initial step in identifying how they play a role in infectious keratitis, particularly fungal. The results further support the use of equine ocular tissues as a model for human fungal keratitis. Studies of the TLR expression together with their cytokine profile induced during equine fungal keratitis may help further clarify the pathogenesis of the disease and possibly lead to the development of new treatment protocols for both equines and humans.

[Quality of life in men with arterial hypertension.] / Calidad de vida en el varón con hipertensión arterial.

OBJECTIVE: Systemic arterial hypertension is a Public Health problem, being the most important modifiable risk factor for morbidity and mortality, and the main single contributor to death and disability from all causes. Our objective was to estimate health-related quality of life using the MINICHAL questionnaire, to identify the problems with the greatest impact and the associated risk factors. METHODS: An observational prevalence study of a sample of 262 hypertensive men was carried out. Sociodemographic variables, comorbidity, degree of control and treatment, cardiovascular risk, clinical data and target organ lesions were collected. The following questionnaires were applied: MINICHAL, International Score of Prostate Symptoms and International Index of Erectile Function. Quantitative variables are expressed as mean (standard deviation) and median. The qualitative variables are expressed as absolute value and percentage with the estimation of its 95% confidence interval. The comparison of means was carried out by means of the Student's T test or the Mann Whitney test, as appropriate after checking the normality of the variables with the Kolmogorov-Smirnov test. The association between qualitative variables was estimated using the Chi square statistic or Fisher's exact test. To determine variables that are associated or not with the presence of worse quality of life, multivariate logistic regression was performed, adjusting for those variables that were significant in the univariate analysis or clinically relevant. RESULTS: Of the 262 hypertensive men included in the study, 4.6% declared that neither hypertension nor treatment affected their quality of life. Only 58% compared 52.7% presented a good quality of life through the domains "mental state" with respect to "somatic manifestations" of the MINICHAL questionnaire, with prostate symptoms being the one that had a considerable negative impact on both domains.The multivariate logistic regression analysis showed that the sum of the antihypertensive medication and the moderate-severe degree of prostatic symptoms had an independent effect to predict worse quality of life in the "mental state" domain, while the sum of concomitant medication, HDL-cholesterol levels, a pathological electrocardiogram and filling symptoms had an effect on the domain "somatic manifestations". CONCLUSIONS: When analyzing the "mental state" and the "somatic manifestations" of the MINICHAL questionnaire, only half of them presented a good quality of life.

OBJETIVO: La hipertensión arterial sistémica es un problema de Salud Pública, siendo el factor de riesgo modificable más importante para la morbimortalidad. Nuestro objetivo fue estimar la calidad de vida relacionada con la salud mediante el cuestionario MINICHAL, identificar los problemas con mayor impacto y los factores de riesgo asociados. METODOS: Se realizó un estudio observacional de prevalencia de una muestra de 262 hombres hipertensos. Se recogieron variables sociodemográficas, de comorbilidad, grado de control y tratamiento, riesgo cardiovascular, datos clínicos y lesiones de órgano diana. Se aplicaron los siguientes cuestionarios: MINICHAL, Puntuación Internacional de Síntomas prostáticos e Índice Internacional de Función eréctil. Las variables cuantitativas se expresan como media (desviación típica) y mediana. Las variables cualitativas se expresan como valor absoluto y porcentaje con la estimación de su 95% intervalo de confianza. La comparación de medias se realizó por medio de la T de Student o test de Mann Whitney, según procedía tras comprobar la normalidad de las variables con el test de Kolmogorov-Smirnov. La asociación entre variables cualitativas se estimó por medio del estadístico Chi cuadrado o test exacto de Fisher. Para determinar las variables que se asocian o no a la presencia de disfunción eréctil se realizó regresión logística multivariada, ajustando por aquellas variables que resultaron significativas en el análisis univariante o clínicamente relevantes. RESULTADOS: De 262 hombres hipertensos incluidos en el estudio, el 4,6% declaró que ni la hipertensión ni el tratamiento afectaban a su calidad de vida. Solo el 58% frente al 52,7% presentaron buena calidad de vida mediante los dominios "estado mental" respecto a "manifestaciones somáticas" del cuestionario MINICHAL, siendo la sintomatología prostática la que tuvo un impacto negativo considerable en ambos dominios. El análisis de la regresión logística multivariada mostró que la suma de la medicación antihipertensiva y el grado moderado-severo de sintomatología prostática tenían efecto independiente para predecir peor calidad de vida en el dominio "estado mental", mientras que la suma de la medicación concomitante, los niveles de HDL-colesterol, un electrocardiograma patológico y la sintomatología de llenado tenían efecto en el dominio "manifestaciones somáticas". CONCLUSIONES: Al analizar el "estado mental" y las "manifestaciones somáticas" del cuestionario MINICHAL, solo la mitad de ellos presentaron una buena calidad de vida.

Identification of anti-horn fly vaccine antigen candidates using a reverse vaccinology approach.

BACKGROUND: The horn fly, Haematobia irritans irritans, causes significant production losses to the cattle industry. Horn fly control relies on insecticides; however, alternative control methods such as vaccines are needed due to the fly's capacity to quickly develop resistance to insecticides, and the pressure for eco-friendly options. METHODS: We used a reverse vaccinology approach comprising three vaccine prediction and 11 annotation tools to evaluate and rank 79,542 translated open reading frames (ORFs) from the horn fly's transcriptome, and selected 10 transcript ORFs as vaccine candidates for expression in Pichia pastoris. The expression of the 10 selected transcripts and the proteins that they encoded were investigated in adult flies by reverse transcription polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Then, we evaluated the immunogenicity of a vaccine candidate in an immunization trial and the antigen's effects on horn fly mortality and fecundity in an in vitro feeding assay. RESULTS: Six of the ten vaccine candidate antigens were successfully expressed in P. pastoris. RT-PCR confirmed the expression of all six ORFs in adult fly RNA. One of the vaccine candidate antigens, BI-HS009, was expressed in sufficient quantity for immunogenicity and efficacy trials. The IgG titers of animals vaccinated with BI-HS009 plus adjuvant were significantly higher than those of animals vaccinated with buffer plus adjuvant only from days 42 to 112, with a peak on day 56. Progeny of horn flies feeding upon blood from animals vaccinated with BI-HS009 plus adjuvant collected on day 56 had 63% lower pupariation rate and 57% lower adult emergence than the control group (ANOVA: F (1, 6) = 8.221, P = 0.028 and F (1, 6) = 8.299, P = 0.028, respectively). CONCLUSIONS: The reverse vaccinology approach streamlined the discovery process by prioritizing possible vaccine antigen candidates. Through a thoughtful process of selection and in vivo and in vitro evaluations, we were able to identify a promising antigen for an anti-horn fly vaccine.

Differential induction of MyD88- and TRIF-dependent pathways in equine monocytes by Toll-like receptor agonists.

Our understanding of the innate immune response in the horse has been limited by a lack of definitive data concerning cell signaling in response to microbial products. Toll-like receptors (TLRs) recognize conserved molecular motifs of microbes and elicit immune responses through their coupling with intracellular adaptor molecules, particularly MyD88 and TRIF. To provide a more definitive characterization of TLR signaling in the horse, the objectives of this study were to: (1) characterize the responses of equine monocytes to TLR ligands that signal through MyD88, TRIF or both in other species, and (2) determine the profiles of gene expression initiated utilizing these adaptor molecules. Monocytes were used to establish concentration response curves for Escherichia coli lipopolysaccharide (LPS; TLR4 ligand) and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam(3)CSK(4); TLR2 ligand) based on expression of procoagulant activity (PCA) and production of tumor necrosis factor-alpha (TNF-alpha); effects of polyinosine-polycytidylic acid (Poly I:C; TLR3 ligand) were determined by quantifying expression of mRNA for interferon-beta (IFN-ss). Expression of genes associated with the MyD88- (TNF-alpha, IL-1ss, IL-6 and IL-10) and TRIF-dependent pathways (IFN-ss, IP-10, RANTES and TRAF1) were measured at intervals spanning 20 h. LPS and Pam(3)CSK(4) induced significantly higher expression of TNF-alpha, IL-1ss, and IL-10 than did Poly I:C. Poly I:C induced significantly higher expression of IFN-ss, IP-10 and RANTES than did either the TLR2 or TLR4 ligands. High concentrations of E. coli LPS did not significantly increase expression of genes associated with the TRIF-dependent pathway. The results of this study suggest that equine monocytes utilize a common intracellular pathway in response to TLR2 and TLR4 ligands, but a distinct pathway in response to TLR3 ligands.

Erectile dysfunction in patients with arterial hypertension. Cardiovascular risk and impact on their quality of life. / Disfunción eréctil en pacientes con hipertensión arterial. Riesgo cardiovascular e impacto en su calidad de vida.

OBJECTIVES: Estimate the frequency of erectile dysfunction in patients with essential hypertension and associated variables, degree of control, cardiovascular risk and the impact on quality of life. MATERIAL AND METHODS: Type of study: Observational study of prevalence in men with essential hypertension. MEASUREMENTS: Sociodemographic and comorbidity variables were collected from each patient (age, Charlson index, dyslipidaemia and prostatic hyperplasia), degree of control of essential hypertension and treatment, cardiovascular risk and metabolic syndrome. The erectile dysfunction was diagnosed by the International Index of Erectile Function (IIEF-15). Quality of life questionnaires were carried out in essential hypertension (MINICHAL), and the international scale of prostatic symptoms (IPSS). RESULTS: The study included 262 hypertensive men with an average age of 65.84years. Erectile dysfunction was presented in 46.1%, being severe in 54.9%. The bivariate analysis shows an independent association between erectile dysfunction and the variables: age, Charlon index, dyslipidaemia, benign prostatic hypertrophy, diastolic blood pressure, years of diagnosis of hypertension, number of treatments, Regicor and Framingham-Wilson, glycaemia, creatinine and GPT, glomerular filtration through the MDRD formula, irritative symptomatology (IPSS) and somatic manifestations (MINICHAL). The final multivariate model found association with age, presentation of dyslipidaemia, prostatic hyperplasia and metabolic syndrome. CONCLUSIONS: Erectile dysfunction is significantly associated with age, dyslipidaemia, benign prostatic hypertrophy and metabolic syndrome.

A comparison of equine and bovine sera as sources of lipopolysaccharide-binding protein activity in equine monocytes incubated with lipopolysaccharide.

Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using LPS-induced expression of procoagulant activity (PCA) by equine monocytes as a readout and (2) evaluate the use of commercial equine serum as a source of LBP activity using LPS concentration response and time course studies to validate the response. Monocytes were isolated from eight horses and incubated with five different serum preparations in the presence or absence of Escherichia coli LPS. The sera tested were heat-inactivated fetal bovine serum (HI-FBS), pooled commercial equine serum (CES), heat-inactivated pooled commercial equine serum (HI-CES), autologous equine serum (AES), and heat-inactivated autologous equine serum (HI-AES). In the absence of LPS, monocytes from half of the horses in the study had increased expression of PCA when incubated with HI-FBS alone; PCA was unaffected by incubation with the other sera. There was a four-fold increase in PCA when monocytes were incubated with LPS in the presence of CES, HI-CES or AES compared to LPS without serum. The combination of HI-FBS and LPS increased PCA 20-fold compared to LPS without serum. The HI-AES serum lacked significant LBP activity. Whereas maximal expression of PCA was induced by 1ng/ml of LPS in the absence of serum, inclusion of 1% CES reduced the LPS concentration required for maximal PCA to 30pg/ml. Monocytes incubated with LPS in the presence of CES had increased PCA at 3h and peaked at 6h. In conclusion, monocytes from many horses are directly stimulated by HI-FBS, suggesting that HI-FBS is not an optimal source of LBP for in vitro studies of LPS with equine monocytes. In contrast, CES and AES are effective sources of LBP activity for such studies, as they do not directly induce activation. Although the heat inactivation process did not affect the LBP activity in CES, it ablated LBP activity in AES. Consequently, investigators are advised to utilize either CES or AES in future studies, but not heat-inactivated AES.

Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes.

OBJECTIVE: To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. SAMPLE POPULATION: Venous blood samples obtained from 19 healthy horses. PROCEDURES: Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay. RESULTS: Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, and IL-10 and decreased LPS-induced expression of IL-6. CONCLUSIONS AND CLINICAL RELEVANCE: The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.

Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors.

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Analysis of the gene regulatory program induced by the homeobox transcription factor distal-less 3 in mouse placenta.

Dlx3, a homeodomain transcription factor, is essential for placental development in the mouse. The Dlx3(-/-) mouse embryo dies at embryonic d 9.5-10 putatively due to placental failure. To develop a more comprehensive understanding of the gene profile regulated by Dlx3, microarray analysis was used to determine differences in gene expression within the placenta of Dlx3(+/+) and Dlx3(-/-) mice. Array analysis revealed differential expression of 401 genes, 33 genes in which signal to log ratio values of null/wild-type were lower than -0.5 or higher than 0.5. To corroborate these findings, quantitative real-time PCR was used to confirm differential expression for 11 genes, nine of which displayed reduced expression and two with enhanced expression in the Dlx3(-/-) mouse. Loss of Dlx3 resulted in a marked reduction (>60%) in mRNA expression of placental growth factor (Pgf), a member of the vascular endothelial growth factor family. Consistent with these results, Pgf secretion from placental explants tended to be reduced in the Dlx3(-/-) mice, compared with wild type. To investigate mechanisms of Dlx3 regulation of Pgf gene transcription, we cloned 5.2 kb of the Pgf 5' flanking sequence for use in reporter gene assays. Expression of the Pgf promoter luciferase reporter containing at least three Dlx3 binding sites was increased markedly by overexpression of Dlx3 supporting the conclusion that Dlx3 may have a direct effect on Pgf promoter activity. These studies provide a novel view of the transcriptome regulated by Dlx3 in mouse placenta. Dlx3 is specifically required for full expression and secretion of Pgf in vivo. Moreover, in vitro studies support the conclusion that Dlx3 is sufficient to directly modulate expression of the Pgf gene promoter in placental cells.

A novel approach to a rabies vaccine based on a recombinant single-cycle flavivirus vector.

The RepliVax® vaccine (RV) platform is based on flavivirus genomes that are rationally attenuated by deletion. These single-cycle RV vaccine candidates targeting flavivirus pathogens have been demonstrated to be safe, highly immunogenic, and efficacious in animal models, including non-human primates. Here we show utility of the technology for delivery of a non-flavivirus immunogen by engineering several West Nile-based RV vectors to express full-length rabies virus G protein. The rabies virus G protein gene was incorporated in place of different West Nile structural protein gene deletions. The resulting RV-RabG constructs were demonstrated to replicate to high titers (8 log10 infectious particles/ml) in complementing helper cells. Following infection of normal cells, they provided efficient rabies virus G protein expression, but did not spread to surrounding cells. Expression of rabies virus G protein was stable and maintained through multiple rounds of in vitro passaging. A sensitive neurovirulence test in 2-3 day old neonatal mice demonstrated that RV-RabG candidates were completely avirulent indicative of high safety. We evaluated the RV-RabG variants in several animal models (mice, dogs, and pigs) and demonstrated that a single dose elicited high titers of rabies virus-neutralizing antibodies and protected animals from live rabies virus challenge (mice and dogs). Importantly, dogs were protected at both one and two years post-immunization, demonstrating durable protective immunity. The data demonstrates the potential of the RepliVax® technology as a potent vector delivery platform for developing vaccine candidates against non-flavivirus targets.

Prognostic value of plasma L-lactate concentration measured cow-side with a portable clinical analyzer in Holstein dairy cattle with abomasal disorders.

BACKGROUND: L-Lactate has been used as a prognostic indicator for ill humans and animals. A portable analyzer that measures L-lactate could help veterinarians decide to proceed with correction of a displaced abomasum. HYPOTHESES: The likelihood of a dairy cow with a displaced abomasum remaining in the herd can be predicted by lactate concentration and other variables. ANIMALS: Thirty-four healthy early-lactation dairy cows, and 131 cows with abomasal displacements (DA) presented to Cornell University, Ithaca, NY. METHODS: Plasma L-lactate was measured using a commercial analyzer (i-STAT). A cow had a positive outcome (PO) if she remained in the herd 30 days after surgical correction of the displaced abomasum and a negative outcome (NO) if she was culled or died in that time. A multivariable model with physical examination and clinicopathologic variables for predicting NO for cows with right-sided abomasal displacements was constructed. RESULTS: The median plasma L-lactate was 0.54 mM/L (interquartile range, 0.42-0.74) in healthy lactating Holstein cows. In cows with right-sided displaced abomasa, median plasma L-lactate concentrations were higher in cows with NO (5.88 mM/L) versus PO (3.23 mM/L) (P = .002). In a multivariable model, which identified chloride, heart rate, and L-lactate as the best fitting variables for cows with right-sided displacements, the probability of NO increased as L-lactate increased. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma L-lactate concentration might be a useful predictor of productive outcomes in cows with right-sided abomasal disorders.

Safety and serological response to a matrix gene-deleted rabies virus-based vaccine vector in dogs.

Dogs account for the majority of human exposures and deaths due to rabies virus (RABV) worldwide. In this report, we show that a replication-deficient RABV-based vaccine in which the matrix gene is deleted (RABV-ΔM) is safe and induces rapid and potent VNA titers after a single inoculation in dogs. Average VNA titers peaked at 3.02 or 5.11 international units (IU/ml) by 14 days post-immunization with a single dose of 10(6) or 10(7) focus forming units (ffu), respectively, of RABV-ΔM. By day 70 post immunization, all dogs immunized with either dose of vaccine showed VNA titers >0.5IU/ml, the level indicative of a satisfactory immunization. Importantly, no systemic or local reactions were noted in any dog immunized with RABV-ΔM. The elimination of dog rabies through mass vaccination is hindered by limited resources, requirement for repeat vaccinations often for the life of a dog, and in some parts of the world, inferior vaccine quality. Our preliminary safety and immunogenicity data in dogs suggest that RABV-ΔM might complement currently used inactivated RABV-based vaccines in vaccination campaigns by helping to obtain 100% response in vaccinated dogs, thereby increasing overall vaccination coverage.

Differential induction of Toll-like receptor gene expression in equine monocytes activated by Toll-like receptor ligands or TNF-α.

Toll-like receptors (TLRs) function as sentinels for the innate immune system, detecting microbial ligands during infection and inflammation. Previous studies indicate that activation of these receptors on equine monocytes leads to discrete pro- and anti-inflammatory responses that are mediated through the induction of specific cytokine genes. However, less is known regarding the regulation of TLR gene expression in these cells. Therefore, we investigated the effects of ligands recognized by TLR2, 3 or 4 upon TLR2, 3 and 4 gene expression by equine monocytes. We determined that incubation of monocytes with TLR2 and 4 ligands, which signal through the intracellular adaptor protein MyD88, induces expression of the TLR2 and 4 genes, but not the TLR3 gene. Conversely, incubation with a TLR3 ligand, which recruits the TRIF adaptor protein, selectively induces expression of the TLR3 gene, but not TLR2 or 4 genes. Furthermore, incubation of these cells with TNF-α, the pro-inflammatory cytokine that is a hallmark of TLR activation, does not affect expression of the three TLR genes. These findings suggest that exposure of equine monocytes to microbial ligands but not to endogenous inflammatory mediators may initiate responses that alter the horse's sensitivity to other microbial components during infections.