Evaluating differences in milk production, reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows.

The objective was to assess differences in productive and reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows located in Western and Southern states of the U.S.A. This retrospective cohort study included data from 3 experiments conducted in 9 dairies. Vaginal discharge was evaluated twice within 12 DIM and scored on a 5-point scale. The highest score observed for each cow was used for group assignment (VD group) as follows: VD 1 and 2 (VD 1/2; n = 1,174) = clear mucus/lochia with or without flecks of pus; VD 3 (n = 1,802) = mucopurulent with < 50% pus; VD 4 (n = 1,643) = mucopurulent with ≥50% of pus or non-fetid reddish/brownish mucous, n = 1,643; VD 5 = fetid, watery, and reddish/brownish, n = 1,800. All VD 5 cows received treatment according to each herd's protocol. Rectal temperature was assessed in a subset of VD 5 cows, and subsequently divided into Fever (rectal temperature ≥39.5°C; n = 334) and NoFever (n = 558) groups. A smaller proportion of cows with VD 5 (67.6%) resumed ovarian cyclicity compared with VD 1/2 (76.2%) and VD 4 (72.9%) cows; however, a similar proportion of VD5 and VD 3 (72.6%) cows resumed ovarian cyclicity. A smaller proportion of VD 5 (85.8%) cows received at least one artificial insemination (AI) compared with VD 1/2 (91.5%), VD 3 (91.0%), or VD 4 (91.6%) cows. Although we did not detect differences in pregnancy at first AI according to VD, fewer cows with VD 5 (64.4%) were pregnant at 300 DIM than cows with VD 1/2 (76.5%), VD 3 (76.2%), or VD 4 (74.7%). Hazard of pregnancy by 300 DIM was smaller for VD 5 compared with VD 1/2, VD 3, or VD 4 cows. A greater proportion of VD 5 cows were removed from the herd within 300 DIM compared with other VD groups. There was 760 kg lesser milk production within 300 DIM for VD 5 compared with VD 2, VD 3, and VD 4, whereas VD 2, VD 3, and VD 4 had similar milk production. We did not detect an association between fever at diagnosis of VD 5 and reproductive performance or milk production. A greater proportion of VD 5 cows without fever were removed from the herd by 300 DIM compared with VD 5 cows with fever. Differences in productive and reproductive performance, and removal of the herd were restricted to fetid, watery, and reddish/brownish vaginal discharge, which was independent of fever.

Molecular profiling in bee venom allergy: clinical and therapeutic characterization in a Portuguese cohort.

Summary: Background. Bee venom allergy (BVA) can trigger local and systemic allergic reactions, including anaphylaxis. Recently, the molecular sensitization profile has gained importance in the reaction's stratification and venom immunotherapy (VIT). Methods. Retrospective analysis of patients with hypersensitivity to BVA, confirmed by specific sIgE to Apis mellifera ≥0.35 kU/L and/or positive skin tests to bee venom commercial extract, evaluated in specialized consultation. Demographic, clinical, and laboratory data (including molecular Api m 1, 4, and 10) were analyzed, looking for risk factors associated with the severity of the index reaction and reactions during VIT. Results. 93 patients were included (55.9% male; median age of 46 years), 57.3% with atopic comorbidities, and 23.4% with cardiovascular comorbidities. The median specific IgE to Apis mellifera was 6.7 kU/L (IQR 1.0-20.3) kU/L. Regarding the molecular profile, the median IgE to Api m 1 was 0.5 kU/L (57.5% positive out of all measurements); Api m 4 - 0.01 kU/L (11.9% positive), and Api m 10 - 0.3 kU/L (50.0% positive). No patient was monosensitized to Api m 4. The median age of the most severe sting reaction was 36 (IQR 26-48) years, with a median severity (Müeller scale) of 3 (IQR 2-3). Forty-seven patients (50.5%) underwent VIT, with 35.6% of reactions recorded. Allergic reactions during VIT were recorded in 35.6% of cases. The severity of the index reaction correlated positively with older ages (p=0.040; r=0.249), in contrast to monosensitization to Api m 1, which was an independent predictor of milder reactions (p=0.015). Sensitization to Api m 10 was associated with a higher likelihood of reactions during VIT (p=0.038) but potentially less systemic reactions at re-stings (p=0.097). Conclusions. Molecular sensitization profile appears to be relevant not only to the severity of index reactions but also during VIT. Studies of a large cohort of patients with molecular profiles are essential to validate these results and improve the clinical and therapeutic approach to BVA.

Colchicine reduces the activation of NLRP3 inflammasome in COVID-19 patients.

OBJECTIVE: To evaluate whether colchicine treatment was associated with the inhibition of NLRP3 inflammasome activation in patients with COVID-19. METHODS: We present a post hoc analysis from a double-blinded placebo-controlled randomized clinical trial (RCT) on the effect of colchicine for the treatment of COVID-19. Serum levels of NOD-like receptor protein 3 (NLRP3) inflammasome products-active caspase-1 (Casp1p20), IL-1ß, and IL-18-were assessed at enrollment and after 48-72 h of treatment in patients receiving standard-of-care (SOC) plus placebo vs. those receiving SOC plus colchicine. The colchicine regimen was 0.5 mg tid for 5 days, followed by 0.5 mg bid for another 5 days. RESULTS: Thirty-six patients received SOC plus colchicine, and thirty-six received SOC plus placebo. Colchicine reduced the need for supplemental oxygen and the length of hospitalization. On Days 2-3, colchicine lowered the serum levels of Casp1p20 and IL-18, but not IL-1ß. CONCLUSION: Treatment with colchicine inhibited the activation of the NLRP3 inflammasome, an event triggering the 'cytokine storm' in COVID-19. TRIAL REGISTRATION NUMBERS: RBR-8jyhxh.

Key elements in hypersensitivity reactions to chemotherapy: experience with rapid drug desensitization in gynaecological cancer in a Tertiary Hospital.

Summary: Rapid drug desensitization (RDD) is a procedure performed when no alternative drug is considered equally effective. The aim of our study is to describe the experience with RDD to cytostatics in patients being treated for gynaecological cancer in a tertiary hospital, over a period of 5 years. In this paper, we review 22 cases and 107 episodes of RDD; 86.3% of patients had advanced disease and the mortality rate at the time of data collection was 50.0%. RDD was performed on 81.8% patients for platinum, 13.6% for taxanes, and 4.5% for anthracyclines. The reintroduction of antineoplastic drugs in all patients with a previous history of immediate hypersensitivity reaction demonstrated the safety and efficacy of this procedure. There was serious complication (anaphylaxis) in only one case.

The economic cost of metritis in dairy herds.

The objective of this study was to estimate the cost of metritis in dairy herds. Data from 11,733 dairy cows from 16 different farms located in 4 different regions of the United States were compiled for up to 305 d in milk, and 11,581 cows (2,907 with and 8,674 without metritis) were used for this study. Metritis was defined as fetid, watery, red-brownish vaginal discharge that occurs ≤21 d in milk. Continuous outcomes such as 305-d milk production, milk sales ($/cow), cow sales ($/cow), metritis treatment costs ($/cow), replacement costs ($/cow), reproduction costs ($/cow), feeding costs ($/cow), and gross profit per cow ($/cow) were analyzed using mixed effect models using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Gross profit was also compared using the Kruskal-Wallis test. Dichotomous outcomes such as pregnant and culling by 305 d in milk were analyzed using the GLIMMIX procedure of SAS. Time to pregnancy and culling were analyzed using the PHREG procedure of SAS. Models included the fixed effects of metritis, parity, and the interaction between metritis and parity, and farm as the random effect. Variables were considered significant when P ≤ 0.05. Metritis cost was calculated by subtracting the gross profit of cows with metritis from the gross profit of cows without metritis. A stochastic analysis was performed with 10,000 iterations using the observed results from each group. Milk yield and proportion of cows pregnant were lesser for cows with metritis than for cows without metritis, whereas the proportion of cows leaving the herd was greater for cows with metritis than for cows without metritis. Milk sales, feeding costs, residual cow value, and gross profit were lesser for cows with metritis than for cows without metritis. Cow sales and replacement costs were greater for cows with metritis than for cows without metritis. The mean cost of metritis from the study herds was $511 and the median was $398. The stochastic analysis showed that the mean cost of a case of metritis was $513, with 95% of the scenarios ranging from $240 to $884, and that milk price, treatment cost, replacement cost, and feed cost explained 59%, 19%, 12%, and 7%, respectively, of the total variation in cash flow differences. In conclusion, metritis caused large economic losses to dairy herds by decreasing milk production, reproduction, and survival in the herd.

Economic comparison between ceftiofur-treated and nontreated dairy cows with metritis.

The objective was to investigate the economic effect of treating dairy cows with metritis using ceftiofur-free acid or leaving them untreated at the time of diagnosis. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis (d 0). Data from 875 dairy cows (506 primiparous and 369 multiparous) from 1 herd in northern Florida that had been part of a larger study evaluating different treatments for metritis were used for the economic analysis. Holstein cows with metritis had been randomly assigned to: Ceftiofur (CEF, n = 239) = subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Untreated (UNT, n = 233) = no treatment applied at metritis diagnosis. Both groups could receive escape therapy if condition worsened. A group of nonmetritic healthy cows (NMET; n = 403) from the same cohort was randomly selected for comparison. Continuous outcomes such as 300-d milk production (kg/cow), milk sales ($/cow), cow sales ($/cow), treatment cost by 60 days in milk ($/cow), reproduction cost ($/cow), replacement cost ($/cow), feeding cost ($/cow), and gross profit per cow ($/cow) were analyzed using the ANOVA (MIXED procedure of SAS version 9.4). Dichotomous outcomes such as pregnancy and culling by 300 d were analyzed using logistic regression (GLIMMIX procedure of SAS). Models included the fixed effects of treatment, parity, and the interaction between treatment and parity. A stochastic analysis was performed with 10,000 iterations using the observed results from each group. The CEF treatment resulted in greater treatment cost by 60 DIM than UNT ($112 vs. $37), but resulted in a greater proportion of pregnant cows (71 vs. 61%) and decreased culling by 300 DIM (29 vs. 39%) compared with UNT. Gross profit was lesser for UNT than NMET ($2,969 vs. $3,426), and CEF was intermediate ($3,219). The stochastic analysis showed that the mean difference in gross profit between UNT and NMET was -$457; saleable milk (49%) and replacement cost (24%) accounted for most of the variation. The mean difference in gross profit between CEF and NMET group was -$207; saleable milk (82%) and initial metritis treatment cost (9%) accounted for most of the variation. The mean difference in gross profit between the UNT and the CEF group was -$250; replacement cost (41%) and cow sales (31%) accounted for most of the variation. In summary, metritis caused large economic losses when left untreated, and CEF reduced those losses by improving fertility, reducing culling and replacement cost, and reducing milk yield losses.

Using chitosan microparticles to treat metritis in lactating dairy cows.

The main objective of this study was to evaluate the efficacy of intrauterine administration of chitosan microparticles (CM) in curing metritis in dairy cows. A secondary objective was to evaluate the effects of metritis treatments on milk yield, survival, and reproductive performance. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis. Holstein cows (n = 826) with metritis from 3 dairies located in northern Florida were blocked by parity (primiparous or multiparous) and, within each block, randomly assigned to one of 3 treatments: CM (n = 276) = intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water at the time of metritis diagnosis (d 0), 2 (d 2), and 4 (d 4) d later; ceftiofur (CEF; n = 275) = subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Control (CON; n = 275) = no treatment applied at metritis diagnosis. All groups could receive escape therapy if condition worsened. Cure was considered when vaginal discharge became mucoid and not fetid. A group of nonmetritic (NMET; n = 2,436) cows was used for comparison. Data were analyzed by generalized linear mixed and Cox's proportional hazard models. Cows in CM and CON had lesser risk of metritis cure on d 12 than cows in CEF (58.6 ± 5.0 vs. 61.9 ± 4.9% vs. 77.9 ± 3.9, respectively). The proportion of cows culled within 60 days in milk (DIM) was greater for cows in CM than for cows in CEF and CON (21.5 ± 2.7 vs. 9.7 ± 1.9 vs. 11.3 ± 2.0%, respectively). Treatment did not affect rectal temperature or plasma nonesterified fatty acids, ß-hydroxybutyrate, and haptoglobin concentrations. Milk yield in the first 60 DIM differed for all treatments, and it was lowest for CM (35.8 ± 0.3 kg/d), followed by CON (36.8 ± 0.3 kg/d) and CEF (37.9 ± 0.3 kg/d). The hazard of pregnancy up to 300 DIM was lesser for CM than CEF (hazard ratio = 0.62; 95% CI: 0.50-0.76), for CM than CON (hazard ratio = 0.77; 95% CI: 0.62-0.95) and for CON than CEF (hazard ratio = 0.80; 95% CI: 0.65-0.99). Culling was greater, and milk yield and fertility were lesser for CEF than NMET. In summary, CM did not improve the cure of metritis, and was detrimental to milk yield, survival, and fertility compared with CON. In contrast, CEF increased the cure of metritis, milk yield, and fertility compared with CM and CON. Finally, the negative effects of metritis on milk yield culling and fertility could not be completely reversed by CEF.

Fructose 1,6-bisphosphate inhibits osteoclastogenesis by attenuating RANKL-induced NF-κB/NFATc-1.

BACKGROUND: Although some glycolytic intermediates have been shown to modulate several cell type formation and activation, the functional role of fructose 1,6-bisphosphate (FBP) on osteoclastogenesis is still unknown. METHODS: Osteoclastogenesis was evaluated on bone marrow preosteoclasts cultured with M-CSF - 30 ng/ml, RANKL - 10 ng/ml, and two concentrations of FBP (100 and 300 µM). TRAP-positive stained cells were counted, and osteoclastogenic marker genes expression were evaluated by qPCR. Osteoclasts resorption capacity was evaluated by the expression of specific enzymes and capacity to resorb a mineralized matrix. The NF-κB activation was detected using RAW 264.7, stably expressing luciferase on the NF-κB responsive promoter. RESULTS: We show that FBP, the product of the first stage of glycolysis, inhibited RANKL-induced osteoclasts differentiation and TRAP activity. The treatment of preosteoclasts with FBP attenuated osteoclast fusion and formation, without affecting cell viability. Moreover, the inhibition of several osteoclastogenic marker genes expression (TRAP, OSCAR, DC-STAMP, Integrin αv, NFATc1) by FBP correlates with a reduction of mineralized matrix resorption capacity. The mechanism underlying FBP-inhibition of osteoclastogenesis involves NF-κB/NFATc1 signaling pathway inhibition. CONCLUSION: Altogether these data show a protective role of a natural glycolytic intermediate in bone homeostasis that may have therapeutic benefit for osteolytic diseases.

The signal transducer and activator of transcription 6 (STAT-6) mediates Th2 inflammation and tissue damage in a murine model of peanut-induced food allergy.

INTRODUCTION: Food allergies are inflammatory conditions mediated by Th2 and probably STAT-6 dependent immune responses. OBJECTIVE AND DESIGN: Here we investigated the role of Signal Transducer and Activator of Transcription 6 (STAT-6) in development of inflammation in peanut allergy. METHODS: To induce food allergy, wild-type (WT) and mice deficient for STAT-6 (Stat6-/-) were sensitized with peanut proteins and challenged with peanut seeds. RESULTS: WT animals lost weight and refused the peanut diet, in contrast to Stat6-/- mice, which had a better maintenance of body weight and more regular seeds' consumption. The augmented peanut-specific IgG, IgG1 and IgE in the allergic WT was abolished in Stat6-/- animals that also presented increased IgG2a. There was an overall reduction in the gut mediators in the absence of STAT-6, including those related to inflammatory and Th2 responses, in contrast to a rising counter regulatory and Th1 reaction in Stat-6-/- mice. These animals had IFN-γ and IL-10 similar to WT after the four-week challenge. Most interestingly, Stat-6-/- mice had no intestinal damage, in contrast to WT animals, which had inflammatory infiltrate, tissue destruction, epithelial exulceration, edema, congestion and loss of villous architecture in the small gut segments. CONCLUSIONS: STAT-6 plays an important role in the establishment of the Th2 inflammatory responses and intestinal damage in peanut allergy.

Expanding the genetic spectrum of ANOS1 mutations in patients with congenital hypogonadotropic hypogonadism.

STUDY QUESTION: What is the prevalence and functional consequence of ANOS1 (KAL1) mutations in a group of men with congenital hypogonadotropic hypogonadism (CHH)? SUMMARY ANSWER: Three of forty-two (7.1%) patients presented ANOS1 mutations, including a novel splice site mutation leading to exon skipping and a novel contiguous gene deletion associated with ichthyosis. WHAT IS KNOWN ALREADY: CHH is characterized by lack of pubertal development and infertility, due to deficient production, secretion or action of GnRH, and can be associated with anosmia/hyposmia (Kallmann syndrome, KS) or with a normal sense of smell (normosmic CHH). Mutations in the anosmin-1 (ANOS1) gene are responsible for the X-linked recessive form of KS. STUDY DESIGN, SIZE, DURATION: This cross-sectional study included 42 unrelated men with CHH (20 with KS and 22 with normosmic CHH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were screened for mutations in the ANOS1 gene by DNA sequencing. Identified mutations were further investigated by RT-PCR analysis and multiplex ligation-dependent probe amplification (MLPA) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Hemizygous mutations were identified in three (7.1%) KS cases: a novel splice acceptor site mutation (c.542-1G>C), leading to skipping of exon 5 in the ANOS1 transcript in a patient with self-reported normosmia (but hyposmic upon testing); a recurrent nonsense mutation (c.571C>T, p.Arg191*); and a novel 4.8 Mb deletion involving ANOS1 and eight other genes (VCX3B, VCX2, PNPLA4, VCX, STS, HDHD1, VCX3A and NLGN4X) in KS associated with ichthyosis. LIMITATIONS, REASONS FOR CAUTION: Objective olfactory testing was not performed in all cases of self-reported normosmia and this may have underestimated the olfactory deficits. WIDER IMPLICATIONS OF THE FINDINGS: This study further expands the spectrum of known genetic defects associated with CHH and suggests that patients with self-reported normal olfactory function should not be excluded from ANOS1 genetic testing. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Portuguese Foundation for Science and Technology. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Physiological strain to prolonged exercise bouts at the walk-run transition speeds depends on locomotion mode in healthy untrained men.

This study compared the physiological strain induced by prolonged walking and running performed at the walk-run transition speed (WRTS) in healthy untrained men. Twenty volunteers (age: 28 ± 5.01 years; height: 174.0 ± 0.3 cm; body mass: 74.5 ± 0.6 kg) underwent the following: (a) ramp-incremental maximal cardiopulmonary exercise test (CPET); (b) specific protocol to detect the WRTS; and (c) two 30-min walking and running bouts at WRTS (mean ± SD: 6.9 ± 0.06 km/h). Expired gases were collected during exercise bouts via the metabolic cart. A significant effect of locomotion mode (F = 4.8, P < 0.001) was observed with running resulting in higher cardiorespiratory responses than walking at the WRTS (oxygen uptake: mean difference = 0.26 L/min; pulmonary ventilation: mean difference = 5.53 L/min; carbon dioxide output: mean difference = 0.32 L/min; heart rate: mean difference = 13 beats/min; total energy expenditure: mean difference = 59 kcal). The rating of perceived exertion was similar across locomotion modes (mean difference = 0.3; P = 0.490). In conclusion, running promoted greater cardiorespiratory responses than walking at the WRTS in untrained healthy men. These data might have practical impact on aerobic training performed at intensities corresponding to WRTS.

Multivariate statistics applied to the reaction of common bean plants to parasitism by Meloidogyne javanica.

The availability of common bean cultivars tolerant to Meloidogyne javanica is limited in Brazil. Thus, the present study aimed to evaluate the reactions of 33 common bean genotypes (23 landrace, 8 commercial, 1 susceptible standard and 1 resistant standard) to M. javanica, employing multivariate statistics to discriminate the reaction of the genotypes. The experiment was conducted in a greenhouse using a completely randomized design with seven replicates. The seeds were sown in 1-L pots containing autoclaved soil and sand in a 1:1 ratio (v:v). On day 19, after emergence of the seedlings, the plants were treated with inoculum containing 4000 eggs + second-stage juveniles (J2). At 60 days after inoculation, the seedlings were evaluated based on biometric and parasitism-related traits, such as number of galls, final nematode population per root system, reproduction factor, and percent reduction in the reproduction factor of the nematode (%RRF). The data were subjected to analysis of variance using the F-test. The Mahalanobis generalized distance was used to obtain the dissimilarity matrix, and the average linkage between groups was used for clustering. The use of multivariate statistics allowed groups to be separated according to the resistance levels of genotypes, as observed in the %RRF. The landrace genotypes FORT-09, FORT-17, FORT-31, FORT-32, FORT-34 and FORT-36 presented resistance to M. javanica; thus, these genotypes can be considered potential sources of resistance.

Expression and activity of NOD1 and NOD2/RIPK2 signalling in mononuclear cells from patients with rheumatoid arthritis.

OBJECTIVES: The aim of this study was to analyse the expression and function of nucleotide-binding oligomerization domain (NOD)1 and NOD2 in isolated cells of patients with rheumatoid arthritis (RA). METHOD: mRNA expression levels of NOD1, NOD2, and receptor-interacting serine/threonine kinase 2 (RIPK2) genes were determined by quantitative polymerase chain reaction (qPCR) in peripheral blood mononuclear cells (PBMCs) and synovial fluid T cells (SFTCs) isolated from RA and osteoarthritis (OA) patients. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in plasma and cell culture supernatants. The stimulatory effect of RA SF was assessed by an in-vitro NOD2 activation assay using nuclear factor kappa B (NF-κB) luciferase-transfected cells. RESULTS: A significantly higher level of NOD2 and RIPK2 mRNA expression, but not NOD1, was observed on PBMCs and SFTCs isolated from RA patients compared to the OA control group. In addition, the NOD2 pathway up-regulation was functional, as stimulation of PBMCs with muramyl dipeptide (MDP) induced the production of higher amounts of tumour necrosis factor (TNF)-α, interleukin (IL)-8, and IL-1ß compared with OA PBMCs. Incubation of PBMCs from healthy donors with recombinant TNF-α or RA serum induced the expression of NOD2 mRNA. Finally, SF isolated from RA patients is able to activate the NF-κB signalling pathway in HEK293T-transfected cells in a NOD2-dependent manner. CONCLUSIONS: Our findings suggest that NOD2/RIPK2 signalling is up-regulated in immune cells of RA patients. Moreover, it seems that there is a NOD2 agonist in the SF of RA patients. Therefore, NOD2/RIPK2 activation can modulate the innate immune response and may play a role in the perpetuation of the inflammatory response in RA.

Clinical response after chitosan microparticle administration and preliminary assessment of efficacy in preventing metritis in lactating dairy cows.

The objectives were to evaluate the clinical response to intrauterine administration of chitosan microparticles (CM) and to assess efficacy for preventing metritis in dairy cows. Holstein cows (n=104; 40 primiparous and 64 multiparous) at increased risk for metritis (cows that had abortion, dystocia, twins, stillbirth, or retained placenta) were randomly assigned to 1 of 2 treatments at 1d in milk (DIM; 24h postpartum): CM group (n=52), daily intrauterine infusion of 8g of CM dissolved in 40mL of sterile water for 5d; control (CON) group (n=52), daily intrauterine infusion of 40mL of sterile water for 5d. Clinical response was assessed by evaluation of parameters associated with inflammation (rectal temperature and plasma haptoglobin concentration) and metabolism [plasma nonesterified fatty acid (NEFA) and ß-hydroxybutyrate (BHB) concentrations] up to 14 DIM, and daily milk yield up to 30 DIM. Uterine discharge pH was evaluated at 4, 7, 10, and 14 DIM as an indicator of bacterial load and acid byproduct production. The cumulative incidence of metritis was evaluated up to 4, 7, 10, and 14 DIM. Continuous and dichotomous outcomes were evaluated with mixed linear and logistic regression analysis, respectively. Treatment with CM did not affect rectal temperature (39.17±0.04 vs. 39.14±0.04°C), haptoglobin (1.10±0.05 vs. 1.07±0.05mg/mL), NEFA (0.64±0.04 vs. 0.63±0.04mmol/L), BHB (0.61±0.03 vs. 0.57±0.03mmol/L), or milk yield (30.3±0.92 vs. 30.1±0.97kg/d) compared with CON. An interaction between treatment and time showed that NEFA concentrations were lower for CM than CON at 10 DIM (0.46±0.06 vs. 0.64±0.06mmol/L). Treatment with CM resulted in greater uterine discharge pH than CON (6.91±0.03 vs. 6.83±0.02). Cows that developed metritis had increased concentrations of haptoglobin and BHB, and decreased uterine discharge pH and milk yield. Treatment with CM resulted in decreased incidence of metritis up to 7 DIM compared with CON (46.2 vs. 65.4%); however, no differences were found at 4 (11.5 vs. 17.3%), 10 (61.5 vs. 73.1%), and 14 DIM (63.5 vs. 73.1%) for CM versus CON, respectively. In conclusion, CM did not alter clinical parameters of cows at risk for metritis, and may merit further investigation for prevention of metritis. However, the duration of treatment may have to be extended to effectively reduce the incidence of metritis during the high-risk period.

Acute Hypotensive Response to Continuous and Accumulated Isocaloric Aerobic Bouts.

Evidence indicates that chronic reductions in blood pressure (BP) due to aerobic exercise depend on the ability to induce post-exercise hypotension (PEH) after each training bout. The purpose of this study was to investigate PEH after isocaloric bouts of continuous and accumulated running. 10 healthy pre-hypertensive men (aged 27.6±3.5 years) performed the following bouts of exercise: a) A continuous bout (CONT) expending a total of 400 kcal; and b) An accumulated bout split into 2×200 kcal (INTER1 and INTER2) to total 400 kcal at 75% of oxygen uptake reserve. BP, mean arterial pressure (MAP) and heart rate variability were monitored 10 min before and 60 min after control and all exercise conditions. The decrease in MAP over time after continuous (400 kcal) and accumulated (2×200 kcal) bouts of exercise was more pronounced than during control (mean diff between 1.6 and 5.4 mmHg, P≤0.01), although the magnitude of change was similar between continuous and accumulated bouts (mean diff=0.1 mmHg, P=0.79). Concomitant to the PEH, sympathovagal balance was inversely related to changes in MAP after isocaloric bouts performed continuously and cumulatively (r=- 0.72 and-0.85, P=0.019 and 0.002, respectively). In conclusion, BP decreased to similar levels after continuous and accumulated acute aerobic exercise matched for total energy expenditure. Our findings also indicate that the recovery pattern of cardiac autonomic activity may have an important role in eliciting PEH.

Umbilical cord blood CD34(+) stem cells and other mononuclear cell subtypes processed up to 96 h from collection and stored at room temperature maintain a satisfactory functionality for cell therapy.

BACKGROUND AND OBJECTIVES: Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96 h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation. MATERIALS AND METHODS: Twenty UCB units were analyzed at 24 and 96 h after collection, frozen for 6 months, thawed and re-evaluated. MNCs were analyzed by flow cytometry, viability by 7-AAD and clonogenic assays (CFU) were performed. RESULTS: After 96 h of storage, no substantial loss of MNC was found (median 7.320 × 10(6 ) × 6.05 × 10(6) ). Percentage and viability CD34(+) cells, B-cell precursors and mesenchymal stem cells were not affected. However, mature B and T lymphocytes as well as granulocytes had a substantial loss. CFU growth was observed in all samples. Prefreezing storage of 96 h was associated with a relative loss of colony formation (median 12%). Postthaw, this loss had a median of 49% (24 h samples) to 56% (96 h samples). CONCLUSION: The delay of 96 h before UCB processing is possible, without a prohibitive impairment of CD34(+) loss in number and functionality.

Influence of working length and apical preparation size on apical bacterial extrusion during reciprocating instrumentation.

AIM: To evaluate the influence of reciprocating single-file instrumentation with different working lengths (WL) and apical preparation sizes on apical bacterial extrusion. METHODOLOGY: Sixty-eight human single-rooted pre-molars were used. Conventional access cavities were prepared, and the root canals were contaminated with an Enterococcus faecalis suspension and incubated at 37°C for 30 days. Teeth were then divided into four groups of 15 specimens each (Reciproc size 25, .08 taper and Reciproc size 40, .06 taper instruments were used at the foramen; Reciproc size 25, .08 taper and Reciproc size 40, .06 taper instruments were used 1 mm short of the foramen). Positive and negative controls consisted of four infected and four uninfected pre-molars that were instrumented according to each experimental group. Bacteria extruded from the apical foramen during instrumentation were collected into vials containing 0.9% NaCl. The microbiological samples were then incubated in a brain-heart agar medium for 24 h. The resulting bacterial titre, in colony-forming units (CFU) per mL, was determined, and these data were analysed using a Wilcoxon matched-pairs signed rank test and a Kruskal-Wallis H-test. The level of significance was set at α = 0.05. RESULTS: No growth was observed in the negative control group. All positive controls demonstrated bacterial growth after the experimental time interval. No significant difference was found in the number of CFU amongst all experimental groups (P = 0.95). CONCLUSIONS: This study showed that the WL and the apical preparation size did not have a significant effect on bacterial extrusion when performing reciprocating instrumentation.

Diagnosis of prolactinoma in two male-to-female transsexual subjects following high-dose cross-sex hormone therapy.

Male-to-female transsexual persons use oestrogens + antiandrogens to adapt their physical bodies to the female sex. Doses are usually somewhat higher than those used by hypogonadal women receiving oestrogen replacement. Particularly in cases of self-administration of cross-sex hormones, doses may be very high. Oestrogens are powerful stimulators of synthesis and release of prolactin and serum prolactin levels are usually somewhat increased following oestrogen treatment. Prolactinomas have been reported in male-to-female transsexual persons, both after use of high and conventional doses of oestrogens but remain rare events. We report two new cases of prolactinomas in male-to-female transsexual persons, one in a 41-year-old subject who had used nonsupervised high-dose oestrogen treatment since the age of 23 years and another one in a 42 year old who had initiated oestrogen treatment at the age of 17 years. Their serum prolactin levels were strongly increased, and the diagnosis of a pituitary tumour was confirmed by imaging techniques. Both cases responded well to treatment with cabergoline treatment whereupon serum prolactin normalised. Our two cases are added to the three cases of prolactinomas in the literature in persons who had used supraphysiological doses of oestrogens.

Can Heart Rate Variability be used to Estimate Gas Exchange Threshold in Obese Adolescents?

This study investigated the agreement and reliability of oxygen uptake (V̇O2), V̇O2 reserve (V̇O2 R), heart rate (HR) and power output at intensities corresponding to the gas exchange threshold (GET) and heart rate variability threshold (HRVT) during maximal cardiopulmonary exercise testing (CPET) in obese and eutrophic adolescents. A further aim was to establish whether the HRVT was able to detect changes in cardio-respiratory fitness in obese adolescents after 3 months of recreational soccer practice. First, 25 obese and 10 eutrophic adolescents (ages 12-17) visited the laboratory twice to perform cycling CPET to test the reliability of CPET outcomes at GET and HRVT. Furthermore, the level of agreement between GET and HRVT was determined for a subgroup of 10 obese adolescents after performing a 3-month recreational soccer program. No significant difference was found for V̇O2, %V̇O2 R, HR and power output at the GET and HRVT (P>0.05), which were equally able to detect improvements in aerobic fitness after the soccer intervention. Correlations between GET and HRVT for V̇O2 and %V̇O2 R ranged from 0.89 to 0.95 (P<0.001) and test-retest reliability ranged from 0.59 to 0.82 (P<0.006). Overall, HRVT seems to be a reliable alternative for prescribing aerobic exercise intensity in obese adolescents.

Utility of a Non-Exercise VO2max Prediction Model for Designing Ramp Test Protocols.

This study investigated the validity of determining the final work rates of cycling and walking ramp-incremented maximal cardiopulmonary exercise tests (CPETs) using a non-exercise model to predict maximal oxygen uptake VO2max and the American College of Sports Medicine ACSM's metabolic equations. The validity of using this methodology to elicit the recommended test duration of between 8 and 12 min was then evaluated. First, 83 subjects visited the laboratory once to perform a cycling (n=49) or walking (n=34) CPET to investigate the validity of the methodology. Second, 25 subjects (cycling group: n=13; walking group: n=12) performed a CPET on 2 separate days to test the reliability of CPET outcomes. Observed VO2max was 1.0 ml·kg(-1)·min(-1) lower than predicted in the cycling CPET (P=0.001) and 1.4 ml·kg(-1)·min(-1) lower in the walking CPET (P=0.001). Only one of the 133 conducted CPETs was outside the test duration range of 8-12 min. Test-retest reliability was high for all CPET outcomes, with intraclass correlation coefficients of 0.90 to 0.99. In conclusion, the non-exercise model is a valid and reliable method for establishing the final work rate of cycling and walking CPETs for eliciting test durations of between 8 and 12 min.