Multiresidue Determination of Growth Promoters in Bovine Serum using QuEChERS and UHPLC-Q-ORBITRAP: Validation and In Vivo Study.

A reliable method using a QuEChERS approach and liquid chromatography coupled to Q-Orbitrap mass spectrometry was optimized and validated for the quantification of 20 growth promoters in bovine serum. The recoveries ranged from 91.4-114.1%, relative standard deviations varied between 0.3-4.0%, and CCα values were between 0.023-0.350 µg L-1. The developed method was applied in an in vivo study using steers, which were intramuscularly treated with commercial injections containing stanozolol. A rapid metabolization was observed, with a detection window ranging from 3 to 10 days. The stability of incurred stanozolol was confirmed after 240 days at -20 °C and also after 5 freeze-thaw cycles. To the best of our knowledge, this is the first work in which an in vivo study was performed to support the monitoring of stanozolol in bovine serum. In addition, the use of Q-Orbitrap high-resolution mass spectrometry allows for retrospective analysis from a surveillance perspective.

Glycans as shapers of tumour microenvironment: A sweet driver of T-cell-mediated anti-tumour immune response.

Essentially all cells are covered with a dense coat of different glycan structures/sugar chains, giving rise to the so-called glycocalyx. Changes in cellular glycosylation are a hallmark of cancer, affecting most of the pathophysiological processes associated with malignant transformation, including tumour immune responses. Glycans are chief macromolecules that define T-cell development, differentiation, fate, activation and signalling. Thus, the diversity of glycans expressed at the surface of T cells constitutes a fundamental molecular interface with the microenvironment by regulating the bilateral interactions between T-cells and cancer cells, fine-tuning the anti-tumour immune response. In this review, we will introduce the power of glycans as orchestrators of T-cell-mediated immune response in physiological conditions and in cancer. We discuss how glycans modulate the glyco-metabolic landscape in the tumour microenvironment, and whether glycans can synergize with immunotherapy as a way of rewiring T-cell effector functions against cancer cells.

Genital Mycoplasmas and Biomarkers of Inflammation and Their Association With Spontaneous Preterm Birth and Preterm Prelabor Rupture of Membranes: A Systematic Review and Meta-Analysis.

Genital mycoplasmas (GM), such as Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum are commonly associated with spontaneous preterm labor (SPTL), spontaneous preterm birth (PTB), and preterm prelabor rupture of membranes (PPROM). This study determined the association between GM and such adverse pregnancy outcomes. We searched for studies published 1980-2019 in MEDLINE, EMBASE, and Web of Science. Studies were eligible when GM was detected during pregnancy. We included 93 and 51 studies in determining the prevalence and the inflammatory biomarkers associated with GM, respectively, using the "metafor" package within R. The protocol was registered with PROSPERO (registration no. CRD42016047297). Women with the studied adverse pregnancy outcomes had significantly higher odds of presence with GM compared to women who delivered at term. For PTB, the odds ratios were: M. hominis (OR: 2.25; CI: 1.35-3.75; I 2: 44%), M. genitalium (OR: 2.04; CIL 1.18-3.53; I 2: 20%), U. parvum (OR: 1.75; CI: 1.47-2.07; I 2: 0%), U. urealyticum (OR: 1.50; CI: 1.08-2.07; I 2: 58%). SPTL had significantly higher odds with M. hominis (OR: 1.96; CI: 1.19-3.23; I 2: 1%) or U. urealyticum (OR: 2.37; CI: 1.20-4.70; I 2: 76%) compared to women without SPTL. Women with PPROM had significantly higher odds with M. hominis (OR: 2.09; CI: 1.42-3.08; I 2: 0%) than women without PPROM. However, our subgroup analysis based on the diagnostic test and the sample used for detecting GM showed a higher prevalence of GM in maternal samples than in fetal samples. GM presence of the cervix and vagina was associated with lower odds of PTB and preterm labor (PTL). In contrast, GM presence in the AF, fetal membrane, and placenta was associated with increased odds of PTB and PTL. However, genital mycoplasmas may not elicit the massive inflammation required to trigger PTB. In conclusion, GM presence in the fetal tissues was associated with significantly increased odds of PTB and PTL.

In Vivo Administration of Stanozolol in Cattle: Depletion and Stability Studies Using UHPLC-Q-Orbitrap.

An in vivo study was performed in order to evaluate the depletion time of stanozolol and its main metabolites using naturally incurred urine sample collected after the administration of intramuscular injections in 12 steers. A stability study was also carried out to investigate the influence of the storage period and the freeze-thaw cycles. A fast parent drug metabolization was observed, because within 6 h after drug administration, the signal of the metabolite 16ß-hydroxystanozolol was predominant. After the second drug administration, a detection window of 17 days was obtained. The stability was studied using ANOVA, in which a storage condition of -20 °C proved stable during 240 days, which was also confirmed after 5 freeze-thaw cycles.

The Role of the Iron Protoporphyrins Heme and Hematin in the Antimalarial Activity of Endoperoxide Drugs.

Plasmodium has evolved to regulate the levels and oxidative states of iron protoporphyrin IX (Fe-PPIX). Antimalarial endoperoxides such as 1,2,4-trioxane artemisinin and 1,2,4-trioxolane arterolane undergo a bioreductive activation step mediated by heme (FeII-PPIX) but not by hematin (FeIII-PPIX), leading to the generation of a radical species. This can alkylate proteins vital for parasite survival and alkylate heme into hematin-drug adducts. Heme alkylation is abundant and accompanied by interconversion from the ferrous to the ferric state, which may induce an imbalance in the iron redox homeostasis. In addition to this, hematin-artemisinin adducts antagonize the spontaneous biomineralization of hematin into hemozoin crystals, differing strikingly from artemisinins, which do not directly suppress hematin biomineralization. These hematin-drug adducts, despite being devoid of the peroxide bond required for radical-induced alkylation, are powerful antiplasmodial agents. This review addresses our current understanding of Fe-PPIX as a bioreductive activator and molecular target. A compelling pharmacological model is that by alkylating heme, endoperoxide drugs can cause an imbalance in the iron homeostasis and that the hematin-drug adducts formed have strong cytocidal effects by possibly reproducing some of the toxifying effects of free Fe-PPIX. The antiplasmodial phenotype and the mode of action of hematin-drug adducts open new possibilities for reconciliating the mechanism of endoperoxide drugs and for malaria intervention.

A Quantitative and Confirmatory Method Employing Liquid Chromatography Coupled to Hybrid High-Resolution Mass Spectrometry and QuEChERS for the Determination of Thirty-Seven Growth Promoter Residues in Bovine Urine.

In this work, 37 growth promoters were quantitatively determined in bovine urine using a QuEChERS approach with acetonitrile, NaCl, and MgSO4:PSA for sample extraction. The analytes were separated and detected by liquid chromatography coupled to hybrid high-resolution mass spectrometry. The method was validated in accordance with the Decision 657/2002/EC guidelines, in which recoveries fell within the range 84-113%, relative standard varied between 2 and 32%, and detection limit between 0.1 and 2.5 µg L-1. An adequate performance was evidenced during a proficiency test evaluation, and the developed method has been applied to routine analysis of growth promoters in Brazil. A highlight is the easiness of sample extraction combined with a quantitative determination of forbidden drugs using high-resolution mass spectrometry, which enables retrospective analysis in a surveillance perspective.

Ovulation suppression protects against chromosomal abnormalities in mouse eggs at advanced maternal age.

The frequency of egg aneuploidy and trisomic pregnancies increases with maternal age. To what extent individual approaches can delay the "maternal age effect" is unclear because multiple causes contribute to chromosomal abnormalities in mammalian eggs. We propose that ovulation frequency determines the physiological aging of oocytes, a key aspect of which is the ability to accurately segregate chromosomes and produce euploid eggs. To test this hypothesis, ovulations were reduced using successive pregnancies, hormonal contraception, and a pre-pubertal knockout mouse model, and the effects on chromosome segregation and egg ploidy were examined. We show that each intervention reduces chromosomal abnormalities in eggs of aged mice, suggesting that ovulation reduction delays oocyte aging. The protective effect can be partly explained by retention of chromosomal Rec8-cohesin that maintains sister chromatid cohesion in meiosis. In addition, single-nucleus Hi-C (snHi-C) revealed deterioration in the 3D chromatin structure including an increase in extruded loop sizes in long-lived oocytes. Artificial cleavage of Rec8 is sufficient to increase extruded loop sizes, suggesting that cohesin complexes maintaining cohesion restrict loop extrusion. These findings suggest that ovulation suppression protects against Rec8 loss, thereby maintaining both sister chromatid cohesion and 3D chromatin structure and promoting production of euploid eggs. We conclude that the maternal age effect can be delayed in mice. An implication of this work is that long-term ovulation-suppressing conditions can potentially reduce the risk of aneuploid pregnancies at advanced maternal age.

Can Serum be a Match for Urine in the Regulatory Analysis of Boldenone in Cattle? A Systematic Comparison Between Detection Window, Stability, and Enzymatic Hydrolysis.

This work involved a systematic comparison between serum and urine for the monitoring of anabolic androgenic steroids in livestock. Incurred samples were collected over 120 days from crossbred steers treated with intramuscular injections containing boldenone undecylenate. Independent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were used for the assessment of the respective detection windows, which were larger for serum samples. Both matrices presented adequate performance in terms of long-term stability, assessed using an isochronous approach during 196 days at -20 °C and for five freeze-thaw cycles. The effectiveness of the enzymatic hydrolysis reaction using Helix pomatia juice was also compared. The calculated concentrations in serum samples were not statistically influenced by the deconjugation reaction. On the other hand, urine hydrolysis conditions were studied using a 33 Box-Behnken Design, in which a central point condition led to a satisfactory deconjugation performance. It could be observed that serum exhibited equivalent or better performance than urine for most of the evaluated criteria; thus, its inclusion in the regulatory analysis of boldenone in cattle is supported.

Impact of positive biphasic pressure during low and high inspiratory efforts in Pseudomonas aeruginosa-induced pneumonia.

BACKGROUND: During pneumonia, normal alveolar areas coexist adjacently with consolidated areas, and high inspiratory efforts may predispose to lung damage. To date, no study has evaluated different degrees of effort during Biphasic positive airway pressure (BIVENT) on lung and diaphragm damage in experimental pneumonia, though largely used in clinical setting. We aimed to evaluate lung damage, genes associated with ventilator-induced lung injury (VILI) and diaphragmatic injury, and blood bacteria in pressure-support ventilation (PSV), BIVENT with low and high inspiratory efforts in experimental pneumonia. MATERIAL AND METHODS: Twenty-eight male Wistar rats (mean ± SD weight, 333±78g) were submitted Pseudomonas aeruginosa-induced pneumonia. After 24-h, animals were ventilated for 1h in: 1) PSV; 2) BIVENT with low (BIVENTLow-Effort); and 3) BIVENT with high inspiratory effort (BIVENTHigh-Effort). BIVENT was set at Phigh to achieve VT = 6 ml/kg and Plow at 5 cmH2O (n = 7/group). High- and low-effort conditions were obtained through anaesthetic infusion modulation based on neuromuscular drive (P0.1). Lung mechanics, histological damage score, blood bacteria, and expression of genes related to VILI in lung tissue, and inflammation in diaphragm tissue. RESULTS: Transpulmonary peak pressure and histological damage score were higher in BIVENTHigh-Effort compared to BIVENTLow-Effort and PSV [16.1 ± 1.9cmH2O vs 12.8 ± 1.5cmH2O and 12.5 ± 1.6cmH2O, p = 0.015, and p = 0.010; median (interquartile range) 11 (9-13) vs 7 (6-9) and 7 (6-9), p = 0.021, and p = 0.029, respectively]. BIVENTHigh-Effort increased interleukin-6 expression compared to BIVENTLow-Effort (p = 0.035) as well as expressions of cytokine-induced neutrophil chemoattractant-1, amphiregulin, and type III procollagen compared to PSV (p = 0.001, p = 0.001, p = 0.004, respectively). Tumour necrosis factor-α expression in diaphragm tissue and blood bacteria were higher in BIVENTHigh-Effort than BIVENTLow-Effort (p = 0.002, p = 0.009, respectively). CONCLUSION: BIVENT requires careful control of inspiratory effort to avoid lung and diaphragm damage, as well as blood bacteria. P0.1 might be considered a helpful parameter to optimize inspiratory effort.

Glycans as Immune Checkpoints: Removal of Branched N-glycans Enhances Immune Recognition Preventing Cancer Progression.

Tumor growth is accompanied with dramatic changes in the cellular glycome, such as the aberrant expression of complex branched N-glycans. However, the role of this protumoral N-glycan in immune evasion and whether its removal contributes to enhancement of immune recognition and to unleashing an antitumor immune response remain elusive. We demonstrated that branched N-glycans are used by colorectal cancer cells to escape immune recognition, instructing the creation of immunosuppressive networks through inhibition of IFNγ. The removal of this "glycan-mask" exposed immunogenic mannose glycans that potentiated immune recognition by DC-SIGN-expressing immune cells, resulting in an effective antitumor immune response. We revealed a glycoimmune checkpoint in colorectal cancer, highlighting the therapeutic efficacy of its deglycosylation to potentiate immune recognition and, thus, improving cancer immunotherapy.

Magnetic targeting increases mesenchymal stromal cell retention in lungs and enhances beneficial effects on pulmonary damage in experimental silicosis.

Silicosis is a pneumoconiosis caused by inhaled crystalline silica microparticles, which trigger inflammatory responses and granuloma formation in pulmonary parenchyma, thus affecting lung function. Although systemic administration of mesenchymal stromal cells (MSCs) ameliorates lung inflammation and attenuates fibrosis in experimental silicosis, it does not reverse collagen deposition and granuloma formation. In an attempt to improve the beneficial effects of MSCs, magnetic targeting (MT) has arisen as a potential means of prolonging MSC retention in the lungs. In this study, MSCs were incubated with magnetic nanoparticles and magnets were used for in vitro guidance of these magnetized MSCs and to enhance their retention in the lungs in vivo. In vitro assays indicated that MT improved MSC transmigration and expression of chemokine receptors. In vivo, animals implanted with magnets for 48 hours had significantly more magnetized MSCs in the lungs, suggesting improved MSC retention. Seven days after magnet removal, silicotic animals treated with magnetized MSCs and magnets showed significant reductions in static lung elastance, resistive pressure, and granuloma area. In conclusion, MT is a viable technique to prolong MSC retention in the lungs, enhancing their beneficial effects on experimentally induced silicosis. MT may be a promising strategy for enhancing MSC therapies for chronic lung diseases.

Wapl releases Scc1-cohesin and regulates chromosome structure and segregation in mouse oocytes.

Cohesin is essential for genome folding and inheritance. In somatic cells, these functions are both mediated by Scc1-cohesin, which in mitosis is released from chromosomes by Wapl and separase. In mammalian oocytes, cohesion is mediated by Rec8-cohesin. Scc1 is expressed but neither required nor sufficient for cohesion, and its function remains unknown. Likewise, it is unknown whether Wapl regulates one or both cohesin complexes and chromosome segregation in mature oocytes. Here, we show that Wapl is required for accurate meiosis I chromosome segregation, predominantly releases Scc1-cohesin from chromosomes, and promotes production of euploid eggs. Using single-nucleus Hi-C, we found that Scc1 is essential for chromosome organization in oocytes. Increasing Scc1 residence time on chromosomes by Wapl depletion leads to vermicelli formation and intra-loop structures but, unlike in somatic cells, does not increase loop size. We conclude that distinct cohesin complexes generate loops and cohesion in oocytes and propose that the same principle applies to all cell types and species.

Artemisinin-(Iso)quinoline Hybrids by C-H Activation and Click Chemistry: Combating Multidrug-Resistant Malaria.

A substantial challenge worldwide is emergent drug resistance in malaria parasites against approved drugs, such as chloroquine (CQ). To address these unsolved CQ resistance issues, only rare examples of artemisinin (ART)-based hybrids have been reported. Moreover, protein targets of such hybrids have not been identified yet, and the reason for the superior efficacy of these hybrids is still not known. Herein, we report the synthesis of novel ART-isoquinoline and ART-quinoline hybrids showing highly improved potencies against CQ-resistant and multidrug-resistant P. falciparum strains (EC50 (Dd2) down to 1.0 nm; EC50 (K1) down to 0.78 nm) compared to CQ (EC50 (Dd2)=165.3 nm; EC50 (K1)=302.8 nm) and strongly suppressing parasitemia in experimental malaria. These new compounds are easily accessible by step-economic C-H activation and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reactions. Through chemical proteomics, putatively hybrid-binding protein targets of the ART-quinolines were successfully identified in addition to known targets of quinoline and artemisinin alone, suggesting that the hybrids act through multiple modes of action to overcome resistance.

Simultaneous Extraction of Pesticides and Polycyclic Aromatic Hydrocarbons in Brazilian Cachaça Using a Modified QuEChERS Method Followed by Gas Chromatography Coupled to Tandem Mass Spectrometry Quantification.

A modified QuEChERS method was optimized for simultaneous extraction of 93 pesticides and 6 polycyclic aromatic hydrocarbons (PAHs) in cachaça. The procedure employed 20 mL of sample, 10 mL of dichloromethane, 1 g of NaCl, and 6 g of MgSO4. The methods were validated in accordance with pesticide tolerances set by the National Health Surveillance Agency of Brazil and government guidelines of Brazil and the European Union. The linearity of all curves was adequate, with calculated tr higher than the critical value, at the 95% confidence level. For pesticides, recoveries ranged between 86.7 and 118.2%, relative standard deviation (RSD) ≤ 20%, at least at two concentration levels, and limit of detection (LOD) and limit of quantitation (LOQ) were 2.5 and 10.0 µg L-1, respectively. For PAHs, recoveries ranged between 84.8 and 111.5%, RSD was between 6.2 and 27.3%, LOD and LOQ were 0.25 and 1.0 µg L-1, respectively. The combined standard uncertainty was lower than 50% of the relative expanded uncertainty value at concentration levels of greater relevance in both methods. Analyses of five commercial samples detected the presence of 9 pesticides (10.0-128.0 µg L-1) and 6 PAHs (2.0-4.0 µg L-1), indicating the need for a specific legislation for Brazilian cachaça.

Glycans as Key Checkpoints of T Cell Activity and Function.

The immune system is highly controlled and fine-tuned by glycosylation, through the addition of a diversity of carbohydrates structures (glycans) to virtually all immune cell receptors. Despite a relative backlog in understanding the importance of glycans in the immune system, due to its inherent complexity, remarkable findings have been highlighting the essential contributions of glycosylation in the regulation of both innate and adaptive immune responses with important implications in the pathogenesis of major diseases such as autoimmunity and cancer. Glycans are implicated in fundamental cellular and molecular processes that regulate both stimulatory and inhibitory immune pathways. Besides being actively involved in pathogen recognition through interaction with glycan-binding proteins (such as C-type lectins), glycans have been also shown to regulate key pathophysiological steps within T cell biology such as T cell development and thymocyte selection; T cell activity and signaling as well as T cell differentiation and proliferation. These effects of glycans in T cells functions highlight their importance as determinants of either self-tolerance or T cell hyper-responsiveness which ultimately might be implicated in the creation of tolerogenic pathways in cancer or loss of immunological tolerance in autoimmunity. This review discusses how specific glycans (with a focus on N-linked glycans) act as regulators of T cell biology and their implications in disease.

Analysis of chromosomes from mouse oocytes and mammalian cultured cells by light microscopy.

As carriers of the genetic material, chromosomes are of prime interest in the life sciences. Although all aspects of chromosome biology should ideally be studied in living cells, the isolation of chromosomes can greatly facilitate their analysis. This can be achieved by lysing mitotic or meiotic cells under conditions where their content, including their chromosomes, is spread out on the surface of microscopy glass slides. Here we describe three such chromosome spreading techniques, which have been instrumental in analyzing chromosomes from either mouse oocytes or mammalian cultured cells in mitosis. For both chromosomes from oocytes and mitotic cells, we describe immunofluorescence protocols that enable the visualization of proteins with specific antibodies. For mitotic chromosomes, we also provide a classic protocol for Giemsa staining. This protocol cannot be used to localize proteins but is useful to determine structural features of chromosomes, such as sister chromatid cohesion and chromosome condensation. The question of how chromosome nondisjunction during the meiotic division causes aneuploidy is of great interest in oocyte chromosome research. Because we have found that ploidy in mouse oocytes can be determined more reliably in fixed cells than in spread chromosomes, we also describe a protocol for the in situ fixation and immunofluorescence analysis of chromosomes in mouse oocytes.

Cytokine and Adhesion Molecule Expression Induced by Different Strains of Staphylococcus aureus in Type 1 Diabetic Rats: Role of Insulin.

Introduction:Staphylococcus aureus may provoke peritonitis and death, especially in immunocompromized individuals such as diabetic patients. We evaluated the role of insulin in S. aureus-induced peritoneal infection in diabetic and non-diabetic rats. Materials/Methods: Alloxan-diabetic male Wistar rats and their respective controls received intraperitoneal injections of different strains of S. aureus or sterile phosphate-buffered saline. After 3 days of infection, the first set of diabetic and non-diabetic rats received 4 and 1 IU, respectively, of neutral protamine Hagedorn insulin and were analyzed 8 h later. The second set of diabetic and non-diabetic rats received 4 and 1 IU, respectively, of insulin 2 h before intraperitoneal infection and a half dose of insulin at 5 p.m. for the next 2 days and were analyzed 16 h later. The following measurements were performed: (a) number of cells in the peritoneal lavage fluid (PeLF), white blood cell count, and blood glucose; (b) serum insulin and corticosterone; (c) cytokine levels in the PeLF; (d) expression of adhesion molecules in the vascular endothelium; and (e) microbicidal activity. Results: Diabetic rats showed an increased number of polymorphonuclear leukocytes (PMNs) and increased concentrations of CINC-1, IL-4, and IFN-γ in the PeLF after infection with the ATCC 25923 or N315 αHL+ strain. The mesenteric expression of PECAM-1 was increased after infection with the N315 HLA+ strain. ICAM-1 expression was increased with ATCC infection. Treatment of diabetic rats with a single dose of insulin restored CINC-1 levels in the PeLF for both strains; however, PMN migration, IL-4, and IFN-γ were restored in rats infected with the ATCC strain, whereas the PeLF concentrations of CINC-2, IL-1ß, and IL-4 were increased in N315-infected animals. Insulin restored PMN migration and CINC-2 levels in the PeLF in ATCC-infected rats. After multiple treatments with insulin, the levels of IL-1ß, IL-6, and IFN-γ were increased in the PeLF of diabetic rats after infection with either strain, and CINC-2 levels were restored in N315-infected animals. Conclusion: These results suggest that insulin distinctively modulates cytokine production or release, PMN leukocyte migration, and adhesion molecule expression during the course of peritonitis induced by different strains of S. aureus.

Can γ-radiation modulate hemagglutinating and anticoagulant activities of PpyLL, a lectin from Phthirusa pyrifolia?

Blood coagulation and platelet-dependent primary homeostasis are important defense mechanisms against bleeding and novel inhibitors have been researched to obtain pharmacological and clinical applications. In this work, the PpyLL, a lectin obtained from Phthirusa pyrifolia, was characterized in terms of its molecular structure and biological functions (anticoagulant, antiplatelet agreggation and hemagglutinating activities) in presence or absence of Gamma radiation exposure. Results revealed a lectin with secondary-structure content by approximately 49% of ß-sheet, 20% of ß-turn and 31% of disordered structure. Irradiation effect demonstrated possible different sites of function by lectin on anticoagulant and hemagglutinating activities, once a decrease about 80% was observed when compared the activities under 0.5kGy of exposition to gamma radiation. An emphatic discussion about the use of gamma radiation as a possible modulator of the lectin activity was made, and once the ionizing radiation affected differently the anticoagulation and hemagglutinating activities, we speculated that the results are determined by selective molecular damages in different binding sites. PpyLL biological activities and gamma radiation modulation could be considered for future researches in biomedical field aiming possible medical applications.

IDENTIFICATION OF BACTERIA IN BLOOD CULTURES FROM CLINICALLY ILL CAPTIVE ANTILLEAN MANATEES (TRICHECHUS MANATUS MANATUS).

Between September 2001 and March 2013, 62 bacterial cultures (37 aerobic and 25 anaerobic) were performed on 37 blood samples from 23 Antillean manatees ( Trichechus manatus manatus) that were kept in captivity at the Brazilian National Center for Research and Conservation of Aquatic Mammals (CMA) in Pernambuco (CMA-PE) and Alagoas (CMA-AL), Brazil. All of the animals sampled exhibited clinical signs at the time of sampling including abscesses (n = 8), debilitation and anorexia (n = 22), and profound lethargy-moribundity (n = 7). The 4 animals with profound lethargy-moribundity died shortly after sampling of unknown causes. Bacteria were isolated from 15/37 (40.5%) and aerobic blood cultures from 13/23 animals (56.5%). None of the anaerobic cultures were positive. Aeromonas caviae , Aeromonas hydrophila , Aeromonas sp., Escherichia coli , Leclercia adecarboxylata , Pantoea agglomerans , Pseudomonas aeruginosa , Pseudomonas stutzeri , Pseudomonas sp., Sphingomonas paucimobilis , coagulase-negative Staphylococcus, and Staphylococcus epidermidis were each found in only one animal; Staphylococcus spp. was found in two; and Vibrio fluvialis in four. Thirteen samples had only one bacteria isolated, one sample had two bacteria, and one sample had three bacteria isolated. Regarding sex, age group, and origin among the manatees examined, 54.5% (6/11) of the females, 58.3% (7/12) of the males, 40% (2/5) of the calves, 66.7% (8/12) of the juveniles, 50% (3/6) of the adults, 55.5% (10/18) at CMA-PE, and 60% (3/5) at CMA-AL were found to be positive for bacterial growth during at least one sampling time. All Antillean manatees were clinically ill. Regarding clinical signs, bacteria were found in 50% (11/22) of blood samples of the animals showing debilitation and anorexia, 1 of 8 (12.5%) of blood samples of the animals showing abscesses, and 3 of 7 (42.9%) of blood samples of the animals showing profound lethargy-moribundity.

Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP.

Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin αIIbß3 through interactions with the KGD/KGE sequence motif in huPK. Integrin αIIbß3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS473, ERK1/2, and p38 MAPK, and to Ca2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and αIIbß3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis.