Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Effects of six carbohydrate sources on dog diet digestibility and post-prandial glucose and insulin response

The effects of six extruded diets with different starch sources (cassava flour, brewer`s rice, corn, sorghum, peas or lentils) on dog total tract apparent digestibility and glycemic and insulinemic response were investigated. The experiment was carried out on thirty-six dogs with six dogs per diet in a completely randomized design. The diets containing brewer`s rice and cassava flour presented the greatest digestibility of dry matter, organic matter and gross energy (p < 0.05), followed by corn and sorghum; pea and lentil diets had the lowest. Starch digestibility was greater than 98% in all diets and was greater for brewer`s rice and cassava flour than for lentils and peas diets (p < 0.05). Dogs` immediate post-prandial glucose and insulin responses (AUC <= 30 min) were greater for brewer`s rice, corn, and cassava flour diets (p < 0.05), and later meal responses (AUC >= 30 min) were greater for sorghum, lentil and pea diets (p < 0.05). Variations in diet digestibility and post-prandial response can be explained by differences in chemical composition of each starch source including fibre content and starch granule structure. The nutritional particularities of each starch ingredient can be explored through diet formulations designed to modulate glycemic response. However, more studies are required to support these.

Short communication: Identification of subclinical cow mastitis pathogens in milk by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Subclinical mastitis is a common and easily disseminated disease in dairy herds. Its routine diagnosis via bacterial culture and biochemical identification is a difficult and time-consuming process. In this work, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows bacterial identification with high confidence and speed (1 d for bacterial growth and analysis). With the use of MALDI-TOF MS, 33 bacterial culture isolates from milk of different dairy cows from several farms were analyzed, and the results were compared with those obtained by classical biochemical methods. This proof-of-concept case demonstrates the reliability of MALDI-TOF MS bacterial identification, and its increased selectivity as illustrated by the additional identification of coagulase-negative Staphylococcus species and mixed bacterial cultures. Matrix-assisted laser desorption-ionization mass spectrometry considerably accelerates the diagnosis of mastitis pathogens, especially in cases of subclinical mastitis. More immediate and efficient animal management strategies for mastitis and milk quality control in the dairy industry can therefore be applied.

In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen wand plasma fibronectin

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the UpL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.

LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules

The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. (C) 2009 Elsevier Masson SAS. All rights reserved.

Decay chain differential equations: Solution through matrix algebra

A matricial method to solve the decay chain differential equations system is presented. The quantity of each nuclide in the chain at a time t may be evaluated by analytical expressions obtained in a simple way using recurrence relations. This method may be applied to problems of radioactive buildup and decay and can be easily implemented computationally. (C) 2009 Elsevier B.V. All rights reserved.

Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells

Objectives: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. Methods: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). Results: The recombinant leptospiral protein of 95 kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P < 0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. Conclusion: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Hematopoietic response of rats exposed to the impact of an acute psychophysiological stressor on responsiveness to an in vivo challenge with Listeria monocytogenes: Modulation by Chlorella vulgaris prophylactic treatment

In this study, we investigated the hematopoietic response of rats pretreated with CV and exposed to the impact of acute escapable, inescapable or psychogenical stress on responsiveness to an in vivo challenge with Listeria monocytogenes. No consistent changes were observed after exposure to escapable footshock. Conversely, the impact of uncontrollable stress (inescapable and psychogenical) was manifested by an early onset and increased severity and duration of myrelossuppression produced by the infection. Small size CFU-CM colonies and increased numbers of clusters were observed, concurrently to a greater expansion in the more mature population of bone marrow granulocytes. No differences were observed between the responses of both uncontrollable stress regimens. CV prevented the myelossuppression caused by stress/infection due to increased numbers of CFU-GM in the bone marrow. Colonies of cells tightly packed, with a very condensed nucleus; in association with a greater expansion in the more immature population of bone marrow granulocytes were observed. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected/stressed rats treated with the algae. CV treatment restored/enhanced the changes produced by stress/infection in total and differential bone marrow and peripheral cells counts. Further studies demonstrated that INF-gamma is significantly reduced, whereas IL-10 is significantly increased after exposure to Uncontrollable stress. Treatment with CV significantly increased INF-gamma levels and diminished the levels of IL-10. Uncontrollable stress reduced the protection afforded by CV to a lethal dose of L. monocytogenes, with survival rates being reduced from (50%) in infected rats to 20% in infected/stressed rats. All together, our results suggest Chlorella treatment as an effective tool for the prophylaxis of post-stress myelossupression, including the detrimental effect of stress on the course and outcome of infections. (C) 2008 Elsevier Inc. All rights reserved.

Single early prenatal lipopolysaccharide exposure prevents subsequent airway inflammation response in an experimental model of asthma

Aims: There has been emerging interest in the prenatal determinants of respiratory disease. In utero factors have been reported to play a role in airway development, inflammation, and remodeling. Specifically, prenatal exposure to endotoxins might regulate tolerance to allergens later in life. The present study investigated whether prenatal lipopolysaccharide (LPS) administration alters subsequent offspring allergen-induced inflammatory response in adult rats. Main methods: Pregnant Wistar rats were treated with LPS (100 mu g/kg, i.p.) on gestation day 9.5 and their ovariectomized female offspring were sensitized and challenged with OVA later in adulthood. The bronchoalveolar lavage (BAL) fluid, peripheral blood, bone marrow leukocytes and passive cutaneous anaphylaxis were evaluated in these 75-day-old pups. Key findings: OVA sensitized pups of NaCl treated rats showed an increase of leucocytes in BAL after OVA challenge. This increase was attenuated, when mothers were exposed to a single LPS injection early in pregnancy. Thus, LPS prenatal treatment resulted in (1) lower increased total and differential (macrophages, neutrophils, eosinophils and lymphocytes) BAL cellularity count; (2) increased number of total, mononuclear and polymorphonuclear cells in the peripheral blood; and (3) no differences in bone marrow cellularity or passive cutaneous anaphylaxis. Significance: In conclusion, female pups treated prenatally with LPS presented an attenuated response to experimentally-induced asthma. We observed reduced immune cell migration from peripheral blood to the lungs, with no effect on the production of bone marrow cells or antibodies. It was suggested that inflammatory events such as exposure to LPS in early fetal life can attenuate allergic inflammation in the lung, which is a common symptom in asthma. (C) 2011 Elsevier Inc. All rights reserved.

13,14-Dihydro-15-Keto Prostaglandin F(2)alpha Release in Response to Oxytocin Challenge Early Post-Partum in Anoestrous Nelore Cows Submitted to Temporary Calf Removal and Progesterone Priming

The study evaluated, in early post-partum anoestrous Nelore cows, if the increase in plasma oestradiol (E2) concentrations in the pre-ovulatory period and/or progesterone priming (P4 priming) preceding ovulation, induced by hormonal treatment, reduces the endogenous release of prostaglandin PGF(2)alpha and prevents premature lysis of the corpus luteum (CL). Nelore cows were subjected to temporary calf removal for 48 h and divided into two groups: GPE/eCG group (n = 10) and GPG/eCG group (n = 10). Animals of the GPE/eCG group were treated with a GnRH agonist. Seven days later, they received 400 ID of eCG, immediately after PGF(2)alpha treatment, and on day 0, 1.0 mg of oestradiol benzoate (EB). Cows of the GPG/eCG group were similarly treated as those of the GPE/eCG group, except that EB was replaced with a second dose of GnRH. All animals were challenged with oxytocin (OT) 9, 12, 15 and 18 days after EB or GnRH administration and blood samples were collected before and 30 min after OT. Irrespective of the treatments, a decline in P4 concentration on day 18 was observed for cows without P4 priming. However, animals exposed to P4 priming, treated with EB maintained high P4 concentrations (8.8 +/- 1.2 ng/ml), whereas there was a decline in P4 on day 18 (2.1 +/- 1.0 ng/ml) for cows that received GnRH to induce ovulation (p < 0.01). Production of 13,14-dihydro-15-keto prostaglandin F(2)alpha (PGFM) in response to OT increased between days 9 and 18 (p < 0.01), and this increase tended to be more evident in animals not exposed to P4 priming (p < 0.06). In conclusion, the increase in E2 during the pre-ovulatory period was not effective in inhibiting PGFM release, which was lower in P4-primed than in non-primed animals. Treatment with EB promoted the maintenance of elevated P4 concentrations 18 days after ovulation in P4-primed animals, indicating a possible beneficial effect of hormone protocols containing EB in animals with P4 priming.

Poor ovarian response in patients younger than 35 years: Is it also a qualitative decline in ovarian function?

Objective. To investigate whether poor response to controlled ovarian stimulation (COS) is due to a qualitative decline in ovarian function. Methods. This retrospective cohort study included 436 patients younger than 35-years old, undergoing COS for intracytoplasmic sperm injection (ICSI). Patients with four or fewer MII oocytes after COS (poor-responder group, PR, n = 52) were age-matched with normoresponder patients (NR, n = 364). Results. Although similar duration of stimulation (10.5 +/- 0.4 and 9.3 +/- 0.8 days; p = 0.1358), increased doses of gonadotrophins (2510 +/- 865 and 2253 +/- 572 IU; p = 0.0061) were used in the PR. The results show a increased chance of cycle ending of PR (PR: 26.9% and NR: 3.1%; p < 0.0001). Although the lower total number of oocytes retrieved (2.4 +/- 1.4 and 16.2 +/- 9.3; p < 0.0001), equal rate of fertilization (70.2% and 72.0%, p = 0.1190) and high quality embryos were obtained (50.0% and 45.2%; p = 0.4895), resulting in similar implantation (14.5% and 19.7%; p = 0.2246) and abortion (10.0% and 15.4%; p = 1.00) rates, respectively. A trend towards increased pregnancy rate per embryo transfer in NR group was noted (PR: 26.3% and NR: 42.2%; p = 0.0818). Conclusions. Low ovarian response could be associated mainly with a quantitative rather than a qualitative decline in ovarian function. Therefore, even if the ovarian response to stimulation is low, patients aged <= 35 years should process to oocyte retrieval.

Differences in thermoregulatory ability between slick-haired and wild-type lactating Holstein cows in response to acute heat stress

Animals inheriting the slick hair gene have a short, sleek, and sometimes glossy coat. The objective of the present study was to determine whether slick-haired Holstein cows regulate body temperature more effectively than wild-type Holstein cows when exposed to an acute increase in heat stress. Lactating slick cows (n = 10) and wild-type cows (n = 10) were placed for 10 h in an indoor environment with a solid roof, fans, and evaporative cooling or in an outdoor environment with shade cloth and no fans or evaporative cooling. Cows were exposed to both environments in a single reversal design. Vaginal temperature, respiration rate, surface temperature, and sweating rate were measured at 1200, 1500, 1800, and 2100 h (replicate 1) or 1200 and 1500 h (replicate 2), and blood samples were collected for plasma cortisol concentration. Cows in the outdoor environment had higher vaginal and surface temperatures, respiration rates, and sweating rates than cows in the indoor environment. In both environments, slick-haired cows had lower vaginal temperatures (indoor: 39.0 vs. 39.4 degrees C; outdoor 39.6 vs. 40.2 degrees C; SEM = 0.07) and respiration rate (indoor: 67 vs. 79 breaths/min; outdoor 97 vs. 107 breaths/min; SEM = 5.5) than wild-type cows and greater sweating rates in unclipped areas of skin (indoor: 57 vs. 43 g.h(-1)/m(2); outdoor 82 vs. 61 g.h(-1)/m(2); SEM = 8). Clipping the hair at the site of sweating measurement eliminated the difference between slick-haired and wild-type cows. Results indicate that slick-haired Holstein cows can regulate body temperature more effectively than wild-type cows during heat stress. One reason slick-haired animals are better able to regulate body temperature is increased sweating rate.

Expression of Extracellular Matrix Proteins in Human Dental Pulp Stem Cells Depends on the Donor Tooth Conditions

Introduction: Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. An important source of adult stem cells is the dental pulp. In dentistry, regenerative strategies are of importance because of hard dental tissue damage especially as result of caries lesions, trauma, or iatrogenic procedures. The regeneration of dental tissues relies on the ability of stem cells to produce extracellular (ECM) proteins encountered in the dental pulp tissue. Thus, the aim of this study was to analyze the expression and distribution of proteins encountered in dental pulp ECM (type I collagen, fibronectin, and tenascin) in stem cells. Methods: Human immature dental pulp stem cells (hIDPSCs) from deciduous (DL-1 and DL-4 cell lines) and permanent (DL-2) teeth were used. The distribution of ECM proteins was observed using the immunofluorescence technique. The gene expression profile was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Positive reactions for all ECM proteins were observed independently of the hIDPSCs analyzed. Type I collagen appeared less evident in DL-2 than in other hIDPSCs. Fibronectin and tenascin were less clear in DL-4. The RT-PCR reactions showed that type I collagen was lesser expressed in the DL-2 cells, whereas fibronectin and tenascin were similarly expressed in all hIDPSCs. Conclusions: The distribution and expression of ECM proteins differ among the hIDPSCs. These differences seemed to be related to the donor tooth conditions (deciduous or permanent, retained or erupted, and degree of root reabsorption). (J Endod 2010;36:826-831)

Expression of extracellular matrix proteins in adenomatoid odontogenic tumor

Altered expression of extracellular matrix (ECM) components has been reported in several pathologies; however, few ECM proteins have been evaluated in adenomatoid odontogenic tumor (AOT). The aim of this study was to analyze the expression and distribution of the ECM proteoglycans: biglycan and decorin; and glycoproteins: osteonectin, osteopontin, bone sialoprotein and osteocalcin in the AOT. Three-micrometer sections from paraffin-embedded specimens were evaluated employing a streptavidin-biotin immunohistochemical method with the antibodies against the proteins previously cited. Only the osteonectin was expressed in the epithelial cells. The eosinophilic amorphous material and the connective tissue showed expression of all components studied. The calcification foci expressed only osteopontin. In conclusion, the low expression of the components studied in neoplastic epithelial cells suggests that the epithelial cells act probably as stimulators of the expression by the stroma, which in turn can act as agonist or antagonist of the tumor growth. These results suggest that the components studied probably have a key role in the biological behavior of the AOT.

Treatment of infrabony defects with or without enamel matrix proteins: A 24-month follow-up randomized pilot study

Objective: To evaluate a comparison of open-flap debridement (OFD) with or without the use of enamel matrix proteins (EMP) for the treatment of infrabony defects. Method and Materials: Ten volunteers (38 infrabony defects) were randomized to receive OFD + EMP (test site) and OFD (control site). Clinical outcomes included mean changes in Plaque Index, Gingival Index, probing pocket depth (PPD), relative attachment level (RAL), gingival recession, width of keratinized tissue, and dental mobility at baseline and at 24 months. Results: A significant reduction of 4.21 +/- 0.97 mm was observed in PPD for the OFD + EMP group (from 6.30 +/- 0.99 mm to 2.09 +/- 0.97 mm) and of 3.28 +/- 1.23 mm for the OFD group (from 6.13 +/- 0.88 mm to 2.85 +/- 1.42 mm) (P < .001). The reduction in PPD was statistically significantly greater for OFD + EMP compared to OFD (P = .03). The mean RAL decreased from 13.26 +/- 1.88 mm to 7.57 +/- 2.05 mm for the OFD + EMP group (a gain of 5.69 +/- 1.96 mm) and from 13.37 +/- 1.71 mm to 8.13 +/- 1.34 min (P < .001) for the OFD group (a gain of 5.24 +/- 1.55 mm). Gingival recession was higher it) the OFD + EMP group than in the OFD group. The mean keratinized tissue significantly decreased from 4.41 +/- 1.39 mm to 3.63 +/- 1.54 mm for OFD flap group (P < .01). Conclusion: Both treatment modalities were efficient in improving RAL and PPD. Within groups, there was a significant reduction in keratinized tissue for OFD and a significant postoperative recession for the OFD + EMP group. Infrabony defects treated with OFD + EMP showed significantly more PPD reduction when compared to OFD. (Quintessence Int 2010;41:125-134)