Mesenchymal Stem Cells Derived From Canine Umbilical Cord Vein-A Novel Source for Cell Therapy Studies

The canine model provides a large animal system to evaluate many treatment modalities using stem cells (SCs). However, only bone marrow ( BM) protocols have been widely used in dogs for preclinical approaches. BM donation consists of an invasive procedure and the number and differentiation potential of its mesenchymal stem cells (MSCs) decline with age. More recently, umbilical cord was introduced as an alternative source to BM since it is obtained from a sample that is routinely discarded. Here, we describe the isolation of MSCs from canine umbilical cord vein (cUCV). These cells can be obtained from every cord received and grow successfully in culture. Their multipotent plasticity was demonstrated by their capacity to differentiate in adipocytic, chondrocytic, and osteocytic lineages. Furthermore, our results open possibilities to use cUCV cells in preclinical trials for many well-characterized canine model conditions homologs to human diseases.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Sedentary subjects have higher PAI-1 and lipoproteins levels than highly trained athletes

Physical exercise protects against the development of cardiovascular disease, partly by lowering plasmatic total cholesterol, LDL-cholesterol and increased HDL-cholesterol levels. In addition, it is now established that reduction plasmatic adiponectin and increased C-reactive protein (CRP) and plasminogen activator inhibitor-1 (PAI-1) levels play a role in the maintenance of an inflammatory state and in the development of cardiovascular disease. This study aimed to examine plasma lipid profile and inflammatory markers levels in individual with sedentary lifestyle and/or highly trained athletes at rest. Methods: Fourteen male subjects (sedentary lifestyle n = 7 and highly trained athletes n = 7) were recruited. Blood samples were collected after an overnight fast (similar to 12 h). The plasmatic lipid profile (Triglycerides, HDL-cholesterol, LDL-cholesterol, total cholesterol, LDL-oxidized and total cholesterol/HDL-c ratio), glucose, adiponectin, C - reactive protein and PAI-1 levels were determined. Results: Total cholesterol, LDL-cholesterol, TG and PAI-1 levels were lower in highly trained athletes group in relation to sedentary subjects (p < 0.01). In addition, we observed a positive correlation between PAI-1 and total cholesterol (r = 0.78; p < 0.0009), PAI-1 and LDL-c (r = 0.69; p < 0.006) and PAI-1 and TG levels (r = 0.56; p < 0.03). The plasma concentration of adiponectin, CRP, glucose, HDL-cholesterol and total cholesterol/HDL-c ratio levels were not different. These results indicate that lifestyle associated with high intensity and high volume exercise induces changes favourable in the lipid profile and PAI-1 levels and may reduce risk cardiovascular diseases.

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background: Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. Methodology/Principal Findings: In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. Conclusions/Significance: These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

The effect of low-level laser irradiation (In-Ga-Al-AsP-660 nm) on melanoma in vitro and in vivo

Background: It has been speculated that the biostimulatory effect of Low Level Laser Therapy could cause undesirable enhancement of tumor growth in neoplastic diseases. The aim of the present study is to analyze the behavior of melanoma cells (B16F10) in vitro and the in vivo development of melanoma in mice after laser irradiation. Methods: We performed a controlled in vitro study on B16F10 melanoma cells to investigate cell viability and cell cycle changes by the Tripan Blue, MTT and cell quest histogram tests at 24, 48 and 72 h post irradiation. The in vivo mouse model (male Balb C, n = 21) of melanoma was used to analyze tumor volume and histological characteristics. Laser irradiation was performed three times (once a day for three consecutive days) with a 660 nm 50 mW CW laser, beam spot size 2 mm(2), irradiance 2.5 W/cm(2) and irradiation times of 60s (dose 150 J/cm(2)) and 420s (dose 1050 J/cm(2)) respectively. Results: There were no statistically significant differences between the in vitro groups, except for an increase in the hypodiploid melanoma cells (8.48 +/- 1.40% and 4.26 +/- 0.60%) at 72 h postirradiation. This cancer-protective effect was not reproduced in the in vivo experiment where outcome measures for the 150 J/cm(2) dose group were not significantly different from controls. For the 1050 J/cm(2) dose group, there were significant increases in tumor volume, blood vessels and cell abnormalities compared to the other groups. Conclusion: LLLT Irradiation should be avoided over melanomas as the combination of high irradiance (2.5 W/cm(2)) and high dose (1050 J/cm(2)) significantly increases melanoma tumor growth in vivo.

The Spleen CD4(+) T Cell Response to Blood-Stage Plasmodium chabaudi Malaria Develops in Two Phases Characterized by Different Properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-Ab(-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-alpha and IFN-gamma production depend mostly on conventional CD4(+) T cells. IFN-gamma is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-gamma and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in Sao Paulo state, Brazil. We show that different viral genotypes entered Sao Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in Sao Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.

VEGF Promotes Malaria-Associated Acute Lung Injury in Mice

The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies.

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads

A novel solid phase extraction technique is described where DNA is bound and eluted from magnetic silica beads in a manner where efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the beta-globin gene. By effectively controlling the movement of the solid phase in the presence of a static sample, the issues associated with reproducibly packing a solid phase in a microchannel and maintaining consistent flow rates are eliminated. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 0.6 mu L of blood at a concentration which is suitable for PCR amplification. In addition, the technique presented here requires inexpensive, common laboratory equipment, making it easily adopted for both clinical point-of-care applications and on-site forensic sample analysis.

Atenolol blunts blood pressure increase during dynamic resistance exercise in hypertensives

center dot Dynamic resistance exercise promotes a sizeable increase in blood pressure during its execution in non medicated hypertensives. WHAT THIS STUDY ADDS center dot Atenolol not only decreases blood pressure level but also mitigates the increase of blood pressure during dynamic resistance exercise in hypertensive patients. An increase in blood pressure during resistance exercise might be at least in part attributed to an increase in cardiac output. AIMS This study was conducted to determine whether atenolol was able to decrease BP level and mitigate BP increase during dynamic resistance exercise performed at three different intensities in hypertensives. METHODS Ten essential hypertensives (systolic/diastolic BP between 140/90 and 160/105 mmHg) were blindly studied after 6 weeks of placebo and atenolol. In each phase, volunteers executed, in a random order, three protocols of knee-extension exercises to fatigue: (i) one set at 100% of 1 RM; (ii) three sets at 80% of 1 RM; and (iii) three sets at 40% of 1 RM. Intra-arterial radial blood pressure was measured throughout the protocols. RESULTS Atenolol decreased systolic BP maximum values achieved during the three exercise protocols (100% = 186 +/- 4 vs. 215 +/- 7, 80% = 224 +/- 7 vs. 247 +/- 9 and 40% = 223 +/- 7 vs. 252 +/- 16 mmHg, P < 0.05). Atenolol also mitigated an increase in systolic BP in the first set of exercises (100% = +38 +/- 5 vs. +54 +/- 9; 80% = +68 +/- 11 vs. +84 +/- 13 and 40% = +69 +/- 7 vs. +84 +/- 14, mmHg, P < 0.05). Atenolol decreased diastolic BP values and mitigated its increase during exercise performed at 100% of 1 RM (126 +/- 6 vs. 145 +/- 6 and +41 +/- 6 vs. +52 +/- 6, mmHg, P < 0.05), but not at the other exercise intensities. CONCLUSIONS Atenolol was effective in both reducing systolic BP maximum values and mitigating BP increase during resistance exercise performed at different intensities in hypertensive subjects.

Finger blood pressure during leg resistance exercise

Blood pressure (BP) assessment during resistance exercise can be useful to avoid high BP, reducing cardiovascular risk, especially in hypertensive individuals. However, non-invasive accurate technique for this purpose is not available. The aim of this study was to compare finger photoplethysmographic (FPP) and intra-arterial BP values and responses during resistance exercise. Eight non-medicated hypertensive subjects (5 males, 30-60 years) were evaluated during pre-exercise resting period and during three sets of the knee extension exercise performed at 80% of 1RM until fatigue. BP was measured simultaneously by FPP and intra-arterial methods. Data are mean +/- SD. Systolic BP was significantly higher with FPP than with intra-arterial: at pre-exercise (157 +/- 13 vs. 152 +/- 10 mmHg; p < 0.01) and the mean (202 +/- 29 vs. 198 +/- 26 mmHg; p < 0.01), and the maximal (240 +/- 26 vs. 234 +/- 16 mmHg; p < 0.05) values achieved during exercise. The increase in systolic BP during resistance exercise was similar between FPP and intra-arterial (+ 73 +/- 29 vs. + 71 +/- 18 mmHg; p = 0.59). Diastolic BP values and increases were lower with FPP. In conclusion, FPP provides similar values of BP increment during resistance exercise than intra-arterial method. However, it overestimates by 2.6 +/- 6.1% the maximal systolic BP achieved during this mode of exercise and underestimates by 8.8 +/- 5.8% the maximal diastolic BP.

Clinic and ambulatory blood pressure responses after resistance exercise

Queiroz, ACC, Gagliardi, JFL, Forjaz, CLM, and Rezk, CC. Clinic and ambulatory blood pressure responses after resistance exercise. J Strength Cond Res 23(2): 571-578, 2009-This study investigated clinic and ambulatory blood pressure (BP) responses after a single bout of low-intensity resistance exercise in normotensive subjects. Fifteen healthy subjects underwent 2 experimental sessions: control-40 minutes of seated rest, and exercise-6 resistance exercises, with 3 sets of as many repetitions as possible until moderate fatigue, with an intensity of 50% of 1-repetition maximum (1RM). Before and for 60 minutes after interventions, clinic BP was measured by auscultatory and oscillometric methods. Postintervention ambulatory BP levels were also measured for 24 hours. In comparison with preintervention values, clinic systolic BP, as measured by the auscultatory method, did not change in the control group, but it decreased after exercise (-3.7 +/- 1.6 mm Hg, p < 0.05). Diastolic and mean BP levels increased after intervention in the control group (+3.4 +/- 1.0 and +3.0 +/- 0.8 mm Hg, respectively, p, 0.05) and decreased in the exercise group (-3.6 +/- 1.7 and -3.4 +/- 1.4 mm Hg, respectively, p < 0.05). Systolic and mean oscillometric BP levels did not change after interventions either in the control or exercise sessions, whereas diastolic BP increased after intervention in the control group (+5.0 +/- 1.7 mm Hg, p < 0.05) but not change after exercise. Ambulatory BP behaviors after interventions were similar in the control and exercise sessions. Significant and positive correlations were observed between preexercise values and postexercise clinic and ambulatory BP decreases. In conclusion, in the whole sample, a single bout of low-intensity resistance exercise decreased postexercise BP under clinic, but not ambulatory, conditions. However, considering individual responses, postexercise clinic and ambulatory hypotensive effects were greater in subjects with higher preexercise BP levels.

Effects of recovery type after a judo match on blood lactate and performance in specific and non-specific judo tasks

The objective of the present study was to verify if active recovery (AR) applied after a judo match resulted in a better performance when compared to passive recovery (PR) in three tasks varying in specificity to the judo and in measurement of work performed: four upper-body Wingate tests (WT); special judo fitness test (SJFT); another match. For this purpose, three studies were conducted. Sixteen highly trained judo athletes took part in study 1, 9 in study 2, and 12 in study 3. During AR judokas ran (15 min) at the velocity corresponding to 70% of 4 mmol l(-1) blood lactate intensity (similar to 50% (V) over dotO(2) peak), while during PR they stayed seated at the competition area. The results indicated that the minimal recovery time reported in judo competitions (15 min) is long enough for sufficient recovery of WT performance and in a specific high-intensity test (SJFT). However, the odds ratio of winning a match increased ten times when a judoka performed AR and his opponent performed PR, but the cause of this phenomenon cannot be explained by changes in number of actions performed or by changes in match`s time structure.