Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1). B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis. (c) 2008 Elsevier Ltd. All rights reserved.

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar-gel immunodiffusion (ACID) test for canine brucellosis, were used as the control panel for B. cants infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies. (C) 2010 Elsevier Ltd. All rights reserved.

One-step reverse transcriptase polymerase chain reaction for the diagnosis of respiratory syncytial virus in children

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory tract infections in infants and children. Rapid diagnosis is required to permit appropriate care and treatment and to avoid unnecessary antibiotic use. Reverse transcriptase (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been considered important tools for virus detection due to their high sensitivity and specificity. In order to maximize use-simplicity and minimize the risk of sample cross-contamination inherent in two-step techniques, a RT-PCR method using only a single tube to detect HRSV in clinical samples was developed. Nasopharyngeal aspirates from 226 patients with acute respiratory illness, ranging from infants to 5 years old, were collected at the University Hospital of the University of Sao Paulo (HU-USP), and tested using IFA, one-step RT-PCR, and semi-nested RT-PCR. One hundred and two (45.1%) samples were positive by at least one of the three methods, and 75 (33.2%) were positive by all methods: 92 (40.7%) were positive by one-step RT-PCR, 84 (37.2%) by IFA, and 96 (42.5%) by the semi-nested RT-PCR technique. One-step RT-PCR was shown to be fast, sensitive, and specific for RSV diagnosis, without the added inconvenience and risk of false positive results associated with semi-nested PCR. The combined use of these two methods enhances HRSV detection. (C) 2007 Elsevier B.V. All rights reserved.

Diagnosis of limb-girdle muscular dystrophy 2A by immunohistochemical techniques

The Western blot technique is currently the standard detection method for suspected limb girdle muscular dystrophy (LGMD) 2A (calpainopathy). This is the first report in the English literature of the successful application of immunohistochemical techniques to support a diagnosis of LGMD 2A. This approach is straightforward and appears to be reasonably specific. We propose that immunohistochemical methods should be re-evaluated for the screening of undiagnosed patients with suspected LGMD 2A.

Akt Expression is Diminished by Physical Inactivity in Early Stages of Development in Soleus Muscle of Rats

Metabolic Syndrome is a group of conditions related to obesity and physical inactivity. Little is known about the role of physical inactivity, in early stages of development, in the susceptibility to insulin resistant phenotype induced by high fat diet. Akt plays a key role in protein synthesis and glucose transport in skeletal muscle and has been regulated by muscle activity. The objective of present study was to determine the effect of early physical inactivity on muscle growth and susceptibility to acquire a diabetic phenotype and to assess its relationship with Akt expression. Forty Wistar male rats were distributed in two groups (standard group, Std) and movement restriction (RM). Between days 23 and 70 after birth, RM group was kept in small cages that did not allow them to perform relevant motor activity. From day 71 to 102 after birth, 10 rats of each group were fed with hyperlipidic diet (groups Std-DAG and RM-DAG). No differences were observed in total body weight although DAG increased epididymal fat pad weight. RM decreased significantly the soleus weight. Insulin-mediated glucose uptake was lower in RM-DAG group. Akt protein levels were lower in RM groups. Real time RT-PCR analysis showed that movement restriction decreased mRNA levels of AKT1 in soleus muscle, regardless of supplied diet. These findings suggest that early physical inactivity limits muscle`s growth and contributes to instauration of insulin resistant phenotype, which can be partly explained by dysregulation of Akt expression.

Effects of long-term diabetes on the structure and cell proliferation of the myometrium in the early pregnancy of mice

P>It is known that the development of diabetic complications in human pregnancy is directly related to the severity and the duration of this pathology. In this study, we developed a model of long-term type 1 diabetes to investigate its effects on the cytoarchitecture, extracellular matrix and cell proliferation during the first adaptation phase of the myometrium for pregnancy. A single dose of alloxan was used to induce diabetes in mice prior to pregnancy. To identify the temporal effects of diabetes the mice were divided into two groups: Group D1 (females that became pregnant 90-100 days after alloxan); Group D2 (females that became pregnant 100-110 days after alloxan). Uterine samples were collected after 168 h of pregnancy and processed for light and electron microscopy. In both groups the histomorphometric evaluation showed that diabetes promoted narrowing of the myometrial muscle layers which was correlated with decreased cell proliferation demonstrated by PCNA immunodetection. In D1, diabetes increased the distance between muscle layers and promoted oedema. Contrarily, in D2 the distance between muscle layers decreased and, instead of oedema, there was a markedly deposition of collagen in the myometrium. Ultrastructural analysis showed that diabetes affects the organization of the smooth muscle cells and their myofilaments. Consistently, the immunoreaction for smooth muscle alpha-actin revealed clear disorganization of the contractile apparatus in both diabetic groups. In conclusion, the present model demonstrated that long-term diabetes promotes significant alterations in the myometrium in a time-sensitive manner. Together, these alterations indicate that diabetes impairs the first phenotypic adaptation phase of the pregnant myometrium.

Comparison of signal-to-cutoff values in first, second, and third generation enzyme immunoassays for the diagnosis of HTLV-1/2 infection in ""at-risk"" individuals from Sao Paulo, Brazil

Data obtained during routine diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) and 2 (HTLV-2) in ""at-risk"" individuals from Sao Paulo, Brazil using signal-to-cutoff (S/C) values obtained by first, second, and third generation enzyme immunoassay (EIA) kits, were compared. The highest S/C values were obtained with third generation EIA kits, but no correlation was detected between these values and specific antibody reactivity to HTLV-1, HTLV-2, or untyped HTLV (p = 0.302). In addition, use of these third generation kits resulted in HTLV-1/2 false-positive samples. In contrast, first and second generation EIA kits showed high specificity, and the second generation EIA kits showed the highest efficiency, despite lower S/C values. Using first and second generation EIA kits, significant differences in specific antibody detection of HTLV-1, relative to HTLV-2 (p = 0.019 for first generation and p < 0.001 for second generation EIA kits) and relative to untyped HTLV (p = 0.025 for first generation EIA kits), were observed. These results were explained by the composition and format of the assays. In addition, using receiver operating characteristics (ROC) analysis, a slight adjustment in cutoff values for third generation EIA kits improved their specificities and should be used when HTLV ""at-risk"" populations from this geographic area are to be evaluated. (C) 2009 Elsevier B.V. All rights reserved.

Vimentin Expression and Myofibroblast Infiltration Are Early Markers of Renal Dysfunction in Kidney Transplantation: An Early Stage of Chronic Allograft Dysfunction?

Introduction. The objective of this study was to show the morphologic characteristics of allograft renal biopsies in renal transplant patients with stable renal function, which can potentially be early markers of allograft dysfunction, after 5 years of follow-up. Methods. Forty-nine renal transplant patients with stable renal function were submitted to renal biopsies and simultaneous measurement of serum creatinine (Cr). Histology was evaluated using Banff scores, determination of interstitial fibrosis by Sirius red staining and immunohistochemical study of proximal tubule and interstitial compartment (using cytokeratin, vimentin, and myofibroblasts as markers). Biopsies were evaluated according to the presence or absence of the epitheliomesenchymal transition (EMT). The interstitial presence of myofibroblasts and tubular presence of vimentin was also analyzed simultaneously. Renal function was measured over the follow-up period to estimate the reduction of graft function. Results. Median posttransplant time at enrollment was 105 days. Patients were followed for 64.3 +/- 8.5 months. The mean Cr at biopsy time was 1.44 +/- 0.33 mg/dL, and after the follow-up it was 1.29 +/- 0.27 mg/dL. Nine patients (19%) had a reduction of their graft function. Eleven biopsies (22%) had tubulointerstitial alterations according to Banff score. Seventeen biopsies (34%) presented EMT. Fifteen biopsies (32%) had high interstitial expression of myofibroblasts and tubular vimentin. Using Cox multivariate analysis, HLA and high expression of interstitial myofibroblasts and tubular vimentin were associated with reduction of graft function, yielding a risk of 3.3 (P = .033) and 9.8 (P = .015), respectively. Conclusion. Fibrogenesis mechanisms occur very early after transplantation and are risk factors for long-term renal function deterioration.

Early Phase of Allergic Airway Inflammation in Diabetic Rats: Role of Insulin on the Signaling Pathways and Mediators

Background: Allergic lung inflammation is impaired in diabetic rats and is restored by insulin treatment. In the present study we investigated the effect of insulin on the signaling pathways triggered by allergic inflammation in the lung and the release of selected mediators. Methods: Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and matching controls were sensitized by s.c. injections of ovalbumin (OA) in aluminium hydroxide, 14 days before OA (1 mg/0.4 ml) or saline intratracheal challenge. A group of diabetic rats were treated with neutral protamine Hagedorn insulin (NPH, 4 IU, s.c.), 2 h before the OA challenge. Six hours after the challenge, bronchoalveolar lavage (BAL) was performed for mediator release and lung tissue was homogenized for Western blotting analysis of signaling pathways. Results: Relative to non-diabetic rats, the diabetic rats exhibited a significant reduction in OA-induced phosphorylation of the extracellular signal-regulated kinase (ERK, 59%), p38 (53%), protein kinase B (Akt, 46%), protein kinase C (PKC)-alpha (63%) and PKC-delta (38%) in lung homogenates following the antigen challenge. Activation of the NF-kappa B p65 subunit and phosphorylation of I kappa B alpha were almost suppressed in diabetic rats. Reduced expression of inducible nitric oxide synthase (iNOS, 32%) and cyclooxygenase-2 (COX-2, 46%) in the lung homogenates was also observed. The BAL concentration of prostaglandin (PG)-E(2), nitric oxide (NO) and interleukin (IL)-6 was reduced in diabetic rats (74%, 44% and 65%, respectively), whereas the cytokine-induced neutrophil chemoattractant (CINC)-2 concentration was not different from the control animals. Treatment of diabetic rats with insulin completely or partially restored all of these parameters. This protocol of insulin treatment only partially reduced the blood glucose levels. Conclusion: The data presented show that insulin regulates MAPK, PI3K, PKC and NF-kappa B pathways, the expression of the inducible enzymes iNOS and COX-2, and the levels of NO, PGE(2) and IL-6 in the early phase of allergic lung inflammation in diabetic rats. It is suggested that insulin is required for optimal transduction of the intracellular signals that follow allergic stimulation. Copyright (C) 2010 S. Karger AG, Basel

Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Study of Blood Porphyrin Spectral Profile for Diagnosis of Chronic Renal Failure

The progression to end-stage renal failure is independent of the initial pathogenic mechanism. Metabolic acidosis is a common consequence of chronic renal failure that results from inadequate ammonium excretion and decreased tubular bicarbonate reabsorption. Protoporphyrin IX (PpIX) is the immediate metabolic precursor of the heme molecule. The purpose of this study was to evaluate the levels of erythrocytes protoporphyrin IX at an animal model during progressive renal disease. A total of 36 eight-week-old male Wistar rats were divided into six groups: Normal, 4 and 8 weeks after 5/6 nephrectomy (NX). Renal function was evaluated by creatinine clearance and plasma creatinine levels. The autofluorescence of erythrocytes porphyrin of healthy and NX rats was analyzed using fluorescence spectroscopy. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and NX rats autofluorescence shape occurred in the 600-700 nm spectral region. A correlation was observed between emission band intensity at 635 nm and progression of renal disease.

Cordierite-bearing lavas from Jaguarao, southern Brazil: Petrological evidence for crustal melts during early rifting of Gondwana

The Jaguarao stratoid dacites (Rio Grande do Sul, Brazil) are limited in areal extent, are comprised of about 3.2 km(3) of preserved erupted material, and outcrop only in areas of the region underlain by mylonitic and ultramylonitic rocks. They are S-type volcanic rocks containing cordierite, orthopyroxene, plagioclase, and ilmenite as liquidus phases, and partially melted granite, gneiss, and migmatite enclaves that are very similar to the Precambrian basement rocks. The Jaguarao lavas have distinct geochemical signatures and Sr-Nd isotopes with respect to other volcanic rocks of the region. Available geochronological data for Jaguarao dacites range between 157 +/- 5 Ma and 139.6 +/- 7.4 Ma. Considering the errors, the younger ages obtained for Jaguarao lavas overlap the 138-128 Ma age of rocks of the Serra Geral Group, and thus indicate that the dacites were erupted prior to the break-up of Gondwana in this region. Petrographic, mineralogical, and petrochemical data, as well as the tectonic context of the Jaguarao lavas, suggest that magma genesis was linked, at least in part, to friction melts. The dacitic magma was generated by partial melting reactions involving biotite breakdown in a dominantly quartz-feldspathic source terrane, leaving a granulite facies residue in subsurface. These melts were probably generated as a consequence of crustal thinning linked to simple shear extension just prior to Gondwana break-up and rifting of the southern Atlantic Ocean. (C) 2009 International Association for Gondwana Research. Published by Elsevier B.V. All rights reserved.

A putative gnetalean gymnosperm Cariria orbiculiconiformis gen. nov et spec. nov from the Early Cretaceous of northern Gondwana

Cariria orbiculiconiformis gen. nov. et spec. nov., a gymnosperm with gnetoid characters is described from the upper Aptian Crato Formation of the Araripe Basin in northeastern Brazil. Gross-morphology and anatomical details have been studied and characters have been discussed in respect to various seed plants. Several of these characters fit best with those of Gnetales and their putative fossil allies. However, the fossil plant cannot be assigned to any known extinct or extant group of seed plants in their current circumscription. Stem gross-morphology, xylotomical characters and epidermal features indicate a gnetophytic relationship, whereas characters of the reproductive organs are rather distinct from those found in extant taxa. The reproductive unit of the new taxon represents a triple organ consisting of two dichasial ovulate structures and one median pollen-producing structure containing smooth, monosulcate, boat-shaped pollen in-situ. Each ovulate structure consists of two distinct pairs of bracts, a sterile one at the base and a fertile one forming a terminal orbicular capsule. Stiff processes found in the apex of the ovulate structure may represent micropylar tubes of seeds, as seen in the Bennettitales-Erdtmanithecales-Gnetales group. C orbiculiconiformis gen. nov. et spec. nov. was ans herbaceous or semi-shrub-like plant that may have been adapted to the r-strategy in a stressful environment. (C) 2011 Elsevier B.V. All rights reserved.

Cearania heterophylla gen. nov et sp nov., a fossil gymnosperm with affinities to the Gnetales from the Early Cretaceous of northern Gondwana

Vegetative and fertile shoots of a shrub-like seed plant from the late Aptian Crato Formation of Brazil are described as Cearania heterophylla Kunzmann, Mohr and Bernardes-de-Oliveira, gen. nov. et sp. nov. Anatomical details of the axes, epidermal features and separate ovulate and pollen producing organs indicate the gymnospermous nature of this plant. The vascular tissue of the axes includes tracheids with bordered pits and fiber tracheids. Vegetative shoots comprising at least three branching orders bear opposite-decussately arranged ovate to lanceolate, dorsiventrally flattened, parallelodromous, rather thick leaves that vary tremendously in size. The amphistomatic leaves bear (brachy-)paracytic stomatal complexes arranged in simple longitudinal files. The ovulate structure is interpreted as a terminally attached single globular ovule/seed surrounded by at least five to six lanceolate bracts. A terminally attached pollen-cone like structure grows on a lateral leafy shoot. The unusual character combination may indicate that the fossils belong to a hitherto unknown group with affinities to ephedroid Gnetales. Sterile shoots formerly often described as Podozamites, Nageiopsis or Lilites that are at least partly congeneric with C. heterophylla Kunzmann, Mohr and Bernardes-de-Oliveira, gen. nov. et sp. nov. had a wide geographic distribution during the Early Cretaceous. (C) 2009 Elsevier B.V. All rights reserved.