The expression of TLR9 in human cutaneous leishmaniasis is associated with granuloma

P>The Toll-like receptor (TLR) signalling pathway is the first system that defends against Leishmania. After recognising Leishmania as nonself, TLRs trigger NF-kappa B expression. NF-kappa B proceeds to the nucleus and promotes the transcription of pro-inflammatory cytokines. TLR9 is thus an important factor in the induction of an effective immune response against Leishmania. We examined the pattern of TLR9 expression in 12 patients with cutaneous leishmaniasis caused by Leishmania braziliensis detected by polymerase chain reaction. Normal skin was analysed as a negative control. TLR9 expression was examined in the dermis and epidermis by immunohistochemical analysis of paraffin-embedded biopsy tissue. TLR9 expression was primarily observed in the granuloma. The protein was detected in a few cells in the dermis. A lower expression level was detected in the epidermis of patients with leishmaniasis when compared with normal skin. The presence of TLR9 in the skin of patients with cutaneous leishmaniasis is associated with granuloma and expressed by macrophages.

Effects of (-)-RO363 at human atrial beta-adrenoceptor subtypes, the human cloned beta(3)-adrenoceptor and rodent intestinal beta(3)-adrenoceptors

1 Chronic treatment of patients with beta-blockers causes atrial inotropic hyperresponsiveness through beta(2)-adrenoceptors, 5-HT4 receptors and H-2-receptors but apparently not through beta(1)-adrenoceptors despite data claiming an increased beta(1)-adrenoceptor density from homogenate binding studies. We have addressed the question of beta(1)-adrenoceptor sensitivity by determining the inotropic potency and intrinsic activity of the beta(1)-adrenoceptor selective partial agonist (-)-RO363 and by carrying out both homogenate binding and quantitative beta-adrenoceptor autoradiography in atria obtained from patients treated or not treated with beta-blockers. In the course of the experiments it became apparent that (-)-RO363 also may cause agonistic effects through the third atrial beta-adrenoceptor. To assess whether (-)-RO363 also caused agonistic effects through beta(3)-adrenoceptors we studied its relaxant effects in rat colon and guinea-pig ileum, as well as receptor binding and adenylyl cyclase stimulation of chinese hamster ovary (CHO) cells expressing human beta(3)-adrenoceptors. 2 beta-Adrenoceptors were labelled with (-)-[I-125]-cyanopindolol. The density of both beta(1)- and beta(2)-adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenate binding. The affinities of (-)-RO363 for beta(1)-adrenoceptors (pK(i) = 8.0-7.7) and beta(2)-adrenoceptors (pK(i) = 6.1-5.8) were not significantly different in the two groups. 3 (-)-RO363 increased atrial force with a pEC(50) of 8.2 (beta-blocker treated) and 8.0 (non-beta-blocker treated) and intrinsic activity with respect to (-)-isoprenaline of 0.80 (beta-blocker treated) and 0.54 (non-beta-blocker treated) (P<0.001) and with respect to Ca2+ (7 mM) of 0.65 (beta-blocker treated) and 0.45 (non-beta-blocker treated) (P<0.01). The effects of (-)-RO363 were resistant to antagonism by the beta(2)-adrenoceptor antagonist, ICI 118,551 (50 nM). The effects of 0.3-10 nM (-)-RO363 were antagonized by 3-10 nM of the beta(1)-adrenoceptor selective antagonist CGP 20712A. The effects of 20-1000 nM (-)-RO363 were partially resistant to antagonism by 30-300 nM CGP 20712A. 4 (-)-RO363 relaxed the rat colon, partially precontracted by 30 mM KCl, with an intrinsic activity of 0.97 compared to (-)-isoprenaline. The concentration-effect curve to (-)-RO363 revealed 2 components, one antagonized by (-)-propranolol (200 nM) with pEC(50)=8.5 and fraction 0.66, the other resistant to (-)-propranolol (200 nM) with pEC(50)=5.6 and fraction 0.34 of maximal relaxation. 5 (-)-RO363 relaxed the longitudinal muscle of guinea-pig ileum, precontracted by 0.5 mu M histamine, with intrinsic activity of 1.0 compared to (-)-isoprenaline and through 2 components, one antagonized by (-)-propranolol (200 nM) with pEC(50)=8.7 and fraction 0.67, the other resistant to (-)-propranolol with pEC(50)=4.9 and fraction 0.33 of maximal relaxation. 6 (-)-RO363 stimulated the adenylyl cyclase of CHO cells expressing human beta(3)-adrenoceptors with pEC(50)=5.5 and intrinsic activity 0.74 with respect to (-)-isoprenaline (pEC(50)=5.9). (-)-RO363 competed for binding with [I-125]cyanopindolol at human beta(3)-adrenoceptors transfected into CHO cells with pK(i)=4.5. (-)-Isoprenaline (pk(i)=5.2) and (-)-CGP 12177A (pK(i)=6.1) also competed for binding at human beta(2)-adrenoceptors. 7 We conclude that under conditions used in this study, (-)-RO363 is a potent partial agonist for human beta(1)- and beta(3)-adrenoceptors and appears also to activate the third human atrial beta-adrenoceptor. (-)-RO363 relaxes mammalian gut through both beta(1)- and beta(3)-adrenoceptors. (-)-RO363, used as a beta(1)-adrenoceptor selective tool, confirms previous findings with (-)-noradrenaline that beta(1)-adrenoceptor mediated atrial effects are only slightly enhanced by chronic treatment of patients with beta-blockers. Chronic treatment with beta(1)-adrenoceptor-selective blockers does not significantly increase the density of human atrial beta(1)- and beta(2)-adrenoceptors.

Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single-pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C-terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co-localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re-expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.

Modulation of human cardiac function through 4 beta-adrenoceptor populations

In human heart there is now evidence for the involvement of four beta-adrenoceptor populations, three identical to the recombinant beta(1)-, beta(2)- and beta(3)-adrenoceptors, and a fourth as yet uncloned putative beta-adrenoceptor population, which we designate provisionally as the cardiac putative beta(4)-adrenoceptor. This review described novel features of beta-adrenoceptors as modulators of cardiac systolic and diastolic function. We also discuss evidence for modulation by unoccupied beta(1)- and beta(2)-adrenoceptors. Human cardiac and recombinant beta(1)- and beta(2)-adrenoceptors are both mainly coupled to adenylyl cyclase through Gs protein, the latter more tightly than the former. Activation of both human beta(1)- and beta(2)-adrenoceptors not only increases cardiac force during systole but also hastens relaxation through cyclic AMP-dependent phosphorylation of phospholamban and troponin I, thereby facilitating diastolic function. Furthermore, both beta(1) and beta(2)-adrenoceptors can mediate experimental arrhythmias in human cardiac preparations elicited by noradrenaline and adrenaline. Human ventricular beta(3)-adrenoceptors appear to be coupled to a pertussis toxin-sensitive protein (Gi?). beta(3)-Adrenoceptor-selective agonists shorten the action potential and cause cardiodepression, suggesting direct coupling of a Gi protein to a K+ channel. In a variety of species, including man, cardiac putative beta(4)-adrenoceptors mediate cardiostimulant effects of non-conventional partial agonists, i.e. high affinity beta(1)- and beta(2)-adrenoceptor blockers that cause agonist effects at concentrations considerably higher than those that block these receptors. Putative beta(4)-adrenoceptors appear to be coupled positively to a cyclic AMP-dependent cascade and can undergo some desensitisation.

Collagen is reduced and disrupted in human aneurysms and dissections of ascending aorta

In ascending aorta aneurysms, there is an enlargement of the whole vessel, whereas aortic dissections (ADs) are characterized by the cleavage of the wall into 2 sheets at the external half. We searched if alterations in collagen could be related to these diseases. Sections of aortas from 14 case patients with acute dissections, 10 case patients with aneurysms, and 9 control subjects were stained with picrosirius. Slides were analyzed under polarized microscopy to evaluate the structure of collagen fibers. The proportion of collagen was calculated in each half of the medial layer by color detection in a computerized image analysis system. Collagen appearance under polarized light was consistent with collagenolysis. The mean collagen proportions at the inner and outer halves, respectively, were 0.50 +/- 0.13 and 0.40 +/- 0.08 in the control group, 0.20 +/- 0.10 and 0.18 +/- 0.12 in the AD group, and 0.33 +/- 0.12 and 0.19 +/- 0.12 in the aneurysm group. The AD (P < .01) and control (P = .04) groups had less collagen at the external half, no difference was found in the aneurysm group (P = .71). In both halves, there was less collagen in the case patients than in the control subjects (all P < .01), but at the internal half, the decrease was significantly greater in the case patients with aneurysms than in those with dissections (P = .03; at the external half, P = .99). Aortic dissections and aneurysms show a decrease in collagen content that could be related to a weakness of the wall underlying the diseases, but the locations of the decrease differ: in dissections, it is situated mostly at the external portion of the media (site of cleavage), whereas in aneurysms, it is more diffuse, consistent with the global enlargement. (c) 2008 Elsevier Inc. All rights reserved.

Pressure-Frequency Sensing Subxiphoid Access System for Use in Percutaneous Cardiac Electrophysiology: Prototype Design and Pilot Study Results

We have designed, built, and tested an early prototype of a novel subxiphoid access system intended to facilitate epicardial electrophysiology, but with possible applications elsewhere in the body. The present version of the system consists of a commercially available insertion needle, a miniature pressure sensor and interconnect tubing, read-out electronics to monitor the pressures measured during the access procedure, and a host computer with user-interface software. The nominal resolution of the system is <0.1 mmHg, and it has deviations from linearity of <1%. During a pilot series of human clinical studies with this system, as well as in an auxiliary study done with an independent method, we observed that the pericardial space contained pressure-frequency components related to both the heart rate and respiratory rate, while the thorax contained components related only to the respiratory rate, a previously unobserved finding that could facilitate access to the pericardial space. We present and discuss the design principles, details of construction, and performance characteristics of this system.

Cortical Thickness Reduction of Normal Appearing Cortex in Patients with Polymicrogyria

OBJECTIVE To examine cortical thickness and volumetric changes in the cortex of patients with polymicrogyria, using an automated image analysis algorithm. METHODS Cortical thickness of patients with polymicrogyria was measured using magnetic resonance imaging (MRI) cortical surface-based analysis and compared with age-and sex-matched healthy subjects. We studied 3 patients with disorder of cortical development (DCD), classified as polymicrogyria, and 15 controls. Two experienced neuroradiologists performed a conventional visual assessment of the MRIs. The same data were analyzed using an automated algorithm for tissue segmentation and classification. Group and individual average maps of cortical thickness differences were produced by cortical surface-based statistical analysis. RESULTS Patients with polymicrogyria showed increased thickness of the cortex in the same areas identified as abnormal by radiologists. We also identified a reduction in the volume and thickness of cortex within additional areas of apparently normal cortex relative to controls. CONCLUSIONS Our findings indicate that there may be regions of reduced cortical thickness, which appear normal from radiological analysis, in the cortex of patients with polymicrogyria. This finding suggests that alterations in neuronal migration may have an impact in the cortical formation of the cortical areas that are visually normal. These areas are associated or occur concurrently with polymicrogyria.

An overview of the immunopathology of human paracoccidioidomycosis

We review here the advances in the understanding of the immunopathology of human paracoccidioidomycosis (PCM). Its investigation must take in account the intriguing natural history of the mycosis and its agent, providing clues to the mechanisms that lead to development of disease (unbalanced host-parasite relationship?) or to the clinically silent, chronic carrier state (balanced host-parasite relationship?), in exposed people living in endemic areas. Although the literature on this subject has progressed notably, the overall picture of what are the mechanisms of susceptibility or resistance continues to be fragmentary. Major advances were seen in the description of both the cytokines/chemokines associated to the different outcomes of the host-parasite interaction, and the fungus-monocyte/macrophage interaction, and cytokines released thereof by these cells. However, relatively few studies have attempted to modify, even in vitro, the patients` unbalanced immune reactivity. Consequently, the benefits of this improved knowledge did not yet reach clinical practice. Fortunately, the previous notion of the immune system as having two nearly independent arms, the innate and adaptive immunities, leaving a large gap between them, is now being overcome. Immunologists are now trying to dissect the connections between these two arms. This will certainly lead to more productive results. Current investigations should address the innate immunity events that trigger the IL-12/IFN-gamma axis and confer protection against PCM in those individuals living in endemic areas, who have been infected, but did not develop the mycosis.

An isolated perfused human placental lobule model for multiple indicator dilution studies

We describe a method for multiple indicator dilution studies in the isolated perfused human placental lobule developed to investigate the relationships between changes in pressure and flow and solute clearance. A peripheral lobule of a human placenta is perfused with a tissue culture-based medium and the perfusate oxygen tension, arterial and venous pressures, pH and perfusion temperature continuously monitored by a computerized system. Flow rates are readily changed. Bolus injections of vascular, extracellular and water space markers, and study compounds can be made into either maternal or fetal circulations, and precisely timed outflow fractions can be collected with computer-controlled fraction collectors, allowing simultaneous determination of concentration-time profiles of each marker. (C) 1997 Elsevier Science Inc.

Long-Term Astroglial Reaction and Neuronal Plasticity in the Subcortical Visual Pathways After a Complete Ablation of Telencephalon in Pigeons (Columba livia)

This paper analyzes the astroglial and neuronal responses in subtelencephalic structures, following a bilateral ablation of the telencephalon in the Columba livia pigeons. Control birds received a sham operation. Four months later the birds were sacrificed and their brains processed for glial fribillary acid protein (GFAP) and neurofilament immunohistochemistry, markers for astrocytes and neurons, respectively. Computer-assisted image analysis was employed for quantification of the immunoreactive labeling in the nucleus rotundus (N.Rt) and the optic tectum (OT) of the birds. An increased number of GFAP immunoreactive astrocytes were found in several subregions of the N.Rt (p .001), as well as in layers 1, 2cd, 3, and 6 of the OT (p .001) of the lesioned animals. Neurofilament immunoreactivity decreased massively in the entire N.Rt of the lesioned birds; however, remaining neurons with healthy aspect showing large cytoplasm and ramified branches were detected mainly in the periphery of the nucleus. In view of the recently described paracrine neurotrophic properties of the activated astrocytes, the data of the present study may suggest a long-lasting neuroglial interaction in regions of the lesioned bird brain far from injury. Such events may trigger neuronal plasticity in remaining brain structures that may lead spontaneous behavior recovery as the one promoted here even after a massive injury.

Early keratocyte apoptosis after epithelial scrape injury in the human cornea

Animal studies in mice, rats, rabbits, pigs and hens demonstrated that anterior keratocytes undergo programmed cell death or apoptosis after corneal epithelial injury. Many other wound healing changes subsequently follow the keratocyte apoptosis response. This study evaluated early keratocyte apoptosis after corneal epithelial scrape injury in human eyes scheduled for enucleation for malignancy. Two eyes had corneal epithelial scrape 1 h prior to the enucleation and another eye served as a control and had no corneal scrape prior to enucleation. One additional eye was enucleated, washed with balanced salt solution, and then had the corneal epithelium scraped 1 h prior to processing for analysis. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and confirmed by transmission electron microscopy (TEM). Anterior keratocyte apoptosis was detected in the three corneas that had epithelial scrape injury, but not in the control unwounded cornea. This study confirmed that keratocyte apoptosis is also an early response to corneal epithelial injury in humans and showed that tears are not essential for keratocyte apoptosis to occur in response to epithelial injury. (C) 2009 Elsevier Ltd. All rights reserved.

Impact of Nervous System Hyperalgesia on Pain, Disability, and Quality of Life in Patients With Knee Osteoarthritis: A Controlled Analysis

Objective. Refractory, disabling pain associated with knee osteoarthritis (OA) is usually treated with total knee replacement. However, pain in these patients might be associated with central nervous sensitization rather than peripheral inflammation and injury. We evaluated the presence of hyperalgesia in patients scheduled for a total knee replacement due to knee osteoarthritis with refractory pain, and we assessed the impact of pressure pain threshold measurements (PPT) on pain, disability, and quality of life of these patients. Methods. Sixty-two female patients were compared with 22 age-matched healthy controls without reported pain for the last year. PPT was measured at the lower extremities subcutaneous dermatomes, over the vastus medialis, adductor longus, rectus femoris, vastus lateralis, tibialis anterior, peroneus longus, iliacus, quadratus lumborum and popliteus muscles and at the supraspinous ligaments from L1-L5, over the L5-S1 and S1-S2 sacral areas and at the pes anserinus bursae and patellar tendon. Results. Patients with knee OA had significantly lower PPT over all evaluated structures versus healthy control subjects (P < 0.001). Lower PPT values were correlated with higher pain intensity, higher disability scores, and with poorer quality of life, except for the role-emotional and general health status. Combined PPT values over the patellar tendon, at the S2 subcutaneous dermatome and at the adductor longus muscle were the best predictors for visual analog scale and Western Ontario and McMaster Universities Osteoarthritis Index pain scores. Conclusion. Patients with pain due to osteoarthritis who were scheduled for total knee replacement showed hyperalgesia of nervous system origin that negatively impacted pain, knee functional capacity, and most aspects of quality of life.

In situ apoptosis of adaptive immune cells and the cellular escape of rabies virus in CNS from patients with human rabies transmitted by Desmodus rotundus

The aim of the current study was to investigate the apoptosis of neurons, astrocytes and immune cells from human patients that were infected with rabies virus by vampire bats bite. Apoptotic neurons were identified by their morphology and immune cells were identified using double immunostaining. There were very few apoptotic neurons present in infected tissue samples, but there was an increase of apoptotic infiltrating CD4+ and TCD8+ adaptive immune cells in the rabies infected tissue. No apoptosis was present in NK, macrophage and astrocytes. The dissemination of the human rabies virus within an infected host may be mediated by viral escape of the virus from an infected cell and may involve an anti-apoptotic mechanism, which does not kill the neuron or pro-apoptosis of TCD4+ and TCD8+ lymphocytes and which allows for increased proliferation of the virus within the CNS by attenuation of the adaptive immune response. (C) 2011 Elsevier B.V. All rights reserved.

Classification model based on Raman spectra of selected morphological and biochemical tissue constituents for identification of atherosclerosis in human coronary arteries

This study presents the results of Raman spectroscopy applied to the classification of arterial tissue based on a simplified model using basal morphological and biochemical information extracted from the Raman spectra of arteries. The Raman spectrograph uses an 830-nm diode laser, imaging spectrograph, and a CCD camera. A total of 111 Raman spectra from arterial fragments were used to develop the model, and those spectra were compared to the spectra of collagen, fat cells, smooth muscle cells, calcification, and cholesterol in a linear fit model. Non-atherosclerotic (NA), fatty and fibrous-fatty atherosclerotic plaques (A) and calcified (C) arteries exhibited different spectral signatures related to different morphological structures presented in each tissue type. Discriminant analysis based on Mahalanobis distance was employed to classify the tissue type with respect to the relative intensity of each compound. This model was subsequently tested prospectively in a set of 55 spectra. The simplified diagnostic model showed that cholesterol, collagen, and adipocytes were the tissue constituents that gave the best classification capability and that those changes were correlated to histopathology. The simplified model, using spectra obtained from a few tissue morphological and biochemical constituents, showed feasibility by using a small amount of variables, easily extracted from gross samples.

Lung Pathology in Fatal Novel Human Influenza A (H1N1) Infection

Rationale: There are no reports of the systemic human pathology of the novel swine H1N1 influenza (S-OIV) infection. Objectives: The autopsy findings of 21 Brazilian patients with confirmed S-OIV infection are presented. These patients died in the winter of the southern hemisphere 2009 pandemic, with acute respiratory failure. Methods: Lung tissue was submitted to virologic and bacteriologic analysis with real-time reverse transcriptase polymerase chain reaction and electron microscopy. Expression of toll-like receptor (TLR)-3, IFN-gamma, tumor necrosis factor-alpha, CD8(+) T cells and granzyme B(+) cells in the lungs was investigated by immunohistochemistry. Measurements and Main Results: Patients were aged from 1 to 68 years (72% between 30 and 59 yr) and 12 were male. Sixteen patients had preexisting medical conditions. Diff use alveolar damage was present in 20 individuals. in six patients, diffuse alveolar damage was associated with necrotizing bronchiolitis and in five with extensive hemorrhage. There was also a cytopathic effect in the bronchial and alveolar epithelial cells, as well as necrosis, epithelial hyperplasia, and squamous metaplasia of the large airways. There was marked expression of TLR-3 and IFN-gamma and a large number of CD8(+) T cell sand granzyme B(+) cells within the lung tissue. Changes in other organs were mainly secondary to multiple organ failure. Conclusions: Autopsies have shown that the main pathological changes associated with S-OIV infection are localized to the lungs, where three distinct histological patterns can be identified. We also show evidence of ongoing pulmonary aberrant immune response. Our results reinforce the usefulness of autopsy in increasing the understanding of the novel human influenza A (H1N1) infection.