Ophthalmic alterations in horses with leptospirosis by serovar Icterohaemorrhagiae in Rio de Janeiro, Brazil

The objective of the study was to determine the association between clinical ophthalmic alterations and seroreactivity to leptospirosis by serovar Icterohaemorrhagiae in horses in Rio de Janeiro, Brazil. A total of 199 horses were studied. A microscopic agglutination test (MAT) was used to detect specific anti-Leptospira antibodies in blood serum. A total of 107 (53.8%) horses were seroreactive (titres > 200); 54 had high (> 800) titres, of which 44 were against serovar Icterohaemorrhagiae. Forty-two out of these 44, plus 40 seronegative horses (titers < 100) were given detailed ophthalmic examinations. Epiphora, ocular congestion, blepharospasm, photophobia, and peripapillary focal depigmentation were the most frequent alterations in seroreactive horses. Most ocular alterations were significantly more frequent in seroreactive horses. Horses seroreactive for leptospirosis (serovar Icterohaemorrhagiae) had a significantly higher prevalence of ophthalmic alterations than seronegative horses, providing additional evidence for an association between leptospirosis and equine uveitis.

Alterations in The States and Contents of Consciousness: Empirical and Theoretical Aspects

The main purpose of the present doctoral thesis is to investigate subjective experiences and cognitive processes in four different types of altered states of consciousness: naturally occurring dreaming, cognitively induced hypnosis, pharmacologically induced sedation, and pathological psychosis. Both empirical and theoretical research is carried out, resulting in four empirical and four theoretical studies. The thesis begins with a review of the main concepts used in consciousness research, the most influential philosophical and neurobiological theories of subjective experience, the classification of altered states of consciousness, and the main empirical methods used to study consciousness alterations. Next, findings of the original studies are discussed, as follows. Phenomenal consciousness is found to be dissociable from responsiveness, as subjective experiences do occur in unresponsive states, including anaesthetic-induced sedation and natural sleep, as demonstrated by post-awakening subjective reports. Two new tools for the content analysis of subjective experiences and dreams are presented, focusing on the diversity, complexity and dynamics of phenomenal consciousness. In addition, a new experimental paradigm of serial awakenings from non-rapid eye movement sleep is introduced, which enables more rapid sampling of dream reports than has been available in previous studies. It is also suggested that lucid dreaming can be studied using transcranial brain stimulation techniques and systematic analysis of pre-lucid dreaming. For blind judges, dreams of psychotic patients appear to be indistinguishable from waking mentation reports collected from the same patients, which indicates a close resemblance of these states of mind. However, despite phenomenological similarities, dreaming should not be treated as a uniform research model of psychotic or intact consciousness. Contrary to this, there seems to be a multiplicity of routes of how different states of consciousness can be associated. For instance, seemingly identical time perception distortions in different alterations of consciousness may have diverse underlying causes for these distortions. It is also shown that altered states do not necessarily exhibit impaired cognitive processing compared to a baseline waking state of consciousness: a case study of time perception in a hypnotic virtuoso indicates a more consistent perceptual timing under hypnosis than in a waking state. The thesis ends with a brief discussion of the most promising new perspectives for the study of alterations of consciousness.

Utilization of microsatellites for the analysis of genomic alterations in colorectal cancers in Brazil

Two different pathogenetic mechanisms are proposed for colorectal cancers. One, the so-called "classic pathway", is the most common and depends on multiple additive mutational events (germline and/or somatic) in tumor suppressor genes and oncogenes, frequently involving chromosomal deletions in key genomic regions. Methodologically this pathway is recognizable by the phenomenon of loss of heterozygosity. On the other hand, the "mutator pathway" depends on early mutational loss of the mismatch repair system (germline and/or somatic) leading to accelerated accumulation of gene mutations in critical target genes and progression to malignancy. Methodologically this second pathway is recognizable by the phenomenon of microsatellite instability. The distinction between these pathways seems to be more than academic since there is evidence that the tumors emerging from the mutator pathway have a better prognosis. We report here a very simple methodology based on a set of tri-, tetra- and pentanucleotide repeat microsatellites allowing the simultaneous study of microsatellite instability and loss of heterozygosity which could allocate 70% of the colorectal tumors to the classic or the mutator pathway. The ease of execution of the methodology makes it suitable for routine clinical typing

Genetic alterations in head and neck squamous cell carcinomas

The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations) and tumor suppressor gene inactivation (loss of function mutations), leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC) development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease.

Infrequent p53 gene alterations in ulcerative colitis

The purpose of this study was to determine whether point mutations and loss of the p53 gene take place in ulcerative colitis which is histologically negative for dysplasia. DNA was extracted from 13 frozen rectal or colon biopsies and blood samples. Ulcerative colitis was classified histologically as active (10 cases) and inactive (3 cases). Exons 5-8 were amplified by PCR, treated with exonuclease and shrimp alkaline phosphatase and sequenced by the dideoxy chain termination method with the Sequenase Version 2.0 DNA sequencing kit. PCR products of intron 6 and exon 4 were digested with MspI and AccII, respectively, for RFLP analysis. No p53 gene mutation was detected in these cases. The number of informative patients for loss of heterozygosity (LOH) at the p53 intron 6 was high, 11 out of 12 (92%), whereas no LOH was observed. LOH affecting p53 exon 4 was not detected in lesions from 5 of 12 patients (42%). In ulcerative colitis, tumor progression is similar to that in sporadic colon cancer, and other oncogenes and tumor suppressor genes are likely to be mutated before the p53 gene.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5%) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

Muscle sympathetic nerve activity and hemodynamic alterations in middle-aged obese women

To study the relationship between the sympathetic nerve activity and hemodynamic alterations in obesity, we simultaneously measured muscle sympathetic nerve activity (MSNA), blood pressure, and forearm blood flow (FBF) in obese and lean individuals. Fifteen normotensive obese women (BMI = 32.5 ± 0.5 kg/m²) and 11 age-matched normotensive lean women (BMI = 22.7 ± 1.0 kg/m²) were studied. MSNA was evaluated directly from the peroneal nerve by microneurography, FBF was measured by venous occlusion plethysmography, and blood pressure was measured noninvasively by an autonomic blood pressure cuff. MSNA was significantly increased in obese women when compared with lean control women. Forearm vascular resistance and blood pressure were significantly higher in obese women than in lean women. FBF was significantly lower in obese women. BMI was directly and significantly correlated with MSNA, blood pressure, and forearm vascular resistance levels, but inversely and significantly correlated with FBF levels. Obesity increases sympathetic nerve activity and muscle vascular resistance, and reduces muscle blood flow. These alterations, taken together, may explain the higher blood pressure levels in obese women when compared with lean age-matched women.

Heart rate and body weight alterations in juvenile specimens of the tropical land snail Megalobulimus sanctipauli during dormancy

The time course of heart rate and body weight alterations during the natural period of dormancy were determined in active feeding and dormant juvenile specimens of Megalobulimus sanctipauli. In both groups, heart rate markedly decreased during the first 40 days of dormancy, tending to stabilize thereafter. This time period coincided with the decrease in environmental temperature during autumn-winter. At the end of the dormancy period, surviving active feeding and dormant snails showed a significant decrease in heart rate which, however, was significantly greater in the latter group. Total body weight decreased concomitantly with heart rate in dormant snails but remained constant in active feeding snails. Body hydration induced significant increases in weight and heart rate in surviving dormant snails. Feeding following hydration promoted a new significant increase in heart rate but not in weight. These results indicate that the decrease in heart rate observed in juvenile specimens of M. sanctipauli during dormancy may be due to at least three factors: 1) decrease in environmental temperature during autumn-winter, 2) starvation which leads to the depletion of endogenous fuel reserves and to a probable decrease in hemolymph nutrient levels, and 3) dehydration which leads to a probable decrease in hemolymph volume and venous return and/or to an increase in hemolymph osmolarity.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58% reduction of tyrosine hydroxylase in the substantia nigra, and a 35% reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 ± 32.5 to 58 ± 16.5 ng/mg protein (47.2% reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 ± 44 protein in control and 175 ± 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Rarity of DNA sequence alterations in the promoter region of the human androgen receptor gene

The human androgen receptor (AR) gene promoter lies in a GC-rich region containing two principal sites of transcription initiation and a putative Sp1 protein-binding site, without typical "TATA" and "CAAT" boxes. It has been suggested that mutations within the 5'untranslated region (5'UTR) may contribute to the development of prostate cancer by changing the rates of gene transcription and/or translation. In order to investigate this question, the aim of the present study was to search for the presence of mutations or polymorphisms at the AR-5'UTR in 92 prostate cancer patients, where histological diagnosis of adenocarcinoma was established in specimens obtained from transurethral resection or after prostatectomy. The AR-5'UTR was amplified by PCR from genomic DNA samples of the patients and of 100 healthy male blood donors, included as controls. Conformation-sensitive gel electrophoresis was used for DNA sequence alteration screening. Only one band shift was detected in one individual from the blood donor group. Sequencing revealed a new single nucleotide deletion (T) in the most conserved portion of the promoter region at position +36 downstream from the transcription initiation site I. Although the effect of this specific mutation remains unknown, its rarity reveals the high degree of sequence conservation of the human androgen promoter region. Moreover, the absence of detectable variation within the critical 5'UTR in prostate cancer patients indicates a low probability of its involvement in prostate cancer etiology.

Effects of chronic ethanol treatment on monoamine levels in rat hippocampus and striatum

We studied the effects of ethanol on concentrations of noradrenaline (NE), dopamine (DA) and serotonin (5-HT) and their metabolites in rat hippocampus and striatum. Ethanol (2 or 4 g/kg, po, from a 20% aqueous solution) was administered daily to male Wistar rats (4-13 per group) for 30 days and animals were sacrificed 30 min or 48 h after the last administration. Monoamines were measured by HPLC and considered significant at P < 0.05. A 47% increase in 5-HT levels was observed in the hippocampus with 4 g/kg ethanol in the 30-min protocol. Ethanol (2 and 4 g/kg) decreased DA (2114.5 ± 126.4 and 1785.1 ± 234.2 ng/g wet tissue, respectively) and 3,4-dihydroxyphenylacetic acid (DOPAC, 1477.6 ± 132.1 and 1218.8 ± 271.7 ng/g wet tissue, respectively) levels, while the higher dose also decreased NE (159.8 ± 13.5), 5-HT (228.0 ± 46.8) and 5-hydroxy-3-indoleacetic acid (5-HIAA, 304.4 ± 37.2 ng/g wet tissue), in the striatum after a 48-h withdrawal as compared to controls (DA: 3063.9 ± 321.3; DOPAC: 2379.6 ± 256.0; NE: 292.8 ± 50.2; 5-HT: 412.4 ± 36.2; 5-HIAA: 703.9 ± 61.4 ng/g wet tissue). In the 30-min protocol, ethanol (2 or 4 g/kg) decreased striatal NE (66 and 70%) and DA (50 and 36%) levels. On the other hand, increases were seen in 5-HIAA (146 and 153%) and 5-HT (59 and 86%) levels. Ethanol (2 g/kg, po) increased the homovanillic acid (HVA)/DA ratio (129%) in the striatum in the 30-min protocol, while at the higher dose it increased the HVA/DA ratio in the 48-h protocol (61%). These results indicate alterations in monoamines, mainly in the striatum, after chronic ethanol, which are influenced by dose and by the length of time after the last drug administration.

Mechanisms underlying gas exchange alterations in an experimental model of pulmonary embolism

The aim of the present study was to determine the ventilation/perfusion ratio that contributes to hypoxemia in pulmonary embolism by analyzing blood gases and volumetric capnography in a model of experimental acute pulmonary embolism. Pulmonary embolization with autologous blood clots was induced in seven pigs weighing 24.00 ± 0.6 kg, anesthetized and mechanically ventilated. Significant changes occurred from baseline to 20 min after embolization, such as reduction in oxygen partial pressures in arterial blood (from 87.71 ± 8.64 to 39.14 ± 6.77 mmHg) and alveolar air (from 92.97 ± 2.14 to 63.91 ± 8.27 mmHg). The effective alveolar ventilation exhibited a significant reduction (from 199.62 ± 42.01 to 84.34 ± 44.13) consistent with the fall in alveolar gas volume that effectively participated in gas exchange. The relation between the alveolar ventilation that effectively participated in gas exchange and cardiac output (V Aeff/Q ratio) also presented a significant reduction after embolization (from 0.96 ± 0.34 to 0.33 ± 0.17 fraction). The carbon dioxide partial pressure increased significantly in arterial blood (from 37.51 ± 1.71 to 60.76 ± 6.62 mmHg), but decreased significantly in exhaled air at the end of the respiratory cycle (from 35.57 ± 1.22 to 23.15 ± 8.24 mmHg). Exhaled air at the end of the respiratory cycle returned to baseline values 40 min after embolism. The arterial to alveolar carbon dioxide gradient increased significantly (from 1.94 ± 1.36 to 37.61 ± 12.79 mmHg), as also did the calculated alveolar (from 56.38 ± 22.47 to 178.09 ± 37.46 mL) and physiological (from 0.37 ± 0.05 to 0.75 ± 0.10 fraction) dead spaces. Based on our data, we conclude that the severe arterial hypoxemia observed in this experimental model may be attributed to the reduction of the V Aeff/Q ratio. We were also able to demonstrate that V Aeff/Q progressively improves after embolization, a fact attributed to the alveolar ventilation redistribution induced by hypocapnic bronchoconstriction.

Cardiac and pulmonary alterations in symptomatic and asymptomatic dogs infectednaturally with Leishmania (Leishmania) chagasi

Fifteen symptomatic and seven asymptomatic dogs infected naturally with Leishmania chagasi were examined in order to identify the presence of parasites and changes in heart and lung. Histopathological, cytological, and immunohistochemical analyses were performed on samples of heart and lung tissues. An inflammatory reaction characterized by inflammatory mononuclear, perivascular and intermuscular infiltrates was observed in both symptomatic and asymptomatic animals on histopathological analysis of the heart. In the lung, there was thickening of the alveolar septa due to congestion, edema, inflammatory infiltrate, and fibroblast proliferation. A focal reaction was observed although a diffuse reaction was present in both groups. On cytological examination, heart and lung imprints revealed amastigotes in two symptomatic animals and heart imprints were found in 1 asymptomatic dog. Immunoperoxidase staining showed amastigotes in the lung and heart of only 1 of 6 symptomatic animals examined. Within the ethical principles and limits of this research, it can be inferred that the study of heart and lung alterations in canine visceral leishmaniasis is increasingly important for understanding the problem related to humans. Dogs with visceral leishmaniasis were a good experimental model, since infection was caused by the same agent and the animals developed clinical, pathological and immunological alterations similar to those observed in humans.

Plasma concentrations and placental immunostaining of interleukin-10 and tumornecrosis factor-α as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

Interleukin-10 (IL-10) appears to be the key cytokine for the maintenance of pregnancy and inhibits the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α). However, there are no studies evaluating the profile of these cytokines in diabetic rat models. Thus, our aim was to analyze IL-10 and TNF-α immunostaining in placental tissue and their respective concentrations in maternal plasma during pregnancy in diabetic rats in order to determine whether these cytokines can be used as predictors of alterations in the embryo-fetal organism and in placental development. These parameters were evaluated in non-diabetic (control; N = 15) and Wistar rats with streptozotocin (STZ)-induced diabetes (N = 15). At term, the dams (100 days of life) were killed under anesthesia and plasma and placental samples were collected for IL-10 and TNF-α determinations by ELISA and immunohistochemistry, respectively. The reproductive performance was analyzed. Plasma IL-10 concentrations were reduced in STZ rats compared to controls (7.6 ± 4.5 vs 20.9 ± 8.1 pg/mL). The placental scores of immunostaining intensity did not differ between groups (P > 0.05). Prevalence analysis showed that the IL-10 expression followed TNF-α expression, showing a balance between them. STZ rats also presented impaired reproductive performance and reduced plasma IL-10 levels related to damage during early embryonic development. However, the increased placental IL-10 as a compensatory mechanism for the deficit of maternal regulation permitted embryo development. Therefore, the data suggest that IL-10 can be used as a predictor of changes in the embryo-fetal organism and in placental development in pregnant diabetic rats.