Rheumatic heart disease: proinflammatory cytokines play a role in the progression and maintenance of valvular lesions

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.     

Bcl-x(L)-mediated changes in metabolic pathways of breast cancer cells: from survival in the blood stream to organ-specific metastasis

Bcl-x(L) protein plays a role in breast cancer dormancy, promoting survival of cells in metastatic foci by counteracting the proapoptotic signals in the microenvironment. The aim of this study was to identify phenotypes mediated by Bcl-x(L) in breast cancer cells that enhance in vivo survival of clinical metastases. 435/Bcl-x(L) or 435/Neo human breast cancer cells were injected into the inguinal mammary gland of nude mice, and tumors, metastases in lymph node, lung, and bone, and bloodstream surviving cells were examined. Proteomic analysis identified 17 proteins that were overexpressed (more than twofold) or underexpressed (less than twofold) in metastases. A protein interaction program allowed us to functionally associate peroxiredoxin 3, peroxiredoxin 2, carbonyl reductase 3, and enolase 1, suggesting a role for cellular responses to oxidative stress in metastasis organ selection. The prediction included proteins involved in redox systems, kinase pathways, and the ATP synthase complex. Furthermore, the interaction of redox proteins with enolase 1 suggests a connection between glycolysis and antioxidant pathways, enabling achievement of a high metastatic activity. In conclusion, Bcl-x(L) mediates a phenotype in which redox pathways and glycolysis are coupled to protect breast cancer metastatic cells during transit from the primary tumor to the metastatic state.     

Electrophysiological analysis of a sensorimotor integration task

The present experiment aimed at investigating electrophysiologic changes observed as beta band asymmetry, by Quantitative Electroencephalography (qEEG), when individuals performed a reaching motor task (catching a ball in free fall). The sample was composed of 23 healthy individuals, of both sexes, with ages varying between 25 and 40 years old. All the subjects were right handed. A two-way ANOVA was applied for the statistical analysis, to verify the interaction between task moment (i.e., 2 s before and 2 s after ball's fall) and electrode (i.e., frontal, central and temporal regions). The first analysis compared electrodes placed over the somatosensory cortex. Central sites (C3–C4) were compared with temporal regions (T3–T4). The results showed a main effect for moment and position. The second analysis was focused over the premotor cortex, which was represented by the electrodes placed on the frontal sites (F3–F4 versus F7–F8), and a main effect was observed for position. Taken together, these results show a pattern of asymmetry in the somatosensory cortex, associated with a preparatory mechanism when individuals have to catch an object during free fall. With respect to task moment, after the ball's fall, the asymmetry was reduced. Moreover, the difference in asymmetry between the observed regions were related to a supposed specialization of areas (i.e., temporal and central). The temporal region was associated with cognitive processes involved in the motor action (i.e., explicit knowledge). On the other hand, the central sites were related to the motor control mechanisms per se (i.e., implicit knowledge). The premotor cortex, represented by two frontal regions (i.e., F3–F4 versus F7–F8), showed a decrease on neural activity in the contralateral hemisphere (i.e., to the right hand). This result is in agreement with other experiments suggesting a participation of the frontal cortex in the planning of the apprehension task. This sensorimotor paradigm may contribute to the repertoire of tasks used to study clinical conditions such as depression, alzheimer and Parkinson diseases.     

Cervical cancer screening program of Paraná: cytohistological correlation results after five years

Since its inception in November 1997, the Cervical Cancer Screening Program of Paraná (CCSPP), Brazil, has resulted in the cytological screening of 2,244,158 women, the coverage of the female population increasing from 43% to 86%. One thousand six hundred one cases screened by cytology, submitted to colposcopy, and subjected to treatment were selected. Cytopathological results were compared with those obtained on the basis of histological analyses of the loop electrical excision procedure specimens, and were subjected to statistical analyses. The data obtained were then compared with cytohistological correlation results from the first year of the program. Considering the exact correlation between cytological and histological diagnoses, the correlation index increased from 53.34% in the first year to 67.3% at the end of 5 yr of the program. Variations that occurred in each diagnostic category are discussed. This study demonstrates a significant improvement in the concordance between cytological and histological results for the 5-yr period compared with the first year of the CCSPP.     

Importance of anesthesia for the genesis of neurogenic pulmonary edema in spinal cord injury

There are reports describing both provocation and inhibition of neurogenic pulmonary edema by anesthetic drugs. Therefore, we compared the effect of two types of anesthesia on the formation of neurogenic pulmonary edema in rats with balloon-induced acute spinal cord injury. Animals with sham procedure (group 1) were anesthesized by intraperitoneal sodium pentobarbital. In the experimental groups, rats were submitted to acute spinal cord lesion by insufflations of a balloon in the epidural space at T8 for 1 min (group 3 under i.p. sodium pentobarbital and group 2 under i.p. xylazine-ketamine anesthesia). In rats with pentobarbital anesthesia, systolic blood pressure doubled the baseline value during compression, whereas this effect was less pronounced in the ketamine-xylazine group. The pulmonary index (100 x wet lung weight/body weight) was 0.395 (+/-0.018) in sham-operated rats, rose to 0.499 (+/-0.060) in group 2, and was maximum under pentobarbital anesthesia (0.639+/-0.14; p=0.0018). Histologic examination of the spinal cord showed parenchymal ruptures and acute hemorrhage. Comparison of the pulmonary index with histologic slides of lung parenchyma revealed that relevant intra-alveolar edema occurred only for index values above 0.55. On electron microscopy, endothelial alterations, and damage of the alveolar lining cells were found. Our study indicates that neurogenic pulmonary edema caused by spinal cord injury is less pronounced in rats under xylazine-ketamine anesthesia, when compared with pentobarbital.     

Transient elevation of spontaneous release at the frog neuromuscular junction following acetylcholine iontophoresis

Miniature end-plate currents (m.e.p.c.s) were recorded from frog neuromuscular junctions using a two-electrode voltage clamp. The m.e.p.c. frequency was temporarily elevated following 10 s iontophoretic applications of acetylcholine (ACh) when the junctions were clamped at 100 mV. This post-ACh burst of quanta was also observed at unclamped junctions. At –100 mV, the intensity of the burst was proportional to the amount of current flowing across the end-plate during ACh iontophoresis, but usually did not reach its peak until the end-plate receptor channels were almost completely closed. Addition of 0.5 M TTX to the Ringer's solution, or total replacement of sodium with quanidine, lithium, or methylamine did not inhibit the burst. No burst was observed in Ca²⁺-free, EGTA solutions, or in Ca²⁺-free solutions containing 2 mM Mn²⁺. Sr²⁺ effectively substituted for Ca²⁺. Addition of 2 mM Co²⁺ or Mn²⁺ to normal Ringer's did not inhibit the burst. Presynaptic muscarinic receptors did not obviously contribute to the burst, since it was not blocked by atropine, nor produced by oxotremorine or pilocarpine. The ACh analogs carbachol and acetyl--methylcholine also produced the burst. The burst was highly dependent on the muscle membrane potential during the period of ACh iontophoresis, becoming more intense at potentials negative to –100 mV and disappearing at –60 mV. The critical importance of the post-synaptic membrane potential suggests that the burst may be due to an action of the muscle end-plate on the motor nerve terminal, possibly by the movement of an anionic substance through open end-plate receptor channels, but this hypothesis does not account for the delay of the burst until near the end of the ACh-induced end-plate current.     

Identification of orchard grass (Dactylis glomerata) pollen allergens following electrophoretic transfer to nitrocellulose

Orchard grass (cocksfoot) pollen extracts, fractionated by polyacrylamide gradient electrophoresis or SDS gel electrophoresis were electroblotted onto nitrocellulose membranes and probed with sera from orchard grass pollen-allergic patients and 125I-anti-human IgE. The IgE-binding components of the pollen were detected by autoradiography. Elution studies showed that allergens could be extracted immediately and continuously over a 3-hour period. Two fractions of MWs 28,000 and 30,000 could be detected only after 20 min extraction. SDS-PAGE separations gave the better resolution revealing 19 electrophoretically-separate components, 13 of which bound human IgE. All of the IgE-binding components had MWs in the range 14,000 - 70,000. Three of the bands bound IgE from more than 85% of the serum samples. Following gradient gel electrophoresis, IgE binding was exhibited by 10 bands in the range MW 5,000 to greater than 669,000. The technique used allows one to quantitatively examine patients' sera for allergen-specific IgE antibodies and to identify the clinically important allergens. Results revealed numerous allergenic components over a wide MW range while patterns of IgE binding with different patients' sera demonstrated a great diversity of IgE antibody responses. This study demonstrates the suitability of the electroblotting technique combined with autoradiography for the investigation of allergenic components of grass pollen extracts and hence has application to extract standardization and immunotherapy. Such studies can be carried out rapidly, economically and with a high degree of sensitivity.     

A re-examination of ryegrass (Lolium perenne) pollen allergens

Electroblotting of crude ryegrass pollen extracts and purified group I, II and III allergens identified 14 IgE-binding components, 8 of which were previously unrecognized. In addition to allergen groups I, II and III, which are already regarded as clinically important, and on the basis of the frequencies and intensities of IgE binding with sera from 42 ryegrass pollen-allergic patients, proteins with molecular weights (MWs) of 60, 32, 30 and 28 kD were identified as allergens of possible major clinical importance. Six other pollen components with MWs ranging from 23 to 80 kD and which reacted with IgE antibodies in the sera of 33-50% of patients, should also be viewed as proteins with potential clinical relevance for at least a proportion of the patients. The electrophoretic separation patterns of ryegrass pollen extracts in both alkaline and acid gels and IgE-probed membrane transfers produced in this study should serve as useful reference patterns for standardization purposes. In addition, the identification of the complete allergen recognition pattern by individual patients will permit safer and more effective diagnosis and therapy of ryegrass pollen sensitivities.     

Detection of house dust mite allergens and frequency of IgE binding following electroblotting and enzyme immunoassay

Mite allergens, fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 45 mite atopic sera and an enzyme immunostaining assay for IgE. More than 20 different mite components bound IgE and almost every serum showed a different pattern of binding. 7 of the 20 components were bound by half of the sera and 70% were bound by at least 20% of the sera. These results demonstrate a greater number of house dust mite allergens and a far greater diversity of the IgE antibody response to mite allergens than has previously been described.     

Standardization of rye-grass pollen (Lolium perenne) extract. An immunochemical and physicochemical assessment of six candidate international reference preparations

Six candidate extracts of Lolium perenne (rye-grass) pollen have been studied in 6 laboratories using a variety of immunochemical and physicochemical techniques. Radioallergosorbent test inhibition, crossed immunoelectrophoresis, crossed radio-immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with immunoblot, thin-layer isoelectric focusing and enzyme-linked immunosorbent assay inhibition were used to evaluate each of the coded extracts. The source materials were also studied for identity and possible contamination by light microscopy. On the basis of these data, the Rye-Grass Working Party recommended to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies that the extract coded C be chosen as the candidate international reference preparation.