Role of non-nitric oxide non-prostaglandin endothelium-derived relaxing factor(s) in bradykinin vasodilation

The most conspicuous effect of bradykinin following its administration into the systemic circulation is a transient hypotension due to vasodilation. In the present study most of the available evidence regarding the mechanisms involved in bradykinin-induced arterial vasodilation is reviewed. It has become firmly established that in most species vasodilation in response to bradykinin is mediated by the release of endothelial relaxing factors following the activation of B2-receptors. Although in some cases the action of bradykinin is entirely mediated by the endothelial release of nitric oxide (NO) and/or prostacyclin (PGI2), a large amount of evidence has been accumulated during the last 10 years indicating that a non-NO/PGI2 factor accounts for bradykinin-induced vasodilation in a wide variety of perfused vascular beds and isolated small arteries from several species including humans. Since the effect of the non-NO/PGI2 endothelium-derived relaxing factor is practically abolished by disrupting the K+ electrochemical gradient together with the fact that bradykinin causes endothelium-dependent hyperpolarization of vascular smooth muscle cells, the action of such factor has been attributed to the opening of K+ channels in these cells. The pharmacological characteristics of these channels are not uniform among the different blood vessels in which they have been examined. Although there is some evidence indicating a role for KCa or KV channels, our findings in the mesenteric bed together with other reports indicate that the K+ channels involved do not correspond exactly to any of those already described. In addition, the chemical identity of such hyperpolarizing factor is still a matter of controversy. The postulated main contenders are epoxyeicosatrienoic acids or endocannabinoid agonists for the CB1-receptors. Based on the available reports and on data from our laboratory in the rat mesenteric bed, we conclude that the NO/PGI2-independent endothelium-dependent vasodilation induced by BK is unlikely to involve a cytochrome P450 arachidonic acid metabolite or an endocannabinoid agonist.

Detection of point mutations by non-isotopic single strand conformation polymorphism

We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens.

The photodynamic and non-photodynamic actions of porphyrins

Porphyrias are a family of inherited diseases, each associated with a partial defect in one of the enzymes of the heme biosynthetic pathway. In six of the eight porphyrias described, the main clinical manifestation is skin photosensitivity brought about by the action of light on porphyrins, which are deposited in the upper epidermal layer of the skin. Porphyrins absorb light energy intensively in the UV region, and to a lesser extent in the long visible bands, resulting in transitions to excited electronic states. The excited porphyrin may react directly with biological structures (type I reactions) or with molecular oxygen, generating excited singlet oxygen (type II reactions). Besides this well-known photodynamic action of porphyrins, a novel light-independent effect of porphyrins has been described. Irradiation of enzymes in the presence of porphyrins mainly induces type I reactions, although type II reactions could also occur, further increasing the direct non-photodynamic effect of porphyrins on proteins and macromolecules. Conformational changes of protein structure are induced by porphyrins in the dark or under UV light, resulting in reduced enzyme activity and increased proteolytic susceptibility. The effect of porphyrins depends not only on their physico-chemical properties but also on the specific site on the protein on which they act. Porphyrin action alters the functionality of the enzymes of the heme biosynthetic pathway exacerbating the metabolic deficiencies in porphyrias. Light energy absorption by porphyrins results in the generation of oxygen reactive species, overcoming the protective cellular mechanisms and leading to molecular, cell and tissue damage, thus amplifying the porphyric picture.

Increased training prevents the impairing effect of intra-amygdala infusion of the non-NMDA receptor antagonist CNQX on inhibitory avoidance expression

Intra-amygdala infusion of the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) prior to testing impairs inhibitory avoidance retention test performance. Increased training attenuates the impairing effects of amygdala lesions and intra-amygdala infusions of CNQX. The objective of the present study was to determine the effects of additional training on the impairing effects of intra-amygdala CNQX on expression of the inhibitory avoidance task. Adult female Wistar rats bilaterally implanted with cannulae into the border between the central and the basolateral nuclei of the amygdala were submitted to a single session or to three training sessions (0.2 mA, 24-h interval between sessions) in a step-down inhibitory avoidance task. A retention test session was held 48 h after the last training. Ten minutes prior to the retention test session, the animals received a 0.5-µl infusion of CNQX (0.5 µg) or its vehicle (25% dimethylsulfoxide in saline). The CNQX infusion impaired, but did not block, retention test performance in animals submitted to a single training session. Additional training prevented the impairing effect of CNQX. The results suggest that amygdaloid non-NMDA receptors may not be critical for memory expression in animals given increased training.

Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases

Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis) and non-autoimmune (subacute thyroiditis) disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.

Application of isotope-selective non-dispersive infrared spectrometry for the evaluation of the 13C-urea breath test: comparison with three concordant methods

The aim of this work was to compare the performance of isotope-selective non-dispersive infrared spectrometry (IRIS) for the 13C-urea breath test with the combination of the 14C-urea breath test (14C-UBT), urease test and histologic examination for the diagnosis of H. pylori (HP) infection. Fifty-three duodenal ulcer patients were studied. All patients were submitted to gastroscopy to detect HP by the urease test, histologic examination and 14C-UBT. To be included in the study the results of the 3 tests had to be concordant. Within one month after admission to the study the patients were submitted to IRIS with breath samples collected before and 30 min after the ingestion of 75 mg 13C-urea dissolved in 200 ml of orange juice. The samples were mailed and analyzed 11.5 (4-21) days after collection. Data were analyzed statistically by the chi-square and Mann-Whitney test and by the Spearman correlation coefficient. Twenty-six patients were HP positive and 27 negative. There was 100% agreement between the IRIS results and the HP status determined by the other three methods. Using a cutoff value of delta-over-baseline (DOB) above 4.0 the IRIS showed a mean value of 19.38 (minimum = 4.2, maximum = 41.3, SD = 10.9) for HP-positive patients and a mean value of 0.88 (minimum = 0.10, maximum = 2.5, SD = 0.71) for negative patients. Using a cutoff value corresponding to 0.800% CO2/weight (kg), the 14C-UBT showed a mean value of 2.78 (minimum = 0.89, maximum = 5.22, SD = 1.18) in HP-positive patients. HP-negative patients showed a mean value of 0.37 (minimum = 0.13, maximum = 0.77, SD = 0.17). IRIS is a low-cost, easy to manage, highly sensitive and specific test for H. pylori detection. Storing and mailing the samples did not interfere with the performance of the test.

Serum cytokine levels in autoimmune and non-autoimmune hyperthyroid states

Although the role of interleukin-2 (IL-2) and interferon gamma (gIFN) is still poorly understood in hyperthyroid diseases, it is reasonable to assume that these cytokines may be present at higher levels in Graves' disease (GD) than in other primarily non-autoimmune thyroid diseases. In order to look for an easy method to distinguish GD from primarily non-autoimmune causes of hyperthyroidism, we compared 13 healthy individuals with 21 treated and untreated hyperthyroid GD patients and with 19 patients with hyperthyroidism due to other etiologies: 7 cases of multinodular goiter, 5 cases of excessive hormone replacement and 7 cases of amiodarone-associated hyperthyroidism. All patients presented low TSH levels and a dubious clinical thyroid state. We found a good correlation between TSH and serum IL-2 levels (r = 0.56; P<0.01). Serum IL-2 (P<0.01) and gIFN (P<0.01) levels were lower in the hyperthyroid group of patients than in control subjects, suggesting a depressed TH1 pattern in the T-cell subset of hyperthyroid patients. GD had normal IL-2 levels, while patients with other forms of thyrotoxicosis presented decreased IL-2 levels (P<0.05). There was no difference between treated and untreated GD patients. We suggest that the direct measurement of serum IL-2 level may help to confirm hyperthyroidism caused by GD.

Amifostine (WR-2721), a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY) (7 g/m2), used to mobilize peripheral blood progenitor cells (PBPC) and to reduce tumor burden. We enrolled 29 patients, 22 (75.9%) affected by aggressive and 7 (24.1%) by indolent non-Hodgkin's lymphoma (NHL), who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients) affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0.04). None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage) colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively). Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.

Photodynamic therapy: a new concept in medical treatment

A new concept in the therapy of both neoplastic and non-neoplastic diseases is discussed in this article. Photodynamic therapy (PDT) involves light activation, in the presence of molecular oxygen, of certain dyes that are taken up by the target tissue. These dyes are termed photosensitizers. The mechanism of interaction of the photosensitizers and light is discussed, along with the effects produced in the target tissue. The present status of clinical PDT is discussed along with the newer photosensitizers being used and their clinical roles. Despite the promising results from earlier clinical trials of PDT, considerable additional work is needed to bring this new modality of treatment into modern clinical practice. Improvements in the area of light source delivery, light dosimetry and the computation of models of treatment are necessary to standardize treatments and ensure proper treatment delivery. Finally, quality assurance issues in the treatment process should be introduced.

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 ± 304; C to F1 (C x A) = 3899 ± 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 ± 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 ± 287; B to B = 2759 ± 170, and A to A = 2835 ± 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration.

Acute effect of different antidepressants on glycemia in diabetic and non-diabetic rats

Diabetic patients have a 20% higher risk of depression than the general population. Treatment with antidepressant drugs can directly interfere with blood glucose levels or may interact with hypoglycemic agents. The treatment of depression in diabetic patients must take into account variations of glycemic levels at different times and a comparison of the available antidepressant agents is important. In the present study we evaluated the interference of antidepressants with blood glucose levels of diabetic and non-diabetic rats. In a first experiment, male adult Wistar rats were fasted for 12 h. Imipramine (5 mg/kg), moclobemide (30 mg/kg), clonazepam (0.25 mg/kg), fluoxetine (20 mg/kg) sertraline (30 mg/kg) or vehicle was administered. After 30 min, fasting glycemia was measured. An oral glucose overload of 1 ml of a 50% glucose solution was given to rats and blood glucose was determined after 30, 60 and 90 min. Imipramine and clonazepam did not change fasting or overload glycemia. Fluoxetine and moclobemide increased blood glucose at different times after the glucose overload. Sertraline neutralized the increase of glycemia induced by oral glucose overload. In the second experiment, non-diabetic and streptozotocin-induced diabetic rats were fasted, and the same procedures were followed for estimation of glucose tolerance 30 min after glucose overload. Again, sertraline neutralized the increase in glycemia after glucose overload both in diabetic and non-diabetic rats. These data raise the question of whether sertraline is the best choice for prolonged use for diabetic individuals, because of its antihyperglycemic effects. Clonazepam would be useful in cases with potential risk of hypoglycemia.

Is pancreas development abnormal in the non-obese diabetic mouse, a spontaneous model of type I diabetes?

Despite extensive genetic and immunological research, the complex etiology and pathogenesis of type I diabetes remains unresolved. During the last few years, our attention has been focused on factors such as abnormalities of islet function and/or microenvironment, that could interact with immune partners in the spontaneous model of the disease, the non-obese diabetic (NOD) mouse. Intriguingly, the first anomalies that we noted in NOD mice, compared to control strains, are already present at birth and consist of 1) higher numbers of paradoxically hyperactive ß cells, assessed by in situ preproinsulin II expression; 2) high percentages of immature islets, representing islet neogenesis related to neonatal ß-cell hyperactivity and suggestive of in utero ß-cell stimulation; 3) elevated levels of some types of antigen-presenting cells and FasL+ cells, and 4) abnormalities of extracellular matrix (ECM) protein expression. However, the colocalization in all control mouse strains studied of fibroblast-like cells (anti-TR-7 labeling), some ECM proteins (particularly, fibronectin and collagen I), antigen-presenting cells and a few FasL+ cells at the periphery of islets undergoing neogenesis suggests that remodeling phenomena that normally take place during postnatal pancreas development could be disturbed in NOD mice. These data show that from birth onwards there is an intricate relationship between endocrine and immune events in the NOD mouse. They also suggest that tissue-specific autoimmune reactions could arise from developmental phenomena taking place during fetal life in which ECM-immune cell interaction(s) may play a key role.

Eradication of Helicobacter pylori infection in patients with duodenal ulcer and non-ulcer dyspepsia and analysis of one-year reinfection rates

Helicobacter pylori (HP) infection is endemic worldwide. The proposed treatment is expensive and there are few reports regarding reinfection rates in Brazil. The aim of this study was to compare the eradication rates obtained with two therapeutic options and to evaluate reinfection one year after treatment. This was a prospective randomized trial with 55 patients. Thirty-nine patients had active duodenal ulcer (DU) and 16 non-ulcer dyspepsia (NUD), and all tested positive for HP. Diagnosis was based on at least two positive tests: ultrarapid urease test, histology and/or culture. Patients were randomized to two groups: group OMC treated with 40 mg omeprazole (once a day), 500 mg metronidazole and 250 mg clarithromycin (twice daily) for 7 days, or group NA treated with 300 mg nizatidine (once a day) and 1000 mg amoxicillin (twice daily) for 14 days. Those patients in whom HP was eradicated were followed up for one year to evaluate reinfection. Twenty-five patients were randomized for OMC and 30 for NA. HP eradication occurred in 20/25 patients (80%) treated with OMC and 13/30 (43%) treated with NA (P = 0.01). After reallocation because of initial treatment failure, the overall eradication rate was 44/51 patients (86%). After an average follow-up of one year, we evaluated 34 patients (23 with DU and 11 with NUD). Reinfection occurred in 3/34 patients (7.6%). We conclude that OMC is effective for HP eradication, and that NA should not be used. Reinfection occurs in 7.6% of the patients in the first year after eradication.

Behavioral correlates of the activity of serotonergic and non-serotonergic neurons in caudal raphe nuclei

We investigated the behavioral correlates of the activity of serotonergic and non-serotonergic neurons in the nucleus raphe pallidus (NRP) and nucleus raphe obscurus (NRO) of unanesthetized and unrestrained cats. The animals were implanted with electrodes for recording single unit activity, parietal oscillographic activity, and splenius, digastric and masseter electromyographic activities. They were tested along the waking-sleep cycle, during sensory stimulation and during drinking behavior. The discharge of the serotonergic neurons decreased progressively from quiet waking to slow wave sleep and to fast wave sleep. Ten different patterns of relative discharge across the three states were observed for the non-serotonergic neurons. Several non-serotonergic neurons showed cyclic discharge fluctuations related to respiration during one, two or all three states. While serotonergic neurons were usually unresponsive to the sensory stimuli used, many non-serotonergic neurons responded to these stimuli. Several non-serotonergic neurons showed a phasic relationship with splenius muscle activity during auditory stimulation. One serotonergic neuron showed a tonic relationship with digastric muscle activity during drinking behavior. A few non-serotonergic neurons exhibited a tonic relationship with digastric and/or masseter muscle activity during this behavior. Many non-serotonergic neurons exhibited a phasic relationship with these muscle activities, also during this behavior. These results suggest that the serotonergic neurons in the NRP and NRO constitute a relatively homogeneous population from a functional point of view, while the non-serotonergic neurons form groups with considerable functional specificity. The data support the idea that the NRP and NRO are implicated in the control of somatic motor output.

Intercellular contact-dependent survival of human A549, NCI-H596 and NCI-H520 non-small cell lung carcinoma cell lines

In the present study, we examined the relationship between cell phenotype and cell survival of three human non-small cell lung carcinoma cell lines (A549, NCI-H596 and NCI-H520). Cells in exponential growth at various densities were incubated for 24 h at 37ºC in a 5% CO2 humidified atmosphere and then exposed to UV radiation for 1 min (256 nm, 40 W, source-to-target distance 100 cm). After two days the surviving cells were quantified by sulforhodamine ß staining and DNA fragmentation assay. The differences in UV sensitivity at 60 x 10³ cells/cm² among the cell lines were not related to the proliferative state of the cells but to the extent of intercellular contact. In contrast to A549 and NCI-H596, irradiated NCI-H520 cells presented lower DNA fragmentation and an aggregated cell culture phenotype even prior to confluence, suggesting that a contact-effect mechanism provides further protection against UV radiation.