Evaluation of the cytogenetic effects of I-131 preceded by recombinant human thyrotropin (rhTSH) in peripheral lymphocytes of Wistar rats

The present study was carried out to investigate the cytogenetic effects of therapeutic exposure to radioiodine preceded by rhTSH in an animal model. Three groups of Wistar rats (n = 6) were used: one group was treated only with I-131 (11.1 MBq/animal); the other two groups received rhTSH (1.2 mu g/rat of either Thyrogen or rhTSH-IPEN, respectively) 24 h before administration of radioiodine. The percentage of lymphocytes with chromosome aberrations and the average number of aberrations and of dicentrics per cell were determined on blood samples collected 24 h, 7 and 30 days after administration of I-131. The data show that the treatment with radioiodine alone or associated with rhTSH resulted in a greater quantity of chromosome alterations in relation to basal values after 24 h, with a gradual decline after 7 and 30 days of treatment. An increase in chromosome alterations was also seen after rhTSH treatment alone. Neither of the treatments, i.e., with I-131 alone or associated with hormone, resulted in an aneugenic effect or influenced the kinetics of cellular proliferation in rat blood lymphocytes. There was no significant difference between the cytogenetic effects of Thyrogen and rhTSH-IPEN treatment. These data suggest that the treatment with radioiodine, associated or not with rhTSH, affects to a limited extent a relatively small number of cells although the occurrence of late stochastic effects could not be discarded.

Assessment of factors that confound MRI and neuropathological correlation of human postmortem brain tissue

In spite of considerable technical advance in MRI techniques, the optical resolution of these methods are still limited. Consequently, the delineation of cytoarchitectonic fields based on probabilistic maps and brain volume changes, as well as small-scale changes seen in MRI scans need to be verified by neuronanatomical/neuropathological diagnostic tools. To attend the current interdisciplinary needs of the scientific community, brain banks have to broaden their scope in order to provide high quality tissue suitable for neuroimaging- neuropathology/anatomy correlation studies. The Brain Bank of the Brazilian Aging Brain Research Group (BBBABSG) of the University of Sao Paulo Medical School (USPMS) collaborates with researchers interested in neuroimaging-neuropathological correlation studies providing brains submitted to postmortem MRI in-situ. In this paper we describe and discuss the parameters established by the BBBABSG to select and to handle brains for fine-scale neuroimaging-neuropathological correlation studies, and to exclude inappropriate/unsuitable autopsy brains. We tried to assess the impact of the postmortem time and storage of the corpse on the quality of the MRI scans and to establish fixation protocols that are the most appropriate to these correlation studies. After investigation of a total of 36 brains, postmortem interval and low body temperature proved to be the main factors determining the quality of routine MRI protocols. Perfusion fixation of the brains after autopsy by mannitol 20% followed by formalin 20% was the best method for preserving the original brain shape and volume, and for allowing further routine and immunohistochemical staining. Taken to together, these parameters offer a methodological progress in screening and processing of human postmortem tissue in order to guarantee high quality material for unbiased correlation studies and to avoid expenditures by post-imaging analyses and histological processing of brain tissue.

Infusion of Recombinant Human Tissue Plasminogen Activator Through the Superior Mesenteric Artery in the Treatment of Acute Mesenteric Venous Thrombosis

Acute mesenteric venous thrombosis is an uncommon condition that is usually treated with systemic anticoagulation. Catheter-directed thrombolysis through the superior mesenteric artery may be a viable adjunct to treat this morbid condition. In the present article, we have described a case of superior mesenteric venous thrombosis treated with catheter-directed infusion of tissue plasminogen activator through the superior mesenteric artery.

Methimazole and propylthiouracil equally cross the perfused human term placental lobule

Propylthiouracil (PTU) is widely believed to cross the placenta less freely than methimazole (MMI) and is therefore regarded as the preferred drug for treatment of hyperthyroidism in pregnancy. Clinical studies comparing the two drugs show, however, no differences in maternal or fetal thyroid function. We investigated transfer from the maternal to the fetal circuit in the isolated perfused term human placental lobule of low and high doses of PTU (4 mu g/mL and 40 mu g/mL) and MMI(1.5 mu g/mL and 15 mu g/mL) in protein-free perfusate and low doses of both drugs with addition of 40 g/L of bovine albumin. Both drugs readily crossed the placenta, reaching equilibrium in all experiments in about 2 h. Drug concentrations in the two circuits fitted a two compartmental model. Transfer kinetics for the two drugs were similar, nonsaturable, and unaffected by addition of albumin. Clearances (mL.min(-1).g(-1), means +/- SD) of PTU from maternal to fetal circuits were: 0.229 +/- 0.110, 0.216 +/- 0.065, and 0.170 +/- 0.032; and for transfer of MMI: 0.165 +/- 0.025, 0.232 +/- 0.153, and 0.174 +/- 0.009 (for low doses without, low doses with, and high doses without albumin, respectively). Clearances of PTU from fetal to maternal circuits were: 0.147 +/- 0.072, 0.109 +/- 0.014, and 0.116 +/- 0.028; and for transfer of MMI: 0.095 +/- 0.029, 0.122 +/- 0.088, and 0.12 +/- 0.005 (in the same experiments). There was no significant difference between drugs or drug doses and no effect of addition of albumin. We conclude that PTU and MMI have similar placental transfer kinetics.

Expression of recombinant human follicle stimulating hormone mRNA in Chinese hamster ovary cells under different promoters

Levels of recombinant human follicle stimulating hormone (r-hFSH) mRNA expressed under butyrate and zinc treatment were compared in two CHO-K1 derived cell lines. In King cells under the metallothionein promoter, butyrate induced the increase in both r-hFSH productivity (q(FSH)) and mRNA levels proportionally. In the presence of 1 mM butyrate and 40 mu M zinc, a 4-fold increase in q(FSH) and mRNA levels was achieved as compared to zinc (40) alone; this wasa approximately 6 times higher than in serum free medium. In Darren cells under the beta-actin promotor butyrate induced an increase in q(SFH) but not in mRNA levels.

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.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas

Background Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2. Objectives To search for mutations and genomic instability in sporadic and inherited BCCs. Methods DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database. Results Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g. 2885G>C in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g. 2839T>G in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1. Conclusion Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.

What needs to be done? Occupational therapy responsibilities and challenges regarding human rights

Background: Addressing human rights issues brings forth ethical and political responsibilities for occupational therapists and requires new epistemological and educational approaches. The way occupational therapists have faced these challenges has depended upon historical, cultural and social contexts. Aim and method: By means of literature review and historical analysis, this paper reflects on how occupational therapists have dealt with human matters issues and on the contemporary changes within the profession. Results and discussion: The paper portrays how Latin American occupational therapists have engaged in social transformation by choosing not to transform ethical and political problems into technical matters. Taking into account experiences and views from South Africa, Brazil and Chile, the paper outlines the importance of developing political literacy and interdisciplinary professional/postprofessional education to prepare the new generation of occupational therapists to engage in social transformation. Addressing issues of invisibility and lack of access to human rights, the paper reflects on the need of developing conceptual tools and strategies for change, and discusses the transformations being produced in contemporary occupational therapy. Conclusion: Occupational therapists and scientists need to be attentive to human rights issues. They also need to answer the call for interconnectedness in the present-day complex societies, and engage in networking and a cross-bordering dialogue. Nevertheless, although necessary and welcome, international cooperation requires a permanent exercise of cultural sensitivity, political awareness and self-awareness.

Bioactivation of Phenytoin by Human Cytochrome P450: Characterization of the Mechanism and Targets of Covalent Adduct Formation

The cytochrome P450-dependent covalent binding of radiolabel derived fi om phenytoin (DPH) and its phenol and catechol metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) and 5-(3',4'-dihydroxyphenyl)-5-phenylhydantoin (CAT), was examined in liver microsomes. Radiolabeled HPPH and CAT and unlabeled CAT were obtained from microsomal incubations and isolated by preparative HPLC. NADPH-dependent covalent binding was demonstrated in incubations of human liver microsomes with HPPH. When CAT was used as substrate, covalent adduct formation was independent of NADPH, was enhanced in the presence of systems generating reactive oxygen species, and was diminished under anaerobic conditions or in the presence of cytoprotective reducing agents. Fluorographic analysis showed that radiolabel derived from DPH and HPPH was selectively associated with proteins migrating with approximate relative molecular weights of 57-59 kDa and at the dye front (molecular weights < 23 kDa) on denaturing gels. Lower levels of radiolabel were distributed throughout the molecular weight range. In contrast, little selectivity was seen in covalent adducts formed from CAT. HPPH was shown to be a mechanism-based inactivator of P450, supporting the contention that a cytochrome P450 is one target of covalent binding. These results suggest that covalent binding of radiolabel derived from DPH in rat and human Liver microsomes occurs via initial P450-dependent catechol formation followed by spontaneous oxidation to quinone and semiquinone derivatives that ultimately react with microsomal protein. Targets for covalent binding may include P450s, though the catechol appears to be sufficiently stable to migrate out of the P450 active site to form adducts with other proteins. In conclusion, we have demonstrated that DPH can be bioactivated in human liver to metabolites capable of covalently binding to proteins. The relationship of adduct formation to DPH-induced hypersensitivity reactions remains to be clarified.

Characterisation of the recently cloned rat noradrenaline transporter - Comparison with bovine and human transporters

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [3H]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET.

Generation and phenotypic characterization of new human ovarian cancer cell lines with the identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells

This study describes a simple method for long-term establishment of human ovarian tumor lines and prediction of T-cell epitopes that could be potentially useful in the generation of tumor-specific cytotoxic T lymphocytes (CTLs), Nine ovarian tumor lines (INT.Ov) were generated from solid primary or metastatic tumors as well as from ascitic fluid, Notably all lines expressed HLA class I, intercellular adhesion molecule-1 (ICAM-1), polymorphic epithelial mucin (PEM) and cytokeratin (CK), but not HLA class II, B7.1 (CD80) or BAGE, While of the 9 lines tested 4 (INT.Ov1, 2, 5 and 6) expressed the folate receptor (FR-alpha) and 6 (INT.Ov1, 2, 5, 6, 7 and 9) expressed the epidermal growth factor receptor (EGFR); MAGE-1 and p185(HER-2/neu) were only found in 2 lines (INT.Ov1 and 2) and GAGE-1 expression in 1 line (INT.Ov2). The identification of class I MHC ligands and T-cell epitopes within protein antigens was achieved by applying several theoretical methods including: 1) similarity or homology searches to MHCPEP; 2) BIMAS and 3) artificial neural network-based predictions of proteins MACE, GAGE, EGFR, p185(HER-2/neu) and FR-alpha expressed in INT.Ov lines, Because of the high frequency of expression of some of these proteins in ovarian cancer and the ability to determine HLA binding peptides efficiently, it is expected that after appropriate screening, a large cohort of ovarian cancer patients may become candidates to receive peptide based vaccines. (C) 1997 Wiley-Liss, Inc.

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.

Relative dispersion of intravascular transit times during isolated human limb perfusions for recurrent melanoma

Aims We have characterized the relative dispersion of vascular and extravascular markers in the limbs of three patients undergoing isolated limb perfusions with the cytotoxic melphalan for recurrent malignant melanoma both before and after melphalan dosing. Methods A bolus of injectate containing [Cr-51] labelled red blood cells, [C-14]-sucrose and [H-3]-water was injected into an iliac or femoral artery and outflow samples collected at 1 s intervals by a fraction collector. The radioactivity due to each isotype was analysed by either gamma [Cr-51] or beta [C-14 and H-3] counting. The moments of the outflow fraction-time profiles were estimated by a nonparametric (numerical integration) method and a parametric model (sum of two inverse Gaussian functions). Results The availability, mean transit time and normalised variance (CV2) obtained for labelled red blood cells, sucrose and water were similar before and after melphalan dosing and with the two methods of calculation but varied between the patients. Conclusions The vascular space is not well-stirred but characterized by a CV2 similar that reported previously for in situ rat hind limb and rat liver perfusions. A flow-limited blood-tissue exchange was observed for the permeating indicators. Administration of melphalan did not influence the distribution characteristics of the indicators.

Induction of smooth muscle cell nitric oxide synthase by human leukaemia inhibitory factor: Effects in vitro and in vivo

We have previously shown that human leukaemia inhibitory factor (hLIF) inhibits perivascular cuff-induced neointimal formation in the rabbit carotid artery. Since nitric oxide (NO) is a known inhibitor of smooth muscle growth, NO synthase (NOS) activity in the presence of hLIF was examined in vivo and in vitro. In rabbit aortic smooth muscle cell (SMC) culture, significant NOS activity was observed at 50 pg/ml hLIF, with maximal activity at 5 ng/ml. In the presence of the NOS inhibitor L-NAME, hLIF-induced activation of NOS was greatly decreased, however it was still 63-fold higher than in control (p < 0.05). SMC-DNA synthesis was significantly reduced (-47%) following incubation with hLIF plus L-arginine, the substrate required for NO production (p < 0.05), with no effect observed in the absence of L-arginine. Silastic cuff placement over the right carotid artery of rabbits resulted in a neointima 19.3 +/- 5.4% of total wall cross-sectional area, which was increased in the presence of L-NAME (27.0 +/- 2.0%; p < 0.05) and reduced in the presence of L-arginine (11.3 +/- 2.0%; p < 0.05). The effect of L-arginine was ameliorated by co-administration of L-NAME (16.4 +/- 1.5%). However, administration of L-NAME with hLIF had no effect on the potent inhibition of neointimal formation by hLIF (3.2 +/- 2.5 vs. 2.1 +/- 5.4%, respectively). Similarly, with hLIF administration, NOS activity in the cuffed carotid increased to 269.0 +/- 14.0% of saline-treated controls and remained significantly higher with coadministration of L-NAME (188.5 +/- 14.7%). These results indicate that hLIF causes superinduction of NO by SMC, and that it is, either partially or wholly, through this mechanism that hLIF is a potent inhibitor of neointimal formation in vivo and of smooth muscle proliferation in vitro.