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Until recent years, main biologic markers of inflammation used in current practice were limited to erythrocyte sedimentation rate, fibrinogen, and serum protein electrophoresis. A better understanding of inflammatory mechanisms and improvement of laboratories technologies helped in better understanding of the role and potential usefulness of inflammatory reaction proteins. Arrival of proteic profile and, more recently, the development of automation, still improved analysis of variations of different inflammatory reaction proteins. These proteins are then analyzed as an element of a "functional biological system", with known and so expected kinetics and ranges. The analyze of proteic profile combines the analyze of proteins variations, with elected but not exclusive associations, as Immunoglobulins and Complement, Orosomucoid and Haptoglobin, or Albumin and Transferrin. In Internal Medicine, proteic profile may help in solving daily problems. These problems may be so schematized: when the fundamental pathology is not yet known in an unraveling check-up, facing clinical symptoms, with a normal or fewly disrupted usual biologic panel, proteic profile may help to choose investigations necessary for the diagnosis; in the follow-up of patients treated for known inflammatory pathology facing new symptoms, part has to be done between complication of the disease and/or of the treatment, new pathology associated or unefficiency of the treatment. We report herein part of our experience of proteic profile in an Internal Medicine department, from some particularly demonstrative case reports: congenital or acquired abnormality of iron metabolism, with normal usual iron panel (iron deficiency, hemochromatosis); severe evolutive inflammatory or infectious disease with normal erythrocyte sedimentation rate (temporal arteritis, infectious endocarditis).

Sublobular nodules of hepatocytes free of iron or exhibiting much less iron than the surrounding parenchyma, referred to in this study as iron-free-foci, are frequently found in the livers of patients with genetic hemochromatosis complicated by hepatocellular carcinoma. To test the hypothesis that such nodules are preneoplastic lesions, iron-free foci were sought in the initial liver biopsy specimens of 185 patients with untreated and uncomplicated genetic hemochromatosis. Iron-free foci were found in 14 (7.6%) patients, all men, aged from 38 to 76 yr, with heavy iron overload and with fibrosis or cirrhosis. Twelve patients with iron-free foci were followed for 0.9 to 15 yr (7 +/- 6 yr). In six (50%), HCC developed, compared with 2 (8%) from a control group consisting of 24 patients without IFF matched according to age, sex, degree of fibrosis, liver iron amount and follow-up duration. The mean number of iron-free foci per iron-free foci-positive specimen was 3.2 +/- 2.1. Ten patients had dysplastic aspects in their iron-free foci, and four had intrahepatocytic iron-positive inclusions at the periphery of iron-free foci. Proliferative cell nuclear antigen was positive in 75% of iron-free foci and in 24% +/- 21% of hepatocyte nuclei in iron-free foci. This study clearly demonstrates that iron-free foci are proliferative lesions and strongly suggests that such nodules are preneoplastic foci. Therefore the finding of IFF in the initial liver biopsy specimen from a patient with genetic hemochromatosis should lead to regular screening for hepatocellular carcinoma.

The antioncogenes, or tumor suppressor genes, as negative regulators of cell division, stand in contrast to oncogenes. For most human cancers, the more frequently mutated genes are the antioncogenes, the principal exception being the leukemias and lymphomas. Persons heterozygous for germ-line mutations in antioncogenes are strongly predisposed to one or more kinds of cancer, and most dominantly inherited cancer is attributable to such heterozygosity. Seven antioncogenes have been cloned through the study of these persons, and several others have been mapped. An eighth one was mapped and cloned through the investigation of tumors and is not yet known in hereditary form. Three dominantly inherited forms of cancer are not attributable to mutations in antioncogenes. The corresponding nonhereditary forms of most cancers generally reveal abnormalities of the same antioncogenes that are found in the hereditary forms but may also show additional ones. Some cancers, especially the embryonal tumors of children, have a small number of antioncogene mutations; some others, such as most sarcomas, have more, and the common carcinomas have the most, reflecting a hierarchy of controls over growth of stem cell populations. Still more members of this gene category remain to be mapped and cloned through the study of cancer families and of tumors. The genes that have been cloned act at diverse points in the signal transduction pathway in cells, from the outer cell membranes to sites of gene transcription, in some cases as negative regulators of oncogene expression.

The recent identification of the Huntington's disease (HD) gene, enabled us to synthesize oligopeptides corresponding with the carboxy-terminal end of the predicted HD-gene (IT15) product. Immunobiochemcial studies with polyclonal antibodies directed against this synthetic peptide (position 3114-3141) on lymphoblastoid cells from normal individuals and patients with Huntington disease, revealed the presence of a protein (huntingtin) with a molecular mass of approximately 330 kDa. Immunocytochemical studies showed a cytoplasmic localization of huntingtin in various cell types including neurons. In most of the neuronal cells the protein was also present in the nucleus. No difference in molecular mass or intracellular localization was found between normal and mutant cells.

The effects of daily oral administration of a high dose of 10 mg norethisterone acetate (NET-Ac.)/kg/day over 14 weeks on serum lipid and lipoprotein parameters as well as on blood coagulation were investigated in female monkeys (M. fascicularis). Measurements of lipids and lipoprotein cholesterol were performed in weeks--5 and -1 before treatment and in weeks 4, 8 and 12 after treatment. In addition, various blood coagulation and fibrinolytic parameters were determined in weeks 11-14 after treatment with NET-Ac. Furthermore, the serum levels of norethisterone (NET) were determined in order to monitor the real systemic compound exposure and revealed that Cmax and AUC (0-3 h) values reached for norethisterone in this experiment in monkeys were about 25 times higher than those obtained after an oral contraceptive dose of NET-Ac. in women. The results of lipid and lipoprotein cholesterol determinations showed decreases in serum total lipids, phospholipids, triglycerides and total cholesterol associated with similar decreases in HDL-, LDL- and VLDL-cholesterol fractions after NET-Ac.-treatment in monkeys. These effects were observed from week 4 onwards and maintained their magnitude up to week 12 after treatment. Since both HDL- and LDL-cholesterol fractions decreased, the HDL/LDL-ratio remained almost unchanged. Thus, the results obtained in this study after high-dose treatment with NET-Ac. in monkeys did not indicate any changes of lipid and lipoprotein parameters which in humans are supposed to be associated with an increased risk of cardiovascular lesions, namely a decrease in HDL- and increase in LDL-cholesterol fractions. The results of blood coagulation and fibrinolytic parameters showed increased antithrombin-III and plasminogen levels besides minor changes in other parameters, thus indicating that NET-Ac.-treatment does not contribute to an increased risk of cardiovascular thrombotic events in the cynomolgus monkey.

1. Fibrinogenase was isolated from Candida albicans NH-1 by DEAE-Cellulose, Sephadex G-75 and Sephadex G-100 column chromatographies. 2. The purified fibrinogenase gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 3. The enzyme preparation had a molecular weight of 13,000, isoelectric point of pH 4.2 and possessed 117 amino acid residues. 4. The purified fibrinogenase possessed capillary permeability-increasing activity. 5. The enzyme hydrolyzed fibrinogen, casein, hide powder azure, azocoll hydrolytic activities and also hydrolyzed the oxidized B chain of insulin. The cleavage sites in the oxidized B chain of insulin were identified as Asp(3)-Glu(4), Glu(13)-Ala(14), Ala(14)-Leu(15), Tyr(16)-Leu(17), Arg(22)-Gly(23), Phe(25)-Tyr(26) and Tyr(26)-Thr(27). 6. Fibrinogenase activity of this preparation was inhibited by alpha 2-macroglobulin antithrombin-III, o-phenanthroline, disodium ethylenediaminetetra acetic acid and dithiothreitol.

<h4>Background</h4>Protein C and antithrombin III (AT-III) are endogenous anticoagulants that regulate thrombotic processes. This experimental study examined the effect of recombinant tissue-type plasminogen activator (rt-PA) thrombolysis on the activity of these natural anticoagulants.<h4>Methods</h4>Blood clots were produced in the femoral artery segments of six rabbits with endothelial damage and stenosis (group A). Five rabbits with only surgical dissection served as controls (group B). Both groups received an infusion of rt-PA at 30 micrograms/kg/min for 60 min. The plasma activity of activated protein C (APC) and AT-III was determined using chromogenic assays. Blood samples were taken at baseline (t1), after surgical dissection with (group A) or without (group B) thrombosis induction (t2), after rt-PA infusion (t3), and 1 h later (t4).<h4>Results</h4>In group A rabbits, the mean (+/- SEM) APC activity rose significantly (P = 0.02) from 2.2 +/- 0.3 and 3.7 +/- 1.5 mOD units/min at t1 and t2, respectively, to 59.3 +/- 17.8 mOD units/min at t3 and decreased to 8.0 +/- 3.4 mOD units/min at t4. In group B rabbits, the mean (+/- SEM) APC activity rose (P = 0.01) from 1.1 +/- 0.1 and 1.2 +/- 0.1 mOD units/min at t1 and t2 to 17.1 +/- 3.2 mOD units/min at t3 and decreased to 2.4 +/- 0.2 at t4, (P = 0.011 for comparison between groups using two-way analysis of variance). The mean APC value at t3 was higher for group A than for group B (P = 0.06). A slight but statistically significant decrease (P < 0.01) in AT-III activity was noted in both groups, from 92 +/- 3% and 96 +/- 2% at t1 in groups A and B, respectively, to 79 +/- 3% and 86 +/- 2% at t4 (P = 0.028 for comparison between groups).<h4>Conclusion</h4>These data show a transient rise in APC and a slight decrease in AT-III activity during rt-PA thrombolysis. Whether these changes influence thrombolysis outcome remains to be determined.

Heparin coating of an extracorporeal device may reduce blood activation. To evaluate protein and platelet interaction with Duraflo II surface treatment of the cardiopulmonary bypass (CPB) circuit, thirty coronary artery bypass grafting patients were randomly divided into two groups (Duraflo II, n = 15 or Control, n = 15). Binding of antibodies against factor XII, antithrombin III (ATIII) and platelet receptor GpIb were significantly higher (p < 0.01) on the Duraflo II treated surface. Hageman Factor fragment (HFf or factor XIIf) activity observed in the fluid phase tended to be lower in the treated circuits, although complexes of factor XIIf-C1 inhibitor (C1INH) increased by the same extent in both groups. Thrombin was effectively bound on the Duraflo II surface while thrombin-antithrombin III formation was reduced during the first phase of CPB. As expected, this thrombin inhibiting capacity remained higher on the Duraflo II, although in the arterial line a reduction of 10% per hour was observed. This indicated loss of functional bound heparin of the Duraflo II surface. During the second phase of CPB, after release of the aortic crossclamp, factor XII and thrombin were strongly activated in both groups indicated by a sharp increase in concentrations of T/AT complex as well as FPA. Platelet numbers tended to increase more in the control group during CPB than in the Duraflo II group, likely by interaction of platelet GpIb receptors on the Duraflo II treated surface (p < 0.01). However, platelet activation assessed by beta-TG was similar in both groups of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Low molecular weight heparins are well established in the prophylaxis of deep vein thrombosis in patients with general surgery, in high risk patients undergoing elective hip surgery or emergency surgery and also in patients with an enhanced risk of thrombosis who are treated in medical wards. There are, however, many possibilities for improving prophylaxis and treatment with LMWH. The mechanisms by which low molecular weight heparins and also unfractionated heparin inhibit thrombus formation are not fully understood. The inhibition of thrombin formation and local effects at the endothelial level may be more important than antithrombin-III mediated effects on factor IIa and on factor Xa. For most low molecular weight heparins the most effective dose regimens to be used in patients at high risk have not yet been established. Low molecular weight heparins may be more effective in the treatment of deep venous thrombosis than unfractionated heparin. In the therapeutic studies published so far the major intention was to show that low molecular weight heparins can prevent the progression of deep venous thrombosis and pulmonary embolism to the same extent as unfractionated heparin. Extended treatment regimens, however, may lead to a relevant thrombus reduction. Outpatient treatment for a longer period of time with results not far from those obtained with thrombolysis seem possible especially in elderly patients. Low molecular weight heparins in their present form or modified low molecular weight heparins may be useful for long-term treatment of patients with atherosclerosis with the aim of regression of atherosclerotic lesions. New forms of application, e.g. inhalation, may render long-term treatment more feasible.

The relationship between T-cell activation and early events in the replication cycle of simian immunodeficiency virus (SIV) was analyzed in resting T lymphocytes from macaques. We used the polymerase chain reaction to detect an early product of reverse transcription (R/U5) and almost complete viral DNA (long terminal repeat/gag). We found that SIV can enter resting T lymphocytes and initiate replication but that the reverse transcription process is not efficient and proceeds slowly in resting cells. Cross-linking the CD3/T-cell receptor complex with monoclonal antibodies, unlike cross-linking either the CD28 or CD2 accessory receptor and like phorbol myristate acetate, induced a rapid increase in viral R/U5 DNA detected within 3 to 6 h postinfection. Anti-CD3 or phorbol myristate acetate induced replication of full-length viral DNA within 6 to 9 h postinfection, but full-length SIV DNA was not detectable at earlier time points. We then compared various inhibitors of T-cell activation for their effects on viral initiation and complete replication. Cyclosporin A, an inhibitor of a distal step in T-cell activation, blocked anti-CD3-induced T-cell proliferation and completion of SIV DNA replication but had no effect on induced increases in SIV R/U5 DNA. By contrast, initial SIV DNA synthesis was partially blocked by inhibitors of very early steps in T-cell activation, including herbimycin A, an inhibitor of protein tyrosine kinases, and by two different inhibitors of protein kinase C, H7 and staurosporine. Since resting T cells do not efficiently complete SIV DNA synthesis and cyclosporin A can block the formation of complete viral DNA induced in activated T cells, a cellular factor(s) present in activated T cells appears to be required for the formation of full-length SIV DNA.

Reduced expression and/or allelic loss of the putative tumor suppressor gene DCC has been demonstrated in colorectal, gastric, pancreatic, esophageal, breast, and hematological malignancies. We examined the expression of the DCC gene in 22 tissue samples from human gliomas (glioblastoma multiforme, oligodendroglioma, and mixed oligodendroglioma/astrocytoma). Seven of 8 glioblastomas multiforme (88%) had reduced or absent DCC expression, and 8 of the other 14 tumors underexpressed DCC when compared to normal brain tissue. These results demonstrate that reduced expression of DCC occurs in human malignant gliomas and may be part of a common genetic pathway leading to neoplastic transformation and/or tumor progression.

Oncogenic activation of c-Myb in both avian and murine systems often involves N-terminal truncation. In particular, the first of three DNA-binding repeats in c-Myb has been largely deleted during the genesis of the v-myb oncogenes of avian myeloblastosis virus and E26 avian leukemia virus. This finding suggests that the first DNA-binding repeat may have an important role in cell growth control. We demonstrate that truncation of the first DNA-binding repeat of c-Myb is sufficient for myeloid transformation in culture, but deletion of the N-terminal phosphorylation site and adjacent acidic region is not. Truncation of the first repeat decreases the ability of a Myb-VP16 fusion protein to trans activate the promoter of a Myb-inducible gene (mim-1) involved in differentiation. Moreover, truncation of the first repeat decreases the ability of the Myb protein to bind DNA both in vivo and in vitro. These results suggest that N-terminal mutants of c-Myb may transform by regulating only a subset of those genes normally regulated by c-Myb.

Although treatment of intact adult male rats with the pure antiandrogen flutamide or a luteinizing hormone-releasing hormone (LHRH) agonist alone leads to partial inhibition of ventral prostate weight, maximal inhibition is achieved by combination of the two drugs. Potentializing effects of the two compounds were observed even on prostatic ornithine decarboxylase activity. Because LHRH agonists are widely used to achieve medical castration in men treated for prostate cancer, it is of interest to observe that in the dog, known for being the best model for studies of the action of LHRH agonists, flutamide does not interfere with the potent desensitizing action of the LHRH agonist on pituitary LH secretion, thus supporting the combined use of flutamide with an LHRH agonist for maximal androgen blockade without loss of efficiency of the LHRH agonist. Because prostate cancer is known to show a high degree of heterogeneity of its sensitivity to androgens, we analyzed the effect of combined antiandrogen therapy on parameters more sensitive to androgens than ventral prostatic weight itself. In agreement with its pure antiandrogenic characteristics, flutamide alone has no stimulatory effect on the intraprostatic level of mRNA encoding the C1 or C3 component of prostatic binding protein (PBP), whereas cyproterone acetate (CPA), megestrol acetate (MEG), and, especially, medroxyprogesterone acetate (MPA) markedly stimulate PBP-C1 and PBP-C3 mRNA levels, an effect reversed by flutamide, thus further supporting the intrinsic androgenic activity of all these steroidal derivatives. Similar androgenic effects of the steroidal derivatives were observed on prostatic ornithine decarboxylase activity. Androgen-sensitive Shionogi tumor cells were then used to assess the antiandrogenic/androgenic properties of flutamide and the above-indicated steroidal derivatives. MPA, MEG, CPA as well as spironolactone-stimulated cell proliferation under both in vivo and in vitro conditions, thus illustrating the intrinsic androgenic activity of all these compounds. Flutamide was inactive by itself and reversed the stimulatory effect of all other compounds, thus indicating its pure antiandrogenic activity. Although castration reduces intraprostatic dihydrotestosterone (DHT) to undetectable levels in the rat and guinea pig, the concentration remains at about 50% of the value found in intact men after castration, thus indicating an important contribution of the adrenals to DHT in the human prostate, a finding that requires the addition of an antiandrogen to block the action of this important amount of DHT remaining after castration.

Slow, rhythmic membrane potential (Vm) fluctuations occur spontaneously in cortical neurons of urethane-anesthetized rats, and likely underlie EEG activity in the same low-frequency (1-4 Hz or delta) range. Nucleus basalis (NB) stimulation elicits neocortical activation, simultaneously modifying Vm and EEG fluctuations, by way of cortical muscarinic ACh receptors (Metherate et al., 1992). To investigate the nature of spontaneous fluctuations and their modification by NB stimulation, we have obtained intracellular recordings from auditory cortex using the whole-cell recording technique in vivo. Spontaneous Vm fluctuations appeared to contain three components whose polarity and time course resembled the EPSP, putative Cl(-)-mediated IPSP, and putative K(+)-mediated, long-lasting IPSP elicited by thalamic stimulation. The spontaneous, long-lasting hyperpolarization, whose rhythmic occurrence appeared to set the slow-wave rhythm, was associated with an increased conductance that could shunt the thalamocortical EPSP. We hypothesized that spontaneous Vm fluctuations arise from intermixed rapid depolarizations, rapid Cl(-)-mediated hyperpolarizations, and long-lasting, K(+)-mediated hyperpolarizations. NB-mediated cortical activation might then result from muscarinic suppression of K+ permeability, allowing the rapid depolarizations and Cl- fluxes to continue uninterrupted. Tests of this hypothesis showed that (1) intracellular blockade of K+ channels by rapid diffusion of Cs+ from the recording pipette resulted in suppression of spontaneous, long-lasting hyperpolarizations, mimicking the effect of NB stimulation, and reducing shunting of the thalamocortical EPSP; (2) effects of Cs+ and NB stimulation suggested overlapping, or converging, mechanisms of action; however, there were important differential effects on the spontaneous, long-lasting hyperpolarizations and the K(+)-mediated IPSP; and (3) modifying Cl- fluxes with intracellular picrotoxin or high intracellular Cl- concentrations resulted in spontaneous and NB-elicited large-amplitude depolarizations. We conclude that spontaneous, long-lasting hyperpolarizations are K+ fluxes, but are not "spontaneous" K(+)-mediated IPSPs. Since NB-mediated reduction of spontaneous hyperpolarizations implies muscarinic suppression of a K+ conductance, the spontaneous hyperpolarizations more likely result from the calcium-activated K+ current, IK(Ca). Finally, Cl- fluxes form an important component of activated Vm fluctuations that acts to restrain excessive depolarization.

A tamoxifen resistant cell line (clone 9) has been isolated from the tamoxifen sensitive, hormone responsive MCF-7 breast carcinoma cell line after transfection with mixed cDNA libraries, followed by tamoxifen selection in the presence of oestrogens. Transfection was confirmed by Southern analysis with vector probes. Clone 9 in several-fold more resistant to tamoxifen and other anti-oestrogens than wild type cells when cultured either as a monolayer or as colonies in soft agar but retains oestrogen receptors. Clone 9 was less responsive to 17-beta-oestradiol than were wild type MCF-7. In addition to showing in vitro tamoxifen resistance, clone 9 was also tamoxifen resistant in vivo when xenografted into the nude mouse. Culture medium conditioned by clone 9 cells stimulated quiescent cells of the same clone as well as wild type cells, whereas medium conditioned by wild type MCF-7 was inhibitory to both, suggesting that clone 9 may be secreting an autocrine growth factor. Clone 9 provides a novel model for further investigation of the mechanism of anti-oestrogen resistance that occurs without loss of oestrogen receptors. Preliminary results suggest that an autocrine growth stimulatory mechanism may be one pathway of such resistance.

Iron overload is a major cause of morbidity and mortality in thalassemia major patients. All chronic liver diseases may be associated with such endocrine symptoms as diabetes mellitus, testicular failure or hypothyroidism. We studied 15 thalassemic patients (12 men and 3 women; age range: 10-50 years, mean: 22.5 years). All patients received blood transfusions, but only some were treated with iron chelation. Seven patients were splenectomized. MRI was performed with an 0.5 T superconducting magnet, using SE T1- and T2-weighted and IR sequences. We used these data with Bloch's equation to calculate T1 and T2 values. Quantitative analysis was made by calculating signal intensity and relaxation times in 8 hepatic regions of interest: marked reduction in hepatic signal intensity and a negative relationship between T1 and serum ferritin (r = 0.646, p < 0.01) and between T2 and serum ferritin (r = 0.688, p < 0.01) were observed. Moreover, a negative relationship was found between hepatic signal intensity and aspartic aminotransferase (r = 0.524, p < 0.05). Our results confirm the value of MRI in the diagnosis and evaluation of hepatic iron overload but an accurate quantitative analysis can be made only when hepatic iron levels are between 1 and 2 mg/g of liver. Even though it is below statistical significance, the negative relationship between signal intensity and aspartic aminotransferase suggests that hepatic hemochromatosis can influence pituitary-thyroid axis and interfere with peripheral hormone metabolism.