CD3 hyporesponsiveness and in vitro apoptosis are features of T cells from both malignant and nonmalignant secondary lymphoid organs.

There is a dogma in tumor immunology that tumor-infiltrating lymphocytes (TIL) are defective based on their lack of antitumoral efficacy in vivo and on impaired response to in vitro functional tests. However, TIL have been compared usually with peripheral blood T lymphocytes, raising doubts on the conclusions drawn. Therefore, we compared TIL from B cell non-Hodgkin's lymphomas (NHL) with T cells from nonmalignant secondary lymphoid organs. NHL-TIL were unresponsive to activation by immobilized anti-CD3 mAb, although bypassing T cell receptor (TCR)/CD3 signaling led to proliferation. The poor proliferative responses of NHL-TIL could not be explained by quantitative defects in TCRzeta expression. NHL-TIL underwent marked spontaneous apoptosis in vitro with loss of approximately 50% of cells after 24 h of culture. This was associated with downregulation of the antiapoptotic Bcl-xL and Bcl-2 proteins, whereas viable NHL-TIL maintained their expression. IL-2, anti-CD3/IL-2, and manipulation of the Fas/Fas-ligand death pathway had no effect on NHL-TIL survival. Apoptosis was not due to increased cell cycling, as NHL-TIL were quiescent, nonproliferating cells. T cells from inflammatory, nonmalignant tissues gave similar functional results to NHL-TIL, suggesting the existence of factors common to the microenvironment of these diverse pathologies. Thus, the quiescent, anergic phenotype of NHL-TIL cannot be attributed solely to tumor factors, but rather is a feature of T cells from chronic inflammatory lesions.

Fluorouracil combined with the pure (6S)-stereoisomer of folinic acid in high doses for treatment of patients with advanced colorectal carcinoma: a phase I-II study.

BACKGROUND: Potentiation of the antitumor activity of fluorouracil (5-FU) by folinic acid has been demonstrated in patients with colorectal adenocarcinoma. Modulation is due to the interaction of thymidylate synthase, fluorodeoxyuridine monophosphate, and methylene tetrahydrofolate, which leads to the formation of a stable ternary complex with concomitant enzyme inactivation. Folinic acid consists of a mixture of equal parts of two stereoisomers differing in chirality at the C-6 carbon of the pteridine ring. Only the levorotatory (6S)-stereoisomer of folinic acid is transformed into active folate cofactors. However, the (6R)-stereoisomer of folinic acid is not inert; it was shown to interfere with the (6S) form at the cellular level. PURPOSE: The possibility of a deleterious effect of the unnatural stereoisomer on the modulation of 5-FU led us to carry out a phase I-II study of 5-FU combined with the (6S)-stereoisomer of folinic acid given in high doses for treatment of patients with advanced colorectal carcinoma. We also determined the plasma pharmacokinetics of folates after intravenous (IV) injection of (6S)-folinic acid at the dose used in this study. METHODS: Treatment consisted of 5-FU (350-550 mg/m2 per day by IV infusion for 2 hours) and (6S)-folinic acid (100 mg/m2 per day by IV bolus injection) given for 5 consecutive days; the treatment was repeated every 21 days. Twenty-five patients with advanced colorectal carcinoma, who had had no prior chemotherapy, were evaluated for antitumor activity. The quantity of folates in plasma was measured using a microbiological assay. RESULTS: The median follow-up time was 9 months (range, 3.5-15.2 months). The response rate was 52% (complete response, 12%; partial response, 40%). The median time to disease progression for responding patients was 9.2 months (range, 5.9-15+ months). The estimated probability of survival at 12 months was 73%. Palliative improvement in quality of life was achieved in most patients who had symptoms due to the tumor before the start of treatment. The dose-limiting toxic effects were grade 3 diarrhea, dermatitis, and oral mucositis. Grade 4 toxicity did not occur. Myeloid toxicity was minor. After IV injection, (6S)-folinic acid was rapidly cleared from plasma (mean half-lives: alpha = 7.2 minutes and beta = 126 minutes). The mean concentration of the unchanged compound 2 hours after injection was 5.8 mumol/L. CONCLUSION: The (6S)-form of folinic acid potentiates the antitumor effect of 5-FU given concomitantly. IMPLICATION: Our results justify a more complete exploration of the pure active stereoisomer as a modulator of the fluoropyrimidines.

Modulation of 1-beta-D-arabinofuranosylcytosine metabolism by thymidine in human acute leukemia.

Twenty-seven patients with acute leukemia have been treated by sequential 6-day courses of thymidine (30 g/m2 by i.v. continuous infusion, days 1 and 4) and 1-beta-D-arabinofuranosylcytosine (ara-C) (200 mg/m2 by i.v. continuous infusion, days 2,3,5, and 6). Of 25 evaluable patients 4 achieved a complete remission: one of 9 for acute myelogenous leukemia; and 3 of 14 in the blastic crisis of chronic myelocytic leukemia. Six minor responses were also observed. Toxicity was mainly hematological and did not appear to be higher than that expected from ara-C alone. However, thymidine infusions gave rise to headache and somnolence. The clinical benefit of such treatment seems to be limited to the blastic crisis of chronic myelocytic leukemia. Parallel cytokinetic and biochemical studies were performed in order to assess the cytokinetic and metabolic changes induced by both drugs and to correlate them with the clinical response. Recruitment of cells into the S-phase fraction was observed following the first thymidine infusion in the two complete responders and in three of the five nonresponders studied. In contrast to this high pretherapeutic levels of S-phase fraction were observed in most minor responders and in some nonresponders with further decrease following the thymidine infusion. Recruitment of cells into S phase therefore appeared to be an important but not sufficient factor for prediction of complete response to ara-C. Responders in contrast to most nonresponders were characterized by a higher intracellular level of ara-C and its metabolites following the first 24-h infusion of the drug. Deoxythymidine triphosphate and deoxycytidine triphosphate pools were also measured before and during treatment in order to assess if nucleotide pool variations induced by the administration of thymidine can in fact correlate with the intracellular alteration in ara-C metabolism and with clinical response. The level of deoxycytidine triphosphate pools before treatment showed marked interpatient variations but did not correlate with response. As expected, thymidine infusion induced a rise in the deoxythymidine triphosphate pool and a decrease in deoxycytidine triphosphate. The pools, however, generally returned promptly to the pretherapeutic level 24 h after the end of the infusion of thymidine. There were no significant differences between responders and nonresponders in the modulation of these pools.

Treatment of advanced colorectal and gastric adenocarcinomas with 5-fluorouracil and high-dose folinic acid.

We report the results of an expanded trial of 5-fluorouracil (5-FU) combined with high-dose folinic acid for treatment of patients with advanced colorectal or advanced gastric adenocarcinoma. In each treatment course, the patients received both 5-FU (340 to 400 mg/m2/d by intravenous (IV) infusion for a period of 15 minutes) and folinic acid (200 mg/m2/d by IV bolus) for 5 consecutive days, with a 21-day interval between courses. Eighty-six patients with colorectal carcinoma were evaluated. The combined complete and partial response rates were 39% for 54 patients who did not receive prior chemotherapy and 22% for 32 patients who had previously received chemotherapy. Four patients who were previously resistant to 5-FU attained objective responses. The median time to disease progression for the 28 responders was 10 months. The median survival time of responders was 19.5 months, and the probability of their being alive at 2 years was 40%. Of 27 patients with gastric adenocarcinoma, 13 (48%) responded to therapy. Their median time to disease progression was 5.5 months. The median survival time of responders was 11 months, and their probability of being alive at 15 months was 30%. Toxicity was within acceptable limits. Toxic effects included stomatitis, diarrhea, conjunctivitis, skin rash, and mild myeloid hypoplasia. In a separate study, plasma concentrations of L-folates greater than 10(-5) mol/L were achieved after a rapid single IV injection of 200 mg/m2 of folinic acid. Comparisons of our results with those reported in previous studies on 5-FU administered as a single agent suggest that, in advanced colorectal and gastric adenocarcinoma, folinic acid administered in high doses enhances the effectiveness of 5-FU administered concomitantly. Furthermore, some colorectal tumors that were previously resistant to 5-FU become sensitive to this drug. The survival of the patients who responded to therapy was markedly improved over that observed in reported series of untreated patients with advanced colorectal and gastric adenocarcinomas.

Erroneous results of 3H-thymidine incorporation are related to position of thymidine residues in oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) targeted to complementary mRNA sequences have proved to be a powerful approach in assessing the function and the role of unique genes in cell proliferation, differentiation, or transformation. Despite their importance in the development of future therapies, little is known about their fate after uptake by cells. Here, we have examined the contribution of individual nucleotide residues from synthetic nonspecific ODNs on assays commonly used to measure cell proliferation. A dramatic decrease of the 3H-thymidine (3H-T) incorporation was obtained with nonspecific ODNs, while no effect on cell proliferation was observed as assessed by three other techniques. We demonstrate that the presence and position of thymidine in the ODNs directly interfere with the intracellular thymidine pool, leading to faulty data of 3H-T incorporation. As an alternative method, we used 3H-deoxyuridine (3H-dU), which is integrated more efficiently in DNA than thymidine. We observed that 3H-dU incorporation was also decreased. In conclusion, caution should be exercised in the interpretation of the results of 3H-T and 3H-dU incorporation in the presence of oligodeoxynucleotides.

Homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency revealed by stroke in adult siblings.

Three patients from a single family of six siblings had homocystinemia and homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency and had severe recurrent strokes in adult life. Two of the patients died 1 year after clinical onset.

Adenoviral transduction of human 'clinical grade' immature dendritic cells enhances costimulatory molecule expression and T-cell stimulatory capacity.

The therapeutic use of dendritic cells (DC) in antigen-specific anti-tumor vaccines, requires sufficient numbers of functional DC, the preparation of which should comply with the code of Good Manufacturing Practice. In addition, the expression of tumor specific antigen should be possible in these DC. As a preclinical step, the method reported here was developed in healthy volunteers. Monocytes (Mo) were isolated by leukapheresis from 12 donors, purified by elutriation and then cultured for 6 days in sealed bags in AIM-V serum free medium with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13). Between 6x10(8) and 1x10(9) immature DC (iDC) could be differentiated from one leukapheresis. Cells displayed a characteristic iDC phenotype (CD1a(+), CD14(-), CD80(+), CD86(+), HLA DR(+), CD83(-)), and had potent allogeneic and antigen dependent autologous T cell-stimulatory capacity. Moreover, iDC could be further differentiated into mature DC by CD40 ligation as assessed by CD83 expression and the upregulation of HLA-DR and costimulatory molecules. After infection with a recombinant adenovirus encoding for beta-galactosidase (betaGal), 50% to 80% of iDC expressed betaGal without toxicity. Adenovirus infection increased the expression of both costimulatory molecules and CD83, and also increased allogeneic stimulatory capacity. Thus, the method developed here allows us to use large numbers of functional iDC as will be required for therapeutic uses in man. These DC can express a transgenic protein.

Mechanism of inhibition of DNA ligase in Ara-C treated cells.

The activity of DNA ligase, the enzyme involved in ligation of DNA fragments and also in DNA repair is inhibited by Ara-C. The exposure of two human leukemic cell lines, K562 and HL-60 to 10(-5) M Ara-C for 3 h, induces a decrease of DNA ligase activity by 40% in K562 and 92% in HL-60. This decreased activity is due to an inhibition by Ara-CTP of the ligase-adenylate complex generation, the crucial step in the action of this enzyme. The activity of the semi-purified ligase as well as the formation of ligase-adenylate complex are decreased in the presence of Ara-CTP. These results demonstrate that Ara-C via its active form Ara-CTP inhibits DNA ligase activity through the inhibition of the ligase-adenylate complex. Other inhibitors of DNA synthesis, such as hydroxyurea, do not exert the same inhibitory effect. The inhibition of DNA ligase activity may be partly responsible for the cytotoxicity of Ara-C.

Evidence for a transition between singlet and triplet states in the electrochemical reduction of 2,2'-4,4'-tetranitrobiphenyl.

Two different dianionic species, close in energy, result from the electroreduction of 2,2'-4,4'-tetranitrobiphenyl, 1. The first-formed species has biradical characteristics, such as a triplet EPR spectrum, but it slowly evolves to another EPR-silent species, essentially described as a quinoid form. DFT calculations also support the existence of two dianionic states of the dianion, singlet and triplet, differing by less than 10 kcal mol(-1) (in the gas phase); the quinoid singlet state is more stable.

A study of the mechanisms of cytotoxicity of Ara-C on three human leukemic cell lines.

The main biochemical determinants involved in cytosine arabinoside (Ara-C) metabolism were studied in one lymphoblastic (Reh) and two myeloid (HL60 and K562) human leukemic cell lines exhibiting various sensitivities to Ara-C, Reh being the most and HL60 the least sensitive. The level of intracellular Ara-C accumulation and Ara-CTP formation was far more important in Reh cells than in myeloid cell lines but was not closely related to deoxycytidine kinase activity or to deoxycytidine triphosphate pool size. The level of Ara-C incorporated into DNA was similar in the three cell lines. Ara-CTP formation correlated better with the cytotoxicity to clonogenic cells than did Ara-C incorporation into DNA. DNA polymerase alpha was moderately inhibited to various degrees, depending on the cell line; this moderate inhibition does not seem sufficient to explain the inhibition of DNA synthesis. The activity of DNA ligase, the enzyme joining the Okazaki fragments, which was not detected in Reh cells, was strongly inhibited by Ara-C in HL60 and to a lesser degree, in K562 cells. The inhibition of DNA ligase probably also contributes to the inhibition of DNA synthesis and, thus, to the cytotoxic effect of Ara-C and may explain the smaller size of DNA fragments observed following Ara-C treatment. The variations in each critical determinant observed in these three cell lines increase the complexity and plurality of the mechanisms of Ara-C action.

Electrostatic and electrophilic catalysis in the reductive cleavage of alkyl aryl ethers. The influence of ion pairing on the regioselectivity

Electrophilic and electrostatic catalysis have been identified as distinct contributions that affect the reactivity of radical anions in the reductive cleavage of alkyl aryl ethers. Two modes of mesolytic scission of these radical anions are possible: homolytic (dealkylation, a thermodynamically favored but kinetically forbidden process) and heterolytic (dealkoxylation). From our studies (alkali metal reductions, electrochemical studies, use of substrates with a preformed positive charge in certain positions of their structure) it can be concluded that the heterolytic scission is very much dependent on the electrophilic assistance by the counterion and it is only observed in contact ionic pairs with unsaturated cations (electrophilic catalysis). On the other hand, the homolytic scission is observed in solvent-separated ionic pairs, and it is especially efficient when the pair has a controlled topology with a tetralkylammonium cation (saturated cation) near the oxygen atom. The effect of the cation has, in this case, electrostatic origin (electrostatic catalysis), probably lowering the barrier of the intramolecular pi-sigma electron transfer process and thus reducing the kinetic control of the reaction in such a way that the thermodynamically more favorable process is produced.

Synthesis and photochemical reactions of nitroestrogens: possible photoaffinity labels of zero length.

The syntheses of 3,4-dimethoxy-1,3,5(10)-estratrien-17-one and 4-bromo-3-methoxy-2-nitro-1,3,5(10)-estratrein-17-one are described and their photoreactions with amines and hydroxide ion studied. The possible usefulness of these new steroids as photoaffinity labels of zero length is discussed.

Research on HIV sexual risk: social relations-based approach in a cross-cultural perspective.

In opposition to individual-oriented approaches to sexual risk taking, new theoretical approaches have emerged that strive to encompass all of the social relations--that is to say, the connections that structure the components of relations between groups and go beyond simple adaptations to a given interaction--that prevail in a sexual relationship. This paper examines the strengths and weaknesses of such an approach for understanding HIV risk-related behaviour. The analysis is based on data from European surveys of sexual behaviour in the general population, with special attention paid to the data from the Belgian survey, which used a relation-based approach to risk-related behaviour. The analysis shows that sexual behaviour and preventive behaviour patterns of men and women appear to be linked to women's status in a given society. The data also tend to show that the social and preventive contexts also help structure the relations that develop between partners when it comes to negotiating about using a condom. Finally, the same people behave differently depending on the relationship's context. In particular, the balance of power within the couple, which is strongly interconnected with gender relations issues, and even characteristics of the former relationship help explain these behaviour differences.

Aminoglycoside-induced ototoxicity.

One of the major side effects of aminoglycoside antibiotics (AG) is ototoxicity. The authors review the literature revealing many controversies on every aspect of this side-effect. Although epidemiological studies have to face the problem of reliable evaluation techniques, the incidence of cochleo- and vestibulotoxic side-effects has been estimated at 7.5% for each. Netilmicin appears to be less ototoxic. No definite risk factors can be proposed, although age, length of therapy, bacteremia, fever, liver and renal dysfunction are probably very important parameters. Most pathological changes at the cochlear level follow a clear spatial sequence, showing unspecific, degenerative lesions, involving every structure of the cochlea. This makes it impossible to draw etiopathological conclusions. Recent pharmacokinetic studies have rejected the 'accumulation theory' of AGs in perilymph, while also in endolymph no accumulation can be found. Only a few data are available on inner ear tissue levels. Among the different pharmacodynamic hypotheses on the action of AGs, binding of the drug to acidic glycosaminoglycans in the stria vascularis, and interference by the drug with phosphoinositide metabolism in the hair cells seem to be of major importance.

The solitary schwannoma of the eighth cranial nerve. An immunohistochemical study of the cochlear nerve-tumor interface.

Given recent controversy concerning hearing preservation surgery of the acoustic neurinoma, an immunohistochemical study was undertaken to investigate the cochlear nerve-tumor interface. Ten intact medium-sized acoustic neurinomas were studied by means of classic staining procedures and an immunohistochemical technique using monoclonal mouse antibodies to human neurofilaments. Our observations indicate that the cochlear nerve is histologically involved in the tumoral process in those cases in which macroscopically visible adherences between the cochlear nerve and the tumor are present. We were not able to discern a clear cleavage plane. Six of the 10 specimens showed tumoral invasion of the cochlear nerve. Several therapeutic attitudes are discussed in view of these observations and reports from the international literature. In conclusion, the principle of hearing preservation surgery is rejected in favor of total tumor removal in every case in which surgery is indicated.

Dehiscence of the facial canal: developmental aspects.

In a series of human fetuses, the course of the facial canal in the temporal bone was investigated by the use of light and scanning electron microscopy. The normal development of the facial canal was correlated to clinical aspects of facial nerve dehiscences. Our observations demonstrate a more complex way of facial canal development not limited to the 'simple' ossification of the otic capsule. Endochondral ossification of the otic capsule does not virtually change the shape of the primitive facial sulcus. The fibrous layers surrounding the facial nerve seem to be responsible for the final architecture of the facial canal and not the otic capsule ossification by itself. The time sequence of their histological development is equally important and permitted us to distinguish three phases in facial canal development. The role of disturbances in epigenetic control for the initiation of dehiscences is discussed.

A pharyngeal hairy polyp causing neonatal airway obstruction: a case study.

A case of neonatal respiratory distress due to an oropharyngeal hairy polyp is reported with its MRI assessment. The place of hairy polyps in the differential diagnosis of upper respiratory tract obstruction is discussed. Early and rapid diagnosis and treatment may be lifesaving for the newborn.

Histological study of the thin replacement membrane of human tympanic membrane perforations.

A histological study was done on the thin, nearly transparent replacement membrane of tympanic membrane perforations. Human tympanic membranes that were rejected for transplantation, were studied by light and electron microscopy. The abrupt reduction in thickness at the margin of the covered perforation, is entirely due to the reduction of the lamina propria. Even in the thinnest parts of the replacement membrane, a lamina propria is present, separated by continuous basement membranes from the epithelium and mucosa, and measuring no more than some 2-3 microns in thickness. This lamina propria consists of fibrils and interfibrillar matrix, but fibroblasts appear to be lacking. The epithelial layer does not contain basal cells, confirming the thesis that the upper layers are not generated by in situ proliferation, but that they have migrated from the periphery.

Normal growth pattern of the middle ear cleft in the human fetus.

The normal growth pattern of the middle ear cleft was studied on macro- and histological sections of the human fetus. When compared with adult temporal bones, the inclination of the tympanic ring remains unaltered throughout fetal development. Expansion of the middle ear cleft is caused by intrinsic growth and by lateral displacement of its constituent elements: tympanic ring, otic capsule and squamous bone. Not only are the two mutually different modes of growth movement quite dissimilar in magnitude and direction, but so is their resultant vector for each constituent; this dissimilarity in growth movement leads to a characteristic change in shape of the middle ear cleft and to curvature of the tympanic membrane. The intrinsic growth of these elements is simultaneously accompanied by remodelling of their shape; lateral displacement of the squamous bone and tympanic ring is caused by the pressure of the enlarging brain. The consequences of developmental disturbances on the normal growth pattern are discussed.

Early ossification within the human fetal otic capsule: morphological and microanalytical findings.

Besides the use of conventional techniques such as light and polarization microscopy, the present paper proposes the combined use of transmission electron microscopy, secondary and backscattered electron imaging, energy dispersive X-ray analysis and computed tomography for the diagnostic evaluation of ear pathology in the human fetus. These methods were used to revisit the primary calcification front of the fetal otic capsule between 16 and 23 weeks gestational age. Ultramicroscopic evaluation demonstrates similar fetal bone formation to that found in other bones of the human fetus. The formation of the endosteal and periosteal layers is a typical example of early intra-membranous ossification. The enchondral layer is made up of fibrillar bone, laid down around the calcified cartilage remnants. Microchemical analysis indicates a significantly higher Ca/P ratio in the endochondral layer with respect to the endosteum and periosteum. The consequences of a lower Ca/P ratio in the endosteal layer are discussed in view of calcium homeostasis and inner ear function.