Fatal Epstein-Barr virus primo infection in a 25-year-old man treated with azathioprine for Crohn's disease.

We report a case of Epstein-Barr virus (EBV) primo infection with the development of successive infectious mononucleosis, hemophagocytic lymphohistiocytosis, and B-cell lymphoproliferative disorder in a patient treated with azathioprine for Crohn's disease. This case report suggests that specific EBV-related clinical and virological management should be considered when treating a patient with inflammatory bowel disease with azathioprine.

[Frequency of associated malignancies in cutaneous lymphomas: a retrospective study of 86 cases]. / Fréquence des cancers associés dans les lymphomes cutanés. Etude rétrospective de 86 cas.

INTRODUCTION: The initial staging and follow-up of cutaneous lymphomas is far from being standardized. In this retrospective study, we describe the results of systematic laboratory investigations dedicated to a better definition of the TNM stage for the detection of associated malignancies. PATIENTS AND METHODS: This was a retrospective, descriptive, single centre study, including all cases of cutaneous lymphomas seen in the department of dermatology, university hospital of Reims, between 1987 and 2001. Data systematically recorded for each patient included clinical, biological, histological and molecular (cutaneous or circulating T or B clone) findings, imaging (thoracic and abdominal computed tomography scan; or chest X-ray and abdominal ultrasound tomography) and bone marrow histology (for B-cell cutaneous lymphomas only). RESULTS: In cutaneous T-cell lymphomas (n=63 including 47 mycosis fongoides), imaging revealed deep lymph nodes in 4 cases, a carcinoma of the kidney in one case, and a benign tumour in 6 cases. A T-cell clone was detected in the skin in 19/33 cases and in peripheral blood in 17/31 cases. In cutaneous B-cell lymphomas (n=23), imaging showed splenomegaly in 2 cases, a B-cell clone was detected in 3/12 cases in the skin, and bone marrow histology was normal in 21/22 cases. Among patients with cutaneous T-cell lymphomas, 14/63 (22 p. 100) had an associated malignancy. In 8/14 cases, the diagnosis of the associated malignancy was made prior to that of the cutaneous T-cell lymphoma. In 4 cases, the interval between the previous malignancy and the diagnosis of lymphoma was

Upregulation of the netrin receptor (DCC) gene during activation of b lymphocytes and modulation by interleukins.

The DCC (deleted in colon cancer) gene has a brain restricted high expression pattern. It encodes a transmembrane protein of the immunoglobulin superfamily identified as the netrin-1 receptor. It might be a member of the so called "brain-lymphoid" molecules, which control key cell surface events. To test this hypothesis we have assessed the DCC mRNA level in human normal and malignant myeloid and lymphoid cells. A high mRNA content has been observed only in mature B cells at the secreting or presecreting stage. Expression of DCC was also assessed in the anti-CD40 model of immunopoiesis. Activation of purified tonsillar B cells by anti-CD 40 antibody strongly increased the DCC mRNA level and this effect was dramatically enhanced by the association of IL-2 + IL-10, which is a potent and selective in vitro inducer of the B cell memory phenotype. In contrast no effect has been detected after activation of T cells by anti-CD3. These data suggest that the DCC encoded netrin receptor is involved in B cell immunopoiesis.

Value of autologous stem cell transplantation with purged bone marrow as first-line therapy for follicular lymphoma with high tumor burden: a GOELAMS phase II study.

This prospective phase II study was undertaken to evaluate the efficacy and toxicity of early intensive therapy followed by purged autologous bone marrow transplantation (ABMT) in patients with follicular lymphoma with high tumor burden. All patients received the VCAP regimen (vindesine, cyclophosphamide, doxorubicin and prednisone) as conventional chemotherapy and DHAP as second-line therapy. Twenty-nine consecutive patients were included in the study. Twenty-seven patients were grafted, seven in first complete remission (CR) and 20 in first partial remission (PR). Preparative therapy consisted of cyclophosphamide and total body irradiation (TBI) in all the patients. With a median follow-up of 6 years, the actuarial overall survival is 64% and the actuarial event-free survival is 55%. Two treatment-related early deaths were observed. Eleven patients were informative for serial PCR analysis of minimal residual disease after ABMT: two relapsed, four remained disease-free with PCR positivity and five were disease-free with PCR negativity. These encouraging results lay the basis of future prospective randomized trials comparing autologous stem cell transplantation as front-line treatment with conventional chemotherapy for patients with bad prognostic factors.

Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro.

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the deltaF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1beta and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured deltaF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl- concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl- concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.

[Modulators of leukocytic functions]. / Les modificateurs des fonctions leucocytaires.

Polymorphonuclear neutrophils (PMN) and monocytes play a role in vascular diseases. Animal experimental models, using deleukocytation or injection of anti-CD11b-anti-CD18 monoclonal antibodies (inhibition of leukocytic adhesion and of interaction with the endothelial cell) have confirmed this role in the ischemia-reperfusion syndrome and in myocardial infarction. In man, increased production of oxygen radicals, PMN release of elastase, increased monocyte formation of tissue factor (TF) and integrins have been noted in coronary artery disease, in chronic arteriopathy of the lower limbs and in association with vascular risk factors such as diabetes. Pharmacological alteration of leukocyte hyperactivity therefore seems to be justified. Pentoxifylline, used with good effect in arteriopathy of the lower limbs, affects numerous leukocytic functions: diminution in adherence and in PMN production of free radicals, diminution in the formation of TF and cytokines (TNF). Gingkolides reduce leukocytic interactions and platelet activation through an anti-PAF (Platelet Activation Factor) action. Aspirin may interfere with free radicals and inhibit TF formation. alpha-tocopherol blocks the activation, by free radicals, of the transcription factor NF k B. By altering the TNF and IL-1 cytokines, leukocytic activation can be controlled. Other cytokines (IL-4, IL-10) have an immunosuppressive action and reduce the formation of TF. The pharmacological targets are therefore multiple. Their use in vascular diseases is only at a very preliminary stage.

Hypoxia reduces endothelin production by rat alveolar type II cells in primary culture.

The purpose of the study was to describe endothelin (ET) production and to characterize the effect of hypoxia on preproendothelin-1 (preproET-1) messenger ribonucleic acid (mRNA) expression and ET secretion by rat type II pneumocytes in vitro. Rat type II pneumocytes were incubated in a sealed chamber containing a normoxic (21% O2) or hypoxic (1% O2) atmosphere for increasing durations. Immunoreactive ET (irET) was measured in cell supernatants using a radioimmunoassay. Rat preproET-1 mRNA was detected by Northern blot. Rat type II pneumocytes expressed preproET-1 mRNA, contained irET and secreted irET in a time-dependent manner. ET secretion was dependent on de novo ribonucleic acid (RNA) and protein synthesis. Hypoxia decreased irET secretion by 27% and reduced the steady-state level of preproET-1 mRNA by 60% whereas intracellular irET concentration was unchanged. Inhibition was partially reversible with the return to a normoxic atmosphere. Inhibition of nitric oxide synthesis did not prevent the inhibitory effect of hypoxia. In conclusion, rat type II pneumocytes in primary culture secreted immunoreactive endothelin and expressed preproendothelin-1 messenger ribonucleic acid. Hypoxia reversibly reduced endothelin-1 production through a reduction of the steady-state preproendothelin-1 messenger ribonucleic acid level. Nitric oxide synthesis did not mediate the inhibitory effect of hypoxia.

A recombinant human scFv anti-Rh(D) antibody with multiple valences using a C-terminal fragment of C4-binding protein.

Monomeric recombinant molecules prove generally unsatisfactory for in vivo use. Most biological systems are indeed multivalent either structurally, associating different chains, or functionally, when cross-linked by their ligands. Mimicking natural molecules for immune intervention implies the need for multimerizing systems to create multivalent molecules capable of interfering with physiological processing. A multivalent anti-Rh(D) recombinant protein has been designed by reconstructing the antibody binding site of a human monoclonal anti-Rh(D) antibody as a single chain Fv mini antibody, then multimerizing it by inserting at its C-terminal end the C-terminal part of the C4 binding protein (C4bp) alpha chain, which is responsible for the octamer multimerization of that molecule. This soluble multivalent recombinant molecule was functional, bound red blood cells (RBCs), agglutinated them, and did not activate complement. This demonstration model opens the way for future in vivo use of multivalent molecules associating antibody valences and other functional molecules for cell targeting, imaging, or removal of cells such as Rh(D)-positive RBCs for preventing Rh alloimmunization.

Factor V Leiden: detection in whole blood by ASA PCR using an additional mismatch in antepenultimate position.

Factor V Leiden mutation was initially detected in thrombophilic patients and relatives by PCR RFLP (Restriction Fragment Length Polymorphism) according to Bertina (1). This technique presents some drawbacks and the current trend is to simplify the diagnosis. We describe a technique of Allele Specific Amplification (ASA) which is optimized in terms of reliability: an additional mismatch in antepenultimate position enables to obtain the same specificity as PCR RFLP. Furthermore, coamplification of internal control warrants an optimal sensitivity. All the PCR have been simplified: the DNA extraction improvement allows to analyse the genotype with only a few microliters of whole blood whatever the anticoagulant and the procedure of preservation (freezing, dried blood spots, storage at +4 degrees C for several days). This technique saves time. Moreover, full automation of the ASA technique may be shortened thanks to the lack of extraction and the positive/negative reading of the PCR signal.

Inhibition of human TNF alpha and LT in cell-free extracts and in cell culture by antisense oligonucleotides.

Antisense oligonucleotides (ODN), complementary to mRNA of human tumor necrosis factor alpha (TNF alpha) and lymphotoxin (LT) were tested for their ability to inhibit TNFs. TNFs production was studied in cell-free systems including wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL). All ODN were effective in WGE at low concentration (0.2 microM), except those targeted to the 3' region of TNF alpha mRNA. A short ODN complementary to a common region between TNF alpha and LT inhibited both TNFs. In contrast, high ODN concentration (50 microM) was needed to inhibit LT mRNA translation in RRL, whereas no clear inhibition of TNF alpha was observed unless RNase H was added to the translation mixture. ODN effects on TNFs production by stimulated cell line in culture were also investigated. Three ODN-one located in the 5'-untranslated region, one spanning the AUG initiation codon and one downstream of this AUG-were the most effective sequences to decrease TNF alpha production. Two ODN targeted to the AUG initiation codon of LT were also able to inhibit its production. In conclusion we confirm the role of RNase H in cell free systems, and we found that there is no correlation between ODN efficiency in a cell-free system nor in cell culture. Efficient ODN could be used for in vitro investigation of the role of TNF alpha and LT in mechanism in which they are involved.

Differential regulation of TNF alpha, IL-1 beta, IL-6, IL-8, TNF beta, and IL-10 by pentoxifylline.

Pentoxifylline (PTX) is a methylxanthine drug known to inhibit the production of tumor necrosis factor-alpha (TNF alpha), which plays a key role in inflammation. Recent studies also revealed that other cytokines may be inhibited by PTX. We investigated PTX effects on production and mRNA expression of TNF alpha, IL-1 beta, IL-6, IL-8, TNF beta and IL-10. Cytokine release was studied in 1/10 diluted whole blood culture (WB) and in peripheral blood mononuclear cell (PBMC) culture. Cytokine production was triggered in both culture systems by endotoxin (LPS) or by phorbol ester (PMA) plus phytohemagglutinin (PHA). Our results showed that expression and production of TNF alpha and TNF beta were inhibited by PTX in a dose-dependent manner. Moreover, we observed that depending on the way of activating cells, PTX induced an up- or a down-regulation (in PMA + PHA or LPS stimulated cells, respectively) for IL-1 and IL-6 release. We also noted that the effects of PTX on IL-6, IL-8 and IL-10 production were different in WB and in PBMC culture. In conclusion PTX acts on cytokine in a complex manner depending on cellular environment and on the method of activation.

Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products.

The aims of this study were (a) to determine if rat alveolar type II (ATII) cells and human pulmonary epithelial-derived cells (A549 cell line) could generate IL-6 in vitro, (b) to characterize the cytokine regulation of IL-6 gene and protein expression in these cells, and (c) to detect the in vivo expression of immunoreactive IL-6 by human ATII cells. Rat ATII cells in primary culture secreted bioactive IL-6 and immunostained with an anti-IL-6 antiserum. Spontaneous IL-6 secretion by rat ATII cells amounted to 5,690 +/- 770 pg/ml/10(6) cells (n = 12) and was fivefold higher than spontaneous rat alveolar macrophages IL-6 secretion (1,052 +/- 286 pg/ml/10(6) cells, n = 8, P = 0.001). Rat alveolar macrophage conditioned media (CM) increased IL-6 secretion by rat ATII cells through the effect of IL-1 and TNF. IL-6 gene expression and IL-6 secretion by A549 cells was induced by IL-1 beta, TNF alpha, and by human alveolar macrophages and THP1 cells CM. Induction was abolished when CM were preincubated with anti-IL-1 beta and anti-TNF alpha antibody. The combination of IFN gamma and LPS induced the expression of IL-6 mRNA by A549 cells whereas LPS alone had no effect. Immunohistochemical staining evidenced the expression of immunoreactive IL-6 by hyperplastic ATII cells in fibrotic human lung, a condition in which alveolar macrophages are known to be activated. ATII cells in normal human lung did not express immunoreactive IL-6. Our findings demonstrate that ATII cells may be an important source of IL-6 in the alveolar space thereby participating to the regulation of the intra-alveolar immune response.

Use of chromosome painting for marker chromosome identification in two children with congenital disorders.

Identification of supernumerary de novo marker chromosomes was considered up to now as difficult and sometimes impossible with classical cytogenetical banding methods. The determination of their chromosomal origin is now easier with fluorescent in situ hybridisation techniques and enables an exact correlation between chromosomal aberration and phenotypic features to be established. The authors describe the use of chromosome painting with chromosome 13 and 18 Whole library DNA probe for identification of supernumerary markers in tow patients with congenital disorders. Cytogenetic examination in the first cave revealed a mosaicism with a ring chromosome 13 but clinical findings were different from the classical "ring 13 syndrome', and chromosome painting revealed in an extra--dicentric 13 chromosome (mos : 47, XX, -13, +r (13) +dic (13) / 46, XX, r (13) / 45, XX, -13 / 48, XX, -13, +r (13), (12) dic (13) / 47, XX, -13, + (2) r (13), R-banding pattern on prometaphases and chromosome painting in the second case confirmed the marker to be a 18 p isochromosome (47, XX, +i (18p)). The feasibility and the usefulness of chromosome painting in ascertainment of the possible genetic significance of markers is discussed.

Involvement of tumor necrosis factor alpha in intracellular multiplication of Legionella pneumophila in human monocytes.

We investigated the role of tumor necrosis factor alpha (TNF-alpha) in human peripheral monocytes infected with Legionella pneumophila in vitro. Exogenous TNF-alpha significantly inhibited the intracellular multiplication of the bacterium. This effect was concentration and time dependent and was abrogated by anti-TNF antibodies. TNF-alpha levels in the culture supernatants were low but were enhanced by the addition of gamma interferon. When monocytes were cultured and infected in the presence of pentoxyphilline, a potent inhibitor of TNF-alpha synthesis, the intracellular bacterial growth was enhanced. The effect of pentoxyphilline was concentration and time dependent and was due to the inhibition of TNF-alpha production, as shown by Northern (RNA) blot hybridization of total RNA. In addition, the pentoxyphilline partially abolished the inhibitory effect of gamma interferon on bacterial intracellular multiplication. These results suggest that gamma interferon inhibits, at least partially, the intracellular multiplication of L. pneumophila by enhancing TNF-alpha synthesis.

Cytogenetic changes in 67 cranial and spinal meningiomas: relation to histopathological and clinical pattern.

The cytogenetic analysis of 67 meningiomas (58 intracranial and 9 spinal tumors) identified chromosomal abnormalities in 63% of cases. When chromosomes involved in numerical and structural changes with a frequency of more than one standard deviation above the mean were considered, distinct cytogenetic patterns could be identified according to sex, anatomical location and histology. The chromosomes more frequently affected were 1, 2, 3, 4, 8, 14, 15, 19, 22, Y. No conclusion could be drawn regarding the prognostic significance of these karyotypic alterations.

Increased frequency of the long (S) allotype of CR1 (the C3b/C4b receptor, CD35) in patients with systemic lupus erythematosus.

The allotypic forms of the C3b/C4b receptor (CR1, CD35) differ in length, in the number of expressed C3b binding sites and thus in their ability to mediate the processing of circulating C3- and C4-bearing immune complexes. We have used a combination of three informative restriction fragment length polymorphisms (RFLPs) to assess the frequencies of the F (most frequent allele comprised of four long homologous repeats (LHR)), S (five LHR) and F' (three LHR) alleles of the C3b/C4b receptor (CR1, CD35) in a French population of patients with systemic lupus erythematosus (SLE) (n = 63) and healthy controls (n = 158). A significantly higher frequency of the S phenotype was observed among patients (51%) as compared with controls (26%). The F' allele was found in 2/61 patients and 1/85 healthy controls, indicating the rare occurrence of the short CR1 allele in SLE. This allele is also extremely rare in the normal population. The overrepresentation of the S long allele among patients is indicative of a genetic linkage between CR1 and susceptibility to SLE.