Synthesis and evaluation of hybrid molecules targeting the vinca domain of tubulin.

Some hybrids of vinca alkaloids and phomopsin A, linked by a glycine pattern, have been synthesized in one or two steps, by an insertion reaction and shown to inhibit microtubule assembly. These compounds have been elaborated in order to interact with both the "vinca site" and the "peptide site" of the vinca domain in tubulin. Two out of three hybrids are potent inhibitors of microtubules assembly and they present good cytotoxicity against different cell lines. Molecular modelling studies show that they could bind, within the vinca domain, in similar spatial regions as those of vinca and phomopsin thanks to the flexibility provided by the glycine linker used to elaborate these hybrids.

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues--a potential cosmetic role.

The naturally occurring tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10⁻¹¹-10⁻7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10⁻5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo-cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10⁻¹¹-10⁻7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin-1, occludin, ZO-1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two-fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10⁻¹¹-10⁻7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.

Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification.

Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.

Ultrastructural immunoperoxidase demonstration of autologous albumin in the alveolar capillary membrane and in the alveolar lining material in normal rats.

The location of autologous serum albumin within the alveolar-capillary membrane was studied in the rat under physiological conditions using antialbumin antibodies labeled with peroxidase. Albumin was detected in the lung interstitium, and in numerous pinocytic vesicles within endothelial cells and type I alveolar epithelial cells. The immunoreaction was also positive at the level of plasmalemmal membranes of both cell types and in the alveolar lining material.

Indoor airborne asbestos pollution: from the ceiling and the floor.

Electron microscopic measurements of the concentrations of airborne asbestos were carried out inside and outside an office building having ceilings sprayed with a crocidolite-containing material and floors covered with vinyl-chrysotile tiles. Under normal conditions in this building, constructed 10 years ago, the two asbestos-containing materials released fibers into the air. This is the first measurement of elevated (up to 170 nanograms per cubic meter) concentrations of indoor airborne asbestos associated with the weathering of asbestos floor tiles during their service life. Asbestos flooring is used in a large number of buildings and represents the third largest use of asbestos fibers in the United States and in Europe, ranking after roofing and asbestos-cement pipe.

Asbestos: scientific developments and implications for public policy.

Asbestos is a commercial term for a group of fibrous minerals often associated with the development of pulmonary interstitial fibrosis (asbestosis), lung cancer, and malignant mesothelioma in occupationally exposed individuals. The pathogenicity of different forms of asbestos varies--long, thin amphibole fibers are most pathogenic, particularly in the induction of mesothelioma. Available data do not support the concept that low-level exposure to asbestos is a health hazard in buildings and schools. The concentration of asbestos fibers in air, type of asbestos, and size of fibers must be considered in evaluation of potential health risks.

MICA compatibility and immunization in third kidney transplantations.

Significantly lower graft survival has been observed among recipients of a third (G3) compared with a first or second kidney transplantation. Because patients awaiting G3 are largely HLA immunized, they are usually transplanted with a high HLA match. Moreover, their rate of acute rejection episodes is similar to a first or second transplantation. Since major histocompatibility complex class I related chain A (MICA) molecules have been proposed as new targets for antibody recognition, we were interested to type donors and recipients for MICA alleles and to study MICA immunization of these patients. Forty-three pairs of donors and recipients were typed for MICA alleles using Luminex technology (LABtype RSSO). MICA alleles showed strong linkage disequilibrium with the B locus: some 4-digit alleles were preferentially associated with a given MICA allele. A greater frequency of patients with 2 MICA mismatches (MM) was observed among patients with rejection (40%), whereas all the graft losses were observed in patients with 0 or 1 MICA MM. MICA immunization was studied using sera from 52 patients collected on day 0 and after transplantation using a Luminex assay (LABScreen). MICA immunization was less frequent than HLA immunization, and MICA donor-specific antibody (DSA) was equally present in functional and failed grafts. These observations confirmed the potential role of MICA immunization in rejection, whereas the poor graft survival among third transplantations could not be explained by MICA incompatibility or immunization.

Specificity of T cells invading the skin during acute graft-vs.-host disease after semiallogeneic bone marrow transplantation.

The mechanisms responsible for skin lesions during acute graft-vs.-host disease (aGVHD) after allogeneic bone marrow transplantation (BMT) are poorly understood. The exact role of various effector cell populations and "major" (particularly HLA-DP) or "minor" antigens as target molecules is not known. To investigate the nature of cells responsible for tissue injury, we cultured T cells from skin biopsy first with interleukin 2 (IL-2) alone and then in polyclonal activation conditions to avoid in vitro antigenic sensitization before specificity testing. We applied this method to two biopsies performed during aGVHD after semiallogeneic BMT and obtained cytotoxic T cells against four graft mismatches: CD8+ T cells against HLA-A2.2 and HLA-B27 and CD4+ T cells against HLA-DP101 and HLA-DP401. This demonstrates that T cells with documented specificity can be obtained from an aGVHD lesion without antigenic selection. Moreover, these data directly implicate DP as a potential target antigen for aGVHD.

[Post-transfusion acute lung injury (Trali) after plasma infusion in a patient having a constitutional thrombotic microangiopathy]. / Syndrome d'oedème pulmonaire lésionnel aigu post-transfusionnel (Trali) après perfusion de plasma frais congelé au cours d'une microangiopathie thrombotique congénitale.

INTRODUCTION: Transfusion-related acute lung injury is a post-transfusion interstitial lung injury. CASE REPORT: We reported a post-transfusion acute lung injury in a 23-years old woman having a chronic thrombotic microangiopathy related to an ADAMTS 13 constitutional deficiency receiving monthly plasma infusion for six years. The temporal relationship between the lung injury and the infusion of fresh frozen plasma led to the diagnosis of transfusion-related acute lung injury. The finding in the donor of the transfused plasma of an anti-HLA class II antibody recognizing HLA-DR52 present on leucocytes of the recipient suggests a causal relationship between this antigen-antibody conflict and the triggering of the TRALI. This chronic pathologic state requiring monthly plasma transfusions for thrombotic accident prevention raises the question of the selection of plasma obtained from non-immunized donors. CONCLUSION: The occurrence of a post transfusion pulmonary edema without cardio-vascular overload, must lead to consider a TRALI especially in predisposing clinical situations. In the case reported the role of constitutional ADAMTS 13 deficiency in genesis of TRALI is considered.

Pleural carcinogenic potency of mineral fibers (asbestos, attapulgite) and their cytotoxicity on cultured cells.

The carcinogenicity of several samples of mineral fibers was tested following injection of 20 mg in the pleural cavity of noninbred Sprague-Dawley rats. Three samples of chrysotile asbestos (mean length: 3.2, 2.1, and 1.2 micron) induced mesotheliomas at a rate of 48, 52, and 19%, respectively. The first sample was acid leached prior to intrapleural injection; in that group, the percentage of mesotheliomas was reduced to 25%. Treatment with amosite and crocidolite resulted in the occurrence of 57 and 56% of mesotheliomas. Acid-treatment of amphiboles did not significantly modify the percentage of mesotheliomas. When the Stanton's fiber dimensions were taken into consideration to correlate with mesothelioma incidence, the observed number of mesotheliomas in the chrysotile-treated animals was much lower than that expected, suggesting that other fiber parameters (chemistry, physicochemistry) play a role in the carcinogenicity. Attapulgite fibers (mean length: 0.77 micron) did not induce tumor, and the mean survival time was of the same order as that observed in the control groups. The injection of quartz resulted in no mesothelioma but did result in 6 malignant histiocytic lymphomas (17%) and 2 malignant schwannomas (6%). In vitro experiments did not show strong correlation between cytotoxicity and the carcinogenic potency of these minerals, but the qualitative cellular responses might give some indications on the fiber's potency. In addition, the in vitro effects of the fibers seem to be modulated by their size.

Intrapleural production of interleukin 6 during mesothelioma and its modulation by gamma-interferon treatment.

In vivo production of monokines was analyzed in 17 human malignant pleural mesotheliomas. High concentrations of interleukin 6 (IL-6) were detected in pleural effusions, contrasting with low levels of IL-1 beta and tumor necrosis factor alpha. This production arose from malignant cells, as shown by immunochemical analysis of pleural cells and by production of IL-6 by mesothelial cell lines. Intrapleural administration of recombinant human gamma-interferon to six patients led to a marked decrease in intrapleural IL-6 concentrations in all cases. This treatment was associated with in situ activation of macrophages and cytotoxic T-lymphocytes, as indicated by increased intrapleural neopterin and soluble CD8 concentrations. In vitro gamma-interferon had no effect on the production of IL-6 by mesothelial cell lines but decreased the growth of 3 of 6 mesothelioma cell lines. These results indicate that systemic manifestations of malignant mesothelioma, including fever, cachexia, and thrombocytosis may be related to the production of IL-6 by malignant cells, and that local gamma-interferon infusion may reduce this production by stimulating antitumoral immunity and/or by directly decreasing the proliferation of malignant cells.

Lysosomal and cytosolic sialidases in rabbit alveolar macrophages: demonstration of increased lysosomal activity after in vivo activation with bacillus Calmette-Guerin.

Sialidase activity was assayed in homogenized rabbit alveolar macrophages using a fluorogenic substrate: sodium 4-methylumbelliferyl-alpha-D-neuraminate. After differential centrifugation one acid-active enzyme (optimum pH 4.2) was detected in the 16,000 X g pellet that contained lysosomes, mitochondria and peroxisomes. A second activity, with an optimum pH of 5.4, was found in the cytosolic fraction. The acid-active sialidase accounted for more than 95% of the total sialidase activity in crude homogenate. When alveolar macrophages were collected from rabbits stimulated with bacillus Calmette-Guerin (BCG), the acid-active sialidase specific activity was increased 2.5-fold whereas other lysosomal enzymes such as N-acetylglucosaminidase and beta-galactosidase were stable. The cytosolic sialidase activity did not change.

Inhibition of acid sialidase by inorganic sulfate.

Sulfated glycosaminoglycans are known to inhibit mammalian acid-active sialidase. Although the inhibition depends clearly on the presence of sulfate groups on these macromolecules, there was no information on the intrinsic inhibitory potency of inorganic sulfate. In this study, we demonstrate that inorganic sulfates inhibit acid-active Mu-Neu5Ac sialidase of U937 cells. This inhibition was found to be reversible and it appeared to be of the mixed competitive type. Sulfate-induced inhibition was also observed in other cells as well as with other substrates such as sialyl lactose and bovine mixed brain gangliosides. We conclude that the intrinsic inhibitory potency of sulfate groups may be significantly involved in the inhibition of acid-active sialidase by sulfated glycosaminoglycans. In addition, inorganic sulfate by its apparent potency to selectively inhibit acid sialidases might constitute an interesting tool for the characterisation of the minor forms of sialidases occurring in mammalian cells.

CFTR gene mutations in adults with disseminated bronchiectasis.

The severity and type of clinical manifestations are variable in patients with cystic fibrosis (CF). The respiratory syndromes in these patients consist of lung infections associated with disseminated bronchiectasis (DB), asthma, and chronic obstructive pulmonary disease. To investigate the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in chronic pulmonary disease in adults, we studied 32 DB patients with a clinically isolated respiratory syndrome. Careful analysis of all the CFTR gene exons and their flanking regions revealed a significantly increased frequency of CFTR gene mutations in these patients. Thirteen CFTR gene mutations were identified in sixteen different alleles. Six of these mutations, which have previously been reported as CF defects, were found on nine alleles. A further four, two of which had not previously been described (D192N and 406-2 AdeltaC), are potentially disease-causing mutations. We also identified three rare substitutions (R31C, L997F, T1220I), which could be involved in mild CFTR gene disease. Four patients were compound heterozygotes, one carried two CFTR gene mutations (possibly allelic) and six were heterozygous for a mutation. These results indicate that CFTR gene mutations may play a role in bronchiectatic lung disease, possibly in a multifactorial context. These findings have implications for genetic counselling of DB patients and their families.

Quantitation of elastin in human urine and rat pleural mesothelial cell matrix by a sensitive avidin-biotin ELISA for desmosine.

A specific enzyme-linked immunosorbent assay (ELISA) was developed for the determination of desmosine, a cross-linked amino acid specific to fibrous elastin. Competition between solid phase-bound desmosine-protein conjugate and free desmosine for binding to monospecific anti-desmosine antiserum constituted the underlying principle of the assay. The conjugation of desmosine to different protein carriers was carried out with the 1-ethyl-3-(dimethylamino-propyl)carbodiimide (ECDI); rabbits were immunized with desmosine-bovine serum albumin and micro-titer plates were coated with desmosine-egg albumin. An avidin-biotin peroxidase system was used to reveal anti-desmosine antibodies bound to the desmosine-protein conjugate. As both conjugates revealed new non-specific common epitopes on the carrier proteins, prior absorption of the anti-desmosine antiserum on rabbit albumin polymerized with ECDI was required to remove the antibodies directed against these neo-antigens. The absorption procedure resulted in an increased specificity and sensitivity. Values ranging from 0.07 to 4 ng of desmosine/well could be detected and this sensitivity was greater than that obtained in previous immunoassays for desmosine. In order to assess the specificity of the test, samples containing aminoacids and urine hydrolysates were included in an assay. Some cross-reactivity was observed with the desmosine precursor lysinonorleucine and the desmosine isomer isodesmosine but, in contrast the very low cross-reactivity observed with collagen hydrolysate was similar to that exhibited by albumin hydrolysate. Analysis of urine samples from 118 normal male volunteers showed, firstly, that urinary creatinine measurement was a good indicator of the amount of urine which could be safely introduced in the assay without risk of non-specific interference by other organic compounds and, secondly, that the desmosine/creatinine ratio was a reliable index for an in vivo assessment of degraded elastin excretion. The assay also allowed quantitation of elastin fiber biosynthesis in the connective tissue matrix of cultured rat pleural mesothelial cells. This ELISA for demosine is a simple technique which should be useful for further in vivo or in vitro investigations of fibrous elastin tissue metabolism.

Preparative two-step purification of human IL-2 by HPLC and hydrophobic affinity chromatography.

Interleukin 2 (IL-2) has been purified by a protocol using gel filtration high performance liquid chromatography (HPLC) and hydrophobic affinity chromatography with blue-trisacryl M. Peripheral blood lymphocytes or tonsillar lymphocytes were stimulated with phytohemagglutinin (PHA). Serum free conditioned medium (CM) containing IL-2, other lymphokines and residual PHA molecules was analyzed after 3 variations of ammonium sulfate (AS) precipitation: (1) precipitation of CM with 50% AS yielded a precipitate containing most of the residual PHA but also a fraction of IL-2. (2) Precipitation with direct 80% AS of crude CM yielded both IL-2 and residual PHA. (3) A double step procedure (50% AS followed by 80% AS) yielded a precipitate containing IL-2 but free of residual lectin. HPLC purification of these various AS-precipitated materials or of lyophilized crude CM yielded 2 peaks with mitogenic activity as assayed with the CTLL2 murine clone or IL-2-dependent human Con A-stimulated lymphoblasts. IFN was easily separated from IL-2 and PHA, but BCGF still copurified with IL-2. Peak I (25 kDa) was enriched 400-fold for IL-2 while peak II (68 kDa) contained the residual PHA. The IL-2-containing fractions eluted from HPLC were further purified by blue-trisacryl M chromatography. The IL-2 eluted with 0.4 M NaCl. The entire protocol (HPLC followed by blue-trisacryl) led routinely to 8000-fold IL-2 enrichment. Preparative HPLC directly applied to lyophilized crude (CM) enriched IL-2 activity 400-fold with yield averaging 60% of the IL-2 input. The final material was free from interferon and IL-1, but BCGF still copurified with IL-2. The 2-step purified material (HPLC and blue-trisacryl) gave 2 bands in SDS-PAGE both of which contained IL-2.

Anticarbohydrate autoantibodies to sialidase-treated erythrocytes and thymocytes in serum from patients with pulmonary sarcoidosis.

PURPOSE: The presence of immunoglobulins and complement in sarcoid granulomata and bronchoalveolar lavage from patients with sarcoidosis suggests that humoral mechanisms may be of importance in granuloma formation. To test this hypothesis, we examined the possibility that antibodies to specific tissue carbohydrates causing alterations and/or dysfunction of immunocompetent cells might be present during sarcoidosis. Because we had previously shown the presence of sialidase activity in bronchoalveolar lavage from these patients, we have looked for the presence of antibodies that recognize sialidase-treated erythrocytes (mostly antigalactose) in the serum of patients with sarcoidosis. Since thymocytes are spontaneously recognized by peanut agglutinin, a lectin that binds galactose, the reactivity of serum from sarcoidosis patients with normal or neuraminidase-treated thymocytes has also been studied. PATIENTS AND METHODS: Serum samples were obtained from the venous blood of patients with biopsy-proven sarcoidosis, most of whom had no extrathoracic symptoms. The mean patient age was 31 years, with a range from 21 to 57 years. There were 12 women and 19 men, and 10% of the patients were smokers. Sarcoidosis was classified as recent if symptoms had been present for less than 1 year and chronic if symptoms had been present for longer than this. Control serum samples were obtained from patients with idiopathic pulmonary fibrosis (n = 9) and from healthy volunteers (n = 15). Furthermore, serum from patients who had previously had sarcoidosis but in whom cures had been achieved was also studied (n = 6). RESULTS: Sialidase-treated erythrocytes were lysed in autologous serum upon incubation at 37 degrees C providing that the serum came from a patient with active disease. Serum from either normal volunteers or patients with resolved sarcoidosis had no significant cytotoxic activity. Lysis proceeded through activation of the classical complement pathway following fixation of autoantibodies. These antibodies were predominantly of the IgM class. They were able to agglutinate neuraminidase-treated thymocytes, whereas untreated thymocytes did not fix the antibodies. Carbohydrate inhibition experiments demonstrated that these antibodies are mostly galactose specific. As this sugar is located immediately below the sialic acid residues in the carbohydrate moiety of membrane glycoconjugates, it is unmasked following sialidase treatment. CONCLUSION: Since galactose has been shown to be present on the membrane of certain subsets of immunocompetent cells (e.g., lymphocytes and macrophages either spontaneously or after stimulation), it is possible that antigalactose antibodies may affect the metabolism of these cells, leading to some of the immune dysfunctions that are observed during sarcoidosis.

Alternating radiotherapy and chemotherapy schedules in small cell lung cancer, limited disease.

Sixty-three evaluable patients with limited small cell lung carcinoma were entered into two pilot studies alternating 6 cycles of combination chemotherapy (Doxorubicin 40 mg/m2 d 1; VP16213 75 mg/m2 d 1, 2, 3; Cyclophosphamide 300 mg/m2 d 3, 4, 5, 6; and Methotrexate 400 mg/m2 d 2--plus folinic acid rescue--or Cis-Platinum 100 mg/m2 d 2) with 3 courses of mediastinal radiotherapy as induction treatment. The first course of radiotherapy started 10 days after the second cycle of chemotherapy; there was a 7 day rest between chemotherapy and radiotherapy courses. This 6 month induction treatment was followed by a maintenance chemotherapy. The total mediastinal radiation dose was increased from 4500 rad in the first study to 5500 rad in the second. Both protocols obtained a complete response (CR) rate of greater than 85% (with fiberoptic bronchoscopy and histological verification). Local control at 2 years was 61% in the first study and 82% in the second. Relapse-free survival at 2 years was 32 and 37%, respectively. Toxicity was acceptable. We conclude that our results justify further clinical research in alternating radiotherapy and chemotherapy schedules.

The influence of HLA A-B-DR matching on cytomegalovirus disease after renal transplantation. Evidence that HLA-DR7-matched recipients are more susceptible to cytomegalovirus disease.

We examined the prevalence of cytomegalovirus infectious episodes, as defined by clinical, virological, and serological criteria (i.e., CMV disease), in 660 kidney graft recipients; 109 patients (16.5%) developed the disease, and 551 did not. No significant statistical link between CMV disease prevalence and a given HLA-A, -B, or -DR allele was observed. However, patients with HLA-DR7 matched grafts were statistically more frequently found (P < 0.01) in the group of recipients who developed CMV disease as compared with the group who did not develop CMV disease. Furthermore, among patients who developed CMV disease, a significant increase of HLA-DR7 matched over DR7 mismatched patients was noted, whereas no difference between matched and mismatched recipients for the other HLA-DR alleles was found. No difference in the severity of graft failure, often observed during, or immediately after, the CMV episode, was noted between patients matched or mismatched for HLA-DR7. Our data suggest that donor/recipient matching for HLA-DR7 is associated with increased CMV disease.

Effect of protein A immunoadsorption on panel lymphocyte reactivity in hyperimmunized patients awaiting a kidney graft.

Six hyperimmunized patients awaiting a kidney graft underwent immunoadsorption of separated plasma through a protein-A column to remove HLA antibodies. This procedure was partially limited by constant and rapid antibody resynthesis in spite of strong immunosuppression with cyclophosphamide and prednisolone. Only two patients could be grafted with previously positive--currently negative crossmatches. The first died of infection on day 40, having developed early chronic vascular rejection and the other returned to hemodialysis on day 285 after the development of transplant glomerulopathy. However, immunoadsorption seems to be effective in removing HLA antibodies having a titer below 1/2. Such extremely hyperimmunized patients should probably be excluded from the immunoadsorption program.