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Biomedical subjects

S Muller

Publications and source records attributed to S Muller.

At least 163 records · Page 9Linked to original sources
PubMed1997

Action of metformin on erythrocyte membrane fluidity in vitro and in vivo.

The lipid domains of the cell membrane are believed to be one of the sites where biguanides exert their antihyperglycemic effect. We have examined the effects of metformin on the membrane fluidity of intact erythrocytes in vivo and in vitro. Membrane fluidity was measured by monitoring changes in the anisotropy of the fluorescent probe 6-antroyloxystearic acid (6-AS). The erythrocyte membranes from patients with non-insulin dependent diabetes mellitus treated with metformin were more fluid than those from non-insulin dependent diabetes mellitus patients treated by diet or healthy controls. There was no correlation between membrane fluidity and the plasma lipids or the parameters of metabolic control, suggesting that the high fluidity is an effect of metformin itself. Incubation of erythrocytes from healthy controls and diabetic patients treated by diet or glibenclamide with metformin in vitro confirmed that metformin increases the fluidity of erythrocyte membranes. In vitro metformin did not alter the fluidity of membranes from diabetic patients treated with metformin, perhaps because the basal high fluidity due to their in vivo interaction with plasma metformin could be increased no further. Since insulin appears to be required for the antihyperglycemic effect of metformin, the effect of insulin on membrane fluidity was also evaluated. Insulin generally had a small fluidizing effect on erythrocytes in vitro. The fluidizing action of both insulin and metformin could represent a membrane event common to the hormone and drug leading to additive or synergistic effects in vivo.

Adult↗
PubMed1997

In vivo T helper cell response to retro-inverso peptidomimetics.

Peptide analogues containing reversed peptide bonds between each residue along the peptide sequence (retro-inverso modification) have been analyzed for their antigenic and in vivo immunogenic properties in the MHC II and Th cell response context. Two antigenic peptides were selected for this study, namely peptide 103-115 of poliovirus VP1, which is involved in the production of Abs that neutralize the infectivity of the virus, and peptide 435-446 from the third constant region of mouse heavy chain IgG2a allopeptide gamma 2ab, which mimics a corneal Ag implicated in autoimmune keratitis. In a competition assay performed in vitro using reference hybridomas of known MHC class II restriction, both retro-inverso analogues bound (although more weakly in our test) to I-Ad and/or I-Ed class II molecules. However, in both cases, this lower affinity was apparently largely compensated in vivo, as a T cell response (with IL-2 secretion), equivalent to that obtained with the wild-type peptides, was observed following immunization of BALB/c mice with the retro-inverso analogues. Moreover, these T cells proliferated and produced IL-2 in response to the cognate peptides. It is concluded that the T cell receptors of T cells primed in vivo with the retro-inverso analogues readily cross-react with parent and retro-inverso analogue-MHC complexes. The approach of using pseudopeptides containing changes involving the backbone, and not the orientation of side chains, may thus be promising to design potent immunogens for class II-restricted T cells.

Amino Acid Sequence↗
PubMed1997

[Benefits of early nephrological management in chronic renal failure].

OBJECTIVES: We evaluated whether early nephrological referral of patients with chronic renal failure (CRF) resulted in improved condition of patients at initiation of maintenance dialysis and in better outcome on dialysis. PATIENTS AND METHODS: We prospectively recorded clinical status, laboratory parameters, length of hospital stay and outcome of 900 CRF patients who started maintenance dialysis at Necker hospital between January 1989 and December 1996. We compared patients who benefited regular nephrological follow-up, and patients who were referred in emergency conditions at the ultimate stage of CRF. RESULTS: Among the 900 patients, 731 (81.2%) had regular nephrological follow-up, including 632 (70.2%, group IA) with optimal preparation to dialysis and 99 (11%, group IB) whose clinical course was complicated due to heavy comorbidity, whereas 169 (18.8%, group II) had no previous nephrological management. Over the 8-year observation period, the proportion of the latter group did not decrease. Late referred patients had higher blood pressure level, more frequent fluid overload, higher serum levels of urea, creatinine, uric acid and phosphate, and lower levels of bicarbonate, calcium, albumin and creatinine clearance that did well-prepared patients. Mean (+/- SD) hospital stay was 29.7 +/- 15.8 days in the former compared to only 4.8 +/- 3.3 days (p < 0.001) in the latter. Early deaths within 3 months of dialysis initiation were more frequent (7.1 vs 1.6%, p < 0.05) and less patients subsequently were able to be treated out-center (20.1 vs 40.7%, p < 0.05) in group II than in group IA. The overcost induced by late referral may be estimated at 0.25 million French francs per patient. CONCLUSION: An unjustified late nephrological referral of CRF patients still is observed in nearly 20% of cases. Such late referral is detrimental to both patients in terms of altered quality of life and long hospital stay, and to the collectivity due to heavy overcost. Closer cooperation between family physicians and nephrologists is needed to provide optimal management and allow timely preparation to maintenance dialysis of CRF patients.

Female↗
PubMed1997

Isolation of histones and related chromatin structures from spermatozoa nuclei of a dasyurid marsupial, Sminthopsis crassicaudata.

The spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata, have two distinct nuclear regions: uniformly electron-dense chromatin (C1) in the interior and fissured chromatin (C2) at the periphery. To investigate whether the differences in morphology are due to incorporation of different packaging proteins, spermatozoa nuclear proteins were characterised by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) and fractionated by reverse-phase high-pressure liquid chromatography (HPLC). The main protein component was protamine I, but a complete histone complement (H1, H2A, H2B, H3, and H4) was also detected. Immunocytochemistry showed localisation of H4, H2B, and H2A histones to the periphery of the nuclei, a region that corresponded to the C2 chromatin. The fissures in the chromatin of this region disappeared following incubation with fish protamines, indicating that the nucleohistone C2 region may be incompletely condensed relative to nucleoprotamines. This observation is consistent with the view that 60% of phosphodiester charges remain negative in nucleohistone DNA, whereas all DNA charges are neutralised in highly compact nucleoprotamines. Treatment of spermatozoa with micrococcal nuclease showed that the C1 chromatin was resistant to digestion, whereas the C2 region was cleaved into 30- to 38-nm agglomerates and 11-nm nucleosomal-size structures. Thus, this study demonstrates that spermatozoa nuclei of this marsupial species contain peripherally localised histones, and the nucleohistone chromatin accounts for the different morphology of the C2 region compared with the rest of the nucleus.

Amino Acid Sequence↗
PubMed1997

High susceptibility to collagen-induced arthritis in mice lacking IFN-gamma receptors.

Collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis, is induced in DBA/1 (H-2q) mice following immunization with type II collagen (CII) in CFA. Since we have previously shown that IFN-gamma exerts a biphasic effect during the evolution of CIA in DBA/1 mice, we analyzed the development of this disease in mice with a disruption of the IFN-gamma receptor gene (IFN-gammaR(0/0)). Mutant mice were interbred with the DBA/1 strain to yield IFN-gammaR(0/0) mice expressing the H-2q haplotype. In three consecutive experiments, IFN-gammaR(0/0) male mice were found to exhibit severe clinical and histologic arthritis with an average incidence of 88.5 vs 94.1% for the wild DBA/1 strain. Notably, onset of clinical symptoms occurred significantly earlier than in DBA/1 mice. Although of a lower magnitude than in males, CIA also developed early in IFN-gammaR(0/0) female mice and with higher clinical severity than in control DBA/1 females. Immunization of knockout mice with CII resulted in the generation of CII-specific T cells belonging to the Th1 phenotype that recognize the same immunodominant peptides as do DBA/1 mice. CIA in IFN-gammaR(0/0) mice was associated with a down-regulation of the CII-specific IgG response, and this impairment was essentially due to a strong reduction of Abs of the IgG2a isotype. Taken together, our findings provide evidence that IFN-gammaR deficiency in DBA/1 mice leads to the occurrence of severe CIA with an accelerated onset compared with that in wild-type mice, indicating that the proinflammatory action of IFN-gamma has been bypassed in the IFN-gammaR(0/0) mice.

Animals↗
PubMed1997

Membrane fluidity and oxygen diffusion in cholesterol-enriched erythrocyte membrane.

This work studied the effect of cholesteryl hemisuccinate incorporation on membrane fluidity and on the kinetics of oxygen diffusion at different depths in the erythrocyte membrane. Cholesterol concentration in the membrane was expressed as the cholesterol-protein ratio (C/Pt). The membrane fluidity, as assessed by a fluorescence polarization method with diphenyl-hexatriene and 1-(4-trimethylamino)-6-phenylhexa-1,3,5-triene, decreased as the C/Pt ratio increased. Time-resolved fluorescence spectroscopy of pyrene dodecanoic acid (PDA) under an increasing C/Pt ratio in the erythrocyte membrane revealed enhanced oxygen diffusion in the middle of the membrane bilayer (in which PDA was incorporated), which was not the case with pyrene butyric acid (PBA) incorporated in the internal part of the membrane surface. It has generally been accepted that increased membrane fluidity reduces the physical barrier to oxygen permeation. Such conflicting observations on oxygen permeation in the rigidified erythrocyte membrane could be due to variations in oxygen solubility (preferential partitioning) in different polarity microdomains (cholesterol and phospholipid partitions).

Cholesterol↗
PubMed1997

Pseudopeptide TASP inhibitors of HIV entry bind specifically to a 95-kDa cell surface protein.

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KPsi(CH2N)PR]-TASP construct, Psi(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KPsi(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KPsi(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KPsi(CH2N)PR]-TASP, the p95.TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KPsi(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KPsi(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.

Flow Cytometry↗
PubMed1997

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers.

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Animals↗
PubMed1997

An ELISA for detection of apoptosis.

We describe a simple and convenient enzyme-linked immunosorbent assay (ELISA) for the detection of apoptosis in tissue culture. An early event in apoptosis is DNA fragmentation followed by release of nucleosomes into the cytoplasm. Our sandwich assay uses a pair of monoclonal antibodies specific for two nucleosomal epitopes to capture and detect cytoplasmic nucleosomes onto the ELISA plate. Our assay is about 500 times more sensitive than the detection of apoptotic DNA ladder by agarose electrophoresis and is especially suited for the testing of large numbers of samples.

Apoptosis↗
PubMed1997

Accessibility of epitopes on the 52-kD Ro/SSA protein (Ro52) and on the RoRNP associated Ro52 protein as determined by anti-peptide antibodies.

The Ro ribonucleoprotein particle (RoRNP) is the target of a variety of specific anti-protein autoantibodies produced by patients with systemic autoimmune diseases. RoRNPs, the function of which remains elusive, appear to be rather heterogeneous in terms of their molecular composition. Among several Ro proteins, a protein of 52 kD (Ro52) has been identified, but its association with RoRNPs remains questionable. In this study, we first mapped the Ro52 regions that are accessible at the surface of the isolated protein, by means of antibodies raised in rabbits, against 39 overlapping synthetic peptides covering the whole Ro52 molecule and using several different methods, including various ELISA formats and Western blotting (based on the use of recombinant Ro52) and immunoprecipitation of in vitro translated Ro52. Then, the whole set of anti-peptide antibodies was used to attempt to immunoprecipitate RoRNPs from a cell extract of K562 cells. RoRNPs, especially Ro(hY3)RNPs, were effectively immunoprecipitated by certain anti-peptide antibodies, indicating that Ro52 protein is associated with at least a subset of RoRNP particles. As expected, a number of epitopes available on the isolated Ro52 molecule are no longer accessible when Ro52 is associated with the RoRNP. Conversely, neotopes, among them at least one corresponding to a previously characterized epitope recognized by patients' antibodies, are only present at the surface of the particle and not detectable on the isolated Ro52 protein.

Animals↗
PubMed1997

Induction of anti-polycation antibodies in H-2s mice by immunization with nuclear antigens.

Following administration of certain chemicals (heavy metals or lupus-inducing drugs), H-2s mice produce autoantibodies reacting with various nuclear antigens such as fibrillarin in the nucleolus and histones in chromatin. In the present study, we have immunized A.SW (H-2s) mice and their congenic counterparts A.BY (H-2b) mice with bovine thymus nuclei in Freund's adjuvant. As was previously observed with lupus-prone mice, such active immunization did not elicit antinuclear antibodies in any of the experimental groups. Surprisingly, the A.SW immunized with nuclei in adjuvant developed high titers of IgG antibodies that reacted exclusively with synthetic polycations. We obtained several monoclonal IgG antibodies from these mice and verified that these polycation-reactive antibodies were not directed against a specific nuclear antigen. The genetic analysis of the monoclonal antibodies further confirmed their clonal diversity. The mechanisms leading to the appearance of antibodies reactive with highly basic molecules in A.SW mice may be related to their predisposition to produce autoantibodies to cationic nuclear antigens (fibrillarin, histones) during chemically-induced autoimmunity.

Amino Acid Sequence↗
PubMed1997

Synthetic lipopeptides incorporated in liposomes: in vitro stimulation of the proliferation of murine splenocytes and in vivo induction of an immune response against a peptide antigen.

Amphiphilic lipopeptides, such as Pam3CysAlaGly and Pam3CysSerSer, were synthesized and incorporated into liposomes, and their ability to induce the proliferation of BALB/c mouse splenocyte was tested in vitro. When compared to monophosphoryl lipid A (MPL) the following potency order was found: liposomal lipopeptides > liposomal MPL > free (emulsified) lipopeptides. These results strongly depend on the size of the vesicles used: a mitogenic effect was observed only with lipopeptides incorporated within vesicles of diameter < or = 100 nm while lipopeptides in larger vesicles (diameter approximately 300 nm) gave no response. This may be related to the necessity for the liposome-associated lipopeptides to be endocytosed to reach putative intracellular targets. As immunoadjuvanticity seems to be linked to B-lymphocyte activation, the lipopeptides represent attractive alternatives to MPL for the realization of completely synthetic liposome-based peptide vaccine formulations. This was borne out by showing that Pam3CysAlaGly and Pam3CysSerSer, when incorporated in small unilamellar vesicles carrying a covalently conjugated synthetic peptide of sequence IRGERA, corresponding to an epitope of the C-terminal region of histone H3, were able to induce a potent and long-lasting immune response.

Adjuvants, Immunologic↗
PubMed1997

Interaction between certain major histocompatibility complex class II and T-cell receptor V beta alleles promotes the antibody production to extractable nuclear antigen-related peptides.

Our objective was to study the interaction between major histocompatibility complex (MHC) class II and T-cell receptor (TCR) alleles in the recognition of extractable nuclear antigen-derived peptides in 32 patients with systemic lupus erythematosus and 173 of their family members. MHC genes were analyzed using sequence specific oligonucleotides, and TCR beta-chain gene polymorphism using restriction fragment-length polymorphism. One dominant peptide (as defined by enzyme-linked immunosorbent assay autoantibody reactivity) was identified in each antigen studied: peptide 1-20 in Sm-D, peptide 35-58 in U1-RNP-A, and peptide 304-324 in the Ro/SSA 60 Kd protein. None of the MHC class II and TCR beta haplotypes was directly associated with any of the autoantibodies. Twenty-six subjects had antibodies to the peptide Sm-D1-20; nine of them were DRB1*0101/DQB1*0501. Among subjects with this haplotype, the number of responders was higher (p < 0.028, p corrected, pc = 0.336) in those with the 2-25-9 TCR beta haplotype than in the remainder. Conversely, the number of DRB1*04/DQB1*0302 responders was lower (p < 0.030, pc = 0.360) among subjects with the 23-20-9 TCR beta haplotype than in those without. The odds ratios (OR) were 4.23 and 0.21, respectively. Of the 54 subjects positive for anti-U1-RNP-A 35-38, 13 were DRB1*0101/DQB1*0501 and eight DRB1*04/DQB1*0302. The percentage of responders was higher (p < 0.041, pc = 0.492, OR = 3.48) in the former group of subjects with the 2-25-9 TCR beta haplotype, and lower (p < 0.02, pc = 0.024, OR = 0.09) in the latter with the 23-20-9 TCR beta haplotype. Three of the 12 anti Ro/SSA 60Kd 304-324-positive subjects were DRB1*0101/DQB1*0501. All had the 2-25-9 TCR beta haplotype (p < 0.046, pc = 0.552, OR = 6.29) and none the 23-20-9 (p < 0.031, pc = 0.372, OR = 0.10). The same combinations of genes were associated with high/low response toward the three peptides. These data provide evidence for an interplay of the MHC class II and TCR beta alleles in the control of specific autoantibody response to well-defined nuclear Ag peptides.

Adolescent↗
PubMed1997

Fluence rate effects on photodynamic therapy of B16 pigmented melanoma.

In vivo experiments were performed to evaluate the effect of fluence rate on the efficiency of Zn(II)-2,3 naphthalocyanine (ZnNc) photosensitization of B16 pigmented melanoma subcutaneously transplanted in C57B1/6 mice. The tumour was irradiated with 774 nm light at 24 h after an injection of liposome--which incorporated ZnNc (0.5 mg kg-1 b.w.). A total light dose of 360 J cm-2 was delivered at fluence rates of 260, 320, 380, 440 and 500 mW cm-2. Separate groups of mice utilized to monitor tumour temperature changes during irradiation without or after anaesthesia. Tumour response was determined by measuring the mean tumour diameter of the treated towards the untreated animals for a period of 21 days following PDT, as well as the percentage of cured animals. The most promising result (40% complete tumour response) was obtained with anaesthetized mice following 380 mW cm-2. Higher dose rates (440 and 500 mW cm-2) led to less promising results for both anaesthetized and non anaesthetized mice. These results outline the potential of PDT with longer wavelengths for the treatment of highly pigmented tumour tissues. The optimal fluence rate for tumour treatment should be chosen in order to avoid inflammatory effects (tissue swelling) and oxygen suppression with sublethal injury to the tumour cells.

Animals↗
PubMed1997

Si(IV)-methoxyethylene-glycol-naphthalocyanine: synthesis and pharmacokinetic and photosensitizing properties in different tumour models.

A Si(IV)-naphthalocyanine bearing two methoxyethylenglycol axial ligands to the centrally coordinated metal ion (SiNc) was prepared by chemical synthesis and assayed for the phototherapeutic activity after administration in a Cremophor formulation to C57BI/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma or B16 pigmented melanoma. Pharmacokinetic studies indicate that the maximal accumulation in the tumour occurs at 24 h after intraperitoneal injection of 0.5 mg kg-1 of SiNc, although the naphthalocyanine concentration in the Lewis lung carcinoma (0.70 microgram g-1) is significantly larger than that in the B16 pigmented melanoma (0.15 microgram g-1). This results in a higher selectivity of tumour targeting in the case of the lung carcinoma. Photodynamic therapy (782 nm, 370 mW cm-2, 360 J cm-2) at 24 h after SiNc injection causes an efficient tumour response for Lewis lung carcinoma (50% lower tumour diameter on day 19 post-treatment as compared to untreated controls) while the pigmented melanoma shows only a minor response regarding the rate of tumour growth.

Animals↗
PubMed1997

Sclerosing mucoepidermoid carcinoma of the parotid.

Two cases of mucoepidermoid carcinoma of the parotid gland associated with extensive central sclerosis and peripheral lymphoid response are reported. This unusual but distinctive variant of mucoepidermoid carcinoma can be difficult to recognize and may be confused with chronic sialoadenitis or even metastasis to an intraparotid lymph node.

Adolescent↗