Biomedical subjects

M Fontaine

Publications and source records attributed to M Fontaine.

At least 37 records · Page 2Linked to original sources

PubMed1998

Metabolic studies in a patient with severe carnitine palmitoyltransferase type II deficiency.

Here we report on a patient with severe ("non-classic") carnitine palmitoyltransferase type II (CPT II) deficiency. Hypoglycemia prompted by an infectious episode and associated with non-ketotic dicarboxylic aciduria orientated diagnosis towards beta-oxidation deficiency disorders. Blood carnitine levels revealed a secondary carnitine deficiency that was responsive to oral L-carnitine supplementation. Blood acylcarnitine profiles were abnormal and included acetyl (C2:0), butyryl/isobutyryl (C4:0), isovaleryl/2-methylbutyryl (C5:0), hexanoyl (C6:0), myristoyl (C14:0), palmitoyl (C16:0), hexadecenoyl (C16:1), oleyl (C18:1) and stearoyl (C18:0) carnitine. In urine, excess excretion of dicarboxylylcarnitines, mainly dodecanedioylcarnitine, was noticed. Upon carnitine supplementation, C8 to C12 fatty acylcarnitines, with decanoylcarnitine as well as C10 to C14 dicarboxylylcarnitines being prominent, were observed in urine. Biochemical measurements disclosed a severe reduction of mitochondrial CPT II activity (7% of normal values). Correlations of metabolic findings in the patient and physiological roles of CPT II are briefly discussed.

Carnitine↗

PubMed1998

The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis.

The complement anaphylatoxins C5a and C3a are released at the inflammatory site, where they contribute to the recruitment and activation of leukocytes and the activation of resident cells. The distribution of the receptor for C5a (C5aR) has been well studied; however, the receptor for C3a (C3aR) has only recently been cloned, and its distribution is uncharacterized. Using a specific affinity-purified anti-C3aR peptide Ab and oligonucleotides for reverse transcriptase-PCR analysis, C3aR expression was characterized in vitro on myeloid and nonmyeloid cells and in vivo in the brain. C3aR was expressed by adult astrocytes, astrocyte cell lines, monocyte lines THP1 and U937, neutrophils, and monocytes, but not by K562 or Ramos. C3aR staining was confirmed by flow cytometry, confocal imaging, and electron microscopy analysis. A 65-kDa protein was immunoprecipitated by the anti-C3aR from astrocyte and monocyte cell lysates. Our results at the protein level were confirmed at the mRNA level. Using reverse transcriptase-PCR, Southern blot, and sequencing we found that C3aR mRNA was expressed by fetal astrocytes, astrocyte cell lines, and THP1, but not by K562 or Ramos. The astrocyte C3aR cDNA was identical with the reported C3aR cDNA. C3aR expression was not detected in normal brain sections. However, a strong C3aR staining was evident in areas of inflammation in multiple sclerosis and bacterial meningitis. In meningitis, C3aR was abundantly expressed by reactive astrocytes, microglia, and infiltrating cells (macrophages and neutrophils). In multiple sclerosis, infiltrating lymphocytes did not express C3aR, but a strong staining was detected on smooth muscle cells (pericytes) surrounding blood vessels.

Adult↗

PubMed1998

Effects of Lorenzo's Oil on peroxisomes in healthy mice.

We investigated peroxisomal alterations in mice treated with different doses of Lorenzo's Oil (a therapy for X-linked adrenoleukodystrophy patients) for up to 100 days. Hepatic erucic acid levels were already significantly increased 2.2-fold and 2.6-fold in mice treated with 10% and 20% Lorenzo's Oil for 21 days, respectively. No lipidosis was found in liver, myocardium and kidney of any of the treated mice. While hepatic catalase, lauroyl-CoA oxidase and glycolate oxidase, and renal catalase activities were not induced by either diet, myocardial catalase activity was increased in most groups. This suggests that the mechanism of the effect of Lorenzo's Oil in X-linked adrenoleukodystrophy patients may not be a direct effect on the peroxisomes.

Acyl-CoA Dehydrogenase↗

PubMed1998

Expression of a functional anaphylatoxin C3a receptor by astrocytes.

Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand-binding studies, functional responses, and RNA analysis by RT-PCR. Uptake of 125I-C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High-affinity binding sites were abundantly expressed (20,000-80,000 sites per cell) with an estimated K(D) of 1-2 nM. Low-affinity binding sites with a K(D) of 209 nM were largely expressed (n > or = 4 x 10(6) sites per cell) and probably did not reflect a receptor-mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT-PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti-C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca2+] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C-terminus Arg considerably decreased the [Ca2+] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.

Anaphylatoxins↗

PubMed1998

A prospective study of the predictive value of polymerase chain reaction assay for cytomegalovirus in asymptomatic kidney transplant recipients.

Cytomegalovirus (CMV) infection carries the potential for high morbidity in transplant recipients. The institution of pre-emptive therapy prior to the onset of clinical disease on the basis of CMV-polymerase chain reaction (PCR) is very attractive. We prospectively studied 52 asymptomatic kidney transplant recipients to test the hypothesis that serial CMV-PCR assays during the first 3 months post-transplant would identify patients at risk for CMV disease. Twenty-three patients (44.2%) had positive CMV-PCR tests at least once; 2 (8.6%) developed CMV. None of the 29 patients continuously negative for CMV-PCR developed CMV disease. CMV-PCR status did not influence patient and graft survival or the incidence of acute rejection. We conclude that while a substantial number of kidney transplant recipients become positive for CMV-PCR in the early post-transplant period, only a minority will develop CMV disease. Negative CMV-PCR assay is an accurate negative predictor for CMV disease but the value of CMV-PCR as a guide for pre-emptive anti-CMV therapy in kidney transplant recipients appears limited.

Adult↗

PubMed1997

Intrasplenic hepatocyte allotransplantation in dalmation dogs with and without cyclosporine immunosuppression.

Hepatocyte allotransplantation has been performed successfully in several small animal models for the amelioration of inborn metabolic errors. Before a human clinical trial of hepatocyte allotransplantation can be attempted, preliminary experience in a large animal model is needed. We transplanted isolated mongrel hepatocytes into the spleen of dalmatians in the attempt to cure their inborn error of uric acid metabolism. Of 10 dalmatian recipients, two that received 9-10 x 10(9) mongrel hepatocytes died early after surgery of acute portal hypertension and hemorrhage. The eight long-term survivors received 5-6 x 10(9) hepatocytes and were randomized either to no treatment or to oral cyclosporine (CsA). Levels of CsA were adjusted to maintain trough levels between 400 and 800 ng/ml. In the four nonimmunosuppressed dalmatians, a reproducible average reduction in urinary uric acid excretion (UUAEx) of 23.7% was achieved; values returned to baseline within 14 days. In the CsA-immunosuppressed dalmatians, the average decline in UUAEx was 30%. The partial correction of the metabolic defect persisted for an average of 25 days in three immunosuppressed dogs, whereas in one dog, the partial correction lasted for over 90 days. No change in UUAEx was observed in two dalmatians that underwent sham laparotomy and intrasplenic injection of saline solution; CsA given alone to dalmatians did not modify UUAEx. We conclude that the dalmatian dog is a valuable large animal model for studies of the role of hepatocyte transplantation in the cure of inborn hepatic metabolic errors.

Animals↗

PubMed1997

Complement classical pathway expression by human skeletal myoblasts in vitro.

Human myoblasts express immunological properties in vitro and we have previously reported that they produce Complement (C) components of the alternative pathway. Myoblasts activate the classical pathway but are fully protected against C attack by the expression of major C regulators. In order to fully understand the relationship between myoblasts and C, we here report the biosynthesis of C components of the classical pathway by skeletal muscle cells. Human myoblasts in vitro produced C1q, C1r, C1s, C2 and C4 constitutively and all syntheses were upregulated after stimulation with IFN-gamma. We suggest that human myoblasts may constitute a local source of C and therefore C could be implicated in inflammatory or physiopathological processes developed in skeletal muscle.

Blotting, Western↗

PubMed1997

Expression of clusterin and C4 mRNA during rat peripheral nerve regeneration.

The complement system (C) is a major piece of the inflammatory processes triggered after tissue injury. Since implication of the C has been demonstrated during neurodegeneration and Wallerian degeneration, without being clearly explained, we investigated the expression of C4 and clusterin mRNA, at the lesion site, after rat sciatic nerve crush injury. This pilot study was then realized over 28 days during peripheral nerve regeneration. We determined mRNA expression levels in naive control animals (N) and 2, 7, 14 and 28 days (D) after crush experiment, by using semi-quantitative RT-PCR. We observed a basic constitutive expression of both mRNAs in group N. Clusterin mRNA level increased between D2 and D7 to reach 2.5-fold the basic level of expression (N) at D7 and D14, and slightly decreased until D28. C4 mRNA underwent a rapid and marked increase and represented 2-3-fold the N level from D2 to D14, then it decreased until D28 to return to the basic level of expression (N). These preliminary data exhibit very interesting individual variations in mRNA expression and show that a peripheral nerve trauma can stimulate the expression of C4 and clusterin mRNA at the lesion site.

Actins↗

PubMed1997

Characterization of rat C5a anaphylatoxin receptor (C5aR): cloning of rat C5aR cDNA and study of C5aR expression by rat astrocytes.

Complement system activation within the central nervous system (CNS) is involved in demyelinating and neurodegenerative disorders, but the role of complement in the pathogenic process or in the repair remains unclear. Besides the direct lytic effects of complement on target cells (oligodendrocytes or neurons), complement can exert other functions through interaction of complement fragments with specific receptors. The C5a anaphylatoxin, an inflammatory peptide which is formed during complement activation, might play a role in the CNS pathogenesis, and activation and recruitment of glial cells by binding to its receptor (C5aR) on CNS cells. Using degenerate primers corresponding to homologous regions between human and mouse C5aR cDNAs, we have cloned a rat C5aR cDNA probe from rat monocytes RNAs after RT-PCR experiment. The rat C5aR probe isolated by this procedure allowed us to clone the rat C5aR cDNA-coding sequence using a library screening cloning strategy. This probe was also used to study the expression of the C5aR mRNA in the rat CNS. Northern blotting and RT-PCR experiments demonstrated the constitutive expression of C5aR mRNA in brain, spleen, kidney and lung. This transcript was also observed in primary culture of rat astrocytes. Microfluorimetry experiments demonstrated that C5aR expressed by astrocytes in culture is functional since the addition of C5a induced a dose-dependent increase of intracellular calcium concentration. The expression of the C5aR by astrocytes suggests new roles for the C5a anaphylatoxin in reactive astrogliosis to CNS injuries.

Amino Acid Sequence↗

PubMed1997

Colonic xanthelasma due to glyceride accumulation associated with an adenoma.

The case is reported of a 66-year-old woman who presented with endoscopic and histological features of multiple lipid deposits in the mucosa of the sigmoid colon associated with an adenoma. Associated clinical features were abdominal pain and diarrhea. Colectomy led to the complete resolution of symptoms. Biochemical analysis disclosed the presence of glycerides in the mucosa. The pathogenesis of lipid deposits and the possible link with the formation of an adenoma is discussed.

Adenomatous Polyps↗

PubMed1997

[Ethics, bioethics and medical sciences].

The aim of bioethics is to define a wise conduct for humans with regard to their environments, whether living or inanimate. However, owing to their diversity, bioethics can only deal with general problems such as biodiversity. Within the framework of bioethics as a whole, different sectorial bioethics must therefore exist to deal with problems specific to certain environments, for example the Oceans and Seas, the Forests. General bioethics and sectorial bioethics have an important contribution to make to medical sciences but official regulations should be proposed only after an attentive investigation has been made. For instance, the preservation of an apparently threatened biodiversity or the revival of a seriously damaged biodiversity must be the subject of a thorough preliminary scientific study and, if legislative decisions are taken, a very careful scientific control of their consequences must be carried out. One example is given: the decree on the protection of Larids and its impact, with regard to an abusive proliferation of certain gull populations having varied effects on public health. Sectorial bioethics can also have obvious consequences on medical sciences. Thus various harmful attacks on coral reefs (contrary to the concepts of thalassoethics) can lead to the death of corals and the appearance of ciguatera. Thalassoethics, by inciting pollution control, should help to improve the conditions of thalassotherapy. Forest ethics, particularly concerning management, can reduce the greenhouse effect and its consequences on health, as well as protecting plant and animal species inhabiting the ecosystem and bringing new chemical bodies to inspire original pharmacological research. Thus the links between general or sectorial bioethics and medical sciences must always be very close.

Bioethics↗

PubMed1996

Metabolic studies in twin brothers with 2-methylacetoacetyl-CoA thiolase deficiency.

We report clinical and biological investigations in two patients (twin brothers) with 2-methylacetoacetyl-CoA thiolase deficiency. Main clinical features included important staturo-ponderal delay, frequent infectious rhinopharyngitis episodes and an acute metabolic acidosis at the age of 4 years, this metabolic decompensation being adequately halted by bicarbonate supplementation. Since that age, patients developed rather favorably, however, with persistence of the staturo-ponderal delay. Organicaciduria typical of 2-methylacetoacetyl-CoA thiolase deficiency was recorded consisting of excessive excretion of tiglylglycine, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-methylglutaconate, adipate and 2-methylacetoacetate. Blood carnitine levels were altered in patients with increased total and esterified carnitine concentrations and enhanced acyl/free carnitine ratios. Determination of acylcarnitine profiles showed that patients excreted excessive amounts of several acylcarnitines in urine including propionyl, butyryl, isobutyryl, isovaleryl, 2-methylbutyryl and tiglyl-carnitine, the latter acylcarnitine being prominent with, in one of the patients, occurrence of a previously undescribed isomer of this carnitine ester, possibly 2-ethylacrylyl-carnitine. Excretion of these acylcarnitines in urine was increased in response to L-carnitine although, as a whole, this therapy resulted in a less important stimulation of esterified carnitine removal in urine from patients than in the case of supplemented controls. Biochemical investigations on cultured skin fibroblasts confirmed 2-methylacetoacetyl-CoA thiolase deficiency. Through the present report on this rare disease in two siblings, we would like to underline that acylcarnitines can be used in the diagnosis of 2-methylacetoacetyl-CoA thiolase deficiency, a view supported by acylcarnitine profiles further determined in another patient with proven oxothiolase deficiency, adding this pathology to the list of beta-oxidation disorders that may be screened successfully through determination of acylcarnitine profiles in body fluids.

Acetyl-CoA C-Acyltransferase↗

PubMed1996

Acylcarnitine removal in a patient with acyl-CoA beta-oxidation deficiency disorder: effect of L-carnitine therapy and starvation.

Carnitine levels and acylcarnitine profiles in a patient with mild multiple acyl-CoA dehydrogenase deficient beta-oxidation were compared with control results. Whereas blood and urine total carnitine levels were moderately decreased, blood esterified carnitine levels in the patient were about 2-fold higher than in controls. Urinary acylcarnitine profiles presented with a larger variety of carnitine esters than in controls and included propionylcarnitine, butyrylcarnitine, 2-methylbutyrylcarnitine, hexanoylcarnitine and octanolycarnitine. Total carnitine levels in body fluids were similarly affected by chronic oral L-carnitine administration in patient and controls. By contrast, esterified carnitine level increase was 2-fold more important in controls than in patient. Whereas no qualitative changes in urinary acylcarnitine profiles were induced by L-carnitine therapy in controls, several alterations of these profiles were observed in the patient. The effect of starvation on metabolites was also studied, especially beta-oxidation rates assessed by free fatty acids to 3-hydroxybutyric acid ratios in blood from the patient in the untreated and L-carnitine treated states. In the L-carnitine-supplemented patient, the effect of starvation on the time course of carnitine levels and acylcarnitine profiles could also be documented. The ability of chronic oral L-carnitine administration to remove relatively less important amounts of acylcarnitines in the patient than in controls is further discussed, as well as qualitative alterations of acylcarnitine profiles induced by this therapy in the pathological condition.

Acyl-CoA Dehydrogenase↗

PubMed1996

Human skeletal myoblasts spontaneously activate allogeneic complement but are resistant to killing.

The complement (C) system has previously been implicated in several diseases of muscle. We here report that human myoblasts or rhabdomyosarcoma cell lines spontaneously activate C through the classical pathway, causing release of anaphylatoxins and coating of myoblasts with opsonic C fragments but without causing cell killing. Survival of myoblasts is a consequence of the abundant expression of the membrane C regulatory molecules MCP and CD59, and neutralization of CD59 renders cells susceptible to C killing. The decay-accelerating factor was expressed at a very low level. Myoblasts and rhabdomyosarcoma lines also abundantly express the fluid-phase regulators C1-inhibitor, factor H, C4 binding protein, S-protein, and clusterin and secrete a soluble form of CD59. Expression of membrane and fluid-phase regulators is enhanced by either IFN-gamma or TNF-alpha. Although myoblasts resist C killing, spontaneous activation of C on these cells may have important consequences in inflammatory diseases of muscle where the generation of anaphylactic and opsonic fragments will recruit and activate inflammatory cells. C activation on myoblasts may also have consequences for the use of these cells as vehicles for gene delivery. Inhibition of C using soluble complement receptor I (sCR1) efficiently protected myoblasts from C attack in vitro, and this agent, already being tested in therapy of several C-mediated diseases, might be of value in inflammatory muscle disease and in improving the efficiency of gene delivery.

Cell Death↗

PubMed1996

Identification and characterization of complement C3 receptors on human astrocytes.

Astrocytes express C components and have been implicated as a major source of intrathecal C. To ascertain the effects of C activation on these cells, we have evaluated the expression of CR1, CR2, and CR3 (CD35, CD21, and CD11b/CD18) in primary fetal astrocytes and astrocyte cell lines. None of the astrocyte cells tested expressed CR3, whereas primary astrocytes and one of four astrocyte cell lines expressed CR1 (220 kDa), as assessed at the protein and mRNA level. Primary fetal astrocytes and all four astrocyte cell lines expressed CR2 (155 kDa). Expression of CR2 by astrocytes was confirmed at mRNA level by reverse-transcriptase PCR, using different combinations of seven specific CR2 oligonucleotides, and by partial sequencing of the astrocyte CR2 cDNA. Astrocyte CR2 cDNA presented 100% homology with the lymphocyte CR2 cDNA between the position 181 bp to 600 bp and position 1017 bp to 1347 bp. An alternative splicing pattern of exon 11, reported previously in B cells, was observed in astrocyte CR2 cDNA. Astrocyte CR2 was functional, in that it specifically bound C3d and the EBV surface protein gp340, and the binding was blocked specifically with polyclonal anti-CR2. Scatchard analysis of membrane expression of CR2 on astrocytes revealed 2000 functional sites per cell with a Kd (3 x 10(-7) M) identical with that of CR2 on B cell (Raji).

Astrocytes↗

PubMed1996

Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that binds iC3b and CD23.

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short consensus repeats (SCR) 1-2, SCR 4-5 and SCR 9-11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1-2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.

B-Lymphocytes↗

PubMed1996

Use of rhodamine 123 to examine the functional activity of P-glycoprotein in primary cultured brain microvessel endothelial cell monolayers.

The fluorescent dye, rhodamine 123, was used to evaluate the functional activity of the P-glycoprotein (P-gp) efflux transport system in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers. Rhodamine 123 accumulation was increased significantly in BBMEC monolayers treated with the P-gp modifying agent, cyclosporin A (CSA). Rhodamine 123 accumulation was also increased by other P-gp modifying agents. The rank effectiveness of these agents in increasing rhodamine 123 accumulation in BBMEC monolayers was CSA = dipyridamole > verapamil = quinidine. The maximal increase in rhodamine 123 accumulation in CSA treated. BBMEC monolayers was approximately 3 fold greater than in control monolayers and was qualitatively similar to that observed with 3H-vincristine. Comparison of functional activity with the biochemical expression of P-gp in BBMEC monolayers and in an established tumor cell line that over-expresses P-gp indicate that functional activity may be a more descriptive measure of the importance of this drug efflux system than protein expression. Furthermore, these studies suggest that accumulation of rhodamine 123 in BBMEC monolayers can be used to quantitatively examine P-gp activity in the blood-brain barrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins.

Two human neuroblastoma cell lines activated the classical pathway of complement in serum. Activation caused the opsonisation of these cells with complement fragments but with moderate cell killing. Neuroblastoma expressed regulators MCP and CD59 but did not express DAF or CR1. Neutralisation of CD59 rendered the cells susceptible to killing. Neuroblastoma also expressed C1-inhibitor, factor H, clusterin and S-protein. Expression of several regulators was enhanced by incubation with cytokines. Complement inhibition using soluble CRI markedly reduced opsonisation and killing of neuroblastoma. Our results suggest that complement might play a role in neuronal loss and that treatment with complement inhibitors might be of therapeutic value.

Antigens, CD↗