Retrovirus-mediated correction of the metabolic defect in cultured Farber disease cells.

Farber disease is a rare severe lysosomal storage disorder due to a deficient activity of the enzyme acid ceramidase (AC). Patients have granulomas along with lipid-laden macrophages that accumulate in a number of tissues, leading to multiple diverse clinical symptoms. There is no therapy for the disorder and most patients succumb to the disease in early childhood. The severity of the disease progression seems to correlate with the amount of the accumulated ceramide substrate. Since the cDNA for human AC has been elucidated we sought to establish if genetic transfer of this sequence would lead to enzymatic and, especially, functional correction of the catabolic defect in Farber patient cells. To do this, a novel amphotropic recombinant retrovirus was constructed that engineers transfer of the human AC cDNA. On infection of patient fibroblasts, AC enzyme activity in cell extracts was completely restored. Further, substrate-loading assays of intact living cells showed a fully normalized catabolism of lysosomal ceramide. Lastly, as reported for some other corrected enzymatic defects of lysosomes, metabolic cooperativity was seen, in that functionally corrected patient fibroblasts secreted AC that was taken up through the mannose 6-phosphate receptor and used by uncorrected fibroblasts as well as recipient Farber lymphoblastoid cells. This overall transduction and uptake scenario for Farber disease allows future treatment of this severe disorder to be envisioned using gene transfer approaches.

Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL.

1. Oxidized low density lipoproteins (LDL) are toxic to cultured endothelial cells. Mildly oxidized LDL, characterized by relatively low levels of TBARS and only minor modifications of apoB, were obtained by using 2 experimental model systems of oxidation, namely oxidation by u.v. radiation or ferrylmyoglobin (a two electron oxidation product from the reaction of metmyoglobin with H2O2). 2. Toxic concentrations of mildly oxidized LDL induce apoptosis (programmed cell death) of cultured endothelial cells, as shown by typical morphological features, by the in situ TUNEL procedure and by DNA fragmentation revealed on gel electrophoresis. This apoptosis is calcium-dependent and subsequent to the intense and sustained cytosolic [Ca2+]i peak elicited by oxidized LDL. 3. Five naturally occurring phenolic compounds present in food and beverages were able to prevent, in a concentration-dependent manner, the apoptosis of endothelial cells induced by oxidized LDL. Among the compounds tested, caffeic acid was the most effective. Under the conditions used, the protective effect of caffeic acid (IC50 8.3+/-2.1 micromol l[-1]) in the prevention of apoptosis induced by oxidized LDL was significantly higher than that of the other compounds tested (IC50s were 12.4+/-3.2, 14.1+/-4.1, 20.4+/-4.4 and 72.6+/-9.2 micromol l(-1) for ferulic, protocatechuic, ellagic and p-coumaric acids, respectively). 4. The anti-apoptotic effect of caffeic acid results from the addition of two effects, (i) the antioxidant effect which prevents LDL oxidation and subsequent toxicity ('indirect' protective effect); (ii) a 'direct' cytoprotective effect, acting at the cellular level. 5. Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL. 6. In conclusion, phenolic acids (caffeic and ferulic acids being the most potent of the compounds tested under the conditions used) exhibit a potent cytoprotective effect of cultured endothelial cells against oxidized LDL. In addition to antioxidant effect delaying LDL oxidation, caffeic acid acts as a cytoprotective agent, probably by blocking the intracellular signalling triggered by oxidized LDL and culminating in the sustained calcium rise which is involved in oxidized LDL-induced apoptosis.

Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies.

Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human prosaposin deficiency.

Cholesteryl ester storage disease: relationship between molecular defects and in situ activity of lysosomal acid lipase.

The molecular defects in the LIPA gene encoding the lysosomal acid lipase (LAL) were investigated in two unrelated patients affected with cholesteryl ester storage disease (CESD), an autosomal recessive disorder associated with LAL-deficient activity. In cell lysates from both patients there was a severely reduced LAL activity. In a female patient, nucleotide sequencing of amplified LAL genomic DNA or reverse-transcribed mRNA demonstrated that she was a compound heterozygote for two previously reported mutations, a G --> A transition at position -1 of the exon 8 splice donor site, resulting in skipping of the complete exon 8, and a C923 --> T substitution leading to the replacement of His274 to Tyr. The second, male CESD patient was heterozygous for the splice junction mutation and a yet undescribed C --> T substitution at position 233, which introduces a premature in-frame termination codon. The functional consequences of these genetic alterations were evaluated for the first time by studying the catabolic turnover of radiolabeled cholesteryl oleate in intact cells. A lower in situ residual LAL activity was found in cells carrying the stop codon mutation than in cells having the His274 --> Tyr substitution. Since the severely reduced LAL activity was seen in cells from an adult patient with a mild CESD, we conclude that there is no simple direct correlation between the LAL molecular lesions and the biochemical and clinical phenotypes.

A simple method for screening for Farber disease on cultured skin fibroblasts.

Farber disease is an inborn lysosomal storage disorder characterized by accumulation of ceramide in the patient's tissues due to the deficient activity of acid ceramidase. Currently, confirmation of the diagnosis is performed in an extremely limited number of laboratories. We therefore developed a procedure which does not require any particular sphingolipid substrate and is based on the quantitation of ceramide levels in cultured skin fibroblasts. In the method we devised, the ceramide present in cellular lipid extracts subjected to mild alkaline hydrolysis was quantified using the commercially available diacylglycerol kinase kit. We show that both primary cultures of skin fibroblasts and SV40-transformed fibroblasts derived from a series of patients with Farber disease exhibit ceramide excess as compared to their normal counterparts (2345-17 153 pmol/mg cell protein in Farber cells vs. 432-1298 pmol/mg cell protein in controls). Use of this simple method should greatly facilitate the biochemical diagnosis of Farber disease.

Subclass distribution of IgG antibodies to the rat oesophagus stratum corneum (so-called anti-keratin antibodies) in rheumatoid arthritis.

Serum IgG, labelling the stratum corneum of the rat oesophagus epithelium, so-called anti-keratin antibodies (AKA) constitute the most specific marker for the diagnosis of rheumatoid arthritis. In this study, we investigated 31 IgG AKA-positive rheumatoid sera and 21 control sera from patients with non-rheumatoid inflammatory rheumatic diseases. The serum level of IgG1,2,3 and 4 was determined by radial immunodiffusion and the subclass distribution of IgG AKA by a three-step semi-quantitative immunofluorescence assay using standard monoclonal antibodies specific for each of the four human IgG subclasses. In the rheumatoid sera, the serum level of IgG1 was found to be significantly increased and the level of IgG2 significantly decreased with regard to the control sera, while the levels of IgG3 and 4 as well as total IgG were in the normal range. IgG1,2,3, and 4 AKA were detected in 27 (87%), 6 (19%), 4 (13%) and 11 (35%) of the 31 rheumatoid sera, respectively, and were found to be independent of the clinical and biological indices of the disease. In spite of inter-individual heterogeneity, two predominant profiles were distinguished: IgG1 (alone) and IgG(1 + 4), which together represented 18 sera (58%). The large predominance of IgG1 AKA and the quasi-absence of IgG2 AKA suggest that the recognized antigen may be partly comprised of protein. Moreover, the high frequency of occurrence of IgG4 AKA might result from chronic exposure to the eliciting antigen, which could be a genuine autoantigen since we demonstrated that it is also present in the stratum corneum of human epidermis.

The spontaneous development of immune complex type glomerular lesions in outbred mice is dependent on environmental factors and sex.

To investigate the role of environmental factors in the spontaneous development of immune complex glomerulonephritis in the mouse, follow-up studies of various immunological parameters and of kidney lesions have been done in male and female outbred OF1 mice housed from birth to four months of age in two different conditions: (a) in a conventional animal house; (b) in a "controlled" animal house designed for long-term studies on nude mice. Female mice housed under conventional conditions develop mild to intense glomerular lesions with granular deposits of immunoglobulins and C3 after two months of age. The appearance of these lesions is correlated with a sharp increase of various IgG subclasses levels at two months of age. Female mice housed under "controlled" conditions do not show such an increase and have only scarce to mild lesions until four months of age. In male mice, similar observations are done but the differences in immunological parameters and in histopathological findings between the two groups of mice according to the housing conditions are less noticeable. The environmental factors which are involved in the development of these immune mediated lesions are not known; it is hypothetized that infectious agents play a role. This study emphasizes the importance of housing conditions and of sex in immunopathological investigations in the mouse and the possible bias these conditions may introduce, in some experiments.

Polyclonal activation of lymphocytes induced in the mouse by acebutolol, a beta blocking agent.

Acebutolol, a beta blocking agent, was injected at a dose of 1 mg/day, every day for 6 months in C57B1/6 and OF1 mice. No antinuclear antibodies were induced but a transient induction of anti-single stranded DNA antibodies and an increase of IgM, IgA and IgG2a levels were observed during the first 3 weeks in some treated mice as compared to control mice. This effect was more pronounced in C57B1/6 than in OF1 mice and in the C57B1/6 mice, was more frequently observed in female than in male mice. It was further found by cellular studies done in C57B1/6 female mice, that daily i.p. or oral administration of Acebutolol induces a transient polyclonal stimulation of lymphocytes. It is concluded that certain beta blockers might possess some in vivo effects on the immune system.

Cardiovascular activity of a deproteinized blood extract.

The cardiovascular effects of the deproteinized blood extract were investigated in anaesthetized dogs: respiration, general metabolism and systemic haemodynamics were tested and the changes in these parameters after the administration of the drug were studied. At 2 ml kg-1 i.v., the deproteinized blood extract induced a transient drop in arterial blood pressure and bradycardia. On the other hand, the total peripheral resistance and the elastic resistance, of the arteries were decreased and this effect was of longer duration. Moreover, the deproteinized blood extract stimulated the myocardium contractile force and dc/dt and increased the cardiac performances. The mechanism of action of this drug is discussed.

[Role of alpha and beta adrenoceptors in the regulation of adrenergic neurotransmission in the carotid sinus]. / Rôle des alpha et des bêta adrénocepteurs dans la régulation de la neurotransmission adrénergique au niveau du sinus carotidien.

The experiments carried out showed the presence- in sympathetic nerve endings ot the carotid sinus- of alpha and beta adrenoceptors which by means of respective negative and positive feedback processes, modulated NA release induced by a sympathetic nerve stimulation. Similarly, Pgs acted by means of a negative feedback mechanism to regulated adrenergic neuro-transmission in carotid sinus but they could not be considered as the chemical mediators of either alpha or beta adrenoceptors.

[Effect of stimulation of alpha and beta presynaptic adrenoceptors on the liberation of noradrenaline produced by electric stimulation in rabbit carotid sinus]. / Influence de la stimulation des alpha et des beta adrénocepteurs présynaptiques sur la libération de la noradrénaline produite par l'excitation électrique au niveau du sinus carotidien du Lapin.

The experiments carried out on the isolated carotid sinus in the rabbit demonstrated that alpha and beta adrenoceptors are capable of detecting the circulating catecholamines and the catecholamines of the synaptic cleft in order to modulate the release of noradrenaline from the sympathetic nerve endings in response to a potential action.