Human acid ceramidase gene: novel mutations in Farber disease

Farber disease is an autosomal recessive disorder caused by lysosomal acid ceramidase (AC) deficiency. It commonly manifests during the first few months after birth with a unique triad of painful and progressive deformed joints, subcutaneous nodules, and progressive hoarseness. In order to understand the molecular mechanism(s) of pathogenesis of Farber disease, we isolated and characterized a full-length human AC gene, mapped its chromosomal location, determined the tissue-specific expression, and analyzed mutations in Farber disease patients. We also studied the AC-mRNA expression in gastrointestinal tumors and adjoining normal tissues. In addition, we determined the pattern of tissue-specific AC-mRNA expression in the adult mouse and during fetal development. Our results show that human AC gene consists of 14 exons and 13 introns spanning approximately 26.5 kb of genomic DNA. It is mapped to human chromosome 8p22-21.2, a region often disrupted in several cancers. The AC-mRNA is expressed in the mouse fetus from the seventh day of gestation. Interestingly, while the AC-mRNA is expressed in all segments of the normal gastrointestinal tract, none of the gastrointestinal tumor tissues had any AC-mRNA expression. We also uncovered four novel mutations in Farber disease patients that were not previously reported. Taken together, our results not only attest to the physiological importance of AC but also uncover several new mutations in Farber disease that may advance our knowledge towards establishing a genotype-phenotype correlation in this disease.     

Molecular analysis of acid ceramidase deficiency in patients with Farber disease

Farber disease is a rare, autosomal recessively inherited sphingolipid storage disorder due to the deficient activity of lysosomal acid ceramidase, leading to the accumulation of ceramide in cells and tissues. Here we report the identification of six novel mutations in the acid ceramidase gene causing Farber disease: three point mutations resulting in single amino acid substitutions, one intronic splice site mutation resulting in exon skipping, and two point mutations also leading to occasional or complete exon skipping. Of interest, these latter two mutations occurred in adjacent nucleotides and led to abnormal splicing of the same exon. Expression of the mutated acid ceramidase cDNAs in COS-1 cells and subsequent determination of acid ceramidase residual enzyme activity demonstrated that each of these mutations was the direct cause of the acid ceramidase deficiency in the respective patients. In contrast, two known polymorphisms had no effect on acid ceramidase activity. Metabolic labeling studies in fibroblasts of four patients showed that even though acid ceramidase precursor protein was synthesized in these individuals, rapid proteolysis of the mutated, mature acid ceramidase occurred within the lysosome.     

In vivo delivery of human acid ceramidase via cord blood transplantation and direct injection of lentivirus as novel treatment approaches for Farber disease

Farber disease is a rare lysosomal storage disorder (LSD) caused by a deficiency of acid ceramidase (AC) activity and subsequent accumulation of ceramide. Currently, there is no treatment for Farber disease beyond palliative care and most patients succumb to the disorder at a very young age. Previously, our group showed that gene therapy using oncoretroviral vectors (RV) could restore enzyme activity in Farber patient cells. The studies described here employ novel RV and lentiviral (LV) vectors that engineer co-expression of AC and a cell surface marking transgene product, human CD25 (huCD25). Transduction of Farber patient fibroblasts and B cells with these vectors resulted in overexpression of AC and led to a 90% and 50% reduction in the accumulation of ceramide, respectively. Vectors were also evaluated in human hematopoietic stem/progenitor cells (HSPCs) and by direct in vivo delivery in mouse models. In a xenotransplantation model using NOD/SCID mice, we found that transduced CD34(+) cells could repopulate irradiated recipient animals, as measured by CD25 expression. When virus was injected intravenously into mice, soluble CD25 was detected in the plasma and increased AC activity was present in the liver up to 14 weeks post-injection. These findings suggest that vector and transgene expression can persist long-term and offer the potential of a lasting cure. To our knowledge, this is the first report of in vivo testing of direct gene therapy strategies for Farber disease.     

Farber disease in a patient from China

Farber disease (FD) is a rare lysosomal storage disorder caused by mutation of the ASAH1 gene. Classic symptoms of FD include subcutaneous nodules, joint pain and hoarseness. Most patients die during childhood. Here we report a 25‐year‐old female FD patient with rare osteolytic changes of bilateral hands and toes. Genetic analysis revealed novel compound heterozygous mutations in the ASAH1 gene (c.427T>G and c.358G>C). Further research is needed to elucidate the pathophysiological course.     

Leukocyte migration inhibitory factor (LMIF) profile in primary and secondary immunodeficiency disease.

Lymphocytes from patients with primary and secondary immunodeficiency disease were tested for capacity to produce LMIF after mitogen and antigen stimulation as well as for ability to stimulate and respond in unidirectional MLC-LMIF assay. Different patterns of immune abnormality in vitro were detectable when Con A and Candida albicans antigen were used. In addition, significant abnormalities in LMIF responding and stimulatory capacity were demonstrated in patients with Hodgkin's disease. LMIF production after stimulation with different agents allows for a better characterization of cellular defects in immunodeficiency disease.     

Disseminierte Lipogranulomatose (M. Farber) mit Hydrops fetalis

Zusammenfassung Wir berichten von einem weiblichen Frühgeborenen der 29. Schwangerschaftswoche mit schwerem Hydrops fetalis, das 3 Tage nach Geburt an den Folgen einer disseminierten intravasalen Gerinnung (DIC) verstarb. Autoptisch imponierten Anasarka, bilaterale Pleuraergüsse, Aszites und eine Hepatosplenomegalie, wobei makroskopisch stecknadelkopfgro?e wei?e Granulome an der Oberfl?che von Leber, Milz und Lungen auffielen. Lichtmikroskopisch fanden sich Speichermakrophagen des RHS besonders in Leber, Milz, Knochenmark, den lymphatischen Organen, den Kopfspeicheldrüsen, der Schilddrüse und im Nebennierenmark. Gro?hirn, Herz, Nieren, Intestinum und Plazenta waren nicht betroffen. Eine Atrophie der lymphatischen Kompartimente in Milz, Lymphknoten und Thymus sowie ein schwerer Leberschaden sind vermutlich sekund?re Folgen der Erkrankung. Die Diagnose M. Farber wurde biochemisch durch den Nachweis von Ceramidablagerungen in der Milz gesichert wie auch durch die Anreicherung von radioaktivem Ceramid in Fibroblastenkulturen nach Zusatz von markiertem Glucosylceramid. Ultrastrukturell wurden entsprechende Speicherorganellen in Histiozyten wie auch in Endothelzellen und Gallengangsepithelien der Leber gefunden. Unseres Wissens ist dies die erste Beschreibung eines M. Farber bei einem Frühgeborenen mit Hydrops fetalis.      

Leukocyte activation and function-associated antigens in inflammatory disease

Expression of adhesion molecules, CD11a, CD11b and CD18, and of the function-associated molecules CD3, CD4, CD8, CD16, CD19, CD56 and CD57 was assayed on peripheral blood leukocytes from normal control subjects (n=10), and from patients with adult periodontitis PD (n=9), ankylosing spondylitis AS (n=11) and rheumatoid arthritis RA (n=14). A novel rapid fixation leukocyte preparation technique was used which prevents artefactural up-regulation of surface antigens.In RA patients, the percentage of CD18+ lymphocytes was decreased and that of CD11b+ neutrophils was increased. On lymphocytes the mean fluorescence intensity (MFI) of both CD11b and CD18 was decreased whereas that of CD57 was increased. In AS patients the percentages of CD11b+ lymphocytes and neutrophils were increased and CD18+ lymphocytes and neutrophils were decreased. On lymphocytes the MFIs of CD11b and CD18 were decreased, whilst that of CD16 was increased. On neutrophils the MFI for CD18 was increased. No significant differences (p<0.01) were=" seen=" for=" the=" periodontitis=" patients.=" it=" is=" suggested=" that=" the=" antigen=" expression=" on=" peripheral=" blood=" cells=" from=" ra=" and=" as=" patients=" is=" consistent=" with=" leukocyte=">     

Leukocyte activation and function-associated antigens in inflammatory disease

Expression of adhesion molecules, CD11a, CD11b and CD18, and of the function-associated molecules CD3, CD4, CD8, CD16, CD19, CD56 and CD57 was assayed on peripheral blood leukocytes from normal control subjects (n=10), and from patients with adult periodontitis PD (n=9), ankylosing spondylitis AS (n=11) and rheumatoid arthritis RA (n=14). A novel rapid fixation leukocyte preparation technique was used which prevents artefactural up-regulation of surface antigens.In RA patients, the percentage of CD18+ lymphocytes was decreased and that of CD11b+ neutrophils was increased. On lymphocytes the mean fluorescence intensity (MFI) of both CD11b and CD18 was decreased whereas that of CD57 was increased. In AS patients the percentages of CD11b+ lymphocytes and neutrophils were increased and CD18+ lymphocytes and neutrophils were decreased. On lymphocytes the MFIs of CD11b and CD18 were decreased, whilst that of CD16 was increased. On neutrophils the MFI for CD18 was increased. No significant differences (p<0.01) were seen for the periodontitis patients. It is suggested that the antigen expression on peripheral blood cells from RA and AS patients is consistent with leukocyte activation.     

Leukocyte Ig-like receptor complex (LRC) in mice and men

Here, we compare the architecture of membrane receptors with extracellular Ig-like domains located within the leukocyte Ig-like receptor complex (LRC) of humans and mice. The receptors can be classified broadly into four groups, based on the homology of their Ig-like domains and gene architecture. Receptors in the first group are characterized by the presence of the Ig constant type 2-1 (IgC2-1) and variant Ig (vlg) domains, and include the leukocyte Ig-like receptors (LILRs) and murine paired Ig-activating receptors (PIRs). The second group of receptors possess an IgC2-2 domain and comprise the killer-cell Ig-like receptors (KIRs) and platelet collagen receptor glycoprotein VI (GPVI). The third group consists of receptors with IgC2-1, and IgC2-3 or IgC2-4 domains, and includes the receptor for IgA Fc (FCAR), NKp46 and murine Ly94. The fourth group, with a single extracellular IgC2-1 domain, consists of the leukocyte-associated Ig-like receptors (LAIRs). The genomic organization of and evolutionary associations between these receptors and their domains are examined.     

Human Leukocyte Migration Inhibitory Factor (LIF)

The activity of leukocyte migration inhibitory factor (LIF) obtained from Sephadex-G-100-chromatographed supernatants of concanavalin-A-stimulated human lymphocytes was suppressed by two synthetic serine esterase and serine protease inhibitors (di-isopropylfluorophosphate (DFP) and phenylmethylfulfonyl fluoride (PMSF)). LIF activity was also reduced by the naturally occurring protease inhibitors soybean trypsin inhibitor and aprotinin. The observed effect of DFP and PMSF was irreversible, since elimination of the inhibitors by dialysis did not restore LIF activity. The effect of PMSF was dose-, time-, and temperature-dependent, and hydrolytic products of PMSF as well as sodium fluoride were inactive in blocking LIF. These results suggest that LIF may act as a serine esterase or a serine protease, or both of these, and that this putative enzyme is present in an activated form in supernatants from mitogen-stimulated mononuclear cells.     

Human Leukocyte Migration Inhibitory Factor (LIF)

Previously reported experiments suggested that an esterase or a protease, or both, might participate in the expression of human leukocyte migration inhibitory factor (LIF). To clarify this further, a wide variety of simple esters were tested for the ability to protect LIF against inactivation by the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF), alpha-N-benzoyl-L-arginine ethylester (BAEE), a typical trypsin substrate, and bis-p-nitrophenyl phosphate (BNPP), a phosphodiester, were the only esters capable of retaining LIF activity in the presence of PMSF. Agents chemically closely related to these esters were inactive. Moreover, the protection afforded by BAEE and BNPP was the kind that would be anticipated if the esters and irreversible inhibitor competed for the same site on LIF. BAEE and BNPP also protected against inactivation by di-isopropylfluorophosphate (DFP), another irreversible serine esterase inhibitor. In addition, LIF-treated leukocytes partly escaped migration inhibition in the presence of BAEE and BNPP, respectively. These results indicate that human LIF contains a serine residue necessary for lymphokine activity. It is still not proved, however, that LIF as an enzyme is capable of hydrolyzing BAEE and BNPP, although it seems highly possible. The substrate specificities of a putative LIF enzyme are discussed on the basis of the chemical structure of BAEE and BNPP.     

Human Leukocyte Migration Inhibitory Factor (LIF)

Previous findings suggesting an esterase and protease nature of human leukocyte migration inhibitory factor (LIF) were extended by testing the ability of different protease and esterase inhibitors to reduce LIF activity. The serine-specific inhibitors phenylmethylsulfonyl fluoride (PMSF) and physostigmine (eserine) markedly reduced LIF activity, whereas the histidine-specific inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) were inactive. That LIF might act as an esterase and a protease was further strengthened by the ability of pralidoxime methansulfonate (2-PAM) to reestablish LIF activity of supernatants previously inactivated by PMSF. Furthermore, the arginine amides N-benzoyl-L-arginine-p-nitroanilide (BApNA) and N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (BPVApNA) were shown to satisfy the substrate specificities of the putative LIF enzyme. Indeed, BPVApNA seemed to possess a particularly strong affinity for LIF, indicating its potential role in an enzymatic LIF assay.     

Human Leukocyte Migration Inhibitory Factor (LIF)

Recently reported experiments suggest that human leukocyte migration inhibitory factor (LIF) has properties of an esterase and a protease with substrate specificities directed against arginine esters and amides. Also reported previously, the synthetic phosphodiester bis-p-nitrophenyl phosphate (BNPP) but not various phosphomonoesters preserve LIF activity in the presence of the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF). In this paper I demonstrate that guanosine 3'5'-cyclic monophosphoric acid (3',5'-cGMP), a naturally occurring phosphodiester, at concentrations in excess of 10(-5)M also protects LIF against PMSF inactivation. The effect seems specific for the diester bond, its position in the nucleotide, and the guanine base. The possibility that LIF may be a multifunctional or an allosteric enzyme regulated by 3',5'-cGMP is discussed.     

Role of plasma homocysteine and lipoprotein (a) in coronary artery disease

This study looks at the possible role of some non-traditional risk factors for premature coronary artery disease (CAD) and assesses the presence of relationship between these factors and the traditional cardiovascular risk factors. The study subjects (n=45) are divided into three groups comprising 15 premature CAD patients without traditional cardiovascular risk factors (group I); 15 premature CAD patients with one or more traditional cardiovascular risk factors (group II); and 15 healthy normal control subjects matched for age and sex (group III). Estimation of plasma homocysteine (Hcy) and plasminogen activator inhibitor-1 (PAI-1) is performed by enzyme-linked immunosorbent assay (ELISA); plasma folic acid by radioimmunoassay; plasma lipoprotein a (Lpa) by turbidimetry; and plasma lipids by colorimetry. Results showed a significant association between elevated Hcy and low folate levels and premature CAD in both patient groups. Also, a significant association was seen between elevated PAI-1 and CAD in the two patient groups, and between CAD and high levels of Hcy and triglycerides, as well as a low level of high-density lipoprotein cholesterol. Lpa showed significant association with premature CAD only in group II. Thus, Hcy, folic acid and PAI-1 might serve as independent risk factors for premature CAD in patients both with and without traditional coronary risk factors. However, Lpa might confer an additional coronary risk factor only in the presence of traditional risk factors.     

Function of Junctional Adhesion Molecules (JAMs) in Leukocyte Migration and Homeostasis

Homeostasis is a word widely used in the scientific community to refer to the property of a system to maintain its uniformity and functionality. In living organisms, the word refers to the concept enunciated 150 years ago by C. Bernard by which external variations must be compensated for in order to maintain internal conditions compatible with life. This is especially true in the case of highly dynamic system such as the hematopoietic system that requires the coordinated control of cell proliferation and death within specialized microenvironments that are anatomically distinct. As a consequence, hematopoietic cell adhesion and migration must be tightly controlled in order for hematopoietic cells to reach and to be maintained in appropriate microenvironments. The junctional adhesion molecules (JAMs) are adhesion molecules that belong to the immunoglobulin superfamily (IgSf) and that have been initially identified as important players controlling vascular permeability and leukocyte transendothelial migration. This involves the regulated localization of the JAMs at lateral endothelial cell/cell borders and their interaction with leukocyte integrins. More recently, some of the JAM family members have also been found to be expressed by stromal cells and to regulate chemokine secretion within lymphoid organs, acting not only on leukocyte transendothelial migration, but also on hematopoietic cell retention within specialized microenvironments. This review summarizes recent progress in understanding the role of the JAMs in leukocyte adhesion and migration to tentatively draw an integrated view of the homeostatic function of the JAMs within the hematopoietic system.     

Spinal muscular atrophy and Farber disease due to ASAH1 variants: A case report

Genetic variations in the ASAH1 gene are associated with a spectrum of disorders ranging from Farber disease (FD) to spinal muscular atrophy with or without progressive myoclonic epilepsy (SMA‐PME). FD presents most commonly in infants with subcutaneous joint nodules, progressive arthritis and granulomas of the larynx and epiglottis leading to a hoarse cry. SMA‐PME is characterized by childhood onset progressive weakness due to motor neuron disease followed by progressive epilepsy, tremor, and sensorineural hearing loss. We present a case of a 4‐year‐old boy with phenotypic features of both FD and SMA who was found to have two previously unreported heterozygous variants in the ASAH1 gene.