Mucolipidosis I: Increased sialic acid content and deficiency of an α-N-acetylneuraminidase in cultured fibroblasts

Extracts of fibroblasts derived from a patient with mucolipidosis I exhibited a fivefold increase in sialic acid content as compared to those of normal cells. About 80% of this sialic acid was linked to other molecules. Using neuraminlactose as a substrate, mucolipidosis I fibroblasts were found to be severely deficient in an “acid” α-N-acetylneuraminidase. Since other lysosomal hydrolase activities were normal, we hypothesize that the basic metabolic lesion in mucolipidosis I lies in a defective degradation of sialic acid-containing compounds due to the genetic deficiency of a neuraminidase.     

Relationship between α-L-fucosidase deficiency in plasma and α-L-fucosidase activity in leukocytes

Summary After testing a population sample of 185 hospitalized Italian children for the plasma -L-fucosidase deficiency and establishing an approximate threshold value between heterozygotes and wild-type homozygotes, we analyzed by two statistical methods the distribution of the two genotypes. The results obtained by probit analysis agree with threshold and average values expected on the basis of the Hardy-Weinberg equilibrium.In addition, the level of -fucosidase in leukocytes of 12 individuals with deficiency of -fucosidase in plasma was found to be significantly lower than that of 61 controls (P<0.005). These results indicate that the mutation(s) causing a deficiency of -fucosidase in plasma is (are) also expressed in leukocytes.     

Human β-galactosidase and α-neuraminidase deficient mucolipidosis: Genetic complementation analysis of the neuraminidase deficiency

Summary Human -galactosidase and -neuraminidase deficient mucolipidosis [ML(gal^-neur^-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal^-neur^-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular -galactosidase and -neuraminidase levels in heterokaryons. The lack of human -galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal^-neur^-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect may occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal -galactosidase and -neuraminidase.     

β-Galactosidase deficiency in juvenile and adult patients

Summary Six juvenile and adult patients with progressive neurological diseases and -galactosidase deficiency were reported. Any diseases known to date were denied. These cases together with ten case reports in the literature were reviewed and were classified into three groups from clinical and biochemical points. Group 1 patients were characterized by progressive ataxia and myoclonus with gargoyle changes and macular cherry-red spots. In this syndrome -galactosidase activity seems to be secondarily affected by other biochemical defects. A group 2 patient showed similar neurological manifestations without gargoyle changes or macular cherry-red spots. Patients with these clinical features not associated with -galactosidase deficiency have also been described in the literature. Group 3 patients had progressive pyramidal and extrapyramidal disease without gargoyle changes or macular cherry-red spots. These cases may represent juvenile and adult type G_M1-gangliosidosis. Accumulation of G_M1 has not yet been demonstrated.     

β-carotene improves fecal dysbiosis and intestinal dysfunctions in a mouse model of vitamin A deficiency

Vitamin A deficiency (VAD) results in intestinal inflammation, increased redox stress and reactive oxygen species (ROS) levels, imbalanced inflammatory and immunomodulatory cytokines, compromised barrier function, and perturbations of the gut microbiome. To combat VAD dietary interventions with β-carotene, the most abundant precursor of vitamin A, are recommended. However, the impact of β-carotene on intestinal health during VAD has not been fully clarified, especially regarding the VAD-associated intestinal dysbiosis. Here we addressed this question by using Lrat^?/-Rbp^?/? (vitamin A deficient) mice deprived of dietary preformed vitamin A and supplemented with β-carotene as the sole source of the vitamin, alongside with WT (vitamin A sufficient) mice. We found that dietary β-carotene impacted intestinal vitamin A status, barrier integrity and inflammation in both WT and Lrat^?/-Rbp^?/? (vitamin A deficient) mice on the vitamin A-free diet. However, it did so to a greater extent under overt VAD. Dietary β-carotene also modified the taxonomic profile of the fecal microbiome, but only under VAD. Given the similarity of the VAD-associated intestinal phenotypes with those of several other disorders of the gut, collectively known as Inflammatory Bowel Disease (IBD) Syndrome, these findings are broadly relevant to the effort of developing diet-based intervention strategies to ameliorate intestinal pathological conditions.     

Deficiency of two red-cell flavin enzymes in a population in Sardinia: was glutathione reductase deficiency specifically selected for by malaria?

In two areas in Italy where malaria was endemic--in the Po delta and Maremma on the west coast--we have found a high prevalence of an inherited flavin-deficient red cell in the normal population, suggesting selection by malaria. This study in Sardinia enabled a direct comparison of red-cell activities of FAD-dependent glutathione reductase (EGR) and FMN-dependent pyridoxine phosphate (PNP) oxidase in an ethnically homogeneous population, between two coastal villages where malaria was endemic from 300 B.C. and two mountain villages with no history of malaria. Both enzyme activities were significantly lower on the coast, and it did not seem that this could be explained by possible small differences in dietary riboflavin. As was thought to be the case in Ferrara and Grosseto, it is probable that a genetically controlled flavin-deficient red cell was selected for by malaria. Low EGR apoenzyme activity was more common on the coast, usually explaining the accompanying low basic EGR activity, and may also have been selected for by malaria. This adds to evidence from others that the mechanism of defence of a flavin-deficient red cell against malaria may be through EGR deficiency. It could also play a part in the protection given by heterozygous beta-thalassemia. The multifactorial protection of the population against malaria is discussed.     

Mesenchymal stromal cells and fibroblasts: a case of mistaken identity?

The plastic-adherent fibroblast-looking cells that can be isolated and culture-expanded from bone marrow and many other tissues are widely known as mesenchymal stromal cells (MSC). In addition to their fibroblast-like morphology, they are characterized by a panel of cell-surface markers and their potential to differentiate into bone, fat and cartilage. Based on their intriguing immunomodulatory and regenerative properties, MSC are being investigated as cellular therapeutics for a variety of clinical indications. However, many questions regarding the true identity and functionality of these cells in vivo remain unanswered. Fibroblasts, known for a much longer time but still poorly characterized, are also considered to be a ubiquitous stromal element of almost all tissues and are believed to play a role in tissue homeostasis. Despite the presence of MSC and fibroblasts in almost all tissues, similar morphology and other shared characteristics, the exact relationship between MSC and fibroblasts has remained undetermined. In this review, based on recent and old, but often neglected, literature it is suggested that ex vivo culture-expanded MSC and fibroblasts are indistinguishable by morphology, cell-surface markers, differentiation potential and immunologic properties.     

Evidence for the deficiency of β-glucosidase-activating factor in fibroblasts of patients with I-cell disease

Summary Reduced activity of -glucosidase was shown in the cultured skin fibroblasts of four patients with I-cell disease when the enzyme was tested without the use of detergents. In the presence of taurocholate and triton X100 -glucosidase activity was normal. This suggested a deficiency of a -glucosidase-activating factor in I-cell fibroblasts rather than of the enzyme itself. The deficiency of -glucosidase activity was corrected to some extent by mixing cell lysates, and more effectively by cocultivation and fusion of I-cell disease and Gaucher fibroblasts. These results present evidence for the presence of a -glucosidase-activating factor in normal and Gaucher fibroblasts. In fibroblasts of patients with I-cell disease this activator is probably deficient, as is the case for most lysosomal enzymes.     

Expression of TGF-β3 in isolated fibroblasts from foreskin

Background:

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s).

Methods:

We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

Conclusions:

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.Key Words: Fibroblasts, Gene expression, TGF-β3     

The α4β1 integrin and the EDA domain of fibronectin regulate a profibrotic phenotype in dermal fibroblasts

Prompt deposition of fibronectin-rich extracellular matrix is a critical feature of normal development and the host-response to injury. Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. We now report using human dermal fibroblast cultures that the EDA domain of fibronectin or EDA-derived peptides modeled after the C–C′ loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the α4 integrin. Our data indicate that the interaction between the α4β1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition.     

The salvaged blood syndrome: a sequel to mechanochemical activation of platelets and leukocytes?

Disseminated intravascular coagulopathy (DIC) and/or increased vascular permeability in the lungs (ARDS) or systemic circulation (anasarca) has been seen in an occasional patient following the administration of washed autologous red cells. We have found that platelets and leukocytes, activated by the process of salvage, can contaminate such red cell suspensions. Apparently, activation begins with the mechanical deposition of platelets on the centrifuge bowl wall during the cell-concentration phase of blood salvage if there has been substantial prior dilution of the salvaged blood with saline. Electron microscopy of the deposit reveals activated, degranulated platelets lining the inner surface of the bowl. There is a preferential "homing" of specific leukocyte types to local regions of the deposit. On the basis of morphological evidence, we hypothesize that these mechanically activated platelets release leukoattractant substances, including arachidonate-rich phospholipids which trigger the oxidative burst enzymatic pathway in exposed phagocytic cells. These cells, when reinfused, cause increased vascular permeability. This presents clinically as ARDS or anasarca, whereas DIC results from reinfused platelet phospholipid plus accompanying cellular debris.     

Anxiety and depression in children and adults: influence of serotonergic and neurotrophic genes?

There are two major hypotheses regarding the etiology of anxiety and depression: the mono‐amine hypothesis and the hypothesis of an abnormal stress response acting partly via reduced neurogenesis. Association studies have focused on genes involved in these processes, but with inconclusive results. This study investigated the effect of 45 single nucleotide polymorphisms (SNPs) in genes encoding for serotonin receptors 1A, 1D, 2A, catechol‐O‐methyltransferase (COMT), tryptophane hydroxylase type 2 (TPH2), brain derived neurotrophic factor (BDNF), PlexinA2 and regulators of G‐protein‐coupled signaling (RGS) 2, 4, 16. Anxious depression (A/D) symptoms were assessed five times in 11 years in over 11 000 adults with 1504 subjects genotyped and at age 7, 10, 12 and during adolescence in over 20 000 twins with 1078 subjects genotyped. In both cohorts, a longitudinal model with one latent factor loading on all A/D measures over time was analysed. The genetic association effect modeled at the level of this latent factor was 60% and 70% heritable in the children and adults, respectively, and explained around 50% of the total phenotypic variance. Power analyses showed that the samples contained 80% power to detect an effect explaining between 1.4% and 3.6% of the variance. However, no SNP showed a consistent effect on A/D. To conclude, this longitudinal study in children and adults found no association of SNPs in the serotonergic system or core regulators of neurogenesis with A/D. Overall, there has been no convincing evidence, so far, for a role of genetic variation in these pathways in the development of anxiety and depression.     

Incorporation and degradation of GM1 ganglioside and asialoGm1 ganglioside in cultured fibroblasts from normal individuals and patients with β-galactosidase deficiency

The uptake and degradation of G_M1 ganglioside (G_M1) and asialoG_M1 ganglioside (G_A1) were studied in cultured fibroblasts from normal individuals and patients with β-galactosidase deficiency, using the lipid-loading test. The glycolipids were incorporated from the media into the fibroblasts and the terminal galactose was hydrolyzed in normal cells. The hydrolysis rates of G_A1 were 80–86% of normal on the 3rd day after loading, while G_M1 was hydrolyzed slowly; 35–54% on the 14th day. In infantile G_M1 gangliosidosis and I-cell disease, little G_M1 and G_A1 was hydrolyzed on any day of culture, while fibroblasts from patients with adult G_M1 gangliosidosis, Morquio disease type B and galactosialidosis hydrolyzed the lipids at nearly normal rates. The intracellular accumulation of the glycolipids, on the basis of protein content, was abnormally high in the case of infantile G_M1 gangliosidosis and I-cell disease, but normal in the other disorders examined. These observations indicate that the in situ metabolism of G_M1 and G_A1 is probably normal in fibroblasts from patients with adult G_M1 gangliosidosis, Morquio disease type B and galactosialidosis, although in vitro β-galactosidase activities in these disorders are very low. The results are compatible with findings that G_M1 and G_A1 do not accumulate in the somatic organs of patients with adult G_M1 gangliosidosis and galactosialidosis. In I-cell disease, however, the results of the loading test did not agree with the finding that there is little accumulation of glycolipids in postmortem tissues.     

Differential expression, function and response to inflammatory stimuli of 11β-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Expression, activity and function of 11β-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11β-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-α or IL-1β (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-γ was without effect, and there was no difference in 11β-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production – a characteristic feature of synovial derived fibroblasts – was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11β-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11β-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.     

Latent Trypanosoma brucei gambiense foci in Uganda: a silent epidemic in children and adults?

Trypanosoma brucei gambiense sleeping sickness follows a long asymptomatic phase and persists in ancient foci from which epidemic clinical disease arises. A putative focus of T. b. gambiense infections has been identified, initially in mothers and young children, on the Lake Albert shoreline of Western Uganda leading to mass screening of 6207 individuals in September 2008. T. b. gambiense infections were identified by Card Agglutination Test for Trypanosomiasis (CATT) and sub-species-specific PCR although parasitological methods failed to confirm any patent trypanosome infections. In April 2009, CATT positives were re-visited; diagnosis of individuals by CATT and PCR was unstable over the two time points and parasites remained undetected, even using mini Anion Exchange Centrifugation Technique (mAECT). These observations suggest the possibility of a silent focus of disease, where all infected individuals are in a latent stage, and highlight our limited understanding of the local natural history and disease progression of T. b. gambiense in children and adults.     

Steroid 17β-hydroxysteroid dehydrogenase deficiency in man: an inherited form of male pseudohermaphroditism

Sixty-eight males with testicular 17β-hydroxysteroid dehydrogenase deficiency (17β-HSD) were identified among a highly inbred Arab population in Israel, and 45 studied over the last 15 years. The founders of this defect originated in the mountainous region of present Lebanon and Syria, but most of the families now live in Jerusalem, Hebron, the Tel-Aviv area, and in particular Gaza, where the frequency of affected males is estimated at 1 in 100 to 150. Affected individuals (46,XY) are born with ambiguity of the genitalia and reared as females until puberty. Thereafter marked virilization occurs, leading in many cases to the spontaneous adoption of a male gender role. Adults develop a male habitus with abundant body hair and beard, and the phallus and testes enlarge to adult proportions. Gender reassignment was possible only when enough erectile tissue was present at birth and developed into a normal size penis with systemic testosterone. male genitoplasty was performed in 15 children and 8 post-pubertal patients, and female genitoplasty in 2 children and 4 post-pubertal patients. In adults the defect is characterized by markedly increased concentrations of 4-androstendione (4-A) with borderline low to normal testosterone (T) levels, and a high 4-A/T ratio. Dihydrotestosterone (DHT) concentrations were either moderately decreased, normal, or high, and dehydroepiandrosterone levels were high. The estrogen pathway was also impaired, even though both estrone and estradiol-17β levels were elevated. Children had low basal levels of all androgens, but the defect could be demonstrated after prolonged stimulation with human chorionic gonadotropin. LH and FSH levels were very high after puberty, and normal in childhood. However, an over-response to gonadotropin-releasing hormone was found at all ages.

Studies in testicular tissue revealed various abnormalities in steroid metabolism. Tissue from pre-pubertal patients metabolized progesterone (P) only to 4-A, while tissue from post-pubertal patients metabolized P to 16- and 16β-hydroxyprogesterone (5.4- to 10.3-fold greater production), 17-hydroxyprogesterone (5.4- to 8-fold smaller production), 4-A and T. 4-A was also metabolized to T, indicating that 17β-HSD was no longer deficient. Flow studies with equimolar concentrations of [¹⁴C]P and [³H]pregnenolone showed that the 5-ene pathway was the preferential one for androgen biosynthesis. Both in vivo and in vitro studies indicate that the severity of testicular 17β-HSD deficiency changes with age. Whereas the enzyme activity is absent in childhood, there is a progressive restoration after puberty. Androgen production increases progressively to normal so that T and DHT concentrations are sufficiently high to gradually induce somatic and genital virilization, thus enabling an adequate male gender function.     

Androgen deficiency in male patients diagnosed with ANCA-associated vasculitis: a cause of fatigue and reduced health-related quality of life?

Introduction

Low testosterone levels in men are associated with fatigue, limited physical performance and reduced health-related quality of life (HRQOL); however, this relationship has never been assessed in patients with anti-neutrophil cytoplasmic antibodies (ANCA) -associated vasculitides (AAV). The aim of this study was to assess the prevalence of androgen deficiency and to investigate the role of testosterone in fatigue, limited physical condition and reduced HRQOL in men with AAV.

Methods

Male patients with AAV in remission were included in this study. Fatigue and HRQOL were assessed by the multi-dimensional fatigue inventory (MFI)-20 and RAND-36 questionnaires.

Results

Seventy male patients with a mean age of 59 years (SD 12) were included. Scores of almost all subscales of both questionnaires were significantly worse in patients compared to controls. Mean total testosterone and free testosterone levels were 13.8 nmol/L (SD 5.6) and 256 pmol/L (SD 102), respectively. Androgen deficiency (defined according to Endocrine Society Clinical Practice Guidelines) was present in 47% of patients. Scores in the subscales of general health perception, physical functioning and reduced activity were significantly worse in patients with androgen deficiency compared to patients with normal androgen levels. Testosterone and age were predictors for the RAND-36 physical component summary in multiple linear regression analysis. Testosterone, age, vasculitis damage index (VDI) and C-reactive protein (CRP) were associated with the MFI-20 subscale of general fatigue.

Conclusions

This study showed that androgen deficiency was present in a substantial number of patients with AAV. Testosterone was one of the predictors for physical functioning and fatigue. Testosterone may play a role in fatigue, reduced physical performance and HRQOL in male patients with AAV.     

Ganglioside GM1 metabolism in living human fibroblasts with β-galactosidase deficiency

Summary The uptake and catabolism of [³H-ceramide]-G_M1 was followed in living fibroblasts from patient with different forms of -galactosidase deficiency. Gangliosides are identified according to the nomenclature of Svennerholm (1963). A total inability to metabolize the ingested substrate was found in infantile G_M1-gangliosidosis whereas cells from an adult G_M1-gangliosidosis variant showed a slower rate of degradation, compared with controls. Morquio B fibroblasts had a comparable catabolism of G_M1 as controls. Fibroblasts from different types of galactosialidosis, a recessive disease associated with a coexistent -galactosidase/neuraminidase deficiency all showed degradation of ingested G_M1. In view of the molecular defect in this disease, this catabolism must be due to the 10–20% of monomeric -galactosidase molecules present in the lysosomes. Unexpectedly, in these cells an impaired metabolism of G_M3 was found. The same finding was observed when cells with an isolated neuraminidase deficiency (mucolipidosis I) were loated with G_M1. A hypothesis is presented to explain these results.     

Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48 h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72 h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48 h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0 mg/L F⁻. The Cbfa1 protein level of the group treated with 10 mg/L F⁻ at 72 h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48 h in the group treated with 10.0 mg/L F⁻ than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻ groups at 2 h, and in the 0.001 and 0.1 F⁻ groups at 4 h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0 mg/L F⁻ groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.