Management goals for type 1 Gaucher disease: An expert consensus document from the European working group on Gaucher disease.

Gaucher Disease type 1 (GD1) is a lysosomal disorder that affects many systems. Therapy improves the principal manifestations of the condition and, as a consequence, many patients show a modified phenotype which reflects manifestations of their disease that are refractory to treatment. More generally, it is increasingly recognised that information as to how a patient feels and functions [obtained by patient- reported outcome measurements (PROMs)] is critical to any comprehensive evaluation of treatment. A new set of management goals for GD1 in which both trends are reflected is needed. To this end, a modified Delphi procedure among 25 experts was performed. Based on a literature review and with input from patients, 65 potential goals were formulated as statements. Consensus was considered to be reached when ≥75% of the participants agreed to include that specific statement in the management goals. There was agreement on 42 statements. In addition to the traditional goals concerning haematological, visceral and bone manifestations, improvement in quality of life, fatigue and social participation, as well as early detection of long-term complications or associated diseases were included. When applying this set of goals in medical practice, the clinical status of the individual patient should be taken into account.

On fragmenting, densely mineralised acellular protrusions into articular cartilage and their possible role in osteoarthritis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26-µm resolution high contrast X-ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re-imaged with 6-µm resolution X-ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self-healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

B cell lymphoma and myeloma in murine Gaucher's disease.

Multiple myeloma and B cell lymphoma are leading causes of death in Gaucher's disease but the nature of the stimulus driving the often noted clonal expansion of immunoglobulin-secreting B cells and cognate lymphoid malignancy is unknown. We investigated the long-term development of B cell malignancies in an authentic model of non-neuronopathic Gaucher's disease in mice: selective deficiency of ß-glucocerebrosidase in haematopoietic cells [Gba(tm1Karl/tm1Karl)Tg(Mx1-cre)1Cgn/0, with excision of exons 9-11 of the murine GBA1 gene, is induced by poly[I:C]. Mice with Gaucher's disease showed visceral storage of ß-glucosylceramide and greatly elevated plasma ß-glucosylsphingosine [median 57.9 (range 19.8-159) nm; n = 39] compared with control mice from the same strain [median 0.56 (range 0.04-1.38) nm; n = 29] (p < 0.0001). Sporadic fatal B cell lymphomas developed in 11 of 21 GD mice (6-24 months) but only two of eight control animals developed tumours by age 24 months. Unexpectedly, most mice with overt lymphoma had absent or few Gaucher cells but local inflammatory macrophages were present. Eleven of 39 of Gaucher mice developed monoclonal gammopathy, but in the control group only one animal of 25 had clonal immunoglobulin abnormalities. Seven of 10 of the B cell lymphomas were found to secrete a monoclonal paraprotein and the lymphomas stained intensely for pan-B cell markers; reactive T lymphocytes were also present in tumour tissue. In the Gaucher mouse strain, it was notable that, as in patients with this disease, CD138(+) plasma cells frequently surrounded splenic macrophages engorged with glycosphingolipid. Our strain of mice, with inducible deficiency of ß-glucocerebrosidase in haematopoietic cells and a high frequency of sporadic lethal B cell malignancies, faithfully recapitulates human Gaucher's disease: it serves as a tractable model to investigate the putative role of bioactive sphingolipids in the control of B cell proliferation and the pathogenesis of myelomatosis-the most prevalent human cancer associated with this disorder.

Lysosomal delivery of therapeutic enzymes in cell models of Fabry disease.

The success of enzymatic replacement in Gaucher disease has stimulated development of targeted protein replacement for other lysosomal disorders, including Anderson-Fabry disease, which causes fatal cardiac, cerebrovascular and renal injury: deficiency of lysosomal α-Galactosidase A induces accumulation of glycosphingolipids. Endothelial cell storage was the primary endpoint in a clinical trial that led to market authorization. Two α-Galactosidase A preparations are licensed worldwide, but fatal outcomes persist, with storage remaining in many tissues. We compare mechanisms of uptake of α -Galactosidase A into cells relevant to Fabry disease, in order to investigate if the enzyme is targeted to the lysosomes in a mannose-6-phosphate receptor dependent fashion, as generally believed. α -Galactosidase A uptake was examined in fibroblasts, four different endothelial cell models, and hepatic cells in vitro. Uptake of europium-labeled human α -Galactosidase A was measured by time-resolved fluorescence. Ligand-specific uptake was quantified in inhibitor studies. Targeting to the lysosome was determined by precipitation and by confocal microscopy. The quantity and location of cation-independent mannose-6-phosphate receptors in the different cell models were investigated using confocal microscopy. Uptake and delivery of α -Galactosidase A to lysosomes in fibroblasts is mediated by the canonical mannose-6-phosphate receptor pathway, but in endothelial cells in vitro this mechanism does not operate. Moreover, this observation is supported by a striking paucity of expression of cation independent mannose-6-phosphate receptors on the plasma membrane of the four endothelial cell models and by little delivery of enzyme to lysosomes, when compared with fibroblasts. If these observations are confirmed in vivo, alternative mechanisms will be needed to explain the ready clearance of storage from endothelial cells in patients undergoing enzyme replacement therapy.

Acute intermittent porphyria: fatal complications of treatment.

Acute neurovisceral attacks of porphyria can be life threatening. They are rare and notoriously difficult to diagnose clinically, but should be considered, particularly in female patients with unexplained abdominal pain, and associated neurological or psychiatric features or hyponatraemia. The diagnosis might be suggested by altered urine colour and can be confirmed by finding an elevated porphobilinogen concentration in fresh urine protected from light. Severe attacks require treatment with intravenous haem arginate and supportive management with safe drugs, including adequate analgesia. Intravenous glucose in water solutions are contraindicated as they aggravate hyponatraemia, which can prove fatal.

Immune signatures underlying post-acute COVID-19 lung sequelae.

Severe coronavirus disease 2019 (COVID-19) pneumonia survivors often exhibit long-term pulmonary sequelae, but the underlying mechanisms or associated local and systemic immune correlates are not known. Here, we have performed high-dimensional characterization of the pathophysiological and immune traits of aged COVID-19 convalescents, and correlated the local and systemic immune profiles with pulmonary function and lung imaging. We found that chronic lung impairment was accompanied by persistent respiratory immune alterations. We showed that functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)­specific memory T and B cells were enriched at the site of infection compared with those of blood. Detailed evaluation of the lung immune compartment revealed that dysregulated respiratory CD8+ T cell responses were associated with the impaired lung function after acute COVID-19. Single-cell transcriptomic analysis identified the potential pathogenic subsets of respiratory CD8+ T cells contributing to persistent tissue conditions after COVID-19. Our results have revealed pathophysiological and immune traits that may support the development of lung sequelae after SARS-CoV-2 pneumonia in older individuals, with implications for the treatment of chronic COVID-19 symptoms.

Dietary modifications in patients receiving miglustat.

Weight loss and gastrointestinal disturbances are often seen during miglustat therapy for lysosomal storage diseases. A retrospective analysis of data from a mixed group of patients treated with miglustat at two UK centres was performed to evaluate the effect of two different dietary interventions on body weight and gastrointestinal tolerability during the initial 6 months of miglustat therapy. Neurological outcomes in these patients are not discussed herein. Data were analysed from a total of 29 patients with varied neurolipidoses (21 children/adolescents; 8 adults). Negative mean changes in body weight were seen in children/adolescents on an unmodified diet (-8.1%), and in adults (-4.1%) and children/adolescents (-5.2%) on a low-lactose diet. Patients on the low-disaccharide diet showed a positive mean change in body weight (+2.0%), although there was high variability in this group. Non-parametric sub-analysis of median body-weight change in children/adolescents also showed high variability both within and between diet groups, with no statistically significant difference between the effects of different diets on body weight (p = 0.062). The low-lactose diet reduced gastrointestinal disturbances; single small doses of loperamide were required in some patients. Patients on the low-disaccharide diet showed the lowest frequency of gastrointestinal effects. In conclusion, simple dietary modifications allowed the maintenance of body-weight gain in line with normal growth potential during miglustat therapy in young patients with lysosomal storage diseases, and reduced gastrointestinal disturbances.

A novel HEXB mutation and its structural effects in juvenile Sandhoff disease.

Mutations in HEXB, encoding the beta-subunit common to hexosaminidases A and B, cause the neurodegenerative condition, Sandhoff disease. A homozygous missense HEXB mutation (p. D459A) was discovered in six patients with a rare juvenile variant: we show that this disrupts a salt bridge between aspartate D459 and arginine 505 at the subunit interface; R505 mutations are reported in late-onset Sandhoff disease. Identification of D459A contributes to diagnosis and molecular understanding of attenuated Sandhoff disease variants.

Haemochromatosis: an inherited metal and toxicity syndrome.

A newly-identified major histocompatibility Class I-like gene, HFE (originally HLA-H) located approximately 3.5 Mb telomeric to the Class I cluster on chromosome 6p 21.3 harbours mutations in haemochromatosis. Two of these, Cys282Tyr (C282Y) and His63Asp (H63D, a minor determinant) have diagnostic utility as approximately 90% of adults are homozygous or compound heterozygotes for these alleles. The pathophysiological role of HFE is unclear: it is expressed as a surface molecule on many cells and the C282Y mutation disrupts interactions with beta 2-microglobulin, thus preventing surface expression. Lately, there has been experimental evidence that HFE protein interacts with the transferrin-receptor, affecting receptor turnover or its affinity for ligand.

Management of non-neuronopathic Gaucher disease with special reference to pregnancy, splenectomy, bisphosphonate therapy, use of biomarkers and bone disease monitoring.

Enzyme replacement was introduced as treatment for non-neuronopathic Gaucher disease more than 15 years ago. To ensure the best use of this costly ultra-orphan agent, a systematic disease management approach has been proposed by an international panel; this includes the development, by consensus, of achievable treatment goals. Here we critically review these goals and monitoring guidelines and incorporate emerging experience of the disease in the therapeutic era, as well as contemporary clinical research. This review makes recommendations related specifically to the management of pregnancy; the appropriate use of splenectomy and bisphosphonate treatment; the relevance of biochemical markers to disease monitoring; and the use of semi-quantitative methods for assessing bone marrow infiltration. In addition, we identify key areas for development, including the requirement for a validated index of disease severity; the need to correlate widely used biomarkers with long-term disease outcomes, and the desirability of establishing agreed standards for monitoring of bone disease particularly in infants and children with Gaucher disease.

Isolation and characterization of a mutant liver aldolase in adult hereditary fructose intolerance. Identification of the enzyme variant by radioassay in tissue biopsy specimens.

Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l-phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values. To identify a putative enzyme mutant in tissue extracts, aldolase B was purified from human liver by affinity chromatography and monospecific antibodies were prepared from antiserum raised in sheep. Immunodiffusion gels showed a single precipitin line common to pure enzyme and extracts of normal liver and intestine, but no reaction with extracts of brain, muscle, or HFI liver. However, weak positive staining for aldolase in hepatocyte and enterocyte cytosol was demonstrated by indirect immunofluorescence of HFI tissues. This was abolished by pretreatment with pure enzyme protein. Accordingly, a specific radioimmunoassay (detection limit 7.5 ng) was established to quantify immunoreactive aldolase B in human biopsy specimens. Extracts of tissue from affected patients gave 10-25% immunoreactive enzyme in control samples; immunoreactive aldolase in intestinal extracts from four heterozygotes was reduced (to 55%) when compared with seven samples from normal control subjects (P < 0.05). In extracts of HFI tissues, there was a sevenfold reduction in apparent absolute specific activity (1.02 vs. 8.82 U/mg) of immunoreactive fructose-l-phosphate aldolase B, but the apparent specific activity in heterozygotes (7.71 U/mg) was only slightly impaired. Displacement radioimmunotitration of aldolase B in liver supernatants showed a significant (P < 0.005) decrease in antibody avidity for immunoreactive protein in HFI tissue when compared with the pure enzyme or extract of normal control liver. Immunoaffinity chromatography on antialdolase B-Sepharose facilitated isolation and purification of enzyme from liver biopsy specimens. Active aldolase in normal liver, with substrate activity ratios and Michaelis constants identical to biochemically purified human enzyme, could be recovered from antibody columns. Chromatography on monospecific Fab' antialdolase B enabled pure enzyme protein to be retrieved quantitatively from normal control and HFI liver: direct chemical assay showed 1.88 and 1.15 mg aldolase protein/g of tissue, respectively. This confirmed that the catalytic properties of the HFI aldolase were profoundly impaired with specific activities of fructose-l-phosphate cleavage of 7.21 and 0.07 U/mg, respectively. Radioimmunoassay gave estimates of 7.66 and 1.18 U/mg, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that immunopurified aldolase from HFI liver possessed a single subunit size similar to material from control liver extracts: M(r) 39,100 vs. 37,900+/-700 (SD) D, respectively. Electrofocusing under denaturing conditions of aldolase isolated in parallel from control and HFI liver revealed the same complement of subunits and, despite qualitative differences in distribution of bands during degradation, no additional charged species. Fructose phosphate aldolase deficiency in hereditary fructose intolerance is attended by the synthesis of an immunoreactive, but functionally and structurally modified enzyme variant that results from a restricted genetic mutation.

Biosynthesis of the transferrin receptor in rabbit reticulocytes.

These studies were performed to determine whether the reticulocyte can synthesize its own transferrin receptor and, if so, whether synthesis is subject to translational control by intracellular heme. Reticulocytosis (20-35%) was produced by bleeding rabbits and the washed cells were incubated for 1-4 h at 37 degrees C in buffered nutritional medium containing L-[35S]methionine. After washing and detergent lysis in the presence of protease inhibitors, supernatant reticulocyte extracts were analyzed for transferrin receptors by immunoprecipitation with specific ovine receptor antibody raised against denatured rabbit transferrin receptor. Immunoprecipitates were analyzed by SDS-gel electrophoresis and fluorography. Antibody, but not preimmune sheep immunoglobin, consistently precipitated a 35S-labeled protein with an Mr of 90,000 (reduced), coincident with bona fide receptor subunits purified by ligand-affinity chromatography. Incorporation of radioactive methionine was exclusively associated with receptor in reticulocyte stroma, and nascent receptor was not detected on free polyribosomes. Incorporation of radioactivity in the receptor moiety accounted for 0.1-0.2% of total incorporation into TCA insoluble cell protein. Treatment of the cells with 40 micrograms/ml cycloheximide markedly inhibited amino acid incorporation into the receptor, thus indicating de novo synthesis of receptor protein. On treatment of reticulocytes with 4,6 dioxoheptanoate to induce heme deficiency by diminishing the formation of intracellular heme, synthesis of the receptor was inhibited by greater than 50%; synthesis was restored to control rates on addition of 50 microM exogenous hemin. These findings indicate that the reticulocyte retains receptor mRNA and that synthesis of the receptor in erythroid cells is subject to translational regulation by intracellular heme.

Comparing effectiveness of experimental and implemented bycatch reduction measures: the ideal and the real.

Fishers, scientists, and resource managers have made substantial progress in reducing bycatch of sea turtles, seabirds, and marine mammals through physical modifications to fishing gear. Many bycatch-avoidance measures have been developed and tested successfully in controlled experiments, which have led to regulated implementation of modified or new fishing gear. Nevertheless, successful bycatch experiments may not translate to effective mitigation in commercial fisheries because experimental conditions are relaxed in commercial fishing operations. Such a difference between experimental results and real-world results with fishing fleets may have serious consequences for management and conservation of protected species taken as bycatch. We evaluated preimplementation experimental measures and postimplementation efficacy from primary and gray literature for three case studies: acoustic pingers that warn marine mammals of the presence of gill nets, turtle excluder devices that reduce bycatch of turtles in trawls, and various measures to reduce seabird bycatch in longlines. Three common themes to successful implementation of bycatch reduction measures are long-standing collaborations among the fishing industry, scientists, and resource managers; pre- and postimplementation monitoring; and compliance via enforcement and incentives.

Iron binding proteins and influx of iron across the duodenal brush border. Evidence for specific lactotransferrin receptors in the human intestine.

The ability of a range of homologous transferrin-like proteins to donate iron to pieces of human duodenal mucosa, was examined with an in vitro incubation technique. In contrast to serum transferrin and ovotransferrin, only lactotransferrin was able to yield its iron to intestinal tissue, but in an autologous system this protein was unable to donate iron to human reticulocyte preparations. Studies with 125I-labelled lactotransferrin and lactotransferrin dual-labelled with 59Fe and 125I, indicated that the intact protein is excluded from entry into the enterocytes. The experiments suggest that iron may be transported across the brush border after delivery to specific protein binding sites at the cell surface.

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

Classification and genetic features of neonatal haemochromatosis: a study of 27 affected pedigrees and molecular analysis of genes implicated in iron metabolism.

Neonatal haemochromatosis (NH) is a severe and newly recognised syndrome of uncertain aetiology, characterised by congenital cirrhosis or fulminant hepatitis and widespread tissue iron deposition. NH occurs in the context of maternal disease including viral infection, as a complication of metabolic disease in the fetus, and sporadically or recurrently, without overt cause, in sibs. Although an underlying genetic basis for NH has been suspected, no test is available for predictive analysis in at risk pregnancies. As a first step towards an understanding of the putative genetic basis for neonatal haemochromatosis, we have conducted a systematic study of the mode of transmission of this disorder in a total of 40 infants born to 27 families. We have moreover carried out a molecular analysis of candidate genes (beta(2)-microglobulin, HFE, and haem oxygenases 1 and 2) implicated in iron metabolism. No pathogenic mutations in these genes were identified that segregate consistently with the disease phenotype in multiplex pedigrees. However, excluding four pedigrees with clear evidence of maternal infection associated with NH, a pedigree showing transmission of maternal antinuclear factor and ribonucleoprotein antibodies to the affected infants, and two families with possible matrilineal inheritance of disease in maternal half sibs, a large subgroup of the affected pedigrees point to the inheritance of an autosomal recessive trait. This included 14 pedigrees with affected and unaffected infants and a single pedigree where all four affected infants were the sole offspring of consanguineous but otherwise healthy parents. We thus report three distinct patterns of disease transmission in neonatal haemochromatosis. In the differentiation of a large subgroup showing transmission of disease in a manner suggesting autosomal recessive inheritance, we also provide the basis for further genome wide studies to define chromosomal determinants of iron storage disease in the newborn.

Biomarkers in lysosomal storage diseases: a review.

UNLABELLED: A biomarker is generally an analyte that indicates the presence or extent of a biological process, which is itself directly linked to the clinical manifestations and outcome of a particular disease. An ideal biomarker provides indirect but ongoing and specific determinations of disease activity. These characteristics emphasize the value of surrogate biomarkers for non-invasive and detailed monitoring to demonstrate the efficacy of orphan drugs in clinical trials. The emergence of novel laboratory methods has facilitated the search for biomarkers in lysosomal storage diseases (LSDs), by allowing the systematic identification of molecules whose expression is altered as a result of the primary storage pathology. In Gaucher disease, for example, a chemokine, CCL18, has been identified as a biomarker for clinical development that reflects disease severity and response to treatment. CONCLUSION: New methods for the identification of novel biomarkers have the potential to provide mechanistic insights into the molecular pathogenesis of LSDs, including Fabry disease and Gaucher disease.

Substrate reduction therapy for lysosomal storage diseases.

UNLABELLED: The treatment of disordered lipoprotein metabolism with the statin class of drugs is one of the most striking successes in the field of applied medical science: here the use of selective inhibitors of the first committed step of cholesterol biosynthesis, in a complex and highly regulated pathway, leads to improved outcome from a common lipid storage disease that is a blight on whole populations--atherosclerosis. By the same token, substrate reduction is an emerging therapeutic strategy for the arcane field of the lysosomal storage diseases (LSDs). Reduced biosynthesis of glucosylceramide is postulated to allow correction of the imbalance between formation and breakdown of glycosphingolipids; the therapeutic effect of substrate reduction depends upon the presence of residual hydrolytic activity towards those accumulated glycosphingolipid substrates derived from glucosylceramide. First pioneered in the laboratory by Norman Radin, this approach has now been introduced into the clinic: based on the ability to inhibit uridine diphosphate glucosylceramide transferase, the semi-selective iminosugar, N-butyldeoxynojirimycin, is licensed for the treatment of type 1 Gaucher disease. CONCLUSION: Inhibition of substrate formation has wide application in the treatment of LSDs. Decreased glucosylceramide biosynthesis has therapeutic potential in glycosphingolipidoses other than Gaucher disease, and offers promise in several neurodegenerative storage disorders that are currently beyond the reach of other procedures. The results of ongoing clinical trials of miglustat in type 3 Gaucher disease, Niemann-Pick disease type C and GM2 gangliosidosis are eagerly awaited.

Clinical evaluation of biomarkers in Gaucher disease.

UNLABELLED: Novel or candidate biomarkers require thorough evaluation to establish their utility in a clinical setting. This paper describes an evaluation of several established enzyme markers of Gaucher disease and a newly-described chemokine, pulmonary and activation-regulated chemokine (PARC). The ability of the biomarkers to rank patients with Gaucher disease in order of disease severity and organ bulk, and to reflect changes in key clinical parameters in response to enzyme replacement therapy were evaluated. PARC concentrations were found to be reliably correlated with visceral disease and with key clinical responses to enzyme replacement in an unbiased manner. Unlike chitotriosidase and serum angiotensin-converting enzyme activity, genetic variation in serum PARC did not appear to influence its utility as a biomarker. CONCLUSION: For each new candidate biomarker of lysosomal storage diseases, a similar clinical evaluation will be required, though the approach will need to be modified according to the clinical features and natural history of each disorder.