[Clinical Characteristics of CD4-CD56+ Blastic Plasmacytoid Dendritic Cell Neoplasm].

OBJECTIVE: To explore the clinical manifestations, pathological features, immunophenotype, as well as diagnosis, treatment and prognosis of patients with CD4-CD56+ blastic plasmacytoid dendritic cell neoplasm (BPDCN), in order to further understand the rare disease. METHODS: The clinical data, laboratory examinations and treatment regimens of two patients with CD4-CD56+ BPDCN in the First Affiliated Hospital of Wannan Medical College were retrospectively analyzed. RESULTS: The two patients were both elderly males with tumor involved in skin, bone marrow, lymph nodes, etc. Immunohistochemical results of skin lesions showed that both CD56 and CD123 were positive, while CD4, CD34, TdT, CD3, CD20, MPO and EBER were negative. Flow cytometry of bone marrow demonstrated that CD56, CD123, and CD304 were all positive, while specific immune markers of myeloid and lymphoid were negative. Two patients were initially very sensitive to acute lymphoblastic leukemia or lymphomatoid chemotherapy regimens, but prone to rapid relapse. The overall survival of both patients was 36 months and 4 months, respectively. CONCLUSION: CD4-CD56+ BPDCN is very rare and easily misdiagnosed as other hematological tumors with poor prognosis. Acute lymphoblastic leukemia or lymphomatoid therapy should be used first to improve the poor prognosis.

[Clinical Characteristics of Patients with Myeloid Sarcoma].

OBJECTIVE: To investigate the clinical characteristics, diagnosis and treatment methods of patients with myeloid sarcoma(MS)． Methods: The clinical data, laboratory examination, clinical pathology and treatment methods of 15 patients with MS treated in the First Affiliated Hospital of Wannan Medical College from June 2012 to January 2020 were retrospectively analyzed. RESULTS: Among the 15 cases of MS, including eight males and seven females, the middle age of patients were 53（19 to 72）. Among the 15 patients with MS, 4 showed solitary MS, while 11 showed secondary MS. Immunohistochemical results showed that MPO+（12/15）、CD68+（3/6）、Lys+（3/3）、CD34+（6/14）、TdT+（0/9）、CD43+（13/13）、CD117+（6/10）、CD15+（7/10）、CD3+（1/15）、CD20+（0/15）. 6 of 13 patients were survival till follow-up date．The median overall survival (OS) time was 16 months (1-88 months)．Conclusion: Myeloid sarcoma is rare and often secondary from acute myeloid leukemia(AML) and chronic myeogenous leukemia(CML). Isolated MS can easily be misdiagnosed as lymphoma. Treatment response should be evaluated in combination with bone marrow examination, PET/CT and other imagines．Systematic chemotherapy and hematopoietic stem cell transplantation are the main method to treat MS.

[Imatinib as Second-Line Treatment Drug for CML Patients Intolerant to Nilotinib].

OBJECTIVE: To explore the individualized treatment for patient with chronic phase chronic myeloid leukemia(CML-CP). METHODS: The clinical data and treatment process of one CML-CP patient which intolerated to nilotinib were analyzed. RESULTS: Nilotinib was given to the patient once the diagnosis of CML-CP was set. Although major molecular remission (MMR) and complete cytogenetic remission (CCyR) were obtained during treatment for 3 months, a grade 3-4 hepatotoxicity appeared in the course of treatment.With drug reduction and symptomatic treatment, nilotinib was discontinued after 3 withdrawals and replaced with imatinib in January 11, 2015. The patients achieved MMR and CCyR at 7 months after imatinib replacement. At present, the patient tolerated well without any adverse events. CONCLUSION: Imatinib can be used as a second-line treatment drug for CML patients who was intolerant to nilotinib, and with less adverts, good effect and so on.

LAL (Lysosomal Acid Lipase) Promotes Reverse Cholesterol Transport In Vitro and In Vivo.

OBJECTIVE: To explore the role of LAL (lysosomal acid lipase) in macrophage cholesterol efflux and whole-body reverse cholesterol transport. APPROACH AND RESULTS: Immortalized peritoneal macrophages from lal-/- mice showed reduced expression of ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1), reduced production of the regulatory oxysterol 27-hydroxycholesterol, and impaired suppression of cholesterol synthesis on exposure to acetylated low-density lipoprotein when compared with lal+/+ macrophages. LAL-deficient mice also showed reduced hepatic ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8) expression compared with lal+/+ mice. LAL-deficient macrophages loaded with [3H]-cholesteryl oleate-labeled acetylated low-density lipoprotein showed impaired efflux of released [3H]-cholesterol to apoA-I (apolipoprotein A-I), with normalization of [3H]-cholesteryl ester levels and partial correction of ABCA1 expression and cholesterol efflux to apoA-I when treated with exogenous rhLAL (recombinant human LAL protein). LAL-deficient mice injected intraperitoneally with lal-/- macrophages cholesterol loaded and labeled in the same way exhibited only 1.55±0.35% total injected [3H]-cholesterol counts appearing in the feces for 48 h (n=30), compared with 5.38±0.92% in lal+/+ mice injected with labeled lal+/+ macrophages (n=27), P<0.001. To mimic the therapeutic condition of delivery of supplemental LAL in vivo, injection of labeled lal-/- macrophages into lal+/+ mice resulted in a significant increase in reverse cholesterol transport (2.60±0.46% of 3H-cholesterol counts in feces at 48 hours [n=19]; P<0.001 when compared with injection into lal-/- mice). CONCLUSIONS: These results indicate a critical role for LAL in promoting both macrophage and whole-body reverse cholesterol transport and the ability of supplemental LAL to be taken up and correct reverse cholesterol transport in vivo.

Neuronopathic Gaucher disease: dysregulated mRNAs and miRNAs in brain pathogenesis and effects of pharmacologic chaperone treatment in a mouse model.

Defective lysosomal acid ß-glucosidase (GCase) in Gaucher disease causes accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) that distress cellular functions. To study novel pathological mechanisms in neuronopathic Gaucher disease (nGD), a mouse model (4L;C*), an analogue to subacute human nGD, was investigated for global profiles of differentially expressed brain mRNAs (DEGs) and miRNAs (DEmiRs). 4L;C* mice displayed accumulation of GC and GS, activated microglial cells, reduced number of neurons and aberrant mitochondrial function in the brain followed by deterioration in motor function. DEGs and DEmiRs were characterized from sequencing of mRNA and miRNA from cerebral cortex, brain stem, midbrain and cerebellum of 4L;C* mice. Gene ontology enrichment and pathway analysis showed preferential mitochondrial dysfunction in midbrain and uniform inflammatory response and identified novel pathways, axonal guidance signaling, synaptic transmission, eIF2 and mammalian target of rapamycin (mTOR) signaling potentially involved in nGD. Similar analyses were performed with mice treated with isofagomine (IFG), a pharmacologic chaperone for GCase. IFG treatment did not alter the GS and GC accumulation significantly but attenuated the progression of the disease and altered numerous DEmiRs and target DEGs to their respective normal levels in inflammation, mitochondrial function and axonal guidance pathways, suggesting its regulation on miRNA and the associated mRNA that underlie the neurodegeneration in nGD. These analyses demonstrate that the neurodegenerative phenotype in 4L;C* mice was associated with dysregulation of brain mRNAs and miRNAs in axonal guidance, synaptic plasticity, mitochondria function, eIF2 and mTOR signaling and inflammation and provides new insights for the nGD pathological mechanism.

Assessment and regression analysis on instant catapult steam explosion pretreatment of corn stover.

Instant catapult steam explosion (ICSE) offers enormous physical force on lignocellulosic biomass due to its extremely short depressure duration. In this article, the response surface methodology was applied to optimize the effect of working parameters including pressure, maintaining time and mass loading on the crystallinity index and glucose yield of the pretreated corn stover. It was found that the pressure was of essential importance, which determined the physical force that led to the morphological changes without significant chemical reactions, and on the other hand the maintaining time mainly contributed to the thermo-chemical reactions. Furthermore, the pretreated biomass was assessed by scanning electron microscope, X-ray diffraction and Fourier transform infrared spectra to understand mechanisms underlying the ICSE pretreatment.

Reversal of advanced disease in lysosomal acid lipase deficient mice: a model for lysosomal acid lipase deficiency disease.

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TG) and cholesteryl esters (CE) in lysosomes. Mutations of the LIPA gene lead to Wolman disease (WD) and cholesterol ester storage disease (CESD). The disease hallmarks include hepatosplenomegaly and extensive storage of CE and/or TG. The effects of intravenous investigational enzyme therapy (ET) on survival and efficacy were evaluated in Lipa knock out, lal-/- mice with advanced disease using recombinant human LAL (rhLAL). Comparative ET was conducted with lower doses (weekly, 0.8 and 3.2mg/kg) beginning at 16 weeks (study 1), and with higher dose (10mg/kg) in early (8-weeks), middle (16-weeks) and late (24-weeks) disease stages (study 2). In study 1, rhLAL extended the life span of lal-/- mice in a dose dependent manner by 52 (0.8 mg/kg) or 94 (3.2mg/kg) days. This was accompanied by partial correction of cholesterol and TG levels in spleen and liver. In study 2, the high dose resulted in a significant improvement in organ size (liver, spleen and small intestine) and tissue histology as well as significant decreases in cholesterol and TG in all three groups. In the treated livers and spleens the cholesterol and TG levels were reduced to below treatment initiation levels indicating a reversal of disease manifestations, even in advanced disease. ET diminished liver fibrosis and macrophage proliferation. These results show that LAL deficiency can be improved biochemically and histopathologically by various dosages of ET, even in advanced disease.

Multiple pathogenic proteins implicated in neuronopathic Gaucher disease mice.

Gaucher disease, a prevalent lysosomal storage disease (LSD), is caused by insufficient activity of acid ß-glucosidase (GCase) and the resultant glucosylceramide (GC)/glucosylsphingosine (GS) accumulation in visceral organs (Type 1) and the central nervous system (Types 2 and 3). Recent clinical and genetic studies implicate a pathogenic link between Gaucher and neurodegenerative diseases. The aggregation and inclusion bodies of α-synuclein with ubiquitin are present in the brains of Gaucher disease patients and mouse models. Indirect evidence of ß-amyloid pathology promoting α-synuclein fibrillation supports these pathogenic proteins as a common feature in neurodegenerative diseases. Here, multiple proteins are implicated in the pathogenesis of chronic neuronopathic Gaucher disease (nGD). Immunohistochemical and biochemical analyses showed significant amounts of ß-amyloid and amyloid precursor protein (APP) aggregates in the cortex, hippocampus, stratum and substantia nigra of the nGD mice. APP aggregates were in neuronal cells and colocalized with α-synuclein signals. A majority of APP co-localized with the mitochondrial markers TOM40 and Cox IV; a small portion co-localized with the autophagy proteins, P62/LC3, and the lysosomal marker, LAMP1. In cultured wild-type brain cortical neural cells, the GCase-irreversible inhibitor, conduritol B epoxide (CBE), reproduced the APP/α-synuclein aggregation and the accumulation of GC/GS. Ultrastructural studies showed numerous larger-sized and electron-dense mitochondria in nGD cerebral cortical neural cells. Significant reductions of mitochondrial adenosine triphosphate production and oxygen consumption (28-40%) were detected in nGD brains and in CBE-treated neural cells. These studies implicate defective GCase function and GC/GS accumulation as risk factors for mitochondrial dysfunction and the multi-proteinopathies (α-synuclein-, APP- and Aß-aggregates) in nGD.

Gaucher disease: chemotactic factors and immunological cell invasion in a mouse model.

Gaucher disease results from mutations in GBA1 that cause functional disruption of the encoded lysosomal enzyme, acid ß-glucosidase. The consequent excess accumulation of glucosylceramide and glucosylsphingosine in lysosomes is central to the disease pathogenesis with classical involvement of macrophage (Mфs) lineage cells of visceral organs, bone, or brain. Several studies have implicated the increased secretion of chemokines and infiltration of a variety of immunological cells into tissues of Gaucher disease patients. Trafficking of immunological cells to the sites of inflammation requires the presence of chemokines. Although increases of different immunological cells and several chemokines are present in Gaucher disease, the specific chemoattractants that cause the increased influx of immunological cells are not fully defined. Here, increased levels of I-309, MCP-5, CXCL-2, CXCL-9, CXCL-10, CXCL-11, CXCL-13, and their corresponding leukocytes, i.e., MOs (monocytes), Mфs, dendritic cells (DCs), polymorphonuclear neutrophils (PMNs), and T, and B cells were identified in the circulation of mice with Gba1 mutations (D409V/null). Sera from D409V/null mice contained chemoattractants for a variety of immunological cells as shown by ex vivo chemotaxis studies and by flow cytometry. Enhanced chemotaxis towards 9V/null sera was found for 9V/null lung-, spleen-, liver-, and bone marrow-derived Mфs (CD11b(+) F480(+)), PMNs (Gr1(high) CD11b(+)), DCs (CD11c(+) CD11b(+)), T lymphocytes (CD3(+) TCRB(+)), and B lymphocytes (B220(+) CD19(+)). These data support these chemotactic factors as causative to increased tissue infiltration of leukocytes in Gaucher disease.

Ubiquitous transgene expression of the glucosylceramide-synthesizing enzyme accelerates glucosylceramide accumulation and storage cells in a Gaucher disease mouse model.

Gaucher disease is a lysosomal storage disease caused by defective activity of acid ß-glucosidase (GCase), which leads to the accumulation of its major substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in many cells. To modulate cellular substrate concentration in viable mouse models of Gaucher disease (Gba1 mutants), a novel mouse model was created with enhanced glycosphingolipid biosynthesis. This was accomplished by cross-breeding Gba1 mutant mice with mice expressing a transgene (GCStg) containing the mouse glucosylceramide synthase (GCS, Ugcg) cDNA driven by the ROSA promoter, yielding GCStg/Gba1 mice. The GCStg rescued Ugcg null mice from embryonic lethality. GCStg/Gba1 mice showed 2-3 fold increases in tissue GCS activity as well as accelerated GlcCer accumulation and the appearance of lipid-laden CD68 positive macrophages in visceral organs. Although GlcCer/GlcSph concentrations were elevated in the brain, there was no neurodegenerative phenotype up to 1 yr of age conceivably due to the greater residual GCase hydrolytic activity in the brains than in the visceral tissues of 9V/null mice. These studies provide 'proof of principle' for threshold substrate flux that modifies phenotypic development in Gaucher disease and other lysosomal storage diseases.

Gaucher disease: transcriptome analyses using microarray or mRNA sequencing in a Gba1 mutant mouse model treated with velaglucerase alfa or imiglucerase.

Gaucher disease type 1, an inherited lysosomal storage disorder, is caused by mutations in GBA1 leading to defective glucocerebrosidase (GCase) function and consequent excess accumulation of glucosylceramide/glucosylsphingosine in visceral organs. Enzyme replacement therapy (ERT) with the biosimilars, imiglucerase (imig) or velaglucerase alfa (vela) improves/reverses the visceral disease. Comparative transcriptomic effects (microarray and mRNA-Seq) of no ERT and ERT (imig or vela) were done with liver, lung, and spleen from mice having Gba1 mutant alleles, termed D409V/null. Disease-related molecular effects, dynamic ranges, and sensitivities were compared between mRNA-Seq and microarrays and their respective analytic tools, i.e. Mixed Model ANOVA (microarray), and DESeq and edgeR (mRNA-Seq). While similar gene expression patterns were observed with both platforms, mRNA-Seq identified more differentially expressed genes (DEGs) (∼3-fold) than the microarrays. Among the three analytic tools, DESeq identified the maximum number of DEGs for all tissues and treatments. DESeq and edgeR comparisons revealed differences in DEGs identified. In 9V/null liver, spleen and lung, post-therapy transcriptomes approximated WT, were partially reverted, and had little change, respectively, and were concordant with the corresponding histological and biochemical findings. DEG overlaps were only 8-20% between mRNA-Seq and microarray, but the biological pathways were similar. Cell growth and proliferation, cell cycle, heme metabolism, and mitochondrial dysfunction were most altered with the Gaucher disease process. Imig and vela differentially affected specific disease pathways. Differential molecular responses were observed in direct transcriptome comparisons from imig- and vela-treated tissues. These results provide cross-validation for the mRNA-Seq and microarray platforms, and show differences between the molecular effects of two highly structurally similar ERT biopharmaceuticals.

Fine-tuning of xylose metabolism in genetically engineered Saccharomyces cerevisiae by scattered integration of xylose assimilation genes.

Manipulation of multiple genes is a common experience in metabolic engineering and synthetic biology studies. Chromosome integration of multiple genes in one single position is always performed, however, there is so far no study on the integration of multiple genes separately in various positions (here in after referred to as "scattered integration") and its effect on fine-tuning of cellular metabolism. In this study, scattered integration of the xylose assimilation genes PsXR, PsXDH and ScXK was investigated in Saccharomyces cerevisiae, and transcription analysis of these genes as well as their enzyme activities were compared with those observed when the genes were integrated into one single site (defined as "tandem integration" here). Not only notable differences in transcription levels and enzyme activities were observed when the genes were integrated by the two strategies, but also change of the cofactor preference of PsXR gene was validated. Xylose fermentation was further studied with the strains developed with these strategies, and elevated xylose utilization rate was obtained in the scattered integration strain. These results proved that by positioning multiple genes on different chromosomes, fine-tuning of cellular metabolism could be achieved in recombinant S. cerevisiae.

Substrate compositional variation with tissue/region and Gba1 mutations in mouse models--implications for Gaucher disease.

Gaucher disease results from GBA1 mutations that lead to defective acid ß-glucosidase (GCase) mediated cleavage of glucosylceramide (GC) and glucosylsphingosine as well as heterogeneous manifestations in the viscera and CNS. The mutation, tissue, and age-dependent accumulations of different GC species were characterized in mice with Gba1 missense mutations alone or in combination with isolated saposin C deficiency (C*). Gba1 heteroallelism for D409V and null alleles (9V/null) led to GC excesses primarily in the visceral tissues with preferential accumulations of lung GC24∶0, but not in liver, spleen, or brain. Age-dependent increases of different GC species were observed. The combined saposin C deficiency (C*) with V394L homozygosity (4L;C*) showed major GC18:0 degradation defects in the brain, whereas the analogous mice with D409H homozygosity and C* (9H;C*) led to all GC species accumulating in visceral tissues. Glucosylsphingosine was poorly degraded in brain by V394L and D409H GCases and in visceral tissues by D409V GCase. The neonatal lethal N370S/N370S genotype had insignificant substrate accumulations in any tissue. These results demonstrate age, organ, and mutation-specific quantitative differences in GC species and glucosylsphingosine accumulations that can have influence in the tissue/regional expression of Gaucher disease phenotypes.

Ex vivo and in vivo effects of isofagomine on acid ß-glucosidase variants and substrate levels in Gaucher disease.

Isofagomine (IFG) is an acid ß-glucosidase (GCase) active site inhibitor that acts as a pharmacological chaperone. The effect of IFG on GCase function was investigated in GCase mutant fibroblasts and mouse models. IFG inhibits GCase with K(i) ∼30 nM for wild-type and mutant enzymes (N370S and V394L). Fibroblasts treated with IFG at µM concentrations showed enhancement of WT and mutant GCase activities and protein levels. Administration of IFG (30 mg/kg/day) to the mice homozygous for GCase mutations (V394L, D409H, or D409V) led to increased GCase activity in visceral tissues and brain extracts. IFG effects on GCase stability and substrate levels were evaluated in a mouse model (hG/4L/PS-NA) that has doxycycline-controlled human WT GCase (hGCase) expression driven by a liver-specific promoter and is also homozygous for the IFG-responsive V394L GCase. Both human and mouse GCase activity and protein levels were increased in IFG-treated mice. The liver-secreted hGCase in serum was stabilized, and its effect on the lung and spleen involvement was enhanced by IFG treatment. In 8-week IFG-treated mice, the accumulated glucosylceramide and glucosylsphingosine were reduced by 75 and 33%, respectively. Decreases of storage cells were correlated with >50% reductions in substrate levels. These results indicate that IFG stabilizes GCase in tissues and serum and can reduce visceral substrates in vivo.

Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies.

Parkinson's disease (PD), an adult neurodegenerative disorder, has been clinically linked to the lysosomal storage disorder Gaucher disease (GD), but the mechanistic connection is not known. Here, we show that functional loss of GD-linked glucocerebrosidase (GCase) in primary cultures or human iPS neurons compromises lysosomal protein degradation, causes accumulation of α-synuclein (α-syn), and results in neurotoxicity through aggregation-dependent mechanisms. Glucosylceramide (GlcCer), the GCase substrate, directly influenced amyloid formation of purified α-syn by stabilizing soluble oligomeric intermediates. We further demonstrate that α-syn inhibits the lysosomal activity of normal GCase in neurons and idiopathic PD brain, suggesting that GCase depletion contributes to the pathogenesis of sporadic synucleinopathies. These findings suggest that the bidirectional effect of α-syn and GCase forms a positive feedback loop that may lead to a self-propagating disease. Therefore, improved targeting of GCase to lysosomes may represent a specific therapeutic approach for PD and other synucleinopathies.

Acid ß-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing.

OBJECTIVE: Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α-synuclein (SNCA). A loss in lysosomal acid-ß-glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk. METHODS: We analyzed the effects of wild-type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols. RESULTS: We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293-SNCA cells increased GCase activity, as expected (ie, to 167% in MES-SNCA, 128% in PC12-SNCA, and 233% in HEK293-SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293-SNCA cells high GCase activity was associated with SNCA reduction by ≤32% (p = 0.009). Inhibition of cellular GCase activity (to 8-20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA-induced SNCA accumulation could be pharmacologically reversed in D409V-expressing PC12-SNCA cells by rapamycin, an autophagy-inducer (≤40%; 10µM; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, ≥20%), we recorded an age-dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p = 0.019). In young Gaucher disease mice (V394Lgba+/+//prosaposin[ps]-null//ps-transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, ≤10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week-old cathepsin D-deficient mice. INTERPRETATION: Our results demonstrate that GBA mutants promote SNCA accumulation in a dose- and time-dependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders.

Global gene expression profile progression in Gaucher disease mouse models.

BACKGROUND: Gaucher disease is caused by defective glucocerebrosidase activity and the consequent accumulation of glucosylceramide. The pathogenic pathways resulting from lipid laden macrophages (Gaucher cells) in visceral organs and their abnormal functions are obscure. RESULTS: To elucidate this pathogenic pathway, developmental global gene expression analyses were conducted in distinct Gba1 point-mutated mice (V394L/V394L and D409 V/null). About 0.9 to 3% of genes had altered expression patterns (≥ ± 1.8 fold change), representing several categories, but particularly macrophage activation and immune response genes. Time course analyses (12 to 28 wk) of INFγ-regulated pro-inflammatory (13) and IL-4-regulated anti-inflammatory (11) cytokine/mediator networks showed tissue differential profiles in the lung and liver of the Gba1 mutant mice, implying that the lipid-storage macrophages were not functionally inert. The time course alterations of the INFγ and IL-4 pathways were similar, but varied in degree in these tissues and with the Gba1 mutation. CONCLUSIONS: Biochemical and pathological analyses demonstrated direct relationships between the degree of tissue glucosylceramides and the gene expression profile alterations. These analyses implicate IFNγ-regulated pro-inflammatory and IL-4-regulated anti-inflammatory networks in differential disease progression with implications for understanding the Gaucher disease course and pathophysiology.

Comparative therapeutic effects of velaglucerase alfa and imiglucerase in a Gaucher disease mouse model.

Gaucher disease type 1 is caused by the defective activity of the lysosomal enzyme, acid beta-glucosidase (GCase). Regular infusions of purified recombinant GCase are the standard of care for reversing hematologic, hepatic, splenic, and bony manifestations. Here, similar in vitro enzymatic properties, and in vivo pharmacokinetics and pharmacodynamics (PK/PD) and therapeutic efficacy of GCase were found with two human GCases, recombinant GCase (CHO cell, imiglucerase, Imig) and gene-activated GCase (human fibrosarcoma cells, velaglucerase alfa, Vela), in a Gaucher mouse, D409V/null. About 80+% of either enzyme localized to the liver interstitial cells and <5% was recovered in spleens and lungs after bolus i.v. injections. Glucosylceramide (GC) levels and storage cell numbers were reduced in a dose (5, 15 or 60 U/kg/wk) dependent manner in livers (60-95%) and in spleens ( approximately 10-30%). Compared to Vela, Imig (60 U/kg/wk) had lesser effects at reducing hepatic GC (p = 0.0199) by 4 wks; this difference disappeared by 8 wks when nearly WT levels were achieved by Imig. Anti-GCase IgG was detected in GCase treated mice at 60 U/kg/wk, and IgE mediated acute hypersensitivity and death occurred after several injections of 60 U/kg/wk (21% with Vela and 34% with Imig). The responses of GC levels and storage cell numbers in Vela- and Imig-treated Gaucher mice at various doses provide a backdrop for clinical applications and decisions.

Multi-system disorders of glycosphingolipid and ganglioside metabolism.

Glycosphingolipids (GSLs) and gangliosides are a group of bioactive glycolipids that include cerebrosides, globosides, and gangliosides. These lipids play major roles in signal transduction, cell adhesion, modulating growth factor/hormone receptor, antigen recognition, and protein trafficking. Specific genetic defects in lysosomal hydrolases disrupt normal GSL and ganglioside metabolism leading to their excess accumulation in cellular compartments, particularly in the lysosome, i.e., lysosomal storage diseases (LSDs). The storage diseases of GSLs and gangliosides affect all organ systems, but the central nervous system (CNS) is primarily involved in many. Current treatments can attenuate the visceral disease, but the management of CNS involvement remains an unmet medical need. Early interventions that alter the CNS disease have shown promise in delaying neurologic involvement in several CNS LSDs. Consequently, effective treatment for such devastating inherited diseases requires an understanding of the early developmental and pathological mechanisms of GSL and ganglioside flux (synthesis and degradation) that underlie the CNS diseases. These are the focus of this review.

Neuronopathic Gaucher disease in the mouse: viable combined selective saposin C deficiency and mutant glucocerebrosidase (V394L) mice with glucosylsphingosine and glucosylceramide accumulation and progressive neurological deficits.

Gaucher disease is caused by defective acid beta-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C-/-) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities approximately 30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred approximately 48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C-/- alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease.