Lysosomes Of Macrophages Research Articles

Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis.

Sepsis, a condition caused by severe infections, affects more than 30 million people worldwide every year and remains the leading cause of death in hospitals1,2. Moreover, antimicrobial resistance has become an additional challenge in the treatment of sepsis3, and thus, alternative therapeutic approaches are urgently needed2,3. Here, we show that adoptive transfer of macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs) can be applied for the treatment of multidrug-resistant bacteria-induced sepsis in mice with immunosuppression. The MACs are constructed by transfection of vitamin C lipid nanoparticles that deliver antimicrobial peptide and cathepsin B (AMP-CatB) mRNA. The vitamin C lipid nanoparticles allow the specific accumulation of AMP-CatB in macrophage lysosomes, which is the key location for bactericidal activities. Our results demonstrate that adoptive MAC transfer leads to the elimination of multidrug-resistant bacteria, including Staphylococcus aureus and Escherichia coli, leading to the complete recovery of immunocompromised septic mice. Our work provides an alternative strategy for overcoming multidrug-resistant bacteria-induced sepsis and opens up possibilities for the development of nanoparticle-enabled cell therapy for infectious diseases.

Dissecting the localization of lipopolysaccharide-responsive and beige-like anchor protein (LRBA) in the endomembrane system

Introduction and objectivesLRBA deficiency is caused by loss of LRBA protein expression, due to either homozygous or compounds heterozygous mutations in LRBA. LRBA deficiency has been shown to affect vesicular trafficking and autophagy. To date, LRBA has been observed in the cytosol, Golgi apparatus and some lysosomes in LPS-stimulated murine macrophages. The objectives of the present study were to study the LRBA localization in organelles involved in vesicular traffic, phagocytosis, and autophagy in mononuclear phagocytes (MP). Materials and methodsWe analyzed LRBA colocalization with different endosomes markets using confocal microscopy in MP. We used the autophagy inhibitors to determine the role of LRBA in formation, maturation or degradation of the autophagosome. ResultsLRBA intracellular trafficking depends on the activity of the GTPase ADP ribosylation factor-1 (ARF) in MP. LRBA was identified in early, late endosomes but did not colocalize strongly with lysosomal markers. Although LRBA appears not to be recruited during the phagocytic cargo uptake, it greatly colocalized with the microtubule-associated protein 1A/1B-light chain 3 (LC3) under a steady state and this decreased after the induction of autophagy flux. Although the use of inhibitors of lysosome fusion did not restore the LRBA/LC3 colocalization, inhibitors of either early to late endosomes trafficking or PI3K pathway did. ConclusionsTaken together, our results show that LRBA is located in endomembrane system vesicles, mainly in the early and late endosomes. Although LRBA appears not to be involved in the phagocytic uptake, it is recruited in the early steps of the autophagy flux.

Sphingosine kinase-2 prevents macrophage cholesterol accumulation and atherosclerosis by stimulating autophagic lipid degradation

Atherosclerosis is the major cause of ischemic coronary heart diseases and characterized by the infiltration of cholesterol-accumulating macrophages in the vascular wall. Although sphingolipids are implicated in atherosclerosis as both membrane components and lipid mediators, the precise role of sphingolipids in atherosclerosis remains elusive. Here, we found that genetic deficiency of sphingosine kinase-2 (SphK2) but not SphK1 aggravates the formation of atherosclerotic lesions in mice with ApoE deficiency. Bone marrow chimaera experiments show the involvement of SphK2 expressed in bone marrow-derived cells. In macrophages, deficiency of SphK2, a major SphK isoform in this cell type, results in increases in cellular sphingosine and ceramides. SphK2-deficient macrophages have increases in lipid droplet-containing autophagosomes and autolysosomes and defective lysosomal degradation of lipid droplets via autophagy with an impaired luminal acidic environment and proteolytic activity in the lysosomes. Transgenic overexpression of SphK1 in SphK2-deficient mice rescued aggravation of atherosclerosis and abnormalities of autophagosomes and lysosomes in macrophages with reductions of sphingosine, suggesting at least partial overlapping actions of two SphKs. Taken together, these results indicate that SphK2 is required for autophagosome- and lysosome-mediated catabolism of intracellular lipid droplets to impede the development of atherosclerosis; therefore, SphK2 may be a novel target for treating atherosclerosis.

Sickle Cell Hemoglobin Impairs Lysosomal Processing in Macrophages Leading to Inflammasome Activation and Inflammation

BACKGROUND : Sickle cell anemia (SCA) is a chronic hemolytic disease that is characterized by chronic presence of low-level plasma hemoglobin (Hb, 3-10 µM). Plasma Hb can reduce NO availability, induce endothelium damage and activate coagulation cascade and inflammation leading to development of vasculopathies. The Hb scavenging proteins, haptoglobin and hemopexin, are depleted in SCA resulting in increased levels of circulating Hb. Hb is recognized by macrophages/monocytes scavenger receptors and acquired by endocytosis resulting in degradation of Hb in lysosomes. Single nucleotide mutation in the β- globin gene (HbS) promotes its polymerization at low pH which is characteristic for lysosomes. HYPOTHESIS: We hypothesize that HbS forms polymers inside macrophage lysosome impairing its processing by lysosomal enzymes and causing lysosome injury and inflammation. METHODS: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior the sample collection. Whole blood samples were obtained from five SCA patients and five normal control subjects. Human THP-1 promonocytic cells were differentiated into macrophages with PMA and treated with either purified HbS and HbA (Sigma-Aldrich) or whole red blood cell lysate for 3 hrs. After washing, the cells were further cultured for 24-72 hrs. Conditioned media was collected at different time points and cytokine levels were measured by BioPlex Kit (BioRad). Trypan blue assays were used to measure cell proliferation. Lysosome dye (LysoTraker red DND99, Molecular Probe), plasma membrane dye (CellMask deep red plasma membrain stain, Thermo-Fisher Scientific), DAPI (Sigma-Aldrich) and phalloidin-FITC (Sigma-Aldrich) were used for visualization of cell structures. LPS treatment (1µg/mL) was used for generation of M1 macrophages; IL-4 treatment (5µg/mL) was used for generation of M2 macrophages. RESULTS: Treatment of THP1- derived macrophages with lysates of red blood cells obtained from SCA patients induced accumulation of lysosomes, induced apoptosis and stimulated cell proliferation. No significant accumulation of lysosomes, apoptosis and proliferation were observed after treatment of macrophages with lysates of red blood cells obtained from control subjects with normal hemoglobin. Treatment of macrophages with purified HbS but not HbA also induced lysosome accumulation and macrophages proliferation. No activation of inflammation was observed at 24 hrs after treatment with either HbS or HbA proteins. HbS treatment induced a delayed, 72 hrs post treatment, activation of inflammasome evidenced by IL-18 and IL-1β production and secretion of pro-inflammatory cytokines (IL-2, IL-6 and TNF-α). HbS treatment also induced secretion of M-CSF and GM-CSF growth factors. CONCLUSION: Lysosomal processing of HbS was delayed in human macrophages, leading to lysosome accumulation, inflammasome activation, macrophage proliferation and inflammation. This observation can explain long term chronic inflammation observed in patients with SCA. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Disclosures No relevant conflicts of interest to declare.

TFEB activation in macrophages attenuates postmyocardial infarction ventricular dysfunction independently of ATG5-mediated autophagy.

Lysosomes are at the epicenter of cellular processes critical for inflammasome activation in macrophages. Inflammasome activation and IL-1β secretion are implicated in myocardial infarction (MI) and resultant heart failure; however, little is known about how macrophage lysosomes regulate these processes. In mice subjected to cardiac ischemia/reperfusion (IR) injury and humans with ischemic cardiomyopathy, we observed evidence of lysosomal impairment in macrophages. Inducible macrophage-specific overexpression of transcription factor EB (TFEB), a master regulator of lysosome biogenesis (Mϕ-TFEB), attenuated postinfarction remodeling, decreased abundance of proinflammatory macrophages, and reduced levels of myocardial IL-1β compared with controls. Surprisingly, neither inflammasome suppression nor Mϕ-TFEB-mediated attenuation of postinfarction myocardial dysfunction required intact ATG5-dependent macroautophagy (hereafter termed "autophagy"). RNA-seq of flow-sorted macrophages postinfarction revealed that Mϕ-TFEB upregulated key targets involved in lysosomal lipid metabolism. Specifically, inhibition of the TFEB target, lysosomal acid lipase, in vivo abrogated the beneficial effect of Mϕ-TFEB on postinfarction ventricular function. Thus, TFEB reprograms macrophage lysosomal lipid metabolism to attenuate remodeling after IR, suggesting an alternative paradigm whereby lysosome function affects inflammation.

Ratiometric sensing lysosomal pH in inflammatory macrophages by a BODIPY-rhodamine dyad with restrained FRET

Inflammation, as the pathophysiological response of body to harmful stimuli, leads to changes in cellular microenvironment. To research pH changes in lysosomes of macrophages during inflammation, we designed a FRET (Fӧrster resonance energy transfer) based probe, BDP-RhB. The probe showed good lysosome targeting ability, wide response range of pH from 8.0 to 4.0 with significant ratio (I582/I518) change from 0.6 to 3.4, and good reversibility and sustainability. By applying BDP-RhB, we found a decrease of lysosomal pH of macrophages during inflammation.

2546 Rare Case of Duodenal Melanosis in a 72-Year-Old Male

INTRODUCTION: Pseudomelanosis (PM) is a rare condition of unknown etiology. Prevalence of this condition is not well documented however, there have been several case reports. It is described as speckled black pigmentation of intestinal mucosa. Underlying pathogenesis and significance of PM is not well understood. Reports have associated it with medications, including anti-hypertensives and iron supplements and systemic diseases such as chronic renal disease, iron deficiency and diabetes mellitus. It is most commonly discovered as an incidental finding during endoscopy and frequently seen in the duodenum, however, few cases have also reported it in the stomach and jejunum. CASE DESCRIPTION/METHODS: A 72-year-old male with multiple co-morbidities including diabetes, hypertension, peripheral arterial disease and chronic kidney disease presented with epigastric pain, nausea and hematemesis. There was no melena or hematochezia. Last colonoscopy 10-years ago was significant for diverticulosis and hemorrhoids. His home medications included aspirin, clopidogrel, amlodipine, hydralazine, hydrochlorothiazide, atorvastatin, levothyroxine, furosemide and baclofen. On evaluation, digital rectal exam was significant for dark stools and hemoglobin decrease from 11.4 to 9.2 over 8 months. Upper endoscopy showed normal appearing esophagus and gastric mucosa. Dark pigmentation of the duodenal mucosa was noted and biopsied. Pathology findings were consistent with intestinal pseudomelanosis. Patient's symptoms resolved without any additional intervention. He was discharged on pantoprazole and continued on aspirin, but his clopidogrel was discontinued. DISCUSSION: Prognosis of PM is not well understood, but it is considered a benign condition. Unlike melanosis coli, which is a similar finding of black/brown speckles in the colon that has been associated with colon cancer. Histochemical studies have shown the pigments found in PM are likely secondary to accumulation of iron sulfide, ferrous sulfate or lipomenin within the lysosomes of macrophages in lamina propria of small intestine. Current theories suggest PM develops secondary to impaired iron transport coupled with sulfur from anti-hypertensive medications. In our patient, his medical conditions and anti-hypertensive medications likely contributed to this benign finding. Although, considered a benign condition, it is imperative to identify this condition during endoscopy to exclude other serious conditions.

Hemozoin produced by mammals confers heme tolerance.

Free heme is cytotoxic as exemplified by hemolytic diseases and genetic deficiencies in heme recycling and detoxifying pathways. Thus, intracellular accumulation of heme has not been observed in mammalian cells to date. Here we show that mice deficient for the heme transporter SLC48A1 (also known as HRG1) accumulate over ten-fold excess heme in reticuloendothelial macrophage lysosomes that are 10 to 100 times larger than normal. Macrophages tolerate these high concentrations of heme by crystallizing them into hemozoin, which heretofore has only been found in blood-feeding organisms. SLC48A1 deficiency results in impaired erythroid maturation and an inability to systemically respond to iron deficiency. Complete heme tolerance requires a fully-operational heme degradation pathway as haplo insufficiency of HMOX1 combined with SLC48A1 inactivation causes perinatal lethality demonstrating synthetic lethal interactions between heme transport and degradation. Our studies establish the formation of hemozoin by mammals as a previously unsuspected heme tolerance pathway.

Rab20 is critical for bacterial lipoprotein tolerization-enhanced bactericidal activity in macrophages during bacterial infection.

Bacterial cell wall component-induced tolerance represents an important protective mechanism during microbial infection. Tolerance induced by the TLR2 agonist bacterial lipoprotein (BLP) has been shown to attenuate the inflammatory response, and simultaneously to augment antimicrobial function, thereby conferring its protection against microbial sepsis. However, the underlying mechanism by which BLP tolerance augments bactericidal activity has not been fully elucidated. Here, we reported that the induction of BLP tolerance in murine macrophages upregulated the expression of Rab20, a membrane trafficking regulator, at both the mRNA and protein levels upon bacterial infection. The knockdown of Rab20 with Rab20 specific siRNA (siRab20) did not affect the phagocytosis of Escherichia coli (E. coli), but substantially impaired the intracellular killing of the ingested E. coli in BLP-tolerized macrophages. Furthermore, Rab20 was associated with GFP-E. coli containing phagosomes, and BLP tolerization resulted in the enhanced maturation of GFP-E. coli-containing phagosomes associated with Rab20 and strong lysosomal acidification. The knockdown of Rab20 substantially diminished lysosome acidification and disturbed the fusion of GFP-E. coli containing phagosomes with lysosomes in BLP-tolerized macrophages. These results demonstrate that Rab20 plays a critical role in BLP tolerization-induced augmentation of bactericidal activity via promoting phagosome maturation and the fusion of bacteria containing phagosomes with lysosomes.

Localization of active endogenous and exogenous β-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts.

β‐Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor‐targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol‐derived activity‐based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre‐labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme.

Acoustic tweezing cytometry for mechanical phenotyping of macrophages and mechanopharmaceutical cytotripsy

Macrophages are immune cells responsible for tissue debridement and fighting infection. Clofazimine, an FDA-approved antibiotic, accumulates and precipitates as rod-shaped, crystal-like drug inclusions within macrophage lysosomes. Drug treatment as well as pathophysiological states could induce changes in macrophage mechanical property which in turn impact their phenotype and function. Here we report the use of acoustic tweezing cytometry as a new approach for in situ mechanical phenotyping of macrophages and for targeted macrophage cytotripsy. Acoustic tweezing cytometry applies ultrasound pulses to exert controlled forces to individual cells via integrin-bound microbubbles, enabling a creep test for measuring cellular mechanical property or inducing irreversible changes to the cells. Our results revealed that macrophages with crystal-like drug inclusions became significantly softer with higher cell compliance, and behaved more elastic with faster creep and recovery time constants. On the contrary, phagocytosis of solid polyethylene microbeads or treatment with soluble clofazimine rendered macrophages stiffer. Most notably, application of ultrasound pulses of longer duration and higher amplitude in ATC actuated the integrin-bound microbubbles to mobilize the crystal-like drug inclusions inside macrophages, turning the rod-shaped drug inclusions into intracellular microblender that effectively destructed the cells. This phenomenon of acoustic mechanopharmaceutical cytotripsy may be exploited for ultrasound activated, macrophage-directed drug release and delivery.

An Expandable Mechanopharmaceutical Device (1): Measuring the Cargo Capacity of Macrophages in a Living Organism.

Clofazimine (CFZ) is an FDA-approved, poorly soluble small molecule drug that precipitates as crystal-like drug inclusions (CLDIs) which accumulate in acidic cytoplasmic organelles of macrophages. In this study, we considered CLDIs as an expandable mechanopharmaceutical device, to study how macrophages respond to an increasingly massive load of endophagolysosomal cargo. First, we experimentally tested how the accumulation of CFZ in CLDIs impacted different immune cell subpopulations of different organs. Second, to further investigate the mechanism of CLDI formation, we asked whether specific accumulation of CFZ hydrochloride crystals in lysosomes could be explained as a passive, thermodynamic equilibrium phenomenon. A cellular pharmacokinetic model was constructed, simulating CFZ accumulation driven by pH-dependent ion trapping of the protonated drug in the acidic lysosomes, followed by the precipitation of CFZ hydrochloride salt via a common ion effect caused by high chloride concentrations. While lower loads of CFZ were mostly accommodated in lung macrophages, increased CFZ loading was accompanied by organ-specific changes in macrophage numbers, size and intracellular membrane architecture, maximizing the cargo storage capabilities. With increasing loads, the total cargo mass and concentrations of CFZ in different organs diverged, while that of individual macrophages converged. The simulation results support the notion that the proton and chloride ion concentrations of macrophage lysosomes are sufficient to drive the massive, cell type-selective accumulation and growth of CFZ hydrochloride biocrystals. CLDIs effectively function as an expandable mechanopharmaceutical device, revealing the coordinated response of the macrophage population to an increasingly massive, whole-organism endophagolysosomal cargo load.

NEW INSIGHTS INTO RARE METABOLIC DISEASES IN PAEDIATRIC PRACTICE

Rare diseases are those that affect at most one in 2000 persons worldwide. According to the date around 6000-7000 rare diseases have been described until now. Although there are so many differences between rare diseases patents that are affected are facing with the more or less same difficulties. Before a final diagnose they usually spend a few years losing a precious time for the treatment. The aim of this review article is to give insight into a several rare diseases that are successfully treated by enzyme replacement therapy (ERT). A complete search of the Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library, MEDLINE and Pub Med up to January 2018 was carried out. Fabry disease (FD) is a progressive, X-linked inherited disorder of glycosphingolipid metabolism due to deficient or absent lysosomal a-galactosidase A activity. Gaucher disease is characterized by a deficiency of acid β-glucosidase and accumulation of glucosylceramide within lysosomes of tissue macrophages. The accumulation of glucosylceramide is responsible for the manifestations of Gaucher disease, including hepatomegaly, splenomegaly, thrombocytopenia, and anemia. MPS I characterized the defect in the gene for α-L-iduronidase (IUDA), an enzyme that degrades glycosaminoglycans (GAGs) in lysosomes. Pompe disease is a rare, progressive, debilitating and often fatal neuromuscular disorder resulting from the deficiency of a lysosomal enzyme, acid alpha-glucosidase (GAA). Enzyme testing on Dried Blood Spots (DBS) is a 1st tier/screening test for all four above mentioned metabolic rare diseases. An enzyme replacement therapy is the only effective treatment patients with Fabry disease, Pompe disease, Gausher disease and MPS I.

Antioxidants inhibit low density lipoprotein oxidation less at lysosomal pH: A possible explanation as to why the clinical trials of antioxidants might have failed

Oxidised low density lipoprotein (LDL) was considered to be important in the pathogenesis of atherosclerosis, but the large clinical trials of antioxidants, including the first one using probucol (the PQRST Trial), failed to show benefit and have cast doubt on the importance of oxidised LDL. We have shown previously that LDL oxidation can be catalysed by iron in the lysosomes of macrophages. The aim of this study was therefore to investigate the effectiveness of antioxidants in preventing LDL oxidation at lysosomal pH and also establish the possible mechanism of oxidation. Probucol did not effectively inhibit the oxidation of LDL at lysosomal pH, as measured by conjugated dienes or oxidised cholesteryl esters or tryptophan residues in isolated LDL or by ceroid formation in the lysosomes of macrophage-like cells, in marked contrast to its highly effective inhibition of LDL oxidation at pH 7.4. LDL oxidation at lysosomal pH was inhibited very effectively for long periods by N,N'-diphenyl-1,4-phenylenediamine, which is more hydrophobic than probucol and has been shown by others to inhibit atherosclerosis in rabbits, and by cysteamine, which is a hydrophilic antioxidant that accumulates in lysosomes. Iron-induced LDL oxidation might be due to the formation of the superoxide radical, which protonates at lysosomal pH to form the much more reactive, hydrophobic hydroperoxyl radical, which can enter LDL and reach its core. Probucol resides mainly in the surface monolayer of LDL and would not effectively scavenge hydroperoxyl radicals in the core of LDL. This might explain why probucol failed to protect against atherosclerosis in various clinical trials. The oxidised LDL hypothesis of atherosclerosis now needs to be re-evaluated using different and more effective antioxidants that protect against the lysosomal oxidation of LDL.

Plasma chitotriosidase activity versus plasma glucosylsphingosine in wide spectrum of Gaucher disease phenotypes – A statistical insight

Deficiency of beta-glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Macrophages activated by accumulated GlcCer secrete chitotriosidase. Plasma chitotriosidase activity is significantly elevated in patients with active GD and has been suggested to indicate total body Gaucher cell load.There are two biomarkers used to assess the severity of GD - chitotriosidase has been measured for over 20 years, and deacylated GlcCer, known as glucosylsphingosine (GlcSph) is thought to be even more adequate, as it is almost a direct storage substrate.In this paper we focused entirely on statistical analysis, performing a thorough search of possible relations, dependencies and differences in the levels of these two biomarkers in a cohort of 64 Polish GD patients. We found that the treatment of GD with enzyme replacement therapy (ERT) changes the distribution of the disease biomarkers; their levels follow a normal distribution only in untreated patients.The variable “disease biomarker level” was found dependent of the binary variable “treated with ERT or not”. It was found independent of the following variables: “disease type”, “splenectomized or not”, and “heterozygous for 24-bp duplication for CHIT1 variant” or “CHIT1 wild type”.An almost perfect linear correlation (coefficient of determination R2 = 0.99) between the chitotriosidase activity and GlcSph level was revealed in splenectomized patients.

Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device

Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructing sophisticated molecular devices inside the cells of living organisms. Here, using experimental and computational approaches, we probed the relationship between the biological mechanisms mediating lysosomal ion homeostasis and the self-assembly of a weakly basic small molecule building block (clofazimine) into a functional, mechanopharmaceutical device (intracellular Crystal-Like Drug Inclusions – “CLDIs”) in macrophage lysosomes. Physicochemical considerations indicate that the intralysosomal stabilization of the self-assembled mechanopharmaceutical device depends on the pHmax of the weakly basic building block and its affinity for chloride, both of which are consistent with the pH and chloride content of a physiological lysosomal microenvironment. Most importantly, in vitro and in silico studies revealed that high expression levels of the vacuolar ATPase (V-ATPase), irrespective of the expression levels of chloride channels, are necessary and sufficient to explain the cell-type dependent formation, stabilization, and biocompatibility of the self-assembled mechanopharmaceutical device within macrophages.

WORLDSymposium™ 2018 Program.

Deficiency of beta-glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Macrophages activated by accumulated GlcCer secrete chitotriosidase. Plasma chitotriosidase activity is significantly elevated in patients with active GD and has been suggested to indicate total body Gaucher cell load.There are two biomarkers used to assess the severity of GD - chitotriosidase has been measured for over 20 years, and deacylated GlcCer, known as glucosylsphingosine (GlcSph) is thought to be even more adequate, as it is almost a direct storage substrate.In this paper we focused entirely on statistical analysis, performing a thorough search of possible relations, dependencies and differences in the levels of these two biomarkers in a cohort of 64 Polish GD patients. We found that the treatment of GD with enzyme replacement therapy (ERT) changes the distribution of the disease biomarkers; their levels follow a normal distribution only in untreated patients.The variable “disease biomarker level” was found dependent of the binary variable “treated with ERT or not”. It was found independent of the following variables: “disease type”, “splenectomized or not”, and “heterozygous for 24-bp duplication for CHIT1 variant” or “CHIT1 wild type”.An almost perfect linear correlation (coefficient of determination R2 = 0.99) between the chitotriosidase activity and GlcSph level was revealed in splenectomized patients.

Glucosylsphingosine Causes Hematological and Visceral Changes in Mice-Evidence for a Pathophysiological Role in Gaucher Disease.

Glucosylceramide and glucosylsphingosine are the two major storage products in Gaucher disease (GD), an inherited metabolic disorder caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The build-up of glucosylceramide in the endoplasmic reticulum and prominent accumulation in cell lysosomes of tissue macrophages results in decreased blood cell and platelet counts, and skeletal abnormalities. The pathological role of the deacylated form of glucosylceramide, glucosylsphingosine (lyso-Gb1), a recently identified sensitive and specific biomarker for GD, is not well investigated. We established a long-term infusion model in C57BL/6JRj mice to examine the effect of lyso-Gb1 on representative hallmark parameters of GD. Mice received lyso-Gb1 at a dosage of 10 mg·kg−1 per day as a continuous subcutaneous administration, and were routinely checked for blood lyso-Gb1 levels using liquid chromatography-multiple reaction monitoring mass spectrometry (LC/MRM-MS) measurements at four-weekly intervals throughout treatment. The C57BL/6JRj mice showed a stable increase of lyso-Gb1 up to->500-fold greater than the normal reflecting concentrations seen in moderately to severely affected patients. Furthermore, lyso-Gb1 accumulated in peripheral tissues. The mice developed hematological symptoms such as reduced hemoglobin and hematocrit, increased spleen weights and a slight inflammatory tissue response after eight weeks of treatment. The above findings indicate a measurable visceral and hematological response in treated mice that suggests a role for lyso-Gb1 in the development of peripheral signs of GD.

Gastric and Duodenal Pseudomelanosis in a Patient With Chronic Kidney Disease and Oral Iron Supplementation

A 74 year-old lady has a history of hypertension (HTN), diabetes mellitus (DM), mild chronic kidney disease, coronary artery disease, cerebrovascular accident, osteoarthritis, Iron deficiency anemia and gastroesophageal reflux disease. Her medications include ferrous sulphate and hydralazine. She had an upper GI endoscopy for evaluation of iron deficiency anemia and refractory GERD. Endoscopy showed dark, pigmented, granular appearing mucosa in gastric antrum, lesser curvature of gastric body and duodenal bulb (pseudomelanosis). Pathology from gastric biopsies showed reactive gastropathy and gastric siderosis with deposits of granular, darkly pigmented material showing stromal predominant deposition with presence in macrophages and epithelium. This material stained positive with Prussian blue stain and was negative for Melan-A stain. Pseudomelanosis of stomach and duodenum is associated with various clinical conditions including HTN, DM, chronic kidney disease, gastrointestinal hemorrhage and use of various medications including ferrous sulphate, furosemide, hydralazine, propranolol and hydrochlorothiazide. Differential diagnosis includes hemosiderosis/hemochromatosis, brown bowel syndrome and malignant melanoma, which could be differentiated by using special stains and immunohistochemical markers. Histologically, iron pigments are deposited in lysosomes of macrophages in lamina propria. These pigments are detected by Prussian blue Stain and Fontana-Masson stains of macrophages in the lamina propria. It had been seen mostly in older women similar to our patient. Most cases are asymptomatic, however dyspeptic symptoms have been described occasionally. Long term prognosis and consequences of this condition is unknown. No specific treatment is recommended.Figure: EGD revealed Pseudomelanosis in the stomach.Figure: This material stains positive in areas with a Prussian blue iron immunohistochemical stain and is negative for Melan-A stain.Figure: Deposits of granular, darkly pigmented material displays a nonspecific stromal and macrophage predominant pattern.

Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494.

Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment.