Extracellular lysosome-associated membrane protein-1 (LAMP-1) mediates autoimmune disease progression in the NOD model of type 1 diabetes

Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4(+)CD25(+)) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-gamma-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-gamma-driven responses.     

Age-dependent increase in lysosome-associated membrane protein 1 and early-onset behavioral deficits in APPSL transgenic mouse model of Alzheimer's disease

Amyloid precursor protein (APP) is strongly related to the onset of Alzheimer's disease. It possesses cleavage sites for β- and γ-secretases, and the resulting cleaved products (amyloid-β peptides) are capable of causing neurotoxicity. Such cleavage is promoted by the Swedish and London mutations (APPSwe/Lon) inside the APP gene. Here, we characterized APPSL transgenic mice (APPSL-Tg) to determine the effects of this mutation. We observed that both the amount of insoluble amyloid-β and the ratio of amyloid-β 42/40 increased promptly in the brain during 6–16 months of age. Amyloid-β plaques were observed in whole brain sections at 12 months. In contrast, the spatial memory assessed by the Morris water maze task was already impaired at 3 months, which suggested that the APPSL-Tg mice may represent an early-onset model of familial Alzheimer's disease. Furthermore, the levels of LAMP-1, a marker protein of lysosome, increased in the brain at 28 months. Such LAMP-1 protein was detected around the amyloid-β plaques at the hippocampal regions of the APPSL-Tg mice. Our results suggested that the increase in LAMP-1 was enhanced by the accumulation of amyloid-β occurring during aging. Our findings coincided with the pathological hallmarks of Alzheimer's disease.     

Intracellular transport of acid β-glucosidase and lysosome-associated membrane proteins is affected in Gaucher's disease (G202R mutation)

Gaucher's disease (GD) is caused by an inherited deficiency of acid β-glucosidase with storage of glucosylceramides in the lysosomes of macrophages. This study identifies a G202R mutation in the acid β-glucosidase gene in an infant with severe neuronopathic (type 2) GD and only slightly reduced acid β-glucosidase activity. Western blot analysis, pulse chase experiments, and the thin frozen section immunogold method were used to analyse the implications of this mutation on the pathogenesis, clinical heterogeneity and diagnostic evaluation of GD. The results show that acid β-glucosidase persists in the patient's fibroblasts as a mannose-rich polypeptide in the endoplasmic reticulum and is not transported to the lysosomes. By contrast, high expression of the lysosome-associated membrane proteins LAMP-1 and LAMP-2, saposin C, and cathepsin D was observed in the patient's lysosomes. Immunogold labelling of the integral membrane proteins LAMP-1 and LAMP-2 increases significantly at the cell surface of Kupffer cells and fibroblasts as well as at the apical membrane of hepatocytes. In addition, LAMP-1 and LAMP-2 associate with the bilayer of stored glucosylceramide. It is concluded that defective intracellular transport of mutant acid β-glucosidase from the endoplasmic reticulum to lysosomes leads to a more severe clinical phenotype than the residual enzyme activity may indicate. Furthermore, the detection of LAMP in the tubular bundles of undigested glucosylceramides, as well as their increased concentration at the surfaces of the affected cells, suggests that these proteins play a role in the storage or removal of substrate in GD. Intracellular targeting of acid β-glucosidase and LAMP contributes to the broad phenotypic heterogeneity of GD. Copyright © 1999 John Wiley & Sons, Ltd.     

Effect of anti-macrophage inflammatory protein-1alpha on leukocyte trafficking and disease progression in experimental autoimmune uveoretinitis

This study has enabled us to identify the influence of the chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), on leukocyte behavior at the blood-retina barrier in vivo and its link with the inflammatory process and disease pathogenesis. MIP-1alpha has not previously been thought to be effective under conditions of physiological shear flow. However, short-term anti-MIP-1alpha treatment inhibited leukocyte slowing and accumulation and subsequent extravasation of leukocytes at the blood-retina barrier in animals with experimental autoimmune uveoretinitis. This was effective predominantly in the post-capillary venules which have been shown to be the main site of passage of leukocytes across the blood-retina barrier. Long-term anti-MIP-1alpha treatment also prevented decreased leukocyte velocity and reduced disease severity as measured clinically, histologically and in terms of blood-retina barrier breakdown.     

Immune dysregulation and autoreactivity correlate with disease severity in SARS-CoV-2-associated multisystem inflammatory syndrome in children

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Toxicity of human recombinant interleukin-2 in rats. Pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement

To characterize the subacute toxicologic and pathologic effects of human recombinant interleukin-2 (rIL-2) in rats, rIL-2 was administered to rats at doses ranging from 3.0 to 150 X 10(6) units/kg/day for 14 to 16 days, in once or twice daily dosing regimens by intravenous or intraperitoneal dosing routes. The hematologic, clinical chemical, gross pathologic and histopathologic findings were determined. Rats given rIL-2 had dose- and regimen-related incidences of hemolytic anemia, lymphocytosis, neutrophilia, eosinophilia, thrombocytopenia, increased hepatic transaminases, hyperbilirubinemia, hypoalbuminemia, ascites and pleural effusions, and prerenal azotemia. Marked lymphoid infiltration of the liver and eye were associated with hepatocyte necrosis and retinal damage. Eosinophilic infiltration of the adrenal medulla was associated with medullary cell necrosis. Other histopathologic changes included infiltration of multiple tissues by eosinophils and/or lymphocytes, lymphoid hyperplasia and splenic extramedullary erythropoiesis and eosinopoiesis. Hematologic, clinical chemistry, and histopathologic changes were of markedly greater severity in rats receiving rIL-2 twice daily as compared with those receiving the same total dose of rIL-2 once daily. Mortality in severely affected rats was secondary to severe anemia and/or hepatic damage. The results of these studies demonstrate that the major pathologic changes associated with rIL-2 administration in rats are characterized by marked tissue infiltration with lymphocytes and eosinophils. Many of the adverse effects of rIL-2 administration to rats are similar to those reported in humans receiving rIL-2 immunotherapy and the results confirm that the rat is an appropriate species in which to study selected aspects of rIL-2 toxicity.     

CXC-chemokines KC and macrophage inflammatory protein-2 (MIP-2) synergistically induce leukocyte recruitment to the central nervous system in rats

Intracisternal injection of the CXC-chemokines KC or macrophage inflammatory protein (MIP)-2 induced a pleocytosis in the cerebrospinal fluid (CSF) of rats in a dose dependent way. MIP-2 was much more potent than KC. The concurrent injection of both chemokines revealed a profound synergistic effect on leukocyte recruitment into CSF.     

bcl-2 proto-oncogene and Epstein–Barr virus latent membrane protein-1 expression in AIDS-related lymphoma

The expression of bcl-2 protein and Epstein–Barr virus (EBV) latent membrane protein 1 (LMP-1) was investigated in 18 cases of lymphoma occurring in the acquired immunodeficiency syndrome (AIDS). EBV small RNAs were detectable in tumour cells in all cases by in situ hybridization with EBER oligonucleotides. LMP-1 expression was detected in 61% of the cases, and 55% were positive for bcl-2. Dual expression of LMP-1 and bcl-2 was found in 8/18 (44%) cases, while five cases (28%) expressed either LMP-1 or bcl-2 and five expressed neither. Thus, there was an inconsistent relationship between the presence of EBV and the expression of bcl-2. One LMP-1 negative case was found to express bcl-2 in reactive lymphocytes but not in lymphoma cells. No clinical features were found to correlate statistically with LMP-1 or bcl-2 expression in the tumour cells. However, CD4 counts at diagnosis were significantly lower in bcl-2 positive cases ( P < 0.05). The respective roles of EBV LMP-1 and the expression of bcl-2 in lymphogenesis in AIDS patients remains complex and is not yet fully understood.     

Abnormal erythrocyte and lymphocyte chemiluminescence in chronic granulomatous disease: evidence for a generalized membrane defect

Concanavalin A (soluble)- and/or opsonized zymosan (particulate)-stimulated luminol-dependent chemiluminescence was demonstrated in normal human lymphocytes, erythrocytes, and in the membrane (ghost) fractions of polymorphonuclear neutrophils and erythrocytes. By contrast, seven patients with chronic granulomatous disease (five boys with the X-linked type and two female variants) had diminished luminescent responses to con A and/or opsonized zymosan in their lymphocytes, erythrocytes, erythrocyte membranes, as well as neutrophil membranes, suggesting generalization of the deficiency in the neutrophil oxidase system to other cells.     

Bone regeneration by recombinant human bone morphogenetic protein-2 and a novel biodegradable carrier in a rabbit ulnar defect model

The effects of recombinant human bone morphogenetic protein (rhBMP)-2 and a novel carrier, PLGA-coated gelatin sponge (PGS), on bone defect repair was examined. A 1.5 cm unilateral segmental bone defect was created in the ulnar diaphysis of a Japanese white rabbit. In an initial study, defects were either treated with PGS impregnated with various concentrations of rhBMP-2 (0, 0.1, 0.4 and 1 mg/cm(3)) or left untreated. Defect healing was assessed by radiographic union rate, and biomechanical properties of regenerated bones were determined at 16 weeks postoperatively. In a second study, defects were implanted with PGS with or without rhBMP-2, and histologically observed at postoperative weeks 8 and 16. Radiographic union rate increased the dose-dependently at an early time point. All defects treated with rhBMP-2 (0.4 and 1 mg/cm(3)) were radiographically repaired. Mechanical properties of regenerated bones were restored in a dose-dependent manner. Neither ulnae left untreated nor implanted PGS alone showed radiographic union. Longitudinal alignment of lamellar structure was observed histologically at 16 weeks, indicating that remodeling of regenerated bone was complete. Implanted PGS was almost completely resorbed by 8 weeks, and no abnormalities were observed in the surrounding soft tissue. These results suggest that PGS is a promising carrier for rhBMP-2.     

Pulmonary involvement in type 1 Gaucher disease: functional and exercise findings in patients with and without clinical interstitial lung disease

Pulmonary disease is a well-known complication of Type 1 Gaucher disease (GD), although its incidence is not well established and its severity varies. The purpose of this study was to determine the frequency and extent of pulmonary involvement in patients with GD. Pulmonary involvement was assessed by history, physical examination and chest radiograph in 150 consecutive patients with Type 1 GD presenting at a specialized center for genetic diseases. Five patients were noted to have clinical evidence of pulmonary involvement. Full pulmonary function tests were performed in these five patients and in an additional 13 patients randomly selected from the remaining 145. Many of the 18 patients also underwent radionuclide body imaging with 67 Gallium citrate and 111Indium-tagged leucocyte scans, as well as incremental cardiorespiratory exercise tests. Lung biopsies were available in two patients with lung disease, and a second examination of lung tissue was performed in one of these two patients post-mortem. Clinical lung disease was detected in five patients. All five had dyspnea, diffuse infiltrates, restrictive impairment and low single breath CO diffusing capacity (DLCOSB). Two of these patients underwent exercise testing and showed abnormalities consistent with lung disease (ventilatory limitation, excessive ventilation and increased dead space) as well as decreased VO2 max. and anaerobic threshold (AT). In contrast, in the other 13 patients, physical examination, chest radiographs and pulmonary function were normal (except for a low DLCOSB in one patient). Responses on exercise testing (performed in six of the 13 patients) were consistent with a circulatory impairment (decreased VO2 max. and AT). Our study found that <5% of patients with Type 1 GD have clinical interstitial lung disease. In addition, we found that some patients, without evident lung involvement, may experience limitations in physical exertion and are easily fatigued; this is attributable to impaired circulation.     

Expression and distribution of amyloid precursor protein-like protein-2 in Alzheimer's disease and in normal brain.

Amyloid precursor-like protein-2 (APLP-2) belongs to a family of homologous amyloid precursor-like proteins. In the present study we report on the expression and distribution of APLP-2 in fetal and adult human brain and in brains of patients with Alzheimer's disease. We demonstrate that APLP-2 mRNAs encoding isoforms predicted to undergo post-translational modification by chondroitin sulfate glycosaminoglycans are elevated in fetal and aging brains relative to the brains of young adults. Immunocytochemical labeling with APLP-2-specific antibodies demonstrates APLP-2 immunoreactivity in cytoplasmic compartments in neurons and astrocytes, in large part overlapping the distribution of the amyloid precursor protein. In Alzheimer's disease brain, APLP-2 antibodies also label a subset of neuritic plaques. APLP-2 immunoreactivity is particularly conspicuous in large dystrophic neurites that also label with antibodies specific for APP and chromogranin A. In view of the age-dependent increase in levels of chondroitin sulfate glycosaminoglycan-modified forms of APLP-2 in aging brain and the accumulation of APLP-2 in dystrophic presynaptic elements, we suggest that APLP-2 may play roles in neuronal sprouting or in the aggregation, deposition, and/or persistence of beta-amyloid deposits.     

Histopathology,ultrastructure, and clinical phenotypes in thin glomerular basement membrane disease variants

Recent genetic studies indicate that Alport syndrome and thin glomerular basement membrane disease (TMD) may both be due to COL4A3, COL4A4, and COL4A5 mutations, but there is continuing uncertainty concerning the diagnosis and management of patients without classic family history and symptoms. We examined kidney pathology and collagen alpha 3 to alpha 5(IV) expression in a series of 16 patients who presented with overlapping signs between TMD and Alport nephritis. All patients presented with hematuria, and 11 also had proteinuria, of whom 5 had nephrotic range proteinuria. Only 9 had family history of hematuria. In 9 of 16 (60%) we found premature glomerulosclerosis in the renal biopsies. Three of 16 had predominantly wide, lamellated glomerullar basement membranes (GBM), and in these, alpha 3 to alpha 5(IV) was absent in glomeruli or skin, diagnostic of Alport nephritis. One patient (12) had a very wide GBM with intramembranous lucencies but no lamellation. Skin biopsy was collagen alpha 5(IV) positive. Nine of 16 patients had predominantly thin GBM by electron microscopy, and 3 had thin and slightly lamellated GBM. Collagen alpha 3 to alpha 5(IV) expression in the kidney or skin biopsy was present in all of the latter 12 patients. Three patients had end-stage renal disease, 7 patients had hypertension, and 1 patient had chronic renal failure. We found that of the 16 patients with presumed TMD, 3 had X-linked Alport nephritis, 2 appeared to have autosomal recessive Alport nephritis, and the remaining patients had either an Alport or a TMD variant. The latter had histologic and/or clinical evidence of progressive renal disease, including premature glomerulosclerosis, hypertension, sustained proteinuria, and either thin or slight GBM lamellation focally, and preserved alpha 3 to alpha 5(IV) expression. These patients have a TMD variant, but an Alport variant with a potentially transmissible severe defect different from benign hematuria cannot be excluded.     

Antibody responses to Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) and expression of LMP1 in juvenile Hodgkin's disease

Clinical isolates of human herpesvirus 7 (HHV-7) from the saliva of healthy individual were investigated for genetic variations in the regions of two immediate-early (IE) genes, the glycoprotein B (gB) and glycoprotein H (gH) genes, and in R2-repeat. The genomic DNA of 24 isolates from citizens of Thailand, Japan, and the United States was amplified to detect size variations in the IE-1 and IE-2 loci, but none was observed, suggesting that there was no deletion or insertion in these genes, in contrast with an IE gene of human herpesvirus 6 (HHV-6). The sequences of the gB gene from isolates acquired from 5 Japanese and 8 Thai subject were then compared with those of American strains JI and RK with respect to codons that are known to differentiate gB alleles. All the isolates were found to have gB allele C except for the JI strain, which has allele F. Variability was also observed in five specific gH codons, resulting in 6 different groups. The HHV-7 isolates might be classified into two major genetic variants by combining their gB and gH allelic groupings. In the present study, only JI belonged to variant 1, while the rest of the isolates appeared to belong to variant 2. In the R2-repeat region, size heterogeneities were observed among the 24 isolates, due to different repeat numbers (17, 15, 14, 13, or 12 repeats). Therefore, we used the R2-repeat to identify the origins of isolates in a study of HHV-7 transmission, and found HHV-7 to be transmitted within a family from both mothers and fathers to their children.     

Universal screening for Clostridioides difficile in a tertiary hospital: risk factors for carriage and clinical disease

ObjectivesThe role of asymptomatic carriers in Clostridioides difficile infection (CDI) epidemiology is not fully understood. Our aim was to evaluate CD carriage prevalence on admission, associated risk factors, and the risk of developing CDI.MethodsA 10-week surveillance program for CD carriage of all medical patients admitted to the Sheba Medical Centre was implemented, utilizing an admission rectal swab PCR. Healthcare facility-onset CDI (HO-CDI) was recorded and divided into HO-CDI diagnosed in CD carriers and non-carriers.ResultsA total of 4601 admissions were recorded in 3803 patients; 2368 patients had technically analysable rectal swabs, of whom 81 (3.4%) were CD carriers. A multivariate logistic regression model showed that previous hospitalization, old age (>85 years) and low Norton scores were significant independent predictors of CD carriage. Carriers were more likely to receive antimicrobial therapy during hospitalization than non-carriers were. The incidence of HO-CDI in non-carriers was 4.6 cases per 10 000 patient-days; the incidence of HO-CDI in carriers was 76.7 cases per 10 000 patient-days (RR 16.6, 95% CI 4.0–69.1, p .002).ConclusionsIn a prospective study, the rate of CD carriage on admission in medical patients was 3.4%. CD carriers were older, frailer, and more likely to have been hospitalized recently. HO-CDI incidence was significantly higher among CD carriers than among non-carriers, with at least a third of CDI in screened patients developing in carriers. Targeted screening of high-risk groups for CD carriage should be further considered.     

Mild cognitive impairment as predictor for Alzheimer's disease in clinical practice: effect of age and diagnostic criteria

BACKGROUND: We investigated whether the predictive accuracy of mild cognitive impairment (MCI) for Alzheimer-type dementia (AD) in a clinical setting is dependent on age and the definition of MCI used. METHOD: Non-demented subjects older than 40 (n=320) who attended a memory clinic of a university hospital were reassessed 5 years later for the presence of AD. MCI was diagnosed according to the criteria of amnestic MCI, mild functional impairment (MFI), ageing-associated cognitive decline (AACD), and age-associated memory impairment (AAMI). The main outcome measure was the area under the curve (AUC) of a receiver operating characteristic (ROC) curve. Analyses were conducted on the entire sample and on subgroups of subjects aged 40-54, 55-69 and 70-85 years. RESULTS: A diagnosis of AD at follow-up was made in 58 subjects. Four of them were in the 40-54 age group, 29 in the 55-69 age group and 25 in the 70-85 age group. The diagnostic accuracy in the entire sample was low to moderately high with AUCs ranging from 0.56 (AACD) to 0.75 (amnestic MCI). A good predictive accuracy with an AUC >0.80 was only observed in subjects aged 70-85 using the criteria of amnestic MCI (AUC=0.84). CONCLUSIONS: The predictive accuracy of MCI for AD is dependent on age and the definition of MCI used. The predictive accuracy is good only for amnestic MCI in subjects 70-85 years. As subjects with prodromal AD are often younger than 70, the usefulness of MCI as predictor of AD in clinical practice is limited.     

Detection of Epstein–Barr virus nucleic acid sequences and protein in nodal T-cell lymphomas: relation between latent membrane protein-1 positivity and clinical course

Forty-six nodal T-cell lymphomas, classified according to the updated Kiel classification, were investigated for the presence of Epstein–Barr virus (EBV) DNA by polymerase chain reaction (PCR), EBER 1 and 2 (EBER 1/2) and latent membrane protein-1 (LMP-1) expression. A combination of RNA in situ hybridization and immunohistochemistry was used to establish the phenotype of the Epstein–Barr virus harbouring cells. In 21 of 45 cases Epstein–Barr virus DNA sequences could be detected with the polymerase chain reaction. In 15 cases (14 of 21 EBV PCR positive cases), EBER 1/2 positive cells could be demonstrated. As judged by morphology, EBER 1/2 expression was found in non neoplastic and neoplastic lymphoid cells. Double staining revealed that more than 80% of the EBER 1/2 harbouring cells, lacked B-, T- or histiocytic markers, suggesting down regulation of T- and B-cell markers by Epstein–Barr virus. In eight of 15 cases some EBER 1/2 positive T-cells (CD3, CD45RO, CD43) morphologically resembling tumour cells were found. In nine of 14 cases tested EBER 1/2 positive non-neoplastic B-cells (CD20) were seen. Based on in situ hybridization results, four patterns of EBER 1/2 positive cells were found, i.e. single cells (≤1 per medium power field (mpf), n = 3), scattered (1–25/mpf, n = 4), clustered (26–100/mpf, n = 5) and diffuse (≥100/mpf, n = 3). In eight of 15 cases a clustered or diffuse pattern of EBER 1/2 positive cells was found and these lymphomas were therefore considered to be strongly associated with Epstein–Barr virus. In these lymphomas LMP-1 expression was found to be associated with an aggressive clinical course and hepatosplenomegaly.     

博卡病毒VP_2基因在昆虫细胞sf9中表达及临床标本筛查

目的 表达人博卡病毒(HBoV)VP_2蛋白,用于临床标本HBoV的筛查.方法 将VP_2基因重组于杆状病毒基因组,重组的杆状病毒感染昆虫细胞sf9,用HA抗体进行Western Blot鉴定重组融合VP_2蛋白表达.通过电镜观察重组蛋白颗粒.以VP_2蛋白作为抗原对176例临床样本进行免疫印迹筛查.结果 在昆虫细胞中VP_2蛋白的表达量约占细胞总蛋白的60%以上,VP_2蛋白相对分子质量为60×10~3.电镜观察博卡病毒VP_2蛋白形成病毒样颗粒(VLP),其大小在20mm左右;采用免疫印迹技术从临床标本中筛查出4例患者HBoV阳性,阳性率达到2.28%(4/176).结论 本研究成功的在昆虫细胞中表达了博卡病毒VP_2蛋白;表达VP_2蛋白具有一定抗原性,为开展人博卡病血清学研究方法建立提供基础.