Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling

In stimulated neutrophils, the majority of tyrosine phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserved and solubilized tyrosine phosphorylated proteins but also retained their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and hypertonic buffers containing non-ionic detergents resulted in the solubilisation of a significant fraction of tyrosine phosphorylated proteins. Furthermore, we observed that in monosodium urate crystals-stimulated neutrophils, Lyn activity was enhanced in the soluble fraction recovered from the hypertonic fraction, but not from that of the first hypotonic lysis. The distribution of tyrosine phosphorylated proteins between the NP-40 soluble and insoluble fractions was both substrate- and agonist-dependent. In neutrophils stimulated with fMet-Leu-Phe, MSU crystals or by CD32 ligation, the tyrosine phosphorylated proteins were mostly insoluble. On the other hand, in GM-CSF-treated cells, the phosphoproteins were more equally distributed between the two fractions. The results of this study provide a new experimental procedure for the investigation of tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types.     

Autoimmune diseases in myelodysplastic syndrome favors patients survival: A case control study and literature review

Background

We conducted a monocentric retrospective study of patients with myelodysplastic syndromes (MDS) and autoimmune or inflammatory disorders (AIMs) and a literature review. We analyzed the association with subgroups of the WHO 2016 MDS classification and patient's survival in a case control study. Risk factors associated with survival were analyzed by uni- and multivariate analysis.

Molecular characterization of a 1p36 chromosomal duplication and in utero interference define ENO1 as a candidate gene for polymicrogyria

While chromosome 1p36 deletion syndrome is one of the most common terminal subtelomeric microdeletion syndrome, 1p36 microduplications are rare events. Polymicrogyria (PMG) is a brain malformation phenotype frequently present in patients with 1p36 monosomy. The gene whose haploinsufficiency could cause this phenotype remains to be identified. We used high-resolution arrayCGH in patients with various forms of PMG in order to identify chromosomal variants associated to the malformation and characterized the genes included in these regions in vitro and in vivo. We identified the smallest case of 1p36 duplication reported to date in a patient presenting intellectual disability, microcephaly, epilepsy, and perisylvian polymicrogyria. The duplicated segment is intrachromosomal, duplicated in mirror and contains two genes: enolase 1 (ENO1) and RERE, both disrupted by the rearrangement. Gene expression analysis performed using the patient cells revealed a reduced expression, mimicking haploinsufficiency. We performed in situ hybridization to describe the developmental expression profile of the two genes in mouse development. In addition, we used in utero electroporation of shRNAs to show that Eno1 inactivation in the rat causes a brain development defect. These experiments allowed us to define the ENO1 gene as the most likely candidate to contribute to the brain malformation phenotype of the studied patient and consequently a candidate to contribute to the malformations of the cerebral cortex observed in patients with 1p36 monosomy.Subject terms: Gene regulation, Genetics research     

Biopsy of breast microcalcifications using an 11-Gauge vacuum-assisted device: roles and challenges for the pathologist

Stereotactic 11-Gauge vacuum-assisted biopsy provides a valuable tool in the diagnosis of mammographically detected breast microcalcifications. However, this new diagnostic technology presents some limitations and requires a close collaboration between radiologists, pathologists and physicians. The aim of this work is to propose a practical approach in the management of large core biopsies and to summarize the different difficulties faced by the pathologist in the management and histological interpretation of specimens issuing from this device.     

Does disclosure of emotions facilitate recovery from bereavement? Evidence from two prospective studies

Two longitudinal studies assessed whether disclosure of emotions facilitates recovery from bereavement. Study 1 tested prospectively over a 2-year period whether the extent to which bereaved persons talked about their loss to others and disclosed their emotions was associated with better adjustment to the loss of a marital partner. There was no evidence that disclosure facilitated adjustment. Study 2 randomly assigned recently bereaved individuals either to the Pennebaker writing task (J. W. Pennebaker & S. K. Beall, 1986) or to no-essay control conditions. The writing task did not result in a reduction of distress or of doctors visits either immediately after the bereavement or at a 6-month follow-up. Beneficial effects were not demonstrated for bereaved persons who had suffered an unexpected loss or who at the time of the study still expressed a high need for emotional disclosure.     

A longitudinal and prospective study of Epstein-Barr virus load in AIDS-related non-Hodgkin lymphoma.

BACKGROUND: Epstein-Barr virus (EBV) may be causally associated with non-Hodgkin Lymphoma (NHL) in HIV-infected patients. OBJECTIVES: To compare EBV load in whole blood in AIDS-NHL patients, HIV non-AIDS patients and non-HIV-infected persons, and to prospectively measure EBV load in whole blood in AIDS-NHL patients. STUDY DESIGN: Longitudinal and prospective study. RESULTS: We observed no statistical difference in EBV load between AIDS-NHL (3.69log(10) copies/mL [interquartile range (IQR): 2.89-4.27]) and HIV non-AIDS patients (3.08log(10) copies/mL [IQR: 1.29-3.57]) but AIDS-NHL patients had significantly higher EBV loads than HIV-negative controls (1.19log(10) copies/mL [IQR: 0.00-3.29]). We noticed an inverse correlation between CD4+ lymphocytes count and EBV load in patients with AIDS-NHL (r(2)=0.41, P=0.01). In the longitudinal study, the mean EBV load three months after NHL diagnosis decreased significantly (mean difference=-1.69log(10) copies/mL [95% confidence interval: -0.32; -3.04]; P=0.03) under chemotherapy but was still elevated in patients with relapses or no response to chemotherapy. CONCLUSION: Although EBV load seems a suboptimal marker for the diagnosis of AIDS-NHL, we observed a significant decrease of EBV load in patients treated with chemotherapy and a strong association between NHL outcome and EBV load in whole blood.     

Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses

Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-alpha/beta and -lambda. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-alpha/beta and -lambda were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-beta and -lambda were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-alpha/beta and -lambda production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.     

Structural and functional analysis of a new desmin variant causing desmin‐related myopathy

Desmin‐related myopathy is a familial or sporadic disease characterized by skeletal muscle weakness and cardiomyopathy as well as the presence of intracytoplasmic aggregates of desmin‐reactive material in the muscle cells. Previously, two kinds of deletions and eight missense mutations have been identified in the desmin gene and proven to be responsible for the disorder. The present study was conducted to determine structural and functional defects in a pathogenic desmin variant that caused a disabling disorder in an isolated case presenting with distal and proximal limb muscle weakness and cardiomyopathy. We identified a novel heterozygous Q389P desmin mutation located at the C‐terminal part of the rod domain as the causative mutation in this case. Transfection of desmin cDNA containing the patient’s mutation into C2.7, MCF7, and SW13 cells demonstrated that the Q389P mutant is incapable of constructing a functional intermediate filament network and has a dominant negative effect on filament formation. We conclude that Q389P mutation is the molecular event leading to the development of desmin‐related myopathy. Hum Mutat 18:388–396, 2001. © 2001 Wiley‐Liss, Inc.