Ligation of erythrocyte CR1 induces its clustering in complex with scaffolding protein FAP-1

The primary identified function of complement receptor 1 (CR1/CD35) on primate erythrocytes is to bind complement-tagged inflammatory particles including microbes and immune complexes. When erythrocytes circulate through liver and spleen, sinusoidal phagocytes remove CR1-adherent particles and erythrocytes return to the circulation. This process of immune adherence clearance is important for host defense and prevention of autoimmunity. CR1 was previously described as clustered in the human erythrocyte membrane, which was thought to be necessary for binding complement-opsonized particles. In contrast, we demonstrate that on erythrocytes CR1 is not clustered, but dispersed, and able to bind complement-tagged particles. When fresh erythrocytes are solubilized by nonionic detergent, CR1 partitions to the cytoskeleton fraction. Using a PDZ-peptide array, CR1's cytoplasmic tail, which contains 2 PDZ-motifs, binds PDZ domains 2, 3, and 5 of Fas-associated phosphatase 1 (FAP-1), a scaffolding protein. We show that FAP-1, not previously recognized as an erythroid protein, is expressed on circulating erythrocytes. CR1 and FAP-1 coimmunoprecipitate, which confirms their molecular association. Disperse CR1 on erythrocytes may be advantageous for capturing immune-complexes, while ligation-induced CR1 clustering may prevent ingestion of the erythrocyte during the immune-complex transfer to the macrophages by keeping the opsonic stimulus localized thus preventing phagocyosis.     

Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants

BACKGROUND AND AIMS: Resistance is a major challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). We investigated the mechanisms of resistance in patients with progressive GISTs with primary KIT mutations and the efficacy of the kinase inhibitor PKC412 for the inhibition of imatinib-resistant mutants. METHODS: We performed a cytogenetic analysis and screened for mutations of the KIT and PDGFRA kinase domains in 26 resistant GISTs. KIT autophosphorylation status was assessed by Western immunoblotting. Imatinib-resistant GIST cells and Ba/F3 cells expressing these mutant proteins were tested for sensitivity to imatinib and PKC412. RESULTS: Six distinct secondary mutations in KIT were detected in 12 progressive tumors, with V654A and T670I found to be recurrent. One progressive tumor showed acquired PDGFRA -D842V mutation. Amplification of KIT or KIT / PDGFRA was found in 2 patients. Eight of 10 progressive tumors available for analysis showed phosphorylated KIT. Two remaining progressive tumors lost KIT protein expression. GIST cells carrying KIT -del557-558/T670I or KIT -InsAY502-503/V654A mutations were resistant to imatinib, while PKC412 significantly inhibited autophosporylation of these mutants. Resistance to imatinib and sensitivity to PKC412 of KIT -T670I and PDGFRA -D842V mutants was confirmed using Ba/F3 cells. CONCLUSIONS: This study shows the high frequency of KIT/PDGFRA kinase domain mutations in patients with secondary resistance and defines genomic amplification of KIT / PDGFRA as an alternative cause of resistance to the drug. In a subset of patients, cancer cells lost their dependence on the targeted tyrosine kinase. Our findings show the sensitivity of the imatinib-resistant KIT -T670I and KIT -V654A and of PDGFRA -D842V mutants to PKC412.     

When to suspect and how to diagnose pulmonary lymphangioleiomyomatosis

Abstract The objective of this study was to present clinical and radiological data of eight women with histologically proven lymphangioleiomyomatosis (LAM) diagnosed between 1984 and 1994, and to suggest a diagnostic strategy when LAM is suspected. A review of case reports, including results of biopsies, lung function and radiological procedures was undertaken. The mean age of the women at start of symptoms was 36 years, and the mean age at time of diagnosis 42 years. The most frequent presenting complaint was dyspnea, either in conjunction with pneumothorax (3), chylothorax (2) or on exertion (2). All patients had airflow limitation and markedly reduced gas transfer. Five patients had 16 episodes of pneumothorax. In seven patients multiple cysts were observed on the surface of the lung during thoracotomy while computerized tomography (CT) scans revealed numerous cysts evenly distributed throughout the lung parenchyma. The procedures that confirmed the diagnosis included transbronchial lung biopsy (4), open lung biopsy (2), thoracoscopy (1), thoracotomy (3) and autopsy (1). Three specimens had to be revised before the histological diagnosis was confirmed. It was concluded that the important clues to a diagnosis of LAM are recurrent episodes of pneumothoraces in fertile women, progressive air-flow limitation, markedly reduced gas transfer and characteristic findings on thoracic CT scans. A specific request to the pathologist to stain lung tissue specimens for smooth muscle cells is mandatory.     

Impaired survival of peripheral T cells, disrupted NK/NKT cell development, and liver failure in mice lacking Gimap5

The loss of Gimap5 (GTPase of the immune-associated protein 5) gene function is the underlying cause of lymphopenia and autoimmune diabetes in the BioBreeding (BB) rat. The in vivo function of murine gimap5 is largely unknown. We show that selective gene ablation of the mouse gimap5 gene impairs the final intrathymic maturation of CD8 and CD4 T cells and compromises the survival of postthymic CD4 and CD8 cells, replicating findings in the BB rat model. In addition, gimap5 deficiency imposes a block of natural killer (NK)- and NKT-cell differentiation. Development of NK/NKT cells is restored on transfer of gimap5(-/-) bone marrow into a wild-type environment. Mice lacking gimap5 have a median survival of 15 weeks, exhibit chronic hepatic hematopoiesis, and in later stages show pronounced hepatocyte apoptosis, leading to liver failure. This pathology persists in a Rag2-deficient background in the absence of mature B, T, or NK cells and cannot be adoptively transferred by transplanting gimap5(-/-) bone marrow into wild-type recipients. We conclude that mouse gimap5 is necessary for the survival of peripheral T cells, NK/NKT-cell development, and the maintenance of normal liver function. These functions involve cell-intrinsic as well as cell-extrinsic mechanisms.     

Investigation of susceptibility loci identified in the UK rheumatoid arthritis whole-genome scan in a further series of 217 UK affected sibling pairs

OBJECTIVE: A previous whole-genome scan (WGS) of 182 UK rheumatoid arthritis (RA) affected sibling pair (ASP) families suggested linkage to HLA and 11 other chromosome regions. Replication of such findings in an independent cohort can help to distinguish true linkages from false-positive linkages. Since RA is a heterogeneous disease, some loci may be linked only in subsets of patients. Thus, the aim of this study was to investigate in an additional set of RA ASP families linkage to regions showing deviation in expected allele-sharing ratios in the UK WGS and to perform subset analysis on the combined cohort. METHODS: Twenty loci were investigated for linkage in 217 Caucasian UK RA ASPs. Stratification analysis was performed on the combined cohort of 377 RA ASP families to account for sex, RA severity, and the shared epitope (SE). RESULTS: None of the regions of linkage identified in the initial WGS achieved statistical significance in the second cohort. In contrast, after stratification analysis, 14 regions showed nominal evidence of linkage (logarithm of odds score >0.8) in one or more subgroups. In particular, the strength of evidence for linkage to chromosome 16p was increased in subsets of ASPs with younger age at disease onset (LOD score 2.38) and for linkage to chromosome 6q in female-female ASPs (LOD score 2.31) and in ASPs in which both siblings had 2 copies of the SE (LOD score 3.03). CONCLUSION: These results support the evidence for heterogeneity of RA. This information will inform the future design of association-based investigations as the search for disease genes in the linked regions begins.     

Effect of age on in vivo rates of mitochondrial protein synthesis in human skeletal muscle

A progressive decline in muscle performance in the rapidly expanding aging population is causing a dramatic increase in disability and health care costs. A decrease in muscle endurance capacity due to mitochondrial decay likely contributes to this decline in muscle performance. We developed a novel stable isotope technique to measure in vivo rates of mitochondrial protein synthesis in human skeletal muscle using needle biopsy samples and applied this technique to elucidate a potential mechanism for the age-related decline in the mitochondrial content and function of skeletal muscle. The fractional rate of muscle mitochondrial protein synthesis in young humans (24 ± 1 year) was 0.081 ± 0.004%·h⁻¹, and this rate declined to 0.047 ± 0.005%·h⁻¹ by middle age (54 ± 1 year; P < 0.01). No further decline in the rate of mitochondrial protein synthesis (0.051 ± 0.004%·h⁻¹) occurred with advancing age (73 ± 2 years). The mitochondrial synthesis rate was about 95% higher than that of mixed protein in the young, whereas it was approximately 35% higher in the middle-aged and elderly subjects. In addition, decreasing activities of mitochondrial enzymes were observed in muscle homogenates (cytochrome c oxidase and citrate synthase) and in isolated mitochondria (citrate synthase) with increasing age, indicating declines in muscle oxidative capacity and mitochondrial function, respectively. The decrease in the rates of mitochondrial protein synthesis is likely to be responsible for this decline in muscle oxidative capacity and mitochondrial function. These changes in muscle mitochondrial protein metabolism may contribute to the age-related decline in aerobic capacity and muscle performance.     

A model of lytic, latent, and reactivating varicella-zoster virus infections in isolated enteric neurons

Because human primary afferent neurons are not readily obtained, we sought to develop a model in which the lytic, latent, and reactivating phases of varicella-zoster virus (VZV) infection were recapitulated in neurons from an animal source. Enteric neurons were obtained from the small intestine of adult guinea pigs and from the bowel of fetal mice. Latency was established when these neurons were infected by cell-free VZV in the absence of fibroblasts or other cells of mesodermal origin. In contrast, lytic infection ensued when fibroblasts were present or when the enteric neurons were infected by cell-associated VZV. Latency was associated with the expression of a limited subset of viral genes, the products of which were restricted to the cytoplasm. Lysis was associated with the expression of viral glycoproteins, nuclear translocation of latency-associated gene products, and rapid cell death. Reactivation was accomplished by expressing VZV open reading frame (ORF) 61p or herpes simplex virus ICP0 in latently infected neurons. Isolated enteric neurons from guinea pigs and mice recapitulate latent gene expression in human cranial nerve and dorsal root ganglia. Expression of latency-associated VZV gene products was detected in 88% of samples of adult human intestine, suggesting that VZV not only infects enteric neurons but also is latent in the human enteric nervous system. This in vitro model should facilitate further understanding of latency and reactivation of VZV.     

Maternal HLA class II compatibility in men with systemic lupus erythematosus

OBJECTIVE: Maternal-fetal cell transfer during pregnancy can lead to long-lasting microchimerism, which raises the question of whether microchimerism sometimes contributes to autoimmune disease later in life. In an experimental model, transfusion of parental lymphocytes homozygous for major histocompatibility complex alleles results in systemic lupus erythematosus (SLE). We identified male patients with SLE and healthy male subjects and their mothers in order to investigate the mother-son HLA relationship in SLE risk. Male subjects were selected in order to avoid confounding due to fetal microchimerism, which may occur in women. METHODS: HLA genotyping for DRB1, DQA1, and DQB1 was conducted for sons and their mothers. Thirty men with SLE and their mothers were compared with 76 healthy men and their mothers. RESULTS: Sons with SLE were HLA-identical with their mothers (bidirectionally compatible) for the basic HLA-DRB1 groups encoded by DRB1*01 through DRB1*14 more often than were healthy sons (odds ratio [OR] 5.0, P = 0.006). Each DRB1 group contains multiple allelic variants; male patients with SLE and their mothers often were identical for both DRB1 allelic variants (OR 3.2, P = 0.08). For DQA1 and DQB1, the ORs were 2.3 (P = 0.08) and 2.0 (P = 0.21), respectively. When analysis was limited to male subjects with SLE-associated HLA genes (encoding HLA-DR2 or HLA-DR3), the differences further increased for DRB1 basic groups (OR 7.2, P = 0.01), DRB1 alleles (OR 15.0, P = 0.018), DQA1 6.4 (P = 0.006), and DQB1 (OR 5.7, P = 0.027). No increase in (unidirectional) compatibility of the mother from the son's perspective was observed at any locus. CONCLUSION: We observed increased bidirectional HLA class II compatibility of male SLE patients and their mothers compared with healthy men and their mothers. This observation implies that maternal microchimerism could sometimes be involved in SLE and therefore merits further investigation.