Absence of specific cell-surface binding of tissue plasminogen activator in uterine cells

Tissue plasminogen activator (tPA), an arginine-specific serine protease, is an oestrogen-regulated protein in uterine and breast cancer tissue. It contains a domain which shares homology with epidermal growth factor (EGF). The aim of the present study was to determine whether specific tPA receptors or EGF receptors mediate the binding of tPA to cells and whether tPA possesses intrinsic mitogenic activity. The binding of 125I-labelled tPA to rat uterine and liver membranes was shown to be non-specific and could not be displaced by unlabelled tPA or EGF. Furthermore, acid washing of cell membranes did not unmask specific tPA-binding sites. In contrast, 125I-labelled EGF binding to both rat uterine and liver membranes was displaced in a dose-dependent manner by unlabelled EGF, and Scatchard analysis of the binding data revealed dissociation constant (Kd) values of 2.4 and 0.71 nM respectively. Unlabelled tPA (up to 20,000-fold excess) did not displace 125I-labelled EGF binding to these membranes. A study of the binding of 125I-labelled tPA and 125I-labelled EGF to endometrial carcinoma cells (Ishikawa), cervical carcinoma cells (HOG-1) and vulval carcinoma cells (A431) showed that up to a 100-fold excess of EGF or a 1000-fold excess of tPA did not displace 125I-labelled tPA binding to these cells. In contrast, 125I-labelled EGF binding was displaced by unlabelled EGF (Kd values for Ishikawa and HOG-1 cells were 2.72 and 1.92 nM respectively) but not by unlabelled tPA (1000-fold excess).(ABSTRACT TRUNCATED AT 250 WORDS)     

Distortion correction for diffusion‐weighted MRI tractography and fMRI in the temporal lobes

Single shot echo‐planar imaging (EPI) sequences are currently the most commonly used sequences for diffusion‐weighted imaging (DWI) and functional magnetic resonance imaging (fMRI) as they allow relatively high signal to noise with rapid acquisition time. A major drawback of EPI is the substantial geometric distortion and signal loss that can occur due to magnetic field inhomogeneities close to air‐tissue boundaries. If DWI‐based tractography and fMRI are to be applied to these regions, then the distortions must be accurately corrected to achieve meaningful results. We describe robust acquisition and processing methods for correcting such distortions in spin echo (SE) EPI using a variant of the reversed direction k space traversal method with a number of novel additions. We demonstrate that dual direction k space traversal with maintained diffusion‐encoding gradient strength and direction results in correction of the great majority of eddy current‐associated distortions in DWI, in addition to those created by variations in magnetic susceptibility. We also provide examples to demonstrate that the presence of severe distortions cannot be ignored if meaningful tractography results are desired. The distortion correction routine was applied to SE‐EPI fMRI acquisitions and allowed detection of activation in the temporal lobe that had been previously found using PET but not conventional fMRI. Hum Brain Mapp, 2010. © 2010 Wiley‐Liss, Inc.     

Arthritis as presenting manifestation of pure neuritic leprosy--a rheumatologist's dilemma

OBJECTIVES: Leprosy classically presents with cutaneous and neurological manifestations. In diagnosed cases of leprosy, rheumatological involvement varies from 1% to 70%. A primary articular presentation without cutaneous manifestations is not yet known. Herein, we present our experience of five cases of leprosy that presented with predominant articular involvement in the absence of cutaneous manifestations. METHODS: The study was conducted in the Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences located in the state of Uttar Pradesh, one of the nine endemic states in India. Case records of patients with a definite diagnosis of leprosy were screened for the presenting manifestations, pattern of articular involvement, tenosynovitis, neurological signs and symptoms. Reports of nerve conduction study (NCS), nerve and synovial biopsy and other diagnostic tests were retrieved from laboratory records. Available radiographs were examined for evidence of juxta-articular osteopenia and erosions. RESULTS: Case records of 11,740 patients were screened, of which 28 had a diagnosis of leprosy. Twenty patients had presented with rheumatological complaints primarily. Five of the patients who presented with inflammatory arthritis with/without tenosynovitis (n = 4) and tenosynovitis alone (n = 1) had pure neuritic leprosy. All of these patients had thickened peripheral nerves and abnormal NCS. Sural nerve biopsy confirmed the diagnosis of leprosy in all these cases. CONCLUSION: A combination of tenosynovitis and thickened nerves in association with symmetric polyarthritis should raise a suspicion of leprosy even in the absence of cutaneous features.     

Human leukocyte antigen (HLA) class II association with rheumatic heart disease in Pakistan

BACKGROUND AND AIM of the study: Rheumatic heart disease (RHD) is widespread in Pakistan. Specific alleles of the human leukocyte antigen (HLA) system are associated with RHD in various world populations. The study aim was to investigate the involvement of HLA class II alleles in genetic susceptibility to RHD in patients with relatively homogeneous clinical manifestations, in Pakistan. METHODS: Blood samples were collected from 114 unrelated patients (94 females, 20 males) with rheumatic mitral valve disease, predominantly mitral stenosis, as assessed by echocardiography. The control group comprised 109 unrelated, ethnically matched, healthy individuals (60 females, 49 males) with normal echocardiograms. Genomic DNA was extracted from venous blood using a standard phenol/chloroform extraction procedure. HLA-DRB, -DQA1, and -DQB1 alleles were typed using polymerase chain reaction with sequence-specific primers. HLA allele and haplotypes frequencies were then calculated. RESULTS: A significantly higher frequency of DRB1*07 was observed in patients as compared to controls (one-way parametric analysis of variance, F = 4.84, p = 0.028; OR = 1.76, p = 0.039). No alleles for the HLA-DQA1 or -DQB1 loci were associated with the disease. HLA-DRB1*07-DQA1*0501-DQB1*02, the only haplotype that differed significantly between patients and controls (one-way parametric Anova, F = 4.866, p = 0.028; OR = 7.33, p = 0.06), did not exhibit significant linkage disequilibrium. CONCLUSION: These results show that HLA-DRB1*07, associated with RHD in various world populations, is also associated with RHD in the Pakistani population. The validation of HLA associations with RHD, which is observed in different world populations, may lead to the development of a cost-effective strategy in the primary prevention of this disease.     

Absence of major accumulation of mitochondrial ND5 mutations in Parkinson patient muscle

The pathogenesis of the selective loss of dopaminergic neurons in idiopathic Parkinson's disease (PD) has not been understood up to now. Respiratory chain dysfunction and accumulation of mitochondrial DNA deletions to biochemically relevant levels have been observed in the dopaminergic neurons. However, respiratory chain defects have also been reported in other tissues, pointing to a generalized component of oxidative stress in PD. Recently, somatic point mutations in a narrow region of the complex I polypeptide ND5 (codons 120 - 150) were suggested to separate PD patients from age-matched controls, using frontal cortex homogenates. OBJECTIVE: The present study intended to analyze whether those recently described ND5 mutations may also generally occur in skeletal muscle tissue of PD patients, in which complex I dysfunction had been measured earlier with biochemical approaches. MATERIAL: Skeletal muscle biopsy samples of 5 PD individuals with a previously characterized biochemical complex I defect and of 5 age-matched controls were used. METHOD: DNA was extracted from the muscle samples. The relevant ND5 region was PCR-cloned using a high fidelity Pfu polymerase and a low number of PCR cycles (15). Amean number of 96 clones were randomly selected from the ampicillin plates and sequenced by the dye terminator method to allow the detection of low abundance mutations with a sensitivity around 1%. RESULTS: Mutations between codons 120 and 150 were only slightly more frequent in PD versus controls (60 versus 40% of samples affected), while this ratio had been 100 versus 12.5% in frontal cortex. CONCLUSIONS: In contrast to results reported for PD frontal cortex, low-level ND5 mutations between codons 120 and 150 do not accumulate severely in biochemically affected skeletal muscle samples of PD patients.     

Induction of monocyte chemoattractant protein-1 expression by angiotensin II in the pancreatic islets and beta-cells

Angiotensin II (AngII), the principal hormone of the renin-angiotensin system, is actively generated in the pancreas and has been suggested as a key mediator of inflammation. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of mononuclear cells into the pancreatic islets. In this study, we investigated the potential molecular basis for the role of AngII in islet inflammation through studying its effect on MCP-1. AngII significantly increased the expression of MCP-1 mRNA and protein in the RINm5F beta-cell line and activated MCP-1 promoter. AngII-MCP-1 mRNA induction was inhibited by an AngII type 1 receptor antagonist but was unchanged by an AngII type 2 receptor antagonist. AngII-MCP-1 induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a MAPK signaling mechanism. AngII activated the phosphorylation of ERK1/2 but not p38 or c-Jun NH(2)-terminal MAPKs. Inhibition of ERK1/2 activation reduced the AngII-induced MCP-1 synthesis. In nonobese diabetic mice pancreata, the temporal pattern of angiotensin-converting enzyme expression correlated well with progression of insulitis and beta-cell destruction. Immunostaining of pancreatic serial sections show colocalization of angiotensin-converting enzyme with MCP-1 in beta-cells in the islets. In freshly isolated islets from normoglycemic mice, AngII alone and in combination with IL-1beta elicited an inflammatory response by stimulation of MCP-1. Our data suggest a positive autocrine/paracrine action for the local pancreatic AngII-generating system during insulitis and provide the first insight into an AngII-initiated signal transduction pathway that regulates MCP-1 as a possible inflammatory mechanism in the islets.