Adeno-associated virus-mediated vascular endothelial growth factor gene therapy in skeletal muscle before transplantation promotes revascularization of regenerating muscle

Successful clinical transplantation of whole skeletal muscles can be limited by impaired muscle revascularization and regeneration. The aim of this study was to enhance the revascularization (and hence speed of regeneration) of transplanted whole muscles by transducing muscles with the vascular endothelial growth factor (VEGF) gene before transplantation, using a recombinant adeno-associated virus (rAAV). The rAAV encoding VEGF and green fluorescent protein (GFP) (rAAV.VEGF.GFP) was injected into the tibialis anterior muscles of adult BALB/c mice. One month after injection whole muscle autotransplantation was performed. Muscles were sampled 7 days after autografting. GFP expression was examined as an indicator of persistent transgene expression after grafting, and immunohistochemistry was used to identify VEGF, blood vessels, and newly formed myotubes. After grafting, GFP expression persisted only in a few surviving myofibers in the periphery of rAAV.VEGF.GFP-pretreated muscles, although abundant VEGF expression was seen in myogenic cells in all grafted muscles. Quantitative analysis demonstrated that, although only small numbers of rAAV.VEGF.GFP-transduced myofibers were present, whole muscle grafts preinjected with rAAV.VEGF.GFP were significantly more vascular than saline-injected and uninjected control muscle grafts. Furthermore, rAAV.VEGF.GFP-injected whole muscle transplants were further advanced in terms of regeneration (myotube formation) compared with the uninjected control muscle transplants. This study clearly shows that rAAV-mediated VEGF expression persists only in myofibers that survive the necrosis induced by muscle transplantation; however, this amount of VEGF results in significantly increased revascularization and regeneration of whole muscle transplants.     

Clear cell adenocarcinoma with endobronchial polypoid growth

Clear cell adenocarcinoma of the lung is extremely rare. On radiography, a 45-year-old female with fever was found to have an abnormal shadow in the left lower lung field. Bronchoscopy revealed a polypoid tumor in the left bronchus. On biopsy, the tumor was determined to be adenocarcinoma. Preoperative examination found no tumors outside of the lung. The patient underwent left lower lobectomy with bronchial wedge resection. The tumor had completely obstructed and dilated the left lower bronchus, but had not invaded the tissue outside the bronchial wall. Microscopically, the cytoplasm of the tumor cells contained abundant glycogen, and the tumor had solid and glandular structures. The tumor was diagnosed as clear cell adenocarcinoma of the lung.     

The effect of exhaustive exercise on the antioxidant enzyme system in skeletal muscle from calcium-deficient rats

 The aim of the current study was to elucidate the synergism of dietary calcium restriction and exhaustive exercise in the antioxidant enzyme system of rat soleus muscle, and to investigate the involvement of neutrophils in exercise-induced muscle damage. Forty-eight male Wistar rats were assigned to the following groups: control (C) or calcium-restricted [1 month (1 M) or 3 months (3 M)]. Each group was subdivided into acutely exercised or non-exercised groups. Soleus muscle from each rat was analysed to determine the levels of antioxidant enzymes [Mn-superoxide dismutase (SOD), Cu,Zn-SOD, glutathione peroxidase (GPX), and catalase (CAT)]. Dietary calcium restriction resulted in calcium deficiency and upregulated the antioxidant enzymes examined except GPX. Conversely, exhaustive exercise significantly decreased GPX and CAT, but not SODs activities in the calcium-restricted (1 M and/or 3 M) rats. Contents of immunoreactive Mn-SOD and Cu,Zn-SOD were only increased in the 3 M rats. During calcium restriction, the mRNA expression of both forms of SOD showed initial upregulation, followed by downregulation. Exhaustive exercise significantly increased the mRNA expressions only in the 3 M rats. Moreover, exhaustive exercise markedly increased myeloperoxidase activity in soleus muscles from the 1 M and 3 M rats compared with the C rats, and significantly enhanced the ability of neutrophils to generate superoxide in the 3 M rats. The results demonstrate that dietary calcium restriction upregulates certain antioxidant enzyme activities in rat soleus muscle, indicating an enhanced resistance to potential increases in intracellular reactive oxygen species. The results also suggest that exhaustive exercise may cause oxidative damage in soleus muscle of calcium-deficient rats through the activation of neutrophils. Received: 4 August 1997 / Received after revision: 29 September 1997 / Accepted: 26 November 1997     

Coping with dental treatment: The potential impact of situational demands

Coping strategies and anxiety responding of dental patients were studied in order to test the generalizability of previous findings based on volunteer blood donors. State and trait coping measures were administered once, and a process coping scale was administered at three points throughout treatment. Self-report, behavioral observation, and psychophysiological measures of anxiety were sampled for the same periods as process coping. Findings included the replication of a negative relationship between avoidant coping and patient anxiety ratings. Fluctuations in coping were evident across periods, and impact of situational demands and constraints was introduced as an explanation for these variations. A method for direct assessment of coping consistency was introduced. On the basis of the replicable associations with anxiety measures, the ability to detect changes in coping within a situation, and the ability to provide direct evidence of coping consistency, the use of process methodology for coping assessment is encouraged.This research was conducted while the first author was supported by funding from the Medical Research Council of Canada.Portions of this research were presented at the annual convention of the Society of Behavioral Medicine, Philadelphia, 1984.     

SV40-related T-antigen expression in human meningiomas with normal and G-22-monosomic karyotype.

Six of 16 meningiomas tested in early subcultures by indirect immunofluorescence showed SV40 T-antigen. Two different antisera specific for T-antigen were used. One serum gave a positive reaction with six tumours and the other with only two. In one T-antigen positive meningioma, the typical nuclear fluorescence changed, beginning with the second subculture, into an unusual brilliant granular pattern irregularly distributed over the nuclei. In six meningiomas, a specific chromosome aberration (monosomy G 22) was established. However, up to now, no clear correlation between karyotype and T-antigen expression could be found: cells from three meningiomas with positive reactions had normal karyotypes, whereas those from three tumours with typical chromosome loss showed no T-antigen.     

HLA-B27-restricted T cells from patients with ankylosing spondylitis recognize peptides from B*2705 that are similar to bacteria-derived peptides

Ankylosing spondylitis (AS) is an inflammatory systemic disease affecting the spine, sacroiliacal and peripheral joints. Although the aetiology of AS remains unknown, the strong association with the HLA-B27 allele might reflect directly a detrimental effect of the HLA-B27 molecule itself, resulting from its potential capability to present 'arthritogenic' peptides to CD8+ T cells. Because some forms of SpA are triggered by enterobacterial infection, such arthritogenic peptides might originate from autologous and/or bacterial proteins triggering cross-reactive CD8+ T cell clones. Intriguingly, two peptides from the second extracellular domain of HLA-B*2705 share sequence homologies with several enterobacterial antigens, exhibit the HLA-B27-binding-motif, and are presented by HLA-B*2705 itself. The objective of this study was to examine the clonal T cell reactivity against these peptides in patients with AS. To this end, we screened peripheral blood lymphocytes (PBL) of 26 patients with AS and 24 healthy donors for TNF-alpha-producing cells using ELISPOT assays. PBL and synovial fluid-derived lymphocytes (SFL) of peptide-responsive patients were then stimulated and cultured with the relevant peptide and control peptides in vitro. Antigen-specific T cell lines (TCL) were identified by standard chromium release assays. Clonal analysis was performed subsequently applying TCRB-CDR3 spectratyping. Among eight peptides tested, only the HLA-B27 168-176 peptide LRRYLENGK was recognized by PBL from B27+ AS patients but not from B27+ healthy controls (P=0.001). LRRYLENGK-specific T cell clones used preferentially the TCRBV5S1 and the BV14 segment. These results suggest that an HLA-B27-derived peptide with homology to bacterial peptides may play a role in AS.     

Imbalance of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases: a novel approach for explaining the parenchymal liquefaction of the septic spleen?

OBJECTIVE: The causal pathophysiological mechanisms involved in the parenchymal liquefaction of the septic spleen are still far from clear. The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodelling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain chronic inflammatory diseases. METHODS: This study focuses on a search for alterations in the balance between MMP-1 (interstitial collagenase) and TIMP-1 by means of immunostaining, by immunoblotting, and by gel zymography. RESULTS: We found a deregulation of the balance between MMP-1 and TIMP-1 in the septic spleen in favor of the active form of MMP-1. CONCLUSION: Our findings suggest that active MMP-1 is involved in collagenolytic extracellular matrix breakdown in the septic spleen.     

Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss

The pathogenesis of progressive bile duct loss in primary biliary cirrhosis remains unclear. In this study, the involvement of cellular senescence of biliary epithelial cells was examined in liver tissue samples from patients with primary biliary cirrhosis (n = 33), and compared with control diseased and normal livers (n = 83). In addition, cellular senescence was induced by oxidative stress in cultured mouse biliary epithelial cells. Biliary epithelial cells in small bile ducts in primary biliary cirrhosis, especially those in patients presenting with chronic non-suppurative cholangitis, frequently expressed senescence-associated beta-galactosidase, and senescence-associated p16(INK4) and p21(WAF1/CIP). In contrast, senescence-associated markers were rarely expressed in small bile ducts in control livers. The infiltration of myeloperoxidase-positive inflammatory cells into biliary epithelial cell layers was closely associated with the cellular senescence of biliary epithelial cells in early-stage PBC. Cellular senescence of cultured mouse biliary epithelial cells was induced by treatment with H2O2 via the p38MAPK-dependent pathway and nitric oxide-augmented H2O2-induced cellular senescence. Oxidative stress- and nitric oxide-mediated cellular senescence may be involved in bile duct lesions, which are followed by progressive bile duct loss in primary biliary cirrhosis.