Overexpression of CD39/nucleoside triphosphate diphosphohydrolase-1 decreases smooth muscle cell proliferation and prevents neointima formation after angioplasty.

BACKGROUND: Growing evidence implicates the involvement of extracellular nucleotides in the regulation of platelet, leukocyte, endothelial cell (EC) and vascular smooth muscle cell (VSMC) phenotype and function. Within the quiescent vasculature, extracellular nucleotides are rapidly hydrolyzed by CD39, the dominant endothelial nucleoside triphosphate diphosphohydrolase (NTPDase-1). However, vascular CD39/NTPDase-1 activity is lost in EC activated by oxidative stress or proinflammatory mediators, and upon denudation of the endothelium following balloon injury. The consequent increase in extracellular nucleotide concentrations triggers signaling events leading to prothrombotic responses and increased VSMC proliferation. OBJECTIVES: To investigate the effect of overexpressed CD39/NTPDase-1 in injured aorta. METHODS: Using adenoviral-mediated gene transfer we expressed CD39/NTPDase-1 in mechanically denudated rat aortas. We measured intima formation by morphometry and VSMC proliferation by the [(3)H]-thymidine incorporation assay. RESULTS: Targeted expression of CD39 in injured vessels increased NTPDase activity (from 2.91 +/- 0.31 to 22.07 +/- 6.7 nmols Pi mg(-1) protein, 4 days after exposure to the adenovirus) and prevented the formation of neointima. The thickness of the intimal layer in injured aortas exposed to Ad-CD39 was 26.2 +/- 3.9 microm vs. 51.8 +/- 6.1 microm and 64.4 +/- 22.2 microm (P < 0.001) in vessels treated with Ad-beta-gal and saline, respectively. Moreover, targeted expression of CD39/NTPDase-1 caused a 70% (P < 0.01) decrease in proliferation of VSMC isolated from transduced rat aortas as compared with VSMC derived from control vessels. CONCLUSIONS: The presented data suggest that increasing CD39/NTPDase-1 activity in VSMC could represent a novel therapeutic approach for the prevention of stenosis associated with angioplasty and other occlusive vascular diseases.

Block of slow axonal transport and axonal growth by brefeldin A in compartmented cultures of rat sympathetic neurons.

Disruption of the Golgi by brefeldin A (BFA) has been reported to block fast axonal transport and axonal growth. We used compartmented cultures of rat sympathetic neurons to investigate its effects on slow axonal transport. BFA (1 micro g/ml) applied to cell bodies/proximal axons for 6-20 h disrupted the Golgi, reversibly blocked axonal growth, and reversibly blocked anterograde transport of all proteins, including tubulin. The retrograde transport of nerve growth factor (NGF) was also blocked. The phosphorylation of Erk1 and Erk2 in response to NGF was unaffected after 6 h of treatment with BFA, suggesting that the block of axonal transport was specific and direct. Consistent with its principal site of action at the Golgi, no effects were observed when BFA was applied only to the distal axons. Block of fast anterograde and retrograde axonal transport is consistent with the role of the Golgi in supplying transport vesicles. Block of slow axonal transport was surprising, and further results indicated that transport of tubulin en route along the axon was arrested by application of BFA to the cell bodies, suggesting that a continuous supply of anterograde transport vesicles from the Golgi is required to maintain slow axonal transport of cytoskeletal proteins.

Donor DNA can be detected in recipient tissues during rejection of allograft.

The main source of donor DNA in recipients of allograft are "passenger" cells. It is claimed that they are responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have observed that besides cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of the allograft. In this study, we provide evidence for the presence in the recipient of both DNA in "passenger cells" and free DNA in tissues at the terminal stage of rejection. Male BN (RT1 n) rat heart or skin was transplanted to female LEW (RT1 l) rats followed by a vascularized bone marrow in a hindlimb transplant. In another group, heart and skin were transplanted followed by immediate i.v. infusion of donor-type bone marrow cells. CsA was given in a dose of 17 mg/kg body weight for 30 days, then the rats were followed up until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and day 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, as well as lymphoid organs, at the time of rejection of allograft, but not when the rats were maintained on CsA. Taken together, donor DNAwas detected in recipient tissues at the time of heart or skin rejection. It appeared to be released from cells of rejecting grafts and not from "passenger" cells, representing only a minor cellular mass compared with the graft.

Mechanism of antinephritic effect of proteinase inhibitors in experimental anti-GBM glomerulopathy.

We have previously documented amelioration of rat autologous anti-GBM nephritis with the antiproteolytic drugs epsilon-aminocaproic acid (EACA) and aprotinin, given from the day of induction or later in the course of disease. In the present study we investigated potential mechanisms of this effect by assessing interactions of the drugs with proteinase-dependent generation of superoxide anion in glomeruli, and their influence on both GBM degradation in vitro and activity of glomerular proteolytic enzymes. Release of O2- by enzymatically disrupted glomeruli, isolated from nephritic control or EACA/aprotinin-treated rats, was measured with the ferricytochrome reduction method and its activity was correlated with proteinuria and glomerular cellularity at the early phase of the disease. The hydroxyproline release assay was used to quantitate degradation of rat GBM in vitro by leukocyte proteinases stimulated by phorbol myristate acetate (PMA), in the presence or absence of EACA and aprotinin. Finally, the activities of elastase, cathepsins B and L, and plasmin, together with collagenase-like activity, were assessed fluorimetrically in homogenates of glomeruli isolated from control and antiproteolytic-drug-treated nephritic rats. EACA and aprotinin notably inhibited production of superoxide by nephritic glomeruli (by 47% and 66%, respectively), and this effect was not significantly correlated with proteinuria or glomerular hypercellularity at the early stage of disease. On the other hand, generation of O2- by glomeruli of untreated nephritic rats was notably correlated with total glomerular cell counts and numbers of macrophages infiltrating glomeruli. PMA-stimulated neutrophils and macrophages caused degradation of isolated rat GBM in vitro, markedly attenuated in the presence of EACA (P<0.0005) and, to a lesser extent, by addition of aprotinin (P<0.01). The activity of elastase was significantly reduced in glomeruli of nephritic rats treated with EACA or aprotinin (both P<0.001), while activities of remaining proteinases were not appreciably affected. The beneficial influence of proteinase inhibitors on rat anti-GBM disease may be due, at least in part, to abrogation of superoxide generation in nephritic glomeruli. EACA and aprotinin also have potential to interfere with digestion of GBM, and both these effects may be related to suppression of glomerular elastase.

Factors affecting reactivation of Epstein-Barr virus infection after kidney allograft transplantation.

OBJECTIVES: Reactivation of Epstein-Barr virus (EBV) infection in renal transplant recipients may cause significant morbidity and mortality. To evaluate factors associated with activation of EBV replication we followed prospectively a group of 65 recipients of cadaveric kidney for 12 months. METHODS: Sera were collected periodically from these patients and analyzed for the presence of specific anti-EBV antibodies. Control group consisted of renal (n=35) and healthy blood donors (n=35). Enzyme-linked immunoassays based on recombinant EBV proteins were used to detect the following antibody specificities: early antigen (EA) IgA, IgM, and IgG, nuclear antigen (EBNA) IgG. RESULTS: During first year after transplantation, primary infection developed in 4 (6.15%) recipients and reactivation occurred in 18 (27.7%) recipients. Analysis did not show the association of reactivation with type of basic immunosuppressive therapy, prophylactic or therapeutic use of anti-lymphocyte antibodies, as well as acute rejection episodes. There was a borderline association (p=0.068) between the incidence of CMV infection and EBV reactivation. CONCLUSIONS: Our data suggest casual relationship between CMV infection and EBV reactivation.

DNA from rejecting allografts can be detected in recipient nonlymphoid tissues.

The main source of donor DNA in recipients of allograft are "passenger" cells. They are claimed to be responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have noticed that beside of the cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of allograft. In this study we provide evidence for presence in the recipient of both, DNA in "passenger cells" and free DNA in tissues at terminal stage of rejection. Male BN (RTIn) rat heart or skin were transplanted to female LEW (RTII) rats followed by a vascularized bone marrow in hind-limb transplant. CsA was given in a dose of 17mg/kg b.w. for 30 days, then rats were followed until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, beside of lymphoid organs, at the time of rejection of allograft but not when rats were maintained on CsA. Taken together, donor DNA can be detected in recipient tissues at the time of heart or skin rejection. It seems to be released from cells of rejecting grafts and not from "passenger" cells representing only a minor cellular mass compared with the graft.

Tolerance induction following allogeneic vascularized bone marrow transplantation--the possible role of microchimerism.

We have noticed that bone marrow transplanted in a vascularized limb graft, providing a continuous supply of donor bone marrow cells (BMC), may prolong the survival time of a skin graft from the same donor. The question arises whether the microchimerism raised plays a role in the prolonged survival of skin allografts. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection process of transplanted skin. Brown Norway (BN) rats served as donors and Lewis rats as recipients of VBMTx and free skin flap allografts. A hind limb was transplanted, followed by a full-thickness skin graft on the dorsum. Cellular microchimerism was investigated in recipients of VBMTx and skin grafts in blood, spleen, mesenteric lymph node, and bone marrow with the monoclonal antibody OX27 directed against MHC class I polymorphic RT1 on BN cells and quantitatively analyzed in a FACStar. In the VBMTx group, the free skin flap survived 70 days after weaning off cyclosporine A (CsA). An intravenous infusion of BMC in suspension equivalent to that grafted in the hind limb did not prolong skin graft survival after cessation of CsA therapy. Donor-derived cells could be detected in VBMTx recipients as long 70 days after weaning off CsA but not in recipients of i.v. suspension BMC grafting.

[Urinary excretion of extracellular matrix proteins in insulin dependent diabetes mellitus]. / Wydalanie z moczem bialek macierzy zewnatrzkomórkowej w cukrzycy insulinozaleznej.

The purpose of the study was to assess urinary excretion of extracellular matrix proteins and proteolytic enzymes in 12 subjects with IDDM with albuminuria, 12 subjects with IDDM without microalbuminuria and 10 normal healthy subjects. Urinary excretion of FN was significantly higher in subjects with IDDM and albuminuria as compared to patients with IDDM without microalbuminuria and healthy subjects (223.6 +/- 143.2 vs. 103.2 +/- 59.7 vs. 58.3 +/- 12.0 ng/mg creatinine, p < 0.01). Urinary level of type IV collagen was significantly elevated in subjects with IDDM and albuminuria as compared to IDDM without microalbuminuria and healthy subjects of cathepsin B was significantly higher in diabetic patients with albuminuria as compared to patients without microalbuminuria and healthy subjects (0.82 +/- 0.53 vs. 0.25 +/- 0.17 vs. 0.22 +/- 0.05 mlU/mg creatinine, p < 0.01). Urinary activity of plasmin was significantly elevated in diabetic patients with albuminuria as compared to subjects without microalbuminuria and healthy control (0.477 +/- 0.37 vs. 0.194 +/- 0.09 vs. 0.21 +/- 0.02 mlU/mg creatinine, p < 0.01). Our data indicate that increase in the urinary excretion of extracellular matrix proteins may be the useful tool for monitoring glomerular injury.

The gallium scan: problems and misuse in examination of patients with suspected infection.

Gallium citrate Ga 67 (67Ga) has been used for almost ten years as a means of detecting inflammatory lesions in febrile patients. We have reviewed 80 cases from Milwaukee County General Hospital (1977 through 1979) in which 67Ga scanning was performed to detect inflammatory lesions in patients by suspected infection. Fifty scans also were available for review by the nuclear medicine staff. We found the sensitivity of 67Ga scans at our institution to be 90%, but specificity to be only 64%. In five (6%) of the cases, 67Ga scanning was the most important means of establishing a diagnosis. Of the 50 scans available for review, only 26 scans (52%) were interpreted in the same way as the original reading. Twenty-two (27%) of all scans in retrospect should not have been ordered, either because of the fever was gone and the patient's condition was improving or the diagnosis was already made. We conclude that 67Ga scans can be useful to detect inflammatory lesions but that they are frequently used inappropriately by physicians at our hospital, they are difficult to interpret, and the specificity is lower than the sensitivity.

Evaluation of postresuscitation left ventricular global and segmental function by radionuclide ventriculography in sudden coronary death survivors of prehospital cardiac arrest: Correlation to subsequent short-term prognosis.

Previous data have shown that patients with significant left ventricular (LV) dysfunction, marked abnormalities in resting hemodynamics, and multiple vessel coronary artery disease (CAD) are at increased risk for sudden cardiac death. However, in-hospital assessment of ventricular function in the early postresuscitation period of out-of-hospital cardiac arrest has not been evaluated and related to short-term outcome. By using bedside radionuclide ventriculography (RNV) we evaluated LV ejection fraction (LVEF) and wall motion in 36 nonconsecutive patients within 24 hours of their episode of out-of-hospital cardiac arrest. There were 24 men and 12 women with mean age of 62.3 years (range 21 to 85 years). Total mortality of the entire group was 38.8% at 4 weeks. Eighteen patients had LVEF less than 0.30 and 18 had LVEF greater than 0.30. Mortality in the low LVEF group was 55.5% at 4 weeks versus 22.2% in patients with LVEF greater than 0.30 (p less than 0.05). Patients with normal LV wall motion had no short-term mortality (none of seven), while patients having abnormal LV wall motion had a significantly higher short-term mortality (14 of 29) (p less than 0.05). We conclude that out-of-hospital cardiac arrest survivors have a high incidence of severe LV dysfunction in the early postresuscitation period and that a significantly higher early mortality is seen in the group with LVEF less than 0.30. In addition, abnormalities of LV wall motion by RNV examination demonstrated poorer prognosis in the first 4 weeks than patients with normal LV wall motion.

Bone scintigraphy in the reflex sympathetic dystrophy syndrome.

Sixty-four consecutive patients were studied for possible reflex sympathetic dystrophy syndrome (RSDS). They were divided into five groups, based upon specific clinical criteria, and the radiographic and scintigraphic findings in each group were examined. Osteoporosis was the most common radiographic abnormality, present in 69% of subjects with definite, probable, or possible RSDS, as compared with 21% opf those with RSDS. Scintigraphic abnormalities were noted in 60% of RSDS patients but in only 7% of the others. These findings included increased blood flow and enhanced periarticular radionuclide activity in the affected extremity. Of 11 patients with serial scintigraphy, six (55%) demonstrated a return to normal, symmetrical patterns following successful therapy. The scan may reflect an active, potentially reversible disorder of local blood flow in RSDS. Furthermore, the scintigraphic patterns may be useful in the diagnosis and in predicting which pattients are likely to respond to systemic steroid therapy.

The reflex sympathetic dystrophy syndrome (RSDS). III. Scintigraphic studies, further evidence for the therapeutic efficacy of systemic corticosteroids, and proposed diagnostic criteria.

Sixty-four patients were evaluated prospectively for a reflex sympathetic dystrophy syndrome (RSDS), using quantitative clinical measurements, high-resolution roentgenography and scintigraphy. Five separate groups were identified by their clinical features, allowing us to distinguish patients with definite or incomplete forms of the RSDS as well as 16 patients with other disorders. Scintigraphy was found to be a useful diagnostic study that may also provide a method of predicting therapeutic response. Systemic corticosteroid therapy proved to be a highly effective mode of treatment for up to 90 percent of the patients with the RSDS.

Unilateral increased radioactivity in the lower extremities on routine 99mTc-pyrophosphate bone imaging.

Unilateral increased activity in the lower extremities was noted in 5 patients who underwent routine 99mTc-pyrophosphate bone imaging for metastatic evaluation. In three patients a large pelvic mass or retroperitoneal hematoma was noted on the affected side. Clinical evidence of lymphatic obstruction was apparent in two cases with pelvic mass. In the fourth case, venous disease (thrombophlebitis) was noted and the fifth case showed osteomyelitis. The possible mechanism for this phenomenon may include obstruction of the lymphatic or venous circulation and inflammation. Other causes of soft tissue uptake of 99mTc-PYP are reviewed. The above mentioned conditions must be excluded when asymmetrical activity of 99mTc-PYP is seen on routine bone imaging.

Lung disease in reformed drug addicts: diagnostic and physiologic correlations.

Pulmonary morphologic alterations secondary alterations secondary to intravenous drug abuse have been previously identified by histopathologic study of autopsy and biopsy material. Recent investigative efforts have been directed toward defining functional abnormalities that would allow for early clinical diagnosis. For this reason, 12 asymptomatic addicts were studied with roentgenography, spirometry, single breath diffusing capacity (DLco), 99Tc perfusion and 133 Xe ventilation scanning. Nine of the twelve were heavy cigarette smokers. None reported historical evidence of chronic bronchitis or right-sided endocarditis. The DLco was the only significant routine pulmonary function abnormality (p less than .001) observed in these patients. The DLco was significantly reduced even after correction for smoking habits (p less than .001). The decrease in DLco correlated with the number of perfusion defects detected by 99Tc scanning (r = -.560). There was no correlation between DLco and the number of defects on the 133Xe ventilation scan, age, pack-years smoked, or other routine pulmonary function tests. Further, there was no correlation between the number of perfusion and the number of ventilation defects. The data suggest that the decrease in DLco found in these addicts is the result of reduction of the pulmonary capillary bed secondary to vascular or interstitial granulomatosis.