Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.     

Expression of platelet-derived growth factor and its receptors by two pre-B acute lymphocytic leukemia cell lines

Platelet-derived growth factors (PDGF) are potent regulators of cell proliferation. The three isoforms of PDGF AA, AB, and BB are encoded by two genes: PDGF A and PDGF B. The v-sis oncogene is homologous to the PDGF-B gene. v-sis can transform cells that express the appropriate PDGF receptors. Two different types of receptors, PDGF-alpha and PDGF- beta, also encoded by two genes, have been identified. We show that two cell lines. SMS-SB and NALM-6, both derived from pre-B-cell acute lymphocytic leukemias, express the PDGF-A chain gene, and one of them, SMS-SB, releases PDGF-A chains into the media. The SMS-SB cells also express the PDGF-beta receptor, whereas NALM-6 cells express the PDGF- alpha receptor and bind PDGF. This extends the possible targets for PDGF to the B-cell lineage lymphocytes.     

Response to 2'-deoxycoformycin after failure of interferon-alpha in nonsplenectomized patients with hairy cell leukemia

Two patients with hairy cell leukemia with massive splenomegaly and severe pancytopenia were treated with recombinant alpha-A interferon (IFN-alpha-2a). There was no significant response to a trial of IFN- alpha-2a (11 and 20 weeks) with respect to blood counts or spleen size. Subsequent treatment with 2'-deoxycoformycin (dCF) for 8 consecutive weeks (4 mg/m2/wk) resulted in normalization of spleen size and a normalization of peripheral blood counts and bone marrow in one patient. The second patient demonstrated a reduction in spleen size and improved blood counts following 9 weeks of dCF therapy but eventually became refractory. This demonstrates that dCF is non-cross-resistant with interferon and confirms the efficacy of dCF in nonsplenectomized patients.     

The effects of dietary supplementation with n-3 polyunsaturated fatty acids in patients with rheumatoid arthritis: a randomized, double blind trial

STUDY OBJECTIVE: To determine the effect of dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA) on disease variables in patients with rheumatoid arthritis. DESIGN: Multicenter, randomized, placebo controlled, double blind. SETTING: Three Danish hospital Departments of Rheumatology. PATIENTS: Fifty-one patients with active rheumatoid arthritis. INTERVENTION: Random allocation to 12 weeks of treatment with either six n-3 PUFA capsules (3.6 g) or six capsules with fat composition as the average Danish diet. MAIN RESULTS: Significant improvement of morning stiffness and joint tenderness. No significant effect on the four other assessed clinical parameters. No serious side effects. CONCLUSIONS: Dietary supplementation with n-3 PUFA in patients with rheumatoid arthritis improved two out of six patient reported disease parameters. Further studies are needed to clarify the more precise role of n-3 PUFA in the treatment of rheumatoid arthritis.     

Aortic saddle embolus. A twenty-year experience.

Clinical experience with aortic saddle embolus (ASE) is not extensive due to the relative infrequent lodging of emboli at the aortic bifurcation. During the period 1962-1982, 26 patients (mean age, 56 years) were treated at the UCLA Medical Center for ASE and followed from 2 to 158 months (mean, 45 months). These cases were reviewed in order to identify features of diagnosis, anticoagulation, and operation which impact on results. All 26 patients presented with bilateral lower extremity ischemia with or without extension of clot to the iliac bifurcation. Ninety-six per cent of emboli were of cardiac origin and one-third occurred in patients who had previous symptoms of chronic lower extremity ischemia. Rest pain and motor/sensory deficits were main complaints in 92% of the patients, but did not become manifest until more than 6 hours, unlike more distal emboli which have an earlier presentation. Preoperative angiography, even in the patient with a history of claudication, has a small role in planning the surgical approach to patients with ASE and, although performed in 11 patients, it influenced operation in only two. Operation within the "golden period" of 6 hours after embolization did not significantly influence outcome after ASE, since 20 patients were operated on more than 6 hours after embolization, with results similar to six patients who were operated on less than 6 hours after embolization. Early high-dose heparinization, used in all patients and maintained for a mean of 12 days, may have contributed to this effect. In 22 patients (85%) Forgarty catheter extraction via bilateral groin approaches was used with a mortality of 14%; only one death was directly attributed to the catheter embolectomy. In 15% of patients, a direct approach on the aorta was selected with a zero mortality rate. Postoperative functional result was excellent with an amputation rate of only 2% (one limb). Re-embolization occurred in seven patients (27%) after discharge, five of whom had not been maintained on Coumadin and two who were not anticoagulated adequately. The authors conclude that the keys to successful treatment of ASE include high dose heparin which is maintained through the perioperative period, embolectomy without preoperative angiography, and maintenance of long-term oral anticoagulation.     

Molecular characterization of CTX‐M‐15‐producing clinical isolates of Escherichia coli reveals the spread of multidrug‐resistant ST131 (O25:H4) and ST964 (O102:H6) strains in Norway

Nationwide, CTX‐M‐producing clinical Escherichia coli isolates from the Norwegian ESBL study in 2003 (n=45) were characterized on strain and plasmid levels. Bla_CTX‐M allele typing, characterization of the genetic environment, phylogenetic groups, pulsed field gel electrophoresis (PFGE), serotyping and multilocus sequence typing were performed. Plasmid analysis included S1‐nuclease‐PFGE, polymerase chain reaction‐based replicon typing, plasmid transfer and multidrug resistance profiling. Bla_CTX‐M‐15 (n=23; 51%) and bla_CTX‐M‐14 (n=11; 24%) were the major alleles of which 18 (78%) and 6 (55%), respectively, were linked to ISEcp1. Thirty‐two isolates were of phylogenetic groups B2 and D. Isolates were of 29 different XbaI‐PFGE‐types including six regional clusters. Twenty‐three different O:H serotypes were found, dominated by O25:H4 (n=9, 20%) and O102:H6 (n=9, 20%). Nineteen different STs were identified, where ST131 (n=9, 20%) and ST964 (n=7, 16%) were dominant. Bla_CTX‐M was found on ≥100 kb plasmids (39/45) of 10 different replicons dominated by IncFII (n=39, 87%), FIB (n=20, 44%) and FIA (n=19, 42%). Thirty‐nine isolates (87%) displayed co‐resistance to other classes of antibiotics. A transferable CTX‐M phenotype was observed in 9/14 isolates. This study reveals that the majority of CTX‐M‐15‐expressing strains in Norway are part of the global spread of multidrug‐resistant ST131 and ST‐complex 405, associated with ISEcp1 on transferrable IncFII plasmids.     

Assessing the validity of body mass index standards in early postmenopausal women

OBJECTIVE: To examine the relationship between percentage of total body fat (%Fat) and body mass index (BMI) in early postmenopausal women and to evaluate the validity of the BMI standards for obesity established by the NIH. RESEARCH METHODS AND PROCEDURES: Three hundred seventeen healthy, sedentary, postmenopausal women (ages, 40 to 66 years; BMI, 18 to 35 kg/m(2); 3 to 10 years postmenopausal) participated in the study. Height, weight, BMI, and %Fat, as assessed by DXA, were measured. Receiver operating characteristic analysis was performed to evaluate the ability of BMI to discriminate obesity from non-obesity using 38%Fat as the criterion value. RESULTS: A moderately high relationship was observed between BMI and %Fat (r = 0.81; y = 1.41x + 2.65) with a SE of estimate of 3.9%. Eighty-one percent of other studies examined fell within 1 SE of estimate as derived from our study. Receiver operating characteristic analysis showed that BMI is a good diagnostic test for obesity. The cutoff for BMI corresponding to the criterion value of 38%Fat that maximized the sum of the sensitivity and specificity was 24.9 kg/m(2). The true-positive (sensitivity) and false-positive (1--specificity) rates were 84.4% and 14.6%, respectively. The area under the curve estimate for BMI was 0.914. DISCUSSION: There is a strong association between %Fat and BMI in postmenopausal women. Current NIH BMI-based classifications for obesity may be misleading based on currently proposed %Fat standards. BMI >25 kg/m(2) rather than BMI >30 kg/m(2) may be superior for diagnosing obesity in postmenopausal women.     

Preliminary study of the behavioral effects of LBS-neuron implantation on seizure susceptibility following middle cerebral artery occlusion in the rat

Neural transplantation is a promising treatment strategy that can restore the motor, sensory and cognitive functions in the rat middle cerebral artery occlusion (MCAO) model of stroke. In particular, neuronal cells derived from a human teratocarcinoma cell line, called hNT neurons or LBS neurons (clinical grade preparation), are effective in improving behavioral recovery after stroke. In the elderly, epilepsy is a common sequelae of stroke, especially if the infarction involves cerebral cortex. However, the effect of implanting neural cells on seizure susceptibility in the MCAO model has not yet been determined. The purpose of this study was to determine the susceptibility to pentylenetetrazol (PTZ)-induced seizures in normal, MCAO-lesioned and MCAO-lesioned rats in which the LBS neurons were injected. Adult, male Sprague-Dawley rats were subjected to 60 min of MCAO using the intraluminal filament technique followed 3–4 weeks later by transplantation of 80,000 LBS-neurons into the ipsilateral cortex. Susceptibility to PTZ-induced seizures was tested 4–6 weeks post-transplant at doses of 35, 50 and 70 mg/kg, administered subcutaneously. Latency to the first ictal response, latency to first generalized seizure, duration of the first generalized seizure, and the number of generalized seizures in an hour post-PTZ treatment observation period was determined. Even though there was a tendency for groups that underwent MCAO to be more susceptible to seizures, there were no statistically significant differences between the groups and no differences between MCAO alone and MCAO animals in which cells had been implanted. While grafted cells were identified in all but one injected animal, the results suggest that the grafts may not have been healthy either from immunological rejection or PTZ-induced injury. These results suggest that while placing cells within the cortex does not reduce seizure susceptibility, it also does not increase the incidence of seizures. Further investigations are warranted.