Treatment of bone loss in proximal femurs of postmenopausal osteoporotic women with AGN1 local osteo-enhancement procedure (LOEP) increases hip bone mineral density and hip strength: a long-term prospective cohort study.

This first-in-human study of AGN1 LOEP demonstrated that this minimally-invasive treatment durably increased aBMD in femurs of osteoporotic postmenopausal women. AGN1 resorption was coupled with new bone formation by 12 weeks and that new bone was maintained for at least 5-7 years resulting in substantially increased FEA-estimated femoral strength. INTRODUCTION: This first-in-human study evaluated feasibility, safety, and in vivo response to treating proximal femurs of postmenopausal osteoporotic women with a minimally-invasive local osteo-enhancement procedure (LOEP) to inject a resorbable triphasic osteoconductive implant material (AGN1). METHODS: This prospective cohort study enrolled 12 postmenopausal osteoporotic (femoral neck T-score ≤ - 2.5) women aged 56 to 89 years. AGN1 LOEP was performed on left femurs; right femurs were untreated controls. Subjects were followed-up for 5-7 years. Outcomes included adverse events, proximal femur areal bone mineral density (aBMD), AGN1 resorption, and replacement with bone by X-ray and CT, and finite element analysis (FEA) estimated hip strength. RESULTS: Baseline treated and control femoral neck aBMD was equivalent. Treated femoral neck aBMD increased by 68 ± 22%, 59 ± 24%, and 58 ± 27% over control at 12 and 24 weeks and 5-7 years, respectively (p < 0.001, all time points). Using conservative assumptions, FEA-estimated femoral strength increased by 41%, 37%, and 22% at 12 and 24 weeks and 5-7 years, respectively (p < 0.01, all time points). Qualitative analysis of X-ray and CT scans demonstrated that AGN1 resorption and replacement with bone was nearly complete by 24 weeks. By 5-7 years, AGN1 appeared to be fully resorbed and replaced with bone integrated with surrounding trabecular and cortical bone. No procedure- or device-related serious adverse events (SAEs) occurred. CONCLUSIONS: Treating femurs of postmenopausal osteoporotic women with AGN1 LOEP results in a rapid, durable increase in aBMD and femoral strength. These results support the use and further clinical study of this approach in osteoporotic patients at high risk of hip fracture.

The X-ray Surface Forces Apparatus: Structure of a Thin Smectic Liquid Crystal Film Under Confinement.

An x-ray surface forces apparatus for simultaneously measuring forces and structures of confined complex fluids under static and flow conditions is described. This apparatus, combined with an intense synchrotron x-ray source, allows investigation of molecular orientations within a thin liquid crystal film confined between two shearing mica surfaces 3900 angstroms apart. The layer-forming smectic liquid crystal 8CB (4-cyano-4'-octylbiphenyl) adopted a series of distinct planar layer orientations, including the bulk flow-forbidden b orientation.

Calcium dependency and free calcium concentrations during insulin secretion in a hamster beta cell line.

Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 mM Ca2+ triggered a full first-phase insulin response. These data suggest that high glucose generates a non-Ca2+ signal that turns over rapidly and provide direct evidence that K+ triggers insulin release by drawing extracellular Ca2+ into the beta cell through verapamil-sensitive Ca2+ channels. However, an increase [Ca2+]i is not the primary signal that evokes glucose-stimulated insulin release in this beta cell line.

Proton magnetic resonance spectroscopic imaging in children with recurrent primary brain tumors.

PURPOSE: Proton magnetic resonance spectroscopic imaging ((1)H-MRSI) is a noninvasive technique for spatial characterization of biochemical markers in tissues. We measured the relative tumor concentrations of these biochemical markers in children with recurrent brain tumors and evaluated their potential prognostic significance. PATIENTS AND METHODS: (1)H-MRSI was performed on 27 children with recurrent primary brain tumors referred to our institution for investigational drug trials. Diagnoses included high-grade glioma (n = 10), brainstem glioma (n = 7), medulloblastoma/peripheral neuroectodermal tumor (n = 6), ependymoma (n = 3), and pineal germinoma (n = 1). (1)H-MRSI was performed on 1. 5-T magnetic resonance imagers before treatment. The concentrations of choline (Cho) and N-acetyl-aspartate (NAA) in the tumor and normal brain were quantified using a multislice multivoxel method, and the maximum Cho:NAA ratio was determined for each patient's tumor. RESULTS: The maximum Cho:NAA ratio ranged from 1.1 to 13.2 (median, 4.5); the Cho:NAA ratio in areas of normal-appearing brain tissue was less than 1.0. The maximum Cho:NAA ratio for each histologic subtype varied considerably; approximately equal numbers of patients within each tumor type had maximum Cho:NAA ratios above and below the median. Patients with a maximum Cho:NAA ratio greater than 4.5 had a median survival of 22 weeks, and all 13 patients died by 63 weeks. Patients with a Cho:NAA ratio less than or equal to 4.5 had a projected survival of more than 50% at 63 weeks. The difference was statistically significant (P =.0067, log-rank test). CONCLUSION: The maximum tumor Cho:NAA ratio seems to be predictive of outcome in children with recurrent primary brain tumors and should be evaluated as a prognostic indicator in newly diagnosed childhood brain tumors.

Perifusion of a clonal cell line of Simian virus 40-transformed beta cells. Insulin secretory dynamics in response to glucose, 3-isobutyl-1-methylxanthine, and potassium.

A perifusion system for the study of insulin secretory dynamics of a clonal, Simian virus 40-transformed hamster pancreatic beta cell line (HIT cells) is described. After a change from glucose-free to higher glucose levels in the perifusate, insulin secretion increased rapidly in a dose-dependent manner. The pattern of glucose-stimulated insulin release was monophasic and was not sustained during a continued glucose stimulus. Perifusing the cells with low glucose (0.3 mg/ml) before a glucose stimulus of 3.5 mg/ml resulted in more rapid insulin release with lower peak secretory rates than those seen after a glucose-free period. The combined stimulus of high glucose and 100 microM 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, significantly enhanced the acute insulin secretory response and also resulted in a biphasic secretory pattern that was sustained throughout the 60-min stimulation period. Insulin secretion stimulated by IBMX required a nonstimulatory level of glucose in the perifusing media, and, if this requirement was met, the immediate release of insulin was similar to that evoked by high glucose alone. High potassium (40 mM) also triggered a monophasic release of insulin. These studies demonstrate that glucose or high K+, which depolarizes the plasma membrane, and IBMX, an agent presumed to increase intracellular cyclic AMP levels, can signal the acute release of insulin from these beta cells. This cell line is a unique model system for studying the mechanism of insulin secretion.

Induction of islet cytodifferentiation by fetal mesenchyme in adult pancreatic ductal epithelium.

Recombinant tissue consisting of adult ductal epithelium isolated from pancreas and fetal mesenchyme was transplanted subcutaneously in the inguinal region of nude mice or epididymal fat pads of rats with a tissue chamber device for short-term (8-day) or long-term (6- to 12-wk) duration. We found that recombinant tissue underwent morphogenesis and cytodifferentiation, thereby forming islets that contained cells immunocytochemically positive for insulin and glucagon. Islet cytodifferentiation occurred in approximately 20% of the recombinants. In recombinants that developed into islets, the tissue was always in close association with an extracellular matrix, nerves, and blood vessels. Controls consisting of mesenchyme alone or duct epithelium alone showed no evidence of morphogenesis of cytodifferentiation. Pancreatic rudiments were also implanted to serve as positive controls. This is the first demonstration of islet cytodifferentiation from adult duct epithelium.

Effect of rise in cAMP levels on Ca2+ influx through voltage-dependent Ca2+ channels in HIT cells. Second-messenger synarchy in beta-cells.

With a glucose-responsive beta-cell line (HIT cells), we tested the hypothesis that the cytosolic free-Ca2+ level ([Ca2+]i) is an intracellular signal through which a rise in cyclic AMP (cAMP) levels is transmitted to potentiate glucose-stimulated insulin secretion. In these cells, glucose stimulates the acute release of insulin without increasing [Ca2+]i or altering cAMP content. Either forskolin or 3-isobutylmethylxanthine (IBMX) potentiated glucose-stimulated insulin secretion and increased cAMP levels. At either a submaximal glucose concentration or maximally stimulatory glucose concentration, both IBMX and forskolin triggered a rapid rise in [Ca2+]i (1.9- and 1.5-fold increase over basal levels, respectively). Similarly, glucagon stimulated a 1.3-fold increase in [Ca2+]i over basal levels. The effect on [Ca2+]i required glucose and was secondary to Ca2+ influx through voltage-dependent Ca2+ channels because it was blocked by either chelation of extracellular Ca2+ with EGTA or by the Ca2+-channel blockers verapamil and nimodipine. Verapamil also inhibited IBMX potentiation of glucose-stimulated insulin secretion and the IBMX-induced rise in [Ca2+]i in a dose-dependent manner with IC50s of 2 x 10(-5) and 4 x 10(-6) M, respectively. We conclude that in the beta-cell, a rise in cAMP levels increases Ca2+ influx through voltage-dependent Ca2+ channels and that this represents a mechanism by which cAMP potentiates glucose-stimulated insulin secretion in beta-cells.

Increase in cAMP levels in beta-cell line potentiates insulin secretion without altering cytosolic free-calcium concentration.

With an SV40-transformed hamster beta-cell line (HIT cells) as a model system, we tested the hypothesis that a rise in cAMP levels potentiates insulin release by an effect on the cytosolic free-Ca2+ concentration ([Ca2+]i). Intracellular cAMP levels were measured by radioimmunoassay, and [Ca2+]i was monitored with the fluorescent Ca2+ indicator quin 2. Insulin secretion was followed in static incubations or perifusion of the cells. In perifusion, both high glucose and depolarization of the beta-cell with 40 mM K+ trigger a monophasic pattern of insulin release without altering the HIT cell cAMP content. Addition of either the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the adenylate cyclase activator forskolin dramatically increased the cellular cAMP content, potentiated the burst phase of insulin release, and coupled the immediate phase of insulin secretion to a sustained secretory response. However, increases in cellular cAMP content were not associated with a change in [Ca2+]i. Thus, the potentiation of insulin secretion by a rise in cAMP in the HIT cell is not mediated by a release of stored Ca2+. Either a glucose-generated signal or a rise in [Ca2+]i triggered by high K+ can synergize with a rise in cAMP to couple the burst or immediate release of insulin evoked by either secretagogue to the sustained release of insulin.

Marrow harvesting for autologous marrow transplantation.

The results and complications of 224 marrow collections from 200 patients with malignant disease who underwent marrow aspiration and storage for subsequent autologous marrow transplantation (AMT) were analyzed. The median age of the patients was 35 years (range 1-68); 131 patients had hematologic malignancies and 69 had solid tumors. Thirty-one patients proceeded directly to AMT after marrow aspiration at a median of 4.5 days (range 0-10). A further 75 patients received AMT a median of 3.0 months (12 days-10 years) after marrow aspiration. The remaining 94 patients had marrow stored but not infused. When a second aspiration was performed from the same patient within seven weeks, the yield of marrow nucleated cells was significantly reduced (p less than 0.02). A negative linear correlation was observed between CFU-C/kg harvested and the day to achieve a posttransplant blood neutrophil count greater than 500/cmm (r = -0.3092, p less than 0.05). A total of 36 (17.4%) complications associated with marrow aspiration were observed including two (0.97%) life-threatening episodes. Postoperative fever accounted for 23 of 34 episodes of minor complications. There was no increased risk of serious complications with decreased time from aspiration to transplant. It was concluded that the morbidity and mortality from autologous marrow aspiration did not differ significantly from that observed in normal donors.

Venocclusive disease of the liver after chemoradiotherapy and autologous bone marrow transplantation.

We determined the incidence of venocclusive disease of the liver (VOD) in 96 recipients of autologous bone marrow transplants (BMT) to be 9.4%, a figure less than that reported for allogeneic transplantation. The development of VOD was compared in a cohort of 21 autologous BMT recipients and in 56 randomly chosen, comparably conditioned, concurrent allogeneic BMT recipients. One of these 21 (4.8%) autologous recipients developed VOD, versus 14 of 56 (25%) allogeneic recipients (P less than 0.05). Logistic regression analysis confirmed pretransplant hepatocellular dysfunction as a risk factor for VOD, and suggested that the use of methotrexate and/or cyclosporine contributes to the development of VOD after chemoradiation therapy. However, a graft-versus-host reaction cannot be excluded as a cause of the higher incidence of VOD in allogeneic recipients.

Allogeneic bone marrow transplantation for Philadelphia-chromosome positive acute lymphoblastic leukemia.

Allogeneic bone marrow transplantation (BMT) produced remission in three patients with Philadelphia-chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL) in relapse. Two patients had remissions which lasted longer than two years. Since the prognosis of Ph1-positive ALL treated with conventional therapy is poor, BMT is indicated in first remission in this disease.

Bronchiolitis obliterans complicating bone marrow transplantation.

A 19-year-old woman with extensive, persistent chronic graft versus host disease (GVHD), following an HLA-identical bone marrow graft for acute leukemia, developed rapidly progressive airflow obstruction 140 days post-transplantation (PT) and presented clinically with persistent cough, inspiratory rales, bronchospasm and exertional dyspnea. Pulmonary function tests (PFT) showed rapidly evolving severe airflow obstruction and hypoxemia without restrictive ventilatory defect. Open lung biopsy on the 204th day PT confirmed focal bronchiolitis obliterans. On the 381st day PT, she remained clinically stable. Chest x-ray film showed mild overinflation, but was otherwise unremarkable. PFT's continued to show very severe airflow obstruction without restrictive ventilatory defect. The etiology of the obliterative bronchiolitis might be explained on the basis of a direct immunologic reaction mediated by GVHD or possibly a joint viral-GVHD interaction. Awareness and further detailed documentation and analysis of this unusual respiratory syndrome associated with marrow transplant recipients may help clarify the role of GVHD in the development of lung disease in recipients of marrow grafts.

Engraftment in 86 with patients lymphoid malignancy after autologous marrow transplantation.

The kinetics of marrow engraftment was retrospectively analysed in 55 patients with malignant lymphoma (ML) and 31 patients with acute lymphoblastic leukemia (ALL) after marrow-ablative therapy followed by autologous bone marrow transplantation. Thirty-eight percent of patients with ML, most of whom were transplanted in relapse and 13% of patients with ALL, mostly transplanted in remission, showed failed or delayed engraftment. Analysis of the total patient group showed that failure to recover platelet counts was significantly correlated with detection of disease in the marrow early after transplantation (p less than 0.001). Platelet recovery was also correlated with survival (p = 0.0001), disease-free survival (p = 0.0001), and the probability of relapse (p = 0.02). In those patients achieving engraftment, multivariate regression analysis failed to reveal any single in vitro test of marrow nucleated cell or progenitor cell numbers that significantly influenced time to achieve recovery of either granulocyte or platelet counts.

Allogeneic and syngeneic marrow transplantation following high dose dimethylbusulfan, cyclophosphamide and total body irradiation.

Fifty-eight patients received an allogeneic or syngeneic marrow transplant following conditioning with high doses of dimethylbusulfan (DMB), cyclophosphamide (CY) and total body irradiation (TBI). Thirty-two patients had either chronic myeloid leukemia (CML) in accelerated phase or blast transformation, or acute leukemia after first relapse. The actuarial survival of these 32 patients at 3 years was 12% compared with 25% for a group of 206 patients with similar diagnoses prepared for transplantation with CY and TBI alone. This reduced survival was associated with a greater incidence of early non-leukemic deaths, in particular as a result of severe hepatic veno-occlusive disease. The incidence of leukemic relapse was not different in the two groups. Of 13 patients with CML in chronic phase who received syngeneic transplants following DMB, CY and TBI, nine are alive in hematologic and cytogenetic remission from 3.9 to 9.4 (median 6.2) years post-transplant.

Immunoisolation of adult porcine islets for the treatment of diabetes mellitus. The use of photopolymerizable polyethylene glycol in the conformal coating of mass-isolated porcine islets.

Functional porcine islets, free of known pathogens, can serve as a source of insulin producing cells for the treatment of experimentally induced insulin dependent Diabetes Mellitus. Porcine islets can be conformally coated (microencapsulated) with a covalently linked, stable permselective membrane while maintaining islet viability and function. The PEG conformal coating is immunoprotective in a discordant xenograft animal model (porcine islets to rat).

Nitroblue tetrazolium (NBT) activity in human leucocytes after freezing.

Human peripheral blood leucocytes (neutrophil-rich) were collected either with heparin or acid citrate dextrose, frozen with dimethyl sulphoxide at a controlled rate, stored in liquid nitrogen at--196 degrees C and reconstituted with a solution containing dextran. After reconstitution, 20.2% of cells (in absolute numbers 1 in 5 fresh cells) showed a strongly positive nitroblue tetrazolium (NBT) reaction. The quantitative NBT test confirmed the synthesis of formazan/10(6) reconstituted neutrophilsa s15% of the fresh capacity. A slow titration reconstitution method for cells did not improve the functional capacity of thawed leucocytes as judged by the NBT test. When comparing anticoagulants, heparin increased the post-reconstitution cell yields after freezing and increased the absolute number of reconstituted cells capable of developing a positive NBT reaction.

The survival of cryopreserved human bone marrow stem cells.

Two methods for cryopreservation of bone marrow stem cells were compared using bone marrow obtained from 36 patients. Included in this group were 21 persons with the diagnosis of leukaemia including 14 either with acute myeloid or lymphoblastic leukaemia in remission following intensive remission induction chemotherapy. After freeze-preservation and reconstitution, all marrow samples were tested for nucleated cell (NC) recovery and grown on agar to assess colony forming units (CFUC) and cluster forming units in culture (CluFUc). A slow dilution reconstitution method using freezing media containing AB negative plasma resulted in recovery of 85% of the CFUc activity of fresh marrow. This result was significantly better than the 47% CFUc recovery obtained when freezing media without plasma and a rapid dilution reconstitution technique were used. NC recoveries following slow dilution (51%) and rapid dilution (44%) were not significantly different. CluFUc were disproportionately reduced compared with CFUc although yielding similar results with both methods (26% and 32%). No correlation was found for either method between CFUc and NC recovery or between CFUc and CluFUc recovery in cryopreserved bone marrow.

Airway complications from laryngoscopy and panendoscopy.

Laryngoscopy and panendoscopy can cause airway complications. To determine the risk to the airway from reintubation following general anesthesia in otolaryngology patients, we examined recovery room and anesthesia records at the Albany Veterans Administration Medical Center covering a 10-year period. From this information we determined the incidence of recovery room reintubation and studied airway risk factors associated with otolaryngologic endoscopy. From 1975 to 1984, 10,060 surgical patients were intubated at the Albany VA Medical Center. Only 17 patients (0.17%) required reintubation. Of 1,365 otolaryngology patients intubated during the same period, 324 had laryngoscopy and 302 had panendoscopy. Significantly, four laryngoscopy patients (1.2%) and nine panendoscopy patients (3%) required recovery room intubation. Nine endoscopy patients needed reintubation within 1 hour of extubation. We conclude that the risk of postoperative airway compromise is significantly greater among patients who underwent diagnostic laryngoscopy and panendoscopy than among patients who had general anesthesia for other reasons.

Preparation of leucocyte poor blood for transfusion.

Three methods for the preparation of leucocyte poor red cells for transfusion have been evaluated. A new filtration (IMUGARD) method produced an easily prepared high quality leucocyte-poor preparation but which was not suitable for prolonged storage. A simple centrifugation method was suitable for the preparation of a sterile long life leucocyte-poor preparation and could be used for the majority of patients. A dextran sedimentation method was efficient in leucocyte removal but with the loss of a significant proportion of red cells.

Therapeutic plasmapheresis by continuous flow cnetrifugation.

This report reviews the experience at Auckland Hospital with continuous flow centrifugation as a mean of carrying out therapeutic plasmapheresis. Over a three year period from August 1975, 26 patients, most with dysproteinaemias or Goodpasture's syndrome, were treated with a total of 260 therapeutic procedures.