Incidence and costs of severe hypoglycaemia requiring attendance by the emergency medical services in South Central England.

AIMS: The aim was to estimate the incidence of severe hypoglycaemia requiring emergency ambulance assistance, its management and associated costs. METHODS: A retrospective observational study used routinely collected data for a 1-year period from December 2009 to November 2010 from the South Central Ambulance Service National Health Service Trust, UK. The main outcome was episodes reported by ambulance personnel and costs were estimated from published data. RESULTS: During the 1-year study period, 398,409 emergency calls were received, of which 4081 (1.02%) were coded as hypoglycaemia. The overall numbers (and annual rate) of hypoglycaemia recorded among people ≥ 15 years with presumed diabetes was 3962 (2.1%), but for those aged 15-35 years was 516 (7.5%) and for those aged ≥ 65 years was 1886 (1.9%). Of those attended, 1441 (35.3%) were taken to hospital. The estimated total cost of initial ambulance attendance and treatment at scene was £553,000; if transport to hospital was necessary, the additional ambulance costs were £223,000 plus emergency department costs of £140,000; and the cost of primary care follow-up was estimated as £61,000. The average cost per emergency call was £263. The estimated annual cost of emergency calls for severe hypoglycaemia is £13.6m for England. CONCLUSIONS: Our estimates suggest prevalence of severe hypoglycaemia attended by the emergency services is high in younger age groups and lower for older age groups, although the absolute numbers of severe events in older age groups contribute substantially to the overall costs of providing emergency assistance for hypoglycaemia.

Comparative stability of sodium tetradecyl sulphate (STD) and polidocanol foam: impact on vein damage in an in-vitro model.

OBJECTIVES: To compare the half-life of STD and polidocanol air-based foams and the damage they inflict upon human great saphenous vein in an in-vitro model. METHODS: The time for the volume of 3% STD and polidocanol foams to reduce by 10% (T(90)) and 50% (T(50)) was recorded in an incubator at 37 °C. Segments of proximal GSV harvested during varicose vein surgery were filled with foam for 5 or 15 min. Histological analysis determined percentage endothelial cell loss and depth of media injury. RESULTS: Median (±IQR) T(90) and T(50) for polidocanol were 123.3 s (111.7-165.6) and 266.3 s (245.6-383.1) versus 102.03 s (91.1-112) and 213.13 s (201-231.6) for STD (T(90)p = 0.008, T(50)p = 0.004). Median endothelial loss with polidocanol was; 63.5% (62.2-82.8) and 85.9% (83.8-92.5) versus 86.3% (84.8-93.7) and 97.64% (97.3-97.8) for STD after 5 and 15 min (p = 0.076 and p = 0.009). The median depth and % media thickness injured were 0 µm (0-0 µm) and 0% for both assessments with polidocanol versus 37.4 µm (35.3-45.8 and 43.4 µm (42.1-46.7) and 3.5% (3.1-3.6) and 5.3% (3.7-6.0) after 5 and 15 min for STD (p < 0.01 for all comparisons). CONCLUSION: Although polidocanol foam shows greater stability than STD foam perhaps remaining in the vein for longer, endothelial cell loss and damage to the media were significantly greater with STD.

Remodeling of an acellular collagen graft into a physiologically responsive neovessel.

Surgical treatment of vascular disease has become common, creating the need for a readily available, small-diameter vascular graft. However, the use of synthetic materials is limited to grafts larger than 5-6 mm because of the frequency of occlusion observed with smaller-diameter prosthetics. An alternative to synthetic materials would be a biomaterial that could be used in the design of a tissue-engineered graft. We demonstrate that a small-diameter (4 mm) graft constructed from a collagen biomaterial derived from the submucosa of the small intestine and type I bovine collagen has the potential to integrate into the host tissue and provide a scaffold for remodeling into a functional blood vessel. The results obtained using a rabbit arterial bypass model have shown excellent hemostasis and patency. Furthermore, within three months after implantation, the collagen grafts were remodeled into cellularized vessels that exhibited physiological activity in response to vasoactive agents.

Vitreous cryopreservation maintains the function of vascular grafts.

Avoidance of ice formation during cooling can be achieved by vitrification, which is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth. We show that a vitrification approach to storing vascular tissue results in markedly improved tissue function compared with a standard method involving freezing. The maximum contractions achieved in vitrified vessels were >80% of fresh matched controls with similar drug sensitivities, whereas frozen vessels exhibited maximal contractions below 30% of controls and concomitant decreases in drug sensitivity. In vivo studies of vitrified vessel segments in an autologous transplant model showed no adverse effects of vitreous cryopreservation compared with fresh tissue grafts.

Comparative in vitro effects of cyclophosphamide derivatives on murine bone marrow-derived stromal and hemopoietic progenitor cell classes.

We investigated the in vitro effects of ASTA-Z-7595, ASTA-Z-7557, ASTA-Z-7654, and 4-hydroperoxycyclophosphamide (4HC) on murine stromal fibroblastoid colony-forming units, committed hemopoietic progenitors (erythroid burst-forming units and granulocyte/macrophage colony-forming units), and pluripotent hemopoietic stem cells assayed by the spleen colony-forming unit (CFU-s) assay. In general, the drugs showed a time-and dose-dependent effect on colony-forming unit survival, and the relative toxicities were in the order in which the drugs are listed above. We found a relative sparing of day 12 CFU-s compared with day 7 CFU-s and committed hemopoietic and stromal progenitors, although colony size of day 12 CFU-s was reduced. Our results support two possible mechanisms for delayed or inadequate hemopoietic reconstitution in clinical studies using bone marrow purged with 4-hydroperoxycyclophosphamide or ASTA-Z-7557, i.e., damage to (a) transplantable stromal cells or (b) the hemopoietic stem cells.

Hemopoiesis on skin-derived fibroblasts in vitro.

Bone marrow fibroblasts have been shown to have a role in the support and regulation of hemopoiesis, both in vivo and in vitro. In this study we examine the ability of skin-derived fibroblasts to interact with hemopoiesis in vitro. Murine skin and bone marrow-derived fibroblasts were similar with respect to their abilities to support granulopoiesis and release colony-stimulating activity. Detailed analysis of skin fibroblast cultures 1 week after seeding with stromal cell-depleted bone marrow demonstrated that both multipotential hemopoietic stem cells and erythroid stem cells were maintained, while granulocyte/macrophage colony-forming units far exceeded inoculum values. Immunostaining demonstrated the presence of foci of T200 positive hemopoietic cells on the surface of the fibroblasts with less frequent scattered M1/70 and F4/80 positive macrophages. The majority of cells (greater than 90%) released from the stromal layer were of the granulocytic series. These findings demonstrate that the hemopoietic regulatory properties previously attributed to bone marrow-derived fibroblasts are not unique to fibroblasts derived from hemopoietic tissues.

Enhanced tissue strength in cryopreserved, collagen-based blood vessel constructs.

Traditional cryopreservation methods do not adequately preserve complex natural or engineered multicellular tissues due to the ice formation in the extracellular matrices. Vitrification is an alternate ice-free method for cryopreservation. This study compares the effects of vitrification and conventional cryopreservation on an engineered blood vessel construct. Collagen-based vascular constructs were used as models in this study. Tubular constructs were cut into rings and distributed into fresh, frozen, and vitrified groups for evaluation of mechanical properties and cell viability. Passive mechanical tests revealed enhanced tissue strength after both freezing and vitrification. Cryosubstitution studies of frozen and vitrified constructs revealed negligible ice in the vitrified specimens and extensive ice formation in the extracellular matrix of frozen specimens. Morphological changes associated with ice formation were visible within tissues preserved using traditional cryopreservation but not in tissue preserved using vitrification. The metabolic assay results indicated that vitrified tissue had similar viability to fresh controls. These results suggest that the increased tissue strength after cryopreservation may relate to thermal property change during preservation that cross-link collagen in tissue-engineered blood vessels. Further development of this cryopreservation method is necessary to minimize the alteration in material property and maintain cell viability of the constructs.

Vitrification of tissue engineered pancreatic substitute.

Despite significant advances, some critical issues remain for the long-term storage of an engineered pancreas. In this study we employed a tissue engineered pancreatic substitute model-insulin-secreting betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate beads-to demonstrate that a prototype vitrification method can prevent ice formation and maintain cell viability/function. The results showed that the structure of the frozen samples was distorted by ice crystals throughout the matrix. In marked contrast, the vitrified samples appeared to be free of ice. Morphologic studies demonstrated extensive fractures and vacuolation in frozen specimens while there were no fractures in vitrified TEPSs. Both vitrified and frozen constructs showed some vacuolization compared to the control samples. Frozen beads showed a significantly decreased viability compared to fresh controls and the VS55 group (P < .001). There was no significant difference between the vitrified and fresh samples. Vitrification using the VS55 protocol shows similar viability and secretion properties to the control group of fresh beads. Vitrification using the PEG 400 protocol resulted in slightly lower viability and secretion properties relative to the control group; conventional freezing resulted in even significantly lower viability and secretion properties. These results combine to demonstrate feasibility of vitrification as a storage method for a tissue engineered pancreas.

Preservation of embryonic kidneys for transplantation.

BACKGROUND: Long-term storage of embryonic kidneys is crucial for the organization of transplantation and organ banking. In this study, we investigated the effects of controlled-rate freezing and ice-free vitrification on metanephroi (MN) viability. METHODS: Metanephroi isolated from 15-day (E15) timed pregnant Lewis rats were either: (i) frozen, using a DMSO/FCS/RPMI solution and a controlled freezing rate of -0.3 degrees C/min, from -10 degrees to -40 degrees C; or (ii) cryopreserved in an ice-free state by rapid cooling to -100 degrees C in cryoprotectant (VS55), followed by vitrification to -120 degrees C. After cryopreservation, the metanephroi were stored at -135 degrees C for 48 hours. After storage the MN were rewarmed, resuspended in culture media, and their viability was assessed using the AlamarBlue assay and histology (light microscopy, TEM, and cryosubstitution). RESULTS: There was statistically no difference in embryonic kidney metabolic activity of either of the cryopreserved MN groups relative to the control untreated group. However, cryosubstitution demonstrated the presence of significant ice formation during controlled-rate freezing, yet in contrast the amount of ice was significantly reduced by vitrification. This was confirmed by TEM, where vacuolation of the cytoplasm of controlled-rate frozen metanephroi was observed, whereas vitrified metanephroi had little cytoplasmic disruption. However, vitrified metanephroi showed mitochondrial and nuclear injury at the cellular level. CONCLUSIONS: There is a need for long-term storage of organs to make MN transplantation a reality. This study demonstrates that standard freezing methods are unsuitable for this purpose. Vitrification yielded more promising results, but further development is required.

Hepatic function in hypothermically stored porcine livers: comparison of hypothermic machine perfusion vs cold storage.

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.

Quantitation of stromal and hemopoietic progenitors in spleen and femoral marrow derived from Steel (Slj/+ and Sl/Sld) mice and their normal littermates.

In vitro analyses of stromal fibroblast colony-forming units (CFUF) and hemopoietic progenitors (erythroid burst-forming units, BFUE; granulocyte/macrophage colony-forming units, CFUGM) in hemopoietic organs of 'Steel' (Slj/+ and Sl/Sld) mice and their hematologically normal littermates were performed. CFUF incidence was not significantly different from +/+ controls in hemopoietic organs from either 'Steel' allele. There was, however, a tendency for the CFUF content to be above normal in 'Steel' spleens. BFUE incidence and absolute numbers were found to be significantly reduced in both Sl/Sld and Slj/+ spleens. We confirm previous reports of a reduced femoral BFUE content and reduced splenic and femoral CFUGM content in Sl/Sld mice. The total body content of BFUE and CFUGM was 21 and 50 percent of +/+ littermates, respectively, in Sl/Sld mice and 68 and 64 percent in Slj/+ mice. These results indicate that the microenvironmental defect in Slj/+ and Sl/Sld mice a) involves hemopoietic progenitors committed to both the erythrocytic and granulocyte/macrophage differentiation pathways; b) is greater in spleen than in femoral marrow; and c) cannot be explained by a deficiency in stromal progenitor content of the hemopoietic organs.

Hemopoiesis in demineralized bone allografts.

Demineralized allogeneic bone matrix (DBM), implanted in muscle, induces the formation of an ossicle within which histologically recognizable hemopoietic tissue develops. Analyses of rabbit ossicle marrow in a methylcellulose culture system demonstrated the presence of committed hemopoietic precursors; colony-forming units in culture (CFUC), erythroid colony-forming units (CFUE) and erythroid burst-forming units (BFUE) by six weeks post-implantation. The time courses of colony and burst formation by progenitor cells of ossicle and femoral marrow were similar. Induction of hemolytic anemia by phenylhydrazine hydrochloride at six weeks post-DBM implantation showed that the ossicle marrow was responsive to systemic erythropoietic stimuli. The DBM implant may provide a unique model for studying the development of hemopoietic microenvironments within bone.

Effects of burst-promoting activity (BPA) on hemoglobin biosynthesis in culture.

We have discovered that human and rabbit bone marrow conditioned media (BMCM) promote the growth of bursts in cultures containing erythropoietin. We then demonstrated that the measurement of 59Fe incorporation into heme was a more quantitative assay for burst-promoting activity (BPA) than counting burst numbers. Furthermore, we have observed that the use of low (less than 15%) concentrations of fetal calf serum is a convenient way of reducing endogenous sources of BPA for studies of the BPA in the test samples. When 59Fe incorporation and cell numbers of individual rabbit erythropoietic bursts were analyzed simultaneously, BPA caused a shift in the cumulative frequency distributions without changes in their shapes. These results indicated that BPA augments hemoglobin synthesis by stimulating cell proliferation during the early phase of burst formation. In addition, human BPA increased the relative proportion of fetal hemoglobin in the hemoglobins synthesized by adult circulating BFU-e. BPA appears to augment cell proliferation of early precursors which are committed to produce F cells.

Splenic accumulation of stromal progenitor cells in response to bacterial lipopolysaccharides.

An analysis was made of fibroblastoid colony-forming units (CFUF) in spleens of mice treated with bacterial lipopolysaccharide (LPS). Increased CFUF numbers were observed on days 2, 3 and 4 after treatment with 10, 30 and 100 micrograms of LPS. The splenic CFUF accumulation occurred at the same time as spleen hemopoiesis increased, however, the CFUF returned to normal and subnormal numbers on day 5, while the spleen cellularity was still elevated on day 21 after LPS treatment. The mechanism of splenic CFUF accumulation appeared to involve CFUF migration via the blood from other sites of hemopoiesis rather than increased CFUF plating efficiency in vitro or enhanced CFUF proliferation. The results suggest a relationship between the LPS-induced fibroblastoid progenitor cell increase and a requirement for splenic microenvironments to support LPS-induced hemopoiesis.

Radiation damage to femoral hemopoietic stroma measured by implant regeneration and quantitation of fibroblastic progenitors.

Radiation damage to femoral hemopoietic stroma in mice was measured 6 weeks after irradiation and reconstitution with syngeneic bone marrow by quantitation of fibroblastic progenitor cells (CFUF), and by assaying hemopoietic progenitors and CFUF numbers in regenerated subcutaneous femur implants. A dose-dependent effect of radiation on both preimplantation CFUF numbers and implant regeneration was observed. Changes in bone marrow cell (BMC) graft size did not alter these stromal parameters. Therefore, transplanted CFUF, which were present in the BMC graft, did not alter either the CFUF content of femurs or their regenerative capacity. The strong correlation between CFUF numbers and implant regenerative capacity after irradiation, leads us to suggest a function for CFUF in femoral implant stromal regeneration.

Regulation of in vitro myelopoiesis by a hemopoietic stromal fibroblastic cell line.

An adherent cell line (AP63) derived from murine spleen was characterized as fibroblastic, and several of its properties distinguished it from other adherent cells (i.e., macrophages and endothelial cells). The ability of the AP63 cells to regulate in vitro myelopoiesis was investigated. Medium conditioned by the cell line (CM) induced granulocyte-macrophage (GM) colonies, thus demonstrating the production of colony-stimulating activity by AP63 cells. A relatively large proportion of these colonies had a "tight" morphology and contained many early myeloid cells and cells capable of secondary cluster and colony formation. CM also contained a prostaglandinlike inhibitor of colony formation. Furthermore, AP63 cells inhibited GM colony formation by bone marrow cells in their immediate vicinity, whereas colony formation was stimulated at greater distances. These observations may reflect in vivo regulatory properties of hemopoietic stromal fibroblasts with respect to proliferation and differentiation of GM progenitor cells.

Hemopoiesis on purified bone-marrow-derived reticular fibroblast in vitro.

Murine bone-marrow-derived reticular fibroblast cultures were tested for the ability to support hemopoiesis and release hemopoietic growth factors in vitro. The reticular fibroblast cultures employed in these studies were 95%-100% pure on the basis of Mac-1, F4/80, MIV-51, T200, antifactor VIII and ER-TR7 monoclonal antibody staining. Further support for the fibroblastic nature of these cells was obtained from collagen and laminin analyses. Addition of stromal cell-depleted bone marrow cell suspensions to flasks of confluent reticular fibroblasts resulted in production and release of granulocytes, monocyte-macrophages, and granulocyte-monocyte progenitors from the adherent layer for 4-8 weeks. Pluripotent spleen colony-forming units were detected during the first four weeks. Assay of reticular fibroblast conditioned medium for hemopoietic growth factors demonstrated production of granulocyte-macrophage colony-stimulating activity and stem-cell-activating factor. We did not detect any erythroid burst-promoting activity. These results suggest that reticular fibroblasts may play a role in the maintenance of pluripotent stem cells and in the proliferation and differentiation of cells committed to the granulocyte-monocyte lineage.

Characterization of fibroblastic stromal cells from murine bone marrow.

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.

An in vitro analysis of murine hemopoietic fibroblastoid progenitors and fibroblastoid cell function during aging.

We determined the number of fibroblastoid progenitors (fibroblastoid colony-forming units, CFU-F) in femurs and spleens derived from (CBA X C57BL)F1 mice of different ages. The femoral CFU-F population size increased from 350 at 1 week of age and plateaued at approximately 1900 CFU-F at 8 weeks of age. The mean incidence of CFU-F per 10(6) femoral marrow nucleated cells decreased from 82 at 8 weeks of age to 55 at 70 weeks of age; however, due to an increase in femur cellularity, there was no decrease in the CFU-F population size. The splenic CFU-F population decreased from 1700 at 1 week of age to 180 at 8 weeks of age; no further change was observed in mice up to 70 weeks' old. Analysis of colony-stimulating activity production by fibroblastoid colonies derived from young (6 weeks) and aged (70 weeks) mouse femoral marrow demonstrated no difference. These results indicate that there is no change in CFU-F numbers or fibroblastoid cell colony-stimulating activity production associated with the age-related increase in hemopoietic organ cellularity and hemopoietic progenitor content observed in this mouse strain. There were, however, major changes in the CFU-F population sizes during development of both femoral marrow and spleen in the first 2 months after birth.

Recovery of hemopoietic stromal progenitor cells after lethal total-body irradiation and bone marrow transplantation in mice.

The recovery of fibroblastic colony-forming units (CFU-F) in murine bone marrow hemopoietic stroma was studied during eighteen months after 9 Gy lethal total-body irradiation and reconstitution with syngeneic bone marrow cells. After an initial depletion, CFU-F numbers increased from 10% of normal values at three months to 40% at 18 months after treatment, irrespective of graft size and presence of CFU-F in the graft. Fourteen months after treatment 35% of all CFU-F present in the recipients' bone marrow was donor-derived independent of graft size. When mice were treated with high-dose lipopolysaccharide-W three months after irradiation and bone marrow transplantation, CFU-F numbers decreased to hardly detectable levels within one day, and then recovered to normal numbers four weeks later--whereas radiation control mice still had low CFU-F numbers. These data suggest that after lethal total-body irradiation the stroma still contained viable fibroblastic cells that had lost their in vitro colony-forming capacity as a result of radiation damage. In consequence there was no need for replacement of these fibroblastic cells by donor-derived or host-derived CFU-F. Only depletion of CFU-F from the bone marrow, as was induced with lipopolysaccharide, stimulated repopulation of the stroma with colony-forming fibroblastic cells.