Results of the pilot study of DIADEM: a comprehensive disease management programme for type 2 diabetes.

AIMS: The purpose of the study was to evaluate the feasibility and preliminary clinical effects of the DIADEM disease management programme for type 2 diabetic patients. METHODS: The study was performed at two test sites (Cardiff, UK: Aachen, Germany) including 137 and 166 patients, respectively. In 16 study centres any patients with type 2 diabetes capable of communicating by phone and able to perform blood pressure, blood glucose or urine glucose self-measurements were included. The maximum programme duration was 6 months at Cardiff and 4 months at Aachen, during which patients were assessed for glycaemic control, cardiovascular risk profile and the presence of complications of diabetes. Data were entered via the internet to a central server. RESULTS: At entry into the programme the patient group in Cardiff had significantly lower mean age (60.3+/-9.4 years versus 64.9+/-8.7 years, p<0.001) and duration of diabetes (6.1+/-5.7 years versus 7.4+/-7.0 years, p<0.05) than in Aachen, however body mass index (31.6+/-5.2 kg/m(2) versus 29.5+/-4.9 kg/m(2), p<0.01), HbA1c (7.7+/-1.2% versus 7.1+/-1.2%, p<0.001) and systolic blood pressure (138.4+/-15.1 mmHg versus 133.5+/-11.5 mmHg, p<0.001) were significantly higher. In contrast, total cholesterol (4.7+/-1.0 mmol/l versus 5.5+/-1.1 mmol/l, p<0.001) was significantly lower in Cardiff compared to Aachen. Following entry into the programme highly significant improvements in HbA1c (Cardiff from 7.7% to 7.1%, p<0.001; Aachen from 7.2% to 6.8%, p<0.05) and total cholesterol concentrations (Cardiff: 4.66-4.46 mmol/l; Aachen: 5.33-5.15 mmol/l; both p<0.05) were observed. There were no significant changes in blood pressure at either site. CONCLUSIONS: Intensive diabetes care was delivered to DIADEM patients and relevant and significant improvements in diabetes care were achieved demonstrating that an IT-based diabetes disease management service can improve care for patients with type 2 diabetes.

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

The biodegradation of hydroxyapatite bone graft substitutes in vivo.

Hydroxyapatite (HA) ceramics are widely used for bone reconstruction. They are osteoconductive and serve as structural scaffolds for the deposition of new bone. Generally, scaffold materials should be degradable as they affect the mechanical properties of the reconstructed bone negatively. Degradation by osteoclasts during the bone remodelling process is desirable but often does not take place. In the current study we analysed by light microscopy the degradation of two granular HA implants in critically sized defects in the mandibula of Goettingen mini-pigs five weeks after implantation. Bio-Oss consists of sintered bovine bone and NanoBone is a synthetic HA produced in a sol-gel process in the presence of SiO2. We found that both biomaterials were degraded by osteoclasts with ruffled borders and acid phosphatase activity. The osteoclasts created resorption lacunae and resorptive trails and contained mineral particles. Frequently, resorption surfaces were in direct contact with bone formative surfaces on one granule. Granules, especially of NanoBone, were also covered by osteoclasts if located in vascularised connective tissue distant from bone tissue. However, this usually occurred without the creation of resorption lacunae. The former defect margins consisted of newly formed bone often without remnants of bone substitutes. Our results show that the degradation of both biomaterials corresponds to the natural bone degradation processes and suggest the possibility of complete resorption during bone remodelling.

Bone functions and the requirements for bone grafts and substitutes in the orofacial region.

Bone is the largest calcium storage, has distinctive plasticity and adaptability and is part of the supporting tissue. An adequate composition is thus necessary. The bone matrix consists of organic and anorganic structures. Osteoblasts, osteoclasts and osteocytes are responsible for bone formation, resorption and metabolism. The periosteum, endosteum and bone tissue are a functional unit and provide protection, nutrition and growth. Bone is subject to continuous remodelling.

Critical considerations on the diagnostic appraisal, adaptation and remodelling of bone graft substitutes.

The diagnostic assessment of skeletal defects has a long-standing tradition. As a result of the development of new bone grafting materials, the demands on diagnostic assessment have also increased. The mode and quality of diagnostic appraisal are crucial to further clinical use and outcome prediction. Alongside traditional clinical and biological techniques, molecular biological methods have gained a broad scope of application and will be used even more frequently in the future.

The survival and proliferation of fibroblasts on orthodontic miniscrews with different surface treatment: an in vitro study.

It is of fundamental importance for prosthodontic and orthodontic applications that there is a short osseointegration time of dental implants without inflammation of the surrounding tissue. In addition to the chemical properties of the implant material, the surface morphology is an equally critical parameter. The objective of this work was to study the effect of two simple surface treatments on the survival and proliferation of fibroblasts. Three groups of orthodontic miniscrews (Mondeal) were used. One group was given an airflow (EMS, Schweiz) treatment, the second was sand-blasted in the area of the threading and a third group served as a control. After preparation sterilised screws were cultured in vitro with fibroblasts (L-929). The metabolic cell activity on the implant surface was determined after 24, 48 and 120 hours using the alamarBlue assay and a count of DAPI labelled fibroblasts was performed with a fluorescence microscope. After 24 hours, but not at 48 hours and 120 hours, the metabolic activity of the fibroblasts was slightly decreased for the airflow screw group. Generally, no significant difference was found regarding metabolic activity and proliferation of fibroblasts within the different groups.

Wound management after the application of bone grafting substitutes in the orofacial region.

Surgical dressing after the application of bone grafting material depends on the type and size of the defect. A complete and tension-free wound closure has proved to be successful. In this context the infection problem needs special attention. Bone graft substitutes with an adequate surface structure, porosity and chemical properties, in combination with sufficient blood circulation, hold osteoconductive potential. They serve as a guide rail for the osteoblast-induced formation of new bone tissue, which at best may lead to complete replacement of the grafting material.

Biomechanical hearts: muscular blood pumps, performed in a 1-step operation, and trained under support of clenbuterol.

BACKGROUND: As shown previously in goats, clenbuterol increased the power of electrically conditioned skeletal muscle ventricles (SMVs) of clinically relevant size (150 mL), which were constructed around a mock system. They pumped against a pressure of 60 to 70 mm Hg immediately during surgery and up to several months after, finally at >1 L/min. SMVs without clenbuterol administration failed. Thus, we expected that clenbuterol-supported SMVs might become integrated into the circulation by a 1-step operation instead of the 2-step procedure required up to now. METHODS AND RESULTS: In adult Boer goats (n=5), latissimus dorsi muscle was wrapped around a polyurethane chamber of 150 mL that was connected to the descending aorta. This muscular flow-through pumping chamber containing a stabilizing inner layer (called a biomechanical heart [BMH]) was formed and immediately made to work against a systemic load with the support of clenbuterol (5x150 microg/wk). During surgery, the mean stroke volume of BMHs was 53.8+/-22.4 mL. One month after surgery, in peripheral arterial pressure, the mean diastolic (P(MD)) and minimal diastolic (P(min)) pressures of BMH-supported heart cycles differed significantly from unsupported ones (P(MD)=+2.9+/-1.1 mm Hg [P<0.04], P(min)=-2.4+/-0.9 mm Hg [P<0.04]). After BMH-supported heart contractions, the subsequent maximal rate of pressure generation, dP/dt(max), increased by 20.5+/-8.1% (P<0.02). One BMH, catheterized 132 days after surgery, shifted a volume of 34.8 mL per beat and 1.4 L/min with a latissimus dorsi muscle of 330 g. Depending on duration of training, the percentage of myosin heavy chain type 1 ranged between 31% and 100%. CONCLUSIONS: Under support of clenbuterol, BMHs of a clinically relevant size can be trained effectively in the systemic circulation after a 1-step operation and offer the prospect of a sufficient volume shift and probably unloading of the left ventricle.

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells.

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.

Postsynaptic receptor occupancy during evoked transmission at striatal GABAergic synapses in vitro.

The effect of benzodiazepines (BZs) on GABA(A)-ergic synaptic responses depends on the control receptor occupancy: the BZ-induced enhancement of receptor affinity can lead to greater peak amplitudes of quantal responses only when, under normal conditions, receptors are not fully saturated at peak. Based on this fact, receptor occupancy at the peak of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) has been assessed in various mammalian neuronal preparations. To use the same principle with compound (or multiquantal), action potential-evoked IPSCs, complications introduced by quantal asynchrony in conjunction with the BZ-induced increase in the decay time of the quantal responses have to be overcome. We used a simple analytic convolution model to calculate expected changes in the rise time and amplitude of postsynaptic currents when the decay time constant, but not the peak amplitude, of the underlying quantal responses is increased, this being the expected BZ effect at saturated synapses. Predictions obtained were compared with the effect of the BZ flunitrazepam on IPSCs recorded in paired pre- and postsynaptic whole cell voltage-clamp experiments on striatal neurons in cell culture. In 22 pairs, flunitrazepam (500 nM) reliably prolonged the decay of IPSCs (49 +/- 19%, mean +/- SE) and in 18 of 22 cases produced an enhancement in their peak amplitude that varied markedly between 3 and 77% of control (26.0 +/- 5.3%). The corresponding change in rise time, however (+0.38 +/- 0.11 ms, range -0.8 to +1.3 ms) was far smaller than calculated for the observed changes in peak amplitude assuming fixed quantal size. Because therefore an increase in quantal size is required to explain our findings, postsynaptic GABA(A) receptors were most likely not saturated during impulse-evoked transmission at these unitary connections. The peak amplitudes of miniature IPSCs in these neurons were also increased by flunitrazepam (500 nM, +26.8 +/- 6.6%), and their decay time constant was increased by 26.3 +/- 7.3%. Using these values in our model led to a slight overestimate of the change in compound IPSC amplitude (+28 to +30%).

Clenbuterol-supported dynamic training of skeletal muscle ventricles against systemic load: a key for powerful circulatory assist?

BACKGROUND: The profound loss of power that occurs in skeletal muscle after electrical conditioning has been the major limiting factor in its clinical application. This study investigates a 3-fold approach for chronic conditioning of skeletal muscle ventricles (SMVs) combining electrical transformation, dynamic training against systemic load, and pharmacological support with clenbuterol. METHODS AND RESULTS: In 10 adult male goats, SMVs were constructed from latissimus dorsi muscle wrapped around an intrathoracic training device with windkessel characteristics. SMVs were stimulated electrically and trained dynamically by shifting volume against systemic load. Group 1 goats were controls (n=5), and group 2 goats (n=5) were supported with clenbuterol (150 microg 3 times a week). SMV dynamics were recorded weekly over 5 to 8 months: peak pressure (P(max)), stroke volume (SV), volume displacement per minute (VD), stroke work per day (SW/d), and maximum rates of pressure generation, +dP/dt(max), and decay, -dP/dt(max). In group 1, after 149.5+/-2.7 days (n=4), data were P(max)=70.8+/-4.7 mm Hg, SV=3.2+/-1.2 mL, VD=62.3+/-21.1 mL/min, SW/d=0.8+/-0.4 kJ, +dP/dt(max)=64+/-13 mm Hg/s, and -dP/dt(max)=156+/-32 mm Hg/s. These parameters were significantly improved (P<0.007) in the clenbuterol-treated group 2 after 151+/-2.7 days: P(max)=176.2+/-43.8 mm Hg, SV=23.3+/-6.1 mL, VD=568.2+/-186.1 mL/min, SW/d=9.1+/-2.2 kJ, +dP/dt(max)=1134+/-267 mm Hg/s, and -dP/dt(max)=1028+/-92 mm Hg/s. In 2 SMVs of group 2, VD increased to 1090 and 1235 mL/min after 202 and 246 days of training, respectively. At termination, myosin heavy chains were totally transformed into myosin heavy chain-1 in all SMVs. CONCLUSIONS: This clenbuterol-supported dynamic training provides powerful SMVs that may have important clinical implications for the treatment of end-stage heart failure by muscular blood pumps.

Sr2+-dependent asynchronous evoked transmission at rat striatal inhibitory synapses in vitro.

1. At striatal inhibitory synapses in cell culture, replacement of extracellular Ca2+ with Sr2+ desynchronized inhibitory postynaptic currents (IPSCs), reducing their peak amplitude and producing a succession of late, asynchronous synaptic events (late release). In the averaged IPSC waveform this resulted in an increase in both the fast and the slow decay time constant as well as in the time to peak. 2. Rapid removal of extracellular Sr2+ during late release was without effect on the time course of the averaged IPSC. Thus, late release is not dependent on continuous Sr2+ influx, but must be related to the way in which Sr2+, as opposed to Ca2+, interacts with constituents of the intracellular space. 3. After application of the membrane-permeant acetoxymethyl ester (AM) form of the Ca2+-chelator BAPTA, Sr2+-induced late release was greatly reduced and the kinetics of the Sr2+-dependent IPSC approached those of the Ca2+-dependent response. EGTA AM had a similar but less pronounced effect. 4. Using rapid solution exchange, we stimulated synapses first in Sr2+- or Ca2+- and 100-300 ms afterwards in Ca2+-containing solution. Paired-pulse facilitation of late release was the same whether the conditioning pulse induced a presynaptic influx of Sr2+ or of Ca2+. 5. It is concluded that Sr2+-mediated asynchrony is probably due to a less efficient intraterminal buffering of Sr2+ as opposed to Ca2+, allowing for Sr2+ ions to activate release in an area less confined to the immediate vicinity of the presynaptic Ca2+ channel. This hypothesis explains both the action of endogenous buffers and the apparent lack of specific facilitatory interaction between Ca2+-mediated and Sr2+-induced late release.

Production of trypsin by cells of the exocrine pancreas is paralleled by the expression of the KH protein vigilin.

Vigilin, a protein with a continuous series of 14 KH motifs, forms part of a multiprotein complex containing tRNA. Several lines of evidence have suggested that vigilin expression is enhanced in those cells which were actively engaged in protein synthesis. Accordingly, we show here by immunoelectronmicroscopy a close association of vigilin with the rough endoplasmic reticulum in rat pancreatic cells. Histological examination of these cells furthermore demonstrates the highest intensity of vigilin staining in the perinuclear, intranuclear, and basolateral regions where the endoplasmic reticulum is mainly amassed. In vivo challenge of starving rats fed prior to sacrifice raised in parallel the protein levels of both trypsin and vigilin when compared to unchallenged animals and was associated with enhanced expression of the vigilin gene. In contrast, in human and rat cell lines of pancreatic tumors with a constitutively high expression of vigilin no further stimulation by cholecystokinin treatment could be achieved. Our data provide circumstantial evidence that vigilin may play a crucial role in the ability of an organ, e.g., pancreas, to cope with the physiological demand to upregulate protein synthesis.

Expression of vigilin in the uterus of ovariectomized steroid-treated rats and during the estrous cycle.

The expression of vigilin in the uterus of rats was investigated by immunoblotting and immunohistochemistry and compared to the ultrastructural features of the endometrial cells. Vigilin could not be detected in the uteri of ovariectomized rats. Administration of estrogen, alone or in combination with progesterone, significantly stimulated the expression of vigilin, mainly in the luminal and glandular epithelial cells. Ultrastructurally, these cells show the morphological features of an increased protein synthesis. Untreated mature rats demonstrate a cyclic pattern of vigilin expression with high levels during the estrogen-dominated proestrus and early estrus stages and low levels at metestrus. The down-regulation of vigilin starts with the occurrence of apoptosis and autophagocytosis in the epithelium, but precedes the vanishing of the secretory granules. At diestrus the vigilin expression is intermediate and the vigilin staining of the epithelial cells is reduced. However, the endometrial fibroblasts show a faint staining. Morphologically, these fibroblasts are characterized by large euchromatic nuclei and dilated cisternae of the rough endoplasmic reticulum. The results suggest that in the uterus of rats the expression of vigilin is stimulated by estrogen. Under the experimental conditions chosen no influence of progesterone on vigilin expression was detected.

[Neurovascular infrahyoid muscle fascia flap for tongue and pharynx reconstruction. Postoperative histologic long-term follow-up]. / Der neurovaskuläre infrahyoidale Muskelfaszien-lappen zur Zungen-und Pharynxrekonstruktion.

After surgical treatment of cancer, reconstruction of large defects in the oral cavity or pharynx with myocutaneous island flaps will often lead to cicatrical shrinkage of the transplant. To avoid further loss of function we used a neurovascular infrahyoid muscle flap for reconstruction of large muscular defects in the mouth or pharynx. The advantages of this neurovascular flap were voluntary muscular contractions and reduced shrinkage of the flap's volume compared with myocutaneous island flaps alone. In this study we examined the characteristics of the transplanted muscles at 6 weeks, 7 months and 11 months after surgery. Biopsies were taken from the transplanted infrahyoid muscles and examined with light and electron microscopy. To identify nerves an immunofluorescence method was used. In the specimens taken several months postoperatively no atrophy or degeneration of the musculature could be seen. As a possible consequence of tenectomy an irregular arrangement of the myofibrils was noted. Nerves were found in all biopsies that served as further evidence for a functioning musculature.

PCNA-immunoreactivity in the uterus of rats after treatment with the antiestrogen tamoxifen.

We used an antibody to the proliferating cell nuclear antigen (PCNA) to investigate the effect of the long-term administration of tamoxifen on proliferative activity in the uterus of mature rats. Untreated cycling and ovariectomized rats served as controls. The PCNA labelling indices (PI) and the mitotic indices (MI) were estimated for the luminal and glandular epithelium and for the stromal fibroblasts. A strong correlation was found for PI and MI in the luminal and in the glandular epithelium, and a lower, but also significant correlation, for the endometrial stroma cells. Tamoxifen treatment decreased the PI of the luminal epithelial cells and of the stroma as much as ovariectomy. In both of these groups, the proportion of anti-PCNA positive cells in the glandular epithelium was significantly higher than in the luminal epithelium. These data indicate that tamoxifen has a strong antiproliferative effect on the uterus of mature rats, and that this antiestrogenic action is cell type specific.

Immunohistochemical visualization of the enteric nervous system using antibodies against protein gene product (PGP) 9.5.

Both the investigation of the enteric nervous system and the diagnosis of its pathological changes require reliable staining methods. In order to assess the potential of protein gene product (PGP) 9.5 as a marker for the enteric nervous system, we examined its immunoreaction in whole-mount preparations of the guinea pig and porcine small intestine, using a rabbit polyclonal antiserum. The immunohistochemical technique reveals the fundamental architectural features of the ganglionic and aganglionic plexuses. Furthermore, it enables a reproducible and differentiated visualization of the enteric nerve cells to be made, so that the various nerve cell types can be morphologically identified.

Morphology of the rat uterus after long-term treatment with progesterone antagonists.

The effects of long-term treatment with the progesterone antagonists ZK 98.299 and ZK 112.993 on the uterus of intact mature rats were investigated with light and electron microscopy. After 3-4 weeks treatment with both progesterone antagonists, the uterine luminal epithelium showed ongoing mitotic activity, increased apoptosis and invasion by granulocytes. Many uteri showed metaplastic areas with stratified squamous epithelium. Basically, the same changes occurred, but to a lesser extent, in the glandular epithelium. At the ultrastructural level, the epithelial cells displayed the morphological features of a certain degree of differentiation. The dissociation of collagen fibres, infiltration by granulocytes and dilatation of small vessels were observed in the subepithelial connective tissue. The myometrium increased in thickness and electron microscopic examination revealed hypertrophic myocytes with a well developed granular endoplasmic reticulum. Most of the morphological reactions may be regarded as due to the direct inhibitory action of progesterone antagonists at the level of the different uterine tissues and the resulting unopposed action of estrogen. The metaplastic changes and the suppression of the anti-proliferative action of progesterone on uterine epithelial cells should be taken into account when treating women in their reproductive years with these drugs for long periods of time, as may be necessary for the endocrine treatment of mammary cancer and endometriosis.

[Importance of the cell membrane for the sequestration of aged erythrocytes]. / Die Bedeutung der Membran für die Sequestration gealterter Erythrocyten.

The erythrocyte membrane is an important cell organelle which determines the intravital life time of the cell in a decisive way. Their mechanic properties are closely connected with the metabolism of the cells. Increasing disturbance of the cellular metabolism especially in the course of biological aging leads to enhanced cell rigidity. It is supposed that the decreased elasticity of the aged erythrocytes in critical regions of the blood circulation (capillaries with a diameter of 2.5 to 3.0 micrometers) leads to pressure induced vesiculation and spherocytosis. It is possible that also IgG-receptors are demarked by this process. After passing a threshold concentration as immunobiological signals increased irreversible binding of IgG induces the sequestration of the erythrocytes by the erythrocyte destructing system.