Adsorption and release properties of growth factors from biodegradable implants.

The present investigation was performed to study the adsorption behavior of growth factors and their release characteristics from biodegradable implants in an in vitro study. We investigated the stability of growth factors administered on various scaffolds. We used porous tricalcium phosphate ceramics (alpha-TCP), a neutralized glass-ceramics (GB9N), a composite (polylactid/-glycolid/GB9N), and solvent dehydrated human bone as carriers. Block shaped scaffolds (sized: 7 x 7 x 10 mm) were loaded with 5 microg of either bone morphogenetic protein (rxBMP-4), basic fibroblast growth factor (rh-bFGF), or vascular endothelial growth factor (rh-VEGF) solved in 150 microL PBS. The growth factors were labeled with Iodine125 (I-125) for detecting the adsorbed and released amount of growth factors by counting the samples for total I-125 activity. We observed that the adsorption of these growth factors seems to depend on two different parameters: first on the nature of the tested material, and second on the growth factors on their own. The release kinetics of the growth factors from the biodegradable implants can be described as a two phase process-a very rapid release during the first hours by an elution of not adsorbed protein, followed by a specific release, which depends upon the chemical/physical interaction of the material and the growth factor used. Analyzing the eluted proteins on SDS-PAGEs rh-VEGF was degraded into a smaller fragment with a size of around 15 kDa, while rxBMP-4 and rh-bFGF showed a complete degradation into fragments smaller than 3 kDa after more than 3 days. Although this in vitro study suggests that biodegradable implants might be successfully used as carriers for osteogenic growth factors, the different release kinetics as well as the alteration of their molecular structure including loss of biological activity should be considered.

Inadequate suspension of neutral protamine Hagendorn (NPH) insulin in pens.

BACKGROUND: Neutral protamine Hagedorn (NPH) insulin is one of the most commonly used insulins in insulin pens. NPH in pen cartridges is in a two-phase solution with either a solvent or a short-acting insulin, and needs adequate mixing for complete resuspension. We assessed whether NPH insulin is accurately resuspended by patients and the association of suspension errors with diabetes control. METHODS: 109 patients (39 with type 1 diabetes) who had received conventional diabetic education had the NPH content of their cartridges measured by an optical system; a control cartridge was designated as 100%. A questionnaire was used to assess clinical details and insulin suspension habits. After the information about residual insulin error was known, all 109 patients were instructed to resuspend their insulin by rolling and tipping the pen 20 times. 52 patients were randomly selected to have cartridges re-analysed 3 months or 6 months later and to complete another questionnaire. FINDINGS: Only 10 (9%) of 109 patients tipped and rolled their pen more than ten times. NPH insulin content ranged from 5% to 214% and varied by more than 20% in 71 (65%) of 109 cartridges. There was no relation between inadequate suspension and the frequency of hypoglycaemic episodes (r=0.2, p=0.08). For all patients, there was a correlation between the absolute error of NPH suspension and cycles of rolling and tipping the pen (r=-0.23, p<0.05). After education on resuspending the pen's contents, data were available from 44 of 52 patients; suspension errors decreased in 35 (80%), were unchanged in three (7%), and increased in six (13%). The 35 patients with improved NPH insulin suspension had fewer mean hypoglycaemic episodes per month compared with the previous period (0.4 [SD 0.1] vs 1.0 [0.3], p<0.05). Mean HbA1c values in patients with improved suspension quality did not differ from baseline (8.4% [0.3] vs 8.9% [0.4], p=0.07). Mixing of NPH insulin by a mechanical device showed that at least 20 cycles were necessary before complete resuspension was obtained. INTERPRETATION: Inadequate NPH insulin suspension is common. We recommended that patients tip pens that contain NPH insulin at least 20 times, since inadequate mixing may impair diabetes control.

Neurofilament phosphorylation and axon diameter in the squid giant fibre system.

Electron energy loss spectroscopic analysis of squid giant axons in a phosphorus energy window yielded bright signals, which were shown to originate from highly phosphorylated neurofilaments. The frequency and distribution of these signals were analysed at defined intervals in cross-sections of the giant axon, starting from its origin in the stellate ganglion and extending distally along the stellar nerve. The analysis revealed a proximodistal gradient of increasing neurofilament phosphorylation. Within the stellate ganglion and for some distance beyond, the increase in frequency of signals correlated with the widening of the neurofilament meshwork and the radial growth of the axon. This agrees with the hypothesis that neurofilament phosphorylation regulates axon calibre by affecting interfilament spacing. In distal axon domains where the axon diameter diminished, contrary to expectations, the spacing of signals increased and the signals were significantly larger. Hyperphosphorylation apparently compensated for a diminishing supply of neurofilament protein. Contrary to predictions, the presynaptic terminal of the giant synapse contained a distinct and highly phosphorylated neurofilament meshwork. We conclude that the growth of the axon diameter is a function of neurofilament phosphorylation, interfilament spacing and neurofilament density. A mature and highly phosphorylated neurofilament cytoskeleton completely filled the presynaptic terminal of the giant synapse.

Videodensitometric analysis of electron spectroscopic micrographs--a tool for detection of biologically relevant elements with high resolution.

Electron energy-loss spectroscopic imaging (ESI) yields high-resolution, element-sensitive images. However, ESI suffers from difficulties in distinguishing element-specific and background contributions. New methods have therefore been introduced which use grey-level measurements in micrographic images for a more accurate detection of element distributions. A videodensitometric method allowed the detection of low phosphorus levels in axoplasmic neurofilaments of squid giant axons. Here we further verify these results by investigating the relationship of videodensitometry and electron energy-loss spectroscopy (EELS), particularly considering the peculiarities of these methods in terms of automatic background correction and representation of the results. Six biological specimens and two nonbiological specimens were examined both by EELS and by videodensitometry. In all cases comparable results were obtained. The overlapping PL2,3 and SL2,3 ionization edges could clearly be recognized individually by both methods, and controls showed that mass density variations within the specimens did not impair elemental analysis. Additional evidence supporting the detection of phosphorylation sites in squid neurofilaments was obtained in both EELS and videodensitometric measurements of neurofilament-enriched pellets and of aggregated axoplasmic particles. Thus, video-densitometry appears to be a useful tool for an improved exploration of the full imaging capabilities of energy filtering electron microscopy.

High-resolution imaging of protein phosphorylation in squid axons and synapse by electron energy loss spectroscopy.

We demonstrate that clusters of phosphorus atoms can be detected in energy loss spectroscopic images (ESI) of cytoskeletal proteins of squid axons. In series of images taken at four energy windows below and three windows above the phosphorus P-L2,3 ionization edge, signal-to-background intensity differences were analyzed by videodensitometry. A distinct increase of relative intensities was recorded above the phosphorus edge in neurofilaments of the peripheral giant axon and in those of the presynaptic terminal. A high level of neurofilament phosphorylation in the peripheral axon supports previous biochemical and immunochemical results, but our finding of phosphorylated neurofilaments in the presynaptic axon conflicts with these studies. Our method may be advantageous for analysis with high elemental and spatial resolution of the phosphorylation state of cytoskeletal protein molecules in situ.

Early changes of intracranial pressure, perfusion pressure, and blood flow after acute head injury. Part 1: An experimental study of the underlying pathophysiology.

The present study examines intracranial pressure (ICP), cerebral perfusion pressure (CPP), and cerebral circulation immediately after experimental head injury in an animal model. The underlying systemic hemodynamic changes were also observed. To produce a standardized head injury, a fluid-percussion device was applied to the dura at the midline of 10 piglets. Seven other nontraumatized animals served as a control group. Hemodynamic parameters as well as ICP and CPP were recorded on-line, one value every 1.4 seconds. Cerebral blood flow (CBF) and cerebral vascular resistance (CVR) were measured three times using a microsphere technique. Immediately after head injury, the traumatized animals showed a sudden increase in ICP, with a maximum of 40 torr at 3 to 5 minutes, while there was a pronounced decrease in CPP from 85 to 40 torr. The CBF in the various brain areas fell from 55 to 22 ml/min/100 gm within 5 minutes after the impact, and CVR increased to 300% of control values within 90 minutes. The findings of this study demonstrated that cerebral circulation is critically jeopardized within a few minutes after trauma. This, in combination with a subsequent increase in CVR, makes the early development of ischemic brain damage very likely. In traumatized patients, treatment prior to hospital admission must therefore be directed at prevention of this fatal course.

Diphenylhydantoin (DPH) blocks HIV-receptor on T-lymphocyte surface.

Previous reports have shown the capacity of diphenylhydantoin (DPH) to attach to the membranes of lymphatic cells as a hapten and thus exert an unspecific influence on their ability to express certain recognition molecules. This led us to the hypothesis, that DPH might as well serve to manipulate the t-helper-lymphocytes in a way that the mode of infection of these cells by the HIV might be blocked. In order to verify this hypothesis, we exposed normal control lymphocytes as well as lymphocytes from DPH-treated patients (3 X 100-150 mg DPH/day, Phenhydan, for a minimum of 10 days) to radioactively labeled HIV (125I). Remaining radioactivity was assessed using a gamma-counter and measured 64.000-92.000 counts/min (n = 24, mean 80.000) for the control lymphocytes, while remaining radioactivity for the DPH-treated lymphocytes ranged between 2000 and 7000 counts/min (n = 24, mean 4.000, p less than 0.001). These results and similar experiments obtained with FITC-labeled HIV led us to the conclusion that DPH inhibits HIV recognition of T-lymphocytes and therefore might be used in therapy and prophylaxis of AIDS.

An experimental model for measuring intestinal permeability.

In the nonanesthetized rat, the jejunal permeability to four simultaneously administered molecules, mannitol, phenol red, inulin and PVP, was measured by analyzing blood, serum, urine and duodenal fluid for these compounds. Of the molecules which had entered the body, approximately 50% were found in the urine, another 50% in the extracellular space and only about 1% were excreted into the duodenal juice. The intracellular content of the molecules is not accounted for in these numbers. The rate of permeation decreased with increasing molecular weight of the substances. EDTA (25 mmol/l) and deoxycholate (5 mmol/l) increased the jejunal permeability for these molecules but not for mannitol. The alterations in mucosal cell turnover and morphology induced by hydroxyurea did not change jejunal permeability for mannitol and phenol red at any time. 24 and 48 h following hydroxyurea, jejunal permeability for inulin and PVP was decreased.

Extracorporeal irradiation of dog blood: the effects of a radiostrontium irradiator on blood stem cells (CFU-C).

The radiation sensitivity of dog blood stem cells was measured in vitro and in an extracorporeal circulation passing through a radiation field. It was established that the calculated D0 was as low as 0.45 Gy. Investigating the cell killing rate in our equipment (Buchler type 90Sr device for extracorporeal irradiation), we found an overkill situation; the dose delivered was in excess of that which would be required for the total eradication of all stem cells in the peripheral blood passing through the radiation field. Various other types of devices used for extracorporeal irradiation of blood are also reviewed.

Metabolism of dehydroepiandrosterone (DHA) in the mature perfused human placenta (I).

A design for the perfusion of one (monoperfusion) or two (parallelperfusion) cotyledos of one placenta was developed for studies of the metabolism of the precursor steroid dehydroepiandrosterone (DHA). Several parameters are used as viability criteria: vascular resistance, glucose and oxygen consumption, lactate/pyruvate ratio, activity of lactate-dehydrogenase (LDH) in the perfusate, extent of perfusion by dye infusion, and morphological description by electron microscopy. A dosage of 2 mg DHA with 2.5 microCi 14C-radioactive labelled marker is given for testing the metabolizing capacity of the placenta. The labelled metabolites DHA, androstenedione (A), testosterone (T), estrone (Oe 1), and estradiol-17 beta (Oe 2) are separated by thin-layer chromatography and measured by scanning and measurements of scraped radioactive spots by scintillation counting. The steroidogenesis is evaluated with the concentrations of Oe 1 at 15 min (Oe 1.15'), Oe 1 + Oe 2 at 90 min (Oe tot, 90'), total aromatization rate (from perfusate and homogenate after 120 min). Results comparable to DHA are found using DHA-sulphate (DHA-S) as precursor, higher amounts of estrogens are metabolized from A. Mature placentas metabolize DHA in relation to the initial DHA concentration: optimal aromatization is found at 250-350 pmol DHA/ml/g, decreased aromatization at higher or lower concentrations. Oe 1 represents the main placental metabolite.

The influence of estradiol and adriamycin on the RNA biosynthesis in breast cancer.

The extent of the relationship between hormone dependence and cytostatica sensitivity of the tumors was examined for primary breast cancers under standardized in vitro conditions. The effects of estradiol and adriamycin on the RNA biosynthesis in the tumor cells were compared on the basis of the 3H-uridine incorporation in a short-term test system. Estradiol particularly influenced the RNA metabolism of higly differentiated carcinomas, whereas adriamycin exhibited the greatest inhibitory effect on the RNA synthesis in the tumor cells of undifferentiated carcinomas. An exact, unequivocal separation of the breast cancers investigated into hormone-sensitive, cytostatica-resistant and hormone-independent, cytostatica-sensitive tumors, however, was not possible under the in vitro conditions chosen.

[Cytostatic effects on nucleic acid synthesis in primary tumors and lymph node metastases of human breast cancer (author's transl)]. / Nukleinsäuresynthese und deren Beeinflussung durch Cytostatica bei Primärtumor und Lymphknotenmetastase von Mammacarcinomen.

Nucleic acid synthesis and influence of cytostatic agents (adriamycin, cyclophosphamide) were investigated in 73 cases of human breast cancer and in 19 cases of correlating lymph node metastases. An interrelationship between nucleic acid synthesis, proliferation and tumor stage could not be proven at the time of the operation. The labeling index, expressing the proliferation of tumor cells, and the incorporation of radiolabeled nucleic acid precursors (3H-thymidine, 3H-uridine) as an indicator of nucleic acid synthesis were higher in the investigated lymph node metastases than in the primary tumor. Nucleic acid synthesis was suppressed in primary and metastatic lesions in the same way by cytostatic agents.

[Effect of estradiol on RNA biosynthesis of human breast carcinoma cells in vitro]. / Einfluss von Ostradiol auf die RNS-Biosynthese von menschlichen Mammacarcinomzellen in vitro.

The possibility of differentiating estrogen-sensitive human breast cancer using incorporation studies with labeled uridine as a precursor of RNA metabolism is described. The purpose of this study was to explore inadequate function of the estrogen receptor as an alternative or supplementary aid in selecting patients for hormonal manipulation. The disadvantage of the test is that only hormone dependence of a proliferating tumor cell population can be evaluated. Highly differentiated breast cancer cells exhibit the greatest estrogen sensitivity. The hormone-dependent tumors of premenopausal women show an increase in RNA synthesis, whereas uridine incorporation appeared to be inhibited in postmenopausal women.

Incorporation studies of nucleic acid precursors in gastric cancer: an attempt in individualized chemotherapy.

Measurements of the rate of incorporation of radioactively labeled nucleic acid precursors into the DNA and RNA of gastric carcinoma cell suspensions indicated variable rates of proliferation for the tumors. The rate of incorporation generally correlates to the cytological level of differentiation of the carcinoma. Reduced differentiation of the tumors showed a corresponding increase in the rate of proliferation. Knowing the proliferation-dependent effect of most cytostatica, this results in a resistance to cytostatica of highly differentiated gastric cancers. The nucleic acid synthesis of proliferatively active tumors could only be partially inhibited by the cytostatica tested (5-fluorouracil, adriamycin). Carcinomas with metabolic possibility for compensation of the active mechanism of the cytostatica were biochemically resistant. Due to the resulting methodical problems and unaccountable patient-dependent causes of resistance, a conclusive statement about cytostatica-sensitive tumors is difficult to make in incorporation studies.

Pretherapeutic cytostatic agent sensitivity testing of breast cancer. / Prätherapeutische Cytostatica Sensibilität-Testung des Mammacarcinoms.

With and without addition of cytostatic agents, incorporation of radiolabeled precursors into the nucleic acid of breast cancer cells was determined in short-term culture. The labeling index, a measure of tumor proliferation, depended largely on cytologic differentiation. Incorporation of radiolabeled precursors in moderately and low differentiated breast cancers was significantly high, whereas only in a part of these tumors was incorporation inhibited by adriamycin and cyclophosphamide. Therefore a group of drug sensitive and metabolic drug resistant tumors could be defined in vitro. Primary resistance against the tested cytostatic agents has been proved in low proliferating and high differentiated breast cancer cells.

[Functional and metabolic changes of the mucosa during the occlusion of the small bowel of the rat (author's transl)]. / Untersuchungen zu Funktion und Stoffwechsel der Mucosa beim mechanischen Dünndarmileus der Ratte

In this study we investigated the function of the obstructed small bowel of the rat, the behaviour of the mucosal enzymes, the metabolic changes of the small bowel wall and the morphology of the mucosa. We found a decrease of passive transport of 3H-Antipyrine which was equal after 24 and 48 hrs. The active transport of 14C-Glucose was found to be progressively inhibited after occlusion. The metabolic enzymes SDH, G-6-PDH, and GOT remained unchanged, LDH was increased after 48 hrs, which can be explained by enzyme induction. The lactate-pyruvate ratio in the tissue of the obstructed bowel was 3 times as high as in the controls. The brush-border enzymes maltase and especially the alkaline phosphatase are decreased with progressive obstruction, which probably is caused by diffusion into the lumen. By electron-microscopy there are no changes in the brush-border membrane but a swelling of mitochondria which is caused by hypoxia.

Transport studies in the small bowel obstruction of the rat.

In the mechanically obstructed small bowel of the rat the decrease of passive transport of antipyrine was found to be equal after 24 and 48 h. The active transport of glucose was found to be progressively inhibited after occlusion.