Angle-stable interlocking nailing in a canine critical-sized femoral defect model for bone regeneration studies: In pursuit of the principle of the 3R's.

Introduction: Critical-sized long bone defects represent a major therapeutic challenge and current treatment strategies are not without complication. Tissue engineering holds much promise for these debilitating injuries; however, these strategies often fail to successfully translate from rodent studies to the clinical setting. The dog represents a strong model for translational orthopedic studies, however such studies should be optimized in pursuit of the Principle of the 3R's of animal research (replace, reduce, refine). The objective of this study was to refine a canine critical-sized femoral defect model using an angle-stable interlocking nail (AS-ILN) and reduce total animal numbers by performing imaging, biomechanics, and histology on the same cohort of dogs. Methods: Six skeletally mature hounds underwent a 4 cm mid-diaphyseal femoral ostectomy followed by stabilization with an AS-ILN. Dogs were assigned to autograft (n = 3) or negative control (n = 3) treatment groups. At 6, 12, and 18 weeks, healing was quantified by ordinal radiographic scoring and quantified CT. After euthanasia, femurs from the autograft group were mechanically evaluated using an established torsional loading protocol. Femurs were subsequently assessed histologically. Results: Surgery was performed without complication and the AS-ILN provided appropriate fixation for the duration of the study. Dogs assigned to the autograft group achieved radiographic union by 12 weeks, whereas the negative control group experienced non-union. At 18 weeks, median bone and soft tissue callus volume were 9,001 mm3 (range: 4,939-10,061) for the autograft group and 3,469 mm3 (range: 3,085-3,854) for the negative control group. Median torsional stiffness for the operated, autograft treatment group was 0.19 Nm/° (range: 0.19-1.67) and torque at failure was 12.0 Nm (range: 1.7-14.0). Histologically, callus formation and associated endochondral ossification were identified in the autograft treatment group, whereas fibrovascular tissue occupied the critical-sized defect in negative controls. Conclusion: In a canine critical-sized defect model, the AS-ILN and described outcome measures allowed refinement and reduction consistent with the Principle of the 3R's of ethical animal research. This model is well-suited for future canine translational bone tissue engineering studies.

[Comparative studies of the activity of disaccharidases in the mucosa of the small intestines in dystrophic, formerly hypotrophic-born young children and of patients with flat mucosa of various etiology]. / Vergleichende Untersuchungen über die Aktivitäten von Disaccharidasen in der Dünndarmschleimhaut von dystrophen, ehemals hypotroph geborenen Kleinkindern und von Patienten mit flacher Mucosa unterschiedlicher Genese.

The disaccharidase activities of the mucous membrane of the small intestine were determined in formerly hypotrophic children who showing a dystrophy with a morphological normal mucosa (n = 36), patients with a flat mucosa caused by enteral protein intolerances of different genesis (n = 27), patients with a morphologically and functionally normal mucosa (n = 51). In about half of the former small-for-date children were shown activities lower than the simple standard deviation of the normal value, for lactase n = 17, for sucrase n = 14, for maltase n = 12. Some children showed pathologically reduced activities even below the double standard deviation for the normal value: lactase n = 8, sucrase n = 5, maltase n = 3.

[Care of patients with celiac disease in adolescence and young adulthood]. / Betreuung von Zöliakie-Patienten im Adoleszenten- und jungen Erwachsenenalter.

60 patients suffering from coeliac disease were continuously cared for during a longer period. In these patients the diagnosis was established on the basis of the commonly used ESPGAN criteria in their childhood. Another biopsy was performed in adolescence and early adulthood. In 26 patients the diagnosis coeliac disease was confirmed. These patients are recommended to carry on keeping to a strict diet. 4 patients show a normal mucosa (type I) while following normal nutrition for more than 2 up to 5 years in adulthood although in their childhood the diagnosis of coeliac disease was established. While being put an a diet or following a short lasting normal nutrition, respectively, 17 patients have a morphologically intact mucosa of the small intestine, which is, however, in nearly all cases functionally damaged. In these patients the diagnosis is to be clarified definitely by means of a final biopsy of the small intestine after an at least 2-year-lasting exposition to gluten. 13 patients refused a biopsy in adulthood.

Nitrogen Fixation in Continuous Culture with NH(4)Cl-Containing Media.

N balance and N dilution were determined from growth of Azospirillum brasilense Sp7 and two unidentified gram-negative nitrogen-fixing microorganisms in continuous culture supplied with NH(4)Cl. At the 1.1 and 2.2 mM NH(4)Cl steady states (N-to-C ratios of 1:68 and 1:34, respectively), the organisms grew with NH(4)Cl and N(2) as N sources simultaneously under carbon limitation. No ammonium could be detected in the supernatant of these cultures.

Preclinical and clinical testing of radiolabelled monoclonal antibodies for immunoscintigraphy.

Immunoscintigraphy is a new method for in vivo diagnosis of diseases using monoclonal antibodies. Emphasis is placed on diagnosis of malignant tumors although the range of application includes a number of non-malignant diseases. To date, no European country has issued clear guidelines on the testing and registration of those monoclonal antibodies labelled with a radionuclide and used for diagnostic. This involves the risk of overregulation which would considerably reduce the applicability of the method. This holds particularly true since the complications initially anticipated with the use of such compounds did not occur. The conduct of immunoscintigraphy has evolved during the last few years. For reasons of applicability, but also and mainly for reasons of radiation hygiene, I-131 and finally also In-111 were abandoned as labelling nuclides and replaced by Tc-99m. The protein amount involved was reduced. Some false estimations, which were due to particularities or artifacts of the iodinated antibodies used at the beginning, had to be corrected: the representation of the liver in a scintigram is part of the physiological distribution of antibodies; and fragments of antibodies normally do not present the anticipated kinetic advantages. The clinical results obtained with colon carcinomas show that not only recurrencies and metastases, but also primary tumors can be detected with equally high sensitivity. In contrast, radioimmunotherapy does not yet seem as successful, at least against solid tumors.

Influence of the placenta on fetal plasma insulin and neonatal hepatic enzyme induction in the rat.

In order to study the placental role in controlling glucose metabolism and neonatal induction of tyrosine aminotransferase in rat liver, a comparison was made between the artificially delivered-rat feto-placental unit and the newborn littermates. The immunoreactive serum insulin in the feto-placental unit remains as high as in utero for at least 1 h whereas in the newborn rat it decreases rapidly after delivery. From Caesarian section to 6 h later, in contrast to the newborn littermates, the feto-placental unit remains hyperglycaemic. Upon glucagon injection the feto-placental unit shows a delay in the increase of the hepatic tyrosine aminotransferase activity in comparison with the newborn rat.

Root-Zone-Specific Oxygen Tolerance of Azospirillum spp. and Diazotrophic Rods Closely Associated with Kallar Grass.

The effect of oxygen on N(2)-dependent growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) was studied. To enable precise comparison, bacteria were grown in dissolved-oxygen-controlled batch and continuous cultures. Steady states were obtained from about 1 to 30 muM O(2), some of them being carbon limited. All strains needed a minimum amount of oxygen for N(2)-dependent growth. Nitrogen contents between 10 and 13% of cell dry weight were observed. The response of steady-state cultures to increasing O(2) concentrations suggested that carbon limitation shifted to internal nitrogen limitation when N(2) fixation became so low that the bacteria could no longer meet their requirements for fixed nitrogen. For Azospirillum lipoferum Rp5, increase of the dilution rate resulted in decreased N(2) fixation in steady-state cultures with internal nitrogen limitation. Oxygen tolerance was found to be strain specific in A. lipoferum with strain Sp59b as a reference organism. Oxygen tolerance of strains from Kallar grass was found to be root zone specific. A. halopraeferens Au 4 and A. lipoferum Rp5, predominating on the rhizoplane of Kallar grass, and strains H6a2 and BH72, predominating in the endorhizosphere, differed in their oxygen tolerance profiles. Strains H6a2 and BH72 still grew and fixed nitrogen in steady-state cultures at O(2) concentrations exceeding those which absolutely inhibited nitrogen fixation of both Azospirillum strains. It is proposed that root-zone-specific oxygen tolerance reflects an adaptation of the isolates to the microenvironments provided by the host plant.

Close association of azospirillum and diazotrophic rods with different root zones of kallar grass.

The populations of diazotrophic and nondiazotrophic bacteria were estimated in the endorhizosphere and on the rhizoplane of Kallar grass (Leptochloa fusca) and in nonrhizosphere soil. Microaerophilic diazotrophs were counted by the most-probable-number method, using two semisolid malate media, one of them adapted to the saline-sodic Kallar grass soil. Plate counts of aerobic heterotrophic bacteria were done on nutrient agar. The dominating N(2)-fixing bacteria were differentiated by morphological, serological, and physiological criteria. Isolates, which could not be assigned to a known species, were shown to fix nitrogen unequivocally by N(2) incorporation. On the rhizoplane we found 2.0 x 10 diazotrophs per g (dry weight) of root, which consisted in equal numbers of Azospirillum lipoferum and Azospirillum-like bacteria showing characteristics different from those of known Azospirillum species. Surface sterilization by NaOCI treatment effectively reduced the rhizoplane population, so that bacteria released by homogenization of roots could be regarded as endorhizosphere bacteria. Azospirillum spp. were not detected in the endorhizosphere, but diazotrophic, motile, straight rods producing a yellow pigment occurred with 7.3 x 10 cells per g (dry weight) of root in the root interior. In nonrhizosphere soil we found 3.1 x 10 nitrogen-fixing bacteria per g. Diazotrophs were preferentially enriched in the Kallar grass rhizosphere. In nonrhizosphere soil they made up 0.2% of the total aerobic heterotrophic microflora, on the rhizoplane they made up 7.1%, and in the endorhizosphere they made up 85%. Owing to high numbers in and on roots and their preferential enrichment, we concluded that diazotrophs are in close association with Kallar grass. They formed entirely different populations on the rhizoplane and in the endorhizosphere.

Investigations on the mechanism of photodynamic action of different psoralens with DNA.

Investigations on the photodynamic action of psoralens with DNA were performed, using experimental techniques of fluorescence lifetime and NMR-CIDNP, as well as SCF-MO and CNDO molecular orbital calculations. It has been shown that the formation of a biradical through the triplet state is the decisive step for psoralen dimer formation, as well as for cyclobutane addition with thymine, while singlet oxygen production is responsible for enzyme inactivation (e.g., lysozyme and trypsin). The molecular orbital calculations, in agreement with experimental results, indicate that the differences in biological effectivity of different psoralens are based on variations in triplet formation probability.

Electric field effects on bacteria and yeast cells.

Comparative studies were carried out describing the lethal effects of electric pulses on GRAM-negative bacteria, GRAM-positive bacteria, and yeast cells. Microorganisms are killed by the pulse treatment without visible morphological destruction. The observed survival rates are figured as functions of the field strength E and the treatment time t (pulse number X time constant) revealing three explicit parameters as sufficient to explain the kinetics of the results. These parameters are determined by the species of microorganism used and moreover depend on the physiological properties of the microbial population. GRAM-positive bacteria and yeasts were found to be less sensitive to electric pulse treatment than GRAM-negative bacteria, when low pulse numbers are applied. Treatment with high pulse numbers reveals survival rates below 1% for all microorganisms examined. Cells from the logarithmic growth phase are killed in markedly higher percentage than cells harvested from the stationary growth phase. The obtained results as well as further studies confirm the hypothesis of an electric induced selective damage of inner cell membranes.

Killing of bacteria with electric pulses of high field strength.

Bacteria of the type E. coli K12 have been treated in experiments using high-voltage pulses of short time (microseconds) as a killing agent. The role of different experimental parameters has been studied: kind of electrolyte, concentration, length of pulses, field strength, pH and temperature. Electrolytes with bivalent cations were found to reduce the lethal action. the relative rate of killed bacteria was shown to be mainly governed by the field strength and the treatment time, which is defined by the product of pulse number and decay time constant. From the obtained results a function has been developed which enables the precalculation of the killing rate for E. coli, provided that certain limits of experimental conditions are considered. No correlation between the applied electric energy and the lethal effect could be found.

Lethal effects of high-voltage pulses on E. coli K12.

The lethal effects of high-voltage capacitor-discharges in suspensions of E. coli K12 with varying electrolytes have been examined. A reduction of more than 99.9% of living cells, dependent on the applied voltage could be proved. The bactericidal action is assumed to be due to direct effects of high electric fields. Electrolytically produced chlorine was shown to act as an additional toxic agent, when chloride is present in the treated medium. The relative survival rate of bacteria has been found to depend also on the concentration of cells during pulse treatment.