Autologous Mesenchymal Stromal Cells Immobilized in Plasma-Based Hydrogel for the Repair of Articular Cartilage Defects in a Large Animal Model.

The treatment of cartilage defects in trauma injuries and degenerative diseases represents a challenge for orthopedists. Advanced mesenchymal stromal cell (MSC)-based therapies are currently of interest for the repair of damaged cartilage. However, an approved system for MSC delivery and maintenance in the defect is still missing. This study aimed to evaluate the effect of autologous porcine bone marrow MSCs anchored in a commercially available polyglycolic acid-hyaluronan scaffold (Chondrotissue®) using autologous blood plasma-based hydrogel in the repair of osteochondral defects in a large animal model. The osteochondral defects were induced in twenty-four minipigs with terminated skeletal growth. Eight animals were left untreated, eight were treated with Chondrotissue® and eight received Chondrotissue® loaded with MSCs. The animals were terminated 90 days after surgery. Macroscopically, the untreated defects were filled with newly formed tissue to a greater extent than in the other groups. The histological evaluations showed that the defects treated with Chondrotissue® and Chondrotissue® loaded with pBMSCs contained a higher amount of hyaline cartilage and a lower amount of connective tissue, while untreated defects contained a higher amount of connective tissue and a lower amount of hyaline cartilage. In addition, undifferentiated connective tissue was observed at the edges of defects receiving Chondrotissue® loaded with MSCs, which may indicate the extracellular matrix production by transplanted MSCs. The immunological analysis of the blood samples revealed no immune response activation by MSCs application. This study demonstrated the successful and safe immobilization of MSCs in commercially available scaffolds and defect sites for cartilage defect repair.

Bioassay-directed chemical analysis and detection of mutagenicity in ambient air of the coke oven.

In the present study, we summarize the results of studies on the mutagenic potential of the main fractions and subfractions of extractable organic material (EOM) in the ambient air at the workplaces of the coke oven. The objective of our experiments was to apply the Bioassay-Directed Chemical Analysis (with the use of the Ames test) for the identification of the differences in the mutagenicity of these fractions, in relationship to the complex mixture of EOM in occupational air. From the evaluation of results, it is possible to deduce the following conclusions: (1) The comparison of the mutagenicity in the main fractions (basic, acidic, neutral) demonstrates the existence of differences in mutagenic potential. Of the total mutagenicity, 20.4% is in the basic fraction, 25.4% in the acidic fraction and 54.2% in the neutral fraction. (2) In general, 90.1% of the mutagenicity found in the basic, acidic and neutral fractions together was associated with the requirement of metabolic activation in vitro (+S9). In the case of the neutral fraction, it was 51.8%. (3) These results also suggest that frameshift mutations are the major component (53.8%) of the total mutagenicity of the main fractions. (4) With regards to the mutagenicity of organic compounds in the neutral fraction it appeared that genotoxicants of its subfractions (slightly and moderately polar and aromatic) play the main role. Carcinogenic aromatic hydrocarbons (PAH) and genotoxic nitrocompounds play an important role as determinants of the mutagenic potential of complex mixtures of harmful compounds in ambient air. This is confirmed first by the results of short-term bacterial tests.

Long-term use of intramuscular insulin therapy in a type I diabetic patient with subcutaneous insulin resistance.

We studied a 26-year-old Type 1 diabetic patient who experienced recurrent episodes of ketoacidosis and who was unresponsive to subcutaneous insulin, but normally responsive to intravenous insulin as demonstrated by insulin challenge test. Attempts at intravenous and intraperitoneal insulin administration were complicated by recurrent septicaemia. We therefore investigated the hypoglycaemic effect of intramuscular insulin administration in this patient. After intramuscular injection of NPH and Ultralente human insulin (0.1 U kg-1), the lowest plasma glucose levels occurred 1 and 7 h later, respectively; the hypoglycaemic effect lasted approximately 2 and 12 h, respectively. We based insulin therapy on intramuscular NPH as a fast-acting insulin and Ultralente as an intermediate-acting insulin using four injections a day. During the next 24 months, the patient was hospitalized for 4 weeks versus 56 weeks in the 20 months preceding intramuscular insulin administration, and was able to resume full-time work. HbAlC decreased from 11.7% to 8.7% (normal range: 4.2-5.9%). Thus, long-term intramuscular insulin therapy is a feasible alternative to intravenous or intraperitoneal insulin in patients with well-demonstrated resistance to subcutaneous insulin.

Calcium is essential in normalizing intolerance to glucose that accompanies vitamin D depletion in vivo.

Vitamin D is essential for normal insulin secretion in vivo and in vitro. The differential effect of calcium and of the vitamin D endocrine system in the insulin response to secretagogues is still a subject of debate. To study the roles of calcium and the vitamin D system in the in vivo insulin response, GTT and insulin sensitivity tests were conducted in rats presenting vitamin D depletion and hypocalcemia or vitamin D depletion supplemented with calcium alone for 3, 7, or 14 days, vitamin D3 (6.5 nmol/day x 7 days), or 1,25(OH)2D3 (28 pmol/day x 7 days). Serum calcium was 1.28 +/- 0.04 mM in hypocalcemic vitamin D-depleted rats, 1.47 +/- 0.06 (NS), 1.8 +/- 0.2 (P < 0.0002), and 2.04 +/- 0.07 (P < 0.0001) mM after 3, 7, or 14 days, respectively, of calcium supplementation; vitamin D3- or 1,25(OH)2D3-supplemented animals had serum calcium of 2.61 +/- 0.04 or 2.48 +/- 0.05 mM (P < 0.0001 vs. hypocalcemic vitamin D-depleted rats). Rats with hypocalcemia and vitamin D depletion had significantly higher glucose concentrations (P < 0.0005) and lower insulin response during GTT than all other groups (P < 0.001). Differences in insulin sensitivity could not account for differences in response because exogenous insulin administration led to a similar drop in glucose concentrations in all groups, with the nadir averaging 51.7 +/- 2.6% of initial values. To distinguish between calcium and the vitamin D system in the GTT response, rats were treated with a nonhypercalcemic analogue of 1,25(OH)2D3, OCT (28 pmol/day x 4-7 days) with or without dietary calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of antibodies to submitochondrial particles, F1-ATPase complex and liver specific lipoprotein in liver diseases using ELISA]. / Detekce protilátek proti submitochondriálnym cásticím, F1-ATP-ázovému komplexu a jaternímu specifickému lipoproteínu u jaterních onemocnení pomocí ELISA.

ELISA was elaborated for the determination of antibodies against liver antigen complexes--submitochondrial particles, F1-ATPase and liver specific lipoprotein. The parameters achieved so far allow to use the assay as an undemanding complementary laboratory technique in diagnosing and monitoring hepatopathies of autoimmune origin. Cross reactivity between individual antigen complexes was recorded in the majority of sera from positively reacting patients. The preliminary results show that individual antigen complexes have similar antigen structures bound to high-molecular membrane complexes, some of which, however, can be solubilized on maintaining antigen activity in ELISA.

Vitamin D depletion retards the normal regeneration process after partial hepatectomy in the rat.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the hormone of the vitamin D3 (D3) endocrine system, has been shown to influence malignant and normal cell proliferation/differentiation, while insulin (I) is known to be essential for liver growth. To investigate the influence of D3 on liver regeneration, the effect of the D status was studied in D-depleted rats (D-) pretreated with: G1, placebo (D-, hypocalcemic); G2, oral calcium only (D-, normocalcemic); G3, D3; and G4, 1,25-(OH)2D3. Two thirds hepatectomy (HX) or sham operation was performed, and regeneration was studied for 3 weeks. I response to glucose challenge and the hepatic I receptor were also studied. Cell volume, DNA, and RNA were not affected by pretreatment. After HX, the pattern of [3H]thymidine incorporation into DNA (P less than 0.003) and the cell labeling index (P less than 0.0001) were highly influenced by pretreatment and suggestive of an earlier appearance of the S phase of the cell cycle in the 1,25-(OH)2D3-treated compared to the D- hypocalcemic group. Furthermore, the mitotic index revealed a significant effect of pretreatment (P less than 0.01), with peak mitosis 24 h after HX in D3-treated and 1,25-(OH)2D3-treated rats compared to 30-36 h after HX in the D- groups. Liver weight restitution was impaired in D- rats (P less than 0.009) and is illustrated by the estimated time required to achieve 70% recovery of the resected liver mass, which was found to be 186 and 300 h in G1 and G2, and 154 and 107 h in G3 and G4. G1 rats had significantly higher glucose concentrations (fasting as well as after glucose injection) and reduced I secretion when challenged with glucose (P less than 0.001); they also had an upregulation in hepatic I receptor number (P less than 0.005) compared to calcium or D3-treated rats, while 1,25-(OH)2D3 led to a liver I receptor number similar to that found in hypocalcemic D- rats; the affinity of the I receptor was, however, only slightly changed by pretreatment (P less than 0.08). Our data indicate that in D depletion, hypocalcemia retards DNA synthesis and liver mass recovery, while normocalcemia contributes to DNA synthesis, but fails to sustain mitosis and compensatory liver growth to a level comparable to that found after D3 and/or 1,25-(OH)2D3 repletion. The observation that both D3 and 1,25-(OH)2D3 significantly promoted normal liver recovery after partial HX illustrates the role of the D endocrine system in normal cell physiology in vivo.

[Serum lysozyme and copper levels in rats after the administration of dust]. / Obsah lyzozýmu a medi v sérech potkanu po podání prachu.

Changes in the content of lysozyme and copper were studied in the blood serum of rats in four time intervals (1, 2, 12, and 24 weeks) after administration of 50 mg TiO2, Sio2 or coal dust and the copper content was also studied 12 weeks after administration of 3 industrial dusts. The obtained results were supplemented by histopathological examinations and in the 12-week interval by the determination of the lung wet weight. The lysozyme content was statistically significantly increased compared to controls practically over the whole time course with differences in the level of the response to SiO2 in comparison with the response to TiO2 and coal. With the exception of the first interval, the serum copper level was statistically significantly increased only after quartz dust administration. The obtained results were compared with literary data and findings reported from clinical practice.

Blood glucose measurements during symptomatic episodes in patients with suspected postprandial hypoglycemia.

Postprandial (reactive) hypoglycemia is a controversial disorder because its symptoms are not specific, the presence of symptoms often correlates weakly with blood glucose concentrations measured after oral glucose administration, and glucose levels have rarely been measured when symptoms have developed spontaneously. In this study, we measured blood glucose in 28 patients with suspected postprandial hypoglycemia while they were experiencing their typical symptoms, in capillary blood collected on filter paper. Five patients (18 percent) had symptoms of "hypoglycemia" that were associated with blood glucose levels of 2.8 mmol per liter (50 mg per deciliter) or less, and eight other patients (29 percent) had symptoms associated with glucose levels of 2.9 to 3.3 mmol per liter on at least one occasion. Six (5 percent) of 132 reported symptomatic episodes that were associated with blood glucose levels of 2.8 mmol per liter or less, and 16 (12 percent) with levels of 2.9 to 3.3 mmol per liter. In 140 measurements made in 17 normal subjects, blood glucose levels were never found to be less than 2.8 mmol per liter, and levels of 2.9 to 3.3 mmol per liter were found only twice. No specific symptom was associated with low glucose levels in the patients with suspected hypoglycemia. The relief of symptoms by ingesting food was more often associated with low than with normal blood glucose levels: 86 percent of episodes in which blood glucose values were less than or equal to 3.3 mmol per liter were relieved by eating, as compared with 53 percent of episodes in which values were more than 3.3 mmol per liter (P less than 0.007). No correlation was found between plasma glucose levels measured after oral glucose administration and blood glucose levels measured during symptoms. We conclude that postprandial hypoglycemia is infrequent, even in a referral population, and that recording blood glucose measurements during spontaneously occurring symptomatic episodes is necessary to establish the diagnosis since symptoms are not specific and oral glucose-tolerance testing is not helpful.

Suppression of T4 secretion in a metastatic follicular carcinoma.

Despite total thyroidectomy, a patient with metastatic follicular carcinoma of the thyroid remained biologically euthyroid three months after stopping thyroxine (T4) therapy. Thyroid hormone production was investigated by means of a modified tri-iodothyronine (T3) suppression test, in which serum T4 was used as a suppression marker. After three weeks of oral T3 (Cytomel) therapy (50 micrograms/day), the serum T4 decreased from normal (108 nmol/L) to undetectable values. However, even though suppressive therapy was effective in preventing TSH dependent hormone secretion by the tumor, it did not prevent tumor growth and the eventual death of the patient.

The use of the DENTACRYL rapid (Spofa) resin for preparation of the rat tracheobronchial casts.

We have made tracheobronchial casts of the rat lungs using DENTACRYL Rapid (Spofa)--the synthetic methacrylic resin of Czechoslovak origin. The details of the method are fully described. The differences between preparations of the intact rat tracheobronchial trees and those of the rats after quartz instillation are illustrated. These differences are analogous to the commonly described changes in bronchograms.

Effects of continuous subcutaneous insulin infusion versus multiple injections on insulin receptors in insulin-dependent diabetics.

We compared continuous subcutaneous insulin infusion (CSII) versus multiple injections (MI) in the treatment of insulin-dependent diabetes mellitus (IDDM) to assess the effect of glucose control on monocyte insulin receptors. Each IDDM patient (n = 8) was treated for 2 mo by MI (HS Ultralente and AC boluses of regular insulin) and for 2 mo by CSII in a randomized fashion. Prestudy preprandial/postprandial blood glucose levels were 199 +/- 33/261 +/- 28 mg/dl and improved to 124 +/- 12/156 +/- 13 mg/dl during MI and to 115 +/- 11/151 +/- 11 mg/dl during CSII. Glycosylated hemoglobin before the study was 10.1 +/- 0.5% and decreased to 8.8 +/- 0.4 and 8.3 +/- 0.3% during MI and CSII, respectively. The specific 125I-labeled insulin binding to circulating monocytes in a group of nonobese controls (n = 17) was 4.6 +/- 0.2%. In our poorly controlled diabetics during conventional therapy, the 125I-insulin binding was decreased to 3.7 +/- 0.3 (P less than .025). This was not significantly affected by MI despite good glucose control (4.0 +/- 0.3%). With CSII, however, good glucose control was associated with normalization of 125I-insulin binding to monocytes (4.7 +/- 0.27%). The affinity of the insulin receptors was normal before the study and was not affected by either MI or CSII. In conclusion, these observations demonstrate that in IDDM, intensive therapy by MI and CSII resulted in similar good glucose control, but only CSII resulted in normalization of insulin receptors on circulating monocytes.

[Incidence of the 1/29 Robertsonian translocation in the cattle population in Czechoslovakia]. / Výskyt Robertsonovy translokace 1/29 v populaci skotu CSR.

We studied the inheritance of a serious chromosomal aberration--Robertsonian translocation 1/29 (R.t.1/29) in the female and male progeny of three successive generations after five heterozygous sires, carriers of this chromosomal aberration. Of the total number of 43 progenies that were examined cytogenetically, we found in ten sons of the second successive generation R.t.1/29 in 50% and in 26 daughters in 53%. In the third successive generation we found out this chromosomal aberration in one of two bullocks and in none of five heifers. In all cases we recorded the diploid chromosome number 59,XYt or 59,XXt in heterozygous constitution, in 100% of all examined mitoses. The translocation is of a monocentric type. The results including the data on the fertility of sires do not, however, demonstrate explicitly the reduced fertility of sires with this aberration, or of their progeny, as mentioned by some researchers.

[The character of chromosome banding in the karyotype of the roebuck]. / Charakter prouzkování chromozómu v karyotypu srnce obecného.

We examined the karyotype in five individuals of roe-deer (Capreolus capreolus), coming from Southern Moravia. We subjected to examination the cells of bone marrow, lymphocytes of peripheral blood and germ sexual cells of roebuck. In all examined animals and in all examined tissues we found out a diploid number of seventy chromosomes. All autosomes were acrocentric, sex chromosomes had two arms. During the C-banding the centromere regions of all autosomes stained deep--dark; this demonstrated a great amount of constitutive heterochromatin. Chromosome X stained negatively during the C-banding. The pattern of G-banding was used to establish homologous pairs of chromosomes.

Characteristics of patients with normal T3 and T4 and a low TSH response to TRH.

The nature of the thyroid disorder presented by patients with normal T4 and T3 but blunted TSH response to TRH has not been clarified. In this study, we compared thyroid function tests in 16 such patients with those of 14 controls and 10 hyperthyroid patients. Basal total T4, free T4, total T3, iodine uptake and cholesterol of the study group were similar to controls but significantly (P less than 0.001) lower than in hyperthyroid patients, except for cholesterol which was higher. In contrast, the basal TSH, increase in TSH after TRH stimulation, and decrease of T4 during T3 suppression tests were similar to data obtained in hyperthyroid patients but significantly (P less than 0.001) lower than in controls. Pulse rate was mid-way between the control and the hyperthyroid group. Thyroid stimulating antibody (TSAb) was measured with human thyroid cells in culture; the assay was positive in four subjects in the 16-patient group and in all hyperthyroid patients tested. TSH stimulation test showed a hyporesponse in iodine uptake in the four patients with positive TSAb (26 +/- 29%), as well as in hyperthyroid patients (6 + 5%). However, there was a hyper-response to TSH (213 +/- 52%) in the remaining 12 patients in the group, none of whom had TSAb. Thus TSAb is not seen as responsible for the thyroid disorder in the majority of patients with normal T3 and T4 and absent or blunted TSH response to TRH; surprisingly, most of these patients have thyroid hypersensitivity to TSH. These two characteristics, absence of TSAb and hypersensitivity to TSH, delineate a thyroid disorder clearly different from Graves' disease.

Effect of trimebutine on intestinal motility and plasma motilin in the dog.

In a previous report, trimebutine was shown to induce premature periods of phase III activity in fasting dogs, and its action was blocked by naloxone. In this study, we observed that trimebutine (5 mg X kg-1 iv) could induce premature phase IIIs in canine small intestine during interdigestive and digestive periods; trimebutine-induced phase IIIs were migrating along the small intestine faster than spontaneous activity fronts and; trimebutine-induced phase IIIs were accompanied by sharp rises in concentrations of plasma motilin. To further elucidate the trimebutine-stimulatory mechanism, we verified its effects on the release of various circulating peptides that influence intestinal motility: short-interval blood sampling during trimebutine infusion revealed that plasma motilin increases induced by trimebutine preceded the beginning of phase III in proximal duodenum; and gastrin and insulin postprandial releases were abolished by trimebutine. Therefore, trimebutine, by its simultaneous but opposite effects on various peptides that individually carry positive (e.g., motilin) or negative (e.g., gastrin and insulin) influences on the generation of activity fronts, could alter the equilibrium between stimulatory and inhibitory forces in such a way that, in some circumstances (e.g., postprandial period), stimulatory mechanisms become predominant.

Late increase in serum 1,25-dihydroxyvitamin D one month after surgery for adenomatous hyperparathyroidism.

This study was designed to follow the evolution of serum 1,25(OH)2D after surgery for primary hyperparathyroidism. Ten patients were studied before and for up to 85 d after removal of a single parathyroid adenoma. Blood and 24 h urine were obtained at various time points, for the measurement of serum or urinary phosphate and calcium indices. Before surgery, serum calcium (2.91 +/- 0.06 mmol/l; mean +/- SEM), parathyroid hormone (354 +/- 47 pg/ml) and 1,25(OH)2D (61.2 +/- 7.8 pg/ml) were elevated while serum phosphate (1.01 +/- 0.07 mmol/l) tended to be low. Relative hypoparathyroidism was evident for up to 5 d after surgery with the lowest value for serum parathyroid hormone (41 +/- 16 pg/ml) on day 1, serum calcium (2.12 +/- 0.06 mmol/l) on day 3 and highest value for serum phosphate (1.41 +/- 0.13 mmol/l) on day 5. As expected, serum 1,25(OH)2D levels decreased to 35.9 +/- 4.2 pg/ml 24 h after surgery. Stabilization of serum and urinary parameters to normal values was seen between day 5 and day 27; the only exception was serum 1,25(OH)2D, which increased again at day 27 to 57.6 +/- 5.0 pg/ml, a value as high as that before surgery. It was still elevated at day 50 (58.3 +/- 4.3 pg/ml), but returned towards normal values in three out of four patients (44 +/- 3.9 pg/ml) by day 80. No variation in 25(OH)D or 24,25(OH)2D was seen throughout the study. 1,25(OH)2D values could be related to serum parathyroid hormone values before surgery (r = 0.659, P less than 0.05) but not after. The secondary increase in serum 1,25(OH)2D could not be related to variations in serum calcium, phosphate, parathyroid hormone or diet. Further studies will be required to explain this phenomenon.

[The karyotype and character of chromosome bands in mountain sheep in Czechoslovakia]. / Karyotyp a charakter prouzkování chromozómu muflona v CSR.

Thirty-one moufflon (Ovis musimon) coming from eight localities in Bohemia and Moravia were examined. All animals had a diploid number of chromosomes without any numerical or structural aberrations from the standard set. On the basis of G-banding, all chromosomes were arranged into homologous pairs. C-banding was used to prove the localization of constitutive heterochromatin in the centromeric regions of the majority of chromosomes. Nucleolus organizers, demonstrated by means of A-staining, were located telometrically on the chromosomes in three big two-arm pairs and two acrocentric pairs. The number of active nucleolus organizers in individual cells ranged from 2 to 7. When these results were compared with the published data on the karyotype of sheep, the two forms were found to have much in common. It can be supposed that the karyotypes of moufflon and domestic sheep differ in the frequency of the occurrence of Ag-positive sections with nucleolus organizers.