Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo.

Bone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model. OBJECTIVE: The objective of this study was to characterise ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep. DESIGN: oBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation capacity. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. RESULTS: Expanded oBMSC were positive for CD44 and CD146 and negative for CD45. The common adipogenic induction ingredient, 3-Isobutyl-1-methylxanthine (IBMX), was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not. CONCLUSION: The sensitivity of oBMSC, compared to human BMSC, to IBMX in standard adipogenic assays highlights species-associated differences. Micro-pellets manufactured from oACh were more effective than micro-pellets manufactured from oBMSC in the repair of osteochondral defects in sheep. While oBMSC can be driven to form cartilage-like tissue in vitro, the effective use of these cells in cartilage repair will depend on the successful mitigation of hypertrophy and tissue integration.

Transforming growth factor-beta stimulates human bone marrow-derived mesenchymal stem/stromal cell chondrogenesis more so than kartogenin.

A previous study identified kartogenin (KGN) as a potent modulator of bone marrow mesenchymal stem/stromal cell (BMSC) chondrogenesis. This initial report did not contrast KGN directly against transforming growth factor-beta 1 (TGF-ß1), the most common growth factor used in chondrogenic induction medium. Herein, we directly compared the in vitro chondrogenic potency of TGF-ß1 and KGN using a high resolution micropellet model system. Micropellets were cultured for 7-14 days in medium supplemented with TGF-ß1, KGN, or both TGF-ß1 + KGN. Following 14 days of induction, micropellets exposed to TGF-ß1 alone or TGF-ß1 + KGN in combination were larger and produced more glycosominoglycan (GAG) than KGN-only cultures. When TGF-ß1 + KGN was used, GAG quantities were similar or slightly greater than the TGF-ß1-only cultures, depending on the BMSC donor. BMSC micropellet cultures supplemented with KGN alone contracted in size over the culture period and produced minimal GAG. Indicators of hypertrophy were not mitigated in TGF-ß1 + KGN cultures, suggesting that KGN does not obstruct BMSC hypertrophy. KGN appears to have weak chondrogenic potency in human BMSC cultures relative to TGF-ß1, does not obstruct hypertrophy, and may not be a viable alternative to growth factors in cartilage tissue engineering.

Sheep as a model for evaluating mesenchymal stem/stromal cell (MSC)-based chondral defect repair.

Osteoarthritis results from the degradation of articular cartilage and is one of the leading global causes of pain and immobility. Cartilage has a limited capacity for self-repair. While repair can be enhanced through surgical intervention, current methods often generate inferior fibrocartilage and repair is transient. The development of tissue engineering strategies to improve repair outcomes is an active area of research. While small animal models such as rodents and rabbits are often used in early pre-clinical work, larger animals that better recapitulate the anatomy and loading of the human joint are required for late-stage preclinical evaluation. Because of their physiological similarities to humans, and low cost relative to other large animals, sheep are routinely used in orthopedic research, including cartilage repair studies. In recent years, there has been considerable research investment into the development of cartilage repair strategies that utilize mesenchymal stem/stromal cells (MSC). In contrast to autologous chondrocytes derived from biopsies of articular cartilage, MSC offer some benefits including greater expansion capacity and elimination of the risk of morbidity at the cartilage biopsy site. The disadvantages of MSC are related to the challenges of inducing and maintaining a stable chondrocyte-like cell population capable of generating hyaline cartilage. Ovine MSC (oMSC) biology and their utility in sheep cartilage repair models have not been reviewed. Herein, we review the biological properties of MSC derived from sheep tissues, and the use of these cells to study articular cartilage repair in this large animal model.

A randomized clinical trial of a standard versus vegetarian diet for weight loss: the impact of treatment preference.

BACKGROUND: With obesity rampant, methods to achieve sustained weight loss remain elusive. OBJECTIVE: To compare the long-term weight-loss efficacy of 2 cal and fat-restricted diets, standard (omnivorous) versus lacto-ovo-vegetarian, and to determine the effect of a chosen diet versus an assigned diet. DESIGN, SUBJECTS: A randomized clinical trial was conducted with 176 adults who were sedentary and overweight (mean body mass index, 34.0 kg/m(2)). Participants were first randomly assigned to either receive their preferred diet or be assigned to a diet group and second, were given their diet of preference or randomly assigned to a standard weight-loss diet or a lacto-ovo-vegetarian diet. Participants underwent a university-based weight-control program consisting of daily dietary and exercise goals plus 12 months of behavioral counseling followed by a 6-month maintenance phase. MEASUREMENTS: Percentage change in body weight, body mass index, waist circumference, low- and high-density lipoprotein, glucose, insulin and macronutrient intake. RESULTS: The program was completed by 132 (75%) of the participants. At 18 months, mean percentage weight loss was greater (P=0.01) in the two groups that were assigned a diet (standard, 8.0% (s.d., 7.8%); vegetarian, 7.9% (s.d., 8.1%)) than in those provided the diet of their choice (standard, 3.9% (s.d., 6.1%); vegetarian, 5.3% (s.d., 6.2%)). No difference was observed in weight loss between the two types of diet. Over the 18-month program, all groups showed significant weight loss. CONCLUSIONS: Participants assigned to their dietary preference did not have enhanced treatment outcomes. However, all groups lost weight with losses ranging from 4 to 8% at 18 months.

High sensitivity of basophils predicts side-effects in venom immunotherapy.

BACKGROUND: Systemic side-effects of venom immunotherapy (VIT) represent a considerable problem in the treatment of patients allergic to Hymenoptera venom. We examined the hypothesis whether basophil responsiveness might be connected with the adverse reactions to VIT. METHODS: Basophil surface expression of activation marker CD63 induced by different concentrations of honeybee and wasp venom (0.1 and 1 mug/ml) was measured by flow cytometry in 34 patients with history of systemic anaphylactic reactions to Hymenoptera sting just before rush honeybee or wasp VIT. RESULTS: Eleven of 34 patients had systemic anaphylactic reaction (Mueller grades I-III) and one patient a large local reaction to VIT. In those 12 patients, median percentage of activated basophils after stimulation with VIT-specific venom in concentration of 0.1 microg/ml was 99% (range: 17-195) of value reached with stimulation with 1 microg/ml. Side-effects occurred in all patients with 0.1/1 ratios over 92% (eight of 12). In contrast, in 22 patients with no side-effects, the median 0.1/1 ratio was 25% (range: 2-92). These concentration-dependent activation ratios were significantly different between the groups with and without side reactions (P < 0.0001). We also show significant positive correlation of the occurrence/clinical grade of the side-effects with individual ratios of CD63 basophil response (r = 0.73, P < 0.0001). CONCLUSION: The results suggest that increased basophil sensitivity to allergen-specific in vitro stimulation is significantly associated with major side-effects of VIT.

[Chloroquine-induced drug hypersensitivity alveolitis]. / Chloroquin-induzierte arzneimitteltoxische Alveolitis.

This case history presents a 41-years-old man with chronic discoid erythematosus who was treated with chloroquine. After 2 weeks of treatment he developed dyspnea, purulent sputum, high fever, diffuse papular rash, and diffuse peripheral homogeneous infiltrates on chest x-ray. Rapid recovery occurred after discontinuing chloroquine. Subsequent peroral provocation with chloroquine caused symptoms suggestive of hypersensitivity pneumonitis. Analysis of bronchoalveolar lavage fluid showed eosinophilia and a reduced CD4/CD8 ratio. Although treatment with chloroquine is often complicated by side effects, in a literature search we could not find any report on acute pneumonitis.

Bronchoscopic removal of foreign bodies in adults: experience with 62 patients from 1974-1998.

The authors reviewed their experience with therapeutic bronchoscopy for removal of tracheobronchial foreign bodies in the adult. Bronchoscopy records and collection of foreign bodies in the endoscopic department were retrospectively examined. Among 37,466 bronchoscopies performed between 1974-1998, 62 (0.2%) were performed for the removal of tracheobronchial foreign bodies. Medical history was suggestive of foreign body aspiration in 33 patients and the chest radiograph was suggestive in 10 patients. The procedure was performed with the flexible bronchoscope in 42 patients (68%), rigid bronchoscope in 4 (6%), and with both in 16 (26%) patients. Foreign bodies were found in the right bronchial tree on 42 occasions, in the left on 20 and in the trachea once. In 39 patients, inflammatory granulations were found around the foreign body. The origins of the foreign bodies included: bone fragments (n=31), vegetable (n=10), broncholith (n=8), a part of dental prosthesis (n=7), endodontic needle (n=2), a metallic (n=2), or plastic (n=1) particle, a tracheostomy tube (n=1) and a match (n=1). In one patient, 2 foreign bodies were found. The foreign bodies were successfully removed in all but 2 patients (3%). The most useful instruments for removal were alligator forceps and the wire basket. Foreign bodies in the tracheobronchial system are rare in adults. They can be successfully removed in the majority of patients under either flexible or rigid bronchoscopy.

Double-blind, randomised, placebo-controlled study of two concentrations of azelastine eye drops in seasonal allergic conjunctivitis or rhinoconjunctivitis.

This double-blind, randomised, placebo-controlled study was carried out to assess the efficacy and safety of 0.025% and 0.05% azelastine eye drops twice daily administered for 14 days to patients with seasonal allergic conjunctivitis or rhinoconjunctivitis. A total of 278 patients were recruited and 226 patients were evaluable for per protocol analysis. The target parameter was the response rate. Four eye symptoms, including the main symptom (itching) were recorded by patients in diaries and eight symptoms were assessed by physicians before and after seven and 14 days of treatment. Severity of symptoms was measured on a four-point scale. The response rates for itching (improvement of at least one score point within the first three days) according to patient assessment were 43% for placebo, 52% for 0.025% and 56% for 0.05% azelastine (NS). However, a more objective assessment of the three main eye symptoms by physicians showed a concentration-dependent improvement in response rate compared with placebo (a decrease of > or = 3 points from a baseline total score of > or = 6), which reached statistical significance for 0.05% azelastine on Day 7 (p < 0.002). In the evaluable patient population, the scores of the three main eye symptoms as well as of all eight recorded eye symptoms, as assessed by the physician, were significantly (p < 0.05) lower in the 0.05% azelastine eye drops group in comparison with the placebo group at Day 7. Inefficacy was the cause of withdrawal in five and three patients on 0.025% and 0.05% azelastine, respectively, and in six patients on placebo. Adverse drug effects, mainly a mild, transient irritation and a bitter or unpleasant taste, were reported by 14% (0.025%), 20% (0.05%) and 15% (placebo) of the patients. No serious side-effects occurred. Azelastine eye drops are effective and well tolerated at a concentration of 0.05% for the treatment of seasonal allergic conjunctivitis.

[Gold salt alveolitis in 3 patients with rheumatoid arthritis]. / Goldsalzalveolitis bei drei Patienten mit rheumatoider Arthritis.

BACKGROUND: When the characteristic symptoms for an interstitial pulmonary disease arise in patients with rheumatoid arthritis, a drug-induced alveolitis should be considered in the differential diagnosis. In such cases, the administration of the drug and gold salts should be stopped. PATIENTS AND METHODS: The cases of three patients with rheumatoid arthritis (RA) who had been treated with gold salts for 2 months (A), 23 months (B), and 36 months (C) are presented. The total dose of sodium aureothiomalate amounted to 280 mg for patient A, 1150 mg for patient B, and 2190 mg for patient C. Clinical signs, X-rays of the lungs, pulmonary function tests, and laboratory tests were evaluated for the three patients while, for patient A BAL as well as provocation tests were additionally performed before and after therapy. In this case, the histological picture of the lungs is presented; biopsies were taken during the first BAL. RESULTS: The clinical complaints of all 3 patients were similar, with the alveolitis being observed as diffuse in one case and above all in the upper regions in two cases on radiology. This led to differing degrees of diffusion disorders in the lungs. In patient A, the diagnosis was made in the stage of progressive fibrotic alveolitis and was treated with D-penicillamine. All 3 patients received steroids over 3-6 months and the gold salts were stopped. Because of the long duration and doubtful differential diagnosis for patient A with either rheumatoid lung or gold salt alveolitis, a provocation test with sodium aureothiomalate was performed. All 3 patients had blood eosinophilia while, in case A, a thrombopenia was also found. CONCLUSIONS: A gold salt alveolitis can occur as a side effect of gold salts in addition to skin vasculitis and hematological disorders. When the gold salt administration is not stopped a fibrotic alveolitis can develop. The provocation test can be diagnostically useful to distinguish between a rheumatoid lung and gold salt alveolitis.

[Branhamella catarrhalis respiratory tract infections]. / Infekcije respiracionog trakta Branhamellom catarrhalis.

Forty-six hospitalised patients with respiratory tract infections (RTI) in whom Branhamella catarrhalis had been isolated were evaluated. On average they were 70 years old, there were 39 males and 7 females, 89% of them were smokers. In 85% Branhamella catarrhalis caused exacerbation of chronic bronchitis and in 15% the pneumonia. Most cases of this infection were detected in December and January. In vitro 78% of strains formed beta lactamasis. All 46 isolations were sensitive to cyprofloxacin, 43 (93%) to the combination of amoxicillin with clavulanic acid, 41% (89.1%) to erithromycin, 44 (95.6%) to gentamycin and only 26 (56.5%) to trimetoprim-sulphametoxasol. In the therapy amoxicillin with clavulanic acid, macrolids and kinolons were successful while trimetroprim-sulphametoxasol was less effective.

[Effect of salbutamol administered directly by a nebulizer or by means of a spacer on bronchodilation]. / Bronhodilatacioni efekat salbutamola aplikovanog direktno rasprsivacem i preko spacera.

During three successive days in 14 patients with bronchial asthma after application of Salbutamol directly from the metered-dose inhaler (MDI) or indirectly by spacer the changes of FEF and FEV1 were measured. For inhalation through spacer the double blind crossover trial with placebo was used. Out of 12 patients three of them responded with better flows (FEV1, FEF) after inhalation via MDI. In 7 of them there were no significant differences between ways of application while in two of them the flows by spacer were significantly better. Tachypnea, low lung volumes as well as poor co-operation of the patient, especially elderly, should speak in favour of the use of spacer. However, in young, co-operative asthmatic patients the MDI is suitable. Further controlled studies of new applications of inhalation therapy should be studied.

[Comparison of amoxicillin and amoxiclav in the therapy of respiratory infections]. / Uporedenje amoksicilina i amoksiklava u terapiji respiracionih infekcija (RI).

Randomly hospitalized patients with respiratory tract infections admitted to three pulmonary departments of the Golnik Institute for Pulmonary Diseases and Tuberculosis were enrolled in an open, comparative clinical study of Amoksiklav and Amoxicillin. A group of 26 patients with a mean age of 64.5 years presenting with pneumonia (13), exacerbation of chronic bronchitis (12) and bronchiectasis (1) were given Amoskilav, while another 20 patients with a mean age of 61.4 years presenting with pneumonia (9), exacerbation of chronic bronchitis (5), bronchiectasis (5) and sinusitis (1) received Amoxicillin. The efficacy of treatment was assessed by bacteriological findings of respiratory tract specimens, sputum and blood leucocytosis, macroscopic purulence of sputum and the presence of fever. The bacteriological findings are shown in detail. Leucocytosis and macroscopic purulence of sputum significantly improved on Amoksiklav therapy (p less than 0.05) while with Amoxicillin there was no significant improvement. With respect to the presence of fever, there was no significant difference between Amoksiklav and Amoxicillin. The overall clinical and bacteriological response was very good and good in 88.5% of patients treated with Amoksiklav compared to 75% of those receiving Amoxicillin. Additionally, 1000 pathogenic strains were tested for their response to Amoksiklav and Amoxicillin. Amoksiklav proved superior against strains of Branhamella catarrhalis, E. coli, coagulase-negative staphylococci and K. pneumoniae (p less than 0.01).

[Comparison of in vitro activity of ciprofloxacin and pefloxacin against strains isolated from clinical material]. / Uporedna in vitro aktivnost ciprofloksacina i pefloksacina prema sojevima izolovanim iz klinickog materijala.

The susceptibility of 392 strains of pathogenic bacteria isolated from clinical material of hospitalized patients in Institute for Chest Diseases Golnik were studied. 100 strains of Escherichia coli, 56 strains of Enterobacter spp., 40 strains of Proteus mirabilis, 50 strains of Gram negative nonfermentative bacteria, 46 strains of Pseudomonas aeruginosa and 100 strains of Staphylococcus aureus were tested. Minimal inhibitory concentrations of ciprofloxacin and pefloxacin were determined by agar dilution test. The susceptibility of tested strains is greater to ciprofloxacin, except susceptibility of strains of Staphylococcus aureus, which is greater to pefloxacin. Strains of nonfermentative bacteria have lower susceptibility to both quinolones.

[Pulmonary hypertension in diffuse scleroderma and the CREST syndrome]. / Plucna hipertenzija kod difuzne sklerodermije i CREST sindroma.

In 22 patients with systemic sclerosis (SS) i.e. 16 patients with diffuse scleroderma and 6 patients with CREST syndrome the presence of pulmonary hypertension (PH) was investigated by microcatheter. Manifest PH was found in 3 patients with diffuse scleroderma (19%) and in 3 patients with CREST syndrome (50%). Latent PH was found in 8 patients with diffuse scleroderma (50%) and in 2 patients with CREST syndrome (33%). In our patients the occurrence of PH in CREST syndrome was more frequent than in diffuse scleroderma which is in accordance with the data from the literature. Isolated PH (without simultaneous involvement of pulmonary interstitium) was found in most patients with CREST syndrome (but in 1 case) and in 3 out of 11 patients with diffuse scleroderma.