Edaravone attenuates disease severity of experimental auto-immune encephalomyelitis and increases gene expression of Nrf2 and HO-1.

The aim of this study was to evaluate therapeutic potential of edaravone in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) and to expand the knowledge of its mechanism of action. Edaravone (6 mg/kg/day) was administered intraperitoneally from the onset of clinical symptoms until the end of the experiment (28 days). Disease progression was assessed daily using severity scores. At the peak of the disease, histological analyses, markers of oxidative stress (OS) and parameters of mitochondrial function in the brains and spinal cords (SC) of mice were determined. Gene expression of inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha was determined at the end of the experiment. Edaravone treatment ameliorated EAE severity and attenuated inflammation in the SC of the EAE mice, as verified by histological analysis. Moreover, edaravone treatment decreased OS, increased the gene expression of the Nrf2 and HO-1, increased the activity of the mitochondrial complex II/III, reduced the activity of the mitochondrial complex IV and preserved ATP production in the SC of the EAE mice. In conclusion, findings in this study provide additional evidence of edaravone potential for the treatment of multiple sclerosis and expand our knowledge of the mechanism of action of edaravone in the EAE model.

Increased Number of Mast Cells in Epicardial Adipose Tissue of Cardiac Surgery Patients With Coronary Artery Disease.

Chronic inflammation of adipose tissue is associated with the pathogenesis of cardiovascular diseases. Mast cells represent an important component of the innate defense system of the organism. In our work, we quantified mast cell number in epicardial adipose tissue (EAT), subcutaneous adipose tissue (SAT), and right atrial myocardium (RA) in patients undergoing open heart surgery (n=57). Bioptic samples of EAT (n=44), SAT (n=42) and RA (n=17) were fixed by 4 % paraformaldehyde and embedded into paraffin. An anti-mast cell tryptase antibody was used for immunohistochemical detection and quantification of mast cells. We also demonstrated immunohistochemically the expression of CD117 and chymase markers. In EAT of patients with coronary artery disease (CAD), higher incidence of mast cells has been found compared to patients without CAD (3.7±2.6 vs. 2.1±1.2 cells/mm(2)). In SAT and RA, there was no difference in the number of mast cells in CAD and non-CAD patients. Mast cells in SAT, EAT and RA expressed CD117 and chymase. An increased incidence of mast cells in EAT of CAD patients may indicate the specific role of these inflammatory cells in relation to EAT and coronary arteries affected by atherosclerosis.

[Surgical Treatment of Congenital Cleft Foot - Case Study and Literature Review]. / Operacní lécení vrozeného rozstepu nohy ­ kazuistika a prehled literatury.

Congenital cleft foot is a deformity characterised by genetic heterogeneity and a high degree of phenotypic variability. This together with its very low incidence is the reason for rather controversial opinions on the treatment. The authors present a case study of a boy with unilateral cleft foot classified as type III by Blauth-Borisch and type II by Abraham et al., who underwent a surgery at the age of 12 months. The defect was closed by rectangular soft tissue flaps, the intermetatarsal ligament connecting the first metatarsal head and the third metatarsal head was reconstructed with local fibrous tissue flap. The present hypermobility with extension position of the first ray were stabilised by the Kirschner wire inserted along the axis of the first ray from the dorsum of the talus through the middle of its head to the medial chondrogenic tarsal bones and further through the first metatarsal bone and the corrected metatarsophalangeal joint of the hallux. At the age of 6 years and 7 months, due to dynamic abductovalgus foot deformity, lengthening calcaneal osteotomy was also performed. Favourable clinical and radiographic outcomes of the used surgical technique were observed by the authors. The cleft foot as such shall be treated after a thorough evaluation of the patient and his/her necessary monitoring during the childhood. Key words: ectrodactyly, cleft foot, split hand/foot malformation.

[Development of Hip Joints Treated Non-operatively for Developmental Dysplasia of the Hip by Overhead Traction - Clinical and Radiographic Assessment after Reaching Skeletal Maturity]. / Vývoj kycelních kloubu konzervativne lécených pro kycelní dysplazii metodou overhead trakce ­ klinické a rentgenologické hodnocení po dosazení skeletální zralosti.

PURPOSE OF THE STUDY The purpose of the retrospective study was to analyse the success rate and safety of overhead traction in treating decentered hip joints. In patients treated strictly non-operatively, i.e. with no open reduction and no operative management of residual dysplasia performed, acetabular development was assessed once they have reached the skeletal maturity. MATERIAL AND METHODS The restrospective study included a total of 40 patients treated by overhead traction in the period 1999-2002 for developmental dysplasia of the hip (ultrasound classes III.A, III.B, IV according to Graf classification). The study group consisted of 35 girls and 5 boys. 10 patients were affected bilaterally. The two-phase traction was applied during the hospital stay and concluded by arthrography of the treated hip joint under general anaesthesia to confirm its concentric reduction and stability within the safe zone. To allow healing, bilateral plaster hip spica cast was applied for 6 weeks, followed by Pavlik harness. The Salter diagnostic criteria were used for avascular necrosis. The acetabular development in non-operatively treated patients was assessed after reaching skeletal maturity according to the Severin criteria. The radiographic parameters (center-edge angle of Wiberg, Sharp angle, Tönnis angle, acetabular coverage of the femoral head, grade of osteoarthritis according to Tönnis classification) were statistically compared with the control group of 18 healthy contralateral hip joints. RESULTS By overhead traction the closed reduction of 43 hips (86%) was successfully achieved. In the group of hips with successful closed reduction, avascular necrosis of the femoral head developed in 1 case (2.3%). In preschool age, surgical correction of the residual acetabular dysplasia or subluxation was performed in 7 hip joints (16%) in the group of successful closed reductions. According to the Severin criteria for the evaluation of radiographic results, the findings were favourable (Severin Ia, Ib) after reaching the skeletal maturity in 29 of 43 (67%) hip joints, in which closed reduction was originally successfully achieved. No statistically significant difference in the studied radiographic parameters was found between the group of treated hip joints and the control group. DISCUSSION Despite the general consensus regarding the beneficial effects of early diagnosis of developmental dysplasia of the hip in the form of better treatment outcomes and reduced risk of complications in the world literature, the authors of individual studies do not agree as to the used treatment method, timing and duration of treatment. Neither they agree as regards the importance of traction therapy in decentered hip joints. Our study brings information primarily on the acetabular development in patients treated by traction, commenced early, namely before the age of 6 months. CONCLUSIONS Based on our experience, the overhead traction is an efficient treatment option in managing decentered hip joints (ultrasound types III.A, III.B, IV according to Graf). It is a safe method provided the safe zone principles are adhered to during the traction treatment as such as well as during the retention phase with the plaster hip spica applied. Development of the treated hip joint shall be regularly followed up until the skeletal maturity is reached. Key words: developmental dysplasia of the hip, closed reduction, overhead traction.

Some Possibilities to Study New Prophylactics against Nerve Agents.

Nerve agents belong to the most dangerous chemical warfare agents and can be/were misused by terrorists. Effective prophylaxis and treatment is necessary to diminish their effect. General principles of prophylaxis are summarized (protection against acetylcholinesterase inhibition, detoxification, treatment "in advance" and use of different drugs). They are based on the knowledge of mechanism of action of nerve agents. Among different examinations, it is necessary to test prophylactic effectivity in vivo and compare the results with protection in vitro. Chemical and biological approaches to the development of new prophylactics would be applied simultaneously during this research. Though the number of possible prophylactics is relatively high, the only four drugs were introduced into military medical practice. At present, pyridostigmine seems to be common prophylactic antidote; prophylactics panpal (tablets with pyridostigmine, trihexyphenidyl and benactyzine), transant (transdermal patch containing HI-6) are other means introduced into different armies as prophylactics. Scavenger commercionally available is Protexia®. Future development will be focused on scavengers, and on other drugs either reversible cholinesterase inhibitors (e.g., huperzine A, gallantamine, physostigmine, acridine derivatives) or other compounds.

[Mid-Term Outcomes of Reconstruction of Charcot Foot Neuroarthropathy in Diabetic Patients]. / Strednedobé výsledky rekonstrukce Charcotovy neuropatické artropatie nohy u diabetiku.

PURPOSE OF THE STUDY The aim of this study was to evaluate the mid-term outcomes of the surgical reconstruction of Charcot Foot Neuroarthropathy in diabetic patients with failed conservative treatment and indicated for a below-knee amputation. MATERIAL AND METHODS In the period from 2010 to 2015 the surgical reconstruction of inactive, chronic Charcot Foot Neuroarthropathy classified as type II and III by Sanders and Frykbeg was performed in 16 patients with failed conservative treatment. All these patients were by the diabetes centres initially indicated for a below-knee amputation. The performed evaluation focused on the clinical outcome (limb preservation, walking in footwear, full weight-bearing capability, the radiographic result (talar-first metatarsal angle, calcaneal inclination - negative, neutral, positive), complications (associated and not associated with the surgery). RESULTS The mean follow-up period was 4.7 years (2.5-7.5 years). From the original group of 16 patients indicated for a below-knee amputation following the failure of conservative treatment, the amputation was performed in one patient only. After the surgical reconstruction 15 patients were able to fully weight-bear when achieving plantigrade foot position, of whom 9 wore regular footwear and 6 customised diabetic footwear. The talar-first metatarsal angle was corrected from the mean 30 degrees (20-45) to the mean 5 degrees (0-10). The calcaneal inclination was corrected from the negative preoperative value in all the cases to neutral in 5 patients and positive in 10 patients. The surgery-associated complications were the following: infectious complications - positive preoperative cultivation in 10 out of 16 patients, secondary healing of the surgical wound in 7 patients, the need of additional ablation of plantar prominence of tarsal skeleton in 2 patients, screw prominence in 2 patients with the need of extraction - all healed without complications. In one case a lower limb amputation was performed due to secondary limb ischemia. The complications not associated with the surgery consisted of a soft tissue injury due to neuropathy. DISCUSSION Conservative treatment remains the basic approach to Charcot foot neuroarthropathy which is often associated with a long-term off-loading of the affected limb on a wheelchair, repeated hospital stays, changes of wound dressing of plantar ulcers. These are stated as the most frequent indications for a major amputation. Nonetheless, even the major amputation is accompanied by complications. The candidate for a reconstruction surgery should be a cooperating, compensated, informed diabetic patient with Charcot foot neuroarthropathy, either instable or stable, but non-plantigrade. It is necessary to diagnose and treat the impairment of leg blood supply and osteomyelitis, and to provide an appropriate rehabilitation. CONCLUSIONS Of the original 16 indications for a lower limb amputation in diabetic patients with Charcot foot neuroarthropathy, only one amputation was performed. Positive mid-term outcomes of surgical reconstruction justify further development of this method, bearing in mind the necessity of careful indication and preoperative preparation of the patient in cooperation with diabetologists. Precise and accurate surgical technique and relevant postoperative care are essential to minimise the potential complications. Key words:Charcot arthropathy, diabetic neuropathy, rocker - bottom foot deformity, foot reconstruction.

Metronidazole Attenuates the Intensity of Inflammation in Experimental Autoimmune Uveitis.

Autoimmune uveitis is a serious sightthreatening disease that in many cases fails to respond to conventional immunosuppressive or biological therapy. Experimental models used in research allow more detailed study of pathogenesis of the autoimmune process and testing new therapeutic strategies. Recent results show that infection can trigger autoimmune diseases, and some commensal microorganisms are essential in causing disease activity. The aim of this work was to assess the effect of broadspectrum antibiotics - combination of metronidazole and ciprofloxacin or metronidazole alone - on the intensity of intraocular inflammation in experimental autoimmune uveitis (EAU). EAU was induced in mouse strain C57BL/6J by interphotoreceptor retinoid- binding protein in complete Freund's adjuvant and pertussis toxin. The grade of uveitis was assessed clinically and histologically in haematoxylin and eosin- stained tissues. Lymphocytes and macrophages were detected in cryosections using the immunoperoxidase method with antibodies. The therapy was commenced one week before EAU induction and continued throughout the experiment. In addition, metronidazole treatment was also started two weeks before EAU induction. Antibiotics significantly reduced the intensity of uveitis compared to the control group (P < 0.05). The effects of combination of ciprofloxacin and metronidazole and of metronidazole alone were similar when the therapy started one week before EAU induction (P < 0.05). Metronidazole commenced two weeks before EAU induction and throughout the experiment suppressed the intensity of EAU with even higher statistical significance (P < 0.0001). It can be assumed that the high protective effect of metronidazole on EAU intensity may be due not only to its antimicrobial effect, but also to its immunomodulatory activity.

[Tear of the Distal Biceps Brachii Tendon - Correlation of Ultrasound and Operative Findings, Surgical Therapy Results]. / Ruptura distální slachy musculus biceps brachii ­ korelace sonografických a operacních nálezu, výsledky operacní terapie.

PURPOSE OF THE STUDY When treating tears of the distal biceps brachii muscle tendon, we repeatedly noticed a difference between the preoperative ultrasound findings and the operative findings. The aim of the study was to retrospectively correlate these findings in order to determine the sensitivity of the ultrasound examination in everyday orthopaedic practice. Moreover, we compared the results and complications of surgical treatment through two operative techniques used at our department. MATERIAL AND METHODS In the 2004-2016 period 20 patients underwent a surgery at our department for total tear of the distal tendon of the biceps brachii muscle. In 18 patients an ultrasound examination was performed preoperatively. In 3 patients it was repeated. Therefore, there were a total of 21 ultrasound observations made in this group of patients. Excluded were the cases of chronic tendinoses or inveterated tears. The group was divided into two sub-groups. The first sub-group was composed of patients in whom the preoperative ultrasound examination was performed by a radiologist-specialist in the musculoskeletal system, the second sub-group was composed of patients examined by a non-specialist. The ultrasound findings were compared with the operative findings. With the use of a formula for the calculation of sensitivity of the test, the sensitivity of the ultrasound examination was determined for proper recognition of a complete tear of the tendon concerned. Also, we compared the results and complications of the two operative methods applied: the technique using bone anchor vs. the Boyd-Anderson technique of transosseous reinsertion. RESULTS The sensitivity of the ultrasound examination was 91% in examinations performed by a radiologist-specialist and 40% in examinations performed by no-specialists. Both the surgical techniques brought very good results in our group of patients. The reported complications included 2 cases of temporary radial nerve palsy, 1 case of formation of heterotopic ossifications. DISCUSSION The sensitivity of ultrasound is adequate according to the literature. In our group of patients, the same applied only to examinations performed by a radiologist-specialist in the musculoskeletal system. This is because the ultrasound examination of the distal biceps tendon is a highly specialised examination. When performed by a non-specialist, the result of examination obtained in our observations is rather misleading, thus could lead to an improper method of treatment. Partial tears of this tendon are very rare according to the literature. Indirect signs of the partial tear presence at this location detected by ultrasound resulted in most cases in an incorrect diagnosis, therefore the description of a partial tear visualised by the ultrasound should be reserved exclusively for cases when intact fibrils are clearly detected during the examination. For unclear cases, the MRI scan is indicated. The results of both the surgical techniques of reinsertion applied were very good. The method using the bone anchors is technically easier to perform. Nonetheless, it has its specifics. CONCLUSIONS To diagnose correctly the tear of the distal biceps muscle tendon it is essential to perform a thorough clinical examination and to obtain the medical history of the patient, especially the mechanism of injury. Sonography can be beneficial only provided the examination is carried out by a specialist in the musculoskeletal system, with the use of appropriate device and under standard conditions. For surgical treatment of this injury we prefer the technique using a bone anchor, namely particularly since it is technically easier to perform. The functional results are very good. Key words: distal biceps tendon, elbow, tendon tear, ultrasound, suture anchors.

The concept of hybrid molecules of tacrine and benzyl quinolone carboxylic acid (BQCA) as multifunctional agents for Alzheimer's disease.

Novel tacrine-benzyl quinolone carboxylic acid (tacrine-BQCA) hybrids were designed based on multi-target directed ligands (MTLDs) paradigm, synthesized and evaluated in vitro as inhibitors of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). Tacrine moiety is represented herein as 7-methoxytacrine, 6-chlorotacrine or unsubstituted tacrine forming three different families of seven members, i.e. 21 compounds in overall. Introducing BQCA, a positive modulator of M1 muscarinic acetylcholine receptors (mAChRs), the action of novel compounds on M1 mAChRs was evaluated via Fluo-4 NW assay on the Chinese hamster ovarian (CHO-M1WT2) cell line. All the novel tacrine-BQCA hybrids were able to block the action of hAChE and hBChE in micromolar to nanomolar range. The hAChE kinetic profile of 5p was found to be mixed-type which is consistent with our docking experiments. Moreover, selected ligands were assessed for their potential hepatotoxicity on HepG2 cell line and presumable permeation through the blood-brain barrier by PAMPA assay. Expected agonistic profile towards M1 mAChRs delivered by BQCA moiety was not confirmed. From all the hybrids, 5o can be highlighted as non-selective cholinesterase inhibitor (hAChE IC50 = 74.5 nM; hBChE IC50 = 83.3 nM) with micromolar antagonistic activity towards M1 mAChR (IC50 = 4.23 µM). A non-selective pattern of cholinesterase inhibition is likely to be valuable during the onset as well as later stages of AD.

[Bacteriuria and Symptomatic Urinary Tract Infections during Antimicrobial Prophylaxis in Patients with Short-Term Urinary Catheters - Prospective Randomised Study in Patients after Joint Replacement Surgery]. / Bakteriurie a symptomatický mocový infekt pri antimikrobiální profylaxi krátkodobe zavedených mocových katétru ­ prospektivní randomizovaná studie u pacientu po implantaci kloubní náhrady.

PURPOSE OF THE STUDY A very serious complication following joint replacement surgery is periprosthetic joint infection that can be caused by a urinary tract infection. Insertion of an indwelling urinary catheter constitutes a risk factor that may result in urinary tract infections. The aim of this prospective randomised study was to compare the occurrence of significant bacteriuria and symptomatic urinary tract infections during antibiotic prophylaxis at the time of removal of an indwelling urinary catheter by cotrimoxazole in two doses and with no administration of antibiotics. We also monitored the incidence of potential periprosthetic infection following the endoprosthesis implantation. The findings of preoperative urine tests were compared with the declared negative preoperative examination. MATERIAL AND METHODS The study included patients indicated for a total hip or knee replacement with a negative urine culture as a part of the preoperative testing. Where leukocyteria was detected, urine culture by mid-stream clean catch urine was obtained. The second part included patients, in whom an indwelling urinary catheter had to be inserted postoperatively for urine retention and/or monitoring of fluid balance and who were divided into two groups on a rota basis. No antibiotics were administered to the first group, whereas Cotrimoxazol 960 mg tablets p.o. was administered to the second group, 14 and 2 hours before the removal of the catheter. The urine culture test was performed 4 hours after the removal of the indwelling urinary catheter, in both the groups. The test was repeated after 14 days and a questionnaire was filled in to report urinary tract complications. Considered as significant bacteriuria by urinalysis was the laboratory finding of > 10x4 CFU/ml in case of a single pathogen or > 10x5 in case of multiple pathogens. The results were statistically processed by Fischer's exact test with the level of significance = 0.05. RESULTS In the first part of the study leukocyturia was detected by a test strip in 112 of the total of 478 patients. In 10 women, significant bacteriuria was found. Altogether 50 women and 50 men were randomly assigned to the second part of the study. The indwelling urinary catheter was in place for 4 days on average. In men, no statistically significant difference was detected in significant bacteriuria findings, in women a statistically significant difference of p = 0.00162 was found after the removal and after 14 days the borderline of statistical significance of p = 0.0507 was achieved, but no symptomatic urinary tract infection was present. In the period from 20 to 32 months following the total joint replacement, no periprosthetic infection caused by urinary tract infection was reported. DISCUSSION There is enough evidence to prove the correlation between the symptomatic urinary tract infection and periprosthetic infection. On the other hand, asymptomatic bacteriuria is a common finding in patients before the planned hip of knee joint replacement and its treatment is not recommended. No consensus has been achieved as yet regarding the method of antibiotic prophylaxis for an inserted urinary catheter. Antibiotics are administered throughout the period of catheterisation by an indwelling urinary catheter, during its removal, or are not administered at all. In our study antibiotics were administered during the removal of an indwelling urinary catheter and a statistically significant difference was found in women. It concerned, however, only a higher incidence of asymptomatic bacteriuria not treated by antibiotics, which in the next follow-up period did not lead to periprosthetic infection. CONCLUSIONS Despite the negative pre-operative urine culture, frequent incidence of leukocyturia and symptomatic urinary infections were detected in a fairly high number of cases. Therefore, we recommend asking the patients during the hospital admission process specifically about the urinary infection symptoms. The results of our study show that antibiotic prophylaxis during the removal of indwelling urinary catheters placed for a short-term is unnecessary. Key words: endoprosthesis, urinary catheter, bacteriuria, urinary tract infection.

[The Clinical Signs of Experimental Autoimmune Uveitis]. / Klinické projevy experimentální autoimunitní uveitidy.

INTRODUCTION: Autoimmune uveitis is a sight threatening disease which in many cases fails to respond to conventional immunosuppressive or biological therapy. The research in experimental models of autoimmune uveitis helps to find new therapeutical strategies. The aim of this study is to present the clinical and histological signs of experimental autoimmune uveitis (EAU) in mice. METHODS: EAU was induced in C57BL/6 mice by subcutaneous application of IRBP (interphotoreceptor retinoid binding protein) in complete Freunds adjuvant and intraperitoneal application of pertussis toxin. Clinical evaluation of uveitis was performed in vivo using special imaging system with otoscope. Histological evaluation of uveitis was performed at day 35 post induction of EAU on hematoxylin and eosin stained frozen sections. Clinical and histological grading was used to assess the inflammation intensity of EAU. RESULTS: The intensity of inflammation is depicted on representative fundus images and histological images of retina at day 35 post induction. CONCLUSION: The model of EAU is robust and reproducible and allows us to study the immunopathological mechanisms of inflammation and its regulation. The inflammatory signs in our model are similar to findings of posterior uveitis of autoimmune etiology in humans, thus we may apply our experimental results in human medicine.

Sirtuin 1 modulation in rat model of acetaminophen-induced hepatotoxicity.

Sirtuin 1 (SIRT1) is involved in important biological processes such as energy metabolism and regulatory functions of the cell cycle, apoptosis, and inflammation. Our previous studies have shown hepatoprotective effect of polyphenolic compound resveratrol, which is also an activator of SIRT1. Therefore, the aim of our present study was to clarify the role of SIRT1 in process of hepatoprotection in animal model of drug-induced liver damage. Male Wistar rats were used for both in vivo and in vitro studies. Hepatotoxicity was induced by single dose of acetaminophen (APAP). Some rats and hepatocytes were treated by resveratrol or synthetic selective activator of sirtuin 1 (CAY10591). The degree of hepatotoxicity, the activity and expression of the SIRT1 were determined by biochemical, histological and molecular-biological assessments of gained samples (plasma, liver tissue, culture media and hepatocytes). Resveratrol and CAY attenuated APAP-induced hepatotoxicity in vivo and in vitro. Moreover, both drugs enhanced APAP-reduced SIRT1 activity. Our results show that modulation of the SIRT1 activity plays a role in hepatoprotection. Synthetic activators of SIRT1 would help in understanding the role of SIRT1 and are therefore a major boost towards the search for specific treatment of liver disease.

[Evaluation of the importance of a ready-made, gentamicin-impregnated spacer in relation to bacteriological findings in patients with periprosthetic joint infections]. / Zhodnocení významu ready-made spaceru s gentamicinem ve vztahu k bakteriologickým nálezum u pacientu s infekcí kloubní náhrady.

BACKGROUND: Periprosthetic infection is a serious complication in total hip and knee arthroplasty. The complex therapeutic approach within two-stage reimplantation includes the use of antibiotic-impregnated spacers (temporary joint replacements). The aim of this paper was to evaluate bacteriological findings in selected patients with periprosthetic infection in whom a ready-made gentamicin-impregnated spacer was used to treat the infection. MATERIALS AND METHODS: Between 2008 and 2012, a ready-made, gentamicin-impregnated cement spacer was used in 24 patients to treat periprosthetic hip or knee infection within two-stage reimplantation. All components of the prosthesis and periprosthetic tissue samples were sent for microbiological examination at the first revision surgery, while at the second revision surgery, the spacer and surrounding tissue samples were sent in. In six patients with an inserted knee spacer, the level of gentamicin in the joint fluid was measured. Subsequently, the patients were regularly monitored. RESULTS: Twenty-two (92%) of 24 patients were bacteriologically positive by culture. The most commonly detected causative agents were coagulase-negative staphylococci. Other isolates were Staphylococcus aureus, Corynebacterium, anaerobic bacteria, and Salmonella serotype Enteritidis. Nineteen (76%) of 25 primary pathogens were gentamicin sensitive. Spacers from two patients were culture positive for coagulase-negative staphylococci that tested resistant to gentamicin. During the follow-up of at least two years, none of the patients developed another periprosthetic infection. CONCLUSION: The success rate of two-stage-reimplantation total hip and knee arthroplasty using ready-made, gentamicin-impregnated spacers was 100 % in our cohort of patients; no other periprosthetic infection was reported during the follow-up of at least two years. From the bacteriological results, it appears that the ready-made, gentamicin-impregnated spacer only covers 76 % of the range of the causative agents. The solution would be to use a spacer impregnated with a combination of vancomycin and gentamicin that would be effective against all cultured species.

Mycophenolate mofetil and cyclophosphamide treatments suppress inflammation intensity in an experimental model of autoimmune uveitis.

In human, autoimmune uveitis is a leading cause of visual disability and ranks with diabetic retinopathy as a major source of blind registrations in developed countries. Since most cases of non-infectious uveitis are considered to be autoimmune or at least immune-mediated, the management of such patients has rested on appropriate immunosuppression. Some patients, however, despite maximal immunotherapy, fail to respond or are seriously intolerant of the drug therapies. Since its establishment 20 years ago, the model of experimental autoimmune uveoretinitis has served as a useful template for novel therapeutic approaches. The aim of our study was to compare the efficacy of mycophenolate mofetil and cyclophosphamide and golimumab treatment in the mouse model of experimental autoimmune uveitis. The intensity of intraocular inflammation was evaluated histologically in the treatment and control groups. Experimental autoimmune uveitis has been induced in mouse strain C57BL/6 by subcutaneous application of interphotoreceptor retinoid binding protein in complete Freund's adjuvant and pertussis toxin. The treatment was commenced on the day of uveitis induction. Cyclophosphamide was applied intraperitoneally in a single dose (100 mg/kg), mycophenolate mofetil intraperitoneally daily (30 mg/kg or 50 mg/kg), golimumab subcutaneously weekly (70 mg/kg). Sham intraperitoneal injection of a placebo (aqua pro injectione) and untreated mice with experimental autoimmune uveitis served as controls. The results show statistically significant suppression of experimental uveitis both with mycophenolate mofetil and with cyclophosphamide, and thus support its use in human medicine.

Associations between cardiac fibrosis and permanent atrial fibrillation in advanced heart failure.

Atrial fibrosis is considered as the basis in the development of long-standing atrial fibrillation (AF). However, in advanced heart failure (HF), the independent role of fibrosis for AF development is less clear since HF itself leads to atrial scarring. Our study aimed to differentiate patients with AF from patients without AF in a population consisting of patients with advanced HF. Myocardial samples from the right atrial and the left ventricular wall were obtained during heart transplantation from the explanted hearts of 21 male patients with advanced HF. Long-standing AF was present in 10 of them and the remaining 11 patients served as sinus rhythm controls. Echocardiographic and hemodynamic measurements were recorded prior to heart transplantation. Collagen volume fraction (CVF), transforming growth factor-beta (TGF-beta), and connective tissue growth factor (CTGF) expression in myocardial specimens were assessed histologically and immunohistochemically. The groups were well matched according to age (51.9+/-8.8 vs. 51.3+/-9.3 y) and co-morbidities. The AF group had higher blood pressure in the right atrium (13.6+/-7.7 vs. 6.0+/-5.0 mmHg; p=0.02), larger left atrium diameter (56.1+/-7.7 vs. 50+/-5.1 mm; p=0.043), higher left atrium wall stress (18.1+/-2.1 vs. 16.1+/-1.7 kdynes/m(2); p=0.04), and longer duration of HF (5.0+/-2.9 vs. 2.0+/-1.6 y, p=0.008). There were no significant differences in CVF (p=0.12), in CTGF (p=0.60), and in TGF-beta expression (p=0.66) in the atrial myocardium between the two study groups. In conclusions, in advanced HF, atrial fibrosis expressed by CVF is invariably present regardless of occurrence of AF. In addition to atrial wall fibrosis, increased wall stress might contribute to AF development in long-standing AF.

[Leukocyte esterase testing for examination of exudate associated with skeletal system diseases. Comparison with cytological and microbiological examinations]. / Porovnání vysetrení výpotku pohybového aparátu pomocí leukocytární esterázy s jejich cytologickým a kultivacním vysetrením.

PURPOSE OF THE STUDY: Leukocyte esterase is an enzyme in neutrophils from which it is released into exudate; its detection by colorimetric test strips indicates the presence of neutrophils. This is a rapid method to find whether exudate is of infectious or non-infectious aetiology. The aim of the study was to determine the sensitivity and specificity of leukocyte esterase testing with use of AUTION Sticks (Arkray) for examination of exudates obtained in inflammatory diseases of the skeletal system. MATERIAL AND METHODS: Exudates associated with skeletal system diseases were collected from 45 patients in the period from July 1st to December 31 st , 2012. Aspirates obtained under sterile conditions were examined for leukocyte esterase; cytological and microbiological examinations were also carried out. For the detection of leukocyte esterase, a drop of aspirate was placed on the reagent zone of a test strip and the resulting colour reaction was read after 90 minutes. Changes in colour were compared with a reference strip provided by the manufacturer. The results were assessed on a five-shade scale as follows: 0, no colour change; 1 to 4, gradual change from light pink to deep purple. The results were compared with those of cytological and microbiological examinations. Shade 4 on the strip corresponded to a positive cytological finding of bacterial infection, and shades 3 and 4 correlated with a positive microbial finding. The sensitivity and specificity of leukocyte esterase testing were statistically evaluated for both comparisons. RESULTS: Based on the results of cytological and microbiological examinations, an infectious aetiology of exudate was diagnosed in 21 (44.4%) and non-infectious aetiology in 24 (63.6%) patients. With leukocyte esterase reagent strips when shade 4 was taken as a positive result, the sensitivity and specificity of examination was assessed as 0.6190 and 0.9583, respectively. When taking both shade 3 and shade 4 for a positive result, sensitivity and specificity were 0.8571 and 0.8750, respectively. Shades 0 and 1 corresponded to the number of leukocytes in exudate that was no higher than 2 x 109/ml. DISCUSSION: The detection of leukocyte esterase is a quick and easy examination. It is useful for readily excluding or confirming an infectious aetiology of exudate and can, to some extent, substitute a cytological examination. It can also help to make a quick decision whether one- or two-stage joint reimplantation should be performed and thus eliminate the need of intra-operative histological examination of frozen tissue samples. A drawback of the method was that exudate samples contaminated with blood interfered with an assessment of colour shades. However, this can be avoided by centrifugation of the sample and use of a supernatant free from erythrocytes. CONCLUSIONS: Diagnosing infectious aetiology of joint exudate or exudate from an abscess using leukocyte esterase reagent strips appears, according to our results, to be a promising, semi-quantitative method with high specificity and sensitivity which is rapid, simple and affordable. It can be useful particularly in out-patient institutions for a quick diagnosis of arthritis; intraoperatively, it can serve as an additional method to other exudate examinations.

[Bone healing capacity in patients undergoing total hip arthroplasty]. / Schopnost kostní reparace u pacientu podstupujících implantaci TEP kycelního kloubu.

PURPOSE OF THE STUDY: To identify some characteristics of bone repair capacity in elderly patients who undergo total hip arthroplasty, which requires good healing ability of bone for implant osteointegration and bone defect repair, particularly if revision arthroplasty is necessary. MATERIAL AND METHODS: In a group of 27 patients (mean age, 70 ± 7 years; range, 60 to 81 years) a coincidence of osteoarthritis and osteopenia/ osteoporosis was assessed, and mesenchymal stem cells (MSC) were isolated and their numbers, viability and proliferative capacity were evaluated. The MSC populations were examined for their behaviour on bone tissue scaffolds used in orthopaedic surgery for treatment of bone lesions. Each patient underwent bone densitometry examination before total hip arthroplasty. Bone marrow was harvested intra-operatively from the trochanteric region of the femur. From a portion of bone marrow, MSCs were isolated and cultured, and a mononuclear cell concentrate was obtained. Either whole bone marrow or a mononuclear cell concentrate was applied to selected matrices (allograft, demineralised bone matrix, porous beta-tricalcium phosphate (-TCP), pressed hydroxyapatite or calcium sulphate). The production of new collagen and extracellular mineralized matrix were first assessed in expansion medium and, when the production was low, differentiation medium was used. RESULTS: A coincidence of osteoarthritis and osteopenia/osteoporosis was found in 50% of the patients. All were women with a low body mass index and had been post-menopausal for an average of 23 years. The isolated MSCs contained a high percentage of viable cells (mean, 95%). The mesenchymal cells of patients with osteopenia, as compared with those having normal bone density, showed markedly lower numbers of fibroblastic colony forming units (CFU-F) per ml and had a lower proliferative capacity because the population doubling time during the first four passages was much longer. Of the scaffolds tested, allografts showed the most marked collagen and extracellular mineralized matrix production in expansion medium with either whole bone marrow or a monocyte concentrate; porous -TCP was the best of bone graft substitutes in collagen and extracellular mineralized matrix production by both whole bone marrow and a monocyte concentrate, but this was only in differential medium. DISCUSSION: The coincidence of ostearthritis with osteopenia/osteoporosis was found in a higher number of our patients than is reported in the literature. Also, a lower MSC proliferative capacity and a low number of CFU-F/ml in the patients with low bone density were interesting findings. Better bone regeneration would generally be achieved with higher MSC numbers and the use of growth factors for stimulation of osteoinduction and angiogenesis. Bone marrow harvesting for MSC isolation, cultivation and subsequent transplantation is currently feasible only in an experiment. A bone marrow aspirate can be applied, but it may not provide a sufficient number of MSCs. In addition to autologous bone grafts, the best collagen production was on allografts. In bone graft substitutes, the porous structure played an important role because on a non-porous material (calcium sulphate) the formation of collagen was very low. There was no difference in collagen and extracellular mineralized matrix production between whole bone marrow and monocyte concentrates. CONCLUSIONS: Elderly patients have reduced bone healing capacity also because of osteopenia/osteoporosis that occurs more often than it is generally diagnosed, including its coincidence with osteoarthritis. The mesenchymal stem cells isolated from osteopenic bone give a lower number of CFU-F/ml and have a lower proliferative capacity. Of the matrices for new bone formation, allografts showed the best results because collagen was produced already in expansion medium. Of the graft substitutes, porous -TCP was the best, but with collagen production in differential medium. The use of bone marrow aspirate is currently a method of choice in order to increase MSC numbers at the site of bone healing. The use of growth factors is an expensive treatment. To achieve the goal of reliable promotion of osteogenesis with cultured MSC transplantation and use of composite materials with pro-osteogenic and pro-angiogenic factors will still require many experimental and clinical studies.

The branching pattern of villous capillaries and structural changes of placental terminal villi in type 1 diabetes mellitus.

Maternal diabetes is associated with changes of the placental structure. These changes include great variability of vascularity manifested by strikingly hypovascular as well as hypervascular terminal villi. In this paper, normal placental terminal villi and pathological villi of type 1 diabetic placentas were compared concerning the structure of villous stroma, spatial arrangement of villous capillary bed and quantitative assessment of capillary branching pattern. Formalin fixed and paraffin embedded specimens of 14 normal and 17 Type 1 diabetic term placentas were used for picrosirius staining, vimentin and desmin immunohistochemistry and confocal microscopy. 3D models of villi and villous capillaries were constructed from stacks of confocal optical sections. Hypervascular as well as hypovascular villi of diabetic placenta displayed changed structure of villous stroma, i.e. the collagen envelope around capillaries looked thinner and the network of collagen fibers seemed less dense. The desmin immunocytochemistry has shown that stromal cells of hypervascular as well as hypovascular villi appeared nearly or completely void of desmin filaments. In comparison with normal villi, capillaries of hypovascular villi had a smaller diameter and displayed a markedly wavy course whereas in hypervascular villi numerous capillaries occurred in reduced stroma and often had a large diameter. The quantitative assessment of capillary branching has shown that villous capillaries are more branched in diabetic placentas. It is concluded that type 1 maternal diabetes enhances the surface area of the capillary wall by elongation, enlargement of diameter and higher branching of villous capillaries and disrupts the stromal structure of terminal villi.

[Venous thromboembolism prophylaxis after total hip arthroplasty]. / Prevence tromboembolické nemoci po implantaci totální endoprotézy kycelního kloubu.

PURPOSE OF THE STUDY The authors compare the frequency of thromboembolic disease in the patients receiving prophylactic therapy for 10 days with that in the patients having a prolonged course of preventive treatment recommended for up to 35 days after total hip arthroplasty (THA). MATERIAL The group first evaluated comprised patients undergoing total hip replacement in 2005 and 2006 when enoxaparin was administered for 10 days after surgery. Patients with revision THA were not included. The other group included patients operated on in 2008 who received preventive treatment for 35 post-operative days either with enoxaparin for the whole time, or with enoxaparin for 10 days of hospital stay and then warfarin up to the 35th day after surgery. Patients under going revision THA and those taking other prophylactic agents were not evaluated. METHODS The evaluation of the 2005/6 group included retrospective reviews of medical records, questionnaires sent to the patients and information received from their general practitioners. The 2008 group assessment was based on the information obtained at the patient's follow-up visit at 3 months and completed with data from the questionnaires and medical notes. We focused on the records of distal or proximal deep vein thrombosis in lower extremities and of pulmonary embolism including cases with a fatal outcome. Complications associated with pharmacological prevention were also recorded. RESULTS In the 2005/6 group including 478 patients with an average age of 67.2 years, 23 (4.8 %) patients developed thrombo- embolic disease within 3 months of surgery, six patients had pulmonary embolism of which two of them died. The thromboembolic complication developed at a median of 30.5 post-operative days, i.e., after patient discharge from hospital. In the 2008 group comprising 289 patients with an average age of 63.8 years, three patients (1 %) developed thromboembolic disease within 3 months of surgery. Of them, one woman had deep vein thrombosis in relation to a high factor VIII level; and one developed pulmonary embolism with no fatal outcome. Only the minority of patients (6.9 %) continued enoxaparin therapy, the majority (93.1 %) preferred conversion to warfarin after discharge from hospital. Of the total number of 289 patients evaluated, complications associated with prophylactic treatment were recorded in 52 patients (18 %), name- ly, in 35 patients (12.1 %) it was difficult to establish the correct dosage of warfarin, in 13 patients (4.5 %) warfarin caused minor bleeding or dyspepsia and in 4 patients (1.4 %) major bleeding was recorded. DISCUSSION In our study the patients receiving a 10-day prophylactic therapy showed a slightly higher occurrence of thromboembolic disease within 3 months of THA surgery (4.8 %) than the patients reported by Eikelboom et al. (4.3 %). The results of prolonged prophylactic treatment for at least 35 days were similar, thromboembolic disease was found in 1 % of the patients. The development of thromboembolic event was recorded in our study at a median of 30.5 post-operative days, as compared with a median of 17 days in the study by Liebermann et al. In both studies, most of the thromboembolic complications developed after the patients had been discharged from hospital. The majority of patients chose warfarin for prolonged preventive treatment. There was a high complication rate (18 %) due to not finding the correct warfarin dosage or because of its overdose CONCLUSIONS Patients undergoing THA are at high risk of developing deep vein thrombosis. The risk can be markedly reduced by prolonged pharmacological ophylaxis It is recommended that the use of warfarin for this prolonged therapy should be care- fully considered, because its optimal dose is difficult to establish and its overdose may cause serious complications.

Pericyte coverage of fetoplacental vessels in pregnancies complicated by Type 1 diabetes mellitus.

Placentas from pregnancies complicated by Type 1 diabetes mellitus (DM 1) display altered vascular morphology and function. Here we studied the extent of pericyte coverage in microvessels of normal pregnancies and pregnancies complicated by DM 1. We used smooth muscle actin (SMA) as a marker for quantitation of pericyte coverage in placental capillaries. The extent of pericyte coverage around the vessel circumference was 38 ± 11% in normal vs. 33 ± 10% in DM 1 pregnancies. We found that there is no statistically significant difference in the extent of pericyte coverage around the capillary circumference between DM 1 and normal pregnancies.