Metabolic cardio- and reno-protective effects of empagliflozin in a prediabetic rat model.

The mechanisms behind the cardiovascular and renal benefits of empagliflozin is not fully understood. The positive impact of the medication on cardiovascular mortality can not be solely attributed to its antidiabetic effect, with a metabolic mechanism possibly involved. To investigate the metabolic effects of empagliflozin treatment (10 mg/kg/day for 6 weeks), we used an adult male rat model with serious vascular complications associated with metabolic syndrome and prediabetes. Impaired glucose tolerance, severe albuminuria and impaired insulin sensitivity were induced by intragastric administration of methylglyoxal and high sucrose diet feeding for four months. Although empagliflozin decreased body weight, non-fasting glucose and insulin, glucagon levels remained unchanged. In addition, empagliflozin increased adiponectin levels (+40%; p < 0.01) and improved skeletal muscle insulin sensitivity. Increased non-esterified fatty acids (NEFA) in empagliflozin-treated rats is understood to generate ketone bodies. Empagliflozin increased ß-hydroxybutyrate levels in serum (+66%; p < 0.05) and the myocardium (30%; p < 0.01), suggesting its possible involvement as an alternative substrate for metabolism. Empagliflozin switched substrate utilisation in the myocardium, diverting glucose oxidation to fatty acid oxidation. Representing another favorable effect, empagliflozin also contributed to decreased uric acid plasma levels (-19%; p < 0.05). In the kidney cortex, empagliflozin improved oxidative and dicarbonyl stress parameters and increased gene expression of ß-hydroxybutyrate dehydrogenase, an enzyme involved in ketone body utilisation. In addition, empagliflozin decreased microalbuminuria (-27%; p < 0.01) and urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion (-29%; p < 0.01). Our results reveal the important systemic metabolic effect of empagliflozin on alterations in substrate utilisation and on increased ketone body use in prediabetic rats. Improved oxidative and dicarbonyl stress and decreased uric acid are also possibly involved in the cardio- and reno-protective effects of empagliflozin.

Needleless electrospun and centrifugal spun poly-ε-caprolactone scaffolds as a carrier for platelets in tissue engineering applications: A comparative study with hMSCs.

The biofunctionalization of scaffolds for tissue engineering is crucial to improve the results of regenerative therapies. This study compared the effect of platelet-functionalization of 2D electrospun and 3D centrifugal spun scaffolds on the osteogenic potential of hMSCs. Scaffolds prepared from poly-ε-caprolactone, using electrospinning and centrifugal spinning technology, were functionalized using five different concentrations of platelets. Cell proliferation, metabolic activity and osteogenic differentiation were tested using hMSCs cultured in differential and non-differential medium. The porous 3D structure of the centrifugal spun fibers resulted in higher cell proliferation. Furthermore, the functionalization of the scaffolds with platelets resulted in a dose-dependent increase in cell metabolic activity, proliferation and production of an osteogenic marker - alkaline phosphatase. The effect was further promoted by culture in an osteogenic differential medium. The increase in combination of both platelets and osteogenic media shows an improved osteoinduction by platelets in environments rich in inorganic phosphate and ascorbate. Nevertheless, the results of the study showed that the optimal concentration of platelets for induction of hMSC osteogenesis is in the range of 900-3000 × 109 platelets/L. The study determines the potential of electrospun and centrifugal spun fibers with adhered platelets, for use in bone tissue engineering.

Restoration of severely damaged endodontically treated premolars: The influence of the endo-core length on marginal integrity and fatigue resistance of lithium disilicate CAD-CAM ceramic endocrowns.

OBJECTIVES: To explore fatigue limits of ceramic endocrowns for premolars. METHODS: Forty-eight devitalized premolars were cut at the CEJ. They were restored with standardized CAD-CAM lithium disilicate reinforced ceramic restorations (IPS e.max CAD, Ivoclar-Vivadent) and divided into four Groups (n=12): overlays (Group A, no endo-core, negative control), endocrowns with an endo-core of 2mm (Group B), 4mm (GroupC) and crowns with post and core (Group D, positive control). All specimens were first submitted to thermo-mechanical cyclic loading (TCML)(1.7Hz, 49N, 600000 cycles, 1500 thermo-cycles). Margins were analysed before and after the loading. Survived specimens were then submitted to cyclic isometric stepwise loading (5Hz, 200N to 1200N) until completion of 105000 cycles or failure. In case of fracture, fragments were analysed using SEM and failure mode was determined. Results of stepwise loading were statistically analysed by Kaplan-Meier life survival analysis and log rank test (p=0.05). RESULTS: All the specimens survived the TCML test except four specimens of Group A (early restorations' debonding). No difference in percentages of closed margins was found between endocrowns (Groups B, C) and crowns (Group D). After the stepwise test, differences in survival within the groups were not statistically significant. Most of restorations experienced non-reparable fracture. CONCLUSIONS: Endocrowns with both 2-mm and 4-mm long endo-cores displayed outcomes after fatigue equivalent to classical crowns. Results of this study discourage the use of flat overlays with only adhesive retention. CLINICAL SIGNIFICANCE: When restoring extremely destroyed devitalized premolars, adhesive strategies should be coupled to a macro-mechanical retention in the root.

[Comparison between the Range of Movement Canine Real Cervical Spine and Numerical Simulation - Computer Model Validation]. / Porovnání rozsahu pohybu reálné krcní pátere psa s pocítacovou simulací ­ validace pocítacového modelu.

PURPOSE OF THE STUDY In developing new or modifying the existing surgical treatment methods of spine conditions an integral part of ex vivo experiments is the assessment of mechanical, kinematic and dynamic properties of created constructions. The aim of the study is to create an appropriately validated numerical model of canine cervical spine in order to obtain a tool for basic research to be applied in cervical spine surgeries. For this purpose, canine is a suitable model due to the occurrence of similar cervical spine conditions in some breeds of dogs and in humans. The obtained model can also be used in research and in clinical veterinary practice. MATERIAL AND METHODS In order to create a 3D spine model, the LightSpeed 16 (GE, Milwaukee, USA) multidetector computed tomography was used to scan the cervical spine of Doberman Pinscher. The data were transmitted to Mimics 12 software (Materialise HQ, Belgium), in which the individual vertebrae were segmented on CT scans by thresholding. The vertebral geometry was exported to Rhinoceros software (McNeel North America, USA) for modelling, and subsequently the specialised software Abaqus (Dassault Systemes, France) was used to analyse the response of the physiological spine model to external load by the finite element method (FEM). All the FEM based numerical simulations were considered as nonlinear contact statistic tasks. In FEM analyses, angles between individual spinal segments were monitored in dependence on ventroflexion/ /dorziflexion. The data were validated using the latero-lateral radiographs of cervical spine of large breed dogs with no evident clinical signs of cervical spine conditions. The radiographs within the cervical spine range of motion were taken at three different positions: in neutral position, in maximal ventroflexion and in maximal dorziflexion. On X-rays, vertebral inclination angles in monitored spine positions were measured and compared with the results obtain0ed from FEM analyses of the numerical model. RESULTS It is obvious from the results that the physiological spine model tested by the finite element method shows a very similar mechanical behaviour as the physiological canine spine. The biggest difference identified between the resulting values was reported in C6-C7 segment in dorsiflexion (Δφ = 5.95%), or in C4-C5 segment in ventroflexion (Δφ = -3.09%). CONCLUSIONS The comparisons between the mobility of cervical spine in ventroflexion/dorsiflexion on radiographs of the real models and the simulated numerical model by finite element method showed a high degree of results conformity with a minimal difference. Therefore, for future experiments the validated numerical model can be used as a tool of basic research on condition that the results of analyses carried out by finite element method will be affected only by an insignificant error. The computer model, on the other hand, is merely a simplified system and in comparison with the real situation cannot fully evaluate the dynamics of the action of forces in time, their variability, and also the individual effects of supportive skeletal tissues. Based on what has been said above, it is obvious that there is a need to exercise restraint in interpreting the obtained results. Key words: cervical spine, kinematics, numerical modelling, finite element method, canine.

Fatigue behavior of resin-modified monolithic CAD-CAM RNC crowns and endocrowns.

OBJECTIVE: To evaluate the influence of different types of modifications with resin on fatigue resistance and failure behavior of CAD-CAM resin nano ceramic (RNC) restorations for maxillary first premolars. METHODS: Sixty standardized resin composite root dies received CAD-CAM RNC endocrowns (n=30) and crowns (n=30) (Lava Ultimate, 3M Espe). Restorations were divided into six groups: full anatomic endocrowns (group A) and crowns (group D), buccal resin veneered endocrowns (group B) and crowns (group E) and buccal resin veneered endocrowns (group C) and crowns (group F) with a central groove resin filling. A nano-hybrid resin composite was used to veneer the restorations (Filtek Supreme, 3M Espe). All specimens were first submitted to thermo-mechanical cyclic loading (1.7Hz, 49N, 600000 cycles, 1500 thermo-cycles) and then submitted to cyclic isometric stepwise loading (5Hz) until completion of 105000 cycles or failure after 5000 cycles at 200N, followed by 20000 cycles at 400N, 600N, 800N, 1000N and 1200N. In case of fracture, fragments were analyzed using SEM and modes of failure were determined. Results were statistically analyzed by Kaplan-Meier life survival analysis and log rank test (p=0.05). RESULTS: The differences in survival between groups were not statistically significant, except between groups D and F (p=0.039). Endocrowns fractured predominantly with a mesio-distal wedge-opening fracture (82%). Partial cusp fractures were observed above all in crowns (70%). Analysis of the fractured specimens revealed that the origin of the fracture was mainly at the occlusal contact points of the stepwise loading. SIGNIFICANCE: Veneering of CAD-CAM RNC restorations has no influence on their fatigue resistance except when monolithic crowns are modified on their occlusal central groove.

Neurological Deficits of an Rps19(Arg67del) Model of Diamond-Blackfan Anaemia.

Diamond-Blackfan anaemia is a rare disease caused by insufficient expression of ribosomal proteins and is characterized by erythroid hypoplasia often accompanied by growth retardation, congenital craniofacial and limb abnormalities. In addition, Diamond-Blackfan anaemia patients also exhibit a number of behavioural abnormalities. In this study we describe the behavioural effects observed in a new mouse mutant carrying a targeted single amino acid deletion in the ribosomal protein RPS19. This mutant, created by the deletion of arginine 67 in RPS19, exhibits craniofacial, skeletal, and brain abnormalities, accompanied by various neurobehavioural malfunctions. A battery of behavioural tests revealed a moderate cognitive impairment and neuromuscular dysfunction resulting in profound gait abnormalities. This novel Rps19 mutant shows behavioural phenotypes resembling that of the human Diamond-Blackfan anaemia syndrome, thus creating the possibility to use this mutant as a unique murine model for studying the molecular basis of ribosomal protein deficiencies.

MMP-19 deficiency causes aggravation of colitis due to defects in innate immune cell function.

Matrix metalloproteinases (MMPs) are potential biomarkers for disease activity in inflammatory bowel disease (IBD). However, clinical trials targeting MMPs have not succeeded, likely due to poor understanding of the biological functions of individual MMPs. Here, we explore the role of MMP-19 in IBD pathology. Using a DSS-induced model of colitis, we show evidence for increased susceptibility of Mmp-19-deficient (Mmp-19(-/-)) mice to colitis. Absence of MMP-19 leads to significant disease progression, with reduced survival rates, severe tissue destruction, and elevated levels of pro-inflammatory modulators in the colon and plasma, and failure to resolve inflammation. There was a striking delay in neutrophil infiltration into the colon of Mmp-19(-/-) mice during the acute colitis, leading to persistent inflammation and poor recovery; this was rescued by reconstitution of irradiated Mmp-19(-/-) mice with wild-type bone marrow. Additionally, Mmp-19-deficient macrophages exhibited decreased migration in vivo and in vitro and the mucosal barrier appeared compromised. Finally, chemokine fractalkine (CX3CL1) was identified as a novel substrate of MMP-19, suggesting a link between insufficient processing of CX3CL1 and cell recruitment in the Mmp-19(-/-) mice. MMP-19 proves to be a critical factor in balanced host response to colonic pathogens, and for orchestrating appropriate innate immune response in colitis.

Transgenic reporter mice with promoter region of murine LRAT specifically marks lens and meiosis spermatocytes.

Lecithin:retinol acyltransferase (LRAT) is the major enzyme responsible for retinol esterification in the mammalian body. LRAT exhibits specific activity in the cells with active retinol metabolism where it converts retinols into retinyl esters, which represents the major storage form of retinol. Besides hepatic stellate cells in the liver, LRAT appears to have a key physiologic role in several other tissues. In this study, we generated a transgenic reporter mouse expressing green fluorescence protein (EGFP) under the control of region containing -1166 bps from promoter upstream from the putative transcriptional start site and 262 bps downstream of this start. Transgenic reporter mice exhibited specific expression in eyes and testes. In eyes, expression of EGFP-reporter is found in lens and lens epithelium and fibers from embryo to adulthood. In testes, LRAT-EGFP reporter is expressed both in Sertoli and in spermatocytes marking initiation of spermatogenesis in prepubertal mice. Our data show that the examined LRAT regulatory region is sufficient to achieve strong and selective expression in the eye and testes but not in liver and other organs.

Differential expression and processing of matrix metalloproteinase 19 marks progression of gastrointestinal diseases.

Matrix metalloproteinases (MMPs), responsible for extracellular matrix remodelling and processing of numerous soluble and cell-surface proteins, appear to play important roles in pathogenesis of gastrointestinal diseases. MMPs influence migration of inflammatory cells, mucosal destruction, matrix deposition and degradation. In this study, we analysed the expression of MMP-19 in the main forms of gastrointestinal diseases including inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, and colorectal carcinoma. We identified prominent MMP-19 expression in unaffected areas of intestinal epithelia and macrophages but not in other cells or tissues. Abundant expression of MMP-19 was also found in the endothelium of blood and lymphatic vessels of inflamed intestinal tissue. High MMP-19 immunoreactivity was also associated with macrophages in inflamed areas and myenteric plexuses. In comparison to the intestinal epithelium, all these cell types and compartments appeared to express MMP-19 irrespective of the disease pathogenesis and progression. Intestinal epithelia exhibited striking differential immunoreactivity for MMP-19. While immunoreactivity of monoclonal antibody recognizing the propeptide domain declined in virtually all IBD and colorectal carcinoma samples, other polyclonal antibodies against the hinge region and propetide domain did not show such an obvious decrease. Additional Western blotting analysis revealed that the antibodies against MMP-19 recognize differently processed forms of this MMP. The disappearance of immunoreactivity of the monoclonal anti-propeptide domain antibody does not mean down-regulation of MMP-19, but processing of the immature form. As this processing likely leads to the activation of this MMP, the differential staining pattern may be an important sign of disease progression.

ADAM10/17-dependent release of soluble c-Met correlates with hepatocellular damage.

The signalling pathway elicited by hepatocyte growth factor (HGF) and its receptor c-Met is indispensable for liver development and regeneration. It has been described that c-Met is released from the cell surface by a disintegrin and metalloprotease 10 (ADAM10) resulting in a soluble c-Met form known as sMet. Using the human hepatocellular HepG2 and hepatic stellate cell LX2 lines we show that sMet is released from the cell surface of liver cells by both ADAM17 and ADAM10, with ADAM17 appearing to be the major proteinase. Moreover, using a mouse model of 3,5-diethoxycarbonyl- 1,4-dihydroxycollidine (DDC)-induced hepatobiliary obstruction we show that serum levels of sMet correlate well with the liver damage state and consecutive regeneration as well as with established markers of liver damage such as alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin. However, sMet exhibited remarkably better correlation with liver damage and inflammation than did serum tumour necrosis factor α (TNF-α), whose shedding is also mediated by ADAM proteolytic activity. Our results indicate that the proteolytic activity of ADAM10/17 is essential for regulating HGF/c-Met signalling during acute liver damage and following regeneration and that the differential serum levels of sMet together with expression of c-Met/HGF might be a useful indicator not only for damage, but also for ongoing liver regeneration.

The effect of chronic alcohol administration on bone mineral content and bone strength in male rats.

Alcohol use has been identified as a risk factor for the development of osteoporosis. Eight male Wistar rats at two months of age were alcoho-fed (7.6 g 95 % ethanol/kg b.w. per day) to evaluate the effects of long-term administration (three months) of alcohol in drinking water. We have used a dose which is considered to be comparable to a dose of 1 liter of wine or 2.5 liters of 12(°) beer used in male adults daily. The bones were tested mechanically by a three-point bending test in a Mini Bionix (MTS) testing system. The bones from alcohol-fed rats were characterized by a reduction in bone density as well as in ash, calcium and phosphate content. In alcohol-fed rats the reduction in bone mineral density (10 %) was reflected by about 12 % reduction of mechanical strength of femur (158+/-5.5 vs. 178+/-3.2 N/mm(2)). Alcohol significantly altered femoral cortical thickness. In our experiment alcohol itself did not exert any antiandrogenic effect and it did not produce changes in the weight of seminal vesicles. Liver function test (GGT, ALP, AST) did not differ between alcohol-fed rats and control rats. Alcohol-induced bone loss is associated with increased bone resorption and decreased bone formation. These results document the efficacy of alcohol at the dose of 7.6 g 95 % ethanol/kg b.w. to cause bone loss and loss of bone mechanical strength in intact rats. The results of the present study may be interpreted as supporting the hypothesis of alcohol as a risk factor for osteoporosis.

Erratum in Int J Biol Markers 2007; 22 (4): 265-73. Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

Errata Corrige In the article 'Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma' by Velinov N et al, which was published in the October-December issue of the International Journal of Biological Markers (Int J Biol Markers 2007; 22 (4): 265-73), an author and his affiliation were omitted, namely G. Mishev. We reprint herewith the names of all authors together with their affiliations:N. Velinov1, D. Aebersold2*, N. Haeni1*, R. Hlushchuk1,4, G. Mishev1, F. Weinstein1, R. Sedlacek3, V. Djonov1,4 1 Institute of Anatomy, University of Bern, Bern 2 Department of Radiation Oncology, University of Bern, Inselspital, Bern - Switzerland 3 Institute of Biochemistry, University of Kiel, Kiel - Germany 4 Institute of Anatomy, University of Fribourg, Fribourg - Switzerland *The contribution of both authors was equivalent.

Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

AIMS: To evaluate the expression of matrix metalloproteinase-19 (MMP-19) in oropharyngeal squamous cell carcinoma along with its association with structural features of invasiveness. To investigate whether MMP-19 expression correlates with lymphatic or systemic metastasis and prognosis in patients who have received definitive radiotherapy. METHODS AND RESULTS: The histological evaluation of the invasive front was based on Bryne's malignancy grading system. We correlated the immunohistochemical expression pattern with morphological parameters which characterize tumor invasiveness such as keratinization, nuclear polymorphism, invasion pattern, and the host inflammatory response. Local immunoreactivity for MMP-19 was positively correlated with tumor invasiveness as reflected in its structural characteristics and the degree of nuclear polymorphism, and negatively correlated with the inflammatory response of the host. No correlation existed between MMP-19 expression and clinicopathological features (TNM stage, grade of differentiation) or a patient''s outcome and prognosis. CONCLUSIONS: This latter finding probably reflects the unique change for MMPs from high immunoreactivity within healthy tissue areas and non-invasive tumor parts, through absence in the least invasive neoplastic regions, to strong re-expression at a highly invasive front of the same tumor. Our findings indicate that MMP-19 can be used as a marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

The effect of chronic nicotine administration on bone mineral content and bone strength in normal and castrated male rats.

Tobacco, containing nicotine as the principal pharmacologically active chemical, has been identified as being a risk factor for the development of osteoporosis. Thirty-two male Wistar rats of two months of age were castrated or sham operated to evaluate the effects of long-term administration (four months) of nicotine in drinking water (9.0 mg/kg/day). The presence of cotinine in urine confirmed successful delivery of nicotine. The bones were tested mechanically by a three point bending test in a Mini Bionix (MTA) testing system. The bones from castrated rats were characterized by a reduction in bone density as well as ash, calcium and phosphate content. Castration significantly altered mechanical properties of bone (9%) and femoral cortical thickness. When intact rats were treated with a high dose of nicotine, nicotine had negative effect on tibial bone density as well as ash, calcium, phosphate content and significantly altered the mechanical properties of bone (12%) and femoral cortical thickness compared to intact animals. Nicotine itself does not exert any anti-androgenic effect and does not produce changes in the weight of seminal vesicles. Nicotine-induced bone loss is associated with high bone turnover in the male rats as expressed by increased TrACP and B-ALP. When castrated rats were treated with the high dose of nicotine the changes in bone density resulting from castration were not further potentiated. These results document the efficacy of nicotine at high doses to cause bone loss and loss of bone mechanical strength in intact rats. The results of the present study may be interpreted as supporting the hypothesis of nicotine as a risk factor for osteoporosis.

Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

AIMS: To evaluate the expression of matrix metalloproteinase-19 (MMP-19) in oropharyngeal squamous cell carcinoma along with its association with structural features of invasiveness. To investigate whether MMP-19 expression correlates with lymphatic or systemic metastasis and prognosis in patients who have received definitive radiotherapy. METHODS AND RESULTS: The histological evaluation of the invasive front was based on Brynes malignancy grading system. We correlated the immunohistochemical expression pattern with morphological parameters which characterize tumor invasiveness such as keratinization, nuclear polymorphism, invasion pattern, and the host inflammatory response. Local immunoreactivity for MMP-19 was positively correlated with tumor invasiveness as reflected in its structural characteristics and the degree of nuclear polymorphism, and negatively correlated with the inflammatory response of the host. No correlation existed between MMP-19 expression and clinicopathological features (TNM stage, grade of differentiation) or a patient''s outcome and prognosis. CONCLUSIONS: This latter finding probably reflects the unique change for MMPs from high immunoreactivity within healthy tissue areas and non-invasive tumor parts, through absence in the least invasive neoplastic regions, to strong re-expression at a highly invasive front of the same tumor. Our findings indicate that MMP-19 can be used as a marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

[Treatment of deep cartilage defects of the knee with autologous chondrocyte transplantation on a hyaluronic Acid ester scaffolds (Hyalograft C)]. / Osetrení hlubokých defektu chrupavky kolena transplantací autologních chondrocytu fixovaných na nosici z esteru kyseliny hyaluronové (Hyalograft C).

PURPOSE OF THE STUDY: The treatment of chondral defects by transplantation of autologous chondrocytes has recently shown further development. Various biomaterials are used as carriers facilitating attachment and even distribution of chondrocytes in the defect. Since 2003 Hyalograft C, hyaluronan-based scaffolds, has been used, in a clinical study, for implantation of autologous chondrocytes in the treatment of deep chondral lesions of the knee at our department. MATERIAL: Eight patients (7 men and 1 woman; average age, 31 years) followed up for at least 9 months were evaluated. The lesions with an average size of 3.9 cm2 were localized on femoral condyles. METHODS: The outcome of surgery was evaluated on the basis of the IKDC Subjective Knee Evaluation Form, Knee Injury and Osteoarthritis Outcome Score (KOOS) and Lysholm knee score. The patients underwent MR examination preoperatively and at 3, 6 and 12 months after surgery. The newly-formed cartilage was assessed by International Cartilage Repair Society (ICRS) visual scores at second-look arthroscopy carried out at 9 to 12 months following transplantation. Consistency of the new cartilage developing in the defect and that of healthy cartilage around the defect was compared by means of a special indentation probe in three patients. A biopsy sample was collected from the grafted site for histological, histochemical and immunohistochemical examination. RESULTS: All patients reported improvement in knee function on average at 10 months after surgery. The average IKDC subjective score increased from 46 points preoperatively to 74 points postoperatively. The KOOS evaluation showed pain relief and improved function. In quality of life evaluation the average score of 35 points before surgery increased to 70 points after it. The average Lysholm knee score was 61 points before and 83 points after surgery. MR findings correlated well with arthroscopic findings. Second-look arthroscopy showed a normal appearance of the newly-formed cartilage in six, and an abnormal appearance in two patients. The average ICRS visual score was 9.4 points. No graft failure was recorded. The newly-produced tissue had the histological characteristics of a mixed hyaline and fibrous cartilage in seven patients, and of hyaline-like cartilage in one patient. DISCUSSION: The ICRS visual repair assessment of the newly-formed tissue showed that our results were better than the one-year outcomes reported by Bartlett et al. (11 patients after transplantation of a collagen bilayer seeded with chondrocytes), but worse than the results of an Italian multi-center study (55 patients with Hyalograft C-based grafts followed up on average for 14 months). At almost one year, implantation of on a Hyalograft C resulted in the production of mixed cartilage incorporated well in the subchondral bone. Only one patient had mature hyaline cartilage. One year is too short to allow for complete remodeling of the newly formed cartilage into a mature hyaline cartilage. This is in agreement with other studies suggesting that the new cartilage continues to mature and remodel for a time longer than one year. CONCLUSIONS: Based on our results we suggest that the use of Hyalograft C is a safe and effective option for treatment of deep chondral defects of the knee; it is particularly useful in patients in whom the primary defect treatment has failed. The application of Haylograft C is relatively quick and easy; this is convenient when surgery involves more than one procedure (ligament reconstruction, osteotomy). However, a definite evaluation of this method will be possible only after long-term results are available. Key words: deep cartilage defects, chondral defects, cartilage repair, autologous chondrocyte transplantation, hyaluronan- based scaffold, Hyalograft C, cartilage repair assessment, ICRS.

Proliferation-based T-cell selection for immunotherapy and graft-versus-host-disease prophylaxis in the context of bone marrow transplantation.

Graft-versus-host disease (GvHD) caused by alloreactive T cells within the graft is a major drawback of allogeneic BMT, but depletion of T cells leads to higher rates of relapse, opportunistic infections and graft failure. Therefore, selective removal of GvHD-inducing alloreactive T cells might be beneficial. We describe here the separation of alloresponsive T cells, based on carboxyfluorescein succimidyl ester labeling, in vitro allostimulation and FACS-sorting. In vivo effects of the separated cell populations were investigated in the context of allogeneic BMT in murine models: in vitro resting T cells were shown to survive in the allogeneic host and retain immunoreactivity against 'third-party' antigens. As demonstrated in two different transplantation models, elimination of proliferating cells significantly reduces GvHD but offers no advantages to using T-cell-depleted bone marrow alone concerning engraftment and tumor control. Transplanting T cells that proliferate in response to tumor antigens in vitro may narrow down the spectrum of antigens recognized by T cells and therefore reduce GvHD while maintaining graft-facilitating function and tumor control. Therefore, selecting tumor-reactive T cells on the basis of their proliferative response in vitro may be beneficial for the recipient, less time consuming than T-cell cloning and still reduce the extent of GvHD.