Osmosensing, osmosignalling and inflammation: how intervertebral disc cells respond to altered osmolarity.

Intervertebral disc (IVD) cells are naturally exposed to high osmolarity and complex mechanical loading, which drive microenvironmental osmotic changes. Age- and degeneration-induced degradation of the IVD's extracellular matrix causes osmotic imbalance, which, together with an altered function of cellular receptors and signalling pathways, instigates local osmotic stress. Cellular responses to osmotic stress include osmoadaptation and activation of pro-inflammatory pathways. This review summarises the current knowledge on how IVD cells sense local osmotic changes and translate these signals into physiological or pathophysiological responses, with a focus on inflammation. Furthermore, it discusses the expression and function of putative membrane osmosensors (e.g. solute carrier transporters, transient receptor potential channels, aquaporins and acid-sensing ion channels) and osmosignalling mediators [e.g. tonicity response-element-binding protein/nuclear factor of activated T-cells 5 (TonEBP/NFAT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)] in healthy and degenerated IVDs. Finally, an overview of the potential therapeutic targets for modifying osmosensing and osmosignalling in degenerated IVDs is provided.

Characterization of Porcine Granulosa Cell Line AVG-16.

Commercially available, but not yet characterized, the AVG-16 granulosa cell line was established from granulosa cells of medium porcine follicles. To examine the suitability of the AVG-16 cell line for studying the molecular mechanism of action of various environmental oestrogens, we investigated: 1/ cell morphology (by standard haematoxylin and eosin (HE) staining); 2/ basal and follicle-stimulating hormone (FSH) or luteinizing hormone (LH)-stimulated steroid hormone (progesterone; P4 and 17ß-oestradiol; E2) secretion (by radioimmunoassay) and 3/ expression of receptors involved in the regulation of granulosa cell function: FSH receptor (FSHR), LH receptor (LHR), oestrogen receptor α (ERα), oestrogen receptor ß (ERß) and aryl hydrocarbon receptor (AhR). mRNA and protein expression was determined by RT-PCR and fluorescence immunocytochemistry, respectively. The secretion of P4 and E2 by AVG-16 cells was in the range of steroid hormone secretion by porcine cultured primary granulosa cells. Neither FSH (100 ng/ml) nor LH (100 ng/ml) affected P4 and E2 secretion by AVG-16 cells. The presence of FSHR and LHR at both mRNA and protein level was not demonstrated in the cells. However, AVG-16 cells were found to express mRNA and protein of ERα, ERß and AhR. The results of our study showed that AVG-16 cells possess the capability of steroid hormone production, and both oestrogen receptors and AhR are present in these cells. Therefore, AVG-16 cells may serve as an unlimited source of homogenous porcine granulosa cells useful for studying the effects of environmental oestrogens on ovarian physiology.

Transcriptomic profiling of mare endometrium at different stages of endometrosis.

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-ß1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members' gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-ß1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis.

The value of ventricular gradient for predicting pulmonary hypertension and mortality in hemodialysis patients.

Pulmonary hypertension (PHT) is associated with increased mortality in hemodialysis (HD) patients. The ventricular gradient optimized for right ventricular pressure overload (VG-RVPO) is sensitive to early changes in right ventricular overload. The study aimed to assess the ability of the VG-RVPO to detect PHT and predict all-cause and cardiac mortality in HD patients. 265 selected HD patients were enrolled. Clinical, biochemical, electrocardiographic, and echocardiographic parameters were evaluated. Patients were divided into normal and abnormal VG-RVPO groups, and were followed-up for 3 years. Abnormal VG-RVPO patients were more likely to be at high or intermediate risk for PHT, were older, had longer HD vintage, higher prevalence of myocardial infarction, higher parathormone levels, shorter pulmonary flow acceleration time, lower left ventricular ejection fraction, higher values of left atrial volume index, left ventricular mass index, and peak tricuspid regurgitant velocity. Both all-cause and CV mortality were higher in abnormal VG-RVPO group. In multivariate Cox analysis, VG-RVPO remained an independent and strong predictor of all-cause and CV mortality. In HD patients, abnormal VG-RVPO not only predicts PHT, but also all-cause and CV mortality.

Evidence-Based Network Modelling to Simulate Nucleus Pulposus Multicellular Activity in Different Nutritional and Pro-Inflammatory Environments.

Initiation of intervertebral disc degeneration is thought to be biologically driven. This reflects a process, where biochemical and mechanical stimuli affect cell activity (CA) that compromise the tissue strength over time. Experimental research enhanced our understanding about the effect of such stimuli on different CA, such as protein synthesis or mRNA expression. However, it is still unclear how cells respond to their native environment that consists of a "cocktail" of different stimuli that might locally vary. This work presents an interdisciplinary approach of experimental and in silico research to approximate Nucleus Pulposus CA within multifactorial biochemical environments. Thereby, the biochemical key stimuli glucose, pH, and the proinflammatory cytokines TNF-α and IL1ß were considered that were experimentally shown to critically affect CA. To this end, a Nucleus Pulposus multicellular system was modelled. It integrated experimental findings from in vitro studies of human or bovine Nucleus Pulposus cells, to relate the individual effects of targeted stimuli to alterations in CA. Unknown stimulus-CA relationships were obtained through own experimental 3D cultures of bovine Nucleus Pulposus cells in alginate beads. Translation of experimental findings into suitable parameters for network modelling approaches was achieved thanks to a new numerical approach to estimate the individual sensitivity of a CA to each stimulus type. Hence, the effect of each stimulus type on a specific CA was assessed and integrated to approximate a multifactorial stimulus environment. Tackled CA were the mRNA expressions of Aggrecan, Collagen types I & II, MMP3, and ADAMTS4. CA was assessed for four different proinflammatory cell states; non-inflamed and inflamed for IL1ß, TNF-α or both IL1ß&TNF-α. Inflamed cell clusters were eventually predicted in a multicellular 3D agent-based model. Experimental results showed that glucose had no significant impact on proinflammatory cytokine or ADAMTS4 mRNA expression, whereas TNF-α caused a significant catabolic shift in most explored CA. In silico results showed that the presented methodology to estimate the sensitivity of a CA to a stimulus type importantly improved qualitative model predictions. However, more stimuli and/or further experimental knowledge need to be integrated, especially regarding predictions about the possible progression of inflammatory environments under adverse nutritional conditions. Tackling the multicellular level is a new and promising approach to estimate manifold responses of intervertebral disc cells. Such a top-down high-level network modelling approach allows to obtain information about relevant stimulus environments for a specific CA and could be shown to be suitable to tackle complex biological systems, including different proinflammatory cell states. The development of this methodology required a close interaction with experimental research. Thereby, specific experimental needs were derived from systematic in silico approaches and obtained results were directly used to enhance model predictions, which reflects a novelty in this research field. Eventually, the presented methodology provides modelling solutions suitable for multiscale approaches to contribute to a better understanding about dynamics over multiple spatial scales. Future work should focus on an amplification of the stimulus environment by integrating more key relevant stimuli, such as mechanical loading parameters, in order to better approximate native physiological environments.

Differential regulation of TRP channel gene and protein expression by intervertebral disc degeneration and back pain.

Intervertebral disc (IVD) degeneration and consequent low back pain (LBP) are common and costly pathological processes that require improved treatment strategies. Transient Receptor Potential (TRP) channels constitute a family of multimodal ion channels that have recently emerged as contributors to disc pathologies and were thus proposed as potential therapeutic targets, although limited data on their presence and function in the IVD exist. The purpose of this study was to determine the mRNA and protein expression of TRP channels in non-degenerated and degenerated human IVD tissue (with different pain intensity and chronicity) using gene array, conventional qPCR and immunohistochemistry. We could demonstrate that 26 out of 28 currently known TRP channels are expressed in the IVD on the mRNA level, thereby revealing novel therapeutic candidates from the TRPC, TRPM and TRPML subfamilies. TRPC6, TRPM2 and TRPML1 displayed enhanced gene and protein expression in degenerated IVDs as compared to non-degenerated IVDs. Additionally, the gene expression of TRPC6 and TRPML1 was influenced by the IVD degeneration grade. Pain intensity and/or chronicity influenced the gene and/or protein expression of TRPC6, TRPM2 and TRML1. Interestingly, decreased gene expression of TRPM2 was observed in patients treated with steroids. This study supports the importance of TRP channels in IVD homeostasis and pathology and their possible application as pharmacological targets for the treatment of IVD degeneration and LBP. However, the exact function and activation of the highlighted TRP channels will have to be determined in future studies.

Malnutrition Risk in Kidney Recipients Treated With Mycophenolate Mofetil Is Associated With IMPDH1 rs2278294 Polymorphism.

BACKGROUND: Malnutrition is known to increase morbidity and mortality in renal transplant recipients, whereas little is known about genetic predisposition to low body mass index (BMI) in the transplant setting. Inosine monophosphate dehydrogenase (IMPDH) regulates intracellular fat accumulation, pre-adipicytes maturation, and is a target of mycophenolic acid (MPA) used as a standard immunosuppressant. We hypothesized that MPA may interfere with fat tissue formation and weight gain in kidney transplant recipients and this process may be modified by IMPDH1 or IMPDH2 (genes encoding constitutive and inducible IMPDH) small nucleotide polymorphism variants. STUDY DESIGN: In an observational longitudinal study of kidney transplant recipients treated with mycophenolate mofetil, genetic factors were IMPDH1 (rs2278294, rs2278293) and IMPDH2 (rs11706052) allelic variants, the main outcome was the time-dependent change in BMI, and secondary outcomes were occurrence of BMI below 18.5 or 20 kg/m2. RESULTS: In a study group of 190 patients, no association was found between BMI changes and rs11706052 and rs2278293 variants. In terms of rs2278294, we found that allele G was associated with significantly slower BMI gain in a dominant model of inheritance. Concerning secondary endpoints, none of the AA carriers were underweight at 6 months post-implantation, while at least 2% of G allele carriers were underweight. From the first post-transplant year, all AA carriers had BMI above 20 kg/m2, while among G allele carriers at least 10% had BMI < 20 kg/m2 by generalized estimating equations. CONCLUSION: Based on our results, we postulate that MPA derivates influence post-transplant BMI and potentially also body fat content. In consequence, genotyping rs2278294 would potentially allow clinicians to personalize MPA treatment.

Glucocorticosteroids as antioxidants in treatment of asthma and COPD. New application for an old medication?

Inhaled corticosteroids (ICS) are the standard of care in asthma and are widely used in the treatment of patients with COPD. The influence of steroids on inflammatory processes has long been established since glucocorticoids and their receptor belong to the regulatory network involved in inhibition of several inflammatory pathways. Inflammatory processes are usually accompanied by an increased oxidative burden followed by a depletion of antioxidants. Therefore, the effects of steroids on antioxidant status have been investigated revealing possible positive effects on the reduced antioxidant enzyme activity. Nevertheless, the mechanisms of this modulation have not been fully elucidated yet. It is possible that antioxidant enzyme activity is regulated at the level of transcription. Additionally, because of the fact that antioxidant enzymes are trace element dependent, steroids may affect their activity through influence on trace element accumulation. This review summarizes the effects of steroids on the antioxidant enzymes activity in vitro and in vivo in relation to asthma and COPD.

Influence of pre- and post-slaughter factors on the reduced glutathione content of beef muscles.

The aim of this experiment was to assess the effect of certain factors (muscle anatomy, paternal breed, diet, age at slaughter, castration, process of meat aging and grilling) on the content of reduced glutathione (GSH) in beef. The research material included selected beef muscles acquired from steers and bulls obtained by crossing Polish Holstein-Friesian cows with meat breed bulls (Limousin, Charolais, Hereford). An analysis of ante-mortem factors such as the castration, slaughter age, and fattening of the animals showed no significant effect on the content of GSH (α=0,05). On the other hand, the paternal breed of animals was observed to have a significant effect on GSH content. In the study, GSH content significantly increased during meat aging. In contrast, grilling caused a loss approximately 40% of GSH content. Based on the study, it can be concluded that the distribution of GSH in anatomical beef muscles is uneven.

Comparación de seguridad y efectividad entre los accesos radiales derecho e izquierdo en la intervención coronaria percutánea / Comparison of safety and effectiveness between the right and left radial artery approach in percutaneous coronary intervention

Introducción y objetivos: Existen pocos estudios que comparen los accesos por la radial izquierda (ARI) y por la radial derecha en intervenciones coronarias percutáneas (ICP) en población general y practicadas por cirujanos con diferentes grados de experiencia en intervencionismo. El objetivo de nuestro estudio es comparar la seguridad y el beneficio clínico con cada acceso en pacientes no seleccionados con síndrome coronario agudo (SCA) y angina estable (AE). Métodos: Para evitar los posibles sesgos de un estudio no aleatorizado, se usó la puntuación de propensión para comparar ambos accesos radiales. Se recogieron datos de 18.716 pares con AE y 46.241 con SCA sometidos a ICP con implante de stent entre 2014 y 2017, en 151 centros terciarios con cardiología intervencionista en Polonia (registro nacional de Polonia [ORPKI]). Resultados: No se encontraron diferencias en cuanto a mortalidad y complicaciones periprocedimiento en AE. El ARI se asoció con mayores dosis de radiación independientemente de la presentación clínica (AE, 1.067,0±947,10 frente a 1.007,4±983,5 mGy; p=0,001; SCA, 1.212,7±1.005,5 frente a 1.053,5±1.029,7 mGy; p=0,001). En los pacientes con SCA, el ARI se asoció con mayor cantidad de contraste (174,2±75,4 frente a 167,2±72,1ml; p=0,001). Además, en los pacientes con SCA y ARI, las complicaciones periprocedimiento como disección coronaria (el 0,16 frente al 0,09%; p=0,008), fenómeno de no reflow (el 0,65 frente al 0,49%; p=0,005) y hemorragia en el sitio de punción (el 0,09 frente al 0,05%; p=0,04) resultaron más frecuentes. No hubo diferencias en la mortalidad entre los 2 grupos (p=0,90). Conclusiones: Los resultados que se presentan podrían estar en relación con una menor experiencia en el ARI. Ambos accesos son seguros en los pacientes con AE, pero el ARI se asoció con una mayor frecuencia de complicaciones periprocedimiento de ICP en el SCA (AU)

Introduction and objectives: There is a paucity of data comparing the left radial approach (LRA) and right radial approach (RRA) for percutaneous coronary intervention (PCI) in all-comers populations and performed by operators with different experience levels. Thus, we sought to compare the safety and clinical outcomes of the RRA and LRA during PCI in “real-world” patients with either stable angina or acute coronary syndrome (ACS). Methods: To overcome the possible impact of the nonrandomized design, a propensity score was calculated to compare the 2 radial approaches. The study group comprised 18 716 matched pairs with stable angina and 46 241 with ACS treated with PCI and stent implantation between 2014 and 2017 in 151 tertiary invasive cardiology centers in Poland (the ORPKI Polish National Registry). Results: The rates of death and periprocedural complications were similar for the RRA and LRA in stable angina patients. A higher radiation dose was observed with PCI via the LRA in both clinical presentations (stable angina: 1067.0±947.1 mGy vs 1007.4±983.5 mGy, P=.001; ACS: 1212.7±1005.5 mGy vs 1053.5±1029.7 mGy, P=.001). More contrast was used in LRA procedures but only in ACS patients (174.2±75.4mL vs 167.2±72.1mL, P=.001). Furthermore, periprocedural complications such as coronary artery dissection (0.16% vs 0.09%, P=.008), no-reflow phenomenon (0.65% vs 0.49%, P=.005), and puncture site bleeding (0.09% vs 0.05%, P=.04) were more frequently observed with the LRA in ACS patients. There was no difference in mortality between the 2 groups (P=.90). Conclusions: Our finding of poorer outcomes with the LRA may be related to lower operator experience with this approach. While both the LRA and RRA are safe in the setting of stable angina, the LRA was associated with a higher rate of periprocedural complications during PCI in ACS patients (AU)

Fever, human herpesvirus-6 (HHV-6) seroconversion, and acute rejection episodes as a function of the initial seroprevalence for HHV-6 in renal transplant recipients.

Human herpesvirus-6 (HHV-6) is an opportunistic viral pathogen of emerging clinical significance in immunocompromised patients. We performed a seroepidemiological survey to test the relation between seroprevalence among donors and recipients for HHV-6 at three endpoints. Before transplantation sera obtained from cadaveric donors and from potential recipients were tested for IgG antibodies against HHV-6 using an enzyme-linked immunoassay. The group of recipient sera, including samples obtained before as well as 2, 4, 12, and 48 weeks after transplantation, were tested for anti-HHV-6 IgM antibodies using an indirect immunofluorescence assay. The statistical analysis was performed with the Cox proportional hazards models. The HHV-6 seronegative group (n = 11) compared with the HHV-6 seropositive group (n = 109) showed twice the risk of HHV-6 IgM seroconversion (RR = 2.24; P < .04), with a greater risk of fever, namely 3.8, which was on the verge of statistical significance. The opposite trend toward an association with acute rejection episodes was observed among HHV-6 seronegative patients (RR = 1.81). The presence of IgG antibody in the sera of donors to IgG seropositive recipients had no association with the occurrence of IgM seroconversion. In contrast, IgM antibodies to HHV-6 appeared in four of five seronegative patients who received allografts from IgG seropositive donors. These preliminary data suggest that the effects seem to be the consequence of HHV-6 transmission through a renal allograft.

Human Pooled Immunoglobulin as Treatment of Active Antibody-Mediated Rejection of Transplanted Kidney.

BACKGROUND: Antibody-mediated rejection (ABMR) has emerged as the leading cause of renal graft loss. The optimal treatment protocol in ABMR remains unknown. This study aimed to assess the efficacy of intravenous immunoglobulin (IVIG) for treatment of ABMR in renal recipients. METHODS: Thirty-nine ABO-compatible cross-match-negative renal recipients with biopsy-proven ABMR composed the study group. Pulses of methylprednisolone (MP) and appropriate enhancement of net state of immunosuppression were applied in all individuals; 17/39 recipients were administered IVIG (IVIG group); the remaining 22/39 patients, identified to be nonadherent or unsatisfactorily immunosuppressed, were kept on the initial treatment (MP group). Serum creatinine concentration was obtained at each of 10 intended visits, and glomerular filtration rate (GFR) was estimated with the use of the standard Modification of Diet in Renal Disease (MDRD) formula. Generalized linear mixed model was used for statistical analysis. RESULTS: Renal function (modeled as linear slope of MDRD-based GFR change over time, separately for the pre- and post-intervention periods) improved significantly in IVIG-treated recipients. Pre-intervention slopes were -0.72 and -0.46 mL/min/mo for IVIG and MP groups, respectively (P = NS), whereas post-intervention the slopes changed to -0.03 and -0.47 mL/min/mo (IVIG and MP, respectively; P < .005). Within-group changes of slopes at the time of intervention were 0.69 and -0.01 mL/min/mo in IVIG (P < .01) and MP (P = NS) groups, respectively. The relative slope change (pre- to post-intervention) was 0.7 mL/min/mo in favor of the IVIG group (P < .033). None of the classic immunologic or nonimmunologic graft function predictors influenced GFR during 12 months of follow-up. CONCLUSIONS: IVIG improved graft function in renal recipients diagnosed with ABMR.

Association of MYH9 rs3752462 and rs5756168 Polymorphisms With Transplanted Kidney Artery Stenosis.

Allelic variants of the MYH9 gene, encoding myosin nonmuscle heavy chain type IIA, have been shown to correlate with diminished glomerular filtration rates and end-stage kidney disease in individuals of Caucasian ancestry. Myosin nonmuscle heavy chain type IIA is expressed during development as well as in injured vessels and kidney structures. We hypothesized that MYH9 risk variants may correlate with kidney artery injury and dysfunctional healing, such as transplant renal artery stenosis (TRAS). Our study aimed at evaluating the association of MYH9 risk allelic variants (rs4821480, rs4821481, rs3752462, rs11089788, rs136211, rs5756168, rs2032487, and rs2239784) with TRAS, defined as >50% renal artery lumen reduction. Genotyping was performed with the use of custom Taqman genotyping assays on DNA samples (n = 295) from white deceased-donor kidney transplant recipients and genomic DNA from the corresponding donors. Statistical analysis was performed with the use of Kaplan-Meier estimates, log-rank tests, and proportional hazard Cox models. Recipients carrying TT in rs5756168 experienced diminished risk of TRAS (hazard ratio [HR], 0.31; P < .009), whereas organs carrying CC in rs3752462 were exposed to excessive TRAS risk (HR, 2.54; P < .047). In multivariate stepwise analysis TRAS was 10.9-fold increased in kidneys originating from rs3752462 CC, whereas the risk was decreased 3.45-fold (adjusted HR, 0.29) in recipients carrying rs5756168 TT (P < .007 and P < .033, respectively). Intracranial bleeding or trauma compared with other mechanisms of donor death diminished TRAS risk by 87% and 91%, respectively (P < .030 and P < .017). Our study is the first to identify genetic predisposition to transplant renal artery stenosis.

Targeting Alpha Toxin and ClfA with a Multimechanistic Monoclonal-Antibody-Based Approach for Prophylaxis of Serious Staphylococcus aureus Disease.

UNLABELLED: Staphylococcus aureus produces numerous virulence factors, each contributing different mechanisms to bacterial pathogenesis in a spectrum of diseases. Alpha toxin (AT), a cytolytic pore-forming toxin, plays a key role in skin and soft tissue infections and pneumonia, and a human anti-AT monoclonal antibody (MAb), MEDI4893*, has been shown to reduce disease severity in dermonecrosis and pneumonia infection models. However, interstrain diversity and the complex pathogenesis of S. aureus bloodstream infections suggests that MEDI4893* alone may not provide adequate protection against S. aureus sepsis. Clumping factor A (ClfA), a fibrinogen binding protein, is an important virulence factor facilitating S. aureus bloodstream infections. Herein, we report on the identification of a high-affinity anti-ClfA MAb, 11H10, that inhibits ClfA binding to fibrinogen, prevents bacterial agglutination in human plasma, and promotes opsonophagocytic bacterial killing (OPK). 11H10 prophylaxis reduced disease severity in a mouse bacteremia model and was dependent on Fc effector function and OPK. Additionally, prophylaxis with 11H10 in combination with MEDI4893* provided enhanced strain coverage in this model and increased survival compared to that obtained with the individual MAbs. The MAb combination also reduced disease severity in murine dermonecrosis and pneumonia models, with activity similar to that of MEDI4893* alone. These results indicate that an MAb combination targeting multiple virulence factors provides benefit over a single MAb neutralizing one virulence mechanism by providing improved efficacy, broader strain coverage, and protection against multiple infection pathologies. IMPORTANCE: Alternative strategies to broad-spectrum antibiotics are required to combat the antibiotic resistance epidemic. Previous attempts at active or passive immunization against Staphylococcus aureus targeting single antigens have failed in clinical trials despite positive preclinical data. To provide broad disease and isolate coverage, an effective immunization strategy likely must target multiple virulence mechanisms of the pathogen. Herein, we tested a multimechanistic MAb combination targeting alpha toxin (AT) and clumping factor A (ClfA) that neutralizes AT-mediated cytotoxicity, blocks fibrinogen binding by ClfA, prevents bacterial agglutination, targets the bacteria for opsonophagocytic killing, and provides broad isolate coverage in a lethal-bacteremia model. Although each MAb alone was effective in bacteremia against some individual isolates, the MAb combination provided improved protection against other isolates. These results illustrate the importance of targeting multiple virulence mechanisms and highlight the potential for an MAb combination targeting AT and ClfA to effectively prevent S. aureus disease.

The interrelationship between markers of inflammation and oxidative stress in chronic obstructive pulmonary disease: modulation by inhaled steroids and antioxidant.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is accompanied by both airway and systemic inflammation and by oxidative stress. This study aimed to characterise the relationship between oxidative stress and inflammatory components in induced sputum and blood. MATERIAL & METHODS: We studied blood and sputum samples from stable COPD patients (mean FEV1 60.5+/-7.5% predicted) at baseline (no treatment) and after 10 weeks treatment with either inhaled steroid, fluticasone propionate (FP) (1000 microg/d) or 10 weeks treatment with N-acetylcysteine (600mg/d) (NAC). We assessed the inflammatory markers (IL-8, ECP, sICAM-1, NE) in sputum and serum and we compared them with blood markers of oxidative stress (SOD, GPx, TEAC, albumin, vitamin E and A). RESULTS: At baseline blood sICAM-1 correlated with IL-8 levels (P<0.01, r = 0.62) and negatively with GPx (P<0.01, r = -0.63) and with TEAC (P<0.05, r = -0.53). TEAC correlated positively with GPx (P<0.01, r = 0.70). Correlation between sICAM and IL-8 disappeared after NAC treatment. The correlation between sICAM and GPx disappeared after FP treatment. The correlation between TEAC and GPx was maintained after both NAC and FP. CONCLUSIONS: The relationship between markers of inflammation, adhesion and antioxidant capacity is significantly modulated by treatment with N-acetylcysteine or inhaled corticosteroids.

Serum TGF-beta1 correlates with chronic histopathological lesions in protocol biopsies of kidney allograft recipients.

INTRODUCTION: Transforming growth factor-beta (TGF-beta) is a well-known profibrotic factor playing a role in chronic kidney allograft nephropathy. Cyclosporine (CsA)-sparing immunosuppressive regimens may improve long-term graft function. Our aim was to study the influence of immunosuppressive treatment with versus without calcineurin inhibitors on serum TGF-beta levels and histological changes in protocol biopsies of kidney allograft recipients. PATIENTS AND METHODS: In this prospective, randomized study of 42 low-rejection risk patients we randomized two groups: group A: mycophenolate mofetil (MMF), prednisone, daclizumab, and reduced CsA dose for 7 months (5 mg per kg per day) followed by complete withdrawal (n = 21); and group B: normal CsA dose (10 mg per kg per day adjusted according to C2 levels), MMF, prednisone, and no daclizumab (n = 21). METHODS: In both groups we performed histological assessments (Banff 97) and measured serum TFG-beta levels before as well as, at 3 and 12 months after transplantation. RESULTS: We found a relationship between immunosuppressive regimen and the TGF-beta concentration over 1 year of observation. Before transplant the TGF-beta1 levels did not differ between the groups (P = .29); at 3 months they were 33 +/- 9 vs 49 +/- 15 pg per mL, respectively, in groups A and B (P = .08), and at 12 months they were 39.5 +/- 4 versus 55.5 +/- 11 pg per mL, respectively, in groups A and B (P = .03). Protocol biopsies at 12 months in group B showed chronic tubular lesions more pronounced than in group A. TGF-beta1 concentrations were significantly higher among group B than A. We conclude that TGF-beta1 concentration may predict the development of kidney graft fibrosis; early CsA withdrawal may achieve a reduction in chronic tubular and interstitial injury of cadaveric kidney allografts.

The impact of immune responses on lung cancer and the development of new treatment modalities.

OBJECTIVE: This presentation covers predominantly review data in relation with immune responses initiating and accompanying lung carcinogenesis or- on the contrary-contributing to novel therapeutic developments. Occasionally, personal findings will be considered. RESULTS 1 OF IMMUNE DEFICIENCY: It is known for several decades that cancer incidence (several sites) is increased in subjects receiving immunosuppressive therapy, e.g. to avoid transplant rejection, or suffering from AIDS. We have observed that in areas heavily polluted by industrial activities, resulting in immune deficiency, cancer incidence is increased, notably for lung cancer. On the other hand, neoplastic cells are able to escape the host's immune responses by inducing apoptosis of the effector T lymphocytes. Apoptosis in T-cells is triggered by the interaction of the membrane receptor Fas with its normal ligand Fas L, or an activating antibody. Now lung carcinoma cells have been shown to express Fas L, enabling them to destroy cytolytic T cells. RESULTS 2 OF IMMUNE TREATMENT: It is well over a century ago that interest in the immunotherapy of cancer was aroused by the observation of tumour regressions concomitant with bacterial infection, an observation leading to the development of 'Coley's toxin', a mixture of killed bacteria (presently known to act through the presence of TNF-alpha). Since these long-standing empirical attempts, a lasting search for immune control of cancer has been initiated, comprising such different approaches as active non-specific immunotherapy, active specific immunotherapy, approaches based on the use of monoclonal antibodies, as well as those depending on cellular immunity and the development of adoptive immunotherapy, and the use of peptide vaccines. These different approaches will be described and their results presented. CONCLUSION: Present state-of-the-art will be discussed and new pathways for development evoked; better understanding of immune mechanisms is opening new avenues for improved treatment efficacy.

Environmental genotoxicity and cancer risk in humans: a combined evaluation correlating the results of the Tradescantia micronucleus assay in the field and human biomarker assessments in serum. I. The TRAD-MCN assay.

It is well documented that environmental pollution from industrial activity, sewage farms, hazardous waste sites, incinerators, etc, contributes to the overall cancer risk and that this contribution can be considerable under certain circumstances. It is important, therefore, to identify the level of genotoxic activity in the environment and to relate it to biomarkers of cancer risk in humans. After reviewing a range of cytogenetic assays, we have selected the Tradescantia micronucleus assay (TRAD-MCN) developed by Ma et al to be used in indoor and field evaluations. The meiotic pollen mother cells of T clone 4430 are particularly sensitive to chemical pollutants; the buds are exposed for 6-8 h. We describe assays made down wind from a coal-fired power station and from the vicinity of two waste sites. Statistically significant results were obtained at 200 m and 600 m down wind from the power station; higher levels of micronucleus frequencies (MN) were found in foggy rather than dry conditions. Similarly, in the vicinity of two waste sites the MN frequencies were significantly increased in both dry and foggy conditions up to 1.5 km down wind; this was despite previous efforts to rehabilitate the sites. The TRAD-MCN assay is sensitive, reproducible, easy to perform, well standardized, inexpensive and undemanding in equipment. We propose that it be the primary test for genotoxicity evaluation and mapping followed, in suspicious areas, by human biomarker assays.

Use of higher plants in the biomonitoring of environmental genotoxic pollution.

Genotoxicity is recognised as being the first step in carcinogenesis. Hence the identification ambient genotoxicity represents an important step in cancer risk assessment even if non-genotoxic mechanisms also occur. Genotoxicity can be assessed after exposure of populations to chemical or physical agents, as cytogenetic alterations, mutations or production of DNA/protein adducts. Well defined higher plants represent an excellent basis for cytogenetic evaluations after exposure to genotoxic pollutants, especially that the maturation of their gametes (meiosis) follows the same patterns as in animals and humans. We present a description of the Tradescantia Micronucleus Assay (TRAD-MCN) and results of a series of field evaluations after environmental pollution (urban settings, industrial sites, landfills). A significant correlation is observed between the intensity of the pollution and the ratio of micronuclei appearing at the tetrad stage of meiosis. The method is easy, requiring no special equipment, reproducible, rather inexpensive. It allows the establishment of "genotoxicity maps" and the follow-up monitoring of the polluted sites. In environmental monitoring, we consider the TRAD-MCN assay as the first-line procedure presenting the additional advantage of not involving human populations primary evaluations, thus avoiding psychological stress.

Polyomavirus BK infection.

Because it is an important factor affecting renal transplant function, BK infections are significant problem in posttransplant. BK nephropathy develops in 5% of renal allograft recipients, in most cases within the first year after the procedure. The gold standard for BK nephropathy diagnosis is still immunohistochemical staining for large T antigen in graft biopsy specimens. The aim of the present study was to evaluate the incidence of and factors influencing BK nephropathy in our renal allograft population. Among 89 renal or pancreas/kidney allograft recipients, BKV DNA was detected in 1 or more serum samples in 17 patients but BK nephropathy was diagnosed in only 1 case. Plasmacytic tubulitis was an exclusive feature in PCR-positive patients with 2 (20%) cases but no such findings in the PCR-negative group. In 40% of patients in the PCR-positive group at least 1 rejection episode was diagnosed versus 22% in the PCR-negative group. There were no significant differences in both groups according to total ischemia time, immunosuppressive treatments, or mean serum creatinine at 1 year after transplantation.