Predicting the risk for corneal graft rejection by aqueous humor analysis.

PURPOSE: Cytokine patterns determined in the aqueous humor before penetrating keratoplasty (PK) may enable us to predict immune reactions (IR). We therefore analyzed 6 cytokines in the aqueous humor of patients before PK. By prospective clinical follow-up, we tested whether patients who developed an IR would present different preoperative cytokine patterns compared to patients without IR. METHODS: We analyzed 18 samples of aqueous humor from 18 patients undergoing PK. The following cytokines were analyzed by cytometric bead array: interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 10 (IL-10), tumor-necrosis-factor α (TNF-α), and interferon γ (INF-γ). Seven patients presented with signs of IR during follow up. We performed Cox proportional hazards analysis to determine significant predictors for IR. We iteratively eliminated all co-variates with p values over 0.1 from the survival model (backward selection). RESULTS: Our final Cox model included the hazardous factors IL-4 (p=0.043) and INF-γ (p=0.059), protective factors IL-2 (p=0.081), IL-5 (p=0.028), and age at time of surgery (p=0.029). We performed a linear discriminant analysis based on these coefficients. The resulting function was: (-9.979*IL5) + (9.262*IL4) + (-3.928*IL2) + (1.709*IFN-γ) + (-0.183*age). A median of -4.97 separated patients with and without IR with no classification error. CONCLUSIONS: We demonstrate that cytokine levels in the aqueous humor can be predictive for IR. Our method allowed an almost 100% separation between patients with and without IR. This finding has the potential to improve the aftercare of PK fundamentally. However, our results need to be confirmed in a larger prospective cohort.

Distinct cytokine pattern in aqueous humor during immune reactions following penetrating keratoplasty.

PURPOSE: Cytokine patterns in the aqueous humor before penetrating keratoplasty might permit us to predict the development of immune reactions. We therefore analyzed ten different cytokines in the aqueous humor of patients before and following penetrating keratoplasty. METHODS: We collected aqueous humor from patients undergoing cataract extraction or penetrating keratoplasty (baseline, n=197), from patients undergoing cataract extraction after penetrating keratoplasty without signs of an endothelial immune reaction (acceptors, n=33), and from patients who underwent irrigation of the anterior chamber due to endothelial immune reactions (rejectors, n=25). We determined interleukin (IL)-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (INF)-gamma, and tumor necrosis factor-alpha in the aqueous humor, using cytometric bead arrays. RESULTS: We found statistically significant differences for IL-10 and INF-gamma among the study groups. We observed no significant differences concerning all other cytokines evaluated in this study. CONCLUSIONS: Elevated levels of INF-gamma emphasize its important role in immune reactions following penetrating keratoplasty. Antagonizing INF-gamma might be an option when treating immune reactions. High levels of IL-10 during endothelial immune reactions may reflect a downregulation of the delayed-type hypersensitivity. Perhaps this is a donor-specific activation of the immune privilege in the anterior chamber as an attempt to prevent damage during immune reactions. We plan to investigate, in upcoming prospective trials, whether the analysis of cytokine patterns in the aqueous humor before penetrating keratoplasty will enable us to predict the occurrence of immune reactions.

Active transforming growth factor-beta2 is increased in the aqueous humor of keratoconus patients.

PURPOSE: The secretion of transforming growth factor-beta2 (TGF-beta2) into the aqueous humor was identified to be important for the maintenance of the immunological privilege in the anterior chamber of the eye. Therefore, prognosis of penetrating corneal grafts might depend on intraocular levels of TGF-beta2. In this study, we determined active TGF-beta2 levels in eyes with pathological corneas prior to penetrating keratoplasty (PK). METHODS: Anterior chamber puncture was performed in 38 cataract patients without PK (group I), in 15 keratoconus eyes undergoing PK (group II), and in 66 eyes with various indications for PK (group III). About 0.05-0.1 ml of aqueous humor were harvested from each patient. Analysis of active TGF-beta2 was started via ELISA within three h following puncture. RESULTS: Average active TGF-beta2 concentration was 39.9+/-24.3 pg/ml (median 41.1 pg/ml) in group I, 78.9+/-53.8 pg/ml (median 85.8 pg/ml) in group II, and 31.5+/-37.7 pg/ml (median 23.6 pg/ml) in group III. The differences between group II and groups I and III were statistically significant (p<0.001). CONCLUSIONS: The highest levels of active TGF-beta2 were found in keratoconus eyes. This finding might partly explain the excellent prognosis of keratoconus eyes regarding PK. Further (prospective) studies are being carried out to discover whether active TGF-beta2 might serve as predictive parameter for immune reactions following PK.

Proteomics highlights decrease of matricellular proteins in left ventricular assist device therapy†.

OBJECTIVES: We investigated the impact of mechanical unloading with a left ventricular assist device (LVAD) on the myocardial proteome. METHODS: We collected 11 patient-matched samples of myocardial left ventricular tissue of patients with non-ischaemic dilate cardiomyopathy, harvested at time of LVAD implant ('pre-LVAD') and heart transplant ('post-LVAD'). Samples were studied by quantitative proteomics. Further we performed histological assessment of deposited collagens and immune infiltration in both pre- and post-LVAD samples. RESULTS: A core set of >1700 proteins was identified and quantified at a false discovery rate <1%. The previously established decrease post-LVAD of alpha-1-antichymotrypsin was corroborated. We noted a post-LVAD decrease of matricellular proteins and proteoglycans such as periostin and versican. Also, proteins of the complement system and precursors of cardiac peptide hormones were decreased post-LVAD. An increase post-LVAD was evident for individual proteins linked to the innate immune response, proteins involved in diverse metabolic pathways, and proteins involved in protein synthesis. Histological analysis did not reveal significant alterations post-LVAD of deposited collagens or immune infiltration. The proteomic data further highlighted a pronounced inter-patient heterogeneity with regards to the impact of LVAD therapy on the left ventricular myocardial proteome. Finally, the proteomic data showed differential proteolytic processing in response to LVAD therapy. CONCLUSIONS: Our findings underline a strong impact of LVAD therapy on the left ventricular myocardial proteome. Together with previous studies, protein markers of LVAD therapy such as alpha-1-antichymotrypsin are becoming apparent. Further, matricellular proteins are emerging as important components in response to LVAD therapy.

Determination of active TGF-beta2 in aqueous humor prior to and following cryopreservation.

Transforming growth factor-beta2 (TGF-beta2) is one of the most important immunosuppressive cytokines in the anterior chamber of the eye. It is secreted as a complex with latency-associated peptide as an inactive precursor. Only the activated form of TGF-beta2 can bind to its receptor and induce signaling. To date, the concentration of active TGF-beta2 in aqueous humor was exclusively determined using samples that had been preserved at -80 degrees C. Quantitative measurements of the activated form directly after sampling have not yet been taken. The aim of this study was to investigate the effect of cryopreservation on the concentration of active TGF-beta2 in the aqueous humor. Samples of aqueous humor were drawn from patients with either cataract or a corneal disorder for determination of TGF-beta2 using a Sandwich-ELISA. In group I (n=30, patients with corneal disorders or cataract), one part of each sample was tested for active TGF-beta2 directly after sampling, whereas the remaining material was stored at -80 degrees C for later analysis. Group II consisted of patients undergoing a simple cataract extraction (n=38), and active TGF-beta2 levels were determined within 3 h after sampling. In Group III (n=34, patients with corneal disorders or cataract), active TGF-beta2 was determined within 3 h after puncture, as were total TGF-beta2 levels after acidic activation for each sample. The average level of active TGF-beta2 in the aqueous humor of group I analyzed directly after sampling was 35+/-31 (median 37) pg/ml. In contrast, frozen samples from the same patients showed an average concentration of 155+/-103 (median 152) pg/ml. The average level of active TGF-beta2 in aqueous humor of 38 cataract (group II) eyes was 40+/-24 (median 41) pg/ml determined within 3 h after puncture. The average level of total TGF-beta2 in group III was 1,654+/-631 (median 1,542) pg/ml compared to 33+/-39 (median 28) pg/ml of active TGF-beta2 determined directly after sampling, yielding a ratio of 2% of active to total TGF-beta2. Levels of active TGF-beta2 in aqueous humor determined directly after sampling were 4.4 fold lower than those measured in frozen samples. Thus, samples meant for determining active TGF-beta2 levels should not be kept frozen.

Genetic and epidemiological risk factors in the development of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the chronic lung disease of preterm infants and still represents a major burden of prematurity. Several clinical risk factors for the onset of the disease are already known. In addition, some candidate genes have recently been identified. We set out to determine clinical as well as genetic risk factors for the development of BPD in the German population. 155 infants born with a gestational age < or = 28 at the tertiary neonatal Centre, Freiburg, were recruited. Clinical data were recorded from hospital charts. 47 children developed moderate or severe BPD. For genetic analyses, 37 polymorphisms within sixteen genes were genotyped on all children. The strongest epidemiological risk factor for BPD was birth weight, followed by low gestational age. Genetic association was detected with single polymorphisms within Tumour necrosis factor alpha, Toll like receptor 10 and vascular endothelial growth factor. The former two genes showed also association with BPD in haplotype analyses. In conclusion, association of BPD was far more convincingly found with a few clinical factors than with genetic polymorphisms. This underscores the genetic complexity of the disease. Furthermore, the identification of predisposing genetic polymorphisms might be hampered by the complex interaction between clinical and genetic factors.

Contribution of different carbon sources to isoprene biosynthesis in poplar leaves.

This study was performed to test if alternative carbon sources besides recently photosynthetically fixed CO2 are used for isoprene formation in the leaves of young poplar (Populus x canescens) trees. In a 13CO2 atmosphere under steady state conditions, only about 75% of isoprene became 13C labeled within minutes. A considerable part of the unlabeled carbon may be derived from xylem transported carbohydrates, as may be shown by feeding leaves with [U-13C]Glc. As a consequence of this treatment approximately 8% to 10% of the carbon emitted as isoprene was 13C labeled. In order to identify further carbon sources, poplar leaves were depleted of leaf internal carbon pools and the carbon pools were refilled with 13C labeled carbon by exposure to 13CO2. Results from this treatment showed that about 30% of isoprene carbon became 13C labeled, clearly suggesting that, in addition to xylem transported carbon and CO2, leaf internal carbon pools, e.g. starch, are used for isoprene formation. This use was even increased when net assimilation was reduced, for example by abscisic acid application. The data provide clear evidence of a dynamic exchange of carbon between different cellular precursors for isoprene biosynthesis, and an increasing importance of these alternative carbon pools under conditions of limited photosynthesis. Feeding [1,2-13C]Glc and [3-13C]Glc to leaves via the xylem suggested that alternative carbon sources are probably derived from cytosolic pyruvate/phosphoenolpyruvate equivalents and incorporated into isoprene according to the predicted cleavage of the 3-C position of pyruvate during the initial step of the plastidic deoxyxylulose-5-phosphate pathway.