Prognosis of sentinel node staged patients with primary cutaneous melanoma

Background

This study investigated survival probabilities and prognostic factors in sentinel lymph node biopsy (SLNB) staged patients with cutaneous melanoma (CM) with the aim of defining subgroups of patients who are at higher risk for recurrences and who should be considered for adjuvant clinical trials.

Methods

Patients with primary CM who underwent SLNB in the Department of Dermatology, University of Tuebingen, Germany, between 1996 and 2009 were included into this study. Survival probabilities and prognostic factors were evaluated by Kaplan-Meier and multivariate Cox proportional hazard models.

Results

1909 SLNB staged patients were evaluated. Median follow-up time was 44 months. Median tumor thickness was 1.8 mm, ulceration was present in 31.8% of cases. The 5-year Overall Survival (OS) was 90.3% in SLNB negative patients (IB 96.2%, IIA 87.0%, IIB 78.1%, IIC 72.6%). Patients with micrometastases (stage IIIA/B) had a 5-year OS rate of 70.9% which was clearly less favorable than for stages I–II. Multivariate analysis revealed tumor thickness, ulceration, body site, histopathologic subtype and SLNB status as independent significant prognostic factors.

Conclusion

Survival rates of patients with primary CM in stages I–II were shown to be much more favorable than previously reported from non sentinel node staged collectives. For future clinical trials, sample size calculations should be adapted using survival probabilities based on sentinel node staging.     

Accuracy of Non-Enhanced CT in Detecting Early Ischemic Edema Using Frequency Selective Non-Linear Blending

Purpose

Ischemic brain edema is subtle and hard to detect by computed tomography within the first hours of stroke onset. We hypothesize that non-enhanced CT (NECT) post-processing with frequency-selective non-linear blending (“best contrast”/BC) increases its accuracy in detecting edema and irreversible tissue damage (infarction).

Methods

We retrospectively analyzed the NECT scans of 76 consecutive patients with ischemic stroke (exclusively middle cerebral artery territory—MCA) before and after post-processing with BC both at baseline before reperfusion therapy and at follow-up (5.73±12.74 days after stroke onset) using the Alberta Stroke Program Early CT Score (ASPECTS). We assessed the differences in ASPECTS between unprocessed and post-processed images and calculated sensitivity, specificity, and predictive values of baseline NECT using follow-up CT serving as reference standard for brain infarction.

Results

NECT detected brain tissue hypoattenuation in 35 of 76 patients (46.1%). This number increased to 71 patients (93.4%) after post-processing with BC. Follow-up NECT confirmed brain infarctions in 65 patients (85.5%; p = 0.012). Post-processing increased the sensitivity of NECT for brain infarction from 35/65 (54%) to 65/65 (100%), decreased its specificity from 11/11 (100%) to 7/11 (64%), its positive predictive value (PPV) from 35/35 (100%) to 65/69 (94%) and increased its accuracy 46/76 (61%) to 72/76 (95%).

Conclusions

This post-hoc analysis suggests that post-processing of NECT with BC may increase its sensitivity for ischemic brain damage significantly.     

Establishment,Characterization and Chemosensitivity of Three Mismatch Repair Deficient Cell Lines from Sporadic and Inherited Colorectal Carcinomas

Background

Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts.

Methodology

MMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo.

Principal Findings

Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-ras^wt, B-raf^mut, HROC87: K-ras^wt, B-raf^mut), whereas the HROC113 cell line (K-ras^mut, B-raf^wt) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM⁺) tumor cells, showing surface tumor marker expression (CEACAM⁺). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity.

Conclusions

These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.     

Impact of aggressiveness of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">F. culmorum</Emphasis> isolates on yield parameters and mycotoxin production in wheat

Plant-associated isolates from Fusarium graminearum and F. culmorum were inoculated on wheat in field experiments in 2007 and 2008 to ascertain their influence on fungal colonization of the ears, as well as mycotoxin contamination (deoxynivalenol, DON; nivalenol, NIV; zearalenone, ZEA) and yield parameters in the mature crop after inoculation with or without irrigation. The isolates were assigned to four different groups of aggressiveness on the basis of pathogenic symptom development and mycotoxin production in vitro. Increased levels of trichothecene-producing Fusarium DNA in the ears indicated a successful inoculation of the plants, which resulted in increased DON content in the wheat kernels in 2007. Dry conditions at anthesis markedly suppressed fungal colonization as well as mycotoxin accumulation. However, due to precipitation during the ripening period, yield and thousand-kernel weight were similar whether or not irrigation was applied at the time of inoculation. The level of aggressiveness among the isolates as determined in vitro was not reflected in the field experiment. The activity of the extracellular invertase in developing ears increased as a plant response to pathogen infection, especially when the plants were irrigated at the time of inoculation. In 2008, the Fusarium inoculation of wheat heads did not cause fungal growth and mycotoxin contamination in the grain, because of the dry weather conditions that occurred over the entire period of anthesis and ripening. The risk of future mycotoxin contamination in grains was discussed based on climate change prognosis.     

Osteopenia due to enhanced cathepsin K release by BK channel ablation in osteoclasts

Background

The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified.

Methodology/Principal Findings

We found, that in juvenile bone the large conductance, voltage and Ca²⁺-activated (BK) K⁺ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K⁺ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK^−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK^−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca²⁺ and triiodthyronine as well as osteoclastogenesis were not altered in BK^−/− females.

Conclusion/Significance

Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK^−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.     

The RTP site shared by the HIV-1 Tat protein and the 11S regulator subunit alpha is crucial for their effects on proteasome function including antigen processing

Nuclease A (NucA) from Anabaena sp. is a non-specific endonuclease able to degrade single and double-stranded DNA and RNA. The endonucleolytic activity is inhibited by the nuclease A inhibitor (NuiA), which binds to NucA with 1:1 stoichiometry and picomolar affinity. In order to better understand the mechanism of inhibition, the solution structure of NuiA was determined by NMR methods. The fold of NuiA is an alpha-beta-alpha sandwich but standard database searches by DALI and TOP revealed no structural homologies. A visual inspection of alpha-beta-alpha folds in the CATH database revealed similarities to the PR-1-like fold (SCOP nomenclature). The similarities include the ordering of secondary structural elements, a single helix on one face of the alpha-beta-alpha sandwich, and three helices on the other face. However, a major difference is in the IV helix, which in the PR-1 fold is short and perpendicular to the I and III helices, but in NuiA is long and parallel to the I and III helices. Additionally, a strand insertion in the beta-sheet makes the NuiA beta-sheet completely antiparallel in organization. The fast time-scale motions of NuiA, characterized by enhanced flexibility of the extended loop between helices III and IV, also show similarities to P14a, which is a PR-1 fold. We propose that the purpose of the PR-1 fold is to form a stable scaffold to present this extended structure for biological interactions with other proteins. This hypothesis is supported by data that show that when NuiA is bound to NucA significant changes in chemical shift occur in the extended loop between helices III and IV.     

Calcium Signaling Controls Pathogenic Th17 Cell-Mediated Inflammation by Regulating Mitochondrial Function

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A “new” genetic polymorphism of a human serum protein: inter-alpha-trypsin-inhibitor

Summary A new genetic polymorphism of a human serum glycoprotein, the inter--trypsin-inhibitor (ITI), has been demonstrated by population and family studies. Sera were examined after neuraminidase treatment by isoelectric focusing on agarose gels followed by immunoblotting or by immunfixation with specific ITI-antiserum. Using this method, three common ITI phenotypes 1, 1–2 and 2, as well as two further rare ITI types 1–3 and 2–3 were disclosed. Genetically, these phenotypes are controlled by three allelic genes that determine a total of six phenotypes. These alleles are designated ITI^*1, ITI^*2 and ITI^*3. The homozygous form of the third allele ITI^*3 has not been found, as yet. The frequencies of ITI were examined in two population samples from Southern Germany (n=248) and from Tyrol, Austria (n=124). The gene frequencies of the common alleles ITI^*1 and ITI^*2 were 0.575 and 0.417, respectively, in Southern Germany, and 0.577 and 0.423, respectively, in Tyrol, Austria. The third allele ITI^*3 was found only in the sample from Southern Germany, thus far, and was calculated to be 0.008.     

Female mice carrying a defective Alox15 gene are protected from experimental colitis via sustained maintenance of the intestinal epithelial barrier function

Lipoxygenases (ALOXs) are involved in the regulation of cellular redox homeostasis. They also have been implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators and play a role in the pathogenesis of inflammatory diseases, which constitute a major health challenge owing to increasing incidence and prevalence in all industrialized countries around the world. To explore the pathophysiological role of Alox15 (leukocyte-type 12-LOX) in mouse experimental colitis we tested the impact of systemic inactivation of the Alox15 gene on the extent of dextrane sulfate sodium (DSS) colitis. We found that in wildtype mice expression of the Alox15 gene was augmented during DSS-colitis while expression of other Alox genes (Alox5, Alox15b) was hardly altered. Systemic Alox15 (leukocyte-type 12-LOX) deficiency induced less severe colitis symptoms and suppressed in vivo formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the major Alox15 (leukocyte-type 12-LOX) product in mice. These alterations were paralleled by reduced expression of pro-inflammatory gene products, by sustained expression of the zonula occludens protein 1 (ZO-1) and by a less impaired intestinal epithelial barrier function. These results are consistent with in vitro incubations of colon epithelial cells, in which addition of 12S-HETE compromised enantioselectively transepithelial electric resistance. Consistent with these data transgenic overexpression of human ALOX15 intensified the inflammatory symptoms. In summary, our results indicate that systemic Alox15 (leukocyte-type 12-LOX) deficiency protects mice from DSS-colitis. Since exogenous 12-HETE compromises the expression of the tight junction protein ZO-1 the protective effect has been related to a less pronounced impairment of the intestinal epithelial barrier function.