CD200 is induced by ERK and is a potential therapeutic target in melanoma

Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM.     

A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair

Mismatch repair contributes to genetic stability, and inactivation of the mammalian pathway leads to tumor development. Mismatch correction occurs by an excision-repair mechanism and has been shown to depend on the 5′ to 3′ hydrolytic activity exonuclease 1 (Exo1) in eukaryotic cells. However, genetic and biochemical studies have indicated that one or more Exo1-independent modes of mismatch repair also exist. We have analyzed repair of nicked circular heteroduplex DNA in extracts of Exo1-deficient mouse embryo fibroblast cells. Exo1-independent repair under these conditions is MutLα-dependent and requires functional integrity of the MutLα endonuclease metal-binding motif. In contrast to the Exo1-dependent reaction, we have been unable to detect a gapped excision intermediate in Exo1-deficient extracts when repair DNA synthesis is blocked. A possible explanation for this finding has been provided by analysis of a purified system comprised of MutSα, MutLα, replication factor C, proliferating cell nuclear antigen, replication protein A, and DNA polymerase δ that supports Exo1-independent repair in vitro. Repair in this system depends on MutLα incision of the nicked heteroduplex strand and dNTP-dependent synthesis-driven displacement of a DNA segment spanning the mismatch. Such a mechanism may account, at least in part, for the Exo1-independent repair that occurs in eukaryotic cells, and hence the modest cancer predisposition of Exo1-deficient mammalian cells.     

Colorimetric indicators of microbial contamination in corneal preservation medium

PURPOSE: To compare acid-base and oxidation-reduction indicators and to investigate the effect of buffer and temperature on the colorimetric detection of microbial growth in corneal preservation media. METHODS: Corneal preservation media containing gentamicin, without or with HEPES buffer, were prepared with either phenol red or AlamarBlue indicators (AccuMed International, Westlake, OH, U.S.A.). Both media were inoculated with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, Serratia marcescens, or Candida albicans and then incubated at 4 degrees C, 22 degrees C, or 35 degrees C. The pH or percent reduction were determined hourly for eight hours, then daily for one week. RESULTS: The length of time before a confirmed change in pH or reduction occurred varied by microorganism, storage temperature, and buffering capacity. At 4 degrees C, none of the microorganisms caused a detectable pH change in buffered medium within one day after inoculation, although two bacterial species reduced AlamarBlue within four hours. At 22 degrees C and 35 degrees C, all bacteria except P. aeruginosa produced a pH shift within a few hours, and all tested bacterial species reduced AlamarBlue. For bacteria producing detectable pH changes, HEPES-buffered medium took longer to change than medium without HEPES. C. albicans was not detectable in HEPES-buffered medium at any temperature by phenol red and was only detectable by AlamarBlue after 2-3 days at 22 degrees C and 35 degrees C. CONCLUSION: Acidic shifts in refrigerated corneal preservation medium do not occur during contamination by several microorganisms. AlamarBlue, a redox indicator, is more sensitive than phenol red in detecting some bacteria. C. albicans is not reliably detected by pH or redox indicators.     

Longitudinal study of brain function and depletion of iron stores in individual subjects

Seven men living in a controlled research unit showed decrements in iron stores due to repeated phlebotomy. For each subject, weekly measures of cognitive task performance, electroencephalographic (EEG) power and Event-Related Potentials (ERPs) were examined in relation to serum ferritin level. No reliable relationships of ferritin level to cognitive performance ability were observed. Some EEG findings appeared similar to previous observations on EEG data and iron status in cross-sectional research. For four of the seven subjects, asymmetries of EEG power in the occipital electrodes appeared related to the decline in iron status over time.     

Management of unresectable stage III non-small cell lung cancer: the role of combined chemoradiation

Until the late 1980s, thoracic radiation therapy (TRT) was considered the standard of care for patients with stage III disease despite extremely poor 5-year survival rates. Several studies evaluating TRT combined with chemotherapy showed a survival advantage. Based on these data, combined modality therapy became accepted as the standard of care in this group of patients with good performance status and made the treatment of locally advanced non-small cell lung cancer (NSCLC) a multidisciplinary endeavor. Recent studies have shown that concurrent chemoradiotherapy offers a significantly greater survival advantage than sequential chemoradiotherapy and should be considered standard of care in stage III inoperable NSCLC. Although numerous Phase III trials have clearly demonstrated a survival benefit in those patients who receive combined modality therapy, many questions remain. The most effective combination of drugs, their optimal mode of administration, the use of either induction or consolidation therapy in addition to a backbone of concurrent therapy, and the details of TRT, including total dose, fractionation, acceleration, treatment volumes, and tumor targeting remain important issues to define. Although progress has been made in treatment for locally advanced NSCLC, the majority of patients still die within 5 years either from locoregional or distant progression of disease. This article will review the current data regarding treatment of this heterogeneous group of patients. In addition, a brief summary of new molecular therapies and chemotherapeutics will be presented.     

Comparison of the Etest and the NCCLS-approved agar dilution method to detect metronidazole and clarithromycin resistant Helicobacter pylori

Although the NCCLS has approved the agar dilution method as the test of choice for antimicrobial susceptibility testing of Helicobacter pylori, a critical evaluation of this method in clinical trials to detect antibiotic resistance has not been performed. This study compares the Etest and agar dilution methods for detection of metronidazole and clarithromycin resistance in clinical isolates of H. pylori. MIC data were gathered from US-based clinical trials. The Etest was performed on Mueller-Hinton sheep blood agar plates following incubation for 4 days under 12% CO(2). The agar dilution test was performed according to the recently approved NCCLS methodology using aged sheep blood in a Mueller-Hinton agar base. Metronidazole resistance as determined by Etest was significantly higher than that determined by agar dilution (39%; 690/1768 vs. 25. 1%; 367/1465)(P<0.01). Clarithromycin resistance as determined by Etest was higher than that determined by agar dilution, but was not significantly different (12.5%; 209/1671 vs. 10.6%; 150/1414)(P>0.5). Inter-patient metronidazole resistance showed that the MIC values for identical isolates tested by both methods were equivalent in 58% (109/188). Of the 42% with a >2log(2) difference in MIC values, 17. 6% had a change in susceptibility pattern. For clarithromycin, 71.4% (237/332) of the MIC values for identical isolates tested by both methods had equivalent MIC values. Of the MIC values with a >2log(2) difference in MIC values, only 3% showed a change in susceptibility pattern. Intra-patient variability, i.e. paired isolates from the same patient, was assessed only for metronidazole. Of the 1393 paired isolates tested by Etest, 38.8% were shown to be resistant. Almost 69% of the Etest MIC determinations were deemed equivalent and 16.7% had a change in susceptibility pattern. Of the 639 paired isolates tested by agar dilution, 23.9% were resistant to metronidazole. Almost 72% of the agar dilution MIC values were equivalent and 11.3% of the determinations had a change in susceptibility pattern. Clarithromycin resistance rates are similar, when determined by either test method. The Etest yields a significantly higher prevalence of metronidazole resistance among H. pylori compared with the agar dilution method and both methods yield discordant results, when isolates from different parts of the same stomach are compared. Neither method is reliable in determining metronidazole resistance in H. pylori.     

Unilateral eyelid retraction secondary to contralateral ptosis in dysthyroid ophthalmopathy

A patient with dysthyroid eye disease presented with unilateral lid retraction secondary to a contralateral ptosis. While others have reported similar findings in various underlying disease processes, to the best of our knowledge this is the first case in the literature with dysthyroid ophthalmopathy, and the first where the results of surgical management are presented. The importance of testing for secondary lid retraction by manual elevation of the ptotic partner is stressed.     

Basis of the gabamimetic profile of ethanol

This article summarizes the proceedings of a symposium held at the 2005 Research Society on Alcoholism meeting. The initial presentation by Dr. Wallner provided evidence that selected GABA(A) receptors containing the delta subunit display sensitivity to low intoxicating ethanol concentrations and this sensitivity is further increased by a mutation in the cerebellar alpha6 subunit, found in alcohol-hypersensitive rats. Dr. Mameli reported that ethanol affects gamma-aminobutyric acid (GABA) function by affecting neural circuits that influence GABA release. Dr. Parsons presented data from electrophysiological and microdialysis investigations that ethanol is capable of releasing GABA from presynaptic terminals. Dr. Morrow demonstrated that systemic ethanol increases neuroactive steroids in brain, the absence of which alters various functional responses to ethanol. Dr. Criswell presented evidence that the ability of ethanol to increase GABA was apparent in some, but not all, brain regions indicative of regional specificity. Further, Dr. Criswell demonstrated that neurosteroids alone and when synthesized locally by ethanol act postsynaptically to enhance the effect of GABA released by ethanol in a region specific manner. Collectively, this series of reports support the GABAmimetic profile of acutely administered ethanol being dependent on several specific mechanisms distinct from a direct effect on the major synaptic isoforms of GABA(A) receptors.     

Determinism and stochasticity during maturation of the zebrafish antibody repertoire

It is thought that the adaptive immune system of immature organisms follows a more deterministic program of antibody creation than is found in adults. We used high-throughput sequencing to characterize the diversifying antibody repertoire in zebrafish over five developmental time points. We found that the immune system begins in a highly stereotyped state with preferential use of a small number of V (variable) D (diverse) J (joining) gene segment combinations, but that this stereotypy decreases dramatically as the zebrafish mature, with many of the top VDJ combinations observed in 2-wk-old zebrafish virtually disappearing by 1 mo. However, we discovered that, in the primary repertoire, there are strong correlations in VDJ use that increase with zebrafish maturity, suggesting that VDJ recombination involves a level of deterministic programming that is unexpected. This stereotypy is masked by the complex diversification processes of antibody maturation; the variation and lack of correlation in full repertoires between individuals appears to be derived from randomness in clonal expansion during the affinity maturation process. These data provide a window into the mechanisms of VDJ recombination and diversity creation and allow us to better understand how the adaptive immune system achieves diversity.