An Exhaustion-Like Phenotype Constrains the Activity of CD4+ T Cells Specific for a Self and Melanoma Antigen

While the immune system has the capacity to recognize and destroy melanoma, tolerance mechanisms often hinder the development of effective anti-tumor immune responses. Since many melanoma antigens are self proteins expressed in normal melanocytes, self antigen exposure before tumor development can negatively impact the function of T cells specific for these self/tumor antigens. However, the contribution of self tolerance to anti-melanoma T cell dysfunction remains largely unexplored. We have previously described a TCR transgenic (Tg) mouse model in which T cells specific for the self/melanoma antigen, tyrosinase-related protein 1 (TRP1), develop in the presence of endogenous TRP1 expression (Ag+) and diminished antigen presentation due to the absence of gamma-interferon-inducible lysosomal thiol reductase (GILT-/-). We show that TRP1-specific T cells from these Ag+GILT-/-Tg mice do not protect from melanoma tumor growth, fail to induce autoimmune vitiligo, and undergo diminished proliferation compared to T cells from Ag-GILT+/+Tg mice. Despite an increased frequency of TRP1-specific Treg cells in Ag+GILT-/-Tg mice compared to Ag-GILT+/+Tg animals, Treg cell depletion only partially rescues the proliferative capacity of T cells from TRP1-expressing mice, suggesting the involvement of additional suppressive mechanisms. An increased percentage of melanoma-specific T cells from Ag+GILT-/-Tg animals express PD-1, an inhibitory receptor associated with the maintenance of T cell exhaustion. Antibody blockade of PD-1 partially improves the ability of TRP1-specific T cells from Ag+GILT-/-Tg mice to produce IL-2. These findings demonstrate that melanoma-specific T cells exposed to a self/melanoma antigen in healthy tissue develop an exhaustion-like phenotype characterized by PD-1-mediated immunosuppression prior to encounter with tumor.     

Molecular Characterization of Melanoma Cases in Denmark Suspected of Genetic Predisposition

Both environmental and host factors influence risk of cutaneous melanoma (CM), and worldwide, the incidence varies depending on constitutional determinants of skin type and pigmentation, latitude, and patterns of sun exposure. We performed genetic analysis of CDKN2A, CDK4, BAP1, MC1R, and MITFp.E318K in Danish high-risk melanoma cases and found CDKN2A germline mutations in 11.3% of CM families with three or more affected individuals, including four previously undescribed mutations. Rare mutations were also seen in CDK4 and BAP1, while MC1R variants were common, occurring at more than twice the frequency compared to Danish controls. The MITF p.E318K variant similarly occurred at an approximately three-fold higher frequency in melanoma cases than controls. To conclude, we propose that mutation screening of CDKN2A and CDK4 in Denmark should predominantly be performed in families with at least 3 cases of CM. In addition, we recommend that testing of BAP1 should not be conducted routinely in CM families but should be reserved for families with CM and uveal melanoma, or mesothelioma.     

Behavioural Effects of Tourism on Oceanic Common Dolphins,Delphinus sp., in New Zealand: The Effects of Markov Analysis Variations and Current Tour Operator Compliance with Regulations

Common dolphins, Delphinus sp., are one of the marine mammal species tourism operations in New Zealand focus on. While effects of cetacean-watching activities have previously been examined in coastal regions in New Zealand, this study is the first to investigate effects of commercial tourism and recreational vessels on common dolphins in an open oceanic habitat. Observations from both an independent research vessel and aboard commercial tour vessels operating off the central and east coast Bay of Plenty, North Island, New Zealand were used to assess dolphin behaviour and record the level of compliance by permitted commercial tour operators and private recreational vessels with New Zealand regulations. Dolphin behaviour was assessed using two different approaches to Markov chain analysis in order to examine variation of responses of dolphins to vessels. Results showed that, regardless of the variance in Markov methods, dolphin foraging behaviour was significantly altered by boat interactions. Dolphins spent less time foraging during interactions and took significantly longer to return to foraging once disrupted by vessel presence. This research raises concerns about the potential disruption to feeding, a biologically critical behaviour. This may be particularly important in an open oceanic habitat, where prey resources are typically widely dispersed and unpredictable in abundance. Furthermore, because tourism in this region focuses on common dolphins transiting between adjacent coastal locations, the potential for cumulative effects could exacerbate the local effects demonstrated in this study. While the overall level of compliance by commercial operators was relatively high, non-compliance to the regulations was observed with time restriction, number or speed of vessels interacting with dolphins not being respected. Additionally, prohibited swimming with calves did occur. The effects shown in this study should be carefully considered within conservation management plans, in order to reduce the risk of detrimental effects on common dolphins within the region.     

Malassezia Intra-Specific Diversity and Potentially New Species in the Skin Microbiota from Brazilian Healthy Subjects and Seborrheic Dermatitis Patients

Malassezia yeasts are part of the resident cutaneous microbiota, and are also associated with skin diseases such as seborrheic dermatitis (SD). The role these fungi play in skin diseases and why they are pathogenic for only some individuals remain unclear. This study aimed to characterize Malassezia microbiota from different body sites in healthy and SD subjects from Brazil. Scalp and forehead samples from healthy, mild SD and severe SD subjects were collected. Non-scalp lesions from severe SD patients were also sampled. 5.8S rDNA/ITS2 amplicons from Malassezia sp. were analyzed by RFLP and sequencing. Results indicate that Malassezia microbiota did not group according to health condition or body area. Phylogenetic analysis revealed that three groups of sequences did not cluster together with any formally described species, suggesting that they might belong to potential new species. One of them was found in high proportions in scalp samples. A large variety of Malassezia subtypes were detected, indicating intra-specific diversity. Higher M. globosa proportions were found in non-scalp lesions from severe SD subjects compared with other areas, suggesting closer association of this species with SD lesions from areas other than scalp. Our results show the first panorama of Malassezia microbiota in Brazilian subjects using molecular techniques and provide new perspectives for further studies to elucidate the association between Malassezia microbiota and skin diseases.     

Historical Invasion Records Can Be Misleading: Genetic Evidence for Multiple Introductions of Invasive Raccoons (Procyon lotor) in Germany

Biological invasions provide excellent study systems to understand evolutionary, genetic and ecological processes during range expansions. There is strong evidence for positive effects of high propagule pressure and the associated higher genetic diversity on invasion success, but some species have become invasive despite small founder numbers. The raccoon (Procyon lotor) is often considered as a typical example for such a successful invasion resulting from a small number of founders. The species’ largest non-native population in Germany is commonly assumed to stem from a small number of founders and two separate founding events in the 1930s and 1940s. In the present study we analyzed 407 raccoons at 20 microsatellite loci sampled from the invasive range in Western Europe to test if these assumptions are correct. Contrary to the expectations, different genetic clustering methods detected evidence for at least four independent introduction events that gave rise to genetically differentiated subpopulations. Further smaller clusters were either artifacts or resulted from founder events at the range margin and recent release of captive individuals. We also found genetic evidence for on-going introductions of individuals. Furthermore a novel randomization process was used to determine the potential range of founder population size that would suffice to capture all the alleles present in a cluster. Our results falsify the assumption that this species has become widespread and abundant despite being genetically depauperate and show that historical records of species introductions may be misleading.     

Cytokine Profiles at Admission Can Be Related to Outcome in AIDS Patients with Cryptococcal Meningitis

Cryptococcal meningitis (CM) remains as common life-threatening AIDS-defining illness mainly in resource-limited settings. Previous reports suggested that baseline cytokine profiles can be associated to fungal burden and clinical outcome. This study aimed to evaluate the baseline cytokine profiles in AIDS patients with CM and its relation with the outcome at weeks 2 and 10. Thirty AIDS patients with CM diagnosed by cerebrospinal fluid (CSF) Cryptococcus neoformans positive culture, India ink stain and cryptococcal antigen test were prospectively evaluated. As controls, 56 HIV-infected patients without CM and 48 non-HIV individuals were included. Baseline CSF and sera levels of IL-2, IL-4, IL-8, IL-10, IL-12p40, IL-17A, INF-γ and TNF-α were measured by ELISA. Of 30 CM patients, 24 (80%) were male, median age of 38.1. The baseline CSF high fungal burden and positive blood culture were associated with a positive CSF culture at week 2 (p = 0.043 and 0.029). Most CSF and sera cytokines presented higher levels in CM patients than control subjects (p < 0.05). CSF levels of IL-8, IL-12p40, IL-17A, TNF-α, INF-γ and sera TNF-α were significantly higher among survivors at weeks 2 and 10 (p < 0.05). Patients with increased intracranial pression exhibited CSF IL-10 high levels and poor outcome at week 10 (p = 0.032). Otherwise, baseline CSF log10 IFN-γ and IL-17A were negatively correlated with fungal burden (r = -0.47 and -0.50; p = 0.0175 and 0.0094, respectively). The mortality rate was 33% (10/30) at week 2 and 57% (17/30) at week 10. The severity of CM and the advanced immunodeficiency at admission were related to a poor outcome in these patients. Otherwise, the predominant Th1 cytokines profile among survivors confirms its pivotal role to infection control and would be a prognostic marker in cryptococcal meningitis.     

Mitochondrial Dynamics Protein Drp1 Is Overexpressed in Oncocytic Thyroid Tumors and Regulates Cancer Cell Migration

Oncocytic cell tumors are characterized by the accumulation of morphologically abnormal mitochondria in their cells, suggesting a role for abnormal mitochondrial biogenesis in oncocytic cell transformation. Little is known about the reason for the dysmorphology of accumulated mitochondria. The proteins regulating the morphology of mitochondria, the "mitochondria-shaping" proteins, can modulate their size and number; however, nothing is known hitherto about a possible involvement of mitochondrial dynamics in oncocytic cell transformation in tumors. Our aim was to assess the status of the mitochondria morphology and its role in oncocytic cell transformation. We therefore evaluated the expression pattern of the main mitochondrial fusion and fission proteins in a series of thyroid cell tumor samples, as well as in thyroid tumor cell lines, with and without oncocytic cell features. The expression of mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1 and Fis1) proteins were evaluated by immunohistochemistry (IHC) in a series of 88 human thyroid tumors. In vitro studies, for comparative purposes and to deepen the study, were performed using TPC1 - a papillary thyroid carcinoma derived cell line—and XTC.UC1, an oncocytic follicular thyroid carcinoma-derived cell line. Both IHC and in vitro protein analyses showed an overall increase in the levels of "mitochondrial-shaping" proteins in oncocytic thyroid tumors. Furthermore, overexpression of the pro-fission protein Drp1 was found to be associated with malignant oncocytic thyroid tumors. Interestingly, genetic and pharmacological blockage of Drp1 activity was able to influence thyroid cancer cells’ migration/invasion ability, a feature of tumor malignancy. In this study we show that unbalanced mitochondrial dynamics characterize the malignant features of thyroid oncocytic cell tumors, and participate in the acquisition of the migrating phenotype.     

The role of thioredoxin reductase 1 in melanoma metabolism and metastasis

Although significant progress has been made in targeted and immunologic therapeutics for melanoma, many tumors fail to respond, and most eventually progress when treated with the most efficacious targeted combination therapies thus far identified. Therefore, alternative approaches that exploit distinct melanoma phenotypes are necessary to develop new approaches for therapeutic intervention. Tissue microarrays containing human nevi and melanomas were used to evaluate levels of the antioxidant protein thioredoxin reductase 1 (TR1), which was found to increase as a function of disease progression. Melanoma cell lines revealed metabolic differences that correlated with TR1 levels. We used this new insight to design a model treatment strategy that creates a synthetic lethal interaction wherein targeting TR1 sensitizes melanoma to inhibition of glycolytic metabolism, resulting in a decrease in metastases in vivo. This approach holds the promise of a new clinical therapeutic strategy, distinct from oncoprotein inhibition.