31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

A five-year survey of dematiaceous fungi in a tropical hospital reveals potential opportunistic species.

Dematiaceous fungi (black fungi) are a heterogeneous group of fungi present in diverse environments worldwide. Many species in this group are known to cause allergic reactions and potentially fatal diseases in humans and animals, especially in tropical and subtropical climates. This study represents the first survey of dematiaceous fungi in Malaysia and provides observations on their diversity as well as in vitro response to antifungal drugs. Seventy-five strains isolated from various clinical specimens were identified by morphology as well as an internal transcribed spacer (ITS)-based phylogenetic analysis. The combined molecular and conventional approach enabled the identification of three classes of the Ascomycota phylum and 16 genera, the most common being Cladosporium, Cochliobolus and Neoscytalidium. Several of the species identified have not been associated before with human infections. Among 8 antifungal agents tested, the azoles posaconazole (96%), voriconazole (90.7%), ketoconazole (86.7%) and itraconazole (85.3%) showed in vitro activity (MIC ≤ 1 µg/mL) to the largest number of strains, followed by anidulafungin (89.3%), caspofungin (74.7%) and amphotericin B (70.7%). Fluconazole appeared to be the least effective with only 10.7% of isolates showing in vitro susceptibility. Overall, almost half (45.3%) of the isolates showed reduced susceptibility (MIC >1 µg/mL) to at least one antifungal agent, and three strains (one Pyrenochaeta unguis-hominis and two Nigrospora oryzae) showed potential multidrug resistance

Identification and Characterization of a Rare Fungus, Quambalaria cyanescens, Isolated from the Peritoneal Fluid of a Patient after Nocturnal Intermittent Peritoneal Dialysis.

Peritonitis is the leading complication of peritoneal dialysis, which is primarily caused by bacteria rather than fungi. Peritonitis is responsible for approximately 18% of the infection-related mortality in peritoneal dialysis patients. In this paper, we report the isolation of a rare fungus, Quambalaria cyanescens, from the peritoneal fluid of a man after he switched from continuous ambulatory peritoneal dialysis to nocturnal intermittent peritoneal dialysis. Based on the morphological examination and multigene phylogeny, the clinical isolate was confirmed as Q. cyanescens. This pathogen exhibited low sensitivity to all tested echinocandins and 5-flucytosine. Interestingly, morphological characterization revealed that Q. cyanescens UM 1095 produced different pigments at low temperatures (25°C and 30°C) on various culture media. It is important to monitor the emergence of this rare fungus as a potential human pathogen in the tropics. This study provides insight into Q. cyanescens UM 1095 phenotype profiles using a Biolog phenotypic microarray (PM). Of the 760 nutrient sources tested, Q. cyanescens UM 1095 utilized 42 compounds, and the fungus can adapt to a broad range of osmotic and acidic environments. To our knowledge, this is the first report of the isolation of Q. cyanescens from peritoneal fluid, revealing this rare fungus as a potential human pathogen that may be misidentified using conventional methods. The detailed morphological, molecular and phenotypic characterization of Q. cyanescens UM 1095 provides the basis for future studies on its biology, lifestyle, and potential pathogenicity

Identification and characterization of Daldinia eschscholtzii isolated from skin scrapings, nails, and blood

Background Daldinia eschscholtzii is a filamentous wood-inhabiting endophyte commonly found in woody plants. Here, we report the identification and characterization of nine D. eschscholtzii isolates from skin scrapings, nail clippings, and blood. Methods The nine isolates were identified based on colony morphology, light microscopy, and internal transcribed spacer (ITS)-based phylogeny. In vitro antifungal susceptibility of the fungal isolates was evaluated by the Etest to determine the minimum inhibitory concentration (MIC). Results The nine isolates examined were confirmed as D. eschscholtzii. They exhibited typical features of Daldinia sp. on Sabouraud Dextrose Agar, with white felty colonies and black-gray coloration on the reverse side. Septate hyphae, branching conidiophore with conidiogenous cells budding from its terminus, and nodulisporium-like conidiophores were observed under the microscope. Phylogenetic analysis revealed that the nine isolates were clustered within the D. eschscholtzii species complex. All the isolates exhibited low MICs against azole agents (voriconazole, posaconazole, itraconazole, and ketoconazole), as well as amphotericin B, with MIC of less than 1 µg/ml. Discussion Early and definitive identification of D. eschscholtzii is vital to reducing misuse of antimicrobial agents. Detailed morphological and molecular characterization as well as antifungal profiling of D. eschscholtzii provide the basis for future studies on its biology, pathogenicity, and medicinal potential

Genome Anatomy of <i>Pyrenochaeta unguis-hominis</i> UM 256, a Multidrug Resistant Strain Isolated from Skin Scraping

<div><p><i>Pyrenochaeta unguis-hominis</i> is a rare human pathogen that causes infection in human skin and nail. <i>P</i>. <i>unguis-hominis</i> has received little attention, and thus, the basic biology and pathogenicity of this fungus is not fully understood. In this study, we performed in-depth analysis of the <i>P</i>. <i>unguis-hominis</i> UM 256 genome that was isolated from the skin scraping of a dermatitis patient. The isolate was identified to species level using a comprehensive multilocus phylogenetic analysis of the genus <i>Pyrenochaeta</i>. The assembled UM 256 genome has a size of 35.5 Mb and encodes 12,545 putative genes, and 0.34% of the assembled genome is predicted transposable elements. Its genomic features propose that the fungus is a heterothallic fungus that encodes a wide array of plant cell wall degrading enzymes, peptidases, and secondary metabolite biosynthetic enzymes. Antifungal drug resistance genes including <i>MDR</i>, <i>CDR</i>, and <i>ERG11/CYP51</i> were identified in <i>P</i>. <i>unguis-hominis</i> UM 256, which may confer resistance to this fungus. The genome analysis of <i>P</i>. <i>unguis-hominis</i> provides an insight into molecular and genetic basis of the fungal lifestyles, understanding the unrevealed biology of antifungal resistance in this fungus.</p></div

Microscopic features of dematiaceous fungi.

<p>(A-D) Macroconidia of <i>Curvularia</i>, <i>Bipolaris</i>, <i>Exserohilum</i> and <i>Alternaria</i> (E) Arthroconidia of <i>Neosyctalidium</i> (F) Globose chain conidia and ramoconidia of <i>Cladosporium</i> (G) conidia of <i>Daldinia</i> (H) Dark conidia of <i>Nigrospora</i> (I) <i>Chaetomium</i> perithecium covered with long setae and dark ascospores (J) Spine-like conidiophore and hyaline conidia of <i>Exophiala</i> (K) <i>Ochroconis</i> two-celled clavate conidia with cylindrical conidiophore. <i>Bars</i> 20 µm.</p

<i>In vitro</i> susceptibility of dematiaceous fungal isolates to antifungal agents, grouped according to MIC^a categories.

a<p>minimum inhibitory concentration.</p>b<p>Number of fungal isolates.</p>c<p>Amphotericin B.</p>d<p>Ketoconazole.</p>e<p>Fluconazole.</p>f<p>Itraconazole.</p>g<p>Voriconazole.</p>h<p>Posaconazole.</p>i<p>Anidulafungin.</p>j<p>Caspofungin.</p><p>MIC categories:</p><p>Category A: ≤1 µg/mL (FLC: ≤1 µg/mL).</p><p>Category B: >1–32 µg/mL (FLC: >1–256 µg/mL).</p><p>Category C: >32 µg/mL (FLC: >256 µg/mL).</p

Bayesian phylogenetic tree of <i>Pyrenochaeta</i> sp. based on the combined genes of ITS, SSU and LSU sequenced data.

<p>The phylogenetic tree were constructed with 12 <i>Pyrenochaeta</i> species. The tree is rooted with <i>C</i>. <i>hispidulum</i> and <i>S</i>. <i>terrestris</i> as outgroup. The numbers on the nodes indicate Bayesian posterior probabilities.</p