Development of Allogeneic NK Cell Adoptive Transfer Therapy in Metastatic Melanoma Patients: In Vitro Preclinical Optimization Studies

Natural killer (NK) cells have long been considered as potential agents for adoptive cell therapy for solid cancer patients. Until today most studies utilized autologous NK cells and yielded disappointing results. Here we analyze various modular strategies to employ allogeneic NK cells for adoptive cell transfer, including donor-recipient HLA-C mismatching, selective activation and induction of melanoma-recognizing lysis receptors, and co-administration of antibodies to elicit antibody-dependent cell cytotoxicity (ADCC). We show that NK cell activation and induction of the relevant lysis receptors, as well as co-administration of antibodies yield substantial anti-cancer effects, which are functionally superior to HLA-C mismatching. Combination of the various strategies yielded improved effects. In addition, we developed various clinically-compatible ex vivo expansion protocols that were optimized according to fold expansion, purity and expression of lysis receptors. The main advantages of employing allogeneic NK cells are accessibility, the ability to use a single donor for many patients, combination with various strategies associated with the mechanism of action, e.g. antibodies and specific activation, as well as donor selection according to HLA or CD16 genotypes. This study rationalizes a clinical trial that combines adoptive transfer of highly potent allogeneic NK cells and antibody therapy.     

The clinical significance of class III (suspicious) urine cytology

I. Sternberg, R. Rona, S. Olsfanger, S. Lew and I. Leibovitch The clinical significance of class III (suspicious) urine cytology Background: Urine cytology, combined with cystoscopy, is the mainstay of the diagnosis and surveillance of urothelial carcinoma (UC). While classes I and II urine cytology are considered benign and classes IV and V are considered malignant the clinical significance of class III urine cytology is unclear. We evaluated the positive predictive value of class III urine cytology for concurrent and subsequent UC. Methods: The records of all class III urine cytology cases during a 3‐year period were retrospectively reviewed for the presence of concurrent and subsequent UC, determined by cystoscopy and histological confirmation. Results: Of 111 cases, 54 (48.7%) were associated with concurrent UC and 14 (12.6%) with subsequent UC after an initial evaluation negative for malignancy, with a mean time to diagnosis of 10.8 months. Of 27 cases of class III urine cytology with no prior history of UC, 13 (48.1%) had concomitant UC and none had subsequent UC. Of 84 cases of class III urine cytology with a prior history of UC, 41 (48.8%) had a concomitant diagnosis of UC and 14 (16.7%) developed UC during their follow‐up, leading to a total of 55 (65.5%) cases of UC. Conclusions: Patients with class III urine cytology and a prior history of UC should undergo a full initial evaluation of their urinary tract, and should be followed vigorously if this evaluation is negative for malignancy. Patients without a prior diagnosis of UC and class III urine cytology should also undergo a full initial evaluation, while further larger studies are needed to elucidate the need for further follow‐up in such patients.     

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent K_m of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.     

Spatial Differences in East Scotia Ridge Hydrothermal Vent Food Webs: Influences of Chemistry,Microbiology and Predation on Trophodynamics

The hydrothermal vents on the East Scotia Ridge are the first to be explored in the Antarctic and are dominated by large peltospiroid gastropods, stalked barnacles (Vulcanolepas sp.) and anomuran crabs (Kiwa sp.) but their food webs are unknown. Vent fluid and macroconsumer samples were collected at three vent sites (E2, E9N and E9S) at distances of tens of metres to hundreds of kilometres apart with contrasting vent fluid chemistries to describe trophic interactions and identify potential carbon fixation pathways using stable isotopes. δ¹³C of dissolved inorganic carbon from vent fluids ranged from −4.6‰ to 0.8‰ at E2 and from −4.4‰ to 1.5‰ at E9. The lowest macroconsumer δ¹³C was observed in peltospiroid gastropods (−30.0‰ to −31.1‰) and indicated carbon fixation via the Calvin-Benson-Bassham (CBB) cycle by endosymbiotic gamma-Proteobacteria. Highest δ¹³C occurred in Kiwa sp. (−19.0‰ to −10.5‰), similar to that of the epibionts sampled from their ventral setae. Kiwa sp. δ¹³C differed among sites, which were attributed to spatial differences in the epibiont community and the relative contribution of carbon fixed via the reductive tricarboxylic acid (rTCA) and CBB cycles assimilated by Kiwa sp. Site differences in carbon fixation pathways were traced into higher trophic levels e.g. a stichasterid asteroid that predates on Kiwa sp. Sponges and anemones at the periphery of E2 assimilated a proportion of epipelagic photosynthetic primary production but this was not observed at E9N. Differences in the δ¹³C and δ³⁴S values of vent macroconsumers between E2 and E9 sites suggest the relative contributions of photosynthetic and chemoautotrophic carbon fixation (rTCA v CBB) entering the hydrothermal vent food webs vary between the sites.     

Selectively bred oysters can alter their biomineralization pathways,promoting resilience to environmental acidification

Commercial shellfish aquaculture is vulnerable to the impacts of ocean acidification driven by increasing carbon dioxide (CO₂) absorption by the ocean as well as to coastal acidification driven by land run off and rising sea level. These drivers of environmental acidification have deleterious effects on biomineralization. We investigated shell biomineralization of selectively bred and wild‐type families of the Sydney rock oyster Saccostrea glomerata in a study of oysters being farmed in estuaries at aquaculture leases differing in environmental acidification. The contrasting estuarine pH regimes enabled us to determine the mechanisms of shell growth and the vulnerability of this species to contemporary environmental acidification. Determination of the source of carbon, the mechanism of carbon uptake and use of carbon in biomineral formation are key to understanding the vulnerability of shellfish aquaculture to contemporary and future environmental acidification. We, therefore, characterized the crystallography and carbon uptake in the shells of S. glomerata, resident in habitats subjected to coastal acidification, using high‐resolution electron backscatter diffraction and carbon isotope analyses (as δ¹³C). We show that oyster families selectively bred for fast growth and families selected for disease resistance can alter their mechanisms of calcite crystal biomineralization, promoting resilience to acidification. The responses of S. glomerata to acidification in their estuarine habitat provide key insights into mechanisms of mollusc shell growth under future climate change conditions. Importantly, we show that selective breeding in oysters is likely to be an important global mitigation strategy for sustainable shellfish aquaculture to withstand future climate‐driven change to habitat acidification.     

Analysis of the Role of Interleukin 6 Receptor Haplotypes in the Regulation of Circulating Levels of Inflammatory Biomarkers and Risk of Coronary Heart Disease

Variants at the interleukin 6 receptor (IL6R) gene regulate inflammation and are associated with risk of coronary heart disease (CHD). The aim of the present study was to investigate the effects of IL6R haplotypes on circulating levels of inflammatory biomarkers and risk of CHD. We performed a discovery analysis in SHEEP, a myocardial infarction (MI) case control study (n = 2,774) and replicated our results in two large, independent European populations, PROCARDIS, a CHD case control study (n = 7,998), and IMPROVE (n = 3,711) a prospective cardiovascular cohort study. Two major haplotype blocks (rs12083537A/G and rs4075015A/T—block 1; and rs8192282G/A, rs4553185T/C, rs8192284A/C, rs4240872T/C and rs7514452T/C—block 2) were identified in the IL6R gene. IL6R haplotype associations with C-reactive protein (CRP), fibrinogen, IL6, soluble IL6R (sIL6R), IL6, IL8 and TNF-α in SHEEP, CRP and fibrinogen in PROCARDIS and CRP in IMPROVE as well as association with risk of MI and CHD, were analyzed by THESIAS. Haplotypes in block 1 were associated neither with circulating inflammatory biomarkers nor with the MI/CHD risk. Haplotypes in block 2 were associated with circulating levels of CRP, in all three study populations, with fibrinogen in SHEEP and PROCARDIS, with IL8 and sIL6Rin SHEEP and with a modest, non significant, increase (7%) in MI/CHD risk in the three populations studied. Our results indicate that IL6R haplotypes regulate the circulating levels of inflammatory biomarkers. Lack of association with the risk of CHD may be explained by the combined effect of SNPs with opposite effect on the CHD risk, the sample size as well as by structural changes affecting sIL6R stability in the circulation.