Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC).

Background: Neoadjuvant anti-PD-1 may improve outcomes for patients with resectable NSCLC and provides a critical window for examining pathologic features associated with response. Resections showing major pathologic response to neoadjuvant therapy, defined as ≤10% residual viable tumor (RVT), may predict improved long-term patient outcome. However, %RVT calculations were developed in the context of chemotherapy (%cRVT). An immune-related %RVT (%irRVT) has yet to be developed. Patients and methods: The first trial of neoadjuvant anti-PD-1 (nivolumab, NCT02259621) was just reported. We analyzed hematoxylin and eosin-stained slides from the post-treatment resection specimens of the 20 patients with non-small-cell lung carcinoma who underwent definitive surgery. Pretreatment tumor biopsies and preresection radiographic 'tumor' measurements were also assessed. Results: We found that the regression bed (the area of immune-mediated tumor clearance) accounts for the previously noted discrepancy between CT imaging and pathologic assessment of residual tumor. The regression bed is characterized by (i) immune activation-dense tumor infiltrating lymphocytes with macrophages and tertiary lymphoid structures; (ii) massive tumor cell death-cholesterol clefts; and (iii) tissue repair-neovascularization and proliferative fibrosis (each feature enriched in major pathologic responders versus nonresponders, P < 0.05). This distinct constellation of histologic findings was not identified in any pretreatment specimens. Histopathologic features of the regression bed were used to develop 'Immune-Related Pathologic Response Criteria' (irPRC), and these criteria were shown to be reproducible amongst pathologists. Specifically, %irRVT had improved interobserver consistency compared with %cRVT [median per-case %RVT variability 5% (0%-29%) versus 10% (0%-58%), P = 0.007] and a twofold decrease in median standard deviation across pathologists within a sample (4.6 versus 2.2, P = 0.002). Conclusions: irPRC may be used to standardize pathologic assessment of immunotherapeutic efficacy. Long-term follow-up is needed to determine irPRC reliability as a surrogate for recurrence-free and overall survival.

Analysis of natural product regulation of cannabinoid receptors in the treatment of human disease.

The organized, tightly regulated signaling relays engaged by the cannabinoid receptors (CBs) and their ligands, G proteins and other effectors, together constitute the endocannabinoid system (ECS). This system governs many biological functions including cell proliferation, regulation of ion transport and neuronal messaging. This review will firstly examine the physiology of the ECS, briefly discussing some anomalies in the relay of the ECS signaling as these are consequently linked to maladies of global concern including neurological disorders, cardiovascular disease and cancer. While endogenous ligands are crucial for dispatching messages through the ECS, there are also commonalities in binding affinities with copious exogenous ligands, both natural and synthetic. Therefore, this review provides a comparative analysis of both types of exogenous ligands with emphasis on natural products given their putative safer efficacy and the role of Δ9-tetrahydrocannabinol (Δ9-THC) in uncovering the ECS. Efficacy is congruent to both types of compounds but noteworthy is the effect of a combination therapy to achieve efficacy without unideal side-effects. An example is Sativex that displayed promise in treating Huntington's disease (HD) in preclinical models allowing for its transition to current clinical investigation. Despite the in vitro and preclinical efficacy of Δ9-THC to treat neurodegenerative ailments, its psychotropic effects limit its clinical applicability to treating feeding disorders. We therefore propose further investigation of other compounds and their combinations such as the triterpene, α,ß-amyrin that exhibited greater binding affinity to CB1 than CB2 and was more potent than Δ9-THC and the N-alkylamides that exhibited CB2 selective affinity; the latter can be explored towards peripherally exclusive ECS modulation. The synthetic CB1 antagonist, Rimonabant was pulled from commercial markets for the treatment of diabetes, however its analogue SR144528 maybe an ideal lead molecule towards this end and HU-210 and Org27569 are also promising synthetic small molecules.

Mitogenomic and microsatellite variation in descendants of the founder population of Newfoundland: high genetic diversity in an historically isolated population.

The island of Newfoundland, the first of England's overseas colonies, was settled from the 17th century onward by restricted numbers of English, Irish, and French immigrants, in small "outport" communities that have maintained geographic, religious, and linguistic isolation to the latest generations. To measure the extent of modification and loss of genetic variation through founder effect, drift, and inbreeding in this historically isolated population, we analyzed the complete mitochondrial DNA (mtDNA) genomes and 14 microsatellite loci from each of 27 individuals with matrilineal ancestries extending to the colonial period. Every individual has a unique mtDNA genome sequence. All but one of these genomes are assignable to one of five major (H,J,K,T, and U) or minor (I) European haplogroups. The possibility of homoplasy at single nucleotide polymorphism (SNP) sites that define subtypes within the H haplogroup is discussed. Observed haplogroup proportions do not differ significantly from those of western Europeans or between English and Irish Newfoundlanders. The exceptional individual is a member of haplogroup A2, who appears to be the descendant of a Mi'kmaq First Nations mother and a French father, a common marriage pattern in the early settlement of Newfoundland. Microsatellite diversity is high (HE = 0.763), unstructured with respect to mtDNA haplotype or ethnicity, and there is no evidence of linkage disequilibrium. There is a small but significant degree of inbreeding (FIS = 0.0174). Collection of whole mtDNA genome data was facilitated by the use of microarray sequencing, and we describe a simple algorithm that is 99.67% efficient for sequence recovery.

Pyruvate kinase deficiency as a cause of extreme hyperbilirubinemia in neonates from a polygamist community.

Neonatal hemolytic jaundice is a risk factor for kernicterus. Pyruvate kinase (PK) deficiency is a rare cause of neonatal hemolytic jaundice, with a prevalence estimated at 1 case per 20,000 births in the United States, but with a higher prevalence among the Amish communities in Pennsylvania and Ohio. We discovered four neonates with PK deficiency born in a small community of polygamists. All four had early, severe, hemolytic jaundice. PK deficiency should be considered in neonates with early hemolytic, Coombs-negative, non-spherocytic jaundice, particularly in communities with considerable consanguinity. Such cases should be recognized early and managed aggressively to prevent kernicterus.

B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis.

BACKGROUND: The life cycle of most eukaryotic organisms includes a meiotic phase, in which diploid parental cells produce haploid gametes. During meiosis a single round of DNA replication is followed by two rounds of chromosome segregation. In the first, or reductional, division (meiosis I), which is unique to meiotic cells, homologous chromosomes segregate from one another, whereas in the second, or equational, division (Meiosis II) sister centromeres disjoin. Meiotic DNA replication precedes the initiation of recombination by programmed Spo11-dependent DNA double-strand breaks. Recent reports that meiosis-specific cohesion is established during meiotic S phase and that the length of S phase is modified by recombination factors (Spo11 and Rec8) raise the possibility that replication plays a fundamental role in the recombination process. RESULTS: To address how replication influences the initiation of recombination, we have used mutations in the B-type cyclin genes CLB5 and CLB6, which specifically prevent premeiotic replication in the yeast Saccharomyces cerevisiae. We find that clb5 and clb5 clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in chromatin accessibility, heteroallelic recombination, and SC formation. The severity of these phenotypes in each mutant reflects the extent of replication impairment. CONCLUSIONS: This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in DNA replication but also in other key events of meiotic prophase. Links between the function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on subsequent events are discussed.

Disruption and functional analysis of six ORFs on chromosome XV: YOL117w, YOL115w ( TRF4), YOL114c, YOL112w ( MSB4), YOL111c and YOL072w.

We have carried out the systematic disruption of six ORFs on chromosome XV, of Saccharomyces cerevisiae using the long flanking homology technique to replace each with the KanMX cassette; we have also constructed plasmids containing replacement cassettes and cognate clones for each ORF. Disruption of three of the ORFs-YOL117w, YOL114c, and YOL112w (also known as MSB4)-does not result in any noteworthy phenotype with respect to temperature or nutritional requirements, but yol112w mutants with an additional disruption of YNL293w, which encodes a protein similar to Yol112w, exhibit a slow growth phenotype. The protein specified by YOL114c shares similarity with the human DS-1 protein. Disruption of YOL115w confers slow growth, cold sensitivity and poor sporulation; this ORF has been described elsewhere as TRF4, which encodes a topoisomerase I-related protein. Cells with disruptions of YOL111c, whose product is weakly similar to the human ubiquitin-like protein GdX, are slightly impaired in mating. Mutants disrupted for YOL072w, the predicted product of which is unrelated to any protein of known function, grow slowly, are cold-sensitive and sporulate with reduced efficiency.

Disruption and functional analysis of seven ORFs on chromosome IV: YDL057w, YDL012c, YDL010w, YDL009c, YDL008w (APC11), YDL005c (MED2) and YDL003w (MCD1).

In the context of the EUROFAN project, we have carried out the systematic disruption of seven ORFs on chromosome IV of Saccharomyces cerevisiae using the long flanking homology technique to replace each ORF with the KanMX cassette. Targeted disruption of YDL057w, YDL012c, or YDL010w with YDL009c (the two ORFs overlap) confers no overt defects in haploid growth on a variety of media at different temperatures, in mating, or in the sporulation of diploids homozygous for the disruption. By contrast, YDL008w and YDL003w disruptants are non-viable. The product of YDL008w (elsewhere identified as APC11) is a component of the anaphase promoting complex. YDL003w (also termed MCD1) is a homologue of Schizosaccharomyces pombe rad21, an essential gene implicated in DNA double-strand break repair and nuclear organization in fission yeast. In budding yeast, this ORF has been shown by several laboratories to encode a protein involved in sister chromatid cohesion and chromosome condensation. The remaining ORF, YDL005c (also termed MED2), encodes a component of the transcriptional activator complex known as Mediator. Disruption of YDL005c confers a modest slow growth phenotype on rich medium and a more severe phenotype on minimal medium, aberrant cellular morphology, and mating defects; diploids homozygous for the disruption cannot sporulate.

Recombination at work for meiosis.

In sexually reproducing organisms, homologous recombination increases genetic diversity in gametes and ensures proper chromosome segregation. Recent publications have provided details of the molecular intermediates and proteins involved, the control of the distribution of recombination events at the chromosomal level, and the surveillance mechanisms that coordinate recombination with the meiotic cell cycle.

Nucleotide sequences of nuclear tRNA(Cys) genes from Nicotiana and Arabidopsis and expression in HeLa cell extract.

We have recently characterized Nicotiana cytoplasmic (cyt) tRNA(GCA)Cys as a novel UGA suppressor tRNA. Here we have isolated its corresponding (NtC1) and a variant (NtC2) gene from a genomic library of Nicotiana rustica. Both tRNA(Cys) genes are efficiently transcribed in HeLa cell nuclear extract and yield mature cyt tRNAs(Cys). Sequence analysis of the upstream region of the RAD51 single-copy gene of the Arabidopsis thaliana genome revealed a cluster of three tRNA(Cys) genes which have the same polarity and comprise highly similar flanking sequences. Of the three Arabidopsis tRNA(Cys) genes only one (i.e. AtC2) appears to code for a functional gene which exhibits an almost identical nucleotide sequence to NtC1. These are the first sequenced nuclear tDNAs(Cys) of plant origin.

Induction by cells of immune responses to intestinal epithelial cell-associated components (ECAC): transfer with cultured murine mesenteric and popliteal/axillary lymph node cells.

Although systemic and mucosal immune responses to intestinal epithelial self-antigens occur in several human disorders, there is no model system with which to study the physiology and regulation of the underlying cellular events. Therefore, we undertook to induce an immune response to purified epithelial macromolecules in the Lewis rat; characterize in vitro the reactive cells; and then transfer with immunocytes this antiepithelial reactivity to naive syngeneic rats, identifying the fine specificity and site of humoral and cell-mediated immunity induced in the cell recipient. Donor animals sensitized systemically (via footpad) or locally in gut mucosa (via Peyer's patches) to syngeneic or xenogeneic epithelial antigens generated specific immunoglobulin and were found to have T lymphocytes in the draining nodal areas (including the mesenteric nodes) which were (a) antigen-specific, having a [3H]thymidine uptake in the presence of antigen 30-fold the control; (b) generally of the Thelper/inducer subclass (W3/25+) which, upon further culture, developed phenotype surface markers for activation (IL-2R+); (c) able to induce an antigen-specific humoral and cell-mediated responses upon intravenous injection into naive syngeneic hosts; and (d) demonstrable in gut-associated lymphoid tissue (mesenteric lymph nodes) and, to a lesser extent in spleen, of the cell recipient. Further, lymphocytes cloned from reactive mesenteric lymph node cells demonstrated specificity for a gel-purified subfraction of epithelial antigen, designated P1, containing highly conserved organ-specific macromolecules thought to be autoantigenic for gut.

Evaluation of 60 chemicals in a preliminary developmental toxicity test.

The number of chemicals in commerce which have not been evaluated for potential developmental toxicity is large. Because of the time and expense required by conventional developmental toxicity tests, an abbreviated assay is needed that will preliminarily evaluate otherwise untested chemicals to help prioritize them for conventional testing. A proposed short-term in vivo assay has been used in a series of studies in which a total of 60 chemicals were tested. Some were independently tested two or four times each. In this preliminary test, pregnant mice were dosed during mid-pregnancy and were then allowed to deliver litters. Litter size, birth weight, and neonatal growth and survival to postnatal day 3 were recorded as indices of potential developmental toxicity. Results in this assay and conventional mouse teratology tests were generally concordant. Conventional data were available for 14 chemicals (ten teratogens, one fetotoxin, three nonteratogens), of which 11 (nine teratogens, one fetotoxin, one nonteratogen) produced evidence of developmental toxicity. This included conventional data for three chemicals (ethylene glycol, diethylene glycol dimethyl ether, and triethylene glycol dimethyl ether) that were untested before the present study. As high priority candidates for conventional testing on the basis of results here, all were subsequently studied in a standard teratology assay and were confirmed to be teratogenic in mice. Additionally, one of them (ethylene glycol) plus a fourth high priority candidate for conventional study (diethylene glycol monomethyl ether) were subsequently tested in rats and were found to be teratogenic in that species.

Biofeedback in strokes.

The effectiveness of biofeedback therapy, using the electromyograph, was compared with conventional physiotherapy treatments in eleven hemiparetic patients with severe disability of the upper limb. These were divided randomly into two groups, a group of six patients receiving biofeedback treatments and five receiving physiotherapy treatments with conventional techniques. The results showed greater improvements in the biofeedback group in most fields of testing, and the patients in this group had a greater degree of control over the patterns of movement in the upper limb and the relaxation of spastic muscle groups. It was concluded that a wide range of hemiparetic patients should benefit in some degree by treatment of this form.