Immunoregulatory functions of VISTA.

Utilization of negative checkpoint regulators (NCRs) for cancer immunotherapy has garnered significant interest with the completion of clinical trials demonstrating efficacy. While the results of monotherapy treatments are compelling, there is increasing emphasis on combination treatments in an effort to increase response rates to treatment. One of the most recently discovered NCRs is VISTA (V-domain Ig-containing Suppressor of T cell Activation). In this review, we describe the functions of this molecule in the context of cancer immunotherapy. We also discuss factors that may influence the use of anti-VISTA antibody in combination therapy and how genomic analysis may assist in providing indications for treatment.

Fat4-Dchs1 signalling controls cell proliferation in developing vertebrae.

The protocadherins Fat4 and Dchs1 act as a receptor-ligand pair to regulate many developmental processes in mice and humans, including development of the vertebrae. Based on conservation of function between Drosophila and mammals, Fat4-Dchs1 signalling has been proposed to regulate planar cell polarity (PCP) and activity of the Hippo effectors Yap and Taz, which regulate cell proliferation, survival and differentiation. There is strong evidence for Fat regulation of PCP in mammals but the link with the Hippo pathway is unclear. In Fat4(-/-) and Dchs1(-/-) mice, many vertebrae are split along the midline and fused across the anterior-posterior axis, suggesting that these defects might arise due to altered cell polarity and/or changes in cell proliferation/differentiation. We show that the somite and sclerotome are specified appropriately, the transcriptional network that drives early chondrogenesis is intact, and that cell polarity within the sclerotome is unperturbed. We find that the key defect in Fat4 and Dchs1 mutant mice is decreased proliferation in the early sclerotome. This results in fewer chondrogenic cells within the developing vertebral body, which fail to condense appropriately along the midline. Analysis of Fat4;Yap and Fat4;Taz double mutants, and expression of their transcriptional target Ctgf, indicates that Fat4-Dchs1 regulates vertebral development independently of Yap and Taz. Thus, we have identified a new pathway crucial for the development of the vertebrae and our data indicate that novel mechanisms of Fat4-Dchs1 signalling have evolved to control cell proliferation within the developing vertebrae.

DYNC1H1 mutation alters transport kinetics and ERK1/2-cFos signalling in a mouse model of distal spinal muscular atrophy.

Mutations in the gene encoding the heavy chain subunit (DYNC1H1) of cytoplasmic dynein cause spinal muscular atrophy with lower extremity predominance, Charcot-Marie-Tooth disease and intellectual disability. We used the legs at odd angles (Loa) (DYNC1H1(F580Y)) mouse model for spinal muscular atrophy with lower extremity predominance and a combination of live-cell imaging and biochemical assays to show that the velocity of dynein-dependent microtubule minus-end (towards the nucleus) movement of EGF and BDNF induced signalling endosomes is significantly reduced in Loa embryonic fibroblasts and motor neurons. At the same time, the number of the plus-end (towards the cell periphery) moving endosomes is increased in the mutant cells. As a result, the extracellular signal-regulated kinases (ERK) 1/2 activation and c-Fos expression are altered in both mutant cell types, but the motor neurons exhibit a strikingly abnormal ERK1/2 and c-Fos response to serum-starvation induced stress. These data highlight the cell-type specific ERK1/2 response as a possible contributory factor in the neuropathological nature of Dync1h1 mutations, despite generic aberrant kinetics in both cell types, providing an explanation for how mutations in the ubiquitously expressed DYNC1H1 cause neuron-specific disease.

Replication-timing boundaries facilitate cell-type and species-specific regulation of a rearranged human chromosome in mouse.

In multicellular organisms, developmental changes to replication timing occur in 400-800 kb domains across half the genome. While examples of epigenetic control of replication timing have been described, a role for DNA sequence in mammalian replication-timing regulation has not been substantiated. To assess the role of DNA sequences in directing developmental changes to replication timing, we profiled replication timing in mice carrying a genetically rearranged Human Chromosome 21 (Hsa21). In two distinct mouse cell types, Hsa21 sequences maintained human-specific replication timing, except at points of Hsa21 rearrangement. Changes in replication timing at rearrangements extended up to 900 kb and consistently reconciled with the wild-type replication pattern at developmental boundaries of replication-timing domains. Our results are consistent with DNA sequence-driven regulation of Hsa21 replication timing during development and provide evidence that mammalian chromosomes consist of multiple independent units of replication-timing regulation.

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

Specific Composition of Lipid Phases Allows Retaining an Optimal Thylakoid Membrane Fluidity in Plant Response to Low-Temperature Treatment.

Thylakoid membranes isolated from leaves of two plant species, the chilling tolerant (CT) pea and chilling sensitive (CS) runner bean, were assessed for the composition of lipids, carotenoids as well as for the arrangement of photosynthetic complexes. The response to stress conditions was investigated in dark-chilled and subsequently photo-activated detached leaves of pea and bean. Thylakoids of both species have a similar level of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), but different sulfoquinovosyldiacylglycerol to phosphatidylglycerol (PG) ratio. In pea thylakoid fraction, the MGDG, DGDG and PG, have a higher double bond index (DBI), whereas bean thylakoids contain higher levels of high melting point PG. Furthermore, the lutein to the ß-carotene ratio is higher in bean thylakoids. Smaller protein/lipid ratio in pea than in bean thylakoids suggests different lipid-protein interactions in both species. The differences between species are also reflected by the course of temperature-dependent plots of chlorophyll fluorescence pointing various temperatures of the lipid phase transitions of pea and bean thylakoids. Our results showed higher fluidity of the thylakoid membrane network in pea than in bean in optimal temperature conditions. Dark-chilling decreases the photochemical activity and induces significant degradation of MGDG in bean but not in pea leaves. Similarly, substantial changes in the arrangement of photosynthetic complexes with increase in LHCII phosphorylation and disturbances of the thylakoid structure take place in bean thylakoids only. Changes in the physical properties of bean thylakoids are manifested by the conversion of a three-phase temperature-dependent plot to a one-phase plot. Subsequent photo-activation of chilled bean leaves caused a partial restoration of the photochemistry and of membrane physical properties, but not of the photosynthetic complexes arrangement nor the thylakoid network structure. Summarizing, the composition of the thylakoid lipid matrix of CT pea allows retaining the optimal fluidity of its chloroplast membranes under low temperatures. In contrast, the fluidity of CS bean thylakoids is drastically changed, leading to the reorganization of the supramolecular structure of the photosynthetic complexes and finally results in structural remodeling of the CS bean thylakoid network.

Defining the Signature of VISTA on Myeloid Cell Chemokine Responsiveness.

The role of negative checkpoint regulators (NCRs) in human health and disease cannot be overstated. V-domain Ig-containing Suppressor of T-cell Activation (VISTA) is an Ig superfamily protein predominantly expressed within the hematopoietic compartment and has been studied for its role in the negative regulation of T cell responses. The findings presented in this study show that, unlike all other NCRs, VISTA deficiency dramatically impacts on macrophage cytokine and chemokine production, as well as the chemotactic response of VISTA-deficient macrophages. A select group of inflammatory chemokines, including CCL2, CCL3, CCL4, and CCL5, was strikingly elevated in culture supernatants from VISTA KO macrophages. VISTA deficiency also altered chemokine receptor recycling and profoundly disrupted myeloid chemotaxis. The impact of VISTA deficiency on chemotaxis in vivo was apparent with the reduced ability of both KO macrophages and MDSCs to migrate to the tumor microenvironment. This is the first demonstration of an NCR impacting on myeloid mediator production and chemotaxis, and will guide the use of anti-VISTA therapeutics to manipulate the chemotaxis of inflammatory macrophages or immunosuppressive MDSCs in inflammatory diseases and cancer.

Nuclear morphometry as a tool of limited capacity for distinguishing renal oncocytoma from chromophobe carcinoma.

The principal types of renal tumors include malignant clear cell renal cell carcinoma, chromophobe carcinoma (ChRCC), papillary carcinoma and benign oncocytoma (RO) and adenoma. Both oncocytoma and chromophobe carcinoma are characterized by a solid growth pattern of cell with abundant cytoplasm and in some cases may be difficult to distinguish based on histology only. The material for the study consisted of 58 chromophobe carcinomas and 16 oncocytomas. At least 100 nuclei per case were segmented from images of DAPI-stained slides. The geometric and texture features were extracted and used for analysis. Significant differences between RO and ChRCC were found in all the analyzed parameters, however overlapping of the features exists. None of the constructed models permitted to classify cases in concordance with diagnoses.

Evaluation of HER2/neu gene amplification in patients with invasive breast carcinoma. Comparison of in situ hybridization methods.

One of the prognostic and predictive factors in invasive breast carcinomas is determination of the HER2/neu proto-oncogene amplification or HER2 protein overexpression. HER2 amplification/overexpression is associated with a more aggressive disease course, greater likelihood of recurrence and generally poor prognosis. The authors compared the specificity, simplicity of a given procedure and method standardization, the simplicity of evaluation the results of each in situ hybridization method and time needed for performing the test. Sixty-three cases of infiltrating breast carcinoma from surgically excised tumors and core needle biopsies were included in the study. The first step was the determination of HER2 status by immunohistochemistry. The patients with moderate (2+) and strong (3+) overexpression of HER2 protein were chosen for determining HER2 amplification by three methods of in situ hybridization: FISH, CISH and in situ hybridization with silver autometallography. The statistical analysis revealed a good agreement between IHC and ISH methods and among ISH methods. The results indicate that all in situ hybridization methods are equivalent tools for evaluating HER2 gene amplification in archival material. There is no clear answer which method is the best assay to determine HER2 marker status, although the authors present some advantages and disadvantages of all the described techniques and a proposed algorithm for choosing a method for a given laboratory.

CDH1 gene promoter hypermethylation in gastric cancer: relationship to Goseki grading, microsatellite instability status, and EBV invasion.

Hypermethylation of the CDH1 promoter region seems to be the most common epigenetic mechanism in this gene silencing in gastric cancer. In this study, CDH1 promoter hypermethylation was observed in 54.8% (46/84) of the analyzed sporadic gastric carcinomas. We introduce a new relation: clustering of Goseki grading into 3 grade was determined by CDH1 promoter hypermethylation. The percentage of methylation in Goseki III cancers was significantly higher (83%) when compared with other grades; the lowest proportion was detected in IV (36%) and II (38%) groups, whereas grade I demonstrated typical percentage of promoter hypermethylation. A novel polymorphism R732R in exon 14 of the CDH1 gene was detected by mutational analysis. Additionally, all cases with the MSI-high phenotype revealed CDH1 promoter hypermethylation. In MSI-low and MSS gastric cancers, this percentage was lower, reaching 71% and 41%, respectively. Moreover, the methylation status was correlated with the LOH phenotype. We detected CDH1 promoter hypermethylation in all EBV-positive gastric cancers (5/5), whereas methylation in the EBV-negative group occurred in 58% of cases. We also report that "methylated" tumors were slightly larger than "nonmethylated," whereas the second group revealed a higher probability of longer patient survival, though these relationships were not statistically significant. These results suggest that downregulation of E-cadherin, caused by promoter hypermethylation, in sporadic gastric carcinomas may be associated with a worse prognosis and specific tumor phenotype.

Tissue microarray FISH applied to colorectal carcinomas with various microsatellite status.

Colorectal carcinoma is etiopathologically heterogenic. It may develop through a sequence of mutations leading to chromosome instability or be a result of defects in DNA repair mechanisms manifested by microsatellite instability of varying degrees. Colorectal carcinoma can thus be classified into microsatellite-stable (MSS), highly microsatellite unstable (MSI-H) and intermediate low-level microsatellite unstable (MSI-L) groups. Fluorescent hybridization in situ (FISH) is a method of detecting specific sequences of nucleic acids that is based on specific bonding of a fluorescent marker-associated probe and specific DNA fragment. The material consisted of 146 non-selected cases of colorectal carcinoma patients operated on at First Chair of General Surgery, Collegium Medicum, Jagiellonian University in Cracow, Poland. Following a standard histopathological evaluation, tissue microarrays were prepared using a Tissue MicroArray Builder, and FISH was performed employing probes specific for chromosomes 1, 8, 17 and 18. Microsatellite instability was evaluated in frozen material using the PCR reaction with gel and capillary electrophoresis. The mean number of signals obtained for chromosome 1 in the entire material was 2.06, while the corresponding mean values in the MSS group equaled 2.07, in the MSI-L group - 2.07, and in the MSI-H group - 2.01. The mean number of signals for chromosome 17 in the entire material was 2.1, in the MSS group - 2.11, in the MSI-L group - 2.13, and in the MSI-H group - 2.01. The number of signals for chromosome 18 in the entire material was 2, in the MSS group - 2, in the MSI-L group - 2, and in the MSI-H group - 2. The means number of signals for chromosome 8 in the entire material was 2.07, in the MSS group - 2.08, in the MSI-L group - 2.01, and in the MSI-H group - 2. These differences are not sufficient for distinguishing colorectal carcinoma molecular forms.

A New VISTA on combination therapy for negative checkpoint regulator blockade.

Negative checkpoint regulators function to restrain T cell responses to maintain tolerance and limit immunopathology. However, in the setting of malignancy, these pathways work in concert to promote immune-mediate escape leading to the development of a clinically overt cancer. In the recent years, clinical trials demonstrating the efficacy of blocking antibodies against these molecules have invigorated the field of immunotherapy. In this review, we discuss the current understanding on established NCR blockade and how strategic combination therapy with anti-VISTA antibody can be used to target multiple non-redundant NCR pathways.

Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis.

Skeletal shape varies widely across species as adaptation to specialized modes of feeding and locomotion, but how skeletal shape is established is unknown. An example of extreme diversity in the shape of a skeletal structure can be seen in the sternum, which varies considerably across species. Here we show that the Dchs1-Fat4 planar cell polarity pathway controls cell orientation in the early skeletal condensation to define the shape and relative dimensions of the mouse sternum. These changes fit a model of cell intercalation along differential Dchs1-Fat4 activity that drives a simultaneous narrowing, thickening and elongation of the sternum. Our results identify the regulation of cellular polarity within the early pre-chondrogenic mesenchyme, when skeletal shape is established, and provide the first demonstration that Fat4 and Dchs1 establish polarized cell behaviour intrinsically within the mesenchyme. Our data also reveal the first indication that cell intercalation processes occur during ventral body wall elongation and closure.

Comparison of efficacy and safety of first-line palliative chemotherapy with EOX and mDCF regimens in patients with locally advanced inoperable or metastatic HER2-negative gastric or gastroesophageal junction adenocarcinoma: a randomized phase 3 trial.

The aim of the study was to compare efficacy and safety of first-line palliative chemotherapy with (EOX) epirubicin/oxaliplatin/capecitabine and (mDCF) docetaxel/cisplatin/5FU/leucovorin regimens for untreated advanced HER2-negative gastric or gastroesophageal junction adenocarcinoma. Fifty-six patients were randomly assigned to mDCF (docetaxel 40 mg/m(2) day 1, leucovorin 400 mg/m(2) day 1, 5FU 400 mg/m(2) bolus day 1, 5FU 1000 mg/m(2)/d days 1 and 2, cisplatin 40 mg/m(2) day 3) or EOX (epirubicin 50 mg/m(2) day 1, oxaliplatin 130 mg/m(2) day 1, capecitabine 1250 mg/m(2)/d days 1-21). The primary endpoint was overall survival. The median overall survival was 9.5 months with EOX and 11.9 months with mDCF (p = 0.135), while median progression-free survival was 6.4 and 6.8 months, respectively (p = 0.440). Two-year survival rate was 22.2 % with mDCF compared to 5.2 % with EOX. Patients in the EOX arm had more frequent reductions in chemotherapy doses (34.5 vs. 3.7 %; p = 0.010) and delays in subsequent chemotherapy cycles (82.8 vs. 63.0 %; p = 0.171). There was no statistically significant difference in the rates of grade 3-4 adverse events (EOX 79.3 vs. mDCF 61.5 %; p = 0.234). As compared with the mDCF, the EOX regimen was associated with more frequent nausea (34.5 vs. 15.4 %), thromboembolic events (13.8 vs. 7.7 %), abdominal pain (13.8 vs. 7.7 %) and grades 3-4 neutropenia (72.4 vs. 50.0 %), but lower incidences of anemia (44.8 vs. 61.5 %), mucositis (6.9 vs. 15.4 %) and peripheral neuropathy (6.9 vs. 15.4 %). In conclusion, the mDCF regimen was associated with a statistically nonsignificant 2.4-month longer median overall survival without an increase in toxicity. This trial is registered at ClinicalTrials.gov, number NCT02445209.

Regulation of neuronal migration by Dchs1-Fat4 planar cell polarity.

Planar cell polarity (PCP) describes the polarization of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for postnatal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood [1]. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways [1-3]. Fz-PCP is conserved in vertebrates, but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 and DCHS1, key components of Fat-PCP signaling, cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration [4]. Here, we investigate the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor (FBM) neurons as a model. We find that Fat4 and Dchs1 are expressed in complementary gradients and are required for the collective tangential migration of FBM neurons and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM neurons and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM neuron migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM neuron polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. Our results also identify Fat-PCP as a novel neuronal guidance system and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes.

Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate.

Non-syndromic cleft lip/palate (NSCL/P) is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88×10(-2)-5.02×10(-9)). This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show for the first time that cellular defences against DNA damage may take part in determining the susceptibility to NSCL/P. These results are in accordance with the hypothesis of aetiological overlap between this malformation and cancer, and suggest a new pathogenic mechanism for the disease.

Mouse cytoplasmic dynein intermediate chains: identification of new isoforms, alternative splicing and tissue distribution of transcripts.

BACKGROUND: Intracellular transport of cargoes including organelles, vesicles, signalling molecules, protein complexes, and RNAs, is essential for normal function of eukaryotic cells. The cytoplasmic dynein complex is an important motor that moves cargos along microtubule tracks within the cell. In mammals this multiprotein complex includes dynein intermediate chains 1 and 2 which are encoded by two genes, Dync1i1 and Dync1i2. These proteins are involved in dynein cargo binding and dynein complexes with different intermediate chains bind to specific cargoes, although the mechanisms to achieve this are not known. The DYNC1I1 and DYNC1I2 proteins are translated from different splice isoforms, and specific forms of each protein are essential for the function of different dynein complexes in neurons. METHODOLOGY/PRINCIPAL FINDINGS: Here we have undertaken a systematic survey of the dynein intermediate chain splice isoforms in mouse, basing our study on mRNA expression patterns in a range of tissues, and on bioinformatics analysis of mouse, rat and human genomic and cDNA sequences. We found a complex pattern of alternative splicing of both dynein intermediate chain genes, with maximum complexity in the embryonic and adult nervous system. We have found novel transcripts, including some with orthologues in human and rat, and a new promoter and alternative non-coding exon 1 for Dync1i2. CONCLUSIONS/SIGNIFICANCE: These data, including the cloned isoforms will be essential for understanding the role of intermediate chains in the cytoplasmic dynein complex, particularly their role in cargo binding within individual tissues including different brain regions.

Renal papillary carcinoma classification into subtypes may be reproduced by nuclear morphometry.

OBJECTIVE: To analyze relationships between nuclear features of papillary renal cell carcinoma (PapRCC) subtypes. STUDY DESIGN: The material for the study consisted of 53 cases, of which 29 were type 1, 17 type 2 and 7 intermediate. At least 100 nuclei per case were segmented from images of 4',6-diamidino-2-phenylindole-stained slides. The geometric and texture features were extracted and used for analysis. RESULTS: In analysis of variance, it was shown that both individual cases and tumor types differ in the majority of the parameters. On nonsupervised expectation-maximization clustering, it was possible to classify the nuclei into separate categories, but PapRCC classes were not reproduced. The neural network classified the nuclei with sensitivity >0.6 and specificity >0.75. Analyzing the results for individual cases, the nuclei of type 1 cases were properly classified in 74-91%, nuclei of type 2 cases in 58-80% and nuclei of intermediate cases in 53-70%. CONCLUSION: Our findings show that PapRCC subtypes are distinct enough to be reproduced by image analysis.

TDP-43 is a culprit in human neurodegeneration, and not just an innocent bystander.

In 2006 the protein TDP-43 was identified as the major ubiquitinated component deposited in the inclusion bodies found in two human neurodegenerative diseases, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenesis of both disorders is unclear, although they are related by having some overlap of symptoms and now by the shared histopathology of TDP-43 deposition. Now, in 2008, several papers have been published in quick succession describing mutations in the TDP-43 gene, showing they can be a primary cause of amyotrophic lateral sclerosis. There are many precedents in neurodegenerative disease in which rare single-gene mutations have given great insight into understanding disease processes, which is why the TDP-43 mutations are potentially very important.