Understanding how the immune system affects cancer development and progression has been one of the most challenging questions in immunology. Research over the past two decades has helped explain why the answer to this question has evaded us for so long. We now appreciate that the immune system plays a dual role in cancer: It can not only suppress tumor growth by destroying cancer cells or inhibiting their outgrowth but also promote tumor progression either by selecting for tumor cells that are more fit to survive in an immunocompetent host or by establishing conditions within the tumor microenvironment that facilitate tumor outgrowth. Here, we discuss a unifying conceptual framework called "cancer immunoediting," which integrates the immune system's dual host-protective and tumor-promoting roles.

To develop a simple, relatively noninvasive small-animal model of reversible regional cerebral ischemia, we tested various methods of inducing infarction in the territory of the right middle cerebral artery (MCA) by extracranial vascular occlusion in rats. In preliminary studies, 60 rats were anesthetized with ketamine and different combinations of vessels were occluded; blood pressure and arterial blood gases were monitored. Neurologic deficit, mortality rate, gross pathology, and in some instances, electroencephalogram and histochemical staining results were evaluated in all surviving rats. The principal procedure consisted of introducing a 4-0 nylon intraluminal suture into the cervical internal carotid artery (ICA) and advancing it intracranially to block blood flow into the MCA; collateral blood flow was reduced by interrupting all branches of the external carotid artery (ECA) and all extracranial branches of the ICA. In some groups of rats, bilateral vertebral or contralateral carotid artery occlusion was also performed. India ink perfusion studies in 20 rats documented blockage of MCA blood flow in 14 rats subjected to permanent occlusion and the restoration of blood flow to the MCA territory in six rats after withdrawal of the suture from the ICA. The best method of MCA occlusion was then selected for further confirmatory studies, including histologic examination, in five additional groups of rats anesthetized with halothane. Seven of eight rats that underwent permanent occlusion of the MCA had resolving moderately severe neurologic deficits (Grade 2 of 4) and unilateral infarcts averaging 37.6 +/- 5.5% of the coronal sectional area at 72 hours after the onset of occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Colon carcinoma is the second most common cause of death from cancer. The isolation and characterization of tumorigenic colon cancer cells may help to devise novel diagnostic and therapeutic procedures. Although there is increasing evidence that a rare population of undifferentiated cells is responsible for tumour formation and maintenance, this has not been explored for colorectal cancer. Here, we show that tumorigenic cells in colon cancer are included in the high-density CD133+ population, which accounts for about 2.5% of the tumour cells. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumour in immunodeficient mice, whereas CD133- cells did not form tumours. Such tumours were serially transplanted for several generations, in each of which we observed progressively faster tumour growth without significant phenotypic alterations. Unlike CD133- cells, CD133+ colon cancer cells grew exponentially for more than one year in vitro as undifferentiated tumour spheres in serum-free medium, maintaining the ability to engraft and reproduce the same morphological and antigenic pattern of the original tumour. We conclude that colorectal cancer is created and propagated by a small number of undifferentiated tumorigenic CD133+ cells, which should therefore be the target of future therapies.

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4-ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that are approximately 21 nucleotides in length and control many developmental and cellular processes in eukaryotic organisms. Research during the past decade has identified major factors participating in miRNA biogenesis and has established basic principles of miRNA function. More recently, it has become apparent that miRNA regulators themselves are subject to sophisticated control. Many reports over the past few years have reported the regulation of miRNA metabolism and function by a range of mechanisms involving numerous protein-protein and protein-RNA interactions. Such regulation has an important role in the context-specific functions of miRNAs.

DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.

Emerging infectious diseases, such as SARS and Zika, present a major threat to public health^1-3. Despite intense research efforts, how, when and where new diseases appear are still the source of considerable uncertainly. A severe respiratory disease was recently reported in the city Wuhan, Hubei province, China. Up to 25th of January 2020, at least 1,975 cases have been reported since the first patient was hospitalized on the 12th of December 2019. Epidemiological investigation suggested that the outbreak was associated with a seafood market in Wuhan. We studied one patient who was a worker at the market, and who was admitted to Wuhan Central Hospital on 26th of December 2019 experiencing a severe respiratory syndrome including fever, dizziness and cough. Metagenomic RNA sequencing⁴ of a bronchoalveolar lavage fluid sample identified a novel RNA virus from the family Coronaviridae, designed here as WH-Human-1 coronavirus. Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) previously sampled from bats in China. This outbreak highlights the ongoing capacity of viral spill-over from animals to cause severe disease in humans.

Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 'helpless' CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.

Motile cells extend a leading edge by assembling a branched network of actin filaments that produces physical force as the polymers grow beneath the plasma membrane. A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion. Signaling pathways converging on WASp/Scar proteins regulate the activity of Arp2/3 complex, which mediates the initiation of new filaments as branches on preexisting filaments. After a brief spurt of growth, capping protein terminates the elongation of the filaments. After filaments have aged by hydrolysis of their bound ATP and dissociation of the gamma phosphate, ADF/cofilin proteins promote debranching and depolymerization. Profilin catalyzes the exchange of ADP for ATP, refilling the pool of ATP-actin monomers bound to profilin, ready for elongation.

A bacterial gene (neo) conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion. Whereas normal cells are killed by the antibiotic G418, those that acquire and express neo continue to grow in the presence of G418. In the course of the selection, neo DNA becomes associated with high molecular weight cellular DNA and is retained even when cells are grown in the absence of G418 for extended periods. Since neo provides a marker for dominant selections, cell transformation to G418 resistance is an efficient means for cotransformation of nonselected genes.

Schizophrenia is a highly heritable disorder. Genetic risk is conferred by a large number of alleles, including common alleles of small effect that might be detected by genome-wide association studies. Here we report a multi-stage schizophrenia genome-wide association study of up to 36,989 cases and 113,075 controls. We identify 128 independent associations spanning 108 conservatively defined loci that meet genome-wide significance, 83 of which have not been previously reported. Associations were enriched among genes expressed in brain, providing biological plausibility for the findings. Many findings have the potential to provide entirely new insights into aetiology, but associations at DRD2 and several genes involved in glutamatergic neurotransmission highlight molecules of known and potential therapeutic relevance to schizophrenia, and are consistent with leading pathophysiological hypotheses. Independent of genes expressed in brain, associations were enriched among genes expressed in tissues that have important roles in immunity, providing support for the speculated link between the immune system and schizophrenia.

The elucidation of the human genome sequence has made it possible to identify genetic alterations in cancers in unprecedented detail. To begin a systematic analysis of such alterations, we determined the sequence of well-annotated human protein-coding genes in two common tumor types. Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of approximately 90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention, and open fertile avenues for basic research in tumor biology.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or para-articular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.

Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells. We show that siRNA expression mediated by this vector causes efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Stable expression of siRNAs using this vector mediates persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time. Therefore, the pSUPER vector constitutes a new and powerful system to analyze gene function in a variety of mammalian cell types.

Interleukin 17 (IL-17) has been linked to autoimmune diseases, although its regulation and function have remained unclear. Here we have evaluated in vitro and in vivo the requirements for the differentiation of naive CD4 T cells into effector T helper cells that produce IL-17. This process required the costimulatory molecules CD28 and ICOS but was independent of the cytokines and transcription factors required for T helper type 1 or type 2 differentiation. Furthermore, both IL-4 and interferon-gamma negatively regulated T helper cell production of IL-17 in the effector phase. In vivo, antibody to IL-17 inhibited chemokine expression in the brain during experimental autoimmune encephalomyelitis, whereas overexpression of IL-17 in lung epithelium caused chemokine production and leukocyte infiltration. Thus, IL-17 expression characterizes a unique T helper lineage that regulates tissue inflammation.

Although there are many commercially available statistical software packages, only a few implement a competing risk analysis or a proportional hazards regression model with time-dependent covariates, which are necessary in studies on hematopoietic SCT. In addition, most packages are not clinician friendly, as they require that commands be written based on statistical languages. This report describes the statistical software 'EZR' (Easy R), which is based on R and R commander. EZR enables the application of statistical functions that are frequently used in clinical studies, such as survival analyses, including competing risk analyses and the use of time-dependent covariates, receiver operating characteristics analyses, meta-analyses, sample size calculation and so on, by point-and-click access. EZR is freely available on our website (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html) and runs on both Windows (Microsoft Corporation, USA) and Mac OS X (Apple, USA). This report provides instructions for the installation and operation of EZR.

By analyzing the effects of single base substitutions around the ATG initiator codon in a cloned preproinsulin gene, I have identified ACCATGG as the optimal sequence for initiation by eukaryotic ribosomes. Mutations within that sequence modulate the yield of proinsulin over a 20-fold range. A purine in position -3 (i.e., 3 nucleotides upstream from the ATG codon) has a dominant effect; when a pyrimidine replaces the purine in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Single base substitutions around an upstream, out-of-frame ATG codon affect the efficiency with which it acts as a barrier to initiating at the downstream start site for preproinsulin. The optimal sequence for initiation defined by mutagenesis is identical to the consensus sequence that emerged previously from surveys of translational start sites in eukaryotic mRNAs. The mechanism by which nucleotides flanking the ATG codon might exert their effect is discussed.

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.

A simple method for generating cDNA libraries from submicrogram quantities of mRNA is described. It combines classical first-strand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis [Okayama, H., and Berg, P., Mol. Cell. Biol. 2 (1982) 161-170]. Neither the elaborate vector-primer system nor the classical hairpin loop cleavage by S1 nuclease are used. cDNA thus made can be tailed and cloned without further purification or sizing. Cloning efficiencies can be as high as 10(6) recombinants generated per microgram mRNA, a considerable improvement over earlier methods. Using the fully sequenced 1300 nucleotide-long bovine preproenkephalin mRNA, we have established by sequencing that the method yields faithful full-length transcripts. This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.

The outbreak of a novel betacoronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development, we determined a 3.5 Å-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.

Chronic activation of the immune system is a hallmark of progressive HIV infection and better predicts disease outcome than plasma viral load, yet its etiology remains obscure. Here we show <em>that</em> circulating microbial products, probably derived from the gastrointestinal tract, are a cause of HIV-related systemic immune activation. Circulating lipopolysaccharide, which we used as an indicator of microbial translocation, was significantly increased in chronically HIV-infected individuals and in simian immunodeficiency virus (SIV)-infected rhesus macaques (P <or= 0.002). We show <em>that</em> increased lipopolysaccharide is bioactive in vivo and correlates with measures of innate and adaptive immune activation. Effective antiretroviral therapy seemed to reduce microbial translocation partially. Furthermore, in nonpathogenic SIV infection of sooty mangabeys, microbial translocation did not seem to occur. These data establish a mechanism for chronic immune activation in the context of a compromised gastrointestinal mucosal surface and provide new directions for therapeutic interventions <em>that</em> modify the consequences of acute HIV infection.

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells--cancer stem cells--possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 x 10(4) unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.