Composite scoring system and optimal tumor budding cut-off number for estimating lymph node metastasis in submucosal colorectal cancer.

BACKGROUND: Tumor budding is associated with lymph node (LN) metastasis in submucosal colorectal cancer (CRC). However, the rate of LN metastasis associated with the number of tumor buds is unknown. Here, we determined the optimal tumor budding cut-off number and developed a composite scoring system (CSS) for estimating LN metastasis of submucosal CRC. METHODS: In total, 395 patients with histologically confirmed T1N0-2M0 CRC were evaluated. The clinicopathological characteristics were subjected to univariate and multivariate analyses. The Akaike information criterion (AIC) values of the multivariate models were evaluated to identify the optimal cut-off number. A CSS for LN metastasis was developed using independent risk factors. RESULTS: The prevalence of LN metastasis was 13.2%. Histological differentiation, lymphatic or venous invasion, and tumor budding were associated with LN metastasis in univariate analyses. In multivariate models adjusted for histological differentiation and lymphatic or venous invasion, the AIC value was lowest for five tumor buds. Unfavorable differentiation (odds ratio [OR], 8.16; 95% confidence interval [CI], 1.80-36.89), lymphatic or venous invasion (OR, 5.91; 95% CI, 2.91-11.97), and five or more tumor buds (OR, 3.01; 95% CI, 1.21-7.69) were independent risk factors. In a CSS using these three risk factors, the rates of LN metastasis were 5.6%, 15.5%, 31.0%, and 52.4% for total composite scores of 0, 1, 2, and ≥ 3, respectively. CONCLUSIONS: For the estimation of LN metastasis in submucosal CRC, the optimal tumor budding cut-off number was five. Our CSS can be utilized to estimate LN metastasis.

Immunogenicity after outbreak response immunization activities among young healthcare workers with secondary vaccine failure during the measles epidemic in Korea, 2019.

BACKGROUND: Despite high vaccination coverage, measles outbreaks have been reported in measles elimination countries, especially among healthcare workers in their 20 and 30 s. This study was designed to identify measles-susceptible individuals and to evaluate whether primary or secondary vaccine failure occurred during measles outbreak response immunization (ORI) activities. METHODS: The study population was divided into three groups as follows: natural immunity group (Group 1), vaccine-induced immunity group (Group 2), and vaccine failure group (Group 3). We evaluated the immunogenicity of measles among healthcare workers using three methods-enzyme-linked immunoassays, plaque reduction neutralization tests, and avidity assays. The results were assessed at baseline, 4 weeks after, and 6 months after the completion of measles-mumps-rubella (MMR) vaccination. RESULTS: In total, 120 subjects were enrolled, with 40 subjects in each group. The median age of Group 3 was 29 years, which was significantly lower than that of the other groups. The baseline negative measles virus (MeV) IgG in Group 3 increased to a median value of 165 AU/mL at 4 weeks after ORI and was lower than that in Groups 1 and 2. The median neutralizing antibody titer was highest in Group 1, and this was significantly different from that in Group 2 or Group 3 at 4 weeks (944 vs. 405 vs. 482 mIU/mL, P = 0.001) and 6 months (826 vs. 401 vs. 470, P = 0.011) after ORI. The rates of high MeV avidity IgG were highest in Group 2, and these were significantly different from those in Groups 1 or 3 at 4 weeks (77.5 vs. 90% vs. 88.6%, P = 0.03) and 6 months (81 vs. 94.8 vs. 82.1%, P = 0.01) after ORI. CONCLUSIONS: Considering the MeV-neutralizing antibodies and IgG avidity after MMR vaccination in measles-susceptible group, vaccine failure is inferred as secondary vaccine failure, and further data regarding the maintenance of immunogenicity are needed based on long-term data. The MeV-neutralizing antibody levels were highest in the natural immunity group, and the primary vaccine-induced immunity group showed the highest rates of high MeV IgG avidity.

Motor neuronal repletion of the NMJ organizer, Agrin, modulates the severity of the spinal muscular atrophy disease phenotype in model mice.

Spinal muscular atrophy (SMA) is a common and often fatal neuromuscular disorder caused by low levels of the Survival Motor Neuron (SMN) protein. Amongst the earliest detectable consequences of SMN deficiency are profound defects of the neuromuscular junctions (NMJs). In model mice these synapses appear disorganized, fail to mature and are characterized by poorly arborized nerve terminals. Given one role of the SMN protein in orchestrating the assembly of spliceosomal snRNP particles and subsequently regulating the alternative splicing of pre-mRNAs, a plausible link between SMN function and the distal neuromuscular SMA phenotype is an incorrectly spliced transcript or transcripts involved in establishing or maintaining NMJ structure. In this study, we explore the effects of one such transcript-Z+Agrin-known to be a critical organizer of the NMJ. We confirm that low SMN protein reduces motor neuronal levels of Z+Agrin. Repletion of this isoform of Agrin in the motor neurons of SMA model mice increases muscle fiber size, enhances the post-synaptic NMJ area, reduces the abnormal accumulation of intermediate filaments in nerve terminals of the neuromuscular synapse and improves the innervation of muscles. While these effects are independent of changes in SMN levels or increases in motor neuron numbers they nevertheless have a significant effect on the overall disease phenotype, enhancing mean survival in severely affected SMA model mice by ∼40%. We conclude that Agrin is an important target of the SMN protein and that mitigating NMJ defects may be one strategy in treating human spinal muscular atrophy.

A pathogenic S250F missense mutation results in a mouse model of mild aromatic l-amino acid decarboxylase (AADC) deficiency.

Homozygous mutations in the aromatic l-amino acid decarboxylase (AADC) gene result in a severe depletion of its namesake protein, triggering a debilitating and often fatal form of infantile Parkinsonism known as AADC deficiency. AADC deficient patients fail to produce normal levels of the monoamine neurotransmitters dopamine and serotonin, and suffer a multi-systemic disorder characterized by movement abnormalities, developmental delay and autonomic dysfunction; an absolute loss of dopamine is generally considered incompatible with life. There is no optimal treatment for AADC deficiency and few truly good models in which to investigate disease mechanisms or develop and refine therapeutic strategies. In this study, we introduced a relatively frequently reported but mildly pathogenic S250F missense mutation into the murine Aadc gene. We show that mutants homozygous for the mutation are viable and express a stable but minimally active form of the AADC protein. Although the low enzymatic activity of the protein resulted in only modestly reduced concentrations of brain dopamine, serotonin levels were markedly diminished, and this perturbed behavior as well as autonomic function in mutant mice. Still, we found no evidence of morphologic abnormalities of the dopaminergic cells in mutant brains. The striatum as well as substantia nigra appeared normal and no loss of dopamine expressing cells in the latter was detected. We conclude that even minute levels of active AADC are sufficient to allow for substantial amounts of dopamine to be produced in model mice harboring the S250F mutation. Such mutants represent a novel, mild model of human AADC deficiency.

Host Cell Mimic Polymersomes for Rapid Detection of Highly Pathogenic Influenza Virus via a Viral Fusion and Cell Entry Mechanism.

Highly pathogenic avian influenza virus (HPAIV) infections have occurred continuously and crossed the species barrier to humans, leading to fatalities. A polymerase chain reaction based molecular test is currently the most sensitive diagnostic tool for HPAIV; however, the results must be analyzed in centralized diagnosis systems by a trained individual. This requirement leads to delays in quarantine and isolation. To control the spread of HPAIV, rapid and accurate diagnostics suitable for field testing are needed, and the tests must facilitate a differential diagnosis between HPAIV and low pathogenic avian influenza virus (LPAIV), which undergo cleavage specifically by trypsin- or furin-like proteases, respectively. In this study, a differential avian influenza virus rapid test kit is developed and evaluated in vitro and using clinical specimens from HPAIV H5N1-infected animals. It is demonstrated that this rapid test kit provides highly sensitive and specific detection of HPAIV and LPAIV and is thus a useful field diagnostic tool for H5N1 HPAIV outbreaks and for rapid quarantine control of the disease.

Comparison of the virulence of three H3N2 canine influenza virus isolates from Korea and China in mouse and Guinea pig models.

BACKGROUND: Avian-origin H3N2 canine influenza virus (CIV) has been the most common subtype in Korea and China since 2007. Here, we compared the pathogenicity and transmissibility of three H3N2 CIV strains [Chinese CIV (JS/10), Korean CIV (KR/07), and Korean recombinant CIV between the classic H3N2 CIV and the pandemic H1N1 virus (MV/12)] in BALB/c mouse and guinea pig models. The pandemic H1N1 (CA/09) strain served as the control. RESULTS: BALB/c mice infected with H1N1 had high mortality and obvious body weight loss, whereas no overt disease symptoms were observed in mice inoculated with H3N2 CIV strains. The viral titers were higher in the group MV/12 than those in groups JS/10 and KR/07, while the mice infected with JS/10 showed higher viral titers in all tissues (except for the lung) than the mice infected with KR/07. The data obtained in guinea pigs also demonstrated that group MV/12 presented the highest loads in most of the tissues, followed by group JS/10 and KR/07. Also, direct contact transmissions of all the three CIV strains could be observed in guinea pigs, and for the inoculated and the contact groups, the viral titer of group MV/12 and KR/07 was higher than that of group JS/10 in nasal swabs. These findings indicated that the matrix (M) gene obtained from the pandemic H1N1 may enhance viral replication of classic H3N2 CIV; JS/10 has stronger viral replication ability in tissues as compared to KR/07, whereas KR/07 infected guinea pigs have more viral shedding than JS/10 infected guinea pigs. CONCLUSIONS: There exists a discrepancy in pathobiology among CIV isolates. Reverse genetics regarding the genomes of CIV isolates will be helpful to further explain the virus characteristics.

Molecular and phylogenetic analyses of influenza B viruses isolated from pediatric inpatients in South Korea during the 2011-2012 winter season.

Influenza B virus remains a major cause of respiratory diseases worldwide. Because of limited epidemiological and genetic data, the local and global transmission patterns of influenza B virus are not fully understood. Here we report the molecular and phylogenetic characterization of 163 influenza B virus isolates from pediatric inpatients with influenza-like illness in the winter of 2011-2012 in South Korea. Analysis of haemagglutinin and neuraminidase genes of the influenza B isolates revealed that both B/Victoria (62 %) and B/Yamagata lineages (38 %) co-circulated during that influenza season, and a considerable number of the isolates carried several amino acid substitutions in the four major antigenic epitopes of their haemagglutinin protein.

Rapid virulence shift of an H5N2 avian influenza virus during a single passage in mice.

Influenza A viruses must undergo adaptation to acquire virulence in new host species. In mouse models, host adaptation for virulence is generally performed through 5 to 20 lung-to-lung passages. However, highly pathogenic avian influenza viruses (e.g., H5N1 and H7N7 subtypes) have been observed to acquire virulence in mice after only a few in vivo passages. In this study, a low-pathogenic avian influenza H5N2 virus, A/Aquatic Bird/Korea/CN2/2009, which was a prevalent subtype in South Korea in 2009, was serially passaged in mice to evaluate its potential to become highly pathogenic. Unexpectedly, the virus became highly pathogenic in mice after a single lung-to-lung passage, resulting in 100% lethality with a mean death time (MDT) of 6.1 days postinfection (DPI). Moreover, the pathogenicity gradually increased after subsequent in vivo passages with an MDT of 5.2 and 4.2 DPI after the second and third passage, respectively. Our molecular analysis revealed that two amino acid changes in the polymerase complex (a glutamate-to-lysine substitution at position 627 of PB2 and a threonine-to-isoleucine substitution at position 97 of PA) were associated with the increased pathogenicity; the PB2 E627K mutation was responsible for the initial virulence conversion (0 to 100% lethality), while the PA T97I mutation acted as an accessory for the increased virulence.

Attenuation of the virulence of a recombinant influenza virus expressing the naturally truncated NS gene from an H3N8 equine influenza virus in mice.

Equine influenza virus (EIV) causes a highly contagious disease in horses and other equids. Recently, we isolated an H3N8 EIV (A/equine/Kyonggi/SA1/2011) from a domestic horse in South Korea that exhibited symptoms of respiratory disease, and found that the EIV strain contained a naturally mutated NS gene segment encoding a truncated NS1 protein. In order to determine whether there was an association between the NS gene truncation and viral virulence, a reverse genetics system was applied to generate various NS gene recombinant viruses using the backbone of the H1N1 A/Puerto Rico/8/1934 (PR/8) virus. In a mouse model, the recombinant PR/8 virus containing the mutated NS gene of the Korean H3N8 EIV strain showed a dramatically reduced virulence: it induced no weight loss, no clinical signs and no histopathological lesions. However, the mice infected with the recombinant viruses with NS genes of PR/8 and H3N8 A/equine/2/Miami/1963 showed severe clinical signs including significant weight loss and 100% mortality. In addition, the levels of the pro-inflammatory cytokines; IL-6, CCL5, and IFN-γ, in the lungs of mice infected with the recombinant viruses expressing a full-length NS1 were significantly higher than those of mice infected with the virus with the NS gene from the Korean H3N8 EIV strain. In this study, our results suggest that the C-terminal moiety of NS1 contains a number of virulence determinants and might be a suitable target for the development of a vaccine candidate against equine influenza.

Virulence of a novel reassortant canine H3N2 influenza virus in ferret, dog and mouse models.

An outbreak of a canine influenza virus (CIV) H3N2 reassortant derived from pandemic (pdm) H1N1 and CIV H3N2 in companion animals has underscored the urgent need to monitor CIV infections for potential zoonotic transmission of influenza viruses to humans. In this study, we assessed the virulence of a novel CIV H3N2 reassortant, VC378, which was obtained from a dog that was coinfected with pdm H1N1 and CIV H3N2, in ferrets, dogs, and mice. Significantly enhanced virulence of VC378 was demonstrated in mice, although the transmissibility and pathogenicity of VC378 were similar to those of classical H3N2 in ferrets and dogs. This is notable because mice inoculated with an equivalent dose of classical CIV H3N2 showed no clinical signs and no lethality. We found that the PA and NS gene segments of VC378 were introduced from pdmH1N1, and these genes included the amino acid substitutions PA-P224S and NS-I123V, which were previously found to be associated with increased virulence in mice. Thus, we speculate that the natural reassortment between pdm H1N1 and CIV H3N2 can confer virulence and that continuous surveillance is needed to monitor the evolution of CIV in companion animals.

Canine susceptibility to human influenza viruses (A/pdm 09H1N1, A/H3N2 and B).

We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009) and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naïve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection occurring in dogs since 2008. Furthermore, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.

Viral dominance of reassortants between canine influenza H3N2 and pandemic (2009) H1N1 viruses from a naturally co-infected dog.

BACKGROUND: Since avian-origin H3N2 canine influenza virus (CIV) was first identified in South Korea in 2008, the novel influenza virus has been reported in several countries in Asia. Reverse zoonotic transmission of pandemic H1N1 (2009) influenza virus (pH1N1) has been observed in a broad range of animal species. Viral dominance and characterization of the reassortants of both viruses was undertaken in the present study. FINDINGS: Here we describe the viral dominance of 23 CIV reassortants between pH1N1 and canine H3N2 influenza viruses from a naturally co-infected dog. These results indicate that the M gene of pandemic H1N1 and the HA gene of canine H3N2 are predominant in the reassortants. Furthermore, unlike the original canine H3N2 virus, some reassortants showed high pathogenicity in mice. CONCLUSIONS: This study suggests that continuous monitoring of influenza infection in companion animals may be necessary to investigate the potential of the emergence of novel influenza viruses.

Comparative pathology of pigs infected with Korean H1N1, H1N2, or H3N2 swine influenza A viruses.

BACKGROUND: The predominant subtypes of swine influenza A virus (SIV) in Korea swine population are H1N1, H1N2, and H3N2. The viruses are genetically close to the classical U.S. H1N1 and triple-reassortant H1N2 and H3N2 viruses, respectively. Comparative pathogenesis caused by Korean H1N1, H1N2, and H3N2 SIV was evaluated in this study. FINDINGS: The H3N2 infected pigs had severe scores of gross and histopathological lesions at post-inoculation days (PID) 2, and this then progressively decreased. Both the H1N1 and H1N2 infected pigs lacked gross lesions at PID 2, but they showed moderate to severe pneumonia on PID 4, 7 and 14. The pigs infected with H1N1 had significant scores of gross and histopathological lesions when compared with the other pigs infected with H1N2, H3N2, and mock at PID 14. Mean SIV antigen-positive scores were rarely detected for pigs infected with H1N2 and H3N2 from PID 7, whereas a significantly increased amount of viral antigens were found in the bronchioles and alveolar epithelium of the H1N1infected pigs at PID 14. CONCLUSIONS: We demonstrated that Korean SIV subtypes had different pulmonary pathologic patterns. The Korean H3N2 rapidly induced acute lung lesions such as broncho-interstitial pneumonia, while the Korean H1N1 showed longer course of infection as compared to other strains.

Influenza Virus-Derived CD8 T Cell Epitopes: Implications for the Development of Universal Influenza Vaccines.

The influenza virus poses a global health burden. Currently, an annual vaccine is used to reduce influenza virus-associated morbidity and mortality. Most influenza vaccines have been developed to elicit neutralizing Abs against influenza virus. These Abs primarily target immunodominant epitopes derived from hemagglutinin (HA) or neuraminidase (NA) of the influenza virus incorporated in vaccines. However, HA and NA are highly variable proteins that are prone to antigenic changes, which can reduce vaccine efficacy. Therefore, it is essential to develop universal vaccines that target immunodominant epitopes derived from conserved regions of the influenza virus, enabling cross-protection among different virus variants. The internal proteins of the influenza virus serve as ideal targets for universal vaccines. These internal proteins are presented by MHC class I molecules on Ag-presenting cells, such as dendritic cells, and recognized by CD8 T cells, which elicit CD8 T cell responses, reducing the likelihood of disease and influenza viral spread by inducing virus-infected cell apoptosis. In this review, we highlight the importance of CD8 T cell-mediated immunity against influenza viruses and that of viral epitopes for developing CD8 T cell-based influenza vaccines.

Selective Suppression of Integrin-Ligand Binding by Single Molecular Tension Probes Mediates Directional Cell Migration.

Cell migration interacting with continuously changing microenvironment, is one of the most essential cellular functions, participating in embryonic development, wound repair, immune response, and cancer metastasis. The migration process is finely tuned by integrin-mediated binding to ligand molecules. Although numerous biochemical pathways orchestrating cell adhesion and motility are identified, how subcellular forces between the cell and extracellular matrix regulate intracellular signaling for cell migration remains unclear. Here, it is showed that a molecular binding force across integrin subunits determines directional migration by regulating tension-dependent focal contact formation and focal adhesion kinase phosphorylation. Molecular binding strength between integrin αvß3 and fibronectin is precisely manipulated by developing molecular tension probes that control the mechanical tolerance applied to cell-substrate interfaces. This data reveals that integrin-mediated molecular binding force reduction suppresses cell spreading and focal adhesion formation, attenuating the focal adhesion kinase (FAK) phosphorylation that regulates the persistence of cell migration. These results further demonstrate that manipulating subcellular binding forces at the molecular level can recapitulate differential cell migration in response to changes of substrate rigidity that determines the physical condition of extracellular microenvironment. Novel insights is provided into the subcellular mechanics behind global mechanical adaptation of the cell to surrounding tissue environments featuring distinct biophysical signatures.

Hairless plays a role in formation of inner root sheath via regulation of Dlx3 gene.

The Hairless (Hr), a transcription factor, is expressed in the suprabasal cell layer of the interfollicular epidermis and the lower portion of the hair follicle epithelium, where its expression is dependent on the hair cycle. Recently, we reported a new Hr mutant mouse, Hr(Hp), in which the hairless protein (HR) was overexpressed. In this study, we documented abnormal formation of inner root sheath (IRS), suppressed expression of Dlx3, and IRS keratins in the Hr(Hp)/Hr(Hp) skin. We also found that HR down-regulated Dlx3 mRNA expression through suppression of Dlx3 promoter activity. In addition, we showed that Dlx3 regulated the expression of IRS keratins. Our results demonstrate that regulation of Dlx3 by HR affects the IRS keratin expression, thus modulating the formation of IRS of hair follicle.

Complete genome sequence of an avian-origin H3N2 canine influenza virus isolated from dogs in South Korea.

An avian-origin Korean H3N2 canine influenza virus (CIV) strain, designated A/canine/Korea/01/2007 (H3N2), was isolated from nasal swabs of pet dogs exhibiting severe respiratory syndrome in 2007. In the present study, we report the first complete genome sequence containing 3' and 5' noncoding regions (NCRs) of H3N2 CIV, which will provide important insights into the molecular basis of pathogenesis, transmission, and evolution of CIV.

Puzzling inefficiency of H5N1 influenza vaccines in Egyptian poultry.

In Egypt, efforts to control highly pathogenic H5N1 avian influenza virus in poultry and in humans have failed despite increased biosecurity, quarantine, and vaccination at poultry farms. The ongoing circulation of HP H5N1 avian influenza in Egypt has caused >100 human infections and remains an unresolved threat to veterinary and public health. Here, we describe that the failure of commercially available H5 poultry vaccines in Egypt may be caused in part by the passive transfer of maternal H5N1 antibodies to chicks, inhibiting their immune response to vaccination. We propose that the induction of a protective immune response to H5N1 is suppressed for an extended period in young chickens. This issue, among others, must be resolved and additional steps must be taken before the outbreaks in Egypt can be controlled.

Diet-induced obesity dramatically reduces the efficacy of a 2009 pandemic H1N1 vaccine in a mouse model.

BACKGROUND: Obesity, a risk factor for increased severity of diverse diseases, is believed to have negative impact on vaccine efficacy. Recently, mortality has emerged as an outcome of pandemic influenza A virus subtype H1N1, necessitating development of effective vaccine strategies. Here we investigated effects of diet-induced obesity on vaccine-induced immune responses and protective efficacy against pandemic H1N1 influenza virus. METHODS: Diet-induced obese and lean C57BL/6J mice were immunized with commercial monovalent 2009 H1N1 vaccine, and antigen-specific antibody responses and neutralizing activities were observed. Following vaccination, mice were challenged with homologous H1N1 virus, and pathogenesis and mortality were examined. RESULTS: Vaccine-induced H1N1-specific antibody responses and neutralizing activities were markedly reduced in obese mice. Consistent with antibody responses, lung virus titers were significantly higher in obese mice than in lean controls after challenge. In addition, obese group showed greatly increased expression of proinflammatory cytokines and chemokines in lung tissue, severe lung inflammation, and higher eventual mortality rate (100%) compared with that among lean control mice (14%). CONCLUSIONS: Our results show that prophylactic immune responses and protectiveness induced by 2009 H1N1 vaccine could be extremely compromised in diet-induced obesity. These results suggest that novel vaccination strategies for high-risk groups, including the obese population, are required.

Muscle: an independent contributor to the neuromuscular spinal muscular atrophy disease phenotype.

Spinal muscular atrophy (SMA) is a pediatric-onset neuromuscular disorder caused by insufficient survival motor neuron (SMN) protein. SMN restorative therapies are now approved for the treatment of SMA; however, they are not curative, likely due to a combination of imperfect treatment timing, inadequate SMN augmentation, and failure to optimally target relevant organs. Here, we consider the implications of imperfect treatment administration, focusing specifically on outcomes for skeletal muscle. We examine the evidence that muscle plays a contributing role in driving neuromuscular dysfunction in SMA. Next, we discuss how SMN might regulate the health of myofibers and their progenitors. Finally, we speculate on therapeutic outcomes of failing to raise muscle SMN to healthful levels and present strategies to restore function to this tissue to ensure better treatment results.