Ebola virus-induced eye sequelae: a murine model for evaluating glycoprotein-targeting therapeutics.

BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.

Mycobacterial biotin synthases require an auxiliary protein to convert dethiobiotin into biotin.

Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.

Comparing the efficacy of combined versus single immune cell adaptive therapy targeting colorectal cancer.

PURPOSE: Colorectal cancer (CRC) is the most frequent cancer with limited therapeutic achievements. Recently, adoptive cellular immunotherapy has been developed as an antitumor therapy. However, its efficacy has not been tested in CRC. This study investigated the ability of an immune cell cocktail of dendritic cells (DCs), T cells, and natural killer (NK) cells to overcome immunological hurdles and improve the therapeutic efficacy of cell therapy for CRC. METHODS: CRC lysate-pulsed monocyte-derived DCs (Mo-DCs), CRC antigen-specifically expanded T cells (CTL), and in vitro-expanded NK cells were cultured from patient peripheral blood mononuclear cells (PBMC). The ability of the combined immune cells to kill autologous tumor cells was investigated by co-culturing the combined immune cells with patient-derived tumor cells. RESULTS: The Mo-DCs produced expressed T cell co-stimulating molecules like CD80, CD86, human leukocyte antigen (HLA)-DR and HLA-ABC, at high levels and were capable of activating naive T cells. The expanded T cells were predominantly CD8 T cells with high levels of CD8 effector memory cells and low levels of regulatory T cells. The NK cells expressed high levels of activating receptors and were capable of killing other cancer cell lines (K562 and HT29). The immune cell cocktail demonstrated a higher ability to kill autologous tumor cells than single types. An in vivo preclinical study confirmed the safety of the combined immune cell adaptive therapy showing no therapy-related death or general toxicity symptoms. CONCLUSION: The results suggested that combined immune cell adaptive therapy could overcome the limited efficacy of cell immunotherapy.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

Background: It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Methods: Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. Results: E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. Conclusion: E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Pulmonary Artery Measurements as Postnatal Prognostic Tool in Right Congenital Diaphragmatic Hernia.

BACKGROUND: Right-sided congenital diaphragmatic hernia (RCDH) is a rare and often fatal congenital anomaly, primarily attributed to lung hypoplasia, which is associated with small branch pulmonary artery (PA). This study investigated whether postnatal PA measurements obtained through echocardiography are associated with mortality or the extracorporeal membrane oxygenation (ECMO) requirement in neonates with RCDH. METHODS: A retrospective study was conducted on neonates with RCDH born between 2008 and 2022. Echocardiography was performed on the day of birth. The diameter of the main PA (MPA) was measured at the maximal dimension, and the diameters of the left PA (LPA) and right PA (RPA) were measured at the bifurcation. The primary outcome was mortality or ECMO requirement. Parameters, including the LPA:MPA ratio, RPA:MPA ratio, Nakata index, McGoon ratio, and ejection fraction (EF), were analyzed and compared with the observed-to-expected lung-to-head ratio (o/e LHR), initial blood gas, and defect size as predictive values. RESULTS: Among 39 neonates with RCDH, 25 (64.1 %) survived without ECMO. The non-survivor or ECMO group exhibited lower o/e LHR, reduced EF, smaller LPA and RPA diameters, and larger MPA diameter than survivors. Lower LPA:MPA ratio, Nakata index, McGoon ratio, and higher initial PaCO2 were associated with adverse outcomes. Notably, the LPA:MPA ratio showed the highest predictive capability (area under the curve, 0.983; p < 0.001). CONCLUSION: The LPA:MPA ratio is a promising postnatal predictor of mortality or ECMO requirement in neonates with RCDH. Additionally, Nakata index, McGoon ratio, and initial PaCO2 are significantly correlated with outcomes. LEVEL OF EVIDENCE: This is a level III. TYPE OF STUDY: Prognostic study.

Outcomes at 18-24 Months of Infants with Birth Weight under 500 g Born in Korea during 2013-2017: A Nationwide Cohort Study.

INTRODUCTION: This study aimed to investigate the outcomes of infants at 18-24 months born in the Korean Neonatal Network with a birth weight <500 g. METHODS: The anthropometric and neurodevelopmental data of infants with a birth weight <500 g at a gestational age of ≥22 weeks who were registered in the Korean Neonatal Network 2013-2017 and followed up at a corrected age of 18-24 months were reviewed. Neurodevelopmental impairment was defined as the presence of any of the following: (1) cerebral palsy; (2) severe visual impairment; (3) hearing impairment; or (4) cognitive impairment. Cognitive impairment was defined as (1) a Bayley Scales of Infant Development-II Mental Development Index score <70; and (2) Bayley Scales of Infant and Toddler Development-III Cognitive and Language Composite scores <85. Cognitive testing was performed for infants with suspected problems upon clinician's referral to developmental specialists. RESULTS: At a median corrected age of 20 months, 26/52 (50%) of included infants had neurodevelopmental impairment. Cerebral palsy, severe visual impairment, wearing of glasses, hearing impairment, and cognitive impairment occurred in 22%, 0%, 8%, 5%, and 57% of the included infants, respectively. The proportions of infants with <2 standard deviations of weight, length, and head circumference were 54%, 52%, and 56%, respectively. The majority (70%) of infants were rehospitalized, and the most common cause was respiratory problems. CONCLUSION: Half of infants with a birth weight <500 g in Korea may exhibit neurodevelopmental impairment and growth retardation at a corrected age of 18-24 months. Multidisciplinary follow-up along with continuous rehabilitation will be needed to improve neurological and physical development in this special population.

Early echocardiographic pulmonary artery measurements as prognostic indicators in left congenital diaphragmatic hernia.

BACKGROUND: To predict whether the left pulmonary artery (LPA) to the main pulmonary artery (MPA) ratio measured by echocardiography in left congenital diaphragmatic hernia (CDH) was related to death or need for extracorporeal membrane oxygenation (ECMO). METHODS: This retrospective study analyzed neonates with left CDH born between 2018 and 2022 in a single tertiary medical institution. Echocardiography was performed immediately after birth. The diameter of the LPA was measured at the bifurcation, and the diameter of the MPA was measured at the maximal dimension during the systolic phase. The Nakata index, McGoon ratio, and ejection fraction (EF) were analyzed and compared with the LPA: MPA ratio as predictive values. RESULTS: Seventy-two neonates with left CDH were included, 19 (26.4%) died or needed ECMO, and 53 (73.6%) survived without ECMO. The lower observed/expected lung-to-head ratio, lower EF, lower LPA: MPA ratio, lower RPA: MPA ratio, lower Nakata index, and lower McGoon ratio were associated with death or need for ECMO. By multivariate analysis, lower LPA: MPA ratio, RPA: MPA ratio, and Nakata index were independent postnatal risk factors for death or need for ECMO. Among the measurements, the LPA: MPA ratio had the highest area under the curve (0.957) with a sensitivity of 84.2% and specificity of 96.3% at a cut-off value of 31.2%. CONCLUSION: In patients with left CDH, the LPA: MPA ratio measured by echocardiography could be used as an independent postnatal predictor of death or need for ECMO.

Evaluation of a New Chemiluminescent Immunoassay-Based Interferon-Gamma Release Assay for Detection of Latent Tuberculosis Infection.

Background and Objectives: This study aimed to evaluate the performance of a new chemiluminescent immunoassay-based tuberculosis (TB) interferon-gamma release assay (IGRA), AdvanSureI3 TB-IGRA (LG Chem Ltd., Seoul, Republic of Korea), for detecting latent tuberculosis infection in comparison with T-SPOT.TB (Oxford Immunotec, Oxford, UK). Materials and Methods: Between June 2021 and December 2021, 125 non-duplicate blood specimens were collected from adult volunteers; each subject received both tests concurrently. Total agreement and Cohen's kappa coefficient (κ) were used to calculate concordance. The Jonckheere-Terpstra test was used to examine the correlation between interferon-gamma (IFN-γ) levels in AdvanSureI3 TB-IGRA and spot counts in T-SPOT.TB. Results: The IGRA findings of the two assays revealed 90.8% (95% confidence interval [CI] = 84.2-94.8) total agreement with κ of 0.740 (95% CI = 0.595-0.885), showing substantial agreement between the two tests. Additionally, the amount of IFN-γ in AdvanSureI3 TB-IGRA increased with the spot counts in T-SPOT.TB (p < 0.001). Conclusions: Our research revealed that the results of the AdvanSureI3 TB-IGRA were comparable to those of T-SPOT.TB.

Neonatal Zika virus infection causes transient perineuronal net degradation.

Perineuronal nets (PNNs) form a specialized extracellular matrix that predominantly surrounds parvalbumin (PV)-expressing GABAergic inhibitory interneurons and help regulate neuronal activity. Their formation early in the postnatal period is regulated by neuronal signaling and glial activation raising concerns that part of the long-term effects ascribed to perinatal viral infections could be mediated by altered PNN formation. Previously, we developed a model of neonatal Zika virus (ZIKV) infection where mice have lifelong neurological sequelae that includes motor disfunction and reduced anxiety coupled with a persistent low-grade expression in proinflammatory markers despite resolving the acute infection. Here, we demonstrate that ZIKV infection to P1 neonatal mice results in a reduction of PNN formation during the acute disease with significant reduction in Wisteria floribunda agglutinin (WFA) staining at the peak of infection [15 days post infection (dpi)] that persisted after the symptoms resolved (30 dpi). At 60 dpi, when there is residual inflammation in the CNS, the number of WFA+ cells and the level of WFA staining as well as levels of aggrecan and brevican in the brains of convalescent mice were not different from those in uninfected controls, however, there was increased frequency of PNNs with an immature phenotype. Over time the impact of the perinatal infection became less evident and there were no clear differences in PNN morphology between the groups at 1 year post infection. Of note, the reduction in PNNs during acute ZIKV infection was not associated with decreased mRNA levels of aggrecan or brevican, but increased levels of degraded aggrecan and brevican indicating increased PNN degradation. These changes were associated with increased expression of matrix metalloproteinase 12 (MMP12) and MMP19, but not MMP9, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) or ADAMTS5. Together our findings indicate that infection at the time of PNN development interferes with PNN formation, but the nets can reform once the infection and inflammation subside.

Mycobacterial Regulatory Systems Involved in the Regulation of Gene Expression Under Respiration-Inhibitory Conditions.

Mycobacterium tuberculosis is the causative agent of tuberculosis. M. tuberculosis can survive in a dormant state within the granuloma, avoiding the host-mounting immune attack. M. tuberculosis bacilli in this state show increased tolerance to antibiotics and stress conditions, and thus the transition of M. tuberculosis to the nonreplicating dormant state acts as an obstacle to tuberculosis treatment. M. tuberculosis in the granuloma encounters hostile environments such as hypoxia, nitric oxide, reactive oxygen species, low pH, and nutrient deprivation, etc., which are expected to inhibit respiration of M. tuberculosis. To adapt to and survive in respiration-inhibitory conditions, it is required for M. tuberculosis to reprogram its metabolism and physiology. In order to get clues to the mechanism underlying the entry of M. tuberculosis to the dormant state, it is important to understand the mycobacterial regulatory systems that are involved in the regulation of gene expression in response to respiration inhibition. In this review, we briefly summarize the information regarding the regulatory systems implicated in upregulation of gene expression in mycobacteria exposed to respiration-inhibitory conditions. The regulatory systems covered in this review encompass the DosSR (DevSR) two-component system, SigF partner switching system, MprBA-SigE-SigB signaling pathway, cAMP receptor protein, and stringent response.

Anti-PD-L1 Antibody and/or 17ß-Estradiol Treatment Induces Changes in the Gut Microbiome in MC38 Colon Tumor Model.

PURPOSE: 17ß-Estradiol (E2) supplementation suppresses MC38 tumor growth by downregulating the expression of programmed death-ligand 1 (PD-L1). This study aims to figure out the gut microbiota that respond to anti-PD-L1 and/or estrogen treatment in MC38 colon cancer model. Materials and Methods: A syngeneic colon tumor model was developed by injection of MC38 cells into C57BL/6 background male and female mice. Three days before MC38 cells injection, E2 was supplemented to male mice daily for 1 week. Male and female mice with MC38 tumors (50-100 mm3) were injected with anti-PD-L1 antibody. Fresh feces were collected 26 days after injection of MC38 cells and 16S rRNA metagenomics sequencing of DNA extracted from feces was used to assess gut microbial composition. RESULTS: At the taxonomic family level, Muribaculaceae was enriched only in the MC38 male control group. In male mice, linear discriminant analysis effect size analysis at the species level revealed that the four microorganisms were commonly regulated in single and combination treatment with anti-PD-L1 and/or E2; a decrease in PAC001068_g_uc and PAC001070_s (family Muribaculaceae) and increase in PAC001716_s and PAC001785_s (family Ruminococcaceae). Interestingly, in the anti-PD-L1 plus E2 group, a decrease in opportunistic pathogens (Enterobacteriaceae group) and an increase in commensal bacteria (Lactobacillus murinus group and Parabacteroides goldsteinii) were observed. Furthermore, the abundance of Parabacteroides goldsteinii was increased in both males and females in the anti-PD-L1 group. CONCLUSION: Our results suggest that gut microbial changes induced by the pretreatment of estrogen before anti-PD-L1 might contribute to treatment of MC38 colon cancer.

Changes in Gut Microbiome upon Orchiectomy and Testosterone Administration in AOM/DSS-Induced Colon Cancer Mouse Model.

PURPOSE: Sex hormones are known to affect the gut microbiota. Previously, we reported that endogenous and exogenous testosterone are associated with colorectal cancer (CRC) development and submucosal invasion. In the present study, we investigated whether the gut microbiota is affected by orchiectomy (ORX) and testosterone propionate (TP) administration using an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CRC mouse model. MATERIALS AND METHODS: Gut microbiota was evaluated by means of 16S rRNA gene sequencing of stool DNA extracted from feces that were obtained at 13 weeks after AOM injection (from 22-week-old animals) and stored in a gas-generating pouch. RESULTS: The increase in microbial diversity (Chao1 and Phylogenetic Diversity index) and Firmicutes/Bacteroidetes (F/B) ratio upon AOM/DSS treatment in ORX mice was significantly decreased by TP supplementation. The ratio of commensal bacteria to opportunistic pathogens was lower in the TP-administered females and ORX mice than in the AOM/DSS group. Opportunistic pathogens (Mucispirillum schaedleri or Akkermansia muciniphila) were identified only in the TP group. In addition, microbial diversity and F/B ratio were higher in male controls than in female and ORX controls. Flintibacter butyricus, Ruminococcus bromii, and Romboutsia timonensis showed similar changes in the male control group as those in the female and ORX controls. CONCLUSION: In conclusion, testosterone determines the dysbiosis of gut microbiota, which suggests that it plays a role in the sex-related differences in colorectal carcinogenesis.

Occult metastasis to the superficial level VI lymph nodes in papillary thyroid carcinoma.

BACKGROUND: This study aimed to evaluate the incidence and risk factors of occult metastasis to superficial level VI, defined as the space anterior to the strap muscles, including the lymph nodes between the sternocleidomastoid and sternohyoid muscles and suprasternal space lymph nodes. METHODS: We studied 129 patients with papillary thyroid carcinoma who underwent thyroidectomy and neck dissection, including superficial level VI dissection. RESULTS: Of the 129 patients, 62 (48%) had lymph nodes in the harvested specimens of superficial level VI, and the mean number of lymph nodes retrieved was 1.9 ± 1.2. Occult metastasis to superficial level VI occurred in four patients (3.1%). No significant risk factors of superficial level VI occult metastasis were noted in multivariate analysis. CONCLUSIONS: Occult metastasis to superficial level VI was rare in patients with papillary thyroid carcinoma. Therefore, prophylactic dissection of superficial level VI may not be necessary for primary papillary thyroid carcinoma.

BSL2-compliant lethal mouse model of SARS-CoV-2 and variants of concern to evaluate therapeutics targeting the Spike protein.

Since first reported in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rapidly acquiring mutations, particularly in the spike protein, that can modulate pathogenicity, transmission and antibody evasion leading to successive waves of COVID19 infections despite an unprecedented mass vaccination necessitating continuous adaptation of therapeutics. Small animal models can facilitate understanding host-pathogen interactions, target selection for therapeutic drugs, and vaccine development, but availability and cost of studies in BSL3 facilities hinder progress. To generate a BSL2-compatible in vivo system that specifically recapitulates spike protein mediated disease we used replication competent, GFP tagged, recombinant Vesicular Stomatitis Virus where the VSV glycoprotein was replaced by the SARS-CoV-2 spike protein (rVSV-SARS2-S). We show that infection requires hACE2 and challenge of neonatal but not adult, K18-hACE2 transgenic mice (hACE2tg) leads to productive infection of the lungs and brains. Although disease progression was faster in SARS-CoV-2 infected mice, infection with both viruses resulted in neuronal infection and encephalitis with increased expression of Interferon-stimulated Irf7, Bst2, Ifi294, as well as CxCL10, CCL5, CLC2, and LILRB4, and both models were uniformly lethal. Further, prophylactic treatment targeting the Spike protein (Receptor Binding Domain) with antibodies resulted in similar levels of protection from lethal infection against rVSV-SARS2-S and SARS-CoV-2 viruses. Strikingly, challenge of neonatal hACE2tg mice with SARS-CoV-2 Variants of Concern (SARS-CoV-2-α, -ß, ϒ, or Δ) or the corresponding rVSV-SARS2-S viruses (rVSV-SARS2-Spike-α, rVSV-SARS2-Spike-ß, rVSV-SARS2-Spike-ϒ or rVSV-SARS2-Spike-Δ) resulted in increased lethality, suggesting that the Spike protein plays a key role in determining the virulence of each variant. Thus, we propose that rVSV-SARS2-S virus can be used to understand the effect of changes to SARS-CoV-2 spike protein on infection and to evaluate existing or experimental therapeutics targeting spike protein of current or future VOC of SARS-CoV-2 under BSL-2 conditions.

Combination treatment with 17ß-estradiol and anti-PD-L1 suppresses MC38 tumor growth by reducing PD-L1 expression and enhancing M1 macrophage population in MC38 colon tumor model.

17ß-estradiol (E2) is known to have a protective effect in colorectal cancer (CRC); thus, E2 may be effective for cancer immunotherapy in CRC. The aim of this study is to evaluate the effect of combination therapy with E2 and anti-programmed cell death receptor-1 ligand (PD-L1) antibodies, and the effects of sex and estrogen on colon tumor growth, PD-L1 expression, and tumor-associated cell populations in an MC38 colon tumor model. Male mice showed increased MC38 colon tumor growth and PD-L1 expression in tumor sections as well as higher proportion of cancer-associated fibroblasts (CD45-CD31-CD140a+), PD-L1-expressing tumor cells (CD45-CD274+) and tumor-associated macrophages (TAMs) (CD11b+F4/80+CD274+) compared to female mice. E2 treatment prior to MC38 injection significantly reduced these phenomena in male mice. Furthermore, co-treatment with E2 and anti-PD-L1 antibodies significantly inhibited MC38 tumor growth and reduced PD-L1-expressing cells in male mice compared to treatment with either E2 or anti-PD-L1 antibodies alone. Combination treatment with E2 and anti-PD-L1 decreased TAM population (CD11b+F4/80+) in the tumor mass while increasing M1 TMAs (CD11b+F4/80+CD86+). These results suggest that estrogen inhibits MC38 tumor growth by downregulating PD-L1 expression and regulating tumor-associated cell populations. Furthermore, estrogen boosted the effect of anti-PD-L1 antibody in the MC38 tumor model.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

NK cells require immune checkpoint receptor LILRB4/gp49B to control neurotropic Zika virus infections in mice.

Immune cells express an array of inhibitory checkpoint receptors that are upregulated upon activation and limit tissue damage associated with excessive response to pathogens or allergens. Mouse leukocyte immunoglobulin like receptor B4 (LILRB4), also known as glycoprotein 49B (gp49B), is an inhibitory checkpoint receptor constitutively expressed in myeloid cells and upregulated in B cells, T cells, and NK cells upon activation. Here, we report that expression of LILRB4, which binds Zika virus (ZIKV), was increased in microglia and myeloid cells infiltrating the brains of neonatal mice with ZIKV-associated meningoencephalitis. Importantly, while C57BL/6 mice developed transient neurological symptoms but survived infection, mice lacking LILRB4/gp49B (LILRB4 KO) exhibited more severe signs of neurological disease and succumbed to disease. Their brains showed increased cellular infiltration but reduced control of viral burden. The reduced viral clearance was associated with altered NK cell function in the absence of LILRB4/gp49B. In naive animals, this manifested as reduced granzyme B responses to stimulation, but in ZIKV-infected animals, NK cells showed phenotypic changes that suggested altered maturation, diminished glucose consumption, reduced IFN-γ and granzyme B production, and impaired cytotoxicity. Together, our data reveal LILRB4/gp49B as an important regulator of NK cell function during viral infections.

Transcultural Adaptation and Validation of the Korean Version of the Vocal Tract Discomfort Scale.

OBJECTIVES: The present study translated the Vocal Tract Discomfort Scale (VTDS) into Korean (K-VTDS) and evaluated its reliability and validity. STUDY DESIGN: This was a prospective study. METHODS: The VTDS was first translated into Korean and validated. One hundred and fifty-nine patients with voice disorders were divided into three different diagnostic groups (functional, structural, and neurologic voice disorder) and 131 vocally healthy adults were also included. All participants completed the K-VTDS and the Korean version of the Voice Handicap Index (K-VHI) and Korean versions of the Voice-Related Quality of Life (K-VRQOL). The internal consistency of the K-VTDS was analyzed through Cronbach's α coefficient. The VTDS score differences related to the diagnostic groups were assessed with t test and analysis of variance. We assessed the correlation between the K-VTDS, the K-VHI, and the K-VRQOL using Pearson's correlation analysis. RESULTS: High internal consistency and the test-retest reliability of the K-VTDS were found. The voice disorder group had significantly higher K-VTDS scores for the subscales and total scores than those in the healthy group (P < 0.001). The K-VTDS scores for the subscale of frequency and total scores were highest in the functional voice disorder group. We found a significant difference in frequency and total score of the K-VTDS between the functional voice disorder group and the structural voice disorder group (P < 0.05). We observed a strong positive correlation among the scores for the subscales of frequency and severity, and total scores in the K-VTDS. The K-VTDS showed moderate correlation with the K-VHI and the K-VRQOL. CONCLUSION: The K-VTDS is a reliable and valid instrument for voice assessment for voice disorders in Korean-speaking patients.

Potential Role of Heme Oxygenase-1 in the Resolution of Experimentally Induced Colitis through Regulation of Macrophage Polarization.

BACKGROUND/AIMS: Heme oxygenase-1 (HO-1) plays a central role in cellular defense against inflammatory insults, and its induction in macrophages potentiates their efferocytic activity. In this study, we explored the potential role of macrophage HO-1 in the resolution of experimentally induced colitis. METHODS: To induce colitis, male C57BL/6 mice were treated with 2% dextran sulfate sodium (DSS) in the drinking water for 7 days. To investigate efferocytosis, apoptotic colon epithelial CCD 841 CoN cells were coincubated with bone marrow-derived macrophages (BMDMs). RESULTS: Administration of the HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blunted the resolution of DSS-induced intestinal inflammation and expression of the proresolving M2 macrophage marker CD206. BMDMs treated with apoptotic colonic epithelial cells showed significantly elevated expression of HO-1 and its regulator Nrf2. Under the same experimental conditions, the proportion of CD206-expressing macrophages was also enhanced. ZnPP treatment abrogated the upregulation of CD206 expression in BMDMs engulfing apoptotic colonic epithelial cells. This result was verified with BMDMs isolated from HO-1-knockout mice. BMDMs, when stimulated with lipopolysaccharide, exhibited increased expression of CD86, a marker of M1 macrophages. Coculture of lipopolysaccharide-stimulated BMDMs with apoptotic colonic epithelial cell debris dampened the expression of CD86 as well as the pro-inflammatory cytokines in an HO-1-dependent manner. Genetic ablation as well as pharmacologic inhibition of HO-1 significantly reduced the proportion of efferocytic BMDMs expressing the scavenger receptor CD36. CONCLUSIONS: HO-1 plays a key role in the resolution of experimentally induced colitis by modulating the polarization of macrophages.