Single-cell T-cell receptor repertoire profiling in dogs.

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.

Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Microbiological Quality and Antimicrobial Resistance of Commercial Probiotic Products for Food-Producing Animals.

Probiotics have been popularly used in livestock production as an alternative to antibiotics. This study aimed to investigate the microbiological quality and phenotypic and genotypic antimicrobial resistance of bacteria in probiotic products sold for food animals. A total of 45 probiotic products were examined for the number of viable cells, species, and antimicrobial susceptibility; the contamination of Escherichia coli and Salmonella; and the presence of 112 genes encoding resistance to clinically important antimicrobials and transferability of AMR determinants. The results showed that 29 of 45 products (64.4%) were incorrectly labeled in either number of viable cells or bacterial species. None of the tested products were contaminated with E. coli and Salmonella. A total of 33 out of 64 bacterial isolates (51.6%) exhibited resistance to at least one antimicrobial agent. Of the 45 products tested, 16 (35.5%) carried AMR genes. Almost all AMR genes detected in probiotic products were not correlated to the AMR phenotype of probiotic strains formulated in the products. Three streptomycin-resistant Lactobacillus isolates could horizontally transfer their AMR determinants. The findings demonstrated that the probiotic products could serve as reservoirs for the spread of AMR genes and may not yield benefits to animals as claimed. The need for the adequate quality control of probiotic products is highlighted.

Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells.

Cytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed "surface shearing" experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewisx. Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells. IMPORTANCE: Campylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

Nanoscale details of mitochondrial constriction revealed by cryoelectron tomography.

Mitochondria adapt to changing cellular environments, stress stimuli, and metabolic demands through dramatic morphological remodeling of their shape, and thus function. Such mitochondrial dynamics is often dependent on cytoskeletal filament interactions. However, the precise organization of these filamentous assemblies remains speculative. Here, we apply cryogenic electron tomography to directly image the nanoscale architecture of the cytoskeletal-membrane interactions involved in mitochondrial dynamics in response to damage. We induced mitochondrial damage via membrane depolarization, a cellular stress associated with mitochondrial fragmentation and mitophagy. We find that, in response to acute membrane depolarization, mammalian mitochondria predominantly organize into tubular morphology that abundantly displays constrictions. We observe long bundles of both unbranched actin and septin filaments enriched at these constrictions. We also observed septin-microtubule interactions at these sites and elsewhere, suggesting that these two filaments guide each other in the cytosolic space. Together, our results provide empirical parameters for the architecture of mitochondrial constriction factors to validate/refine existing models and inform the development of new ones.

Prevalence and Associated Factors of optrA-Positive-Enterococcus faecalis in Different Reservoirs around Farms in Vietnam.

Linezolid is an antibiotic of last resort for the treatment of infections caused by Gram-positive bacteria, including vancomycin-resistant enterococci. Enterococcus faecalis, a member of enterococci, is a significant pathogen in nosocomial infections. E. faecalis resistance to linezolid is frequently related to the presence of optrA, which is often co-carried with fex, phenicol exporter genes, and erm genes encoding macrolide resistance. Therefore, the common use of antibiotics in veterinary might promote the occurrence of optrA in livestock settings. This is a cross-sectional study aiming to investigate the prevalence of optrA positive E. faecalis (OPEfs) in 6 reservoirs in farms in Ha Nam province, Vietnam, and its associated factors and to explore genetic relationships of OPEfs isolates. Among 639 collected samples, the prevalence of OPEfs was highest in flies, 46.8% (51/109), followed by chickens 37.3% (72/193), dogs 33.3% (17/51), humans 18.7% (26/139), wastewater 16.4% (11/67) and pigs 11.3%, (14/80). The total feeding area and total livestock unit of the farm were associated with the presence of OPEfs in chickens, flies, and wastewater. Among 186 OPEfs strains, 86% were resistant to linezolid. The presence of optrA was also related to the resistant phenotype against linezolid and levofloxacin of E. faecalis isolates. Close genotypic relationships identified by Pulsed Field Gel Electrophoresis between OPEfs isolates recovered from flies and other reservoirs including chickens, pigs, dogs, and wastewater suggested the role of flies in the transmission of antibiotic-resistant pathogens. These results provided warnings of linezolid resistance although it is not used in livestock.

An alternative mechanism for skeletal muscle dysfunction in long-term post-viral lung disease.

Chronic lung disease is often accompanied by disabling extrapulmonary symptoms, notably skeletal muscle dysfunction and atrophy. Moreover, the severity of respiratory symptoms correlates with decreased muscle mass and in turn lowered physical activity and survival rates. Previous models of muscle atrophy in chronic lung disease often modeled chronic obstructive pulmonary disease (COPD) and relied on cigarette smoke exposure and LPS stimulation, but these conditions independently affect skeletal muscle even without accompanying lung disease. Moreover, there is an emerging and pressing need to understand the extrapulmonary manifestations of long-term post-viral lung disease (PVLD) as found in COVID-19. Here, we examine the development of skeletal muscle dysfunction in the setting of chronic pulmonary disease caused by infection due to the natural pathogen Sendai virus using a mouse model of PVLD. We identify a significant decrease in myofiber size when PVLD is maximal at 49 days after infection. We find no change in the relative types of myofibers, but the greatest decrease in fiber size is localized to fast-twitch-type IIB myofibers based on myosin heavy chain immunostaining. Remarkably, all biomarkers of myocyte protein synthesis and degradation (total RNA, ribosomal abundance, and ubiquitin-proteasome expression) were stable throughout the acute infectious illness and chronic post-viral disease process. Together, the results demonstrate a distinct pattern of skeletal muscle dysfunction in a mouse model of long-term PVLD. The findings thereby provide new insights into prolonged limitations in exercise capacity in patients with chronic lung disease after viral infections and perhaps other types of lung injury.NEW & NOTEWORTHY Our study used a mouse model of post-viral lung disease to study the impact of chronic lung disease on skeletal muscle. The model reveals a decrease in myofiber size that is selective for specific types of myofibers and an alternative mechanism for muscle atrophy that might be independent of the usual markers of protein synthesis and degradation. The findings provide a basis for new therapeutic strategies to correct skeletal muscle dysfunction in chronic respiratory disease.

CIViCdb 2022: evolution of an open-access cancer variant interpretation knowledgebase.

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC's functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing >3200 variants in >470 genes from >3100 publications.

An intervention pilot to facilitate harm reduction service decentralization in Vietnam.

INTRODUCTION: Harm reduction services, including methadone maintenance therapy (MMT), have been decentralized to Vietnam's community health care settings. This study aims to pilot test an intervention to facilitate decentralized harm reduction service delivery in Vietnam. METHODS: The research team conducted an intervention pilot between August 2020 and May 2021 with six community MMT distribution sites in Thai Nguyen Province of Vietnam. We recruited five commune health workers (CHW) from each center (N = 30). In-person intervention training included content to correct misconceptions about harm reduction and reduce stigmatizing attitudes toward patients who use drugs and teach CHWs to self-examine and improve their service provision process. The study team developed a web-based platform to streamline CHW's patient monitoring and referral efforts. The team assessed intervention outcomes at baseline, 3-, and 6-months. CHWs in the intervention group provided acceptability ratings and feedback on the intervention at 6-months. RESULTS: CHWs in both intervention and control groups had similar background characteristics and outcome measures at baseline. CHWs in the intervention group, compared to those in the control group, showed a significantly higher level of improvement in adherence to service delivery protocol at 3-months. CHW in the intervention group had a significantly lower level of management-related stress compared to the control group at 6-months, although the intervention effect measured by the difference in change from baseline was not statistically significant. CHWs who participated in the final focus group reported high acceptability of the intervention. CONCLUSION: This intervention pilot demonstrated acceptability and promising outcomes on community-based harm reduction service delivery. Similar intervention strategies can be applied to enhance the decentralization of other chronic disease treatment services.

An alternative mechanism for skeletal muscle dysfunction in long-term post-viral lung disease.

Chronic lung disease is often accompanied by disabling extrapulmonary symptoms, notably skeletal muscle dysfunction and atrophy. Moreover, the severity of respiratory symptoms correlates with decreased muscle mass and in turn lowered physical activity and survival rates. Previous models of muscle atrophy in chronic lung disease often modeled COPD and relied on cigarette smoke exposure and LPS-stimulation, but these conditions independently affect skeletal muscle even without accompanying lung disease. Moreover, there is an emerging and pressing need to understand the extrapulmonary manifestations of long-term post-viral lung disease (PVLD) as found in Covid-19. Here, we examine the development of skeletal muscle dysfunction in the setting of chronic pulmonary disease using a mouse model of PVLD caused by infection due to the natural pathogen Sendai virus. We identify a significant decrease in myofiber size when PVLD is maximal at 49 d after infection. We find no change in the relative types of myofibers, but the greatest decrease in fiber size is localized to fast-twitch type IIB myofibers based on myosin heavy chain immunostaining. Remarkably, all biomarkers of myocyte protein synthesis and degradation (total RNA, ribosomal abundance, and ubiquitin-proteasome expression) were stable throughout the acute infectious illness and chronic post-viral disease process. Together, the results demonstrate a distinct pattern of skeletal muscle dysfunction in a mouse model of long-term PVLD. The findings thereby provide new insight into prolonged limitations in exercise capacity in patients with chronic lung disease after viral infections and perhaps other types of lung injury.

Evaluating the Safety and Quality of Life of Colorectal Cancer Patients Treated by Autologous Immune Enhancement Therapy (AIET) in Vinmec International Hospitals.

(1) Colorectal cancer (CRC) is an increasingly prevalent disease with a high mortality rate in recent years. Immune cell-based therapies have received massive attention among scientists, as they have been proven effective as low-toxicity treatments. This study evaluated the safety and effectiveness of autologous immune enhancement therapy (AIET) for CRC. (2) An open-label, single-group study, including twelve patients diagnosed with stages III and IV CRC, was conducted from January 2016 to December 2021. Twelve CRC patients received one to seven infusions of natural killer (NK)-cell and cytotoxic T-lymphocyte (CTL). Multivariate modelling was used to identify factors associated with health-related quality-of-life (HRQoL) scores. (3) After 20−21 days of culture, the NK cells increased 3535-fold, accounting for 85% of the cultured cell population. Likewise, CTLs accounted for 62.4% of the cultured cell population, which was a 1220-fold increase. Furthermore, the QoL improved with increased EORTC QLQ-C30 scores, decreased symptom severity, and reduced impairment in daily living caused by these symptoms (MDASI-GI report). Finally, a 14.3 ± 14.1-month increase in mean survival time was observed at study completion. (4) AIET demonstrated safety and improved survival time and HRQoL for CRC patients in Vietnam.

Differential Cytotoxicity of Curcumin-Loaded Micelles on Human Tumor and Stromal Cells.

Although curcumin in the form of nanoparticles has been demonstrated as a potential anti-tumor compound, the impact of curcumin and nanocurcumin in vitro on normal cells and in vivo in animal models is largely unknown. This study evaluated the toxicity of curcumin-loaded micelles in vitro and in vivo on several tumor cell lines, primary stromal cells, and zebrafish embryos. Breast tumor cell line (MCF7) and stromal cells (human umbilical cord vein endothelial cells, human fibroblasts, and human umbilical cord-derived mesenchymal stem cells) were used in this study. A zebrafish embryotoxicity (FET) assay was conducted following the Organisation for Economic Co-operation and Development (OECD) Test 236. Compared to free curcumin, curcumin PM showed higher cytotoxicity to MCF7 cells in both monolayer culture and multicellular tumor spheroids. The curcumin-loaded micelles efficiently penetrated the MCF7 spheroids and induced apoptosis. The nanocurcumin reduced the viability and disturbed the function of stromal cells by suppressing cell migration and tube formation. The micelles demonstrated toxicity to the development of zebrafish embryos. Curcumin-loaded micelles demonstrated toxicity to both tumor and normal primary stromal cells and zebrafish embryos, indicating that the use of nanocurcumin in cancer treatment should be carefully investigated and controlled.

Dual roles of oxostephanine as an Aurora kinase inhibitor and angiogenesis suppressor.

The Aurora kinases, including Aurora A, B and C, play critical roles in cell division. They have been found overexpressed in a number of types of cancer and may thus be potential targets in cancer therapy. Several Aurora kinase inhibitors have been identified and developed. Some of these have been used in clinical trials and have exhibited certain efficacy in cancer treatment. However, none of these has yet been applied clinically due to the poor outcomes. Oxostephanine is an aporphine alkaloid isolated from several plants of the genus Stephania. This compound has been reported to inhibit Aurora kinase activity in kinase assays and in cancer cells. The present study aimed to investigate the real­time effects of oxostephanine extracted from Stephania dielsiana Y.C. Wu leaves on the growth of an ovarian cancer cell line (OVCAR­8, human ovarian carcinoma); these effects were compared to those of the well­known Aurora kinase inhibitor, VX­680. The effects of oxostephanine on stromal cells, as well as endothelial cells were also examined. The results demonstrated that oxostephanine was an Aurora kinase inhibitor through the prevention of histone H3 phosphorylation at serine 10, the mislocalization of Aurora B and the induction of aneuploidy. Moreover, this substance was selectively cytotoxic to human umbilical vein endothelial cells (hUVECs), whereas it was less cytotoxic to human fibroblasts and umbilical cord­derived mesenchymal stem cells. In addition, this compound significantly attenuated the migration and tube formation ability of hUVECs. Taken together, the present study demonstrates that oxostephanine plays dual roles in inhibiting Aurora kinase activity and angiogenesis. Thus, it may have potential for use as a drug in cancer treatment.

Colibactin-Producing Escherichia coli Induce the Formation of Invasive Carcinomas in a Chronic Inflammation-Associated Mouse Model.

BACKGROUND: Escherichia coli producing the genotoxin colibactin (CoPEC or colibactin-producing E. coli) abnormally colonize the colonic mucosa of colorectal cancer (CRC) patients. We previously showed that deficiency of autophagy in intestinal epithelial cells (IECs) enhances CoPEC-induced colorectal carcinogenesis in ApcMin/+ mice. Here, we tested if CoPEC trigger tumorigenesis in a mouse model lacking genetic susceptibility or the use of carcinogen. METHODS: Mice with autophagy deficiency in IECs (Atg16l1∆IEC) or wild-type mice (Atg16l1flox/flox) were infected with the CoPEC 11G5 strain or the mutant 11G5∆clbQ incapable of producing colibactin and subjected to 12 cycles of DSS treatment to induce chronic colitis. Mouse colons were used for histological assessment, immunohistochemical and immunoblot analyses for DNA damage marker. Results: 11G5 or 11G5∆clbQ infection increased clinical and histological inflammation scores, and these were further enhanced by IEC-specific autophagy deficiency. 11G5 infection, but not 11G5∆clbQ infection, triggered the formation of invasive carcinomas, and this was further increased by autophagy deficiency. The increase in invasive carcinomas was correlated with enhanced DNA damage and independent of inflammation. Conclusions: CoPEC induce colorectal carcinogenesis in a CRC mouse model lacking genetic susceptibility and carcinogen. This work highlights the role of (i) CoPEC as a driver of CRC development, and (ii) autophagy in inhibiting the carcinogenic properties of CoPEC.

Nuclear trafficking of bacterial effector proteins.

Bacterial pathogens can subvert host responses by producing effector proteins that directly target the nucleus of eukaryotic cells in animals and plants. Nuclear-targeting proteins are categorised as either: "nucleomodulins," which have epigenetic-modulating activities; or "cyclomodulins," which specifically interfere with the host cell cycle. Bacteria can deliver these effector proteins to eukaryotic cells via a range of strategies. Despite an increasing number of reports describing the effects of bacterial effector proteins on nuclear processes in host cells, the intracellular pathways used by these proteins to traffic to the nucleus have yet to be fully elucidated. This review will describe current knowledge about how nucleomodulins and cyclomodulins enter eukaryotic cells, exploit endocytic pathways and translocate to the nucleus. We will also discuss the secretion of nuclear-targeting proteins or their release in bacterial membrane vesicles and the trafficking pathways employed by each of these forms. Besides their importance for bacterial pathogenesis, some nuclear-targeting proteins have been implicated in the development of chronic diseases and even cancer. A greater understanding of nuclear-targeting proteins and their actions will provide new insights into the pathogenesis of infectious diseases, as well as contribute to advances in the development of novel therapies against bacterial infections and possibly cancer.

Molecular typing of conjunctivitis-causing adenoviruses in Hanoi, Vietnam from 2017 to 2019 and complete genome analysis of the most prevalent type (HAdV-8).

Adenoviral conjunctivitis is a common epidemic worldwide. In Vietnam, up to 80,000 patients are infected with adenoviral conjunctivitis annually. However, there are few investigations on the pathogenic adenoviruses that cause conjunctivitis. In total, 120 eye-swab samples were collected from patients with viral conjunctivitis symptoms in Hanoi, Vietnam from 2017 to 2019. Human adenoviruse (HAdV) was detected in 67 samples (55.83%) using polymerase chain reaction amplification of at least one of three HAdV-specific marker genes (hexon, penton, and fiber). Of the 67 HAdV samples, 46 samples could be analyzed by all three marker genes. DNA sequence analysis and phylogenetic tree building based on the three marker genes from the 46 HAdV samples revealed five different HAdV types associated with conjunctivitis in Hanoi, including HAdV-3 (4.3%), HAdV-4 (2.2%), HAdV-8 (89.1%), HAdV-37 (2.2%), and a potential recombinant type between types HAdV-8 and HAdV-3 (2.2%). This showed that HAdV-8 was the most common type identified in Hanoi. Complete genome analysis of HAdV-8 isolated from a Vietnamese patient (VN2017) using Sanger sequencing revealed 34 unique nucleotide changes, indicating that the adenovirus continuously accumulates new mutations. Hence, continuous surveillance of HAdV-8 changes in Vietnam is necessary in the future.

Application of a Sulfoxonium Ylide Electrophile to Generate Cathepsin X-Selective Activity-Based Probes.

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.

Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-Producing Escherichia coli in ApcMin/+ Mice.

BACKGROUND & AIMS: Colibactin-producing Escherichia coli (CoPEC) colonize the colonic mucosa of a higher proportion of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC. METHODS: Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. ApcMin/+ mice and ApcMin/+ mice with IEC-specific disruption of Atg16l1 (ApcMin/+/Atg16l1ΔIEC) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by immunohistochemical staining, immunoblot, and quantitative reverse transcription polymerase chain reaction for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction. RESULTS: Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. Under uninfected condition, ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, after infection with 11G5, ApcMin/+/Atg16l1ΔIEC mice developed more and larger tumors, with a significant increase in mean histologic score, than infected ApcMin/+ mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected ApcMin/+/Atg16l1ΔIEC mice vs 11G5-infected ApcMin/+ mice. CONCLUSION: Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in 11G5-infected ApcMin/+ mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.

Ocular delivery of proteins and peptides: Challenges and novel formulation approaches.

The impact of proteins and peptides on the treatment of various conditions including ocular diseases over the past few decades has been advanced by substantial breakthroughs in structural biochemistry, genetic engineering, formulation and delivery approaches. Formulation and delivery of proteins and peptides, such as monoclonal antibodies, aptamers, recombinant proteins and peptides to ocular tissues poses significant challenges owing to their large size, poor permeation and susceptibility to degradation. A wide range of advanced drug delivery systems including polymeric controlled release systems, cell-based delivery and nanowafers are being exploited to overcome the challenges of frequent administration to ocular tissues. The next generation systems integrated with new delivery technologies are anticipated to generate improved efficacy and safety through the expansion of the therapeutic target space. This review will highlight recent advances in formulation and delivery strategies of protein and peptide based biopharmaceuticals. We will also describe the current state of proteins and peptides based ocular therapy and future therapeutic opportunities.

Chaetocin enhances dendritic cell function via the induction of heat shock protein and cancer testis antigens in myeloma cells.

Dendritic cells (DC)-based vaccines are considered useful in cancer immuno-therapy, and the interactions of DC and dying tumor cells are important and promising for cancer immunotherapy. We investigated whether chaetocin could be used to induce death of myeloma cells, for loading onto DCs can affect DCs function. In this study, we show that the dying myeloma cells treated with chaetocin resulted in the induction of heat shock protein (HSP) 90, which was inhibited by antioxidant N-acetyl cysteine, and showed an increase in the expression of MAGE-A3 and MAGE-C1/CT7. DCs loaded with chaetocin-treated dying myeloma cells produced low levels of IL-10 and enhanced the cross presentation of DCs. Additionally, these DCs most potently inhibited regulatory T cells, induced Th1 polarization and activated myeloma-specific cytotoxic T lymphocytes compared with DCs loaded with UVB-irradiated dying myeloma cells. These results suggest that the pretreatment of myeloma cells with chaetocin can enhance DC function through the up-regulation of HSP90 and cancer testis antigens in dying myeloma cells and can potently induce the Th1 polarization of DCs and myeloma-specific cytotoxic T lymphocytes.