De Novo Epigenetic Programs Inhibit PD-1 Blockade-Mediated T Cell Rejuvenation.

Immune-checkpoint-blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA-methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells, and approaches to reverse these programs improved T cell responses and tumor control during ICB. These data establish de novo DNA-methylation programming as a regulator of T cell exhaustion and barrier of ICB-mediated T cell rejuvenation.

Lung γδ T Cells Mediate Protective Responses during Neonatal Influenza Infection that Are Associated with Type 2 Immunity.

Compared to adults, infants suffer higher rates of hospitalization, severe clinical complications, and mortality due to influenza infection. We found that γδ T cells protected neonatal mice against mortality during influenza infection. γδ T cell deficiency did not alter viral clearance or interferon-γ production. Instead, neonatal influenza infection induced the accumulation of interleukin-17A (IL-17A)-producing γδ T cells, which was associated with IL-33 production by lung epithelial cells. Neonates lacking IL-17A-expressing γδ T cells or Il33 had higher mortality upon influenza infection. γδ T cells and IL-33 promoted lung infiltration of group 2 innate lymphoid cells and regulatory T cells, resulting in increased amphiregulin secretion and tissue repair. In influenza-infected children, IL-17A, IL-33, and amphiregulin expression were correlated, and increased IL-17A levels in nasal aspirates were associated with better clinical outcomes. Our results indicate that γδ T cells are required in influenza-infected neonates to initiate protective immunity and mediate lung homeostasis.

Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves.

Escherichia coli field isolates from calves were characterized and categorized into the most significant diarrheagenic pathotypes using polymerase chain reaction (PCR) assays with different specific primers. The used PCR systems were designed to detect sequences representing the group-specific virulence genes encoding fimbriae f5 (K99), Shiga toxins (stx1 and stx2), heat-stable enterotoxins (st), heat-labile enterotoxins (lt), intimin (eae), hemolysin (hylA), and EAEC heat-stable enterotoxin (astA). In the present work, a total of 150 E. coli field isolates were recovered from 150 fecal swabs collected from 100 diarrheic and 50 apparently healthy in-contact cattle and buffalo calves under 3 months old. Out of these 150 isolated E. coli, 106 isolates from 77 diarrheic and 29 in-contact calves harbored one or more of the investigated virulence genes. The pathotyping of the isolates could classify them into shigatoxigenic E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) with a 30.7, 2.7, 12.7, and 7.3% distribution, respectively. Meanwhile, the detection rates of f5, stx1, stx2, st, lt, eae, hylA, and astA genes were 17.3, 27.3, 6.7, 10, 37.3, 17.7, 9.3, and 20.7%, respectively. These virulence genes were found either single or in different combinations, such as stx/eae, stx/st/f5, eae/st/f5, or st/lt/f5. Four attaching-effacing shigatoxigenic E. coli isolates (AE-STEC) harboring stx/eae were retrieved from diarrheic calves. Although none of the stx-or eae-positive isolates was verified as O157:H7, STEC isolates detected in apparently healthy calves have potential pathogenicity to humans highlighting their zoonotic importance as reservoirs. Atypical combinations of ETEC/STEC and ETEC/EPEC were also detected in percentages of 14.7 and 2.7%, respectively. Most of these atypical combinations were found more in buffalo calves than in cattle calves. While STEC and EPEC isolates were detected more in cattle calves than in buffalo calves, ETEC isolates were the same in the two species. The pathogenic E. coli infection in calves was recorded to be higher in the first weeks of life with the largest numbers of virulence factor-positive isolates detected at the age of 4 weeks. Histopathological examination of five intestinal samples collected from four dead buffalo calves revealed typical attaching and effacing (AE) lesion which was correlated with the presence of intimin encoding virulence gene (eae). Other lesions characterized by hemorrhagic enteritis, shortening and fusion of intestinal villi and desquamation of the lining epithelium of intestinal mucosa had also been detected.

Endoplasmic reticulum (ER) stress and hypoxia response pathways interact to potentiate hypoxia-inducible factor 1 (HIF-1) transcriptional activity on targets like vascular endothelial growth factor (VEGF).

Cells respond to suboptimal microenvironments by activating stress signaling pathways, like the unfolded protein response and hypoxia-induced transcription factors HIF-1/2, to restore homeostasis. Both cytoprotective pathways have been well studied in isolation at the biochemical and molecular levels. Mounting evidence reveals that they can be activated simultaneously in tumor cells and, likely, in other tissues experiencing inadequate microenvironments and that they share some transcriptional targets, like the proangiogenic factor VEGFA. However, the potential interaction between these pathways is poorly understood. Cell culture experiments revealed that as a consequence of unfolded protein response activation, ATF4 bound to the human VEGFA promoter and activated its transcription, whereas HIF-1 did so in response to hypoxia. When both pathways were activated together, VEGFA transcripts were induced to a higher level than when either stress was applied alone. Surprisingly, this was not due to the combined actions of the stress pathway-specific transcription factors. Instead, we found that endoplasmic reticulum stress potentiated HIF-1 activity to transactivate VEGF expression as well as another well characterized target, BNIP3. These data reveal an unexpected interaction between two important cytoprotective responses that are likely to have significant consequences in environmentally compromised tissues and tumor cells.

DNAJB1-PRKACA fusion neoantigens elicit rare endogenous T cell responses that potentiate cell therapy for fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 T cells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC.

Spatiotemporal development of the human T follicular helper cell response to Influenza vaccination.

We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.

Regulated release of ERdj3 from unfolded proteins by BiP.

DnaJ proteins often bind to unfolded substrates and recruit their Hsp70 partners. This induces a conformational change in the Hsp70 that stabilizes its binding to substrate. By some unknown mechanism, the DnaJ protein is released. We examined the requirements for the release of ERdj3, a mammalian ER DnaJ, from substrates and found that BiP promoted the release of ERdj3 only in the presence of ATP. Mutations in ERdj3 or BiP that disrupted their interaction interrupted the release of ERdj3. BiP mutants that were defective in any step of the ATPase cycle were also unable to release ERdj3. These results demonstrate that a functional interaction between ERdj3 and BiP, including both a direct interaction and the ability to stimulate BiP's ATPase activity are required to release ERdj3 from substrate and support a model where ERdj3 must recruit BiP and stimulate its high-affinity association with the substrate through activation of ATP hydrolysis to trigger its own release from substrates. On the basis of similarities among DnaJs and Hsp70s, this is likely to be applicable to other Hsp70-DnaJ pairs.

The role of Musca domestica and milk in transmitting pathogenic multidrug-resistant Escherichia coli and associated phylogroups to neonatal calves.

Escherichia coli, as a global source of antimicrobial resistance, is a serious veterinary and public health concern. The transmission of pathogenic multidrug-resistant (MDR) E. coli within diarrheic calves and its correlation with Musca domestica and milk strains have been investigated. In total, 110, 80, and 26 E. coli strains were obtained from 70 rectal swabs from diarrheic calves, 60 milk samples and 20 M. domestica, respectively. Molecular pathotyping of E. coli revealed the presence of pathogenic E. coli with a higher percentage of shigatoxigenic strains within diarrheic calves and M. domestica at 46.4% and 34.6%, respectively. Phenotypic antimicrobial resistance revealed higher ß-lactams resistance except for cefquinome that exhibited low resistance in M.domestica and milk strains at 30.8% and 30%, respectively. The extended-spectrum cephalosporin (ESC) resistant strains were detected within fecal, M. domestica, and milk strains at 69.1%, 73.1%, and 71.3%, respectively. All E. coli strains isolated from M. domestica exhibited MDR, while fecal and milk strains were harboring MDR at 99.1% and 85%, respectively. Molecular detection of resistant genes revealed the predominance of the blaTEM gene, while none of these strains harbored the blaOXA gene. The highest percentages for blaCTXM and blaCMYII genes were detected in M. domestica strains at 53.8% and 61.5%, respectively. Regarding colistin resistance, the mcr-1 gene was detected only in fecal and milk strains at 35.5% and 15%, respectively. A high frequency of phylogroup B2 was detected within fecal and M. domestica strains, while milk strains were mainly assigned to the B1 phylogroup. Pathogenic E. coli strains with the same phenotypic and genotypic antimicrobial resistance and phylogroups were identified for both diarrheic calves and M. domestica, suggesting that the possible role of M. domestica in disseminating pathogenic strains and antimicrobial resistance in dairy farms.

Immunological, Histopathological, Molecular Identification and Genotyping of Toxoplasma gondii in Small Ruminants in Egypt.

<b>Background and Objective:</b> Toxoplasmosis is an infective zoonotic disease caused by protozoan <i>Toxoplasma gondii </i>(<i>T. gondii</i>).<i> </i>Molecular identification of <i>T. gondii</i> followed by studying the hereditary variety range of <i>T. gondii </i>isolates in Egypt was investigated. <b>Materials and Methods:</b> Blood samples were acquired from 138 live ewes and 212 she-goats from 5 governorates of Egypt, also the blood and its related tissue samples (uterus, diaphragm, heart and thigh muscles from each animal) were collected from slaughtered 180 ewes and 206 she-goats from Cairo and Giza abattoirs. <b>Results:</b> Using ELISA, the total seropositivity of live ewes and she-goats was 26.8 and 21.2%, respectively, while it was 16.6 and 33% in slaughtered ewes and she-goats, respectively. <i>T. gondii</i> tissue cysts with the associated characteristic histopathological changes were detected in different organs. Twenty-eight <i>T. gondii</i> isolates were confirmed using PCR, while among 24 milk samples from seropositive live ewes and she-goats, only 12.5 and 6.25%, were positive using PCR, respectively. Genotyping using multiple nested PCR (n-PCR) combined with restriction enzyme analysis (RFLP) of the surface antigen 2 (SAG2) gene confirmed 26 isolates (92.8%) as type II and 2 (7.1%) as type III. <b>Conclusion:</b> Type II and III are the most common <i>T. gondii</i> genotypes in Egyptian small ruminants with additional importance for public health in Egypt. Further studies are needed on the role of milk in the transmission of toxoplasmosis.

BiP mutants that are unable to interact with endoplasmic reticulum DnaJ proteins provide insights into interdomain interactions in BiP.

The heat shock protein (Hsp)70 family of molecular chaperones interacts with unfolded proteins through a C-terminal substrate-binding domain (SBD) that is controlled by nucleotide binding to the N-terminal domain. The ATPase cycle is regulated by cochaperones, including DnaJ proteins that accelerate ATP hydrolysis to stabilize the Hsp70-substrate complex. We found that R197 in hamster BiP, which resides at the surface of the nucleotide-binding domain, is critical for both association with endoplasmic reticulum DnaJ proteins and interaction with the SBD. Decreasing the positive charge at this residue enhanced basal ATPase activity, destabilized interaction with the SBD, and reduced substrate release both in vitro and in vivo. Mutation of three glutamic acids in the SBD mimicked many of these effects. Our data provide insights into communications between the two domains and suggest a mechanism by which DnaJ proteins increase ATP hydrolysis.

Evaluation of different diagnostic methods for diagnosis of Lumpy skin disease in cows.

Viral isolation, polymerase chain reaction (PCR), dot blot hybridization (DBH), and indirect enzyme-linked immunosorbent assay (iELISA) were used for the diagnosis of lumpy skin disease in clinically infected, fevered, and apparently normal dairy cows. Lumpy skin disease virus (LSDV) was isolated from skin biopsies and blood samples collected from clinically infected cows in percentages of 72% and 20%, respectively. The virus recovered from blood samples collected from fevered cows in percentage of 33.3%. Both PCR and DBH detected viral DNA in 100% of skin biopsies collected from clinically infected cows whereas the detection rates in blood samples collected from clinically infected animals were 100% and 84% using PCR and DBH, respectively. Viral DNA was detected in blood samples collected from fevered cows using PCR and DBH in percentages of 77.8% and 66.6%, respectively. Only 19.1% of blood samples collected from in-contact cows was positive for both of PCR and DBH. Detection rates of antibodies against LSDV using iELISA in serum samples collected from clinically infected and fevered cows were 56% and 11.1%, respectively, whereas all in-contact cows had no antibodies against the virus.

Serological evaluation for the current epidemic situation of foot and mouth disease among cattle and buffaloes in Egypt.

AIM: The present study was aimed to investigate the epidemic situation of foot-and-mouth disease (FMD) in Egypt from 2016 to 2018 based on the detection of FMD virus (FMDV) in carrier or previously infected animals, by determination of antibodies against non-structural protein (NSP), implementation a pilot study on circulating FMDV serotypes and assure the efficacy of locally produced inactivated trivalent vaccine. MATERIALS AND METHODS: A total of 1500 sera were collected from apparent healthy vaccinated cattle and buffaloes from three Egyptian geographical sectors, representing ten governorates. Determination of FMD antibodies against NSP was carried out using 3ABC enzyme-linked immunosorbent assay (ELISA) test. Serotyping of the circulating FMDV and assure the vaccine efficacy was performed using solid-phase competitive ELISA. RESULTS: The 3ABC ELISA test revealed 26.4% and 23.7% positive for FMDV-NSP antibodies in cattle and buffalo sera, respectively. The highest positivity was in Delta Sector among both cattle 42.3% and buffaloes 28.8%. Serotyping of FMDV-positive NSP sera in El-Qalyubia Governorate for the circulating FMDV serotypes O, A, and Southern African Territories (SAT) 2 was 52.2%, 17.4%, and 30.4% in cattle and 31.8%, 27.3%, and 40.9% in buffaloes, respectively. The overall protection level due to the vaccination program was 62.1 and 60.9% in cattle and buffaloes, respectively, while the protective level of the FMDV serotypes O, A, and SAT2 included in the inactivated trivalent vaccine was 73.9, 84.6, and 63.8% in cattle and 72.3, 82.3, and 63.5% in buffaloes, respectively. CONCLUSION: The present study recommended full determination for the immunogenic relationship between the vaccine strains and the field strains to attain maximum protection against the circulating viruses.

A population of proinflammatory T cells coexpresses αß and γδ T cell receptors in mice and humans.

T cells are classically recognized as distinct subsets that express αß or γδ TCRs. We identify a novel population of T cells that coexpress αß and γδ TCRs in mice and humans. These hybrid αß-γδ T cells arose in the murine fetal thymus by day 16 of ontogeny, underwent αß TCR-mediated positive selection into CD4+ or CD8+ thymocytes, and constituted up to 10% of TCRδ+ cells in lymphoid organs. They expressed high levels of IL-1R1 and IL-23R and secreted IFN-γ, IL-17, and GM-CSF in response to canonically restricted peptide antigens or stimulation with IL-1ß and IL-23. Hybrid αß-γδ T cells were transcriptomically distinct from conventional γδ T cells and displayed a hyperinflammatory phenotype enriched for chemokine receptors and homing molecules that facilitate migration to sites of inflammation. These proinflammatory T cells promoted bacterial clearance after infection with Staphylococcus aureus and, by licensing encephalitogenic Th17 cells, played a key role in the development of autoimmune disease in the central nervous system.

Pediatric patients with acute lymphoblastic leukemia generate abundant and functional neoantigen-specific CD8+ T cell responses.

Cancer arises from the accumulation of genetic alterations, which can lead to the production of mutant proteins not expressed by normal cells. These mutant proteins can be processed and presented on the cell surface by major histocompatibility complex molecules as neoepitopes, allowing CD8+ T cells to mount responses against them. For solid tumors, only an average 2% of neoepitopes predicted by algorithms have detectable endogenous antitumor T cell responses. This suggests that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphoblastic leukemia (ALL) have tumor-associated neoepitope-specific CD8+ T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. These responses include a public neoantigen from the ETV6-RUNX1 fusion that is targeted in seven of nine tested patients. We characterized phenotypic and transcriptional profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at the single-cell level and found a heterogeneous population that included highly functional effectors. Moreover, we observed immunodominance hierarchies among the CD8+ TILs restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in pediatric ALL despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. Furthermore, these data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens.

Brucellosis: Evolution and expected comeback.

Brucellosis is a serious infectious disease which causes great direct and indirect economic loses for animal holders worldwide such as the reduction of milk and meat production through abortions/culling of positive reactors, the expense of disease control/eradication and farmers compensation. Although the disease was eradicated from most of the industrial countries, it remains one of the most common zoonotic diseases in developing countries being responsible for more than 500,000 new cases yearly. Brucella is considered to be a bioterrorism organism due to its low infectious doses (10-100 bacteria), capability of persistence in the environment, rapid transmission via different routes including aerosols, and finally due to its difficult treatment by antibiotics.There are many reasons to believe that a new comeback of brucellosis may occur in near future. This expectation is supported by the recent discovery of new atypical Brucella species with new genetic properties and the recent reports of (man to man) disease transmission as will be discussed later. The development of new concepts and measurements for disease control is urgently required. In the present review, the evolution of Brucella and the different factors favoring its comeback are discussed.

Metabolic signaling directs the reciprocal lineage decisions of αß and γδ T cells.

The interaction between extrinsic factors and intrinsic signal strength governs thymocyte development, but the mechanisms linking them remain elusive. We report that mechanistic target of rapamycin complex 1 (mTORC1) couples microenvironmental cues with metabolic programs to orchestrate the reciprocal development of two fundamentally distinct T cell lineages, the αß and γδ T cells. Developing thymocytes dynamically engage metabolic programs including glycolysis and oxidative phosphorylation, as well as mTORC1 signaling. Loss of RAPTOR-mediated mTORC1 activity impairs the development of αß T cells but promotes γδ T cell generation, associated with disrupted metabolic remodeling of oxidative and glycolytic metabolism. Mechanistically, we identify mTORC1-dependent control of reactive oxygen species production as a key metabolic signal in mediating αß and γδ T cell development, and perturbation of redox homeostasis impinges upon thymocyte fate decisions and mTORC1-associated phenotypes. Furthermore, single-cell RNA sequencing and genetic dissection reveal that mTORC1 links developmental signals from T cell receptors and NOTCH to coordinate metabolic activity and signal strength. Our results establish mTORC1-driven metabolic signaling as a decisive factor for reciprocal αß and γδ T cell development and provide insight into metabolic control of cell signaling and fate decisions.

Molecular biological tools applied for identification of mastitis causing pathogens.

The molecular diagnostic tools became the gold standard of mastitis diagnosis in the last few years. They enable rapid, qualitative, quantitative and large scale diagnosis. In addition to their role in diagnosis, they can identify pathogens at the subspecies level which is necessary for the epidemiological studies. They are increasingly used in mastitis control programs through identification of suitable candidates for vaccine production and through the selection of mastitis resistant cattle breeds. The present molecular techniques are continuously improved and new techniques are developed in order to provide higher sensitivity and specificity and to minimize the costs. The present work aims to provide an overview of the modern molecular tools, discuss why they replaced the traditional tools and became the new gold standard in mastitis diagnosis through comparing both traditional and molecular tools, explore the prospective of the molecular diagnostic techniques in mastitis diagnosis and control and to explore new horizons of using molecular assays in near future.