Influenza A virus (IAV) is known to cause mild to severe respiratory illness. Under some conditions, the infection can lead to pneumonia (viral or bacterial), acute respiratory distress syndrome, and other complications that can be fatal, especially in vulnerable populations such as the elderly, young children, and individuals with underlying health conditions. Despite previous studies, little is known about the host immune response and neuroimmune interactions in IAV infection. Using RNA sequencing, we performed transcriptomic analysis of murine lung tissue 21 days post infection (dpi) with IAV (H1N1) in order to find the differentially expression genes (DEGs) related to the host immune response and neuroimmune interactions inside the lung during recovery. Among 792 DEGs, 434 genes were up-regulated, whereas 358 genes were down-regulated. The most prominent molecular functions of the up-regulated genes were related to the immune response and tissue repair, whereas a large proportion of the down-regulated genes were associated with neural functions. Although further molecular/functional studies need to be performed for these DEGs, our results facilitate the understanding of the host response (from innate immunity to adaptive immunity) and neuroimmune interactions in infected lungs at the recovery stage of IAV infection. These genes might have potential uses as mechanistic/diagnostic biomarkers and represent possible targets for anti-IAV therapies.

Lymphoma is a cancer arising from B or T lymphocytes that are central immune system ‎components. It is one of the three most common cancers encountered in the canine; ‎lymphoma affects middle-aged to older dogs and usually stems from lymphatic tissues, ‎such as lymph nodes, lymphoid tissue, or spleen. Despite the advance in the management of ‎canine lymphoma, a better understanding of the subtype and tumor aggressiveness is still ‎crucial for improved clinical diagnosis to differentiate malignancy from hyperplastic ‎conditions and to improve decision-making around treating and what treatment type to use. ‎This study aimed to evaluate a potential novel biomarker related to iron metabolism, ‎embryonic haemoglobin (HBE), for early diagnosis. Archived samples in combination with ‎prospective samples collected from dogs with and without lymphoma were used in this ‎study for the retrospective analyses of this tumor based on the same biomarker amplified by ‎real-time quantitative polymerase chain reaction. The HBE mRNA was aberrantly expressed ‎in canine B and T cell lymphoma compared to the normal lymph node tissue and ‎hyperplastic lymph nodes. In conclusion, this study identified a novel potential biomarker for improving lymphoma ‎diagnosis and treatment in dogs. Further studies with larger sample sizes are needed to ‎confirm the suitability of this biomarker for canine lymphoma diagnosis.

The central nervous system (CNS) influences the immune system generally by regulating the systemic concentration of humoral substances (e.g., cortisol and epinephrine), whereas the peripheral nervous system (PNS) communicates specifically with the immune system according to local interactions/connections. An imbalance between the components of the PNS might contribute to pathogenesis and the further development of certain diseases. In this review, we have explored the "thread" (hardwiring) of the connections between the immune system (e.g., primary/secondary/tertiary lymphoid tissues/organs) and PNS (e.g., sensory, sympathetic, parasympathetic, and enteric nervous systems (ENS)) in health and disease in vitro and in vivo. Neuroimmune cell units provide an anatomical and physiological basis for bidirectional crosstalk between the PNS and the immune system in peripheral tissues, including lymphoid tissues and organs. These neuroimmune interactions/modulation studies might greatly contribute to a better understanding of the mechanisms through which the PNS possibly affects cellular and humoral-mediated immune responses or vice versa in health and diseases. Physical, chemical, pharmacological, and other manipulations of these neuroimmune interactions should bring about the development of practical therapeutic applications for certain neurological, neuroimmunological, infectious, inflammatory, and immunological disorders/diseases.

Background Influenza A virus (IAV) is the only influenza virus causing flu pandemics (i.e., global epidemics of flu disease). Influenza (the flu) is a highly contagious disease that can be deadly, especially in high-risk groups. Worldwide, these annual epidemics are estimated to result in about 3 to 5 million cases of severe illness and in about 290,000 to 650,000 respiratory deaths. We intend to reveal the effect of IAV infection on the host′s metabolism, immune response, and neurotoxicity by using a mouse IAV infection model. Methods 51 metabolites of murine blood plasma (33 amino acids/amino acid derivatives (AADs) and 18 metabolites of the tryptophan pathway) were analyzed by using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry with Electrospray Ionization at the acute (7 days post-infection (dpi)), resolution (14 dpi), and recovery (21 dpi) stages of the virus infection in comparison with controls. Results Among the 33 biogenic amino acids/AADs, the levels of five amino acids/AADs (1-methylhistidine, 5-oxoproline, α-aminobutyric acid, glutamine, and taurine) increased by 7 dpi, whereas the levels of ten amino acids/AADs (4-hydroxyproline, alanine, arginine, asparagine, cysteine, citrulline, glycine, methionine, proline, and tyrosine) decreased. By 14 dpi, the levels of one AAD (3-methylhistidine) increased, whereas the levels of five amino acids/AADs (α-aminobutyric acid, aminoadipic acid, methionine, threonine, valine) decreased. Among the 18 metabolites from the tryptophan pathway, the levels of kynurenine, quinolinic acid, hydroxykynurenine increased by 7 dpi, whereas the levels of indole-3-acetic acid and nicotinamide riboside decreased. Conclusions Our data may facilitate understanding the molecular mechanisms of host responses to IAV infection and provide a basis for discovering potential new mechanistic, diagnostic, and prognostic biomarkers and therapeutic targets for IAV infection.

Coronavirus disease 2019 (COVID-19) is an extremely contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early disease recognition of COVID-19 is crucial not only for prompt diagnosis and treatment of the patients, but also for effective public health surveillance and response. The reverse transcription-polymerase chain reaction (RT-PCR) is the most common method for the detection of SARS-CoV-2 viral mRNA and is regarded as the gold standard test for COVID-19. However, this test and those for antibodies (IgM and IgG) and antigens have certain limitations (e.g., by yielding false-negative and false-positive results). We have developed an RNA fluorescence in situ hybridization (FISH) method for high-sensitivity detection of SARS-CoV-2 mRNAs in HEK 293T cell cultures as a model. After transfection of HEK 293T cells with plasmids, Spike (S)/envelope (E) proteins and their mRNAs were clearly detected inside the cells. In addition, hybridization time could be reduced to 2 hours for faster detection when probe concentration was increased. Our approach might thus significantly improve the sensitivity and specificity of SARS-CoV-2 detection and be widely applied for the high-sensitivity single-molecular detection of other RNA viruses (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Hepatitis A virus, all influenza viruses, and human immunodeficiency virus (HIV)) in various types of samples including tissue, body fluid, blood, and water. RNA FISH can also be utilized for the detection of DNA viruses (e.g., Monkeypox virus, human papillomavirus (HPV), and cytomegalovirus (CMV)) by detection of their mRNAs inside cells or body fluid.

The central nervous system regulates the immune system through the secretion of hormones from the pituitary gland and other endocrine organs, while the peripheral nervous system (PNS) communicates with the immune system through local nerve-immune cell interactions, including sympathetic/parasympathetic (efferent) and sensory (afferent) innervation to lymphoid tissue/organs. However, the precise mechanisms of this bi-directional crosstalk of the PNS and immune system remain mysterious. To study this kind of bi-directional crosstalk, we performed immunofluorescent staining of neurofilament and confocal microscopy to reveal the distribution of nerve fibers and nerve-immune cell associations inside mouse spleen. Our study demonstrates (i) extensive nerve fibers in all splenic compartments including the splenic nodules, periarteriolar lymphoid sheath, marginal zones, trabeculae, and red pulp; (ii) close associations of nerve fibers with blood vessels (including central arteries, marginal sinuses, penicillar arterioles, and splenic sinuses); (iii) close associations of nerve fibers with various subsets of dendritic cells, macrophages (Mac1 + and F4/80 + ), and lymphocytes (B cells, T helper cells, and cytotoxic T cells). Our data concerning the extensive splenic innervation and nerve-immune cell communication will enrich our knowledge of the mechanisms through which the PNS affects the cellular- and humoral-mediated immune responses in healthy and infectious/non-infectious states.

Neural regulation of the function of the gastrointestinal tract (GIT) relies on a delicate balance of the two divisions of its nervous system, namely, the intrinsic and extrinsic divisions. The intrinsic innervation is provided by the enteric nervous system (ENS), whereas the extrinsic innervation includes sympathetic/parasympathetic nerve fibers and extrinsic sensory nerve fibers. In the present study, we used immunofluorescent staining of neurofilament-heavy (NF-H) to reveal the distribution of nerve fibers and their associations with immune cells inside mouse Peyer's patches (PP), an essential part of gut-associated lymphoid tissue (GALT). Our results demonstrate (1) the presence of an extensive meshwork of NF-H-immunoreactive presumptive nerve fibers in all PP compartments including the lymphoid nodules, interfollicular region, follicle-associated epithelium, and subepithelial dome; (2) close associations/contacts of nerve fibers with blood vessels including high endothelial venules, indicating neural control of blood flow and immune cell dynamics inside the PP; (3) close contacts between nerve fibers/endings and B/T cells and various subsets of dendritic cells ( e.g., B220⁻, B220⁺, CD4⁻, CD4⁺, CD8⁻, and CD8⁺). Our novel findings concerning PP innervation and nerve-immune-cell contacts in situ should facilitate our understanding of bi-directional communications between the PNS and GALT. Since the innervation of the gut, including PP, might be important in the pathogenesis and progression of some neurological, infectious, and autoimmune diseases, e.g., prion diseases and inflammatory bowel disease, better knowledge of PNS-immune system interactions in the GALT (including PP) should benefit the development of potential treatments for these diseases via neuroimmune manipulations.

The peripheral nervous system communicates specifically with the immune system via local interactions. These interactions include the “hardwiring” of sympathetic/ parasympathetic (efferent) and sensory nerves (afferent) to primary (e.g., thymus and bone marrow) and secondary (e.g., lymph node, spleen, and gut-associated lymphoid tissue) lymphoid tissue/organs. To gain a better understanding of this bidirectional interaction/crosstalk between the two systems, we have investigated the distribution of nerve fibres and PNS-immune cell associations in situ in the mouse lymph node by using immunofluorescent staining and confocal microscopy/ three-dimensional reconstruction. Our results demonstrate: i) the presence of extensive nerve fibres in all compartments (including B cell follicles) in the mouse lymph node; ii) close contacts/ associations of nerve fibres with blood vessels (including high endothelial venules) and lymphatic vessels/sinuses; iii) close contacts/associations of nerve fibres with various subsets of dendritic cells (e.g., B220+CD11c+, CD4+CD11c+, CD8a+ CD11c+, and Mac1+CD11c+), Mac1+ macrophages, and B/T lymphocytes. Our novel findings concerning the innervation and nerve-immune cell interactions inside the mouse lymph node should greatly facilitate our understanding of the effects that the peripheral nervous system has on cellular- and humoral-mediated immune responses or vice versa in health and disease.

The central nervous system impacts the immune system mainly by regulating the systemic concentration of humoral substances, whereas the peripheral nervous system (PNS) communicates with the immune system specifically according to local "hardwiring" of sympathetic/parasympathetic (efferent) and sensory (afferent) nerves to the primary and secondary lymphoid tissue/organs (e.g., thymus spleen and lymph nodes). In the present study, we use immunofluorescent staining of neurofilament-heavy to reveal the distribution of nerve fibers and the nerve-immune cell neighborhood inside the mouse thymus. Our results demonstrate (a) the presence of an extensive meshwork of nerve fibers in all thymic compartments, including the capsule, subcapsular region, cortex, cortico-medullary junction and medulla; (b) close associations of nerve fibers with blood vessels (including the postcapillary venules), indicating the neural control of blood circulation and immune cell dynamics inside the thymus; (c) the close proximity of nerve fibers to various subsets of thymocytes (e.g., CD4+, CD8+ and CD4+CD8+), dendritic cells (e.g., B220+, CD4+, CD8+ and F4/80+), macrophages (Mac1+ and F4/80+) and B cells. Our novel findings concerning thymic innervation and the nerve-immune cell neighborhood in situ should facilitate the understanding of bi-directional communications between the PNS and primary lymphoid organs. Since the innervation of lymphoid organs, including the thymus, may play essential roles in the pathogenesis and progression of some neuroimmune, infectious and autoimmune diseases, better knowledge of PNS-immune system crosstalk should benefit the development of potential therapies for these diseases.

Introduction: Haemoglobin is a widely studied protein due to its important roles in physiology and pathology. Aberrant expression of haemoglobins, including primitive globins, have been reported in various sites and disease states and may have utility in some instances as diagnostic and/or prognostic markers. Despite this, robust detection of haemoglobin epsilon in the placenta during development by immunohistochemistry has not been well documented. Aim: To evaluate a polyclonal antibody against human haemoglobin subunit epsilon (HBE) by immunohistochemistry during primitive erythropoiesis in the developing mouse placenta. Methods and results: An immunohistochemistry protocol was developed using a commercially available anti-human haemoglobin subunit epsilon antibody on the mouse placenta at embryonic day 11.5. Strong and specific cytoplasmic staining was observed in primitive erythroid cells within the blood cell islands. By contrast, the placenta endothelium, mesothelium and mesoderm were all immunonegative for epsilon haemoglobin. Conclusions: An immunohistochemistry protocol for the specific detection of epsilon haemoglobin was successfully developed using mouse placenta tissue. This assay has utility as a tool for the study of erythropoiesis during development and/or detecting the ectopic expression of epsilon globins in disease states such as cancer.

The nervous system and the immune system communicate extensively with each other in order to maintain homeostasis and to regulate the immune response. The peripheral nervous system (PNS) communicates specifically with the immune system according to local interactions, including the “hardwiring” of sympathetic/parasympathetic (efferent) and sensory nerves (afferent) to lymphoid tissue and organs. To reveal this type of bidirectional neuroimmune interaction at the microscopic level, we used immunofluorescent staining of glial fibrillary acidic protein (GFAP) coupled with confocal microscopy/3D reconstruction to reveal the distribution of non-myelinating Schwann cells (NMSCs) and their interactions with immune cells inside mouse spleen. Our results demonstrate i) the presence of an extensive network of NMSC processes in all splenic compartments including the splenic nodules, periarteriolar lymphoid sheath (PALS), marginal zone, trabecula, and red pulp; ii) the close association of NMSC processes with blood vessels (including central artries and their branches, marginal sinuses, penicillar arterioles and splenic sinuses); iii) the close “synapse-like” interaction/association of NMSC processes with various subsets of dendritic cells (DCs; e.g., CD4+CD11c+ DCs, B220+CD11c+ DCs, and F4/80+ CD11c+ DCs), macrophages (F4/80+), and lymphocytes (B cells, CD4+ T helper cells). Our novel findings concerning the distribution of NMSCs and NMSC-leukocytes interactions inside mouse spleen should improve our understanding of the mechanisms through which the PNS affects cellular- and humoral-mediated immune responses in a variety of health conditions and infectious/non-infectious diseases.

Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the leading genetic cause of autosomal dominant familial Parkinson’s disease. We previously reported that two mutations within the ROC domain, namely R1441C and A1442P, exhibit increased protein degradation leading to lowered steady state LRRK2 protein levels in HEK293 cells. More recently, the common WD40 domain LRRK2 haplotype, Met2397, which is a risk factor for Crohn’s disease, has been shown to lower steady state protein levels in HEK293 cells. In view of recent evidence implicating LRRK2 and inflamemation in PD, we investigated the effects of Met2397 on LRRK2 expression, and compared them to the Thr2397 variant and other LRRK2 mutants. In this study, we transfected HEK293 cells with plasmid constructs encoding the different LRRK2 variants, and analyzed the resulting protein levels by Western blot and flow cytometry. Here we found that both the Met2397 and Thr2397 haplotypes yield similar levels of LRRK2 protein expression and do not appear to impact cell viability in HEK293 cells, compared to other LRRK mutants. Thus, we have concluded that the Met2397 haplotype is unlikely to play a role in LRRK2 mediated or idiopathic PD.

The thymus is innervated by sympathetic/parasympathetic nerve fibers from the peripheral nervous system (PNS), suggesting a neural regulation of thymic function including T-cell development. Despite some published studies, data on the innervation and nerve-immune interaction inside the thymus remain limited. In the present study, we used immunofluorescent staining of glial fibrillary acidic protein (GFAP) coupled with confocal microscopy/three-dimensional (3D) reconstruction to reveal the distribution of non-myelinating Schwann cells (NMSC) and their interactions with immune cells inside mouse thymus. Our results demonstrate (1) the presence of an extensive network of NMSC processes in all compartments of the thymus including the capsule, subcapsular region, cortex, cortico-medullary junction, and medulla; (2) close associations/interactions of NMSC processes with blood vessels, indicating the neural control of blood flow inside the thymus; (3) the close "synapse-like" association of NMSC processes with various subsets of dendritic cells (DC; e.g., B220+ DCs, CD4+ DCs, and CD8+ DCs), and lymphocytes (B cells, CD4+/CD8+ thymocytes). Our novel findings concerning the distribution of NMSCs and the associations of NMSCs and immune cells inside mouse thymus should help us understand the anatomical basis and the mechanisms through which the PNS affects T-cell development and thymic endocrine function in health and disease.

Background: The cat flea (Ctenocephalides felis) is a blood-feeding ectoparasitic insect and particular nuisance pest of companion animals worldwide. Identification of genes that are differentially expressed in response to feeding is important for understanding flea biology and discovering targets for their control.Methods: C. felis fleas were maintained and fed for 24 h using an artificial rearing system. The technique of suppression subtractive hybridization was employed to screen for mRNAs specifically expressed in fed fleas.Results: We characterized nine distinct full-length flea transcripts that exhibited modulated or de novo expression during feeding. Among the predicted protein sequences were two serine proteases, a serine protease inhibitor, two mucin-like molecules, a DNA topoisomerase, an enzyme associated with GPI-mediated cell membrane attachment of proteins and a component of the insect innate immune response.Conclusions: Our results provide a molecular insight into the physiology of flea feeding. The protein products of the genes identified may play important roles during flea feeding in terms of blood meal digestion, cellular growth/repair and protection from feeding-associated stresses.

Acute T cell leukaemia (T-ALL) is an aggressive tumour arising from immature T cells (a type of lymphocyte or white blood cell that plays a central role in cell-mediated immunity). T-ALL frequently presents with clinical features, such as high cell counts and invasion of the central nervous system.

INTRODUCTION:Connective tissue growth factor (CTGF/CCN2) has been shown previously to be aberrantly expressed in a high proportion of paediatric precursor B cell acute lymphoblastic leukaemia (pre-B ALL), suggesting a potential oncogenic role in this tumour type. We therefore assessed CTGF mRNA transcript diversity in B-lineage ALL using primary patient specimens and cell lines.METHODS:CTGF mRNA expression was evaluated by quantitative real-time PCR and Northern blotting. We performed a structural analysis of CTGF mRNA by nested reverse-transcriptase PCR and examined CTGF protein diversity by immunoblotting.RESULTS:Northern blot analysis of pre-B ALL cell lines revealed short CTGF transcripts that were expressed in association with the active phase of cellular growth. Structural analysis confirmed the synthesis of several novel CTGF mRNA isoforms in B-lineage ALL cell lines that were uniformly characterised by the retention of the coding sequence for the C-terminal (CT) domain. One of these novel spliceforms was expressed in a majority (70%) of primary pre-B ALL patient specimens positive for canonical CTGF mRNA. Evidence that these alternative transcripts have coding potential was provided by cryptic CTGF proteins of predicted size detected by immunoblotting.CONCLUSION:This study identifies for the first time alternative splicing of the CTGF gene and shows that a short CTGF splice variant associated with cell proliferation is expressed in most cases of primary CTGF-positive pre-B ALL. This novel variant encoding only the CT domain may play a role in pre-B ALL tumorigenesis and/or progression.

External parasites represent a considerable nuisance and cause of suffering and economic losses in companion animals, livestock and humans. Among these, the cat flea Ctenocephalides felis is a widespread pest across temperate and tropical areas of the world with particular importance in domestic pets.

The class 1A aldehyde dehydrogenase (ALDH1A) subfamily of genes encode enzymes that function at the apex of the retinoic acid (RA) signalling pathway. We detected aberrant expression of ALDH1A genes, particularly ALDH1A2, in a majority (72%) of primary paediatric T cell acute lymphoblastic leukaemia (T-ALL) specimens. ALDH1A expression was almost exclusive to T-lineage, but not B-lineage, ALL. To determine whether ALDH1A expression may have relevance to T-ALL cell growth and survival, the effect of inhibiting ALDH1A function was measured on a panel of human ALL cell lines. This revealed that T-ALL proliferation had a higher sensitivity to modulation of ALDH1A activity and RA signalling as compared to ALL cell lines of B-lineage. Consistent with these findings, the genes most highly correlated with ALDH1A2 expression were involved in cell proliferation and apoptosis. Evidence that such genes may be targets of regulation via RA signalling initiated by ALDH1A activity was provided by the TNFRSF10B gene, encoding the apoptotic death receptor TNFRSF10B (also termed TRAIL-R2), which negatively correlated with ALDH1A2 and showed elevated transcription following treatment of T-ALL cell lines with the ALDH1A inhibitor citral (3,7-dimethyl-2,6-octadienal). These data indicate that ALDH1A expression is a common event in T-ALL and supports a role for these enzymes in the pathobiology of this disease.

The emergence and rapid rise of flowering plants is one of the most extraordinary and yet still not fully explained phenomena in evolutionary history. Could what Darwin himself called an "abominable mystery" be caused by jumping genes?