Effector Molecules Of Innate Immunity Research Articles

Evaluation of alpha-defensins as a marker of severity in chronic obstructive pulmonary disease.

Defensins are key effector molecules of innate immunity that can contribute towards the diagnosis and monitoring of chronic obstructive pulmonary disease (COPD). The present study was conducted to investigate the role of alpha-defensins in patients with COPD by quantifying serum and sputum samples. A total of 180 patients were enrolled and divided into four groups, and sputum and serum values of alpha-defensins were assessed. The sensitivity, specificity, and accuracy of sputum alpha-defensin as a diagnostic biomarker were evaluated to assess its utility in diagnosing COPD. The mean value of sputum alpha-defensins was found to be statistically significant amongst the four groups (P < 0.001). The highest levels were found in subjects with AECOPD (385.76 ± 116.62 ng/mL). Sputum alpha-defensins were found to be negatively correlated with FEV 1 values (rho = -0.31, P < 0.001). Sputum alpha-defensins can be used as a potential marker for predicting acute exacerbation of COPD. In addition, they could serve as an indicator of disease severity in COPD patients.

Antimicrobial peptide cecropin B functions in pathogen resistance of Mythimna separata.

Mythimna separata (Lepidoptera: Noctuidae) is an omnivorous pest that poses a great threat to food security. Insect antimicrobial peptides (AMPs) are small peptides that are important effector molecules of innate immunity. Here, we investigated the role of the AMP cecropin B in the growth, development, and immunity of M. separata. The gene encoding M. separata cecropin B (MscecropinB) was cloned. The expression of MscecropinB was determined in different developmental stages and tissues of M. separata. It was highest in the prepupal stage, followed by the pupal stage. Among larval stages, the highest expression was observed in the fourth instar. Tissue expression analysis of fourth instar larvae showed that MscecropinB was highly expressed in the fat body and haemolymph. An increase in population density led to upregulation of MscecropinB expression. MscecropinB expression was also upregulated by the infection of third and fourth instar M. separata with Beauveria bassiana or Bacillus thuringiensis (Bt). RNA interference (RNAi) targeting MscecropinB inhibited the emergence rate and fecundity of M. separata, and resulted in an increased sensitivity to B. bassiana and Bt. The mortality of M. separata larvae was significantly higher in pathogen plus RNAi-treated M. separata than in controls treated with pathogens only. Our findings indicate that MscecropinB functions in the eclosion and fecundity of M. separata and plays an important role in resistance to infection by B. bassiana and Bt.

Transcriptome analysis and immune gene expression of channel catfish (Ictalurus punctatus) fed diets with inclusion of frass from black soldier fly larvae.

The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (Ictalurus punctatus) to a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immune-related genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50g/kg) inclusion diet, or a high-frass (300g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1β type a, IL-1β type b, IL-17, IFN-γ, and TNFα), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits.

Abstract 2488: PET imaging of innate immunity activation In Vivo with redox-tuned 4-[18F]fluoro-1-naphthol

Abstract High redox potential reactive oxygen and nitrogen species (ROS/RNS), such as O2 free radicals, superoxide, and hypochlorous acid, generated by activities of the NADPH oxidase-2 (NOX2)/myeloperoxidase (MPO) axis and related enzymes, are key effector molecules of innate immunity in physiological and diseased inflammatory states. Other lower energy species (H2O2, NO) provide adjuvant signaling functions. NOX2- and MPO-derived high energy radicals are known to oxidize naphthol species, wherein the naphthol products bind to proximate proteins and activated myeloid cells. Herein, we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a novel redox-tuned radiopharmaceutical that selectively detects by positron emission tomography (PET) high energy radicals produced by activated innate immunity. The products of human MPO plus H2O2, but not H2O2 alone, rapidly and completely oxidized [18F]4FN. All-trans-retinoic acid-differentiated HL-60 "neutrophil-like" human cells activated with phorbol-12-myristate-13-acetate (PMA) retained [18F]4FN 5-fold over unstimulated cells. 4-ABAH, an MPO-specific inhibitor, or DPI, a broad oxidase inhibitor, blocked cellular retention by &amp;gt;95%. [18F]4FN PET/CT imaging readily discriminated foci of inflammation in vivo in three distinct murine models of acute inflammation: endotoxin-induced whole-body toxic shock, PMA-induced mild contact dermatitis of the ear, and lipopolysaccharide (LPS)-induced ankle arthritis. Mechanistically, in mice in vivo, 4-ABAH reduced inflammation-induced [18F]4FN retention, and Cybb-/- (Nox2-/-) gene-deletion strongly and significantly abrogated PMA-induced [18F]4FN retention. Thus, [18F]4FN shows promise as a robust redox-tuned reporter for imaging activation states of innate immunity by PET/CT, is ready for translation. [18F]4FN PET imaging may find application in a variety of inflammatory states associated with cancer therapy, immunotherapy-related adverse events, as well as other diseases, including arthritis, hepatitis, atherosclerosis, COVID-19, as well as up-staging and monitoring multi-organ inflammation. Citation Format: Seth T. Gammon, Federica Pisaneschi, Vincenzo Paolillo, Sarah Qureshy, David Piwnica-Worms. PET imaging of innate immunity activation In Vivo with redox-tuned 4-[18F]fluoro-1-naphthol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2488.

Expression of human caspase-4 in the gingival epithelium affected with periodontitis: Its involvement in Porphyromonas gingivalis-challenged gingival epithelial cells

ObjectiveImplication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DesignEx vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human β-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. ResultsEx vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. ConclusionsCaspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

Admixture Mapping of Sepsis in European Individuals With African Ancestries.

Sepsis is a severe systemic inflammatory response to infections that is accompanied by organ dysfunction. Although the ancestral genetic background is a relevant factor for sepsis susceptibility, there is a lack of studies using the genetic singularities of a recently admixed population to identify loci involved in sepsis susceptibility. Here we aimed to discover new sepsis loci by completing the first admixture mapping study of sepsis in Canary Islanders, leveraging their distinctive genetic makeup as a mixture of Europeans and African ancestries. We used a case-control approach and inferred local ancestry blocks from genome-wide data from 113,414 polymorphisms genotyped in 343 patients with sepsis and 410 unrelated controls, all ascertained for grandparental origin in the Canary Islands (Spain). Deviations in local ancestries between cases and controls were tested using logistic regressions, followed by fine-mapping analyses based on imputed genotypes, in silico functional assessments, and gene expression analysis centered on the region of interest. The admixture mapping analysis detected that local European ancestry in a locus spanning 1.2 megabases of chromosome 8p23.1 was associated with sepsis (lowest p = 1.37 × 10−4; Odds Ratio [OR] = 0.51; 95%CI = 0.40–0.66). Fine-mapping studies prioritized the variant rs13249564 within intron 1 of MFHAS1 gene associated with sepsis (p = 9.94 × 10−4; OR = 0.65; 95%CI = 0.50–0.84). Functional and gene expression analyses focused on 8p23.1 allowed us to identify alternative genes with possible biological plausibility such as defensins, which are well-known effector molecules of innate immunity. By completing the first admixture mapping study of sepsis, our results revealed a new genetic locus (8p23.1) harboring a number of genes with plausible implications in sepsis susceptibility.

Regulatory Roles of Antimicrobial Peptides in the Nervous System: Implications for Neuronal Aging.

Antimicrobial peptides (AMPs) are classically known as important effector molecules in innate immunity across all multicellular organisms. However, emerging evidence begins to suggest multifunctional properties of AMPs beyond their antimicrobial activity, surprisingly including their roles in regulating neuronal function, such as sleep and memory formation. Aging, which is fundamental to neurodegeneration in both physiological and disease conditions, interestingly affects the expression pattern of many AMPs in an infection-independent manner. While it remains unclear whether these are coincidental events, or a mechanistic relationship exists, previous studies have suggested a close link between AMPs and a few key proteins involved in neurodegenerative diseases. This review discusses recent literature and advances in understanding the crosstalk between AMPs and the nervous system at both molecular and functional levels, with the aim to explore how AMPs may relate to neuronal vulnerability in aging.

Imaging of innate immunity activation in vivo with a redox-tuned PET reporter.

High-redox-potential reactive oxygen species and reactive nitrogen species (ROS/RNS), generated by NADPH oxidase-2 (NOX2), myeloperoxidase (MPO) and related enzymes, are key effector molecules of innate immunity. High-redox-potential radicals are difficult to distinguish by imaging from less potent ROS/RNS functioning as background biological signaling molecules. Here we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a redox-tuned radiopharmaceutical that selectively binds proteins and cells when oxidized by products of human MPO plus H2O2, but not H2O2 alone, and can be detected using positron emission tomography (PET). Activating HL-60 neutrophil-like human cells with phorbol ester (PMA) caused [18F]4FN retention five-fold over unstimulated cells. An MPO-specific inhibitor (4-ABAH) blocked cellular retention by more than 95%. [18F]4FN PET/CT imaging discriminated inflammatory foci in vivo in three murine models of activated innate immunity: endotoxin-induced toxic shock, PMA-induced contact dermatitis and lipopolysaccharide-induced ankle arthritis. 4-ABAH and Cybb-/- (Nox2-/-) gene deletion strongly abrogated [18F]4FN retention in vivo. Thus, [18F]4FN shows promise as a robust reporter of innate immunity activation by PET/CT.

Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase

Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome’s very 3’-end. The synthesis of short abortive leader transcripts (leaderRNAs) has been linked to transcription initiation for some NNS viruses. Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60–80. LeaderRNA synthesis requires hexamer phasing in the 3’-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3’-leader promoter. We further found different genomic 3’-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3’-leader promoter, i.e., at the second nucleotide of the genome 3’-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus.

Comparative molecular characterization and In silico analysis of neck and carbohydrate recognition domain (NCRD) of conglutin in gene in buffalo (Bubalis bubalis) and nilgai (Boselaphus tragocamelus)

Conglutinin, a high molecular-weight C-type lectin belongs to the family of collectins, was originally detected in bovine serum which binds sugar residues in a Ca2+-dependent manner and it is an effector molecules in innate immunity. Total RNA extracted from liver sample of buffalo and nilgai were used to synthesize cDNA. Conglutinin NCR domain cDNA (497 bp) was amplified by proof reading DNA polymerase using primers established for cattle (Bostaurus). The amplicons were ligated to cloning vector (pJET), that was transformed into E. coli DH5α competent cells and the recombinant plasmids (pJET-BuCGN and pJET-NCGN) were characterized by Pst I restriction analysis. The amplicon (497 bp) was released from the recombinant plasmids (pJET-BuCGN and pJET-NCGN) upon Eco RI and Xho I double digestion, sequencing of the recombinant plasmids were carried out. BuCGN was found to be 99.6% similar to NCGN at nucleotde level, and 100% at predicted amino acid level. The predicted proteins of both the species (162 amino acids) were expected to possess five β-sheets in the secondary structure.

Host Directed Therapy Against Infection by Boosting Innate Immunity.

The innate immune system constitutes the first line of defense against invading pathogens, regulating the normal microbiota and contributes to homeostasis. Today we have obtained detailed knowledge on receptors, signaling pathways, and effector molecules of innate immunity. Our research constellation has focused on ways to induce the expression of antimicrobial peptides (AMPs), the production of oxygen species (ROS and NO), and to activate autophagy, during the last two decades. These innate effectors, with different mechanisms of action, constitute a powerful defense armament in phagocytes and in epithelial cells. Innate immunity does not only protect the host from invading pathogens, but also regulates the composition of the microbiota, which is an area of intense research. Notably, some virulent bacteria have the capacity to downregulate innate defenses and can thereby cause invasive disease. Understanding the detailed mechanisms behind pathogen-mediated suppression of innate effectors are currently in progress. This information can be of importance for the development of novel treatments based on counteraction of the downregulation; we have designated this type of treatment as host directed therapy (HDT). The concept to boost innate immunity may be particularly relevant as many pathogens are developing resistance against classical antibiotics. Many pathogens that are resistant to antibiotics are sensitive to the endogenous effectors included in early host defenses, which contain multiple effectors working in cooperation to control infections. Here, we review recent data related to downregulation of AMPs by pathogenic bacteria, induction of innate effector mechanisms, including cytokine-mediated effects, repurposed drugs and the role of antibiotics as direct modulators of host responses. These findings can form a platform for the development of novel treatment strategies against infection and/or inflammation.

Exosomes secreted from microglia during chronic virus infection can induce demyelinating disease

Abstract Theiler’s murine encephalomyelitis virus (TMEV) induced demyelinating disease is a murine model for multiple sclerosis (MS). TMEV persists in microglia and leads to development of chronic progressive demyelinating disease associated with an inflammatory immune response in the central nervous system (CNS) of susceptible mice. TMEV-infected microglia secrete innate immune cytokines, chemokines, and effector molecules associated with development and progression of demyelinating disease. Recently, we have found that TMEV-infected microglia secrete nanovesicles called exosomes that contain and transfer viral RNA to bystander cells in the CNS. Our present studies show that exosomes secreted by microglia during TMEV-induced demyelinating disease (63–98dpi) contain viral RNA/genome that can be taken up by other CNS resident cells, transferring the viral RNA. More importantly, these exosomes could induce the bystander CNS cells to express inflammatory cytokines and chemokines. When administered into naïve mice, exosomes secreted by microglia during TMEV-induced demyelinating disease promoted MS-like pathology including microglia activation and demyelination in the spinal cord. These results suggest that exosomes secreted by infected microglia may directly contribute to the development and progression of demyelinating disease.

Amyloid beta is an early responder cytokine and immunopeptide of the innate immune system.

A molecular level conceptualization of the pathogenesis of Alzheimer's disease (AD) remains elusive with many competing hypotheses, particularly via proteopathic and immunopathic mechanisms. However, these need not be competitive. If amyloid beta (Aβ) is regarded as an “early responder cytokine,” then proteopathic considerations become encompassed within an overarching hybrid proteopathic‐immunopathic mechanism. As argued in this commentary, Aβ is in fact a molecular constituent of the innate immune system. Aβ is an antimicrobial peptide (AMP) functioning not only as a killer peptide, but also as a modulatory immunopeptide. Aβ satisfies the definition of a cytokine, exhibiting interdependency with other cytokines. Aβ also satisfies the functional definition of a chemokine, existing within the AMP‐chemokine spectrum. Aβ, like conventional cytokines, both binds to and is released by microglial cells. Finally, Aβ interacts with the complement and Toll‐like receptor systems analogously to established cytokines. Aβ may thus be regarded as an effector molecule of innate immunity.

Low Evolutionary Selection Pressure in Senescence Does Not Explain the Persistence of Aβ in the Vertebrate Genome.

The argument is frequently made that the amyloid-β protein (Aβ) persists in the human genome because Alzheimer’s disease (AD) primarily afflicts individuals over reproductive age and, therefore, there is low selective pressure for the peptide’s elimination or modification. This argument is an important premise for AD amyloidosis models and therapeutic strategies that characterize Aβ as a functionless and intrinsically pathological protein. Here, we review if evolutionary theory and data on the genetics and biology of Aβ are consistent with low selective pressure for the peptide’s expression in senescence. Aβ is an ancient neuropeptide expressed across vertebrates. Consistent with unusually high evolutionary selection constraint, the human Aβ sequence is shared by a majority of vertebrate species and has been conserved across at least 400 million years. Unlike humans, the overwhelming majority of vertebrate species do not cease reproduction in senescence and selection pressure is maintained into old age. Hence, low selective pressure in senescence does not explain the persistence of Aβ across the vertebrate genome. The “Grandmother hypothesis” (GMH) is the prevailing model explaining the unusual extended postfertile period of humans. In the GMH, high risk associated with birthing in old age has lead to early cessation of reproduction and a shift to intergenerational care of descendants. The rechanneling of resources to grandchildren by postreproductive individuals increases reproductive success of descendants. In the GMH model, selection pressure does not end following menopause. Thus, evolutionary models and phylogenetic data are not consistent with the absence of reproductive selection pressure for Aβ among aged vertebrates, including humans. Our analysis suggests an alternative evolutionary model for the persistence of Aβ in the vertebrate genome. Aβ has recently been identified as an antimicrobial effector molecule of innate immunity. High conservation across the Chordata phylum is consistent with strong positive selection pressure driving human Aβ’s remarkable evolutionary longevity. Ancient origins and widespread conservation suggest the human Aβ sequence is highly optimized for its immune role. We detail our analysis and discuss how the emerging “Antimicrobial Protection Hypothesis” of AD may provide insights into possible evolutionary roles for Aβ in infection, aging, and disease etiology.

The antimicrobial protection hypothesis of Alzheimer's disease

ObjectiveWe explore here a novel model for amyloidogenesis in Alzheimer's disease (AD). This new perspective on AD amyloidosis seeks to provide a rational framework for incorporating recent and seemingly independent findings on the antimicrobial role of β-amyloid and emerging experimental, genetic, and epidemiological data, suggesting innate immune-mediated inflammation propagates AD neurodegeneration. BackgroundAD pathology is characterized by cerebral deposition of amyloid-β protein (Aβ) as β-amyloid. Genetic studies have confirmed the key role of Aβ in AD, revealing that mutation-mediated shifts in the peptides generation lead to early onset familial Alzheimer's disease. However, Aβ generation appears normal for the majority of AD patients, who lack early onset familial Alzheimer's disease mutations. In prevailing models of nonfamilial AD, individual genetics and age-associated changes in brain milieu promote an intrinsically abnormal propensity of Aβ for self-association. However, emerging findings are increasingly inconsistent with characterization of Aβ oligomerization as a nonphysiological and exclusively pathological activity. Recent studies suggest Aβ is an ancient, highly conserved effector molecule of innate immunity. Moreover, Aβ oligomerization and β-amyloid generation appear to be important innate immune pathways that mediate pathogen entrapment and protect against infection. New AD amyloidogenesis modelRecent findings on inflammation-mediated neurodegeneration and the role of Aβ in immunity have led to emergence of the “Antimicrobial Protection Hypothesis” of AD. In this model, β-amyloid deposition is an early innate immune response to genuine, or mistakenly perceived, immunochallenge. Aβ first entraps and neutralizes invading pathogens in β-amyloid. Aβ fibrillization drives neuroinflammatory pathways that help fight the infection and clear β-amyloid/pathogen deposits. In AD, chronic activation of this pathway leads to sustained inflammation and neurodegeneration. Mounting data link elevated brain microbe levels with AD. The Antimicrobial Protection Hypothesis reveals how increased brain microbial burden may directly exacerbate β-amyloid deposition, inflammation, and AD progression. Amyloid cascade hypothesisIn the antimicrobial protection model, the modality of Aβ's pathophysiology is shifted from abnormal stochastic behavior toward dysregulated innate immune response. However, β-amyloid deposition in AD still leads to neurodegeneration. Thus, the new model extends but remains broadly consistent with the Amyloid Cascade Hypothesis and overwhelming data showing the primacy of Aβ in AD pathology.

Pathogenesis of Rickettsial Diseases: Pathogenic and Immune Mechanisms of an Endotheliotropic Infection

Obligately intracytosolic rickettsiae that cycle between arthropod and vertebrate hosts cause human diseases with a spectrum of severity, primarily by targeting microvascular endothelial cells, resulting in endothelial dysfunction. Endothelial cells and mononuclear phagocytes have important roles in the intracellular killing of rickettsiae upon activation by the effector molecules of innate and adaptive immunity. In overwhelming infection, immunosuppressive effects contribute to the severity of illness. Rickettsia-host cell interactions involve host cell receptors for rickettsial ligands that mediate cell adhesion and, in some instances, trigger induced phagocytosis. Rickettsiae interact with host cell actin to effect both cellular entry and intracellular actin-based mobility. The interaction of rickettsiae with the host cell also involves rickettsial evasion of host defense mechanisms and exploitation of the intracellular environment. Signal transduction events exemplify these effects. An intriguing frontier is the array of rickettsial noncoding RNA molecules and their potential effects on the pathogenesis and transmission of rickettsial diseases.

Recombinant rabies virus expressing interleukin-6 enhances the immune response in mouse brain.

Rabies, which is caused by the rabies virus (RABV), is an ancient zoonosis that has a high mortality rate. Previous studies have indicated that recombinant RABV expressing canine interleukin-6 (rHEP-CaIL6), induced more virus-neutralizing antibodies than parental RABV in mice following intramuscular immunization. To investigate the immune response induced in the CNS by rHEP-CaIL6 after intranasal or intracranial administration in mice, the permeability of the blood-brain barrier (BBB), the infiltration of CD3 T cells, and innate immune response-related effector molecules in the CNS were examined. It was observed that infection of rHEP-CaIL6 led to enhanced BBB permeability following intranasal infection. More CD3 T cells infiltrated into the central nervous system (CNS) in mice infected with rHEP-CaIL6 than in those infected with the HEP-Flury strain. Furthermore, rHEP-CaIL6 induced an increased expression of innate immune response-related effector molecules, compared with the parental HEP-Flury strain, within the CNS. Taken together, these findings suggest that rHEP-CaIL6 induced stronger immune responses in mice brains, which is more beneficial for virus clearance. These results may also partly illustrate the role of IL6 in RABV infection.

Molecular Cloning of Cathelicidin-like cDNA from Andrias davidianus

Antimicrobial peptides (AMPs) as part of host defense systems has been widely recognized in most organisms. Cathelicidin is an important family of AMPs acting as multifunctional effector molecules in innate immunity and exists in organisms with cathelicidin-like precursor. Andrias davidianus (A. davidianus) is a unique species in China and the biggest amphibians in the world. With the rapid growth of A. davidianus aquaculture, pathogens of bacteria, virus and fungus were reported, however little is known about antimicrobial peptides derived from A. davidianus. To investigate antimicrobial peptides of cathelicidin-like in A. davidianus, cathelicidin-like precursor gene cloning and bioinformatic analysis was carried out. The results showed that 1106 bp full-length cDNA of cathelicidin-like precursor was obtained, which was including a 35 bp 5' terminal UTR, a 546 bp open reading frame (ORF) and a 525 bp 5' terminal UTR. The cathelicidin-like precursor amino acid (AA) sequence of A. davidianus comprised N-terminal signal peptide (21 AA), highly conserved cathelin domain and C-terminal mature peptide. The cathelicidin-like precursor gene nucleotide sequence showed low identify with other cathelicidin-like sequences, while AA sequence displayed relatively higher similarity with cathelicidin-like isolated from other species. Phylogenetic tree indicated cathelicidinlike precursor of A. davidianus was firstly clade with Tylototrition verrucosus, which also belonged to Caudata, Amphibian. The precursor gene expression was detected by RT-qPCR. The result displayed this gene was abundant expression in A. davidianus skin. According the specificity proteases cleavage and characteristic of cathelicidin, five putative mature cathelicidin were predicted. This study confirms the presence of cathelicidin in A. davidianus. Their results not only reveal innate immune system of A. davidianus but also enlarge the AMP knowledge of urodele amphibians.

Immunohistochemical expression of human β-defensin-2 in verruca vulgaris

Background Verruca vulgaris is the commonest viral cutaneous infection that is caused by human papilloma virus. There are more than 150 genotypically different types of human papilloma virus. Types 1, 2, 4, 27, and 57 were found with verruca vulgaris. Human β-defensins (hBDs), a family of small antimicrobial peptides, are important effector molecules of innate immunity, possessing antimicrobial, anti-inflammatory, and immunomodulatory activities. Aim The aim of this work was to investigate the immunohistochemical expression of hBD-2 in verruca vulgaris lesion in a trial to elucidate its possible role in the pathogenesis of the disease. Patients and methods This study included 15 lesional skin specimens of 15 patients with typical clinically and histopathologically diagnosed verruca vulgaris (group I). Seven specimens of nonlesional normal skin of contralateral sides of seven of these patients (group II) in addition to other seven normal skin specimens from seven healthy subjects of matched age and sex (group III) served as controls. All specimens were studied histopathologically using hematoxylin and eosin stain and immunohistochemically for detection of hBD-2 expression. Results All lesional skin specimens (group I) showed positive expression of hBD-2. Twelve (80%) specimens showed positive expression in all epidermal layers [immunoreactivity (IR) 3]. Two (13.3%) specimens showed positive expression in the stratum corneum only (IR 1), and one (6.7%) specimen showed positive expression in the stratum corneum and stratum granulosum (IR 2). Groups II and III showed no expression of hBD-2 (IR 0). There was highly significant difference between patients and controls (P Conclusion There was positive immunohistochemical expression of hBD-2 in lesional skin specimens taken from patients with verruca vulgaris, whereas there was no expression in normal skin specimens taken from the patients and the volunteers. This may indicate a possible role of hBD-2 in the pathogenesis of verruca vulgaris and also in the immunity against it.

Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors

Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human β-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression.