Defense Peptides Research Articles

Host defense peptides at the crossroad of endothelial cell physiology: Insight into mechanistic and pharmacological implications.

Antimicrobial peptides (AMPs), particularly host defense peptides (HDPs), have gained recognition for their role in host defense mechanisms, but they have also shown potential as a promising anticancer, antiviral, antiparasitic, antifungal and immunomodulatory agent. Research studies in recent years have shown HDPs play a crucial role in endothelial cell function and biology. The function of endothelial cells is impacted by HDPs' complex interplay between cytoprotective and cytotoxic actions as they are known to modulate barrier integrity, inflammatory response and angiogenesis. This biphasic response varies and depends on the peptide structure, its concentration, and the microenvironment. These effects are mediated through key signaling pathways, including MAPK, NF-κB, and PI3K/Akt, which controls responses such as cell proliferation, apoptosis, and migration. In the present review, we have discussed the significance of the intriguing relationship between HDPs and endothelial cell physiology which suggests it potential as a therapeutic agents for the treating wounds, cardiovascular diseases, and inflammation-related endothelial damage.

Biochemistry, Mechanistic Intricacies, and Therapeutic Potential of Antimicrobial Peptides: An Alternative to Traditional Antibiotics.

The emergence of drug-resistant strains of pathogens becomes a major obstacle to treating human diseases. Antibiotics and antivirals are in the application for a long time but now these drugs are not much effective anymore against disease-causing drugresistant microbes and gradually it is becoming a serious complication worldwide. The development of new antibiotics cannot be a stable solution to treat drug-resistant strains due to their evolving nature and escaping antibiotics. At this stage, antimicrobial peptides (AMPs) may provide us with novel therapeutic leads against drug-resistant pathogens. Structurally, antimicrobial peptides are mostly α-helical peptide molecules with amphiphilic properties that carry the positive charge (cationic) and belong to host defense peptides. These positively charged AMPs can interact with negatively charged bacterial cell membranes and may cause the alteration in electrochemical potential on bacterial cell membranes and consequently lead to the death of microbial cells. In the present study, we will elaborate on the implication of AMPs in the treatment of various diseases along with their specific structural and functional properties. This review will provide information which assists in the development of new synthetic peptide analogues to natural AMPs. These analogues will eliminate the limitations of natural AMPs like toxicity and severe hemolytic activities.

Antimicrobial Potential of Scorpion-Venom-Derived Peptides.

The frequent and irrational use of antibiotics by humans has led to the escalating rise of antimicrobial resistance (AMR) with a high rate of morbidity-mortality worldwide, which poses a challenge to the development of effective treatments. A large number of host defense peptides from different organisms have gained interest due to their broad antibacterial spectrum, rapid action, and low target resistance, implying that these natural sources might be a new alternative to antimicrobial drugs. As important effectors of prey capture, defense against other animal attacks, and competitor deterrence, scorpion venoms have been developed as important candidate sources for modern drug development. With the rapid progress of bioanalytical and high throughput sequencing techniques, more and more scorpion-venom-derived peptides, including disulfide-bridged peptides (DBPs) and non-disulfide-bridged peptides (NDBPs), have been recently identified as having massive pharmacological activities in channelopathies, pathogen infections, and cancer treatments. In this review, we summarize the molecular diversity and corresponding structural classification of scorpion venom peptides with antibacterial, antifungal, and/or antiparasitic activity. We also aim to improve the understanding of the underlying mechanisms by which scorpion-venom-derived peptides exert these antimicrobial functions, and finally highlight their key aspects and prospects for antimicrobial therapeutic or pharmaceutical application.

Rumicidins are a family of mammalian host-defense peptides plugging the 70S ribosome exit tunnel

The antimicrobial resistance crisis along with challenges of antimicrobial discovery revealed the vital necessity to develop new antibiotics. Many of the animal proline-rich antimicrobial peptides (PrAMPs) inhibit the process of bacterial translation. Genome projects allowed to identify immune-related genes encoding animal host defense peptides. Here, using genome mining approach, we discovered a family of proline-rich cathelicidins, named rumicidins. The genes encoding these peptides are widespread among ruminant mammals. Biochemical studies indicated that rumicidins effectively inhibited the elongation stage of bacterial translation. The cryo-EM structure of the Escherichia coli 70S ribosome in complex with one of the representatives of the family revealed that the binding site of rumicidins span the ribosomal A-site cleft and the nascent peptide exit tunnel interacting with its constriction point by the conservative Trp23-Phe24 dyad. Bacterial resistance to rumicidins is mediated by knockout of the SbmA transporter or modification of the MacAB-TolC efflux pump. A wide spectrum of antibacterial activity, a high efficacy in the animal infection model, and lack of adverse effects towards human cells in vitro make rumicidins promising molecular scaffolds for development of ribosome-targeting antibiotics.

Development of Low-Toxicity Antimicrobial Polycarbonates Bearing Lysine Residues.

Antibiotic resistance has been threatening public health for a long period, while the COVID pandemic aggravated the scenario. To combat antibiotic resistance strains, host defense peptides (HDPs) mimicking molecules have attracted considerable attention. Herein, we reported a series of polycarbonates bearing cationic lysine amino acid residues that could mimic the mechanism of action of HDPs and possess broad-spectrum antimicrobial activity. Moreover, those polymers had negligible toxicity toward red blood cells and mammalian cells. The membrane-disruption mechanism endows the lysine-containing polycarbonates with low possibility of resistance development and the fast killing kinetics, making them promising candidates for antimicrobial development.

Chemokine Receptor N-Terminus Charge Dictates Reliance on Post-Translational Modifications for Effective Ligand Capture and Following Boosting by Defense Peptides.

The chemokine receptors CCR1 and CCR5 display overlapping expression patterns and ligand dependency. Here we find that ligand activation of CCR5, not CCR1, is dependent on N-terminal receptor O-glycosylation. Release from O-glycosylation dependency is obtained by increasing CCR5 N-terminus acidity to the level of CCR1. Ligand activation of CCR5, not CCR1, drastically improves in the absence of glycosaminoglycans (GAGs). Ligand activity at both CCR1 and CCR5 is boosted by positively charged/basic peptides shown to interact with acidic chemokine receptor N-termini. We propose that receptors with an inherent low N-terminus acidity rely on post-translational modifications (PTMs) to efficiently compete with acidic entities in the local environment for ligand capture. Although crucial for initial ligand binding, strong electrostatic interactions between the ligand and the receptor N-terminus may counteract following insertion of the ligand into the receptor binding pocket and activation, a process that seems to be aided in the presence of basic peptides. Basic peptides bind to the naked CCR1 N-terminus, not the CCR5 N-terminus, explaining the loss of boosting of ligand-induced signaling via CCR5 in cells incapable of glycosylation.

Host Defense Peptides: Exploiting an Innate Immune Component Against Infectious Diseases and Cancer

Host Defense Peptides: Exploiting an Innate Immune Component Against Infectious Diseases and Cancer

Dietary supplementation of Astragalus flavonoids regulates intestinal immunology and the gut microbiota to improve growth performance and intestinal health in weaned piglets.

Astragali Radix (AS) is a widely used herb in traditional Chinese medicine, with calycosin as its main isoflavonoid. Our previous study discovered that calycosin triggers host defense peptide (HDP) production in IPEC-J2 cells. The aim of this study is to investigate the alleviation effects of AS total flavone and AS calycosin on growth performance, intestinal immunity, and microflora in weaned piglets. Sixty-four piglets were assigned randomly to 4 treatment groups, (1) CON: the basal diet, (2) P-CON: the basal diet plus antibiotics (1 g/kg), (3) AS-TF: the basal diet plus AS total flavone at 60 mg/day per piglet, (4) AS-CA: the basal diet plus AS calycosin at 30 mg/day per piglet. Each treatment consists of 4 replicates with 4 piglets per replicate. Results showed that treatment with AS-TF and AS-CA enhanced average daily growth and average daily feed intake compared to the CON group (P < 0.01), while AS-CA significantly reduced the diarrhea rate (P < 0.05). Both AS-TF and AS-CA significantly increased serum immunoglobulin (Ig) A and IgG levels, with AS-CA further boosting intestinal mucosal secretory IgA levels (P < 0.05). Histological analysis revealed improvements in the morphology of the jejunum and ileum and goblet cell count by AS-TF and AS-CA (P < 0.05). Supplementation of AS-TF and AS-CA promoted the expression of several intestinal HDPs (P < 0.05), and the effect of AS-CA was better than that of AS-TF. In addition, the AS-TF and AS-CA regulated jejunal microbial diversity and composition, with certain differential bacteria genera were showing high correlation with serum cytokines and immunoglobulin levels, suggesting that the intestinal flora affected by AS-TF and AS-CA may contribute to host immunity. Overall, AS CA and AS TF all improved growth performance and health, likely by enhancing nutrition digestibility, serum and intestinal immunity, and intestinal microbial composition. They showed the similar beneficial effect, indicating AS CA appears to be a major compound contributing to the effects of AS TF. This study demonstrated the positive effect of AS flavonoids on weaned piglets and provided a scientific reference for the efficient use of AS products.

Investigation on the in vitro effects of resveratrol on peripheral blood mononuclear cells harvested from healthy and atopic dogs

Canine atopic dermatitis (AD) is a common skin disease. Many therapeutic options are available to decrease inflammation and ameliorate pruritus. However, those treatments are associated with side effects, a long lag phase for efficacy, or high expense. Natural plant extracts have been identified as possible, safer alternatives to traditional anti-inflammatory and antipruritic drugs. Resveratrol has been revisited as a new, possible alternative for its numerous beneficial properties. The purpose of this study was to evaluate the in vitro effects of resveratrol on peripheral blood mononuclear cells (PBMC) harvested from healthy and atopic privately-owned dogs.The PBMC harvested from nine healthy and 11 atopic dogs were isolated and exposed to four concentrations (1.5-9 μg/mL) of resveratrol both with or without phytohemagglutinin stimulation. After 24 h cytotoxicity, host defense peptides (HDPs), as well as oxidative stress (catalase and superoxide dismutase), and pro-inflammatory cytokines were assessed. Cytotoxicity was not observed in either group under any experimental conditions. An increase in catalase was only seen in healthy PBMC after exposure to low concentrations of resveratrol (p ≤ 0.03). Resveratrol did not show any effect on canine HDPs. Compared to baseline, there was a significant reduction in monocyte chemotactic protein-1 and interleukin-6 after exposure to 9 μg/mL of resveratrol in unstimulated healthy (p = 0.029) and stimulated atopic (p = 0.0075) PBMC. In conclusion, these data confirm the overall lack of cytotoxicity of resveratrol on healthy and atopic PBMC at the tested concentrations. However, at the concentrations tested, there was only a minimal effect of resveratrol on the secretion of HDPs and pro-inflammatory cytokines.

Incorporating Single-Copper Sites and Host Defense Peptides into a Nanoreactor for Antibacterial Therapy by Bioinspired Design

A sustainable solution to the dramatic spread of antibiotic resistance threatening public health security is the development of antibiotic-free antimicrobial substances. Inspired by natural host defense mechanisms involving amino-terminal copper−nickel binding motif (ATCUN) antimicrobial peptides (AMPs), we have designed and prepared an artificial complex (Cu@G-AMPs) incorporating single-atom Cu catalysts for antibacterial therapy. The substrate of the complex, formed from guanine doped with abundant heteroatoms, anchored single Cu atoms with a coordination number of 2 and an average bond length of 1.91Å. Interestingly, Cu@G-AMPs, exhibiting Fenton-like catalytic activity, caused the inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by generating and delivering reactive oxygen species (ROS) cargo. Mechanistically, the intrinsic stress response system of MRSA underwent an irreversible collapse when Cu@G-AMPs initiated its offensive program associated with non-specific targets. Furthermore, Cu@G-AMPs, which inherited the immunomodulatory properties of AMPs, sequentially carried out the functions of pulling edge closure, stabilizing granulation tissue, promoting collagen fiber proliferation, alleviating inflammation, and promoting neovascularization in wound areas infected by MRSA. Our results show that Cu@G-AMPs will provide a new perspective on untangling the complex regulatory networks that resistant bacteria have cultivated to deactivate commercial antibiotics.

Antibacterial and Anti-Inflammatory Activity of Branched Peptides Derived from Natural Host Defense Sequences.

Antibiotic resistance is a major global health threat, necessitating the development of new treatments and diverse molecules to combat severe infections and preserve the efficacy of existing drugs. Antimicrobial peptides (AMPs) offer a versatile arsenal against bacteria, and peptide structure branching can enhance their resistance to proteases and improve their overall efficacy. A small library of peptides derived from natural host defense peptides and synthesized in a tetrabranched form was selected against E. coli. Six selected branched peptides were further studied for antibacterial activity against a panel of strains, biofilm inhibition, protease resistance, and cytotoxicity. Their structure was predicted computationally and their mechanism of action was investigated by electron microscopy and by using fluorescent dyes. The peptide BAMP2 showed promise in a mouse skin infection model, indicating the potential for local infection treatment.

The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens.

Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.

Complex of Defense Polypeptides of Wheatgrass (Elytrigia elongata) Associated with Plant Immunity to Biotic and Abiotic Stress Factors.

Plant defense polypeptides play a crucial role in providing plants with constitutive immunity against various biotic and abiotic stressors. In this study, we explored a complex of proteins from wheatgrass (Elytrigia elongata) spikelets to estimate their role in the plant's tolerance to various environmental factors. The current research shows that in vitro protein extracts from E. elongata spikelets possess antifungal activity against certain Fusarium species, which are specific cereal pathogens, at concentrations of 1-2 mg/mL. In this study, we reproduced these antifungal activities using a 4 mg/mL extract in artificial fungal infection experiments on wheat grain (Triticum aestivum) under controlled laboratory conditions. Furthermore, the tested extract demonstrated a protective effect on Saccharomyces cerevisiae exposed to hyper-salinity stress at a concentration of 2 mg/mL. A combined scheme of fractionation and structural identification was applied for the estimation of the diversity of defense polypeptides. Defensins, lipid-transfer proteins, hydrolase inhibitors (cereal bifunctional trypsin/alpha-amylase inhibitors from a Bowman-Birk trypsin inhibitor), and high-molecular-weight disease resistance proteins were isolated from the extract. Thus, wheatgrass spikelets appear to be a reservoir of defense polypeptides. Our findings contribute to a deeper understanding of plant defense proteins and peptides and their involvement in the adaptation to various stress factors, and they reveal the regulatory effect at the ecosystem level.

S100A12 inhibits Streptococcus pneumoniae and aids in wound healing of corneal epithelial cells both in vitro and in vivo

Streptococcus pneumoniae, a leading cause of corneal infections worldwide, are extremely aggressive despite antibiotic sensitivity and exhibit increased resistance towards antibiotics. Antimicrobial peptides are often considered as potent alternatives against antibiotic resistance and here we have investigated the possible roles of S100A12, a host defense peptide, in wound healing and S. pneumoniae infection. S100A12 significantly inhibited growth of S. pneumoniae by disruption of membrane integrity along with increased generation of reactive oxygen species. Additionally, S100A12 accelerated cell migration and wound closure in human corneal epithelial cells and in a murine corneal wound model by activation of EGFR and MAPK signaling pathways.

Impact of in ovo administration of xylo- and mannooligosaccharides on broiler chicken gut health

The intestinal mucosa creates a connection between the gut microbiota and the host. This study aimed to modify the gut microbiota of broiler chickens by in ovo stimulation with xylo-oligosaccharide (XOS) and manno-oligosaccharide (MOS) prebiotics and to determine the changes occurring in specific gut segments. Three hundred incubated eggs of Ross 308 broiler chickens on the 12th d of incubation were injected with: saline (control), xylotriose (XOS3), xylotetrose (XOS4), mannotriose (MOS3) or mannotetrose (MOS4). Tissue and digesta samples were collected post-mortem from 8 randomly selected individuals from each group, on d 42 after hatching. Gene expression analysis in the cecum and ileum was performed by RT-qPCR for a panel of genes: innate immune response genes (IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-1β, IFNγ, IFNβ), nutrient sensing and nutrient transport genes (FFAR2, FFAR4, GLUT1, GLUT2, GLUT5), host defence peptides (AvBD1, CATHL2), and barrier function genes (MUC6, CLDN1, TJAP). The relative abundance of bacteria was determined by qPCR for individual bacteria (Akkermansia muciniphilla, Bifidobacterium spp., Clostridium difficile, Escherichia coli, Faecalibacterium prausnitzii, and Lactobacillus spp.). Stimulation with prebiotics caused changes in the abundance of bacteria especially Lactobacillus spp. and Bifidobacterium spp. in the cecum. The abundance of both genera increased in each study group compared to the control group. The highest abundance of Bifidobacterium spp. in the ileum was found in the MOS3 group compared to the control group. There were changes in the XOS4 and MOS3 groups in the expression of: FFAR4, GLUT1, AvBD1, CATHL2, IL-2, IL-12, and IL-17 in the caecum. In conclusion, in ovo administration of prebiotics increased intestinal colonization by bacteria. The prebiotics influenced gene expression levels via changes in the gut microbiota.

StackDPPred: Multiclass prediction of defensin peptides using stacked ensemble learning with optimized features

Host defense or antimicrobial peptides (AMPs) are promising candidates for protecting host against microbial pathogens for example bacteria, virus, fungi, yeast. Defensins are the type of AMPs that act as potential therapeutic drug agent and perform vital role in various biological process. Conventional Experiments to identify defensin peptides (DPs) are time consuming and expensive. Thus, the shortcomings of wet lab experiments are leveraged by computational methods to accurately predict the functional types of DPs. In this paper, we aim to propose a novel multi-class ensemble-based prediction model called StackDPPred for identifying the properties of DPs. The peptide sequences are encoded using split amino acid composition (SAAC), segmented position specific scoring matrix (SegPSSM), histogram of oriented gradients-based PSSM (HOGPSSM) and feature extraction based graphical and statistical (FEGS) descriptors. Next, principal component analysis (PCA) is used to select the best subset of attributes. After that, the optimized features are fed into single machine learning and stacking-based ensemble classifiers. Furthermore, the ablation study demonstrates the robustness and efficacy of the stacking approach using reduced features for predicting DPs and their families. The proposed StackDPPred method improves the overall accuracy by 13.41% and 7.62% compared to existing DPs predictors iDPF-PseRAAC and iDEF-PseRAAC, respectively on validation test. Additionally, we applied the local interpretable model-agnostic explanations (LIME) algorithm to understand the contribution of selected features to the overall prediction. We believe, StackDPPred could serve as a valuable tool accelerating the screening of large-scale DPs and peptide-based drug discovery process.

Structural Classification Insights Into the Plant Defensive Peptides.

This study presents a structural phylogenetic analysis of plant defensive peptides, revealing their evolutionary relationships, structural diversification, and functional adaptations. Utilizing a robust dataset comprising both experimental and predicted structures sourced from the RCSB Protein Data Bank and AlphaFold DB, we constructed a detailed phylogenetic tree to elucidate the distinct evolutionary paths of plant defensive peptide families. Our findings showcase the evolutionary intricacies of defensive peptides, highlighting their diversity and the conservation of key structural motifs critical to their antimicrobial or defensive functions. The results also underscore the adaptive significance of defensive peptides in plant evolution, highlighting their roles in responding to ecological pressures and pathogen interactions.

Mimics of Host Defense Peptides Derived from Dendronized Polylysines for Antibacterial and Anticancer Therapy.

Bacteria in tumor microenvironments promote carcinogenesis and trigger complications, suggesting the significance of intervening in bacterial growth in cancer treatment. Here, dendrimer-derived mimics (DMs) of host defense peptides (HDPs) were designed for antibacterial and anticancer therapy, which feature a dendronized polylysine core and polycaprolactone arms. DMs displayed not only remarkable activities against Staphylococcus aureus and human lung cancer cells, but also exceptional selectivity. The membranolytic mechanism revealed by morphology analysis explained their low susceptibility to induce resistance. Further, the optimized DM inhibited tumor growth in the subcutaneous tumor model when administered via intraperitoneal injection and exhibited negligible toxicity to tissues. Overall, we combined the superiority of dendrimers and the mechanism from HDPs to design agents with dual antibacterial and anticancer activities that possess great potential for clinical oncology therapy.

Novel BRICHOS-related defensin-like antimicrobial peptide from the marine polychaeta &lt;i&gt;Arenicola marina&lt;/i&gt;

To date, polychaetes remain a poorly studied class of invertebrate animals in the context of clarification of their immune system functioning and, in particular, of antimicrobial peptides (AMPs) biodiversity. AMPs, also known as host defense peptides, play a key role in host protection from various pathogens and regulation of the species composition of symbiotic microbes. The study of biosynthesis of AMPs in polychaetes has revealed an interesting pattern, namely so-called BRICHOS domain in the precursor proteins of a number of such peptides. The conserved structure of this domain allows to perform a bioinformatic search for AMP precursors in polychaete transcriptomes. In this work, we found and studied a new BRICHOS-associated AMP from the lugworm Arenicola marina, which represents a structural family of defensin-like peptides stabilized by four disulfide bonds, not previously identified in marine worms. The peptide, designated as AmBRI-44a, contained 44 amino acid residues and was obtained by heterologous expression in Escherichia coli. AmBRI-44a was shown to have a specific activity against a narrow spectrum of Gram-positive bacteria and did not exhibit pronounced cytotoxic effects on eukaryotic cell line HEK293T. A potential mechanism of the antibacterial action of this peptide may be associated with inhibition of bacterial cell wall biosynthesis, as indicated by genetic and phenotypic analysis of selected AmBRI-44a-resistant bacteria Bacillus licheniformis B-511. The results obtained allow us to consider the novel peptide AmBRI-44a as a candidate compound for the development of an antibiotic agent that could potentially be effective in the treatment of infectious diseases mediated by multidrug-resistant Gram-positive bacteria.

Extensive differential DNA methylation between tuberculosis skin test positive and skin test negative cattle

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), represents a significant problem for the agriculture industry as well as posing a risk for human health. Current diagnostic tests for bTB target the cell-mediated immune (CMI) response to infection with M. bovis, primarily through screening of animals with the tuberculin skin test. Epigenetic modifications have been shown to alter the course of the immune response and differentially methylated regions (DMRs) might also influence the outcome of the skin test in cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of a group of cattle identified as test positive for M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay compared to a test negative control group [n = 8/group, total of 16 WGBS libraries]. Although global methylation profiles were similar for both groups across the genome, 223 DMRs and 159 Differentially Promoter Methylated Genes (DPMGs) were identified between groups with an excess of hypermethylated sites in SICTT positive cattle (threshold > 15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB). KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT negative cattle that were IFN-γ positive showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17 F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation is associated with SICTT test results and the degree of methylation could influence effective host immune responses.