Ameliorative Effects of Zingiber officinale Rosc on Antibiotic-Associated Diarrhea and Improvement in Intestinal Function.

The global increase in antibiotic consumption is related to increased adverse effects, such as antibiotic-associated diarrhea (AAD). This study investigated the chemical properties of Zingiber officinale Rosc (ZO) extract and its ameliorative effects using a lincomycin-induced AAD mouse model. Intestinal tissues were evaluated for the expression of lysozyme, claudin-1, and α-defensin-1, which are associated with intestinal homeostasis. The cecum was analyzed to assess the concentration of short-chain fatty acids (SCFAs). The chemical properties analysis of ZO extracts revealed the levels of total neutral sugars, acidic sugars, proteins, and polyphenols to be 86.4%, 8.8%, 4.0%, and 0.8%, respectively. Furthermore, the monosaccharide composition of ZO was determined to include glucose (97.3%) and galactose (2.7%). ZO extract administration ameliorated the impact of AAD and associated weight loss, and water intake also returned to normal. Moreover, treatment with ZO extract restored the expression levels of lysozyme, α-defensin-1, and claudin-1 to normal levels. The decreased SCFA levels due to induced AAD showed a return to normal levels. The results indicate that ZO extract improved AAD, strengthened the intestinal barrier, and normalized SCFA levels, showing that ZO extract possesses intestinal-function strengthening effects.

Electroacupuncture promoted intestinal defensins and rescued the dysbiotic cecal microbiota of high-fat diet-induced obese mice.

Obesity is currently one of the most important challenges to public health worldwide. Acupuncture has been widely used to treat obesity. However, whether acupuncture regulates intestinal innate immunity via intestinal microbiota against obesity remains to be elucidated. In this study, electroacupuncture (EA) effectively reduced body weight and fat accumulation in obese mice persistently fed a high-fat diet. Full-length 16S rDNA sequencing showed dysbiotic microbiota in the cecum of obese mice. The composition and function of the cecal microbiota of obese mice were markedly restored after EA treatment. After 21 d of EA intervention, the expression of defensin alpha 5 (Defa5) was restored to healthy controls, whereas fat digestion and absorption genes including fabp1 were markedly decreased in the jejunum of obese mice. The Defa5 levels were positively correlated with the family Lachnospiraceae and negatively correlated with obesity indexes. EA also reduced tissue inflammation, ameliorated misaligned glucose tolerance, and inhibited key genes for intestinal lipid absorption. In summary, EA exerted an anti-obesity effect by promoting intestinal defensins, rescuing dysbiotic cecal microbiota, and reducing lipid absorption in a synergistic mode. We present for the first time the key role of alpha defensins in the relationship between gut microbiota and disease during electroacupuncture treatment of obesity. The mucosal innate immunity seems to have a stronger ability to shape the microbiota than dietary factors.

Analysis of α-Defensin 5 Secretion in Differentiated Caco-2 Cells: Comparison of Cell Bank Origin.

α-Defensin 5 has a particularly broad antibacterial spectrum; it eliminates pathogenic microorganisms and regulates intestinal flora. Although Caco-2 cells are similar to small intestinal cells, it is unclear whether they secrete α-defensin 5. Therefore, we investigated whether Caco-2 cells secrete α-defensin 5 and determined the secretion mechanism using cells from three cell banks (ATCC, DSMZ, and RIKEN). The Caco-2 cell proliferation rate increased with the number of culture days, irrespective of cell bank origin. On the other hand, the alkaline phosphatase activity, which affects cell differentiation and the mRNA levels of several cytokines, such as interleukin 8 (IL-8), IL-6, IL-1ß, tumor necrosis factor-α (TNF-α), and IL-2, in the Caco-2 cells fluctuated with the number of culture days, and differed for each cell bank. α-Defensin 5 secretion was detected in all three cell bank Caco-2 cells; particularly, the ATCC Caco-2 cells grew linearly depending on the cell culture day as well as the levels of IL-8 and TNF-α mRNA. This suggested that α-defensin 5 secretion in the ATCC Caco-2 cells was associated with fluctuations in the mRNA levels of various cytokines, such as IL-8 and TNF-α. In conclusion, Caco-2 cells may be a simple model for screening health food components and drugs that affect α-defensin 5 secretion.

RTD-1 therapeutically normalizes synovial gene signatures in rat autoimmune arthritis and suppresses proinflammatory mediators in RA synovial fibroblasts.

Rhesus theta defensin-1 (RTD-1), a macrocyclic immunomodulatory host defense peptide from Old World monkeys, is therapeutic in pristane-induced arthritis (PIA) in rats, a model of rheumatoid arthritis (RA). RNA-sequence (RNA-Seq) analysis was used to interrogate the changes in gene expression in PIA rats, which identified 617 differentially expressed genes (DEGs) in PIA synovial tissue of diseased rats. Upstream regulator analysis showed upregulation of gene expression pathways regulated by TNF, IL1B, IL6, proinflammatory cytokines, and matrix metalloproteases (MMPs) involved in RA. In contrast, ligand-dependent nuclear receptors like the liver X-receptors NR1H2 and NR1H3 and peroxisome proliferator-activated receptor gamma (PPARG) were downregulated in arthritic synovia. Daily RTD-1 treatment of PIA rats for 1-5 days following disease presentation modulated 340 of the 617 disease genes, and synovial gene expression in PIA rats treated 5 days with RTD-1 closely resembled the gene signature of naive synovium. Systemic RTD-1 inhibited proinflammatory upstream regulators such as TNF, IL1, and IL6 and activated antiarthritic ligand-dependent nuclear receptor pathways, including PPARG, NR1H2, and NR1H3, that were suppressed in untreated PIA rats. RTD-1 also inhibited proinflammatory responses in IL-1ß-stimulated human RA fibroblast-like synoviocytes (FLS) in vitro and diminished expression of human orthologs of disease genes that are induced in rat PIA synovium. Thus, the antiarthritic mechanisms of systemic RTD-1 include homeostatic regulation of arthritogenic gene networks in a manner that correlates temporally with clinical resolution of rat PIA.

Effects of Protein Versus Carbohydrate Supplementation on Markers of Immune Response in Master Triathletes: A Randomized Controlled Trial.

Objective: This study examines the long-term effects of ingesting hydrolyzed beef protein versus carbohydrate on indirect markers of immunity during 10 weeks of endurance training in master-aged triathletes (n = 16, age 35-60 years). Methods: Participants were randomly assigned to either a hydrolyzed beef protein (PRO, n = 8) or nonprotein isoenergetic carbohydrate (CHO, n = 8) condition, which consisted of ingesting 20 g of each supplement, mixed with water, once a day immediately post workout, or before breakfast on nontraining days. Salivary human neutrophil peptides (HNP1-3) were measured before and after performing an incremental endurance test to volitional exhaustion at both pre and post intervention. Additionally, baseline levels of platelets, neutrophils, eosinophil basophils, monocytes, and lymphocytes were determined at pre and post intervention. Results: No significant changes in baseline concentration and secretion rate of salivary HNP1-3 were observed for either treatment. The CHO group showed a nonsignificant decrease in resting HNP1-3 concentrations following the intervention (p = 0.052, effect size d = 0.53). Protein supplementation demonstrated a significant reduction in lymphocyte counts pre to post intervention (mean [SD]: 2.30 [0.57] vs. 1.93 [0.45] 103/mm3, p = 0.046, d = 0.77), along with a moderate but not statistically significant increase (d = 0.75, p = 0.051) of the neutrophil-to-lymphocyte ratio. Conclusions: In master-aged triathletes, postworkout ingestion of only protein, with no carbohydrate, may not be as effective as carbohydrate alone to attenuate negative long-term changes of some salivary and cellular immunological markers. Future studies should consider the co-ingestion of both macronutrients.

Self-Assembling Myristoylated Human α-Defensin 5 as a Next-Generation Nanobiotics Potentiates Therapeutic Efficacy in Bacterial Infection.

The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human α-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli ( E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.

Reishi mushroom Ganoderma lucidum Modulates IgA production and alpha-defensin expression in the rat small intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Immunoglobulin A (IgA) secretion and alpha-defensins play a role in the innate immune system to protect against infection. Ganoderma lucidum (W.Curt.: Fr.) P. Karst. (Reishi) is a well-known mushroom in traditional Chinese medicine. This study aimed to determine the effects of Reishi on IgA secretion from Peyer's patch (PP) cells and alpha-defensin-5 (RD-5) and RD-6 expression in the rat small intestine. MATERIALS AND METHODS: The rats received an oral injection of 0.5-5mg/kg of Reishi powder (1mL/kg) by sonde. All animals were euthanized 24h after Reishi administration. We examined RD-5, RD-6, and Toll-like receptor (TLR) 4 mRNA levels in the jejunum, ileum, and in Peyer's patches (PP) through quantitative real-time PCR analysis. IgA secretion from PP was measured through enzyme-linked immunosorbent assay of the supernatant after primary culture. RESULTS: Reishi increased IgA secretion in the presence of lipopolysaccharide (LPS) and increased TLR4 mRNA levels, but had no effect on the viability of PP cells. Moreover, Reishi increased RD-5, RD-6, and TLR4 mRNA levels significantly in the ileum in a concentration-dependent manner. CONCLUSIONS: Reishi can induce IgA secretion and increase the mRNA levels of RD-5 and RD-6 in the rat small intestine, through a TLR4-dependent pathway. The present results indicate that Reishi might reduce the risk of intestinal infection.

Effects of vitamin D status on oral health.

Normal humans of all ages have the innate ability to produce vitamin D following sunlight exposure. Inadequate vitamin D status has shown to be associated with a wide variety of diseases, including oral health disorders. Insufficient sunlight exposure may accelerate some of these diseases, possibly due to impaired vitamin D synthesis. The beneficial effects of vitamin D on oral health are not only limited to the direct effects on the tooth mineralization, but are also exerted through the anti-inflammatory functions and the ability to stimulate the production of anti-microbial peptides. In this article, we will briefly discuss the genesis of various oral diseases due to inadequate vitamin D level in the body and elucidate the potential benefits of safe sunlight exposure for the maintenance of oral and general health.

Vitamin D Promotes Pneumococcal Killing and Modulates Inflammatory Responses in Primary Human Neutrophils.

Streptococcus pneumoniae is a major human pathogen and a leading cause of pneumonia, septicemia, and meningitis worldwide. Despite clinical studies linking vitamin D deficiency and pneumonia, molecular mechanisms behind these observations remain unclear. In particular, the effects of vitamin D on neutrophil responses remain unknown. Using pneumococcal strains, primary neutrophils isolated from human blood, and sera from patients with frequent respiratory tract infections (RTIs), we investigated the effects of vitamin D on neutrophil bactericidal and inflammatory responses, including pattern recognition receptors, antimicrobial peptides, and cytokine regulation. We found that vitamin D upregulated pattern recognition receptors, TLR2, and NOD2, and induced the antimicrobial human neutrophil peptides (HNP1-3) and LL-37, resulting in increased killing of pneumococci in a vitamin D receptor-dependent manner. Antibodies targeting HNP1-3 inhibited bacterial killing. Vitamin D supplementation of serum from patients with bacterial RTIs enhanced neutrophil killing. Moreover, vitamin D lowered inflammatory cytokine production by infected neutrophils via IL-4 production and the induction of suppressor of cytokine signaling (SOCS) proteins SOCS-1 and SOCS-3, leading to the suppression of NF-κB signaling. Thus, vitamin D enhances neutrophil killing of S. pneumoniae while dampening excessive inflammatory responses and apoptosis, suggesting that vitamin D could be used alongside antibiotics when treating pneumococcal infections.

Design of a potent antibiotic peptide based on the active region of human defensin 5.

Human defensin 5 (HD5) is a broad-spectrum antibacterial peptide with a C-terminal active region. To promote the development of this peptide into an antibiotic, we initially substituted Glu21 with Arg because it is an electronegative residue located around the active region. Although detrimental to dimer formation, the E21R substitution markedly enhanced the antibacterial activity of HD5 and increased its ability to penetrate cell membranes, demonstrating that increasing the electropositive charge compensated for the effect of dimer disruption. Subsequently, a partial Arg scanning mutagenesis was performed, and Thr7 was selected for replacement with Arg to further strengthen the antibacterial activity. The newly designed peptide, T7E21R-HD5, exhibited potent antibacterial activity, even in saline and serum solutions. In contrast to monomeric E21R-HD5, T7E21R-HD5 assembled into an atypical dimer with parallel ß strands, thus expanding the role of increasing electropositive charge in bactericidal activity and providing a useful guide for further defensin-derived antibiotic design.

Peripheral α-defensins 1 and 2 are elevated in Alzheimer's disease.

Biomarkers enabling the preclinical identification of Alzheimer's disease (AD) remain one of the major unmet challenges in the field. The blood cellular fractions offer a viable alternative to current cerebrospinal fluid and neuroimaging modalities. The current study aimed to replicate our earlier reports of altered binding within the AD-affected blood cellular fraction to copper-loaded immobilized metal affinity capture (IMAC) arrays. IMAC and anti-amyloid-ß (Aß) antibody arrays coupled with mass spectrometry were used to analyze blood samples collected from 218 participants from within the AIBL Study of Aging. Peripheral Aß was fragile and prone to degradation in the AIBL samples, even when stored at -80°C. IMAC analysis of the AIBL samples lead to the isolation and identification of alpha-defensins 1 and 2 at elevated levels in the AD periphery, validating earlier findings. Alpha-defensins 1 and 2 were elevated in AD patients indicating that an inflammatory phenotype is present in the AD periphery; however, peripheral Aß levels are required to supplement their prognostic power.

Glutamine Improves Innate Immunity and Prevents Bacterial Enteroinvasion During Parenteral Nutrition.

BACKGROUND: Patients receiving parenteral nutrition (PN) are at increased risk of infectious complications compared with enteral feeding, which is in part explained by impaired mucosal immune function during PN. Adding glutamine (GLN) to PN has improved outcome in some clinical patient groups. Although GLN improves acquired mucosal immunity, its effect on innate mucosal immunity (defensins, mucus, lysozymes) has not been investigated. METHODS: Forty-eight hours following venous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 10), PN (n = 12), or PN + GLN (n = 13) for 5 days. Small intestine tissue and luminal fluid were collected for mucin 2 (MUC2), lysozyme, cryptdin 4 analysis, and luminal interleukin (IL)-4, IL-10, and IL-13 level measurement. Tissue was also harvested for ex vivo intestinal segment culture to assess tissue susceptibility to enteroinvasive Escherichia coli. RESULTS: In both luminal and tissue samples, PN reduced MUC2 and lysozyme (P < .0001, respectively) compared with chow, whereas GLN addition increased MUC2 and lysozyme (luminal, P < .05; tissue, P < .0001, respectively) compared with PN alone. PN significantly suppressed cryptdin 4 expression, while GLN supplementation significantly enhanced expression. IL-4, IL-10, and IL-13 decreased significantly with PN compared with chow, whereas GLN significantly increased these cytokines compared with PN. Functionally, bacterial invasion increased with PN compared with chow (P < .05), while GLN significantly decreased enteroinvasion to chow levels (P < .05). CONCLUSIONS: GLN-supplemented PN improves innate immunity and resistance to bacterial mucosal invasion lost with PN alone. This work confirms a clinical rationale for providing glutamine for the protection of the intestinal mucosa.

Immunoprotective effect of epigallocatechin-3-gallate on oral anticancer drug-induced α-defensin reduction in Caco-2 cells.

The aim of this study was to determine the effect of interaction between tegafur (FT) and epigallocatechin-3-gallate (EGCG) on the expression of α-defensins (HD-5: human α-defensin 5, HD-6: human α-defensin 6) by using a Caco-2 cell line as a model of human intestinal epithelial cells. This is the first study in which the effect of interaction of an oral anticancer drug and functional food on the innate immune system was examined. α-Defensins are abundant constituents of mouse and human paneth cells and play a role in the innate immune system in intestine. We detected HD-5 and HD-6 mRNA in Caco-2 cells and evaluated the effects of FT and EGCG on these mRNA levels. HD-5 and HD-6 mRNA levels were decreased by exposure to FT. Production of reactive oxygen species (ROS) was induced by exposure to FT as well as H2O2 exposure, and EGCG suppressed FT-induced production of ROS. Furthermore, FT-induced decrease in HD-5 and HD-6 mRNA levels was almost completely suppressed by EGCG. These results indicate that EGCG restored the decrease of α-defensins induced by FT at the transcriptional level in Caco-2 cells, suggesting that EGCG can be used as adjunctive therapy in chemotherapy.

In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment.

Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.

Proteomic bronchiolitis obliterans syndrome risk monitoring in lung transplant recipients.

BACKGROUND: Obliterative bronchiolitis poses a primary obstacle for long-term survival of lung transplant recipients and manifests clinically as bronchiolitis obliterans syndrome (BOS). Establishing a molecular level screening method to detect BOS-related proteome changes before its diagnosis by forced expiratory volume surrogate marker criteria was the main objective of this study. METHODS: Bronchoalveolar lavage was performed in 82 lung transplant recipients (48/34 with/without known BOS development) at different time points between 12 and 48 months after lung transplantation. A mass spectrometry-based method was devised to generate bronchoalveolar lavage fluid proteome profiles that were screened for BOS-specific alterations. Statistically significant marker peptides and proteins were identified and validated by in-gel digestion, tandem mass spectrometric sequencing, and quantitative immunoassays. RESULTS: Among the panel of statistically significant markers were Clara cell protein, calgranulin A, human neutrophil peptides, and the antimicrobial agent histatin. To assess their clinical relevance, a highly sensitive and specific classifier model was developed. Positive BOS classification by monitoring of seven polypeptides correlated strongly with a significant decrease in BOS-free time. Thus, it was possible to detect high-risk patients early on in the pathogenetic process. CONCLUSIONS: Monitoring the bronchoalveolar lavage fluid levels of seven polypeptides detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allows a reliable prediction of early BOS using a Random Forest decision tree-based classifier model. The high accuracy of this robust model and its synergistic potential in combination with established forced expiratory volume-based diagnostics could make it an effective tool to supplement the current diagnostic regime after multicentric validation.

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord.

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

Rapid evolution and diversification of mammalian alpha-defensins as revealed by comparative analysis of rodent and primate genes.

Mammalian alpha-defensins constitute a family of cysteine-rich, cationic antimicrobial peptides produced by phagocytes and intestinal Paneth cells, playing an important role in innate host defense. Following comprehensive computational searches, here we report the discovery of complete repertoires of the alpha-defensin gene family in the human, chimpanzee, rat, and mouse with new genes identified in each species. The human genome was found to encode a cluster of 10 distinct alpha-defensin genes and pseudogenes expanding 132 kb continuously on chromosome 8p23. Such alpha-defensin loci are also conserved in the syntenic chromosomal regions of chimpanzee, rat, and mouse. Phylogenetic analyses showed formation of two distinct clusters with primate alpha-defensins forming one cluster and rodent enteric alpha-defensins forming the other cluster. Species-specific clustering of genes is evident in nonprimate species but not in the primates. Phylogenetically distinct subsets of alpha-defensins also exist in each species, with most subsets containing multiple members. In addition, natural selection appears to have acted to diversify the functionally active mature defensin region but not signal or prosegment sequences. We concluded that mammalian alpha-defensin genes may have evolved from two separate ancestors originated from beta-defensins. The current repertoires of the alpha-defensin gene family in each species are primarily a result of repeated gene duplication and positive diversifying selection after divergence of mammalian species from each other, except for the primate genes, which were evolved prior to the separation of the primate species. We argue that the presence of multiple, divergent subsets of alpha-defensins in each species may help animals to better cope with different microbial challenges in the ecological niches which they inhabit.

Role of human neutrophil peptide-1 as a possible adjunct to antituberculosis chemotherapy.

We report the role of human neutrophil peptide (HNP)-1 as an adjunct to antituberculosis (anti-TB) drugs. The combination of HNP-1, isoniazid, and rifampicin was evaluated against Mycobacterium tuberculosis H(37)Rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (MICs) of these agents. In vitro results revealed >1-log unit reductions even when HNP-1 and anti-TB drugs were used at 1/16 MICs. This combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 MICs of HNP-1 and drugs. HNP-1 used in conjunction with anti-TB drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. The effective therapeutic dosage of drugs could be reduced to half by supplementing HNP-1 in the therapeutic schedule. These results clearly suggest that HNP-1 can be used as adjunct chemotherapy with conventional drugs against TB.

Blood cell gene expression profiling in rheumatoid arthritis. Discriminative genes and effect of rheumatoid factor.

UNLABELLED: To study the pathogenic importance of the rheumatoid factor (RF) in rheumatoid arthritis (RA) and to identify genes differentially expressed in patients and healthy individuals, total RNA was isolated from peripheral blood mononuclear cells (PBMC) from eight RF-positive and six RF-negative RA patients, and seven healthy controls. Gene expression of about 10,000 genes were examined using oligonucleotide-based DNA chip microarrays. The analyses showed no significant differences in PBMC expression patterns from RF-positive and RF-negative patients. However, comparisons of gene expression patterns from all fourteen RA patients and healthy controls identified a subset of discriminative genes. These results were validated by real time reverse transcription polymerase chain reaction (RT-PCR) on another group of RA patients and healthy controls. This confirmed that the following genes had a significantly higher expression in RA patients than in healthy controls: CD14 antigen, defensin alpha-1 and alpha-3 (DEFA), fatty-acid-Coenzyme A ligase, long-chain 2 (FACL), ribonuclease 2 (RNASE2), S100 calcium-binding protein A8 and A12 (S100A8 and S100A12). In contrast, the expression of MHC class II, DQ beta1 (HLA-DQB1) was significantly reduced in RA patients compared to healthy controls. CONCLUSIONS: With the analytical procedure employed, we did not find any indication that RF-positive and RF-negative RA are two fundamentally different diseases. Most of the genes discriminative between RA patients and healthy individuals are known to be involved in immunoinflammatory responses, especially those related to altered phagocytic functions.

The potential of human neutrophil peptides in tuberculosis therapy.

The problems of drug resistance and bacterial persistence in tuberculosis have prompted scientists to search for clues from the latest advances in microbiology and immunology. Recent research on human neutrophil peptides (HNPs) has highlighted their bactericidal action against Mycobacterium tuberculosis and suggested that neutrophils may play a more important defensive role in tuberculosis than previously thought. Human neutrophil peptides belong to a family of antimicrobial and cytotoxic peptides known as 'defensins'. Neutrophils use both oxidative and non-oxidative microbicidal mechanisms to provide the host with innate immunity against microbial infections. Defensins are most abundant among an array of oxygen-independent antimicrobial proteins and peptides in neutrophil granules. Defensins are effective against a wide spectrum of microbes including bacteria, viruses, fungi, spirochetes and mycobacteria. In addition to direct antimicrobial activity, HNPs can potentially influence the inflammatory or immune responses by modulating cytokine production or acting like opsonins or chemotactic factors. HNPs are active against M. tuberculosis grown in vitro or within macrophages. HNPs released by neutrophils recruited in the early lesion could attract monocytes to the site and macrophages may in vivo uptake the extracellular HNPs and kill the intracellular pathogens. As such, HNPs are potential therapeutic agents against tuberculosis. HNPs are also cytotoxic against a wide range of normal mammalian cells; however, there is evidence that defensins may not cause significant cytotoxicity at the therapeutic level. Finally, the clinical application of HNPs must be evaluated in the context of possible drug resistance, as some resistance-associated genes have been identified.