Nonspecific Host Defense Research Articles

Plasminogen activator inhibitor type I may contribute to transient, non-specific changes in immunity in the subacute phase of murine tuberculosis

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths worldwide. Non-specific host defense mechanisms such as the coagulation and fibrinolytic system may give insight in possible new therapeutic targets. Plasminogen activator inhibitor type-1 (PAI-1), an important regulator of inflammation and fibrinolysis, might be of interest as tuberculosis patients have elevated plasma levels of PAI-1. In this study we set out to investigate the role of PAI-1 during tuberculosis in vivo. Wildtype (WT) and PAI-1 deficient (PAI-1−/−) mice were intranasally infected with M. tuberculosis H37rv and sacrificed after 2, 5 and 29 weeks. Five weeks post-infection, bacterial loads in lungs of PAI-1−/− mice were significantly higher compared to WT mice, while no differences were seen 2 and 29 weeks post-infection. At two weeks post-infection increased influx of macrophages and lymphocytes was observed. PAI-1 deficiency was associated with a reduced cytokine response in the lungs; however, upon stimulation with tuberculin purified protein derivative (PPD), PAI-1−/− splenocytes released increased levels of IFN-γ compared to WT. No clear differences were found between PAI-1−/− and WT mice at 29 weeks after infection. In conclusion, these data suggest that PAI-1 contributes to transient, non-specific changes in immunity during the early phase of murine tuberculosis.

Nasal Nitric Oxide and Lifestyle Exposure to Tobacco Smoke

Nitric oxide (NO) is a reactive gas generated by inflammatory cells and mucosal epithelial cells of the nose and paranasal sinuses and is an important mediator in nonspecific host defense against infectious agents. However, NO also mediates physiologic events such as vasodilation, mucus hypersecretion, and mucosal disruption that are associated with inflammatory conditions, and it is a regulator of ciliary beat frequency. In the present study, we hypothesized that lifestyle exposure to tobacco smoke, whether through active smoking or by inadvertent exposure to secondhand tobacco smoke, would result in higher detectable levels of nasal NO (nNO) than are found in well-documented nonsmokers. Nasal NO measurements were obtained concomitant with assays of urine cotinine from well-documented nonsmokers, active smokers, and individuals exposed by lifestyle to secondhand smoke. These parameters were statistically analyzed to determine whether increasing levels of tobacco smoke exposure yield higher concentrations of nNO. Our results and subsequent statistical analyses imply that active smokers who exhibit high urine cotinine levels exhibit significant increases in nNO levels in comparison to both nonsmokers and nonsmokers exposed to secondhand smoke. There is an increased level of nNO associated with tobacco smoke exposure that may contribute to the inflammatory processes characteristic of disease pathogenesis in smokers.

Cell Surface of Lactococcus lactis Is Covered by a Protective Polysaccharide Pellicle

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

Characterization of immune-active peptides obtained from milk fermented byLactobacillus helveticus

The objectives of this research were to confirm the effect of compounds derived from milk fermented by Lactobacillus helveticus (LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were tested in vitro for immuno-modulating activity using murine macrophages (RAW 264.7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-alpha), and Interleukin-1beta (IL1-beta)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from beta-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-alpha showed a direct correlation between protein concentration and TNF-alpha release. The fraction contained four novel peptides, three derived from the hydrolysis of beta-casein and one from the hydrolysis of alpha-lactalbumin. These results confirm that fermentation of milk by Lactobacillus helveticus (LH-2) results in the production of specific peptides capable of modulating macrophage activity.

Effects of bacterial lipopolysaccharide on thermoregulation in green anole lizards ( Anolis carolinensis)

Fever is a non-specific host defense mechanism that comprises part of the innate immune response. Innate immune function is thought to be an important adaptive immunological response to infection because it occurs across a broad diversity of phyla. Some reptiles can mount a febrile response, despite the fact that their internal body temperatures ( T bs) are, to some extent, controlled by the environmental temperatures in which they live. This study was undertaken to determine if LPS would induce fever in green anole lizards ( Anolis carolinensis). Lizards were maintained in thermal gradients (22–45 °C) with a 12-h diurnal cycle. anoles were injected with LPS, pyrogen-free saline, or left untreated, and their T bs were recorded every 15 min using internal cloacal probes. All lizards showed a diurnal periodicity in T b characterized by decreased temperatures during the scotophase (dark hours) and higher temperatures during the photophase (light phase). Anoles injected with LPS exhibited a hypothermic response, relative to untreated and saline-injected animals. The response varied from 2.1 to 4.6 °C lower than control lizards. The hypothermic response was initiated within 12–24 h of LPS injection, and continued for 3 days after treatment. However, the anapyrexic response was observed primarily during scotophases, with photophase hypothermia observed only on the first day after LPS injection.

Effects of a Hyaluronate-Carboxymethylcellulose Membrane (Seprafilm) on Human Polymorphonuclear Neutrophil Functions

Seprafilm (Genzyme Corp., Cambridge, MA) is safe and effective for reducing adhesion formation in abdominal and pelvic surgery. However, the relationships between Seprafilm and postoperative complications, including intra-abdominal abscess formation and intra-abdominal inflammation, remain controversial. Polymorphonuclear neutrophils (PMNs) play crucial roles in the processes of intra-abdominal inflammation, infection, and abscess formation, and PMNs act as the first line in nonspecific host defense against a wide range of pathogens. The present study evaluates the effect of Seprafilm itself on human PMNs in experimental models in vitro. Human PMNs were isolated from blood samples obtained from 14 healthy volunteers. The functions of the isolated cells were assessed by examining their phagocytosis, apoptosis/necrosis rates, cytokine production, and PMN-elastase release with and without Seprafilm in an in vitro experimental model of inflammation and endotoxin-stimulation. There were no significant differences in the PMN phagocytosis, apoptosis/necrosis rates, cytokine production, or PMN-elastase release into the supernatant after coculture with bacteria between assays with and without Seprafilm. In the PMN stimulation models involving tumor necrosis factor-alpha and lipopolysaccharide, there were no significant differences in cytokine production between assays with and without Seprafilm. Furthermore, a similar experiment measuring cytokine production in the presence of well-dissolved Seprafilm produced corresponding results. Our results suggest Seprafilm itself does not affect the functions of isolated human PMNs in in vitro experimental models of inflammation and endotoxin-stimulation.

Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation

Dietary proteins harbour bioactive peptides that can be released by a fermentation process. Fish proteins are a valuable and little-exploited source of potentially active biopeptides. The aim of this research was to evaluate the effects of a commercially available fermented fish protein concentrate (Seacure ®) (FPC) derived from a fermentation process, on the mucosal immune response in a murine model. BALB/c mice received the FPC or the non-fermented powder at different concentrations (0.20, 0.25 or 0.30 mg/ml) for 2, 5 or 7 consecutive days. At the end of each feeding period, histological studies of the gut were carried out and the phagocytic activity of peritoneal macrophages, the number of IgA+ cells in the small intestine lamina propria and bronchial tissue and the number of IL-4+, IL-6+, IL-10+, IFN γ+ and TNF α+ cells in the small intestine lamina propria were determined. Different accumulative doses of FPC did not induce any inflammatory immune response and the normal morphology of the small intestine was not affected. Phagocytic activity of peritoneal macrophages was enhanced following FPC administration at 0.3 mg/ml for 7 consecutive days. The number of IgA+ cells increased in the small intestine lamina propria but not in the bronchial tissue. IL-4, IL-6 and IL-10 were all significantly increased in the lamina propria of the small intestine of animals that received FPC. At the same time, some pro-inflammatory cytokines such as IFN γ and TNF α also increased, but the intestinal homoeostasis was maintained and no tissue damage was observed. We conclude that FPC is an immunomodulating food with a demonstrated capacity to enhance non-specific host defense mechanisms.

Human and bovine lactoferrins in the milk of recombinant human lactoferrin‐transgenic dairy cows during lactation

Seven Friesian human lactoferrin (hLf)-transgenic primiparous dairy cows expressing recombinant hLf (rhLf) in their milk were included in the study. After calving, concentrations of rhLf and bovine LF (bLf) in the milk, somatic cell count and milk yield were determined. The concentration of rhLf was found to be constant, about 2.9 mg/mL, throughout the early lactation period of 3 months. The concentration of bLf in colostrum was higher after calving, but decreased rapidly during the first days of lactation. The mean concentration of bLf was 0.15 mg/mL, but concentrations varied between cows from 0.07 mg/mL to 0.26 mg/mL. Based on that, it may be possible to improve the non-specific host defence mechanism in the mammary gland of dairy cows by enhancing the content of rhLf in the milk.

Adenovirus Infection Dramatically Augments Lipopolysaccharide-Induced TNF Production and Sensitizes to Lethal Shock

We observed a remarkable synergism of adenoviruses and LPS in triggering the production of TNF in intact animals. We found that in mice pre-exposed to adenoviruses, LPS injections generated extremely high levels of TNF with altered kinetics. The elevated TNF synthesis stemmed mostly from posttranscriptional up-regulation of TNF production, although transcription of the TNF gene was also induced. Adenoviruses and LPS exhibited a significant but less dramatic synergism in the induction of IL-6, IFN-gamma, and NO. Only marginal changes were detected in the synthesis of a panel of other cytokines. Different serotypes of the virus showed practically identical effects. As deletion mutants lacking indispensable viral genes or UV inactivated virions exhibited similar activities as the infectious, wild-type virus, it seems unlikely that the viral genome plays any significant role in the phenomenon. Published data indicate that other viruses also show some kind of synergism with LPS, although by different cellular mechanisms. T cells and their IFN-gamma production--crucial in the synergism of influenza viruses and LPS--were dispensable in our experiments. We suggest that the phenomenon is probably a general one: an overlap between different molecular mechanisms detecting bacterial and viral pathogens and inducing mediators of nonspecific cell-mediated host defense. The synergism of viruses and LPS (bacteria) could be a concern in medical practice as well as in gene therapy experiments with high doses of recombinant adenoviruses.

Interaction between infection, nutrition and immunity in tropical medicine

Host nutritional state has an important role in susceptibility to bacteria, parasites, and viral infections. Infection precipitates the appearance of signs and symptoms of nutrition deficiencies in the undernourished; this can aggravate infection evolution. Infection stimulates specific and non-specific host defense mechanisms; these are directly influenced by the nutritional state of micro- and macronutrients. Immune alterations, which are closely related to nutritional status, markedly contribute to a higher susceptibility to infectious agents and can also contribute to worsening nutritional state, forming a vicious cycle.

Effect of Bisphenol A on non-specific immunodefenses against non-pathogenic Escherichia coli

We examined the effect of Bisphenol A (BPA) on non-specific defense in experiments with a non-pathogenic bacterium, Escherichia coli K-12. Mice were pretreated by a subcutaneous route with BPA (5 mg/kg body weight) for 5 consecutive days in the back and 3 days after the last treatment, injected by the intra-peritoneal route with E. coli K-12. BPA pretreatment caused a decrease of T and B cell populations in the spleen of treated mice. After the challenge with E. coli, the activity to eliminate bacteria from the peritoneal cavity in the early stage of infection (within 24 h) was diminished compared with non-treated mice. BPA induced the migration of excess neutrophils into the peritoneal cavity, but reduced their phagocytic activity against E. coli K-12. For macrophages and lymphocytes, BPA reduced the population in the spleen and the accumulation at infection foci. The production of MCP-1 was enhanced by BPA treatment, but that of IL-6 was suppressed after infection. These results suggest that BPA possessed immunotoxicity and reduced the non-specific host defense as an acute toxicity.

Fever: pathological or physiological, injurious or beneficial?

Fever, the body's most manifest sign of infectious illness, is only one of a concatenation of complex, nonspecific host defense responses to infectious pathogens termed the acute-phase response. It develops in most endotherms via the activation of a combination of various autonomic and behavioral mechanisms. It also occurs in many ectotherms, most usually as the result of behavioral processes alone. Although fever may shorten disease duration and improve survival, antipyretic medications are nonetheless routinely prescribed, with apparently negligible adverse effects on the course and outcome of the disease. The popularity of antipyretics is probably due mostly to their moderating effects of the discomfort level and consequent alleviation of the anxiety of afflicted patients and/or their caregivers. But is treatment of fever really indicated? Would letting fever run its natural course be better? Recent data suggest that, while heat/fever may kill some pathogenic microbes, this would not seem to be its principal role. Rather, heat/fever would appear to serve an important adjuvant function by enhancing the effectiveness of certain selective, stimulus-activated adaptive immune responses and thereby helping to compartamentalize the acute-phase response to the infected site. But arguably even more important may be its temporal modulation of the stimulus-induced generation of TNF α, IL-1 β and IL-6 early during the innate immune response, thereby obviating the risk of the potential harmful effects that could result from their dysregulated co-expression.

Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis

Recent studies have shown that fish are able to mount protective immune responses against various parasites. One of the best characterized parasite–host system in this context is the ciliate Ichthyophthirius multifiliis (Ich) parasitizing a range of freshwater fishes. Both specific and non-specific host defence mechanisms are responsible for the protection of fish against challenge infections with this ciliate. The specific humoral components comprise at least specific antibodies. The non-specific humoral elements included are the alternative complement pathway and probably lectins. Cellular factors involved in the specific response are B-cells and putative T-cells. The non-specific effector cells recognized are various leukocytes. In addition, goblet-cells and mast cells (EGC-cells) may have a function. The NCC-cell (suggested analogue to NK-cells in mammals) seems to play a role in the non-specific response. This well documented protective response in freshwater fishes against Ich has urged the development of anti-parasitic vaccines. Indeed, such products based on formalin killed parasites have been developed and found to offer the vaccinated host a satisfactory protection. However, the collection of parasites for vaccine production is extremely laborious. It involves keeping infected fish due to the fact that in vitro propagation of the parasite is still insufficiently developed. Gaining knowledge of amino acid sequences and its encoding DNA-sequences for the protective antigens (i-antigens) in the parasite was a major breakthrough. That achievement made it possible to produce a recombinant protein in E. coli and preliminary results indicated a certain protection of fish vaccinated with this product. Recent work has shown that the free-living and easily cultivated ciliate Tetrahymena can be transformed and express the i-antigen. This path seems to be promising for future development of vaccines against Ich. A novel approach in fish is the development of DNA-vaccines. Successful DNA-vaccination trials have been conducted in fish against viral infections and the technology also makes it possible to develop a DNA-vaccine against Ich. Other approaches to immuno-protection against Ich have been the use of heterologous vaccines. Thus, both bath and injection vaccination using live or killed (un-transformed) Tetrahymena has been reported to offer treated fish a certain level of protection. Such protection could be explained by non-specific reactions and the efficacy and duration of this vaccination type should be further evaluated.

Functional heterogeneity in liver and lung macrophages

Although initially considered merely "scavenger cells" that participate in immunologic responses only after B and T lymphocytes have performed their biological tasks, more recent evidence suggests that macrophages play a key role in host defense as well as in the maintenance of normal tissue structure and function. For macrophages to perform their biological functions, they must be activated. This involves up-regulation of an array of signaling pathways resulting in altered gene expression and increased biochemical and functional activity. Macrophages have been identified in almost all tissues of the body. However, the basal activity of these cells, as well as their ability to respond to inflammatory mediators, varies considerably with their location. In addition, even within a particular tissue, there is evidence of macrophage heterogeneity. The largest populations of macrophages in the body are located in the liver and lung. Because of the unique attributes of these tissues, hepatic and pulmonary macrophages play essential roles not only in nonspecific host defense but also in the homeostatic responses of these tissues. In this review, the functional and biochemical activities of macrophages localized in the liver and lungs are compared. Evidence suggests that these represent distinct cell populations with unique functions and responsiveness to inflammatory agents.

Pathogenesis of enteric Campylobacter infection.

Campylobacter jejuni and related species are important human pathogens, causing acute human enterocolitis, and they are the most common cause of food-borne diarrhoea in many industrialized countries. Previous infection with certain strains of C. jejuni is also linked with the development of the neurological disorder Guillain-Barre syndrome (GBS). Despite its importance as a human pathogen, relatively little is understood of the mechanisms of C. jejuni-associated disease. The recent release of the complete genome sequence of C. jejuni strain NCTC 11168, together with new strategies for random and directed mutagenesis, have allowed a better insight into some of the genetic determinants of C. jejuni virulence. In this review current knowledge on the pathogenesis of C. jejuni infection is summarized, and possible directions of future research indicated.

Role of macrophages and inflammatory mediators in chemically induced toxicity

Macrophages are critical cellular effectors of nonspecific host defense. They are also potent secretory cells releasing an array of mediators including proinflammatory and cytotoxic cytokines and growth factors, bioactive lipids, hydrolytic enzymes and reactive oxygen and nitrogen intermediates, each of which has been implicated in tissue injury. The research in our laboratories has focused on analyzing the role of macrophages in chemically induced injury in the lung and the liver. In both these tissues, a localized accumulation of macrophages is observed following toxicant exposure. This is directly correlated with the generation of cytotoxic inflammatory mediators at these sites. Moreover, when macrophage functioning is blocked, pulmonary and hepatic injury induced by toxicants such as ozone or acetaminophen is prevented. These findings provide direct support for our hypothesis that macrophages contribute to tissue injury. Approaches using pharmacologic inhibitors and transgenic animals are currently being used to evaluate the specific macrophage-derived products involved in the pathogenic process. Our results suggest that the extent to which a particular mediator contributes to injury depends on the nature of the toxicant, the target tissue, and quantities of the mediator produced.

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection.

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Implications of early apoptosis of infected cells as an important host defense

Apoptosis is widely recognized as being a host defense against viral infections, since viruses require live cells. There has been increasing acceptance of the view that apoptosis is also a defense against other intracellular pathogens and even against pathogens that adhere to host cells. An implication of apoptosis being a host defense is a need to reassess to what extent the cell death at infection sites may constitute a protective host response. A concept stressed here is that infected cells are a hazard to other cells and to the individual, so the benefits of early apoptosis are emphasized. Therefore, promoting the survival of infected cells, even though still functional, may carry risks. A further consideration is the possibility that the apoptotic stimulus of nutrient restriction may be acting in infection-induced anorexia to promote apoptosis of infected cells, thereby serving as a non-specific host defense.

Tuftsin-bearing liposomes in treatment of macrophage-based infections

The use of liposomes as drug carriers in treatment of various diseases has been explored extensively for more than 20 years. ‘Conventional’ liposomes, when administered in vivo by a variety of routes, rapidly accumulate in the mononuclear phagocyte system (MPS). The inherent tendency of the liposomes to concentrate in MPS can be exploited in enhancing the non-specific host defence against infections by entrapping in them the macrophage modulators, and as carriers of antibiotics in treatment of intracellular infections that reside in MPS. This must further be enhanced by grafting on the liposome surface the ligands, e.g. tuftsin, that not only binds specifically to the MPS cells but also enhances their natural killer activity. Keeping this in view, we designed and developed tuftsin-bearing liposomes as drug carriers for the treatment of macrophage-based infections and outline these studies in this overview.

Antimicrobial effects of α-MSH peptides

The presence of the ancient anti-inflammatory peptide alpha-melanocyte-stimulating hormone [alpha-MSH (1-13), SYSMEHFRWGKPV] in barrier organs such as gut and skin suggests a role in the nonspecific (innate) host defense. alpha-MSH and and its carboxy-terminal tripeptide (11-13, KPV) were determined to have antimicrobial influences against two major and representative pathogens: Staphylococcus aureus and Candida albicans. alpha-MSH peptides significantly inhibited S. aureus colony formation and reversed the enhancing effect of urokinase on colony formation. Antimicrobial effects occurred over a broad range of concentrations including the physiological (picomolar) range. Small concentrations of alpha-MSH peptides likewise reduced viability and germ tube formation of the yeast C. albicans. Antimicrobial influences of alpha-MSH peptides could be mediated by their capacity to increase cellular cAMP. Indeed, this messenger was significantly augmented in peptide-treated yeast and the potent adenylyl cyclase inhibitor dideoxyadenosine (ddAdo) partly reversed the killing activity of alpha-MSH peptides. Reduced killing of pathogens is a detrimental consequence of therapy with anti-inflammatory drugs. Because alpha-MSH has potent anti-inflammatory effects we determined influences of alpha-MSH on C. albicans and S. aureus killing by human neutrophils. alpha-MSH peptides did not reduce killing but rather enhanced it, likely as a consequence of the direct antimicrobial activity. alpha-MSH peptides that combine antipyretic, anti-inflammatory, and antimicrobial effects could be useful in treatment of disorders in which infection and inflammation coexist.