Pulmonary Defense Research Articles

Pulmonary Haptoglobin and CD163 Are Functional Immunoregulatory Elements in the Human Lung

Background: The acute-phase protein haptoglobin (Hp) and its receptor CD163 serve as immunomodulators and possess anti-inflammatory besides antioxidant functions. Objectives: To further understand the role of the recently described pulmonary Hp (pHp) and its receptor CD163 in case of inflammation and infection, pHp and CD163 were investigated on mRNA and protein level to gain insight into the cellular events taking place upon stimulation with the inflammatory mediators LPS, Pam3, cytokine IL-6 and dexamethasone, and upon infection with respiratory pathogens (Haemophilus influenzae, Streptococcuspneumoniae and Chlamydia pneumoniae) by use of a human ex vivo tissue culture model and cell cultures of A549 and alveolar epithelial cells type II. In addition, pHp and CD163 expression in COPD and sarcoidosis was assessed. Methods: We conducted experiments using 942 ex vivo cultured lung samples applying immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, real-time PCR, RT-PCR, slot and Western immunoblot analyses with tissue lysates and culture supernatants as well as ELISA and cytometric bead array analyses. Results: This study describes for the first time the expression, regulation and secretion of pHp and its receptor CD163 in the human lung. The release of soluble mediators from A549 cell line and human monocyte-derived macrophages was observed indicating that Hp differentially activates the release of soluble mediators and major chemoattractants. Conclusions: The findings indicate a native function of pHp and CD163 as functional pulmonary defense elements due to local expression, regulation and secretion during lung infection and as part of the inflammatory immune response of the respiratory system.

Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway

Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

Altered pulmonary defense system in lung injury induced by didecyldimethylammonium chloride in mice

Didecyldimethylammonium chloride (DDAC), a representative dialkyl-quaternary ammonium compound (QAC), could contaminate working atmospheres when used in disinfectant operation and adversely affect human health. Furthermore, the development of bacteria resistant to DDAC might become public health concern. We postulated that DDAC instillation in the lungs alters pulmonary antioxidant and antimicrobial responses and increases susceptibility to systemic administration of a bacterial component lipopolysaccharide (LPS). Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment. Pulmonary cytotoxicity in recovered bronchoalveolar lavage was evident on Days 1 and 7, and inflammatory cell influx and interleukin-6 expression peaked on Day 7, in association with altered antioxidant and antimicrobial responses, as demonstrated by measuring heme oxygenase-1, glutathione peroxidase 2, lactoferrin, and mouse β-defensin-2 and -3 mRNA in the lung samples. The impaired defense system tended to enhance the inflammatory reaction caused by a systemic administration of LPS; the effect was in association with increased expression of toll-like receptor-4 mRNA. The results suggest that DDAC alters pulmonary defense system, which may contribute to susceptibility to an exogenous infectious agent.

Activities of Antioxidant Enzymes in Relation to Oxidative and Nitrosative Challenges in Childhood Asthma

Background. This study aimed to investigate the relationship between antioxidant enzyme activities, extent of airway inflammation, and systemic oxidative stress in children suffering from atopic asthma. Methods. A total of 35 asthmatic (AG) and 21 healthy children (CG) participated in this study. The volume of fractionated exhaled NO (Feno) was measured with the NIOX test system. The activities of the erythrocyte antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and the total antioxidant capacity (TAC) were determined photometrically. Plasma interleukin (IL)-6 was measured using ELISA; malondialdehyde (MDA) levels were analyzed using HPLC. Results. Compared to healthy controls, asthmatic children exhaled a significantly (p < .001) higher mean volume of Feno, had significantly reduced plasma concentrations of TAC (p = .006), and significantly enhanced levels of MDA (p < .001) and IL-6 (p = .012). SOD (p = .027), CAT (p < .001), and GSH-Px (p = .005) were significantly less active in the asthma group and significantly negatively associated with Feno (SOD/Feno: p = .017; CAT/Feno: p = .008; GSH-Px/Feno: p = .001); the oxidative stress marker MDA showed such correlations in both investigated groups (SOD/MDA: AG: p = .001, CG: p = .381; CAT/MDA: AG: p = .003, CG: p = .020; GSH-Px/MDA: AG: p = .006, CG: p = .011). Furthermore, there was a significant (p< .01) positive correlation between MDA/Feno and MDA/IL-6 observed in both groups. Conclusions. These results indicate that inflammation of the bronchial tree, reflected by increased NO formation in the airways and enhanced systemic oxidative stress, is related to an alteration of antioxidant enzyme activities in childhood asthma. Modulating the activity of antioxidant enzymes may therefore have beneficial effects on pulmonary and systemic antioxidant defense mechanisms and could reduce airway inflammation and oxidative stress in asthmatics.

The IκB Family Member Bcl-3 Coordinates the Pulmonary Defense against Klebsiella pneumoniae Infection

Bcl-3 is an atypical member of the IκB family that has the potential to positively or negatively modulate nuclear NF-κB activity in a context-dependent manner. Bcl-3's biologic impact is complex and includes roles in tumorigenesis and diverse immune responses, including innate immunity. Bcl-3 may mediate LPS tolerance, suppressing cytokine production, but it also seems to contribute to defense against select systemic bacterial challenges. However, the potential role of Bcl-3 in organ-specific host defense against bacteria has not been addressed. In this study, we investigated the relevance of Bcl-3 in a lung challenge with the Gram-negative pathogen Klebsiella pneumoniae. In contrast to wild-type mice, Bcl-3-deficient mice exhibited significantly increased susceptibility toward K. pneumoniae pneumonia. The mutant mice showed increased lung damage marked by neutrophilic alveolar consolidation, and they failed to clear bacteria in lungs, which correlated with increased bacteremic dissemination. Loss of Bcl-3 incurred a dramatic cytokine imbalance in the lungs, which was characterized by higher levels of IL-10 and a near total absence of IFN-γ. Moreover, Bcl-3-deficient mice displayed increased lung production of the neutrophil-attracting chemokines CXCL-1 and CXCL-2. Alveolar macrophages and neutrophils are important to antibacterial lung defense. In vitro stimulation of Bcl-3-deficient alveolar macrophages with LPS or heat-killed K. pneumoniae recapitulated the increase in IL-10 production, and Bcl-3-deficient neutrophils were impaired in intracellular bacterial killing. These findings suggest that Bcl-3 is critically involved in lung defense against Gram-negative bacteria, modulating functions of several cells to facilitate efficient clearance of bacteria.

Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells

Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues.

Seasonal FluMist Vaccination Induces Cross-Reactive T Cell Immunity against H1N1 (2009) Influenza and Secondary Bacterial Infections

T cell epitopes have been found to be shared by circulating, seasonal influenza virus strains and the novel pandemic H1N1 influenza infection, but the ability of these common epitopes to provide cross-protection is unknown. We have now directly tested this by examining the ability of live seasonal influenza vaccine (FluMist) to mediate protection against swine-origin H1N1 influenza virus infection. Naive mice demonstrated considerable susceptibility to H1N1 Cal/04/09 infection, whereas FluMist-vaccinated mice had markedly decreased morbidity and mortality. In vivo depletion of CD4(+) or CD8(+) immune cells after vaccination indicated that protective immunity was primarily dependent upon FluMist-induced CD4(+) cells but not CD8(+) T cells. Passive protection studies revealed little role for serum or mucosal Abs in cross-protection. Although H1N1 influenza infection of naive mice induced intensive phagocyte recruitment, pulmonary innate defense against secondary pneumococcal infection was severely suppressed. This increased susceptibility to bacterial infection was correlated with augmented IFN-γ production produced during the recovery stage of H1N1 influenza infection, which was completely suppressed in mice previously immunized with FluMist. Furthermore, susceptibility to secondary bacterial infection was decreased in the absence of type II, but not type I, IFN signaling. Thus, seasonal FluMist treatment not only promoted resistance to pandemic H1N1 influenza infection but also restored innate immunity against complicating secondary bacterial infections.

Clinical Review: Gene-based therapies for ALI/ARDS: where are we now?

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) confer substantial morbidity and mortality, and have no specific therapy. The accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ALI/ARDS, suggest that the disease may be amenable to gene-based therapies. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS have revealed multiple therapeutic targets for gene-based approaches. Strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ALI/ARDS have all demonstrated promise in preclinical models. Despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ALI/ARDS remains to be realized. Multiple barriers to effective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection efficiency of nonviral delivery methods. Deficits remain in our knowledge regarding the optimal molecular targets for gene-based approaches. Encouragingly, recent progress in overcoming these barriers offers hope for the successful translation of gene-based approaches for ALI/ARDS to the clinical setting.

Quantification and Evaluation of the Role of Antielastin Autoantibodies in the Emphysematous Lung

Chronic obstructive pulmonary disease (COPD) may be an autoimmune disease. Smoking causes an imbalance of proteases and antiproteases in the lung resulting in the generation of elastin peptides that can potentially act as autoantigens. Similar to COPD, Z alpha-1 antitrypsin deficiency (Z-A1ATD) and cystic fibrosis (CF) are associated with impaired pulmonary antiprotease defences leading to unopposed protease activity. Here, we show that there is a trend towards higher bronchoalveolar lavage fluid (BALF) antielastin antibody levels in COPD and Z-A1ATD and significantly lower levels in CF compared to control BALF; the lower levels in CF are due to the degradation of these antibodies by neutrophil elastase. We also provide evidence that these autoantibodies have the potential to induce T cell proliferation in the emphysematous lung. This study highlights that antielastin antibodies are tissue specific, can be detected at elevated levels in COPD and Z-A1ATD BALF despite their being no differences in their levels in plasma compared to controls, and suggests a therapeutic role for agents targeting these autoantibodies in the lungs.

Airway microbiology and host defences in paediatric non-CF bronchiectasis

Bronchiectasis in children without cystic fibrosis is most common in socioeconomically disadvantaged communities. Recurrent pneumonia in early childhood and defective pulmonary defences are important risk factors. These help establish a 'vicious cycle' of impaired mucociliary clearance, infection, airway inflammation and progressive lung injury. Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis and Pseudomonas aeruginosa are the main infecting pathogens. H. influenzae predominates across all ages, while P. aeruginosa is found in older children with advanced disease. It is uncertain whether viruses and upper airway commensal bacteria play an important aetiological role. Overall, the microbiological data are limited however and there are difficulties obtaining reliable respiratory specimens from young children. Bronchiectasis is a complex disorder resulting from susceptibility to pulmonary infection and poorly regulated respiratory innate and adaptive immunity. Airway inflammatory responses are excessive and persist, even once infection is cleared. Improved specimen collection, molecular techniques and biomarkers are needed to enhance management.

Airway Mucus Function and Dysfunction

Pulmonary defense against environmental injury depends on airway mucus, which traps inhaled toxins that are then cleared from the lungs by ciliary beating and cough. This review examines the normal and abnormal formation and clearance of mucus, as well as the therapy of mucus dysfunction.

Oxidation of Z α1-Antitrypsin by Cigarette Smoke Induces Polymerization

The acceleration of chronic obstructive pulmonary disease (COPD) by cigarette smoke (CS) in individuals with severe genetic deficiency of α(1)-antitrypsin (Z-AT [Glu342Lys]) exemplifies the critical importance of gene-environmental interactions to the development of COPD. We investigated the molecular basis for the interaction between Z-AT and CS. Female mice (8-10 wk old) transgenic for normal (M-AT) or Z-AT on CBA background were exposed to four 1R3F cigarettes daily for 5 days. Age and sex matched littermates not exposed to CS were used as controls. Bronchoalveolar lavage fluid and lung homogenates were assessed for inflammatory cells, neutrophil elastase, and AT conformers. Z-AT was purified from plasma, exposed to CS extract, and assessed for the development and temporal relationship between AT conformers. Mice transgenic for Z-AT developed a significant increase in pulmonary polymers after acute CS exposure (P = 0.001). There were also increased neutrophils in CS-Z lungs versus controls (P < 0.001), which were tightly correlated with polymer concentrations (r(2) = 0.93). Oxidation of human plasma Z-AT by CS or N-chlorosuccinimide greatly accelerated polymerization (P = 0.004), which could be abrogated by antioxidants (P = 0.359 versus Z control). Our data show that CS accelerates polymerization of Z-AT by oxidative modification, which, in so doing, further reduces pulmonary defense and increases neutrophil influx into the lungs. These novel findings provide a molecular explanation for the striking observation of premature emphysema in ZZ homozygotes who smoke. Further work is required to assess whether antioxidant therapy may be beneficial in Z-AT-related COPD.

Pulmonary infection defense after lung transplantation: does airway ischemia play a role?

During a classical lung transplantation procedure, there is no attempt to restore the bronchial circulation, giving rise to ischemia of the mucosa with initial loss of ciliated function. This might compromise the ciliary function and the infection defense mechanisms of the airways. Although initially the airways are characterized by ischemia (epithelial sloughing), later on, there seems to be a restoration of the ciliary function and up to now, there is no clear evidence that there is an increased risk of infectious episodes due to the lack of bronchial circulation, although this has not extensively been investigated. Local pulmonary defense mechanisms after lung transplantation without bronchial revascularization seem to be preserved at least in a stable condition. Whether the loss of bronchial circulation has a negative impact when complications develop (such as bronchiolitis obliterans syndrome) remains speculative.

Inhibition by Cigarette Smoke of Nuclear Factor-κB–Dependent Response to Bacteria in the Airway

Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on pulmonary defense are incompletely understood. Based on the observation that interactions between bacteria and host cells result in the expression of critical defense genes regulated by NF-κB, we hypothesized that cigarette smoke alters NF-κB function. In this study, primary human tracheobronchial epithelial cells were treated with cigarette smoke extract (CSE) and exposed to Haemophilus influenzae, and the effects of CSE on bacteria-induced signaling and gene expression were assessed. CSE inhibited high concentrations of induced NF-κB activation and the consequent expression of defense genes that occurred in airway epithelial cells in response to H. influenzae. This decreased activation of NF-κB was not attributable to cell loss or cytotoxicity. Glutathione augmentation of epithelial cells decreased the effects of CSE on NF-κB-dependent responses, as well as the effects on the inhibitor of κB and the inhibitor of κB kinase, which are upstream NF-κB regulators, suggesting the involvement of reactive oxygen species. The relevance of these findings for lung infection was confirmed using a mouse model of H. influenzae airway infection, in which decreased NF-κB pathway activation, keratinocyte chemoattractant (KC) chemokine expression, and neutrophil recruitment occurred in animals exposed to cigarette smoke. The results indicate that although cigarette smoke can cause inflammation in the lung, exposure to smoke inhibits the robust pulmonary defense response to H. influenzae, thereby providing one explanation for the increased susceptibility to respiratory bacterial infection in individuals exposed to cigarette smoke.

New Look at an Old Problem: Bacterial Superinfection after Influenza

This commentary highlights the important role of bacterial superinfection in the morbidity and mortality of influenza virus infection.

Mechanisms of suppression of alveolar epithelial cell GM-CSF expression in the setting of hyperoxic stress

Pulmonary expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) is critically important for normal functional maturation of alveolar macrophages. We found previously that lung GM-CSF is dramatically suppressed in mice exposed to hyperoxia. Alveolar epithelial cells (AEC) are a major source of GM-CSF in the peripheral lung, and in vivo hyperoxia resulted in greatly reduced expression of GM-CSF protein by AEC ex vivo. We now explore the mechanisms responsible for this effect, using primary cultures of murine AEC exposed to hyperoxia in vitro. Exposure of AEC to 80% oxygen/5% CO(2) for 48 h did not induce overt toxicity, but resulted in significantly decreased GM-CSF protein and mRNA expression compared with cells in normoxia. Similar effects were seen when AEC were stressed with serum deprivation, an alternative inducer of oxidative stress. The effects in AEC were opposite those in a murine lung epithelial cell line (MLE-12 cells), in which hyperoxia induced GM-CSF expression. Both hyperoxia and serum deprivation resulted in increased intracellular reactive oxygen species (ROS) in AEC. Hyperoxia and serum deprivation induced significantly accelerated turnover of GM-CSF mRNA. Treatment of AEC with catalase during oxidative stress preserved GM-CSF protein and mRNA and was associated with stabilization of GM-CSF mRNA. We conclude that hyperoxia-induced suppression of AEC GM-CSF expression is a function of ROS-induced destabilization of GM-CSF mRNA. We speculate that AEC oxidative stress results in significantly impaired pulmonary innate immune defense due to effects on local GM-CSF expression in the lung.

Analysis of the Sputum and Inflammatory Alterations of the Airways in Patients with Common Variable Immunodeficiency and Bronchiectasis

INTRODUCTION:Common variable immunodeficiency is characterized by defective antibody production and recurrent pulmonary infections. Intravenous immunoglobulin is the treatment of choice, but the effects of Intravenous immunoglobulin on pulmonary defense mechanisms are poorly understood.OBJECTIVE:The aim of this study was to verify the impact of intravenous immunoglobulin on the physical properties of the sputum and on inflammatory alterations in the airways of patients with Common variable immunodeficiency associated with bronchiectasis.METHOD:The present study analyzed sputum physical properties, exhaled NO, inflammatory cells in the sputum, and IG titers in 7 patients with Common variable immunodeficiency and bronchiectasis with secretion, immediately before and 15 days after Intravenous immunoglobulin. A group of 6 patients with Common variable immunodeficiency and bronchiectasis but no sputum was also studied for comparison of the basal IgG level and blood count. The 13 patients were young (age=36±17 years) and comprised predominantly of females (n=11).RESULTS:Patients with secretion presented significantly decreased IgG and IgM levels. Intravenous immunoglobulin was associated with a significant decrease in exhaled NO (54.7 vs. 40.1 ppb, p<0.05), sputum inflammatory cell counts (28.7 vs. 14.6 cells/mm3, p<0.05), and a significant increase in respiratory mucus transportability by cough (42.5 vs. 65.0 mm, p < 0.05).CONCLUSION:We concluded that immunoglobulin administration in Common variable immunodeficiency patients results in significant improvement in indexes of inflammation of the airways with improvement in the transportability of the respiratory mucus by cough.

Abrupt Weaning Significantly Increases Mortality Following a Secondary Bacterial Respiratory Infection

Secondary bacterial respiratory infections are a major cause of mortality in fall-weaned feedlot calves, and epidemiological studies implicate a variety of stressors as significant contributing factors. Experimental studies have identified stressors that may compromise pulmonary defense mechanisms but there is no evidence that these functional changes alter respiratory disease outcome. We used a model of combined viral and bacterial respiratory disease to determine if nutritional and psychological stressors (abrupt weaning; AW) altered respiratory disease mortality. Mortality was doubled in AW calves challenged with Mannheimia haemolytica four days after a primary bovine herpesvirus-1 (BHV-1) respiratory infection, when compared to calves adapted to weaning (pre-conditioned; PC) for two weeks prior to respiratory challenge. Reduced survival time and decreased lung pathology in the AW group suggested death was due to an acute systemic reaction. Viral shedding did not differ significantly between the two treatment groups. AW calves and all calves developing fatal pneumonia had significantly elevated interferon (IFN)-y levels in nasal secretions and increased systemic proinflammatory responses. Analysis of blood leukocytes revealed significantly increased CD14 and TLR4 gene expression in animals with fatal pneumonia. These analyses support the conclusion that stress enhanced innate immune responses to viral infection without altering the level of BHV-1 infection. These studies provide the first quantitative evidence that stress associated with abrupt weaning contributes significantly to fatal bovine respiratory disease.

Intestinal Colonization with Enterococcus faecium Does Not Influence Pulmonary Defense against Pseudomonas aeruginosa in Mice

BackgroundEnterococci, and especially multiresistant Enterococcus faecium, are increasingly found colonizing hospitalized patients. This increased prevalence of colonization is not only associated with an increased prevalence of infections caused by enterococci, but also by infections with other nosocomial pathogens. In this study we investigated the causality of this observed relationship, by determining the influence of intestinal colonization with E. faecium on pulmonary defense against Pseudomonas aeruginosa.Methodology/Principal FindingsThree groups of mice were tested; 2 groups of mice were pre-treated with vancomycin, of which one group was subsequently treated by oral gavage of vancomycin-resistant E. faecium (VRE). The third group did not receive any pre-treatment. P. aeruginosa pneumonia was induced in all mice. Vancomycin treatment resulted in intestinal gram-negative bacterial overgrowth and VRE treatment resulted in colonization throughout the intestines. All 3 groups of mice were able to clear P. aeruginosa from the lungs and circulation, with comparable lung cytokine responses and lung damage. Mice treated with vancomycin without VRE colonization displayed modestly increased plasma levels of TNF-α and IL-10.ConclusionOvergrowth of E. faecium and/or gram-negative bacteria does not impact importantly on pulmonary defense against P. aeruginosa pneumonia.

Pulmonary antioxidant defences and protein damage during the ageing process of both sexes

The free radical theory holds that the senescence is caused by oxidative damage that results from an imbalance between reactive oxygen and nitrogen species (RONS) and antioxidant defences. Hence, it plays an important role in the field of gerontology. We evaluated, in male and female rats, the activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and total superoxide dismutase (tSOD), as well as oxidative protein damage in pulmonary tissue at 3, 6, 12, and 20 months of age. The results show an increase in the activities of all antioxidant enzymes at 12 months of age in female rats, suggesting an association with the reproductive life cycle. Protein damage in female pulmonary tissues did not change significantly throughout the ageing process. In male rats, the activity of GPx in 20 months of age showed an inter-gender increase, while the tSOD and GPx showed higher activities in 20 months of age in the intra-gender analysis. The male lung showed higher protein damage at 6 months of age. These findings suggest that antioxidant enzymatic activity is connected to the reproductive life cycle.