![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
2 Departments of Nutrition, and 3 Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599
* To whom correspondence should be addressed. E-mail: melinda_beck{at}unc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
-lactone oxidase gene-inactivated mice (gulo–/– mice) require vitamin C supplementation for survival. Five-wk-old male and female gulo–/– mice were provided water or water containing 1.67 mmol/L vitamin C for 3 wk before inoculation with influenza A/Bangkok/1/79. There were no differences in lung influenza virus titers between vitamin C–adequate and –deficient mice; however, lung pathology in the vitamin C–deficient mice was greater at 1 and 3 d after infection but less at d 7 compared with vitamin C–adequate mice. Male vitamin C–deficient mice had higher expression of mRNA for regulated upon activation normal T expressed and secreted (RANTES), IL-1ß, and TNF-
in the lungs at d 1 after infection compared with male controls. However, at d 3 after infection, male vitamin C–deficient mice had less expression of mRNA for RANTES, monocyte chemotactic protein-1 (MCP-1), and IL-12 compared with male controls. None of these differences were observed in female mice. Vitamin C–deficient male mice also had greater nuclear factor-
B activation as early as 1 d after infection compared with male controls. These data suggest that vitamin C is required for an adequate immune response in limiting lung pathology after influenza virus infection.
|
Introduction |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
Although studies of the effect of vitamin C on colds have been reported (15–18), there are few data on the effect of vitamin C on influenza virus infection. Infection with influenza virus causes a great deal of morbidity and mortality worldwide each year. In the United States alone, influenza virus infection results in over 36,000 deaths and 114,000 hospitalizations/year (19). Infection with influenza virus causes damage to both the lungs and airways due predominantly to inflammatory responses. Although the immune response is critical to the recovery from influenza infection, it is also responsible for the lung inflammation that contributes to lung pathology. Administration of vitamin C alone or vitamin C in combination with vitamin E reduces lipid peroxidation and monooxygenase enzyme activation induced by influenza virus infection (20), which may have an effect on lung pathology postinfection.
Vitamin C is synthesized from glucose in the liver of most mammalian species, except for humans, nonhuman primates, guinea pigs, and some fruit bats. These species lack the enzyme L-gulono--lactone oxidase (Gulo),4 which is essential for synthesis of the vitamin C immediate precursor 2-keto-1-gulonolactone. The DNA encoding gulonolactone oxidase (Gulo) in humans has undergone substantial mutation, resulting in the absence of a functional enzyme (21,22). The ability of mice and rats to synthesize vitamin C makes it impossible to study the effects of vitamin C deficiency in these animals and puts limitations to the interpretation of vitamin C supplementation studies due to uncontrolled de novo vitamin C synthesis. However, the creation of gulo–/– mice (23) provides an excellent model for studying vitamin C deficiency and its effect on the immune response to viral infection.
Previous work in our laboratory has demonstrated the importance of selenium (as an antioxidant) and vitamin E in optimizing the immune response to influenza virus infection (24–26). To determine whether vitamin C was also important for immune function, we investigated the effects of vitamin C deficiency on the infection by a mild strain of influenza virus (influenza A Bangkok/1/79) in gulo–/– mice.
|
Materials and Methods |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
Mice.
Gulo–/– mice on a C57BL/6 background were bred in the University of North Carolina at Chapel Hill animal facility, which is fully accredited by the American Association of Laboratory Animal Care. Mice were housed 3–4/cage. All mice were maintained under protocols approved by the Institutional Animal Use and Care Committee of the University of North Carolina at Chapel Hill. For experiments, 5-wk-old male and female gulo–/– mice (4–5 mice/group) were provided water or water containing 1.67 mmol/L vitamin C for 3 wk before inoculation with influenza A/Bangkok/1/79. Mice were fed a commercial nonpurified diet (Lab Diet 5P76). At baseline before infection (d 0) and 1, 3, and 7 d after infection, mice were killed by cervical dislocation and the tissues were collected for the determination of lung pathology, tissue ascorbic acid (the reduced form of vitamin C), glutathione levels, the expression of proinflammatory chemokines and cytokines, and nuclear factor-B (NF-B) activation in the lung.
Infection of mice. Mice were lightly anesthetized with an intraperitoneal injection of ketamine (0.022 mg) and xylazine (0.0156 mg). Following anesthesia, mice were infected intranasally with 10 hemagglutinating units (HAU) of influenza A/Bangkok/1/79 in 0.05 mL sterile PBS.
Tissue ascorbic acid measurement.
Ascorbic acid (the reduced form of vitamin C) was measured by the ,'-dipyridyl method as described (28).
Glutathione measurement. Total glutathione (GS) and reduced glutathione (GSH) were analyzed in tissue extracts prepared in 5% 5-sulfosalicylic acid (S2130, Sigma) using a glutathione reductase–coupled recycling assay (29). GSH disulfide concentrations were calculated in extracts pretreated with 2-vinylpyridine (132292, Aldrich). GSH was calculated by subtracting GSH disulfide from total GS.
Histopathology of lungs.
The left lung was removed and inflated with 4% paraformaldehyde fixative in 0.1 mol/L sodium phosphate buffer (pH 7.2). Sections (6 µm) were fixed in acetone and stained with hematoxylin-eosin. The extent of inflammation was graded without knowledge of the experimental variables by 2 independent investigators. Grading was performed semiquantitatively according to the relative degree (from lung to lung) of inflammatory infiltration. The scoring was as follows: 0, no inflammation; 1+, mild influx of inflammatory cells with inflammatory infiltrates clustered around vessels; 2+, increased inflammation with 
25–50% of the total lung involved; 3+, severe inflammation involving 50–70% of the lung; and 4+, almost all lung tissue contained inflammatory infiltrates.
Western blot.
Cytosol and nuclear protein extracts were prepared from lung tissue using Nuclear/Cytosol Fractionation kit (K266–100, Biovision) following protocols provided by the manufacturer. Extracts were separated by 12% (wt:v) SDS polyacrylamide gels and electrophoretically blotted onto Immobilon-P membranes (Millipore) according to standard procedures. The membranes were blocked with 5% nonfat milk and treated with rabbit anti-mouse inhibitory B (IB) (9242, Cell Signaling Technology) and anti-mouse ß-actin (4967, Cell Signaling Technology) overnight at 4°C. Membranes were then incubated with a secondary goat anti-rabbit IgG conjugated with horseradish peroxidase (7074, Cell Signaling Technology) for 1 h at room temperature and stained with SuperSignal West Pico Chemiluminescent Substrate (Pierce). The chemiluminescent signals were acquired using a 16-bit CCD camera (GeneGnome system, Syngene). Densitometry analysis was conduct using the GeneSnap software (Syngene).
Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed with the Panomics' EMSA kit (AY1030) according to protocols provided by the manufacturer.
Quantitation of viral titers. One-half of the lung was removed and total RNA was isolated using the TRIzol method (Invitrogen). Reverse transcription was carried out using Superscript II First Strand Synthesis kit (11904–018, Invitrogen) and random hexamer primers. Expression of the influenza matrix (M1) gene was determined by quantitative real time PCR (qRT-PCR) as described (30). Fluorescent reporters were detected using Bio-Rad iCycler PCR machine and primers and probes were purchased from Applied Biosystems. The levels of mRNA for glyseraldehyde-3-phosphate dehydrogenase were determined for all samples and were used to normalize expression of the influenza M1 gene. Data were converted to HAU units using real time PCR standards made from the virus stock with known HAU titer.
Quantitation of lung mRNA cytokine levels.
mRNA levels for murine regulated upon activation normal T expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, IL-12, IL-1ß, TNF-, and glyseraldehyde-3-phosphate dehydrogenase were determined using qRT-PCR, as described above. All data are expressed as the ratio to vitamin C supplemented, uninfected controls.
Statistical analysis. The glutathione, ascorbic acid, and lung pathology data were pooled from male and female mice because we found no sex effect at any time point by Student's t test. Ascorbic acid data were log transformed and analyzed for the effect of vitamin C status and time by two-way ANOVA. The Tukey HSD test was used to test the effect of vitamin C status at each time point separately. The glutathione and lung pathology data were analyzed for the effect of vitamin C status and time by the same method but without log transformation. An effect of time was observed in lung pathology data but not for either ascorbic acid or glutathione. Paired t test was used to test the difference in ascorbic acid levels between the lung and the liver. qRT-PCR data between vitamin C–deficient and –adequate groups were analyzed by Kruskal-Wallis test. For qRT-PCR, male and female samples were analyzed separately on different plates. Because there may be possible plate-plate differences, a comparison between male and female data would not be appropriate. Body weight data and Western-blot quantification were analyzed by Student's t test. All statistical analyses were performed with JMP software (SAS). P < 0.05 was considered significant.
|
Results |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
|
Increased lung pathology of vitamin C–deficient mice. At d 7 after infection, vitamin C–deficient mice had significantly greater lung pathology compared with vitamin C–adequate mice. Interestingly, the vitamin C–deficient mice had less lung pathology early after infection, at d 1 and 3 (Fig. 2).
|
Lung proinflammatory chemokine expression. Male vitamin C–deficient gulo–/– mice had lower expression of RANTES mRNA in the lung at 1 d after infection and higher expression of RANTES and MCP-1 mRNA at d 3 after infection compared with male vitamin C–adequate gulo–/– mice (Fig. 3A,C). None of these differences were observed in female mice (Fig. 3B,D).
|
in the lungs 1 d after infection (Fig. 4C,E) and higher expression of mRNA for IL-12 at d 3 after infection (Fig. 4A) compared with male vitamin C–adequate gulo–/– mice. Overall, vitamin C–adequate male mice had a rapid immune response that was delayed in vitamin C–deficient male mice. None of these differences were observed in female mice (Fig. 4B,D,F). We detected no difference in INF , ß, or expression between vitamin C–deficient and vitamin C–adequate mice.
|
B activation.
At d 1 after infection, male vitamin C–deficient gulo–/– mice had a lower level of IB in the cytosolic fractions (Fig. 5A) and a higher level of NF-B in the nuclear fractions of cells in the lung (Fig. 5B) compared with male vitamin C–adequate gulo–/– mice.
|
|
Discussion |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
B (34). Hydrogen peroxide activates NF-B by increasing the phosphorylation of mitogen-activated protein kinase p38 and Akt, components of the protein kinase cascades that lead to IB phosphorylation (33). To our knowledge, this study describes for the first time the immune response to infection with influenza virus in gulo–/– vitamin C–deficient mice. We found that vitamin C–deficient mice had greater lung pathology late after infection compared with vitamin C–adequate controls, although there were no differences in lung viral titers. These results suggest that vitamin C does play a role in reducing influenza virus–induced lung pathology.
A main contributor to influenza-induced lung pathology is the infiltration of inflammatory cells into the lungs. These immune cells produce a variety of chemokines and cytokines that are involved in attracting increased numbers and activation of immune cells to combat the viral infection. Chemokines are potent chemoattractant cytokines and have been considered the main candidate molecules responsible for the selective recruitment of distinct leukocyte populations. Members of the CC-chemokine subfamily, such as MCP-1 and RANTES, preferentially attract monocytes and lymphocytes (35). RANTES and MCP-1 are both involved in several inflammatory disorders of the lung (36,37).
To determine whether vitamin C deficiency could alter the production of cytokines/chemokines by immune cells, we examined the lungs of infected mice for proinflammatory cytokine and chemokine mRNA levels. At d 1 after infection, vitamin C–deficient male mice had higher expression of mRNA for RANTES compared with vitamin C–adequate male mice. However, at d 3 after infection, vitamin C–deficient male mice had less RANTES and MCP-1 mRNA production in the lungs. Female vitamin C–deficient mice had RANTES and MCP-1 mRNA levels similar to vitamin C–adequate female mice. Thus, male mice deficient in vitamin C had an impaired immune response to influenza infection, which may have contributed to increased lung pathology.
Examination of proinflammatory cytokines IL-12, IL-1ß, and TNF- also revealed sex differences. IL-12 is considered a central regulator of immune responses (38). Produced by dendritic cells, macrophages, and neutrophils, IL-12 promotes natural killer (NK) cells and cytotoxic T lymphocyte activity as well as induces the secretion of INF- by NK cells (38). IL-12 mRNA levels were higher at d 3 in male vitamin C–deficient mice compared with male controls. Female vitamin C–deficient mice had a trend toward a higher IL-12 mRNA production at d 7 as well (P = 0.077). The higher IL-12 levels in vitamin C–deficient mice may be a consequence of increased inflammatory cells in the lungs of these mice.
IL-1ß and TNF- are also proinflammatory cytokines. Produced by activated macrophages and monocytes, IL-1ß generates systemic and local responses to infection by inducing fever, activating lymphocytes, and promoting migration of leukocytes into the site of infection (39). TNF- is also produced largely by macrophages and monocytes. TNF- induces a local protective effect by acting on blood vessels to increase vascular permeability to fluid, proteins, and cells, and increasing endothelial adhesiveness for leukocytes and platelets (40). Only male gulo–/– mice demonstrated a difference in mRNA for IL-1ß and TNF- between vitamin C–deficient and –adequate groups. At d 1 after infection, mRNA levels for both of the cytokines were lower in the vitamin C–deficient male mice compared with vitamin C–adequate male mice, which suggests that the less lung pathology at d 1 after infection may be due to the lower levels of inflammatory mediators. However, because female gulo–/– mice also had less pathology at d 1 after infection without the corresponding lower levels of IL-1ß and TNF-, there are likely other inflammatory mediators that contributed to the inflammatory process.
The delay in the development of pathology in the vitamin C–deficient mice may have been due to a slow initial immune response in these animals. The less immune cell infiltration of the vitamin C–deficient mice at 1 and 3 d after infection may be due to less production of chemokines and cytokines at an early stage in the infection as well as changes in the migration ability of inflammatory cells. For example, Ganguly et al. demonstrated a significant reduction in the migration of macrophages in vitro from vitamin C–deficient guinea pigs compared with vitamin C–sufficient guinea pigs. Addition of vitamin C to the cultures partially reversed the reduced migration (41).
How does a deficiency in vitamin C affect the expression of mRNA for cytokines and chemokines? One possibility is the greater oxidative stress in the vitamin C–deficient mice. Alterations in GS and GSH levels in the vitamin C–deficient mice suggest that these mice were oxidatively stressed. Higher levels of ROI are associated with NF-B activation (42,43). NF-B, in turn, activates gene transcription for a number of cytokines and chemokines, including IL-12, RANTES, and MCP-1. We found that lungs from vitamin C–deficient male mice had a lower level of the NF-B inhibitor, IB, in the cytosol and a higher level of NF-B in the nucleus compared with vitamin C–adequate male mice. These results suggest that the greater oxidative stress in the vitamin C–deficient male mice following influenza infection led to greater NF-B activation, which in turn upregulated the transcription of proinflammatory cytokines and chemokines. However, because no differences in proinflammatory mediators were detected in female vitamin C–deficient mice, other possibilities for the greater pathology must be considered (for example, a difference in chemokine receptor expression between male and female).
Vitamin C deficiency did not affect the ability of the immune system to eliminate influenza virus replication in the lungs, reflected by similar viral titers between vitamin C–deficient and –supplemented mice. However, influenza A/Bangkok/1/79 is a human influenza virus that is not mouse adapted and therefore induces a mild lung pathology from which the mice recover. Although a deficiency in vitamin C did not inhibit influenza virus clearance in this study, the possibility that vitamin C deficiency could impair the ability of the host to clear a more virulent mouse-adapted strain of influenza virus cannot be excluded.
This study also demonstrated that vitamin C deficiency affected the immune response of male, but not female, mice. Lung and liver vitamin C levels were not different between male and female gulo–/– mice, suggesting that the sex difference was not due to different tissue vitamin C concentrations. However, we cannot exclude the possibility that immune cell vitamin C concentrations differed between males and females. Sex differences in immune responses are known. Males and females have differences in immune responses to injury and infection, and the gonadal steroid hormones, estrogen and testosterone, are responsible for the differences (44–46). Physiological levels of estrogen stimulate the immure response whereas testosterone suppresses it (47–49). A more recent ex vivo study using whole blood of humans found that men had a higher percentage of TNF-, IL-1ß, and IL-12–producing monocytes compared with women after endotoxin stimulation (50). However, because the pathology in our study was similar between male and female vitamin C–deficient mice despite the clear immunological differences, it is likely that other immune functions may have been impaired in the female mice, such as NK cell activity or changes in proinflammatory mediators that were not examined in this study. To our knowledge, this study is the first to demonstrate a sex difference in the effect of vitamin C deficiency on influenza infection.
We conclude that vitamin C is an important nutrient for adequate immune function and limitation of pathogenesis following influenza virus infection. As shown for other antioxidant nutrients, such as selenium and vitamin E, vitamin C may be important in limiting the increased oxidative stress that occurs during an influenza infection, thus lowering tissue inflammation. Further studies characterizing the sex effect of vitamin C are warranted.
|
FOOTNOTES |
|---|
4 Abbreviations used: EMSA, electrophoretic mobility shift assay; GS, total glutathione; GSH, glutathione; Gulo, gulonolactone oxidase; Gulo, L-gulono--lactone oxidase gene; HAU, hemagglutinating unit; IB, inhibitory B; MCP, monocyte chemotactic protein; NF-B, nuclear factor-B; NK cells, natural killer cells; qRT-PCR, quantitative real time PCR; RANTES, regulated upon activation normal T expressed and secreted; ROI, reactive oxygen intermediates.
Manuscript received 10 May 2006. Initial review completed 7 June 2006. Revision accepted 24 July 2006.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
1. Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, Park JB, Lazarev A, Graumlich JF, et al. Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci USA. 1996;93:3704–9.
2. Washko PW, Wang Y, Levine M. Ascorbic acid recycling in human neutrophils. J Biol Chem. 1993;268:15531–5.
3. Hughes RE, Maton SC. The passage of vitamin C across the erythrocyte membrane. Br J Haematol. 1968;14:247–53.[Medline]
4. Hornig D, Weber F, Wiss O. Studies on the uptake of (I- 14 C)ascorbic acid and (I- 14 C)-dehydroascorbic acid by platelets of guinea pigs. Clin Chim Acta. 1971;33:187–96.[Medline]
5. Bergsten P, Amitai G, Kehrl J, Dhariwal KR, Klein HG, Levine M. Millimolar concentrations of ascorbic acid in purified human mononuclear leukocytes. Depletion and reaccumulation. J Biol Chem. 1990;265:2584–7.
6. Stankova L, Rigas DA, Bigley RH. Dehydroascorbate uptake and reduction by human blood neutrophils, erythrocytes, and lymphocytes. Ann N Y Acad Sci. 1975;258:238–42.[Medline]
7. Evans RM, Currie L, Campbell A. The distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Br J Nutr. 1982;47:473–82.[Medline]
8. Hemila H, Douglas RM. Vitamin C and acute respiratory infections. Int J Tuberc Lung Dis. 1999;3:756–61.[Medline]
9. Leibovitz B, Siegel BV. Ascorbic acid and the immune response. Adv Exp Med Biol. 1981;135:1–25.[Medline]
10. Siegel BV. Enhancement of interferon production by poly(rI)-poly(rC) in mouse cell cultures by ascorbic acid. Nature. 1975;254:531–2.[Medline]
11. Manzella JP, Roberts NJ Jr. Human macrophage and lymphocyte responses to mitogen stimulation after exposure to influenza virus, ascorbic acid, and hyperthermia. J Immunol. 1979;123:1940–4.
12. Rayment SJ, Shaw J, Woollard KJ, Lunec J, Griffiths HR. Vitamin C supplementation in normal subjects reduces constitutive ICAM-1 expression. Biochem Biophys Res Commun. 2003;308:339–45.[Medline]
13. Akaike T, Suga M, Maeda H. Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO. Proc Soc Exp Biol Med. 1998;217:64–73.[Medline]
14. Hemila H. Does vitamin C alleviate the symptoms of the common cold?–a review of current evidence. Scand J Infect Dis. 1994;26:1–6.[Medline]
15. Gorton HC, Jarvis K. The effectiveness of vitamin C in preventing and relieving the symptoms of virus-induced respiratory infections. J Manipulative Physiol Ther. 1999;22:530–3.[Medline]
16. Sasazuki S, Sasaki S, Tsubono Y, Okubo S, Hayashi M, Tsugane S. Effect of vitamin C on common cold: randomized controlled trial. Eur J Clin Nutr. 2006;60:9–17.[Medline]
17. Douglas RM, Chalker EB, Treacy B. Vitamin C for preventing and treating the common cold. Cochrane Database Syst Rev. 2000;2:CD000980.[Medline]
18. Douglas RM, Hemila H, D'Souza R, Chalker EB, Treacy B. Vitamin C for preventing and treating the common cold. Cochrane Database Syst Rev. 2004;4:CD000980.[Medline]
19. Bridges CB, Harper SA, Fukuda K, Uyeki TM, Cox NJ, Singleton JA. Prevention and control of influenza. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep. 2003;52:1–34; quiz CE1–4.[Medline]
20. Tantcheva LP, Stoeva ES, Galabov AS, Braykova AA, Savov VM, Mileva MM. Effect of vitamin E and vitamin C combination on experimental influenza virus infection. Methods Find Exp Clin Pharmacol. 2003;25:259–64.[Medline]
21. Nishikimi M, Fukuyama R, Minoshima S, Shimizu N, Yagi K. Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. J Biol Chem. 1994;269:13685–8.
22. Nishikimi M, Yagi K. Biochemistry and molecular biology of ascorbic acid biosynthesis. Subcell Biochem. 1996;25:17–39.[Medline]
23. Maeda N, Hagihara H, Nakata Y, Hiller S, Wilder J, Reddick R. Aortic wall damage in mice unable to synthesize ascorbic acid. Proc Natl Acad Sci USA. 2000;97:841–6.
24. Beck MA. Antioxidants and viral infections: host immune response and viral pathogenicity. J Am Coll Nutr. 2001;20:384S–8S.
25. Beck MA, Levander OA, Handy J. Selenium deficiency and viral infection. J Nutr. 2003;133:1463S–7S.
26. Beck MA, Williams-Toone D, Levander OA. Coxsackievirus B3-resistant mice become susceptible in Se/vitamin E deficiency. Free Radic Biol Med. 2003;34:1263–70.[Medline]
27. Lennette DA. (1995) General principles for laboratory diagnosis of viral, rickettsial, and chlamydial infections. Washington: American Public Health Association. 7th ed.
28. Omaye ST, Turnbull JD, Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods Enzymol. 1979;62:3–11.[Medline]
29. Anderson ME. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 1985;113:548–55.[Medline]
30. Ward CL, Dempsey MH, Ring CJ, Kempson RE, Zhang L, Gor D, Snowden BW, Tisdale M. Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. J Clin Virol. 2004;29:179–88.[Medline]
31. Akaike T, Noguchi Y, Ijiri S, Setoguchi K, Suga M, Zheng YM, Dietzschold B, Maeda H. Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc Natl Acad Sci USA. 1996;93:2448–53.
32. Morel Y, Barouki R. Repression of gene expression by oxidative stress. Biochem J. 1999;342:481–96.[Medline]
33. Hensley K, Robinson KA, Gabbita SP, Salsman S, Floyd RA. Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med. 2000;28:1456–62.[Medline]
34. Li N, Karin M. Is NF-kappaB the sensor of oxidative stress? FASEB J. 1999;13:1137–43.
35. Schall TJ. Biology of the RANTES/SIS cytokine family. Cytokine. 1991;3:165–83.[Medline]
36. Conti P, DiGioacchino M. MCP-1 and RANTES are mediators of acute and chronic inflammation. Allergy Asthma Proc. 2001;22:133–7.[Medline]
37. Rose CE Jr, Sung SS, Fu SM. Significant involvement of CCL2 (MCP-1) in inflammatory disorders of the lung. Microcirculation. 2003;10:273–88.[Medline]
38. Lamont AG, Adorini L. IL-12: a key cytokine in immune regulation. Immunol Today. 1996;17:214–7.[Medline]
39. Dinarello CA. Interleukin-1 beta, interleukin-18, and the interleukin-1 beta converting enzyme. Ann N Y Acad Sci. 1998;856:1–11.[Medline]
40. Janeway C. Immunobiology 5: the immune system in health and disease. 5th ed. New York: Garland; 2001.
41. Ganguly R, Durieux MF, Waldman RH. Macrophage function in vitamin C-deficient guinea pigs. Am J Clin Nutr. 1976;29:762–5.
42. Kretz-Remy C, Mehlen P, Mirault ME, Arrigo AP. Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression. J Cell Biol. 1996;133:1083–93.
43. Mehlen P, Kretz-Remy C, Preville X, Arrigo AP. Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death. EMBO J. 1996;15:2695–706.[Medline]
44. Wichmann MW, Zellweger R, DeMaso CM, Ayala A, Chaudry IH. Enhanced immune responses in females, as opposed to decreased responses in males following haemorrhagic shock and resuscitation. Cytokine. 1996;8:853–63.[Medline]
45. Zellweger R, Wichmann MW, Ayala A, Stein S, DeMaso CM, Chaudry IH. Females in proestrus state maintain splenic immune functions and tolerate sepsis better than males. Crit Care Med. 1997;25:106–10.[Medline]
46. McGowan JE Jr, Barnes MW, Finland M. Bacteremia at Boston City Hospital: occurrence and mortality during 12 selected years (1935–1972), with special reference to hospital-acquired cases. J Infect Dis. 1975;132:316–35.[Medline]
47. Grossman CJ. Regulation of the immune system by sex steroids. Endocr Rev. 1984;5:435–55.
48. Paavonen T, Andersson LC, Adlercreutz H. Sex hormone regulation of in vitro immune response. Estradiol enhances human B cell maturation via inhibition of suppressor T cells in pokeweed mitogen-stimulated cultures. J Exp Med. 1981;154:1935–45.
49. Sthoeger ZM, Chiorazzi N, Lahita RG. Regulation of the immune response by sex hormones. I. In vitro effects of estradiol and testosterone on pokeweed mitogen-induced human B cell differentiation. J Immunol. 1988;141:91–8.[Abstract]
50. Bouman A, Schipper M, Heineman MJ, Faas MM. Gender difference in the non-specific and specific immune response in humans. Am J Reprod Immunol. 2004;52:19–26.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. A. Sheridan and M. A. Beck The Immune Response to Herpes Simplex Virus Encephalitis in Mice Is Modulated by Dietary Vitamin E J. Nutr., January 1, 2008; 138(1): 130 - 137. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
different from lung in that group, P < 0.05.
