C- Phycocyanin Effect on IFN-γ Production in Patients with Chronic
Egyptian Journal of Medical Microbiology, April 2009 Vol. 18, No. 2 C- Phycocyanin Effect on IFN-γ Production in Patients with Chronic Hepatitis C Infection: In vitro Study Osama M. Basha,* Sahar G. Zaghloul,* Refat A. Sadeq,** Raghda A. Hafez,** Mohammad A.A. El-Mohsen,* Yassin M. El-Ayoty *** * Department of Internal Medicine, ** Department of Microbiology, Faculty of Medicine, Zagazig University, *** Department of Botany, Faculty of Science, Zagazig University, Egypt ABSTRACT Hepatitis C virus (HCV) is a serious global health threat and current medical treatment options are limited. Gama interferon (IFN-γ) is an important pro-inflammatory cytokine with antiviral activity however, the mechanism of its action as anti-hepatitis C treatment remains unclear. Many studies suggest that priming with IFN-γ prior to initiation of INF-γ treatment has a beneficial effect in some cases of chronic hepatitis C infected patients through changing the balance of cytokines in favour of a Th-1 type response in the host. C-phycocyanin is a water-soluble biliprotein from the filamentous Cyanobactrium spirulina platenesis with a potent antioxidant, anti-inflammatory and anti cancerous properties. In this study we investigated the effect of C-phycocyanin on the production of IFN-γ from the peripheral blood mononuclear cells (PBMC) obtained from patients with chronic hepatitis C with different degrees of liver disease severity. We obtained two groups of blood; the first was from healthy volunteers as a control group and the second was from patients with hepatitis C virus infection. Both groups were subjected to phycocyanin and phytoheamaglutinin as stimulators of the release of interferon gamma from the PBMC. We found that in both groups the level of IFN-γ production without addition of phycocyanin or phytoheamaglutinin was less than after addition of 5ul or 10 ul of phycocyanin. Also the results showed that the mean level of IFN-γ production in the diseased group was higher than that of the control group. Among the diseased group the production was much higher in class B subgroup (Child B)sss. The safety of Spirulina and its content C-phycocyanin and its immune stimulatory effects encourage us to recommend its use in vivo study specially in chronic hepatitis C patients who failed to respond to or unfit for the classical interferon therapy. Key wards: Hepatitis C-virus (HCV), C-phycocyanin (C-Pc), Gama interferon (IFN-γ) and interleukin I secretion,(5) antioxidant effects,(6) anticancer(7) and antiviral effects as it affects the replications of several viruses including Herpes simplex type I, human cytomegalo virus, measles, mumps, influenza A virus and HIV-I virus.(8) Spirulina contains mainly two phycobiliproteins, namely C-phycocyanin (CPC) and allophycocyanin (A-PC) approximately at a ratio of 10:1.(9) C-phycocyanin comprises a protein and chromophore. The protein moiety consists of α and β subunits of molecular weight in the range of 18.000 and 20.000, respectively and exist as a complex interacting mixture of trimer, hexamer and decamer aggregates.(10) Many reports proved that C-PC has many pharmacological characteristics including antiinflammatory, anti-oxidant,(11) radical scavenging activities,(12) inhibits cyclooxygenase-2(13) and has anti-tumor activity.(14) Hepatitis C virus infects an estimated 170 million person's world wide.(15) In most infected INTRODUCTION Natural compounds from blue green algae have attracted a great deal of interest as physiologically functional food supplements and several bioactive metabolites have been successfully developed into clinically useful drugs.(1) More than 60 % of anti-cancer and 70 % of anti-infective antibiotics currently in clinical use are natural product or natural product-based.(2) Spirulina has long history of use as food. Its name derives from the spiral or helical nature of its filaments. There are reports that it was used as food in Mexico during Aztec civilization. It is still used as food in the lake Chad area where it is sold as dried bread called dihe.(3) Spirulina has a rich content of protein, vitamins, essential amino acids, minerals and essential fatty acids.(4) Many researches proved that Spirulina and its extracts have many pharmacological benefits as immunmo-dulatory function with stimulatory effects on interferon 81 Egyptian Journal of Medical Microbiology, April 2009 Vol. 18, No. 2 according to Child-Pugh classifications; class A, B and C, each subgroup included 10 samples. All were excluded to be free from renal, cardiac, diabetes mellitus or autoimmune diseases Methods 1. Liver function test: • Serum Alanin amino transferase, Aspartate amino transferase, Alkaline phosphatase using Bio merieux kit. • Total direct and indirect bilirubin and serum albumin using Diamond kits. • Prothrombin time and INR using Diaplstin kit. 2. Detection of viral markers in serum • The presence of Anti-HCV antibodies (IgG) in the serum was determined by qualitative enzyme immunoassay. • Viral nucleic acids extraction and isolation. RNA in the serum was extracted, reverse transcriped and amplified using quantitative (PCR).The results were interperated as copies /ml. 3. Detection of auto antibodies: Antinuclear antibodies are assayed in all cases of HCV group to exclude auto immune hepatitis by indirect immuno-fluorescence. 4. Mononuclear cells (PBMC) isolation: Under sterile safety hood, 5 ml of blood were diluted with equal volume of phosphate buffer solution (Biochrom AG); diluted blood was added over Ficoll-Hypaque solution 1.077 (Biochrom AG) in a ratio of 3:1 for each sample. The tubes were centrifuged at 2000 rpm at room temperature (22°C) for 30 minutes. The buffy coat containing the mononuclear cells was obtained and washed with phosphate buffer at 1:1 volume by centrifugation at 1000 rpm for 10 minutes. The last step was repeated for two extra times but the last time was done with RPMI 1640 media (Furo-lone) ,(5) 5. Extraction and determination of C-PC: C-PC was extracted from blue-green algae according to Bousiba and Richmond(21) as follow: 20 gm of experimental algae was suspended in 200 ml of 0.1 sodium phosphate buffer pH 7.2, containing 100ug/ml lysozome and 10 ml EDTA. The enzymatic disintegration of the cell wall occurred by placing the algae in shaking water path at 30°C for 24 hours. The slurry centrifuged for 1 hour at 10.000 rpm to remove cell debris, yielding a clear supernatant of C-PC. The crude C-PC was centrifuged for 30 hours at 10.000 rpm at 40°C. C-PC was precipitated using ammonium sulphate 75% at pH 7.2 for individuals, this remarkable RNA virus evades the immune system and establishes a chronic infection that can lead to cirrhosis, end stage liver disease and hepatocellular carcinoma. While advances have been made in treating HCV, the current therapy, a combination of pegylated interferon IFN-α and ribavirin is poorly tolerated and is effective in only 50% of genotype I HCV- infected patients.(16) Thus, a great need exist for more effective and better tolerated therapies for HCV. Interferon-γ (INF– γ) is a key player in immune response to virus infection and its efficacy for treatment of hepatitis C patients is under clinical trials. INF– γ exerts multiple roles in the regulation of inflammation and immune responses during HCV infection and studies have suggested that it plays a critical role in the control of HCV infection and seems to be well tolerated.(17) Further researches proved that in addition to the reduction of claudin-1 expression, IFN-γ treatment also led to significant changes in the distribution of claudin-1, CD8, and scavenger receptor class B type I. These receptors are important for virus C to invade the hepatocyte or other susceptible cells.(18,19) Other effects of IFN-γ include enhancement of Th1 cells and inhibition of Th2 cells, enhancement of immune lysis of HCV infected cells, inhibition of hepatic fibrosis by an effect on TGF-beta and has an effect on HCV induced carcinogenesis.(20) In this study, we tried to elucidate an immunmodulatory role of natural safe substances as phycocyanin in patients with chronic viral C hepatitis through stimulation the release of INF–γ from peripheral blood mononuclear cell (PBMC) SUBJECTS, MATERIALS & METHODS This study was performed in Internal Medicine, Microbiology and Immunology Departments, Faculty of Medicine, Zagazig University and included 40 subjects. They were divided into two main groups. Group I: Control group, which included 10 healthy persons (6 males and 4 females their ages ranged from 38 to 62 years) with no history of liver diseases, negative HCV antibodies with normal abdominal ultrasonography. Group II: Included 30 hepatic patients (18 males and 12 females 38 to 62 years) having chronic HCV infection previously diagnosed by polymerase chain reaction (PCR). This group was further subdivided into 3 subgroups 82 Egyptian Journal of Medical Microbiology, April 2009 Vol. 18, No. 2 AST, ALT, INR, serum albumin, WBCs, PLT, and total bilirubin. There was significant positive correlation between the viral load, age, AST, INR albumin and total bilirubin (Fig. 1, 2, 3, 4, 5). 6 hours. The precipitated C-PC was dissolved in phosphate buffer and dialyzed over night at 4°C against the name buffer. 6. Culture and stimulation of the peripheral mononuclear cells(5): 200 ml of 1× 1000.000/ml PBMC were recovered into RPMI 1640 media with 10% (vol/vol) fetal bovine serum (FBs) and 1% (wt/vol) L-glutamine, penicillin (1U/ml) and Streptomycin (0.1 mg/ml) and incubated in 96 wells of tissue culture plate. 20 ul phytoheamaglutinin (PHA) was added to well. 5 ul of phycocyanin, its concentration was 3.5 Mg/50ml were added to another well, and 10 ul of phycocyanin, its concentration was 3.5 Mg/50ml were added to another well. There was another well without phycocyanin or phyto-heamoaglutinin. The culture plates were incubated in the incubator at 37°C, 5% CO2 and optimum humidity. After 24 hours, the supernatant was collected, centrifuged at 1000 rpm for 10 minutes and kept frozen at 20°C. The level of IFN-γ was measured in the cell culture supernatant using ELISA. 7. Interferon gamma assay: (R , D . systemes) This assay employs the quantitative sandwhich enzyme immunoassay technique. A polyclonal antibody specific for IFN-γ had been per-coated into a microplate. Standards and samples are pipetted into the wells and any IFN-γ was bounded by the immobilized antibody. After washing away any unbounded substances, an enzyme-linked polyclonal antibody specific to IFN-γ was added. Following a wash to remove any unbounded antibody enzyme reagent, a substrate solution was added and colors developed in proportion to the amount of IFN-γ bound in the initial step. The color development was stopped and the intensity of the color was measured. 8. Calculation of results Average the duplicate reading for each standard, control, and sample and subtract the average zero standard optical density. ELISA reader [Stat Fox 303, made in Germany] was adjusted at wave length 450 to read the absorbance and four standards were used as calibrator. 70 60 Age 50 40 30 20 10 0 0 500000 1000000 1500000 2000000 2500000 3000000 PCR Figure (1) Correlation between viral load and Age 160 140 120 AST 100 80 60 40 20 0 0 500000 1000000 1500000 2000000 2500000 3000000 PCR Figure (2) Correlation between viral load and AST 3.0 2.5 INR 2.0 1.5 1.0 0.5 0.0 0 500000 1000000 1500000 2000000 2500000 3000000 PCR RESULTS Figure (3) Correlation between viral load and INR Correlation between viral load and other parameters The relation of viral load and each of the following parameters was determined; age, Hb, 83 Egyptian Journal of Medical Microbiology, April 2009 Vol. 18, No. 2 6 350 5 300 250 PLT Alb 4 3 2 200 150 100 1 50 0 0 0 500000 1000000 1500000 2000000 2500000 3000000 0 500 1000 Figure (4) Correlation between viral load and Albumin 2000 2500 Figure (7) Correlation between interferon –γ production level of and PLT 4.5 180 4.0 160 3.5 140 3.0 120 ALT Bil 1500 Interferon γ PCR 2.5 2.0 100 80 60 1.5 40 1.0 20 0.5 0 0.0 0 0 500000 1000000 1500000 2000000 2500000 500 3000000 1000 1500 2000 2500 Interferon γ PCR Figure (8) Correlation between interferon –γ production level of and ALT Figure (5) Correlation between viral load and bilirubin 4.5 4.0 Correlation between IFN –γ and other parameters The relation of IFN –γ and each of the same parameters was determined. There was significant positive correlation between the level of IFN–γ and Hb, PLT, ALT, total bilirubin (Fig. 6,7,8,9). 3.5 Bil 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 1000 1500 2000 2500 Figure (9) Correlation between interferon –γ production level of and bilirubin 16 14 12 HB 500 Interferon γ 18 10 Measurement of γ interferon without addition of phycocyanin or phytohemagglutinin on PBMNC of the two groups: in control group I; it was 67.2 ±109.2. In group II: subgroup A; it was 116.7 ± 128, subgroup B; it was 400.6 ± 304 and subgroup C; it was 175.2 ± 132.4. There is statistically significant difference among the groups K =14.9 and P =0.0018 (Fig. 10). 8 6 4 2 0 0 500 1000 1500 2000 2500 Interferon γ Figure (6) Correlation between interferon –γ production level of and Hb 84 Egyptian Journal of Medical Microbiology, April 2009 450 A B C Control Vol. 18, No. 2 800 A 400 B C Control 700 350 600 300 250 500 200 400 150 300 100 200 50 100 0 A B C Control 0 Figure (10) Measurement of gamma interferon without addition of phycocyanin or phytohemagglutinin of the two groups A A B C C Control Figure (12) Measurement of gamma interferon after addition of 10 µl of phycocyanin on PBMNC of the two groups Measurement of gamma interferon after addition of 5 µl of phycocyanin on PBMNC of the two groups: in control group I; it was 99.87±83.3 In group II: subgroup A; it was 187.6± 148.3, subgroup B; it was 739.6 ± 730.7 and subgroup C; it was 363.7 ± 413.3. There was statistically significant difference among the groups (K =7.65 and P = 0.05) (Fig. 11). 800 B Measurement of γ interferon after addition of phyto-hemagglutinin on PBMNC of the two groups: in control group I; it was 854.67±660 group II: subgroup A; it was 1446.5 ± 521.5, subgroup B; it was 3336 ± 66.4 and subgroup C; it was 2796.2 ± 744. There was statistically significant difference among the groups (K = 31.2 and P = 0.001) (Fig. 13). Control 3500 A B C Control 700 3000 600 2500 500 2000 400 300 1500 200 1000 100 500 0 A B C 0 Control A B C Control Figure (11) Measurement of gamma interferon after addition of 5µl of phycocyanin on PBMNC of the two groups Figure (13) Measurement of gamma interferon after addition of phytohemagglutinin on PBMNC of the two groups Measurement of γ interferon after addition of 10µl of phycocyanin on PBMNC of the two groups: in control group I; it was 142.9±101.8. In group II: subgroup A; it was 217.4 ± 172.3, subgroup B; it was 765.1 ± 791.9 and subgroup C; it was 447.8 ± 418.2. There was statistically significant difference among the groups (K=16.6 and P = 0.001) (Fig. 12). DISCUSSION The overall majority of experimental studies and clinical observations among patients with HCV infection favour immune mediated hepatocellular damage over direct viral cytopathic effect.(22) Recent investigations were done concerning the benefits of gamma interferon in management 85 Egyptian Journal of Medical Microbiology, April 2009 of chronic HCV patient especially who failed to respond to classical interferon therapy.(25) IFN-γ is a unique member of the IFN family. Its receptor, signaling pathway, and cellular effects differ from those of IFN- α and IFN-ß(26). IFN-γ may contribute to HCV clearance in several ways. First, it enhances NK cell activity and induces the expression of inflammatory and potentially antiviral cytokines such as tumor necrosis factor alpha.(27) Second, it facilitates induction and effector function of T cells via upregulation of major histocompatibility complex class I and II proteins and promotes antigen processing via induction of immunoproteasomes.(28) Third, it facilitates T-cell homing from lymph nodes and peripheral blood to the site of infection via induction of T-cellrecruiting chemokines such as IFN-inducible protein 10 (CXCL10), IFN-inducible T-cell chemo-attractant (CXCL11) and monokine induced by IFN- (CXCL9).(29) Further results from confocal microscopy and Western blot analysis showed that in addition to the reduction of CLDN1 expression, IFN-γ treatment also led to significant changes in the distribution of CLDN1, CD81, and scavenger receptor class B type I. The main receptors for viral activation.(30) Many recent of researchers add an evidence for the immuno-modulatory effects of phycobiliproteins.(31) Phycobiliproteins are a small group of highly conserved chromoproteins that constitute the phycobilisome, a macromolecular protein complex whose main function is to serve as a light harvesting complex for the photosynthetic apparatus of cyanobacteria and eukaryotic groups.(33) The most common classes of phycobiliproteins are allophycocyanin, phycocyanin and phycoerythrin all of which are formed of α and β protein subunits and carry different isomeric linear tetrapyrrole prosthetic groups.(34) C-phycocyanin (C-PC) is one of the major biliproteins of spirulina platensis, a blue grean alga, with anti oxidant and radical scavenging properties. It is also known to exhibit anti inflammatory and anti cancer properties. However the main mechanism of action of C-PC is not clearly under stood.(35) PC is composed of two dissimilar and protein subunits of 17 000 and 19 500 Da, respectively, with one bilin chromophore attached to the αsubunit (α84) and two to the subunit (β84, β155).(36) The chemical structure of the bilin chromophores in PC is very similar to bilirubin, Vol. 18, No. 2 which is considered to be a physiologically important antioxidant against reactive species.(37) It inhibits oxidative modification of plasma proteins and aromatic amino acid residues.(38) In our work we found that the mean level of INF –γ in the diseased sub groups (A, B and C) was higher than control group in resting condition. Also, the production of INF –γ by PBMNCs was higher in hepatitis patients than control group after addition of C-PC and phytohemagglutinin .This was in agreement with the results of Missale et al.(39) and Tsai et al. (40) who reported higher concentration of INF gamma in the serum of patients with chronic viral C hepatitis. In contrast to this result Jirillo et al. (41) reported that a significant decrease of INF- γ was reported in patients with viral hepatitis C. These conflicting results may be due to changes in Th1 & Th2 responses among patients with chronic viral hepatitis C. Also it might be explained by the difference in study size. Also, this work showed that INF-γ production was higher in hepatic patient than in control group after addition of 5 µl and 10 µl of C-PC. This adds an evidence for the immunmodulatory effects of phycocyanin and supported by a group of researches from University of California ـDavis(5) who reported that Spirulina and its extracts stimulated the secretion of interlukin-1, LL- 4 and interferon IFN ـγ to nearly 2.0, 3.3, and 13.6 times basal levels respectively. Also they reported that induction of lFN–γ by Spirulina was found to be comparable to that seen after phytohemagglutinin (PHA) stimulation. In spite it was different in our results where induction of IFN ـγ by phytohemag-glutinin was higher than that induced by phycocyanin. It may be corrected by increasing the dose of phycocyanin added to PBMNC. Also in paper presented at the 30th annual meeting of the Japanese Society for immunology. Saeki et al.(42) presented the results of a human study using spirulina extract and reported that lFN–γ secretion activity was enhanced significantly after two weeks of spirulina extract administration. Also he added that, surprisingly, lFN–γ and NK cell activities continued up to 6 months after administration of extract was discontinued. In the present work , the mean level of IFN–γ production was increased in the diseased group after addition of 5µl and 10µl of phycocyanin but the increase was most obvious in child B followed by C & lastly A. Although many human studies showed low level of IFN-γ in chronic HCV infection, our in- 86 Egyptian Journal of Medical Microbiology, April 2009 vitro study showed that PBMNCs were still able to produce IFN–γ, even in advanced stage of liver disease, in resting state and after stimulation by Phycocyanin with different doses. This means that the body does not make enough natural interferon to effectively fight infection and the addition of exogenous help (Phycocyanin that stimulation PBMNCs to produce IFN-γ) may aid in fighting off the infection. The safety of spirulina and the high stimulatory effect of its constituent C-PC on IFN–γ production, encourage for further in-vivo study and on a large scale of people. Vol. 18, No. 2 9. 10. 11. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 12. Borowitzka M., A., (1995): microalgae as sources of pharmaceuticals and other biologically active compounds, J. Appl. Phycol. 7,3-15. Subhashi J., Mahipal S.V., Reddy M.C., et al. (2004): Molecular mechanisms in CPhycocyanin induced apoptosis in human chronic myeloid leukemia cell line-K562, Biochem. Pharmacol. 68 (3) 453-462. Abdulqader G., Barsanti L. and Tredici M. (2000): Harvest of Arthrospira platensis from Lake Kossorom (Chad) and its household usage among the Kanembu. J. Appl Phycol. 12:493-498. Belay A. and Houston M. (2002): IThe Potential Application of Spirulina (Arthrospira) as a nutritional and therapeutic supplement in health management. The Journal of the American Nutraceutical Association Vol. 5, No. 2, Mao TK., Van de Water J. and Gershwin ME. (2000): Effect of spirulina on the secretion of cytokines from peripheral blood mononuclear cells. J. Med Food; 3:135-140. Bhat V.D. and Madhyastha K. M. (2001): Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis: protection against oxidative damage to DNA. Biochem Biophys Res Commun 285, 262-266. Wang H., Liu Y., Gao X et al. (2007): The recombinant B subunit of CPhycocyanin inhibits cell proliferation and induces apoptosis Cancer letters 247, 150158. Ayehunie S., Belay A., Baba TW. and Ruprecht RM. (1998): Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis). J 13. 14. 15. 16. 17. 18. 19. 20. 87 Acquired Immune Deficiency Syndromes and Human Retrovirology. 18:7-12. Bermejo R., Talavera E.M., Alvarez-pez J.M. and Orte J.C. (1997): Chromatographic purification of biliproteins from spirulina platensis: High performance liquid chromatographic separation of their and sub units .J. Chrom. A. 778,441-450. Glazer A.N. and Appl. J. (1994): Phycobiliproteins – a family of valuable, widely used fluorophores. J. Appl. Phycol. 6, 105-112. Romay C., Armesto J., Remirez D., et al. (1998): Antioxidant and anti-inflammatory properties of C-phycocynin from blue-green algae, Inflamm. Res. 47, 1:36-41. Bhat V.B. and Madyastha K.M. (2000): C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro, Biochem Biophys, Res Commun. 275, 1:20-25. Reddy M C., Subhashini J and Mahipal S V. (2003): C-phycocyanin, a selective cyclooxygenase -2 inhibitor. Biochem Biophys Res. Commun. 304, 2:385 -92. Roy KR., Arunasree KM., Reddy NP., et al. (2007): Alteration of mitochondrial membrane potential by Spirulina platensis C-phycocyanin induces apoptosis in the doxorubicinresistant human hepatocellularcarcinoma cell line. HepG2; 47, 3:159-67. Major ME. and Feinstone SM. (1997): The molecular virology of hepatitis C. Hepatology. 25:1527-38. Hadziyannis SJ., Sette H Jr., and Morgan TR., et al. (2004): Peginterferon alfa-2a and ribavirin combination therapy in chronic hepatitis C: A randomized study of treatment duration and ribavirin dose. Ann Inter Med; 140:346-55. Wei X., Jia Z., Lian J., et al. (2009): Journal of Interferon & Cytokine Research. 29, 3:171-178. Shin E., Protzer U., Untergasser A., et al. (2005) : Liver-Directed Gamma Interferon Gene Delivery in Chronic Hepatitis C. Journal of Virology, p. 13412-13420 Vol. 79, No. 21. Rüffer C. and Gerke V. (2004): The Cterminal cytoplasmic tail of claudins 1 and 5 but not its PDZ-binding motif is required for apical localization at epithelial and endothelial tight junctions. Eur J. Cell Biol. 83, 4:135-44. Gattoni A., Parlato A., Vangieri B., et al. (2006): Interferon-gamma: biologic Egyptian Journal of Medical Microbiology, April 2009 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. Vol. 18, No. 2 31. Fairchild C.D. and Glazer A.N. (1994): J. Biol. Chem. 269, 28988-28996. 32. Romay C., Gonzalez R., Ledon N., et al. (2003): C-phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects, Curr Prot Pept Sci 4, 207-216. 33. Glazer A.N. (1988): in Methods Enzymol. (Fleischer, S. and Parker, L. Eds.)Vol. 167, 304-312. 34. Wejestal R., Norkrans G. and Weilm O. (1995): Lymphocyte subsets and beta 2 microglobulin expression in chronic hepatitis C. Clin Exp Immunol 87, 3:340. 35. Reddy C.M., Bhat V.B., Kiranmai G., et al. (2000) Biochem. Biophys. Res Commun 277, 599-603. 36. Turner L., Houghton J.D. and Brown S.B. (1997): Planta 201, 78-83. 37. Stocker R., Yamamoto Y., McDonagh A.F., Glazer A.N. and Ames B.N. (1987): Science, 235, 1043-1046. 38. Neuzil J. and Stocker R. (1994): J. Biol. Chem., 269, 16712-16719. 39. Missale G., Ferrari G. and Fiaccadori F. (1995): Cytokine mediators in acute inflammation and chronic course of viral hepatitis. Ann Ital Med Int 10, I:14. 40. Tsai SL., Liaw YF., Chen MH., et al. (1997): Detection of type 2 like Thelper cells in hepatitis C virus infection: Implications for hepatitis C virus chronicity. Hepatology 25, 2:449. 41. Jirillo E., Greco B., Caradonna L. et al. (1995): Evaluation of cellular immune responses and soluble mediators in patients with chronic hepatitis C virus (cHCV) infection. Immunopharmacol Immunotoxicol; 2: 347–64. 42. Saeki Y., Matsumoto M., Hayashi A., et al. (2000): The effect of Spirulina hot water extract to the basic immune activation. Summary of paper presented at the 30th Annual Meeting of the Japanese Society for Immunology. 14-16. functions and HCV therapy (type I/II) (2 of 2 parts). 157, 5:457 - 68. Boussiba S. and Richmond A.E. (1979): Isolation and characterization of phycocyanin from the blue green algae spirulina platensis. Arch. MicroBiol 120, 155-159. Weber F., Kochs G. and Haller O. (2004): Inverse interference: how viruses fight the interferon system. Viral Immunol 17:498-515. Fan XG., Yakoob J., Fan XJ., Keeling PWN. (1995): Effect of IL-4 on peripheral blood lymphocyte proliferation: implication in immuno-pathogenesis of H. pylori infection. Immunol Lett; 48:45–48. Fan XG., Yakkob J., Fan XJ., et al. (1996): Enhanced Th2 responses: immune mechanism of H. pylori infection. Ir J. Med Sci 165:37–39. Thimme R., Bukh J., Spangenberg H. C., et al. (2002): Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc. Natl. Acad. Sci. USA 99:15661-15668. Biron, C. A. (2001): Interferons and other cytokines, p. 321-351. In D. Knipe and P. Howley (ed.), Fields virology, vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa. Paludan S. R. (2000): Synergistic action of pro-inflammatory agents: cellular and molecular aspects. J. Leukoc. Biol. 67:1825. Fruh K. and Yang Y. (1999): Antigen presentation by MHC class I and its regulation by interferon gamma. Curr. Opin. Immunol 11:76-81. Zeremski M., Petrovic L.M. and Tala A.H. (2006): The role of chemokines as inflammatory mediators in chronic hepatitis C virus infection. Journal of viral hepatitis 14, 10:675-687. Lalor P.F., Shields P., Grant A., and Adams D.H. (2002): Recruitment of lymphocytes to the human liver. Immunol. Cell Biol. 80:52-64. 88 Vol. 18, No. 2 Egyptian Journal of Medical Microbiology, April 2009 ﺗﺄﺛﻴﺮ اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﻋﻠﻰ اﻧﺘﺎج اﻻﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻓﻰ ﻣﺮﺿﻰ اﻻﻟﺘﻬﺎب اﻟﻜﺒﺪى اﻟﻔﻴﺮوﺳﻰ اﻟﻤﺰﻣﻦ )س( :دراﺳﺔ ﺧﺎرج اﻟﺠﺴﻢ اﺳﺎﻣﺔ ﻣﺤﻤﺪ ﺑﺎﺷﺎ * ،ﺳﺤﺮ ﺟﻮدة زﻏﻠﻮل* ،رﻓﻌﺖ ﻋﺒﺪ اﻟﺴﻤﻴﻊ ﺻﺎدق** ،راﻏﺪة ﻋﺒﺪ اﻟﻠﻄﻴﻒ ﺣﺎﻓﻆ**، ﻣﺤﻤﺪﻋﺒﺪ اﻟﻮاﺣﺪ ﻋﺒﺪ اﻟﻤﺤﺴﻦ* ،ﻳﺎﺳﻴﻦ اﻟﻌﻴﻮﻃﻰ ***. *ﻗﺴﻢ اﻟﺒﺎﻃﻨﺔ اﻟﻌﺎﻣﺔ **،ﻗﺴﻢ اﻟﻤﻴﻜﺮو ﺑﻴﻮﻟﻮﺟﻰ واﻟﻤﻨﺎﻋﺔ آﻠﻴﺔ اﻟﻄﺐ *** ﻗﺴﻢ اﻟﻨﺒﺎت آﻠﻴﺔ اﻟﻌﻠﻮم .ﺟﺎﻣﻌﺔ اﻟﺰﻗﺎزﻳﻖ .ﻣﺼﺮ ﻳﻌﺘﺒﺮ اﻵن أآﺜﺮ ﻣﻦ %٦٠ﻣﻦ اﻷدوﻳ ﺔ اﻟﻤ ﻀﺎدة ﻟﻠ ﺴﺮﻃﺎن واﻟﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ ﻣﻨﺘﺠ ﺎت ﻃﺒﻴﻌﻴ ﺔ أو ﻣ ﺸﺘﻘﺔ ﻣ ﻦ ﻣ ﺼﺎدر ﻃﺒﻴﻌﻴﺔ . اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ هﻮ ﻣﺮآﺐ ﻃﺒﻴﻌﻰ ﻳﺤﻀﺮ ﻣﻦ اﻟﻄﺤﺎﻟ ﺐ اﻟﺰرﻗ ﺎء وﻗ ﺪ اﺗ ﻀﺢ ﻟ ﻪ ﺗ ﺄﺛﻴﺮات ﻣﺨﺘﻠﻔ ﺔ آﻤ ﻀﺎد ﻟﻸﻟﺘﻬ ﺎب وﻣ ﻀﺎد ﻟﻸآﺴﺪة .وﺣﺪﻳﺜًﺎ ﻗﺪ ﻇﻬﺮ ﻟﻠﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﻧﺸﺎط ﺿﺪ اﻷورام. اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﻳﻌﺘﺒﺮ ﻣﻦ ﻣﻜﻮﻧﺎت ﺳﺒﻴﺮوﻟﻴﻨﺎ ﺑﻼﺗﻴﻨﺰﻳﺰ واﻟﺘﻰ ﺗﺴﻤﻰ اﻵن أورﺛﺮوﺳﺒﻴﺮا واﻟﺘﻰ ﻃﺎﻟﻤﺎ اﺳﺘﺨﺪﻣﺖ آﻐﺬاء . اﻟﻬﺪف ﻣﻦ اﻟﺪراﺳﺔ -: دراﺳﺔ ﺗﺄﺛﻴﺮ اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﻋﻠﻰ إﻧﺘﺎج اﻹﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻣﻦ ﺧﻼﻳﺎ أﺣﺎدﻳ ﺔ اﻟﻨ ﻮى ﻓ ﻰ ﻣﺮﺿ ﻰ اﻷﻟﺘﻬ ﺎب اﻟﻜﺒ ﺪى اﻟﻔﻴﺮوﺳ ﻰ اﻟﻤﺰﻣﻦ )س( دراﺳﺔ ﺧﺎرج اﻟﺠﺴﻢ . اﻟﻤﺮﺿﻰ وﻃﺮﻳﻘﺔ اﻟﺒﺤﺚ : ﺷ ﻤﻠﺖ اﻟﺪراﺳ ﺔ ﻋﻠ ﻰ ٤٠ﺷﺨ ﺼﺎ ﻣ ﻦ ﻏﻴ ﺮ اﻟﻤ ﺼﺎﺑﻴﻦ ﺑ ﺄﻣﺮاض اﻟﻜﻠ ﻰ واﻟﻘﻠ ﺐ وداء اﻟ ﺴﻜﺮى وﻣ ﺮض ذاﺗ ﻰ اﻟﻤﻨﺎﻋ ﺔ أو ﻳﺘﻨﺎوﻟﻮن أى دواء ﻳﺆﺛﺮ ﻋﻠﻰ ﻣﻨﺎﻋﺔ اﻟﺠﺴﻢ. وﺗﻢ ﺗﻘﺴﻴﻤﻬﻢ إﻟﻰ : ﻣﺠﻤﻮﻋﺔ )أ( وﺷﻤﻠﺖ ﻋﻠﻰ ١٠ :أﺷﺨﺎص ﻣﻦ اﻷﺻﺤﺎء آﻤﺠﻤﻮﻋﺔ ﺿﺎﺑﻄﺔ . ﻣﺠﻤﻮﻋﺔ )ب( وﺷﻤﻠﺖ ﻋﻠﻰ ٣٠ﺷﺨﺺ ﻣﻤﻦ ﻳﻌﺎﻧﻮن ﻣﻦ اﻹﻟﺘﻬﺎب اﻟﻜﺒﺪى اﻟﻔﻴﺮوﺳﻰ اﻟﻤﺰﻣﻦ )س( وﻗﺪ ﺗﻢ ﺗﻘﺴﻴﻢ هﺬﻩ اﻟﻤﺠﻤﻮﻋﺔ ﻋﻠﻰ ﺣﺴﺐ ﺗﻘﺴﻴﻢ ﺗﺸﺎﻳﻠﺪ ﺑﻮف إﻟﻰ ﺛﻼث ﻣﺠﻤﻮﻋﺎت أﺧﺮى : ﻣﺠﻤﻮﻋﺔ ﻓﺮﻋﻴﺔ ) (١وﺗﺸﻤﻞ ﻋﺸﺮة ﻣﺮﺿﻰ ﻣﻦ ﺗﻘﺴﻴﻢ ﺗﺸﺎﻳﻠﺪ ﺑﻮف وهﻰ آﻼس . A ﻣﺠﻤﻮﻋﺔ ﻓﺮﻋﻴﺔ ) (٢وﺗﺸﻤﻞ ﻋﺸﺮة ﻣﺮﺿﻰ ﻣﻦ ﺗﻘﺴﻴﻢ ﺗﺸﺎﻳﻠﺪ ﺑﻮف وهﻰ آﻼس . B ﻣﺠﻤﻮﻋﺔ ﻓﺮﻋﻴﺔ ) (٣وﺗﺸﻤﻞ ﻋﺸﺮة ﻣﺮﺿﻰ ﻣﻦ ﺗﻘﺴﻴﻢ ﺗﺸﺎﻳﻠﺪ ﺑﻮف وهﻰ آﻼس . C آﻞ أﻓﺮاد هﺬا اﻟﺒﺤﺚ واﻟﺬﻳﻦ ﺗﻢ اﺧﺘﻴﺎرهﻢ ﻗﺪ ﺗﻢ ﻓﺤﺼﻬﻢ إآﻠﻴﻨﻴﻜﻴﺎ وﻋﻤﻞ اﻟﻔﺤﻮﺻﺎت اﻟﺮوﺗﻴﻨﻴﺔ ﻟﻬﻢ ﺷﺎﻣﻠﺔ آﻼ ﻣﻦ : -١ﺻﻮرة دم آﺎﻣﻠﺔ . -٢وﻇﺎﺋﻒ اﻟﻜﺒﺪ وﺗ ﺸﻤﻞ )أﻧﻨﺰﻳﻤ ﺎت اﻵﻻﻧ ﻴﻦ أﻣﻴﻨﻮﺗﺮاﻧ ﺴﻔﺮاز– واﻟﻔﻮﺳ ﻔﺎﺗﻴﺰ اﻟﻘﻠ ﻮى واﺳ ﺒﺮﺗﺎت اﻣﻴﻨﻮﺗﺮاﻧ ﺴﻔﺮاز– اﻟﺒﻠﻴ ﺮوﺑﻴﻦ ﺑﺎﻟﺪم آﺎﻣﻞ– واﻷﻟﺒﻮﻣﻴﻦ– وزﻣﻦ اﻟﺒﺮوﺛﺮوﻣﺒﻴﻦ (. -٣اﻷﺷﻌﺔ اﻟﺘﻠﻴﻔﺰﻳﻮﻧﻴﺔ ﻋﻠﻰ اﻟﺒﻄﻦ ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ ﻗﻴﺎس ﻧﺴﺒﺔ اﻹﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻗﺒﻞ وﺑﻌﺪ إﺿﺎﻓﺔ اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ اﻟﻤﻔﺮز ﻣﻦ ﺧﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮى ﺑﻌﺪ ﻋﺰﻟﻬﺎ . أوﺿﺤﺖ ﻧﺘﺎﺋﺞ اﻟﺪراﺳﺔ ﻣﺎ ﻳﻠﻰ : -١ﻓﻰ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﺔ وﻓﻰ آﻞ ﻣﻦ اﻟﻤﺠﻤﻮﻋ ﺎت اﻟﻔﺮﻋﻴ ﺔ :أن ﻣﺘﻮﺳ ﻂ إﻧﺘ ﺎج اﻻﻧﺘﺮﻓﻴ ﺮون ﺟﺎﻣ ﺎ ﻓ ﻰ ﻋ ﺪم إﺿ ﺎﻓﺔ أى ﻣ ﻦ اﻟﻔﻴﻜﻮﺳ ﻴﺎﻧﻴﻦ واﻟﻔﻴﺘ ﻮهﻴﻢ أﺟﻠ ﻮﺗﻴﻨﻴﻦ أﻗ ﻞ ﻣ ﻦ ﻣﺘﻮﺳ ﻂ إﻧﺘ ﺎج اﻻﻧﺘﺮﻓﻴ ﺮون ﺟﺎﻣ ﺎ ﻋﻨ ﺪ إﺿ ﺎﻓﺔ ٥و ١٠ﻣﻴﻜﺮوﻟﺘ ﺮ ﻣ ﻦ اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ وأﻗﻞ ﻣﻦ ﻣﺘﻮﺳﻂ إﻧﺘﺎج اﻹﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻋﻨﺪ إﺿﺎﻓﺔ اﻟﻔﻴﺘﻮهﻴﻢ أﺟﻠﻮﺗﻴﻨﻴﻦ. -٢ﻣﺘﻮﺳ ﻂ إﻧﺘ ﺎج اﻹﻧﺘﺮﻓﻴ ﺮون ﺟﺎﻣ ﺎ ﻓ ﻰ اﻟﻤﺠﻤﻮﻋ ﺔ اﻟ ﻀﺎﺑﻄﺔ أﻗ ﻞ ﻣ ﻦ ﻣﺮﺿ ﻰ اﻹﻟﺘﻬ ﺎب اﻟﻜﺒ ﺪى اﻟﻔﻴﺮوﺳ ﻰ )س( ﻓ ﻰ اﻟﻤﺠﻤﻮﻋﺎت اﻟﻔﺮﻋﻴﺔ اﻟﺜﻼث. -٣ﻣﺴﺘﻮى إﻧﺘﺎج اﻹﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻣﻦ ﺧﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮى آﺎن اﻋﻠﻰ ﻓﻰ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻔﺮﻋﻴﺔ ٢ﻣﻦ اﻟﻤﺠﻤﻮﻋﺎت اﻷﺧﺮى . ﻓﻰ ﺿﻮء اﻟﺪراﺳﺔ اﻟﺤﺎﻟﻴﺔ ﻧﺴﺘﺨﻠﺺ اﻵﺗﻰ: -١ﻓﻰ ﺣﺎﻟﺔ ﻋﺪم إﺿ ﺎﻓﺔ آ ﻼ ﻣ ﻦ اﻟﻔﻴﻜﻮﺳ ﻴﺎﻧﻴﻦ واﻟﻔﻴﺘ ﻮهﻴﻢ أﺟﻠ ﻮﺗﻴﻨﻴﻦ وأﻳ ﻀﺎ ﺑﻌ ﺪ إﺿ ﺎﻓﺔ ٥ﻣﻴﻜﺮوﻟﺘ ﺮ و ١٠ﻣﻴﻜﺮوﻟﺘ ﺮ واﻟﻔﻴﺘ ﻮهﻴﻢ أﺟﻠ ﻮﺗﻴﻨﻴﻦ وﺟ ﺪ أن إﻧﺘ ﺎج اﻻﻧﺘﺮﻓﻴ ﺮون ﺟﺎﻣ ﺎ ﻋ ﻦ ﻃﺮﻳ ﻖ ﺧﻼﻳ ﺎ أﺣﺎدﻳ ﺔ اﻟﻨ ﻮى آ ﺎن اﻋﻠ ﻰ ﻓ ﻰ ﻣﺠﻤﻮﻋ ﺔ ﻣﺮﺿﻰ اﻟﻜﺒﺪ ﻋﻦ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﺔ -٢ﻣﺎزاﻟﺖ اﻟﺨﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮى ﻓﻰ ﻣﺮﺿﻰ اﻹﻟﺘﻬﺎب اﻟﻜﺒﺪى ﻗﺎدرة ﻋﻠﻰ اﻧﺘﺎج اﻻﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻓﻰ ﺣﺎﻟﺔ ﻋ ﺪم إﺿ ﺎﻓﺔ آﻼ ﻣﻦ اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ واﻟﻔﻴﺘﻮهﻴﻢ أﺟﻠﻮﺗﻴﻨﻴﻦ وﻳﺰﻳﺪ ﺑﻌﺪ ﺣﺜﻬﺎ ﺑﺎﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﺑﺠﺮﻋﺎت ﻣﺨﺘﻠﻔﺔ واﻟﻔﻴﺘﻮهﻴﻢ أﺟﻠﻮﺗﻴﻨﻴﻦ . - -٣إﻧﺘﺎج اﻹﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻓﻰ ﺣﺎﻟﺔ ﻋﺪم إﺿﺎﻓﺔ آﻼ ﻣ ﻦ اﻟﻔﻴﻜﻮﺳ ﻴﺎﻧﻴﻦ واﻟﻔﻴﺘ ﻮهﻴﻢ أﺟﻠ ﻮﺗﻴﻨﻴﻦ ﻣ ﻦ ﺧﻼﻳ ﺎ أﺣﺎدﻳ ﺔ اﻟﻨ ﻮى وﺑﻌﺪ ﺣﺜﻬﺎ ﺑﺎﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ واﻟﻔﻴﺘﻮهﻴﻢ أﺟﻠﻮﺗﻴﻨﻴﻦ ﻳﻼﺣﻆ ﻓﻰ ﻣﺮﺿ ﻰ اﻟﻜﺒ ﺪ ﺑ ﺪرﺟﺎت اﻟﻤ ﺮض اﻟﻤﺨﺘﻠﻔ ﺔ ﻓ ﻰ اﻟﻤﺠﻤﻮﻋ ﺎت اﻟﻔﺮﻋﻴﺔ اﻟﺜﻼث . 89 Vol. 18, No. 2 Egyptian Journal of Medical Microbiology, April 2009 ﻓﻰ ﺿﻮء اﻟﺪراﺳﺔ اﻟﺤﺎﻟﻴﺔ ﻧﺴﺘﺨﻠﺺ اﻟﺘﻮﺻﻴﺎت اﻵﺗﻴﺔ -: -١اﻟﺨﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮى ﻟﻬﺎ اﻟﻘﺪرة ﻋﻠ ﻰ إﻧﺘ ﺎج اﻹﻧﺘﺮﻓﻴ ﺮون ﺟﺎﻣ ﺎ ﺧ ﺎرج اﻟﺠ ﺴﻢ وﻧﺤ ﻦ ﻧﻨ ﺼﺢ ﺑﺪراﺳ ﺎت ﻋﻠ ﻰ اﻹﻧ ﺴﺎن ﺑﺎﻋﻄﺎء اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﺛﻢ ﻗﻴﺎس اﻹﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻓﻰ دم ﻣﺮﺿﻰ اﻹﻟﺘﻬﺎب اﻟﻜﺒﺪى اﻟﻔﻴﺮوﺳﻰ اﻟﻤﺰﻣﻦ . -٢إﺟﺮاء ﻣﺤﺎوﻻت ﻹﻋﻄﺎء اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﻣﻊ اﻹﻧﺘﺮﻓﻴﺮون أﻟﻔﺎ ﻓ ﻰ ﻣﺮﺿ ﻰ اﻹﻟﺘﻬ ﺎب اﻟﻜﺒ ﺪى اﻟﻤ ﺰﻣﻦ أو ﻟﻠﻤﺮﺿ ﻰ اﻟﻐﻴ ﺮ ﻣﻨﺎﺳﺒﻴﻦ ﻹﻋﻄﺎء اﻹﻧﺘﺮﻓﻴﺮون أﻟﻔﺎ . -٣وﻷن اﻟﻔﻴﻜﻮﺳﻴﺎﻧﻴﻦ ﻟﺔ اﻟﻘﺪرة ﻋﻠﻰ إﻧﺘﺎج اﻹﻧﺘﺮﻓﻴﺮون ﺟﺎﻣﺎ ﻟﺬﻟﻚ ﻣﻤﻜﻦ اﺳﺘﺨﺪاﻣﺔ آﻐ ﺬاء ﻟﻠﻤﺮﺿ ﻰ واﻷﺻ ﺤﺎء وه ﻮ ﻳﻌﺘﺒﺮ ﻣﻦ إﺣﺪى ﻣﻜﻮﻧﺎت ﺳﺒﻴﺮوﻟﻴﻨﺎ اﻟﺘﻰ ﺗﺘﻤﻴﺰ ﺑﺪرﺟﺔ ﻣﻦ اﻷﻣﺎن . 90
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