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Subpollen particles are rich carriers of major short ragweed allergens and NADH dehydrogenases: quantitative proteomic and allergomic study
Get PDFBackground: Short ragweed (Ambrosia artemisiifolia) allergies affect more than 36 million people annually. Ragweed pollen grains release subpollen particles (SPP) of respirable size upon hydration or a change in air electrical conditions. The aim of this study was to characterize the proteomes and allergomes of short ragweed SPP and total pollen protein extract (TOT), and compare their effects with those of standard aqueous pollen protein extract (APE) using sera from short ragweed pollen-sensitized patients. Methods: Quantitative 2D gel-based and shotgun proteomics, 1D and 2D immunoblotting, and quantitative ELISA were applied. Novel SPP extraction and preparation protocols enabled appropriate sample preparation and further downstream analysis by quantitative proteomics. Results: The SPP fraction contained the highest proportion (94%) of the allergome, with the largest quantities of the minor Amb a 4 and major Amb a 1 allergens, and as unique, NADH dehydrogenases. APE was the richest in Amb a 6, Amb a 5 and Amb a 3, and TOT fraction was the richest in the Amb a 8 allergens (89% and 83% of allergome, respectively). Allergenic potency correlated well among the three fractions tested, with 1D immunoblots demonstrating a slight predominance of IgE reactivity to SPP compared to TOT and APE. However, the strongest IgE binding in ELISA was noted against APE. New allergenic candidates, phosphoglycerate mutase and phosphoglucomutase, were identified in all the three pollen fractions. Enolase, UTP-glucose-1-phosphate uridylyltransferase and polygalacturonase were observed in SPP and TOT fractions as novel allergens of the short ragweed pollen, as previously described. Conclusion and Clinical Relevance: We demonstrated that the complete major (Amb a 1 and 11) and almost all minor (Amb a 3, 4, 5, 6, 8 and 9) short ragweed pollen allergen repertoire as well as NADH oxidases are present in SPP, highlighting an important role for SPP in allergic sensitization to short ragweed.This is the peer‐reviewed version of the article: Smiljanic, K.; Apostolovic, D.; Trifunovic, S.; Ognjenovic, J.; Perusko, M.; Mihajlovic, L.; Burazer, L.; Hage, M. van; Velickovic, T. C. Subpollen Particles Are Rich Carriers of Major Short Ragweed Allergens and NADH Dehydrogenases: Quantitative Proteomic and Allergomic Study. Clinical & Experimental Allergy 2017, 47 (6), 815–828.[https://doi.org/10.1111/cea.12874
Modulation of the specific immune response in Balb/cmice by intranasal application of recombinant H1D2 chimera
No full textBACKGROUND: Group 1 and group 2 allergens from house dust mite are the major elicitors of respiratory allergic diseases and the main candidates for immunotherapy. RESULTS: The potential therapeutic role of a chimera composed of recombinant Der p 2 (D2) linked to Influenza A virus hemagglutinin 1 (H1) for intranasal application was created, expressed and tested in a mouse model. H1D2 and D2 were produced by genetic engineering in Escherichia coli and their primary structure was confirmed by mass fingerprint. Both antigens preserved IgE reactivity in immunoblot with serum from seven house dust mite allergic persons. Balb/c mice were sensitized with D2 allergen in alum and subsequently received H1D2 or D2, intranasally. The reduced levels of serum D2 specific IgE, together with the increased serum specific IgG and IgA were detected in both groups which received H1D2 and D2 intranasally. A higher level of effector CD4+CD25+ spleen lymphocytes was found only in the group of mice which received i.n. H1D2. CONCLUSION: H1D2 chimera can have therapeutic potential in Der p 2 allergic persons as dual vaccine which, beside protective allergen specific, can provide protective antibodies against Influenza A virus hemagglutinin 1. (C) 2016 Society of Chemical Industr
Persistence of Lactobacillus rhamnosus and colonic no production in the rat
No full textINTRODUCTION: Lactobacillus rhamnosus LB64 (Liobif®, produced by Institute ,Torlak", Belgrade)
is a well-known bacterial strain widely used as probiotic with many beneficial and limited unwanted
effects. An important aspect of any probiotic strain is the capacity to colonize the gut. Our objective
was to determine if colonizing capacity of LB correlates with its immunomodulatory properites.
Goal: We studied the persistence of L. rhamnosus LB64 in the gastroinestinal tract of healthy adult
rats after its oral administration during the early postnatal or young adult period as well as its impact
on colonic NO production in colitis deseased rats.
METHODS: Rats of DA strain were given solution containing L. rhamnosus (3 x 10°/ml/kg) during
early postnatal (3.-30. day of life, LB/H20), young adult period (31.-70. day of life, H20/LB) or were
given tap water throughout (3.-70. day of life, H20/H20). Fecal samples (100 mg) were resuspended
and mechanically homogenized. Dilutions were plated on selected media (MRS, Institute Torlak),
incubated for 48h (37°C, limited aeration) and the number of bacteria was expressed as mean CFU/g
of feces. Colitis was induced by intrarectal administration of 40mg/kg TNBS in 50% ethanol and NO
production was determined by Griess reaction in supernatants from colonic tissue seven days later.
RESULT: Both regimes of LB treatment showed a significant presence of LB64 and its extended
persistence in the intestine. However, colitis-induced decrease in colonic NO production was
prevented only in LB/H20 group.
CONCLUSION: Early postnatal treatment with LB has proven to be more effective in restricting
colonic changes induced by colitis in later life. Our results point to the importance of lactic acid
bacteria colonization in early life for shaping local immune response in the adulthood (Supported by
Ministry of Education, Science and Technological Development Republic of Serbia, Grant No
175050.)
Sex and age as determinants of rat T-cell phenotypic characteristics: influence of peripubertal gonadectomy
No full textThe study examined the influence of age, sex and peripubertal gonadectomy on a set of T-cell phenotypic parameters. Rats of both sexes were gonadectomised at the age of 1 month and peripheral blood and spleen T lymphocytes from non-gonadectomised and gonadectomised 3- and 11-month-old rats were examined for the expression of differentiation/activation (CD90/CD45RC) and immunoregulatory markers. Peripheral blood T lymphocytes from non-gonadectomised rats showed age-dependent sexual dimorphisms in (1) total count (lower in female than male 11-month-old rats); (2) CD4+:CD8 + cell ratio (higher in female than male rats of both ages); (3) the proportion of recent thymic emigrants in CD8 + T cells (lower in female than male 3-month-old rats) and (4) the proportions of mature na lt ve and memory/activated cells (irrespective of age, the proportion of na lt ve cells was higher, whereas that of memory/activated cells was lower in females). Gonadectomy influenced magnitudes or direction of these sex differences. Additionally, sex differences in peripheral blood T-lymphocyte parameters did not fully correspond to those observed in T-splenocyte parameters, suggesting the compartment-specific regulation of the major T-cell subpopulations' and their subsets' composition. Furthermore, there was no sexual dimorphism in the proportion of either CD25 + Foxp3 + cells among CD4 + or CD161+ (NKT) cells within CD8 + T lymphocytes. However, there was gonadal hormone-independent age-associated sexual dimorphism in the proportion of CD161 + cells (NKT cells) in CD8 + T splenocytes. Overall, the study revealed age-dependent variations in sexual dimorphisms in T-cell parameters relevant for immune response efficacy and showed that they are T-cell compartment-specific and partly gonadal hormone-related
Liver cystic echinococcosis and human host immune and autoimmune follow-up: A review
Get PDFCystic echinococcosis (CE) is an infectious disease caused by the larvae of parasite Echinococcus granulosus (E. granulosus). To successfully establish an infection, parasite release some substances and molecules that can modulate host immune functions, stimulating a strong anti-inflammatory reaction to carry favor to host and to reserve self-survival in the host. The literature was reviewed using MEDLINE, and an open access search for immunology of hydatidosis was performed. Accumulating data from animal experiments and human studies provided us with exciting insights into the mechanisms involved that affect all parts of immunity. In this review we used the existing scientific data and discuss how these findings assisted with a better understanding of the immunology of E. granulosus infection in man. The aim of this study is to point the several facts that challenge immune and autoimmune responses to protect E. granulosus from elimination and to minimize host severe pathology. Understanding the immune mechanisms of E. granulosus infection in an intermediate human host will provide, we believe, a more useful treatment with immunomodulating molecules and possibly better protection from parasitic infections. Besides that, the diagnosis of CE has improved due to the application of a new molecular tool for parasite identification by using of new recombinant antigens and immunogenic peptides. More studies for the better understanding of the mechanisms of parasite immune evasion is necessary. It will enable a novel approach in protection, detection and improving of the host inflammatory responses. In contrast, according to the "hygiene hypothesis", clinical applications that decrease the incidence of infection in developed countries and recently in developing countries are at the origin of the increasing incidence of both allergic and autoimmune diseases. Thus, an understanding of the immune mechanisms of E. granulosus infection is extremely important
Tetanus vaccination related to the decline of trachoma in the Western World? Anti-tetanus antibodies confer partial protection against ocular chlamydial infection.
No full textSex Differences in Macrophage Functions in Middle-Aged Rats: Relevance of Estradiol Level and Macrophage Estrogen Receptor Expression
No full textThe aim of this study was to examine the influence of sex on age-related changes in phenotype and functional capacity of rat macrophages. The potential role of estradiol as a contributing factor to a sex difference in macrophage function with age was also examined. Thioglycollate-elicited peritoneal macrophages derived from the young (2 months old) and the naturally senescent intact middle-aged (16 months old) male and female rats were tested for cytokine secretion and antimicrobial activity (NO and H2O2 production and myeloperoxidase activity). Serum concentration of estradiol and the expression of estrogen receptor (ER)alpha and ER beta on freshly isolated peritoneal macrophages were also examined. Decreased secretion of IL-1 beta and IL-6 by macrophages from middle-aged compared to the young females was accompanied with the lesser density of macrophage ER alpha expression and the lower systemic level of estradiol, whereas the opposite was true for middle-aged male rats. Macrophages in the middle-aged females, even with the diminished circulating estradiol levels, produce increased amount of IL-6, and comparable amounts of IL-1 beta, TNF-alpha, and NO to that measured in macrophages from the middle-aged males. Age-related changes in macrophage phenotype and the antimicrobial activity were independent of macrophage ER alpha/ER beta expression and estradiol level in both male and female rats. Although our study suggests that the sex difference in the level of circulating estradiol may to some extent contribute to sex difference in macrophage function of middle-aged rats, it also points to more complex hormonal regulation of peritoneal macrophage activity in females
Primena koncepta 3r u produkciji antivenoma na konjima
No full textPoznato je da 3R koncept u radu sa eksperimentalnim životinjama označava: smanjenje broja korišćenih životinja (Reduction), zamenu testova na
životinjama in vitro metodama (Replacement) i poboljšanje tretmana i
manipulacije (Refinement). Ovaj koncept je uspešno primenjen u postupku
produkcije zmijskog protivotrova (antivenoma) u Institutu za virusologiju,
vakcine i serume "Torlak".
Životinje izbora za imunizaciju u proizvodnji antivenoma su često konji
zbog mogućnosti produkcije velike (komercijalne) količine antivenoma što
se ne može lako postići na malim životinjama. Jedan od najvažnijih koraka u
produkciji antivenoma je imunizacija otrovom (venomom) na takav način
da se u organizmu životinje indukuje dugotrajan i visok titar specifičnih
antitela. Standardni imunizacioni protokol podrazumevao je injektovanje
velike količine emulzifikovanog otrova na jedno mesto, što je stvaralo
granulome i/ili rane na mestu ubrizgavanja. Novom šemom imunizacije,
aplikacijom male količine emulzije na mnogo mesta, ovakve neželjene
pojave su prestale ili postale zanemarljive (Refinement).
Nova šema imunizacije podrazumeva primenu doktrine "Mala doza, mala
količina-zapremina, mnogo mesta injektovanja" (" Low dose, low volume,
multi-site immunization "). Na taj način se smanjuje potreba za velikom
količinom otrova, zbog što posledično dovodi do smanjenja potrebe za
nabavkom velikog broja zmija, smanjenja rada sa zmijama, kao i smanjenja
opasnosti za radnika koji radi sa zmijama u više aspekata (kraći rad, manji
umor, smanjena šansa za eksces). Nova šema imunizacije je ujedno poboljšala titar antitela, a time povećala količinu antiseruma koja se može
produkovati u jednom konju, što dalje dovodi do smanjenja broja konja
potrebnih za imunizaciju (Reduction).
Paralelno sa razvijanjem nove imunizacione šeme, u laboratoriji je razvijen
ELISA test za detekciju IgG antitela specifičnih za venom. Indukcija i
povećanje specifičnih antitela, standardno se prati testom potence na
miševima, kojim se meri koliku zaštitu produkovan konjski antiserum pruža
miševima koji su primili letalnu dozu otrova. Pokazali smo da ELISA test može da zameni test potence tokom imunizacionog perioda i perioda
održavanja titra (Replacement).
Novi imunizacioni protokol i razvijen laboratorijski metod za praćenje
kinetike specifičnih antivenom antitela, omogućili su ne samo pojednostavljenje produkcionog postupka i povećanje prinosa proizvoda, već i
znatno poboljšanje dobrobiti eksperimentalnih životinja, naročito konja.
Ovako uspešan pristup konceptu 3R može se primeniti i u proizvodnji
drugih imunih seruma na konjima
The effects of Lactobacillus rhamnosus lb64 on translocation of enteric bacteria and splenic no production during colitis
No full textINTRODUCTION: It has been described that loss of intestinal barrier integrity and consequent
translocation of gut microbiota toward extraintestinal sites contribute to colitis development. As
Lactobacillus rhamnosus LB64 (Liobif®, produced by Institute ,,Torlak", Belgrade) is widely used as
an antidiarrheal drug, we sought to determine if it has potential to affect translocation of gut
microbiota during colitis.
GOAL: We studied the influence of oral administration of LB64 on a) translocation of enteric bacteria
and b) its own potential to translocate from gut to lymphoid organs under physiological conditions
and during colitis. Besides, we have determined splenic nitric oxide (NO) production.
METHODS: Rats of DA strain were given a solution containing LB64 (3 x 10¢/ml/kg) during early
postnatal (3.-30. day of life, LB/H20), young adult period (31.-70. day of life, H20/LB) or were given
LB (LB/LB) or tap water (H20/H20) throughout (3.-70. day of life). Colitis was induced by intrarectal
administration of 40mg/kg TNBS in 50% ethanol, Seven days later, spleen and mesenteric lymph
nodes (MLN) were excized, macerated and plated on Mac Concey agar and MRS (37°C) for 24h and
48h, respectively. NO production was determined by Griess reaction in supernatants of homogenized
spleen tissue.
RESULT: No presence of LB64 was discovered in MLN or spleens of either healthy or colitic rats.
However, both E.coli and Enterococcus spp. were detected in MLN and spleens of rats with colitis but
not in lymphoid organs of healthy rats. Treatments with LB didn't affect colitis-induced enteric
bacteria traslocation, but prevented colitis-induced increase in splenic NO production.
CONCLUSION: Although LB did not affect bacterial translocation during colitis, it exerted antiinflammatory
effect on a systemic level without leaving the gut. Our results suggest
immunomodulatory activity of LB64 (Supported by Ministry of Education, Science and Technological
Development Republic of Serbia, Grant No 175050.)
Postvaccination Accumulation of the Influenza Virus Antigen in the Rat Choroid Plexus
No full textWe examined the accessibility of influenza virus and diphtheria-tetanus toxin (DiTe) antigens to the choroid plexus (CP) within the postvaccination period and the expression of CD11b molecules (by immunohistochemistry). Eighteen Dark Agouti (DA) rats were divided into three groups: (i) animals administered with influenza vaccine (Flu), (ii) animals administered with DiTe vaccine (DiTe), and (iii) nontreated (Contr) animals. The serum antibody titers following influenza and diphtheria-tetanus vaccination were detected by the ELISA test. Immunohistochemical staining revealed a great number of viral antigen-positive and CD11b-positive brain cells in Flu rats compared to a very small number of the respective cells in DiTe animals and no staining in the Contr group. DiTe- and Flu-rats showed significant increases in the serum anti-tetanus toxoid and anti-influenza virus antibody levels compared to those in the Contr group. The results obtained attract attention towards the dynamic role of the CP in the immunosurveillance of the CNS. Based on the viral antigen deposits accumulated in the CP, it has been proposed that the latter can play an active role in modulation of the immune response after influenza vaccine immunization