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Modulation of peritonal murine macrophages functional characteristics by Bet v 1-BanLec chimera
No full textAllergen-specific immunotherapy (ASIT) is suitable treatment for patients with respiratory
allergies. Banana Lectin (BanLec) is mannose-specific protein and can be used to deliver
particular therapeutic to target cells since they are decorated by different glycan structures
1. BanLec binds murine peritoneal macrophages and modulates their functional
characteristics by binding to oligosaccharide structures on TLR2 2. This makes BanLec
attractive protein for ASIT. Combining purified allergens with different vector systems or
adjuvants can improve their immunogenicity 3. The aim of this study was to test
immunomodulatory potential of Bet v 1-BanLec chimera on murine macrophages in terms
of upregulation and secretion of cytokines. The recombinant construct of the major birch
(Betula verrucosa) pollen allergen Bet v 1 and BanLec has been designed and produced by
recombinant DNA technology. Biological activity of the purified construct was confirmed
by binding of Bet v 1-BanLec to a horseradish peroxidase glycoprotein in ELISA. After
incubation murine peritoneal macrophages to different concentrations of Bet v 1-BanLec
we demonstrated its immunomodulatory potential by measuring cytokine level, and level
of expressed genes. Binding of FITC labelled Bet v 1-BanLec chimera to surface
glucoproteins of murine macrophages was confirmed by confocal microscopy. Furhted in
vitro studies will evaluate immunomodulatory potential of BanLec-Bet v 1 for application
in ASIT
High prevalence of anti-thyroglobulin IgE in suspected atopic and non-atopic individuals
No full textThyroglobulin is a large glycoprotein which contains neutral sugars, sialic acid and
iodine. In cow and pig, it also contains an α-gal epitope, which has been pinpointed as
an epitope responsible for delayed immediate‐type hypersensitivity to red meat.
The goal of this study was to test the reactivity of human serum IgE to unmodified
commercial bovine and porcine thyroglobulin in ELISA. Two groups of subjects were
tested; the people who were suspected to be atopic by general practitioners and
professional athletes who declared themselves as not being atopic. Thyroglobulin
specific IgE, IgG and IgG subclasses were tested in ELISA. IgE to commercial meat
extract was also assessed in ELISA. Porcine thyroglobulin was separated according to
charge on a DEAE matrix. The fractions were dot blotted and analyzed for IgE
reactivity.
Among 229 suspected atopic subjects 27 (11.8%) had high IgE level and 41 (17.9%)
had moderate level of thyroglobulin specific IgE. The situation was similar with
professional athletes (n=38) 15,8% high and 23.7% moderate. The suspected atopic
subjects who had high IgE level to thyroglobulin also had statistically higher meat
extract specific IgE, but were not significantly different in any other antibody
class/subclass tested. The IgG subclasses present, specific for thyroglobulin were IgG2
and IgG4.
Subjects who had detectable IgE levels were significantly younger (Hi: intermediary:
low= 13: 13: 27 years; p<0.0002; Kruskal-Wallis). The IgE reactivity to porcine
thyroglobulin is dependent on charge. The reactivity to α-Gal epitope cannot be
assessed by using unmodified commercial bovine or porcine thyroglobulin in ELISA.
The function of the detected anti-thyroglobulin IgE antibodies is unknown, but
considering their preferential distribution in the younger age group they might come
from the pool of natural IgE antibodies
Sexual dimorphism in thymic senescence
No full textThe study showed sexual dimorphism in the kinetics of thymic involution in Dark Agouti rats, so in 24-month-old males prominent thymic fibro-adipose degeneration was found, whereas fibrous changes dominated in thymi of age-matched females. This dimorphism reflected sex-specific constellation of age-related alterations in the expression of the key proadipogenic factors (xanthine oxidase-induced PPARy, STAT3, the transcription factor controlling PPARy downstream adipocyte- differentiation-related gene expression, and IL-6) and TGF-β, the key pro-fibrinogenic factor. The age-related epithelial-mesenchymal transition in thymi of Dark Agouti rats was accompanied by decline in thymopoiesis mirrored in decrease in the frequency of the most mature CD4+CD8-/CD4-CD8+ TCRaβhigh thymocytes and CD4+ and CD8+ recent thymic emigrants in peripheral blood (PB). This was more prominent in males than in females. Irrespective of sex, differentiation/maturation “block” leading to accumulation of the least mature CD45RC+CD2-CD4-CD8- thymocytes, accompanied by decline in the frequency of descendant double positive (DP) TCRaβ- ones was observed with aging. This was followed by opposing changes in the efficacy of positive/negative selection in males and females, which was related to sex-specific alterations in thymic expression of Nur77, a nuclear receptor involved in negative selection, and surface density of CD90 (negative regulator of thymocyte-selection threshold) on thymocytes undergoing selection. Moreover, compared with old females, in age-matched males CD4+CD8-/CD4-CD8+ TCRaβhigh thymocyte ratio was shifted towards the latter. However, CD4+/CD8+ ratio in PB was skewed towards CD8+ T cells in both sexes. Irrespective of sex, with aging the frequency of CD4+CD25+Foxp3+ thymocytes diminished, but that of CD4+CD25+Foxp3+ T regulatory cells (Tregs) in PB increased, most likely due to enhanced expansion of “induced” Tregs. Collectively, the study i) indicates necessity of sex-specific approaches in designing thymus-rejuvenating strategies and ii) warns that changes in the PB T-cell compartment do not necessarily reflect sex-based differences in T-cell generation in thymus
Heart rate variability and ß-adrenergic receptor gene expression in doxorubicin-induced cardiomyopathy
Get PDFAntraciklinski antibiotik doksorubicin pokazao se u višedecenijskoj kliničkoj praksi kao efikasan lek u terapiji brojnih maligniteta. Međutim, njegovu primenu ozbiljno sprečava pojava organske, a posebno srčane toksičnosti. Doksorubicinska kardiomiopatija razvija se najranije 30 dana, a nekada i nakon više godina od terapije doksorubicinom, rezistentna je na postojeću terapiju i ima veliku stopu smrtnosti. Premda su mehanizmi doksorubicinske kardiomiopatije dobro izučeni, još uvek ne postoje efikasna prevencija ni terapija. Zato je primena doksorbicina u kliničkoj praksi prihvatljiva samo uz stalni monitoring rada srca. U kardiološkoj praksi, varijabilitet srčane frekvencije (HRV) i senzitivnost arterijskog baroreceptorskog refleksa (sBRS) koriste se u prognostičke svrhe kod hipertenzije i ishemijske bolesti srca. U ovom radu ispitivani su dijagnostički i prognostički značaj HRV i sBRS za rànō otkrivanje kardiomiopatije prouzrokovane doksorubicinom i upoređivanje sa standardnim metodama – ehokardiografijom i serumskim markerima oštećenja kardiomiocita – srčanim troponinima. S obzirom na to da je uloga β-adrenergičkih receptora (β-AR) u nastanku srčane slabosti dobro poznata, pratili smo i ekspresiju srčanih β1- i β2-AR kod pacova tretiranih doksorubicinom, i poredili sa promenama HRV. Eksperimenti su izvedeni na odraslim mužjacima pacova Wistar soja, kojima je u femoralnu arteriju plasiran polietilenski kateter za registrovanje hemodinamskih parametara i uzorkovanje krvi. Eksperimentalnoj (DOX, n = 50) grupi, aplikovan je doksorubicin u kumulativnoj dozi od 15 mg/kg. Kontrolnoj grupi (CONT, n = 18) aplikovan je fiziološki rastvor u istoj zapremini. Procenjivana je opšta toksičnost doksorubicina i praćeni su hemodinamski ehokardiografski paramentri: pre tretmana, 35. dana (DOX35) i 70. dana (DOX70) nakon aplikacije doksorubicina. HRV analiziran je upotrebom nekoliko metóda: spektralne analize (Hilbert-Huangova transformacija), Poenkareovih dijagrama, aproksimativne entropije i entropijeuzorka...Anthracycline antibiotic doxorubicin, used in clinical practice for several decades, has been shown as effective antineoplastic drug. Unfortunately, its usage is hindered due to multiple organotoxicity and especially cardiac toxicity. Doxorubicin-induced cardiomyopathy may develop 30 days, or even several years after following treatment. It is resistant to treatment, and has very high mortality rate. Although the mechanisms of doxorubicin-induced cardiomyopathy are well studied, there are still no effective treatment and prevention. Hence, the usage of doxorubicin in clinical practice is acceptable merely under close monitoring of cardiac function. In cardiology practice, heart rate variability (HRV) and baroreflex sensitivity (sBRS) are used as prognostic markers in hypertension and ischemic heart disease. Herein, we evaluated and compared diagnostic and prognostic values of HRV and sBRS in doxorubicin-induced cardiomyopathy to standard echocardiographic and serum cardiac troponin T and I methods. In addition, considering the pathophysiological role of β-adrenergic receptors (β-AR) in heart failure, we scrutinised the expression of β1- and β2-AR in doxorubicin-induced cardiomyopathy and its association to HRV changes. Experiments were performed on adult male Wistar rats, replete with femoral arterial catheter for blood pressure recording and blood sampling. Doxorubicin (DOX, n = 50) or saline (CONT, n = 18) were applied to rats in cumulative dose of 15 mg/kg (i.p.). General toxicity, cardiovascular hemodynamics and echocardiography were assessed before treatment, 35 days and 70 days after treatment. HRV and blood pressure variability (BPV) were evaluated by several methods (Hilbert-Huang frequency analysis, Poincaré plots, approximate entropy and sample entropy). The sequence method was used to evaluate sBRS. Levels of cardiac troponin T and I were measured by ECLIA method. β1-AR and β2-AR gene expression was measured by RT-qPCR..
Influence of ageing on sex and strain differences in immune response to inactivated influenza vaccine
No full textEfficacy of the immune response to vaccine depends on genetic background, sex and
age of the recipient. However, mechanisms underlying this phenomenon have not been
elucidated, yet. The study investigated influence of sex and age on serum IgG response
to seasonal trivalent inactivated split influenza vaccine (TIV) in BALB/c and C57BL/6
mice, and mechanisms underlying this response. Total serum IgG responses to
influenza virus type A strains declined with aging, in a strain-specific manner.
Consequently, strain differences (greater IgG responses in BALB/c mice) observed in
young mice (three-month-old) were abrogated in old (eighteen-month-old) ones.
However, irrespective of strain and age, females developed stronger influenza type Aspecific IgG responses than males. Despite age-related decrease in influenza B-specific
serum IgG response, it was comparable between old BALB/c and C57BL/6 mice. The
strain/sex-specific differences in age-related changes in the magnitudes of IgG
responses to TIV correlated with those in number of germinal centre (GC) B
splenocytes. These differences were related to those in B splenocyte and CD4+
splenocyte proliferation in culture upon restimulation with influenza viruses from TIV.
The magnitudes of IgG responses also correlated to T follicular regulatory (Tfr)/T
follicular helper and Tfr/GC B splenocyte ratios across all groups of mice. Aging,
irrespective of influenza virus-specificity, affected serum IgG2a(c)/IgG1 ratios
(reflecting IFN-γ/IL-4 production level ratio) in male BALB/c and female C57BL/6
mice, respectively. Thus, although in young mice of both strains these ratios were
comparable between sexes, in old females they were shifted towards IgG1 when
compared with age-matched males. Consistently, the IFN-γ/IL-4 production level ratios
in splenocyte cultures stimulated with influenza viruses from old females of both strains
were shifted towards IL-4 compared with that in age-matched male cultures. The study
stimulates further research to formulate sex-specific strategies to improve efficacy of
influenza vaccine in elderly
Electrophoretic and mass spectrometry-based characterization of soluble fraction of camel milk proteins upon freeze and spray drying treatment
Get PDFObjective: Camel milk is highly nutritious food with numerous health benefits proposed. Demandfor camel milk has increased worldwide.Production of camel milk powders facilitate its transport,prolonge shelf-life, and also offer an attractive additive for various food products. In this study wecharacterized proteins of soluble fraction of freeze/spray dried camel milk powders.Material and Methods: Whole camel milk powders were prepared by spray drying treatment at sixdifferent inlet temperatures (190°C - 250°C) or by freeze drying. The soluble protein fractions uponthe treatments were analysed by combination of electrophoretic techniques and circular dichroism.Freeze dried camel milk and spray dried at 250°C were analysed by mass spectrometry.Results: SDS-PAGE revealed non-uniform increase in Mw of major protein bands, while nativeelectrophoresis revealed non-uniform decrease in pI values with increased inlet temperature ofspray drying. That indicated occurence of the Maillard reaction. Far-UV circular dichroism spectrashowed no differences in secondary structures between freeze and spray dried samples. Massspectrometry identified α-lactalbumin, glycosylation-dependant cell adhesion molecule 1(GLYCAM1), immunoglobulin heavy chain, peptidoglycan recognition protein and camel serumalbumin as dominant proteins in soluble fraction of camel milk powders. Carboxymethyl-lisyne(CML), well known marker of Maillard reaction in food analysis, was detected on GLYCAM1 and onimmunoglobulin heavy chain.Conclusions: Our results indicate glycation of camel milk proteins via Maillard reaction upon spraydrying treatment which further may affect techno-functional properties of camel milk powders,their shelf-life and nutritional value.Acknowledgments: This work was supported by the Ministry of Education, Science andTechnological Development of the Republic of Serbia, grant number 172024. The project leading tothis application has received funding from the European Union's Horizon 2020 research andinnovation programme under grant agreement No 810752
Natural killer cells as participants in pathogenesis of rat experimental autoimmune encephalomyelitis (EAE): lessons from research on rats with distinct age and strain
Get PDFNatural killer (NK) cells, influencing dendritic cell (DC)-mediated CD4+ lymphocyte priming in draining lymph nodes (dLNs) and controlling spinal cord (SC) infiltration with encephalitogenic CD4+T lymphocytes, modulate EAE (multiple sclerosis model). This study examined their putative contribution to age-related differences in EAE development in Dark Agouti (DA) (exhibiting age-related decrease in EAE susceptibility) and Albino Oxford (AO) (becoming susceptible to EAE with aging) rats. Aging increased NK cell number in dLNs from rats of both strains. In AO rats, but not in DA ones, it also increased the numbers of IFN-gamma-producing NK cells (important for DC activation) and activated/matured DCs, thereby increasing activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated CD25+Foxp3-CD4+ cell number. Aging in DA rats diminished activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated Foxp3-CD4+ cell number. However, MBP-stimulated CD4+ cell proliferation did not differ in dLN cell cultures from young and aged AO rats (as more favorable activated/matured DC/Foxp3-CD4+ cell ratio was abrogated by lower intrinsic CD4+ cell proliferative capacity and a greater regulatory CD25+Foxp3+CD4+ lymphocyte frequency), but was lower in those from aged compared with young DA rats. At SC level, aging shifted Foxp3-CD4+/cytotoxic CX3CR1+ NK cell ratio towards the former in AO rats, so it was less favorable in aged AO rats exhibiting prolonged neurological deficit compared with their DA counterparts. The study showed strain and age differences in number of IFN-gamma-producing NK cells in EAE rat dLNs, and suggested that their pathogenetic relevance depends on frequency and/or activity of other cells involved in CD4+ T cell (auto)immune response
Implementation of the One Health approach to fight arbovirus infections in the Mediterranean and Black Sea Region: Assessing integrated surveillance in Serbia, Tunisia and Georgia
Get PDFBackground In the Mediterranean and Black Sea Region, arbovirus infections are emerging infectious diseases. Their surveillance can benefit from one health inter-sectoral collaboration; however, no standardized methodology exists to study One Health surveillance. Methods We designed a situation analysis study to document how integration of laboratory/clinical human, animal and entomological surveillance of arboviruses was being implemented in the Region. We applied a framework designed to assess three levels of integration: policy/institutional, data collection/data analysis and dissemination. We tested the use of Business Process Modelling Notation (BPMN) to graphically present evidence of inter-sectoral integration. Results Serbia, Tunisia and Georgia participated in the study. West Nile Virus surveillance was analysed in Serbia and Tunisia, Crimea-Congo Haemorrhagic Fever surveillance in Georgia. Our framework enabled a standardized analysis of One Health surveillance integration, and BPMN was easily understandable and conducive to detailed discussions among different actors/institutions. In all countries, we observed integration across sectors and levels except in data collection and data analysis. Data collection was interoperable only in Georgia without integrated analysis. In all countries, surveillance was mainly oriented towards outbreak response, triggered by an index human case. Discussion The three surveillance systems we observed prove that integrated surveillance can be operationalized with a diverse spectrum of options. However, in all countries, the integrated use of data for early warning and inter-sectoral priority setting is pioneeristic. We also noted that early warning before human case occurrence is recurrently not operationally prioritized
Propranolol Impairs Primary Immune Responses in Rat Experimental Autoimmune Encephalomyelitis
No full textObjective: We examined the effect of beta-adrenoceptor (AR) blockade in the preclinical phase of experimental autoimmune encephalomyelitis (EAE), the most commonly used model of multiple sclerosis, on the development of primary CD4+ T-cell responses in draining lymph nodes (dLNs). Methods: CD11b+ cell migration to dLNs, CD4+ T-cell activation/proliferation, and IL-17+ CD4+ (Th17) cell numbers in dLN and spinal cord (SC) were examined in male and female Dark Agouti rats using flow cytometry analysis. Results: Irrespective of sex, in propranolol-treated (PT) rats, migration of CD11b+ antigen-presenting cells from the site of immunization to dLNs was impaired compared with saline-treated controls and consequently the frequency of all CD11b+ cells in dLNs and activated cells among them, too. This correlated with decreased expression of CCL19/21 transcripts in dLNs. Consistently, the frequency of activated/proliferating cells among dLN CD4+ T cells was reduced in PT rats. Additionally, propranolol reduced the number of Th17 cells in dLNs and SC. Consistently, male and female PT rats exhibited a decreased incidence of EAE and prolonged duration of the asymptomatic disease phase. Conclusion: This study suggests that sympathetic dysregulation is involved in the outbreak of clinical EAE. (C) 2019 S. Karger AG, Base