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This study investigated whether immunostimulatory DNA sequences (ISS) induce a transient or sustained inhibition of Th2 responses to inhaled antigen. We sensitized mice with subcutaneous injections to develop a Th2 response to ovalbumin (ova) and then administered a dose of ISS prior to ova inhalation challenge. Mice were then rechallenged with ova by inhalation a second time at varying time points after the first ova inhalation (1 to 8 weeks later) to determine whether the ISS dose administered prior to the first ova inhalation protected against a subsequent second ova inhalation challenge. A single dose of ISS inhibited the Th2 response to the first inhalation of ova antigen, as well as 4 weeks later to the second inhalation of ova. However, ISS did not inhibit a Th2 response to the second inhalation of ova 8 weeks later. The reversible inhibition of Th2 responses at 8 weeks suggests the need for repeated ISS administration at monthly intervals., Depto. de Medicina, Fac. de Medicina, TRUE, pub

Replication complexes (RCs), formed by positive-strand (+) RNA viruses through rearrangements of host endomembranes, protect their replicating RNA from host innate immune defenses. We have shown that two evolutionarily conserved defense systems, autophagy and interferon (IFN), target viral RCs and inhibit viral replication collaboratively. However, the mechanism by which autophagy proteins target viral RCs and the role of IFN-inducible GTPases in the disruption of RCs remains poorly understood. Here, using murine norovirus (MNV) as a model (+) RNA virus, we show that the guanylate binding protein 1 (GBP1) is the human GTPase responsible for inhibiting RCs. Furthermore, we found that ATG16L1 mediates the LC3 targeting of MNV RC by binding to WIPI2B and CAPRIN1, and that IFN gamma-mediated control of MNV replication was dependent on CAPRIN1. Collectively, this study identifies a novel mechanism for the autophagy machinery-mediated recognition and inhibition of viral RCs, a hallmark of (+) RNA virus replication. IMPORTANCE Replication complexes provide a microenvironment important for (+) RNA virus replication and shield it from host immune response. Previously we have shown that interferon gamma (IFNG) disrupts the RC of MNV via evolutionarily conserved autophagy proteins and IFN-inducible GTPases. Elucidating the mechanism of targeting of viral RC by ATG16L1 and IFN-induced GTPase will pave the way for development of therapeutics targeting the viral replication complexes. Here, we have identified GBP1 as the sole GBP targeting viral RC and uncovered the novel role of CAPRIN1 in recruiting ATG16L1 to the viral RC.

Studien utvärderar egenskaper hos mesenkymala stromaceller (MSC) i passage 2 och 3. I ett samarbete mellan Cellcolabs och Karolinska Institutet (KI) genomfördes projektet med Katarina Le Blancs forskargrupp. Genom att studera membranmarkörer från tre friska MSC-donatorer, tillsammans med deras förmåga att differentiera till osteoblaster, adipocyter och kondrocyter, samt deras förmåga att inhibera profileringen av CD8+ och CD4+ T-lymfocyter, och slutligen deras möjlighet att öka uttrycket av Indoleamine 2,3-dioxygenase 1 (IDO1) och interleukin 6 (IL6), kunde en jämförelse mellan passage 2 och 3 göras. I korta drag kunde enbart en tydlig skillnad göras mellan de två passagerna. Skillnaden sågs i förmågan att differentiera till osteoblaster, där passage 3 inte kunde prestera på samma nivå som passage 2. Utöver detta var resultaten för de två typerna jämförbara och antydde inte till några större förändringar mellan passage 2 och 3. För att stärka tillförlitligheten av resultatet bör dock fler MSC-donatorer jämföras., The study evaluates the characteristics and consistency of mesenchymal stromal cells (MSCs) in two different passages, specifically 2 and 3. In a collaboration between Cellcolabs and Karolinska Institutet (KI), the project was conducted with Katarina Le Blanc’s research group. Through studying the surface expression on cells from three distinct MSC donors, along with their differentiation ability into osteoblasts, adipocytes and chondrocytes, their capability to suppress the proliferation of CD8+ and CD4+ T lymphocytes, and finally their possibility to increase the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and interleukin 6 (IL6), a comparison between passage 2 and 3 could be done. It was seen that a clear distinction could be made between the two passages while looking at their ability to differentiate into osteoblasts. The remaining results showed comparable outcomes between passage 2 and 3, suggesting minor changes with the increased passage number. However, to strengthen the reliability of the outcome, more MSC donors should be compared.

AbstraktSyftet med den här berättelsen handlar om Abskopal regression av subkutant implanterat N29 råttgliom som inträffar efter immunisering med syngena IFN-utsöndrande celler och behandling av kontralaterala tumörer med pulserande elektriska fält (PEF) och/eller strålbehandling (RT). Studien utfördes på råttor av Fischer-344-stammen med råttgliom N29-tumörer implanterade subkutant på flanken eller på både det högra behandlade bakbenet och det vänstra obehandlade bakbenet. Pulsade elektriska fält (PEF) gavs med 16 exponentiellt avtagande pulser vid en maximal elektrisk fältstyrka på 1400 V/cm och t1/e= 1 ms. Strålbehandling (RT) gavs med 60Co gammastrålning vid dagliga fraktioner av 5 Gy, till en total absorberad dos av 20 Gy. Djuren fick intraperitoneala injektioner av bestrålade (70 Gy), IFN- genmodifierade N29-tumörceller en gång i veckan under tre veckor. Med enbart IFN, PEF eller RT inträffade ingen signifikant minskning av tumörtillväxthastigheten (TGR) i de vänstra laterala tumörerna. Men i kombinationen av IFN-tumörcellsvaccin med PEF och RT inträffade en mycket signifikant minskning av TGR-värdena i de vänstra laterala tumörerna. Den specifika terapiförbättringen (STE) av kombinationsterapin resulterade i ett oväntat högt STE-värde på 0,70±0,08 mycket signifikant skilt från noll (p < 0,0001). Genom att kombinera immunisering med enbart PEF blir TER-värdet endast cirka 0,2 och AER noll, vilket betyder att det inte finns någon abskopal effekt.Men med alla tre behandlingsmodaliteter kombinerade och alla kombinationer av oberoende behandlingar med PEF, RT eller IFN anses en liknande effekt inträffa på tumörerna genom att kombinera RT med antingen PEF eller immunisering med IFN-utsöndrande celler. Således fungerar PEF som ett in vivo elektrovaccin.Den terapeutiska effekten av elektropulsbehandling med eller utan bleomycin skulle förstärkas ytterligare genom att lägga till Sunitinib en tyrosinkinashämmare i en reducerad dos jämfört med standardrekommendati

Studien utvärderar egenskaper hos mesenkymala stromaceller (MSC) i passage 2 och 3. I ett samarbete mellan Cellcolabs och Karolinska Institutet (KI) genomfördes projektet med Katarina Le Blancs forskargrupp. Genom att studera membranmarkörer från tre friska MSC-donatorer, tillsammans med deras förmåga att differentiera till osteoblaster, adipocyter och kondrocyter, samt deras förmåga att inhibera profileringen av CD8+ och CD4+ T-lymfocyter, och slutligen deras möjlighet att öka uttrycket av Indoleamine 2,3-dioxygenase 1 (IDO1) och interleukin 6 (IL6), kunde en jämförelse mellan passage 2 och 3 göras. I korta drag kunde enbart en tydlig skillnad göras mellan de två passagerna. Skillnaden sågs i förmågan att differentiera till osteoblaster, där passage 3 inte kunde prestera på samma nivå som passage 2. Utöver detta var resultaten för de två typerna jämförbara och antydde inte till några större förändringar mellan passage 2 och 3. För att stärka tillförlitligheten av resultatet bör dock fler MSC-donatorer jämföras., The study evaluates the characteristics and consistency of mesenchymal stromal cells (MSCs) in two different passages, specifically 2 and 3. In a collaboration between Cellcolabs and Karolinska Institutet (KI), the project was conducted with Katarina Le Blanc’s research group. Through studying the surface expression on cells from three distinct MSC donors, along with their differentiation ability into osteoblasts, adipocytes and chondrocytes, their capability to suppress the proliferation of CD8+ and CD4+ T lymphocytes, and finally their possibility to increase the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and interleukin 6 (IL6), a comparison between passage 2 and 3 could be done. It was seen that a clear distinction could be made between the two passages while looking at their ability to differentiate into osteoblasts. The remaining results showed comparable outcomes between passage 2 and 3, suggesting minor changes with the increased passage number. However, to strengthen the reliability of the outcome, more MSC donors should be compared.

The gene RARRES3 uses an unexpected strategy to eliminate the parasite Toxoplasma gondii from human cells.

The gene RARRES3 uses an unexpected strategy to eliminate the parasite Toxoplasma gondii from human cells.

For the zoonotic disease Q fever, serological analysis plays a dominant role in the diagnosis of Coxiella burnetii infection and in pre-screening for past exposure prior to vaccination. A number of studies suggest that assessment of C. burnetii-specific T-cell IFNγ responses may be a more sensitive tool to assess past exposure. In this study, we assessed the performance of a whole blood C. burnetii IFNγ release assay in comparison to serological detection in an area of high Q fever incidence in 2014, up to seven years after initial exposure during the Dutch Q fever outbreak 2007-2010. In a cohort of >1500 individuals from the Dutch outbreak village of Herpen, approximately 60% had mounted IFNγ responses to C. burnetii. This proportion was independent of the Coxiella strain used for stimulation and much higher than the proportion of individuals scored sero-positive using the serological gold standard immunofluorescence assay. Moreover, C. burnetii-specific IFNγ responses were found to be more durable than antibody responses in two sub-groups of individuals known to have sero-converted as of 2007 or previously reported to the municipality as notified Q fever cases. A novel ready-to-use version of the IFNγ release assay assessed in a subgroup of pre-exposed individuals in 2021 (10-14 years post exposure) proved again to be more sensitive than serology in detecting past exposure. These data demonstrate that C. burnetii-induced IFNγ release is indeed a more sensitive and durable marker of exposure to C. burnetii than are serological responses. In combination with a simplified assay version suitable for implementation in routine diagnostic settings, this makes the assessment of IFNγ responses a valuable tool for exposure screening to obtain epidemiological data, and to identify previously exposed individuals in pre-vaccination screens.

The inflammatory environment of demyelinated lesions in multiple sclerosis (MS) patients contributes to remyelination failure. Inflammation activates a cytoprotective pathway, the integrated stress response (ISR), but it remains unclear whether enhancing the ISR can improve remyelination in an inflammatory environment. To examine this possibility, the remyelination stage of experimental autoimmune encephalomyelitis (EAE), as well as a mouse model that incorporates cuprizone-induced demyelination along with CNS delivery of the proinflammatory cytokine IFN-γ were used here. We demonstrate that either genetic or pharmacological ISR enhancement significantly increased the number of remyelinating oligodendrocytes and remyelinated axons in the inflammatory lesions. Moreover, the combined treatment of the ISR modulator Sephin1 with the oligodendrocyte differentiation enhancing reagent bazedoxifene increased myelin thickness of remyelinated axons to pre-lesion levels. Taken together, our findings indicate that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation, suggesting that ISR enhancement may provide reparative benefit to MS patients.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by dysfunction of salivary and lacrimal glands, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Autoantibodies, such as anti-SSA and anti-SSB antibodies, are hallmarks and important diagnostic factors for SS. In our previous study, we demonstrated that SS-like xerostomia was observed in SATB1 conditional knockout (SATB1cKO) mice, in which the floxed SATB1 gene was specifically deleted in hematopoietic cells as early as 4 weeks of age. In these mice, autoantibodies were not detected until 8 weeks of age in SATB1cKO mice, although exocrine gland function reached its lowest at this age. Therefore, other markers may be necessary for the diagnosis of SS in the early phase. Here, we found that mRNA expression of the interferonγ (IFN-γ) gene and the IFN-responsive indoleamine 2,3-dioxygenase (IDO) gene is upregulated in the salivary glands of SATB1cKO mice after 3 and 4 weeks of age, respectively. We detected l-kynurenine (l-KYN), an intermediate of l-tryptophan (l-Trp) metabolism mediated by IDO, in the serum of SATB1cKO mice after 4 weeks of age. In addition, the upregulation of IDO expression was significantly suppressed by the administration of IFN-γ neutralizing antibodies in SATB1cKO mice. These results suggest that the induction of IFN-dependent IDO expression is an initial event that occurs immediately after the onset of SS in SATB1cKO mice. These results also imply that serum l-KYN could be used as a marker for SS diagnosis in the early phases of the disease before autoantibodies are detectable.

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy.

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in αKlotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of αKlotho, a sight that may inspire novel therapeutic approaches.

Infektionen mit H. pylori führen zu Veränderungen im Aufbau und der zellulären Zusammensetzung des Magenepithels. Aktuelle Studien haben gezeigt, dass Stromazellen, die sich in unmittelbarer Nähe der epithelialen Stammzelle befinden, die funktionelle Nische für dieses Kompartiment bilden und wichtige Faktoren für die Regulierung des Stammzellumsatzes und Differenzierung darstellen. Daher konzentriere ich mich bei dieser Arbeit auf die Rolle der Myh11+ Myofibroblasten, die sich sowohl unter als auch zwischen den Epitheldrüsenzellen befinden. Ich fand heraus, dass die Myofibroblasten in der Homöostase einen BMP-Gradienten entlang der Drüsenachse mit erhöhter Expression von Bmp2 im oberen Teil erzeugen, während die Drüsenbasis von Zellen umgeben ist, die Bmp-Inhibitoren produzieren. Basierend auf der Funktionsanalyse mit 3D-Organoidmodellen ist der Bmp-Signalweg ein zentraler Faktor für die rasche Differenzierung von Stammzellen in faveoläre mukusproduzierende Zellen. Darüber hinaus wurde ein auto-parakriner Signalweg gefunden, der zur Bildung von Bmp2 in Epithelzellen führt. Meine Untersuchungen Daten zeigten eine Verringerung des BMP-Signalwegs in H. pylori-infizierten Myofibroblasten. Darüber hinaus führte eine Infektion mit H. pylori nicht nur zum Verlust der stromalen, sondern auch der epithelialen Bmp2-Expression. Diese Beobachtung ging mit einer Zunahme von IFNγ einher, was auf eine Verbindung zwischen beiden Signalwegen hinwies. Tatsächlich zeigten anschließende Experimente mit Organoiden, dass IFNγ den Bmp-Signalweg hemmt und dadurch die terminale Differenzierung blockiert. Zusammenfassend zeigen meine Untersuchungen, dass das Schicksal einer Zelle, die zur Oberfläche der Magendrüse wandert, eine Induktion des BMP-Signalwegs erfährt. Die Reduktion dieses Signals durch IFNγ deutet auf einen Mechanismus hin, der Veränderungen in der zellulären Differenzierung und die Entwicklung von prämalignen epithelialen Läsionen im Verlauf einer H. pylori-Infektion bewirkt., Infection with Helicobacter pylori (H. pylori) leads to alterations of the topology and cellular composition of the gastric epithelium. Recent studies have shown that stromal cells located in close proximity to the epithelial stem cell are creating the niche for this compartment and provide crucial factors that regulate stem cell turnover and fate decisions. Therefore, this thesis focuses on of Myh11+ myofibroblasts that are located beneath and between the epithelial gland cells. I found that during homeostasis, the myofibroblasts generate a BMP gradient along the gland axis with strong expression of Bmp2 in the upper part, whereas the base is surrounded by cells producing BMP inhibitors. Based on functional analysis with 3D organoid models, the BMP gradient occurs to be a main factor responsible for rapid differentiation of stem cells into pit surface mucous cells. Moreover, experiments led me to identify an auto-paracrine feed-forward BMP2 loop in epithelial cells, which further stabilizes BMP signaling once it is activated and induces terminal differentiation. Data presented in this study demonstrated a reduction of BMP signaling in H. pylori-infected myofibroblasts. Furthermore, infection with H. pylori resulted in loss of not only stromal, but also epithelial Bmp2 expression. This observation was accompanied with increase of Interferon γ (IFNγ), which indicated a link between both pathways. Consistently, stimulation of organoids with IFNγ impairs BMP signaling and the BMP2 feed-forward loop, and thereby blocks terminal differentiation. Together, this study shows that the fate of a cell migrating into the surface of the gastric gland is determined by an induction of BMP signaling and stabilized by an auto-paracrine BMP signaling enhancement. Reduction of this signaling by IFNγ revealed a mechanism which contributes to altered cellular differentiation and development of premalignant epithelial alterations in the context of H. pylori infection.

Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in alpha Klotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of alpha Klotho, a sight that may inspire novel therapeutic approaches.

Probiotic bacteria Bacillus subtilis have antiviral and immunomodulatory effects, and therefore drugs containing these bacteria can have sufficient therapeutic potential in the treatment of acute nonobstructive bronchitis in children.Objective: to study the clinical and immunological efficacy of probiotic bacteria Bacillus subtilis in the treatment of acute nonobstructive bronchitis in children.Materials and methods. There were 40 children aged from two to six years followed-up. In the comprehensive treatment of acute bronchitis 14 children were administered the drug Subalinum® containing the bacteria Bacillus subtilis, the other children received only routine treatment. The severity of the patient's condition was estimated according to the BSS (Bronchitis Severity Score) scale. Laboratory examination included: complete blood count, urinalysis, nasal cytosmear, immunoenzyme techniques. Etiological diagnosis was established on the basis of virological methods. The interferon gamma level was detected by means of «Гамма-Інтерферон — ІФА-БЕСТ А-8752 (Gamma-interferon-ELISA-BEST A-8752)» kit, manufactured by the Vector-Best (Вектор-Бест) company; lactoferrin — by using Нuman Lactoferrin NK 329 Edition 06–16 kit, which is manufactured by Hycult Biotech (USA), neutrophilic α-defensins (1–3) — Human HNP NK317 Edition 08–16 reagents manufactured by Hycult Biotech (USA).Results. In all children, there was acute onset of disease, manifested in catarrhal and general inflammatory syndromes. The use of a drug containing Bacillus subtilis in therapy contributed to a duration reduction of the symptoms, an increase in the lactoferrin level in the oropharyngeal fluid by the period of convalescence, and a combination of IFN-g and antimicrobial factors.Conclusions. The use of Subalinum® containing bacteria Bacillus subtilis, is a pathogenetic method of the acute nonobstructive bronchitis treatment in children., Пробиотические бактерии Bacillus subtilis обладают противовирусным и иммуномодулирующим действием, в связи с чем препараты, содержащие данные бактерии, могут обладать достаточным терапевтическим потенциалом при лечении острых простых бронхитов у детей.Цель: изучение клинико-иммунологической эффективности пробиотических бактерий Bacillus subtilis при лечении острого простого бронхита у детей.Материалы и методы. Под наблюдением находилось 40 детей в возрасте от двух до шести лет. 14 детей в комплексной терапии острого бронхита получали препарат «Субалин», содержащий бактерии Bacillus subtilis, остальные дети — только стандартную терапию. Тяжесть состояния больных оценивалась по шкале BSS (Bronchitis severity score). Лабораторное исследование включало: общий анализ крови, мочи, назоцитологические, иммуноферментные методы. Этиологический диагноз устанавливался на основании вирусологических методов исследования. При определении концентрации интерферона гамма использовался набор «Гамма-Интерферон-ИФА-БЕСТ А-8752» фирмы «Вектор-Бест»; лактоферрина — набор Нuman Lactoferrin NK 329 Edition 06-16 производства компании Hycult Biotech (USA), нейтрофильных α-дефензинов (1-3) — реактивы Нuman HNP NK317 Edition 08-16 производства компании Hycult Biotech (USA).Результаты. У всех детей заболевание началось остро и проявлялось катаральным и общевоспалительным синдромами. Применение в терапии препарата, содержащего бактерии Bacillus subtilis, способствовало сокращению продолжительности симптомов заболевания, увеличению содержания лактоферрина в ротоглотковой жидкости к периоду реконвалесценции заболевания и сопряжению продукции IFN-g и антимикробных факторов.Выводы. Применение «Субалина», содержащего бактерии Bacillus subtilis, является патогенетическим методом лечения острого простого бронхита у детей., Пробіотичні бактерії Bacillus subtilis мають противірусну та імуномодулюючу дію, у зв'язку з чим препарати, що містять дані бактерії, можуть мати достатній терапевтичний потенціал при лікуванні гострих простих бронхітів у дітей.Мета: вивчення клініко!імунологічної ефективності пробіотичних бактерій Bacillus subtilis при лікуванні гострого простого бронхіту у дітей.Матеріали і методи. Під спостереженням перебувало 40 дітей віком від двох до шести років. 14 дітей у комплексній терапії гострого бронхіту отримували препарат «Субалін», що містить бактерії Bacillus subtilis, решта дітей отримували лише стандартну терапію. Важкість стану хворого оцінювалася за шкалою BSS (Bronchitis severity score). Лабораторне дослідження включало: загальний аналіз крові, сечі, назоцитологічні, імуноферментні методи. Етіологічний діагноз встановлювався на підставі вірусологічних методів дослідження. При визначенні концентрації інтерферону гамма використовувався набір «Гамма-Інтерферон – ІФА-БЕСТ А-8752» фірми «Вектор-Бест»; лактоферину — набір Нuman Lactoferrin NK 329 Edition 06–16 виробництва компанії Hycult Biotech (USA), нейтрофільних α-дефензінів (1-3) — реактиви Нuman HNP NK317 Edition 08-16 виробництва компанії Hycult Biotech (USA).Результати. У всіх дітей захворювання почалося гостро і проявлялося катаральним і загальнозапальним синдромами. Застосування в терапії препарату, що містить бактерії Bacillus subtilis, сприяло скороченню тривалості симптомів захворювання, підвищенню вмісту лактоферину у ротоглотоковій рідині до періоду реконвалесценції захворювання і поєднанню продукції IFN-g та антимікробних факторів.Висновки. Застосування «Субаліну», що містить бактерії Bacillus subtilis, є патогенетичним методом лікування гострого простого бронхіту у дітей.

Актуальність. Основними етіологічними факторами гострого простого бронхіту у дітей є респіраторнотропні віруси, у зв’язку з чим препарати інтерферону можуть бути використані при лікуванні відповідних респіраторних захворювань. Мета: вивчення клініко-імунологічної ефективності рекомбінантного інтерферону альфа-2b при лікуванні гострого простого бронхіту в дітей. Матеріали та методи. Під наглядом перебувало 49 дітей віком від двох до шести років, хворих на гострий простий бронхіт, 23 дитини отримували препарат Лаферобіон®, що містить рекомбінантний інтерферон ­альфа-2b. При проведенні роботи використовувалися клінічні й лабораторні методи дослідження. Тяжкість стану хворого оцінювалася за Bronchitis severity score (шкалою BSS). Лабораторне дослідження включало загальний аналіз крові, сечі, застосовувались імуноферментні методи. Етіологічний діагноз встановлювався на підставі вірусологічних методів дослідження. При визначенні концентрації інтерферону гамма використовувався набір «Гамма-інтерферон-ІФА-БЕСТ А-8752» фірми «Вектор Бест»; лактоферину — набір Нuman Lactoferrin NK 329 Edition 06-16 виробництва компанії Hycult Biotech (USA), нейтрофільних a-дефензинів 1–3 — реактиви Нuman HNP NK317 Edition 08-16 виробництва компанії Hycult Biotech (USA). Результати. У всіх дітей, хворих на гострий простий бронхіт, захворювання почалося гостро й проявлялося катаральним і загальнозапальним синдромами. Згідно з оцінкою ознак гострого простого бронхіту за шкалою BSS на початку захворювання і в періоді реконвалесценції, застосування в терапії рекомбінантного інтерферону альфа-2b сприяє більш швидкому зменшенню всіх клінічних проявів захворювання й збільшенню вмісту лактоферину в ротоглотковій рідині до періоду реконвалесценції. Висновки. Ректальне призначення рекомбінантного інтерферону альфа-2b Лаферобіон® є етіопатогенетичним методом лікування гострого простого бронхіту в дітей., Background. The main etiological factors of acute simple bronchitis in children are respiratory viruses, and therefore interferon preparations can be used in the treatment of these respiratory diseases. The purpose of the work was to study the clinical and immunological efficacy of recombinant alpha 2b interferon in the treatment of acute simple bronchitis in children. Materials and methods. We have examined 49 children aged 2 to 6 years with acute simple bronchitis; 23 children received Laferobion® containing recombinant alpha 2b interferon. Du­ring the work, clinical and laboratory methods of investigation were used. The severity of the patient’s condition was assessed according to the Bronchitis severity score. The laboratory study included: general blood and urine test, nasocytological, immunoassay methods. The etiological diagnosis was established ­using virological methods of investigation. When determining the concentration of interferon gamma, “Gamma interferon-IFA-BEST A-8752” set manufactured by Vector Best was used; lactoferrin — Нuman Lactoferrin NK 329 Edition 06-16 set manufactured by Hycult Biotech (USA), neutrophil a-defensins 1-3 — Нuman HNP NK317 Edition 08-16 reagents manufactured by Hycult Biotech (USA). Results. In all children with acute simple bronchitis, the disease began acutely and manifested itself with catarrhal and general inflammatory syndromes. According to the evaluation of acute simple bronchitis signs at the onset of the disease and in the period of convalescence ­using Bronchitis severity score, the administration of recombinant alpha 2b interferon promotes a faster reduction of all clinical manifestations of the disease and an increase in the lactoferrin content in the oropharyngeal fluid until the period of convalescence. Conclusions. The rectal administration of recombinant alpha 2b interferon Laferobion® is an etiopathogenetic method for the treatment of acute simple bronchitis in children, Актуальность. Основными этиологическими факторами острого простого бронхита у детей являются респираторнотропные вирусы, в связи с чем препараты интерферона могут быть использованы при лечении соответствующих респираторных заболеваний. Цель: изучение клинико-иммунологической эффективности рекомбинантного интерферона альфа-2b при лечении острого простого бронхита у детей. Материалы и методы. Под наблюдением находилось 49 детей в возрасте от двух до шести лет, больных острым простым бронхитом, 23 ребенка получали препарат Лаферобион®, содержащий рекомбинантный интерферон альфа-2b. При проведении работы использовались клинические и лабораторные методы исследования. Тяжесть состояния больного оценивалась по Bronchitis severity score (шкале BSS). Лабораторное исследование включало общий анализ крови, мочи, применялись иммуноферментные методы. Этиологический диагноз устанавливался на основании вирусологических методов исследования. При определении концентрации интерферона гамма использовался набор «Гамма-интерферон-ИФА-БЕСТ А-8752» фирмы «Вектор Бест»; лактоферрина — набор Нuman Lactoferrin NK 329 Edition 06-16 производства компании Hycult Biotech (USA), нейтрофильных a-дефензинов 1–3 — реактивы Нuman HNP NK317 Edition 08-16 производства компании Hycult Biotech (USA). Результаты. У всех детей с острым простым бронхитом заболевание началось остро и проявлялось катаральным и общевоспалительным синдромами. Согласно оценке признаков острого простого бронхита в начале заболевания и в периоде реконвалесценции по шкале BSS, применение в терапии рекомбинантного интерферона альфа-2b способствует более быстрому уменьшению всех клинических проявлений заболевания и увеличению содержания лактоферрина в ротоглоточной жидкости к периоду реконвалесценции. Выводы. Ректальное назначение рекомбинантного интерферона альфа-2b Лаферобион® является этиопатогенетическим методом лечения острого простого бронхита у детей.

Огляд присвячений проблемі діагностики латентної туберкульозної інфекції (ЛТІ). Наведені дані про існуючі тести для скринінгу на ЛТІ: їх чутливість, специфічність, переваги й недоліки. Більш детально розглянуті можливості застосування квантиферонового тесту для діагностики ЛТІ. Надана інформація щодо методики його проведення, інтерпретації результатів і факторів, що можуть впливати на отримані результати. Розглянуті питання діагностичних можливостей квантиферонового тесту в особливих категорій пацієнтів: дітей, ВІЛ-інфікованих, вагітних тощо. Розглядаються питання хибно-негативних результатів і факторів, що можуть впливати на такі результати., Обзор посвящен проблеме диагностики латентной туберкулезной инфекции (ЛТИ). Приведены данные о существующих тестах скрининга на ЛТИ: их чувствительности, специфичности, преимуществах и недостатках. Более подробно рассмотрены возможности применения квантиферонового теста при диагностике ЛТИ. Представлена информация о методике его проведения, интерпретации результатов и факторах, которые могут влиять на полученные результаты. Рассмотрены вопросы диагностических возможностей квантиферонового теста у особых категорий пациентов: детей, ВИЧ-инфицированных, беременных и пр. Рассматриваются вопросы ложнонегативных результатов и факторов, которые могут влиять на такие результаты., The review deals with the problem of diagnosis of latent tuberculosis infection (LTI). The article presents information about LTI screening tests: their sensitivity, specificity, advantages and disadvantages. The possibilities of using quantiferon test during the diagnosis of LTI are discussed in more detail. The article provides information on the methodology for its implementation, the interpretation of results and factors that may affect the results. The questions about the diagnostic limitations of quantiferon test in special categories of patients: children, HIV-infected persons, pregnant women were discussed. Issues of false negative results and factors that may affect such results are considered.

Type 1 diabetes is characterised by the autoimmune destruction of insulin producing beta-cells in the islets of Langerhans of the pancreas. Immune cells release pro-inflammatory cytokines such as interferon-g (IFN-g), tumour necrosis factor-a (TNF-a) and interleukin- 1b (IL-1b) into the islet microenvironment which activate phosphorylation cascades and gene expression in beta-cells that increase their susceptibility to autoimmune attack and destruction. Protein tyrosine phosphatases (PTPs) regulate phosphorylation based signalling pathways and have previously been shown to negatively regulate IFN-g induced cell death of beta-cells in vitro. We previously showed that during immune infiltration to the islet PTPs, including PTPN1 and PTPN6, are rendered catalytically inactive through oxidation resulting in loss of signal regulation. The overall aim of this thesis is to observe if antioxidant treatment can reduce autoimmune development in the NOD/Lt mouse through reduction of oxidised PTPs and dissect the role of PTPN1 and PTPN6 in the regulation of cytotoxic signalling events in the NIT-1 beta-cell line and isolated NODPI islets in vitro. Chapter 3 studies the effect of the mitochondrial targeted antioxidant mito-TEMPO on insulitis and diabetes development in the NOD/Lt mouse. Delivery of mito-TEMPO through drinking water reduced levels of oxidised PTPs in the pancreas of NOD/Lt mice but had no effect on the development of insulitis, activity or number of CD8+ and CD4+ T- cells in the periphery in NOD/Lt mice or diabetes development in a diabetes transfer model. Chapter 4 describes the regulation of cytokine signalling in NIT-1 cells and isolated NODPI islets by PTPN1. Inhibition of PTPN1 activity reduced IFN-g, TNF-a and IL-1b induced death of NIT-1 cells in vitro. Activation of the IFN-g, TNF-a and IL-1b signalling pathways and downstream transcription of pro-inflammatory gene signatures associated with autoimmune diabetes were also reduced with PTPN1 inactivation. F

Activation of the antigen presentation mechanisms by cancer cells is one of the main pathways used by the immune system for tumor detection and suppression. Induction of the expression of molecules of the Major Histocompatibility complex class II (MHC-II) by the interferon- (IFN) is important for the efficient presentation of tumor antigens. Nevertheless, it has been observed that expression of these molecules is supressed in tissular contexts where the concentration of estradiol (E2) is high. In this work we attempted to explain if the down-regulation exerted by estradiol on the expression of the MHC-II molecules in breast cancer cells was mediated by a silencing effect of the estrogen receptor- (ER) through a possible estrogen receptor binding site (ERBS) in the locus of promoter IV (pIV) of the master regulator of MHC-II expression, the class II transactivator (CIITA). The breast cancer cell line MDA-MB-231 (ER-/ERβ-) and its stable transfectants MC2 (ER+) and VC5 (empty vector) were used as model cell lines. Expression of the MCH-II molecules is controlled by CIITA, and stimulation with IFN activates the transcription of the pIV of CIITA. Stimulation of these cell lines with IFN induced expression of the MCH-II molecules and addition of E2 repressed such expression only in the MC2 cell line, as observed by flow cytometry analysis. Six other breast cancer cell lines were tested, with only the MCF7 cell line showing a significant inhibition. Then we analyzed if the inhibition of the MHC-II expression was due to a down-regulation of CIITA. Protein analysis performed by western blot and mRNA quantification by RT-qPCR both revealed down-regulation of CIITA in the cells exposed to IFN+E2 compared to those treated only with IFN. However, reporter gene analysis did not demonstrate any influence of our candidate ERBS in the inhibition of the activation of CIITA-pIV. ChIP-seq analysis of the VC5 and MC2 cell lines for ER also failed to demonstrate an, Résumé : L’activation des mécanismes de présentation antigénique par les cellules cancéreuses est l’une des voies principales employées par le système immunitaire pour la détection et la suppression des tumeurs. L’induction de l’expression de molécules du complexe majeur d’histocompatibilité de classe II (CMH-II) par l’interféron- (IFN) est importante pour la présentation efficace des antigènes tumoraux. Cependant, il a été observé que l’expression de ces molécules est supprimée dans certains tissus dans lesquels la concentration d’estradiol (E2) est élevée. Dans ce travail, nous avons tenté de déterminer si l’inhibition exercée par l’estrogène (E2) sur l'expression des molécules du CMH-II dans des cellules de cancer du sein est médiée par un effet de silençage du récepteur de l’estrogène- (ER) à travers d’un possible site de liaison de récepteur d'estrogène (ERBS) dans le locus du promoteur IV du régulateur clé de l’expression du CMH-II, CIITA. La lignée cancéreuse mammaire cellulaire de cancer de sein MDA-MB-231 (ER-/ERβ-) et ses transfectants stables MC2 (ER+) et VC5 (vecteur vide) ont été utilisés comme des lignées cellulaires modèles. L'expression des molécules du CMH-II est contrôlée par CIITA, et la stimulation avec l’IFN active la transcription du pIV de CIITA. La stimulation de ces lignées cellulaires avec l’IFN induit l'expression des molécules du CMH-II et l'addition d’E2 réprime de cette expression seulement dans la lignée cellulaire MC2, telle qu'elle est observée par analyse de cytométrie de flux. Six autres lignées de cancer de sein ont été testées et seulement la lignée cellulaire MCF7 montrait une inhibition significative. Ensuite, nous avons analysé si l'inhibition de l'expression du CMH-II était due à une régulation de CIITA. L'analyse des protéines effectuée par Western blot et la quantification de l'ARNm par RT-qPCR quantitative ont révélé une inhibition de CIITA dans les cellules exposées à l’IFN + E2 par rapport à celles traitées seul

Chlamydia pneumoniae has been suggested as a stimulator of the atherosclerotic process. Mice fed a normal diet were infected intranasally with C. pneumoniae and given one intraperitoneal injection of C-14-cholesterol tracer per day for 12 days. Bacteria were demonstrated in the aorta in the early phase of infection and in lungs and liver throughout the study period of 20 days. C-14-cholesterol was not affected in the heart but increased in the blood, liver and aorta on day 4 when the infection was clinically most severe. Furthermore, on day 20 C-14-cholesterol tended to be increased in the aorta. Accordingly, copper-and zinc levels and expressions of the infection biomarkers Cxcl2 and Ifng increased in the liver on day 4 with a tendency of increased of copper, zinc and Ifng on day 20. In mice where bacteria could be cultivated from the lungs, expressions of cholesterol transporters Abca1 and Idol were both increased in the liver on day 4. The increased levels of C-14-cholesterol in blood and aorta together with increased Abca1 and Idol in the liver during C. pneumoniae infection in mice fed a normal diet suggest that this pathogen may have a role in the initiation of the atherosclerotic process.

This thesis has made a significant contribution to future chlamydial research by uncovering the chlamydial pathogenic mechanisms which will potentially help in the development of targeted vaccine against the pathogen. This thesis has made important new contributions to our understanding of Chlamydia pneumoniae specific adaptations to stress responses and has provided new perspectives on the survival of this successful pathogen. This thesis has used two well established microbial stressors and has identified major differences in stress responses between human and animal Chlamydia pneumoniae isolates.

Although photodynamic therapy (PDT) yields very good outcomes in numerous types of superficial solid cancers, some tumors respond suboptimally to PDT. Novel treatment strategies are therefore needed to enhance the efficacy in these therapy-resistant tumors. One of these strategies is to combine PDT with inhibitors of PDT-induced survival pathways. In this respect, the transcription factor nuclear factor ¿B (NF-¿B) has been identified as a potential pharmacological target, albeit inhibition of NF-¿B may concurrently dampen the subsequent anti-tumor immune response required for complete tumor eradication and abscopal effects. In contrast to these postulations, this study demonstrated that siRNA knockdown of NF-¿B in murine breast carcinoma (EMT-6) cells increased survival signaling in these cells and exacerbated the inflammatory response in murine RAW 264.7 macrophages. These results suggest a pro-death and immunosuppressive role of NF-¿B in PDT-treated cells that concurs with a hyperstimulated immune response in innate immune cells.

Els macròfags tenen un paper clau en la inflamació. Durant l’inici del procés inflamatori aquestes cèl·lules s’activen i mostren una potent funció fagocítica i microbicida que pot tenir efectes destructius en els teixits on actua. L’activació dels macròfags implica la inducció de més de quatre-cents gens, i dóna com a resultat més capacitat per eliminar els bacteris i per regular moltes altres cèl·lules a través de l’alliberament de citocines i quimiocines. L’activació excessiva d’aquestes cèl·lules té efectes perjudicials, com ara el xoc sèptic, que pot conduir a la síndrome de disfunció orgànica múltiple i a la mort. En altres situacions la persistència dels resultats de l’activitat proinflamatòria pot contribuir al desenvolupament de processos d’inflamació crònica, com l’artritis reumatoide, la psoriasi i la malaltia inflamatòria de l’intestí. Per evitar aquests efectes indesitjables els macròfags han desenvolupat diversos mecanismes per regular l’excés d’activació, de manera que es condueix a la desactivació dels macròfags i a la resolució de la inflamació.Paraules clau: macròfag, interferó g, inflamació, activació dels macròfags, desactivació dels macròfags., Macrophages play key roles in inflammation. During the onset of the inflammatory process, these phagocytic cells become activated and have destructive effects. Macrophage activation, which involves the induction of more than 400 genes, results in an increased capacity to eliminate bacteria and to regulate many other cells through the release of cytokines and chemokines. However, excessive activation has damaging effects, such as septic shock, which can lead to multiple organ dysfunction syndrome and death. In other situations, persistence of pro-inflammatory activity results in the development of chronic inflammation, such as rheumatoid arthritis, psoriasis and inflammatory bowel disease. To prevent undesirable effects, several mechanisms have evolved to control excess activation, thereby leading to macrophage deactivation and the resolution of inflammation. In this review we will discuss the molecular mechanisms of proliferation, activation and survival of macrophages.Key words: macrophage, interferon gamma, inflammation, macrophageactivation, macrophage deactivation.

PurposeThis study explored the relationships between variations in cytokines genes and depressive symptoms in a sample of patients who were assessed prior to and for six months following breast cancer surgery. Phenotypic differences between Resilient (n = 155) and Subsyndromal (n = 180) depressive symptom classes, as well as variations in cytokine genes were evaluated.MethodPatients were recruited prior to surgery and followed for six months. Growth mixture modeling was used to identify distinct latent classes based on Center for Epidemiological Studies Depression (CES-D) Scale scores. Eighty-two single nucleotide polymorphisms and 35 haplotypes among 15 candidate cytokine genes were evaluated.ResultsPatients in the Subsyndromal class were significantly younger, more likely to be married or partnered, and reported a significantly lower functional status. Variation in three cytokine genes (i.e., interferon gamma receptor 1 (IFNGR1 rs9376268), interleukin 6 (IL6 rs2069840), tumor necrosis factor alpha (TNFA rs1799964)), as well as age and functional status predicted membership in the Subsyndromal versus the Resilient class.ConclusionsA variation in TNFA that was associated with Subsyndromal depressive symptoms in a sample of patients and their family caregivers was confirmed in this sample. Variations in cytokine genes may place these patients at higher risk for the development of Subsyndromal levels of depressive symptoms.

Glioblastoma Multiforme (GBM) is the most common type of brain tumor and it is uniformly fatal. The community standard of treatment for this disease is gross or subtotal resection of the tumor, followed by radiation and temozolomide. At recurrence bevacizumab can be added for increased progression free survival. Many challenges are encountered while trying to devise new drugs to treat GBM, such as the presence of the blood brain barrier which is impermeable to most drugs. Therefore in the past few years attention was turned to immunological means for the treatment of this devastating disease. EGFRvIII targeting has proven a good way to attack glioblastoma cells by using the immune system. Although in still in development, this approach holds the promise as a great first step toward immune-tailored drugs for the treatment of brain cancers.

PurposeThis study explored the relationships between variations in cytokines genes and depressive symptoms in a sample of patients who were assessed prior to and for six months following breast cancer surgery. Phenotypic differences between Resilient (n = 155) and Subsyndromal (n = 180) depressive symptom classes, as well as variations in cytokine genes were evaluated.MethodPatients were recruited prior to surgery and followed for six months. Growth mixture modeling was used to identify distinct latent classes based on Center for Epidemiological Studies Depression (CES-D) Scale scores. Eighty-two single nucleotide polymorphisms and 35 haplotypes among 15 candidate cytokine genes were evaluated.ResultsPatients in the Subsyndromal class were significantly younger, more likely to be married or partnered, and reported a significantly lower functional status. Variation in three cytokine genes (i.e., interferon gamma receptor 1 (IFNGR1 rs9376268), interleukin 6 (IL6 rs2069840), tumor necrosis factor alpha (TNFA rs1799964)), as well as age and functional status predicted membership in the Subsyndromal versus the Resilient class.ConclusionsA variation in TNFA that was associated with Subsyndromal depressive symptoms in a sample of patients and their family caregivers was confirmed in this sample. Variations in cytokine genes may place these patients at higher risk for the development of Subsyndromal levels of depressive symptoms.

Atopic dermatitis is a chronic, pruritic inflammatory dermatosis that affects up to 25% of children and 2% to 3% of adults. This guideline addresses important clinical questions that arise in atopic dermatitis management and care, providing recommendations based on the available evidence. In this third of 4 sections, treatment of atopic dermatitis with phototherapy and systemic immunomodulators, antimicrobials, and antihistamines is reviewed, including indications for use and the risk-benefit profile of each treatment option.

Glioblastoma Multiforme (GBM) is the most common type of brain tumor and it is uniformly fatal. The community standard of treatment for this disease is gross or subtotal resection of the tumor, followed by radiation and temozolomide. At recurrence bevacizumab can be added for increased progression free survival. Many challenges are encountered while trying to devise new drugs to treat GBM, such as the presence of the blood brain barrier which is impermeable to most drugs. Therefore in the past few years attention was turned to immunological means for the treatment of this devastating disease. EGFRvIII targeting has proven a good way to attack glioblastoma cells by using the immune system. Although in still in development, this approach holds the promise as a great first step toward immune-tailored drugs for the treatment of brain cancers.

Background: Smoking may affect cytokine levels, including IFN-γ, IL-6, and IL-10. Objectives: This study aimed to analyze the difference in levels of cytokines (IFN-γ, IL-6 and IL-10) based on the degree of smoking, and how the daily intake of nutrient influence the relationship of smoking with cytokines level. Method: This was a cross sectional study. Study subject consisted of 23 adult, healthy, smoker men. Ethical clearance was issued by Commission of Ethics of Medical and Public Health Research, Faculty of Public Health Diponegoro University. Smoking variabel was obtained from questions. Level of cytokines examined consists of IFN-γ, IL-6 and IL-10 was measured using ELISA (pg/dl). Nutritional intake was measured by method of 2x24-hour recall. The difference of level of IFN-γ, IL-6, IL-10 and daily nutrient intake based on smoking degree was analyzed with Mann Whitney (α 0,05). Result: The result showed no difference in level of IFN-γ, IL-6 and IL-10 found between group of light and heavy smokers. In the group of heavy smokers, daily nutrient intake was higher compare to light smokers. However, the significant difference only found in vitamin C (p = 0.042). Conclusion: Immune response, as measured by level of interferon gamma, interleukin 6 and interleukin 10, do not differ betwen light and heavy smokers due to micronutrient intake

Objective: Interleukin-17A (IL-17A), an inflammatory cytokine, has been implicated in atherosclerosis, in which inflammatory cells within atherosclerotic plaques express IL-17A. However, its role in the development of atheroscelrosis remains to be controversial. Methods and Results: To directly examine the role of IL-17A in atherosclerosis, we generated apolipoprotein E (ApoE)/IL-17A double-deficient (ApoE^[-/-]IL-17A^[-/-]) mice. Mice were fed with high-fat diet (HFD) for either 8 or 16 weeks, both starting at ages of 6-8 weeks. We found that splenic CD4+ T cells produced high amounts of IL-17A in ApoE^[-/-] mice after HFD feeding for 8 weeks. Atherosclerosis was significantly accelerated in HFD-fed ApoE^[-/-]IL-17A^[-/-] mice compared with ApoE^[-/-] mice. Splenic CD4+ T-cells of ApoE^[-/-]IL-17A^[-/-] mice after HFD feeding for 8 weeks, but not for 16 weeks, exhibited increased IFN-γ and decreased IL-5 production. Importantly, formation of vulnerable plaque as evidenced by reduced numbers of vascular smooth muscle cells and reduced type I collagen deposition in the plaque was detected in ApoE^[-/-]IL-17A^[-/-] mice after HFD feeding for 8 weeks. Conclusions: These results suggest that IL-17A regulates the early phase of atherosclerosis development after HFD feeding and plaque stability, at least partly if not all by modulating IFN-γ and IL-5 production from CD4+ T-cells.

Introduction: Papular urticaria caused by the bites of fleas traditionally has been defined as a chronic allergic disease. However, currently no clear relationship has been described between this pathology and common allergic diseases.Objective: The expression of IL-10, IL-4 and IFN-γ as markers of effector T cell responses was examined in skin lesions of patients with papular urticaria by flea bite.Materials and methods: Fourteen skin lesion biopsies were sampled from children with a clinical diagnosis of papular urticaria by flea bite and were compared with 5 healthy skin biopsies of children with no history of the disease. All children were under 12 years old. RNA was extracted with trizol and the expression levels of cytokines were analyzed by real time PCR technique.Results: A wide range in the expression levels of IFN-γ and IL-10 was noted as well as constant low values of IL-4. Three distinct profiles were observed, but which did not correspond to a recognizable pattern among the patients. The samples obtained from healthy tissues showed no expression of any of the cytokines.Conclusions: This is the first characterization of cytokines that mediate the immune response at the site of the skin lesion in children with papular urticaria by flea bite. The data indicated that the local response was mixed and that a single phenotype is not predominant among the patients., Introducción. La urticaria papular por picadura de pulga se conoce como una enfermedad alérgica. Sin embargo, las investigaciones no muestran una clara relación con las enfermedades alérgicas.Objetivo. Estudiar la expresión de IL-10, IL-4 e IFN-γ, como marcadores de la respuesta efectora de células T en lesiones de piel de pacientes con urticaria papular por picadura de pulga.Materiales y métodos. Se incluyeron 14 biopsias de lesiones de piel de niños con diagnóstico de urticaria papular por picadura de pulga y 5 biopsias de piel sana obtenidas de niños sometidos a cirugía por enfermedades no inflamatorias. Todas las muestras se obtuvieron de niños menores de 12 años. Se extrajo ARN con trizol y se cuantificaron los niveles de expresión de las citocinas con la técnica de reacción en cadena de la polimerasa en tiempo real.Resultados. En los pacientes con urticaria papular por picadura de pulga, se encontró amplia diversidad en los niveles de expresión de IFN-γ e IL-10, y valores bajos constantes para IL-4. Se observaron tres perfiles que no corresponden a un patrón común en los pacientes. Las muestras obtenidas de tejidos sanos no presentaron expresión de las citocinas.Conclusiones. Los datos corresponden a la primera descripción de citocinas que median la respuesta inmunitaria en el sitio de la lesión cutánea en niños con con urticaria papular por picadura de pulga, lo cual indica que la respuesta local es mixta ya que no se encuentra predominio de un fenotipo específico en ninguno de los pacientes.

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Evidence suggests that the neurotropic parasite Toxoplasma gondii may play a role in the development of cognitive impairments. My hypothesis was that congenital exposure to T. gondii would lead to detectable age and sex related differences in behavior and neurotransmitter levels in mice. The neurotransmitter dopamine and commonly used anti-schizophrenic agents were evaluated against T. gondii in human fibroblast cells. Dopamine caused a significant increase in tachyzoite numbers at 250 nM but not 100 nM and the drugs valproic acid, fluphenazine, thioridazine and trifluoperazine inhibited T. gondii development. The effects T. gondii infection had on behavior were examined using a congenital mouse model. Previous work demonstrated maternal immune stimulation (MIS) with interferon gamma (INF-g) resulted in decreased fetal mortality from congenital T. gondii infections; therefore I examined the effects of INF- g treatment of mothers to determine if protection from the behavioral effects of T. gondii occurred in their offspring. No differences in concentrations of neurotransmitters in the brains of congenitally infected mice were observed. I found that mice infected with T. gondii developed adult onset behavior impairments with decreased rate of learning, increased activity and decreased memory, indicating cognitive impairment for male mice and not female mice. My findings support the evidence T. gondii is a factor in the development of cognitive impairments. My results for T. gondii exposed male mice are consistent with the convention that males have more cognitive impairments in the prodromal stage of schizophrenia. MIS with IFN-g had a minimal effect on behavior post sexual maturity but had a greater effect on pre sexual maturity female mice which exhibited difficulties with spatial memory, coordination and the ability to process stimuli. The results indicate the behavior alterations from IFN- g are transient. When MIS is given prior to congenital infection with T. go

Das Ubiquitin-Proteasom-System ist an der Degradation cytosolischer Proteine und der Generierung von Antigenen beteiligt, die über MHC Klasse I Moleküle CD8+ T Zellen präsentiert werden. Die Antigenprozessierung wird durch Typ I und II Interferone beeinflusst, welche die Formierung des Immunoproteasoms und des Proteasomen-Aktivators PA28 induzieren und so die katalytische Aktivität des Ubiquitin-Proteasom-Systems qualitativ verändern. In der vorliegenden Arbeit wurde im Zellkulturmodell unter dem Einfluss von IFN gamma die zunehmende Inkorporation der Immunountereinheiten in de novo assemblierende 20S Proteasomen und die daraus resultierende Veränderung der proteolytische Aktivität untersucht. Die Inkorporation der Immunountereinheiten wurde mittels 2D Gelelektrophorese und Western Blots von 20S Proteasomen untersucht, die nach unterschiedlicher Stimulationsdauer mit IFN gamma aus HeLa Zellen isoliert wurden. Es konnte gezeigt werden, dass innerhalb der ersten 24h einer IFN gamma Stimulation die strukturelle Heterogenität des zellulären Proteasomenpools zunimmt, indem sowohl intermediäre als auch Immunoproteasomen assemblieren. In der Nativ-PAGE von Lysaten IFN gamma stimulierter Zellen wurde eine Zunahme des 20S Proteasoms als freier Komplex und in Assoziation mit PA28 beobachtet, während die Menge des zum ATP-abhängigen Abbau von polyubiquitinierten Proteinen notwendigen 26S Proteasoms unverändert blieb. Die Stimulation mit IFN gamma hatte eine Steigerung der gesamtproteasomalen Aktivität zur Folge, die unter Inhibition der Interaktion zwischen 20S Proteasom und PA28 verzögert erfolgte. Die katalytischen Eigenschaften isolierter Proteasomen wurden anhand der Generierung eines immunrelevanten Hepatitis C CTL Epitops des viralen Core Proteins in vitro untersucht. Im Verlauf der IFN gamma Stimulation de novo assemblierte Proteasomen wiesen jeweils unterschiedliche Präferenzen für die Generierung des untersuchten CTL Epitops auf. Eine weitere, proteasomen-spezifische, The ubiquitin proteasome system is responsible for the degradation of cytosolic proteins and the processing of MHC class I restricted antigens. The generation of these antigens is influenced by type I and II interferons which induce the expression of immunoproteasomes and the proteasome activator PA28; and thereby impact the quality of peptides processed by the proteasome system. The adoption of the proteasome system to a proinflammatory environment has been investigated in a cell culture model by isolating proteasomes after different stages of IFN gamma stimulation. The composition of isolated proteasomes was analysed by 2D PAGE and western blot approach. The presented work shows that within 24h of IFN gamma stimulation an increasing heterogeneity of the cellular proteasome pool is observed, resulting from the assembly of both intermediate type proteasomes and immunoproteasomes at the early stage of IFN gamma stimulation. It could be shown by native PAGE of HeLa cell lysates that IFN gamma induces increasing amounts of 20S proteasomes and PA28 associated proteasomes without decreasing the amount of 26S proteasomes that are necessary for the ATP dependent degradation of ubiquitinated proteins; and resulting in an enhanced total proteasomal activity in vitro. This increase in activity was delayed when the interaction of 20S proteasomes and PA28 was inhibited. A comparative analysis of the ability of isolated 20S proteasomes to generate a known hepatitis C virus derived CTL epitope in vitro proved that during early IFN gamma stimulation de novo assembled proteasomes exhibited a structure specific preference to generate the HCV CTL epitope either alone or in combination with the proteasome activator PA28. Within the first 12h of IFN gamma stimulation the epitope was generated with higher efficiency by 20S proteasomes in association with PA28, whereas after 24h the impact of PA28 on the proteasome pool was less pronounced. The presented work shows that IFN gamma induces

Das Ubiquitin-Proteasom-System ist an der Degradation cytosolischer Proteine und der Generierung von Antigenen beteiligt, die über MHC Klasse I Moleküle CD8+ T Zellen präsentiert werden. Die Antigenprozessierung wird durch Typ I und II Interferone beeinflusst, welche die Formierung des Immunoproteasoms und des Proteasomen-Aktivators PA28 induzieren und so die katalytische Aktivität des Ubiquitin-Proteasom-Systems qualitativ verändern. In der vorliegenden Arbeit wurde im Zellkulturmodell unter dem Einfluss von IFN gamma die zunehmende Inkorporation der Immunountereinheiten in de novo assemblierende 20S Proteasomen und die daraus resultierende Veränderung der proteolytische Aktivität untersucht. Die Inkorporation der Immunountereinheiten wurde mittels 2D Gelelektrophorese und Western Blots von 20S Proteasomen untersucht, die nach unterschiedlicher Stimulationsdauer mit IFN gamma aus HeLa Zellen isoliert wurden. Es konnte gezeigt werden, dass innerhalb der ersten 24h einer IFN gamma Stimulation die strukturelle Heterogenität des zellulären Proteasomenpools zunimmt, indem sowohl intermediäre als auch Immunoproteasomen assemblieren. In der Nativ-PAGE von Lysaten IFN gamma stimulierter Zellen wurde eine Zunahme des 20S Proteasoms als freier Komplex und in Assoziation mit PA28 beobachtet, während die Menge des zum ATP-abhängigen Abbau von polyubiquitinierten Proteinen notwendigen 26S Proteasoms unverändert blieb. Die Stimulation mit IFN gamma hatte eine Steigerung der gesamtproteasomalen Aktivität zur Folge, die unter Inhibition der Interaktion zwischen 20S Proteasom und PA28 verzögert erfolgte. Die katalytischen Eigenschaften isolierter Proteasomen wurden anhand der Generierung eines immunrelevanten Hepatitis C CTL Epitops des viralen Core Proteins in vitro untersucht. Im Verlauf der IFN gamma Stimulation de novo assemblierte Proteasomen wiesen jeweils unterschiedliche Präferenzen für die Generierung des untersuchten CTL Epitops auf. Eine weitere, proteasomen-spezifische, The ubiquitin proteasome system is responsible for the degradation of cytosolic proteins and the processing of MHC class I restricted antigens. The generation of these antigens is influenced by type I and II interferons which induce the expression of immunoproteasomes and the proteasome activator PA28; and thereby impact the quality of peptides processed by the proteasome system. The adoption of the proteasome system to a proinflammatory environment has been investigated in a cell culture model by isolating proteasomes after different stages of IFN gamma stimulation. The composition of isolated proteasomes was analysed by 2D PAGE and western blot approach. The presented work shows that within 24h of IFN gamma stimulation an increasing heterogeneity of the cellular proteasome pool is observed, resulting from the assembly of both intermediate type proteasomes and immunoproteasomes at the early stage of IFN gamma stimulation. It could be shown by native PAGE of HeLa cell lysates that IFN gamma induces increasing amounts of 20S proteasomes and PA28 associated proteasomes without decreasing the amount of 26S proteasomes that are necessary for the ATP dependent degradation of ubiquitinated proteins; and resulting in an enhanced total proteasomal activity in vitro. This increase in activity was delayed when the interaction of 20S proteasomes and PA28 was inhibited. A comparative analysis of the ability of isolated 20S proteasomes to generate a known hepatitis C virus derived CTL epitope in vitro proved that during early IFN gamma stimulation de novo assembled proteasomes exhibited a structure specific preference to generate the HCV CTL epitope either alone or in combination with the proteasome activator PA28. Within the first 12h of IFN gamma stimulation the epitope was generated with higher efficiency by 20S proteasomes in association with PA28, whereas after 24h the impact of PA28 on the proteasome pool was less pronounced. The presented work shows that IFN gamma induces

IIn the first study, we investigated the functional implications of prolonged TLR signalling on IFN-γ mediated killing of mycobacteria by murine macrophages in vitro. TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither TNF nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ In the second study, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response. Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The work presented in this thesis provide further insight into the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to susceptibility of the host to infection.