The aim of this research was to examine characteristics and predictors of auto stimulatory behavior in children with autism spectrum disorders (ASD), as well as to determine the presence of auto stimulatory behavior exhibited by children with ASD. The sample included 43 participants diagnosed with ASD. The instrument used was Behavior Problems Inventory (BPI-01, Rojahn et al., 2002), subscale for stereotyped behavior. Our results show that children with ASD from our sample have lower rates of auto stimulatory behavior, with the most common ones being repetitive body movements, arm shaking, bouncing around, running and pacing. The school type has not proven to be a predictor of auto stimulatory behaviors. However, male participants and those with low functioning ASD exhibit more auto stimulatory behaviors than females and those with high functioning ASD. The presence of auto stimulatory behaviors persists through age. Auto-stimulatory behaviors exhibited by children with ASD can be reduced or eliminated, however it is important to use evidence-based interventions with proven benefits for the child. Since stereotypy is usually automatically maintained and therefore is one of the behaviors that is often difficult to reduce, it is recommended that parents are taught on how to implement interventions in their home environment.

Background Adoptive T cell therapy as a treatment for solid tumours is gaining increasing interest. Cancer neoantigens as targets for such therapy is also gaining recognition. Personalised tumour trained lymphocytes (pTTL) is a novel autologous T cell therapy targeting patient-specific neoantigens. A phase I/ II First in Human (FIH) clinical trial of pTTL in Stage IV colorectal cancer (CRC) patients will be initiated in the near future. Methods pTTL is produced through in vitro expansion of T cells derived from regional lymph nodes (RLNs). The T cells derived from RLNs, nodes in anatomical proximity of the tumour, contain a pool of naive and antigen-experienced T cells enriched for tumour-antigen specificity. This enriched population is stimulated during pTTL production with an array of neoantigen epitopes individually designed using PIOR ® , an in house-developed software for neoantigen detec-tion and selection. Selected neoantigens are linked to paramag-netic particles using EpiTCer ® technology. The resulting EpiTCer ® particles are used to stimulate the RLN T cells via phagocytosis and presentation of the neoantigen epitopes by antigen-presenting cells. This process is HLA-independent. Each pTTL product is unique due to the personalised nature of cellular and molecular players (cancer characteristics, immune cell properties and neoantigens are specific to one single individual).

As no data to our knowledge exist, the aim of the study was to describe the national prevalence and characteristics of Danish children and adolescents with severely impaired lung function.

The aim of this research was to examine the frequency of different barriers faced by special education teachers in working with children with autism spectrum disorders (ASD). The sample included 53 participants of both genders and of different ages who were diagnosed with ASD. The instrument used to assess the presence of barriers in teaching refers to the Assessment of Barriers in Teaching (VB-Mapp Barriers Assessment -Sundberg, 2008). In our sample, it was found that all respondents have a pronounced presence of teaching barriers, while the most frequent barriers are prompt dependence, generalization difficulties, socialization problems, escape or avoidance of given tasks and the absence of the tact ability. Having in mind the obtained results, it is highlighted as a necessity to develop an approach that would lead to elimination of the mentioned barriers, as well as to develop a treatment plan to address those teaching barriers in working with children with PSA and consequently enable a higher degree of adoption of new knowledge and skills by children with ASD.

Introduction. Visual-motor integration (VMI) is defined as the degree to which visual perception (VP) and finger-hand movements are well coordinated. The VMI consists of two components: VP and motor coordination (MC). The main goal of our research was to determine whether there are differences in age and gender categories in VMI, VP and MC scores, as well as whether there is a correlation between VMI and school success of younger school-aged children. Methods. Out of 103 student respondents, 52 were female (50.5%), aged 6 to 11 years (8.05 ± 1.44 years), divided into two groups according to age: 6-8 years (first, second and third grade) and 9-11 years (fourth to fifth grade). Data on the level of VMI were obtained by applying the following tests: Beery-Buktenica Developmental Test of VMI, VP test and MC test. Results. In the older age group of subjects, a significant difference was observed in the mean values of the score on the VMI (12.67 ± 1.92), VP (23.69 ± 3.21) and MC (24.34 ± 3.23) tests comparing to the younger group of subjects (9.98 ± 2.12; 20.80 ± 3.2; 19.65 ± 3.82) (p < 0.001), while the difference in the mean values of scores in relation to gender was not observed. A significant, positive and strong correlation was observed between the scores on the VMI, VP and MC test with the success of second to fifth grade students (p < 0.050). Conclusion. Given such a strong correlation between VMI and the success of younger students, we conclude that it is important that VMI disabilities are identified in time, so that these students can be referred for further assessment and receive the necessary support.

The urban microclimate is gradually changing due to climate change, extreme weather conditions, urbanization, and the heat island effect. In such an altered environment, outdoor thermal comfort can have a strong impact on public health and quality of life in urban areas. In this study, three main urban areas in Serbia were selected: Belgrade (Central Serbia), Novi Sad (Northern Serbia), and Niš (Southern Serbia). The focus was on the temporal assessment of OTC, using the UTCI over a period of 20 years (1999–2018) during different seasons. The main aim is the general estimation of the OTC of Belgrade, Novi Sad, and Niš, in order to gain better insight into the bioclimatic condition, current trends and anomalies that have occurred. The analysis was conducted based on an hourly (7 h, 14 h, and 21 h CET) and “day by day” meteorological data set. Findings show the presence of a growing trend in seasonal UTCI anomalies, especially during summer and spring. In addition, there is a notable increase in the number of days above the defined UTCI thresholds for each season. Average annual UTCIs values also show a positive, rising trend, ranging from 0.50 °C to 1.33 °C. The most significant deviations from the average UTCI values, both seasonal and annual, were recorded in 2000, 2007, 2012, 2015, 2017, and 2018.

Introduction. Higher level of knowledge and frequent contacts with peers with disabilities can influence the emergence of more positive attitudes of students towards peers with disabilities. In regard to that, our aim was to test the importance of knowledge, contact frequency and other possible factors influencing attitudes of students toward disabled peers. Methods. The study included 140 students of 4th and 5th grade of primary schools. The research was conducted in the period from December 2020 to March 2021 in two primary schools. The Chedoke McMaster scale was used to examine students' attitudes toward peers with disabilities, while Contact with Disabled Persons Scale and the Children's Knowledge about Handicapped Persons Scale were used to assess frequency of contact and knowledge about disabilities. Results. Girls showed a significantly higher level (25.21±6.21) of frequency of contacts with students with disabilities compared to boys (19.66±7.30) (p=0.043) and higher level of knowledge (27.88±5.88) about disabilities compared to boys (25.50±4.69) (p=0.009). Respondents who attended school together with children with disabilities (31.07 ± 8.41) showed a significantly higher level of frequency of contacts with students with disabilities compared to respondents who did not attend school with peers with disabilities (13.72±6.32) (p=0.001). Conclusion. Higher level of knowledge and frequent contacts with peers with disabilities does not have influence on the emergence of more positive attitudes of students towards peers with disabilities.

Significance Dicer is a ribonuclease III enzyme in biosynthesis of miRNAs, regulators of gene expression involved in macrophage differentiation. We found a specific truncation of Dicer in monocytic cells resulting from apparently constitutive cleavage by a serine protease. Inhibition of this proteolytic truncation, which occurred during macrophage differentiation in presence of TLR ligands or prostaglandin E2, up-regulates full-length Dicer and promotes miR biosynthesis. Regulation of transcription of pri-miRNA is one mode to regulate biosynthesis of mature miRNA. Inhibition of constitutive proteolysis of Dicer, as described here, provides a second layer of regulation, at the level of miRNA processing. Our data provide insights to Dicer and miRNAs in macrophage polarization/differentiation, a key process in the innate immune response. Dicer is a ribonuclease III enzyme in biosynthesis of micro-RNAs (miRNAs). Here we describe a regulation of Dicer expression in monocytic cells, based on proteolysis. In undifferentiated Mono Mac 6 (MM6) cells, full-length Dicer was undetectable; only an ∼50-kDa fragment appeared in Western blots. However, when MM6 cells were treated with zymosan or LPS during differentiation with TGF-β and 1,25diOHvitD3, full-length Dicer became abundant together with varying amounts of ∼170- and ∼50-kDa Dicer fragments. Mass spectrometry identified the Dicer fragments and showed cleavage about 450 residues upstream from the C terminus. Also, PGE2 (prostaglandin E2) added to differentiating MM6 cells up-regulated full-length Dicer, through EP2/EP4 and cAMP. The TLR stimuli strongly induced miR-146a-5p, while PGE2 increased miR-99a-5p and miR-125a-5p, both implicated in down-regulation of TNFα. The Ser protease inhibitor AEBSF (4-[2-aminoethyl] benzene sulfonyl fluoride) up-regulated full-length Dicer, both in MM6 cells and in primary human blood monocytes, indicating a specific proteolytic degradation. However, AEBSF alone did not lead to a general increase in miR expression, indicating that additional mechanisms are required to increase miRNA biosynthesis. Finally, differentiation of monocytes to macrophages with M-CSF or GM-CSF strongly up-regulated full-length Dicer. Our results suggest that differentiation regimens, both in the MM6 cell line and of peripheral blood monocytes, inhibit an apparently constitutive Dicer proteolysis, allowing for increased formation of miRNAs.

ABSTRACT Introduction: In recent years, immunotherapy for the treatment of solid cancer has emerged as a promising therapeutic alternative. Adoptive cell therapy (ACT), especially T cell-based, has been found to cause tumor regression and even cure in a percentage of treated patients. Checkpoint inhibitors further underscore the potential of the T cell compartment in the treatment of cancer. Not all patients respond to these treatments; however, many challenges remain. Areas covered: This review covers the challenges and progress in tumor antigen target identification and selection, and cell product manufacturing for T cell ACT. Tumor immune escape mechanisms and strategies to overcome those in the context of T cell ACT are also discussed. Expert opinion: The immunotherapy toolbox is rapidly expanding and improving, and the future promises further breakthroughs in the T cell ACT field. The heterogeneity of the tumor microenvironment and the multiplicity of tumor immune escape mechanisms pose formidable challenges to successful T cell immunotherapy in solid tumors, however. Individualized approaches and strategies combining treatments targeting different immunotherapeutic aspects will be needed in order to expand the applicability and improve the response rates in future.

Chronic inflammation increases the risk of lung cancer. Macrophages (MO) are important players in inflammation, with regulatory and executive functions. Eicosanoids and exosomes can be both triggers and mediators of these functions. Cysteinyl leukotrienes (CysLTs) are the most potent mediators of broncho-constriction in the lungs, a function exerted via CysLT1 receptor. Their function in asthma is well described, but little is known about CysLTs and lung cancer. In the first study we investigated how the interaction between pulmonary epithelium and leukocytes affects CysLTs formation. Monocytic cells and eosinophils formed LTC4, which was exported and promptly converted to LTD4 by pulmonary epithelial cells in a transcellular manner. The lung cancer cell line A549 expressing γ-glutamyl transpeptidase 1 (GGT-1) showed a high activity. Exosomes released by A549 cells also contained GGT-1 and efficiently converted LTC4 to LTD4. On the other hand, healthy bronchial epithelial cells (PBEC) expressing GGT-5 formed LTD4 12 times more slowly. The results highlight an active role for epithelial cells and their exosomes in biosynthesis of LTD4, which may be of particular relevance in the lung, given that LTD4 is the most potent agonist of CysLT1. MOs can be differentiated from blood monocytes with GM-CSF and M-CSF, resulting in cells primed toward the inflammatory M1and resolving M2-states. A comprehensive analysis of eicosanoid formation in these two in vitro models is missing and our second study focused on this gap. By LC-MS analysis, we observed that both MO phenotypes released pro-resolving lipid mediators (PGE metabolite, LXA4) in resting conditions. When the same cells were incubated (30 min) with bacterial stimuli, there was a shift to pro-inflammatory eicosanoids: M-CSF MOs produced high amounts of LTC4, relevant for M2 functions in asthma. GM-CSF cells expressed the highest levels of cPLA2, 5-LO and FLAP; and in ionophore incubations these cells also produced the highest levels of 5-HETE. However, MCSF MO formed more products apparently due to a better response to bacterial stimuli, demonstrated by enhanced mobilization and activation of cPLA2 and 5-LO. In conclusion, GM-CSF and M-CSF can regulate specific pathways in MOs, and it appears that eicosanoid biosynthesis primarily reflect the cellular response and activation mechanisms, rather than the protein expression profile. In colon cancer a pro-tumorigenic effect of LTD4 but not LTC4 has been demonstrated. A pro-tumorigenic effect has been shown also for exosomes. To extend the findings of our first study, we used pleura exudates from lung cancer patients to isolate primary cancer cells and exosomes. Both cells and exosomes metabolized LTC4 to LTD4, and we also found that exosomes stimulated CysLTs formation in the cancer cells. Cancer cells from all patients expressed CysLT1, and exosomes promoted their migration and survival in a CysLT1 dependent manner, as demonstrated by the inhibition by montelukast (MK) treatment, a CysLT1 antagonist used to treat asthma. In cancer, interactions between the transformed cancer cells and other recruited cell types in the tumor are important. Tumor associated macrophages (TAMs) provide cancer cells with a suitable low-grade inflammation milieu including growth promoting factors. Taken together, the results in this thesis suggest a novel pro-tumorigenic mechanism based on this theme, driven by the exosomes/CysLT1 cascade: TAMs provide LTC4 that lung cancer cells and their exosomes convert to LTD4. Via CysLT1 receptor this promotes survival and migration of the cancer cells. A protective effect in lung cancer has been previously described for MK and our results suggest a possible mechanism for this, driven by the exosomes/ CysLT1 cascade, further encouraging the use of this drug in lung cancer treatment. LIST OF SCIENTIFIC PAPERS I. Lukic, A., Ji, J., Idborg, H., Samuelsson, B., Palmberg, L., Gabrielsson, S., & Rådmark, O. (2016). Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4. Journal of lipid research, 57(9), 1659-1669 II. Lukic, A., Larssen, P., Fauland, A., Samuelsson, B., Wheelock, C. E., Gabrielsson, S., & Radmark, O. (2017). GM-CSF–and M-CSF–primed macrophages present similar resolving but distinct inflammatory lipid mediator signatures. The FASEB Journal, 31(10), 4370-4381. III. Lukic, A., Wahlund, C., Gomez, C., Brodin, D., Samuelsson, B., Wheelock, C. E., Gabrielsson, S., & Radmark, O. Exosomes and malignant cells from lung cancer pleura exudates form LTD4, promoting cell migration and survival in a CysLT1 dependent mechanism. Manuscript Publications not included in this thesis: I. Torregrosa Paredes, P., Esser, J., Admyre, C., Nord, M., Rahman, Q. K., Lukic, A., ... & Scheynius, A. (2012). Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma. Allergy, 67(7), 911-919. II. Basavarajappa, D., Wan, M., Lukic, A., Steinhilber, D., Samuelsson, B., & Rådmark, O. (2014). Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis. Proceedings of the National Academy of Sciences, 111(31), 11371-11376. III. Martinez-Bravo, M. J., Wahlund, C. J., Qazi, K. R., Moulder, R., Lukic, A., Rådmark, O., ... & Gabrielsson, S. (2017). Pulmonary sarcoidosis is associated with exosomal vitamin D–binding protein and inflammatory molecules. Journal of Allergy and Clinical Immunology, 139(4), 1186-1194. LIST OF ABBREVIATIONS AA AERD BAL BEC BLT CD COPD COX cPLA2 CYP CysLT DHA DiHET DiHOME EDP EET EGFR EMT EP EPA EpOME ESCRT FLAP fMLP GGT GM-CSF Arachidonic acid Aspiring exacerbated respiratory disease Bronco-alveolar lavage Bronchial epithelial cells Leukotriene B4 receptor Cluster of differentiation Chronic obstructive pulmonary disease Cyclooxygenase Cytosolic phospholipase A2 Cytochrome P450 Cysteinyl leukotriene Docosahexaenois acid Dihydroxyeicosatrienoic acids Dihydroxyoctadecenoic acid Epoxydocosapentaenoic acid Epoxyeicosatrienoic acid Epithelial growth factor receptor Epithelial to mesothelial transition Prostaglandin E2 receptor Epoxydocosapentaenoic acid Epoxyoctadecenoic acid Endosomal complex required for transport Five lipoxygenase activating protein N-formylmethionyl-leucyl-phenylalanine Gamma-glutamyl transpeptidase Granulocyte macrophage-colony stimulating factor M-CSF GSH GTP HDoHE HETE HODE HpETE HPLC IL IFN-γ LA LC-MS LO LPS Macrophage-colony stimulating factor Glutathione Guanosine Triphosphate Hydroxydocosahexaenoic acid Hydroxyeicosatetraenoic acid Hydroxyoctadecadienoic acid Hydroperoxy eicosatetraenoic acid High-performance liquid chromatography Interleukin Interferon γ Linoleic acid Liquid chromatography-mass spectrometry Lipoxygenase Lipopolysaccharide LT LTC4s LTB4h LX MDSC MHC miRNA MK MM6 mPGES MSC MVB NSCLC PBEC PBMC Leukotriene LTC4 synthase LTB4 hydrolase Lipoxin Myeloid derived suppressor cells Major histocompatibility complex Micro RNA Montelukast Mono Mac 6 Microsomal prostaglandin E2 synthase Mesenchymal stem cells Multivesicular bodies Non-small cell lung cancer Primary BEC Peripheral blood mononuclear cells PE PG PGN PMN PTGIS PUFA RvE/RvD TLR SBC sEH SPM TAM Th TME TNFα Treg TXAS VEGF Pleural exudate Prostaglandin Peptidoglycan Polymorphonuclear neutrophils PGI2 synthase Polyunsaturated fatty acids Resolvins E/D series Toll like receptor Serine Borate Complex soluble Epoxide Hydrolase Specialized proresolving mediators Tumor associated macrophages T helper cell Tumor micro-environment Tumor necrosis factor alpha Regulatory T cell Thromboxane A synthase 1 Vascular endothelial growth factor

M1 and M2 activated macrophages (Mϕs) have different roles in inflammation. Because pathogens may first encounter resting cells, we investigated lipid mediator profiles prior to full activation. Human monocytes were differentiated with granulocyte Mϕ colony‐stimulating factor (GM‐CSF) or Mϕ colony‐stimulating factor (M‐CSF), which are known to prime toward M1 or M2 phenotypes, respectively. Lipid mediators released during resting conditions and produced in response to bacterial stimuli (LPS/N‐formylmethionyl‐leucyl‐phenylalanine or peptidoglycan) were quantified by liquid chromatography‐mass spectrometry. In resting conditions, both Mϕ phenotypes released primarily proresolving lipid mediators (prostaglandin E2 metabolite, lipoxin A4, and 18‐hydroxyeicosapentaenoic acid). A striking shift toward proinflammatory eicosanoids was observed when the same cells were exposed (30 min) to bacterial stimuli: M‐CSF Mϕs produced considerably more 5‐lipoxygenase products, particularly leukotriene C4, potentially linked to M2 functions in asthma. Prostaglandins were formed by both Mϕ types. In the M‐CSF cells, there was also an enhanced release of arachidonic acid and activation of cytosolic phospholipase A2. However, GM‐CSF cells expressed higher levels of 5‐lipoxygenase and 5‐lipoxygenase–activating protein, and in ionophore incubations these cells also produced the highest levels of 5‐hydroxyeicosatetraenoic acid. In summary, GM‐CSF and M‐CSF Mϕs displayed similar proresolving lipid mediator formation in resting conditions but shifted toward different proinflammatory eicosanoids upon bacterial stimuli. This demonstrates that preference for specific eicosanoid pathways is primed by CSFs before full M1/M2 activation.—Lukic, A., Larssen, P., Fauland, A., Samuelsson, B., Wheelock, C.E., Gabrielsson, S., Radmark, O. GM‐CSF– and M‐CSF–primed macrophages present similar resolving but distinct inflammatory lipid mediator signatures. FASEB J. 31, 4370–4381 (2017). www.fasebj.org

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