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Heparan sulfate proteoglycans are important modulators of cellular processes where the negatively charged polysaccharide chains interact with target proteins. The sulfation pattern of the heparan sulfate chains will determine the proteins that will bind and the affinity of the interactions. The N-deacetylase/N-sulfotransferase (NDST) enzymes are of key importance during heparan sulfate biosynthesis when the sulfation pattern is determined. In this chapter, metabolic labeling of heparan sulfate with [(35)S]sulfate or [(3)H]glucosamine in cell cultures is described, in addition to characterization of polysaccharide chain length and degree of N-sulfation. Methods to measure NDST enzyme activity are also presented.

In May 2020, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) published a recommendation report entitled "Recommendation on nonanimal-derived antibodies". In this report, the EURL ECVAM specifically states: "Therefore, taking into consideration the ESAC Opinion on the scientific validity of replacements for animal-derived antibodies, EURL ECVAM recommends that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic and therapeutic applications. The provisions of Directive 2010/63/EU should be respected, and EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking." (1). Here, we are providing the American Association of Pharmaceutical Scientists (AAPS) opinion on the EURL ECVAM recommendation report. In brief, there has been a clear and strong progress in reduction of animal use in the drug discovery and development process, including significant reduction of animal use in production of antibody reagents. Yet, it is proposed that more data need to be generated, shared and discussed within the scientific community before a decision to implement the change to non-animal derived antibodies is made.

In May 2020, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) published a recommendation report entitled "Recommendation on nonanimal-derived antibodies". In this report, the EURL ECVAM specifically states: "Therefore, taking into consideration the ESAC Opinion on the scientific validity of replacements for animal-derived antibodies, EURL ECVAM recommends that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic and therapeutic applications. The provisions of Directive 2010/63/EU should be respected, and EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking." (1). Here, we are providing the American Association of Pharmaceutical Scientists (AAPS) opinion on the EURL ECVAM recommendation report. In brief, there has been a clear and strong progress in reduction of animal use in the drug discovery and development process, including significant reduction of animal use in production of antibody reagents. Yet, it is proposed that more data need to be generated, shared and discussed within the scientific community before a decision to implement the change to non-animal derived antibodies is made.

Executive function deficits are often reported as a specific weakness in preterm children. Yet, executive function development is still not fully understood. In a prospective longitudinal study, 115 preterm born children, ≤31 weeks of gestation, were recruited at birth and subject to neuropsychological assessments at ages 5.5 and 18 years. By applying Miyake and colleagues' integrative framework of executive function to our data, two core components of executive function, working memory and cognitive flexibility, were identified through confirmatory factor analysis. Developmental stability was investigated in a serial multiple mediator structural equation model. Biological, medical, and social factors as well as mental development at 10 months were entered as predictors. Both components of executive function were highly stable from 5.5 to 18 years. Gestational age, intrauterine growth, lack of perinatal medical complications, and female sex were positively related to mental development at 10 months, which together with parental education influenced both core executive functions at 5.5 years. Working memory at 5.5 years mediated outcome in working memory at 18 years. In addition to the mediation of cognitive flexibility at 5.5 years, perinatal medical complications and restricted intrauterine growth had a continued direct negative impact on cognitive flexibility at 18 years. The application of a theoretical framework added to our understanding of executive function development in preterm born children. The study supports early identification of executive deficits among children born preterm, as deficits are unlikely to diminish with maturation.

Analysis of heparan sulfate synthesized by HEK 293 cells overexpressing murine NDST1 and/or NDST2 demonstrated that the amount of heparan sulfate was increased in NDST2- but not in NDST1-overexpressing cells. Altered transcript expression of genes encoding other biosynthetic enzymes or proteoglycan core proteins could not account for the observed changes. However, the role of NDST2 in regulating the amount of heparan sulfate synthesized was confirmed by analyzing heparan sulfate content in tissues isolated from Ndst2(-/-) mice, which contained reduced levels of the polysaccharide. Detailed disaccharide composition analysis showed no major structural difference between heparan sulfate from control and Ndst2(-/-) tissues, with the exception of heparan sulfate from spleen where the relative amount of trisulfated disaccharides was lowered in the absence of NDST2. In vivo transcript expression levels of the heparan sulfate-polymerizing enzymes Ext1 and Ext2 were also largely unaffected by NDST2 levels, pointing to a mode of regulation other than increased gene transcription. Size estimation of heparan sulfate polysaccharide chains indicated that increased chain lengths in NDST2-overexpressing cells alone could explain the increased heparan sulfate content. A model is discussed where NDST2-specific substrate modification stimulates elongation resulting in increased heparan sulfate chain length.

Objective: This study is the first in a series of four empirical investigations to develop International Classification of Functioning, Disability and Health (ICF) Core Sets for Autism Spectrum Disorder (ASD). The objective was to use a systematic review approach to identify, number, and link functional ability and disability concepts used in the scientific ASD literature to the nomenclature of the ICF‐CY (Children and Youth version of the ICF, covering the life span). Methods: Systematic searches on outcome studies of ASD were carried out in Medline/PubMed, PsycINFO, ERIC and Cinahl, and relevant functional ability and disability concepts extracted from the included studies. These concepts were then linked to the ICF‐CY by two independent researchers using a standardized linking procedure. New concepts were extracted from the studies until saturation of identified ICF‐CY categories was reached. Results: Seventy‐one studies were included in the final analysis and 2475 meaningful concepts contained in these studies were linked to 146 ICF‐CY categories. Of these, 99 categories were considered most relevant to ASD (i.e., identified in at least 5% of the studies), of which 63 were related to Activities and Participation, 28 were related to Body functions, and 8 were related to Environmental factors. The five most frequently identified categories were basic interpersonal interactions (51%), emotional functions (49%), complex interpersonal interactions (48%), attention functions (44%), and mental functions of language (44%). Conclusion: The broad variety of ICF‐CY categories identified in this study reflects the heterogeneity of functional differences found in ASD—both with respect to disability and exceptionality—and underlines the potential value of the ICF‐CY as a framework to capture an individual's functioning in all dimensions of life. The current results in combination with three additional preparatory studies (expert survey, focus groups, and clinical study) will provide the scientific basis for defining the ICF Core Sets for ASD for multipurpose use in basic and applied research and every day clinical practice of ASD. Autism Res 2015, 8: 782–794. © 2015 The Authors Autism Research published by Wiley Periodicals, Inc. on behalf of International Society for Autism Research

Main objectives: executive functions are frequently a weakness in children born preterm. We examined associations of executive functions and general cognitive abilities with brain structure in preterm born adolescents who were born with appropriate weight for gestational age and who have no radiological signs of preterm brain injury on neuroimaging. Methods: The Stockholm Neonatal Project (SNP) is a longitudinal, population-based study of children born preterm (Results: significant differences in grey and white matter regional volumes and widespread differences in FA were seen between the two groups. No significant correlations were found between cognitive measures and brain volumes in any group after correction for multiple comparisons. However, there were significant correlations between FA in projection fibres and long association fibres, linking frontal, temporal, parietal, and occipital lobes, and measures of executive function and general cognitive abilities in the preterm born adolescents, but not in the term born adolescents. Overall significance of the study: in persons born preterm, in the absence of perinatal brain injury on visual inspection of MRI, widespread alterations in regional brain tissue volumes and microstructure are present in adolescence/young adulthood. Importantly, these alterations in WM tracts are correlated with measures of executive function and general cognitive abilities. Our findings suggest that disturbance of neural pathways, rather than changes in regional brain volumes, are involved in the impaired cognitive functions.

Cognitive outcome after preterm birth is heterogeneous, and group level analyses may disguise individual variability in development. Using a person-oriented approach, this study investigated individual cognitive patterns and developmental trajectories from preschool age to late adolescence. As part of a prospective longitudinal study, 118 adolescents born preterm, with a birth weight1,500 g, participated in neuropsychological assessments at age 5½ years and at 18 years. At each age, four cognitive indices, two tapping general ability and two tapping executive functions, were formed to reflect each individual's cognitive profile. Cluster analyses were performed at each age separately, and individual movements between clusters across time were investigated. At both 5½ and 18 years, six distinct, and similar, cognitive patterns were identified. Executive functions were a weakness for some but not all subgroups, and verbal ability was a strength primarily among those whose overall performance fell within the normal range. Overall, cognitive ability at 5½ years was highly predictive of ability at age 18. Those who performed at low levels at 5½ years did not catch up but rather deteriorated in relative performance. Over half of the individuals who performed above the norm at 5½ years improved their relative performance by age 18. Among those performing around the norm at 5½ years, half improved their relative performance over time, whereas the other half faced increased problems, indicating a need for further developmental monitoring. Perinatal factors were not conclusively related to outcome, stressing the need for cognitive follow-up assessment of the preterm-born child before school entry.

Staphylococcus aureus is a major pathogen that can cause a broad spectrum of serious infections including skin infections, pneumonia and sepsis. Peritoneal mast cells have been implicated in the host response towards various bacterial insults and to provide mechanistic insight into the role of mast cells in intraperitoneal bacterial infection we here studied the global effects of S. aureus on mast cell gene expression. After co-culture of peritoneal mast cells with live S. aureus we found by gene array analysis that they up-regulate a number of genes. Many of these corresponded to pro-inflammatory cytokines, including interleukin-3, interleukin-13 and tumour necrosis factor-α. The cytokine induction in response to S. aureus was confirmed by ELISA. To study the role of peritoneal mast cells during in vivo infection with S. aureus we used newly developed Mcpt5-Cre(+) × R-DTA mice in which mast cell deficiency is independent of c-Kit. This is in contrast to previous studies in which an impact of mast cells on bacterial infection has been proposed based on the use of mice whose mast cell deficiency is a consequence of defective c-Kit signalling. Staphylococcus aureus was injected intraperitoneally into mast-cell-deficient Mcpt5-Cre(+) × R-DTA mice using littermate mast-cell-sufficient mice as controls. We did not observe any difference between mast-cell-deficient and control mice with regard to weight loss, bacterial clearance, inflammation or cytokine production. We conclude that, despite peritoneal mast cells being activated by S. aureus in vitro, they do not influence the in vivo manifestations of intraperitoneal S. aureus infection.

Mast cells (MCs) are major effector cells contributing to allergic conditions. When activated, they can release large amounts of active proteases, including chymase from their secretory granules. Here we assessed the role of the chymase mouse mast cell protease 4 (mMCP-4) in allergic airway inflammation induced by house-dust mite (HDM) extract. mMCP-4(-/-) mice demonstrated elevated airway reactivity and eosinophilia compared with wild-type (WT) animals, suggesting a protective role for mMCP-4 during the late inflammatory phase of the disease. However, mMCP-4 also contributed to the sensitization phase, as indicated by higher levels of serum immunoglobulin E in mMCP-4(-/-) vs. WT mice and higher levels of cytokines secreted by HDM-restimulated mMCP-4(-/-) vs. WT splenocytes. In line with a contribution of mMCP-4 in the early stages of disease, HDM extract directly induced chymase secretion from MCs. The elevated airway and inflammatory responses of mMCP-4(-/-) mice were associated with a profound increase in the levels of interleukin (IL)-33 in the lung tissue. Moreover, WT MCs degraded IL-33 more efficiently than did MCs lacking mMCP-4. Together, our findings identify a protective role of a MC chymase in a physiologically relevant model for airway inflammation and suggest that chymase-mediated regulation of IL-33 can account for this protective function.

Granzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy.

Here we show that mast cells (MCs) express the metalloproteases of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and that ADAMTS expression is influenced by MC activation. Co-culture of MCs with live Gram-positive bacteria caused a profound induction of ADAMTS-9 and -6, as well as down-regulated expression of ADAMTS-5. Similar patterns were also seen after MC activation with calcium ionophore and by immunoglobulin E receptor crosslinking. Moreover, ADAMTS-5, -6 and -9 were all induced by activation of terminally differentiated murine peritoneal MCs and in a human MC line. ADAMTS-9 up-regulation in response to immunoglobulin E receptor crosslinking was strongly dependent on Gö6976-sensitive protein kinase C and partly dependent on nuclear factor of activated T cells and nuclear factor kappa-light-chain-enhancer of activated B cells, respectively. The expression of ADAMTS-5, -6 and -9 was closely linked to MC maturation, as shown by their strong induction during the differentiation of bone marrow precursor cells into mature MCs. ADAMTS family members have been shown to possess aggrecanase activity. Accordingly, MCs were shown to express aggrecanase activity. Finally, ADAMTS-5 protein was detected in MCs by immunocytochemistry. Taken together, the present study reveals ADAMTS expression by MCs and that MC activation regulates the expression of these proteases, thus implicating the ADAMTS family of proteases in MC function.

T-cell intracellular antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1(-/-)) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1(-/-) mice also had higher levels of Df-specific IgE and IgG(1) in serum and ex vivo restimulated Tia-1(-/-) lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.

Mast cells (MCs) can have either detrimental or beneficial effects on malignant processes but the underlying mechanisms are poorly understood. Here we addressed this issue by examining the interaction between Lewis Lung Carcinoma (LLC) cells and MCs. In vivo, LLC tumors caused a profound accumulation of MCs, suggesting that LLC tumors have the capacity to attract MCs. Indeed, transwell migration assays showed that LLC-conditioned medium had chemotactic activity towards MCs, which was blocked by an antibody towards stem cell factor. In order to gain insight into the molecular mechanisms operative in tumor–MC interactions, the effect of LLC on the MC gene expression pattern was examined. As judged by gene array analysis, conditioned medium from LLC cells caused significant upregulation of numerous cell surface receptors and a pro-angiogenic Runx2/VEGF/Dusp5 axis in MCs, the latter in line with a role for MCs in promoting tumor angiogenesis. Among the genes showing the highest extent of upregulation was Tnfrsf9, encoding the anti-tumorigenic protein 4-1BB, suggesting that also anti-tumorigenic factors are induced. Quantitative RT-PCR analysis showed that 4-1BB was upregulated in a transient manner, and it was also shown that tumor cells induce 4-1BB in human MCs. Immunohistochemical analysis showed that LLC-conditioned medium induced 4-1BB also at the protein level. Together, this study provides novel insight into the molecular events associated with MC–tumor interactions and suggests that tumor cells induce both pro- and anti-tumorigenic responses in MCs.

Mast cells (MC) have pathogenic roles in numerous disorders, and strategies that stabilize MC or induce MC apoptosis are therefore emerging as possible therapeutic regimens. A typical feature of MC is their high content of secretory lysosomes (granules), containing numerous components such as biogenic amines, cytokines, serglycin proteoglycan and proteases. Damage to the secretory lysosomes will thus lead to leakage of these compounds, including the proteases, into the cytosol, and this could potentially trigger apoptosis. Here, we evaluated whether MC are sensitive to cell death induced by secretory lysosome destabilization, induced by the lysosomotropic agent Leu-Leu-OMe (LLME). Human MC were sensitive to LLME-induced cell death. In contrast, fibroblasts and HEK-293 cells were largely resistant. As judged by Annexin V/propidium iodide staining, LLME caused apoptotic cell death, and this was supported by induction of caspase-3-like activity, detection of activated caspase-3 by immunoblot analysis and reduced cell death in the presence of a caspase inhibitor. In support of a role for serglycin in regulating LLME-induced cell death, the survival rate of various cell types correlated negatively with the level of serglycin expression. In summary, this study introduces the concept of using lysosomotropic agents to induce cell death of human MC.

There is a wide variation among humans and mice in airway hyperresponsiveness (AHR) in the absence of allergen sensitization, i.e., naïve AHR. Because mast cell (MC) activation is thought to mediate AHR in atopic asthmatic subjects, we asked whether MCs mediate naïve AHR in A/J mice. We generated an A/J congenic strain lacking c-Kit by introgression of the Wv mutation, which resulted in the elimination of MCs and the abrogation of naïve AHR. Imatinib, which disrupts Kit signaling, also abrogated AHR in A/J mice. Remarkably, introduction of the Vga9 Mitf mutation into the A/J background resulted in the ablation of MCs but did not ameliorate AHR. These results indicate that c-Kit is required for development of AHR in an MC-independent fashion.

Mast cells store an impressive array of preformed compounds (mediators) in their secretory granules. When mast cells degranulate, these are released and have a profound impact on any condition in which mast cell degranulation occurs. The preformed mast cell mediators include well-known substances such as histamine, proteoglycans, proteases, and preformed cytokines, as well as several recently identified compounds. Mast cells have recently been implicated in a large number of novel pathological settings in addition to their well-established contribution to allergic reactions, and there is consequently a large current interest in the molecular mechanisms by which mast cells act in the context of a given condition. In many cases, preformed mast cell mediators have been shown to account for functions ascribed to mast cells, and these compounds are hence emerging as major players in numerous pathologies. In this review we summarize the current knowledge of preformed mast cell mediators.

This article contributes to the ongoing debate on the role of university research for innovation and economic growth, a debate highly influenced by concepts such as Mode 2 and regional innovation systems and clusters. A prominent trend in many EU and OECD countries is to direct research funding towards so-called Centres of Excellence (CoEs) in order to stimulate the industrial output of scientific research. The implementation of the CoE approach is viewed as an attempt to bridge research and innovation policy. By using Sweden as an example and providing an overview and critical discussion concerning Swedish research policy during the period 2001 to 2007 we show that the rhetoric within research policy has changed and become increasingly intertwined with innovation policy. In practice, however, this is not as evident. The study draws on (a) an analysis of policy literature pointing out regulatory and organizational changes concerning the increasing emphasis on linking research to competitive industrial milieus, and (b) a comprehensive database including 110 CoEs, presenting a detailed picture of university-industry collaboration, cross-disciplinarity, and prioritized sectors. We fiind that the CoEs account for a relatively small share of government funding, but may however have a strengthening impact on particular research milieus and industries, especially in the life sciences. Additionally, although contemporary policy rhetoric appears to highlight steering funding to geographically-concentrated milieus, thereby linking leading university research to regional industrial clusters, this has only been manifested in a few cases – notably in the Vinnvaxt programme run by Vinnova, the national agency promoting innovation systems.

Nonselective inhibition of PG synthesis augments inflammation in mouse models of airway disease, but the roles of individual PGs are not completely clarified. To investigate the role of PGE2 in a mouse model of airway inflammation induced by a natural allergen, we used mice lacking the critical terminal synthetic enzyme, microsomal PGE2 synthase (mPGES)-1. Mice lacking mPGES-1 (ptges−/− mice) and wild-type C57BL/6 controls were challenged intranasally with low doses of an extract derived from the house dust mite Dermatophagoides farinae (Der f). The levels of PGE2 in the bronchoalveolar lavage fluids of Der f-treated ptges−/− mice were ∼80% lower than the levels in wild-type controls. Der f-induced bronchovascular eosinophilia was modestly enhanced in the ptges−/− mice. Both Der f-treated strains showed similar increases in serum IgE and IgG1, as well as comparable levels of Th1, Th2, and Th17 cytokine production by Der f-stimulated spleen cells. These findings indicated that mPGES-1–derived PGE2 was not required for allergen sensitization or development of effector T cell responses. Unexpectedly, the numbers of vascular smooth muscle cells and the thickness of intrapulmonary vessels were both markedly increased in the Der f-treated ptges−/− mice. These vascular changes were suppressed by the administration of the stable PGE2 analog 16, 16-dimethyl PGE2, or of selective agonists of the E-prostanoid (EP) 1, EP2, and EP3 receptors, respectively, for PGE2. Thus, mPGES-1 and its product, PGE2, protect the pulmonary vasculature from remodeling during allergen-induced pulmonary inflammation, and these effects may be mediated by more than one EP receptor.