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Aims/hypothesis Rapid and adequate islet revascularisation and restoration of the islet–extracellular matrix (ECM) interaction are significant factors influencing islet survival and function of the transplanted islets in individuals with type 1 diabetes. Because the ECM encapsulating the islets is degraded during islet isolation, understanding the process of revascularisation and engraftment after transplantation is essential and needs further investigation. Methods Here we apply a longitudinal and high-resolution imaging approach to investigate the dynamics of the pancreatic islet engraftment process up to 11 months after transplantation. Human and mouse islet grafts were inserted into the anterior chamber of the mouse eye, using a NOD.ROSA-tomato.Rag2−/− or B6.ROSA-tomato host allowing the investigation of the expansion of host vs donor cells and the contribution of host cells to aspects such as promoting the encapsulation and vascularisation of the graft. Results A fibroblast-like stromal cell population of host origin rapidly migrates to ensheath the transplanted islet and aid in the formation of a basement membrane-like structure. Moreover, we show that the vessel network, while reconstituted by host endothelial cells, still retains the overall architecture of the donor islets. Conclusions/interpretation In this transplantation situation the fibroblast-like stromal cells appear to take over as main producers of ECM or act as a scaffold for other ECM-producing cells to reconstitute a peri-islet-like basement membrane. This may have implications for our understanding of long-term graft rejection and for the design of novel strategies to interfere with this process.

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA−/−MafB+/−, but not MafA−/− islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

Sterile liver inflammation and fibrosis are associated with many liver disorders of different etiologies. Both type 1 and type 2 inflammatory responses have been reported to contribute to liver pathology. However, the mechanisms controlling the balance between these responses are largely unknown. Natural killer T (NKT) cells can be activated to rapidly secrete cytokines and chemokines associated with both type 1 and type 2 inflammatory responses. As these proteins have been reported to accumulate in different types of sterile liver inflammation, we hypothesized that these cells may play a role in this pathological process. We have found that a transgenic NKT (tgNKT) cell population produced in the immunodeficient 2,4αβNOD.Rag2−/− mice, but not in 2,4αβNOD.Rag2+/− control mice, promoted a type 1 inflammatory response with engagement of the NOD-, LRR- and pyrin domain-containing protein-3 (NLRP3) inflammasome. The induction of the type 1 inflammatory response was followed by an altered cytokine profile of the tgNKT cell population with a biased production of anti-inflammatory/profibrotic cytokines and development of liver fibrosis. These findings illustrate how the plasticity of NKT cells modulates the inflammatory response, suggesting a key role for the NKT cell population in the control of sterile liver inflammation.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

Glucagon-like peptide 1 (GLP-1) is produced by L cells in the small intestine in response to ingested glucose and increases insulin release from pancreatic beta cells by activation of its cognate receptor (GLP-1R). Stimulation of this receptor also contributes to increased beta cell survival and regeneration. We have found that pancreatic beta cells from Non Obese Diabetic (NOD) mice express significantly lower levels of GLP-1R than C57BL/6 mice, leaving the NOD beta cells with an impaired response to GLP-1 stimulation. The lower expression appears to be caused by accelerated degradation of GLP-1R in the beta cells, a process that can be reversed by inhibiting trafficking to the lysosome. Importantly, our results appear to translate to the human disease since we also observed significantly lower expression of the GLP-1R in pancreatic islets from donors with type 1 diabetes. These results suggest that beta cell physiology may play a role in susceptibility to autoimmune inflammation.

Aims/hypothesisObesity is associated with glucose intolerance and insulin resistance and is closely linked to the increasing prevalence of type 2 diabetes. In mouse models of diet-induced obesity (DIO) and type 2 diabetes, an increased fat intake results in adipose tissue expansion and the secretion of proinflammatory cytokines. The innate immune system not only plays a crucial role in obesity-associated chronic low-grade inflammation but it is also proposed to play a role in modulating energy metabolism. However, little is known about how the modulation of metabolism by the immune system may promote increased adiposity in the early stages of increased dietary intake. Here we aimed to define the role of type I IFNs in DIO and insulin resistance.MethodsMice lacking the receptor for IFN-α (IFNAR−/−) and deficient in plasmacytoid dendritic cells (pDCs) (B6.E2-2fl/fl.Itgax-cre) were fed a diet with a high fat content or normal chow. The mice were analysed in vivo and in vitro using cellular, biochemical and molecular approaches.ResultsWe found that the development of obesity was inhibited by an inability to respond to type I IFNs. Furthermore, the development of obesity and insulin resistance in this model was associated with pDC recruitment to the fatty tissues and liver of obese mice (a 4.3-fold and 2.7-fold increase, respectively). Finally, we demonstrated that the depletion of pDCs protects mice from DIO and from developing obesity-associated metabolic complications.Conclusions/interpretationOur results provide genetic evidence that pDCs, via type I IFNs, regulate energy metabolism and promote the development of obesity.

The TCF4 gene encodes for the basic helix–loop–helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.

Multiple sclerosis is a neuroinflammatory degenerative disease, caused by activated immune cells infiltrating the CNS. The disease etiology involves both genetic and environmental factors. The mouse genetic locus, Eae27, linked to disease development in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis, was studied in order to identify contributing disease susceptibility factors and potential drug targets for multiple sclerosis. Studies of an Eae27 congenic mouse strain, revealed that genetic variation within Eae27 influences EAE development. The Abl2 gene, encoding the non-receptor tyrosine kinase Arg, is located in the 4,1 megabase pair long Eae27 region. The Arg protein plays an important role in cellular regulation and is, in addition, involved in signaling through the B- and T-cell receptors, important for the autoimmune response. The presence of a single nucleotide polymorphism causing an amino acid change in a near actin-interacting domain of Arg, in addition to altered lymphocyte activation in the congenic mice upon immunization with myelin antigen, makes Abl2/Arg a candidate gene for EAE. Here we demonstrate that the non-synonymous SNP does not change Arg's binding affinity for F-actin but suggest a role for Abl kinases in CNS inflammation pathogenesis by showing that pharmacological inhibition of Abl kinases ameliorates EAE, but not experimental arthritis.

Quinoline-3-carboxamides (Q substances) are small molecule compounds with anti-inflammatory properties. In this study, we used one of these substances, Paquinimod, to treat a novel model for chronic liver inflammation and liver fibrosis, the NOD-Inflammation Fibrosis (N-IF) mouse. We show that treatment of N-IF mice significantly reduced inflammation and resulted in the regression of fibrosis, even when the treatment was initiated after onset of disease. The reduced disease phenotype was associated with a systemic decrease in the number and reduced activation of disease-promoting transgenic natural killer T (NKT)-II cells and their type 2-cytokine expression profile. Paquinimod treatment also led to a reduction of CD115+ Ly6Chi monocytes and CD11b+ F4/80+ CD206+ macrophages.

The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes. We adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets. We demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour. Together, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes.

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.

In diabetes, pancreatic beta-cells play a key role. These cells are clustered within structures called islets of Langerhans inside the pancreas and produce insulin, which is directly secreted into the blood stream. The dense vascularization of islets of Langerhans is critical for maintaining a proper regulation of blood glucose homeostasis and is known to be affected from the early stage of diabetes. The deep localization of these islets inside the pancreas in the abdominal cavity renders their in vivo visualization a challenging task. A fast label-free imaging method with high spatial resolution is required to study the vascular network of islets of Langerhans. Based on these requirements, we developed a label-free and three-dimensional imaging method for observing islets of Langerhans using extended-focus Fourier domain Optical Coherence Microscopy (xfOCM). In addition to structural imaging, this system provides three-dimensional vascular network imaging and dynamic blood flow information within islets of Langerhans. We propose our method to deepen the understanding of the interconnection between diabetes and the evolution of the islet vascular network. (C) 2016 Optical Society of America

This deposit is composed by the main article plus the supplementary materials of the publication. Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders. Vetenskapsrådet (VR) grant: (K2013-67X-07929- 27-3).

Background T1D and AITD are autoimmune disorders commonly occurring in the same family and even in the same individual. The genetic contribution to these disorders is complex making uncovering of susceptibility genes very challenging. The general aim of this study was to identify loci and genes contributing to T1D/AITD susceptibility. Our strategy was to perform linkage and association studies in the relatively genetically homogenous population of northern Sweden. We performed a GWLS to find genomic regions linked to T1D/AITD in families from northern Sweden and we performed an association study in the families to test for association between T1D/AITD and variants in previously published candidate genes as well as a novel candidate gene, CD247. Methods DNA prepared from 459 individuals was used to perform a linkage and an association study. The ABI PRISM Linkage Mapping Set v2.5MD10 was employed for an initial 10-cM GWLS, and additional markers were added for fine mapping. Merlin was used for linkage calculations. For the association analysis, a GoldenGate Custom Panel from Illumina containing 79 SNPs of interest was used and FBAT was used for association calculations. Results Our study revealed linkage to two previously identified chromosomal regions, 4q25 and 6p22, as well as to a novel chromosomal region, 1q23. The association study replicated association to PTPN22, HLA-DRB1, INS, IFIH1, CTLA4 and C12orf30. Evidence in favor of association was also found for SNPs in the novel susceptibility gene CD247. Conclusions Several risk loci for T1D/AITD identified in published association studies were replicated in a family material, of modest size, from northern Sweden. This provides evidence that these loci confer disease susceptibility in this population and emphasizes that small to intermediate sized family studies in this population can be used in a cost-effective manner for the search of genes involved in complex diseases. The linkage study revealed a chromosomal region in which a novel T1D/AITD susceptibility gene, CD247, is located. The association study showed association between T1D/AITD and several variants in this gene. These results suggests that common susceptibility genes act in concert with variants of CD247 to generate genetic risk for T1D/AITD in this population. Electronic supplementary material The online version of this article (doi:10.1186/s12881-016-0333-z) contains supplementary material, which is available to authorized users.

Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis depending on their activation phenotype. Autoimmune type 1 diabetes (T1D) is a chronic proinflammatory condition characterized by unresolved destruction of pancreatic islets. Adoptive cell transfer of macrophages with immunosuppressive properties represents a novel immunotherapy for treatment of such chronic autoimmune diseases. We used a panel of cytokines and other stimuli to discern the most effective regimen for in vitro induction of immunosuppressive macrophages (M2r) and determined interleukin (IL)-4/IL-10/transforming growth factor-β (TGF-β) to be optimal. M2r cells expressed programmed cell death 1 ligand-2, fragment crystallizable region γ receptor IIb, IL-10, and TGF-β, had a potent deactivating effect on proinflammatory lipopolysaccharide/interferon-γ–stimulated macrophages, and significantly suppressed T-cell proliferation. Clinical therapeutic efficacy was assessed after adoptive transfer in NOD T1D mice, and after a single transfer of M2r macrophages, >80% of treated NOD mice were protected against T1D for at least 3 months, even when transfer was conducted just prior to clinical onset. Fluorescent imaging analyses revealed that adoptively transferred M2r macrophages specifically homed to the inflamed pancreas, promoting β-cell survival. We suggest that M2r macrophage therapy represents a novel intervention that stops ongoing autoimmune T1D and may have relevance in a clinical setting.

The objective was to investigate the effects of omega-3 fatty acids, known for their anti-inflammatory effects, on time to lymphoma progression and survival in the TLL mouse, a strain genetically prone to developing aggressive T-cell lymphoma. Compared to mice fed a standard diet, TLL mice fed omega-3 (menhaden fish oil) experienced a significant delay in disease progression and were more likely to remain alive and symptom free during the first 8 months of the study. In contrast, omega-6 supplementation (corn oil) did not significantly affect lymphoma progression. Irrespective of diet, all mice eventually progressed, and 1-year survival was not different between the groups. Immunological analysis demonstrated a significantly altered B-cell compartment and fewer NK cells in healthy C57Black6 mice fed omega-3, compared to controls. In conclusion, a diet rich in omega-3 fatty acids delays lymphoma development in the TLL mouse possibly by mechanisms that include complex effects on immune function.

Little is known about target organ-infiltrating NK cells in type 1 diabetes and other autoimmune diseases. In this study, we identified NK cells with a unique phenotype in the pancreas of NOD mice. Pancreatic NK cells, localized to the endocrine and exocrine parts, were present before T cells during disease development and did not require T cells for their infiltration. Furthermore, NK cells, or NK cell precursors, from the spleen could traffic to the pancreas, where they displayed the pancreatic phenotype. Pancreatic NK cells from other mouse strains shared phenotypic characteristics with pancreatic NK cells from NOD mice, but displayed less surface killer cell lectin-like receptor G1, a marker for mature NK cells that have undergone proliferation, and also did not proliferate to the same extent. A subset of NOD mouse pancreatic NK cells produced IFN-γ spontaneously, suggesting ongoing effector responses. However, most NOD mouse pancreatic NK cells were hyporesponsive compared with spleen NK cells, as reflected by diminished cytokine secretion and a lower capacity to degranulate. Interestingly, such hyporesponsiveness was not seen in pancreatic NK cells from the nonautoimmune strain C57BL/6, suggesting that this feature is not a general property of pancreatic NK cells. Based on our data, we propose that NK cells are sentinel cells in a normal pancreas. We further speculate that during inflammation, pancreatic NK cells initially mediate proinflammatory effector functions, potentially contributing to organ-specific autoimmunity, but later become hyporesponsive because of exhaustion or regulation.

V(H) gene family specific polymerase chain reaction (PCR) amplification was performed in 87 B-cell lymphoma samples from 4 different subgroups. No apparent restriction in the VH gene usage was found in follicular lymphomas, lymphoplasmacytoid lymphomas or large B-cell lymphomas, whereas a biased VH1 utilization was shown in patients with chronic lymphocytic leukemia. Eleven of 18 chronic lymphocytic leukemia cases utilized the VH1 gene family, and nucleotide sequencing of the VH1 gene rearrangements revealed that a majority utilized the DP10 (51p1) germline gene, which has been reported to be strongly associated with autoimmune disease. No VH5 or VH6 rearrangements were amplified in the chronic lymphocytic leukemia subgroup, 2 gene families which previously have been found to be over-represented in these patients. In a high proportion (40%) of large B-cell lymphomas, VH gene family-specific PCR failed to amplify any rearrangement. Using primers hybridizing to the framework regions 2 and 3 and Southern blot analysis of the immunoglobulin heavy chain locus, clonal rearrangements were displayed in two-thirds of these PCR negative cases. However, the rearrangement status could not be elucidated in 5 of 35 patients with large B-cell lymphoma.

Objective: To establish whether subtypes of ischemic stroke aggregate within ischemic stroke–affected sibling pairs more than expected by chance alone. Methods: This retrospective family study was based on a pooled analysis of two cohorts of male and female adult sibling pairs with symptomatic ischemic stroke. One hospital-based cohort of 404 individuals (first proband seen August 30, 1999) was recruited from the United States and Canada, and another population-based cohort of 198 individuals (first proband seen April 17, 1997) was recruited from Umea, Sweden. Subtype diagnoses were based on Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Results: Agreement for subtype diagnoses within families was poor (mean ± asymptotic SE κ = 0.17 ± 0.04). Occurrence of one ischemic stroke subtype in a proband was not associated with a greater likelihood of that subtype being the qualifying stroke subtype in the sibling. Comparable levels of agreement were seen when restricting the analysis to same-sex sibling pairs (κ = 0.22 ± 0.05) to sibling pairs in which the proband’s stroke occurred before the age of 65 years (κ = 0.16 ± 0.05) or to pairs in which the proband’s stroke occurred at or after the age of 65 years (κ = 0.19 ± 0.05). Conclusions: The subtype of ischemic stroke in a proband was a poor determinant of the subtype of ischemic stroke in the respective sibling. This suggests that many genetic risk factors for ischemic stroke may not be specific for one subtype.

A recent study found association of one microsatellite and five single nucleotide polymorphisms (SNPs) in intron 3 of the TCF7L2 gene with type 2 diabetes (T2D) in the Icelandic, Danish and American populations. The aim of the present study was to investigate if those SNPs were associated to T2D in two (family- and population-based) cohorts from northern Sweden. We genotyped four of the associated SNPs in a case-control cohort consisting of 872 T2D cases and 857 controls matched with respect to age, sex and geographical origin and in a sample of 59 extended families (148 affected and 83 unaffected individuals). Here, we report replication of association between T2D and three SNPs in the case-control (rs7901695, P=0.003; rs7901346, P=0.00002; and rs12255372, P=0.000004) and two SNPs in the family-based (rs7901695, P=0.01 and rs7901346, P=0.04) samples from northern Sweden. This replication strengthens the evidence for involvement of TCF7L2 in T2D.

Background and Purpose— Taking advantage of low genetic variations in northern Sweden, we performed a genome-wide linkage scan to investigate the susceptibility loci for common forms of stroke. Methods— Fifty-six families, containing multiple cases of stroke and whose data had been previously used to replicate linkage to the phosphodiesterase 4D locus on chromosome 5q, were genotyped in a genome-wide scan. Fine mapping was performed, and subsequently 53 additional families from the same region were genotyped over the candidate regions. Results— Linkage calculations were performed by using 3 different disease models, from a very broad (all stroke cases defined by World Health Organization MONICA criteria) to a narrower (ischemic stroke only) stroke phenotype. With all models, nonparametric multipoint linkage analysis yielded allele-sharing log of the odds (LOD) scores >1.2 at 9 locations: 1p34, 5q13, 7q35, 9q22, 9q34, 13q32, 14q32, 18p11, and 20q13. The highest allele-sharing LOD scores were obtained on chromosomes 5q (previously reported), 1p (LOD=2.09), and 18p (LOD=2.14). Fine mapping resulted in increased allele-sharing LOD scores for chromosome 5q (previously reported) and 9q22 (LOD=1.56), but all others decreased. Combining these initial results with a subsequent analysis of 53 additional families, we obtained the highest allele-sharing LOD scores on chromosomes 5q, 13q, and 18p, although none reached the initial genome-wide allele-sharing LOD scores. Conclusions— Genetic analysis of stroke in families from northern Sweden did not identify any new major stroke loci. This indicates that multiple minor susceptibility loci in addition to the previously known locus on chromosome 5 could contribute to the disease.

Autoimmune diabetes is a consequence of immune-cell infiltration and destruction of pancreatic β-cells in the islets of Langerhans. We analyzed the cellular composition of the insulitic lesions in the autoimmune-prone non-obese diabetic (NOD) mouse and observed a peak in recruitment of plasmacytoid dendritic cells (pDCs) to NOD islets around 8-9 weeks of age. This peak coincides with increased spontaneous expression of type-1-IFN response genes and CpG1585 induced production of IFN-α from NOD islets. The transcription factor E2-2 is specifically required for the maturation of pDCs, and we show that knocking out E2-2 conditionally in CD11c+ cells leads to a reduced recruitment of pDCs to pancreatic islets and reduced CpG1585 induced production of IFN-α during insulitis. As a consequence, insulitis has a less aggressive expression profile of the Th1 cytokine IFN-γ and a markedly reduced diabetes incidence. Collectively, these observations demonstrate a disease-promoting role of E2-2 dependent pDCs in the pancreas during autoimmune diabetes in the NOD mouse.

We present data from a genome-wide scan identifying genetic factors conferring susceptibility to type 2 diabetes. The linkage analysis was based on 59 families from northern Sweden, consisting of a total of 129 cases of type 2 diabetes and 19 individuals with impaired glucose tolerance. Model-free linkage analysis revealed a maximum multipoint logarithm of odds score of 3.19 for D2S2987 at 267.7 cM (P = 0.00058), suggesting that a gene conferring susceptibility to type 2 diabetes in the northern Swedish population resides in the 2q37 region. These data replicate, in a European population, previously identified linkage of marker loci in this region to type 2 diabetes in Mexican Americans. In contrast, no evidence in support of association to the previously identified single nucleotide polymorphisms in the calpain-10 gene was observed in a case-control cohort derived from the same population.

Migraine is a complex, multifactorial disorder for which several loci have been identified in the human genome. We have previously reported linkage to a 10 Mb-region on chromosome 6p12.2-p21.1 in one large Swedish pedigree involving migraine with and without aura. To further investigate this candidate region, a dense set of single nucleotide polymorphic (SNP) markers was used for fine-mapping, decreasing the critical region to 8.5 Mb. Within this region, EFHC1 was recently identified as the disease gene for juvenile myoclonic epilepsy. Migraine and epilepsy has been suggested to share disease mechanisms and therefore EFHC1 is an excellent candidate gene for migraine in this family. Mutation analysis of the gene revealed a disease-segregating polymorphism in the promoter. Association analysis of the polymorphism in a case-control material did not support a role for this gene in migraine pathology. We therefore analyzed five additional candidate genes in the disease-critical region, including MEP1A, RHAG, IL17, SLC25A27 and TNFRSF21. In two of these genes, MEP1A and RHAG, we identified two novel polymorphisms associated with the disease haplotype. The combination of these polymorphisms could not be found in any control individuals, suggesting that they might be involved in genetic predisposition to migraine in this family.

Unprecedented cure after infection with the lethal Plasmodium berghei ANKA was observed in an F2 progeny generated by intercrossing the wild-derived WLA and the laboratory C57BL/6 mouse strains. Resistant mice were able to clear parasitaemia and establish immunity. The observed resistance was disclosed as a combinatorial effect of genetic factors derived from the two parental strains. Genetic mapping of survival time showed that the WLA allele at a locus on chromosome 1 (colocalizing with Berghei resistance 1 (Berr1), a locus associated with resistance to experimental cerebral malaria) increases the probability to resist early death. Also, the C57Bl/6 allele at a novel locus on chromosome 9 (Berr3) confers overall resistance to this lethal Plasmodium infection. This report underlines the value of using wild-derived mouse strains to identify novel genetic factors in the aetiology of disease phenotypes, and provides a unique model for studying parasite clearance and immunity associated with malaria.

The underlying mechanistic causes of immune cell infiltration in the islets of Langerhans and beta cell failure in the non-obese diabetic (NOD) mouse is still to be completely revealed. Substance P (SP) is a substance known to have pro-inflammatory, endocrine, neuromodulatory and trophic effects, and its preferred receptor, the neurokinin receptor 1 (NK-1 R), is reported to be involved in extravasation of granulocytes and in inflammation and tissue derangement. Therefore, we have investigated the expression of NK-1 R during development of insulitis in the NOD mouse. We show that the magnitude of immunoreactivity scoring NK-1 R expression in the islets was increased in the 12-week-old NOD mouse. Expression of NK-1 R co-localized with expression of glucagon. In line with this expression pattern, we did not detect any effect of SP on glucose-induced insulin release. NK-1 R expression was particularly observed in islet cells in association with the clusters of immune cells. Expression of NK-1 R was also demonstrated in a fraction of the infiltrating B and T lymphocytes, as well as on infiltrating macrophages and dendritic cells. The observations show that the level of NK-1 R expression is increased in 12-week-old NOD mice, being correlated with the occurrence of islet mononuclear infiltration. Our data suggest that SP may act as a chemoattractant, contributing to the pathogenic mononuclear infiltration process in the NOD mouse. On the whole, the observations suggest that SP and the NK-1 R to certain extents are involved in the changes that occur during the development of insulitis in the NOD mouse.

About 5% of glioma cases are familial. Most glioma families are not ascribed to the well-known glioma predisposing syndromes. One segregation analysis has supported an autosomal recessive gene in glioma families, which could be studied by homozygosity mapping. The ancestors of seven glioma families from the northern region of Sweden were traced through genealogical databases. A common ancestor and inbreeding were traced to give support to an autosomal recessive gene. Homozygosity mapping was performed with a genome-wide scan of 811 markers with linkage calculations. The families were geographically mapped to see if familial glioma was more common in northern compared with southern Sweden. Three of the seven families were remotely related. Homozygosity mapping did not reveal any allele homozygous for all three families. However, there was a geographical clustering of glioma families in the northern region of Sweden. A non-parametric analysis showed an allele-sharing LOD score of 1.05 for marker D1S196 on chromosome 1q23. Genealogical studies linking glioma families might be a tool for linkage in a small set of families. This study did not support an autosomal recessive gene, implicating a low penetrant dominant gene as a possible explanation to the glioma family clustering.

The innervation of the islets of Langerhans may constitute a first target for the autoimmunity that develops in type 1 diabetes. Here, we report the occurrence of a decrease in general innervation within the islets in the nonobese diabetic (NOD) mouse, and the establishment of strands of Schwann cells, as detected via p75 and S-100 immunoreactivity (IR), and varicose nerve fibers expressing tyrosine kinase A (TrkA) in association with the immune cells. The findings suggest that there are marked attempts for neurotrophins to promote nerve ingrowth and survival for islet tissue and that remodeling of innervation occurs in the continuation of the insulitis process preceding the onset of type 1 diabetes.

Identification of genes associated with pain insensitivity syndromes can increase the understanding of the pathways involved in pain and contribute to the understanding of how sensory pathways relate to other neurological functions. In this report we describe the mapping and identification of the gene responsible for loss of deep pain perception in a large family from northern Sweden. The loss of pain perception in this family is characterized by impairment in the sensing of deep pain and temperature but with normal mental abilities and with most other neurological responses intact. A severe reduction of unmyelinated nerve fibers and a moderate loss of thin myelinated nerve fibers are observed in the patients. Thus the cases in this study fall into the class of patients with loss of pain perception with underlying peripheral neuropathy. Clinically they best fit into HSAN V. Using a model of recessive inheritance we identified an 8.3 Mb region on chromosome 1p11.2-p13.2 shared by the affected individuals in the family. Analysis of functional candidate genes in the disease critical region revealed a mutation in the coding region of the nerve growth-factor beta (NGFB) gene specific for the disease haplotype. This NGF mutation seems to separate the effects of NGF involved in development of central nervous system functions such as mental abilities, from those involved in peripheral pain pathways. This mutation could therefore potentially provide an important tool to study different roles of NGF, and of pain control.

We have identified a large family in the northern part of Sweden with multiple cases of autoimmune diseases, namely type 1 diabetes (T1D), Graves' disease (GD) and Hashimoto's thyroiditis (HT). The family members affected by any of these diseases share a region of 2.4 Mb that comprises among others the CTLA4 gene. We determined that all affected members of the family shared the HLA susceptibility haplotype (DR4-DQA1*0301-DQB1*0302). Analysis of genetic interaction conditioning for HLA haplotype provided strong evidence that the critical region which includes the CTLA4 gene acts together with the HLA locus on the etiology of disease (lodscore 4.20 (theta=0.0). The study of this family allowed us to: (1) reinforce a number of reports on linkage and association of the CTLA4 region to T1D and AITD; (2) demonstrate that a single haplotypic variant in this region constitutes an etiological factor to disease susceptibility in T1D, GD and HT; (3) reveal a strong genetic interaction of the CTLA4 and HLA loci in the genetic architecture of autoimmune disease; (4) emphasise the value of large pedigrees drawn from isolated populations as tools to single out the effect of individual loci in the etiology of complex diseases.

Migraine is the most common type of chronic episodic headache. To find novel susceptibility genes for familial migraine with and without aura, a genomewide screen was performed in a large family from northern Sweden. Evidence of linkage was obtained on chromosome 6p12.2-p21.1, with a maximum two-point lod score of 5.41 for marker D6S452. The patients with migraine shared a common haplotype of 10 Mb between markers D6S1650 and D6S1960.

In this study, a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia with an early onset and high incidence was established and characterized. All tumors analyzed were found to express the alpha,beta T-cell receptor, and the majority of them had a mature, CD3+CD4+CD8- immunophenotype. In a few cases, tumors with a more immature CD3+CD4+CD8+ phenotype were isolated. Expanded phenotyping using a broad panel of lymphocyte differentiation markers confirmed the mature T-cell phenotype of the tumors. Histologic and cell cycle analysis of the tumors revealed an aggressive lymphoblastic malignancy with a very high proliferation rate and widespread engagement of bone marrow and lymphoid as well as nonlymphoid organs. Thus, the TLL mouse strain represents a unique model for the analysis of the oncogenesis and progression of mature T-cell tumors and for the development of therapeutic measures to combat such tumors.

To determine whether the genetic background of the insulin-producing beta cells of the pancreas contributes to autoimmune diabetes susceptibility, we have used a model of the disease based on transferring spleen cells from nonobese diabetic (NOD) C57BL/6 (B6) embryo aggregation (EA) chimeras into B6 and NOD irradiated mice. Insulitis and diabetes could be induced into both B6 and NOD hosts, albeit with low incidence. Cyclophosphamide (CY) treatment, known to accelerate diabetes in prediabetic NOD mice, was found to increase diabetes incidence up to 50-60% in both B6 and NOD mice reconstituted with chimeric splenocytes, while diabetes did not occur in CY-treated B6 mice reconstituted with B6 splenocytes. We conclude that the genetic make-up of the target organ does not affect the final stage of the pathogenesis of insulin-dependent diabetes mellitus.

Twenty-two patients with relapsed or progressive B-cell lymphomas (BCL) were analysed for alterations in the rearrangement status in the immunoglobulin heavy (IgH) chain gene in samples obtained on different occasions during the course of the disease. The analysis was performed using Southern blot hybridization of the IgH gene and polymerase chain reaction (PCR) amplification of the VH gene families combined with single-strand conformation polymorphism (SSCP) analysis. Using Southern blot analysis, we found that all 22 lymphomas displayed clonal IgH rearrangements, and changes during tumour progression occurred in 8 cases. These alterations were mainly observed in cases with follicular or transformed lymphomas. More than one malignant (sub)clone, indicated by more than two rearranged bands, was detected in one case at diagnosis and in three cases at relapse. Outgrowth of subclones with divergent rearrangement patterns in different compartments was also observed in 2 out of 8 cases. PCR-SSCP analysis indicated that all 15 cases studied displayed clonal rearrangements and in 6 cases altered rearrangement patterns were detected in later samples. Southern blotting and PCR-SCCP analysis gave equivalent results. No association was found between time to relapse or survival time and alterations in rearrangement pattern. The present study illustrates that the neoplastic cell clones in BCL often display alterations in their IgH locus, but the significance of this feature remains to be clarified.

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated fromCtla4andCd28and in fact marks a 20-centimorgan region encompassingIdd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

We have compared the B-lymphocyte repertoire in seven IDDM patients with 12 healthy controls by examining the variable heavy (V(H)) gene expression. The V(H) gene representation in the pool of pokeweed mitogen (PWM) stimulated, immunocompetent B cells and in the pool of naturally activated plasma cells (actual repertoire) was analysed by RNA-RNA in situ hybridization. Differences between IDDM patients and normal controls in the relative expression of several V(H) gene families were observed. In IDDM patients, the V(H)3 was significantly underrepresented in the PWM stimulated repertoire. In the actual B cell repertoire the V(H)5 clones were underrepresented among diabetic patients. Moreover, the altered distribution of V(H) gene usage between the PWM stimulated repertoire and the actual repertoire observed in normal controls was found to be less pronounced in the IDDM patients. This observation suggests a defect in the V-gene directed cellular selection occurring between resting, immunocompetent B cells and naturally activated plasma cells. The possible implication of the observed aberrations in the B cell selection process for the pathogenesis of autoimmunity is discussed.

The authors have compared the VH gene utilization patterns among small resting immunocompetent B cells and large naturally activated B lymphocytes of healthy human adults. They employed a non-radioactive RNA in situ hybridization technique that allows detection of VH gene family expression at the single cell level. Pokeweed mitogen stimulated and unmanipulated mononuclear cells from peripheral blood and spleen of unrelated individuals were hybridized to digoxigenin-labelled antisense RNA probes specific for human VH families 1-6 and for the constant region genes C mu and C gamma. The observed VH gene family utilization patterns did not correlate with the genomic complexity of human VH genes. The VH3 gene family was most frequently used among resting B cells in both peripheral blood and spleen. Among naturally activated lymphocytes the VH6 gene was markedly over-represented, while expression of the VH1 and VH3 gene families was decreased. The data show that V-region mediated selection participates in shaping the peripheral antibody repertoire in healthy adults.

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders.

The majority of embryo aggregation (EA) mouse chimeras between non-obese diabetic (NOD) mice and C57BL/6 (B6) mice show clear signs of insulitis frequently accompanied by beta-cell destruction. Less than 5% of these chimeras, however, spontaneously progress to autoimmune diabetes, an incidence far lower than observed in NOD mice. The resistance in chimeras can be accounted for by the target organ chimerism and/or the immune system chimerism. To investigate the mechanism(s) controlling diabetes resistance in these mice, we studied a total of 92 NOD--B6 EA chimeras that showed overt lymphoid chimerism and treated 34 chimeras with cyclophosphamide (CY), a compound known to precipitate an acute form of insulin-dependent diabetes mellitus (IDDM) in pre-diabetic NOD mice, by interfering with regulatory mechanisms. We found that CY-treated EA chimeras displayed an increase in the NOD:B6 lymphocyte ratio and 32% of them developed diabetes that could be adoptively transferred to irradiated NOD or NOD-rag-2-/- mice. These findings suggest that lymphocyte chimerism rather than beta-cell chimerism accounts for diabetes resistance in NOD--B6 EA chimeras and that the susceptibility to CY-induced diabetes may be related to the proportion of NOD versus B6 lymphoid cells.