Suitability of machine learning models for prediction of clinically defined Stage III/IV periodontitis from questionnaires and demographic data in Danish cohorts.

AIM: To evaluate if, and to what extent, machine learning models can capture clinically defined Stage III/IV periodontitis from self-report questionnaires and demographic data. MATERIALS AND METHODS: Self-reported measures of periodontitis, demographic data and clinically established Stage III/IV periodontitis status were extracted from two Danish population-based cohorts (The Copenhagen Aging and Midlife Biobank [CAMB] and The Danish Health Examination Survey [DANHES]) and used to develop cross-validated machine learning models for the prediction of clinically established Stage III/IV periodontitis. Models were trained using 10-fold cross-validations repeated three times on the CAMB dataset (n = 1476), and the resulting models were validated in the DANHES dataset (n = 3585). RESULTS: The prevalence of Stage III/IV periodontitis was 23.2% (n = 342) in the CAMB dataset and 9.3% (n = 335) in the DANHES dataset. For the prediction of clinically established Stage III/IV periodontitis in the CAMB cohort, models reached area under the receiver operating characteristics (AUROCs) of 0.67-0.69, sensitivities of 0.58-0.64 and specificities of 0.71-0.80. In the DANHES cohort, models derived from the CAMB cohort achieved AUROCs of 0.64-0.70, sensitivities of 0.44-0.63 and specificities of 0.75-0.84. CONCLUSIONS: Applying cross-validated machine learning algorithms to demographic data and self-reported measures of periodontitis resulted in models with modest capabilities for the prediction of Stage III/IV periodontitis in two Danish cohorts.

Microvesicles in active lupus nephritis show Toll-like receptor 9-dependent co-expression of galectin-3 binding protein and double-stranded DNA.

Circulating microvesicles (MVs) from patients with systemic lupus erythematosus (SLE) express the type 1 interferon (IFN)-inducible protein galectin-3 binding protein (G3BP), which may enhance their deposition in the glomerular basement membrane. The release of G3BP-expressing MVs from normal peripheral blood mononuclear cells (PBMCs) is induced by Toll-like receptor 9 (TLR-9) ligands, and these vesicles contain autoantibody-accessible double-stranded DNA (dsDNA). This study compares the release of MVs expressing G3BP and dsDNA from PBMCs derived from SLE patients with or without active lupus nephritis (LN) and from healthy donors, and taps further into the potential dependency on IFN-α for their generation and impacts of TLR-7/TLR-9 co-stimulation. PBMCs from 10 healthy donors and 12 SLE patients, six of whom had active LN at study inclusion, were stimulated in-vitro with recombinant human IFN-α and the TLR-9 agonists oligodeoxynucleotide (ODN)2216 or ODN2395 alone or in combination with the TLR-7 agonist gardiquimod. MVs in the supernatants were subsequently isolated by differential centrifugation and their expression of G3BP and dsDNA was quantified by flow cytometry. Stimulation with ODN2395 significantly increased the release of MVs co-expressing G3BP and dsDNA from PBMCs isolated from healthy donors and SLE patients. The expression of G3BP on individual MVs and the proportion of G3BP and dsDNA double-positive MVs released were increased in active LN patients. Neither co-stimulation with gardiquimod nor with the IFN-α inhibitor IN-1 had any effect on the MV release induced by ODN2395. In conclusion, the TLR-9-mediated inducibility of MVs co-expressing G3BP and dsDNA is increased in SLE patients with active LN.

Peptidylarginine deiminase-4 gene polymorphisms are associated with systemic lupus erythematosus and lupus nephritis.

OBJECTIVE: Peptidylarginine deiminase-4 (PAD4) is highly expressed by neutrophils and essential for citrullination occurring during the formation of neutrophil extracellular traps, which have been implicated in the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN). Single-nucleotide polymorphisms (SNPs) in PADI4 influence PAD4 expression and functionality. Here, we investigate whether SNPs in PADI4 influence the risk of SLE or LN. METHOD: Altogether, 234 SLE patients and 484 controls were genotyped for nine PADI4 SNPs known to alter PAD4 functionality and/or expression, or to be associated with other autoimmune diseases, using an in-house multiplex Luminex assay. All analyses were adjusted for age and gender. RESULTS: Heterozygosity for rs1748033, and heterozygosity and homozygosity for rs1635564, were associated with increased occurrence of SLE [odds ratio (OR) 1.55, 95% confidence interval (CI) 1.08-2.23; OR 1.52, 95% CI 1.06-2.19; and OR 2.06, 95% CI 1.08-3.93, respectively]. Homozygosity for rs1635564 was also associated with increased occurrence of LN (OR 3.35, 95% CI 1.2-10.97). Notably, gene dose effects of the rs1635564 variant allele were observed for SLE (p = 0.005) and LN (p = 0.01). Carriage of minor alleles of five other SNPs (rs11203366, rs11203367, rs874881, rs2240340, and rs11203368) was associated with increased occurrence of LN and hypertension. CONCLUSION: The rs1635564 polymorphism of PADI4 is a candidate risk factor for SLE, particularly with renal involvement. Additional PADI4 polymorphisms also conferred increased risk of LN. Overall, these findings support the notion of PAD4 contributing to the pathogenesis of SLE and LN.

In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.

BACKGROUND AND OBJECTIVE: The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs). MATERIAL AND METHODS: Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement. RESULTS: The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1ß, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18). CONCLUSION: Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.

Porphyromonas gingivalis-induced production of reactive oxygen species, tumor necrosis factor-α, interleukin-6, CXCL8 and CCL2 by neutrophils from localized aggressive periodontitis and healthy donors: modulating actions of red blood cells and resolvin E1.

BACKGROUND AND OBJECTIVES: Porphyromonas gingivalis is regarded as a significant contributor in the pathogenesis of periodontitis and certain systemic diseases, including atherosclerosis. P. gingivalis occasionally translocates from periodontal pockets into the circulation, where it adheres to red blood cells (RBCs). This may protect the bacterium from contact with circulating phagocytes without affecting its viability. MATERIAL AND METHODS: In this in vitro study, we investigated whether human peripheral blood neutrophils from 10 subjects with localized aggressive periodontitis (LAgP) and 10 healthy controls release the proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor α (TNF-α), the chemokine (C-X-C motif) ligand 8 (CXCL8; also known as IL-8) and chemokine (C-C motif) ligand 2 (CCL2; also known as monocyte chemotactic protein-1) and intracellular reactive oxygen species (ROS) in response to challenge with P. gingivalis. In addition, the impact of RBC interaction with P. gingivalis was investigated. The actions of resolvin E1 (RvE1), a known regulator of P. gingivalis induced neutrophil responses, on the cytokine and ROS responses elicited by P. gingivalis in cultures of neutrophils were investigated. RESULTS: Upon stimulation with P. gingivalis, neutrophils from subjects with LAgP and healthy controls released similar quantities of IL-6, TNF-α, CXCL8, CCL2 and intracellular ROS. The presence of RBCs amplified the release of IL-6, TNF-α and CCL2 statistically significant in both groups, but reduced the generation of ROS in the group of healthy controls, and showed a similar tendency in the group of subjects with LAgP. RvE1 had no impact on the production of intracellular ROS, TNF-α, IL-6, CXCL8 and CCL2 by neutrophils from either group, but tended to reduce the generation of ROS in subjects with LAgP in the absence of RBCs. CONCLUSIONS: Our data support that binding to RBCs protects P. gingivalis from ROS and concomitantly enhances neutrophil release of proinflammatory cytokines providing a selective advantage for P. gingivalis growth.

Influence of complement on neutrophil extracellular trap release induced by bacteria.

BACKGROUND AND OBJECTIVES: Neutrophil extracellular trap (NET) release has generally been studied in the absence of serum, or at low concentrations of untreated or heat-inactivated serum. The influence of serum complement on NET release therefore remains unclear. We examined the DNA release induced by Staphylococcus aureus and three oral bacteria: Actinomyces viscosus, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum subsp. vincettii. MATERIAL AND METHODS: Bacteria-stimulated NET release from the neutrophils of healthy donors was measured fluorometrically. Various complement containing and complement blocking conditions were used, including heat inactivation of the serum and antibody blockade of complement receptors 1 (CR1, CD35) and 3 (CR3, CD11b/CD18). RESULTS: While the presence of serum markedly enhanced NET release induced by S. aureus, A. actinomycetemcomitans, and to a lesser extent by A. viscosus, there was no enhancement of NET release induced by F. nucleatum. The serum-mediated enhancement of NET release by A. actinomycetemcomitans was neutralized by heat inactivation of serum complement, while this was not the case for S. aureus. Blockade of CR1, significantly reduced NET release induced by S. aureus, A. actinomycetemcomitans and A. viscosus, while blockade of CR3, had no effect. However, opsonization of S. aureus with antibodies may also have contributed to the enhancing effect of serum, independently of complement, in that purified IgG promoted NET release. CONCLUSIONS: In conclusion, complement opsonization promotes NET release induced by a variety of bacteria, including A. actinomycetemcomitans, and CR1 plays a dominant role in the process. Complement consumption or deficiency may compromise NETosis induced by some bacterial species, including A. actinomycetemcomitans. Within biofilms, the complement-inactivating abilities of some bacteria may protect other species against NETosis, while these are more vulnerable when adopting a planktonic lifestyle.

Altered balance between self-reactive T helper (Th)17 cells and Th10 cells and between full-length forkhead box protein 3 (FoxP3) and FoxP3 splice variants in Hashimoto's thyroiditis.

T helper type 17 (Th17) cells play a pathogenic role in autoimmune disease, while interleukin (IL)-10-producing Th10 cells serve a protective role. The balance between the two subsets is regulated by the local cytokine milieu and by the relative expression of intact forkhead box protein 3 (FoxP3) compared to FoxP3Δ2, missing exon 2. Th17 and Th10 cell differentiation has usually been studied using polyclonal stimuli, and little is known about the ability of physiologically relevant self-antigens to induce Th17 or Th10 cell differentiation in autoimmune thyroid disease. We subjected mononuclear cells from healthy donors and patients with Hashimoto's thyroiditis (HT) or Graves' disease (GD) to polyclonal stimulation, or stimulation with human thyroglobulin (TG), human thyroid peroxidase (TPO), or Esherichia coli lipopolysaccharide (LPS). TPO and LPS induced increased differentiation of naive CD4(+) CD45RA(+) CD45R0(-) T cells from HT patients into Th17 cells. Th10 cell proportions were decreased in HT after polyclonal stimulation, but were comparable to those of healthy donors after antigen-specific stimulation. Taken together, our data show that an increased Th17 : Th10 ratio was found in HT patients after stimulation with thyroid-specific self-antigens. We also observed an elevated baseline production of IL-6 and transforming growth factor (TGF)-ß1 and of mRNA encoding FoxP3Δ2 rather than intact FoxP3. This may contribute to the skewing towards Th17 cell responses in HT.

T cell reconstitution in allogeneic haematopoietic stem cell transplantation: prognostic significance of plasma interleukin-7.

Infections and acute graft-versus-host disease (aGVHD) are major causes of treatment-related mortality and morbidity following allogeneic haematopoietic stem cell transplantation (HSCT). Both complications depend on reconstitution of the T-lymphocyte population based on donor T cells. Although it is well established that Interleukin-7 (IL-7) is a cytokine essential for de novo T cell development in the thymus and homoeostatic peripheral expansion of T cells, associations between circulating levels of IL-7 and T cell reconstitution following HSCT have not been investigated previously. We prospectively measured IL-7 levels in 81 patients undergoing myeloablative HSCT with either sibling donor or an unrelated donor. Plasma IL-7 levels peaked at day +7 post-transplant (1.3-82.4 pg/ml), at the time of maximal lymphopaenia. In multivariate analysis, peak levels of IL-7 were significantly higher in patients treated with anti-thymocyte globulin (ATG) compared with those not treated with ATG (P = 0.0079). IL-7 levels at day +7 were negatively associated with T cell counts at day +30 to +60 (at day +60: CD3(+) : ß = -10.6 × 10(6) cells/l, P = 0.0030; CD8(+) : ß = -8.4 × 10(6) cells/l, P = 0.061; CD4(+) : ß = -2.1 × 10(6) cells/l, P = 0.062) in multivariate analyses. In adults, high IL-7 levels were associated with increased risk of grade II-IV aGVHD (OR = 5.4, P = 0.036) and reduced overall survival (P = 0.046). The present data indicate that high plasma levels of IL-7 in the early post-transplant period are predictive for slow T cell reconstitution, increased risk of aGVHD and increased mortality following HSCT.

The complement system and its role in the pathogenesis of periodontitis: current concepts.

Periodontitis is a highly prevalent inflammatory disease in tooth supporting tissues, induced by bacteria growing in a biofilm on tooth surfaces. Components of the complement system are present in the periodontal tissue and the system is activated in periodontitis. Continuous complement activation and modulation by bacteria within the biofilm in periodontal pockets, however, may enhance local tissue destruction, providing the biofilm with both essential nutrients and space to grow. A more profound understanding of the mechanisms involved in complement-derived tissue degradation may facilitate the development of new treatment concepts for periodontitis. Further studies on the role of complement in periodontitis pathogenesis may also contribute to the understanding of why some individuals fail to resolve periodontitis. Here, we review evidence that links complement to the pathogenesis of periodontitis with an emphasis on interaction of complement with bacteria from periodontitis-associated biofilm.

Single nucleotide polymorphisms in genes encoding toll-like receptors 7, 8 and 9 in Danish patients with systemic lupus erythematosus.

Several studies indicate a role for toll-like receptors (TLRs) in the pathogenesis of systemic lupus erythematosus (SLE). We aimed to investigate the risk of SLE and typical clinical and serological manifestations of SLE potentially conferred by selected single nucleotide polymorphisms (SNPs) of genes encoding TLR7, TLR8, and TLR9. Using a multiplexed bead-based assay, we analyzed eight SNPs in a cohort of 142 Danish SLE patients and a gender-matched control cohort comprising 443 individuals. Our results showed an association between the rs3853839 polymorphism of TLR7 and SLE (G vs. C, P = 0.008, OR 1.60, 95 % CI 1.12-2.27 in females; P = 0.02, OR 4.50, 95 % CI 1.18-16.7 in males) confirming recent findings in other populations. Additionally, an association between the rs3764879 polymorphism of TLR8 and SLE (G vs. C, P < 0.05, OR 1.36, 95 % CI 0.99-1.86 in females; P = 0.06, OR 4.00, 95 % CI 0.90-17.3 in males) was found. None of the other investigated SNPs were associated with SLE but several SNPs were associated with clinical and serological manifestations. In summary, a previously shown association between the rs3853839 SNP of TLR7 and SLE in Asian patients was also found in Danish patients. Together with the association of several other SNPs of TLR8 and TLR9 with various clinical and serological manifestations of SLE these findings corroborate the pathogenic significance of TLRs in SLE.

Genetic polymorphisms of dsRNA ligating pattern recognition receptors TLR3, MDA5, and RIG-I. Association with systemic lupus erythematosus and clinical phenotypes.

This study aimed to demonstrate possible associations between genetic polymorphisms in Toll-like receptor 3, interferon induced with helicase C domain 1 (IFIH1) and DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 and systemic lupus erythematosus (SLE), including the phenotypes lupus nephritis and malar rash, as well as the presence of autoantibodies against nucleic acid-containing complexes. Genotyping was carried out in two Danish cohorts [Copenhagen (CPH) and Odense (ODE)] totaling 344 patients and was compared with 641 previously genotyped healthy controls. In the ODE cohort, the patients were only genotyped for the rs1990760 polymorphism of IFIH1. Single nucleotide polymorphisms (SNPs) were determined by a multiplex bead-based assay (CPH cohort) or real-time PCR (ODE cohort). Associations were investigated using the Cochran-Armitage trend test. The odds ratio (OR) for minor allele homozygotes versus major allele homozygotes suggested a protective effect of the IFIH1 rs1990760 SNP for SLE in the ODE cohort [OR 0.52, 95 % confidence intervals (95 % CI) 0.31-0.88, Pcorr. = 0.05] but not in the CPH cohort, although the OR suggested a trend in the same direction, and when combining the two patient cohorts, ORs were 0.57, 95 % CI 0.37-0.88. None of the other investigated polymorphisms showed any association with SLE. Regarding phenotypes, we found a statistically significant association between rs1990760 and malar rash in the CPH cohort, with ORs suggesting a protective effect (OR 0.28, 95 % CI 0.13-0.62 for heterozygotes and OR 0.11, 95 % CI 0.03-0.41 for homozygotes, Pcorr. = 0.0001). There were no significant associations between rs1990760 and presence of anti-dsDNA, anti-U1RNP, or anti-Smith antibodies. Our study supports previous findings of an association between the rs1990760 polymorphism of IFIH1 and SLE and indicates that this SNP may also be associated with malar rash in SLE patients although this finding needs confirmation.

Altered bacterial profiles in saliva from adults with caries lesions: a case-cohort study.

The aim of this study was to learn whether presence of caries in an adult population was associated with a salivary bacterial profile different from that of individuals without untreated caries. Stimulated saliva samples from 621 participants of the Danish Health Examination Survey were analyzed using the Human Oral Microbe Identification Microarray technology. Samples from 174 individuals with dental caries and 447 from a control cohort were compared using frequency and levels of identified bacterial taxa/clusters as endpoints. Differences at taxon/cluster level were analyzed using Mann-Whitney's test with Benjamini-Hochberg correction for multiple comparisons. Principal component analysis was used to visualize bacterial community profiles. A reduced bacterial diversity was observed in samples from subjects with dental caries. Five bacterial taxa (Veillonella parvula, Veillonella atypica, Megasphaera micronuciformis, Fusobacterium periodontium and Achromobacter xylosoxidans) and one bacterial cluster (Leptotrichia sp. clones C3MKM102 and GT018_ot417/462) were less frequently found in the caries group (adjusted p value <0.01) while two bacterial taxa (Solobacterium moorei and Streptococcus salivarius) and three bacterial clusters (Streptococcus parasanguinis I and II and sp. clone BE024_ot057/411/721, Streptococcus parasanguinis I and II and sinensis_ot411/721/767, Streptococcus salivarius and sp. clone FO042_ot067/755) were present at significantly higher levels (adjusted p value <0.01). The principal component analysis displayed a marked difference in the bacterial community profiles between groups. Presence of manifest caries was associated with a reduced diversity and an altered salivary bacterial community profile. Our data support recent theories that ecological stress-induced changes of commensal microbial communities are involved in the shift from oral health to tooth decay.

MicroRNA-regulated non-viral vectors with improved tumor specificity in an orthotopic rat model of hepatocellular carcinoma.

In hepatocellular carcinoma (HCC), tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs (miRNAs) are powerful negative regulators of gene expression and many are downregulated in human HCC. We identified seven miRNAs that are also downregulated in tumors in a rat hepatoma model (P<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these miRNAs. Attenuation of luciferase expression by the corresponding miRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two miRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared with the control vector (6.40 and 0.26%, respectively; P<0.05). In tumor, miR-122 had a nonsignificant trend towards decreased (∼50%) expression, whereas miR-26 had no significant effect on tumor expression. To our knowledge, this is the first work using differentially expressed miRNAs to de-target transgene expression in an orthotopic hepatoma model and to identify miR-26a, in addition to miR-122, for de-targeting liver. Considering the heterogeneity of miRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease.

Glatiramer acetate antibodies, gene expression and disease activity in multiple sclerosis.

BACKGROUND: Glatiramer acetate (GA) treatment suppresses disease activity in multiple sclerosis (MS). The immunological response to treatment may differ in patients who are stable on GA therapy and patients with breakthrough disease activity, but the results of previous studies are inconsistent. OBJECTIVES: We studied the immunological response to GA and its relationship with disease activity. METHODS: Anti-GA antibodies in plasma and the expression of genes encoding cytokines and T-cell-polarizing transcription factors in blood cells were analysed by flow cytometric bead array and polymerase chain reaction (PCR) analysis in 39 untreated and 29 GA-treated relapsing-remitting MS patients. Definition of breakthrough disease was based on the occurrence of relapses, disability progression, or gadolinium (Gd)-enhanced MRI. RESULTS: The expression of T helper type 1 (Th1) and Th17 cytokines and transcription factors was reduced during long-term treatment, but there was no relationship between the expression of cytokines and transcription factors and anti-GA antibodies. High expression of mRNA encoding GATA3 and lymphotoxin-ß (LT-ß) was associated with low disease activity in Gd-enhanced MRI studies. None of the variables studied were associated with clinical disease activity. GA treatment resulted in the development of IgG and IgG4 anti-GA antibodies during the first months of treatment, persisting during long-term treatment. CONCLUSIONS: The observed relationship between the expression of mRNA encoding GATA3 and LT-ß expression and MRI disease activity deserves further analysis in future studies. The development of anti-GA antibodies was observed in all patients treated with GA, but this was not related with measures of cellular immunity, clinical or MRI disease activity.

PET in vivo generators 134Ce and 140Nd on an internalizing monoclonal antibody probe.

The in vivo-generator radionuclides 140Nd (t1/2 = 3.4 d) and 134Ce (t1/2 = 3.2 d) were used to trace a urokinase-type plasminogen activator (uPA)-targeting mouse monoclonal antibody, ATN-291, in U87 MG xenograft tumor-bearing mice. ATN-291 is known to internalize on the uPA/uPA-receptor pair, making it an appropriate targeting vector for investigating the fate of in vivo generator daughters on internalizing probes. Ante-mortem and post-mortem PET imaging at 120 h post-injection gave no indication of redistribution of the positron emitting daughter nuclides 134La and 140Pr from tumor tissue (p > 0.5). The lack of redistribution indicates that the parent radionuclides 134Ce and 140Nd could be considered as long-lived PET-diagnostic matches to therapeutic radionuclides like 177Lu, 161Tb and 225Ac when internalizing bioconjugates are employed.

The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity.

Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRα170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register.

Systemic adverse events following rituximab therapy in patients with Graves' disease.

BACKGROUND AND AIM: Rituximab (RTX) therapy has shown promising results in Graves' disease (GD), with or without ophthalmopathy. We examined the occurrence of adverse events in GD patients treated with RTX. SUBJECTS AND METHODS: Ten patients received RTX and methimazole, while 10 patients received methimazole only. Adverse events were recorded, and the presence of circulating immune complexes (CIC) was measured as IgG, IgM and complement component 3 (C3) depositing on normal monocytes following incubation with patient plasma. RESULTS: Five patients had benign infusion-related adverse events at first infusion. Two patients developed a serum sickness-like reaction 11 days after the first RTX-infusion. One of these patients developed diarrhea, raised orosomucoid levels, lowgrade inflammation in colonoscopic biopsies, and iridocyclitis 1 yr later. At day 14, the most pronounced immunoglobulin/ C3-adherent to the test monocytes, indicative of CIC, was observed in the presence of plasma from these 2 patients (p=0.003 to p=0.01 vs asymptomatic patients). A 3rd patient had recurrent fever and symmetric polyarthritis from day 38, and colonoscopy-verified ulcerative colitis at day 68. This patient had the 3rd highest increase in Ig deposition on monocytes by day 14. The arthralgias persisted in 2 of the patients, despite glucocorticoid rescue therapy. CONCLUSIONS: We report articular adverse events in 3 and gastrointestinal symptoms in 2 out of 10 GD patients who received RTX without concurrent immunosupression. The joint symptoms were related to CIC formation.

COVID-19 as a mediator of interferon deficiency and hyperinflammation: Rationale for the use of JAK1/2 inhibitors in combination with interferon.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) elicits an interferon (IFN) deficiency state, which aggravates the type I interferon deficiency and slow IFN responses, which associate with e.g. aging and obesity. Additionally, SARS-CoV-2 may also elicit a cytokine storm, which accounts for disease progression and ultimately the urgent need of ventilator support. Based upon several reports, it has been argued that early treatment with IFN-alpha2 or IFN-beta, preferentially in the early disease stage, may prohibit disease progression. Similarly, preliminary studies have shown that JAK1/2 inhibitor treatment with ruxolitinib or baricitinib may decrease mortality by dampening the deadly cytokine storm, which - in addition to the virus itself - also contributes to multi-organ thrombosis and multi-organ failure. Herein, we describe the rationale for treatment with IFNs (alpha2 or beta) and ruxolitinib emphasizing the urgent need to explore these agents in the treatment of SARS-CoV-2 - both as monotherapies and in combination. In this context, we take advantage of several safety and efficacy studies in patients with the chronic myeloproliferative blood cancers (essential thrombocythemia, polycythemia vera and myelofibrosis) (MPNs), in whom IFN-alpha2 and ruxolitinib have been used successfully for the last 10 (ruxolitinib) to 30 years (IFN) as monotherapies and most recently in combination as well. In the context of these agents being highly immunomodulating (IFN boosting immune cells and JAK1/2 inhibitors being highly immunosuppressive and anti-inflammatory), we also discuss if statins and hydroxyurea, both agents possessing anti-inflammatory, antithrombotic and antiviral potentials, might be inexpensive agents to be repurposed in the treatment of SARS-CoV-2.

Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy.

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.