Transcriptome alterations in peripheral blood B cells of patients with multiple sclerosis receiving immune reconstitution therapy.

BACKGROUND: Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called "immune reconstitution therapies" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS. OBJECTIVES: The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy. METHODS: B cells were obtained from blood samples of patients with relapsing-remitting MS (n = 50), patients with primary progressive MS (n = 13) as well as healthy controls (n = 28). The patients with relapsing MS received either monthly infusions of natalizumab (n = 29) or a pulsed IRT with alemtuzumab (n = 15) or cladribine (n = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration. RESULTS: Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2, IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction. CONCLUSIONS: We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers.

SNX27 regulates DRA activity and mediates its direct recycling by PDZ-interaction in early endosomes at the apical pole of Caco2 cells.

DRA (downregulated in adenoma, SLC26A3) and NHE3 (Na+/H+ exchanger 3, SLC9A3) together mediate intestinal electroneutral NaCl absorption. Both transporters contain PDZ (postsynaptic density 95, disc large, zonula occludens 1) binding motifs and interact with PDZ adaptor proteins regulating their activity and recycling. SNX27 (sorting nexin 27) contains a PDZ domain and is involved in the recycling of cargo proteins including NHE3. The interaction of SNX27 with DRA and its potential role for the activity and recycling of DRA have been evaluated in this study. SNX27 specifically interacts with DRA via its PDZ domain. The knockdown (KD) of SNX27 reduced DRA activity by 50% but was not accompanied by a decrease of DRA surface expression. This indicates that DRA is trafficked to specific functional domains in the plasma membrane in which DRA is particularly active. Consistently, the disruption of lipid raft integrity by methyl-ß-cyclodextrin has an inhibitory effect on DRA activity that was strongly reduced after SNX27 KD. In differentiated intestinal Caco2 cells, superresolution microscopy and a novel quantitative axial approach revealed that DRA and SNX27 colocalize in rab5-positive early endosomes at the apical pole. SNX27 regulates the activity of DRA in the apical plasma membrane through binding with its PDZ domain. This interaction occurs in rab5-positive early endosomes at the apical pole of differentiated intestinal Caco2 cells. SNX27 is involved in the direct recycling of DRA to the plasma membrane where it is inserted into lipid rafts facilitating increased activity.NEW & NOTEWORTHY SNX27 has a PDZ domain and is involved in the regulation and recycling of transmembrane proteins. The role of SNX27 on the activity and recycling of the intestinal Cl-/HCO3- exchanger DRA has not yet been studied. This study shows that SNX27 directly interacts with DRA in early endosomes at the apical pole of intestinal Caco2 cells and mediates its direct recycling to facilitate high activity in lipid rafts in the apical plasma membrane.

Human adipocytes and CD34+ cells from the stromal vascular fraction of the same adipose tissue differ in their energy metabolic enzyme configuration.

Adipose tissue plays a role in energy storage and metabolic balance and is composed of different cell types. The metabolic activity of the tissue itself has been a matter of research for a long time, but comparative data about the energy metabolism of different cell types of human subcutaneous adipose tissue are sparse. Therefore, we compared the activity of major energy metabolic pathways of adipocytes and CD34+ cells from the stromal vascular fraction (SVF) separated from the same tissue. This CD34+ cell fraction is enriched with adipose tissue-derived mesenchymal progenitors, as they account for the largest proportion of CD34+ cells of the SVF. Adipocytes displayed significantly higher mitochondrial enzyme capacities compared to CD34+ SVF-cells, as shown by the higher activities of isocitrate dehydrogenase and ß-hydroxyacyl-CoA dehydrogenase. Inversely, the CD34+ SVF-cells showed higher capacities for cytosolic carbohydrate metabolism, represented by the activity of glycolysis and the pentose phosphate pathway. Thus, the CD34+ SVF-cells may ensure the provision of pentose phosphates and reduction equivalents for the replication of DNA during proliferation. The data indicate that these two cell fractions of the human adipose tissue vary in their metabolic configuration adapted to their physiological demands regarding proliferation and differentiation in vivo.

A therapy with miglustat, 2-hydroxypropyl-ß-cyclodextrin and allopregnanolone restores splenic cholesterol homeostasis in Niemann-pick disease type C1.

BACKGROUND: Niemann-Pick disease type C1 (NPC1) is an autosomal-recessive lipid-storage disorder with an estimated minimal incidence of 1/120,000 live births. Besides other neuronal and visceral symptoms, NPC1 patients develop spleen dysfunction, isolated spleno- or hepatosplenomegaly and infections. The mechanisms of splenomegaly and alterations of lipid metabolism-related genes in NPC1 disease are still poorly understood. METHODS: Here, we used an NPC1 mouse model to study a splenoprotective effect of a treatment with miglustat, 2-hydroxypropyl-ß-cyclodextrin and allopregnanolone and showed that this treatment has a positive effect on spleen morphology and lipid metabolism. RESULTS: Disease progress can be halted and blocked at the molecular level. Mutant Npc1 (Npc1-/-) mice showed increased spleen weight and increased lipid accumulation that could be avoided by our treatment. Also, FACS analyses showed that the increased number of splenic myeloid cells in Npc1-/- mice was normalized by the treatment. Treated Npc1-/- mice showed decreased numbers of cytotoxic T cells and increased numbers of T helper cells. CONCLUSIONS: In summary, the treatment promotes normal spleen morphology, stabilization of lipid homeostasis and blocking of inflammation, but alters the composition of T cell subtypes.

Allogeneic yet major histocompatibility complex-matched bone marrow transplantation in mice results in an impairment of osteoblasts and a significantly reduced trabecular bone.

Secondary osteopenia following allogeneic bone marrow or stem cell transplantation (BMT or HSCT) is a significant source of morbidity in patients. It is believed to be caused by a number of factors related to the myeloablative conditioning and subsequent therapy regimen. We here aimed to investigate whether the allogeneic bone marrow by itself directly impacts on the bone mass of the patient. We thus performed syn- and allogeneic BMT between two inbred mouse strains, which share an identical major histocompatibility complex background yet differ in their bone phenotypes. BMT was well tolerated, yielded survival rates of 97% and allowed for a regular physiological development. However, allogeneic BMT led to a significant reduction of trabecular bone mass that was independent of strain, sex, immunosuppressive medication, complications resulting from graft versus host disease, underlying bone phenotype and numbers of osteoclasts. Instead, reduced trabecular bone mass correlated with reduced plasma levels of amino-terminal propeptide of type I collagen. Our results suggest that osteopenia following allogeneic BMT is significantly influenced by an impaired osteoblast activity that may stem from a lack of communication between the resident osteoblasts and an allogeneic bone marrow-derived cell type. Elucidating this incompatibility will open new approaches for the therapy of secondary osteopenia.

A unique population of IgG-expressing plasma cells lacking CD19 is enriched in human bone marrow.

Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19(+) PC, CD19(-) PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DR(low)Ki-67(-)CD95(low)CD28(+)CD56(+/-), with increased BCL2 and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin VH rearrangements of CD19(-) BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19(-) PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19(+) and CD19(-) PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection.

Experimental silicosis does not aggravate collagen-induced arthritis in mice.

OBJECTIVES: To investigate the effect of chronic lung inflammation on the incidence and severity of collagen-induced arthritis in mice. METHODS: Chronic lung inflammation in the form of silicosis was induced via intranasal application of silica particles. Immunization with collagen Type II commenced one week later and mice were sacrificed six weeks after booster immunization. Thereafter, silicosis was confirmed via flow cytometry and arthritis was evaluated performing knee and paw histology. RESULTS: Pronounced lung inflammation in the silica-treated compared to PBS-treated control mice was demonstrated by significantly elevated broncho-alveolar lavage (BAL) cell count, attributable to increased numbers of macrophages and granulocytes. Inflammation in the lungs was not associated with elevated PAD2 and PAD4 expression, yet silica treated animals had significantly higher aCCP serum titers. However, lung inflammation did not lead to an increase in the incidence of arthritis, nor did it exacerbate the macroscopic or histologic joint scores. CONCLUSIONS: Chronic lung inflammation resulting from silicosis does not aggravate collagen-induced arthritis in mice.

24-nor-ursodeoxycholic acid ameliorates inflammatory response and liver fibrosis in a murine model of hepatic schistosomiasis.

BACKGROUND & AIMS: Intrahepatic granuloma formation and fibrosis characterize the pathological features of Schistosoma mansoni infection. Based on previously observed substantial anti-fibrotic effects of 24-nor-ursodeoxycholic acid (norUDCA) in Abcb4/Mdr2(-/-) mice with cholestatic liver injury and biliary fibrosis, we hypothesized that norUDCA improves inflammation-driven liver fibrosis in S. mansoni infection. METHODS: Adult NMRI mice were infected with 50 S. mansoni cercariae and after 12 weeks received either norUDCA- or ursodeoxycholic acid (UDCA)-enriched diet (0.5% wt/wt) for 4 weeks. Bile acid effects on liver histology, serum biochemistry, key regulatory cytokines, hepatic hydroxyproline content as well as granuloma formation were compared to naive mice and infected controls. In addition, effects of norUDCA on primary T-cell activation/proliferation and maturation of the antigen-presenting-cells (dendritic cells, macrophages) were determined in vitro. RESULTS: UDCA as well as norUDCA attenuated the inflammatory response in livers of S. mansoni infected mice, but exclusively norUDCA changed cellular composition and reduced size of hepatic granulomas as well as TH2-mediated hepatic fibrosis in vivo. Moreover, norUDCA affected surface expression level of major histocompatibility complex (MHC) class II of macrophages and dendritic cells as well as activation/proliferation of T-lymphocytes in vitro, whereas UDCA had no effect. CONCLUSIONS: This study demonstrates pronounced anti-inflammatory and anti-fibrotic effects of norUDCA compared to UDCA in S. mansoni induced liver injury, and indicates that norUDCA directly represses antigen presentation of antigen presenting cells and subsequent T-cell activation in vitro. Therefore, norUDCA represents a promising drug for the treatment of this important cause of liver fibrosis.

Bone resorption correlates with the frequency of CD5⁺ B cells in the blood of patients with rheumatoid arthritis.

OBJECTIVE: The prevention of bone resorption and subsequent joint destruction is one of the main challenges in the treatment of patients suffering from RA. Various mechanisms have previously been described that contribute to bone resorption in tightly defined cohorts. Here we analysed a cross-sectional cohort of RA patients and searched for humoral and cellular markers in the peripheral blood associated with bone resorption. METHODS: We enrolled 61 consecutive RA patients positive for ACPA. Blood was analysed by flow cytometry to determine the percentages of regulatory T cells and B cell subpopulations. Cytokine (TNF-α, IL-6, IL-10) and ACPA levels as well as the bone resorption marker CTX-1 were determined from the patients' sera. Standard clinical disease parameters were included. RESULTS: Multivariate analyses showed that the percentages of CD5(+) B cells were positively correlated with CTX-1 serum levels. However, neither low-avidity ACPA nor serum IL-6 levels, both known to be produced by CD5(+) cells, were associated with CTX-1 in patients' sera. There was no correlation between CTX-1 levels and clinical parameters or ACPA levels. CONCLUSION: In summary, we found that the CD5(+) B cell population is associated with bone resorption as measured via serum CTX-1 levels in a cross-sectional cohort of RA patients. However, a possible functional link between CD5(+) B cells and bone resorption still needs to be defined.

The presence of FCGR2B promoter or transmembrane region variant alleles leads to reduced serum IL-6 levels in rheumatoid arthritis.

The inhibitory FcγRIIB plays an important role for the peripheral B cell tolerance and plasma cell homeostasis, and any malfunctioning is predicted to result in humoral autoimmunity. An association between rheumatoid arthritis (RA) and FCGR2B promoter and TM region variant alleles, both of which result in a reduced functionality, is only insufficiently elucidated. We here set out to investigate the impact of these variants on disease progression in European RA patients. One hundred and five ACPA-positive RA patients were genotyped for the FCGR2B -386G>C promoter and the 695T>C transmembrane region variants. Moreover, serum titers for IL-6, TNFα, CTX-1 and ACPAs were measured and peripheral blood T cell and B cell populations analyzed for expression of the activation markers CTLA-4 and CD86. The presence of an FCGR2B variant allele results in reduced serum IL-6, a trend toward later disease onset and reduced requirement for biological treatment, but does not seem to aggravate RA. Likewise, the presence of the TM region variant allele is associated with a lower activation state of the Tregs and of naïve and memory B cells. The observation of a malfunctioning FcγRIIb not aggravating RA is counterintuitive at first. However, the etiology of RA is linked to inflammatory episodes, and the lack of B cell inhibition may support an accelerated antibody-mediated clearance of the disease initiating and perpetuating agents. It would thereby shorten inflammatory episodes, postpone the onset of disease and result in a less severe course of RA in carriers of FCGR2B variant alleles.

High bone mass in the STR/ort mouse results from increased bone formation and impaired bone resorption and is associated with extramedullary hematopoiesis.

We here describe the novel high bone mass phenotype in STR/ort mice that leads to increased bone masses of cortical and trabecular bone and is associated with elevated osteoblast activity and impaired osteoclast function alike. Comparison of STR/ort and C57BL/6 mice reveals an increase in trabecular bone volumes of the vertebrae and at femoral metaphysis. In the females, this difference is significant as early as 2 months of age and at 9 months the females by far exceed their age matched males in all parameters measured. The increase in cortical bone mass at femoral diaphysis results from an apposition to the endosteal surface, it is significant for both sexes as early as 1 month of age and leads to bone marrow compression and extramedullary hematopoiesis. Altered activities of both, the osteoblast and the osteoclast contribute to the high bone mass and collectively this phenotype supports a multifactorial pathogenesis. Moreover, the spontaneous development of osteoarthritis in male STR/ort mice is suggestive of a tight correlation between trabecular bone mass and the development of degenerative changes of the articular cartilage.

Vector-based SARS-CoV-2 vaccination is associated with improved T-cell responses in hematological neoplasia.

In order to elucidate mechanisms for severe acute respiratory syndrome coronavirus 2 vaccination success in hematological neoplasia, we, herein, provide a comprehensive characterization of the spike-specific T-cell and serological immunity induced in 130 patients in comparison with 91 healthy controls. We studied 121 distinct T-cell subpopulations and the vaccination schemes as putative response predictors. In patients with lymphoid malignancies an insufficient immunoglobulin G (IgG) response was accompanied by a healthy CD4+ T-cell function. Compared with controls, a spike-specific CD4+ response was detectable in fewer patients with myeloid neoplasia whereas the seroconversion rate was normal. Vaccination-induced CD4+ responses were associated to CD8+ and IgG responses. Vector-based AZD1222 vaccine induced more frequently detectable specific CD4+ responses in study participants across all cohorts (96%; 27 of 28), whereas fully messenger RNA-based vaccination schemes resulted in measurable CD4+ cells in only 102 of 168 participants (61%; P < .0001). A similar benefit of vector-based vaccination was observed for the induction of spike-specific CD8+ T cells. Multivariable models confirmed vaccination schemes that incorporated at least 1 vector-based vaccination as key feature to mount both a spike-specific CD4+ response (odds ratio, 10.67) and CD8+ response (odds ratio, 6.56). Multivariable analyses identified a specific CD4+ response but not the vector-based immunization as beneficial for a strong, specific IgG titer. Our study reveals factors associated with a T-cell response in patients with hematological neoplasia and might pave the way toward tailored vaccination schemes for vaccinees with these diseases. The study was registered at the German Clinical Trials Register as #DRKS00027372.

Senescence determines the fate of activated rat pancreatic stellate cells.

In chronic pancreatitis (CP), persistent activation of pancreatic stellate cells (PSC) converts wound healing into a pathological process resulting in organ fibrosis. Here, we have analysed senescence as a novel mechanism involved in the termination of PSC activation and tissue repair. PSC senescence was first studied in vitro by establishing long-term cultures and by applying chemical triggers, using senescence-associated ß-Galactosidase (SA ß-Gal) as a surrogate marker. Subsequently, susceptibility of PSC to immune cell-mediated cytolysis was investigated employing cocultures. Using the model of dibutyltin dichloride-induced CP in rats, appearance of senescent cells was monitored by immunohistochemistry and immunofluorescence, and correlated with the progression of tissue damage and repair, immune cell infiltration and fibrosis. The results indicated that long-term culture and exposure of PSC to stressors (doxorubicin, H(2) O(2) and staurosporine) induced senescence. Senescent PSC highly expressed CDKN1A/p21, mdm2 and interleukin (IL)-6, but displayed low levels of α-smooth muscle actin. Senescence increased the susceptibility of PSC to cytolysis. In CP, the number of senescent cells correlated with the severity of inflammation and the extension of fibrosis. Areas staining positive for SA ß-Gal overlapped with regions of fibrosis and dense infiltrates of immune cells. Furthermore, a close physical proximity of immune cells and activated PSC was observed. We conclude that inflammation, PSC activation and cellular senescence are timely coupled processes which take place in the same microenvironment of the inflamed pancreas. Lymphocytes may play a dual-specific role in pancreatic fibrogenesis, triggering both the initiation of wound healing by activating PSC, and its completion by killing senescent stellate cells.

Longitudinal Humoral and Cellular Immune Responses Following SARS-CoV-2 Vaccination in Patients with Myeloid and Lymphoid Neoplasms Compared to a Reference Cohort: Results of a Prospective Trial of the East German Study Group for Hematology and Oncology (OSHO).

Purpose: To assess humoral responses longitudinally and cellular immunogenicity following SARS-CoV-2-vaccination in patients with hematologic and oncologic malignancies receiving checkpoint-inhibitors. Methods: This prospective multicenter trial of the East-German-Study-Group-for-Hematology-and-Oncology, enrolled 398 adults in a two (patients; n = 262) to one (controls; n = 136) ratio. Pre-vaccination, day 35 (d35), and day 120 (d120) blood samples were analyzed for anti-spike antibodies and d120 IL-2+IFNγ+TNFα+-CD4+- and CD8+-cells. Laboratories were blinded for patients and controls. Results: Patients belonged to the myeloid (n = 131), lymphoid (n = 104), and checkpoint-inhibitor (n = 17) cohorts. While d35 seroconversion was higher in controls (98%) compared to patients (68%) (p < 0.001), d120 seroconversion improved across all patient cohorts [checkpoint-inhibitors (81% to 100%), myeloid (82% to 97%), lymphoid (48% to 66%)]. CD4+- and CovCD8+-cells in the lymphoid (71%/31%) and control (74%/42%) cohorts were comparable but fewer in the myeloid cohort (53%, p = 0.003 /24%, p = 0.03). In patients with hematologic malignancies, no correlation between d120 humoral and cellular responses was found. A sizeable fraction of lymphoid patients demonstrated T-cell responses without detectable spike-specific-IgGs. Conclusions: Evidence of vaccine-elicited humoral and/or cellular immunogenicity in most patients is provided. Both humoral and cellular responses are crucial to determine which patients will generate/maintain immunity. The findings have implications on public health policy regarding recommendations for SARS-CoV-2 booster doses.

Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study.

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing 9 disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis, we subsequently used canonical correlation analysis of 176 training cases to project the multidimensional space of all 26 immunophenotypic parameters into 36 2-dimensional plots for each possible pairwise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%), and mantle cell lymphoma (95.4%). Burkitt and CD10+ diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD10- diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms.

The impact of HLA-DRB alleles on the subclass titres of antibodies against citrullinated peptides.

OBJECTIVES: The association between HLA-DR haplotypes and RA have been well established. However, the molecular mechanisms of how HLA mediates susceptibility and/or progression of the disease remain elusive. We therefore turned to the RA-specific antibodies directed against citrullinated peptide antigens (ACPAs) and investigated the association between HLA-DRB1 shared epitope (SE) alleles and the IgG subclass titres of cyclic citrullinated peptide (CCP)- and mutated citrullinated vimentin (MCV)-specific antibodies. METHODS: One hundred and twenty-seven RA patients were typed for their HLA-DRB1 haplotypes applying low resolution and alleles potentially carrying the SE were sequenced. All patients' sera were analysed by ELISA for the presence of ACPA and 77 patients positive for CCP-specific antibodies were further analysed for the respective IgG subclasses. Subclass titres were then correlated to the presence of a SE. Finally, all patients were screened for the HLA-DRB4-associated splice variant. RESULTS: We found a gene dosage effect of the HLA-DRB1*04-associated SE on both the MCV- and CCP-specific IgG3 levels. The HLA-DRB4-associated splice variant accumulates in ACPA-negative RA patients. CONCLUSIONS: Both the dose-dependent increase in IgG3 among ACPA and the accumulation of the splice variant in ACPA-negative patients imply differential expression of the HLA alleles as the mechanism contributing to the susceptibility and/or disease progression of RA. The preponderance of IgG3 hints at a skewing towards a Th1 response and is reminiscent of increased signal strengths at the immunological synapse. Likewise, the abrogation of HLA-DRB4 expression due to the splice variant reduces the signal strength and seems to protect from ACPA development.

Cardiomyocyte Transplantation after Myocardial Infarction Alters the Immune Response in the Heart.

We investigated the influence of syngeneic cardiomyocyte transplantation after myocardial infarction (MI) on the immune response and cardiac function. Methods and Results: We show for the first time that the immune response is altered as a result of syngeneic neonatal cardiomyocyte transplantation after MI leading to improved cardiac pump function as observed by magnetic resonance imaging in C57BL/6J mice. Interestingly, there was no improvement in the capillary density as well as infarct area as observed by CD31 and Sirius Red staining, respectively. Flow cytometric analysis revealed a significantly different response of monocyte-derived macrophages and regulatory T cells after cell transplantation. Interestingly, the inhibition of monocyte infiltration accompanied by cardiomyocyte transplantation diminished the positive effect of cell transplantation alone. The number of CD68+ macrophages in the remote area of the heart observed after four weeks was also different between the groups. Transcriptome analysis showed several changes in the gene expression involving circadian regulation, mitochondrial metabolism and immune responses after cardiomyocyte transplantation. Conclusion: Our work shows that cardiomyocyte transplantation alters the immune response after myocardial infarction with the recruited monocytes playing a role in the beneficial effect of cell transplantation. It also paves the way for further optimization of the efficacy of cardiomyocyte transplantation and their successful translation in the clinic.

Integrative modeling of transcriptional regulation in response to antirheumatic therapy.

BACKGROUND: The investigation of gene regulatory networks is an important issue in molecular systems biology and significant progress has been made by combining different types of biological data. The purpose of this study was to characterize the transcriptional program induced by etanercept therapy in patients with rheumatoid arthritis (RA). Etanercept is known to reduce disease symptoms and progression in RA, but the underlying molecular mechanisms have not been fully elucidated. RESULTS: Using a DNA microarray dataset providing genome-wide expression profiles of 19 RA patients within the first week of therapy we identified significant transcriptional changes in 83 genes. Most of these genes are known to control the human body's immune response. A novel algorithm called TILAR was then applied to construct a linear network model of the genes' regulatory interactions. The inference method derives a model from the data based on the Least Angle Regression while incorporating DNA-binding site information. As a result we obtained a scale-free network that exhibits a self-regulating and highly parallel architecture, and reflects the pleiotropic immunological role of the therapeutic target TNF-alpha. Moreover, we could show that our integrative modeling strategy performs much better than algorithms using gene expression data alone. CONCLUSION: We present TILAR, a method to deduce gene regulatory interactions from gene expression data by integrating information on transcription factor binding sites. The inferred network uncovers gene regulatory effects in response to etanercept and thus provides useful hypotheses about the drug's mechanisms of action.

A novel method to efficiently isolate medullary thymic epithelial cells from murine thymi based on UEA-1 MicroBeads.

OBJECTIVE: The central mechanism for establishing a self-tolerant and functional T cell repertoire includes the promiscuous expression of otherwise tissue-restricted proteins by medullary thymic epithelial cells (TEC). We here demonstrate a novel and highly efficient method for isolating this rare key cell type. METHODS: We combined the enrichment of medullary TEC via UEA-1 MicroBeads with the subsequent depletion of residual CD45+ hematopoietic cells via specific size exclusion and compared our results to the standard Percoll enrichment method and isolation procedure via flow cytometric cell sorting. RESULTS: The addition of 2 µl UEA-1 MicroBeads per 108 thymus cells turned out best for optimal enrichment (an average of 22% purity compared to 1.2% for Percoll) and yield (an average of 1.73 × 105 medullary TEC per thymus compared to 5.16 × 104 for Percoll). After depletion of residual CD45+ cells, our method not only reached a purity of 75.5% but also turned out less stressful for the cells as compared to flow cytometric cell sorting. CONCLUSION: We here provide a fast and versatile procedure for enriching medullary TEC that yields higher purity and recovery rates than the standard Percoll enrichment method Our enrichment procedure in combination with CD45+ depletion via specific size exclusion is comparable to the current gold standard flow cytometric cell sorting method. SIGNIFICANCE STATEMENT: We developed a fast and versatile procedure to isolate a high number medullary TEC to investigate the biochemical processes of medullary TEC in more depths.

Periodontal treatment prevents arthritis in mice and methotrexate ameliorates periodontal bone loss.

Recent studies indicate a causal relationship between the periodontal pathogen P. gingivalis and rheumatoid arthritis involving the production of autoantibodies against citrullinated peptides. We therefore postulated that therapeutic eradication P. gingivalis may ameliorate rheumatoid arthritis development and here turned to a mouse model in order to challenge our hypothesis. F1 (DBA/1 x B10.Q) mice were orally inoculated with P. gingivalis before collagen-induced arthritis was provoked. Chlorhexidine or metronidazole were orally administered either before or during the induction phase of arthritis and their effects on arthritis progression and alveolar bone loss were compared to intraperitoneally injected methotrexate. Arthritis incidence and severity were macroscopically scored and alveolar bone loss was evaluated via microcomputed tomography. Serum antibody titres against P. gingivalis were quantified by ELISA and microbial dysbiosis following oral inoculation was monitored in stool samples via microbiome analyses. Both, oral chlorhexidine and metronidazole reduced the incidence and ameliorated the severity of collagen-induced arthritis comparable to methotrexate. Likewise, all three therapies attenuated alveolar bone loss. Relative abundance of Porphyromonadaceae was increased after oral inoculation with P. gingivalis and decreased after treatment. This is the first study to describe beneficial effects of non-surgical periodontal treatment on collagen-induced arthritis in mice and suggests that mouthwash with chlorhexidine or metronidazole may also be beneficial for patients with rheumatoid arthritis and a coexisting periodontitis. Methotrexate ameliorated periodontitis in mice, further raising the possibility that methotrexate may also positively impact on the tooth supporting tissues of patients with rheumatoid arthritis.