Immunomodulatory role of spleen tyrosine kinase in chronic inflammatory and autoimmune diseases.

BACKGROUND: The high prevalence of chronic inflammatory diseases or autoimmune reactions is a major source of concern and affects the quality of life of patients. Chronic inflammatory or autoimmune diseases are associated with many diseases in humans, including asthma, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and cancer. Splenic tyrosine kinase (SYK) is a non-receptor tyrosine kinase that plays an important role in immune receptor signalling in immune and inflammatory responses. METHODS: This is a review article in which we searched for keywords "splenic tyrosine kinase", "inflammation" and "autoimmune diseases" in published literature such as Pubmed and Web of Science to collect relevant information and then conducted a study focusing on the latest findings on the involvement of SYK in chronic inflammatory or autoimmune diseases. RESULTS: This paper reviews the regulation of Fcγ, NF-κB, B cell and T cell-related signalling pathways by SYK, which contributes to disease progression in chronic inflammatory and autoimmune diseases such as airway fibrosis, inflammatory skin disease and inflammatory bowel disease. CONCLUSION: This paper shows that SYK plays an important role in chronic inflammatory and autoimmune diseases. syk targets hematological, autoimmune and other inflammatory diseases and therefore, inhibition of SYK expression or blocking its related pathways may provide new ideas for clinical prevention and treatment of inflammatory or autoimmune diseases.

A gastric cancer patient-derived three-dimensional cell spheroid culture model.

Patients with gastric cancer exhibit considerable genetic and phenotypic heterogeneity, necessitating the establishment of a model for personalized medicine screening. Here, we successfully established three-dimensional cell spheroids (3D cell spheroids) from fresh gastric cancer tissues (n = 30) as in vitro models for further therapeutic screening. Hematoxylin-eosin and immunohistochemical staining of whole spheroids and parental tumor tissues revealed that the 3D cell spheroids recapitulated the parental tissue structure and maintained the histological characteristics of the parental tumor tissue during long-term expansion in vitro. Further, transcriptome sequencing verified that the cell spheroids could recapitulate the gene expression profile characteristics of the parental tumor tissue. Drug susceptibility testing of the 3D cell spheroids demonstrated that these cell spheroids can be used as a reliable model for drug prediction.

CRISPR/Cas9-Induced Mutagenesis of TMS5 Confers Thermosensitive Genic Male Sterility by Influencing Protein Expression in Rice (Oryza sativa L.).

The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5.

Critical roles of RGS16 in the mucosal inflammation of ulcerative colitis.

BACKGROUND: Ulcerative colitis is a chronic and progressive inflammatory disorder. The regulator of the G-protein signaling (RGS) is involved in the pathogenesis of several immune system disorders. RGS16, a member of the RGS protein superfamily, has been shown to play critical roles in several immune system-related diseases. However, the roles of RGS16 in ulcerative colitis remain to be elucidated. METHODS: We analyzed the expression of RGS16 in peripheral blood mononuclear cells (PBMCs) and inflamed mucosa of ulcerative colitis patients using quantitative reverse transcription-PCR, western blotting and immunohistochemistry. We performed Spearman's correlation to analyze the correlation between RGS16 expression and the ulcerative colitis endoscopic index of severity (UCEIS), Mayo index, erythrocyte sedimentation rate (ESR) and serum tumor necrosis factor alpha (TNF-a) and IL-17A levels. Further, PBMCs were stimulated with inflammatory cytokines in vitro . RESULTS: RGS16 expression significantly increased in the colonic mucosa and PBMCs from patients with ulcerative colitis and significantly correlated with the Mayo index, UCEIS, ESR and serum TNF-α and IL-17A levels. TNF-α upregulated RGS16 expression in PBMCs in a dose- and time-dependent manner via the nuclear factor kappa beta (NF-kB) signaling pathway. Moreover, anti-TNF treatment with infliximab significantly decreased RGS16 expression in PBMCs and intestinal mucosa of patients with ulcerative colitis. CONCLUSION: Our study revealed a novel mechanism by which RGS16 expression in ulcerative colitis is positively correlated with disease activity. Thus, RGS16 might serve as a potential therapeutic marker for the treatment of ulcerative colitis.

IRF5 Acts as a Potential Therapeutic Marker in Inflammatory Bowel Diseases.

BACKGROUND: Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn's disease (CD), are chronic inflammatory disorders. As is well known, interferon regulatory factor (IRF) 5 is closely associated with the pathogenesis of various inflammatory diseases. But the exact role of IRF5 in IBD remains unclear. METHODS: In this study, we detected IRF5 expression in peripheral blood mononuclear cells (PBMCs) and inflamed mucosa from IBD patients by immunohistochemistry, western blot, and quantitative real-time polymerase chain reaction. Peripheral blood CD4+ T cells were stimulated with inflammatory cytokines and transfected by lentivirus. RESULTS: In active IBD patients, the expression of IRF5 in PBMCs and inflamed colonic tissues was obviously increased and significantly associated with disease activity. Ectopic overexpression of IRF5 could promote the differentiation of IBD CD4+ T cells into Th1 and Th17 cells by regulating T-bet and RAR related orphan receptor C, whereas knockdown of IRF5 had the opposite effects. Tumor necrosis factor (TNF)-α upregulated expression of IRF5 in CD4+ T cells, but anti-TNF treatment with infliximab could markedly reduce IRF5 expression in CD4+ T cells and intestinal mucosa of CD patients. CONCLUSION: Our study reveals a novel mechanism that IRF5 levels are correlated with disease activity in IBD and might function as a possible marker for the management of IBD via regulating Th1 and Th17 immune responses and cytokine production.

Development of Chromosome Segment Substitution Lines (CSSLs) Derived from Guangxi Wild Rice (Oryza rufipogon Griff.) under Rice (Oryza sativa L.) Background and the Identification of QTLs for Plant Architecture, Agronomic Traits and Cold Tolerance.

Common wild rice contains valuable resources of novel alleles for rice improvement. It is well known that genetic populations provide the basis for a wide range of genetic and genomic studies. In particular, chromosome segment substitution lines (CSSLs) ais a powerful tool for fine mapping of quantitative traits, new gene discovery and marker-assisted breeding. In this study, 132 CSSLs were developed from a cultivated rice (Oryza sativa) cultivar (93-11) and common wild rice (Oryza rufipogon Griff. DP30) by selfing-crossing, backcrossing and marker-assisted selection (MAS). Based on the high-throughput sequencing of the 93-11 and DP30, 285 pairs of Insertion-deletions (InDel) markers were selected with an average distance of 1.23 Mb. The length of this DP30-CSSLs library was 536.4 cM. The coverage rate of substitution lines cumulatively overlapping the whole genome of DP30 was about 91.55%. DP30-CSSLs were used to analyze the variation for 17 traits leading to the detection of 36 quantitative trait loci (QTLs) with significant phenotypic effects. A cold-tolerant line (RZ) was selected to construct a secondary mapping F2 population, which revealed that qCT2.1 is in the 1.7 Mb region of chromosome 2. These CSSLs may, therefore, provide powerful tools for genome wide large-scale gene discovery in wild rice. This research will also facilitate fine mapping and cloning of QTLs and genome-wide study of wild rice. Moreover, these CSSLs will provide a foundation for rice variety improvement.

LRCH1 suppresses migration of CD4+ T cells and refers to disease activity in ulcerative colitis.

Background: Ulcerative colitis (UC) is a chronically remittent and progressive inflammatory disorder. LRCH1 is reported to be involved in the immune-regulation of several diseases. However, the exact roles of LRCH1 in UC are still obscure. Materials and Methods: LRCH1 expression was analyzed in the inflamed mucosa and peripheral blood mononuclear cells (PBMCs) from patients with UC by quantitative RT-PCR and immunohistochemistry. Peripheral blood CD4+ T cells were transfected with lentivirus-expressing LRCH1 (LV-LRCH1) or LV-sh-LRCH1, and cytokine expression was determined by using flow cytometry, quantitative RT-PCR and ELISA. Transfected CD4+ T cells were harvested to examine the capacity of chemotaxis using Transwell plate. Results: LRCH1 expression was highly decreased in colonic mucosa and PBMCs from patients with A-UC, and negatively correlated with disease activity. Up or down regulation of LRCH1 did not affect the differentiation of CD4+ T cells, and the related cytokines expression. Moreover, LRCH1 inhibited migratory capacity of CD4+ T cells toward CXCL12 by PKCα. Conclusion: LRCH1 plays an important role in the pathogenesis of UC, possibly through modulating the migration of CD4+ T cells. Therefore, targeting LRCH1 might serve as a novel therapeutic approach in the management of UC.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase activity by antibodies present in the cerebrospinal fluid of patients with multiple sclerosis.

We have previously shown that B cells and Abs reactive with GAPDH and antitriosephosphate isomerase (TPI) are present in lesions and cerebrospinal fluid (CSF) in multiple sclerosis (MS). In the current study, we studied the effect of anti-GAPDH and anti-TPI CSF IgG on the glycolytic enzyme activity of GAPDH and TPI after exposure to intrathecal IgG from 10 patients with MS and 34 patients with other neurologic diseases. The degree of inhibition of GAPDH activity by CSF anti-GAPDH IgG in the seven MS samples tested varied from 13 to 98%, which seemed to correlate with the percentage of anti-GAPDH IgG in the CSF IgG (1-45%). Inhibition of GAPDH activity (18 and 23%) by CSF IgG was seen in two of the 34 patients with other neurologic diseases, corresponding to the low percentage of CSF anti-GAPDH IgG (1 and 8%). In addition, depletion of anti-GAPDH IgG from CSF IgG, using immobilized GAPDH, removed the inhibitory effect of the IgG on GAPDH. No inhibition of GAPDH activity was seen with CSF samples not containing anti-GAPDH IgG. No inhibition of TPI activity was seen with any purified CSF IgG sample. These findings demonstrate an increased percentage of anti-GAPDH Abs in the CSF of patients with MS that can inhibit GAPDH glycolytic enzyme activity and may contribute to neuroaxonal degeneration.

B cell clonal expansion and somatic hypermutation of Ig variable heavy chain genes in the synovial membrane of patients with osteoarthritis.

Inflammatory mediators have been explored as possible factors in the initiation and/or progression of osteoarthritis (OA). This study shows that synovial infiltration by B lymphocytes is present in almost half of the knee OA cases. The degree of B lymphocyte infiltration is associated with more pronounced synovial inflammation and with the presence of plasma cells and lymphoid follicles in more severe cases. To examine whether these B cells are merely bystanders or could be involved in the pathogenesis of OA, we analyzed the Ig H chain variable region (V(H)) genes of B cells recovered from the synovial membrane of five OA patients with marked B cell infiltration. Sequence analysis of CDR3 regions of rearranged VDJ genes revealed clonal or oligoclonal B cell expansions in all cases. Expanded B cell clones in four of five OA patients showed clustered somatic mutations, occurring mainly in the CDRs and with a high replacement-to-silent ratio (>2.9), indicating that these cells are postgerminal center B cells that had been positively selected through their Ag receptor. These data demonstrate the presence in inflamed knee OA synovium of clonally expanded, Ag-driven B cells that may contribute to the development or progression of the disease.

Triosephosphate isomerase- and glyceraldehyde-3-phosphate dehydrogenase-reactive autoantibodies in the cerebrospinal fluid of patients with multiple sclerosis.

Our previous results revealed that Igs in lesions and single chain variable fragment Abs (scFv-Abs) generated from clonal B cells in the cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) bind to axons in MS brains. To study the axonal Ags involved in MS, we identified the glycolytic enzymes, triosephosphate isomerase (TPI) and GAPDH, using Igs from the CSF and scFv-Abs generated from clonal B cells in the CSF and in lesions from MS patients. Elevated levels of CSF-Abs to TPI were observed in patients with MS (46%), clinically isolated syndrome (CIS) suggestive of MS (40%), other inflammatory neurological diseases (OIND; 29%), and other noninflammatory neurological diseases (ONIND; 31%). Levels of GAPDH-reactive Abs were elevated in MS patients (60%), in patients with CIS (10%), OIND (14%), and ONIND (8%). The coexistence of both autoantibodies was detected in 10 MS patients (29%), and 1 CIS patient (3%), but not in patients with OIND/ONIND. Two scFv-Abs generated from the CSF and from lesions of a MS brain showed immunoreactivity to TPI and GAPDH, respectively. The findings suggest that TPI and GAPDH may be candidate Ags for an autoimmune response to neurons and axons in MS.

Clonal expansion of IgA-positive plasma cells and axon-reactive antibodies in MS lesions.

Immunoglobulin A (IgA), the predominant immunoglobulin class in mucosal secretions, has been found in the cerebrospinal fluid of patients with multiple sclerosis (MS). In this study we examined the infiltration of clonally expanded IgA plasma cells in lesions of MS brains. Sequences of complementarity-determining region 3 of IgA variable heavy chain (V(H)) genes demonstrated the clonal expansion of IgA-bearing plasma cells in MS lesions. Somatic mutations and ongoing intra-clonal mutations occurred in their V(H) genes. Immunohistochemical study demonstrated infiltration of dimer and polymer IgA1- and A2-positive plasma cells in perivascular spaces, in the parenchyma of MS lesions, and in the adjacent white matter. Double immunofluorescence staining showed binding of IgA antibody on axons and walls of microvessels in the areas of chronic active and inactive demyelination. Bielshowsky's silver impregnation revealed axonal damage in these areas. These findings suggest that IgA in the CNS are localized on axons in lesions and may contribute to axonal damage in MS.

Axon reactive B cells clonally expanded in the cerebrospinal fluid of patients with multiple sclerosis.

Demyelination and axonal loss have been described as the histological hallmarks of inflammatory lesions of multiple sclerosis (MS) and are the pathological correlates of persistent disability. However, the immune mechanisms underlying axonal damage in MS remain unknown. Here, we report the use of single chain-variable domain fragments (scFv) from clonally expanded cerebrospinal fluid (CSF) B cells to show the role of an anti-axon immune response in the central nervous system (CNS) in MS. The cellular and subcellular distribution of the antigen(s) recognized by these CSF-derived clonal scFv antibodies (CSFC-scFv Abs) was studied by immunochemical staining of brain tissues obtained at autopsy from patients with MS. Immunochemistry showed specific binding of CSFC-scFv Abs to axons in acute MS lesions. The stained axons showed three major types of axonal pathological changes: 1) linear axons, axonal ovoid formation, and axonal transection were seen in the myelinated white matter adjacent to the lesion; 2) accumulation of axonal ovoid formations and Wallerian degeneration were seen at the border between demyelinated lesions and the adjacent white matter; and 3) Wallerian degeneration occurred at the center and edge of acute demyelinated lesions. These findings suggest a B cell axonal specific immune response in the CNS in MS.

Polyclonal B-cell expansion in the cerebrospinal fluid of patients with pseudotumor cerebri.

To investigate the hypothesis that pseudotumor cerebri (PTC) is associated with humoral immunity, we analyzed immunoglobulin heavy chain variable region (Ig-VH) genes of B cells in the cerebrospinal fluid (CSF) of 10 patients with PTC. Using RT-PCR and sequencing techniques, intrathecal B-cell Ig-VH genes were amplified in 6 of 10 PTC samples. Sequence analysis of complementarity-determining region 3 (CDR 3) and VH genes revealed a polyclonal intrathecal B-cell expansion in these patients. The nucleotide sequences showed that one-third of analyzed sequences had a high replacement to silent nucleotide substitution ratio, indicating an antigen-driven T-cell-dependent intrathecal B-cell proliferation. Moreover, other one-third had germline VH genes without or with a few nucleotide mutations, suggesting a T-cell-independent natural B-cell-mediated humoral immunity in the CNS of these patients. Our results suggest that both T-cell-dependent and T-cell-independent humoral immunity are present in the CSF of PTC.

Intrathecal B-cell clonal expansion, an early sign of humoral immunity, in the cerebrospinal fluid of patients with clinically isolated syndrome suggestive of multiple sclerosis.

The development of somatically mutated memory and plasma B cells is a consequence of T cell-dependent antigen-challenged humoral immunity. To investigate the role of B cell-mediated humoral immunity in the initiation and evolution of multiple sclerosis (MS), we analyzed Ig variable heavy chain genes of intrathecal B cells derived from patients with a first clinical manifestation suggestive of MS. Sequences of Ig variable regions showed that B cells in the cerebrospinal fluid from most of these patients were clonally expanded and carried somatic hypermutated variable heavy chain genes. The mutations showed a high replacement-to-silent ratio and were distributed in a way suggesting that these clonally expanded B cells had been positively selected through their antigen receptor. In comparison, intrathecal B-cell clonal expansion often precedes both oligoclonal IgG bands and multiple magnetic resonance imaging lesions. Clinical follow-up study showed that patients with clonally expanded intrathecal B cells had a high rate of conversion to clinically definite MS. The findings provide direct evidence of recruitment of germinal center differentiated B lymphocytes into the central nervous system during the initiation of MS. These results indicate B cell-mediated immune response in the cerebrospinal fluid is an early event of inflammatory reaction in the central nervous system of MS. This procedure also provides a more sensitive method to evaluate the association of humoral immunity in the evolution of MS.