Rindera graeca (A. DC.) Boiss. & Heldr. (Boraginaceae) In Vitro Cultures Targeting Lithospermic Acid B and Rosmarinic Acid Production.

The in vitro cultures of Rindera graeca, a rare endemic plant, were developed as a sustainable source of phenolic acids. Various shoot and root cultures were established and scaled up in a sprinkle bioreactor. A multiplication rate of 7.2 shoots per explant was achieved. HPLC-PDA-ESI-HRMS analysis revealed the presence of rosmarinic acid (RA) and lithospermic acid B (LAB) as the main secondary metabolites in both the shoot and root cultures. The maximum RA (30.0 ± 3.2 mg/g DW) and LAB (49.3 ± 15.5 mg/g DW) yields were determined in root-regenerated shoots. The strongest free radical scavenging activity (87.4 ± 1.1%), according to 2,2-diphenyl-1-picrylhydrazyl-hydrate assay, was noted for roots cultivated in a DCR medium. The highest reducing power (2.3 µM ± 0.4 TE/g DW), determined by the ferric-reducing antioxidant power assay, was noted for shoots cultivated on an SH medium containing 0.5 mg/L 6-benzylaminopurine. A genetic analysis performed using random amplified polymorphic DNA and start codon targeted markers revealed genetic variation of 62.8% to 96.5% among the investigated shoots and roots. This variability reflects the capacity of cultivated shoots and roots to produce phenolic compounds.

Cytokine and autoantibody profiling related to histopathological features in primary Sjogren's syndrome.

OBJECTIVE: To investigate a potential correlation between circulating cytokine and autoantibody levels and histopathological features in subgroups of patients with primary SS (pSS). METHODS: Minor salivary gland biopsies from a cohort of 141 patients fulfilling the American-European consensus classification criteria for pSS were re-examined and grouped according to focus score (FS) and germinal centre (GC) status; serum samples were analysed for autoantibodies, chemokines and cytokines. RESULTS: Of the 115 available biopsies, 18 (16%) lacked characteristic focal mononuclear cell infiltrates [FS < 1 (FS-)] but patients were positive for Ro/SSA and/or La/SSB. IL-17, IL-1RA, IL-15, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, eotaxin, IFN-alpha and IL-4 levels were significantly increased in the 27 (23%) patients with ectopic GC formation (GC+) in the salivary glands compared with the GC- patients (n = 70). In addition, minor differences in cytokine levels were found when comparing age groups. CONCLUSION: Degenerative changes observed in the minor salivary glands of patients with pSS may represent 'burned out' inflammation. The elevated levels of IL-4 found in these patients may influence the reduced salivary flow observed in GC+ patients. Increased titres of Th17-associated cytokines, IL-17, IL-1beta and the IL-23 subunit IL-12p40, may indicate a higher activity of these cells in GC+ patients. Differences in cytokine levels may be utilized when sub-grouping the SS patients into disease phases and may consequently have implications for treatment.

Role of B cells in Sjögren's syndrome--from benign lymphoproliferation to overt malignancy.

The classical view of B cell biology is that these cells respond to foreign and self antigens and in this way promote protection, primarily by production of antibodies. However, recent studies suggest that B cells have diverse functions within the immune system other than antibody production, which could contribute to autoimmunity. This involves organization of lymphoid tissue, regulation of dendritic cells, antigen presentation, activation of T cells and production of cytokines. Both abnormalities in the distribution of B cell subsets, and recent discovery of clinical benefit after B cell depletion highlight the pivotal role of B cells in autoimmunity. This change in view of the role of B cells will be exemplified in one autoimmune disease namely Sjögren's syndrome.

Non-proliferating plasma cells detected in the salivary glands and bone marrow of autoimmune NOD.B10.H2b mice, a model for primary Sjögren's syndrome.

Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoimmune conditions such as primary Sjögren's syndrome (pSS). Particularly, the long-lived PC subset residing in the bone marrow has shown to continuously produce autoantibodies, whilst remaining unaffected by immunosuppressive treatment. We have previously shown accumulation of potentially long-lived PCs in chronically inflamed salivary glands of pSS patients. In this study, we aimed to characterise the PC compartment in the salivary glands (the target organ for pSS) and bone marrow before the onset of the murine pSS like disease versus advanced diseases progression. Bromodeoxyuridine (BrdU) was incorporated to distinguish the long-lived PCs. Double immunohistochemical staining and immunofluorescence were then conducted on submandibular gland and bone marrow sections from 8- and 40-week-old mice to identify BrdU and CD138. BrdU(+) cells were detected in the submandibular glands of 8-week-old mice, and observed within all focal infiltrates by 40 weeks of age. Most CD138(+) PCs were however BrdU(-) and located predominantly on the periphery of these infiltrates. This observation was verified through immunofluorescence. A comparable staining pattern was observed in the bone marrow of 8- and 40-week-old NOD.B10.H2b mice, where some of the CD138(+) cells also expressed BrdU. Interestingly, megakaryocytes in the bone marrow of NOD.B10.H2b mice were detected in close proximity to CD138(+) cells, illustrating a possible presence of PC survival niches. Our results demonstrate the presence and accumulation of potentially long-lived PCs in NOD.B10.H2b mice as the disease advances.

Distinct phenotypes of plasma cells in spleen and bone marrow of autoimmune NOD.B10.H2b mice.

Long-lived plasma cells (PCs) residing in the bone marrow (BM) are important producers of protective antibodies. However, when reacting against self-antigens, these PCs produce autoantibodies that contribute to progression of autoimmune diseases such as Sjögren's syndrome (SS). By using a murine model of primary SS, the NOD.B10.H2b mice, we characterized phenotype and generation of PCs at different stages of the pSS disease progression. In general, the PC population found in the NOD.B10.H2b mice expressed high amounts of MHCII, IgG, and BrdU. We further demonstrate the presence of both short- and long-lived PCs in autoimmune spleen and in autoimmune BM. A long-lived PC subset was also found in the spleen and BM of non-autoimmune BALB/c mice, which have not been treated with any immunological agent. Quantitative investigation of splenic and BM PCs revealed that in the NOD.B10.H2 mice, splenic PCs migrate not only to the BM but possibly also to the sites of inflammation. Finally, BM in the aged NOD.B10.H2b mice (40-week-old) presented significantly higher quantities of long-lived PCs than BM of BALB/c mice.

Salivary glands of primary Sjögren's syndrome patients express factors vital for plasma cell survival.

INTRODUCTION: The presence of circulating Ro/SSA and La/SSB autoantibodies has become an important marker in the classification criteria for primary Sjögren's syndrome (pSS). Plasma cells producing these autoantibodies are mainly high affinity plasma cells originating from germinal centre reactions. When exposed to the right microenvironment these autoimmune plasma cells become long-lived and resistant to immunosuppressive treatment. Since autoimmune plasma cells have been detected in the salivary glands of SS patients, we wanted to investigate if the glandular microenvironment is suitable for plasma cell survival and if glandular residing plasma cells are the long-lived plasma cell subset. METHODS: Single, double and triple immunohistochemistry as well as immunofluorescence staining was performed on minor salivary gland tissue retrieved from pSS, chronically inflamed and normal subjects. RESULTS: We detected significant numbers of CD138+, non-proliferating, Bcl-2 expressing plasma cells in the salivary glands of pSS patients with high focus score (FS). Furthermore, we demonstrated that CXCL12 and interleukin (IL)-6 survival factors were highly expressed in pSS salivary gland epithelium and by focal mononuclear infiltrating cells. Notably, adipocytes when present in the salivary gland tissue were an important source of CXCL12. We clearly demonstrate that plasma cells are localised in close proximity to CXCL12 and IL-6 expressing cells and thus that the environment of salivary glands with high FS provide factors vital for plasma cell survival. CONCLUSIONS: Plasma cells residing in the salivary glands of pSS patients with high FS showed phenotypic characteristics of the long-lived plasma cell subtype. Furthermore, the pSS salivary gland microenvironment provided niches rich in factors vital for plasma cell survival.

Local and systemic cytokine and chemokine responses after parenteral influenza vaccination.

BACKGROUND AND OBJECTIVE: In this study we investigated the levels of cytokines and chemokines produced locally and systemically after influenza vaccination of patients undergoing tonsillectomy. METHODS: Blood and saliva were collected prior to, and 1 or 2 weeks after vaccination at the time of the tonsillectomy. The cytokine and chemokine concentrations were determined in both unstimulated (whole blood, serum and saliva) and in vitro influenza stimulated peripheral blood mononuclear cell (PBMC) and tonsillar lymphocyte (TMC) cultures. RESULTS: We found that influenza vaccination elicited protective levels of serum haemagglutination inhibition antibodies and a significant local antibody response in the saliva. No significant differences were observed in the cytokine or chemokine levels 1 or 2 weeks post-vaccination in either the serum or saliva. Similarly, no significant differences were found in the gene expression levels in PBMC after vaccination, but interleukin (IL)-2, IL-4, gamma-interferon and transforming growth factor-beta were slightly elevated at 1 week post-vaccination but decreased by 2 weeks post-vaccination. In contrast, increased concentrations of a mixture of type 1, type 2 and inflammatory cytokines were produced 1 and 2 weeks after influenza vaccination by in vitro-stimulated PBMC and TMC. CONCLUSION: We show that cytokine responses can be measured after influenza vaccination in in vitro-stimulated lymphocytes but not directly in the blood or saliva. These results will provide a useful baseline that can be used for comparison of the immune response in human volunteers involved in clinical trials of novel influenza vaccines.

The humoral immune response and protective efficacy of vaccination with inactivated split and whole influenza virus vaccines in BALB/c mice.

Recently the urgency of developing a pandemic influenza vaccine has lead to the re-evaluation of the use of whole virus vaccine. We have compared the humoral immune response and the protective efficacy of whole and split influenza virus vaccines in mice. Whole virus vaccine was more immunogenic particularly after the first dose of vaccine, generally eliciting higher numbers of systemic antibody secreting cells and an earlier and higher neutralising antibody response. Immunisation with one dose of whole virus vaccine more effectively reduced viral shedding upon non-lethal homologous viral challenge, but two doses of split virus vaccine was most effective at limiting viral replication and this was correlated with high influenza specific serum IgG concentrations. The two vaccine formulations induced different T helper profiles particularly after one dose of vaccine; split virus vaccine induced a type 2 bias response, whereas whole virus vaccine elicited a dominant type 1 response.