Khairul Matin

Role of stromal-cell derived factor-1 in the development of autoimmune diseases in non-obese diabetic mice

The chemokine stromal‐cell derived factor‐1 (SDF‐1) controls maturation, trafficking, and homing of certain subsets, lymphoid cells including immunogenic B and T cells, as a ligand of the CXCR4 chemokine receptor. Insulin‐dependent diabetes mellitus (IDDM) and Sjögrens syndrome (SS), both highly regulated autoimmune diseases, develop spontaneously in non‐obese diabetic (NOD) mice. To investigate the role of SDF‐1 in the development of autoimmune diseases, we injected groups of NOD female mice with antibodies to SDF‐1 (anti‐SDF‐1), which resulted in a 30% reduction of diabetes up to 30 weeks of age, delayed average diabetes onset by 10 weeks, and suppressed insulitis. Autoimmune sialoadenitis was evident in anti‐SDF‐1‐injected mice (SDF‐1‐Ig group) at the same level as in all groups of mice, whether injected with non‐specific antibodies or not. In addition, in the SDF‐1‐Ig group, a greater number of immunoglobulin M (IgM)− IgD− B220low CD38+ CD43+ CD23− progenitor B cells and IgM+ IgD+ B220high CD43− CD38+ CD24+ CD23+ mature B cells remained in the bone marrow, whereas infiltration of mature IgM+ B cells was less extensive in peripheral tissues. Our results suggested that anti‐SDF‐1 antibodies injection was effective in inhibiting diabetes and insulitis without affecting autoimmune sialoadenitis or SS in NOD mice. SDF‐1 may be an essential chemokine for trafficking and migration of autoreactive B cells in the development of diabetes.

Effects of electrodeposited poly(ethylene glycol) on biofilm adherence to titanium

Protein-resistant coatings have been studied for inhibiting biofilm formation on implant devices. In this study, titanium (Ti) surfaces were biofunctionalized with poly(ethylene glycol) (PEG) by electrodeposition and were evaluated as biofilm substrates under an oral simulated environment. Streptococcus gordonii, an early colonizer of oral biofilms, was inoculated on Ti and PEG-electrodeposited Ti (PEG-Ti) surfaces and was analyzed quantitatively and topographically. Streptococcus mutans supplemented with sucrose, a late colonizer mainly found in dental plaque, was also used to form biofilms on the surfaces of Ti and PEG-Ti for 20 h followed by sonication as a means of detaching the biofilms. The results indicated that the attachment of S. gordonii on PEG-Ti surfaces was inhibited compared with Ti, and the S. mutans biofilm was easier to be detached from the surface of PEG-Ti than that of Ti. Moreover, the presence of PEG electrodeposited on Ti surface inhibited salivary protein adsorption. The degree of detachment of biofilms from PEG-Ti was associated with the inhibition of the salivary protein adsorption, suggesting weak basal attachment of the biofilms to the electrodeposited surfaces. Therefore, controlling protein adsorption at the initial stage of biofilm formation may be an effective strategy to protect metal surfaces from bacterial contamination not only in dental manipulations but also in orthopedic applications.

Surface Response of Fluorine Polymer-Incorporated Resin Composites to Cariogenic Biofilm Adherence

ABSTRACT Experimental resin composites with incorporated polytetrafluoroethylene (PTFE) particles were developed, which theoretically could improve the surface properties of the materials, including the inhibition of bacterial adherence. To assess the surface properties in relation to biofilm formation and detachment, 23.1% (wt/wt) linear PTFE particles (FL-30) and cross-linked PTFE particles (FC-30) were incorporated into pure resin composites. Pure PTFE plates and pure resin composites without PTFE (F-0) were used as control specimens. Sucrose-dependent Streptococcus mutans biofilms were formed on the specimen blocks inside an oral biofilm reactor for various time periods and analyzed with or without application of driving forces. In addition, water contact angles and surface roughness were measured. The water contact angles of FL-30 (61.2°) and FC-30 (65.8°) were larger than that of F-0 (48.5°). The largest contact angle (107°) was detected on pure PTFE plates. However, the surfaces of FL-30, FC-30, and pure PTFE plates were rougher than that of F-0. Although the surface properties of the materials differed in terms of contact angles and roughness, these factors seemed not to affect biofilm formation on the surfaces within 5 h. Pure PTFE plates harbored almost the same amounts of biofilm as F-0. However, when a very strong driving force was applied, it was clear that there were significantly smaller amounts of biofilms retained on pure PTFE plates, which showed contact angles much higher than those of the other materials. Hydrophobicity of the resin composite was improved by incorporation of PTFE fillers. However, surface resistance against biofilm formation was not improved.

RGD motif enhances immunogenicity and adjuvanicity of peptide antigens following intranasal immunization

The use of peptides for various aspects of medical science has been a significant advance. Peptide-based vaccines are promising, but weak immunogenic potency is impeding the clinical application. We have remarkably enhanced the immunogenicity of peptide antigens by addition of motifs that bind to cell attachment proteins, such as arginine-glysine-aspartate (RGD), to the amino acid sequence. The modified peptides induced antigen-specific serum antibodies by intranasal immunization without adjuvants. RGD, an integrin-binding motif was the strongest, among several molecules tested in this experiment, giving an average of 10 times enhancement of antibody titers when incorporated into several peptide antigens. The peptides also acted as an efficient adjuvant following the intranasal immunization with protein antigens. Our data support the feasibility of developing peptide vaccines and peptide adjuvants for intranasal vaccination.

E2f1 Mutation Induces Early Onset of Diabetes and Sjögren’s Syndrome in Nonobese Diabetic Mice

E2f1 is an important regulator of T cell proliferation, differentiation, and apoptosis that controls the transcription of a group of genes that are normally regulated at the G1 to S phase transition in the cell cycle. Insulin-dependent diabetes mellitus (IDDM) and Sjögren’s syndrome (SS) are highly regulated autoimmune diseases that develop spontaneously in NOD mice. The aim of the present in vivo study was to explore the functional importance of the E2f1 molecule in IDDM and SS, in the context of whole animal physiology and pathophysiology, using E2f1-deficient NOD mice. For the experiment, we produced NOD mice homozygous for a nonfunctional E2f1 allele onto a NOD background. E2f1-deficient NOD mice developed an early and increased onset of diabetes as compared with their littermates. These mice also exhibited a defect in T lymphocyte development, leading to excessive numbers of mature T cells (CD4+ and CD8+), due to a maturation stage-specific defect in the apoptosis of thymocytes and peripheral T cells. We also found that they also exhibited a more rapid and increased entry into the S phase following antigenic stimulation of spleen cells and thymocytes in vitro. Furthermore, E2f1-deficient mice showed a profound decrease of immunoregulatory CD4+CD25+ T cells, while the spleen cells of NOD mice lacking E2f1 showed a significant increase of the proinflammatory cytokine IFN-γ following antigenic stimulation in vitro. Consistent with these observations, E2f1 homozygous mutant NOD mice were highly predisposed to the development of IDDM and SS.

Establishment of an Animal Model Using Recombinant NOD.B10.D2 Mice To Study Initial Adhesion of Oral Streptococci

ABSTRACT An oral biofilm is a community of surface-attached microorganisms that coats the oral cavity, including the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases in patients with dry mouth or Sjögrens syndrome (SS). The purpose of this study was to establish an animal model for studying the initial adhesion of oral streptococci that cause biofilm formation in patients with dry mouth and SS in an attempt to decrease the influence of cariogenic organisms and their substrates. In nonobese diabetogenic (NOD) mice that spontaneously develop insulin-dependent diabetes mellitus (IDDM) and SS, we replaced major histocompatibility complex (MHC) class II (Ag7 Eg7) and class I Db with MHC class II (Ad Ed) and class I Dd from nondiabetic B10.D2 mice to produce an animal model that inhibited IDDM without affecting SS. The adhesion of oral streptococci, including Streptococcus mutans, onto tooth surfaces was then investigated and quantified in homologous recombinant N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice. We found that a higher number of oral streptococci adhered to the tooth surfaces of N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice than to those of the control C57BL/6 and B10.D2 mice. On the basis of our observation, we concluded that these mouse models might be useful as animal models of dry mouth and SS for in vivo biological studies of oral biofilm formation on the tooth surfaces.

Inhibitory effects of MoAbs against a surface protein antigen in real-time adherence in vitro and recolonization in vivo of Streptococcus mutans.

A surface protein antigen (PAc) of Streptococcus mutans, particularly the A‐region of the molecule, has been reported to interact with salivary components on the tooth surface. It might be a candidate antigen inducing the production of antibodies against the adherence of S. mutans to the tooth surface. We investigated the effects of monoclonal antibodies (MoAbs) obtained by immunization of synthetic PAc peptides that completely correspond to the amino acid sequence of part of the A‐region. These MoAbs recognize several core B‐cell epitopes in the sequence. Two (KH5 and SH2) of these antibodies reacted with both S. mutans and Streptococcus sobrinus, but not with Streptococcus sanguis, Streptococcus salivarius, Porphyromonas gingivalis or Lactobacillus casei. They clearly inhibited the real‐time adherence of S. mutans to salivary components in a biosensor. KH5, which showed a real‐time inhibition (71%), also significantly prevented the recolonization of S. mutans on the tooth surface in rats. These results suggested that the core B‐cell epitope (‐Y‐‐‐L‐‐Y‐‐‐‐) recognized by KH5 was the essential sequence in the antigenic epitopes of PAc protein recognized specifically by the inhibitory antibody. Therefore, the amino acid residues were found to be important in the initial attachment of S. mutans to the tooth surface. These results provide for the mechanism of PAc molecule in the initial attachment of S. mutans on the tooth surface and more effective designs for the removal of S. mutans and S. sobrinus from the oral cavity.

An artificial biofilm induced secondary caries model for in vitro studies

BACKGROUND The aim of this study was to establish a new in vitro model for biofilm induced secondary caries studies using an oral biofilm reactor. METHODS An approximately 2 × 3 × 2 mm(3) sized dentino-enamel Class I cavity was prepared in the middle of a square-shaped specimen from the mid-labial portion of bovine incisors. The cavities were partially filled with either Clearfil AP-X with SE-Bond or Clearfil AP-X without any bond. Artificial biofilms were then formed on the resin composite filled surfaces using three species of oral bacteria in an oral biofilm reactor for 20 hours followed by 7- or 30-day incubation periods. RESULTS The lesions were clearly visible on fluorescence microscopy and by scanning electron microscopy in the enamel at the interface of resin restorations in all samples. The data from image analysis showed that the lesion size was largest in the No-bond samples with statistically significant differences (p < 0.05). Demineralization along the cavity wall extended deeper in No-bond compared to SE-Bond samples and penetration was significantly deeper in No-bond 30-day samples. CONCLUSIONS A primary artificial secondary caries model was established using biofilms for in vitro studies and the significance of using a bonding system could also be verified.

Effects of human interleukin-18 and interleukin-12 treatment on human lymphocyte engraftment in NOD-scid mouse

NOD/LtSz‐prkdcscid/prkdcscid (non‐obese diabetic‐severe combine immunodeficiency; NOD‐scid) mice grafted with human peripheral blood lymphoid cells have been used as an in vivo humanized mouse model in various studies. However, cytotoxic human T cells are induced in this model during immune responses, which gives misleading results. To assist in grafting of human lymphocytes without the induction of cytotoxic human T cells, we investigated the effects of T helper type 1 (Th1) and Th2 cytokines on human lymphocyte grafting and migration, as well as the production of immunoglobulin deposited in glomeruli and human immunodeficiency virus‐1 (HIV‐1) infection using NOD‐scid mice. Administration of interleukin‐18 (IL‐18) and IL‐12 enhanced the grafting of human CD4+ and CD8+ T cells in the mice, whereas co‐administration prevented grafting due to interferon‐γ‐dependent apoptosis. Immunoglobulin A (IgA) deposits were observed in mice treated with IL‐18 alone, but not in those given phosphate‐buffered saline, IL‐12 alone, or IL‐18 + IL‐12. A high rate of HIV infection was also observed in the IL‐18‐treated group. Together, these results indicate that IL‐18 may be effective for the grafting and migration of CD4+ and CD8+ T cells, except for the induction of apoptosis and regulation of class‐switching IgA. IL‐18‐administered NOD‐scid mice provide a useful small humanized model for the study of HIV infection and IgA nephropathy.

Potentials of Mouthwashes in Disinfecting Cariogenic Bacteria and Biofilms Leading to Inhibition of Caries

Objectives: The aim of this study was to compare the effects of certain commercially available mouthwashes on cariogenic bacteria and biofilms, following the acquisition of inhibition potentials of caries. Materials and Methods: Mouthwashes containing I) chlorhexidine gluconate (CHG; 0.0005% w/v), II) benzethonium chloride (BTC; 0.01% w/v), III) an essential oil (Listerine), and IV) povidone-iodine (PVP-I; 0.035% w/v) were tested on planktonic cariogenic bacteria, biofilms, and an ex vivo caries model. Bacterial aliquots were inoculated with each solution separately and vortexed for 10 seconds at room temperature. Bacterial viability was subsequently investigated by fluorescence microscopy (FM) after staining with a BacLight viability kit and the number of colony-forming units (CFUs) was counted. Similarly, mouthwash solutions were applied to artificial cariogenic biofilms, and bacterial viability of the biofilms was investigated as stated above. Inhibition potentials of two selected mouthwashes of carious lesions were investigated using biofilm-induced caries and a secondary caries model. In all steps, a phosphate-buffered saline (PBS) solution was included as a control. Results: Planktonic cariogenic bacteria and bacteria embedded in biofilms were killed in remarkably large numbers with Listerine and PVP-I treatment compared to PBS and other gargles. CFU counts also showed significant reduction after treatment with Listerine and PVP-I compared to other solutions (P<0.05). Listerine also displayed significant (P<0.05) inhibition effects in preventing the progression of demineralization. Conclusion: Bactericidal potencies of the mouthwashes varied significantly, suggesting that mouthwashes like Listerine can be useful for the prevention of caries and secondary caries.