Combined Treatment With TGF-ß1, Retinoic Acid, and Lactoferrin Robustly Generate Inducible Tregs (iTregs) Against High Affinity Ligand.

Forkhead box P3-positive (Foxp3+)-inducible Tregs (iTregs) are readily generated by TGF-ß1 at low TCR signaling intensity. TGF-ß1-mediated Foxp3 expression is further enhanced by retinoic acid (RA) and lactoferrin (LF). However, the intensity of TCR signaling required for induction of Foxp3 expression by TGF-ß1 in combination with RA and LF is unknown. Here, we found that either RA or LF alone decreased TGF-ß1-mediated Foxp3 expression at low TCR signaling intensity. In contrast, at high TCR signaling intensity, the addition of either RA or LF strongly increased TGF-ß1-mediated Foxp3 expression. Moreover, decreased CD28 stimulation was more favorable for TGF-ß1/LF-mediated Foxp3 expression. Lastly, we found that at high signaling intensities of both TCR and CD28, combined treatment with TGF-ß1, RA, and LF induced robust expression of Foxp3, in parallel with powerful suppressive activity against responder T cell proliferation. Our findings that TGFß/RA/LF strongly generate high affinity Ag-specific iTreg population would be useful for the control of unwanted hypersensitive immune reactions such as various autoimmune diseases.

Frequency-Domain Reverse-Time Migration with Analytic Green's Function for the Seismic Imaging of Shallow Water Column Structures in the Arctic Ocean.

Seismic oceanography can provide a two- or three-dimensional view of the water column thermocline structure at a vertical and horizontal resolution from the multi-channel seismic dataset. Several seismic imaging methods and techniques for seismic oceanography have been presented in previous research. In this study, we suggest a new formulation of the frequency-domain reverse-time migration method for seismic oceanography based on the analytic Green's function. For imaging thermocline structures in the water column from the seismic data, our proposed seismic reverse-time migration method uses the analytic Green's function for numerically calculating the forward- and backward-modeled wavefield rather than the wave propagation modeling in the conventional algorithm. The frequency-domain reverse-time migration with analytic Green's function does not require significant computational memory, resources, or a multifrontal direct solver to calculate the migration seismic images as like conventional reverse-time migration. The analytic Green's function in our reverse-time method makes it possible to provide a high-resolution seismic water column image with a meter-scale grid size, consisting of full-band frequency components for a modest cost and in a low-memory environment for computation. Our method was applied to multi-channel seismic data acquired in the Arctic Ocean and successfully constructed water column seismic images containing the oceanographic reflections caused by thermocline structures of the water mass. From the numerical test, we note that the oceanographic reflections of the migrated seismic images reflected the distribution of Arctic waters in a shallow depth and showed good correspondence with the anomalies of measured temperatures and calculated reflection coefficients from each XCDT profile. Our proposed method has been verified for field data application and accuracy of imaging performance.

Lactoferrin-derived chimeric peptide (LFch) strongly boosts TGFß1-mediated inducible Treg differentiation possibly through downregulating TCR/CD28 signalling.

We recently reported that lactoferrin (LF) induces Foxp3+ Treg differentiation through binding to TGFß receptor III (TßRIII), and this activity was further enhanced by TGFß1. Generally, a low T-cell receptor (TCR) signal strength is favourable for Foxp3+ Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, containing lactoferricin [aa 17-30] and lactoferrampin [aa 265-284]), along with TGFß1 on Foxp3+ Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFß1-induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFß1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFß1 receptor III (sTßRIII). Consistent with this, the activity of LFch on TGFß1-induced Foxp3 expression was also abrogated by treatment with sTßRIII. Finally, the TGFß1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch robustly enhances TGFß1-induced Foxp3+ Treg differentiation by diminishing TCR/CD28 signal intensity.

Oral administration of Faecalibacterium prausnitzii and Akkermansia muciniphila strains from humans improves atopic dermatitis symptoms in DNCB induced NC/Nga mice.

Atopic dermatitis (AD) is a common inflammatory skin disease, and its pathogenesis is closely associated with microbial homeostasis in the gut, namely the gut-skin axis. Particularly, recent metagenomics studies revealed that the abundance of two major bacterial species in the gut, Faecalibacterium prausnitzii and Akkermansia muciniphila, may play a critical role in the pathogenesis of AD, but the effect of these species in AD has not yet been elucidated. To evaluate the potential beneficial effect of F. prausnitzii or A. muciniphila in AD, we conducted an animal model study where F. prausnitzii EB-FPDK11 or A. muciniphila EB-AMDK19, isolated from humans, was orally administered to 2,5-dinitrochlorobenzene (DNCB)-induced AD models using NC/Nga mice at a daily dose of 108 CFUs/mouse for six weeks. As a result, the administration of each strain of F. prausnitzii and A. muciniphila improved AD-related markers, such as dermatitis score, scratching behavior, and serum immunoglobulin E level. Also, the F. prausnitzii and A. muciniphila treatments decreased the level of thymic stromal lymphopoietin (TSLP), triggering the production of T helper (Th) 2 cytokines, and improved the imbalance between the Th1 and Th2 immune responses induced by DNCB. Meanwhile, the oral administration of the bacteria enhanced the production of filaggrin in the skin and ZO-1 in the gut barrier, leading to the recovery of functions. Taken together, our findings suggest that F. prausnitzii EB-FPDK11 and A. muciniphila EB-AMDK19 have a therapeutic potential in AD, which should be verified in humans.

Lactoferrin Potentiates Inducible Regulatory T Cell Differentiation through TGF-ß Receptor III Binding and Activation of Membrane-Bound TGF-ß.

Lactoferrin (LF) is known to possess anti-inflammatory activity, although its mechanisms of action are not well-understood. The present study asked whether LF affects the commitment of inducible regulatory T cells (Tregs). LF substantially promoted Foxp3 expression by mouse activated CD4+T cells, and this activity was further enhanced by TGF-ß1. Interestingly, blocking TGF-ß with anti-TGF-ß Ab completely abolished LF-induced Foxp3 expression. However, no significant amount of soluble TGF-ß was released by LF-stimulated T cells, suggesting that membrane TGF-ß (mTGF-ß) is associated. Subsequently, it was found that LF binds to TGF-ß receptor III, which induces reactive oxygen species production and diminishes the expression of mTGF-ß-bound latency-associated peptide, leading to the activation of mTGF-ß. It was followed by phosphorylation of Smad3 and enhanced Foxp3 expression. These results suggest that LF induces Foxp3+ Tregs through TGF-ß receptor III/reactive oxygen species-mediated mTGF-ß activation, triggering canonical Smad3-dependent signaling. Finally, we found that the suppressive activity of LF-induced Tregs is facilitated mainly by CD39/CD73-induced adenosine generation and that this suppressor activity alleviates inflammatory bowel disease.

APB-F1, a long-acting feline granulocyte colony-stimulating factor fusion protein, created by exploiting FL335, a chimeric Fab specific for feline serum albumin.

Off-label use of a human granulocyte colony stimulating factor (hG-CSF) has been allowed to treat dogs and cats with neutropenia. However, repeated administration of hG-CSF induces undesirable anti-drug antibody (ADA) responses, implying the necessity of animal-derived G-CSF as a therapeutic reagent, preferably with a long-acting capability. Herein, we generated a recombinant fusion protein by genetically combining FL335, a chimeric Fab specific for feline serum albumin (FSA), and feline G-CSF (fG-CSF), with the ultimate goal of developing a long-acting therapeutic fG-CSF for cats. The resulting FL335-fG-CSF fusion protein, referred to as APB-F1, was produced well as a functional form in a Chinese hamster ovary (CHO) expression system. In in vitro analyses, APB-F1 bound to FSA at high affinity (KD = 400 pM) and possessed 0.78 × 107 U/mg G-CSF biological activity, clearly proving its biological functionality. Pharmacokinetic (PK) and pharmacodynamic (PD) studies using healthy cats revealed that the serum half-life (t1/2) of APB-F1 was increased five times compared with that of fG-CSF (t1/2 = 13.3 h vs. 2.7 h) in subcutaneous (SC) injections. Additionally, APB-F1 induced a profound and sustained increase in white blood cell (WBC) and actual neutrophil count (ANC) up to 10 days, which was far superior to other G-CSF preparations, including filgrastim (Neupogen™) and even pegfilgrastim (Neulasta™). Conclusively, a long-acting fG-CSF with potent in vivo bioactivity was successfully created by using FL335; thus, we provided evidence that our "anti-serum albumin Fab-associated" (SAFA) technology can be applied reliably in developing valuable long-acting biologics in veterinary medicine.

Lactoferrin Induces Tolerogenic Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that initiate both T-cell responses and tolerance. Tolerogenic DCs (tDCs) are regulatory DCs that suppress immune responses through the induction of T-cell anergy and Tregs. Because lactoferrin (LF) was demonstrated to induce functional Tregs and has a protective effect against inflammatory bowel disease, we explored the tolerogenic effects of LF on mouse bone marrow-derived DCs (BMDCs). The expression of CD80/86 and MHC class II was diminished in LF-treated BMDCs (LF-BMDCs). LF facilitated BMDCs to suppress proliferation and elevate Foxp3+ induced Treg (iTreg) differentiation in ovalbumin-specific CD4+ T-cell culture. Foxp3 expression was further increased by blockade of the B7 molecule using CTLA4-Ig but was diminished by additional CD28 stimulation using anti-CD28 Ab. On the other hand, the levels of arginase-1 and indoleamine 2,3-dioxygenase-1 (known as key T-cell suppressive molecules) were increased in LF-BMDCs. Consistently, the suppressive activity of LF-BMDCs was partially restored by inhibitors of these molecules. Collectively, these results suggest that LF effectively causes DCs to be tolerogenic by both the suppression of T-cell proliferation and enhancement of iTreg differentiation. This tolerogenic effect of LF is due to the reduction of costimulatory molecules and enhancement of suppressive molecules.

Activation of p38α in T cells regulates the intestinal host defense against attaching and effacing bacterial infections.

Intestinal infections by attaching and effacing (A/E) bacterial pathogens cause severe colitis and bloody diarrhea. Although p38α in intestinal epithelial cells (IEC) plays an important role in promoting protection against A/E bacteria by regulating T cell recruitment, its impact on immune responses remains unclear. In this study, we show that activation of p38α in T cells is critical for the clearance of the A/E pathogen Citrobacter rodentium. Mice deficient of p38α in T cells, but not in macrophages or dendritic cells, were impaired in clearing C. rodentium. Expression of inflammatory cytokines such as IFN-γ by p38α-deficient T cells was reduced, which further reduced the expression of inflammatory cytokines, chemokines, and antimicrobial peptide by IECs and led to reduced infiltration of T cells into the infected colon. Administration of IFN-γ activated the mucosal immunity to C. rodentium infection by increasing the expression of inflammation genes and the recruitment of T cells to the site of infection. Thus, p38α contributes to host defense against A/E pathogen infection by regulating the expression of inflammatory cytokines that activate host defense pathways in IECs.

Multidisciplinary approach for a patient with dentinogenesis imperfecta and anterior trauma.

Dentinogenesis imperfecta is an inherited dentinal dysplasia involving several risks for orthodontic treatment. This case report describes the multidisciplinary treatment of a 17-year-old girl whose Class II Division 1 malocclusion was complicated by dentinogenesis imperfecta type II and maxillary anterior trauma.

Laser-aided circumferential supracrestal fiberotomy and low-level laser therapy effects on relapse of rotated teeth in beagles.

OBJECTIVE: To investigate the effectiveness and periodontal side effects of laser circumferential supracrestal fiberotomy (CSF) and low-level laser therapy (LLLT) on orthodontically rotated teeth in beagles. MATERIALS AND METHODS: Eighteen mandibular incisors from nine dogs were divided into three groups by treatment (n = 6/group): A, orthodontic couple force application only (control); B, laser CSF following orthodontic couple force application; and C, LLLT following orthodontic couple force application. Both mandibular lateral incisors were rotated for 4 weeks, and the relapse tendency was observed for 4 weeks more without any retainers. The amount of relapse, sulcus depth, and gingival recession were measured at weeks 4 and 8. One-way analysis of variance (ANOVA) and Scheffé's post hoc test were used for data analysis. Tissue specimens were examined at week 8 under light microscopy after hematoxylin-eosin (H&E) and Masson's trichrome staining. RESULTS: The mean percentage of relapse was 41.29% in group A, 14.52% in group B, and 56.80% in group C (P < .001). Four weeks after laser CSF, the sulcus depth increased by 0.67 mm, but no gingival recession was observed. There was no significant difference between groups A and C in terms of sulcus depth and gingival recession. CONCLUSIONS: Laser CSF is an effective procedure to decrease relapse after tooth rotation, causing no apparent damage to the supporting periodontal structures, whereas LLLT on orthodontically rotated teeth without retainers appears to increase the relapse tendency.

Effects of low-level laser therapy after Corticision on tooth movement and paradental remodeling.

BACKGROUND AND OBJECTIVE: Both Corticision and low-level laser therapy (LLLT) are known to affect the rate of tooth movement. Our objective was to investigate the combined effects of Corticision and LLLT on the tooth movement rate and paradental remodeling in beagles. STUDY DESIGN/MATERIALS AND METHODS: The maxillary second premolars (n = 24) of 12 beagles were randomly divided into four groups (n = 6 per group) based on the treatment modality: group A, only orthodontic force (control); group B, orthodontic force plus Corticision; group C, orthodontic force plus LLLT; group D, orthodontic force plus Corticision and LLLT. RESULTS: Ratios of second premolar-to-canine movement were greater by 2.23-fold in group B and 2.08-fold in group C, but 0.52-fold lesser in group D than in group A. The peak velocity was observed at an earlier stage of tooth movement in group B but at a later stage in group C during the 8-week treatment period. At week 8, both tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts on the compression side and proliferating cell nuclear antigen (PCNA)-positive osteoblasts on the tension side increased significantly (P<.05) in group C but decreased in group D. Histomorphometric analysis revealed that the mean apposition length of newly formed mineralized bone during the 8 weeks of treatment significantly increased in both group B (2.8-fold) and group C (2.2-fold). In group D, the labeling lines on lamina dura were thin and discontinuous, but intratrabecular remodeling and lamellation were found to be active. CONCLUSION: Periodic LLLT after Corticision around a moving tooth decreased the tooth movement rate and alveolar remodeling activity.

Effects of Corticision on paradental remodeling in orthodontic tooth movement.

OBJECTIVE: To investigate the biologic effects of Corticision on alveolar remodeling in orthodontic tooth movement. MATERIALS AND METHODS: In this study, 16 cats were divided into 3 groups: group A, only orthodontic force (control); group B, orthodontic force plus Corticision; and group C, orthodontic force plus Corticision and periodic mobilization. Histologic and histomorphometric studies were performed on tissue specimens on days 7, 14, 21, and 28. RESULTS: Extensive direct resorption of bundle bone with less hyalinization and more rapid removal of hyalinized tissue were observed in group B. The accumulated mean apposition area of new bone on day 28 was observed to be 3.5-fold higher in group B than in the control group A. CONCLUSIONS: Corticision might be an efficient procedure for accelerating orthodontic tooth movement accompanied with alveolar bone remodeling.

Severe Class II division 1 malocclusion treated by orthodontic miniplate with tube.

A miniplate with tube (C-tube) was placed in the interdental spaces between both left and right upper second premolars and first molars in a 15-year-8 month-old male patient with a Class II malocclusion who with severe anterior protrusion and lower anterior crowding. The treatment plan consisted of extracting both upper first premolars, en masse retraction of the upper six anterior teeth and lower anterior decrowding. C-tubes were used as substitutes for posterior dental anchorage during upper anterior retraction. The particular design of the C-tubes made it possible to retract fully with minimal gingival irritation. The correct overbite and overjet were obtained by intruding and retracting the maxillary incisors to their proper positions and this correction remained stable for at least 27 months after debonding. Also, facial balance was improved. The active treatment period was 14 months. The application of this new appliance, consideration of case selection, and sequence of treatment are presented.

Protective and anti-arthritic effects of deer antler aqua-acupuncture (DAA), inhibiting dihydroorotate dehydrogenase, on phosphate ions-mediated chondrocyte apoptosis and rat collagen-induced arthritis.

The effect of water extract of deer antler aqua-acupuncture (DAA; Cervi Pantotrichum Cornu) prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe (Nokyong in Korean), a traditional immunosuppressive and immunoactivating Korean herbal acupuncture [Int. Immunopharm. 3 7 (2003) 1001] on rat chondrocyte apoptosis was studied. Terminally differentiated hypertrophic chondrocytes were isolated from rat costochondrial cartilage and cell death was measured in the presence of 3-5 mM phosphate ions (Pi). The effect of 10 microg/ml DAA was compared to that of phosphonoformic acid (PFA), a competitive inhibitor of the Na-Pi co-transport on Pi-induced apoptosis in chondrocytes. A total of 1 mM PFA blocked anion-induced cell death and prevented an increase in the cell Pi content. In a parallel study, we determined that the DAA also protected chondrocytes from death. On the other hand, the effect of DAA was also evaluated as an inhibitor of dihydroorotate dehydrogenase (DHO-DHase) and tested in the rat collagen-induced arthritis (CIA) model. Female 7-week-old Sprague-Dawley rats were used for the evaluation of DAA in the CIA model. Arthritis was evaluated by arthritis score, body weight loss, bone destruction score. DAA was administered by bilateral Shinsu (B23) acupuncture five times per week (10, 20, 30, and 100 microg/kg/day). DAA inhibited rat liver DHO-DHase in vitro with Ki = 843 +/- 43 microg/ml. The anti-proliferative effect of DAA was caused by cell cycle arrest at the S-phase. Treatment with 300 mg/kg/day of DAA completely prevented the development of CIA based on the reduction of the arthritis score. The 50% effective dose (ED50) of DAA on arthritis score was 64 mg/kg. DAA ameliorated body weight loss associated with disease onset. DAA suppressed the development of arthritis, even when it was administered after a booster immunization of collagen. DAA is a novel immunosuppressant which inhibits DHO-DHase and its effects in CIA suggest that it could be useful in the treatment of rheumatoid arthritis.