Principles and Clinical Application of Free-Style Capillary Perforator-Based Flap for Coverage of Facial Skin Cancer Defects.

This study introduces a free-style perforator based island flap (PBIF) for the reconstruction of skin defects. From March 2012 to December 2022, a retrospective investigation was conducted on patients who underwent reconstruction for facial defects due to skin cancer. Data on the patients' gender, age, anesthesia method, diagnosis, defect location, flap size, complications, and follow-up periods were collected. There are several principles for designing the PBIF: finger-pinching method, alignment with the direction of wrinkles, the smaller width and longer length of the flap, and proximal attachment to the muscle. A total of 32 patients were included, with an average age of 63.6 years. Surgeries were performed in various regions, such as the infraorbital area, nose, cheek, philtrum, and the anterior/posterior/inferior auricular regions, with an average flap size of 7.63 cm2. There were no complications, such as venous congestion or vascular insufficiency in the skin flaps, although one case required revisional closure due to flap disruption. The PBIF is a useful and effective method for the restoration of facial defects. This method can provide simple yet aesthetically satisfying results, showing stable outcomes without complex surgeries or complications. This study indicates the potential for this method to be more widely employed in reconstructive surgeries in the future.

Analysis of Operation Duration in Thoracodorsal Artery Perforator Free-Flap Surgery of the Lower Extremities.

PURPOSE: This retrospective study aimed to identify the factors that affect the duration of thoracodorsal artery perforator (TDAP) free-flap surgery and to offer strategies for optimizing the surgical procedure. METHODS: We analyzed 80 TDAP flap surgeries performed by a single surgeon between January 2020 and December 2022, specifically focusing on free flaps used for lower-extremity defects with single-artery and single-vein anastomosis. The operation duration was defined as the time between the surgeon's initial incision and completion of reconstruction. Linear regression analyses were conducted to identify the factors affecting operation duration. RESULTS: The average operative duration was 149 minutes (range, 80-245 minutes). All flaps survived, although 8 patients experienced partial flap loss. The operative duration decreased with increasing patient age and when end-to-end arterial anastomosis was performed. However, the risk increased with larger flap sizes and in patients with end-stage renal disease. CONCLUSIONS: Our study identified several factors and methods that could accelerate TDAP free-flap procedures. These findings offer valuable insights for optimizing surgical processes and improving overall surgical outcomes. Although further research is needed to confirm and expand upon these findings, our study provides important guidance for surgeons in developing effective strategies for TDAP flap surgery.

Reconstruction of a Severe Open Tibiofibular Fracture using an Ipsilateral Vascularized Fractured Fibula with a Thoracodorsal Artery Perforator Free Flap.

The Gustilo IIIB tibiofibular fractures often result in long bone loss and extensive soft tissue defects. Reconstruction of these complex wounds is very challenging, especially when it includes long bone grafts, because the donor site is limited. We describe our experience using a set of chimeric ipsilateral vascularized fibula grafts with a thoracodorsal artery perforator free flap to reconstruct the traumatic tibia defects. A 66-year-old male suffered a severe comminuted tibia fracture and segmented fibula fracture with large soft tissue defects as a result of a traffic accident. He also had an open calcaneal fracture with soft tissue defects on the ipsilateral side. All the main vessels of the lower extremity were intact, and the cortical bone defect of the tibia was almost as large as the fractured fibula segment. We used an ipsilateral vascularized fibula graft to reconstruct the tibia and a thoracodorsal artery perforator flap to resurface the soft tissue, using the distal ends of peroneal vessels as named into sequential chimeric flaps. After 3 weeks, the calcaneal defect was reconstructed with second thoracodorsal artery perforator free flap. Reconstruction was successful and allowed rapid rehabilitation because of reduced donor site morbidity.

Multiple digit resurfacing with a lateral thoracic free flap: Two-stage mitten hand and division procedures.

PURPOSE: The conventional abdominal and groin flaps for resurfacing the defect have several disadvantages, including the risk of flap failure due to accidental traction or detachment, immobilization of the arm before division, and aesthetic dissatisfaction because of the flap bulkiness. The aim of this study was to share our experiences with the free lateral thoracic flap and elucidate the optimal timing of division in complex hand reconstruction, which yielded favorable outcomes in terms of both functionality and aesthetics. METHODS: This article is a retrospective review of multiple digit resurfacing using free tissue transfer from 2012 to 2022. Patients who underwent two-stage operation including mitten hand creation using superthin thoracodorsal artery perforator (TDAp) free flap and secondary division were included. A flap was elevated over the superficial fascia layer the midportion between the anterior border of the latissimus dorsi and pectoralis major muscles and once the pedicle was found, an outline that matched the defect was created. A process named "pushing with pressure and cutting" was carried out before pedicle ligation until all the superficial fat tissue had been removed except for around the perforator. Two cases (18%) involved defects of the entire fingers reconstructed by TDAp flap with anterolateral thigh flap. Six cases (55%) had a super-thin TDAp flap only. In two cases (18%), non-vascularized iliac bone grafting was required for finger lengthening. One case (9%) was resurfaced with a TDAp chimeric flap including a skin paddle with the serratus anterior muscle. The primary outcome was defined as the survival or failure of the flap, while the secondary outcomes associated complications such as infection and partial flap necrosis. A statistical analysis was not performed due to the size of the case series. RESULTS: All 13 flaps survived completely without any complications. Flap dimension ranged from 12 cm × 7 cm to 30 cm × 15 cm. Mitten hand duration prior to division was 41.9 days on average which was essential for the optimal result. During the division procedures, there were nine cases of debulking (82%), six cases of split-thickness skin graft (STSG) (55%), and three cases of Z-plasty performed on the first web space (27%). The mean follow-up period was 20.2 months. Mean Disability of the Arm, Shoulder, and Hand (DASH) Questionnaire score was 10.76. CONCLUSIONS: We resurfaced severe soft tissue defects of multiple fingers with thin to super-thin free flaps, mainly TDAp flaps. Surgeons can restore original hand shape using a two-stage reconstructive strategy of mitten hand creation and proper division timing to create a 3-dimensional hand structure, even in severely injured hands with multiple soft tissue defects of the digits.

Strategy for resurfacing complex extremity defect with chimeric, linked flaps: A review with a 10-year experience at major trauma center.

For the reconstruction of the extensive and/or three-dimensional soft-tissue defect in upper and lower extremities, chimeric flaps composed of multiple flaps or tissues with separate vascular supplies can supply economical use of tissue and superior esthetic results. Herein, we investigated the effectiveness of the thoracodorsal axis chimeric flap through the review the largest collection of long-term data. A retrospective review of all patients who received the thoracodorsal axis chimeric flap in complex three-dimensional defects of extremities between January of 2012 and December of 2021. A total of 55 type I/IP classical chimeric flaps, 19 type II/IIP anastomotic chimeric flaps, five type III perforator chimeric flaps, and seven type IV mixed chimeric flaps were analyzed. As the reconstructed area became proximal, flap dimensions increased significantly. And the optimal flap type depended on the location. The TDAp flap can provide large dimensions of skin paddle with latissimus dorsi and serratus anterior muscles with acceptable donor-site morbidities. The TDAp chimeric flaps constructed by microvascular anastomosis of two free flaps can provide large skin dimensions but also tissues with different properties. These characteristics make it possible to resurface the large and extensive defects, reconstruct the complex distal extremity defects, needing tissues with different properties, and cover the three-dimensional defect, obliterating the dead space. The thoracodorsal axis chimeric flap could be a favorable option for extensive, complex, or three-dimensional defects of the upper and lower extremities based on its reliability of the vascular system.

Reconstruction of soft tissue defect around knee with thoracodorsal artery perforator flap and muscle-sparing latissimus dorsi flap.

INTRODUCTION: Around the knee reconstruction is challenging for reconstructive surgeons. Several methods have been proposed, including perforator and muscle flaps; however, all have advantages and disadvantages. As the success rate of free-flap surgery increases, reconstruction around the knee using this method is becoming increasingly popular. Nevertheless, there are no large-scale case reports in the previous literature using either a thoracodorsal artery perforator flap (latissimus dorsi (LD) perforator flap) or a muscle-sparing latissimus dorsi (msLD) flap for reconstruction around the knee. In this retrospective report, we describe our clinical experiences and present an algorithm regarding recipient vessel choice in free-flap reconstructive surgery of around the knee defects. PATIENT AND METHODS: Fifty-six cases in which a flap from the lateral thoracic area was used to reconstruct an around the knee defect between January 2016 and March 2022 were reviewed. The patients were aged 18-87 years (mean, 52.13 years), and of the 56 patients, 36 were male and 20 were female. Injuries were caused by trauma, contracture, rheumatoid vasculitis, tumor, infection, burns, sunken deformity, and pressure sores. The 56 cases included 22 with a defect including the knee, 14 with a defect below the knee (7 of the primary below-knee amputation [BKA] and 7 of the secondary BKA), 9 involving the distal medial thigh, 8 involving the distal lateral thigh, 2 involving the popliteal area, and 1 involving the middle thigh. Most cases were reconstructed using a single LD perforator flap or msLD flap. Chimeric or supplementary flaps were used when extensive coverage or dead space obliteration was required. The average size of the defect area was 253.6 cm2 (range: 5 × 6-21 × 39 cm2 ). RESULTS: In the cases, the recipient artery used included the following: descending genicular artery (23), superficial femoral artery (14), descending branch of the lateral circumflex femoral artery (14), anterior tibial artery (2), popliteal artery (2), and posterior tibial artery (1). The recipient vein included the greater saphenous vein (24), descending branch of the lateral circumflex femoral vein (14), superficial femoral vein (7), descending genicular vein (6), anterior tibial vein (2), popliteal vein (2), and posterior tibial vein (1). The average flap size was 281.8 cm2 (range: 4 × 8-35 × 19 cm2 ). All flaps survived; however, seven complications occurred, including 2 partial flap losses, 1 arterial insufficiency, 1 hematoma, 1 minor dehiscence, 1 donor-site graft loss, and 1 short BKA. Normal knee range of motion (121-140°) was observed in 34 patients and 16 showed varying degrees of limited range of motion. Motion was not observed in four patients who underwent knee fusion and could not be evaluated in two patients who underwent above-knee amputation. The mean follow-up duration was 24.6 months (range: 4-72 months). CONCLUSION: The LD perforator flap is ideal for the reconstruction of around the knee defects because it enables a long pedicle, large flap, and chimeric design. The msLD flap is ideal because it enables strong stump support, dead-space obliteration, and infection control. Moreover, since the two flaps are distant from the knee, this method is advantageous in terms of postoperative rehabilitation and there is minimal donor-site morbidity due to the thin nature of the LD muscle. In addition, the flap can be elevated in three positions and the operation can be completed without positional changes in various recipient vessel locations. Based on our experience, we conclude that the LD flap has the potential to be used as widely as or in preference to the anterolateral thigh flap in the reconstruction of around the knee defects.

Resurfacing of lower extremities: Versatility of thigh flap as pedicle extension graft with consecutive thoracodorsal artery perforator-based flap.

BACKGROUND: For successful microsurgical reconstruction using free tissue transfer, healthy recipient vessels must be obtained from outside the zone of injury. Securing an appropriate length pedicle length is also essential, and various techniques for lengthening a vascular pedicle have been developed. Herein, we present our experience using the descending branch (DB) of the lateral circumflex femoral vessels (LCFVs) with a thigh flap as an extender graft for consecutive second flap. METHODS: We reconstructed the complex and vessel-depleted defects of nine patients. The mean age was 47.6 years. The defects were located in the lower leg in four patients, in the perineum in two patients and in the forearm in three patients. The two patients who suffered from Fournier's gangrene underwent a pedicled anteromedial thigh (pAMT) flap with the DB of the LCFVs and seven patients, five who suffered high-energy trauma and two who had scar contracture, underwent a free anterolateral thigh (ALT) flap with the distal run-off DB of the LCFVs. In all patients, second consecutive free latissimus dorsi or thoracodorsal artery perforator flaps were prepared and the thoracodorsal vessels of the second flap were anastomosed to the distal DB of the LCFVs. RESULTS: The total length of the thigh flap pedicles measured from both ends of the DB of the LCFVs varied from 15 to 20 cm, which was sufficient for use as a vascular conduit. Of the 18 flaps, 17 survived completely without any complications and 1 pAMT flap showed partial necrosis, which was covered with a perineal perforator-based island flap. The mean follow-up period was 16.7 months. Unfortunately, one patient, who suffered a total amputation below the knee and had replantation surgery, underwent amputation due to venous congestion in the distal leg. However, the previous two flaps survived and were used for coverage of the stump. CONCLUSIONS: Using a thigh flap as a vascular extender graft for second flap may be an alternative option in vessel-depleted reconstructions.

p16INK4a-siRNA nanoparticles attenuate cartilage degeneration in osteoarthritis by inhibiting inflammation in fibroblast-like synoviocytes.

In osteoarthritis (OA), chondrocytes in cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favoring disease progression. Although senescence biomarker p16INK4a expression is known to induce aging by halting the cell cycle, therapeutic applications for p16INK4a targeting are limited. Here, we aimed to reduce cartilage damage and alleviate pain using p16INK4a nanoparticles in OA. The p16INK4a expression of human OA chondrocytes and synoviocytes from patients with knee OA was measured and the levels of p16INK4a, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and matrix metalloproteinase (MMP) 13 were examined. p16INK4a siRNA was encapsulated into poly (lactic-co-glycolic acid) (PLGA) nanoparticles and characterized. The partial medial meniscectomy (pMMx) model was performed for the OA model which was investigated by molecular analysis and behavioral tests. The expression of p16INK4a was increased in the synovium and articular cartilage from OA patients. p16INK4a siRNA-loaded PLGA nanoparticles (p16 si_NP) reduced the levels of TNF-α, IL-1ß, and IL-6 especially in fibroblast-like synoviocytes (FLSs), and MMP13 in chondrocytes. Rhodamine-tagged NPs injected into the mouse knee joints were found mainly in the synovium. p16 si_NP injection in the pMMx model alleviated pain-associated behavior, and reduced cartilage damage and p16INK4a in the synovium, and MMP13, collagen X, and NITEGE in cartilage. The preferential reduction of p16INK4a in FLSs by the application of RNAi nanomedicine could contribute to the recovery of osteoarthritic cartilage and relieve pain, suggesting that p16INK4a may be a viable future therapeutic candidate.

CD14+ monocytes and soluble CD14 of synovial fluid are associated with osteoarthritis progression.

Objectives: This study aims to investigate the role of cluster of differentiation 14 (CD14) expressed monocytes and soluble CD14-mediated pathway in the synovial inflammation of knee osteoarthritis (OA). Patients and methods: Between May 2012 and July 2013, a total of 35 patients with knee OA (9 males, 26 females; mean age: 66.3±8.8 years; range, 52 to 79 years) were included in this cross-sectional study. Synovial fluid was obtained from knee joints of 35 OA patients. The CD14+ monocytes from synovial fluid mononuclear cells (SFMCs) were isolated using the MACS. The fibroblast-like synoviocytes (FLSs) isolated from knee joint tissue were incubated with recombinant CD14 and lipopolysaccharide (LPS) for 24 h. Cytokine profiling was performed with the Luminex® Performance Assay or magnetic bead panel kit. The expression of CD14 and CD16 was analyzed by immunohistochemistry and flow cytometry. Results: The concentration of sCD14 in synovial fluid was correlated with the interleukin-6 (IL-6) level (n=35) (ρ=0.654, p<0.001). The culture supernatants of CD14+ monocytes isolated from SFMC (n=15) showed a correlation between sCD14 and IL-6 (ρ=0.784, p=0.001), along with complement component 3 (ρ=0.756, p=0.010), IL-1b (ρ=0.652, p=0.012), and tumor necrosis factor-alpha (ρ=0.806, p=0.001). Following recombinant CD14 and LPS treatment, OA FLS synergistically enhanced the secretion of IL-6, IL-8, and matrix metalloproteinase 3 (n=3, p<0.05). In five paired-samples from identical patients, the proportions of CD14+ monocytes were significantly elevated in recurred synovial fluid compared to those in initial synovial fluid (p=0.043). When monocyte subsets were analyzed in SFMC (n=26), CD14+CD16+monocytes were abundant (p=0.019) and had higher toll-like receptor 4 expression than CD14+CD16- (p<0.001). Conclusion: Our study results suggest that CD14+ monocytes and the sCD14-mediated pathway play an important role in OA aggravation through inflammatory cytokine secretion.

Elevated APE1/Ref-1 Levels of Synovial Fluids in Patients with Rheumatoid Arthritis: Reflection of Disease Activity.

There is growing evidence that apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) regulates inflammatory responses. Rheumatoid arthritis (RA) is an autoimmune disease, which is characterized with synovitis and joint destruction. Therefore, this study was planned to investigate the relationship between APE1/Ref-1 and RA. Serum and synovial fluid (SF) were collected from 46 patients with RA, 45 patients with osteoarthritis (OA), and 30 healthy control (HC) patients. The concentration of APE1/Ref-1 in serum or SF was measured using the sandwich enzyme-linked immunosorbent assay (ELISA). The disease activity in RA patients was measured using the 28-joint disease activity score (DAS28). The serum APE1/Ref-1 levels in RA patients were significantly increased compared to HC and OA patients (0.44 ± 0.39 ng/mL for RA group vs. 0.19 ± 0.14 ng/mL for HC group, p < 0.05 and vs. 0.19 ± 0.11 ng/mL for OA group, p < 0.05). Likewise, the APE1/Ref-1 levels of SF in RA patients were also significantly increased compared to OA patients (0.68 ± 0.30 ng/mL for RA group vs. 0.31 ± 0.12 ng/mL for OA group, p < 0.001). The APE1/Ref-1 concentration in SF of RA patients was positively correlated with DAS28. Thus, APE1/Ref-1 may reflect the joint inflammation and be associated with disease activity in RA.

Role of Stem Cell-Like Memory T Cells in Systemic Lupus Erythematosus.

OBJECTIVE: Stem cell-like memory T (Tscm) cells are long-lived memory T cells that have multipotent capacity to differentiate into different subsets. However, the role of Tscm cells in autoimmune diseases remains unclear. Here, we performed phenotypic studies to identify Tscm cells in patients experiencing systemic lupus erythematosus (SLE). METHODS: CD4+ and CD8+ Tscm cells were identified in SLE patients and healthy controls (HCs). In in vitro culture systems, CD4+ Tscm cells were induced to differentiate into subsets of T cells, including follicular helper T (Tfh) cells, and cytokine production patterns were assessed after stimulation. After confirming induction of transcription factors for Tfh cells, the capacity of CD4+ Tscm-derived Tfh cells to help B cells was analyzed by measuring antibody secretion. RESULTS: The percentages of CD4+ and CD8+ Tscm cells among the naive CD4+/CD8+ or total CD4+ T cell populations were significantly higher in SLE patients than in HCs. Stimulated Tscm cells from SLE patients could replenish themselves and differentiate into other T lymphocyte subsets, including Tfh cells upon stimulation with T cell receptor. Production of T cell factor 1, which is an inducer of Tfh, was also increased. The differentiated Tfh cells increased antibody production by autologous B cells. CONCLUSION: Taken together, these findings suggest that Tscm cells play a role in the pathogenesis of SLE by maintaining Tfh cells.

HM71224, a novel Bruton's tyrosine kinase inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis.

BACKGROUND: Bruton's tyrosine kinase (Btk) is critical for activation of B cells and myeloid cells. This study aimed to characterize the effects of HM71224, a novel Btk inhibitor, both in vitro and in a mouse model of experimental arthritis. METHODS: The kinase inhibition profile of HM71224 was analyzed. The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation. The in vivo effects of HM71224 were investigated in a mouse model of collagen-induced arthritis (CIA). RESULTS: HM71224 irreversibly bound to and inhibited Btk (IC50 = 1.95 nM). The compound also inhibited the phosphorylation of Btk and its downstream molecules such as PLCγ2, in activated Ramos B lymphoma cells and primary human B cells in a dose-dependent manner. Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß by human monocytes, and osteoclast formation by human monocytes. Finally, HM71224 improved experimental arthritis and prevented joint destruction in a murine model of CIA. CONCLUSIONS: HM71224 inhibits Btk in B cells and monocytes and ameliorates experimental arthritis in a mouse model. Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

SF Treg cells transcribing high levels of Bcl-2 and microRNA-21 demonstrate limited apoptosis in RA.

OBJECTIVE: The aim of this study was to investigate the turnover of Treg cells in the SF of RA patients. METHODS: Treg cells were enumerated in peripheral blood and SF of RA patients and analysed by flow cytometry for expression of the proliferation marker Ki-67 and binding of the apoptosis marker annexin V. Sorted Treg cells of RA patients were analysed for expression of anti-apoptotic regulators Bcl-2 and microRNA-21 (miR-21) by RT-PCR. RESULTS: Treg cells displaying a memory phenotype were abundant in the SF of RA patients. SF Treg cells more frequently expressed the proliferation marker Ki-67 than conventional T cells. Only few SF Treg cells were apoptotic, as indicated by limited annexin V staining of these cells. SF Treg cells displayed high transcription levels of Bcl-2 and miR-21 in comparison with SF conventional T cells and peripheral blood Treg cells. CONCLUSION: Treg cells with a memory phenotype accumulate in the SF of RA patients. These Treg cells have a high proliferative activity and demonstrate little apoptosis. The latter finding could be explained by high transcription of Bcl-2 and miR-21 in SF Treg cells.

Therapeutic effect of anti-C-X-C motif chemokine 10 (CXCL10) antibody on C protein-induced myositis mouse.

INTRODUCTION: C-X-C motif chemokine 10 (CXCL10) is a chemokine that plays a critical role in the infiltration of T cells in autoimmune diseases and is reported to be expressed in muscle tissue of polymyositis. To determine the therapeutic efficacy of CXCL10 blockade, we investigated the role of CXCL10 and the effect of anti-CXCL10 antibody treatment in C protein-induced myositis (CIM), an animal model of polymyositis. METHODS: CIM was induced with human skeletal muscle C protein fragment in female C57BL/6 mice. Immunohistochemistry of CXCL10 and C-X-C motif chemokine receptor 3 (CXCR3) and measurement of serum CXCL10 were performed. Cell surface markers and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in CIM lymph node cells was investigated by flow cytometry. Mice with CIM were treated with anti-CXCL10 antibody or control antibody (anti-RVG1) and the inflammation in muscle tissue was assessed. RESULTS: Immunohistochemistry showed increased expression of CXCL10 and CXCR3 in the inflammatory lesions of muscle in CIM. Especially, CD8+ T cells invading myofiber expressed CXCR3. Serum level of CXCL10 was increased in CIM compared to the level in normal mice (normal mouse, 14.3 ± 5.3 pg/ml vs. CIM, 368.5 ± 135.6 pg/ml, P < 0.001). CXCR3 positivity in CD8+ T cells was increased compared to that of CD4+ T cells in the lymph node cells of CIM (CXCR3+ among CD8+ T cell, 65.9 ± 2.1% vs. CXCR3+ among CD4+ T cell, 23.5 ± 4.7%, P <0.001). Moreover, IFN-γ+ cells were increased among CXCR3+CD8+ T cells compared to CXCR3-CD8+ T cells (CXCR3+CD8+ T cell, 28.0 ± 4.2% vs. CXCR3-CD8+ T cell, 9.5 ± 1.5%, P = 0.016). Migration of lymph node cells was increased in response to CXCL10 (chemotactic index was 1.91 ± 0.45). CIM mice treated with anti-CXCL10 antibody showed a lower inflammation score in muscles than those with anti-RVG1 (median, anti-CXCL10 treatment group, 0.625 vs. anti-RVG1 treatment group, 1.25, P = 0.007). CONCLUSIONS: CXCL10/CXCR3 expression was increased in the inflammation of CIM model and its blockade suppressed inflammation in muscle.

CD70-expressing CD4 T cells produce IFN-γ and IL-17 in rheumatoid arthritis.

OBJECTIVE: CD70-expressing CD4 T cells are enriched in RA and promote autoimmunity via co-stimulatory CD70-CD27 interaction. This study aimed to explore the phenotype and cytokine production of CD70(+) CD4 T cells in RA. METHODS: Peripheral blood mononuclear cells from 32 RA patients were isolated and frequencies of CD70(+) cells within different CD4 T subsets were analysed using flow cytometry. IFN-γ and IL-17 production were compared between the CD70(+) and CD70(-) cells. Expression of master transcription factors T-bet, GATA3 and retinoic acid-related orphan receptor gamma t (RORγt) were examined by real-time PCR. Results are presented as mean (s.e.m.). RESULTS: CD4 T cells of healthy controls rarely expressed CD70 as compared with CD4 T cells of RA patients [mean 0.9% (s.e.m. 0.3%) vs 7.6 (0.6), P < 0.001]. In RA, CD70(+) cells were present within all CD4 T cell subsets, i.e. CD45RA(+)CCR7(+) naive, CD45RA(-)CCR7(+) central memory, CD45RA(-)CCR7(-) effector memory and CD45RA(+)CCR7(-) terminally differentiated effector memory T cells with a mean frequency of 3.9% (s.e.m. 1.1%), 4.0 (0.5), 4.2 (0.7) and 9.4 (4.3), respectively. As compared to CD70(-) CD4 T cells, CD70(+) CD4 T cells produced significantly more IFN-γ and IL-17 after short activation. CD70(+) CD4 T cells preferentially expressed transcription factor RORγt. CONCLUSION: CD70(+) CD4 T cells are enriched in RA and may directly contribute to RA pathogenesis by producing IFN-γ and IL-17. Targeting CD70(+) CD4 T cells might offer new therapeutic opportunities in RA.

CXCR3 polymorphism is associated with male gender and pleuritis in patients with systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease involving multiple organs. Chemokines and their receptors play an important role in the pathogenesis of SLE. Lymphocytes expressing CXCR3, chemokine receptors of CXCL4, 9, 10, and 11, increase in patients with SLE and animal models, particularly in those with skin manifestations and nephritis. We investigated CXCR3 genetic polymorphisms in patients with SLE and their association with clinical manifestations. METHODS: A total of 346 patients with SLE and 540 healthy controls were investigated for CXCR3 intron 1 polymorphisms rs2280964 and rs34334103 by Taqman analysis. RESULTS: rs2280964 and rs34334103 were not associated with all patients with SLE, but rs34334103 showed a significant association with male patients with SLE. Among the clinical manifestations, pleuritis was associated with the rs34334103 polymorphism. CONCLUSION: The CXCR3 polymorphism rs34334103 was associated with male gender and pleuritis in patients with SLE.

Genome-wide association study identifies GIMAP as a novel susceptibility locus for Behcet's disease.

OBJECTIVES: To identify non-major histocompatibility complex susceptible genes that might contribute to Behçet's disease (BD). METHODS: We performed a genome-wide association study using DNA samples from a Korean population consisting of 379 BD patients and 800 controls. A replication study was performed in a Japanese population (363 BD patients and 272 controls). To evaluate the functional implication of the target single nucleotide polymorphisms (SNP), gene expression levels in peripheral T cells, allele-specific modulation of promoter activity and biological effect of mRNA knockdown were investigated. RESULTS: We found a novel association of BD to the GIMAP locus, mapped to chromosome 7q36.1 (rs1608157, p=6.01×10(-8) in a minor allele dominant model; rs11769828, allele based p=1.60×10(-6)). A fine mapping study identified an association with four additional SNP: rs1522596 (OR=1.45, p=7.70×10(-6)) in GIMAP4; rs10266069 (OR=1.32, p=2.67×10(-4)) and rs10256482 (OR=1.27, p=5.27×10(-4)) in GIMAP2; and rs2286900 (OR=1.61, p=3.53×10(-5)) in GIMAP1 areas. Replication study using DNA samples from the Japanese population validated the significant association between BD and the GIMAP locus. The GIMAP4 promoter construct plasmid with the minor allele of rs1608157 displayed significantly lower activity than one with the major allele. Moreover, CD4 T cells from BD patients showed a lower level of GIMAP4 mRNA, and GIMAP4 knockdown was protective against Fas-mediated apoptosis. CONCLUSIONS: These results suggest that a GIMAP cluster is a novel susceptibility locus for BD, which is involved in T-cell survival, and T-cell aberration can contribute to the development of BD.

Characterization of effector memory CD8+ T cells in the synovial fluid of rheumatoid arthritis.

Little is known about the cellular characteristics of CD8(+) T cells in rheumatoid arthritis (RA). We addressed this by investigating whether the frequency of the CD8(+) T cell subsets and their phenotypic characteristics are altered in the peripheral blood and synovial fluid (SF) from patients with RA. In this study, CD8(+) T cells, mainly CD45RA(-) effector memory (EM) CD8(+) T cells, were increased significantly in the SF, but not in the peripheral blood from RA patients, compared with healthy controls. The synovial EM CD8(+) T cells were activated phenotypes with high levels of CD80, CD86, and PD-1, and had a proliferating signature in vivo upon Ki-67 staining, whereas the Fas-positive cells were prone to apoptosis. In addition, EM CD8(+) T cells in the SF were less cytotoxic, as they expressed less perforin and granzyme B. In particular, the proportions of synovial fluid mononuclear cells that were CCR4(+)CD8(+) T cells and IL-4-producing CD8(+) T cells (i.e., Tc2 cells) were significantly higher than those in peripheral blood mononuclear cells of patients with RA and healthy controls. In addition, the number of IL-10-producing CD8(+) suppressor T (Ts) cells increased significantly in the SF of RA patients. Especially, CD8(+) T cells were inversely correlated with disease activity. These findings strongly suggest that EM CD8(+) T cells in the SF are increased, likely because of inflammation, and they may be involved in modulating inflammation, thereby affecting the development and progression of RA.

Copy number variation of ß-defensin genes in Behçet's disease.

OBJECTIVES: Behçet's disease (BD) may be triggered by infectious agents in genetically susceptible persons. Human ß-defensin 2 is an inducible antimicrobial peptide, the level of which can be influenced by copy number (CN) of the DEFB4. We investigated the relationship between copy number variation (CNV) of DEFB4 and BD. METHODS: One hundred and ninety-seven patients with BD and 197 healthy controls were enrolled. After measuring CN of DEFB4 with a paralogue ratio test, the CNV was compared between patients and controls. CNV was also analysed in comparison with the clinical manifestations of BD. RESULTS: The CN of DEFB4 was unimodally distributed among the study subjects with mean CN of 4.57 and standard deviation of 1.28. BD samples had numerically lower CN than controls, but the difference was not statistically significant (4.49 ± 1.21 vs. 4.65 ± 1.36, p=0.245). Regarding the relationship between CN of DEFB4 and clinical manifestations, there was no difference of CNV depending on the clinical manifestations. CONCLUSIONS: We found no significant difference in CNV of DEFB4 between patients with BD and controls. Our results suggest that CNV of DEFB4 may not contribute to the pathogenesis of BD.

Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility.

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, P(meta) = 6.6×10(-8), OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, P(meta) = 2.9×10(-7), OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ~146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (P(meta) = 3.2×10(-7), OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (P(meta) = 3.5×10(-4), OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.