Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K.

Oral lichen planus (OLP) is a chronic inflammatory disease associated with T cell infiltration. The crosstalk between oral epithelium and mucosal T cells was considered to be crucial in the pathogenesis of OLP. Here, we selectively extracted the normal epithelium (NE) and lesional epithelium (LE) of buccal mucosa specimens from three patients with OLP by laser capture microdissection due to identify the pathogenic factors. Cathepsin K (CTSK) was identified as one of common upregulated genes in the LE by DNA microarray. Immunohistochemically, CTSK was distinctly detected in and around the LE, while it was rarely seen in the NE. Recent studies showed that CTSK enhanced Toll-like receptor 9 (TLR9) signaling in antigen-presenting cells, leading to Th17 cell differentiation. TLR9 expression mainly co-localized with CD123+ plasmacytoid dendritic cells (pDCs). The number of RORγt-positive cells correlated with that of CTSK-positive cells in OLP tissues. CD123+ pDCs induced the production of Th17-related cytokines (IL-6, IL-23, and TGF-ß) upon stimulation with TLR9 agonist CpG DNA. Moreover, single cell RNA-sequencing analysis revealed that TLR9-positive pDCs enhanced in genes associated with Th17 cell differentiation in comparison with TLR9-negative pDCs. CTSK could induce Th17-related production of CD123+ pDCs via TLR9 signaling to promote the pathogenesis of OLP.

CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

OBJECTIVE: IgG4-related disease (IgG4-RD) is a fibro-inflammatory condition that can affect multiple organs. We previously demonstrated that TLR7-transgenic C57BL/6 mice showed elevated serum IgG1 levels and inflammation with fibrosis in the salivary glands (SGs), lungs, and pancreas. Moreover, we observed extensive Toll-like receptor 7 (TLR-7)-positive CD163+ M2 macrophage infiltration in SGs from IgG4-RD patients. We undertook this study to examine the fibrotic mechanism via the TLR-7 pathway. METHODS: Gene expression in SGs from human TLR7-transgenic mice and IgG4-RD patients was analyzed using DNA microarrays. We extracted the common up-regulated TLR-7-related genes in SGs from TLR7-transgenic mice and IgG4-RD patients. Finally, we investigated the interaction between CD163+ M2 macrophages and fibroblasts before and after stimulation with the TLR-7 agonist loxoribine. RESULTS: In TLR7-transgenic mice and IgG4-RD patients, IRAK3 and IRAK4 were significantly overexpressed. Real-time polymerase chain reaction validated the up-regulation of only IRAK4 in IgG4-RD patients compared with the other groups (P < 0.05). Interleukin-1 receptor-associated kinase 4 (IRAK4) was strongly detected in and around germinal centers in SGs from patients with IgG4-related dacryoadenitis and sialadenitis alone. Double immunofluorescence staining showed that IRAK4-positive cells were mainly colocalized with CD163+ M2 macrophages in SGs (P < 0.05). After stimulation with loxoribine, CD163+ M2 macrophages exhibited significantly enhanced expression of IRAK4 and NF-κB and increased supernatant concentrations of fibrotic cytokines. Finally, we confirmed that the number of fibroblasts was increased by culture with the supernatant of CD163+ M2 macrophages following stimulation with loxoribine (P < 0.05). CONCLUSION: CD163+ M2 macrophages promote fibrosis in IgG4-RD by increasing the production of fibrotic cytokines via TLR-7/IRAK4/NF-κB signaling.

Oral Squamous Cell Carcinoma Contributes to Differentiation of Monocyte-Derived Tumor-Associated Macrophages via PAI-1 and IL-8 Production.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation and metastasis, as well as anti-tumor immune suppression. Recent studies have shown that tumors enhance the recruitment and differentiation of TAMs, but the detailed mechanisms have not been clarified. We thus examined the influence of cancer cells on the differentiation of monocytes to TAM subsets, including CD163+, CD204+, and CD206+ cells, in oral squamous cell carcinoma (OSCC) using immunohistochemistry, flow cytometry, and a cytokine array. Furthermore, we investigated the effect of OSCC cells (HSC-2, SQUU-A, and SQUU-B cells) on the differentiation of purified CD14+ cells to TAM subsets. The localization patterns of CD163+, CD204+, and CD206+ in OSCC sections were quite different. The expression of CD206 on CD14+ cells was significantly increased after the co-culture with OSCC cell lines, while the expressions of CD163 and CD204 on CD14+ cells showed no change. High concentrations of plasminogen activator inhibitor-1 (PAI-1) and interleukin-8 (IL-8) were detected in the conditioned medium of OSCC cell lines. PAI-1 and IL-8 stimulated CD14+ cells to express CD206. Moreover, there were positive correlations among the numbers of CD206+, PAI-1+, and IL-8+ cells in OSCC sections. These results suggest that PAI-1 and IL-8 produced by OSCC contribute to the differentiation of monocytes to CD206+ TAMs.

Activated M2 Macrophages Contribute to the Pathogenesis of IgG4-Related Disease via Toll-like Receptor 7/Interleukin-33 Signaling.

OBJECTIVE: IgG4-related disease (IgG4-RD) is a unique inflammatory disorder in which Th2 cytokines promote IgG4 production. In addition, recent studies have implicated the Toll-like receptor (TLR) pathway. This study was undertaken to examine the expression of TLRs in salivary glands (SGs) from patients with IgG4-RD. METHODS: SGs from 15 patients with IgG4-RD, 15 patients with Sjögren's syndrome (SS), 10 patients with chronic sialadenitis, and 10 healthy controls were examined histologically. TLR family gene expression (TLR-1 through TLR-10) was analyzed by DNA microarray in the submandibular glands (SMGs). Up-regulation of TLRs was confirmed in SGs from patients with IgG4-RD. Finally, the phenotype of human TLR-7 (huTLR-7)-transgenic C57BL/6 mice was assessed before and after stimulation with TLR agonist. RESULTS: In patients with IgG4-RD, TLR-4, TLR-7, TLR-8, and TLR-9 were overexpressed. Polymerase chain reaction validated the up-regulation of TLR-7 in IgG4-RD compared with the other groups. Immunohistochemical analysis confirmed strong infiltration of TLR-7-positive cells in the SGs of patients with IgG4-RD. Double immunohistochemical staining showed that TLR-7 expression colocalized with CD163+ M2 macrophages. After in vitro stimulation with a TLR-7 agonist, CD163+ M2 macrophages produced higher levels of interleukin-33 (IL-33), which is a Th2-activating cytokine. In huTLR-7-transgenic mice, the focus and fibrosis scores in SMGs, pancreas, and lungs were significantly higher than those in wild-type mice (P < 0.05). Moreover, the concentration of serum IgG, IgG1, and IL-33 in huTLR-7-transgenic mice was distinctly increased upon stimulation with a TLR-7 agonist (P < 0.05). CONCLUSION: TLR-7-expressing M2 macrophages may promote the activation of Th2 immune responses via IL-33 secretion in IgG4-RD.

The diagnostic utility of submandibular gland sonography and labial salivary gland biopsy in IgG4-related dacryoadenitis and sialadenitis: Its potential application to the diagnostic criteria.

Objectives: In this study, we investigated the diagnostic utility of submandibular gland (SMG) sonography and labial salivary gland (LSG) biopsy as a less invasive procedure for diagnosing IgG4-related dacryoadenitis and sialadenitis (IgG4-DS)Methods: Sixty-eight patients with suspected IgG4-DS by presenting swelling of elevated serum IgG (>1747 mg/dl) and/or swelling glands underwent SMG sonography, LSG biopsy and measurement for serum IgG4. SMG sonographic diagnosis was determined by the following characteristic changes; 'hypoechoic areas of a nodal pattern with high vascularity' and/or 'hypoechoic areas of a reticular pattern in the superficial part'.Results: Thirty-one patients were diagnosed with IgG4-DS, 5 with IgG4-RD unaccompanied by lacrimal and salivary gland lesions, 28 with Sjögren's syndrome, and 4 with malignant lymphoma. The sensitivity, specificity, and accuracy of SMG sonography and LSG biopsy were 100%, 83.8%, 91.2% and 64.5%, 73.8%, 75.0%, respectively. Moreover, those of SMG sonography and LSG biopsy combined with serum IgG4 concentration (>135 mg/dl) were 100%, 94.6%, 97.1% and 64.5%, 91.9%, 79.4%, respectively.Conclusion: LSG biopsy needs to be extremely careful to diagnose IgG4-DS because of its low sensitivity. SMG sonography is sufficient for the diagnosis of IgG4-DS, especially when combined with serologic analysis. Thus, SMG sonography could adapt to the diagnostic criteria of IgG4-DS as a non-invasive method.

CD206+ tumor-associated macrophages promote proliferation and invasion in oral squamous cell carcinoma via EGF production.

Tumor-associated macrophages (TAMs) promote tumor progression and inhibit anti-tumor immune response by producing various mediators and preferentially express CD163, CD204, and CD206. However, the role of these TAM subsets in oral squamous cell carcinoma (OSCC) remains unclear. Here we investigated the expression and function of TAM subsets in OSCC, especially in cancer cell proliferation. Biopsy sample from 44 patients with OSCC were examined for the expression of TAM markers and EGF by immunohistochemistry. EGF production of TAM subsets isolated from OSCC patients was assessed by flow cytometry. We also examined the effect of conditioned medium from TAM subsets on the proliferation of OSCC cells. CD163+ cells were detected diffusely all over the tumor and connective tissue area, while CD204+ and CD206+ cells were mainly detected in/around the tumors. Flow cytometric analysis found that CD206+ TAMs strongly produced EGF compared with CD163+ and CD204+ TAMs. Cell proliferation and invasion of OSCC cells cultured with conditioned medium of CD206+ TAMs were strongly enhanced and inhibited by anti-EGFR. The number of CD206+ TAMs positively correlated with worse clinical prognosis. Our results revealed differences in localization and EGF production among these TAM subsets. CD206+ TAMs might play a critical role in the proliferation of OSCC via EGF production.

The expansion in lymphoid organs of IL-4+ BATF+ T follicular helper cells is linked to IgG4 class switching in vivo.

Distinct T follicular helper (TFH) subsets that influence specific class-switching events are assumed to exist, but the accumulation of isotype-specific TFH subsets in secondary lymphoid organs (SLOs) and tertiary lymphoid organs has not been hitherto demonstrated. IL-4-expressing TFH cells are surprisingly sparse in human SLOs. In contrast, in IgG4-related disease (IgG4-RD), a disorder characterized by polarized Ig class switching, most TFH cells in tertiary and SLOs make IL-4. Human IL-4+ TFH cells do not express GATA-3 but express nuclear BATF, and the transcriptomes of IL-4-secreting TFH cells differ from both PD1hi TFH cells that do not secrete IL-4 and IL-4-secreting non-TFH cells. Unlike IgG4-RD, IL-4+ TFH cells are rarely found in tertiary lymphoid organs in Sjögren's syndrome, a disorder in which IgG4 is not elevated. The proportion of CD4+IL-4+BATF+ T cells and CD4+IL-4+CXCR5+ T cells in IgG4-RD tissues correlates tightly with tissue IgG4 plasma cell numbers and plasma IgG4 levels in patients but not with the total plasma levels of other isotypes. These data describe a disease-related TFH subpopulation in human tertiary lymphoid organs and SLOs that is linked to IgG4 class switching.

CD163+CD204+ tumor-associated macrophages contribute to T cell regulation via interleukin-10 and PD-L1 production in oral squamous cell carcinoma.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation, invasion, and metastasis by producing various mediators. Although preclinical studies demonstrated that TAMs preferentially express CD163 and CD204, the TAM subsets in oral squamous cell carcinoma (OSCC) remain unknown. In this study, we examined the expression and role of TAM subsets in OSCC. Forty-six patients with OSCC were analyzed for expression of TAMs in biopsy samples by immunohistochemistry. We examined TAM subsets and their production of immune suppressive molecules (IL-10 and PD-L1) in peripheral blood mononuclear cells from three OSCC patients by flow cytometry. CD163 was detected around the tumor or connective tissue, while CD204 was detected in/around the tumors. Flow cytometric analysis revealed that CD163+CD204+ TAMs strongly produced IL-10 and PD-L1 in comparison with CD163+CD204- and CD163-CD204+ TAMs. Furthermore, the number of activated CD3+ T cells after co-culture with CD163+CD204+ TAMs was significantly lower than that after co-culture with other TAM subsets. In clinical findings, the number of CD163+CD204+ TAMs was negatively correlated with that of CD25+ cells and 5-year progression-free survival. These results suggest that CD163+CD204+ TAMs possibly play a key role in the invasion and metastasis of OSCC by T-cell regulation via IL-10 and PD-L1 production.

Myeloid dendritic cells stimulated by thymic stromal lymphopoietin promote Th2 immune responses and the pathogenesis of oral lichen planus.

Oral lichen planus (OLP) is a chronic inflammatory disease characterized by subepithelial T-cell infiltration. Recent studies reported that specific T helper (Th) subsets, especially Th2 cells, are involved in the pathogenesis of OLP. Thymic stromal lymphopoietin (TSLP) is mainly secreted by epithelial cells and potently activates myeloid dendritic cells (mDCs) to induce Th2-mediated inflammation. Here, we investigated the expression of TSLP and related molecules in OLP. Buccal mucosa specimens from patients with OLP, hyperkeratosis, and ulcer were analyzed by immunohistochemistry for expression of TSLP, its receptor (TSLPR), and inflammatory cells. TSLP was detected in/around the epithelium of patients with OLP and hyperkeratosis, whereas TSLPR, CD11c (mDC), and GATA3 (Th2) were strongly expressed in the subepithelial layer only in OLP patients. Double immunofluorescence staining showed that TSLPR expression mainly co-localized with CD11c. Moreover, the number of CD11c- and GATA-3 positive cells was correlated in OLP patients. In lesions selectively extracted by laser microdissection, the mRNA expression of Th2 (IL-4, MDC, TARC, GATA3)- and Th17 (IL-17, RORγt)-related molecules in OLP patients was significantly higher than in other groups. These results suggest that CD11c+ mDCs expressing TSLPR contribute to aberrant Th2 immune responses and the pathogenesis of OLP via TSLP stimulation.

Interleukin-33 produced by M2 macrophages and other immune cells contributes to Th2 immune reaction of IgG4-related disease.

IgG4-related disease (IgG4-RD) is characterized by elevated serum IgG4 and marked infiltration of IgG4-positive cells in multiple organs. Interleukin-33 (IL-33) is a recently described cytokine that is secreted by damaged epithelial cells, macrophages, and dendritic cells, and potently activates helper T type 2 (Th2) immune responses, which have been suggested to play a major role in IgG4 production of IgG4-RD. Here, we assessed the expression of IL-33 and related molecules in the salivary glands (SGs) of patients with IgG4-RD versus that in patients with Sjögren's syndrome (SS) and controls. Expression of IL-33 and its receptor (ST2) was strongly detected around ectopic germinal centers (GCs) in the SGs from patients with IgG4-RD, whereas IL-33 was expressed only in epithelial cells in patients with SS and controls. Moreover, IL-33 and CD68+/CD163+ macrophages were mainly distributed around ectopic GCs in patients with IgG4-RD. Double immunofluorescence staining showed that IL-33 expression co-localized with CD68+/CD163+ macrophages. Finally, mRNA expression levels of IL-33 showed a positive correlation to those of Th2 cytokines (IL-4 and IL-13) in patients with IgG4-RD. Our data suggest that IL-33 produced by M2 macrophages might contribute to the pathogenesis of IgG4-RD via aberrant activation of Th2 immune responses.

Molecular analysis of fungal populations in patients with oral candidiasis using next-generation sequencing.

Oral candidiasis is closely associated with changes in oral fungal biodiversity and is caused primarily by Candida albicans. However, the widespread use of empiric and prophylactic antifungal drugs has caused a shift in fungal biodiversity towards other Candida or yeast species. Recently, next-generation sequencing (NGS) has provided an improvement over conventional culture techniques, allowing rapid comprehensive analysis of oral fungal biodiversity. In this study, we used NGS to examine the oral fungal biodiversity of 27 patients with pseudomembranous oral candidiasis (POC) and 66 healthy controls. The total number of fungal species in patients with POC and healthy controls was 67 and 86, respectively. The copy number of total PCR products and the proportion of non-C. albicans, especially C. dubliniensis, in patients with POC, were higher than those in healthy controls. The detection patterns in patients with POC were similar to those in controls after antifungal treatment. Interestingly, the number of fungal species and the copy number of total PCR products in healthy controls increased with aging. These results suggest that high fungal biodiversity and aging might be involved in the pathogenesis of oral candidiasis. We therefore conclude that NGS is a useful technique for investigating oral candida infections.

DNA Microarray Analysis of Submandibular Glands in IgG4-Related Disease Indicates a Role for MARCO and Other Innate Immune-Related Proteins.

IgG4-related disease (IgG4-RD) is a novel systemic disease entity characterized by elevated serum IgG4 and tissue infiltration of IgG4-positive plasma cells accompanied by severe fibrosis. Although recent studies demonstrated that innate immune cells including monocytes and macrophages might promote local fibrosis and IgG4 production, the pathological mechanism remains unclear. In this study, we sought to identify the disease-associated genes, especially innate immune molecules. Gene expression was analyzed by DNA microarray in submandibular glands (SMGs) from patients with IgG4-RD (n = 5), chronic sialoadenitis (CS) (n = 3), and controls (n = 3). Differentially expressed genes (DEGs) were validated by real-time polymerase chain reaction (PCR) and immunohistochemical staining in IgG4-RD (n = 18), CS (n = 4), Sjögren syndrome (n = 11), and controls (n = 10). Gene expression patterns in the 3 groups were quite different from each other by the pvclust method and principal components analysis. In IgG4-RD, 1028 upregulated genes and 692 downregulated genes were identified as DEGs (P < 0.05). Gene Ontology (GO) term analysis indicated that the upregulated DEGs in IgG4-RD encoded proteins involved in T/B cell activation and chemotaxis. PCR validated significantly higher expression of macrophage receptor with collagenous structure (MARCO), a pattern-recognition receptor, in IgG4-RD compared with the other groups (P < 0.01). Immunohistochemical analysis confirmed that the expression pattern of MARCO was similar to that of the M2 macrophage marker CD163. MARCO was identified as a disease-associated molecule in IgG4-RD by DNA microarray. Moreover, M2 macrophages might contribute to the initiation of IgG4-RD via MARCO.

The diagnostic utility of labial salivary gland biopsy in IgG4-related disease.

OBJECTIVE: For the definitive diagnosis of IgG4-related disease (IgG4-RD), biopsies of local lesions are recommended so as to exclude other diseases, including lymphoma and cancer. However, performing biopsies of underlying organs is technically difficult. In this study, we examined the diagnostic utility of labial salivary gland (LSG) biopsy as a less invasive procedure. METHODS: Sixty-six patients with suspected IgG4-RD by clinical findings or high serum IgG4 underwent LSG biopsy. We examined the relationship between the number of IgG4-positive plasma cells in LSG and clinical findings. RESULTS: The final diagnosis was 45 patients with IgG4-RD, 12 with Sjögren's syndrome, four with suspected Sjögren's syndrome, three with malignant lymphoma, one with systemic lupus erythematosus, and one with Warthin's tumor. The sensitivity, specificity, and accuracy of LSG biopsy were 55.6%, 100.0%, and 70.0%, respectively. Forty-five IgG4-RD patients were divided into two groups: 1) 25 with lesions of salivary glands (IgG4-RD S+) and 2) 20 without these lesions (IgG4-RD S-). Seventeen of 25 (68.0%) IgG4-RD S + and 8 of 20 (40.0%) IgG4-RD S - patients were positive for LSG biopsy. In the IgG4-RD S - patients, the mean number of affected organs and serum IgG4 in the positive cases for LSG biopsy were significantly higher than in the negative cases. CONCLUSION: A solo LSG biopsy is insufficient for the diagnosis of IgG4-RD because of its low sensitivity. However, LSG biopsy combined with clinical findings, including serum IgG4 and number of affected organs, may contribute towards a diagnosis of IgG4-RD patients with affected underlying organs.

A series of thiazole derivatives bearing thiazolidin-4-one as non-competitive ADAMTS-5 (aggrecanase-2) inhibitors.

A series of thiazole bearing thiazolidin-4-one was discovered via high-throughput screening as non-competitive inhibitors of ADAMTS-5. Compound 31 appeared to give the best ADAMTS-5 inhibition and good selectivity over other metalloproteases.

Effects of low-load resistance training with vascular occlusion on the mechanical properties of muscle and tendon.

The present study aimed to investigate the effects of low-load resistance training with vascular occlusion on the specific tension and tendon properties by comparing with those of high-load training. Nine participants completed 12 weeks (3 days/week) of a unilateral isotonic training program on knee extensors. One leg was trained using low load (20% of 1 RM) with vascular occlusion (LLO) and other leg using high load (80% of 1 RM) without vascular occlusion (HL). Before and after training, maximal isometric knee extension torque (MVC) and muscle volume were measured. Specific tension of vastus lateralis muscle (VL) was calculated from MVC, muscle volume, and muscle architecture measurements. Stiffness of tendon-aponeurosis complex in VL was measured using ultrasonography during isometric knee extension. Both protocols significantly increased MVC and muscle volume of quadriceps femoris muscle. Specific tension of VL increased significantly 5.5% for HL, but not for LLO. The LLO protocol did not alter the stiffness of tendon-aponeurosis complex in knee extensors, while the HL protocol increased it significantly. The present study demonstrated that the specific tension and tendon properties were found to remain following low-load resistance training with vascular occlusion, whereas they increased significantly after high-load training.

Applicability of segmental bioelectrical impedance analysis for predicting trunk skeletal muscle volume.

This study aimed to investigate the validity of using segmental bioelectrical impedance (BI) analysis for estimating skeletal muscle volume (MV) in the trunk, defined as the body segment from the acromion process to the greater trochanter. Using a magnetic resonance imaging (MRI) method, the trunk MV was determined in 28 men (19 approximately 34 yr), divided into validation (n = 20) and cross-validation (n = 8) groups, and used as a reference (MV(MRI)). For BI measurements of the trunk, the source electrodes were placed at the dorsal surface of the third metacarpal bone of both hands and the dorsal surface of the third metatarsal bone of both feet, and the detector electrodes were placed at the acromion process of both shoulders and the greater trochanter of both femurs. Using this arrangement, the BI values of five parts of the trunk, both sides of the upper region, the middle region, and both sides of the lower region, were obtained and then used to calculate the whole trunk BI value and BI index (BI index(TR)). In the validation group, a simple regression analysis of the relationship between BI index(TR) and MV(MRI) showed a significant correlation between the two variables (r = 0.884, P < 0.05) and produced a prediction equation with a SE of estimation of 1,020.3 cm3 (8.5%). In the validation and cross-validation groups, there were no significant differences between the measured and estimated MV without systematic errors. These findings indicate that the segmental BI analysis employed in the present study can be used to estimate trunk MV.

A comparison of three bioelectrical impedance analyses for predicting lean body mass in a population with a large difference in muscularity.

This study tested the hypothesis that, as compared to whole-body bioelectrical impedance (BI) analysis, segmental BI analysis can estimate lean body mass (LBM) more accurately in a population with a large difference in muscularity. In addition to whole-body BI, which determines impedance (Z) between the wrist and ankle, two segmental BI analyses which determine the Z value of every body segment in each of (1) the arms, legs and trunk (distal BI) and (2) the upper arms, upper legs and trunk (proximal BI) were applied to a group of 125 male athletes and 75 non-athletes. The subjects were divided into validation and cross-validation groups. Simple and multiple regression analyses were applied to (length)(2)/Z (BI index) values for the whole-body and each body segment, to develop the prediction equations of LBM measured using air-displacement plethysmography. In the validation group, the SE of estimation was similar in the whole-body (3.4 kg, 5.4%), distal (3.4 kg, 5.5%) and proximal BI (3.3 kg, 5.2%) analyses. However, the whole-body and distal BI analyses produced systematical errors in the estimates of LBM. Moreover, the residuals in the two methods significantly (P < 0.05) correlated with the ratios of BI indices of the upper arms and upper legs to those of the arms and legs, respectively, calculated as variables approximating the relative development of lean tissues at the proximal area of limbs. On the other hand, the proximal BI analysis was validated and cross-validated. Thus, the accuracy of estimating LBM was similar in the whole-body and the two segmental BI analyses. However, the prediction equations derived from the use of the whole-body BI index and a combination of the arms, legs and trunk BI indices produced a systematical error relating to the difference between the limb segments in lean tissue development.