Evaluating treatment practices and challenges in systemic Juvenile Idiopathic Arthritis: a comprehensive survey analysis.

OBJECTIVE: This study aims to assess current diagnostic and management for systemic Juvenile Idiopathic Arthritis (sJIA) among physicians, evaluate the challenges encountered in diagnosis and treatment, and identify the educational needs and professional development engagements of physicians managing sJIA. METHODS: A nationwide survey was conducted from November 2023 to March 2024 across tertiary and secondary pediatric and general hospitals in China. The survey targeted physicians with at least three years of specialty experience, resulting in 310 valid responses from 25 provinces, autonomous regions, and municipalities. The survey collected data on diagnostic practices, treatment approaches, and professional development related to sJIA. Data collection was facilitated through WeChat, and statistical analysis was performed using descriptive statistics. Ethical approval was obtained from the Ethics Committee of Beijing Children's Hospital, with informed consent provided electronically by participants. RESULTS: The survey indicated that all physicians encountered suspected or confirmed cases of sJIA, highlighting its prevalence and the diagnostic challenges associated. Regarding diagnostic standards, 53.9% of physicians used the "Consensus on the Diagnosis and Treatment of sJIA and Macrophage Activation Syndrome," 18.1% followed the International League of Associations for Rheumatology (ILAR) standards, and 24.8% adhered to the Pediatric Rheumatology International Trials Organization (PRINTO) standards. In treatment strategies, glucocorticoids and IL-6 receptor monoclonal antibodies were extensively used, with the latter receiving "excellent" and "satisfactory" ratings of 46.5% and 36.1%, respectively, demonstrating high efficacy and acceptance. Main challenges included high treatment costs, complexity of diagnosis, patient compliance issues, and potential long-term side effects of biologics. Additionally, 126 doctors (40.7%) actively participated in more than three academic conferences or systematic learning courses related to sJIA, indicating a strong demand for ongoing education, particularly in new treatment developments and diagnostic skills. CONCLUSION: The findings emphasize the necessity for standardized diagnosis and customized treatment plans tailored to patient-specific conditions in managing sJIA. Key Points • The survey highlights the prevalence and clinical challenges of sJIA among physicians, emphasizing the importance of vigilant diagnosis, multi-system involvement, and differential diagnosis to improve treatment outcomes and patient quality of life.

Element accumulation in the tracheal and bronchial cartilages of monkeys.

Compositional changes in the tracheal and bronchial cartilages can affect respiratory ventilation and lung function. We aimed to elucidate element accumulation in the tracheal and bronchial cartilages of monkeys and divided it into four sites: the tracheal, tracheal bifurcation, left bronchial, and right bronchial cartilages. The elemental content was analyzed using inductively coupled plasma atomic emission spectrometry. The average calcium content was two to three times higher in the tracheal cartilage than in the other three cartilages. The trends of phosphorus and zinc were similar to those of calcium. The average calcium, phosphorus, and zinc cartilage contents were the highest in the tracheal cartilage and decreased in the following order: the left bronchial, right bronchial, and tracheal bifurcation cartilages. These findings revealed that differences existed in element accumulation between different sites within the same airway cartilage and that calcium, phosphorus, and zinc accumulation mainly occurred in the tracheal cartilage. A substantial direct correlation was observed between age and calcium content in the tracheal and bronchial cartilages and all such monkeys with high calcium content were > four years of age. These results suggest that calcium accumulation occurs in the tracheal and bronchial cartilages after reaching a certain age. An extremely substantial direct correlation was observed between calcium and phosphorus contents in the tracheal and bronchial cartilages. This finding is similar to the previously published calcium and phosphorus correlations in several other cartilages, suggesting that the calcium and phosphorus contents of cartilage exist in a certain ratio.

The nuclear pore protein Nup2 is essential for growth and development, stress response, pathogenicity and deoxynivalenol biosynthesis in Fusarium graminearum.

BACKGROUND: Wheat is an important grain crop that has been under serious threat from Fusarium graminearum. Nup2, a member of the nuclear pore complex, plays an important role in regulating eukaryotic nuclear protein transport and participates in gene regulation. Dissecting the function of nuclear pore proteins in pathogenic fungi may provide effective targets for novel fungicides. RESULTS: Mutants exhibited nutritional growth defects, asexual/sexual developmental abnormalities. Deficiency of FgNup2 resulted in increased resistance of Fusarium graminearum to cell wall disruptors and increased sensitivity to metal ions. Pathogenicity analyses showed that the mutant was significantly less virulent on flowering wheat ears, consistent with the observed decrease in deoxynivalenol (DON) production. Furthermore, we showed that FgNup2 interacts synergistically with FgTri6, a transcription factor of the TRI family, to regulate the expression of toxin-producing genes, which, in turn, affects the biosynthesis of DON and related toxins. CONCLUSION: This study revealed that FgNup2 plays important roles in the growth and development, cell wall integrity, stress response, pathogenicity, and DON synthesis of F. graminearum. © 2024 Society of Chemical Industry.

Long noncoding nuclear enriched abundant transcript 1_2 is a promising biomarker for childhood-onset systemic lupus erythematosus.

Importance: Systemic lupus erythematosus (SLE) is a diffuse connective tissue disease with complex clinical manifestations and prolonged course. The early diagnosis and condition monitoring of SLE are crucial to disease prognosis. Objective: To assess the diagnostic value of long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) in childhood-onset SLE (cSLE). Methods: Fifty-seven children diagnosed with SLE, 40 children diagnosed with juvenile idiopathic arthritis (JIA), and 40 healthy children were included. Peripheral blood samples from each patient were collected. A quantitative polymerase chain reaction was used to confirm the expression of lncNEAT1_1 and lncNEAT1_2 in peripheral blood. Associations among parameters were analyzed using the Mann-Whitney U test or independent sample t-test. Results: The expression of both lncNEAT1_1 and lncNEAT1_2 in patients with cSLE were significantly higher than that of healthy control and patients with JIA. Receiver operating characteristic curves revealed an area under the curve (AUC) of 0.633 (95% confidence interval [CI], 0.524-0.742; P = 0.024) for lncNEAT1_1. The AUC of lncNEAT1_2 was 0.812 (95% CI, 0.727-0.897; P < 0.0001) to discriminate individuals with cSLE from health control and children with JIA with a sensitivity of 0.622 and a specificity of 0.925. Moreover, lncNEAT1_2 expression was higher in patients with cSLE presenting with fever, lupus nephritis, elevated erythrocyte sedimentation rate, active disease activity, and decreased C3 level, compared with those without these conditions. However, no similar correlation was observed for lncNEAT1_1. Interpretation: The expression of lncNEAT1_2 was significantly elevated in children with SLE, especially those with fever, renal involvement, and low C3 levels. These findings suggest that lncNEAT1_2 may represent a potential biomarker for cSLE.

A GITRL-mTORC1-GM-CSF Positive Loop Promotes Pathogenic Th17 Response in Primary Sjögren Syndrome.

OBJECTIVE: Glucocorticoid-induced tumor necrosis factor receptor superfamily-related protein (GITR), with its ligand (GITRL), plays an important role in CD4+ T cell-mediated autoimmunity. This study aimed to investigate the underlying mechanisms of GITRL in primary Sjögren syndrome (pSS). METHODS: Patients with pSS and healthy controls were recruited. Serum GITRL and Th17-related cytokines were determined. RNA sequencing was performed to decipher key signal pathways. Nonobese diabetes (NOD) mice were adopted as experimental Sjögren models and recombinant adeno-associated virus (rAAV) transduction was conducted to verify the therapeutic potentials of targeting GITRL in vivo. RESULTS: Serum GITRL was significantly higher in patients with pSS and showed a positive correlation with leukopenia, thrombocytopenia, autoantibodies, lung involvement, and disease activity. Serum GITRL was correlated with Th17-related cytokines. GITRL promoted the expansion of Th17 and Th17.1 cells. Expansion of granulocyte-macrophage colony-stimulating factor positive (GM-CSF+) CD4+ T cells induced by GITRL could be inhibited by blockade of GITRL. Moreover, GM-CSF could stimulate GITRL expression on monocytes. RNA sequencing revealed mammalian target of rapamycin complexes 1 (mTORC1) might be the key modulator. The increased phosphorylation of S6 and STAT3 and the expansion of Th17 and Th17.1 cells induced by GITRL were effectively inhibited by rapamycin, suggesting a GITRL-mTORC1-GM-CSF positive loop in pathogenic Th17 response in pSS. Administration of an rAAV vector expressing short hairpin RNA targeting GITRL alleviated disease progression in NOD mice. CONCLUSION: Our results identified the pathogenic role of GITRL in exacerbating disease activity and promoting pathogenic Th17 response in pSS through a GITRL-mTORC1-GM-CSF loop. These findings suggest GITRL might be a promising therapeutic target in the treatment of pSS.

[Low disease activity and remission status of systemic lupus erythematosus in a real-world study].

OBJECTIVE: To investigate the rates of low disease activity and clinical remission in patients with systemic lupus erythematosus (SLE) in a real-world setting, and to analyze the related factors of low disease activity and clinical remission. METHODS: One thousand patients with SLE were enrolled from 11 teaching hospitals. Demographic, clinical and laboratory data, as well as treatment regimes were collec-ted by self-completed questionnaire. The rates of low disease activity and remission were calculated based on the lupus low disease activity state (LLDAS) and definitions of remission in SLE (DORIS). Charac-teristics of patients with LLDAS and DORIS were analyzed. Multivariate Logistic regression analysis was used to evaluate the related factors of LLDAS and DORIS remission. RESULTS: 20.7% of patients met the criteria of LLDAS, while 10.4% of patients achieved remission defined by DORIS. Patients who met LLDAS or DORIS remission had significantly higher proportion of patients with high income and longer disease duration, compared with non-remission group. Moreover, the rates of anemia, creatinine elevation, increased erythrocyte sedimentation rate (ESR) and hypoalbuminemia was significantly lower in the LLDAS or DORIS group than in the non-remission group. Patients who received hydroxychloroquine for more than 12 months or immunosuppressant therapy for no less than 6 months earned higher rates of LLDAS and DORIS remission. The results of Logistic regression analysis showed that increased ESR, positive anti-dsDNA antibodies, low level of complement (C3 and C4), proteinuria, low household income were negatively related with LLDAS and DORIS remission. However, hydroxychloroquine usage for longer than 12 months were positively related with LLDAS and DORIS remission. CONCLUSION: LLDAS and DORIS remission of SLE patients remain to be improved. Treatment-to-target strategy and standar-dized application of hydroxychloroquine and immunosuppressants in SLE are recommended.

A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity.

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

Anti-Toxoplasma gondii antibodies as a risk factor for the prevalence and severity of systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease characterized by the presence of numerous autoantibodies. The interaction of infectious agents (viruses, bacteria and parasites) and a genetically susceptible host may be a key mechanism for SLE. Toxoplasma gondii is a widespread intracellular parasite that has been implicated in the pathogenesis of autoimmune diseases. However, the relationship between T. gondii infection and the increased risk of SLE in Chinese populations remains unclear. METHODS: The seroprevalence of T. gondii infection was assessed in 1771 serum samples collected from Chinese individuals (908 healthy controls and 863 SLE patients) from different regions of China using an enzyme-linked immunosorbent assay. Serum autoantibodies and clinical information were obtained and analysed. RESULTS: Our observations revealed a higher prevalence of anti-T. gondii antibodies (ATxA) immunoglobulin G (IgG) in serum samples from SLE patients (144/863, 16.7%) than in those from the healthy controls (53/917, 5.8%; P < 0.0001), indicating a 2.48-fold increased risk of SLE in the ATxA-IgG+ population, after adjustment for age and sex (95% confidence interval [CI] 1.70-3.62, P < 0.0001). ATxA-IgG+ SLE patients also showed a 1.75-fold higher risk of developing moderate and severe lupus symptoms (95% CI 1.14-2.70, P = 0.011) compared to ATxA-IgG- patients. Relative to ATxA-IgG- patients, ATxA-IgG+ patients were more likely to develop specific clinical symptoms, including discoid rash, oral ulcer, myalgia and alopecia. Seven antibodies, namely anti-ribosomal RNA protein (rRNP), anti-double stranded DNA (dsDNA), anti-cell membrane DNA (cmDNA), anti-scleroderma-70 (Scl-70), anti-cardiolipin (CL), anti-beta2-glycoprotein-I (B2GPI) and rheumatoid factor (RF), occurred more frequently in ATxA-IgG+ patients. When combined with anti-dsDNA and RF/anti-rRNP/anti-cmDNA/ESR, ATxA-IgG significantly increased the risk for severe lupus. CONCLUSIONS: Our results suggest that ATxA-IgG may be a significant risk factor for SLE prevalence and severity in Chinese populations.

Temporally-coordinated bivalent histone modifications of BCG1 enable fungal invasion and immune evasion.

Bivalent histone modifications, including functionally opposite H3K4me3 and H3K27me3 marks simultaneously on the same nucleosome, control various cellular processes by fine-tuning the gene expression in eukaryotes. However, the role of bivalent histone modifications in fungal virulence remains elusive. By mapping the genome-wide landscape of H3K4me3 and H3K27me3 dynamic modifications in Fusarium graminearum (Fg) during invasion, we identify the infection-related bivalent chromatin-marked genes (BCGs). BCG1 gene, which encodes a secreted Fusarium-specific xylanase containing a G/Q-rich motif, displays the highest increase of bivalent modification during Fg infection. We report that the G/Q-rich motif of BCG1 is a stimulator of its xylanase activity and is essential for the full virulence of Fg. Intriguingly, this G/Q-rich motif is recognized by pattern-recognition receptors to trigger plant immunity. We discover that Fg employs H3K4me3 modification to induce BCG1 expression required for host cell wall degradation. After breaching the cell wall barrier, this active chromatin state is reset to bivalency by co-modifying with H3K27me3, which enables epigenetic silencing of BCG1 to escape from host immune surveillance. Collectively, our study highlights how fungal pathogens deploy bivalent epigenetic modification to achieve temporally-coordinated activation and suppression of a critical fungal gene, thereby facilitating successful infection and host immune evasion.

High killing rate of nematode and promotion of rice growth by synthetic volatiles from Bacillus strains due to enhanced oxidative stress response.

The plant parasitic nematode Aphelenchoides besseyi is a major pest that poses serious threats to different vegetables and crop plants. In the present study, volatiles isolated from Bacillus spp. were utilized as green biocontrol agents to overcome nematodes. In in vitro experiment, Bacillus spp. GBSC56, SYST2, and FZB42 showed the strongest nematicidal activity with killing rates of 80.78%, 75.69%, and 60.45%, respectively, as compared with control. The selected synthetic volatile organic compounds (VOCs), namely albuterol, benzaldehyde (BDH), 1,2-benzisothiazol-3(2H)-one (1,2-HIT), dimethyl disulfide (DMDS), 2-undecanone (2-UD), and 1,3-propanediole (1,3-PD), exhibited strong nematicidal activity, with A. besseyi killing rate of 85.58%, 82.65%, 81.75%, 80.36%, 84.45%, and 82.36%, respectively, at 400 µg/mL. Microscopic analysis proved that the rapid mortality was due to the production of reactive oxygen species (ROS). Molecular docking attributed this ROS production to the nematicidal effect of synthetic VOCs on NADH DEHYDROGENASE SUBUNIT 2, which is known to play a critical role in the suppression of ROS in nematode models. In a greenhouse experiment, the Bacillus strains GBSC56, SYST2, and FZB42 and their synthetic VOCs significantly improved the physiological parameters in terms of growth promotion traits. In addition, selected genes related to growth promotion and defense genes showed a significant upregulation of their expression in rice seedlings treated with those synthetic VOCs. Overall, these findings revealed that the selected Bacillus strains and their synthetic VOCs possess high potential against A. besseyi. Moreover, this study also sheds new light on the mechanisms by which specific Bacillus nematicidal VOCs influence important genes involved in rice plant growth promotion and could effectively be used to suppress plant parasitic nematodes.

Addition of venetoclax to myeloablative conditioning regimens for allogeneic hematopoietic stem cell transplantation in high-risk AML.

BACKGROUND: Venetoclax monotherapy is an effective option for patients with acute myeloid leukemia (AML). Venetoclax has also been used in non-myeloablative conditioning allogeneic hematopoietic stem cell transplantation (allo-HSCT) for high-risk AML with a tolerable toxicity profile. However, the efficacy and safety of a venetoclax-containing myeloablative conditioning (MAC) allo-HSCT regimen for high-risk AML have not been evaluated. OBJECTIVE: To evaluate the safety and efficacy of a MAC regimen containing venetoclax for high-risk AML. STUDY DESIGN: From 25 February 2021 to 4 September 2022, a total of 31 patients with high-risk AML who underwent allo-HSCT and a MAC regimen with venetoclax were analyzed. RESULTS: At the time of transplantation, 21 patients were in first complete remission (CR1), 4 were in a second complete remission (CR2), and 6 in non-remission (NR). Twenty-four patients (77.4%) were minimal residual disease (MRD)-positive before transplant. The FLT3-ITD gene mutation was present in 51.6% of patients. NUP98 rearrangement, MLL rearrangement or MLL-PTD and DEK::CAN fusion genes were found in 5 (16.1%), 7(22.6%) and 2 (6.5%) patients, respectively. Twenty-nine (93.6%) patients underwent haploidentical allo-HSCT. The median follow-up time was 278 days (range: 52-632 days). The 100-day cumulative incidence of grade 3 to 4 acute graft-versus-host disease (aGVHD) was 16.1% (95%CI, 7.2-36.0%). The 180-day cumulative incidence of moderate to severe chronic graft-versus-host disease (cGVHD) was 7.1% (95%CI, 1.9-26.9%). Cumulative incidence of 100-day cytomegalovirus (CMV) viraemia and 100-day Epstein-Barr virus (EBV) viraemia was 61.6% (95%CI, 46.5-81.4%) and 3.2% (95%CI, 0.4-22.2%), respectively. The 600-day overall survival (OS) and leukemia-free survival (LFS) were 80.9% (95%CI, 63.5-93.6%) and 81.3% (95%CI, 64.2-93.7%), respectively. The 600-day relapse incidence (RI) and non-relapse mortality (NRM) was 6.9% (95%CI, 1.8-26.3%) and 11.7% (95%CI, 3.9-35.0%). CONCLUSION: Our study shows that the addition of venetoclax to a MAC allo-HSCT was feasible, safe and effective for high-risk AML patients.

Genetic Dissection and Germplasm Selection of the Low Crude Fiber Component in Brassica napus L. Shoots.

BACKGROUND: Brassica napus is one of the most important oil crops in the world, and B. napus shoots are nutrient-rich fresh vegetables. The crude fiber (CF) component is one of the most important factors affecting the taste quality of B. napus shoots, but the factors underlying the desirable low-CF trait remain poorly understood. METHODS: In this study, a high-density single-nucleotide polymorphism (SNP) map was used to map quantitative trait loci (QTLs) for five CF-related traits in a recombinant inbred population. RESULTS: A total of 49 QTLs were obtained in four environments, including eleven, twelve, eight, twelve and six QTLs for content of neutral detergent fiber, acid detergent fiber, acid detergent lignin, hemicellulose and cellulose, respectively. The phenotypic variation explained by single QTL ranged from 4.62% to 14.76%. Eight of these QTLs were further integrated into four unique QTLs, which controlled two different traits simultaneously. Five CF-component-related candidate genes were identified, among which BnaC03g07110D and BnaC07g21271D were considered to be the most likely candidate genes. In addition, five lines with low CF content were selected, which can be used as excellent germplasm resources in breeding. CONCLUSIONS: The QTLs identified in this study will contribute to our understanding of the genetic mechanism of CF and can be used as targets for reducing CF content in B. napus shoots. In addition, this study also provided excellent germplasm resources for low CF content breeding.

Early onset is an indication of the severity of DADA2 disease.

OBJECTIVE: To find indicators of disease severity and factors of early remission in patients with deficiency of adenosine deaminase 2 (DADA2). METHODS: We enrolled six DADA2 patients from six families. Direct sequencing of adenosine deaminase 2 gene (ADA2) was performed by Sanger analysis. A literature review was conducted for articles regarding paediatric DADA2. RESULTS: We found that more organs were involved in early-onset (≤1 year of age) than in late-onset (>1 year of age) DADA2 patients had high level inflammatory responses, such as elevated ESR, SF, serum amyloid A and CRP. Disease severity was not significantly different from missense and frameshift mutation. Early administration of TNF inhibitor might result in better remission and reduce recurrence. In the literature, four articles describing 51 paediatric DADA2 patients were identified. We also found that fever, stroke, peripheral nervous system involvement, hypogammaglobulinaemia and hypertension were more frequent in early onset DADA2 patients. CONCLUSION: Early-onset DADA2 may be more severe. Early administration of TNF inhibitor can effectively reduce recurrence and quickly alleviate the disease.

Clinical characteristics of 1020 childhood-onset systemic lupus erythematosus: data from a health centre in China.

OBJECTIVES: Childhood-onset systemic lupus erythematosus (cSLE) is a multisystem autoimmune disease characterised and presents partially differently from adults. A large cSLE cohort study is lacking in China. The present study aimed to determine the clinical characteristics in a large population of patients with cSLE, and compare with adult-onset SLE (aSLE) in an SLE cohort of China. METHODS: The retrospective study included patients with cSLE diagnosed at the Beijing Children's hospital between July 2006 and October 2020. All patients met at least 4 of ACR classification criteria for SLE. In addition, data including demographic, clinical and serologic data were collected. Our data were compared with other cSLE cohorts and Chinese aSLE cohorts. RESULTS: A total of 1020 patients were included in this study, comprising 808 female and 212 male patients (female to male ratio, 3.8:1). The mean age at diagnosis of lupus was 11.1 years (range 1.0-17.2). It took on average 6 months (range 0.1-132) from first symptoms to cSLE diagnosis and over 12 months in 12% of patients. The most common primary manifestations at onset were rash (37.2%), fever (33.4%), nephropathy (14.2%) and arthritis (13.6%). The most common clinical manifestations were rash (67.9%) and fever (57.5%). 59.4% of patients had haematological involvement, 46.0% had lupus nephritis, 33.2% had arthritis. cSLE was more active and associated with more inflammation than aSLE patients. CONCLUSIONS: This study is a large single-centre study on cSLE from China and clarifies the clinical phenotype and autoantibody spectrum of cSLE. The clinical manifestations and autoantibody spectrum of cSLE are diverse, with regional and populational differences.

An anatomical study of the tibial nerve branches innervating the posterior tibial artery.

The arteries of the lower limbs are innervated by vascular branches (VBs) originating from the lumbar sympathetic trunk and branches of the spinal nerve. Although lumbar sympathectomy is used to treat nonreconstructive critical lower limb ischemia (CLLI), it has limited long-term effects. In addition, the anatomical structure of tibial nerve (TN) VBs remain incompletely understood. This study aimed to clarify their anatomy and better inform the surgical approach for nonreconstructive CLLI. Thirty-six adult cadavers were dissected under surgical microscopy to observe the patterns and origin points of VBs under direct vision. The calves were anatomically divided into five equal segments, and the number of VB origin points found in each was expressed as a proportion of the total found in the whole calf. Immunofluorescence staining was used to identify the sympathetic nerve fibers of the VBs. Our results showed that the TN gave off 3-4 VBs to innervate the posterior tibial artery (PTA), and the distances between VBs origin points and the medial tibial condyle were: 24.7 ± 16.3 mm, 91.7 ± 66.1 mm, 199.6 ± 52.0 mm, 231.7 ± 38.5 mm, respectively. They were mainly located in the first (40.46%) and fourth (31.68%) calf segments, and immunofluorescence staining showed that they contained tyrosine hydroxylase-positive sympathetic nerve fibers. These findings indicate that the TN gives off VBs to innervate the PTA and that these contain sympathetic nerve fibers. Therefore, these VBs may need to be cut to surgically treat nonreconstructable CLLI.