Noonan syndrome and Noonan-like syndrome with loose anagen hair: rare phenotypes may emerge during follow-up.

Background: Noonan syndrome (NS) and Noonan-like syndrome with loose anagen hair (NS/LAH) are neurodevelopmental syndromes resulting from germline mutations in genes that participate in the rat sarcoma/mitogen-activated protein kinases (RAS/MAPK) pathway. The aim of this retrospective study was to describe common and rare manifestations of NS and NS/LAH. Methods: We collected and analyzed clinical and genetic data from 25 patients with NS and NS/LAH. Results: The patients' median age was 6.3 years (range, 1-13 years), and the male-to-female ratio was 18:7. In total, 19 patients had NS caused by a mutation in PTPN11. Another causative gene was found in six patients, including two patients with a SHOC2 mutation, one patient with a KRAS mutation, one patient with an LZTR1 mutation, one patient with a BRAF mutation, and one patient with a PPP1CB mutation. Short stature was detected in 100% of the patients. This study provides an overview of the clinical features of NS, including unique facial features, short stature, congenital heart defects, and other manifestations. Notably, systemic lupus erythematosus (SLE) was found in two SHOC2-positive patients. One patient had a posterior urethral valve, which is very rare in NS patients. Conclusions: Our study identified several clinical features that were previously poorly related to NS, including SLE. We concluded that SHOC2-related NS is associated with a particularly high risk of SLE, which may have a significant impact on quality of life, and a posterior urethral valve is a novel phenotype. These findings could be helpful in enhancing the understanding of the clinical spectrum of NS.

Validity of A Novel Simulator for Percutaneous Transforaminal Endoscopic Discectomy.

BACKGROUND: Percutaneous transforaminal endoscopic discectomy (PTED) has steep learning curves and a high incidence of complications, but currently, efficient and economical training methods are lacking. This study aimed to validate a novel simulator for PTED. METHODS: The simulated PTED included puncturing and establishing the working channel (PEWC) and endoscopic discectomy, with the PEWC being the tested module. Eleven experts and 21 novices were included and introduced to the simulator and tasks; all participants completed the PEWC. Outcomes included: total operation time, number of fluoroscopy for positioning the working sheath, number of spinal risk region invasion, Global Rating Scale (GRS) and a modified GRS, etc. The Mann-Whitney U test was used to compare 2 groups. Spearman's correlation coefficient analyzed continuous variables. RESULTS: Experts outperformed novices in total operation time (P = 0.001), requiring fewer number of fluoroscopies for positioning the working sheath (P = 0.003). Additionally, experts had a lower number of spinal risk region invasions (P = 0.016) and higher scores on both the GRS (P < 0.001) and modified GRS (P < 0.001). PTED experience correlated with GRS scores (P = 0.001) and modified GRS (P < 0.001). The overall realism scored a median of 4 (3.75-5), and educational value had a median of 4 (range 3-5). CONCLUSIONS: This study demonstrates the validity of the novel simulator, revealing significant associations between PTED experience and performance metrics in a simulated PEWC setting. Furthermore, the PEWC module also offers a good realistic design and high education value according to experts.

Paediatric autoimmune diseases with ELANE mutations associated with neutropenia.

OBJECTIVE: To explore the clinical characteristics of autoimmune diseases in children with ELANE mutations. METHODS: Three cases of children with ELANE mutations manifesting as autoimmune diseases, who were under treatment from April 2020 to May 2021, were retrospectively analysed. RESULTS: Among the three children, two were boys aged 15 years and 22 months (cases 1 and 3) respectively, and the other one was a 22-month-old girl (case 2). All the cases had recurrent infections. Case 1 presented with cyclic neutropenia and systemic lupus erythematosus (SLE). Case 2 presented with severe neutropenia and autoimmune haemolytic anaemia (AHIA). Case 3 presented with severe neutropenia and anti-neutrophil cytoplasm antibodies (ANCA)-associated small vasculitis. Genetic tests showed that they all had heterozygous mutations in the ELANE gene. Case 1 was treated with methylprednisolone and hydroxychloroquine sulphate for 2 years, making neutrophil level return to normal. Case 2 received allogeneic hematopoietic stem cell transplantation and has stopped taking antibiotics, steroids and all the immunosuppressors. Case 3 received subcutaneous injections of granulocyte colony-stimulating factor, oral prednisone and cyclophosphamide. The boy in case 3 has been followed up for one year, and his absolute neutrophil count has increased to 1.56 × 109/L. CONCLUSION: Patients with ELANE mutations, combined with autoimmune diseases, may have recurrent infections. Disease-modifying antirheumatic drugs (DMARDs) are effective for autoimmune diseases. Autoimmune diseases with ELANE mutations associated with neutropenia can be cured through allogeneic hematopoietic stem cell transplantation.

Correlation between CT Score and KL-6: A Severity Assessing in Juvenile Dermatomyositis Associated Interstitial Lung Disease.

Background: There is no radiological measurement to estimate the severity of pediatrics juvenile dermatomyositis (JDM) with interstitial lung disease (ILD). We validated the effectiveness of CT scoring assessment in JDM patients with ILD. Aim: To establish a CT scoring system and calculate CT scores in JDM patients with ILD and to determine its reliability and the correlation with Krebs von den Lungen-6 (KL-6). Methods: The study totally enrolled 46 JDM-ILD patients and 16 JDM without ILD (non-ILD, NILD) patients. The chest CT images (7.0 ± 3.6 years; 32 male and 30 female) were all analyzed. CT scores of six lung zones were retrospectively calculated, included image pattern score and distribution range score. Image pattern score was defined as follows: increased broncho-vascular bundle (1 point); ground glass opacity (GGO) (2 points); consolidation (3 points); GGO with bronchiectasis (4 points); consolidation with bronchiectasis (5 points); and honeycomb lung (6 points). Distribution range score was defined as no infiltrate (0 point); <30% (1 point); 30%-60% (2 points); and ≥60% (3 points). Two pediatric radiologists reviewed all CT images independently. The ROC curve was established, and the optimal cutoff score for severity discrimination was set. Results: The agreement between two observers was excellent, and the ICC was 0.930 (95% CI 0.882-0.959, p < 0.01). CT score and KL-6 level had a positive linear correlation (r = 0.784, p < 0.01). However, the correlation between CT scores of different lung zone and KL-6 level was different. The KL-6 cut off level suggested for JDM with ILD was 209.0 U/ml, with 73.9% sensitivity and 87.5% specificity, and the area under curve was (AUC) 0.864 (p < 0.01). Conclusion: The CT scoring system we established, as a semiquantitative method, can effectively evaluate ILD in JDM-PM patients and provide reliable evidence for treatment.

Sex Comb on Midleg Like-2 Accelerates Hepatocellular Carcinoma Cell Proliferation and Metastasis by Activating Wnt/ß-Catenin/EMT Signaling.

PURPOSE: The purpose of this study was to investigate the influences of sex comb on midleg like-2 (SCML2) on hepatocellular carcinoma (HCC) and potentially related mechanisms. MATERIALS AND METHODS: SCML2 expression in tumor tissues and cells was analyzed using the TCGA database and/or qRT-PCR. The proliferation of HCC cells was detected by CCK-8, colony formation, and EdU assays. The migration and invasion of HCC cells were detected by transwell and wound healing assays. Apoptosis of HCC cells was determined by flow cytometry. Additionally, qRT-PCR and Western blot were used to detect the expression of SCML2 and Wnt/ß-catenin/epithelial-mesenchymal transition (EMT) signaling. A xenograft model in mice was established to verify the in vitro findings. RESULTS: We found that SCML2 was highly expressed in HCC tissues and cells and that high expression of SCML2 was correlated with poor prognosis in HCC patients. SCML2 overexpression promoted proliferation, invasion, and migration and repressed apoptosis of HCC cells. The reverse results were obtained in SCML2-silenced cells. Further, we found that SCML2 activated the Wnt/ß-catenin/EMT pathway. SCML2 silencing reduced the protein levels of Wnt3a, ß-catenin, N-cadherin, Vimentin, and Snail and enhanced E-cadherin protein expression both in vivo and in vitro. CONCLUSION: SCML2 silencing inhibits the proliferation, migration, and invasion of HCC cells by regulating the Wnt/ß-catenin/EMT pathway.

Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma.

Amine oxidase copper containing 1 (AOC1) is a copper-containing amine oxidase that catalyzes the deamination of polyamines. AOC1 functions as an oncogene in human gastric cancer. There is little information available regarding the function of AOC1 in hepatocellular carcinoma (HCC). In the present study, reverse transcription-quantitative PCR was used to detect the expression levels of AOC1 in HCC tissues, and the role of AOC1 in HCC progression was determined using western blot, Cell Counting Kit 8, clone formation, wound-healing and Transwell assays. An AOC1 survival curve was generated with data downloaded from The Cancer Genome Atlas, and Gene Set Enrichment Analysis was performed to investigate the potential biological mechanisms of AOC1 in HCC. AOC1 was found to be upregulated in HCC tissues, which was associated with a poor prognosis. Furthermore, AOC1-knockdown inhibited HCC cell proliferation, migration and invasiveness, suppressed IL-6 expression, as well as decreasing JAK2 and STAT3 phosphorylation. Ultimately, the results of the present study illustrate that AOC1 promoted the proliferation, migration and invasiveness of HCC cells by regulating the IL-6/JAK/STAT3 pathway.

Clinical and genetic characteristics of PSTPIP1-associated myeloid-related proteinemia inflammatory syndrome.

OBJECTIVE: To summarise the clinical and genetic characteristics of three children with PSTPIP1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome. METHODS: This study retrospectively analysed the clinical and genetic data of three children with PAMI syndrome in our hospital between April 2018 and January 2020. RESULTS: One male and two female children were 6 years and 5 months, 8 years and 7 months, and 13 years and 3 months of age. All three patients had a recurrent blood trilineage hypoplasia and splenomegaly. Patient 1 had pyoderma gangrenosum, and Ludwig's angina. Patient 2 had pyogenic arthritis, and pyoderma gangrenosum. Patient 3 had hepatomegaly, pyogenic arthritis, and pulmonary hypertension. Laboratory tests revealed that all three children had elevated C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Patient 1: C-antineutrophilic cytoplasmic antibodies(c-ANCA), positive; antiglobulin test (Coombs), positive. Patient 2: blood zinc, 4.38 mg/L (elevated). Patient 3: Antinuclear antibodies (ANA), 1:100, ß2 glycoprotein I, positive; Coombs test, positive; RF, 28.3 U/ml (elevated); C3, 0.77 g/L (decreased). Genetic testing showed that all 3 patients had PSTPIP1 c.748G > A (p.E250K) spontaneous heterozygous mutations, suggesting the diagnosis of PAMI syndrome. Patient 1 was treated with a combination of methylprednisolone and cyclosporine for 8 months. The patient did not develop new skin lesions. The blood count showed mild neutropenia. The spleen was considerably retracted and the CRP became normal. Patient 2 was treated with etanercept and methylprednisolone. The patient had no further arthralgias and pyoderma gangrenosum showed improvement. The spleen was smaller than before. White blood cells were shown to be approximately 2-3 × 109/L. The haematocrit, platelets, CRP, and AESR were normal. Patient 3 was treated with methylprednisolone, methotrexate, and infliximab 4 times. The patient's joint symptoms disappeared gradually and the liver retracted markedly. The pulmonary artery pressure returned to normal. Moreover, Coombs test result was negative. CRP and AESR were lower than before. CONCLUSION: PAMI syndrome can manifest as pyogenic arthritis, pyoderma gangrenosum, acne, and trilineage hypoplasia, as well as autoimmune diseases. Glucocorticoid and immunosuppressive therapy are partially effective and cytokine antagonists can be used in refractory cases. Whole-exome genetic testing is helpful to confirm diagnosis.

LINC00238 inhibits hepatic carcinoma progression by activating TMEM106C­mediated apoptosis pathway.

The present study aimed to explore the regulatory mechanism of long intergenic non­protein coding (LINC)00238 in hepatocellular carcinoma (HCC). LINC00238 expression in HCC tissues and cell lines was measured using reverse transcription­quantitative PCR. LncTar was used to predict the binding sites between LINC00238 and transmembrane protein 106C (TMEM106C). Survival analysis of LINC00238, TMEM106C and activating transcription factor 3 (ATF3) in patients with HCC was performed based on TCGA data. The proliferation, apoptosis, migration, and invasion of HCC cells were measured by 3­(4,5­dimethylthiazol­2­yl)­5­(3­carboxymethoxyphenyl)­2­(4­sulfophenyl)­2H­tetrazolium assay, flow cytometer, wound healing and Transwell assays, respectively. LINC00238 promoted apoptosis and inhibited proliferation, migration and invasion of HCC cells. LINC00238 was downregulated in HCC. TMEM106C was a target of LINC00238 and TMEM106C expression was negatively regulated by LINC00238. TMEM106C suppressed the apoptosis pathway and decreased the expression of caspase­7, tissue inhibitor of metalloproteinase 2, programmed cell death 4 and ATF3. Notably, ATF3 was the upstream promoter of LINC00238 and positively regulated LINC00238 expression. In conclusion, LINC00238 inhibited HCC progression by inhibiting TMEM106 expression and activating the TMEM106C­mediated apoptosis pathway.

Kinesin family member 2C promotes hepatocellular carcinoma growth and metastasis via activating MEK/ERK pathway.

The current work was intended to explore the function and mechanism of Kinesin family member 2C (KIF2C) in hepatocellular carcinoma (HCC). In this study, KIF2C expression was at a high level in HCC and indicated poor prognosis. Silencing KIF2C significantly suppressed the proliferation, migration, and invasion in HCC cells. Furthermore, silencing KIF2C markedly decreased the expression of Snail, Vimentin, p-MEK, and p-ERK, but increased E-cadherin expression in HCC cells. Moreover, we also found that MEK/ERK inhibitor U0126 could enhance the impact on cell proliferation, migration, and invasion induced by silencing KIF2C in HCC. On the contrary, MEK/ERK activator PAF could weaken the impact induced by silencing KIF2C in HCC. Thus, our findings indicate that KIF2C can promote the proliferation, migration, and invasion by activating MEK/ERK pathway in HCC.

Analysis of five cases of monogenic lupus related to primary immunodeficiency diseases.

OBJECTIVE AND DESIGN: We studied five cases of PID-related monogenic lupus to explore the characteristics. MATERIAL OR SUBJECTS: Among 42 cases of PID patients between 2017-2020, 5 patients were diagnosed as PID-related monogenic lupus, including 2 males and 3 females, with age range from 2 years 3 months to 13 years old. TREATMENTS: DMARDs, biological agents and stem cell transplantation were used to treat different patients. METHODS: We collected the clinical observation indicators, auxiliary examination and treatment of the five patients. RESULTS: Patient 1 was diagnosed with monogenic lupus secondary to severe combined immunodeficiency and received prednisone and methotrexate treatment. Patient 2 was diagnosed with monogenic lupus secondary to activated phosphoinositide 3-kinase δ syndrome. Allogeneic stem cell transplantation was conducted. Patient 3 was diagnosed with monogenic lupus secondary to RAS-associated lymphoproliferative disease. The child was treated with prednisone and rituximab. Patient 4 was diagnosed with monogenic lupus secondary to PSTPIP1-associated myeloid-related proteinaemia inflammatory syndrome. The child was given methylprednisolone, methotrexate, and infliximab. Patient 5 was diagnosed with monogenic lupus secondary to A20 haploinsufficiency. The child was treated with methylprednisolone and infliximab. CONCLUSIONS: Multiple PIDs can lead to monogenic lupus. Different PID-related monogenic lupus has different suitable targeted drugs.

Knockdown of CENPW Inhibits Hepatocellular Carcinoma Progression by Inactivating E2F Signaling.

AIM: This study aimed to evaluate the effects of centromere protein W (CENPW, also known as CUG2) in hepatocellular carcinoma (HCC). METHODS: CENPW expression in HCC tissues and cells was detected by RT-qPCR assay. CCK-8 and colony formation assay were used to assess cell proliferation. Wound healing and Transwell assay was used to detect cell migration and invasion, respectively. The flow cytometry was used to analyze the cell cycle distribution and apoptosis. RESULTS: CENPW expression was upregulated in HCC tissues and cells. Knockdown of CENPW inhibited cell proliferation, migration, and invasion and induced the G0/G1 phase arrest and cell apoptosis in HCC cells, which might involve the E2F signaling regulation. CONCLUSION: CENPW acted as an oncogenic role in HCC progression via activation E2F signaling. Our findings may provide new insights into the studying mechanisms of HCC.

Clinical characteristics and genetic analysis of A20 haploinsufficiency.

PURPOSE: To evaluate the clinical and genetic characteristics of 3 children with Haploinsufficiency of A20 (HA20). METHODS: The clinical and genetic testing data of 3 children with HA20 treated at Capital Institute of Pediatrics (CIP) between August 2016 and October 2019 were retrospectively analysed. RESULT: Patient 1 presented with arthritis and inflammatory bowel disease, patient 2 presented with axial spinal arthritis and lupus-like syndrome, and patient 3 presented with recurrent oral ulcers, gastrointestinal ulcers, and perianal abscesses. Regarding laboratory tests, patients were found to have elevated white blood cell (WBC) count, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). The CRP and ESR was reported to be high in all the patients. The WBC was reported to be high in patient 1 and 3. Patient 2 was positive for antinuclear antibodies, anti-Sjögren's syndrome antigen A, dsDNA, rheumatoid factor and Coombs test. Genetic testing showed that all three patients had heterozygous mutation in TNFAIP3 gene. As for the treatment, patient 1 was treated with TNFα antagonist, patient 2 was treated with TNF α antagonist and sulfasalazine, and patient 3 was treated with corticosteroids and thalidomide. Patients 1 and 2 were followed for four and 3 months, respectively. There was an improvement in joint and gastrointestinal symptoms; inflammatory indices and rheumatoid factor (RF) were normal, and dsDNA and Coombs test became negative. Patient 3 was treated at another hospital and showed gradual improvement in oral ulcers and perianal abscesses. CONCLUSION: HA20 is a single-gene auto-inflammatory disease caused by mutation in tumour necrosis factor (TNF)-α-induced protein 3 (TNFAIP3) gene. It may present as Behçet-like syndrome and resemble various other autoimmune diseases as well. Corticosteroids and immunosuppressive agents are effective treatments, and cytokine antagonists can be used in refractory cases. Whole-exome genetic testing should be proactively performed for children with early-age onset or Behçet-like syndrome to achieve early diagnosis and accurate treatment.

Gastrointestinal perforation in anti-NXP2 antibody-associated juvenile dermatomyositis: case reports and a review of the literature.

BACKGROUND: To summarize the characteristics of gastrointestinal (GI) perforation in anti-nuclear matrix protein 2 (NXP2) antibody-associated juvenile dermatomyositis (JDM). METHODS: Five patients with GI perforation from a JDM cohort of 120 cases are described. Relevant literature was reviewed. RESULTS: Five patients, including four females and one male, were included in the study. The age of onset of these patients ranged from 3.3 to 9.5 years with the median age of 5.0 years. When these patients were complicated by GI perforation, childhood myositis assessment score (CMAS) ranged from 1 to 5 with the median score of 2. Myositis-specific antibody (MSA) spectrum analysis indicated that the five patients were anti-NXP2 antibody positive. The initial symptoms of GI perforation were progressive abdominal pain and intermittent fever. Two patients also presented with ureteral calculus with hydronephrosis and ureteral stricture. Surgery was performed in four patients. One patient failed to undergo a repair as the perforation was high in position. For the other three patients, perforation repair was successful, of which two patients failed due to recurrent perforation. At 24 months postoperative follow-up, one patient was in complete remission on prednisone (Pred) and methotrexate (MTX) treatment, and her ureteral stricture had disappeared. The other four patients died. Adding these cases with 16 other patients described in the literature, the symptom at onset was progressive abdominal pain, which often occurred within 10 months after JDM was diagnosed. Perforation most commonly occurred in the duodenum, although it also occurred at multiple sites or was recurrent. The mortality rate of GI perforation in JDM was 38% (8/21). CONCLUSIONS: All the five perforation cases in our study subjected to MSA analysis were anti-NXP2 antibody positive. The symptom at onset was abdominal pain. The most common site of perforation was the duodenum in the retroperitoneum, and the lack of acute abdominal manifestations prevented early diagnosis. GI perforation may be a fatal complication in JDM, and early diagnosis is very important. More research is needed to determine the pathogenesis and predictive factors of GI perforation in JDM.

Long-term follow-up of autologous hematopoietic stem cell transplantation for refractory juvenile dermatomyositis: a case-series study.

OBJECTIVE: To follow up the refractory juvenile dermatomyositis (JDM) with autologous hematopoietic stem cell transplantation (AHSCT) in a long time and to investigate whether AHSCT is effective and safe to treat refractory JDM. METHODS: We collected the AHSCT and follow-up data of three patients with refractory JDM who received autologous peripheral blood CD34+ cell transplantation in our hospital between June 2004 and July 2015. Those data include: hight, weight, routine blood and urine tests, ESR, CK, ALT, AST, LDH, renal functional tests, lymphocyte subpopulations, HRCT and muscle MRI. The last follow-up was done in June 2017. RESULTS: All three patients had complete remission and could stop prednisone after 3-12 months. None of them relapsed at 144, 113 and 23 months follow-up. Twelve months after their AHSCT, all of their monitoring indexes have returned to normal and they have stopped all medications. Until the date of this article, none of them relapsed or need medicine. CONCLUSION: Our study suggests that AHSCT is safe and effective in treating refractory JDM, and it can provides long term drug-free survival. However, more cases are needed for further confirmation.

[Preparation and biocompatibility of a novel biomimetic osteochondral scaffold: collagen-chitosan/nano-hydroxyapatite-collagen-polylactic acid].

OBJECTIVE: To prepare collagen-chitosan/nano-hydroxyapatite-collagen-polylactic acid (Col-CS/nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. METHODS: PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 : 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 : 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. RESULTS: Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6 degrees C, and the increasing temperature sum of 3 rabbits was less than 1.3 degrees C, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. CONCLUSION: Col-CS/nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.

[Construction of recombinant porcine transforming growth factor beta1 gene lentiviral vector and its expression in bone marrow mesenchymal stem cells].

OBJECTIVE: To construct recombinant lentiviral expression vectors of porcine transforming growth factor beta1 (TGF-beta1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-beta1 gene-modified BMSCs for bone and cartilage tissue engineering. METHODS: The TGF-beta1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated from bone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs were harvested for experiments. BMSCs were then transfected by TGF-beta1 recombinant lentiviral vectors (TGF-beta1 vector group) respectively at multiplicity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected by laser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-beta1 mRNA, TGF-beta1 protein, and collagen type II. RESULTS: Successful construction of recombinant lentiviral vectors of porcine TGF-beta1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-beta1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-beta1 mRNA was significantly higher in TGF-beta1 vector group than in empty vector group and non-transfection group (P < 0.05). Immunocytochemistry results revealed positive expression of TGF-beta1 protein and collagen type II in BMSCs of TGF-beta1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-beta1 protein still could be detected by ELISA in TGF-beta1 vector group. CONCLUSION: TGF-beta1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-beta1 protein can be observed, prompting BMSCs differentiation into chondrocytes.

[Treatment of refractory rheumatism among preschool children with autologous peripheral blood hematopoietic stem cell transplantation].

OBJECTIVE: To investigate the feasibility and safety of autologous peripheral blood hematopoietic stem cell transplantation (auto-PBHSCT) and its therapeutic effect on refractory rheumatism among preschool children. METHODS: Three boys with juvenile rheumatoid arthritis (JRA), juvenile systemic lupus erythematosus (JSLE) and juvenile dermatomyositis (JDM) respectively, 3 to 6 years old with the mean age of 5 years with 3.5 to 22 months course of disease with 14 months on average, received auto-PBHSCT. Their conditions were so severe that conventional therapy failed to control the diseases. The changes of both clinical manifestations and immunologic indexes were observed before and after transplantation with long term following up at specialty clinic of rheumatism. RESULT: The time when neutrophil count >or= 0.5 x 10(9)/L in the 3 children was days +9, +13 and +11 respectively, that of platelet count >or= 20 x 10(9)/L was days +14, +18 and +13 respectively. The cellular immune function remained abnormal with CD4 cells at a low level and CD4/CD8 being inverted. As to the JDM child, the skin rash had disappeared and his muscle tone was improved to grade 5 within one month after the transplantation. The EMG and serum creatase level returned to normal and muscle MRI findings were improved greatly within 2 months after the transplantation. As to the JSLE child, skin rash and proteinuria had disappeared, MRI of brain showed that the pathological changes had been absorbed and EEG returned to normal 3 months after the transplantation, all the autoantibodies turned to negative within 8 months after transplantation. As to the JRA child, the arthritis had been improved remarkably within 3 weeks after auto-PBHSCT. There was no swelling of joints nor movement limitation 3 months post transplantation. The steroids and immunosuppressive drugs were discontinued post transplantation. Cushing syndrome disappeared. Their body heights increased by 10 to 15 cm in the past 18 months, and they all returned to school. There was no relapse during follow-up periods of 25 - 27 months. CONCLUSION: The therapy with auto-PBHSCT for refractory rheumatism among preschool children was remarkably effective in a short-term, yet the safety and long-term effect still need to be further studied.