[Experimental study on the inhibition of the NLRP3 signaling pathway with Shengsan Jiedu Huayu decoction to alleviate inflammatory injury in rats with acute-on-chronic liver failure].

Objective: To observe the therapeutic effect of Shengsan Jiedu Huayu decoction in alleviating inflammatory liver injury in rats with acute-on-chronic liver failure (ACLF) and its effect on the activation intensity for the NLRP3 signaling pathway. Methods: 63 SD rats were randomly divided into a blank group, a model group, and low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction (7.29 g/kg/d, 14.58 g/kg/d, and 29.16 g/kg/d). The ACLF rat model was replicated using carbon tetrachloride combined with d-galactosamine and lipopolysaccharide. Different dose gradients of the Shengsan Jiedu Huayu decoction were used for a five-day intervention treatment, and then rat serum and tissue samples were collected. A biochemical analyzer was used to detect the serum levels of ALT, AST, and TBIL in rats. ELISA was used to detect serum IL-18 and IL-1ß content. HE staining was used to observe histomorphological changes in liver tissue. Immunohistochemistry was used to detect GSDMD expression in liver tissue. Western blot and PCR were used to detect NLRP3, Caspase1, ASC, TLR4, IL-1ß, IL-18 protein, and mRNA expression levels.The groups were compared using analysis of variance and the rank-sum test. Results: Compared with the blank group, the model group's rat liver tissue was severely injured. Serum levels of ALT, AST, and TBIL, inflammatory factors IL-1ß and IL-18, and the GSDMD protein expression level, NLRP3 expression level, TLR4, caspase 1, ASC, IL-1ß, IL-18 protein, and mRNA (P<0.01) were all significantly increased in the model than the blank group (P<0.01). Additionally, compared with the model group, the low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction had improved liver tissue injury in ACLF rats, while the serum levels of ALT, AST, TBIL, IL-1ß, IL-18, liver tissue GSDMD protein, NLRP3, TLR4, caspase 1, and ASC expressions were all lower in the different dose gradients of the Shengsan Jiedu Huayu decoction than the model group, with the most evident reduction in the high-dose group (P<0.01). Conclusion: Shengsan Jiedu Huayu decoction can weaken the activation intensity of the NLRP3 signaling pathway, alleviate liver tissue pathological injury, reduce inflammatory factor release, and alleviate inflammatory liver injury in ACLF rats.

[Diagnostic values of nuclear score combined with cyclin D1 immunocytochemistry in indeterminate thyroid follicular nodules in preoperative fine needle aspiration].

Objective: To assess the feasibility of nuclear score combined with cyclin D1 immunocytochemistry in classifying indeterminate thyroid nodules with fine-needle aspiration (FNA) cytological diagnosis of Bethesda category Ⅲ-Ⅴ. Methods: A consecutive cohort of 118 thyroid FNA specimens with indeterminate diagnosis (TBSRTC category Ⅲ-Ⅴ) and available histopathologic follow-up data were collected between December 2018 and April 2022 at the Department of Pathology, Beijing Hospital, China. These cases were subjected to cytological evaluation and cyclin D1 immunocytochemistry. The optimal cut-off points of a simplified nuclear score and the percentage of cyclin D1-positive cells for the diagnosis of malignancy or low-risk neoplasm were determined using the receiver operating characteristic (ROC) curves and area under the ROC curve (AUC). The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of nuclear score and cyclin D1 immunostaining were evaluated from the crosstabs based on cut-off points. The diagnostic accuracy of simplified nuclear score combined with cyclin D1 immunostaining was estimated using ROC curve analysis. Results: Nuclear grooves, intra-nuclear inclusions and chromatin clearing were more commonly found in malignancy/low-risk neoplasms than benign lesions (P=0.001, P=0.012 and P=0.001 respectively). A cut-off point of≥2 for the simplified nuclear score was sensitive for defining malignancy/low-risk neoplasm, and its PPV, NPV, sensitivity and specificity were 93.6%, 87.5%, 99.0% and 50.0% respectively. A positive cut-off point of 10% positive thyroid cells in cyclin D1 immunostaining demonstrated sensitivity of 88.5%, specificity of 100%, PPV of 100% and NPV of 53.8% for correctly detecting thyroid malignancy or low-risk neoplasm. The sensitivity and PPV of simplified nuclear score combined with cyclin D1 immunostaining were 93.3% and 100%, respectively. Both specificity and NPV were maintained at high levels (100% and 66.7%, respectively). The diagnostic accuracy of simplified nuclear score combined with cyclin D1 immunostaining in detecting thyroid malignancy/low-risk neoplasm was increased to 94.1% compared to using either of them alone. Conclusions: Combing simplified nuclear score and cyclin D1 immunostaining on FNA cytology specimens can increase the diagnostic accuracy in classifying thyroid nodules of indeterminate cytological categories. Thus, this supplementary approach provides a simple, accurate, and convenient diagnostic method for cytopathologists so that may reduce unnecessary thyroidectomies.

[Analysis of recurrent factors and therapeutic effect after laparoscopic inguinal hernia repair].

Objective: To analyze the recurrence factors and reoperation effect of laparoscopic inguinal hernia repair. Methods: A total of 41 patients with recurrence after laparoscopic repair of the inguinal hernia admitted to the Department of Hernia and Abdominal Wall Surgery, Beijing Chaoyang Hospital Affiliated to Capital Medical University from January 2017 to December 2021 were retrospectively analyzed. All patients were males, aging (62±7) years (range: 51 to 75 years). The recurrence intervals were 3 days to 7 years postoperatively. The surgical methods, causes of recurrence, and treatment outcomes of the patients were analyzed. Fisher exact probability method is used to compare the rates. Results: Among all cases, the primary surgical procedures included transabdominal preperitoneal herniorrhaphy (TAPP) in 31 cases and total extraperitoneal herniorrhaphy in 10 cases. The reoperative procedures included the TAPP of 11 cases and the Lichtenstein procedure of 30 cases. The factors of recurrent cases in all patients could be divided into 4 categories, including insufficient mesh coverage in 23 cases, mesh curling in 9 cases, mesh contractuture in 7 cases, and improper mesh fixation in 2 cases. Recurrence, infection, chronic pain, foreign body sensation didn't occur in the followed period of(M(IQR)) 18(24) months(range: 12 to 50 months). There was no statistical difference in the incidence of postoperative seroma between the TAPP and Lichtenstein procedure (3/11 vs. 20.0% (6/30), P=0.68). Conclusions: Postoperative recurrence of laparoscopic inguinal hernia is mostly caused by the lack of mesh coverage. Due to the emphasis on standardized surgical operation, a good outcome could be achieved through reoperation by the TAPP or Lichtenstein procedure.

[Research study on nucleic acid testing of hemostatic stickers used and the surface of the recipients' hands after the inoculation of inactivated SARS-CoV-2 vaccine].

Among the staff of Beijing Chao-Yang Hospital, Capital Medical University, who received the inactivated SARS-CoV-2 vaccine on January 30 in 2021, 28 recipients were selected for this research. Samples for nucleic acid tests were collected from the surface of the recipients' both hands before and after vaccination. The hemostatic stickers used after the inoculation were also collected for nucleic acid tests. The nucleic acid tests of the samples collected from the surface of both hands of the 28 recipients before vaccination were all negative. After vaccination, the nucleic acid tests of the samples collected from the surface of both hands of recipients were positive in 3 cases, and suspicious in 8 cases, with a positive rate of 10.7%. A total of 25 hemostatic stickers used were collected, 24 of them had positive nucleic acid tests, and the rest one had suspicious nucleic acid test result, with a positive rate of 96%. The hemostatic stickers used after the inoculation have the risk of nucleic acid contamination.

[Pulsed electromagnetic fields stimulating osteogenic differentiation and maturation involves primary cilia-PI3K/AKT pathway].

OBJECTIVE: To study whether the pulsed electromagnetic fields (PEMF) promoting rat osteoblasts differentiation and maturation is related to the primary cilia and PI3K/AKT pathway, and to explore the mechanism of PEMF in promoting bone differentiation. METHODS: Enzyme solution was used to obtain newborn SD rats calvarial osteoblasts (ROB), which were processed by 50 Hz 0.6 mT PEMF for 0, 0.5, 1, 1.5 and 2 h, detecting PI3K and AKT protein expression and changes in primary cilia length and incidence; with LY294002 blocking PI3K/AKT signaling pathways we observed whether PEMF promoted osteogenic differentiation of ROB was affected; by interfering IFT88 gene expression by RNAi to inhibit primary cilia we observed whether PI3K/AKT signaling pathway and osteogenic differentiation of ROB was affected. Osteogenic differentiation indexes included alkaline phosphatase (ALP) activity, Real-time PCR and Western blot detection of osteogenic related genes of BMP-2, COL-1 and OSX and calcified nodules number, etc.. RESULTS: After exposure to PEMF for 0, 0.5, 1, 1.5, and 2 h, the protein expression of PI3K and AKT in ROB were increased significantly (P<0.01) and the primary cilia became longer; and the protein expression of PI3K reached the highest level at 0.5 h, as the treatment time of PEMF increased, the PI3K protein expression decreased. AKT showed higher protein expression at 0.5 h and 1.5 h. After blocking the PI3K/AKT signaling pathway with the PI3K blocker LY294002, PEMF could no longer increase ALP activity and the gene expressions of BMP-2, COL-1, OSX which were osteogenically related. However, PEMF could increase the ALP activity and the osteogenically related gene expression in ROB before blocking. After RNAi interfered the primary cilia, PEMF could no longer increase the protein expression of PI3K, which indicated that PEMF could not activate the PI3K/AKT signaling pathway after primary cilia interfering; secondly, the effect of PEMF on enhancing ALP activity disappeared, it also decrease the gene expressions of BMP-2, COL-1, and OSX, and the ability of increasing the calcification nodule formation also disappeared, indicating that the ability of PEMF to promote osteoblast maturation and mineralization disappeared after primary cilia interference. CONCLUSION: PEMF activated the PI3K/AKT signaling pathway through primary cilia on the surface of osteoblasts, then promoted bone formation activity and differentiation.

[Comparison between hybrid surgery and transabdominal preperitoneal surgery in treatment of irreducible inguinal hernia: A case-control study].

Objective: To compare the effects between hybrid surgery and transabdominal preperitoneal surgery in treatment of irreducible inguinal hernia. Methods: A total of 60 patients who underwent laparoscopic inguinal hernia repair between June 2011 and December 2017 were included in the study. Patients were divided into two group: hybrid surgery group (observation group, n=30) and transabdominal preperitoneal group (control group, n=30). The operation time, intraoperative bleeding, hospital stay, hospital cost and complications were analyzed. Results: The operative time of observation group and control group was 45 (35-65) minutes and 50(35-70) minutes, respectively. Intraoperative blood loss of two groups was 10(5-15) ml and 5(2-10) ml. The length of postoperative hospital stay was 2(1-4) days and 2(1-3) days in the two groups, respectively. And the hospitalization cost of two groups was 9 646 (9 066-11 560) yuan and 9 494(8 989-10 660) yuan, respectively. The intraoperative complications occurred in 4 cases in control group, including 1 case of vas deferens injury, 2 cases of spermatic vessel injury and 1 case of inferior epigastric artery injury. No intraoperative complications occurred in observation group. Perioperative complications in observation group and control group included dysuria (6.7% vs 10.0%), scrotum hematoma (3.4% vs 0%), wound pain (46.7% vs 6.7%) and fever (16.7% vs 20.0%). Twelve months of follow-up was completed in all the patients, and no recurrence or infections occurred in the two groups. The incidence of seroma in observation group and control group was 26.7%, 33.3%, respectively. One case of foreign body sensation and one case of chronic pain occurred in control group. The incidence of perioperative wound pain in patients undergoing hybrid surgery was higher than those undergoing transabdominal preperitoneal surgery (P<0.05), but no statistical differences were observed for other variables between the two groups (all P>0.05). Conclusion: Hybrid surgery is safe and feasible for the treatment of irreducible inguinal hernia. Though with a higher incidence of postoperative acute pain, it may have advantages of avoiding injuries of the vas deferens and spermatic vessels.

[Discussion of laparoscopic transabdominal preperitoneal repair for recurrent inguinal hernia].

OBJECTIVE: To explore the surgical techniques and the clinical efficacy of laparoscopic transabdominal preperitoneal repair (TAPP) for recurrent inguinal hernia. METHODS: Clinical data of 367 patients with recurrent inguinal hernia who underwent TAPP repair from Mar. 2009 to Mar. 2015 in Beijing Chao-Yang Hospital of Capital Medical University were analyzed retrospectively. RESULTS: Laparoscopic operations were completed successfully in 365 cases, however, 2 cases were converted to open surgery.The operation time was (55.7±19.3) min (30-100 min) and the hospital stay was (4.9±2.7) d (2-12 d). The incidences of postoperative pain, hydrocele, and urinary retention were 4.1%(15/367), 13.1%(48/367), and 1.3%(5/367) respectively.Other complications such as foreign body sensation, wound infection, and intestinal obstruction.All cases were followed up from 3 to 72 months ((36.5±14.7) months), 2 recurrent cases was observed and no mesh infection and long-term chronic pain were observed. CONCLUSIONS: Laparoscopic TAPP repair has advantages of minimal invasion and few complications, which is safe and effective for recurrent inguinal hernia.

Target replacement strategy for selection of DNA aptamers against the Fc region of mouse IgG.

Aptamers that recognize the IgG Fc region are of great interest because of their wide application as an immunology probing tool, for diagnostics, and as affinity agents for antibody purification. We developed a target replacement strategy as a modification of conventional Systematic Evolution of Ligands by EXponential enrichment (SELEX) in order to efficiently select and identify novel DNA aptamers against the Fc region of mouse IgG. In this new approach, multiple IgG subclasses (IgG1, IgG2a, mouse IgG Fc, and anti-HBs IgG) were sequentially used to select aptamers in one continuous SELEX. After 8 rounds of selection, the aptamers were analyzed using dot blot and an electrophoretic mobility shift assay, which showed universal binding capability to different IgG subclasses. Secondary structure analysis of the aptamers indicated that the stem-loop structure of the aptamers play an important role in binding to the common site in different mouse IgG subclasses. This demonstrated the feasibility of using multiple target replacement SELEX for the selection of aptamers. This target replacement strategy is also expected to be useful for selecting aptamers that bind common regions of molecules other than antibodies.

IFNG and IFNGR1 polymorphisms are associated with tuberculosis: a case-control study.

OBJECTIVE: Previous studies suggested that single-nucleotide polymorphisms (SNPs) of interferon gamma (IFNG) and its receptor IFNGR1 may be involved in the pathogenesis of tuberculosis (TB). We aimed to examine the association of IFNG gene polymorphisms with TB in the Tibetan population and use the machine learning method to establish a clinical prediction model of TB. PATIENTS AND METHODS: A total of 613 TB patients and 603 healthy controls were selected for the study. Associations between SNPs and TB were analyzed using logistic regression, adjusted for sex and age. Clinical data and SNPs were integrated to construct a TB prediction model using random forest (RF) machine learning. RESULTS: For IFNG, rs1861494 CT was a protective factor against TB compared with TT genotype (p = 0.010). The rs1861494 C allele was a protective factor for TB (p = 0.010). For IFNGR1, the rs3799488 C allele reduced the risk of TB by 30% (p < 0.001). rs9376267 CT (p = 0.005) and TT (p = 0.001) genotypes were protective factors for TB. Compared with the rs1327475 GG genotype, the frequency of the GA genotype in the case group significantly differed from the controls (p = 0.013). rs2234711 GA (p < 0.001), AA (p < 0.001) genotype and A (p < 0.001) alleles were also associated with TB. Finally, five markers are identified using the RF model. The area under the curve (AUC) reaches 0.6 in the training set and 0.59 in the test set. CONCLUSIONS: Our study found that IFNG and IFNGR1 gene polymorphisms were associated with TB in a Tibetan population. The results also demonstrate the potential of clinical-SNPs as diagnostic tools for TB.

Titanium-coated mesh versus standard polypropylene mesh in laparoscopic inguinal hernia repair: a prospective, randomized, controlled clinical trial.

OBJECTIVE: We aimed to compare the clinical outcome of titanium-coated mesh and polypropylene mesh in laparoscopic inguinal hernia repair. METHODS: A total of 102 patients who received laparoscopic inguinal hernia repair in January-June 2016 in Beijing Chao-Yang Hospital were enrolled in this study. Patients were randomly divided into two groups, receiving either titanium-coated mesh (n = 50) or standard polypropylene mesh (n = 52). Multiple clinical parameters were collected and analyzed, including clinical manifestations, operative time, intraoperative blood loss, hospital stay, hospital cost, recovery time, and postoperative complications. RESULTS: All procedures were completed. A statistical difference between two groups was not identified in regards to operative time, intraoperative blood loss, hospital stay, and recovery time (P > 0.05). Three cases with seroma and 15 with foreign body sensation were reported in the titanium-coated mesh group; 9 cases with seroma and 17 with foreign body sensation were reported in the standard polypropylene mesh group. There was no significant difference in the incidence of seroma and/or foreign body sensation. A lower hospital cost but longer recovery period was documented in the standard polypropylene mesh group (P < 0.05). No recurrence, infection or chronic pain was observed during 1-year follow-up in both groups. CONCLUSION: Titanium-coated mesh possesses comparable clinical qualities as the standard polypropylene mesh but with a shorter recovery period. Therefore, this mesh is promising for clinical practice though the cost is higher than the standard polypropylene mesh.

Down-regulation of miR-124 target protein SCP-1 inhibits neuroglioma cell migration.

OBJECTIVE: MicroRNAs (miRNAs) have been widely studied as a potential cancer agent, but its efficacy does not get improvement due to poor targeting. miRNAs have been reported to play multiple roles in the development of the tumor. miR-124 is expressed in various tumor. This study aimed to elucidate the expression of miR-124 in neuroglioma cells as well as its related mechanism. MATERIALS AND METHODS: Expression of miR-124 in neuroglioma cells was assessed by Quantitative PCR (q-PCR). Astrocytes (RA cells) were used as control group. The relationship between miR-124 and SCP-1 was explored with bioinformatics tools. Luciferase reporter assay was performed to examine the expression of miR-124 target protein SCP-1. Gene interference technology was used to regulate expression of miR-124 and SCP-1 in neuroglioma cells, and q-PCR was performed to confirm gene interference effects. Migration of miR-124 and SCP-1 in neuroglioma cell was measured by wound healing assay and cell migration test. RESULTS: Compared with control group, the expressions of miR-124 (p=0.0015) and SCP-1 (p=0.0042) were higher in neuroglioma cells. Luciferase reporter assay proved that SCP-1 was the target of miR-124. Wound healing assay and migration test showed down-regulation of SCP-1 inhibited neuroglioma cell migration. Down-regulation of miR-124 didn't influence neuroglioma cell migration movement. CONCLUSIONS: miR-124 and SCP-1 in neuroglioma cell were highly expressed. MiR-124 impeded the progression of neuroglioma via down-regulating SCP-1.

Clinical analysis of 59 children with hand foot and mouth diseases due to enterovirus EV71 and concomitant viral encephalitis.

OBJECTIVE: To analyzed the clinical features of children with HFMD and viral encephalitis and to summarize some treatments. PATIENTS AND METHODS: A total of 59 children with HFMD were included in this study. All children underwent complete blood count, blood biochemical test cerebrospinal fluid examination, chest X-ray and brain MRI. RESULTS: One child died 24 hours after admission due to central respiratory failure with myocardial damage. After the treatment, 58 children had normal temperature, resolved rash, normal complete blood count, biochemical blood tests and cerebrospinal fluid test, respiratory and circulatory symptoms and signs, as well as neurological symptoms, disappeared. The hospitalization time was 12-21 days. After follow-up for 1-3 months, all children were recovered, and without any severe sequelae. CONCLUSIONS: HFMD and the complicated viral encephalitis usually occurred in the children < 3 years old. The clinical manifestations were not typical. Monitoring of the child's clinical symptoms, signs and relevant examinations was required.

Somatic allelic deletion of nm23 in human cancer.

Tumor progression to the metastatic phenotype is accompanied in certain cell types by reduced expression of the nm23 gene. We have localized human nm23-H1 to chromosome 17 by somatic cell hybrid analysis. Regional localization in the CEPH database and in situ hybridization is reported. Somatic allelic deletion of nm23-H1 was observed in human breast, renal, colorectal, and lung carcinoma DNA samples, as compared to DNA from matched normal tissues. A homozygous deletion of nm23-H1 was observed in a lymph node metastasis of a colorectal carcinoma, indicating that nm23-H1 can be recessively inactivated. The data identify nm23-H1 as a novel, independent locus for allelic deletion in human cancer, a characteristic shared with previously described suppressor genes.

Comparison of impact on seizure frequency and epileptiform discharges of children with epilepsy from topiramate and phenobarbital.

OBJECTIVE: To study the impact on seizure frequency and epileptiform discharges of children with epilepsy from topiramate (TPM) and phenobarbital (PB). PATIENTS AND METHODS: Two hundred cases children with epilepsy from August 2010 to August 2013 in our hospital were sampled and randomly divided into two groups. The observation group was treated with TPM while the control group with PB, and then comparing seizure frequency, efficiency, and adverse reactions of two groups. RESULTS: The reduced number of partial seizures, generalized seizures, and total seizures in the observation group were significantly higher than those in the control group, and the rate of cure, markedly effective and total efficiency in observation group were significantly higher than those in the control group. However, the adverse reactions in observation group were significantly lower than those in the control group. Thus, differences were statistically significant (p<0.05). CONCLUSIONS: Compared with PB, TPM showed a better effect on epilepsy treatment with less adverse reactions which were worthy of clinical recommendation.

Effect of glucocorticoid combined with gamma globulin in treatment of children with myasthenia gravis and its effects on immune globulin and complement of children.

OBJECTIVE: To discuss the effects glucocorticoid combined with gamma globulins in the treatment of children with myasthenia gravis and its effects on immune globulin and complement of children. PATIENTS AND METHODS: Clinical data of 70 cases of childhood myasthenia gravis in this hospital were retrospectively analyzed. These cases were randomly divided into observation group and control group. For observation group, there were methylprednisolone and gamma globulins while the only methylprednisolone in the control group. The clinical effects and changes in immune globulin and complement of two groups were observed. RESULTS: The total effective rate for observation group was 94.3% and 74.3% for the control group, and this difference was statistically significant (p < 0.05) The time for relief of symptoms (6.55 ± 1.35 days) and total hospital stay (17.15 ± 3.65 days) in observation group was apparently shorter than the control group, with statistical significance (p < 0.05). CONCLUSIONS: Glucocorticoid and gamma globulin can improve the symptoms and achieve satisfying clinical effects for the treatment of myasthenia gravis in children. Thus, it is valuable for further popularization and application.

Isolation, characterization and chromosomal localization of the human GADD153 gene.

We report the isolation and characterization of the growth arrest and DNA-damage-inducible gene, GADD153, from human cells and show that it is localized in the region 12q13.1-q13.2 on chromosome 12. Comparison of the human gene with the previously described hamster gene revealed a high level of conservation in both the structural and regulatory regions of the genes. Each is composed of four exons with intron/exon junctions maintained at the identical positions. The human Gadd153 protein shares 91% identity with the hamster protein in amino acid sequence, and 78% identity in nucleotide sequence. A 900-bp fragment of 5' flanking sequence from the human gene, when linked to the bacterial cat reporter gene, was found to exhibit promoter activity in HeLa cells which could be further activated by treatment with the DNA alkylating agent, methyl methanesulfonate. Sequence analysis indicated that the human promoter region is relatively G+C-rich and contains putative binding sites for multiple transcription factors, including recognition sites for TATA- and CAAT-binding proteins, six Sp1-binding sites, an activator protein-1 binding site, an E-26-specific sequence-binding protein-1 DNA-binding site, and four interleukin-6 response elements. Many of these sites are also present in an identical position in the hamster gene suggesting they may play an important role in regulating GADD153 expression.

IL-2 inhibits early increases in serum gamma interferon levels associated with graft-versus-host-disease.

We have recently demonstrated that a short course of high-dose IL-2 administered to lethally irradiated mice leads to marked protection from early and late GVHD mortality, especially when T cell-depleted (TCD) host-type bone marrow cells (BMC) are also administered. IL-2 inhibits the GVHD-inducing activity of donor CD4+ cells without inhibiting their graft-vs.-leukemia effects. Since CD4+ T-lymphocytes produce a variety of cytokines, some of which have recently been implicated in the pathogenesis of GVHD, we have studied the possible effect of IL-2 administration on serum levels of various cytokines. Acute GVHD was induced in lethally irradiated B10 mice by bone marrow transplantation (BMT) with MHC-mismatched allogeneic (A/J) BMC and splenocytes. TCD B10 (host-type) BMC were coadministered to maximize the protective effect of IL-2. Serum cytokine levels were compared in recipients of these inocula with or without a protective course of IL-2 treatment. A marked increase in serum IFN-gamma levels was noted on days 3 through 5 post-BMT in GVHD mice compared with syngeneic BMT control recipients. This GVHD-induced rise in serum IFN-gamma was markedly inhibited in IL-2-protected mice. Murine IL-2 levels were only slightly increased in sera of GVHD mice, and were not influenced by treatment with human recombinant IL-2. Serum levels of the monokines TNF-alpha and IL-1 alpha showed variable early elevations in GVHD mice with or without IL-2 treatment, and were not different from levels observed in syngeneic controls. Serum levels of IFN-gamma, IL-1 alpha, and TNF-alpha all declined markedly by day 7 to 8 post-BMT, when GVHD mortality begins. Administration of neutralizing anti-IFN-gamma mAb did not attenuate and tended to accelerate GVHD mortality, and administration of exogenous IFN-gamma did not overcome the protective effect of IL-2 against GVHD. Together, our results indicate that GVHD is associated with high serum levels of several proinflammatory cytokines in the first week post-BMT, but that these levels decline by the time when GVHD mortality begins. IL-2 specifically inhibits the GVHD-associated production of IFN-gamma, but this inhibition in itself does not explain and may even mitigate the protective effect of IL-2 against early GVHD mortality. However, the demonstration that IL-2 markedly inhibits the production of a GVHD-associated cytokine raises the possibility that alterations in the production of as yet undefined cytokines may be responsible for IL-2-induced GVHD protection.

Inhibition of graft-versus-host disease by interleukin-2 treatment is associated with altered cytokine production by expanded graft-versus-host-reactive CD4+ helper cells.

In a fully MHC plus multiple minor antigen-mismatched murine bone marrow transplantation (BMT) model, we have demonstrated that a short course of high dose IL-2, begun on the day of BMT, protects against graft-versus-host disease (GVHD). This inhibitory effect is directed against donor CD4+ cells. To determine whether the mechanism of IL-2-induced GVHD protection involves clonal deletion or anergy of host-reactive donor T helper cells (Th), we performed limiting dilution analyses to measure the frequency of activated Th that reacted to donor, host, and third-party antigens in GVHD control and IL-2-protected mice. Marked and specific expansion of host-reactive Th was observed to a similar extent in GVHD control and IL-2-protected mice by day 5 after BMT, and the number of these cells in the spleen increased by several orders of magnitude between days 3 and 5 after BMT, which suggests that recirculation from other tissues occurred in this period. A high proportion (approximately 80%) of donor T cells expressed CD25 in both GVHD control and IL-2-protected mice on day 4 after BMT, which suggests a high level of bystander T cell activation. Since marked quantitative differences in the GVH response were not observed between GVHD control and IL-2-protected mice, we assessed both groups for qualitative differences in the Th response. Spleen cells isolated in the first 8 days after BMT were cultured with host-type, donor-type, or third-party stimulators or without stimulators, and cytokines were measured in supernatants harvested at 24 hr. GVHD was associated with marked increases in supernatant IFN-gamma levels from day 3 to day 6 after BMT, and with increases in IL-2 levels compared with naive A/J controls or syngeneic BMT controls stimulated with host antigens. Production of these cytokines was specifically induced by host-type antigens. Supernatants from spleens of IL-2-treated mice showed delayed kinetics of IFN-gamma production, and tended to contain higher levels of IL-4 in response to host antigen compared with GVHD controls on days 2 and 4 after BMT. Both IL-4 and IFN-gamma were produced almost exclusively by CD4+ cells in spleens of GVHD control and IL-2-protected mice on day 4. However, no consistent difference was observed between the groups in supernatant IL-2 or IL-10 levels, ruling out a simple Th1 to Th2 switch.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison between 8-prenylnarigenin and narigenin concerning their activities on promotion of rat bone marrow stromal cells' osteogenic differentiation in vitro.

A number of recent studies have suggested that flavonols (a class of phytochemical with many biological activities), might exert protective effects against post-menopausal bone loss. In the present study, we compared naringenin (NG) and 8-prenylnaringenin (PNG), two major naturally occurring flavonols, on in vitro differentiation of osteoblasts and bone resorbing activity, of rat bone marrow stromal cells (BMSCs). Our results indicated that both compounds, at 10(-6)  m, enhanced BMSCs' differentiation. Then effects of the two compounds at 10(-6)  m on ALP activity, osteocalcin secretion and calcium deposition, were compared over a time course. Numbers and areas of colonies stained for ALP (CFU-F(ALP) ) expression, and mineralized bone nodules, were histochemically analysed after 12 days and 16 days osteogenic induction, respectively. Expression of BMP-2, OPG, OSX, RUNX-2 genes and p38MAPK protein were examined using real-time PCR and western blotting, respectively. The data presented indicate that PNG, significantly enhanced the rat BMSCs' differentiation and mineralization through the BMP-2/p38MAPK/Runx2/Osterix signal pathway, greater than did NG. In conclusion, PNG has a more pronounced ability to enhance osteoblast differentiation and mineralization, than NG.

Developmental changes and regional localization of Dspp, Mepe, Mimecan and Versican in postnatal developing mouse teeth.

It has been implicated noncollagenous proteins act as important regulators during odontogenesis. To test the hypothesis that the roles of Dspp, Mepe, Versican and Mimecan in the regulation of odontogenesis may be complementary, comparative investigations on the localization of four proteins were performed by immunohistochemical staining using mouse first molar at different developmental stages as a model. In postnatal 1- day-old mice, all the proteins, excluding Mepe, showed co-expression in young odontoblasts. At postnatal 3, strong immunoreactions for all proteins were detected in odontoblasts. Interestingly, Mepe was present within both cytoplasm and nucleus in odontoblasts. In mice older than 5 days, the expression of Dspp, Mimecan and Versican accumulated in subodontoblastic layer of the coronal pulp at high levels while the co-expression of Mepe and Mimecan significantly existed in predentin. The temporal-spatial specific pattern and unique co-localization of Dspp, Mepe, Mimecan and Versican suggest they play complementary roles during odontogenesis.