Long-term outcomes of fistula-tract laser closure for complex perianal fistulizing Crohn's disease.

BACKGROUND: Fistula-tract laser closure (FiLaC™) has shown promising outcomes in perianal fistulizing Crohn's disease (pfCD). However, most studies assessed a mixed cohort encompassing pfCD and cryptoglandular fistulas during a short follow-up period. This study aimed to evaluate the long-term treatment outcomes of FiLaC™ in patients with complex pfCD. METHODS: Data from patients with complex pfCD who underwent FiLaC™ during deep remission of Crohn's disease between January 2019 and December 2020 were retrospectively analyzed. Patient demographics, surgery history, and medication strategy were registered before surgery. Follow-ups were scheduled at 1, 2, and 3 months after FiLaC™, and at 2-month intervals thereafter. The primary endpoint was clinic healing, while clinic remission/unhealed/recurrence were classified as unhealed. Additionally, adverse events and Wexner fecal incontinence score were documented. RESULTS: Forty-nine patients (40 men and 9 women) with a median age of 26.0 (19.0-35.5) years were included with a median follow-up of 50.0 (39.5-54.0) months. Of these, 31 (63.3%) patients achieved fistula healing, 3 (6.1%) experienced improvement, 3 (6.1%) remained unhealed, and 12 (24.5%) experienced recurrence. Montreal A category was lower in the healed group (P < 0.001). No major complications, such as bleeding or fecal or urinary incontinence, were observed, and pain was transient. The Wexner incontinence score decreased significantly at the last available follow-up, indicating an intact postoperative continence function (P = 0.014). PCDAI scores were significantly higher in the unhealed group (P = 0.041). CONCLUSION: FiLaC™ is an efficient and safe sphincter-saving procedure for patients with complex pfCD.

[X-ray evaluation of pulp calcification in adult permanent teeth after pulpotomy].

OBJECTIVE: To compare the clinical effects of pulpotomy with two kinds of calcium silicate materials, and to evaluate the formation of dentin bridge and pulp calcification after pulpotomy of adult permanent teeth. METHODS: Patients who visited the General Department of Peking University School and Hospital of Stomatology from November 2017 to September 2019 and planned for pulpotomy on permanent premolars and molars with carious exposed pulp were selected. They were randomly divided into two groups. Bioceramic putty material iRoot BP (iRoot group, n=22) and mineral trioxide aggregate MTA (MTA group, n=21) were used as pulp capping agents, respectively. The patients were recalled after one year and two years. The clinical efficacy, dentin bridge index (DBI) and pulp calcification index (PCI) were recorded. Blinding method was used for the patients and evaluators. RESULTS: There was no significant difference in gender, mean age, dentition and tooth position between the two groups (P>0.05). Seven cases were lost during the first year (4 cases in iRoot group and 3 cases in MTA group). In the iRoot group, 1 case had transient sensitivity at the time of 1-year follow-up. The cure rate of the two groups was 100% at the time of 2-year follow-up. The proportion of dentin bridge formation was 38.9% one year after operation, 55.6% two years after operation. The proportion of partial or even complete disappearance of root canal image was 5.6% before operation, 38.9% and 55.6% one and two years after operation, respectively. The difference was statistically significant by rank sum test (P < 0.05). There was no significant difference in dentin bridge formation and pulp calcification between the two groups (P < 0.05). DBI and PCI after operation was as the same as those before operation (44.4% cases of DBI and 25% cases of PCI) or gradually increased (55.6% cases of DBI and 75% cases of PCI). Spearman's nonparametric correlation analysis showed that age was positively correlated with preoperative pulp calcification index (PCI0, P < 0.05), but not with the dentin bridge index (DBI1, DBI2), pulp calcification index (PCI1, PCI2) and the degree of change (DBI2 vs. DBI1, PCI1 vs. PCI0, PCI2 vs. PCI0) 1-year and 2-year after operation (P>0.05). CONCLUSION: According to this study, good clinical effects were obtained within 2-year after pulpotomy of adult permanent teeth with MTA and iRoot. In some cases, the root canal system had a tendency of calcification aggravation, and there was no statistical difference in the development of this trend between the two groups.

[Evaluation of bioceramic putty repairmen iRoot and mineral trioxide aggregate in mature permanent teeth pulpotomy].

OBJECTIVE: To evaluate the clinical characteristics and effectiveness of pulpotomy in mature permanent teeth with bioceramic putty repairmen iRoot and mineral trioxide aggregate (MTA). METHODS: Pulpotomy was performed on mature permanent premolars and molars with carious exposures at the Department of General Dentistry of Peking University School and Hospital of Stomatology, from November 2017 to September 2019. The patients were randomly divided into 2 groups, Group iRoot (n=22) and Group MTA (n=21). In Group iRoot, bioceramic putty repairmen iRoot was used as pulp capping agent, while in Group MTA, mineral trioxide aggregate was used as pulp capping agent. All the patients had signed informed consent forms. The clinical efficacy was evaluated by clinical examinations (temperature and electrical activity test) and imaging examinations 3, 6, and 12 months after surgery. Blinding was used for the patients and evaluators, but due to the obvious differences in the properties of the two pulp capping agents, the blinding method was not used for the treatment provider (the attending physician). RESULTS: There was no significant difference in gender, average age, dentition and tooth position distribution between the two groups (P>0.05). In the study, 7 cases were lost to follow-up 12 months after operation (4 cases in Group iRoot, and 3 cases in Group MTA). One case in each of the two groups had transient sensitivity at the end of the 3-month follow-up, and the pulp vitality was normal at the end of the 6-month follow-up. One case in Group iRoot showed sensitivity at the end of the 12-month follow-up. The success rates of the two groups at the end of 12-month follow-up were 100%, and the cure rates were 94.4% (Group iRoot) and 100% (Group MTA), respectively, and the difference was not statistically significant (P>0.05). No cases in Group iRoot had obvious crown discoloration, while 3 cases in Group MTA had. CONCLUSION: The clinical characteristics and effectiveness of pulpotomy in mature permanent teeth with bioceramic putty repairmen iRoot were similar with MTA. Bioceramic putty repairmen iRoot is an acceptable material when used in pulpotomy of mature permanent teeth. Because it is not easy to cause tooth discoloration after treatment and is convenient to operate, bioceramic putty repairmen iRoot has a better clinical application prospect.

[Effect of ruxolitinib combined with glucocorticoid on cytomegalovirus activation in acute graft-versus-host disease].

Objective: To observe the effect of ruxolitinib combined with glucocorticoid on cytomegalovirus (CMV) activation in patients with acute graft-versus-host disease (aGVHD) . Methods: The clinical data of 195 patients who underwent allogeneic hematopoietic stem cell transplantation in the Department of Hematology of the First Medical Center of the People's Liberation Army General Hospital from August 2018 to September 2020 were retrospectively analyzed. According to the severity of aGVHD, the patients were divided into the non-GVHD group, aGVHD grade Ⅰ group, aGVHD grade Ⅱ-Ⅳ group, and aGVHD grade Ⅲ/Ⅳ group. In addition, they were classified into two subgroups according to the first-line treatment regimen for aGVHD: combined regimen group (ruxolitinib combined with glucocorticoid) and classical regimen group (glucocorticoid alone) . The cumulative incidence of CMV activation, the duration of CMV activation, and the duration of CMV negativity in each subgroup at 90 and 180 days after transplantation were analyzed. The overall survival and disease-free survival rates of patients in both regimens were compared. Results: Sixty-four (32.8%) patients in the group did not develop aGVHD. The numbers of patients with grade Ⅰ, Ⅱ-Ⅳ, and Ⅲ/Ⅳ aGVHD were 30 (15.4%) , 101 (51.8%) , and 14 (7.2%) , respectively. Compared with patients in the classical regimen, no significant difference was observed in the cumulative incidence of CMV activation, duration of CMV activation, and duration of CMV negativity in patients with grade Ⅰ-Ⅳ aGVHD in the combined regimen at 90 and 180 days after transplantation (P>0.05) . Further analysis of patients with grade Ⅱ-Ⅳ and Ⅲ/Ⅳ aGVHD showed that the cumulative incidence of CMV activation, duration of CMV activation, and duration of CMV negativity did not show significant difference between the two treatment regimens (P>0.05) . In addition, there was no significant difference in the overall survival and disease-free survival rates of patients in both regimens (P>0.05) . Conclusion: Ruxolitinib combined with glucocorticoid as the first-line therapy for aGVHD did not increase the risk of CMV activation.

Trichostatin A exerts anti-inflammation functions in LPS-induced acute lung injury model through inhibiting TNF-α and upregulating micorRNA-146a expression.

OBJECTIVE: Acute lung disease is characterized by inflammation. This research aimed to investigate effect of trichostatin A (TSA) on microRNA-146a (miR-146a) and tumor necrosis factor α (TNF-α) in lipopolysaccharide (LPS)-induced alveolar macrophage injury model. MATERIALS AND METHODS: Rat alveolar macrophage, NR8383, was cultured and induced using LPS to establish acute lung injury model in vitro level. Cell Counting Kit-8 (CCK-8) assay was used to determine cell viability of NR8383 cells. TSA was administrated to LPS-induced NR8383 cells. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay was utilized to evaluate TNF-α and miR-146a mRNA expression in LPS and/or TSA treated NR8383 cells. Enzyme-link immunosorbent assay (ELISA) was used to examine TNF-α levels. RESULTS: This study selected 1 ng/ml and 10 ng/ml TSA as the optimal concentrations for treating NR8383 cells. LPS-induced acute lung injury model was successfully established. TSA administration significantly enhanced accounts of LPS-stimulated NR8383 cells. LPS induction significantly increased miR-146a mRNA expression in NR8383 cells compared to NR8383 cells (p<0.05). TSA administration significantly reduced the levels of TNF-α in LPS-induced NR8383 cells compared to those in LPS-induced NR8383 cells (p<0.05). TSA administration significantly enhanced miR-146a expression in LPS-induced NR8383 cells compared to that in LPS-induced NR8383 cells (p<0.05). CONCLUSIONS: TSA administration exerted anti-inflammation functions in LPS-induced acute lung injury model in vitro, which might be triggered by inhibiting TNF-α molecule and upregulating miR-146a expression. The present data hint that TSA could be considered as a potential therapeutic agent for treating acute lung injury.

[The technique and efficacy of complete resection of mesogastrium above the pancreas by the right approach in laparoscopic assisted distal gastrectomy for radical gastrectomy].

Objective: To explore the safety and efficacy of mesogastrium excision above the pancreas by the right approach in radical distal gastrectomy for cancer. Methods: A total of 154 distal gastric cancer patients in the Department of Gastrointestinal Surgery of the first affiliated Hospital of Chongqing Medical University, from October 2013 to October 2014, were included in this random controlled study. These patients were divided into the study group (n=78), who received mesogastrium excision above the pancreas by the right approach, and the control group (n=76), who received mesogastrium excision above the pancreas when by the left approach. Results: No significant differences were observed between the two groups in terms of the number of lymph node, postoperative recover, complications and 3-year survival rate(P>0.05), while significant differences were observed between the two groups in terms of operation time and surgical bleeding(P<0.05). Conclusions: In radical distal gastrectomy for cancer, the safety and efficacy for mesogastrium excision above the pancreas were the same by the right approach, compared to by the left approach, however, the group by the right approach had the advantage on the operation time and surgical bleeding.

[Retrospective comparison of screening criteria for active surveillance for papillary thyroid microcarcinoma].

Objective: To investigate the rationality of management of active surveillance for papillary thyroid microcarcinoma (PTMC) and the main indications for active surveillance for PTMC. Methods: In this study, two criteria were used to evaluate patients with PTMC: low-risk PTMC conditions defined by Kuma hospital and Chinese Association of Thyroid Oncology (CATO) consensus on PTMC management of active surveillance. The patients had received surgical treatment. Clinicopathological characteristics and prognosis of the patients in different groups were compared. Results: A total of 778 patients were enrolled in the study, 565 (72.6%) of them met Kuma screening criteria and only 112 (14.4%) met CATO screening criteria. Kuma low-risk subgroup had lower incidence of cervical lymph node metastasis than Kuma high-risk PTMC subgroup(30.6% vs 47.9%, P<0.05). There were significant differences in multifocal lesions(6.3% vs 16.4%), extrathyroidal extension (1.8% vs 7.5%) and cervical lymph node metastasis(19.6% vs 38.0%) between low-risk and high-risk CATO PTMC subgroups. Patients in the CATO low-risk PTMC subgroup had lower recurrence and longer disease-free survival (DFS) than those in the CATO high-risk PTMC subgroup. But there was no significant difference in recurrence or DFS between Kuma low-risk and high-risk Kuma PTMC subgroups.The Chi-square test of Fisher's exact probabilities test was used to compare clinicopathological characteristics of patients between different groups.Rates of disease-free survival were calculated using the Kaplan-Meier method. Conclusion: CATO screening criteria is relatively strict and may be more suitable for Chinese patients with active surveillance for PTMC.

A retrospective analysis of ovarian stimulation with letrozole in women undergoing artificial insemination by donor.

The aim of this retrospective study was to determine the clinical pregnancy rate in women undergoing letrozole ovarian stimulation and artificial insemination by donor (AID). Between 2012 and 2015, 130 natural cycles, 939 letrozole cycles and 130 letrozole plus gonadotrophin cycles were conducted. Letrozole cycles were divided into three groups according to LH concentration on the day of HCG administration (LH <10 mIU/ml and follicle size ≥18 cm; LH ≤10 to <20 mIU/ml; and LH ≥20 mIU/ml). Pregnancy rates were 17.3%, 22.4% and 26.8%, respectively (P = 0.012). In women given 10 mIU/ml LH or more, logistic regression identified oestradiol (OR 1.002, 95% CI, 1.000 to 1.004, P = 0.029) and leading follicle size (OR 0.861, 95% CI, 0.772 to 0.960, P = 0.007) as significant predictive factors of pregnancy rate; the higher the oestradiol and the smaller the follicles, the better the pregnancy rate. The pregnancy rate was significantly higher in the letrozole plus gonadotrophin group than the letrozole group (P = 0.04). Better pregnancy rates can be achieved if LH surge occurs before HCG administration, especially with higher oestradiol and lower follicle size; treatment with letrozole plus gonadotrophin was significantly more effective than letrozole alone in AID.

Skin compatibility and efficacy of a cosmetic skin care regimen with licochalcone A and 4-t-butylcyclohexanol in patients with rosacea subtype I.

BACKGROUND: Patients with rosacea often show facial sensitivity to cosmetics or skin care products that can influence the severity of symptoms and exacerbate erythema and inflammation. Nevertheless, special skin care is necessary to address cosmetic concerns and reduce the potential side-effects of topical or oral treatment of the disease. Appropriate skin care should comprise gentle cleansing, effective moisturization, soothing actives, UV protection and concealing pigments to help neutralize the appearance of redness. OBJECTIVE: To determine the compatibility and efficacy of a skin care regimen (consisting of a cleanser, a day care with SPF25 and a night care) containing licochalcone A (Lic A), an anti-irritant from the licorice plant Glycyrrhiza inflata, and 4-t-butylcyclohexanol (SymSitive(®) ), a substance which acts as a sensitivity regulator, in female subjects with clinically determined subtype I rosacea. METHODS: Thirty-two test subjects with mild to moderate rosacea used the skin care regimen daily for 8 weeks. Clinical assessment of erythema, subjective irritation and clinical photography were performed at baseline and after 4 and 8 weeks. Additionally, a quality-of-life questionnaire was filled out by the test subjects at baseline and week 8. The subjects completed a self-assessment questionnaire on product properties after 4 and 8 weeks of product use. RESULTS: Clinical assessments and subject response confirmed very good tolerability of the regimen, a statistically significant improvement in clinical grading for erythema and tactile roughness at weeks 4 and 8 and on telangiectasia at week 8 when compared to baseline scores. A statistically significant improvement in facial redness (a*) values, based on the L*a*b* colorimetric system, was determined at week 4 and 8 in comparison to baseline. No difference in corneometric measurement was detected at week 4 and 8 compared to baseline. CONCLUSION: The skin care regimen was found to be highly compatible with the sensitive facial skin of patients with rosacea subtype I and effective in improving signs of rosacea. Therefore, the daily use of skin care products containing LicA and SymSitive(®) in patients with rosacea improves the overall skin appearance and the quality of life of these patients.

MiR-873 regulates ERα transcriptional activity and tamoxifen resistance via targeting CDK3 in breast cancer cells.

miRNAs (microRNAs) are frequently and aberrantly expressed in many cancers. MiR-873 has been revealed to be downregulated in colorectal cancer and glioblastoma. However, its function remains unclear. Here we report that miR-873 is downregulated in breast tumor compared with normal tissue. Enforced expression of miR-873 decreases the transcriptional activity of ER (estrogen receptor)-α but not ERß through the modulation of ERα phosphorylation in ER-positive breast cancer cells. We also found that miR-873 inhibits breast cancer cell proliferation and tumor growth in nude mice. Reporter gene assays revealed cyclin-dependent kinase 3 (CDK3) as a direct target of miR-873. CDK3 was shown to be overexpressed in breast cancer and phosphorylate ERα at Ser104/116 and Ser118. Furthermore, we found that Mir-873 inhibits ER activity and cell growth via targeting CDK3. Interestingly, miR-873 was observed to be downregulated in tamoxifen-resistant MCF-7/TamR cells, while CDK3 is overexpressed in these cells. More importantly, re-expression of miR-873 reversed tamoxifen resistance in MCF-7/TamR cells. Our data demonstrate that miR-873 is a novel tumor suppressor in ER-positive breast cancer and a potential therapeutic approach for treatment of tamoxifen-resistant breast cancer.

Combinatorial treatment of bone marrow stem cells and stromal cell-derived factor 1 improves glycemia and insulin production in diabetic mice.

Transdifferentiation of stem cells into insulin-producing cells for the treatment of diabetes have shown promising but inconsistent results. We examined the potential for attracting bone marrow stem cells (BMSCs) to the pancreas using a chemokine, stromal cell-derived factor 1 (SDF-1). SDF-1 treatment markedly increased the number of GFP labeled BMSCs in the pancreas, but surprisingly, the majority was observed in liver. The liver cells had typical pancreatic endocrine cell gene expression including insulin I, insulin II, PDX-1, somatostatin, and glucagon. Combined treatment with SDF-1 and BMSC transplant reduced hyperglycemia and prolonged the long-term survival of diabetic mice, and a sub group had complete normoglycemia (<150 mg/dl), restored blood insulin levels, and normal glucose tolerance. Our results suggest that SDF-1 could potentially be used to improve the homing of stem cells and ß-cell regeneration. The mechanism appears to involve an increase in insulin producing cells mainly in the liver.

Prevalence of immunoglobulin A deficiency in Chinese blood donors and evaluation of anaphylactic transfusion reaction risk.

OBJECTIVES/AIMS: We investigated the incidence of immunoglobulin A (IgA) deficiency in Chinese population. BACKGROUND: The frequency of IgA deficiency, defined as a serum IgA level of <0.05 mg dL(-1) , has been broadly studied in different ethnic groups. Individuals with IgA deficiency may form specific antibodies against IgA, which can cause an anaphylactic response when the patient receives an IgA-containing blood transfusion. METHODS: A sandwich enzyme-linked immunosorbent assay was performed to screen for IgA deficiency and particle gel immunoassay used for confirmation. IgA antibodies were further detected by the DiaMed anti-IgA test in IgA-deficient blood donors. RESULTS: Of the total 22,609 healthy blood donors screened, only seven cases were confirmed as having IgA deficiency (<0.05 mg dL(-1) ). Another seven cases displayed relative IgA deficiencies, with mean IgA concentrations ranging from 0.39 to 3.70 mg dL(-1) . Anti-IgA was identified in 2 of the 14 IgA-deficient blood donors whose IgA levels were <5 mg dL(-1) . Estimation of the theoretical risk for IgA anaphylactic transfusion reaction was 0.009%. CONCLUSION: The prevalence of IgA deficiency in Chinese is low. However, potential risks exist in performing blood transfusion to IgA-deficient persons, and measures should be taken to reduce IgA anaphylaxis.

Prophylaxis of heterotopic ossification - an updated review.

Heterotopic ossification (HO) is defined as the process by which trabecular bone forms outside of the skeletal structure, occupying space in soft tissue where it does not normally exist. The current popular prophylactic treatment modalities include non-steroidal anti-inflammatory drugs (NSAIDs) and radiation therapy, although the literature remains inconclusive as to which is superior. Additionally, both treatments can lead to adverse effects to the patient. Recently there have been several studies attempting to identify new aspects of the etiology of heterotopic bone formation and introduce new prophylactic modalities with increased efficacy and fewer side effects. For this review, we selectively retrieved articles from Medline published from 1958-2008 on the prophylaxis of HO with the aim of assisting readers in quickly grasping the current status of research and clinical aspects of HO prophylaxis.

Platelet-rich and platelet-poor plasma: development of an animal model to evaluate hemostatic efficacy.

Hemostasis is important for any surgical procedure. One method uses autologous platelet-rich and/or platelet-poor plasma sprayed on the wound site. Although effective, there are little quantitative data available to fully document the extent to which these autologous products function as hemostats. Also, limitations in current animal models make quantitative study of topical hemostats difficult. A porcine partial-thickness skin wound model was developed to compare the hemostatic ability of these treatments with untreated control wounds. Rectangular partial-thickness dermal wounds were created in the back of a pig, which was then sprayed with activated platelet-rich plasma, activated platelet-poor plasma, or left untreated. Bleeding was quantified by two methods: 1) gravimetric measurement of exudate transfer to a sponge over a 15-minute interval, and 2) iron assay of the exudate over this same interval. Values for treated wounds were normalized to those of control wounds to minimize interanimal variability. Both gravimetric and iron assay measurements demonstrated that platelet-rich plasma was effective within 5 minutes after application with normalized bleeding values of approximately 35% and 20%, respectively, of the untreated controls. Corresponding values for platelet-poor plasma were approximately 90% and 65%, respectively, with differences only significant for the iron assay method measured on 10- and 15-minute wound exudate. Although both platelet-rich and platelet-poor plasma demonstrated hemostatic potential, the effect was more robust with the former. Iron assay was a more accurate method of measuring bleeding than gravimetric analysis.

A hypoxia-inducible vigilant vector system for activating therapeutic genes in ischemia.

Hypoxia represents an endogenous pathophysiological signal underlying cell growth, adaptation and death in a variety of diseases, including ischemic heart diseases, stroke and solid tumors. A vigilant vector system depends on a gene switch which can sense the hypoxia signal occurring in ischemic events and turn on/off protective gene expressions when necessary. This system uses the oxygen-dependent degradation domain derived from hypoxia-inducible factor 1alpha as the hypoxia sensor and a double-vector system as signal amplifier. For treating ischemic heart diseases, a cardiac-specific MLC-2v promoter is used to deliver transgenes specifically to the heart. When tested in cardiomyocyte cultures, it produced a rapid and robust gene induction upon exposure to low oxygen. In a mouse model for myocardial infarction, the vigilant vectors turned on therapeutic genes such as heme oxygenase-1 in response to ischemia, significantly reduced apoptosis in the infarct area and improved cardiac functions. The hypoxia-regulated gene transfer afforded by the vigilant vectors may provide a powerful tool for delivering therapeutic proteins specifically to ischemic tissues with optimal physiological control.

Vitreous preservation of articular cartilage grafts.

Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, an ice-free cryopreservation method by vitrification was tested. Osteochondral plugs from New Zealand White rabbits were preserved using either a freezing method or an ice-free vitrification method of cryopreservation. Preserved and fresh control plugs were implanted in the tibial plateau of allogeneic recipients. A modified O'Driscoll grading scale, based on gross pathology, histopathology, and histochemistry, was used to evaluate the explants.The histology of fresh and vitrified explants was essentially the same, while the frozen cryopreserved explants were devoid of chondrocytes and only fibroblastlike cells were observed. The O'Driscoll grading indicated that both fresh and vitrified plugs performed significantly better than frozen plugs (p < or =.05). The results demonstrate the feasibility of vitrification as a storage method for cartilaginous tissues.

Side-chain lactam-bridge conformational constraints differentiate the activities of salmon and human calcitonins and reveal a new design concept for potent calcitonin analogues.

We have recently reported the potent hypocalcemic effects of side-chain lactam-bridged analogues of human calcitonin (hCT) (Kapurniotu, A.; et al. Eur. J. Biochem. 1999, 265, 606-618). To extend these studies, we have now synthesized a new series of (Asp(17), Lys(21)) and (Asp(17), Orn(21)) side-chain bridged salmon calcitonin (sCT) and hCT analogues. The affinities of these analogues for the human calcitonin receptor, hCTR(I1)(-), and for rat-brain membrane receptors were assayed in competitive binding assays, and agonist potencies at the hCTR(I1)(-) receptors were assessed, using a cAMP-responsive gene-reporter assay. The bridged sCT analogues had activities similar to sCT itself. In contrast, an (Asp(17), Orn(21)) side-chain bridged hCT analogue, cyclo(17-21)-[Nle(8), Phe(12), Asp(17), Orn,(21) Tyr(22))-hCT, was 80 and 450 times more active than hCT in the hCTR(I1)(-) and rat-brain receptor binding assays, respectively, and was 90 times more potent than hCT and 16 times more potent than sCT in initiating receptor signaling. An uncyclized, isosteric analogue of this peptide was also more potent than hCT, demonstrating that the cyclization constraint and these single-residue substitutions enhance the activities of hCT in an additive fashion. This study demonstrates that the potency-enhancing effects of lactam-bridge constraints at hCT residues 17-21 are not transferable to sCT. We also show that, in comparison to the hCT analogues, sCT and its analogues are less potent agonists than expected from their hCTR(I1)(-) affinities. This suggests that it may be possible to preserve the efficient signal transduction of hCT while introducing additional receptor affinity-enhancing elements from sCT into our potent lactam-bridged hCT analogue, thereby creating new super-potent, hCT-based agonists.

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.

[Clinical implications of HCV quasispecies heterogeneity in patients with hepatitis C].

OBJECTIVE: To determine the heterogeneity of viral quasispecies and its clinical significance in patients with hepatitis C. METHODS: Quasispecies in the sera from 76 patients infected with hepatitis C virus were detected using single stranded conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVR1). RESULTS: HVR1 was amplified in 72 (94.7%) of the 76 patients. The average number of SSCP bands was 5.8, with a range from 2 to 11. The numbers of quasispecies in acute hepatitis, chronic hepatitis and liver cirrhosis and/or primary hepatocellular carcinoma were 3.1 +/- 1.2, 6.0 +/- 2.3 and 8.4 +/- 4.1, respectively. There was a statistically significant difference among them (P < 0.01). Patients with infection acquired by blood transfusion and i.v. drug use had greater number of quasispecies than those acquired by other transmission pathway (sporadic) (P < 0.05). Patients with genotype 1 a and 1 b infection had increased quasispecies compared with those infected with HCV type 2 and 3(P < 0.05). Increased quasispecies heterogeneity was significantly correlated with serum HCV RNA levels (P < 0.01). CONCLUSIONS: SSCP is a simple, rapid and reliable method for the analysis of HCV viral quasispecies heterogeneity. Quasispecies heterogeneity plays an important role in HCV persistent infection and in the progress of hepatitis C. The duration of HCV infection, HCV genotype and HCV viremia are important determinants for the evolution of HCV quasispecies heterogeneity.