[The value of sinus computed tomography in predicting the response to oral glucocorticoids in chronic rhinosinusitis with nasal polyps].

Objective: To study the value of olfactory cleft scores through computed tomography (CT) in predicting the oral glucocorticoids (GC) sensitivity in chronic rhinosinusitis with nasal polyps. Methods: Fourty-seven consecutive patients with CRSwNP from the Fifth Affiliated Hospital of Sun Yat-sen University between January and March of 2018 were recruited in this prospective, single-blinded study. There were 28 males and 19 females, with age ranging from 17 to 66 years old. After a course of oral prednisone (30 mg/d for 14 d), these patients were subsequently classified into objectively GC-sensitive and -insensitive subgroup according to the change in nasal polyp size score, or subjectively GC-sensitive and -insensitive subgroup according to the change in total nasal symptom score. The following parameters were compared between GC-sensitive and -insensitve subgroups: Lund-Mackay scores, olfactory cleft scores, and blood eosinophil counts and ratio. T test and χ(2) test were used. Multivariate Logistic regression analysis was used for factor prediction and receiver operating characteristic (ROC) curve was used to analyze the predictive ability of those factors. Results: There were 53.2% (25/47) and 61.7% (29/47) of patients objectively and subjectively sensitive to GC therapy, respectively. All data conformed to normal distribution. The olfactory cleft score and the blood eosinophil counts and ratio in objectively GC-sensitive subgroup were significantly higher than those in objectively GC-insensitive subgroup (3.6±1.0 vs 2.2±1.4, (404.4±200.3)/µl vs (209.5±233.1)/µl, (5.25±2.59)% vs (3.17±3.46)%, t value was 3.98, 3.08, respectively, χ(2)=2.35, all P<0.05). The cleft score, the blood eosinophil counts and ratio also showed the same trend in subjectively GC-sensitive and -insensitive subgroup (3.6±1.0 vs 1.9±1.3, (401.4±213.6)/µl vs (171.1±200.2)/µl, (5.39±2.76)% vs (2.48±2.99)%, t value was 5.05, 3.68, respectively, χ(2)=3.40, all P<0.05). Multivariate Logistic regression revealed that olfactory cleft score was an independent risk factor for objective or subjective GC-sensitivity (OR=2.882, 95%CI: 1.301-6.384; OR=2.508, 95%CI: 1.248-5.039). The olfactory cleft score exhibited comparable accuracy with the blood eosinophil ratio as predictor of objective and subjective GC-sensitivity (Area under curve of olfactory cleft score was 0.775, 0.829, respectively). An olfactory cleft score of 3.5 could act as a reliable indicator for predicting the clinical response to GC therapy in CRSwNP. Conclusion: Olfactory cleft score through CT scan has the potential value in predicting GC-sensitivity in CRSwNP patients.

MiR-374b promotes osteogenic differentiation of MSCs by degrading PTEN and promoting fracture healing.

OBJECTIVE: To investigate whether miR-374b can promote the differentiation of MSCs into osteoblasts by mediating PTEN, thus promoting fracture healing. MATERIALS AND METHODS: Primary cultured mouse mesenchymal stem cells were obtained for following experiments. Flow cytometry was used to determine the expression of MSCs surface antigens to identify the purity. Alizarin red staining was used to detect whether MSCs could differentiate into osteoblasts. QRT-PCR was used to detect the expression of osteogenic marker genes as well as miR-374b and PTEN in bone marrow-derived MSCs from fractured mice model. ALP activity detection kit was used to detect ALP activity in cells. Changes of osteogenic proteins in cells were evaluated by Western blot. Bioinformatics methods were used to predict the binding sites between miR-374 and target genes. Luciferase reporter assay was used to confirm whether miR-374 could bind to the target gene. RESULTS: Under normal culture, MSCs grew into a long fusiform shape on the 4th day. After induced in the osteogenic induction medium for seven days, calcified nodules appeared. The results of the detection of MSCs surface antigen markers showed that CD90 was 99.12%, and CD45 was 0.23%. Alizarin red staining showed that MSCs possess the ability to differentiate into osteogenic. The expression level of MiR-374b and PTEN increased significantly in the early stage of fracture in mice, but no significant difference was observed at a later stage. After overexpression of miR-374b, the cell ALP activity, the expression of osteogenesis-related genes and osteogenesis-related proteins was significantly increased, while after knocking out miR-374b, the opposite result was observed. The result of luciferase reporting assay showed that miR-374b can bind to PTEN. As mentioned above, overexpression of miR-374b resulted in upregulation of osteogenic-related genes and proteins, while over-expression of both PTEN and miR-374b could partly reverse the outcomes. CONCLUSIONS: miR-374b can promote osteogenic differentiation of MSCs by degrading PTEN, thereby promoting fracture healing.

Local IL-25 contributes to Th2-biased inflammatory profiles in nasal polyps.

BACKGROUND: IL-25 has been proposed to play a key role in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to evaluate the association of IL-25 with the Th2-biased inflammatory profiles in CRSwNP. METHODS: Nasal polyp (NP) tissues and control uncinate process tissues were collected from 92 patients with CRSwNP, 20 patients with chronic rhinosinusitis without nasal polyps (CRSsNP), and 16 normal control subjects. IL-25 expression was examined using immunohistochemistry and immunofluorescence staining, flow cytometry, RT-qPCR, and ELISA. The inflammatory profiles and clinical characteristics of 2 NP subtypes (IL-25high and IL-25low ) were evaluated, and the effects of IL-25 on Th2 cytokine production in cultured dispersed polyp cells were examined in vitro. RESULTS: The mRNA and protein levels of IL-25 were significantly increased in the polyp tissues compared with the control uncinate process tissues. The IL-25high subtype showed greater computed tomography scores, endoscopic scores, and Th2 response. Exposure to IL-25 activated type 2 innate lymphoid cells and Th2 cells in NP simultaneously which further increased Th2 cytokine production in vitro. CONCLUSIONS: Local IL-25 plays a crucial role in promoting Th2-biased inflammatory profiles in NP and may serve as a promising therapeutic target in CRSwNP patients.

CBL enhances breast tumor formation by inhibiting tumor suppressive activity of TGF-ß signaling.

Casitas B-lineage lymphoma (CBL) protein family functions as multifunctional adaptor proteins and E3 ubiquitin ligases that are implicated as regulators of signaling in various cell types. Recent discovery revealed mutations of proto-oncogenic CBL in the linker region and RING finger domain in human acute myeloid neoplasm, and these transforming mutations induced carcinogenesis. However, the adaptor function of CBL mediated signaling pathway during tumorigenesis has not been well characterized. Here, we show that CBL is highly expressed in breast cancer cells and significantly inhibits transforming growth factor-ß (TGF-ß) tumor suppressive activity. Knockdown of CBL expression resulted in the increased expression of TGF-ß target genes, PAI-I and CDK inhibitors such as p15(INK4b) and p21(Cip1). Furthermore, we demonstrate that CBL is frequently overexpressed in human breast cancer tissues, and the loss of CBL decreases the tumorigenic activity of breast cancer cells in vivo. CBL directly binds to Smad3 through its proline-rich motif, thereby preventing Smad3 from interacting with Smad4 and blocking nuclear translocation of Smad3. CBL-b, one of CBL protein family, also interacted with Smad3 and knockdown of both CBL and CBL-b further enhanced TGF-ß transcriptional activity. Our findings provide evidence for a previously undescribed mechanism by which oncogenic CBL can block TGF-ß tumor suppressor activity.

Effect of stealthy liposomal topotecan plus amlodipine on the multidrug-resistant leukaemia cells in vitro and xenograft in mice.

BACKGROUND: Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy as the over-expressed MDR protein acts as an efflux pump, which leads to a reduction in the uptake of the anticancer agent by tumour cells. We combined topotecan and amlodipine together into the stealthy liposomes, in which amlodipine was applied as a MDR reversing agent to overcome the resistance. MATERIALS AND METHODS: Cytotoxicity, apoptosis and the signalling pathway assays were performed on human chronic myelogenous leukaemia K562, promyelocytic leukaemia HL-60 and MDR HL-60 cells, respectively. Pharmacokinetics and antitumour activity studies were performed on normal Kunming mice and female BALB/c nude mice with MDR HL-60 xenografts, respectively. RESULTS: Topotecan alone was effective in inhibiting the growth of non-resistant leukaemia cells, K562 and HL-60 cells but not the growth of MDR HL-60 cells. The resistance of topotecan in MDR HL-60 cells was potently reversed by the addition of amlodipine. Moreover, amlodipine enhanced the apoptosis-inducing effect of topotecan synergistically. Apoptosis was through activating caspases in a cascade: first, the initiator caspase 8 and then effectors caspase 3/7 (total activity of caspases 3 and 7) were activated. Being encapsulated into the stealthy liposomes with an acidic internal medium, topotecan existed dominantly in an active lactone species, which was reversibly changed from an inactive carboxylate form via a pH-dependent reaction. After administration of stealthy liposomes to mice, the blood exposure of the lactone form was evidently increased and extended. The antitumour effects in the MDR HL-60 xenografted tumour were stealthy liposomal topotecan (SLT) plus amlodipine > SLT > un-encapsulated topotecan > blank control. CONCLUSIONS: The enhanced antitumour activity in the MDR HL-60 cells by the SLT plus amlodipine could be owing to multiple reasons: (a) synergistic apoptosis inducing effect, (b) reversing MDR by amlodipine and (c) increasing the availability of active lactone of topotecan by the stealthy liposomes. The apoptosis induced by amlodipine is through caspase 8 and then the 3/7 signalling pathway.

[Enhancement of immune responses of hepatitis B virus core DNA vaccine by a signal Peptide and a universal helper T lymphocyte epitope].

The human interleukin-2 signal peptide and a potent universal helper T lymphocyte epitope PADRE were spliced to the 5' terminus of hepatitis B viru score HBcAg gene. The modified HBcAg gene was used to construct a DNA vaccine. After the resulted DNA vaccine construct was transfected into COS7 cells, secreted HBcAg was detected in the supernatant by ELISA. BALB/c mice were vaccinated intramuscularly with the modified HBcAg DNA vaccine and the wild-type one. Serum antibodies,T lymphocyte proliferative response and cytotoxic T lymphocyte response of the immunized mice were measured. The results showed that the modified DNA construct induced cellular and humoral immune responses much stronger in vivo than the natural one did, indicating the potential value as a therapeutic vaccine for treatment of chronic hepatitis B.

Retinoic acid causes MEK-dependent RAF phosphorylation through RARalpha plus RXR activation in HL-60 cells.

Retinoic acid (RA) is known to cause the myeloid differentiation of HL-60 human myeloblastic leukemia cells in a process requiring MEK-dependent ERK2 activation. This RA-induced ERK2 activation appears after approximately 4 h and persists until the cells are differentiated and G0 arrested (Yen et al, 1998). This motivates the question of whether RA also activated RAF as part of a typical RAF/MEK/MAPK cascade. Retinoic acid is shown here to also increase the phosphorylation of RAF, but in an unusual way. Surprisingly, increased RAF phosphorylation is first detectable after 12 to 24 hours by phosphorylation-induced retardation of polyacrylamide gel electrophoretic mobility. The RA-induced increased RAF phosphorylation is still apparent after 72 hours of treatment when most cells are differentiated and G0 arrested. There is a progressive dose-response relationship with 10(-8), 10(-7), and 10(-6) M RA. The RA-induced RAF phosphorylation corresponds to increased in vitro kinase activity. Inhibition of MEK with a PD98059 dose which inhibits ERK2 phosphorylation and subsequent cell differentiation also inhibits RAF phosphorylation. RA-induced MEK-dependent RAF phosphorylation is not due to changes in the amount of cellular MEK. The induced RAF phosphorylation, as well as anteceding ERK2 activation, depends on ligand-induced activation of both an RARalpha receptor and an RXR receptor. This and the slow kinetics of activation suggest a need for prior RA-induced gene expression. In summary, RA induces a MEK-dependent prolonged RAF activation, whose slow onset occurs after ERK2 activation but still well before cell cycle arrest and cell differentiation. The RA-induced increased RAF phosphorylation thus differs from typical mitogenic growth factor signaling, features that may contribute to cell cycle arrest and differentiation instead of division as the cellular outcome.

Direct Blue 71 staining of proteins bound to blotting membranes.

A sensitive staining method for protein blots using Direct Blue 71 is described. It is based on the selective binding of dye molecules to proteins in acidic solution and produces bluish violet colored bands. It is a simple and rapid procedure, involving only staining and rinsing steps that occur within 7 min. The sensitivity of this method is 5-10 ng of protein on nitrocellulose (NC) and 10-20 ng on polyvinylidene difluoride (PVDF), which is tenfold better than that of the commonly used Ponceau S staining. Moreover, the staining is reversible for subsequent immunostaining, without impairing immunoreactivity. To remove the dye from the developed bands, changes in pH and hydrophobicity of the solvent are required. Due to its sensitivity, rapidity, simplicity, and low cost, this stain may be more practical than other dye-based stains or metal-based stains for routine laboratory purposes.

A modified Coomassie blue staining of proteins in polyacrylamide gels with Bismark brown R.

A rapid and sensitive protein staining method employing a mixed dye technique has been developed. Solutions of Coomassie brilliant blue R-250 (CB, 0.2%) and Bismark brown R (BBR, 0.05%) were mixed in the volume ratio of 1:0.75 for staining (final molar ratio, 4.5:1). In this staining, BBR inhibits the binding of CB to gel matrix by forming ion-pairing complex with excess CB; therefore, it reduces staining/destaining times and also enhances the staining effect of CB on protein band. As a result, the mixed dye staining enables complete staining/destaining within 20/25 min and detection of up to 25 ng of bovine serum albumin.

Detection of proteins on polyacrylamide gels using calconcarboxylic acid.

We describe here a protein staining method in polyacrylamide gels with a new staining dye, 1-(2-hydroxy-4-sulfo-1-naphthylazo)-2-hydroxy-3-naphthoic acid (calconcarboxylic acid, NN). This method can be performed by both simultaneous and postelectrophoretic staining techniques. Simultaneous staining using 0.01% of NN in upper reservoir buffer eliminates the poststaining step, and thus enables detection of the proteins more rapidly and simply. In poststaining, proteins can be stained by a 30-min incubation of a polyacrylamide gel in 40% methanol/7% acetic acid solution of 0.05% NN. These techniques produced protein staining patterns identical to the ones obtained by the conventional poststaining with Coomassie blue R-250 (CB). NN staining can detect as little as 10 ng of bovine serum albumin by poststaining and 25 ng by simultaneous staining, compared to 50 ng detectable by CB poststaining. In comparing the relationship between band intensity and amount of protein, NN staining gave better linearity than CB staining.

Recurrent pyogenic granulomas: a form of lobular capillary hemangioma.

Two cases of pyogenic granuloma recurred despite what was originally thought to be a sufficient surgical excision. The granulomas presented clinically as polypoid structures, one on the finger and one on the chin. On histologic examination, all showed polypoid configuration with lobules of small vessels and endothelial cell proliferation. The recurrent lesions also showed similar lobules, but interspersed between the collagen bundles in the deeper dermis. Vascular tumors such as the lobular pyogenic granulomas, intravenous capillary hemangiomas, and acquired tufted angiomas were compared to our tumors.

[Comparison of chemical constituents between new and old processed products of rhizoma Typhonii].

Experimental results showed that the original contents of chemical constituents in the new processed products were higher than those in the old one, while the content of assistant alum in the new products was lower than that of the old one. It is thus confirmed that the new processing technology is reasonable.

Segmental absence of small intestinal musculature.

Three instances of segmental absence of small intestinal musculature are described. Based on their clinical and pathological features and on the 12 cases previously described in the English literature, these can be classified into two groups: primary and secondary. In the primary group, the etiology is unknown, the onset of symptoms is acute, and there are no pathologic findings in the remaining layers of the small bowel except for superimposed perforation, or intussusception. In the secondary group, there is a longer history of intestinal symptoms and of multiple surgical procedures. Histologically, there may be ischemic necrosis of remaining layers, fibrosis, calcification, chronic inflammation, and presence of macrophages. These findings indicate secondary destruction of muscle layers due to ischemia and/or infarction, interruption of the blood supply, or trauma.

Female urethral diverticular with clear cell adenocarcinoma.

Clear cell adenocarcinoma in a urethral diverticulum in the female is a very rare and unusual tumor. We recently treated two patients with this tumor. Their presentation, histologic evaluation, and management are reviewed in light of the limited experience in the literature.

Periventricular leukomalacia in infancy: ultrasonic diagnosis and neurological outcome.

During the more than five years from January 1984 to June 1989, twenty-four patients with definite or probable cystic periventricular leukomalacia (PVL) were diagnosed by cranial ultrasonography at Mackay Memorial Hospital. The 24 patients were divided into two groups. Group A comprise two boys and four girls who received longitudinal sonographic follow-ups for leukomalacia. Of these six patients, five were premature and all suffered from severe perinatal insults. In each case, sequences of developmental cystic PVL were observed by serially scanning the brain. High echogenicity was discovered during the initial stages (2 to 7 days) in the periventricular area, and cystic formations were observed between the age of 18 and 60 days. Clinically, only one patient developed normally; four had severe motor dysfunction and poor motor development; and one was lost during follow-up, Group B was composed of 18 patients who visited the out-patient clinic for psychomotor retardation evaluation, and were found through ultrasound to have or possibly have cystic PVL formations at various stages. The clinical work-up revealed that 12 had spastic quadriplegia; 2 had hemiplegia; 3 had spastic displegia; and 1 case had hypotonic cerebral palsy. In infants, PVL is considered to be a much more reliable and important prognostic predictor than intraventricular hemorrhage. Consequently, it is crucial that physicians should screen patients at high risk for PVL, especially those with perinatal insults.

Pulmonary plasma cell granuloma (inflammatory pseudotumor) with invasion of thoracic vertebra.

Plasma cell granulomas (inflammatory pseudotumors) frequently appear as localized benign tumors of the lung in patients under 30 years of age. We describe the case of a 54-year-old woman with a plasma cell granuloma originating in the left lower lobe of lung and extending into the posterior mediastinum with destruction of the T8 vertebral body and pedicle. Distinctive histologic features included granulomatous aggregation of mature plasma cells around small blood vessels and direct invasion of the posterior mediastinum with subsequent dense fibrosis and bony destruction. Immunohistochemical studies revealed polyclonal kappa- and lambda-light chains in the proliferation of plasma cells.