Effect of a Congolese herbal medicine used in sickle cell anemia on the expression of plasminogen activators in human coronary aortic endothelial cells culture

Ethnopharmacological relevance

Aqueous extract of Ceiba pentandra, which is used for the management of sickle cell anemia (SCA) in DR Congo, exhibits antithrombin response by activation of Heparin cofactor II in vitro. This study examines the effect of the plant on the fibrinolytic activity to understand whether it can influence the coagulation–fibrinolysis system, since fibrinolysis disorder is one of the contributing causes of thrombotic crises in SCA.

Materials and methods

Fibrinolysis proteins were determined by enzyme-immunoassay in the conditioned medium of cultured endothelial cells after treatment with the extract. Electrophoresis-zymography and RT-PCR tests were conducted to examine the activity and the RNA synthesis of these proteins, respectively.

Results

It was found that the extract decreased the activity of both tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type-1 (PAI-1). However, it was revealed that this effect was not the result of an inhibition of their biosynthesis by endothelial cells.

Conclusion

From the foregoing, it was revealed that the extract inhibited the secretion of the fibrinolytic proteins without affecting their synthesis by endothelial cells. Thus, the extract may not accelerate the digestion of fibrin clot resulting from thrombotic disorders in SCA.     

Decreased expression of BTG3 was linked to carcinogenesis, aggressiveness, and prognosis of ovarian carcinoma

B-cell translocation gene 3 (BTG3) is a member of the BTG family which inhibits cell proliferation, metastasis, and angiogenesis, and also regulates cell-cycle progression and differentiation in a variety of cell types. However, there is no study to analyze BTG3 expression in epithelial ovarian carcinoma (EOC). Here, we investigated the expression of BTG3 in EOC carcinogenesis and subsequent progression. BTG3 mRNA expression was detected by real-time RT–PCR in ovarian benign and malignant tumors. The expression of BTG3 protein was examined by immunohistochemistry on tissue microarrays containing ovarian normal tissue, benign and borderline epithelial ovarian tumors, and EOCs. Relationships of BTG3 with both EOC clinicopathology and prognosis were analyzed statistically. The expression of BTG3 protein was also evaluated in ovarian normal tissue, benign tumors, and EOCs by western blot. The BTG3 mRNA expression level was higher in ovarian normal tissue and benign tumors than that in borderline, primary, and metastatic carcinoma (p?<?0.05), and was negatively correlated with dedifferentiation and FIGO staging of EOC (p?<?0.05). Using western blot, BTG3 protein was found lower in EOCs compared to the normal and benign tumors (p?<?0.05), and poorly differentiated EOCs showed lower BTG3 expression than well-differentiated and moderately differentiated EOCs (p?<?0.05). Immunohistochemically, BTG3 protein expression was statistically lower in EOCs than normal tissue and benign tumors (p?<?0.05). EOC patients with low BTG3 protein expression showed a higher incidence of metastasis (p?=?0.020), poor differentiation (p?=?0.030), and shorter disease-free time and overall survival time (p?<?0.05). By using Cox’s proportional hazard model, BTG3 protein expression and FIGO staging were independent prognostic factors for both disease-free time and overall survival time of EOCs (p?<?0.05). It was suggested that down-regulated BTG3 expression might play roles in the pathogenesis and aggressiveness of EOC. BTG3 protein expression may be considered as a good marker to indicate the favorable prognosis of EOCs.     

Cyclic changes in the properties of maturing enamel in the bovine permanent incisor as revealed by glyoxal bis(2-hydroxyanil) staining

Staining patterns of the surface and interior of maturing enamel of these tooth germs were examined using the glyoxal bis(2-hydroxyanil) (GBHA) solution which chelates with calcium loosely bound to hydroxyapatite to form insoluble red precipitates. An intense red, band-like pattern of GBHA staining, 1-2 mm wide, first appeared on the incisal portion of lingual enamel surface as complete loops, concentrically arranged; these gradually increased in number. Most of the later-formed bands encircled the entire periphery of the maturing enamel surface. GBHA also revealed reactive areas on the labio-lingual, cut and ground surface of maturing enamel, corresponding exactly to the surface GBHA bands. GBHA did not stain EDTA-etched surface enamel, but did reveal regular staining patterns on the ground surface, disclosed after EDTA treatment. These observations suggest an intimate correlation between the properties of interior and surface enamel, at least with regard to the state of local calcium. The maturation of bovine incisor enamel may start at the lingual aspect of the tooth crown.     

A rapid diagnosis of anaerobic infection in the oro-maxillary region by gas-liquid chromatography

The relationship between volatile fatty acids (VFAs) in pus and infecting bacterial species was examined in order to establish a rapid identification system for anaerobic microorganisms in purulent inflammation in the oro-maxillary region. VFAs were detected by the direct injection of pus into gas-liquid chromatography (GLC). Bacterial examination was carried out by anaerobic culture using blood agar plates. The bacterial identification was carried out mainly according to the VPI manual. Analysis of the direct VFA patterns of each sample resulted in 5 groups. The following bacterial species were the main isolates in each group: Streptococcus intermedius in Group A, Peptostreptococcus micros in Group B, Fusobacterium nucleatum in Group C, Bacteroides gingivalis in Group D, and Peptostreptococcus anaerobius in Group E. The profile of VFAs produced in the PYG culture medium of the above isolated bacteria was compared with the direct VFA patterns. Agreement ratios between direct and PYG VFA patterns were as follows: Group A, 47.1%; Groups B and C, 45.0%; Group D, 87.5%; and Group E, 62.9%. The acetic acid concentration was more than 14 x 10(-4) meq/ml in Group B, the butyric acid concentration was more than 7 x 10(-4) meq/ml in Group C, and the iso-caproic acid concentration was more than 14 x 10(-4) meq/ml in Group E. In these cases, it was found that the agreement ratios between the direct and PYG FVA pattern were high. In Group D, irrespective of the concentration of iso-valeric acid detected, the agreement ratio was very high. The antibiotic susceptibility of the isolates was studied. Efficiency rates of ABPC, PIPC, CCL, CEZ, CMZ, SBT/CPZ, JM, CLDM, MINO and GM were relatively low and resistant rates were high for the gram-negative rods.     

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.     

Physiologically Based Absorption Modeling to Explore the Impact of Food and Gastric pH Changes on the Pharmacokinetics of Alectinib

Alectinib, a lipophilic, basic, anaplastic lymphoma kinase (ALK) inhibitor with very low aqueous solubility, has received Food and Drug Administration-accelerated approval for the treatment of patients with ALK+ non-small-cell lung cancer. This paper describes the application of physiologically based absorption modeling during clinical development to predict and understand the impact of food and gastric pH changes on alectinib absorption. The GastroPlus^? software was used to develop an absorption model integrating in vitro and in silico data on drug substance properties. Oral pharmacokinetics was simulated by linking the absorption model to a disposition model fit to pharmacokinetic data obtained after an intravenous infusion. Simulations were compared to clinical data from a food effect study and a drug-drug interaction study with esomeprazole, a gastric acid-reducing agent. Prospective predictions of a positive food effect and negligible impact of gastric pH elevation were confirmed with clinical data, although the exact magnitude of the food effect could not be predicted with confidence. After optimization of the absorption model with clinical food effect data, a refined model was further applied to derive recommendations on the timing of dose administration with respect to a meal. The application of biopharmaceutical absorption modeling is an area with great potential to further streamline late stage drug development and with impact on regulatory questions.     

Staphylococcal scalded skin syndrome in an extremely low‐birth‐weight neonate: Molecular characterization and rapid detection by multiplex and real‐time PCR of methicillin‐resistant Staphylococcus aureus

Background: Staphylococcal scalded skin syndrome (SSSS), caused by methicillin‐resistant Staphylococcus aureus (MRSA) producing exfoliative toxin (ET), is a life‐threatening infection for neonates in neonatal intensive care units (NICUs). SSSS in extremely low‐birth‐weight (ELBW) neonates is rare. A new class of MRSA (community‐acquired MRSA, CA‐MRSA) has been emerging in the community. The aim of this study was to characterize MRSA from an ELBW neonate with SSSS, and to develop rapid detection methods for SSSS‐associated and emerging pediatric MRSA. Methods: An ELBW infant in the NICU developed SSSS on day 16 after birth. Isolated MRSA was genetically characterized and compared with CA‐MRSA from bullous impetigo (biCA‐MRSA), which is positive for the ET and collagen‐adhesin (CNA) genes in many cases, and the Panton‐Valentine leucocidin (PVL) gene rarely. Specific primers and probes for five virulence genes (for ETA, ETB, ETD, PVL, CNA) were designed for multiplex polymerase chain reaction (PCR) and real‐time PCR. Results: MRSA strain H5 from SSSS exhibited the genotype (ST91, spa416[t375], agr3, SCCmecIVa, CoaI), and possessed the ETB and CNA genes, similar to ST91 biCA‐MRSA (albeit with a divergence). Multiplex PCR detected the ETB and CNA genes of strain H5, and real‐time PCR detected strain H5 at as low as 10² CFU/mL. The assays were 100% specific and 100% sensitive, for the five virulence genes. Conclusion: ETB‐positive ST91 MRSA, which was very similar to ST91 biCA‐MRSA, was isolated from an ELBW infant with SSSS. The multiplex and real‐time PCR assays specifically or quantitatively detected SSSS‐associated and emerging pediatric MRSA.     

Successful unrelated umbilical cord blood cell transplantation without conditioning for a neonate with severe combined immunodeficiency

Taga T, Itoh E, Noda Y, Kato H, Maruo Y, Takano T, Ohta S, Takeuchi Y, Kumaki S. Successful unrelated umbilical cord blood cell transplantation without conditioning for a neonate with severe combined immunodeficiency.
Pediatr Transplantation 2011: 15: E152–E155. © 2010 John Wiley & Sons A/S. Abstract: A neonate was diagnosed as having SCID from his umbilical cord blood cells immediately after birth because his older brother had died of SCID eight months earlier. One locus‐mismatched unrelated umbilical cord blood cell transplantation without conditioning was performed at the age of 30 days. The CD3‐positive cells were detected on day 14 post‐transplantation. There were no peri‐transplantation complications. Four yr after transplantation, the boy is in excellent condition and T and NK cell engraftments are complete. His peripheral B cells with a common gamma chain were not detected by flow cytometry, and he still needs IgG replacement; however, his IgM and IgA levels have gradually increased, and the dosage of IVIG per body weight has gradually decreased. We speculate that the very few B cells that proliferate from transplanted cord blood cells produce gamma globulin. Unrelated cord blood cell transplantation, even though mismatched, without conditioning would be a treatment option for neonates with severe combined immunodeficiency.