Comparison of manual and digital microvascular density counting of RECK expression in glioma

A microvascular density (MVD) counting method for reversion‐inducing cysteine‐rich protein with Kazal motifs (RECK) expression, using a digital image analysis tool, has advantages over manual counting by microscope. Thirty glioma cases with RECK staining were photographed at a magnification of 200× high power field and the photographs in RGB images were analyzed, and stained vessels were captured and were counted automatically. MVD with RECK expression using a digital image analysis tool showed comparable results to those of the manual method. RECK intensity expression could show linear correlation with grades of glioma by the digital method, which was superior compared to the manual method. The present method is recommended to researchers undertaking MVD study for glioma.     

A prospective multicenter phase II study of intrabone marrow transplantation of unwashed cord blood using reduced‐intensity conditioning

Cord blood transplantation (CBT) is associated with delayed hematopoietic recovery and graft failure. To overcome these problems, we conducted a prospective, multicenter phase II study of intrabone marrow transplantation in which patients received reduced‐intensity conditioning without anti‐thymocyte globulin (ATG). The primary endpoint was the probability of full donor engraftment. Forty patients with hematologic malignancies were enrolled. Cord blood (CB) cells were injected without washing into 4 iliac bone sites (2 at each hemipelvis), at which approximately 6 mL of CB was administered at one site with local anesthesia. Full donor engraftment rate was 86.8%. The cumulative incidence of neutrophil and platelet engraftment was 86.4% and 85.5%, respectively. The median time to neutrophil (>0.5 × 10⁹/L) and platelet (2.0 × 10⁹/L) recovery was 17.5 and 44 days, respectively. The probability of severe acute graft‐vs‐host disease (GVHD) was 47.5%. The cumulative incidence of extensive chronic GVHD was 3.0%. The probability of relapse and non‐relapse mortality was 30.4% and 28.0%, respectively. The survival rate at 3 years was 45.6%, although most patients were at an advanced stage. These results suggest that our intrabone marrow‐CBT procedure without using ATG improves hematopoietic recovery and decreases the incidence of chronic GVHD, but does not decrease the incidence of acute GVHD.     

Utility of preoperative imaging diagnosis for a malignant tumor of the mandible: a malignant tumor of the mandible is difficult to discriminate from bisphosphonate-related osteonecrosis of the jaw

We report our experience of a case with a malignant tumor of the mandible in which diagnostic imaging played an important role in the differential diagnosis and therapeutic strategy decisions. The patient was a 78-year-old woman who visited our hospital because of poor healing after tooth extraction. Multiple cytological diagnoses provided class II results, and a histopathological diagnosis of a biopsy also failed to show malignant findings. Therefore, a definitive diagnosis could not be made. Although the patient had a history of osteoporosis treatment, details of her medications were unclear. Therefore, bisphosphonate-related osteonecrosis of the jaw (BRONJ) could not be excluded, causing difficulty in management of the patient’s condition. Eventually, we mainly focused on the diagnostic imaging and planned the therapeutic strategy in accordance with treatment for a malignant tumor. A postoperative histopathological examination of the surgical specimen revealed squamous cell carcinoma. It is sometimes difficult to differentiate among atypical diseases such as malignant tumors of the mandible and BRONJ, based solely on clinical or diagnostic imaging results. However, in the present patient, diagnostic imaging suggested a malignant tumor, and the appropriate treatment could be selected.     

Setting of the soft X-ray field for the calibration of the dosimeters used in mammographic field

In order to produce a cheap and stable X-ray generator system for calibration of dosimeters used in mammographic field, a dummy source of mammographic X-ray was developed using a tungsten (W) target X-ray tube and a molybdenum (Mo) filter. The photon fluence spectra of mammographic equipment were calculated using Birch's formula and aluminum (Al) attenuation curves were derived. The Al attenuation curves of X-rays from W target with Mo filter of various thicknesses were similarly obtained. Comparing the similarities of attenuation curves, the best fit Mo filter thickness was chosen. Consequently, a 0.04 mm thick Mo plus a 4 mm thick poly-methylmethacrylate filter were chosen to be added to W target industrial X-ray tube. The similarity of Al attenuation curves were verified by ionization chamber measurements.     

Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury

Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11-induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O(2) caused TUNEL(+) cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O(2). In A1-null mice, IL-11-induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11- and VEGF-induced cytoprotection.     

Use of sulfonylurea (SU) in the treatment of diabetes mellitus

The main action of SU in the treatment of diabetes mellitus is the stimulation of insulin secretion, and the extrapancreatic action including stimulation of insulin actions in glycogen synthesis and inhibition of glucose production in liver is also reported. The indication of SU in the treatment of diabetes mellitus is for NIDDM usually after diet therapy or suitable exercise therapy. In IDDM and several special cases including diabetic ketoacidosis, severe infection, pregnancy, poor-controlled NIDDM, gangrane, surgery operation, severe renal or hepatic failure et al. insulin therapy should be started. In mild NIDDM, gliclazide, tolbutamide or acetohexamide is used, and in more severe NIDDM glibenclamide is used. The action time of chlorpropamide is very long, usually from 20 to 60 hours, therefore special care should be taken for hypoglycemia. As the causes for secondary failure, transition from NIDDM to IDDM, failure of diet therapy, glucose toxity and others are considered.     

Prenatal diagnosis of persistent cloaca associated with VATER (vertebral defects, anal atresia, tracheo-esophageal fistula, and renal dysplasia)

The cloaca is a single canal from which the urinary, genital, and intestinal tracts arise around gestational weeks 5-6. Persistent cloaca can result from cystic mass formation within the pelvis, which is commonly association with multiple developmental defects. VATER association, which is a spectrum of anomalies, manifested by vertebral defects, anal atresia, tracheo-esophageal fistula with esophageal atresia, and renal dysplasia, arises from abnormalities in mesodermal differentiation. Recently, both conditions have been proposed to represent a continuous spectrum of anomalies, but the pathophysiology concerning the continuity of the development and the clinical condition are still unclear. Since renal failure becomes a serious problem after birth, timely infant delivery is essential to avoid loss of renal function. We report a patient, in whom the overlap between these two conditions was identified, and renal function was lost from one kidney. A polycystic mass was found in the fetal abdomen at 26 weeks of gestation. By ultrasonography, we detected a polycystic left kidney, a single umbilical artery, a ventricular septal defect, an esophageal atresia, ascites, an anal atresia, and a cystic mass with debris behind the bladder. The left kidney was non-functioning and the right kidney showed signs of hydronephrosis at 30 weeks of gestation. We measured the size and the blood flow of renal artery sequentially, and could deliver the fetus before the function was lost from the right kidney. Our observations will help inform future patients where prompt intervention can help improve renal function and infant health.     

Selection of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus by Imipenem

Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates are considered to have emerged from VAN-susceptible S. aureus (VSSA) by spontaneous mutation during VAN exposure. We previously reported that laboratory mutant H14, obtained from VSSA strain ΔIP by exposure to imipenem (IPM), showed overexpression of the vraSR two-component system and a typical hVISA phenotype. In the present study, to elucidate the mechanism of VSSA conversion to hVISA, we further characterized strain H14 by determining its whole-genome sequence, morphology, cell wall synthetic activity, and gene expression. Genome sequencing revealed that H14 harbored a mutated vraS (designated vraS_H14) that caused an amino acid substitution (S₃₂₉→L). This mutation is different from the VraS mutation (N₅→I) identified in representative clinical hVISA strain Mu3. However, H14 exhibited a phenotype similar to that of Mu3, including heterogeneous resistance to VAN, enhanced cell wall synthetic activity, and vraSR overexpression. Replacement of the vraS gene of ΔIP with the mutated vraS_H14 gene confirmed that the S₃₂₉→L substitution was responsible for both the upregulation of vraSR and conversion to the hVISA phenotype. This conversion was also achieved by using the vraS gene of Mu3, which carries a mutation (N₅→I), but not with the native vraS gene of strain N315. Finally, we carried out a study to analyze the appearance of hVISA from VSSA by exposure of ΔIP to selective concentrations of VAN and beta-lactam antibiotics. A total of 8 and 5 hVISA isolates were detected among 50 isolates selected with VAN and IPM, respectively. Among the 13 hVISA mutants, mutation in vraSR was detected only in mutant strain H14, suggesting that additional mutational mechanisms can be responsible for evolution to the hVISA phenotype. We conclude that exposure not only to VAN but also to beta-lactams may select for reduced glycopeptide susceptibility in S. aureus.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a major cause of serious nosocomial infections, and the emergence of virulent MRSA strains in the community is of particular concern (6). Vancomycin (VAN) still serves as the main therapeutic agent for infections caused by multiresistant MRSA strains (17). However, MRSA strains with various degrees of reduced susceptibility to glycopeptides, vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) strains, have emerged among multidrug-resistant MRSA clinical strains (9, 16).Recently, we identified several genes that are overexpressed in VISA strain Mu50 and hVISA Mu3 compared to their levels of expression in their isogenic VAN-susceptible S. aureus (VSSA) strain, strain Mu50Ω (13); and among these, we found the overexpression of the vraSR two-component system (TCS), an upregulator of the S. aureus cell wall biosynthesis pathway (12, 13). We also demonstrated that the vraS gene is overexpressed in ΔIP-H14 (H14), a laboratory-derived hVISA strain obtained by selecting VSSA strain N315ΔIP (ΔIP) with 8 mg/liter of imipenem (IPM) (12) and showed that a single amino acid substitution in VraS was present in H14, Mu3, and Mu50 (10a).In the study described here, we further characterized hVISA strain H14, investigated the role of the vraS mutation on the phenotype of H14, and evaluated the rates of selection of hVISA from VSSA ΔIP following exposure to VAN and beta-lactam antibiotics.