Characterization of cortical neuron outgrowth in two- and three-dimensional culture systems

To improve the ability of regeneration by grafting living cells or by adding growth factor to a lesion site, it is important to find good biomaterials for neuron survival and regeneration. This study focused on two- and three-dimensional cultures in a matrix using biomaterials such as agarose, collagen, fibrin, and their mixtures, because these are considered to be suitable biomaterials for neuron outgrowth. Cortical neurons were dissected from E17 rat embryos and cultured in agarose gel, collagen gel, fibrin glue, and mixtures of collagen and fibrin. Results showed that neurons cultured in collagen gel and fibrin glue had longer periods of survival (more than 3 weeks) and better neurite extension than those observed in agarose gels. As to the survival rate according to the MTT and lactate dehydrogenase assays, fibrin glue was the most suitable biomaterial for neuron survival among the biomaterials examined. With two-dimensional fibrin plating, neuron cells exhibited cell aggregation and stress fibers, but the same results were not observed with collagen gel. There were no differences in neurite extension and survival in the mixtures of collagen and fibrin. The results suggest that collagen and fibrin can provide a suitable substrate for a three-dimensional culture matrix for neuronal survival and differentiation.     

Infected infradiaphragmatic retroperitoneal extralobar pulmonary sequestration: a case report

Infradiaphragmatic extralobar pulmonary sequestration is an extremely rare congenital malformation. It is more frequently diagnosed in the antenatal period due to routine ultrasonic examination of the fetus or in the first 6 months of life, though on rare occasions it is discovered incidentally in adults. A 32-yr-old man presenting with epigastric discomfort and fever was referred. Computed tomographic scanning showed that a 16-cm, multiseptated, dumbbell-shaped, huge cystic tumor was located beneath the diaphragm. On the next day, 850 mL of thick yellowish pus was drained by sonography-guided fine needle aspiration for the purpose of infection control and diagnosis, but no microscopic organisms were found in repeated culture studies. Surgical removal of the cyst was performed through thoracoabdominal incision and most of these pathologic lesions were removed but we could not find the feeding arteries or any fistulous tract to surrounding structures. Histopathologic study revealed that it was extralobar pulmonary sequestration and culture study showed that many WBC and necrotic materials were found but there were no microorganisms in the cystic contents. We report the first case of an infected infradiaphragmatic retroperitoneal extralobar sequestration which was administered a staged management and achieved an excellent clinical course.     

Rapid Cytomegalovirus pp65 Antigenemia Assay by Direct Erythrocyte Lysis and Immunofluorescence Staining

A rapid cytomegalovirus (CMV) pp65 antigenemia assay with direct erythrocyte lysis (DL) with 0.8% NH₄Cl, followed by indirect immunofluorescence staining (IF), was evaluated with 82 blood samples from renal transplant recipients, and the results were compared to those of the conventional antigenemia assay with dextran sedimentation and two-cycle alkaline phosphatase, anti-alkaline phosphatase staining (DS-APAAP). The DL-IF modification gave a higher leukocyte yield compared to DS-APAAP (75.4 versus 54.9%; P < 0.05), with similar leukocyte viability rates of >95%. The DL-IF methodology involved fewer technical steps, and the assay time was shortened from 5 h to less than 3 h. Nineteen of the 82 samples concordantly tested positive for pp65 antigenemia by both assays, and the readings showed a good correlation (r = 0.996; P < 0.01). No discordant results were observed. We conclude that the CMV pp65 antigenemia assay by this novel DL-IF modification is technically simpler, cheaper, and less time-consuming but yields results comparable to those of the conventional DS-APAAP assay. The shortened assay time and increased capacity to handle more samples confer distinct advantages in the rapid diagnosis and prompt treatment of CMV disease in immunosuppressed patients.     

Expression of cytokeratins 7 and 20 in primary carcinomas of the stomach and colorectum and their value in the differential diagnosis of metastatic carcinomas to the ovary

The expressions of cytokeratin (CK) 7 and 20 have been studied in various primary and metastatic carcinomas, and their determination may help distinguish the site of origin of metastatic carcinomas. However, little is known about the factors that determine variations in their expression patterns in primary gastric and colorectal carcinomas. We investigated the expressions of CK7 and CK20 in 289 cases of gastric carcinoma and 225 cases of colorectal carcinoma using a tissue microarray. To evaluate CK7 and CK20 expression patterns of ovarian metastases from gastric or colorectal carcinomas, 54 cases of metastatic carcinomas to the ovary were examined. It was found that 71% (207 of 289) of the gastric carcinomas stained positively for CK7, whereas only 9% (21 of 225) of the colorectal carcinomas proved to be CK7 positive, and that 41% (117 of 289) of the gastric carcinomas and 73% (165 of 225) of the colorectal carcinomas were CK20 positive. The proportion of CK7+/CK20- was highest in the gastric carcinomas at 46% (132 of 289), and was independent of the histologic classification of Lauren (46% of the intestinal type, 45% of the diffuse type). The CK7 and CK20 expression patterns were different in colorectal carcinomas according to histologic grade and location of the tumor. CK7-/CK20+ had the greatest proportion (68%) in colorectal carcinomas, and this was dependent on the tumor's histologic grade (75% of low-grade versus 52% of high-grade) and location (46% of right-sided versus 76% of left-sided). Moreover, 42% (18 of 43) of gastric carcinomas metastatic to the ovary were CK7+/CK20-, whereas 19% (8 of 43) were CK7-/CK20+. All colorectal cancers metastatic to the ovary were CK7-/CK20+, except 1 case that was CK7-/CK20-. In conclusion, the CK7 and CK20 expression patterns in primary gastric carcinomas vary considerably, and those in colorectal carcinomas are associated with histologic grade and tumor location. The CK7-/CK20+ expression pattern is specific for metastatic colorectal carcinomas to the ovary, but has low predictability for colorectal origin in metastatic ovarian carcinoma.     

Antifouling blood purification membrane composed of cellulose acetate and phospholipid polymer

The ideal surface of an artificial blood purification membrane needs hemocompatibility and durability of high performance; it should not adsorb any proteins or cells but should still have high permeability in the desired range of solute size. To improve the anti-fouling property of cellulose acetate (CA) membranes, a CA membrane blended with poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (PMB30) was designed as a blood purification membrane. The polymer solutions for preparing the membrane were prepared using a solvent mixture composed of N, N-dimethylformamide, acetone, 2-propanol or water. The CA and CA/PMB30 blend membranes with an asymmetric and porous structure were prepared by a phase inversion process.The characteristics of the CA/PMB30 blend membrane, such as structural properties, mechanical properties, and solute permeability were examined with attention to changes in the preparation conditions of the membrane. The CA/PMB30 blend membrane had good water and solute permeability and a sharp molecular weight cut-off property. Moreover, the amount of proteins adsorbed on the CA/PMB30 blend membrane surface was less than that of the original CA membrane and a conventional polysulfone membrane. Adhesion and activation of platelets on the CA/PMB30 blend membrane were reduced compared with that on a CA membrane. In addition, the CA/PMB30 blend membrane showed good permselectivity and an antifouling property during a long time ultrafiltration experiment with protein solutions.     

Plurihormonal pituitary adenomas: immunostaining of all pituitary hormones is mandatory for correct classification

AIMS: We studied the clinicopathological characteristics of plurihormonal pituitary adenomas. METHODS AND RESULTS: The study material included 167 plurihormonal adenomas, which consisted of 31% of the surgically removed pituitary adenomas that we collected during a 12-year period. The mean age of patients with plurihormonal adenoma was 45.7 years (range 13-75 years). There were 86 men and 81 women. All tumours were fully classified by immunohistochemical staining for seven pituitary hormones or subunits. Thirty immunohistochemical subtypes of plurihormonal adenomas were recognized. Hormonal symptoms were present in 70% of patients, while serum hormonal levels were increased in 89% of patients. Most patients had symptoms related to only one of the hormones and only 7% of patients had symptoms related to two hormones. The most common hormonal symptom was acromegaly (50%); symptoms related to hyperprolactinaemia ranked second (20%). Double immunostaining of all the possible combinations of the hormones was performed in 30 selected tumours, and they all showed mixtures of hormones in individual adenoma cells in any hormonal combinations studied. The latter finding supported the view that plurihormonal adenomas are monomorphous adenomas. CONCLUSIONS: Plurihormonal adenomas are common pituitary adenomas. Immunohistochemical staining of all pituitary hormones is mandatory for correct classification.     

Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.     

In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1(42) was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 microg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1(42) were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1(19) proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1(19). Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1(42). Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1(42) at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1(42) vaccine for human use.