A specific enzyme-linked immunosorbent assay (ELISA) procedure for detection of heterophile Hanganutziu and Deicher (HD) antibodies

A specific, relatively sensitive, quantitative and standardized enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of heterophile Hanganutziu and Deicher (HD) antibodies which are occasionally elevated in pathologic human sera. The HD antigen-active molecule used for the assay was a ganglioside (N-glycolylneuraminyllactosylceramide, abbreviated as NeuGc-LacCer) previously purified from horse erythrocyte membranes. The test used antigen-coated plastic microtiter plates and anti-human immunoglobulin G (IgG, Fab fragment) conjugated with alkaline phosphatase. Fifty-four normal human sera gave ELISA values ranging from -2 to 2%. Random sera from hospitalized patients were first screened by the horse erythrocyte hemagglutination (HA) test, whereby 5.7% (76 cases) gave abnormal HA titers of 128-4096 compared to titers in normal sera equal to or less than 64. Ninety-seven % of the patients' sera gave abnormal ELISA values (3-200%). They were classified into 3 groups: cancer (42 cases), infection (10 cases), and others (24 cases). The potential value of this ELISA method is discussed.     

Autogenous injectable bone for regeneration with mesenchymal stem cells and platelet-rich plasma: tissue-engineered bone regeneration

We have attempted to regenerate bone in a significant osseous defect with minimal invasiveness and good plasticity, and to provide a clinical alternative to autogenous bone grafts. Platelet-rich plasma (PRP) may enhance the formation of new bone and is nontoxic, nonimmunoreactive, and accelerates existing wound-healing pathways. We have used a combination of PRP as an autologous scaffold with in vitro-expanded mesenchymal stem cells (MSCs) to increase osteogenesis, compared with using the scaffold alone or autogenous particulate cancellous bone and marrow (PCBM). The newly formed bones were evaluated by radiography, histology, and histomorphometric analysis in the defects at 2, 4, and 8 weeks. According to the histological observations, the dog MSCs (dMSCs)/PRP group had well-formed mature bone and neovascularization compared with the control (defect only), PRP, and PCBM groups at 2 and 4 weeks. Histometrically, at 8 weeks newly formed bone areas were 18.3 +/- 4.84% (control), 29.2 +/- 5.47% (PRP), 61.4 +/- 3.38% (PCBM), and 67.3 +/- 2.06% (dMSCs/PRP). There were significant differences between the PCBM, dMSCs/PRP, and control groups. These results demonstrate that the dMSCs/PRP mixture is useful as a osteogenic bone substitute.     

Survey of Hanganutziu and Deicher antibodies in operated patients

The appearance of Hanganutziu and Deicher (HD) antibody in the sera of patients suffering from various diseases, including malignancies of some organs and liver disorders, was investigated by enzyme-linked immunosorbent assay using N-glycolylneuraminyl-lactosylceramide (HD3) and 4-O-acetyl-HD3 as the antigenic molecules. More than 25% of sera from patients suffering from malignancies, cholelithiasis and liver cirrhosis had HD antibody, whereas none of 41 sera from healthy persons had HD antibody. The percentage of HD antibody-positive patients was similar in stages I, II and III of gastric cancer and recurrence cases. Antibody titers of the positive patients in each stage were also not different from those in each other stage. These results indicated that HD antigenic expression on cancerous tissue is not dependent on the cancerous malignancy. The HD antibody level was elevated after surgical removal of cancerous tissues in 5 of 6 patients examined, indicating that tumor growth absorbed the serum antibody. Serum antibody against 4-O-acetyl-HD3 was detected independently of HD3 antibody in some cases; however, in most cases, correlation between the two antibody titers was observed.     

Detection of 4-O-acetyl-N-glycolylneuraminyl lactosylceramide as one of tumor-associated antigens in human colon cancer tissues by specific antibody

Antibody to 4-O-acetyl-N-glycolylneuraminyl lactosylceramide [GM3(Neu4AcGc)] was prepared by immunizing chicken with the glycosphingolipid antigen. The specific antibody was purified by affinity chromatography columns of Octyl-Sepharose linked to the homologous immunogen and its deacetylated analogue [N-glycolylneuraminyl lactosylceramide, GM3(NeuGc)], respectively. The specificity of the purified antibody was confirmed by enzyme-linked immunosorbent assay (ELISA) and inhibition of equine erythrocyte hemagglutination using authentic glycosphingolipids as antigens. The results indicated that the antibody recognized both 4-O-acetyl and N-glycolyl groups of terminal sialic acid residue as the immunodeterminants. The purified specific antibody was applied in the confirmation of the presence of GM3(Neu4AcGc) in ganglioside fractions of human colon cancer tissues, which were suspected to have this antigen by studies of alkaline, periodate or neuraminidase treatment [Higashi et al. (1985) Cancer Res. 45, 3796-3802.], by thin-layer chromatography (TLC)-immunostaining technique.     

Murine thymic plasmacytoid dendritic cells

We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c(+)B220(+) subset expresses Ly6C(high) and MHC class II(low) in contrast with previously described thymic DC (CD11c(+)B220(-) cells). Freshly isolated thymic CD11c(+)B220(+) cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN-alpha following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL-12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN-producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.     

Comparison of merocyanine 540-mediated photodynamic action on leukemia cells between pulsed and continuous wave light sources.

Whether a pulsed laser is superior to a continuous wave (CW) light source in photodynamic therapy (PDT) of cancer is still unclear and contradictory in the literature. Although photosaturation of a sensitizer and oxygen depletion in tumor have been considered to be involved during pulsed laser irradiation, there is a lack of experimental data. In the present work several parameters such as the amount of merocyanine 540 (MC540) in cells, the oxygen concentration in cells, and the amount of photos reaching cells during pulsed laser irradiation, were studied to compare the MC540-mediated PDT effects of a pulsed laser and a CW light source on murine myeloid WEH-3B (JCS) cells in vitro. The results showed that the pulsed laser was less effective at cell inactivation than the CW light under the same irradiation dose. However, when the energy of the pulsed laser was reduced from 0.25 to 0.06 mJ/cm2 while keeping the total irradiation dose unchanged, the photoinactivation of cells was increased significantly. Based on the measurements and calculations for the present experimental conditions, each cell has about 108 MC540 molecules bound (5 microg/ml MC540 for 1 hr) and receives about 109 photos from 0.25 mJ/cm2 of the pulsed laser. The results indicate that the photosaturation of MC540 occurs in the present conditions due to the fact that the photons received by one cell in one laser pulse were much more than the numbers of MC540 molecules bound to one cell. Thus, the photosaturation of the photosensitizer is one of the reasons to explain the different efficiency in cell inactivation between the pulsed laser and CW light.     

Jaundice due to afferent loop obstruction following hepatectomy for a hilar cholangiocarcinoma

A case of jaundice due to obstruction of Roux en Y-limb following hepatectomy for a hilar cholangiocarcinoma is presented. Percutaneous transhepatic biliary drainage improved the jaundice but promoted disseminated intravascular coagulopathy. Our limited experience suggested that afferent loops should be drained directly to prevent reflux of enteric contents into the biliary system. Received: 21 February 1995/Accepted: 24 March 1995     

Adrenocorticotropic hormone and 17-hydroxyprogesterone levels during high-dose glucocorticoid supplement for the management of clitoroplasty of CYP21A2 deficiency

ACTH and 17-hydroxyprogesterone (17OHP) levels during clitoro- and labinoplasty for CYP21A2 deficiency have not been reported. In this study, we measured these levels during surgery. Intensive glucocorticoid supplement (IGS Tx; intravenous bolus injection of daily dose (14.6 to 22.2 mg/m(2)) of hydrocortisone followed by continuous infusion of three times the daily dose for 24 hours) was done to eight patients for surgery. ACTH and 17OHP levels were generally suppressed during operation by this protocol, but mid-surgical elevations of ACTH and 17OHP levels were observed in four out of the eight. In another three patients than the eight as described, oral dexamethasone pretreatment (1 mg/m(2)) was administered for two days before surgery in addition to IGS Tx. ACTH and 17OHP levels were completely suppressed throughout operation, indicating that this kind of additional pretreatment is more effective approach.     

Vitamin A exhibits potent antiamyloidogenic and fibril-destabilizing effects in vitro

Cerebral deposition of amyloid beta-peptide (Abeta) in the brain is an invariant feature of Alzheimer disease (AD). Plasma or cerebrospinal fluid concentrations of antioxidant vitamins and carotenoids, such as vitamins A, C, E, and beta-carotene, have been reported to be lower in AD patients, and these vitamins clinically have been demonstrated to slow the progression of dementia. In this study, we used fluorescence spectroscopy with thioflavin T (ThT) and electron microscopy to examine the effects of vitamin A (retinol, retinal, and retinoic acid), beta-carotene, and vitamins B2, B6, C, and E on the formation, extension, and destabilization of beta-amyloid fibrils (fAbeta) in vitro. Among them, vitamin A and beta-carotene dose-dependently inhibited formation of fAbeta from fresh Abeta, as well as their extension. Moreover, they dose-dependently destabilized preformed fAbetas. The overall activity of the molecules examined was in the order of retinol = retinal > beta-carotene > retinoic acid. Although the exact mechanisms are still unclear, vitamins A and beta-carotene could be key molecules for the prevention and therapy of AD.     

Involvement of TGF-beta signal for peritoneal sclerosing in continuous ambulatory peritoneal dialysis

BACKGROUND: Functional failure of the peritoneal membrane is the most serious problem in long-term continuous ambulatory peritoneal dialysis (CAPD). Transforming growth factor-beta (TGF-ss) is one of the key mediators of fibrosis in some organs, and is thought to be involved in peritoneal alterations. In this study, we examined the role of TGF-beta1/TGF-ss receptors for human peritoneal mesothelial cells (HPMCs) and fibroblasts, and their interactions in CAPD patients. METHODS: HPMCs were cultured for 48 h in a medium containing normal- dose glucose (7 mM), high-dose glucose (30 mM) and mannitol as an osmotic agent, equal to 30 mM glucose. Cell proliferation was observed using the Tetra Color One assay. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). The expression of TGF-ss receptor types I and II was observed by flow cytometry. HPMCs and fibroblasts were co-cultured and assayed using transwell inserts in order to identify the effects of the high-concentration glucose solution. RESULTS: HPMC proliferation was inhibited by the high concentration of glucose but not by mannitol. The inhibition was abrogated by the neutralizing antibody for TGF-beta1. TGF-beta1 was induced by a high concentration of glucose but not by mannitol. The expression of both TGF-ss receptors was augmented in culture with the high concentration of glucose but not with mannitol. In the co-culture assay, the number of HPMCs was decreased and fibroblasts were significantly increased in culture with the high concentration of glucose. CONCLUSIONS: A high concentration of glucose induced a large amount of TGF-beta1 and enhanced the expression of TGF-ss receptors. HPMCs were sensitive to TGF-beta1 in response to a high concentration of glucose. These data suggest that TGF-beta1 from HPMCs exposed to a high concentration of glucose down-regulates the proliferation of HPMCs and accelerates peritoneal fibrosis.