Double antibody enzyme immunoassay for the quantitation of adenosine 3', 5' -cyclic monophosphate (cyclic AMP) and guanosine 3', 5'-cyclic monophosphate (cyclic GMP) in tissue and plasma

A sensitive double antibody enzyme immunoassay for the quantitation of cyclic AMP and cyclic GMP is presented. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide-human serum albumin conjugates. For competitive reaction, antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with beta-D-galactosidase and unlabelled succinylated standard or sample cyclic nucleotides. The antibody-bound enzyme-hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. Activity of the enzyme on the solid phase was fluorometrically determined. The assay system made it possible to ascertain values as low as 5 fmole of cyclic AMP or cyclic GMP. Cyclic nucleotides in plasma could be accurately determined by this method without requiring a deproteinizing reagent as the first step of assay.     

Loss of LMP and TAP expression in human melanoma cells

The goal of this study was to determine the frequency of LMP2, LMP7, TAP1 and/or TAP2 loss in melanoma cell lines and in surgically removed melanoma lesions. Cell extracts from control and IFN-γ treated melanoma cell lines were tested in Western blotting with antisera elicited with LMP2, LMP7, TAP1 and TAP2-specific peptides. LMP2 and LMP7 were not detected in 7 out of 9 cell lines. Expression of both LMP subunits was induced in 5 of the 7 negative cell lines by treatment with IFN-γ. TAP1 and TAP2 were not detected in 3 out of 9 cell lines and were induced in one of them by IFN-γ. Surgically removed lesions were stained in the immunoperoxidase reaction with antibodies purified from the antisera by affinity chromatography on the immunizing peptides. LMP2 and LMP7 were not detected in 20% and 6%, respectively, of 32 primary lesions and in 40% and 12%, respectively, of 25 metastatic lesions. TAP1 and TAP2 were not detected in 15% and 18%, respectively, of 32 primary lesions and in 20% and 24%, respectively, of 25 metastatic lesions. Moreover, the frequency of TAP loss is increased in primary lesions from patients with recurrence of their disease, suggesting a potential role in the course of the disease and a negative impact on the outcome of T cell-based immunotherapy.     

C CELL CARCINOMA OF THE THYROID

Two cases of papillary type of C cell carcinoma of the thyroid were reported. They showed papillary arrangement with Abrovascular stalk in properly Axed tissues. Histochemically argyrophil reaction was positive in the cytoplasm and amyloid deposited in the stroma. Ultrastructurally secretory granules were found in their cytoplasm. The papillary type is not an artifact but one of the histologic variations of this carcinoma. ACTA PATH. SAP. 29: 653–659, 1979.     

Comparison of chemosensitivity tests: clonogenic assay versus MTT assay

When the development of chemotherapeutic agents reaches the clinical trial stage, it is necessary to perform drug sensitivity tests quickly in order to select the most promising agents for the treatment of cancer. In order to assess the possibility of using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a substitute for the human tumor clonogenic assay (HTCA), we evaluated the correlation between the results obtained by these 2 assays in 5 human lung cancer cell lines. The correlation coefficient between the results of the HTCA and the MTT assay was 0.673, indicating a relatively good correlation. The correlation was most prominent in platinum analogues (r = 0.939) and good in anthracyclines/anthracenedione (r = 0.611). However, no significant correlation was observed in vinca alkaloids, etoposide, irinotecan, SN-38 (an active metabolite of irinotecan), and rhizoxin. The results of the MTT assay showed a high degree of correlation with those of the HTCA in predicting the sensitivity of cancer cell lines to platinum analogues, and anthracyclines/anthracenedione. These results suggest that the MTT assay may be more convenient and quickly performed than the HTCA and can replace HTCA in evaluating the effects of anticancer agents, especially the platinum analogues and anthracyclines/anthracenedione.     

Effects of FK506 on rat thymic epithelial cells; immunohistochemical study.

The effects of FK506, a new immunosuppressive agent, on the rat thymus were investigated using the immunoperoxidase technique and flow cytofluorometry using monoclonal antibodies. Flow cytometric analysis of the thymus revealed that the proportion of cells labelled positively with OX7 (Thy-1 antigen), OX8 (CD8, T cytotoxic/suppressor cells) and W3/25 (CD4, T helper cells and macrophages) increased following treatment, with FK506, 1 mg/kg body weight for 14 days. A marked reduction of the thymic medulla following treatment was clearly demonstrated by staining with OX18 (MHC class I) and OX6 (MHC class II). Changes produced by FK506 were also observed in the cortical area of the thymus, being especially marked in the subcapsular area and around the blood vessels by staining with OX6, PKK-1 (alpha-cytokeratin), AB-1040 (type IV collagen), and AB-1220 (laminin). Eventually FK506 treatment resulted in patchy reduction of OX-6, PKK-1, AB-1040 and AB-1220 positive area in the cortex. This evidence suggests that FK506 may impair the thymic microenvironment and subsequently disturb the thymocyte maturation.     

Intraplacental choriocarcinoma with fetomaternal transfusion

Intraplacental choriocarcinoma is very rare, and is usually found only after maternal and fetal metastatic disease is identified. The purpose of this case report is to review the incidence and findings of intraplacental choriocarcinoma. A term placenta was investigated because the newborn was born with severe anemia (Hb 3.0 g/dL). A 2 cm nodule was noted on the surface of the amniotic membrane and grossly resembled an infarction. The tumor was examined microscopically with immunohistochemical staining for the alpha- and beta-human chorionic gonadotropin (alpha-hCG, beta-hCG) subunits, human placental lactogen (hPL) and Ki-67. Microscopically, the tumor consisted of necrotic areas with proliferation of atypical trophoblastic cells and destruction of the villi and capillaries. The cells were positive for the alpha-hCG, beta-hCG subunits, hPL and Ki-67, consistent with intraplacental choriocarcinoma. The mother and newborn were investigated for the presence of metastatic disease. Computed tomography scans and magnetic resonance imaging of the mother and infant were negative for metastatic disease. Choriocarcinoma, limited only to the placenta with no evidence of metastatic disease is very rare. Primary intraplacental choriocarcinoma may frequently be overlooked or missed, and choriocarcinoma may possibly arise in the placenta more often than in retained or persistent trophoblast following pregnancy.     

No evidence of PEG1/MEST gene mutations in Silver‐Russell syndrome patients

Silver‐Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (α and β) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST α coding region, and there were no significant mutations in the 5′‐flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST α were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS. © 2001 Wiley‐Liss, Inc.     

Hypoxia-ischemic insult in neonatal rats induced slowly progressive brain damage related to memory impairment

The present study was designed to determine potential associations between the brain damage induced by hypoxic-ischemic (HI) insult and spatial learning impairment in an eight-arm radial maze task. We first determined the pathological outcomes after 2, 5, 9, and 17 weeks of recovery following the HI insult. The results show that the brain damage progressed from 2 up to 17 weeks of recovery. To clarify the time course of the brain damage changes, we investigated the histological changes of the same individual with magnetic resonance imaging (MRI) after 5, 9, and 57 weeks of recovery following the HI insult. The MRI changes were similar to the histological changes, and the brain damages were exacerbated in the contralateral hemisphere after 57 weeks of recovery following the HI insult. To investigate whether alteration in brain function was correlated with MRI and histological changes, the rats were made to find their way through an eight-arm radial maze was performed at either 7th or 16th weeks of recovery. According to the results, the spatial learning impairments of rats in the maze starting at 16 weeks of recovery were more severe than those at 7 weeks of recovery, indicating that the impairments were progressive and depended on the degree of brain damage. The results of the present study are the first demonstration that the evolutional and specific brain damage following the HI insult is slowly and progressively exacerbated to the contralateral hemisphere and rats who experience the HI are at risk for showing a late impairment of brain function.     

Urinaryα 1 as an index of proximal tubular function in early infancy

Urinary ₁-microglobulin (U-A₁M) was measured in healthy term infants on days 1, 4, 7, 14, 28, 90 and 180 of life. U-A₁M was high until day 14 and declined thereafter. It was significantly correlated with urinary ₂-microglobulin (U-B₂M) throughout the study, but not with serum A₁M on days 1 or 7. Similar to U-B₂M, U-A₁M in the clinically stable term infants with intrauterine growth retardation (n=4–7) was not elevated on days 1–7. In the sick infants who needed immediate resuscitatio at birth (n=4–8), U-A₁M as well as U-B₂M was high on days 1–7 and then decreased to normal levels, suggesting that U-A₁M can be used as a sensitive marker of acute proximal tubular damage and its recovery. These observations indicate that U-A₁M is a useful index of proximal tubular function in early infancy.