Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium

The endometrium is a highly regenerative tissue that plays a crucial role in implantation. We examined the clonal constitution of glandular cells as well as the luminal epithelium of this unique tissue. Using collagenase-based digestion techniques with microscopic manipulation, we isolated individual human endometrial glands and examined their clonality using a polymerase chain reaction-based assay for nonrandom X chromosome inactivation with an X-linked androgen receptor gene. Most of the glands analyzed were composed of monoclonal populations of epithelial cells and one of the glands exhibited a loss of heterogeneity in the androgen receptor gene. In addition, adjacent glands within a 1-mm(2) area shared clonality, suggesting that clonality of the luminal epithelium is regionally defined. The clonality of endometrium was further confirmed in a study of female mice that harbor the green fluorescent protein gene on either the maternal or paternal X chromosome. Fluorescent microscopy of uterine sections revealed that individual endometrial glands consisted completely of either fluorescent or nonfluorescent cells and that the surface epithelium exhibited a clear boundary between these cell types. These findings suggest that single or multiple stem cells with uniform clonality exist on the bottom of each endometrial gland and genetic alterations occurring in such cells may play a critical role in endometrial carcinogenesis. The possible association between area-specific X inactivation of the endometrial surface and the endometrial receptivity of embryo implantation remains to be clarified.     

Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice

Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that plasma membrane cholesterol of macrophages is required for the VirB-dependent internalization of B. abortus and also contributes to the establishment of bacterial infection in mice. The internalization of B. abortus was accelerated by treating macrophages with acetylated low-density lipoprotein (acLDL). Treatment of acyl coenzyme A:cholesterol acyltransferase inhibitor, HL-004, to macrophages preloaded with acLDL accelerated the internalization of B. abortus. Ketoconazole, which inhibits cholesterol transport from lysosomes to the cell surface, inhibited the internalization and intracellular replication of B. abortus in macrophages. The Niemann-Pick C1 gene (NPC1), the gene for Niemann-Pick type C disease, characterized by an accumulation of cholesterol in most tissues, promoted B. abortus infection. NPC1-deficient mice were resistant to the bacterial infection. Molecules associated with cholesterol-rich microdomains, "lipid rafts," accumulate in intracellular vesicles of macrophages isolated from NPC1-deficient mice, and the macrophages yielded no intracellular replication of B. abortus. Thus, trafficking of cholesterol-associated microdomains controlled by NPC1 is critical for the establishment of B. abortus infection.     

Histopathological explanation of the MRI target sign in extra-axial schwannomas

BackgroundTo better understand the nature of magnetic resonance imaging (MRI) findings in schwannomas, especially in the “target sign” of these findings, the histopathological investigation was performed.MethodsThe MRI findings were correlated with histopathological features in 22 samples of schwannomas, which were mostly resected from the extremities. The histopathological analyses included alcian blue staining and immunohistochemical staining for S-100 protein, proliferating cell nuclear antigen (PCNA) and epithelial membrane antigen (EMA).ResultsSeven of the 22 samples of schwannomas of the extremities exhibited target signs including a peripheral zone of homogeneously high signal intensity and a central zone of heterogeneous signal intensity in T2-weighted images. Gadolinium-enhanced T1–weighted images demonstrated a central heterogeneous enhancement and a peripheral ring of homogeneously low signal intensity. Histopathologically, S-100 and PCNA were positive only in the central heterogeneous signal area. In contrast, EMA was only stained on the degenerative epi/perineurium in the peripheral zone.ConclusionIn schwannomas of the extremities showing target sign in T2-weighted images, histopathologically, the peripheral areas were suggested to be mucinous degeneration of the epineurium or perineurium, while the central areas were composed of truly neoplastic cells.     

Anaplastic large cell Ki-1 lymphoma with bone involvement: Report of two cases

Two cases of anaplastic large cell Ki-1 lymphoma involving bone as the most prominent and initial manifestation are reported. The first patient was a 20-year-old male who had back pain and incomplete paraparesis due to vertebral involvement. The second was a 14-year-old girl, whose first clinical signs were fever of unknown origin and sternal bone pain. Radiologically, skeletal lesions were lytic and destructive. Histopathologically, the tumour cells had pleomorphic bizarre nuclei and abundant basophilic cytoplasm. Immunohistochemically, Ki-1 (CD30) reactivity was strongly positive in both cases. Tumour cells were also CD3, CD4, epithelial membrane antigen and interleukin-2 receptor positive in the first case, and CD10, HLA-DR positive in the second case. The former tumour was considered to be of T-cell lineage and the latter of lymphoid progenitor cell origin. Radiation and chemotherapy were temporarily effective. However, both patients died 14 and 7 months after diagnosis, respectively, due to systemic lymph node involvement. These observations suggest that the prognosis for Ki-1 lymphoma involving bone is poorer than indicated in previous reports.     

Dynamic mechanical properties of dental amalgams

The dynamic mechanical properties of two high-copper amalgams (Tytin and Dispersalloy) and two traditional amalgams (Aristalloy and Aristalloy with Zn) were measured over a temperature range of 0-70 degrees C and at frequencies of 0.1, 1, and 10 Hz by means of a DuPont DMA. Values of storage modulus (E') for the amalgams were equivalent to the Young's modulus (E) measured from static mechanical test methods, with Dispersalloy demonstrating the highest moduli. Values of E' decreased with increased temperature. E' of traditional amalgams decreased more rapidly than did those of the Cu-rich amalgams. Values of loss modulus (E") for Tytin were smaller than those of Dispersalloy and the two types of Aristalloy. High values of E" for the traditional amalgams correspond to a greater viscous behavior. Marked differences between the magnitude of tan delta and its temperature coefficients for traditional and high-copper amalgams were observed, which is indicative of differences in visco-elastic behavior between these two amalgam systems.     

Prediction of hepatic graft viability before reperfusion: an analysis of effluent from porcine allografts

Rapid and reliable assessment of hepatic graft viability is important for successful orthotopic liver transplantation (OLTx). OLTx was performed in 11 pairs of pigs via a venovenous bypass. Six of these grafts were transplanted immediately (group A), while the other five were preserved in University of Wisconsin (UW) solution for 24 h and then transplanted (group B). All grafts were flushed with 300 ml of chilled (4°C) Ringer's lactate solution before reperfusion of the graft, when 20 ml of effluent from the graft was collected and the concentrations of ammonia, lactic acid, GOT, and LDH were measured. Four of the six pigs in group A survived longer than 3 days, while the other two pigs died of causes other than graft dysfunction. All five pigs in group B died either of hemoperitoneum or hemodynamic instability due to liver failure. The histology of postperfusion biopsies in group A showed minimal pathological changes, while the grafts in group B revealed moderate to severe ischemic injuries. Ammonia and lactic acid in the effluent of group B were significantly higher than those of group A (1511±216 vs 417±333 g/dl and 114.1±12.2 vs 91.4±12.2 mg/dl, respectively; P<0.05 in both cases). Before reperfusion, the rate of total adenine nucleotides in all of the substances in the graft, which were measured using high performance liquid chromatography (HPLC), inversely correlated with the ammonia levels in the effluent. We conclude that an analysis of the effluent, (i.e. the levels of ammonia and lactic acid), flushed from a hepatic graft before reperfusion could serve as a predictor of hepatic graft viability.     

Contribution of Monoamine Oxidase(MAO) to the Binding of Tertiary Basic Drugs in Isolated Perfused Rat Lung

The effect of tertiary basic drugs on mitochondrial MAO activity and the effect of MAO inhibitors (MAOIs) on basic drug accumulation in the isolated perfused rat lung were studied to clarify the role of MAO in drug binding to lung tissue. In the perfused lung preparation, the inhibition of MAO by basic drugs correlated well with their lipid solubilities and followed competitive kinetics. The inhibitory rank order (imipramine diphenhydramine > quinine > metoclopramide > procainamide) also correlated with their accumulation in the perfused lung. Moreover, MAOI treatment decreased the accumulation of basic drugs in the lung, and the potency of MAOIs to inhibit drug accumulation in the lung correlated with their MAO inhibitory activity. These results indicate that lung MAO has specific binding sites for basic drugs and may function as a drug reservoir.     

Preparation of the antibodies against recombinant human thymidylate synthase for the detection of its intratumoral levels and the application to sensitivity-study of 5-fluorouracil

Thymidylate synthase (TS) is a rate-limiting enzyme in de novo DNA biosynthesis and also a primary target for 5-fluorouracil (5-FU)-based chemotherapy. To estimate the con-elation between the expression of TS in the tumor, clinical response and prognosis in cancer patients treated with 5-FU, we have prepared recombinant human TS (rhTS) protein by culturing of E. coli transfected with the plasmid (pGEX-2TH) encoding the nucleotide sequence of hTScDNA and then obtained a highly purified polyclonal antibody against rhTS protein. Using this anti-TS antibody, it was revealed that the content of TS protein, as determined by Western blot analysis, correlated with the enzyme activity (gamma=0.973) and cytotoxicity of 5-FU, expressed as IC50 value (gamma=0.954) against human colon tumor cells, sensitive to and with acquired-resistance to 5-fluoropyrimidines and other cancer cells. On immunochemical evaluation, it was also confirmed that the tumor cells overexpressing TS proteins were strongly stained by the polyclonal antibody when compared to the cell lines expressing TS to lower extent both in vitro and in vivo conditions. These results indicate that this purified polyclonal antibody to rhTS is applicable to prospective and retrospective clinical studies on immunochemical TS expression in various tumors as a prognostic factor and 5-FU response-predicting parameter.     

A Simple and Rapid Fluorometric Determination Method of α1-Acid Glycoprotein in Serum Using Quinaldine Red

We examined different fluorescent probes suitable for fluorometric determination of ₁-acid glycoprotein (AGP) in serum. Quinaldine red (QR) was shown to bind strongly and selectively to AGP. Taking advantage of the enhanced fluorescence of QR in the presence of AGP, we developed a direct method for the determination of serum AGP without removal of other serum proteins such as albumin. AGP concentrations in serum of healthy volunteers and patients correlated well with results from the conventional single radial immunodiffusion (SRID) method (r = 0.93, slope = 1). The newly developed method is faster and has a larger analytical concentration range than the SRID method. This method can also be used to determine AGP in serum of experimental animals, and it can serve to monitor AGP serum concentrations for pharmacokinetic evaluation of basic drugs.