PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells

PADGEM protein (PADGEM), also known as GMP140, is a platelet alpha- granule membrane protein that is translocated to the external membrane after platelet activation. Although the biosynthesis of this protein was originally thought to be confined to megakaryocytes, the synthesis of PADGEM in endothelial cells was recently demonstrated (McEver et al: Blood 70:1974a, 1987). We now describe the subcellular localization of this protein in endothelial cells. Immunofluorescence staining of permeabilized human umbilical vein endothelial cells with KC4, a well characterized monoclonal antibody to PADGEM, showed positively stained elongated structures similar in distribution and shape to Weibel-Palade bodies. Their identity as Weibel-Palade bodies was confirmed by double label immunofluorescence using KC4 and a polyclonal antiserum to von Willebrand factor (vWf), a protein known to be specifically stored in these organelles. All Weibel-Palade bodies were found to contain PADGEM. In contrast to strong perinuclear staining produced with anti- vWf antibodies, no significant perinuclear staining was obtained with KC4, indicating that relatively little PADGEM is present in the endoplasmic reticulum and in the Golgi apparatus. In endothelial cells treated with secretagogues that stimulate vWf release the elongated structures positive for PADGEM disappeared, further identifying these structures as Weibel-Palade bodies. This observation extends the parallels between Weibel-Palade bodies and alpha-granules and suggests a possible functional association between vWf and PADGEM.     

Occurrence of the t(2;5)(p23;q35) in non-Hodgkin's lymphoma

Primary CD30(Ki-1)-positive anaplastic large-cell lymphoma (ALCL) is considered by some to be a distinct clinicopathologic entity associated with the t(2;5) (p23;q35). However, the specificity of t(2;5) for ALCL has not been carefully studied. Therefore, we performed a detailed analysis of all cases of ALCL with abnormal cytogenetics results in the Nebraska Lymphoma Study Group registry, as well as all other cases of non-Hodgkin's lymphoma with t(2;5) in the registry. We found the t(2;5) in only five of 10 cases of ALCL, four of whom were young patients. However, we also found the t(2;5) in 11 other cases of nonanaplastic lymphoma, including eight children with typical peripheral T-cell lymphomas of various types. The t(2;5) was also found in three older adults with B-cell lymphomas of various types. Thus, the t(2;5) was not specific for CD30+ ALCL. However, t(2;5) may define a clinicopathologic entity in children and young adults characterized by variable morphologies with a T-cell or indeterminate phenotype, CD30-positivity, nodal disease with frequent extranodal involvement, advanced stage, and an excellent response to therapy, including bone marrow transplantation for relapsed disease. The clinical relevance of the t(2;5) in older patients requires further study.     

Advanced primary breast cancer: assessment at mammography of response to induction chemotherapy

The response to induction chemotherapy is an important prognostic factor in patients with nonmetastatic, locally advanced breast carcinomas. Assessment at mammography of the response of 60 breast cancers in 59 women was performed between 1974 and 1986. Responses were excellent in 13 tumors, moderate in 34, and poor in 13 (excellent moderate = 78%). Assessment of response of discrete masses in a fatty breast was easiest; assessment of response of tumor areas that were poorly defined-such as a focal area of architectural distortion or mass in dense breast parenchyma-was more difficult. Of 17 patients with excellent pathologic responses-that is, minimal or no residual tumor-15 (88%) had complete responses (no residual tumor) as determined with mammography, physical examination, or both. Mammography provides information complementary to physical examination and is essential in the accurate assessment of the response to chemotherapy of locally advanced breast cancer.     

Lysosomal abnormalities in degenerating neurons link neuronal compromise to senile plaque development in Alzheimer disease

Antibodies to the lysosomal hydrolases, cathepsins B and D and β-hexosaminidase A, revealed alterations of the endosomal-lysosomal system in neurons of the Alzheimer disease brain, which preceded evident degenerative changes and became marked as atrophy, neurofibrillary pathology, or chromatolysis developed. At the earliest stages of cell atrophy, hydrolase-positive lysosomas accumulated at the basal pole and then massively throughout the perikarya and proximal dendrites of affected pyramidal neurons in Alzheimer prefrontal cortex and hippocampus, far exceeding the changes of normal aging. Secondary lysosomes as well as tertiary residual bodies (lysosomes/lipofuscin) increased implying stimulated, autophagocytosis and lysosomal system activation. Less affected brain regions, such as the thalamus, displayed similar though less extensive alterations. Certain thalamic neurons exhibited a distinctive lysosome-related abnormality characterized by the presence of cell surface blebs of varying size and number filled with intense hydrolase immunoreactivity. At more advanced stages of degeneration in still intact neurons, hydrolase-positive lipofuscin, particularly in the form of abnormal large aggregates, nearly filled the cytoplasm. Similar lipofuscin aggregates were oberved in abundance in the extracellular space following cell lysis and were usually associated with deposits of the β-amyloid protein. Degenerating neurons and their processes were the major source of these aggregates within senile plaques which contained high concentrations of acid hydrolases. We have shown in previous studies that these lysosomal hydrolases in plaques are enzymatically-active. The persistence of lysosomal structures in the brain parenchyma after neurons hyve degenerated is a striking and potentially diagnostic feature of Alzheimer disease which has not been observed, to our knowledge, in other degenerative diseases. The lysosomal response in degerating Alzheimer neurons represents a probable link between an early activation of the lysosomal system in at-risk, normal-appearing neurons and the end-stage contribution of lysosomes to senile plaque formation of emphasizes a slowly progressive disturbance of the lysosomal system throughout the development of Alzheimer disease.     

Immunolocalization of Fas and Fas ligand in the ovaries of women with polycystic ovary syndrome: relationship to apoptosis

Both Fas (APO-1, CD95), an apoptosis-inducing receptor, and its ligand, Fas ligand (FasL, CD95L), have been localized to the ovary. Granulosa cell apoptosis occurs in antral follicular atresia. In polycystic ovary syndrome (PCOS), antral follicles accumulate with some atretic features. The ovarian expression of Fas and FasL was examined in PCOS by immunohistochemistry and correlated with immunodetection of apoptotic cells. Fas immunostaining was present in pre-antral follicle oocytes, some primary and secondary pre-antral follicle granulosa cells, and both granulosa and theca of antral follicles. Thecal staining persisted with advancing atresia, while granulosa staining declined. In antral follicles, abundant Fas-positive cells co-localized with scattered nuclei immunopositive for apoptosis. Ovarian vascular myocytes were strongly Fas-immunopositive. FasL immunostaining was present in pre-antral follicles in oocytes and variably in granulosa. In antral follicles, granulosa and thecal FasL staining increased with advancing atresia. Normal control ovaries showed follicular Fas and FasL staining patterns similar to those in PCOS, but vascular staining was less prominent. In one healthy follicle, Fas immunostaining was seen in the oocyte and weakly in mural granulosa and theca interna. The results suggest that in PCOS, an alteration in Fas-mediated apoptosis, does not cause abnormal folliculogenesis, but may promote ovarian vascular remodelling.     

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations.

We used a colorimetric polymerase chain reaction (PCR)-based assay in kit form to detect directly human immunodeficiency virus type 1 (HIV-1) proviral gag sequences in peripheral blood cells from 68 healthy blood donors, 51 subjects at risk for HIV infection, 122 patients with HIV-1 infection, 11 patients with indeterminate Western blot (immunoblot) results, 4 blood donors HIV-1 positive by enzyme immunoassay, and 13 children born to HIV-1-seropositive mothers. The results obtained in the blood donors and HIV-1-infected patients demonstrated the high degree of diagnostic specificity and sensitivity of the PCR method. HIV-1 infection was excluded in 10 of the 11 patients with indeterminate Western blot results and in all four enzyme immunoassay-positive blood donors. A diagnosis of HIV infection was ruled out by negative PCR results in 5 of 13 children from seropositive mothers, which excluded vertical transmission of the infection in these cases; these children were younger than 3 months and had positive serological results. Two at-risk patients with negative serological results had positive PCR results. All results were confirmed by conventional PCR. In conclusion, colorimetric PCR, which is commercially available in kit form, is an easy and reliable technique that can be used to detect proviral HIV-1 genomes in blood cells, and despite the limitations owing to HIV genome variability, it is useful in the clinical setting for the diagnosis of HIV infection in selected categories of patients.