The development of an automated in situ assay for the detection of human cytomegalovirus in peripheral blood leukocytes.

Human cytomegalovirus (HCMV) infections are common in immunosuppressed patients, especially transplant recipients and patients with AIDS. The utility of an automated in situ hybridization (ISH) assay for the rapid detection of HCMV immediate early mRNA was evaluated using cytospin (Shandon Lipshaw, Inc., Pittsburgh, PA) prepared leukocytes from peripheral blood samples. In this study, the detection of HCMV immediate early protein by immunofluorescent antibody staining of the standard shell vial assay was compared to the detection of HCMV immediate early mRNA in peripheral blood leukocytes using the automated ISH system. Of 135 specimens tested, eight specimens were positive using HCMV ISH compared to seven positive specimens using shell vial assay. Overall, HCMV ISH demonstrated 100% sensitivity and 99% specificity. Since the HCMV ISH assay requires minimal labor, and can be completed in less than 5 h, this method should be evaluated as a potential replacement for shell vial assay for the diagnosis of HCMV infection.

Bcl-2 protein expression associated with resistance to apoptosis in clear cell adenocarcinomas of the vagina and cervix expressing wild-type p53.

BACKGROUND: Clear cell adenocarcinomas (CCAs) of the vagina and cervix are rare tumors that often overexpress wild-type p53. In vitro, expression of protooncogene bcl-2 can block p53-mediated apoptosis. The objective of this study was to determine if bcl-2 is expressed in CCAs and whether this expression is associated with inhibition of apoptosis. METHODS: Twenty-one paraffin-embedded clear cell adenocarcinomas were immunohistochemically stained for bcl-2 (antibody M 887, Dako, Carpinteria, CA) and DNA fragmentation (ApopTag, Oncor, Gaithersburg, MD), a marker for apoptosis. Fifteen tumors were associated with in utero exposure to diethylstilbestrol (DES). Prior p53 gene analysis had indicated the presence of wild-type p53 in each tumor. Human lymphoid tissue containing bcl-2-expressing lymphocytes and DNase I-exposed CCA tissue sections were used as positive controls for the bcl-2 and apoptosis assays, respectively. Expression of bcl-2 and DNA fragmentation was classified (0 to 3+) according to percentage of positive cells and intensity of staining. RESULTS: Expression of bcl-2 was identified in each CCA examined, and was strongly positive (2+ to 3+) in 18 of 21 samples. Despite the presence of wild-type p53, only 4 of 21 tumors showed evidence of apoptosis as assessed through DNA fragmentation. CONCLUSIONS: DNA damage leads to increased intracellular p53 levels. Overexpression of p53 induces apoptosis as a means of protecting organisms from the development of malignancy. CCAs of the vagina and cervix, which contain wild-type p53 genes and often overexpress p53 protein, presumably have evolved mechanisms to avoid p53-induced apoptosis. Our observations are consistent with the hypothesis that overexpression of bcl-2 can inhibit p53-mediated apoptosis and suggest a mechanism by which these rare tumors can arise without mutation of the p53 gene.

Lymphadenopathy, splenomegaly, and altered immunoglobulin production in BCL3 transgenic mice.

The candidate proto-oncogene BCL3 was isolated through its involvement in the t(14;19) found in chronic lymphocytic leukemia and other B-cell neoplasms. As a member of the I kappaB family, BCL3 plays a role in the immune response by interactions with the NF-kappaB family of transcription factors. In order to study the role of BCL3 overexpression in lymphoid malignancies, we generated five lines of E mu-BCL3 transgenic mice. Transgenic animals develop normally but show splenomegaly and an accumulation of mature B cells in lymph nodes, bone marrow and peritoneal cavity. A hyperresponsive immune system is suggested by the follicular hyperplasia and plasmacytosis in lymph nodes of unimmunized animals, increased incidence of antibodies to self-antigens, and a heightened response to cross-linking of surface IgM. Statistically significant decreases in serum IgM and IgG3, but an increase in IgG1 and IgA were also observed. No lymphoid neoplasms have been identified in transgenic animals. The expansion of B cells in vivo is consistent with the overexpression of BCL3 as being one step in the multi-step process of leukemogenesis. The phenotype also suggests that BCL3 plays a part in B cell proliferation and isotype switching.

Morphologic bone marrow changes in patients with posttransplantation lymphoproliferative disorders.

The clinical workup of patients with posttransplantation lymphoproliferative disorders (PTLPDs) frequently involves bone marrow biopsies. However, little is known about the morphologic bone marrow changes in patients with PTLPD. To define the spectrum of morphologic bone marrow changes in such patients, we evaluated the bone marrow biopsy samples of 26 transplant patients with proven extramedullary PTLPD and of 20 transplant patients without PTLPD. Morphologic changes were present in 14 of 26 patients with PTLPD (54%) and consisted of aggregates of lymphoid and plasma cells with variable histologic and cytologic features. Cells expressing Epstein-Barr virus-encoded small transcripts (EBER) were seen in 9 of 13 bone marrow biopsy samples with morphologic changes and in none of the biopsy samples without morphologic changes. Bone marrow changes were significantly more frequent in patients with PTLPD who were younger than 18 years of age (76%) compared with those who were older than 18 years of age (11%). The difference in mortality rates between the patient groups with and without bone marrow changes was statistically not significant, possibly because of the small sample size. The finding that children with PTLPD have an increased incidence of bone marrow changes supports the notion that Epstein-Barr virus-associated PTLPD involves different pathogenetic mechanisms in pediatric patients than in adults.

Expression of bcl-2 and bcl-X in bladder cancer.

PURPOSE: TP53 and RB1 gene mutations in bladder transitional cell carcinoma (TCC) are correlated with grade, stage, recurrence, and survival and may correlate with tumor cell apoptotic potential. Overexpression of the bcl-2 and bcl-X anti-apoptotic genes has been correlated with poor prognosis and chemotherapy resistance in other systems. Similar studies have not been performed in TCC. We thus sought to determine expression of bcl-2 and bcl-X in TCC and correlate these with stage, survival and abnormal pRb or p53 expression. MATERIALS AND METHODS: Forty-two TCC samples (19 Ta and 23 locally advanced tumors) and normal urothelial controls were examined. Immunohistochemistry for p53, pRb, bcl-2 and bcl-X was performed on an automated system using indirect streptavidin biotin/horseradish peroxidase staining. Western immunoblot analysis was performed on bladder cancer cell lines to further characterize bcl-X expression. Recurrence-free and disease-specific survival were retrospectively determined. Kaplan-Meier survival curves were compared using the log rank test, and correlation of abnormal staining with stage and p53 or pRb status was determined using Fisher's exact test. RESULTS: Bcl-2 was expressed in less than 1% of normal urothelial cells, but moderate expression of bcl-x was found in all normal urothelial samples. Only 7.0% of TCC samples (1/19 Ta and 2/23 locally advanced tumors) demonstrated bcl-2 overexpression. Bcl-X overexpression was observed in 45.2% of TCC (8/19 Ta and 11/23 locally advanced tumors). Western blot analysis also revealed that both the long (29 kDa) anti-apoptotic form and short (19 kDa) pro-apoptotic form were overexpressed in bladder cancer cell lines and normal human urothelial cells. Bcl-X overexpression was weakly correlated with normal p53 expression (p = 0.06). There were no correlations of bcl-2 and bcl-X overexpression with abnormal p53, pRb, or tumor stage. There were no differences in recurrence-free or overall survival in patients with abnormal bcl-X staining. CONCLUSIONS: Bcl-2 overexpression is rare in TCC. Bcl-X overexpression is common, likely reflecting its expression pattern in normal urothelium, but is not correlated with stage or abnormal p53 or pRb staining. Within the power limitations of this small study, bcl-X overexpression is not correlated with recurrence or survival.

Role of CD44 in nonpalpable T1a and T1b breast cancer.

Primary infiltrating ductal carcinomas (IDCs) of the breast which measure less than 0.5 cm (T1a lesions) and between 0.5 and 1.0 cm (T1b lesions) are associated with a small risk of nodal metastasis. The role of axillary dissection in T1a and T1b breast cancer is controversial. In the absence of axillary dissection, comparable prognostic information might be obtained by examination of the primary cancer. The adhesion molecule CD44 represents a family of transmembrane proteins that mediate cell-cell and cell-matrix interactions. Previous investigators have correlated expression of CD44 and its isoforms with prognosis in breast cancer. We investigated the value of CD44 isoform expression as a predictor of nodal metastases in nonpalpable T1a and T1b IDC. Monoclonal antibody against the standard form of CD44 (CD44s) and polyclonal antibody directed against the variant isoform (CD44v6) was tested on 34 cases of nonpalpable node-negative infiltrating ductal carcinoma (IDC) less than 1.0 cm and 9 cases of nonpalpable node-positive IDC less than 1.0 cm. The expression of CD44s was significantly decreased in node-positive T1a and T1b IDC versus node-negative T1a and T1b IDC (11% vs 65%). In contrast, 97% of the node-negative IDC and 100% of the node-positive IDC expressed the CD44v6 isoform. We conclude that CD44s expression is significantly altered in T1a and T1b IDC with nodal metastases but that the CD44v6 isoform does not correlate with nodal metastases in nonpalpable stage T1a and T1b IDC.

Gq-protein alpha subunit expression and distribution in pregnant rat myometrial tissues.

OBJECTIVE: Multiple G-protein isoforms play an integral role in signal transduction; the Gq subtype of G-protein alpha subunits is involved in the activation of the phosphatidylinositol signaling pathway. The studies described herein evaluate the expression of Gq, along with Gs and Gi, in pregnant and nonpregnant rat myometrial tissues. METHODS: Myometrium and other tissues were obtained from nonpregnant and timed-pregnant Sprague-Dawley rats. Western blot studies were performed using polyclonal G-protein isoform-specific antibodies. Immunohistochemical studies were performed using the same antibodies with specimens of myometrium, intestine, and skeletal muscle. RESULTS: The Western blot studies confirmed differential expression of all types of G-protein alpha subunit subtypes in rat myometrial tissues. In pregnant rat myometrium, the expression of Gq and Gs was sustained through day 22, whereas, Gi expression decreased on day 20 and remained low through the remainder of gestation. The immunohistochemical studies revealed significant staining for Gq and Gs in the myometrial layers of the pregnant and nonpregnant rat uterus; in contrast, immunostaining for Gi was minimal in nonpregnant myometrium, and even lower in myometrium from pregnant uteri. CONCLUSIONS: These studies have confirmed expression of the Gq, Gi, and Gs alpha subunits in rat myometrial tissue. Immunohistochemistry confirmed that Gq was expressed at high levels in the myometrial layer of the pregnant and nonpregnant uterus. These observations support the hypothesis that Gq expression is critically important for the transduction of hormone signals, such as those responsible for the generation of phasic myometrial contractions.

Characterization of the human apobec-1 gene: expression in gastrointestinal tissues determined by alternative splicing with production of a novel truncated peptide.

In humans, both the expression of apobec-1 and the C to U deamination of apoB mRNA are confined to the small intestine. In order to understand the tissue-restricted pattern of apobec-1 expression, we have isolated the chromosomal gene spanning the human apobec-1 locus. The human apobec-1 gene spans 18 kb and contains five exons, all of which are translated. Transcription initiation, determined by RNase protection and primer extension analyses, is localized to a single start site 34 nt upstream of the open-reading frame in exon 1. A common, but functionally silent, gene polymorphism was detected than changes Ilc80 to MCl. RNase protection and reverse-transcription PCR analysis demonstrated the presence of an exon 2-skipped form of apobec-1 mRNA that arises through use of an alternative splice acceptor. This alternative splicing causes a frame-shift that produces a novel, 36 amino acid peptide. The exon 2-skipped form accounts for approximately 50% of apobec-1 mRNA in the adult small intestine and up to 90% of apobec-1 mRNA in the developing gut. An antipeptide antibody identified the truncated protein in villus cells of the adult small intestine. These data suggest that exon 2-skipping may represent an important control mechanism regulating apobec-1 gene expression in humans.

The clinical significance of extracellular matrix in gangliogliomas.

Based on in vitro studies which demonstrate that collagen IV and laminin inhibit the proliferation and invasiveness of glioma cells, we investigated the clinical significance of these extracellular matrix proteins (ECM) in patients with gangliogliomas, tumors in which ECM is often a prominent feature. Our study compared the relative presence and deposition pattern of collagen IV and laminin in 19 gangliogliomas and in 18 gliomas without ganglion cell differentiation (8 low-grade astrocytomas, 7 anaplastic astrocytomas, and 3 anaplastic mixed gliomas). We also examined whether the presence of collagen IV and laminin correlated with other features often observed in gangliogliomas, including perivascular lymphocytic inflammation, granular bodies, microcalcification, and subarachnoid extension, and whether any of these features were associated with the patient's clinical course. Significant deposition of collagen IV and laminin was found in 9 gangliogliomas (47%), but in none of the other gliomas. The presence of these extracellular proteins in gangliogliomas correlated with both perivascular inflammation (P = 0.003), and involvement of the leptomeninges by tumor (P = 0.008). The duration of symptoms prior to surgical resection was significantly longer for patients whose tumors showed extracellular deposition of collagen IV and laminin than for patients whose tumors lacked deposition of these proteins (mean 13.7 vs 5.1 years; P = 0.02). In addition, the duration of symptoms was significantly longer for patients whose tumors exhibited perivascular inflammation than for patients whose tumors displayed little or no perivascular inflammation (mean 14.8 vs 4.8 years; P = 0.01). These findings suggests that collagen IV and laminin and perivascular inflammation are related to the indolent behavior of gangliogliomas.

The role of p53 mutations in bilateral breast carcinoma.

Mutations of the tumor suppressor gene p53 have been implicated in certain familial cases of breast cancer. We examined a series of 38 cases of nonfamilial bilateral breast cancer using antibodies CM1 and DO7 to p53 wild-type and mutant protein (Novocastra Laboratories) by the avidin-biotin-peroxidase complex method. The two antibodies reacted similarly. Mutant p53 protein was detected in 17 of 76 (22%) tumors but in only 3 of 38 (8%) paired tumors. There were no significant differences in p53 expression between synchronous (< 12 mos) and metachronous tumors (29% vs 17%, P = 0.09) or between first and second tumors (14% vs 26%, P = 0.29). Mutant p53 was detected bilaterally in one metachronous and two synchronous cases, which were amplified and sequenced and two synchronous cases, which were amplified and sequenced by polymerase chain reaction and single strand confirmation polymorphism. One synchronous case showed a bilateral mutation in exon 2-3; the other had a bilateral mutation in exon 8-9. In the metachronous case, a mutation could be demonstrated in only one breast. Analysis of all tumors demonstrated that when p53 protein is overexpressed in the first tumor, there is a 60% probability of overexpression in the second, whereas if absent from the first, it is unlikely to be present in the second. These data suggest that p53 mutations do not play a major role in the pathogenesis of bilateral disease in most women.

In situ dephosphorylation of p53 protein by calf intestinal alkaline phosphatase treatment.

The monoclonal antibody (mAb) 1801 has been reported to identify an N-terminal determinant within the tumor-suppressor protein p53 located between amino acids 32 and 79. This region contains two potential sites for serine phosphorylation at amino acids 33 and 46. Using a novel technique which dephosphorylates proteins in situ in fixed permeabilized cells, we have unmasked determinants in p53 recognized by mAb 1801, allowing additional sites of p53 protein to be detected in immunohistochemically reacted cells. This result indicates that phosphorylation at one or both sites within the determinant recognized by mAb 1801 previously blocked antibody-ligand interaction. It further suggests that in situ dephosphorylation may be of more general use in identifying antibodies which can only bind to epitopes in a particular phosphorylation state.

R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens.

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.

A temporal study of the expression of the capsid, cytoplasmic inclusion and nuclear inclusion proteins of tobacco etch potyvirus in infected plants.

Young leaves of tobacco, systemically infected by tobacco etch potyvirus (TEV), were examined for the presence and distribution of four virus encoded proteins [capsid, cytoplasmic inclusion (CI) and two nuclear inclusion (NI) proteins] at various time periods after inoculation of expanded leaves of the plants. The analyses were carried out by ELISA and by immunogold electron microscopy of thin sections of the leaves. All four proteins were detected simultaneously in the systemic leaves for the first time on the fifth day after inoculation of the expanded leaves. All four proteins increased in concentration until the seventh day and then showed no further increase with the exception of the capsid protein which continued to accumulate. The CI protein was first detected in association with the plasmalemma/cell wall and was subsequently found mostly in the form of pinwheels in the cytoplasm. The two NI proteins were found at all times after infection within the nucleus, although small concentrations were detected in the cytoplasm. These experiments suggest that both the NIa and NIb proteins are transported into the nucleus immediately after synthesis. At the earliest time periods after infection, high concentrations of these proteins (NIa and NIb) were found in their non-inclusion form in the nucleolus. At 14 days after infection, both proteins were found only as inclusions in the nucleus. The capsid protein was found at all stages of infection only in the cytoplasm.

Potato virus Y helper component protein is associated with amorphous inclusions.

The distribution of the helper component (HC) protein of potato virus Y in tissues and cells of infected plants was studied by immunoblotting and immunogold labelling techniques. This HC protein was found in leaf blade and vein tissue but not in the petiole of leaves. In infected cells, the protein was localized in rod-shaped cytoplasmic inclusions known as amorphous inclusions.