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.

Adenoviral-mediated gene transfer of a constitutively active form of the retinoblastoma gene product attenuates neointimal thickening in experimental vein grafts.

PURPOSE: Inappropriate or excessive vascular smooth muscle cell proliferation leads to the development of occlusive lesions in up to 50% of vein grafts. The purpose of this study was to test the hypothesis that induced overexpression of a cytostatic nonphosphorylatable form of the retinoblastoma protein (DeltaRb) would attenuate neointimal thickening in experimental vein grafts. METHODS: A replication-deficient adenovirus vector that encoded a nonphosphorylatable, constitutively active form of DeltaRb was constructed (AdDeltaRb) and contained an NH2-terminal epitope tag from the influenza hemagglutinin molecule (HA). Forty-eight male New Zealand white rabbits underwent surgical exposure of the external jugular vein for transfection with either 3 x 10(10) plaque-forming units/mL AdDeltaRb (n = 16), 3 x 10(10) plaque-forming units/mL control adenovirus (AdBglII, n = 15), or vehicle (n = 17) for 10 minutes at 120 mm Hg. After vector exposure, the vein was excised and interposed end-to-end into the carotid circulation. After 5 days, 12 grafts (four from each group) were excised and assayed for genomic DeltaRb DNA with the polymerase chain reaction or for hemagglutinin molecule expression and localization with immunohistochemistry. The remainder of the grafts (n = 36) were perfusion-fixed after 4 weeks, and 5 microm sections prepared for digital planimetric analysis. RESULTS: Polymerase chain reaction results identified the DeltaRb gene only in the grafts that were transfected with AdDeltaRb. Immunohistochemical analysis results revealed transgene expression in most of the endothelial cells and in many of the smooth muscle cells. After 4 weeks, the grafts that were exposed to AdDeltaRb exhibited a 22% reduction in neointimal thickness (vehicle, 77 +/- 7 microm; AdBglII, 75 +/- 5 microm; AdDeltaRb, 60 +/- 5 microm; P =.05), and medial thickness, luminal diameter, and other parameters were unchanged (medial thickness: vehicle, 72 +/- 10 microm; AdBglII, 85 +/- 7 microm; AdDeltaRb, 69 +/- 9 microm; P = NS; luminal diameter: vehicle, 4.5 +/- 0.2 mm; AdBglII, 4.4 +/- 0.2 mm; AdDeltaRb, 4.7 +/- 0.1 mm; P = NS). CONCLUSION: With this delivery system, adenoviral-mediated gene transfer is highly efficient and induced overexpression of DeltaRb leads to a reduction in vein graft neointimal thickening.

An alternatively spliced form of apobec-1 messenger RNA is overexpressed in human colon cancer.

BACKGROUND & AIMS: Apobec-1 is an RNA-specific cytidine deaminase whose forced overexpression in transgenic animals is associated with hepatic carcinogenesis. Apobec-1 messenger RNA (mRNA) undergoes alternative splicing, generating a catalytically inactive peptide, apobec-T. We have examined apobec-1 gene expression in human gastrointestinal tumors and in colon cancer-derived cell lines. METHODS: Levels of the full-length (apobec-1) and alternatively spliced (apobec-T) mRNAs were measured by RNase protection assay, and apobec-T distribution was determined by immunocytochemical localization. RESULTS: Apobec-1 mRNA was detectable in normal and colon cancer tissue, metastatic nodules, and certain colon cancer-derived cell lines. Apobec-T mRNA abundance was increased an average of 3.5-fold in colon cancers compared with paired control tissue (range, 0.5-14-fold). Immunocytochemical analysis showed apobec-T expression in normal fetal and adult colon and in gastric and small intestinal adenocarcinomas, colonic tubular adenomas, and both primary and metastatic colonic tumors. Overexpression of apobec-T in a tetracycline-responsive cell line decreased cellular proliferation. CONCLUSIONS: Apobec-T is expressed in normal, adenomatous, and cancerous gastrointestinal tissues, and levels of the mRNA encoding this peptide are significantly increased in colon cancer. Although its relationship to colon carcinogenesis has not been defined, the regulated overexpression of apobec-T is associated with an altered growth phenotype.

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.

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.

Genetic immunotherapy for medullary thyroid carcinoma: destruction of tumors in mice by in vivo delivery of adenoviral vector transducing the murine interleukin-2 gene.

A replication defective adenovirus harboring the interleukin-2 gene (AdCMVmIL2) was used for treatment of a mouse medullary thyroid carcinoma (mMTC). We evaluated the antitumor effect and immunological response in the animal model. In small tumors (< or = 30 mm3), intratumor injection of AdCMVmIL2 led to mMTC tumor regression in up to 69% of animals. With large tumors (>30 mm3), almost all treated tumors showed stabilization in size, but did not completely resolve. All mice cured by AdCMVmIL2 treatment failed to develop tumors after reinjection of wild-type mMTC cells, indicating that long-term antitumor immunity developed. Analysis of cytotoxicity indicates that the antitumor effect in cured mice was dependent on cytotoxic T lymphocyte (CTL) activity against the tumor. Histological and immunohistological studies of treated tumors revealed massive CD4+ and CD8+ cell infiltration in AdCMVmIL2 treated tumors, but not in untreated or control virus treated tumors. The data demonstrate the ability of interleukin 2 (IL-2) to elicit specific antitumor immunity and offer hope for this therapy in humans.

Cholecystokinin modulates mucosal immunoglobulin A function.

BACKGROUND: We have established that mucosal immunoglobulin A (IgA) production is highly dependent on cholecystokinin release and is markedly suppressed by glucocorticoids. The purpose of the present study was to examine the role of cholecystokinin on the functional responsiveness of the mucosal IgA system in glucocorticoid treated rats. METHODS: A total of 24 Fischer rats were assigned to three groups of 8 animals each. Animals were injected with vehicle (CON), dexamethasone (DEX) (0.08 mg/150 g), or DEX (0.08 mg/150 gm) and ARL1294KF (500 ng twice daily), a novel and potent long-acting cholecystokinin agonist (DEX+CCK). Animals were treated for 48 hours and killed. Duodenum was harvested, and the total mucosal concentration of cholecystokinin was measured by radioimmunoassay. Mucosal IgA was assayed by quantitation of immunoreactive cells in the ileum. Bacterial adherence was evaluated by quantitative culture of vigorously washed stripped cecal mucosa. Transepithelial electrical resistance, a measure of tight junction permeability, was assessed by mounting strips of adjacent cecal mucosa in Ussing chambers. RESULTS: Glucocorticoid administration resulted in a statistically significant (p < 0.001) decrease in duodenal cholecystokinin, decreased IgA, and impaired mucosal immunity (increased bacterial adherence and decreased tissue resistance). Cholecystokinin administration preserved mucosal immune function in DEX-treated rats. CONCLUSIONS: Cholecystokinin may play an important role in maintaining the functional responsiveness of mucosal immunity during catabolic stress.

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1.

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.

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.

Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT.

The arylhydrocarbon-receptor nuclear translocator (ARNT) is a member of the basic-helix-loop-helix-PAS family of heterodimeric transcription factors which includes the arylhydrocarbon receptor (AHR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the Drosophila single-minded protein (Sim). ARNT forms heterodimeric complexes with the arylhydrocarbon receptor, HIF-1alpha, Sim and the PAS protein Per. In response to environmental pollutants, AHR-ARNT heterodimers regulate genes involved in the metabolism of xenobiotics, whereas ARNT-HIF-1alpha heterodimers probably regulate those involved in the response to oxygen deprivation. By generating a targeted disruption of the Arnt locus in the mouse, we show here that Arnt-/- embryonic stem cells fail to activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond to a decrease in glucose concentration, indicating that ARNT is crucial in the response to hypoxia and to hypoglycaemia. Arnt-/- embryos were not viable past embryonic day 10.5 and showed defective angiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. The defect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reported for mice deficient in vascular endothelial growth factor or tissue factor. On the basis of these findings, we propose a model in which increasing tissue mass during organogenesis leads to the formation of hypoxic/nutrient-deprived cells, the subsequent activation of ARNT, and a concomitant increase in the expression of genes (including that encoding vascular endothelial growth factor) that promote vascularization of the developing yolk sac and solid tissues.

Defective B cell receptor-mediated responses in mice lacking the Ets protein, Spi-B.

Spi-B is a hematopoietic-specific Ets family transcription factor closely related to PU.1. Previous gene targeting experiments have shown that PU.1 is essential for the production of both lymphocytes and monocytes. We have now generated mice with a null mutation at the Spi-B locus. Unlike PU.1 mutant mice, Spi-B-/- mice are viable, fertile and possess mature B and T lymphocytes. However, Spi-B-/- mice exhibit severe abnormalities in B cell function and selective T cell-dependent humoral immune responses. First, although Spi-B-/- splenic B cells respond normally to lipopolysaccharide stimulation in vitro, these B cells proliferate poorly and die in response to B cell receptor (surface IgM) cross-linking. Secondly, Spi-B-/- mice display abnormal T-dependent antigenic responses in vivo and produce low levels of antigen-specific IgG1, IgG2a and IgG2b after immunization. Finally, Spi-B-/- mice show a dramatic defect in germinal center formation and maintenance. In contrast to wild-type animals, germinal centers in Spi-B-/- mice are smaller and short-lived with significantly increased numbers of apoptotic B cells. Taken together, these results demonstrate that Spi-B is essential for antigen-dependent expansion of B cells, T-dependent immune responses and maturation of normal germinal centers in vivo.

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.

MDM2 overexpression in benign and malignant lesions of the human breast.

MDM2 overexpression has been detected in women with benign or cancerous lesions of the breast. Immunohistochemical methods were used to identify overexpression in 11 of 27 benign cases (41%) and 15 of 22 (68%) cancer cases. MDM2 overexpression correlated well with immunohistochemically detected estrogen receptor (ER), suggesting that expression of these proteins was coordinately regulated. To test this, the MCF-10A human breast cell line, negative for both ER and MDM2 expression, was transfected with a wild-type ER. Following transfection, both ER and MDM2 were strongly expressed in two independent clones. Expression of both was reduced in the presence of increasing concentrations of estradiol (10(-10)-10(-8) M) thus, estrogen may be involved in regulation of MDM2 expression in proliferative breast lesions.

No mutations within conserved domain I of the p53 gene in medullary thyroid cancer.

Abnormal mobility shifts in single strand conformational polymorphism (SSCP)-analysis of exons 2 and 3 of the p53 gene in 2 out of 5 medullary thyroid cancers (MTC) prompted us to sequence the corresponding genomic region spanning codons 9-32. We found no mutations of the conserved domain I in the two MTCs that had previously shown mobility shifts, nor in a third MTC case indicating wild-type p53 (in this region). However, we detected a polymorphism consisting of a GC to CG transversion in intron 2 in two of the primary MTCs and one associated blood sample. In addition, we report on a deviation of the published sequence of intron 2 affecting positions 11817-8 and 11874, seen in 7/7 samples analyzed. Our findings corroborate the hypothesis, that MTC carcinogenesis does not require p53 mutations (Yana et al, Jpn J Cancer Res 1992).

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.