Background: We previously observed decreased p53 immunostaining over time in paraffin-embedded sections of ductal carcinoma in situ of the breast of women; these sections had been stored on slides at ...room temperature. This observation suggests that slide storage adversely affects p53-immuno-staining intensity and could result in spurious negative staining for p53 in patient samples. Purpose: The goals of this study were to determine the time course and factors influencing loss of p53 immunoreactivity and to investigate whether a similar loss of reactivity occurs with other antigens commonly used to study breast cancer. Methods: Serial sections cut from 12 formalin-fixed, paraffin-embedded, p53-positive invasive ductal carcinomas of the breast were stored on slides at room temperature or at 4 °C, with or without an additional paraffin coating, for 2, 4, 8, or 12 weeks. For each case, freshly cut slides from the same block (day 0) and stored slides were simultaneously stained for p53 by use of an automated immunostainer. Slides cut from formalin-fixed, paraffin-embedded breast carcinomas and stored for 12 weeks were also stained for factor VIII-related antigen (n = 12), estrogen receptor (ER) (n = 9), and Bcl-2 protein (n = 9). The staining intensity of all slides was assessed by visual microscopic examination and was also quantified by image analysis. Quantitative results were expressed as a percentage (mean ± standard error) of the staining intensity on day 0. Data were analyzed by the Friedman Repeated Measures Analysis of Variance on Ranks, with statistical significance set at two-sided P<.05. Results: The intensity of p53 staining decreased over time in nine (75%) of the 12 cases studied. In three (or 25% of all cases studied) of the nine cases that showed decreased p53 staining, slides stored for 12 weeks were scored as p53 negative. Antigen loss on slides stored at 4 °C was significantly less than that on slides stored at room temperature at all time points (all P<.05). At 12 weeks, the average staining intensity of slides stored at 4 °C was 33.2% ± 9% of that on day 0 compared with 8.4% ± 3% of that on day 0 for slides stored at room temperature (P<.001). Paraffin coating of the sections did not significantly diminish antigen loss at either room temperature or 4 °C, except for slides stored at room temperature for 12 weeks. The intensity of factor VIII staining decreased in nine of 12 cases (average staining intensity, 37.3% ± 6% of that on day 0 at 12 weeks; P =.0001). The intensity of ER and Bcl-2 staining decreased in all nine cases studied at 12 weeks (average staining intensity, 14.0% ± 6% and 21.0% ± 4% of that on day 0, respectively; P =.0001 for each). Conclusions and Implications: Slide storage, particularly at room temperature, results in substantial loss of p53 reactivity, with some p53-positive cases becoming p53 negative after 12 weeks of storage. Substantial loss of immunoreactivity for factor VIII, ER, and Bcl-2 occurs on slides stored at room temperature for 12 weeks. Storage of unstained slides for up to 12 weeks may lead to false-negative immunostaining for p53 and other antigens. J Natl Cancer Inst 1996; 88: 1054–9
Endostatin is a Mr 20,000 COOH-terminal fragment of collagen XVIII that inhibits the growth of several primary tumors. We report here the cloning and expression of mouse endostatin in both ...prokaryotic and eukaryotic expression systems. Soluble recombinant protein expressed in yeast (15-20 mg/L) inhibited the proliferation and migration of endothelial cells in response to stimulation by basic fibroblast growth factor. A rabbit polyclonal antibody was raised that showed positive immunoreactivity to the recombinant protein expressed from both systems. Importantly, the biological activity of the mouse recombinant protein could be neutralized by this antiserum in both endothelial proliferation and chorioallantoic membrane assays. Systemic administration of endostatin at 10 mg/kg suppressed the growth of renal cell cancer in a nude mouse model. The inhibition of tumor growth with soluble yeast-produced protein was comparable to that obtained with non-refolded precipitated protein expressed from bacteria. In addition, two closely related COOH-terminal deletion mutants of endostatin were also tested and showed strikingly differing activity. Collectively, these findings demonstrate the expression of a biologically active form of mouse endostatin in yeast, define a role for the molecule in inhibiting endothelial cell migration, extend its antitumor effects to renal cell carcinoma, and provide a formal proof (via the neutralizing antiserum experiments and the mutant data) that endostatin (and not a possible contaminant) acts as an antiangiogenic agent. Finally, the high level expression of mouse endostatin in yeast serves as an endotoxin free, soluble source of protein for fundamental studies on the mechanisms of tumor growth suppression by angiogenesis inhibitors.
Replication-deficient adenoviruses are known to induce acute injury and inflammation of infected tissues, thus limiting their use for human gene therapy. However, molecular mechanisms triggering this ...response have not been fully defined. To characterize this response, chemokine expression was evaluated in DBA/2 mice following the intravenous administration of various adenoviral vectors. Administration of adCMVbeta gal, adCMV-GFP, or FG140 intravenously rapidly induced a consistent pattern of C-X-C and C-C chemokine expression in mouse liver in a dose-dependent fashion. One hour following infection with 10(10) PFU of adCMVbeta gal, hepatic levels of MIP-2 mRNA were increased >60-fold over baseline. MCP-1 and IP-10 mRNA levels were also increased immediately following infection with various adenoviral vectors, peaking at 6 hr with >25- and >100-fold expression, respectively. Early induction of RANTES and MIP-1beta mRNA by adenoviral vectors also occurred, but to a lesser degree. The induction of chemokines occurred independently of viral gene expression since psoralen-inactivated adenoviral particles produced an identical pattern of chemokine gene transcription within the first 16 hr of administration. The expression of chemokines correlated as expected with the influx of neutrophils and CD11b+ cells into the livers of infected animals. At high titers, all adenoviral vectors caused significant hepatic necrosis and apoptosis following systemic administration to DBA/2 mice. To investigate the role of neutrophils in this adenovirus-induced hepatic injury, animals were pretreated with neutralizing anti-MIP-2 antibodies or depleted of neutrophils. MIP-2 antagonism and neutrophil depletion both resulted in reduced serum ALT/AST levels and attenuation of the adenovirus-induced hepatic injury histologically, confirming that this early injury is largely due to chemokine production and neutrophil recruitment. Our findings further clarify the early immune response against replication-deficient adenoviral vectors and suggest a strategy to prevent adenovirus-mediated inflammation and tissue injury by interfering with chemokine or neutrophil function.
Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the ...formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen α2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209–1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the α3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185–203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395–20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The α3(IV)NC1 inhibited in vivoneovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54–132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185–203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the α3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the α3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.
Picosecond time-resolved x-ray spectroscopy is used to measure the spectral line shift of the 1s2p-1s^{2} transition in He-like Al ions as a function of the instantaneous plasma conditions. The ...plasma temperature and density are inferred from the Al He_{α} complex using a nonlocal-thermodynamic-equilibrium atomic physics model. The experimental spectra show a linearly increasing redshift for electron densities of 1-5×10^{23}cm^{-3}. The measured line shifts are broadly consistent with a generalized analytic line-shift model based on calculations of a self-consistent field ion-sphere model.
Preeclampsia: A renal perspective Karumanchi, S. Ananth; Maynard, Sharon E.; Stillman, Isaac E. ...
Kidney international,
06/2005, Letnik:
67, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Preeclampsia: A renal perspective. Preeclampsia is a syndrome that affects 5% of all pregnancies, producing substantial maternal and perinatal morbidity and mortality. The aim of this review is to ...summarize our current understanding of the pathogenesis of preeclampsia with special emphasis on the recent discovery that circulating anti-angiogenic proteins of placental origin may play an important role in the pathogenesis of proteinuria and hypertension of preeclampsia.
The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in patients with VHL disease and in the majority of patients with sporadic clear cell renal carcinoma (RCC). Overexpression of ...transforming growth factor (TGF) beta 1 has been observed in patients with several cancers, including RCCs, with serum and urine levels correlating inversely with prognosis. We have demonstrated that the VHL tumor suppressor gene product represses TGF- beta 1 mRNA and protein levels ( approximately 3-4-fold) in 786-O RCC cells by decreasing the TGF- beta 1 mRNA half-life. Exogenously added TGF- beta 1 did not suppress the growth of 786-O cells in vitro, nor did the addition of neutralizing antibody (Ab) against TGF- beta have any effect. Indeed, 786-O cells were found to express no TGF- beta type II receptor protein, thus allowing them to escape from the negative growth control of TGF- beta 1. In contrast to the in vitro data, neutralizing Ab to TGF- beta inhibited tumorigenesis and, in some cases, regressed established 786-O tumors in athymic mice. Immunohistochemistry for von Willebrand's factor revealed a 3-4-fold lower tumor microvessel count in the mice treated with TGF- beta Ab compared to controls, suggesting that the Ab was inhibiting angiogenesis. Our findings indicate that TGF- beta 1 is a novel target for the VHL tumor suppressor and that antagonizing its paracrine action may provide novel avenues for treatment of RCCs as well as other tumors that secrete TGF- beta 1.
Offshore wind farms are proposed around the coast of the UK and elsewhere in Europe. These sites tend to be located in shallow coastal waters that often coincide with areas used by over‐wintering ...Common Scoter Melanitta nigra. A large‐scale study was undertaken to ascertain the relationship of the spatial distribution of Common Scoter in Liverpool Bay with prey abundance and environmental and anthropogenic variables that may affect foraging efficiency. The highest numbers of Common Scoter coincided with sites that had a high abundance and biomass of bivalve prey species. There was strong evidence that the maximum observed biomass of bivalves occurred at a mean depth of c. 14 m off the Lancashire coast and at c. 8 m off the north Wales coast. This coincided well with the distribution of Common Scoter at Shell Flat, but less well with the distribution of birds off North Wales. Common Scoters were observed in lowest numbers or were absent from areas in which anthropogenic disturbance (shipping activity) was relatively intense, even when these areas held a high prey biomass. Commercial fishing activities did not appear to contribute to this disturbance.
The human polyomavirus JC (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS that occurs in immunosuppressed individuals. Because ...polyomavirus-induced CNS pathology usually occurs as a result of the reactivation of latent virus, little is known about the disease manifestations of a primary polyomavirus-induced disease in man. To model such a primary infection, SV40-negative rhesus monkeys were immunosuppressed by infection with the virus SHIV-89.6P and then superinfected with the polyomavirus SV40. The animals developed CNS pathology characterized by both demyelination and meningoencephalitis. This observation suggests that a primary polyomavirus infection can be associated with an inflammatory CNS process. These data shed new light on the pathogenic mechanisms of primate polyomaviruses in the immunocompromised host.
Reproductive breast cancer risk factors are hypothesized to act by increasing exposure of the breast to endogenous estrogens,
but few studies have quantitatively examined the association of these ...risk factors with breast tissue composition. This study
is part of a case-control study of breast histological characteristics and breast cancer risk, nested within the Nurses’ Health
Study, a prospective study of 121,700 registered nurses. We studied 300 women who had not been diagnosed with breast cancer,
but for whom we obtained slides from a prior benign breast biopsy. We used a computer-assisted image analysis technique to
assess the proportion of epithelial and fibrous stromal tissue on benign breast biopsy slides, excluding obvious mass lesions.
Mean epithelial proportion was 5.3% (0.1–23%), and mean stromal proportion was 58.7% (3–93%). Women with proliferative breast
disease without atypia had higher epithelial and stromal proportions than women with nonproliferative breast disease ( P < 0.001). Postmenopausal women had a lower epithelial proportion ( P = 0.01), and increasing age at biopsy was associated with decreasing stromal proportion among postmenopausal parous women
( P = 0.004). Among premenopausal women, increasing years since last birth was associated with lower epithelial proportion ( P < 0.001). Other reproductive risk factors were not independently associated with epithelial or stromal proportion. Epithelial
and stromal breast tissue were associated with different factors with the exception of proliferative breast disease, which
was associated with an increase in both epithelial and stromal proportion. The quantitative measurement of epithelial and
stromal proportion may be useful for measuring changes in breast composition.
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