CHEMICAL CONSTITUTION OF ACETONIC EXTRACTS OF NEOPLASTIC HUMAN TISSUES AND RESULTS OF THEIR THERAPEUTICAL UTILIZATION.
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The cytoplasmic extracts of human prostatic tissues yield two classes of "particles" when centrifuged to equilibrium in a sucrose density gradient, one class banding at a density of 1.15-1.18 g/cm3 ("high density particles") and another at a density of 1.07-1.14 g/cm3 ("low density particles"). Both bands display endogenous DNA polymerase activity which is largely resistant to actinomycin D inhibition. The endogenous DNA products synthesized by high density particles give some indication of high molecular weight RNA:DNA complexes. The tissue extracts from normal, hyperplastic, and neoplastic prostate behave similarly in these assays. In addition, explant cultures of hyperplastic and neoplastic prostate either release, or can be induced to release, particles by treatment with bromodeoxyuridine. These particles band at a density of 1.15-1.18 g/cm3 in a sucrose density gradient and possess RNA and associated DNA polymerase activity which utilizes poly(A):oligo(dT). Our results suggest that human prostatic tissues may contain functions analogous in some ways to those of known RNA tumor viruses of other species.
Mechanical or chemical injury to adult rat brain elicited the accumulation in the affected area of trophic activity for cultured parasympathetic, sympathetic, and sensory neurons. Neuronotrophic activity was relatively low both in noninjured brain tissue extracts and in extracts prepared from the tissue surrounding an injury immediately after the lesion was made. However, trophic titers increased considerably over time, first in the brain tissue that formed the walls of the wound and then in the Gelfoam filling the wound cavity. In the tissue adjacent to the injury, trophic titers began to rise immediately after the lesion, reached a maximum 10 days later, and decayed thereafter. In the wound cavity, occupied by Gelfoam, neuronotrophic activity began to increase 6 days postlesion, reached a maximum at day 16 after injury, and decreased at later times. The levels of induced trophic activity appeared to be proportional to the size of the wound. Injury to various brain areas including temporal, entorhinal, occipital, parietal, and frontal cortices, hippocampus, corpus striatum, and cerebellum, all induced a similar increase in neuronotrophic factor(s). Damage to the myelinated fibers of the corpus callosum did not. High trophic titers decayed rapidly with distance from the wound except in areas heavily deafferented by the lesion, where activity also reached high levels. Extracts from all of the above-mentioned brain areas contained toxic activity for cultured spinal cord neurons. The level of neuronotoxic activity was similar both before the lesion and 15 days postlesion, with the possible exception of the corpus callosum. Intraventricular injections of kainic acid at doses which destroy areas CA4, CA3, and part of CA1 of the hippocampus also induced a time-dependent rise of neuronotrophic activity in this structure, comparable to that achieved by mechanical damage. Both kainic acid treatment and mechanical injury cause extensive glial proliferation in the injured and/or deafferented area. The apparent concurrence of glial reaction and increase in neuronotrophic activity suggests that glial cells may be a major source of the induced trophic activity. As an in vivo correlate of cell culture data, the survival of striatal transplants into host cortical wounds was examined. Fragments of embryonic corpus striatum did not survive when transplanted into a freshly made cavity in the entorhinal/occipital cortex of adult rats. Survival was enhanced by introducing a delay between the time at which the wound cavity was made and that at which the striatal tissue was implanted in it.(ABSTRACT TRUNCATED AT 400 WORDS)
OBJECTIVE: The purpose of this study was to quantify the cytokines interleukin (IL)-1 receptor antagonist (RA), IL-6, and transforming growth factor beta(1) found in extracts of inflammatory periapical tissues and to study the effect of a new antibiotic (linezolid) on the levels of these cytokines. STUDY DESIGN: Twenty-two patients with root-filled teeth with persisting periapical pathoses were randomly divided into a control group or an antibiotic group. One tooth from each of the patients was resected at the root-end, and the periapical tissue was collected. IL-1RA, IL-6, and TGF-beta(1) were quantified in tissue extracts through the use of enzyme-linked immunosorbent assays. RESULTS: Measurable amounts of all 3 cytokines were found in all extracts. A statistically significant reduction in IL-1RA per milliliter was observed in the linezolid group in comparison with the control group (P <.05). The difference in variation of IL-1RA in the treatment and control groups was also highly statistically significant (P <.001). CONCLUSIONS: Although drawn from a limited sample size, the results indicate that IL-1RA is a sensitive indicator of the effect of antibacterial treatment on the immune response in periapical inflammatory tissues.
The release of uncombined gonadotropin subunits by rat anterior pituitaries during an in vitro incubation as well as intracellular concentrations were assessed. Uncombined subunits and native gonadotropins were quantitated by radioimmunoassays after samples were subjected to gel filtration on Sephadex G-100 Superfine. Small, but detectable, amounts of uncombined rat LH beta subunit were released under basal conditions. GnRH increased the absolute amount of rLH beta released but did not alter the rLH beta/rLH molar ratio (approximately 0.02). Tissue extracts prepared in aqueous buffer (100,000 x g supernatants) and 0.5% Triton X-100 extracts of the 100,000 x g pellets from the initial homogenization ('pellet extracts') contained larger quantities of uncombined rLH beta as well as elevated rLH beta/rLH molar ratios (approximately 0.10 and approximately 0.20, respectively). Significant amounts of uncombined rLH alpha were released and were present in both tissue and pellet extracts. However, when FSH beta subunit was quantitated in tissue extracts after gel filtration, all of the immunoreactive materials eluted in the position of native rFSH (FSH beta/rFSH molar ratio less than 0.0025). Pellet extracts contained significant amounts of rLH beta, rLH and rLH alpha but lesser amounts of rFSH suggesting that intracellular gonadotropins are not completely extracted when homogenization is performed in aqueous buffers. Thus, rat anterior pituitaries contain and release significant amounts of the uncombined alpha subunit, relatively small amounts of uncombined rLH beta and extremely small amounts of uncombined FSH beta, if any.
BACKGROUND: Carcinosarcomas are comprised of carcinomatous and sarcomatous elements, and their histogenesis remains unclear. The authors examined the serum concentrations of hematopoietic growth factors and performed immunohistochemical studies on an esophageal carcinosarcoma from a patient with marked granulocytosis to determine its histopathogenesis and clonality. METHODS: The authors examined the case of a 63-year-old man with a polypoid tumor of the esophagus associated with marked leukocytosis (131 x 10(9) per liter). Immunohistochemical staining of the esophageal tumor was performed using monoclonal antibodies against granulocyte-colony stimulating factor (G-CSF), keratin, epithelial membrane antigen (EMA), and vimentin. RESULTS: The patient's leukocyte count was increased (124 x 10(9) per liter) on admission. Because mature granulocytes predominantly were increased despite the absence of apparent infection, the patient's serum G-CSF concentration was examined and found to be 286.0 pg/mL and to increase with time. After thoracic esophagectomy was performed, granulocyte count and serum G-CSF concentration rapidly normalized. G-CSF concentration was 50-fold higher in the tumor tissue extract than in the extract from normal esophageal tissue. Microscopic examination of the resected specimens revealed that the tumor was comprised of squamous cell carcinoma (SCC) and spindle-shaped sarcomatous elements, and transitional features were observed within these two components. Immunohistochemical examination disclosed cells that were positive for keratin and EMA in the carcinomatous element and vimentin positive cells in the sarcomatous element. However, both types of tumor cells were positive for G-CSF. CONCLUSIONS: The presence of G-CSF in both SCC cells and spindle-shaped sarcomatous cells indicated that these two components originated from a single clone.
BACKGROUND: Characterization of tissues can be based on the topographical relationship between the cells. Such characterization should be insensitive to distortions intrinsic to the acquisition of biological preparation. In this paper, a method for the robust segmentation of tissues based on the spatial distribution of cells is proposed. MATERIALS AND METHODS: The neighborhood of each cell in the tissue is modeled by the distances to the surrounding cells. Comparison with an example or prototype neighborhood reveals topographical similarity between tissue and prototype. Processing of all cells in the tissue extracts the regions with tissue architecture similar to the given example. RESULTS: Comparison with other topographical-segmentation methods shows that the proposed method is better suited for partitioning tissue architecture. As an example, the quantification of the structural integrity in rat hippocampi after ischemia is demonstrated. In contrast to other methods, the algorithm correlates well with expert evaluation. CONCLUSIONS: The present method reduces the nonbiological variation in the analysis of tissue sections and thus improves confidence in the result. The method can be applied to any field where regular patterns have to be detected, as long as the directional distribution of neighbors may be neglected.
OBJECTIVE: To examine the expression, regulation, and potential roles of bone morphogenetic proteins (BMPs) in arthritic synovium. METHODS: Expression of BMPs in arthritic synovium from patients with rheumatoid arthritis (RA) or spondylarthropathy (SpA) and in noninflamed synovium from patients undergoing diagnostic or therapeutic arthroscopies was studied by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and 2-color immunofluorescence. In vitro regulation of gene expression in fibroblast-like synoviocytes (FLS) was determined by real-time quantitative RT-PCR and immunohistochemistry. We used (3)H-thymidine incorporation after serum deprivation-induced growth arrest to examine effects on FLS proliferation. FLS apoptosis was evaluated by flow cytometry cell cycle analysis. Apoptotic cells in synovial tissue were detected by TUNEL staining. RESULTS: Transcripts of different BMPs, most strikingly BMP-2 and BMP-6, were detected in synovial tissues. By Western blot, BMP-2 and BMP-6 precursor protein was found in RA and SpA synovial tissue extracts, but not in extracts of noninflamed synovial tissue. By immunohistochemistry, BMP-2 and BMP-6 were detected in the hyperplastic lining and the sublining layer of synovium from RA and SpA patients, both in CD90+ cells (FLS) and in some CD68+ cells (macrophages). Proinflammatory cytokines, such as interleukin-1beta and tumor necrosis factor alpha, but not interferon-gamma, enhanced the expression of BMP-2 and BMP-6 transcripts in FLS in vitro. Neither BMP-2 nor BMP-6 affected FLS proliferation. BMP-2 promoted FLS apoptosis, whereas BMP-6 protected against nitric oxide-induced FLS apoptosis. BMP-2-positive apoptotic cells were found in arthritic synovium. CONCLUSION: BMP-2 and BMP-6 are expressed in arthritic synovium and are strongly up-regulated by proinflammatory cytokines. Although BMP signaling has been proposed to be involved in cartilage and bone repair in arthritis, this pathway may be equally important in modulating FLS cell populations in inflamed synovium.
Nuclear magnetic resonance spectroscopy (NMRS) has been used extensively for several decades to elucidate metabolic pathways in biological systems and has led to new insights into cerebral metabolism. Many of these insights have been gained by NMRS using in vitro models, such as tissue extracts, cell cultures or brain slices. Extracts of cells or tissue provide an excellent basis for metabolic studies and facilitate the interpretation of in vivo spectra. (13)C NMRS is at present mostly used for in vitro or animal studies. Conclusions from the epilepsy models presented in this review are that turnover of metabolites is time-dependent in kainate-injected rats with limbic seizures. Early and only temporarily enhanced astrocytic activity is followed by altered metabolism in neurons with an increased turnover of important amino acids such as GABA and glutamate. However, pentylenetetrazole (PTZ) kindling affects astrocytes in younger and glutamatergic neurons in older animals. In the presence of PTZ, phenobarbital decreases labeling of most metabolites in all cell types, except GABAergic neurons, from both labeled precursors in the younger animals. However, in older animals only GABAergic neurons are affected by phenobarbital, as indicated by an increase in GABA labeling. In kaolin-induced hydrocephalus it was shown that astrocyte metabolism is disturbed in the early phase, particularly in the cerebrum. These alterations continue into the chronic period. Only then do the first signs of neuronal metabolic impairment appear, which might explain why dementia is a prominent clinical feature in patients with chronic hydrocephalus.
BACKGROUND: Increase in the number of serotonin (5-HT) releasing neuroendocrine (NE) cells has been shown to be correlated with tumor progression, loss of androgen dependence, and poor prognosis. Serotonin is a well-known mitogen which mediates a wide variety of physiological effects via multiple receptors, of which receptor subtype 1 (5-HTR1) has been identified in prostate cancer (PC) cell lines. Recently, 5-HT has been found to show growth-promoting activity and to be functionally related to oncogenes. MATERIALS AND METHODS: Localization, protein content, and mRNA expression of 5-HTR subtype 1A, 1B, and 1D was studied in prostatic tissue (35 patients), metastases, PC cell lines, a benign prostatic stromal cell line (human prostate cell preparation (hPCP)), and xenografts of PC-3 cells by immunohistochemistry (IHC), Western blotting, and RT-PCR, respectively. The growth-inhibition effect of a 5-HT1A antagonist (NAN-190) on PC cell lines was studied using a bromodeoxyuridine (BrdU) assay. RESULTS: A strong immunoreaction of 5-HTR1A and 1B was demonstrated in high-grade tumor cells (35/35) and a small number of BPH cells, whereas 5-HTR1D was confined to vascular endothelial cells. 5-HTR1A was also demonstrated in PC cells metastasized to lymph node and bone, PC-3, DU145, LNCaP, and in xenografts of PC-3 cells and hPCP. Western blot analysis gave strong bands from PC tissue extracts compared to BPH tissue. Using RT-PCR, 5-HTR1A mRNA was demonstrated in all PC cell lines. An antagonist of 5-HTR1A (NAN-190) inhibited the growth of PC-3, DU145, and LNCaP cells but not of hPCP cells. CONCLUSIONS: This is the first study demonstrating an overexpression of 5-HTR subtypes 1A and 1B in PC cells, especially in high-grade tumors. Moreover, 5-HT stimulates proliferation of PC cells and 5-HTR1A antagonists inhibit proliferation. Thus, we propose that 5-HT has an important role in tumor progression, especially in the androgen-independent state of the disease. The design of specific antagonists for this type of receptor might be useful for the growth control of androgen-independent tumors.
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Protein tyrosine kinase (PTK) activity was assayed in cytosolic extracts from normal breast tissue, benign tumors, and 84 T1-T2, N0-N1 M0, breast carcinomas. Normal breast tissue extracts yielded an average value of 1.9 +/- 1.1 pmol 32P incorporated/min/mg protein, whereas a mean of 12.5 +/- 6.1 was obtained for cancer samples. With a median follow-up of 34 months, in the series of 40 patients classified N-, PTK positive patients presented a significantly smaller 3-year disease free survival than the PTK negative ones. Multivariate analysis shows that PTK activity emerges as a potential prognostic factor in breast cancer (p = 0.02). These preliminary results will be updated on a bigger cohort of patients.
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Twenty Syrian golden hamsters recieved weekly injections of pancreatic cancer inducing DHPN. Their Poly (U) specific serum RNase levels were significantly elevated when compared to the control levels. Following salt fractionation, Poly (U) specific activity was present in both the 40% and 50% salt saturated fractions. Tissue assays showed that Poly (U) specific RNase was present in both pancreas and liver tissue extract, although the liver tissue RNase had a different pH maximum than that of the serum RNase.
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