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Biomedical subjects

C Page

Publications and source records attributed to C Page.

At least 37 records · Page 2Linked to original sources
PubMed2001

Glycosaminoglycans, airways inflammation and bronchial hyperresponsiveness.

Glycosaminoglycans (GAGs) are large, polyanionic molecules expressed throughout the body. The GAG heparin, co-released with histamine, is synthesised by and stored exclusively in mast cells, whereas the closely related molecule heparan sulphate is expressed, as part of a proteoglycan, on cell surfaces and throughout tissue matrices. These molecules are increasingly thought to play a role in regulation of the inflammatory response and heparin, for many years, has been considered to hold potential in the treatment of diseases such as asthma. Heparin and related molecules have been found to exert antiinflammatory effects in a wide range of in vitro assays, animal models and, indeed, human patients. Moreover, the results of studies carried out to date indicate that the antiinflammatory activities of heparin are dissociable from its well-established anticoagulant nature, suggesting that the separation of these characteristics could yield novel antiinflammatory drugs which may be useful in the future treatment of diseases such as asthma

Animals↗
PubMed2001

Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection associated with an inefficient humoral immune response. A study of lymphoid B cells and specific humoral immune response was performed in blood and several lymphoid organs collected from PRRSV experimentally-infected pigs. Groups of specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate of PRRSV, and blood, tonsils, spleen and mediastinal lymph nodes (MLN) were collected at various times postinfection (p.i.) (3-60 days). Lymphoid cells were isolated, immunolabeled for cytofluorometric determination of B cell percentages, used for counting specific anti-PRRSV antibody secreting B cells by an ELISPOT assay, or cultured for metabolic activity. The presence of anti-PRRSV antibodies in the serum of infected pigs was determined using a commercial ELISA assay. Virus detection was performed in all tissues, including lungs, by virus isolation and RT-PCR. The results show that percentages of B cells increased in tonsils as soon as 3 days until 17 days p.i. in PRRSV-infected pigs while they increased in spleen at 3 days p.i. only, due to an increase of larger Ig(high)-producing B cells. Metabolic activity of lymphoid cells from blood and spleen increased at 3 days p.i. only while lymphoid cells from tonsils and MLN transiently decreased at that time and increased thereafter up to 60 days p.i. Anti-PRRSV antibody-secreting B cells occurred in tonsils after 10 days p.i. and strongly increased up to 60 days p.i. However, specific anti-PRRSV-secreting B cells were detected in blood and spleen after 17 days p.i and in MLN only after 45 days p.i. Specific antibodies were detectable in serum at 10 days p.i., reached the maximum level at 45 days and remained high up to 60 days p.i. Infectious virus was detected in lungs and MLN as soon as 3 days p.i., and remained detectable up to 45 days p.i. in tonsils of one pig while viral RNA was detected in most organs up to 60 days p.i. In vitro experiments revealed that inactivated virus induced a stimulation of lymphoid cells isolated from PRRSV-infected pigs while it was cytotoxic for lymphoid cells from control pigs. Taken together, these results indicate that viral infection induced simultaneously a polyclonal activation of B cells, mainly in tonsils, and an exaggerated and prolonged specific humoral immune response due to persistent viral infection in lymphoid organs.

Animals↗
PubMed2001

The zona pellucida-induced acrosome reaction of human spermatozoa involves extracellular signal-regulated kinase activation.

Extracellular signal-regulated kinases (ERKs), belonging to the family of mitogen-activated protein kinases (MAPKs), are cytoplasmic and nuclear serine/threonine kinases involved in the signal transduction of several extracellular effectors. Recent evidence indicates the presence of p21 Ras and the phosphorylation of ERK1 and ERK2, suggesting the occurrence of the Ras/ERK cascade in mammalian spermatozoa. The present article describes the biological role of ERK during the acrosome reaction of human spermatozoa on stimulation with zona pellucida (ZP). The mitogen-activated protein-kinase inhibitor PD098059 was used as a pharmacological tool to study the involvement of extracellular signal-regulated kinases in the induction of the acrosome reaction in human spermatozoa. This compound significantly inhibited the acrosome reaction induced by both ZP and the calcium ionophore A23187. These results suggest that ERKs are involved in the signal transduction pathway through which ZP stimulation works during the process of fertilization.

Acrosome↗
PubMed2000

A modified p53 overcomes mdm2-mediated oncogenic transformation: a potential cancer therapeutic agent.

The antiproliferative activities of wild-type (wt) p53 are inhibited by mdm2 (murine double minute2) oncogene product. We tested growth suppression activity of p53 14/19, an engineered p53 variant, which does not bind mdm2 and is completely resistant to the inhibition by mdm2. p53 14/19, unlike wt p53, suppressed the growth of cancer cells that contain amplified mdm2 oncogene efficiently by direct DNA transfection or adenovirus-mediated gene transfer. In addition, p53 14/19 also inhibited the growth of several different cancer cell lines expressing low levels of mdm2 oncogene product as efficiently as wt p53. We further examined the antioncogenic potencies of p53 14/19 in the rat embryo fibroblast cotransformation assay. Addition of wt p53 failed to cause any significant decrease in ras plus mdm2 foci counts. In contrast, cotransfection of p53 14/19 with ras and mdm2 significantly reduced foci number. In similar experiments, cotransfection of wt p53 or 14/19 p53 resulted in significant inhibition of oncogenic transformation in rat embryo fibroblast mediated by an activated ras plus c-myc, adenovirus E1A, or human papillomavirus E7 oncogenes. Therefore, these results suggest that p53 14/19 modified tumor suppressor gene may be a promising therapeutic agent for human cancers that express abnormally high levels of mdm2 oncogene product.

Adenoviridae↗
PubMed2000

Constitutive activation of stat 3 oncogene product in human ovarian carcinoma cells.

OBJECTIVE: Stat 3 functions in transducing signals from the cell's surface to its nucleus and activation of gene transcription. Aberrations of Stat 3 in breast cancer have raised the possibility of its contribution to oncogenesis. Our goal was to examine ovarian cancer cell lines to determine whether Stat 3 plays a relevant role in ovarian carcinogenesis. METHODS: Protein lysates were extracted from normal ovarian surface epithelial cells and malignant cells. Western blotting techniques were performed with phosphorylation-independent or phosphorylation-specific Stat 3 (tyrosine 705) antibody. Confirmation of Stat 3 activation was determined by a luciferase reporter driven by a promoter containing Stat 3-specific binding sites. Bcl-x(L) and cyclin D(1) were also analyzed by Western blotting. RESULTS: MDAH 2774, OV-1063, Caov-3, and O.C. 22819 expressed high levels of phosphorylated Stat 3. In contrast, A2780 and normal ovarian surface epithelial cells had little Stat 3 phosphorylation recognized. Confirmation of persistent activation of Stat 3 activity was shown by transfection of cells with a Stat 3 luciferase reporter. Potential downstream mediators of Stat 3 including Bcl-x(L) and cyclin D(1) were also evaluated. In cells expressing activated Stat 3, high levels of both Bcl-x(L) and cyclin D(1) were detected, whereas in A2780 cells, which did not express activated Stat 3, only low levels of Bcl-x(L) and cyclin D(1) were expressed. CONCLUSIONS: Constitutive activation of Stat 3 is present in ovarian cancer lines but not in normal ovarian surface epithelial cells. Activation of Stat 3 is a common event during oncogenic transformation upstream to both Bcl-x(L) and cyclin D(1). The relationship of this aberrancy of ovarian carcinoma harboring activated Stat 3 deserves further investigation.

Cell Transformation, Neoplastic↗
PubMed2000

Development of a lyophilization formulation that preserves the biological activity of the platelet-inducing cytokine interleukin-11 at low concentrations.

Recombinant human interleukin-11 (rhIL-11) is a licensed biological therapeutic product in at least one country and is used to combat thrombocytopenia during chemotherapeutic regimens, as well as undergoing clinical trials for a range of other disorders. Following attempts to lyophilize IL-11 at low concentrations, it was clear that a significant loss of recoverable biological activity occurred. Investigation of a variety of factors, including the type of container in which the rhIL-11 was lyophilized, revealed that surface adsorption to glass was a major factor resulting in loss of activity of rhIL-11 in solution (> 40% reduction after 3 h at room temperature), in addition to losses of activity post-lyophilization. To overcome this problem, different formulations containing combinations of human serum albumin (HSA), trehalose and Tween-20 have been investigated. Two formulations were successful in entirely preserving the biological activity of rhIL-11 through lyophilization and subsequent reconstitution (potency estimates of formulated relative to original material being > or =0.97). Accelerated degradation studies, performed at intervals over a six-month period, demonstrated the stability of freeze-dried rhIL-11 using these formulations (predicted annual reduction in potency after storage at -20 degrees C < or =1.4%). In conclusion, we have developed a working combination of excipients (0.5% HSA, 0.1% trehalose and 0.02% Tween-20 in potassium phosphate buffer (pH 7.4)) to formulate a stable rhIL-11 freeze-dried product in glass containers, with no loss in potency. These findings should facilitate development of low dose rhIL-11 products and be an indicator of caution to those using this and other material with similar physical properties, without taking appropriate precautions to avoid losses through adsorption.

Adsorption↗
PubMed2000

Elevated phosphorylation of AKT and Stat3 in prostate, breast, and cervical cancer cells.

We examined whether the persistent activation of AKT or Stat3 oncogene product is present in prostate, breast, and cervical cancer cells. We found that some prostate and breast cancer cell lines express high levels of phosphorylated AKT. Interestingly, the cancer cells, which only express low levels of phosphorylated AKT express high levels of phosphorylated Stat3. AKT or Stat3 is also highly phosphorylated in human papilloma virus-negative cervical cancer cells. Therefore, these results indicate that AKT and Stat3 are highly phosphorylated in some breast, prostate and cervical cancer cells, which may play a role in tumorigenesis of these cancers.

Breast Neoplasms↗
PubMed1999

Overexpression of Bcl-x(L) promotes chemotherapy resistance of mammary tumors in a syngeneic mouse model.

Bcl-x(L), a prosurvival member of the Bcl-2 family that is expressed in many tumors, represses apoptosis induced by chemotherapeutic drugs in vitro. However, the contribution of apoptosis and prosurvival Bcl-2-related proteins to chemotherapy resistance in vivo is unknown and has been challenged by recent results with clonogenic survival assays. To test the ability of Bcl-x(L) to provide chemotherapy resistance to tumors, we transfected the mouse bcl-x(L) gene into the tumorigenic SCK mammary cell line and assessed the response of tumor cells to chemotherapeutic drugs in clonogenic assays and in a syngeneic mouse model. Bcl-x(L) conferred protection on SCK cells against methotrexate at certain drug concentrations, but not at all against 5-fluorouracil in clonogenic survival assays in vitro. Injection of SCK cells transfected with Bcl-x(L) or control plasmid in the mammary fat pads of syngeneic recipient mice resulted in tumors of similar size. However, although the volume of control tumors regressed up to 80% after 4 to 5 days of chemotherapy, SCK tumors expressing Bcl-x(L) did not regress and continued to grow in the presence of methotrexate or 5-fluorouracil. In addition, numbers of apoptotic cells were significantly higher in control tumors as compared to Bcl-x(L)-expressing tumors in animals treated with methotrexate or 5-fluorouracil. These results provide evidence that inhibition of apoptosis through Bcl-x(L) overexpression can promote resistance to chemotherapy in tumors in vivo.

Adenocarcinoma↗
PubMed1999

Endogenous nitric oxide acts as a natural antithrombotic agent in vivo by inhibiting platelet aggregation in the pulmonary vasculature.

Nitric oxide (NO) is a powerful vasodilator and an inhibitor of platelet aggregation in vitro. While the ability of NO to modulate vascular tone in vivo has been proven, only a few studies have assessed its platelet inhibitory activity in vivo. We have employed two complementary animal models of pulmonary platelet thromboembolism to assess the antithrombotic activity of endogenous NO in vivo. The inhibition of nitric oxide synthase (NOS) by L-NAME significantly potentiated while the administration of the NOS substrate L-arginine significantly reduced the accumulation of 111In-labelled platelets in the pulmonary vasculature of rabbits induced by intravenous collagen plus epinephrine. L-NAME or L-arginine did not, however, modify 111In-labelled erythrocyte distribution in lungs and phenylephrine had no effect on platelet accumulation following collagen + adrenaline, suggesting that the effects of L-NAME were not due to vasoconstriction but rather to a direct modification of platelet function. In mice, L-NAME significantly reduced the dose of collagen + adrenaline required to induce thromboembolic mortality, increased the fall in circulating platelets and increased the % of pulmonary vessels occluded by platelet thrombi. The effects of L-NAME were reversed by L-arginine but not by a dose of nicardipine exerting maximal vasodilatation. Phenylephrine did not potentiate collagen + adrenaline-induced mortality. In the pulmonary vasculature in vivo, endogenous NO inhibits collagen + adrenaline-induced aggregation and enhances platelet disaggregation. This natural modulator function of NO is exerted via a direct effect on platelets and not as a result of haemodynamic changes.

Animals↗
PubMed1999

Bioassays for the characterisation and control of therapeutic cytokines; determination of potency.

The primary value of bioassays is that they alone directly assess the biological activity of bioactive substances and products like cytokines. Appropriately designed bioassays reflect the fundamental aspects of the biological activity of a cytokine molecule, including ligand-receptor binding, signal transduction processes (often poorly understood) and the final observed biological effects. Biological assays therefore complement physicochemical and biochemical procedures which normally only assess precise molecular structural features of cytokines. Bioassays provide valuable information concerning the potency of cytokine products. This is essential for evaluating batch-to-batch consistency, appropriate formulations and stability. Bioassay data are crucial at all stages in the development of cytokine products, from early research to final quality control of finished product. However, the type and design of bioassays may differ according to the information required and its intended use. The assays may or may not directly relate to the clinical use of the product. Bioassays can be difficult to perform and time consuming, although this often reflects bad assay choice and/or design. Correct analysis of the assay results is essential if valid data are to be obtained. Standardisation, using correctly calibrated primary and secondary standards, is essential. In vivo bioassays are normally more unreliable than in vitro procedures, but in some cases in vitro systems are either not available or do not address important biological characteristics of a product. Bioassays must be validated for their intended purpose and for the types of samples to be measured. Appropriate statistical analysis should be used to derive the significance and specifications of results. This needs to address both variability in samples and assay performance. Specifications (limits) for product acceptability need to be derived from real data using several batches of cytokine product.

Biological Assay↗
PubMed1998

Bcl-xL is expressed in ovarian carcinoma and modulates chemotherapy-induced apoptosis.

OBJECTIVE: To investigate the role of Bcl-xL in resistance to chemotherapy-induced apoptosis in ovarian carcinoma. METHODS: Two human ovarian carcinoma cell lines were used in this study: A2780 and SKOV3. A2780 cells were transfected with human Bcl-xL or control plasmid alone. Expression of Bcl-xL in single cell clones was analyzed by flow cytometry and protein expression was confirmed by Western blot. For in vitro chemotherapy-induced death assays, cisplatin and Taxol were used. The percentage of apoptotic cells was determined by nuclear propidium iodide staining followed by flow cytometric analysis. Human ascites samples were used to make tumor lysates which were then analyzed for expression of Bcl-xL protein by Western blot. RESULTS: A2780 cells express low levels of endogenous Bcl-xL while SKOV3 cells express high amounts as determined by Western blot. In addition, A2780 cells are sensitive to chemotherapy-induced cell death while SKOV3 cells are resistant. To determine if chemoresistance is mediated by expression of Bcl-xL, A2780 cells were transfected with Bcl-xL or control plasmid. Cells were incubated with either cisplatin or Taxol to induce apoptosis. Bcl-xL-expressing cells were highly resistant to cisplatin and Taxol compared with controls (P < 0.05). All samples of malignant ascites analyzed expressed high levels of Bcl-xL on Western blot. CONCLUSIONS: The results of these studies indicate that Bcl-xL is expressed in ovarian carcinoma, and in A2780 cells functions in a manner analogous to Bcl-2 by inhibiting chemotherapy-induced apoptosis. This may have prognostic significance; previous studies have demonstrated that patients with breast cancer that overexpress Bcl-2 have low-grade, hormonally responsive tumors. In ovarian carcinoma, another Bcl-2 family member, Bcl-xL may be responsible for modulating resistance to chemotherapy-induced apoptosis.

Antineoplastic Agents↗