Human papillomavirus is associated with monoclonal gammopathies of unknown significance

When monoclonal gammopathies arise in persons without evidence of plasma cell malignancy or lymphoproliferative disease, the term monoclonal gammopathy of unknown significance (MGUS) can be used. MGUS is believed to be the preneoplastic phase of lymphoproliferative diseases because many of these patients eventually develop malignant disease, mainly multiple myeloma. We have previously identified human papillomavirus (HPV) in a chronic benign plasma cell tumor of the cervix and in the bone marrow of multiple-myeloma patients. In the following study, we expanded upon our initial observation by analyzing 14 patients with MGUS. Bone marrow biopsies of the patients were analyzed for HPV sequences using polymerase chain reaction (PCR) and in situ hybridization. Normal controls included 26 bone marrow specimens, 24 analyzed by PCR and two by in situ hybridization. A significant association was found to exist between HPV and MGUS (p=0.001). Among 14 patients iwth MGUS, HPV sequences have been identified in 10 of the bone marrow biopsies. These results suggest that HPV can reside in the bone marrow of a premalignant lymphoproliferative disease.     

Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.     

Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.     

Use of comparative reasoning tools for evaluation of the effect of sterilization conditions on fermentations to produce acidic fibroblast growth factor

The effects of various medium sterilization conditions on fermentations of a recombinant, acidic fibroblast growth factor (aFGF) producing Escherichia coli have been studied. Changes in the medium resulting from sterilization were monitored by pH and absorption spectra. This simple experiment provided excellent data for the demonstration of the usefulness of comparative reasoning tools in order to evaluate the effect of sterilization on fermentation performance. The time profiles of the main parameters (e.g., carbon dioxide evolution rate, dissolved oxygen, pH, and aFGF productivity) were simplified into piecewise contiguous linear segments, each of which was sequentially numbered. The length, position, and slope of each tine were then characterized. Application of the comparative reasoning tools confirmed that separate sterilization of the glucose was necessary for the success of the process, despite adding to the cost and complexity. The comparative data analysis also showed that scaleup with longer sterilization holding and cooling times would not be detrimental to aFGF production. (c) 1995 John Wiley & Sons, Inc.     

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.     

Uptake of high-density lipoprotein by Y-organs of the crab,cancer antennarius. II. formal characterization of receptor-mediation with isolated membranes

Y-organs are the ecdysial glands of crustaceans, responsible for synthesis and secretion of ecdysteroid hormones. For this purpose, the glands acquire cholesterol as obligate precursor entirely from circulating high-density lipoprotein (HDL). A preceding study provided evidence for the mechanism of acquisition: Y-organs take up cholesterol bound to HDL by an energy-requiring process, receptor-mediated absorptive endocytosis. The present study characterized the receptors involved utilizing isolated Y-organ membranes. HDL binding was saturable and specific; a dissociation constant (K_d) of 1.08 × 10^?7 M and a binding maximum at equilibrium (B_max) of 70 μg HDL protein/mg membrane protein, were obtained. Binding was decreased by protease and was dependent upon calcium. Y-organs are regulated negatively by a peptide hormone from the eystalks, molt-inhibiting hormone (MIH). Y-organ membranes from de-eyestalked crabs (MIH absent) exhibited the same K_d value as membranes from intact crabs, but a B_max 17% higher. Thus, MIH activity apparently does not change the binding affinity of HDL, but decreases the number of binding sites. These results agree with our previous findings that MIH depresses ecdysteroid synthesis in part by inhibiting cholesterol uptake. Generally, Y-organ cells appear to contain receptors for HDL that are of high affinity and high binding capacity, similar to the characteristics reported for the binding of insect HDL (vitellogenin) to fat bodies and oocytes. © 1995 Wiley-Liss, Inc.     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.     

Interleukin-6 involvement in mesothelioma pathobiology: inhibition by interferon α immunotherapy

A role for interleukin-6 (IL-6) in malignant mesothelioma has been suggested by the clinically presenting symptoms of mesothelioma patients, which include fever, weight loss and thrombocytosis. A murine model of malignant mesothelioma was therefore used to examine the potential role of IL-6 in this cancer type and whether the effect of interferon (IFN) therapy on mesothelioma might be mediated, in part, by regulating IL-6 levels and/or IL-6-induced pathobiology. A panel of human and murine mesothelioma cell lines was assayed for endogenous IL-6 production in a bioassay, and for IL-6-mRNA expression. Four out of 5 human and 5 out of 15 murine mesothelioma cell lines produced moderate to high levels of bioactive IL-6 in vitro. This result was corroborated by mRNA detection. One of the representative murine cell lines, AB22, was chosen for further in vivo studies in the murine mesothelioma model. In AB22-inoculated mice detectable serum IL-6 levels were found to precede macroscopically detectable tumour growth, clinical signs (cachexia, abdominal distension, diarrhoea) and changes in the peripheral lymphoid organs (cell depletion and functional depression). Treatment with anti-IL-6 antibody curtailed the clinical symptoms (P<0.001), as did treatment with recombinant human (rhu) IFN (P<0.001). Neither anti-IL-6 antibody nor rhuIFN had a direct growth-inhibitory effect on the AB22 mesothelioma cell line in vitro, however, in vivo rhuIFN treatment of mice inoculated with AB22 cells attenuated both IL-6 mRNA expression in the tumours and serum IL-6 levels, ameliorated the depression of lymphocyte activities, and enhanced the number of tumour-infiltrating lymphocytes and macrophages. On the basis of these results it is suggested that IL-6 mediates some of these effects, directly or indirectly, and that a combination therapy of rhuIFN and anti-IL-6 antibody may be an improved palliative treatment for patients with malignant mesothelioma.     

DNA strand scission and ADP-ribosyltransferase activity during murine erythroleukemia cell differentiation

The accumulation of DNA strand breaks and activation of ADP-ribosyltransferase (ADPRT) have recently been associated with cellular differentiation. Murine erythroleukemia (MEL) cells undergo erythropoietic differentiation when exposed to dimethyl sulfoxide (Me2SO) and several studies have suggested that DNA strand scission induced by this agent is a prerequisite for expression of the differentiated phenotype. Me2SO induction of MEL cells has also been associated with increases in ADPRT activity in one study, but not in another. We have monitored the effects of Me2SO on DNA strand breaks in preformed and replicating MEL cell DNA. The results clearly demonstrate that DNA fragmentation is not detectable during Me2SO induction of MEL differentiation, even in the presence of 3-aminobenzamide, an inhibitor of ADPRT. Further, these results are consistent with an absence of detectable changes in both endogenous and total potential ADPRT activity during Me2SO-induced MEL differentiation. These findings would argue against Me2SO induction of DNA strand scission and ADPRT in MEL cells undergoing differentiation.     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.