Polymorphic expression of decay-accelerating factor in human colorectal cancer

BACKGROUND: We have previously shown that expression of decay-accelerating factor (DAF), a complement regulatory protein, is enhanced immunohistochemically on the luminal surface of cancer glands in human colorectal cancer and is detected in stool specimens of patients with colorectal cancer. The amount of DAF present in the stools might be influenced by the stability of DAF on the cell surface which is regulated by biochemical properties such as glycosylation of the protein. In the present study, to help elucidate the mechanism for the release of DAF from human colorectal cancers, we biochemically analyzed DAF expression by western and northern blotting by using surgically resected specimens of colorectal cancers. METHODS: Surgically resected colorectal cancer tissues were obtained from 10 patients. Expression of DAF was determined by western and northern blotting, and glycosylation of DAF protein was analyzed with glycosidase digestion. RESULTS: Northern blot analysis demonstrated that the expression of DAF mRNA in colorectal cancer was enhanced two- to threefold compared with normal tissues. In western blotting, expression of DAF protein in the cancer tissue was increased, and heterogeneity in the apparent molecular weight of DAF was observed among patients. When o-linked sugars were removed, this heterogeneity of DAF size diminished. CONCLUSIONS: The polymorphic expression of DAF in colorectal cancer is likely to reflect variability in the o-glycosylation of the protein. We speculate that this variability could affect the stability of DAF on the surfaces of cancer cells and, in turn, the amount of DAF shed into the stools of colorectal cancer patients.     

In vivo evidence that inhibin is a gonadotropin surge-inhibiting/attenuating factor.

Hyperstimulation of ovarian function through exogenous gonadotropin administration suppresses the preovulatory surges of both LH and FSH. This phenomenon has been demonstrated to be the result of a non-steroidal factor released from the ovary that has been designated as either gonadotropin surge-inhibiting factor or attenuating factor (GnSIF/AF). To examine the possibility that inhibin might possess the activity ascribed to this factor, endogenous inhibin was immunoneutralized in female rats under conditions known to stimulate GnSIF/AF activity. Inhibin-like immunoreactivity was found to be significantly elevated in FSH-treated rats prior to the time of the gonadotropin surges. Spontaneous preovulatory surges of both LH and FSH were observed in saline-treated (control) rats that were subsequently treated with either anti-inhibin serum (AS) or normal sheep serum (NS). FSH-treatment completely prevented the occurrence of gonadotropin surges in rats subsequently treated with NS. In FSH-injected rats subsequently treated with AS, however, normal preovulatory surges of both LH and FSH were observed. These results indicate that inhibin may be the factor responsible for the suppression of the preovulatory gonadotropin surges in FSH-treated rats and, at least in this species, may be a GnSIF/AF.     

In vitro and in vivo activity of human recombinant granulocyte-macrophage colony-stimulating factor in dogs

Although it is well documented that human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production and functional activity of human and nonhuman primate granulocytes and macrophages, relatively little is known about its effects on cells obtained from other species. The molecular cloning of the complementary DNA for human GM-CSF has made it possible to determine the cross-reactivity of the purified recombinant human material (rhGM-CSF) on cells of other species. The results presented herein show that specific receptors for human GM-CSF exist on dog bone marrow cells and mature circulating dog granulocytes. The number of the receptors and the apparent binding affinity of the rhGM-CSF to its receptors on granulocytes were similar to those observed either on human or monkey cells. In cultures of dog bone marrow cells, rhGM-CSF was capable of promoting colony formation in a dose-dependent manner. Human GM-CSF also primed dog granulocytes for increased production of reactive oxygen metabolites in response to either phorbolmyristic acetate-or zymosan-activated dog serum. In vivo, s.c. administration to healthy dogs of rhGM-CSF in daily doses of 15, 50, or 150 micrograms/kg body weight over a period of 7-20 days induced a dose-dependent rise of up to a maximum of a fourfold increase in peripheral WBC counts. The rise in WBC counts was mainly due to elevated neutrophil levels, but an increase in the numbers of monocytes and eosinophils was also observed. However, the rhGM-CSF-induced leukocytosis in dogs was not as dramatic as that observed in nonhuman primates. In all rhGM-CSF-treated dogs, circulating platelet counts dropped to nadir levels of about 20%-30% of normal numbers. Dogs that were treated with 150 micrograms/kg rhGM-CSF developed specific antibodies after about 10-12 days of treatment. These antibodies were able to neutralize the effect of rhGM-CSF in in vitro assays. In vivo WBC counts began to decline when specific antibodies developed, but they never dropped below normal levels. Taken together, the results suggest that human GM-CSF does not appear to exhibit absolute species specificity.     

In vivo evidence that cGMP is the second messenger for atrial natriuretic factor.

cGMP generation has been associated with many of the vascular and endocrine actions of atrial natriuretic factor (ANF) in vitro. To examine the role of cGMP as a second messenger for the renal hemodynamic action of ANF in vivo, we measured glomerular filtration rate (GFR) and cGMP concentration in systemic artery, renal vein, and urine as well as in Bowman's space and end-proximal tubule (by free-flow micropuncture) after administration of ANF. ANF increased GFR by 45% and simultaneously induced a greater than 5-fold increase of cGMP concentration in glomerular ultrafiltrate (Bowman's space) when compared to controls. There was no significant increase in either systemic artery or renal vein cGMP concentration. Thus, the source of increased Bowman's space cGMP is not from the blood via filtration but rather from either glomerular mesangial or epithelial cells, which are not in direct contact with the circulation. Although a small amount of tubular handling of cGMP occurred along the length of the nephron, the augmented cGMP production from the glomerulus accounted for most of the 10- to 12-fold higher urinary cGMP excretion observed after ANF administration. Intrarenal arterial infusion of dibutyryl cGMP, but not dibutyryl cAMP, increased GFR in a dose-dependent fashion (from 10 to 1000 microM) by a mechanism similar to that of ANF--an increase in glomerular hydraulic pressure. Thus, ANF markedly stimulated glomerular production of cGMP, which coincided with a marked increase in GFR. Since dibutyryl cGMP itself was capable of increasing GFR, cGMP is the likely second messenger for ANF in vivo.     

In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor.

About 100 derivatives of human recombinant granulocyte colony-stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell-proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.     

In vivo and in vitro evidence for the glycoxidation of low density lipoprotein in human atherosclerotic plaques

Although there have been suggestions that the glycation and oxidation of low density lipoprotein (LDL) might increase its atherogenic potential, little is known about the presence of glycoxidative LDL in human atherosclerotic lesions. We developed specific antibodies against different immunological epitopes of AGE structures, including N(epsilon)-(carboxymethyl)lysine-protein adduct (CML), a glycoxidation product, and structure(s) other than CML (nonCML), and a monoclonal antibody against oxidized phosphatidylcholine (oxPC), as an epitope of oxidized LDL. Immunohistochemical analysis demonstrated that the CML- and oxPC-epitopes were accumulated mainly in macrophage-derived foam cells in atherosclerotic lesions, including fatty streaks and atherosclerotic plaques. On the other hand, the nonCML-epitope and apolipoprotein B were localized mainly in extracellular matrices of atherosclerotic lesions. The CML- and oxPC-epitopes were characterized by a model antigen-generating system using the copper ion-induced peroxidation and/or glucose-induced glycation of LDL. The glycoxidation of LDL caused the formation of CML-epitope with increasing concentrations of copper ion and glucose. It was also formed to some extent in LDL incubated with high concentrations (500 mM) of glucose. However, no CML-epitope was observed in oxidized LDL induced by copper ion alone. On the other hand, the formation of oxPC-epitope in LDL was dependent on copper ion-induced peroxidation, but independent of glucose-induced glycation. The addition of chelators, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, reduced the increase in electrophoretic mobility and TBARS caused by the peroxidation and glycoxidation of LDL, but had no effects on the formation of fructosamine caused by the glycation and glycoxidation of LDL. Chelators as well as aminoguanidine protected the formation of CML-epitope in glycated or glycoxidative LDL. Although the formation of oxPC-epitope was completely inhibited by the addition of chelators, it was partially protected by aminoguanidine. These in vitro results suggest that the glycoxidative modification of LDL may occur in the arterial intima, and may contribute to the development of human atherosclerotic lesions.     

In vivo and in vitro effects of thrombin and plasmin on human factor VIII (AHF).

The relationship between factor VIII (AHF) procoagulant activity and factor VIII-related antigen were examined in patients with disseminated intravascular coagulation (DIC), pulmonary embolism (PE), and coronary artery disease with or without myocardial infarction (MI). It was found that 13 of 13 patients with DIC, 17 of 17 patients with PE, and 10 of 12 patients with MI possessed a significantly elevated factor VIII-related antigen to factor VIII activity ratio (VIII-ratio). The VIII-ratio returned to normal in each of 2 patients with DIC and 1 paitent with PE after treatment with heparin, heparin and alpha-amino-caproic acid, and heparin and coumadin respectively. In contrast, the VIII-ratio was slightly elevated only in 1 of 15 patients with coronary artery insufficiency without MI. In in vitro studies, after treatment of plasma with thrombin or plasmin, factor VIII activity was lost, whereas the amount of factor VIII-related antigen remained the same or was even increased when measured by agarose quantitative immunoelectrophoresis. These observations have led us to conclude that an elevated VIII-ratio is a very sensitive indicator of intravascular coagulation.     

In vivo and in vitro evidence of B cell hyperactivity during Lyme disease

In vitro IgM production by cells from patients with Lyme disease rose during the illness in those studied soon after onset, but fell from elevated levels in those initially studied later than 1 week after onset. Borrelia burgdorferi stimulated normal and patients' cells produced IgM, with cells from acutely ill patients producing the most; production fell during convalescence. Patients with active Lyme disease and those destined for later manifestations often had serum agglutinins for heterologous red blood cells. Thus, there is in vitro and in vivo B cell hyperactivity in Lyme disease caused by B. burgdorferi. Both mitogenic and antigenic stimulation of B cells may induce the humoral response seen in complicated Lyme disease.     

In vivo evidence that endogenous dopamine modulates sympathetic activity in man.

Dopamine receptors type 2 (D2)-like receptor blockers cause an increase in the norepinephrine response to intense physical exercise. However, during intense physical exercise, D2-like antagonists also cause an increase in the epinephrine response, which itself might cause an increase in plasma norepinephrine through the activation of beta2 presynaptic receptors. Therefore, we evaluated the effect of domperidone, a D2-like antagonist, on the norepinephrine response to physical exercise in 6 Addison patients (3 were adrenalectomized and 3 had adrenal tuberculosis). In these patients, the norepinephrine increase observed during exercise was significantly higher after the administration of domperidone than a placebo (F=4,328; P<0.001). Because peripheral plasma norepinephrine does not reflect the sympathetic tone to the heart accurately, we evaluated the effect of domperidone administration (20 mg orally) on the sympathovagal balance, which was measured by the ratio between the high- and low-frequency components of heart rate variability, in 9 normal volunteers in the supine and sitting positions. When compared with placebo, domperidone caused a significant increase in the low/high frequency ratio (P<0.05) in the sitting position without modifying basal and stimulated norepinephrine plasma levels or blood pressure. These data support a role for endogenous dopamine in modulating norepinephrine release by human sympathetic nerves in vivo.     

In vivo and in vitro studies of angiogenin--a potent angiogenic factor.

Angiogenin is a potent blood-vessel-inducing polypeptide with a molecular weight of 14,000 that has a unique ribonucleolytic activity. First isolated from the conditioned medium of tumour cells, angiogenin has since been purified from normal plasma, which suggested that its propensity to induce neovascularization should be strictly controlled. Modulation of that activity might involve interaction of angiogenin with cell-surface receptors and extracellular matrix of endothelial cells, tight-binding inhibition of both its ribonucleolytic activity and cell binding property by ribonuclease inhibitor, as well as the overall influence of divalent copper, a modulator of angiogenesis.     

In vivo and in vitro evidence for slipped mispairing in mammalian mitochondria.

Slipped mispairing between repeated sequences during DNA replication is an important mutagenic event. It is one of several suggested mechanisms thought to be responsible for generating polymorphic regions and large-scale deletions found in mammalian mitochondrial DNA. In the porcine mitochondrial genome, a domain carrying a 10-bp tandemly repeated sequence displays a unique in vivo pattern of repeat copy number polymorphs. Upon passage in Escherichia coli, a recombinant plasmid containing this domain also displays a unique polymorphic pattern that is different from that seen in the animal. To test the hypothesis that these polymorphisms were slippage induced and that the different polymorphic patterns reflected differences in modes of replication, we performed a series of in vitro primer extension reactions. By utilizing either single- or double-stranded templates containing the repeat domain we were able to correlate in vitro generated repeat polymorphism patterns with those seen in the mitochondria or the bacteria, respectively, thus providing experimental evidence that slippage replication is responsible for a major class of mammalian mutations.     

Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.

Human endometrial stromal (ES) cells in culture express PRL, a marker of decidualization, in response to sustained activation of protein kinase A (PKA). Cotreatment with the progestin medroxyprogesterone acetate (MPA) enhanced decidual PRL gene activation in the presence of elevated intracellular cAMP levels. This synergy became apparent, at protein and promoter level, after a lag period of 2 days and increased in a time-dependent manner thereafter. Pretreatment with cAMP advanced the time at which synergy between cAMP and MPA was apparent, suggesting that PKA activation sensitized ES cells to the effects of progestins. Analysis of the progesterone receptor (PR) indicated that PR-A was the predominant form in differentiating ES cells, but its abundance decreased markedly during the course of the decidualization response. The decline in PR levels was of functional relevance, as expression of PR-B or PR-A, by transient transfection, dramatically inhibited the activity of a decidual PRL promoter-reporter construct in response to cAMP. Furthermore, the expression of endogenous PRL protein in response to cAMP or cAMP plus MPA was substantially decreased by constitutive expression of green fluorescence protein-tagged PR, which was localized in the nucleus even in the absence of added ligand. Ligand-independent PR inhibition of the decidual PRL promoter was receptor specific, independent of known PR phosphorylation sites, and required minimally a functional DNA-binding domain. Transient expression of steroid receptor coactivator-1e (SRC-1e), but not SRC-1a, allowed synergy between cAMP and MPA without the requirement of sensitization by pretreatment with cAMP. This raised the possibility that SRC-1e was a component of cAMP-dependent sensitization of ES cells, but there was no evidence of altered messenger RNA expression of either SRC-1 isoform during decidualization. In conclusion, cellular PR levels determine the onset of the decidualization response. Initiation of this process requires elevated intracellular cAMP levels that sensitize ES cells to the actions of progestins through down-regulation of cellular PR levels and possibly via modulation of function of an intermediate factor(s) such as SRC-1e.     

In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair.

This report details the transfer of a human epidermal growth factor (hEGF) expression plasmid to porcine partial-thickness wound keratinocytes by particle-mediated DNA transfer (Accell). After gene transfer an external sealed fluid-filled wound chamber was used to protect the wound, provide containment of the exogenous DNA and expressed peptide, and permit sampling of the wound fluid. Analysis of wound fluid for hEGF and total protein, an indicator of reformation of the epithelial barrier, showed that wounds bombarded with the hEGF plasmid exhibited a 190-fold increase in EGF concentration and healed 20% (2.1 days) earlier than the controls. EGF concentrations in wound fluid persisted over the entire 10-day monitored period, decreasing from 200 pg/ml to 25 pg/ml over the first 5 days. Polymerase chain reaction results showed that plasmid DNA was present in the wound for at least 30 days. These findings demonstrate the possible utility of in vivo gene transfer to enhance epidermal repair.     

Tumor necrosis factor alpha regulates alpha(v)beta5 integrin expression by osteoclast precursors in vitro and in vivo

Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable alpha(v)beta3 integrin, contain abundant alpha(v)beta5 and attach to matrix in an alpha(v) integrin-dependent manner. Furthermore, alpha(v)beta5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-alpha, (TNF) down-regulates alpha(v)beta5 expression by BMMS via attenuation of beta5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the beta5 suppressive effect. The functional implications of TNF-mediated alpha(v)beta5 down-regulation are underscored by the capacity of an alpha(v) inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant alpha(v)beta5 while failing to impact those in which the integrin has been diminished by TNF. Finally, beta5 mRNA in BMMs of wild-type mice administered lipopolysaccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, beta5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the beta5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix attachment of osteoclast precursors and mature osteoclasts are governed by distinct alpha(v) integrins which are differentially regulated by specific cytokines.