J R Stanley
Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation. The results demonstrate the selective destruction of established human colon carcinoma transplants by intravenous injection of either single or fractionated doses of 131I-MAb F(ab')2.
F Buchegger, C Pfister, K Fournier, F Prevel, M Schreyer, S Carrel, J P Mach
We have investigated the role of iron in the intracellular biology of Legionella pneumophila in human monocytes and in the effector arm of cell-mediated immune defense against this intracellular bacterial pathogen. To determine if L. pneumophila intracellular multiplication is iron dependent, we studied the effect of the iron chelator deferoxamine on L. pneumophila infection of monocytes. Deferoxamine at 15 microM completely inhibited L. pneumophila intracellular multiplication. The inhibitory effect of deferoxamine was reversed with equimolar iron-saturated transferrin but not apotransferrin. To examine the potential role of iron in monocyte activation, we investigated the influence of iron-saturated transferrin on L. pneumophila multiplication in IFN gamma-activated monocytes. Iron transferrin, but not apotransferrin, neutralized the capacity of activated monocytes to inhibit L. pneumophila multiplication. To explore a potential mechanism by which activated monocytes might limit the availability of intracellular iron, we examined transferrin receptor expression on nonactivated and activated monocytes cultured in vitro for 5 d. By fluorescence-activated flow cytometry, activated monocytes exhibited markedly fewer transferrin receptors than nonactivated monocytes. By Scatchard analysis of 125I-transferrin binding to monocytes, nonactivated monocytes had 38,300 +/- 12,700 (mean +/- SE) transferrin binding sites, whereas activated monocytes had 10,300 +/- 1,600, a reduction of 73%. Activated and nonactivated monocytes had a similar mean Kd (1.8 +/- 0.2 nM). This study demonstrates that (a) L. pneumophila intracellular multiplication is iron dependent; (b) activated monocytes inhibit L. pneumophila multiplication by limiting the availability of intracellular iron; and (c) transferrin receptors are downregulated on IFN gamma-activated monocytes.
T F Byrd, M A Horwitz
The lytic effect of complement on human erythrocytes has been reported by others to increase when Na+ is substituted for K+ in the external medium. In this paper we have investigated the hypothesis that net loss of K+ through a K+ transport pathway protects erythrocytes from complement-induced colloidosmotic swelling and lysis. Antibody-sensitized human erythrocytes containing different intracellular cation concentrations (nystatin treatment) were exposed to low concentrations of guinea pig serum in media of different cation composition; complement lysis was assessed by the release of hemoglobin and the volume of the surviving cells estimated by their density distribution profiles. Complement-dependent swelling and lysis of erythrocytes (a) were limited by the presence of an outwardly directed K+ electrochemical gradient and (b) were enhanced by carbocyanine, a specific inhibitor of the Ca2+-activated K+ transport pathway, and by absence of Ca2+ in the external medium. We propose that during complement activation a rising cytosolic calcium triggers the Ca2+-activated K+ permeability pathway, the Gardos effect, produces a net K+, Cl- and water loss, and thus limits the colloidosmotic swelling and lysis of erythrocytes.
J A Halperin, C Brugnara, A Nicholson-Weller
We tested the hypothesis that strain-specific immunity occurs after gonococcal infection in a longitudinal study of 227 prostitutes resident in one small community who experienced frequent gonococcal infections. Women were examined and cultured for Neisseria gonorrhoeae at 2-wk intervals. Gonococcal isolates were typed according to protein 1 serovar, auxotype, and beta-lactamase plasmid type, and classified as to serovar and strain. The hypothesis was tested by comparing the predictions of the hypothesis with the observations of the study. Over the 14-mo period of the study, major changes in the prevalence of specific serovars were observed in the gonococcal population infecting these women. Women with HIV infection experienced a higher rate of gonococcal infection (0.56 +/- 0.03 vs. 0.46 +/- 0.04, P less than 0.05, t test) compared with HIV-negative women and were more likely to experience multiple infections with the same strain. The duration of prostitution was inversely related to the frequency of gonococcal infection. Women experiencing an infection with a specific gonococcal serovar were at a 2- to 10-fold reduced risk of reinfection with the same serovar, except for the 1B-1 serovar. The results of the study were consistent with all four predictions of the hypothesis. Infection with a specific gonococcal serovar results in specific but incomplete protection against subsequent infection with the homologous serovar. The mechanism of this protection remains to be determined.
F A Plummer, J N Simonsen, H Chubb, L Slaney, J Kimata, M Bosire, J O Ndinya-Achola, E N Ngugi
We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.
A M Gewirtz, B Calabretta, B Rucinski, S Niewiarowski, W Y Xu
We have identified two distinct beta-myosin heavy chains (MHCs) present in baboon myocardium by electrophoresis in gradient pore gels and by Western blots with anti-MHC MAb. The two beta-MHCs have molecular masses of 210 and 200 kD and share several antigenic determinants including an epitope recognized by a beta-MHC-specific MAb. A fivefold increase in the level of the 200-kD beta-MHC was observed in the hypertrophied left ventricles of baboons with chronic (5.3 +/- 0.7 yr) renal hypertension. A 60% increase (P less than 0.01) in BP and a 100% increase (P less than 0.001) in left ventricular mass to body weight ratio occurred in hypertensive baboons compared with normotensive animals. The Ca2+-activated myosin ATPase activity in hypertrophied left ventricles was decreased by 35% (P less than 0.05) compared with controls. Normal levels of the 200-kD MHC were detected in the right ventricles and intraventricular septa of the hypertensive animals. These data suggest that cardiac MHCs of primates may exist in alternative molecular forms that are indistinguishable by nondenaturing gel electrophoresis and that increased concentration of a second beta-MHC is associated with ventricular hypertrophy (r = 0.55). The functional significance and mechanisms that control the concentration of beta-MHC subspecies remain to be determined.
R D Henkel, J L VandeBerg, R E Shade, J J Leger, R A Walsh
We asked this question: in normal humans, is either a normal dietary intake or normal serum concentration of phosphorus a determinant of the serum concentration of 1,25(OH)2D? In seven normal men whose dietary phosphorus was decreased from 2,300 to 625 mg/d, each intake for 8-9 d, under strictly controlled, normal metabolic conditions, we measured serum concentrations of 1,25(OH)2D daily, and concentrations of phosphorus hourly throughout a 24-h period, before and after restriction. Decreasing dietary phosphorus induced: (a) a 58% increase in serum levels of 1,25(OH)2D; (b) a 35% decrease in serum levels of phosphorus measured in the afternoon; (c) a 12% decrease in the 24-h mean serum level of phosphorus; but, (d) no decrease in morning fasting levels of phosphorus. Serum concentrations of 1,25(OH)2D varied inversely and significantly with 24-h mean concentrations of phosphorus (r = -0.77, P less than 0.001). When these data are combined with those of our prior study in which dietary phosphorus was varied over an extreme range, the relationship between serum levels of 1,25(OH)2D and 24-h mean serum levels of phosphorus is even stronger (r = -0.90, P less than 0.001). In the aggregate, the results demonstrate that in normal men, dietary phosphorus throughout a normal range and beyond, can finely regulate the renal production and serum concentration of 1,25(OH)2D, and provide evidence that this regulation is mediated by fine modulation of the serum concentration of phosphorus.
A A Portale, B P Halloran, R C Morris Jr
The regulation of water and electrolyte homeostasis is multifactorial and includes the heart and kidneys as important regulatory centers. Within the heart, a recently discovered hormone, atrial natriuretic factor (ANF), has been implicated in the maintenance of water and salt balance. Primarily found in mammalian atria, ANF has been detected in low amounts in several tissues, including lungs. A disorder of the ANF system has been demonstrated in genetically cardiomyopathic hamsters, a model for human congestive cardiomyopathy. Atrial ANF gene expression and storage are decreased during development of this disease, while paradoxically, circulating levels of ANF are increased. We have hypothesized that an extracardiac source may contribute to ANF production in these pathological conditions. In this paper we provide evidence that ANF synthesis is stimulated in the lungs of hamsters during development of cardiomyopathy as revealed by increased ANF mRNA and peptide levels. Furthermore, we show that ANF synthesized in lungs is secreted and has identical chromatographic and biological properties to circulating ANF. The increased production of ANF in lungs may be physiologically important in preventing pulmonary edema. Alternatively, during cardiac dysfunction, lungs may play a compensatory role by increasing their contribution to plasma ANF levels.
J Gutkowska, M Nemer, M J Sole, J Drouin, P Sirois
We used cultured human diploid lung fibroblasts as a model system to examine the effects of recombinant IFN-gamma on synthesis of collagen, matrix deposition of newly synthesized collagen, and the expression of cell surface receptors for collagen. Using [3H]proline-labeled cells we found that IFN-gamma resulted in dose-dependent inhibition of fibroblast collagen synthesis. Pulse-chase experiments to analyze compartmentalization of newly synthesized collagen showed that the decrease in collagen synthesis was confined to the soluble pool of procollagen in the medium, while extracellular matrix associated collagen was not changed, indicating that a larger proportion of newly synthesized collagen was deposited into the matrix in IFN-gamma exposed fibroblasts (34.2 vs. 25.3%). This increase in the efficiency of collagen matrix deposition was associated with enhanced expression of a cell surface receptor for collagen as detected by indirect immunofluorescence labeling and analysis by flow cytometry. Fibroblasts (IMR-90) cultured in the presence of IFN-gamma (1,000 U/ml) exhibited a twofold increase in mean linear fluorescence intensity compared with cells cultured under control conditions. The distribution of log fluorescence intensity in both control and IFN-gamma exposed cells was normally distributed about the mean, indicating that discrete subpopulations with respect to receptor expression were not present. Increased fluorescence intensity and log normal distribution of fluorescence intensity also were identified in IFN-gamma-treated lung fibroblasts from a normal adult individual and two strains obtained from patients with pulmonary fibrosis. These results indicate that IFN-gamma modulates fibroblast collagen matrix deposition as well as collagen synthesis. The associated increase in collagen receptors suggests that cytokine-mediated modulation of the cell surface maybe a contributing factor in regulation of fibroblast collagen accumulation in the extracellular matrix or in cellular interaction with collagen-containing matrix. Such an effect could modulate the interaction of fibroblasts with extracellular matrix at sites of inflammation and play an important role in the remodeling of matrix during repair from tissue injury.
J G Clark, T F Dedon, E A Wayner, W G Carter
In the present report we demonstrate that the IL-6 gene is expressed in anti-Ig-activated and neoplastic B cells. After activation with anti-Ig, splenic B cells rapidly expressed IL-6 mRNA with peak expression occurring at 4 h and declining rapidly thereafter. In an attempt to exclude that the IL-6 mRNA expression was in non-B cells, T cells and monocytes were extensively depleted. In this highly purified B cell population, IL-6 mRNA was retained, whereas the expression of the T cell- and monocyte-restricted CD2 and CD14 genes was nearly undetectable. These results are consistent with the conclusion that activated B cells express IL-6 mRNA. Because we found IL-6 mRNA expression in normal activated B lymphocytes, we examined the expression of IL-6 mRNA in B cell neoplasms. 11 of 25 non-Hodgkins B cell lymphomas and 4 of 4 myelomas and plasma cell leukemias expressed IL-6 mRNA, whereas only 1 of 19 B cell leukemias was positive. To exclude that IL-6 mRNA expression in neoplastic B cells was the result of contaminating non-B cells, T cells and monocytes were extensively depleted from the tumor specimens. In the three IL-6-positive tumor samples depleted of T cells and monocytes, IL-6 mRNA expression was retained in all cases. These observations provide support for the idea that the IL-6 gene is expressed in normal activated and neoplastic B cells.
G J Freeman, A S Freedman, S N Rabinowe, J M Segil, J Horowitz, K Rosen, J F Whitman, L M Nadler
To better define the inflammatory infiltrates and kinetics of mediator release during the cutaneous late-phase reaction (LPR), we examined skin biopsies at 8 h, and skin chamber cell counts and mediator release for 12 h after antigen challenge. Compared with the control sites, the antigen-stimulated biopsy sites contained 14 times as many basophils (P less than 0.01) and six times as many eosinophils (P less than 0.001) with one to two fold more mononuclear cells (P less than 0.03) and neutrophils (P less than or equal to 0.01). Similar changes were found in the skin chambers. Although there were neutrophils in the control chamber, they were only twice as numerous in the antigen challenged site (P less than 0.01). Eosinophils were 35-fold (P less than or equal to 0.03) more prevalent in the antigen chamber than the control chamber for hours 8-12 and basophils were noted starting in the eighth hour and were 20-fold (P less than or equal to 0.03) more concentrated in the antigen chamber during the next 4 h. The mononuclear cells were not significantly different between antigen and control blisters. With respect to inflammatory mediators, there was an initial peak of histamine (13.2 +/- 2.9 ng/ml) in the blister fluid at 1 h. The level then fell to approximately 2 ng/ml, followed by a secondary rise starting at the eighth hour and increasing to 9.8 +/- 2.8 ng/ml by the twelfth hour. This secondary increase in histamine correlated significantly (r = 0.81, P less than 0.05) with the observed influx of basophils. PGD2 in the blister fluid rose to 371+/-25 pg/ml during the first 4 h and then slowly decreased to half this level during the last 4 h. Thus, the cutaneous LPR has been shown to manifest a secondary increase in histamine levels and a markedly specific increase in eosinophils and basophils with mediator release apparently being derived from the latter cells.
E N Charlesworth, A F Hood, N A Soter, A Kagey-Sobotka, P S Norman, L M Lichtenstein
We used tuberculous pleuritis as a model to study the compartmentalization and potential immunoregulatory role of 1,25-dihydroxyvitamin D [1,25-(OH)2-D] in human granulomatous disease. In tuberculous pleuritis, mean concentrations of total 1,25-(OH)2-D were elevated in pleural fluid, compared to blood (67 pg/ml vs. 35 pg/ml). Concentrations of albumin, protein and 25-hydroxyvitamin D (25-OH-D) were lower in pleural fluid than blood, suggesting that accumulation of binding proteins does not explain the transpleural gradient of 1,25-(OH)2-D. The mean free 1,25-(OH)2-D concentration in pleural fluid was increased 5.3-fold over that in serum. 1,25-(OH)2-D3 inhibited PPD-induced proliferation of pleural fluid mononuclear cells, antigen-reactive lines and T lymphocyte clones derived from a single cell. Patient-derived PPD-reactive lines expressed a high-affinity intracellular binding moiety for 1,25-(OH)2-D3. Pleural fluid mononuclear cells and PPD-reactive lines did not metabolize 25-OH-D3 to 1,25-(OH)2-D3. The sum of these data suggests that concentration of 1,25-(OH)2-D in pleural fluid of tuberculosis patients is probably due to local hormone production by pleural tissue-based inflammatory cells that are not present in significant numbers in pleural fluid. Elevated concentrations of 1,25-(OH)2-D in pleural fluid may exert receptor-mediated inhibition of antigen-induced proliferation by pleural fluid lymphocytes. Inhibition of lymphocyte proliferation and lymphokine production may prevent tissue destruction from an uncontrolled inflammatory response.
P F Barnes, R L Modlin, D D Bikle, J S Adams
The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(3-44)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. D-Amino acid substitution at either position 1 or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(3-44)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).
L A Frohman, T R Downs, E P Heimer, A M Felix
Removal of the gallbladder is thought to increase formation and pool size of secondary bile acids, mainly deoxycholic acid (DCA), by increased exposure of primary bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA]) to bacterial dehydroxylation in the intestine. We have tested this hypothesis by simultaneous determination of pool size and turnover of DCA, CA, and CDCA in nine women before and at various intervals after removal of a functioning gallbladder. An isotope dilution technique using marker bile acids labeled with stable isotopes (2H4-DCA, 13C-CA, 13C-CDCA) was used. After cholecystectomy, concentration and output of bile acids relative to bilirubin increased (P less than 0.02) in fasting duodenal bile and cholesterol saturation decreased by 27% (P less than 0.05) consistent with enhanced enterohepatic cycling of bile acids. Three months after removal of the gallbladder bile acid kinetics were in a new steady state: pool size and turnover of CDCA were unchanged. Synthesis of CA, the precursor of DCA, was diminished by 37% (P = 0.05), probably resulting from feedback inhibition by continuous transhepatic flux of bile acids. The fraction of CA transferred after 7 alpha-dehydroxylation to the DCA pool increased from 46 +/- 16 to 66 +/- 32% (P less than 0.05). However, this enhanced transfer did not lead to increased input or size of the DCA pool, because synthesis of the precursor CA had decreased.
F Berr, F Stellaard, E Pratschke, G Paumgartner
Tryptase, a neutral protease of human mast cells, is a potentially important indicator of mast cell involvement in various clinical conditions. The current study examined the time course of appearance and disappearance of tryptase in the circulation after an anaphylactic event and the stability of both endogenous and exogenous tryptase in terms of freeze-thawing and temperature. The peak level of tryptase after an experimentally induced systemic anaphylactic reaction occurred 1-2 h after the initiating bee sting in each of three subjects, in contrast to histamine levels which peaked at 5-10 min. In some cases elevated levels of tryptase may not be detected during the initial 15-30 min. Tryptase levels then declined under apparent first order kinetics with a t1/2 of approximately 2h. Similar disappearance kinetics were observed for two subjects presenting in the emergency room with immediate type reactions, one with severe asthma after indomethacin ingestion, the other with systemic anaphylaxis after a bee sting. Histamine levels declined rapidly and were back to baseline by 15-60 min. Measured levels of tryptase in serum or plasma were not diminished by up to four freeze-thaw cycles. Incubation of serum samples taken from subjects with elevated levels of tryptase at 22 and 37 degrees C indicated that greater than 50% of endogenous tryptase was still detected after 4 d. Purified tryptase added to serum or plasma and incubated as above was less stable: approximately 50% of exogenous tryptase in serum and approximately 15% in plasma was detected after 2d of incubation. Therefore, optimally samples should be stored frozen, but even those stored at room temperature for up to 4 d should be satisfactory. The best time to obtain samples for tryptase determinations is 1-2 h after the precipitating event, but depending on the magnitude of the initial response elevated levels of tryptase may be present in the circulation for several hours.
L B Schwartz, J W Yunginger, J Miller, R Bokhari, D Dull
Subacute cutaneous lupus and neonatal lupus are closely associated with the presence of anti-Ro (SSA) autoantibodies, but there is no direct evidence establishing a role for anti-Ro (SSA) in these diseases. After parental injection into mice, IgG from sera containing anti-Ro (SSA) will bind human skin grafted onto the mice. To determine whether the antibody binding is due to anti-Ro (SSA), affinity-purified anti-Ro (SSA) and serum depleted of anti-Ro (SSA) were prepared. After injection into human skin-grafted mice, purified anti-Ro (SSA) antibodies bound an antigen in the human skin graft, while preabsorbing anti-Ro (SSA) serum with Ro (SSA) virtually abolished binding to the human skin graft. Moreover, the pattern of IgG deposition was primarily epidermal and was identical in the human skin-grafted mice injected with purified anti-Ro (SSA) when compared with that found in five patients with subacute lupus (four adults, one neonate). These data directly show that anti-Ro (SSA) antibodies bind to the skin, and support the hypothesis that anti-Ro (SSA) autoantibodies are involved in the disease process that produces subacute cutaneous lupus and neonatal lupus.
L A Lee, K K Gaither, S N Coulter, D A Norris, J B Harley
Little is known regarding regional atrial blood flow responses during varying hemodynamic states in both the normal and hypertrophied atria. This study was undertaken to develop a canine model of chronic atrial hypertrophy and to define in both this group and in normal dogs the regional blood flow response to acute atrial fibrillation and to measure coronary flow reserve. In the 12 dogs with atrial but not ventricular hypertrophy the mean left and right atrial weights were 75 and 47% respectively greater than in the normal group. Blood flow in the normal dogs was less in the appendage than in the non-appendage region for both atria and increased significantly during atrial fibrillation. Similar findings were noted in the hypertrophy group except that during control conditions the left atrial appendage flow was similar to the nonappendage flow. Minimal vascular resistance for the hypertrophy group, 39 +/- 3 was significantly (P less than 0.05) greater when compared to the normal group 28 +/- 2 mmHg/cm3 per min per g. Thus, significant regional blood flow differences occur in both the normal and hypertrophied atria. In addition, atrial hypertrophy does not alter the autoregulatory capacity to the hemodynamic stress of atrial fibrillation but does reduce coronary flow reserve.
R P Bauman, J C Rembert, J C Greenfield Jr
Recombinant tumor necrosis factor (rTNF) and rIFN-gamma induce in the human leukemia cell lines HL-60, ML3, and U937 the accumulation of transcripts of the X chromosome-linked chronic granulomatous disease (X-CGD) gene, encoding the 91-kD heavy chain of cytochrome b-245, a component of the NADPH oxidase of phagocytic cells. The gene is induced within 6 h by either cytokine, and its accumulation is observed upon induction with rIFN-gamma up to 5 d. The combined effect of the two cytokines is more than additive. rIFN-gamma also induces accumulation of X-CGD mRNA in immature myeloid cells from peripheral blood of chronic myeloid leukemia (CML) patients, whereas rTNF has almost no effect. The cells from CML patients constitutively express TNF mRNA, suggesting that endogenously produced TNF may play a role in the effect of rIFN-gamma on these cells. rTNF induces X-CGD gene expression in the myeloid cell lines acting, at least in part, at the transcriptional level, as shown in nuclear run-on experiments. The gene encoding the 22-kD light chain of cytochrome b-245 is constitutively expressed in the human myeloid cell lines and the accumulation of its transcripts is affected by neither rTNF nor rIFN-gamma, rTNF and rIFN-gamma synergistically to induce the cell lines to express the cytochrome b-245 heterodimer (as evaluated by its visible spectrum), and to produce NADPH oxidase activity and H2O2 upon stimulation with phorbol diesters.
M A Cassatella, L Hartman, B Perussia, G Trinchieri
Prosomatostatin (pro-S) and its bioactive posttranslational products, somatostatin-14 (S-14), somatostatin-13 (S-13), and somatostatin-28 (S-28), were measured in human plasma by the use of immunoglobulins to the NH2-terminus of S-28 conjugated with agarose to separate them and, thereafter, by RIA with an antiserum recognizing the COOH-terminus of pro-S, and by specific RIA for the NH2-terminus of S-14 and pro-S. In healthy men, mean basal levels of pro-S were 4 pg equivalent S-14/ml; S-14/S-13 combined were 9 pg equivalent S-14/ml; and S-28 levels were 16 pg/ml. After a 700-kcal meal, pro-S, S-14, and S-14/S-13 did not change, whereas S-28 levels doubled by 120 min and remained elevated for 240 min. To evaluate the origins of these peptides, their levels were compared in peripheral, portal, gastric, and mesenteric veins of anesthetized patients and in patients with total resection of stomach and pancreas before and after nutrient intake. The stomach and small intestine were sources of both peptides; however, most S-28 originated in the small intestine. These findings suggest that, in contrast to S-14, S-28 is a hormone and may modulate postprandial nutrient absorption and use.
J W Ensinck, E C Laschansky, R E Vogel, D A Simonowitz, B A Roos, B H Francis
In an abnormal fibrinogen with severely impaired polymerization of fibrin monomers, we identified a methionine-to-threonine substitution at position 310 of the gamma chain. Furthermore, asparagine at position 308 was found to be N-glycosylated due to a newly formed consensus sequence, asparagine(308)-glycine(309)-threonine(310). The two structural defects in the mutant gamma chain may well perturb the conformation required for fibrin monomer polymerization that is specifically assigned to the D domain of fibrinogen. This alteration also seems to affect the intermolecular gamma chain cross-linking of fibrin and fibrinogen, although the amine acceptor gamma glutamine-398 was found to function normally. These functional abnormalities may well be related to posttraumatic hemorrhage as observed in a 33-yr-old man with moderate hemorrhagic diathesis related to injuries since his early adolescence. The structure of the extra carbohydrate moiety attached to asparagine-308 was found to be identical with those derived from the normal B beta and gamma chains as evidenced by HPLC.
K Yamazumi, K Shimura, S Terukina, N Takahashi, M Matsuda
The present study was designed to elucidate the molecular genetic basis of a familial deficiency of alpha 2-plasmin inhibitor (alpha 2PI). Southern blot hybridization analysis with human alpha 2PI cDNA and genomic DNA probes demonstrated no gross deletion or rearrangement of the gene. By sequencing all the coding exons and exon-intron boundaries of the gene of a homozygote, we identified a single cytidine nucleotide insertion in the exon coding for the carboxyl-terminal region. This frameshift mutation leads to an alteration and elongation of the carboxyl-terminal portion of the deduced amino acid sequence. Synthetic oligonucleotide probes confirmed this frameshift mutation in all the affected family members including both heterozygous parents. In a transient expression assay, the alpha 2PI level in the culture medium of the cells transfected with the mutated alpha 2PI expression vector was very low and only 4% of that of the cells transfected with the normal vector, although the transcript levels and the cellular contents of alpha 2PIs did not differ significantly. Elongation of amino acid sequence in the mutant alpha 2PI was confirmed by an analysis of alpha 2PI in a transient expression experiment. These data indicate that this mutation is the cause of alpha 2PI deficiency in this pedigree.
O Miura, S Hirosawa, A Kato, N Aoki
Complementary DNAs encoding the precursor of human placental short chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) (EC 188.8.131.52) were cloned and sequenced. The cDNA inserts in these clones were 1,852 bases in length combined, and encoded the entire 412-amino acid precursor SCAD (mol wt 44,303). This sequence included the 24-amino acid leader peptide moiety (mol wt 2,576) and 388 amino acids corresponding to the mature protein (mol wt 41,727). The comparison of SCAD and medium chain acyl-CoA dehydrogenase sequences revealed a high degree of homology, suggesting that these enzymes evolved from a common ancestral gene and belong to a gene family. We also studied mutant human SCAD in cultured skin fibroblasts from three patients with hereditary SCAD deficiency. Labeling fibroblast cultures with [35S]-methionine followed by immunoprecipitation with anti-SCAD antibody revealed that a normal size variant SCAD protein was synthesized. In all of the three SCAD-deficient cell lines, the size of variant SCAD mRNA as determined by Northern blotting using one of the normal SCAD cDNA as a probe was also normal, and no difference was observed on Southern blots in the restriction patterns of mutant genomic DNA using EcoRI, TaqI, HincII, and BamHI. These results suggest that the defects in SCAD in these cell lines are caused by a point mutation.
E Naito, H Ozasa, Y Ikeda, K Tanaka
To improve our understanding of the metabolic role of cytokines in protein wasting, we estimated the rates of protein synthesis and degradation in muscle and liver tissues in intact rats treated with several doses of recombinant IL 1 and/or tumor necrosis factor (TNF)/cachectin. Protein breakdown in muscle and liver were derived in vivo from the relationship between [14C]leucine distribution and tissue dilution in reference to circulating leucine. Synthesis was derived from the relationship between [14C]leucine appearance in the protein-bound and free-tissue leucine pools. To specifically relate changes in leucine tracer metabolism to protein dynamics, we separately measured the effect of these cytokines on blood flow to different tissues. The increase in dilution of the tissue-free [14C]leucine by TNF and TNF/IL 1 mixture, but not by IL 1 alone, could not be explained by a hemodynamic effect of these cytokines. Rather, this finding indicated that muscle proteolysis is enhanced by TNF and synergistically augmented by the addition of IL 1. Compatible with these data was the finding that more prolonged infusions of recombinant TNF/cachectin and the combination with IL 1 increased urinary nitrogen excretion. Changes in [14C]leucine dilution in the liver were less pronounced than those in skeletal muscle and consistent with net anabolic effect of TNF on liver protein. We conclude that rats exposed systemically to sublethal doses of TNF respond with increasing muscle and decreasing liver proteolysis, similar to that observed in inflammation and in cancer.
E A Flores, B R Bistrian, J J Pomposelli, C A Dinarello, G L Blackburn, N W Istfan
High-protein diets increase albumin synthesis in rats with Heymann nephritis but albuminuria increases also, causing serum albumin concentration to be suppressed further than in nephrotic animals eating a low-protein diet. Experiments were designed to determine whether dietary protein augmentation directly stimulates albumin synthesis, or whether instead increased albumin synthesis is triggered by the decrease in serum albumin concentration. Evidence is presented that dietary protein augmentation directly stimulates albumin synthesis, accompanied by a proportional increase in steady-state hepatic albumin mRNA concentration (AlbmRNA) and by an increase in AlbmRNA transcription. When the increased albuminuria resulting from dietary protein augmentation is blunted with enalapril, serum albumin concentration is shown to increase in nephrotic rats. Both albumin synthesis and AlbmRNA increase in these animals despite the greater serum albumin concentration. Albumin synthesis correlates inversely with both serum albumin and serum oncotic pressure in nephrotic rats fed 40% protein, but does not correlate with serum albumin concentration in nephrotic rats fed 8.5% protein (LP), even when serum albumin concentration is reduced. Albumin masses are preserved in LP primarily because of reduced albuminuria. Reduced serum oncotic pressure and dietary protein augmentation combine to stimulate albumin synthesis in nephrotic rats at the level of gene transcription.
G A Kaysen, H Jones Jr, V Martin, F N Hutchison
A carboxypeptidase activity was recently identified in highly purified human lung mast cells and dispersed mast cells from skin. Using affinity chromatography with potato-tuber carboxypeptidase inhibitor as ligand, mast cell carboxypeptidase was purified to homogeneity from whole skin extracts. The purified enzyme yielded a single staining band of approximately 34,500 D on SDS-PAGE. Carboxypeptidase enzyme content estimated by determination of specific activity, was 0.5, 5, and 16 micrograms/10(6) mast cells from neonatal foreskin, adult facial skin, and adult foreskin, respectively. Human mast cell carboxypeptidase resembled bovine carboxypeptidase A with respect to hydrolysis of synthetic dipeptides and angiotensin I, but was distinguished from carboxypeptidase A in its inability to hydrolyze des-Arg9 bradykinin. The amino acid composition of human mast cell carboxypeptidase was similar to the composition of rat mast cell carboxypeptidase. The amino-terminal amino acid sequence of mast cell carboxypeptidase demonstrated 65% positional identity with human pancreatic carboxypeptidase B, but only 19% with human carboxypeptidase A. Thus, human mast cell carboxypeptidase is a novel member of the protein family of zinc-containing carboxypeptidases, in that it is functionally similar but not identical to bovine carboxypeptidase A, but has structural similarity to bovine and human pancreatic carboxypeptidase B.
S M Goldstein, C E Kaempfer, J T Kealey, B U Wintroub
Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.
J J Billadello, H L Fontanet, A W Strauss, D R Abendschein
To examine the influence of osteoblast function on aluminum-induced neo-osteogenesis in the mammalian species, we compared the effects of aluminum in sham-operated and thyroparathyroidectomized (TPTX) beagles. TPTX dogs received sufficient calcium carbonate and calcitriol to maintain normal plasma calcium and calcitriol levels, but developed evidence of decreased osteoblast recruitment and activity, including diminished osteoid-covered trabecular bone surface (3.22 +/- 0.21 vs. 10.95 +/- 1.30%) and a decreased osteoblast number (27.8 +/- 8.1 vs. 139.0 +/- 26.0/mm). Administration of aluminum (1.25 mg/kg i.v., three times/wk) increased the serum aluminum levels in both sham (1,087.0 +/- 276.0 vs. 2.7 +/- 0.8 micrograms/liter) and TPTX animals (2,786.0 +/- 569.0 vs. 3.6 +/- 0.8 micrograms/liter) above normal but did not alter the plasma calcium, creatinine, or PTH from control levels in either sham or TPTX dogs. After 8 wk of therapy, however, bone biopsies from sham-operated beagles displayed evidence of neo-osteogenesis including an increased bone volume (47.0 +/- 1.0 vs. 30.4 +/- 0.9%) and trabecular number (4.1 +/- 0.2 vs. 3.2 +/- 0.2/mm). Much of the enhanced volume resulted from deposition of poorly mineralized woven bone (9.9 +/- 2.7%). In contrast, biopsies from aluminum-treated TPTX animals exhibited significantly less evidence of ectopic bone formation. In this regard, bone (35.5 +/- 1.7%) and woven tissue volume (1.4 +/- 0.8%) as well as trabecular number (3.3 +/- 0.1/mm) were significantly less than those of the aluminum-treated controls. These observations illustrate that aluminum reproducibly stimulates neo-osteogenesis and induces a positive bone balance. However, this effect apparently depends on the availability of a functional osteoblast pool which, if depleted by TPTX, limits the expression of aluminum-induced new bone formation.
L D Quarles, H J Gitelman, M K Drezner
We have examined the ability of extracellular ATP to elicit intracellular Ca2+ mobilization in a broad range of human leukocytes at particular stages of hematopoietic differentiation. The average cytosolic [Ca2+] in various leukocyte populations was measured in Fura 2-loaded cell suspensions while the cytosolic [Ca2+] in individual, Indo 1-loaded leukocytes was assayed by flow cytometric methods. Utilizing normal blood- and marrow-derived cells, human leukemic cell lines, and mononuclear cell fractions derived from the blood of patients with various leukemias, we have found that ATP-induced Ca2+ mobilization appears restricted to leukocytes of neutrophil/monocyte ontogeny. Significant ATP-induced increases in cytosolic [Ca2+] were observed in neutrophils, monocytes, and myeloid progenitor cells as immature as myeloblasts, but not in lymphocytes. Extensive characterization of the ATP-induced changes in [Ca2+] observed in the HL-60 promyelocytic cell line have indicated these Ca2+-mobilizing effects of ATP can be correlated with an activation of inositol phospholipid breakdown via the occupation of P2-purinergic receptors Significantly, of the various agonists (FMLP, platelet-activating factor, LTB4, and ATP) which elicit equivalent and maximal Ca2+ mobilization in mature neutrophils and monocytes, ATP was the most efficacious stimulant of Ca2+ mobilization in immature neutrophil/monocyte precursors. Thus, expression of putative P2-purinergic receptors for ATP appears to precede expression of other receptor types known to activate the inositol phospholipid signaling cascades in terminally differentiated phagocytes.
D S Cowen, H M Lazarus, S B Shurin, S E Stoll, G R Dubyak
Approximately 1 out of every 10 eyes undergoing surgery for retinal detachment develops excessive intraocular fibrosis that can lead to traction retinal detachment and ultimate blindness. This disease process has been termed proliferative vitreoretinopathy (PVR). The ability to monitor and grade this fibrotic response accurately within the eye as well as the ability to aspirate vitreous cavity fluid bathing the fibrotic tissue makes this an ideal setting in which to investigate the development of fibrosis. Although laboratory studies have recently shown that transforming growth factor-beta (TGF-beta) can enhance fibrosis, little clinical evidence is yet available correlating the level of this or other growth factors with the degree of fibrosis in a clinical setting. We have found that vitreous aspirates from eyes with intraocular fibrosis associated with PVR have more than three times the amount of TGF-beta (1,200 +/- 300 pM [SEM]) found in eyes with uncomplicated retinal detachments without intraocular fibrosis (360 +/- 91 pM [SEM]). Using an in vitro assay, 84-100% of the TGF-beta activity could be blocked with specific antibodies against TGF-beta 2, whereas only 10-21% could be blocked by specific antibodies against TGF-beta 1. TGF-beta 1 was used in an animal model of traction retinal detachment. Since beta 1 and beta 2 have essentially identical biologic effects and only human beta 1 was available in quantities required, beta 1 was chosen for these in vivo studies. The injection of TGF-beta1 plus fibronectin (FN) but not TGF-beta1 alone into the vitreous cavity of rabbits resulted in the increased formation of intraocular fibrosis and traction retinal detachments as compared to control eyes. In previous studies, intravitreal FN levels were also found to be elevated in eyes with intraocular fibrosis.
T B Connor Jr, A B Roberts, M B Sporn, D Danielpour, L L Dart, R G Michels, S de Bustros, C Enger, H Kato, M Lansing
Glomerular inositol content and the turnover of polyphosphoinositides was reduced by 58% in 1-2 wk streptozotocin diabetic rats. Addition of inositol to the incubation medium increased polyphosphoinositide turnover in glomeruli from diabetic rats to control values. Despite the reduction in inositol content and polyphosphoinositide turnover, protein kinase C was activated in glomeruli from diabetic rats, as assessed by an increase in the percentage of enzyme activity associated with the particulate cell fraction. Total protein kinase C activity was not different between glomeruli from control and diabetic rats. Treatment of diabetic rats with insulin to achieve near euglycemia prevented the increase in particulate protein kinase C. Moreover, incubation of glomeruli from control rats with glucose (100-1,000 mg/dl) resulted in a progressive increase in labeled diacylglycerol production and in the percentage of protein kinase C activity which was associated with the particulate fraction. These results support a role for hyperglycemia per se in the enhanced state of activation of protein kinase C seen in glomeruli from diabetic rats. Glucose did not appear to increase diacylglycerol by stimulating inositol phospholipid hydrolysis in glomeruli. Other pathways for diacylglycerol production, including de novo synthesis and phospholipase C mediated hydrolysis of phosphatidylcholine or phosphatidyl-inositol-glycan are not excluded.
P A Craven, F R DeRubertis
We characterized Fc receptor III (FcR III) on human neutrophils and found it to be heavily glycosylated and polymorphic. In some individuals, FcR III that had been digested with N-glycanase appeared after SDS-PAGE under reducing conditions as two bands with apparent molecular masses of 33 and 29 kD. In other individuals, N-glycanase-treated FcR III appeared as a single band with an Mr of either 33 or 29 kD. After SDS-PAGE of N-glycanase-treated FcR III under nonreducing conditions, the apparent Mr of each structural type was decreased, suggesting the presence of intramolecular disulfide bonds. Digestion of the 33-kD band and the 29-kD band with Staphylococcus aureus V8 protease yielded similar, but not identical, peptide maps. Thus, at least two polymorphic forms of FcR III are expressed on human neutrophils. The structural polymorphism of neutrophil FcR III correlated with previously described antigenic polymorphisms detected by monoclonal antibody Gran 11 and by alloantisera which recognize epitopes of the biallelic, neutrophil antigen (NA) system. Individuals whose neutrophils expressed the two-band structural type of FcR III were NA1NA2 heterozygotes. Individuals whose neutrophils expressed the single 33-kD band structural type were NA2NA2 homozygotes, and individuals whose neutrophils expressed the single 29-kD band structural type were NA1NA1 homozygotes. These findings indicate that antigenic and structural polymorphisms of human neutrophil FcR III are related and can be accounted for by differences at the level of primary protein structure.
P A Ory, I M Goldstein, E E Kwoh, S B Clarkson
The role of oxygenation in the pathogenesis of alcoholic liver injury was investigated in six baboons fed alcohol chronically and in six pair-fed controls. All animals fed alcohol developed fatty liver with, in addition, fibrosis in three. No evidence for hypoxia was found, both in the basal state and after ethanol at moderate (30 mM) or high (55 mM) levels, as shown by unchanged or even increased hepatic venous partial pressure of O2 and O2 saturation of hemoglobin in the tissue. In controls, ethanol administration resulted in enhanced O2 consumption (offset by a commitant increase in splanchnic blood flow), whereas in alcohol fed animals, there was no increase. At the moderate ethanol dose, the flow-independent O2 extraction, measured by reflectance spectroscopy on the liver surface, tended to increase in control animals only, whereas a significant decrease was observed after the high ethanol dose in the alcohol-treated baboons. This was associated with a marked shift in the mitochondrial redox level in the alcohol-fed (but not in control) baboons, with striking rises in splanchnic output of glutamic dehydrogenase and acetaldehyde, reflecting mitochondrial injury. Increased acetaldehyde, in turn, may aggravate the mitochondrial damage and exacerbate defective O2 utilization. Thus impaired O2 consumption rather than lack of O2 supply characterizes liver injury produced by high ethanol levels in baboons fed alcohol chronically.
C S Lieber, E Baraona, R Hernández-Muñoz, S Kubota, N Sato, S Kawano, T Matsumura, N Inatomi
The capacity of interferon-gamma to regulate the generation and release of leukotriene B4 (LTB4) from human alveolar macrophages of normal nonsmoking individuals was evaluated. When alveolar macrophages were incubated for 60 min with heat aggregated IgG (HAIgG), they generated and released 5.7 +/- 1.7 ng of LT B4 per 10(6) cells compared to 1.9 +/- 0.4 ng from cells incubated with buffer alone, P = 0.02. When alveolar macrophages were preincubated with interferon-gamma for 24 h before activation for 60 min with heat-aggregated IgG, the soluble IgG aggregates became a significantly more effective stimulus for LTB4 release, 17.0 +/- 3.9 ng/10(6) cells, P = 0.001, compared to cells incubated in the absence of interferon-gamma and challenged with HAIgG. Interferon-gamma did not alter the response to A23187. This effect of interferon-gamma was both time and dose dependent; it also was specific since neither interferon-alpha nor interferon-beta had a regulatory effect on the release of LTB4 from cells in response to challenge with HAIgG. Preincubation of the alveolar macrophages with interferon-gamma augmented the density of IgG1 receptors by 81.5 +/- 17.3%; neither interferon-alpha nor interferon-beta effected this parameter. Furthermore, monomeric IgG1 blocked HAIgG induced LTB4 release from alveolar macrophages primed with interferon-gamma. Therefore, at least one of the mechanisms by which interferon-gamma primes alveolar macrophages for the production and release of LTB4 in response to stimulation by aggregates of IgG is that of increasing the number of receptors for this stimulus.
J A Rankin, C E Schrader, S M Smith, R A Lewis
The presence of heterogeneous erythroid progenitor cells, contaminant cells, or serum may alter erythroid colony development in vitro. To obtain highly purified colony-forming units-erythroid (CFU-E), we cultured partially purified human blood burst-forming units-erythroid (BFU-E) in methylcellulose with recombinant human erythropoietin (rHuEPO) for 7 d and generated cells that consisted of 30-60% CFU-E, but no BFU-E. A serum-free medium was used that allowed development of the same number of erythroid colonies as serum containing medium, but with a greater percentage of larger colonies. This medium consisted of delipidated crystalline bovine serum albumin, iron saturated transferrin, lipid suspension, fibrinogen, thrombin, Iscove's modified Dulbecco's medium/F-12[HAM], and insulin plus rHuEPO. When CFU-E were cultured in a limiting dilution assay and the percentage of nonresponder wells was plotted against cell concentration, both serum-free cultures and serum-containing cultures yielded overlapping straight lines through the origin indicating that CFU-E development did not depend on accessory cells and that insulin acted directly on the CFU-E. Human recombinant interleukin 3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor had no effect on CFU-E growth, while they markedly enhanced BFU-E growth. Physiological concentrations of recombinant human insulin-like growth factor I (IGF-I) enhanced CFU-E growth in the absence of insulin and, together with rHuEPO in serum-free medium, provided a plating efficiency equal to that of serum-containing medium. Limiting dilution analysis in serum-free medium with IGF-I showed a straight line through the origin indicating that IGF-I also acted directly on the CFU-E and not through an effect on accessory cells. These data demonstrate that CFU-E do not require accessory cells, but do require IGF-I and/or insulin which act directly on the CFU-E.
K Sawada, S B Krantz, E N Dessypris, S T Koury, S T Sawyer
Numerous investigators have suspected that there is a genetic predisposition to rheumatic fever (RF). In this context we have recently produced a series of monoclonal antibodies directed against B cells obtained from RF patients one of which, labeled D8/17, identifies a B cell antigen present in 100% of all RF patients studied. While the highest percentage of positive cells were exhibited by RF probands (33.5% +/- SE), the percentage of cells in unaffected siblings and parents was 14.6 and 13%, respectively. The percentage of positive cells in APSGN probands, unaffected siblings, and parents was 2.96, 3.86, and 2.8%, respectively. A low level of B cells (5-7%) bearing the D8/17 marker was seen in control patients. The segregation pattern of the phenotypes defined by the percentage of D8/17 positive cells within HLA-typed RF families are consistent with an autosomal recessive mode of inheritance not associated with the human MHC system. We postulate that these phenotypes indicate the presence of at least one necessary genetic factor for susceptibility to RF.
A K Khanna, D R Buskirk, R C Williams Jr, A Gibofsky, M K Crow, A Menon, M Fotino, H M Reid, T Poon-King, P Rubinstein
To elucidate the acute metabolic actions of insulin-like growth factor I (IGF-I), we administered a primed (250 micrograms/kg), continuous (5 micrograms/kg.min) infusion of human recombinant (Thr 59) IGF-I or saline to awake, chronically catheterized 24-h fasted rats for 90 min. IGF-I was also infused while maintaining euglycemia (glucose clamp technique) and its effects were compared to those of insulin. IGF-I infusion caused a twofold rise in IGF-I levels and a 75-85% decrease in plasma insulin. When IGF-I alone was given, plasma glucose fell by 30-40 mg/dl (P less than 0.005) due to a transient twofold increase (P less than 0.05) in glucose uptake; hepatic glucose production and plasma FFA levels remained unchanged. IGF-I infusion with maintenance of euglycemia produced a sustained rise in glucose uptake and a marked stimulation of [3-3H]glucose incorporation into tissue glycogen, but still failed to suppress glucose production and FFA levels. IGF-I also produced a generalized 30-40% reduction in plasma amino acids, regardless of whether or not hypoglycemia was prevented. This was associated with a decrease in leucine flux and a decline in the incorporation of [1-14C]leucine into muscle and liver protein (P less than 0.05). When insulin was infused in a dosage that mimicked the rise in glucose uptake seen with IGF-I, nearly identical changes in amino acid metabolism occurred. However, insulin suppressed glucose production by 65% and FFA levels by 40% (P less than 0.001). Furthermore, insulin was less effective than IGF-I in promoting glycogen synthesis. We conclude that (a) IGF-I produces hypoglycemia by selectively enhancing glucose uptake; (b) IGF-I is relatively ineffective in suppressing hepatic glucose production or FFA levels; and (c) IGF-I, like insulin, lowers circulating amino acids by reducing protein breakdown rather than by stimulating protein synthesis. Thus, IGF-I's metabolic actions in fasted rats are readily distinguished from insulin.
R Jacob, E Barrett, G Plewe, K D Fagin, R S Sherwin
We describe the expression and characterization of sodium channels from human brain RNA in the Xenopus oocyte. The expressed channel, studied by whole-cell voltage clamp, reveals characteristic selectivity for sodium as the permeant ion, voltage-dependent gating, and block by nanomolar concentrations of tetrodotoxin. Such channels are not seen in control oocytes injected with solvent only. The anticonvulsant diphenylhydantoin (DPH) inhibits the expressed channel in a voltage- and use-dependent manner, much like the effect seen in primary mammalian neuronal preparations. The inhibition of the expressed human sodium channel by DPH can be described by models previously developed to explain block of Na channels by local anesthetics. The preferential block of Na channels during depolarization helps explain the selectivity of DPH for neurons involved in seizure activity.
G F Tomaselli, E Marban, G Yellen
The possible involvement of platelet-activating factor (PAF) in the pathogenesis of endotoxemia, was investigated by using a binding assay to patients' platelets, complemented with the extraction and chemical characterization of PAF obtained from patients' platelets. Platelets from 12 human volunteers had 281 +/- 63 freely accessible high affinity binding sites (PAF-receptors) per platelet; whereas this number was of 49 +/- 37 PAF-receptors per platelet, n = 14 samples, P less than 0.01, in a group of 13 patients with positive blood culture. A group of patients with respiratory or cardiovascular disturbances and negative blood culture had 253 +/- 74, accessible receptors per platelet (n = 19 samples from 16 patients, P less than 0.01 as compared to septic patients, which was not significantly different when compared to control individuals). Patients with sepsis possessed significant amounts of PAF associated to their platelets, whereas this mediator could not be isolated from platelets of patients with respiratory or cardiovascular disturbances and negative blood culture, nor from platelets of control individuals. PAF was also assayed in whole blood samples and found at high concentrations in sepsis patients. These data indicate that occupancy of PAF receptors in combination with high amounts of platelet-associated PAF, is a common finding in patients with sepsis.
F Lopez Diez, M L Nieto, S Fernandez-Gallardo, M A Gijon, M Sanchez Crespo
Rat adipocyte precursor populations contain clones varying in capacity for replication. In this study we explored factors controlling the frequency of clones of varying replicative capacities (clonal composition). We also explored the relationship between this frequency and fat depot growth. In perirenal and epididymal depots clonal composition was identical bilaterally; perirenal depots contained more extensively replicating clones. Although there were large interanimal differences in clonal composition, variation between animals was always in the same direction for both depots. Clonal composition was unaffected by undernutrition while with animal growth the frequency of the most extensively replicating clones was reduced. Differentiation of precursors occurred in all clones, while differentiation did not occur in skin fibroblasts cloned under identical conditions. Clonal composition and mature fat cell number were related in that fat cell numbers were identical bilaterally in both depots and increased more extensively with growth in perirenal than epididymal tissue. We conclude (a) that clonal composition of adipocyte precursor populations is regulated genetically and by age, (b) that this composition determines, at least in part, the capacity for adipose depot growth.
H Wang, J L Kirkland, C H Hollenberg
We evaluated an elderly patient with a lifelong history of severe bleeding after surgery or trauma and with evidence of persistent hyperfibrinolysis. Routine coagulation studies were normal. Serum plasminogen (40%, normal 72-128%) and alpha 2-antiplasmin (55%, normal 70-145%) activities were decreased. Euglobulin clot lysis was abnormally shortened (50 min) and normalized in vitro with epsilon-aminocaproic acid (EACA). The patient was treated with EACA with prompt cessation of bleeding. Patient tissue-plasminogen activator (t-PA) levels in serum were normal (4.7 ng/ml, control 3.5-7.2) as detected by a two-site immunoradiometric assay (IRMA). Patient fibrinolytic inhibitor activities were assessed by incubating 125I-labeled t-PA with either whole blood or serum followed by SDS-PAGE and autoradiography to identify the resultant protease/protease inhibitor complexes. In comparison to blood samples obtained from normal donors, patient plasma and serum demonstrated reduced binding of a fast-acting plasminogen activator inhibitor to 125I-labeled t-PA. Immunoprecipitation experiments indicated diminished complex formation between type 1 plasminogen activator inhibitor (PAI-1) in patient serum and 125I-labeled t-PA. Low patient PAI-1 activity was confirmed in serum (0.36 U/ml, control 0.87-1.81; n = 3) and in platelet lysates using a functional IRMA to quantitate PAI-1 binding to immobilized t-PA. However, patient serum PAI-1 antigen was within the normal range when analyzed by IRMA (31.8 ng/ml, control 19.6-42.2); this result was confirmed in both serum and platelets by Western blot (n = 3). Mixing experiments using purified PAI-1 as well as patient and control sera did not show evidence for an inhibitor against PAI-1. We conclude that this patient's bleeding diathesis was due to hyperfibrinolysis and defective PAI-1. This patient provides the first demonstration of a link between decreased in vivo PAI-1 activity and disordered hemostasis, and supports a role for PAI-1 in control of vivo fibrinolysis.
R R Schleef, D L Higgins, E Pillemer, L J Levitt
The protein encoded by the protooncogene c-jun, included in the activator protein-1 (AP-1) complex, is probably the critical trans-acting factor controlling transcription of the procollagenase gene which is rate limiting for subsequent synthesis of procollagenase. Therefore, to elucidate possible mechanisms whereby IL-1 stimulates procollagenase synthesis, we measured levels of c-jun and procollagenase mRNA in human serum-starved dermal fibroblasts in response to human recombinant IL-1 beta (hrIL-1 beta). hrIL-1 beta or serum induced rapid increases in c-jun mRNA levels; mRNA levels declined rapidly after hrIL-1 beta and more slowly after exposure to serum. The increases in levels of c-jun mRNA preceded the increases in procollagenase mRNA. Whereas the increases in levels of procollagenase mRNA were blunted by cycloheximide, those of c-jun mRNA were enhanced. We interpret these results as follows: IL-1 or serum induce transcription of c-jun by mechanisms independent of new protein synthesis; c-JUN, the protein product of c-jun in the AP-1 complex, is an essential mediator of the effects of IL-1 or serum in the subsequent induction of expression of the procollagenase gene.
W Conca, P B Kaplan, S M Krane
Endothelin is a newly discovered, potent vasoconstrictor peptide secreted by endothelial cells. The binding of endothelin was studied on cultured human vascular smooth muscle cells obtained from umbilical veins. A single specific binding site for 125I-endothelin was identified, with an apparent Kd of 126 pM and a maximal binding capacity of approximately 10,000 sites per smooth muscle cell. At room temperature the binding was saturable, reached equilibrium at 2 h (using 20 pM endothelin), and was slowly and only partially reversed by unlabeled endothelin. The calcium antagonists nifedipine, nicardipine, and diltiazem did not compete for the same binding site. Conditioned medium from cultured human umbilical vein endothelial cells inhibited the binding of 125I-endothelin dose dependently. This effect was antagonized by anti-endothelin antiserum. We conclude that human umbilical vein smooth muscle cells possess specific binding sites for endothelin, and that human endothelial cells secrete an endothelinlike material.
M Clozel, W Fischli, C Guilly
In Munich-Wistar rats, a micropipette was inserted into a first-order branch of the left main renal artery and continuously infused with human/porcine endothelin (0.4 ng/min). Micropuncture measurements revealed substantial differences within the cortical microcirculation of the same left kidney: SNGFR was some 35% lower in glomeruli exposed to endothelin compared with non-endothelin-perfused glomeruli (P less than 0.005). Similarly, glomerular plasma flow rate was some 38% lower in the endothelin-exposed glomeruli (P less than 0.001). The hypoperfusion and hypofiltration in the endothelin-exposed glomeruli reflected an increase in resistances in the afferent and efferent arterioles. There was no difference in the value of the glomerular capillary ultrafiltration coefficient between the two populations of glomeruli. We also studied kidneys that underwent 25 min of renal artery clamping 48 h before study. Antiendothelin antibody infused into one of the branches of the main renal artery ameliorated the vasoconstriction characteristic of postischemic nephrons: within the cortical microcirculation, the SNGFR in glomeruli exposed to antiendothelin antibody was 27.0 +/- 3.1 nl/min as compared with 17.4 +/- 1.7 measured in glomeruli not perfused with the antibody (P less than 0.001). Similarly, glomerular plasma flow rate was higher in the glomeruli exposed to antiendothelin antibody (128.7 +/- 14.4 nl/min vs. 66.6 +/- 5.6, P less than 0.005). Resistances in both the afferent and efferent arterioles were substantially lower in the antibody-exposed glomeruli. It is, therefore, suggested that endothelin, presumably released from damaged endothelium, may play an important intermediary role in the hypoperfusion and hypofiltration observed in postischemic kidneys.
V Kon, T Yoshioka, A Fogo, I Ichikawa
Expression of transforming growth factor alpha (TGF alpha), and transforming growth factor beta (TGF beta) was assessed in isolated primary rat intestinal epithelial cells as well as a rat intestinal crypt cell-derived cell line (IEC-6). A gradient in TGF beta was present, with high concentrations of a 2.5-kb transcript found in undifferentiated crypt cells and progressively lower amounts of the TGF beta transcript in increasingly differentiated villus cell populations. In contrast, the concentration of 4.5-kb TGF alpha transcript was higher in differentiated villus cells than in mitotically active, undifferentiated populations of crypt epithelial cells. The concentrations of transforming growth factors alpha and beta as determined by radioreceptor binding inhibition assay and direct assessment of transforming growth factor biological activity correlated with Northern blot analysis. Although gradients in the expression of the TGFs were present, equivalent binding was observed in the different intestinal cell populations when assessed with 125I-TGF beta and 125I-EGF (TGF alpha). No EGF transcripts were detected in any intestinal cell population, suggesting that the true ligand of the EGF receptor was TGF alpha. IEC-6 cells expressed both TGF alpha and TGF beta transcripts. In addition to the transcripts identified in the primary intestinal cells, this cell line contained an additional larger TGF alpha transcript (4.8 kb) and smaller TGF beta transcripts (2.2 and 1.8 kb). TGF alpha and TGF beta may play a significant role in the regulation of the balance between proliferative and differentiated cell compartments in the intestinal epithelium through both autocrine and paracrine mechanisms.
S Y Koyama, D K Podolsky
Endothelium-derived relaxing factor has been recently identified as nitric oxide. The purpose of this study was to determine if vasodilator drugs that generate nitric oxide inhibit vascular smooth muscle mitogenesis and proliferation in culture. Three chemically dissimilar vasodilators, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate, dose-dependently inhibited serum-induced thymidine incorporation by rat aortic smooth muscle cells. Moreover, 8-bromo-cGMP mimicked the antimitogenic effect of the nitric oxide-generating drugs. The antimitogenic effect of S-nitroso-N-acetylpenicillamine was inhibited by hemoglobin and potentiated by superoxide dismutase, supporting the view that nitric oxide was the ultimate effector. Sodium nitroprusside and S-nitroso-N-acetylpenicillamine significantly decreased the proliferation of vascular smooth muscle cells. Moreover, the inhibition of mitogenesis and proliferation was shown to be independent of cell damage, as documented by several criteria of cell viability. These results suggest that endogenous nitric oxide may function as a modulator of vascular smooth muscle cell mitogenesis and proliferation, by a cGMP-mediated mechanism.
U C Garg, A Hassid
The human platelet alloantigens, PlA1 and PlA2, comprise a diallelic antigen system located on a component of the platelet fibrinogen receptor, membrane glycoprotein (GP) IIIa. Of the known platelet alloantigens, PlA1, which is carried by 98% of the caucasian population, appears to be the alloantigen that most often provokes neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura. The structural features of the GPIIIa molecule responsible for its antigenicity are as yet unknown. Using the polymerase chain reaction (PcR), we amplified the NH2-terminal region of platelet GPIIIa mRNA derived from PlA1 and PlA2 homozygous individuals. Nucleotide sequence analysis of selected amplified cDNA products revealed a C in equilibrium T polymorphism at base 196 that created a unique Nci I restriction enzyme cleavage site in the PlA2, but not the PlA1 form of GPIIIa cDNA. Subsequent restriction enzyme analysis of cDNAs generated by PcR from 10 PlA1/A1, 5 PlA2/A2, and 3 PlA1/A2 individuals showed that Nci I digestion permitted clear discrimination between the PlA1 and PlA2 alleles of GPIIIa. All PlA2/A2 individuals studied contain a C at base 196, whereas PlA1 homozygotes have a T at this position. This single base change results in a leucine/proline polymorphism at amino acid 33 from the NH2-terminus, and is likely to impart significant differences in the secondary structures of these two allelic forms of the GPIIIa molecule. The ability to perform DNA-typing analysis for PlA phenotype may have a number of useful clinical applications, including fetal testing and determination of the phenotype of severely thrombocytopenic individuals.
P J Newman, R S Derbes, R H Aster