T S Kupper
The effects of a broad array of cytokines, individually and in combination, were determined on separate functions (proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor [GM-CSF] production) and phenotype (expression of class II MHC antigens) of cultured fibroblast-like RA synoviocytes. The following recombinant cytokines were used: IL-1 beta, IL-2, IL-3, IL-4, IFN-gamma, tumor necrosis factor (TNF)-alpha, GM-CSF, and macrophage colony-stimulating factor (M-CSF). Only IFN-gamma induced HLA-DR (but not HLA-DQ) expression. TNF-alpha inhibited IFN-gamma-mediated HLA-DR expression (46.7 +/- 4.1% inhibition) and HLA-DR mRNA accumulation. This inhibitory effect was also observed in osteoarthritis synoviocytes. Only TNF-alpha and IL-1 increased synoviocyte proliferation (stimulation index 3.60 +/- 1.03 and 2.31 +/- 0.46, respectively). IFN-gamma (but none of the other cytokines) inhibited TNF-alpha-induced proliferation (70 +/- 14% inhibition) without affecting the activity of IL-1. Only IL-1 beta and TNF-alpha induced collagenase production (from less than 0.10 U/ml to 1.10 +/- 0.15 and 0.72 +/- 0.24, respectively). IFN-gamma decreased TNF-alpha-mediated collagenase production (69 +/- 19% inhibition) and GM-CSF production but had no effect on the action of IL-1. These data demonstrate mutual antagonism between IFN-gamma and TNF-alpha on fibroblast-like synoviocytes and suggest a novel homeostatic control mechanism that might be defective in RA where very little IFN-gamma is produced.
J M Alvaro-Gracia, N J Zvaifler, G S Firestein
Agonist occupancy of the cloned human serotonin (5-HT)1A receptor expressed in HeLa cells stimulates Na+/K+ ATPase activity as assessed by rubidium uptake. The purpose of the study was to determine which of the receptor-associated signaling mechanisms was responsible for this effect. 5-HT stimulated Na+/K+ ATPase 38% at 2 mM extracellular potassium, an effect characterized by a decrease in apparent K0.5 from 2.8 +/- 0.3 to 1.8 +/- 0.3 mM potassium without a significant change in apparent Vmax. The EC50 for the transport effect was approximately 3 microM 5-HT. The response was pertussis toxin-sensitive but did not involve inhibition of adenylate cyclase, as stimulation of Na+/K+ ATPase by 5-HT was observed in the presence of excess dibutyryl cAMP. Protein kinase C was not required for the response since short-term incubation with the phorbol esters phorbol 12 myristate, 13 acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) did not mimic the 5-HT effect. Moreover, 5-HT increased Na+/K+ ATPase activity after inactivation of protein kinase C by overnight incubation with PMA. 5-HT and the sesquiterpene lactone thapsigargin increased cytosolic calcium in this cell model, and the EC50 for 5-HT corresponded with that for stimulation of Na+/K+ ATPase. Both thapsigargin and A23187, a calcium ionophore, also increased Na+/K+ ATPase activity in a dose-responsive fashion. The response to 5-HT, thapsigargin, and A23187 was blocked by conditions that removed the cytosolic calcium response. By two-dimensional gel electrophoresis, we established evidence for a calcium-sensitive but protein kinase C-independent signaling pathway. We conclude that the 5-HT1A receptor, which we have previously shown to stimulate phosphate uptake via protein kinase C, stimulates Na+/K+ ATPase via a calcium-dependent mechanism. This provides evidence for regulation of two separate transport processes by a single receptor subtype via different signaling mechanisms.
J P Middleton, J R Raymond, A R Whorton, V W Dennis
Expression of insulin-like growth factor I (IGF-I) mRNA by some tumor cell lines of neuroectodermal origin has been described. To further explore the significance of IGF-I mRNA expression in these tumors, a more extensive analysis was performed. Most (9 of 10) neuroectodermal tumor cell lines with a t(11;22) translocation (primitive neuroectodermal tumor [PNET], Ewing's sarcoma, esthesioneuroblastoma) expressed IGF-I mRNA, whereas 0 of 15 cell lines without the translocation (PNET, neuroblastoma) expressed IGF-I. Furthermore, inasmuch as all neuroblastoma (12 of 12) cell lines examined expressed IGF-II RNA, the pattern of IGF expression could distinguish between these closely related tumors. CHP-100, a PNET cell line with the t(11;22) translocation, was shown to secrete both IGF-I protein and an IGF binding protein, IGFBP-2. This cell line also expressed the type I IGF receptor mRNA, and blockade of this receptor by a monoclonal antibody (alpha IR3) inhibited serum-free growth. These data demonstrate that IGF-I expression is a property of neuroectodermal tumors with a t(11;22) translocation and that interruption of an IGF-I autocrine loop inhibits the growth of these tumor cells.
D Yee, R E Favoni, G S Lebovic, F Lombana, D R Powell, C P Reynolds, N Rosen
Activation of complement by Entamoeba histolytica may be initiated by the extracellular 56-kD neutral cysteine proteinase which cleaves the alpha chain of C3. To determine the relationship between the fluid-phase activation of complement and our observation that only strains isolated from patients with invasive disease are resistant to complement-mediated lysis, we investigated the fate of C3 with recent amebic isolates. When 125I-C3 was incubated with trophozoites in serum, C3 in the fluid phase was cleaved to C3b or C3bi, but the alpha chain of the C3 molecules on the cell surface appeared intact. Since the lysis of nonpathogenic strains takes place in the absence of bound C3b, we demonstrated that this reaction occurs by reactive lysis initiated in the fluid phase: (a) the killing of nonpathogenic strains was enhanced when alternative pathway activation was accelerated by the addition of cobra venom factor; (b) non-pathogenic strains were lysed by purified terminal components; and (c) sera incubated with pathogenic E. histolytica produced passive lysis of chicken erythrocytes. These results demonstrate for the first time that complement-sensitive E. histolytica are lysed by activation of the terminal complement components in the fluid phase where the 56-kD neutral cysteine proteinase cleaves C3, and not by the surface deposition of activated C3.
S L Reed, I Gigli
This study examines adaptations in myocardial cytosolic phosphate content and buffering capacity that occur in vivo as a function of development. Phosphate metabolites were monitored in an open chest sheep preparation using a 31P magnetic resonance surface coil over the left ventricle. Newborn lambs (aged 4-9 d, n = 5) underwent exchange transfusion with adult blood to reduce blood-borne 2,3-diphosphoglycerate contamination of the heart monophosphate and phosphomonoester resonances, thus allowing determination of these phosphate concentrations. The blood-exchanged newborns and mature controls (aged 30-60 d, n = 5) were infused with 0.4 N hydrochloric acid to decrease pH from greater than 7.35 to less than 7.00. Simultaneously, intracellular and extracellular pH were determined from the chemical shifts of the respective phosphate peaks and compared to arterial blood pH. Findings were as follows: (a) diphosphoglycerate contribution to the cardiac spectrum was found to be negligible, (b) significant decreases in cytosolic phosphate (P less than 0.03) and phosphomonoester (P less than 0.01) content occurred with maturation, and (c) large decreases in extracellular pH (greater than 0.5 U) in both groups were similarly associated with only small changes in intracellular pH (less than 0.1 U). Change in cytosolic phosphate content implies that alterations occur in the phosphorylation potential with resulting effects on regulation of myocardial respiration, and cardiac energetics.
M A Portman, X H Ning
By cable analysis and intracellular microelectrode impalement in the in vitro perfused renal tubule, we identified alpha- and beta-intercalated (IC) cells along the rabbit distal nephron segments, including the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct in the inner stripe (OMCDi). IC cells were distinguished from collecting duct (CD) cells by a relatively low basolateral membrane potential (VB), a higher fractional apical membrane resistance, and apparent high Cl- conductances of the basolateral membrane. Two functionally different subtypes of IC cells in the CCD were identified based on different responses of VB upon reduction of the perfusate Cl- from 120 to 12 mM: the basolateral membrane of beta-IC cells was hyperpolarized, whereas that of alpha-IC cells was unchanged. This is in accord with the hypothesis that the apical membrane of beta-IC cells contains some Cl(-)-dependent entry processes, possibly a Cl-/HCO3- exchanger. Further characterization of electrical properties of both subtypes of IC cells were performed upon lowering bath or perfusate Cl- from 120 to 12 mM, and raising bath or perfusate K+ from 5 to 50 mM. A 10-fold increase in the perfusate K+ had no effect on VB in both subtypes of IC cells. Upon abrupt changes in Cl- or K+ concentration in the bath, a large or a small depolarization of the basolateral membrane, respectively, was observed in both subtypes of IC cells. The electrical properties of alpha- and beta-IC cells were similar among the distal nephron segments, but their distribution was different: in the CNT, which consists of IC cells and CNT cells, 97.3% (36/37) of IC cells were of the beta type. In the CCD, which consists of IC cells and CD cells, 79.8% (79/99) of IC cells were of the beta-type, whereas in the OMCDi 100% (19/19) were of the alpha type, suggesting that the beta type predominates in the earlier and the alpha type in the later segment.
S Muto, K Yasoshima, K Yoshitomi, M Imai, Y Asano
Rates of hepatic uptake of thyroxine (T4) from dilute solutions of five different plasma T4-binding proteins were measured in the isolated perfused rat liver using an indicator dilution method. For each protein, this rate was compared with the rate of spontaneous dissociation of the T4-protein complex measured in vitro. Proteins studied were human T4-binding globulin (TBG), human T4-binding prealbumin (TBPA), human albumin, rat TBPA, and human albumin isolated from subjects with familial dysalbuminemic hyperthyroxinemia. For each of the five protein-hormone complexes studied, the rate of hepatic uptake of T4 (measured under conditions expected to result in dissociation-limited uptake) closely approximated the rate of spontaneous dissociation of the protein-hormone complex within the hepatic sinusoids. These findings indicate an absence of special cellular mechanisms that facilitate the hepatic uptake of T4 from its plasma binding proteins, and support the view that uptake occurs from the free T4 pool after spontaneous dissociation of T4 from its binding proteins.
C M Mendel, R A Weisiger
Small-cell lung cancer (SCLC) lines are distinguished from non-small-cell lung cancer (NSCLC) lines by their growth in floating aggregates, in contrast to the adherent monolayers formed by NSCLC cells in culture. Of 50 well-characterized SCLC lines recently described by the National Cancer Institute (NCI)-Navy Medical Oncology Branch, only four variant cell lines (SCLC-v) grew as adherent monolayers. One line, NCI-H446, was unique in growing long-term with coexisting floating and surface adherent subpopulations. We have physically segregated these two populations over many passages in vitro to enrich for relatively pure cultures of floating and adherent cells. No differences in c-myc expression, keratin pattern, or cytogenetic appearance were found between the adherent and floating sublines. However, expression of the neuroendocrine marker neuron-specific enolase in the floating cells was three times that found in the adherent cells. The floating subline also had much greater surface expression of neuroendocrine tumor antigens detected by monoclonal antibodies UJ13A and HNK-1, which have been recently shown to detect the neural cell adhesion molecule (NCAM) on SCLC cells. Two other adherent SCLC-v lines were also found to be unreactive with UJ13A and HNK-1, generalizing the association between NCAM expression and the growth of most SCLC cultures as floating aggregates. In conclusion, we have an interesting model to study expression of NCAM as related to the adhesive properties of SCLC cells.
L A Doyle, M Borges, A Hussain, A Elias, T Tomiyasu
Static muscle contraction activates metabolically sensitive muscle afferents that reflexively increase sympathetic nerve activity and arterial pressure. To determine if this contraction-induced reflex is modulated by the sinoaortic baroreflex, we performed microelectrode recordings of sympathetic nerve activity to resting leg muscle during static handgrip in humans while attempting to clamp the level of baroreflex stimulation by controlling the exercise-induced rise in blood pressure with pharmacologic agents. The principal new finding is that partial pharmacologic suppression of the rise in blood pressure during static handgrip (nitroprusside infusion) augmented the exercise-induced increases in heart rate and sympathetic activity by greater than 300%. Pharmacologic accentuation of the exercise-induced rise in blood pressure (phenylephrine infusion) attenuated these reflex increases by greater than 50%. In contrast, these pharmacologic manipulations in arterial pressure had little or no effect on: (a) forearm muscle cell pH, an index of the metabolic stimulus to skeletal muscle afferents; or (b) central venous pressure, an index of the mechanical stimulus to cardiopulmonary afferents. We conclude that in humans the sinoaortic baroreflex is much more effective than previously thought in buffering the reflex sympathetic activation caused by static muscle contraction.
U Scherrer, S L Pryor, L A Bertocci, R G Victor
125I-Serum amyloid P component (SAP), injected intravenously into 10 normal subjects, remained predominantly intravascular with mean (SD) T1/2 (half time) in plasma of 24.5 (5.9) h. The fractional catabolic rate of 68 (19)% of the plasma pool per day was more rapid than other reported human plasma proteins. All radioactivity was excreted in the urine by 14 d. In 16 patients with monoclonal gammopathy or chronic inflammatory diseases, but without amyloidosis, 125I-SAP metabolism was normal. However, among 45 patients with biopsy-proven systemic amyloidosis (25, amyloid A type; 20, amyloid L type), 125I-SAP was cleared from the plasma more rapidly, accumulated in the amyloid deposits, and persisted there. The T1/2 in amyloid, measured directly with 131I-SAP, was 24 d. Repeat studies after 6-18 mo were notably consistent in normals but changed significantly in amyloid patients, generally correlating with clinical signs of disease progression. Measurements of 125I-SAP turnover may thus be of value for diagnosis and monitoring of amyloidosis. Analysis of SAP metabolism in amyloidosis suggests that plasma SAP is in dynamic equilibrium with a very large amyloid pool, and in two autopsies the total mass of SAP in the amyloid deposits was 2,100 and 21,000 mg, respectively.
P N Hawkins, R Wootton, M B Pepys
Anti-CD2-induced T cell proliferation was analyzed in the peripheral blood samples of 31 primary and 8 secondary untreated Sjögren's syndrome patients. Anti-CD2-stimulated PBMC proliferation was very low in about one-third of primary Sjögren's syndrome samples, despite the number of CD2+ cells being similar in primary and secondary Sjögren's syndrome and normal PBMC samples. The depressed response to anti-CD2 was mainly found in anti-Ro+/La+ patients. Experiments on purified T cells demonstrated that a defect at the T cell level was responsible for the anti-CD2 unresponsiveness. Cell proliferation failure was associated with poor IL-2 and IL-2 receptor mRNA expression and, consequently, IL-2 and IL-2 receptor synthesis. Since defective anti-CD2-induced mitogenesis could be reversed by phorbol myristate acetate, but not calcium ionophore A23187, it is probably correlated with impaired protein kinase C activation. Comparison of anti-CD2-triggered PBMC proliferation in treated and untreated patients and a long-term study of nine patients showed that the defect is a stable characteristic in primary Sjögren's syndrome patients, but that it can be reversed by pharmacological immunosuppression.
R Gerli, A Bertotto, E Agea, L Lanfrancone, C Cernetti, F Spinozzi, P Rambotti
The most common deficiency allele of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT) is PI*Z. Some rare deficiency alleles of alpha 1AT produce low but detectable amounts of plasma alpha 1AT (1-20% of normal), which can be differentiated by isoelectric focusing. Others, designated null (QO) alleles, produce no alpha 1AT detectable by routine quantitative methods. We have previously described a method using DNA polymorphisms, haplotypes, and polyacrylamide isoelectric focusing gels, to differentiate various deficiency alleles. Based on haplotypes, we previously identified, in eight patients, five different null alleles, four of which had been previously sequenced. We have now analyzed all 12 null alleles in these eight patients, using allele-specific oligonucleotide probes, and have identified six different null alleles. We have cloned and sequenced one of these, PI*QOludwigshafen, which has a base substitution in exon II, replacing isoleucine 92 in the normal sequence with an asparagine. This substitution of a polar for a nonpolar amino acid occurs in one of the alpha-helices and is predicted to disrupt the tertiary structure. A total of 13 different alpha 1AT deficiency alleles, 6 of them null alleles, have been sequenced to date.
G C Frazier, M A Siewertsen, M H Hofker, M G Brubacher, D W Cox
Prostacyclin (PGI2) has been reported to stimulate activities of acid cholesteryl ester hydrolase (ACEH; EC 126.96.36.199) and neutral cholesteryl ester hydrolase (NCEH; EC 188.8.131.52) in the smooth muscle cells leading to a decrease in intracellular cholesteryl ester. Recently, we have found that the half-life of PGI2 was prolonged through stabilization by HDL. HDL is known to have anti-atherogenic properties, although its precise mechanism has not been fully clarified. We therefore hypothesized that HDL can exert anti-atherogenic action by augmenting PGI2-stimulated increases in the activities of ACEH and NCEH. After incubation with PGI2 and HDL, a cell homogenate was made from which the activities of ACEH and NCEH were assessed. HDL significantly augmented the PGI2-induced increase in the activities of both enzymes. This effect of HDL was abolished in the absence of PGI2. Elevated intracellular levels of cyclic AMP were maintained for longer periods by HDL. The increase in both intracellular cyclic AMP levels and enzyme activities disappeared in the presence of an inhibitor of adenylate cyclase, 2'5'-dideoxyadenosine. Radiolabeled smooth muscle cells demonstrated a significant loss in total cholesterol and cholesteryl ester after treatment with PGI2 and HDL, due to the increase in cholesteryl ester hydrolytic activities. These data suggest that HDL enhanced the PGI2-stimulated hydrolysis of cholesteryl ester and augmented the PGI2-induced reduction of cellular cholesteryl ester content by stabilizing PGI2.
H Morishita, Y Yui, R Hattori, T Aoyama, C Kawai
Studies using cultured cells have shown that gamma interferon (IFN-gamma) induces the expression of Fc gamma RI (the type I Fc receptor for IgG) on human polymorphonuclear neutrophils (PMN) and greatly increases the number of these receptors on human monocytes. Administration of rIFN-gamma in vivo also causes enhanced Fc gamma RI expression on these cell populations. Because streptococcal antigens are potent inducers of IFN-gamma in vitro, we postulated that IFN-gamma would be produced endogenously in vivo in patients with streptococcal infections. Such production of IFN-gamma in vivo, even at low levels, might be expected to induce the expression of Fc gamma RI on monocytes and neutrophils. To evaluate this possibility, we used monoclonal antibody 32 (mAb 32), which is specific for Fc gamma RI, to quantitate the expression of this receptor on human peripheral blood cells. We measured the binding of mAb 32 to monocytes and PMNs isolated from healthy donors and from patients with group A beta-hemolytic streptococcal (GABHS) pharyngitis. PMNs from healthy donors (n = 12) had 700 +/- 600 (mean +/- SD) mAb 32 binding sites. Patients with pharyngitis and negative throat culture for GABHS (n = 11) had 2,100 +/- 1,600 sites on their PMNs. In contrast, the PMNs from patients with documented GABHS pharyngitis (n = 12) had 11,600 +/- 7,500 mAb 32 binding sites on their surface. There was a similar change in the expression of Fc gamma RI on monocytes, with control monocytes having a mean of 19,900 +/- 3,200 mAb 32 binding sites per cell and the GABHS-positive monocytes having 47,500 +/- 21,400 sites. The GABHS-negative throat culture group had a slightly elevated number of Fc gamma RI with a mean of 28,200 +/- 8,400 sites. 10 patients with documented urinary tract infections and three patients with uncomplicated pyelonephritis had no elevation in Fc gamma RI expression. These studies demonstrate that a localized group A streptococcal infection can cause systemic activation of the entire circulating pool of phagocytes, and suggest that a similar level of activation is uncommon in localized gram-negative infections of the urinary tract.
P M Guyre, A S Campbell, W D Kniffin, M W Fanger
We studied cellular processes activated by prostaglandins (PG) that are involved in the protection of gastric chief cell injury estimated in terms of dye exclusion test, release of lactate dehydrogenase (LDH), or 51Cr from prelabeled chief cells. Pretreatment of chief cells with 3 x 10(-6) M PGE2 or PGE1 at 37 degrees C and pH 7.4 for 15 min maximally reduced not only ethanol- but also taurocholic acid-caused LDH release from chief cells. PGs equipotently stimulated increases in the accumulation of diacylglycerol and cyclic AMP without elevating intracellular Ca2+ concentrations in gastric chief cells. The rank order of the potency was equal to that of PGs to reduce the injury. Pretreatment of chief cells with synthetic 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 12-o-tetradecanoyl phorbol 13-acetate (TPA) reduced the injury of chief cells, while 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, failed to reduce the injury and 1-(5-isouinolinylsulfonyl)-2-methylpiperazine (H7) blocked the protective action of PGE2. On the other hand, forskolin and dbcAMP had no effect on ethanol-caused LDH release and diacylglycerol formation in chief cells. These results suggest that PGE2 and PGE1 possess the direct protective action against ethanol- or taurocholic acid-caused injury in chief cells, presumably through the activation of the diacylglycerol/protein kinase C signaling pathway.
Y Konda, H Nishisaki, O Nakano, K Matsuda, K Wada, M Nagao, T Matozaki, C Sakamoto
Cholera toxin (CT) inhibited the in vitro growth of three of four human small-cell lung carcinoma (SCLC) cell lines with a 50% inhibitory concentration of 27-242 ng/ml. Loss of surface membrane ruffling and the capacity of [Tyr4]-bombesin, vasopressin, and fetal calf serum to stimulate increases in intracellular free calcium clearly preceded effects on cellular metabolic activity and cell growth. 125I-[Tyr4]-bombesin binding was unaffected by CT treatment but [Tyr4]-bombesin stimulated phospholipase C activity was decreased in membranes from CT-treated SCLC cells. CT stimulated a rapid but transient increase in intracellular cyclic AMP ([cAMP]i) in SCLC. The effects of CT on susceptible SCLC were not reproduced by elevations of [cAMP]i induced by forskolin or cyclic AMP analogues. GM1 ganglioside, the cellular binding site for CT, was highly expressed in the CT-sensitive but not the CT-resistant SCLC cell lines. In contrast, expression of guanine nucleotide binding protein substrates for ADP-ribosylation by CT was similar. These data demonstrate the existence of a CT-sensitive growth inhibitory pathway in SCLC-bearing GM1 ganglioside. Addition of CT results in decreased responsiveness to several mitogenic stimuli. These results suggest novel therapeutic approaches to human SCLC.
J Viallet, Y Sharoni, H Frucht, R T Jensen, J D Minna, E A Sausville
We compared the activity and physiologic effects of cardiac angiotensin converting enzyme (ACE) using isovolumic hearts from male Wistar rats with left ventricular hypertrophy due to chronic experimental aortic stenosis and from control rats. In response to the infusion of 3.5 X 10(-8) M angiotensin I in the isolated buffer perfused beating hearts, the intracardiac fractional conversion to angiotensin II was higher in the hypertrophied hearts compared with the controls (17.3 +/- 4.1% vs 6.8 +/- 1.3%, P less than 0.01). ACE activity was also significantly increased in the free wall, septum, and apex of the hypertrophied left ventricle, whereas ACE activity from the nonhypertrophied right ventricle of the aortic stenosis rats was not different from that of the control rats. Northern blot analyses of poly(A)+ purified RNA demonstrated the expression of ACE mRNA, which was increased fourfold in left ventricular tissue obtained from the hearts with left ventricular hypertrophy compared with the controls. In both groups, the intracardiac conversion of angiotensin I to angiotensin II caused a comparable dose-dependent increase in coronary resistance. In the control hearts, angiotensin II activation had no significant effect on systolic or diastolic function; however, it was associated with a dose-dependent depression of left ventricular diastolic relaxation in the hypertrophied hearts. These novel observations suggest that cardiac ACE is induced in hearts with left ventricular hypertrophy, and that the resultant intracardiac activation of angiotensin II may have differential effects on myocardial relaxation in hypertrophied hearts relative to controls.
H Schunkert, V J Dzau, S S Tang, A T Hirsch, C S Apstein, B H Lorell
A cystic fibrosis trachea cDNA library was constructed and probed with a synthetic oligonucleotide containing a consensus sequence recently identified in human intestinal mucin. One of the isolated clones, AMN-22, has been characterized extensively. The cDNA sequence of this 884-bp fragment was determined, and revealed a tandem repeat structure rich in threonine and proline residues. The repeating sequence of AMN-22 was similar but not identical to that determined for gut mucin. When examined by Northern analysis, the mRNA hybridizing to AMN-22 is extremely polydisperse in cystic fibrosis (CF) trachea, with apparent message length varying from approximately 2 kb to greater than 10 kb. A similar pattern was observed, with less abundant message, in CF bronchiectatic lung parenchyma. The lung cDNA hybridized to a similarly polydisperse message in ulcerative colitis colon RNA, but did not hybridize to control RNA from U937 lymphoma cells or stomach RNA. Pedigree analysis of restriction digests of genomic DNA revealed a pattern indicating a single polymorphic locus for the mucin gene expressed in the lung and the intestine. Southern analyses of human:mouse somatic cell hybrid cell lines allow a chromosomal localization for the mucin gene to human chromosome II, within the region 11p13-11pTer. Taken together, these data demonstrate that a polymorphic gene encodes a mucin core polypeptide expressed in both lung and intestine.
C Gerard, R L Eddy Jr, T B Shows
The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cofactor. In plasma, rabbit protein S is found in complex with C4b-binding protein. GVQLDLDEAI can inhibit this interaction, resulting in enhancement of the anticoagulant activity of rabbit activated protein C. The effect of the peptide can be blocked by the concurrent addition of human or rabbit C4b-binding protein. When infused into rabbits, GVQLDLDEAI was cleared from the circulation with a half-life of 80 min. This is significantly less rapid than the clearance of similarly sized control peptides (half-life of 15 min), but much more than that of bovine protein S, a much larger protein (half-life of 15 h). Plasma samples removed from the rabbits after infusion with GVQLDLDEAI were found to have increased concentrations of free protein S and to show enhanced anticoagulation by rabbit activated protein C ex vivo in a dose-dependent manner. The concentration for half-maximal effect (5 microM) was very similar to that observed in vitro. These results suggest that the formation of a complex between protein S and C4b-binding protein is important in the regulation of protein S activity in vivo, and that modulation of this interaction allows one to influence the anticoagulant activity of the protein C pathway.
R E Weinstein, F J Walker
The major purpose of this research was to gain insight into the effect of carbohydrate malabsorption on fecal water output. To do this we measured daily fecal output of total carbohydrate, reducing sugars, and organic acids (a product of bacterial fermentation). Normal subjects were studied in their native state and when diarrhea was induced by mechanisms that did and did not involve carbohydrate malabsorption. Patients with malabsorption syndrome were also studied. We concluded that: (a) Excretion of carbohydrate and its breakdown products can be expressed as a single number by converting organic acids to their monosaccharide equivalents. (b) Diarrhea per se causes only a trivial increase in fecal carbohydrate excretion. (c) The molar output of osmotic moieties in feces due to unabsorbed carbohydrate can be determined by adding fecal reducing sugars to organic acids and their obligated cations. This expression parallels almost exactly the effect of increasing doses of lactulose (a nonabsorbable sugar) on fecal water output; one excreted millimole obligates 3.5 g of stool water. This relationship can be used to predict the effect of carbohydrate malabsorption on stool water output in patients with diarrhea. (d) 12 of 19 patients with malabsorption syndrome due to various diseases had excessive fecal excretion of carbohydrate and its breakdown products; of the diseases that cause malabsorption syndrome, combined small and large bowel resection is most likely to result in excessive fecal excretion of carbohydrate and monosaccharide equivalents. In 6 of these 19 patients carbohydrate malabsorption appeared to be the major cause of diarrhea.
H F Hammer, K D Fine, C A Santa Ana, J L Porter, L R Schiller, J S Fordtran
Cellular constituents of the alveolar-capillary wall may be key participants in the recruitment of polymorphonuclear leukocytes to the lung through the generation of the novel neutrophil chemotactic peptide interleukin-8 (IL-8). This interaction appears to occur via the ability of human alveolar macrophage (AM)-derived monokines, tumor necrosis factor (TNF), and interleukin-1 (IL-1) to induce gene expression of IL-8 from pulmonary type II-like epithelial cells (A549). Northern blot analysis demonstrated that steady-state IL-8 mRNA expression, by either TNF- or IL-1 beta-treated A549 cells, occurred in both a dose- and time-dependent fashion. Similarly, extracellular antigenic IL-8, as assessed by specific ELISA, was expressed from TNF- or IL-1 beta-stimulated epithelial cells in a time-dependent fashion with maximal IL-8 antigen detected at 24 h poststimulation. Immunohistochemical staining utilizing rabbit anti-human IL-8 antibody identified immunoreactive, cell-associated IL-8 antigen as early as 8 h post-TNF or IL-1 beta stimulation. A549-generated neutrophil chemotactic bioactivity paralleled IL-8 steady-state mRNA levels. Signal specificity was demonstrated in this system as IL-8 mRNA or protein expression by lipopolysaccharide (LPS)-treated A549 cells was not different from unstimulated cells. Although LPS did not serve as a direct stimulus for the production of IL-8 by type II-like epithelial cells, the condition media from LPS-challenged AM induced a significant expression of IL-8 mRNA by the A549 cells. 24-h conditioned media from LPS-treated cells was as potent as either IL-1 beta or TNF in generating steady-state IL-8 mRNA by A549 cells. Preincubation of LPS-treated AM-conditioned media with anti-human TNF or IL-1 beta neutralizing antibodies resulted in significant abrogation of IL-8 gene expression by A549 pulmonary epithelial cells. These findings demonstrate potential cell-to-cell communication circuits that may be important between AMs and pulmonary epithelial cells during the recruitment phase of acute lung inflammation.
T J Standiford, S L Kunkel, M A Basha, S W Chensue, J P Lynch 3rd, G B Toews, J Westwick, R M Strieter
Tumor necrosis factor-alpha (TNF-alpha) decreased the expression of pulmonary surfactant proteins SP-A and SP-B in human pulmonary adenocarcinoma cell lines. The effect of TNF alpha on SP-A content and mRNA in the pulmonary adenocarcinoma cell line, H441-4, was concentration and time dependent. TNF alpha decreased the cellular content of SP-A to less than 10% of control 48 h after addition. TNF alpha decreased de novo synthesis of SP-A and decreased the accumulation of SP-A in media. SP-A mRNA was decreased within 12 h of addition of TNF alpha, with nearly complete loss of SP-A mRNA observed after 24 h. Inhibitory effects of TNF alpha on SP-A mRNA were dose-related with nearly complete inhibition of SP-A mRNA caused by 25 ng/ml TNF alpha. The effects of TNF alpha on SP-A were distinct from the effects of interferon gamma which increased SP-A content approximately twofold in H441-4 cells. TNF alpha also decreased the content of SP-B mRNA. In contrast to the inhibitory effect of TNF alpha on SP-A and SP-B mRNA, TNF alpha increased mRNA encoding human manganese superoxide dismutase (Mn-SOD). TNF alpha did not inhibit growth, alter cell viability or beta-actin mRNA in either cell line. These in vitro studies demonstrate the marked pretranslational inhibitory effects of the cytokine, TNF alpha, on the expression of pulmonary surfactant proteins, SP-A and SP-B. The results support the concept that macrophage-derived cytokines may control surfactant protein expression.
J R Wispé, J C Clark, B B Warner, D Fajardo, W E Hull, R B Holtzman, J A Whitsett
The present study was designed to test the possibility that T cell receptor genes are associated/linked to those involved in systemic lupus erythematosus (SLE). Genomic DNA was isolated from 31 unrelated Caucasian SLE patients, 34 unrelated Caucasian normals, 5 multiplex American Caucasian SLE families, 9 multiplex Mexican SLE families, and 13 unrelated Mexican normals. The DNA was digested with Pst I, electrophoresed, and transferred to membranes by the Southern blot method. The blots were probed with a cDNA probe for the alpha chain of the T cell receptor. 13 polymorphic RFLP patterns were recognized. 1.3- and 3.0-kb band pairs were observed in 15 of 31 of American Caucasian patients and 4 of 34 American Caucasian controls (chi square, 8.81; P less than 0.002; relative risk, 7); there was no association of any RFLP pattern with Mexican SLE. The cDNA probe was cut with Rsa I, EcoR I, and Ava II into fragments corresponding to the V, J, C, and 3'UT regions. Only the fragment corresponding to the constant region reacted with the 1.3/3.0-kb band pair. These observations suggest that a genetic marker of the constant region of the alpha chain of the T cell receptor is associated with genes involved in SLE.
J G Tebib, J Alcocer-Varela, D Alarcon-Segovia, P H Schur
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3 dramatically decreases parathyroid hormone (PTH) gene transcription. We have now studied the effect of 1,25(OH)2D3 on the 1,25(OH)2D receptor (VDR) in the parathyroid in vivo. Rats were injected with 1,25(OH)2D3 and the parathyroid-thyroid tissue analyzed for PTHmRNA and VDRmRNA. 1,25(OH)2D3 (50 and 100 pmol ip) decreased PTHmRNA at 6 h with a maximum at 48 h (less than 4% of basal), whereas VDRmRNA was increased only after 6 h with a 1.7-fold increase at 24 h. VDRmRNA levels peaked at 25 pmol 1,25(OH)2D3 with a twofold increase. Serum calcium did not affect VDRmRNA. Parathyroid VDRmRNA ran at 2.2 and 4.4 kb, whereas duodenum VDRmRNA had a single band, all of which increased after 1,25(OH)2D3. Weanling rats on a vitamin D-deficient diet for 3 wk had a more intense 2.2-kb transcript, whereas vitamin D-replete rats had a more intense 4.4-kb band. Dispersed parathyroid-thyroid cells were separated by a flow cytometry (FACS) into a parathyroid cell peak containing PTHmRNA and a second peak with cells positive for thyro-globulin mRNA and calcitonin mRNA. VDRmRNA was concentrated in the parathyroid cell peak. In situ hybridization of parathyroid-thyroid and duodenum for VDRmRNA showed its localization to the parathyroid cells and the duodenal mucosa. Therefore, the VDRmRNA in the parathyroid-thyroid tissue represents predominantly parathyroid cell and not C-cell VDRmRNA which is also a 1,25(OH)2D3 target organ. The increased VDR gene expression in the parathyroid cell would amplify the effect of 1,25(OH)2D3 to decrease PTH gene transcription.
T Naveh-Many, R Marx, E Keshet, J W Pike, J Silver
Transforming growth factor beta 1 (TGF-beta 1) is a key regulator of cell growth and differentiation. Under normal physiological conditions, it is made as a biologically latent complex whose significance is unknown. Previous work has indicated that active TGF-beta 1 has a very short plasma half-life in rats (Coffey, R. J., L. J. Kost, R. M. Lyons, H. L. Moses, and N. F. La-Russo. 1987. J. Clin. Invest. 80:750-757). We have investigated the possibility that latent complex formation may extend the plasma half-life of TGF-beta 1 and alter its organ distribution. Radiolabeled latent TGF-beta 1 was formed by noncovalent association of 125I-TGF-beta 1 with the TGF-beta 1 precursor "pro" region from recombinant sources. TGF-beta 1 in this latent complex had a greatly extended plasma half-life (greater than 100 min) in rats compared with active TGF-beta 1 (2-3 min). Whereas active TGF-beta 1 was rapidly taken up by the liver, kidneys, lungs, and spleen and degraded, TGF-beta 1 in the latent complex was largely confined to the circulation, and was less than 5% degraded after 90 min. The pharmacokinetics of TGF-beta 1 in the latent complex were shown to be critically dependent on the degree of sialylation of the complex. The results suggest that formation of latent complexes may switch endogenous TGF-beta 1 from an autocrine/paracrine mode of action to a more endocrine mode involving target organs distant from the site of synthesis.
L M Wakefield, T S Winokur, R S Hollands, K Christopherson, A D Levinson, M B Sporn
We have previously shown that the atrial natriuretic peptide (ANP) content of the anterior hypothalamic region of NaCl-sensitive spontaneously hypertensive rats (SHR-S) is higher than that of Wistar-Kyoto (WKY) rats. ANP has been shown to inhibit neuronal norepinephrine release and to reduce the excitability of hypothalamic neurons. This study tested the hypothesis that blockade of endogenous ANP in the anterior hypothalamus by local microinjection of a monoclonal antibody to ANP (MAb KY-ANP-II) lowers blood pressure in SHR-S. Purified MAb KY-ANP-II (0.055 and 0.55 micrograms) or control mouse IgG in 200 nl saline was microinjected into the anterior hypothalamic area (AHA) of conscious SHR-S and control WKY rats. As a further control, Mab KY-ANP-II (0.55 microgram) was microinjected into the posterior hypothalamic area (PHA) of SHR-S. Anterior hypothalamic microinjection of MAb KY-ANP-II caused significant dose-related decreases in mean arterial pressure (MAP) and heart rate (HR) in SHR-S but not in WKY rats. Control injections of equal volumes of IgG had no effect on MAP or HR. Microinjection of Mab KY-ANP-II into PHA produced no significant alteration in MAP or HR in SHR-S. These data provide the first demonstration that endogenous ANP in a region of brain known to influence cardiovascular function mediates BP and HR control in the rat. These findings suggest that the increased endogenous ANP in the anterior hypothalamus of SHR-S may be involved in the central regulation of BP in the model.
R H Yang, H K Jin, Y F Chen, J M Wyss, S Oparil
In hepatic iron overload, iron-catalyzed lipid peroxidation has been implicated in the mechanisms of hepatocellular injury. Lipid peroxidation may produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which may form aldehyde-protein adducts. We investigated whether lipid peroxidation occurred in rats fed a diet containing 3% carbonyl iron for 5-13 wk, and if this resulted in the formation of MDA- and 4-HNE- protein adducts. Chronic iron feeding resulted in hepatic iron overload (greater than 10-fold) and concomitantly induced a 2-fold increase in hepatic lipid peroxidation. Using an antiserum specific for MDA-lysine protein adducts, we demonstrated by immunohistochemistry the presence of aldehyde-protein adducts in the cytosol of periportal hepatocytes, which co-localized with iron. In addition, MDA- and 4-HNE-lysine adducts were found in plasma proteins of animals with iron overload. Only MDA adducts were detected in albumin, while other plasma proteins including a approximately 120-kD protein had both MDA and 4-HNE adducts. In this animal model of hepatic iron overload, injury occurs primarily in periportal hepatocytes, where MDA-lysine protein adducts and excess iron co-localized.
K Houglum, M Filip, J L Witztum, M Chojkier
The diminished ability of insulin to promote glucose disposal and storage in muscle has been ascribed to impaired activation of glycogen synthase (GS). It is possible that decreased glucose storage could occur as a consequence of decreased glucose uptake, and that GS is impaired secondarily. Muscle glucose uptake in 15 diabetic subjects was matched to 15 nondiabetic subjects by maintaining fasting hyperglycemia during infusion of insulin. Leg muscle glucose uptake, glucose oxidation (local indirect calorimetry), release of glycolytic products, and muscle glucose storage, as well as muscle GS and pyruvate dehydrogenase (PDH) were determined before and during insulin infusion. Basal leg glucose oxidation and PDH were increased in the diabetics. Insulin-stimulated leg glucose uptake in the diabetics (8.05 +/- 1.41 mumol/[min.100 ml leg tissue]) did not differ from controls (5.64 +/- 0.37). Insulin-stimulated leg glucose oxidation, nonoxidized glycolysis, and glucose storage (2.48 +/- 0.27, 0.68 +/- 0.15, and 5.04 +/- 1.34 mumol/[min.100 ml], respectively) were not different from controls (2.18 +/- 0.12, 0.62 +/- 0.16, and 2.83 +/- 0.31). PDH and GS in noninsulin-dependent diabetes mellitus (NIDDM) were also normal during insulin infusion. When diabetics were restudied after being rendered euglycemic by overnight insulin infusion, GS and PDH were reduced compared with hyperglycemia. Thus, fasting hyperglycemia is sufficient to normalize insulin-stimulated muscle glucose uptake in NIDDM, and glucose is distributed normally to glycogenesis and glucose oxidation, possibly by normalization of GS and PDH.
D E Kelley, L J Mandarino
Successful pancreas transplantation in type I diabetic patients restores normal fasting glucose levels and biphasic insulin responses to glucose. However, virtually no data from pancreas recipients are available relative to other islet hormonal responses or hormonal counterregulation of hypoglycemia. Consequently, glucose, glucagon, catecholamine, and pancreatic polypeptide responses to insulin-induced hypoglycemia and to stimulation with arginine and secretin were examined in 38 diabetic pancreas recipients, 54 type I diabetic nonrecipients, and 26 nondiabetic normal control subjects. Glucose recovery after insulin-induced hypoglycemia in pancreas recipients was significantly improved. Basal glucagon levels were significantly higher in recipients compared with nonrecipients and normal subjects. Glucagon responses to insulin-induced hypoglycemia were significantly greater in the pancreas recipients compared with nonrecipients and similar to that observed in control subjects. Glucagon responses to intravenous arginine were significantly greater in pancreas recipients than that observed in both the nonrecipients and normal subjects. No differences were observed in epinephrine responses during insulin-induced hypoglycemia. No differences in pancreatic polypeptide responses to hypoglycemia were observed when comparing the recipient and nonrecipient groups, both of which were less than that observed in the control subjects. Our data demonstrate significant improvement in glucose recovery after hypoglycemia which was associated with improved glucagon secretion in type I diabetic recipients of pancreas transplantation.
P Diem, J B Redmon, M Abid, A Moran, D E Sutherland, J B Halter, R P Robertson
We have developed a murine model of wasting by injecting intracerebrally cells which continuously secrete h-cachectin/TNF (CHO-TNF) to: (a) determine the effects of cachectin/TNF produced continuously in the central nervous system (CNS), and (b) compare the metabolic effects of cachectin/TNF-secreting tumor in the brain to the cachexia caused by CHO-TNF tumor in peripheral tissue (IM). Intracerebral CHO-TNF tumors produced increased serum h-cachectin/TNF levels with lethal hypophagia and weight loss (mean survival time of 11 d); these changes were not observed in association with nonsecretory control brain tumors. The metabolic consequences of intracerebral cachectin/TNF production were indistinguishable from acute, lethal starvation: whole-body lipid content was decreased significantly but protein was conserved. Although intramuscular cachectin/TNF-secreting tumors caused similar increases of serum h-cachectin/TNF levels, profound anorexia did not develop; wasting developed after a longer period of tumor burden (50 d) with classical signs of cachexia (i.e., anemia and depletion of both protein and lipid). These studies provide a reproducible animal model of site-specific cytokine production and suggest that, regardless of serum levels, cachectin/TNF produced locally in brain influences both the rate of development of wasting and its net metabolic effects.
K J Tracey, S Morgello, B Koplin, T J Fahey 3rd, J Fox, A Aledo, K R Manogue, A Cerami
In a family expressing euthyroid hyperthyroxinemia, an increased association of plasma thyroxine (T4) with transthyretin (TTR) is transmitted by autosomal dominant inheritance and is secondary to a mutant TTR molecule with increased affinity for T4. Eight individuals spanning three generations exhibited the abnormality. Although five of eight individuals had elevated total T4 concentrations, all affected individuals were clinically euthyroid and all had normal free T4 levels. Purified TTR from the propositus had an affinity for 125I-T4 three times that of control TTR. Exons 2, 3, and 4 (representing greater than 97% of the coding sequence) of the TTR gene of DNA prepared from the propositus' peripheral blood leukocytes were amplified using the polymerase chain reaction (PCR) and were sequenced after subcloning. Exons 2 and 3 were indistinguishable from normal. In 50% of clones amplified from exon 4, a substitution of adenine (ACC) for guanine (GCC) in codon 109 resulted in the replacement of threonine-for-alanine, a mutation confirmed by amino acid sequencing of tryptic peptides derived from purified plasma TTR. The adenine-for-guanine substitution abolishes one of two Fnu 4H I restriction sites in exon 4. PCR amplification of exon 4 of TTR and restriction digestion with Fnu 4H I confirmed that five affected family members with increased binding of 125I-T4 to TTR are heterozygous for the threonine 109 substitution that increases the affinity of this abnormal TTR for T4.
A C Moses, H N Rosen, D E Moller, S Tsuzaki, J E Haddow, J Lawlor, J J Liepnieks, W C Nichols, M D Benson
Cultured fibroblasts were shown to be capable of catalyzing the conversion of 7 alpha-hydroxy-cholesterol to 7 alpha-hydroxy-4-cholesten-3-one, an important reaction in bile acid synthesis. The apparent Km was approximately 7 mumol/liter and Vmax varied between 3 and 9 nmol/mg protein per h under the assay conditions used. The assay was used to investigate fibroblasts from a patient who presented with a familial giant cell hepatitis and who was found to excrete the monosulfates of 3 beta, 7 alpha-dihydroxy-5-cholenoic acid and 3 beta, 7 alpha, 12 alpha-trihydroxy-5-cholenoic acid in urine (Clayton, P. T., J. V. Leonard, A. M. Lawson, K. D. R. Setchell, S. Andersson, B. Egestad, and J. Sjövall. 1987. J. Clin. Invest. 79:1031-1038). In addition 7 alpha-hydroxy-cholesterol was found to accumulate in the circulation. Cultured fibroblasts from this boy were completely devoid of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase activity. Fibroblasts from his parents had reduced activity, compatible with a heterozygous genotype. The results provide strong evidence for the suggestion that this patient's liver disease was caused by a primary defect in the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase involved in bile acid biosynthesis.
M S Buchmann, E A Kvittingen, H Nazer, T Gunasekaran, P T Clayton, J Sjövall, I Björkhem
To assess the mechanisms responsible for increased gluconeogenesis in noninsulin-dependent diabetes mellitus (NIDDM), we infused [3-14C]lactate, [3-13C]alanine, and [6-3H]glucose in 10 postabsorptive NIDDM subjects and in 9 age- and weight-matched nondiabetic volunteers and measured systemic appearance of alanine and lactate, their release from forearm tissues, and their conversion into plasma glucose (corrected for Krebs cycle carbon exchange). Systemic appearance of lactate and alanine were both significantly greater in diabetic subjects (18.2 +/- 0.9 and 5.8 +/- 0.4 mumol/kg/min, respectively) than in the nondiabetic volunteers (12.6 +/- 0.7 and 4.2 +/- 0.3 mumol/kg/min, respectively, P less than 0.001 and P less than 0.01). Conversions of lactate and alanine to glucose were also both significantly greater in NIDDM subjects (8.6 +/- 0.5 and 2.4 +/- 0.1 mumole/kg/min, respectively) than in nondiabetic volunteers (4.2 +/- 0.4 and 1.8 +/- 0.1 mumol/kg/min, respectively, P less than 0.001 and P less than 0.025). The proportion of systemic alanine appearance converted to glucose was not increased in NIDDM subjects (42.7 +/- 1.9 vs. 44.2 +/- 2.9% in nondiabetic volunteers), whereas the proportion of systemic lactate appearance converted to glucose was increased in NIDDM subjects (48.3 +/- 3.8 vs. 34.2 +/- 3.8% in nondiabetic volunteers, P less than 0.025); the latter increased hepatic efficiency accounted for approximately 40% of the increased lactate conversion to glucose. Neither forearm nor total body muscle lactate and alanine release was significantly different in NIDDM and nondiabetic volunteers. Therefore, we conclude that increased substrate delivery to the liver and increased efficiency of intrahepatic substrate conversion to glucose are both important factors for the increased gluconeogenesis of NIDDM and that tissues other than muscle are responsible for the increased delivery of gluconeogenic precursors to the liver.
A Consoli, N Nurjhan, J J Reilly Jr, D M Bier, J E Gerich
To study chronic catecholamine desensitization, mini-osmotic pumps were implanted subcutaneously to deliver NE, (0.5 micrograms/kg/min) or saline over 3-4 wk in dogs instrumented with left ventricular (LV) pressure gauges and arterial and left atrial pressure catheters. An acute challenge to NE (0.4 micrograms/kg/min) in intact, conscious dogs increased LV dP/dt by 1,531 +/- 208 mmHg/s before NE pumps, and by a similar amount, 1,340 +/- 166 mmHg/s, 3-4 wk after NE pumps. In contrast, an acute challenge to isoproterenol (ISO, 0.4 micrograms/kg/min) increased LV dP/dt by 5,344 +/- 532 mmHg/s before NE pumps, and significantly less (P less than 0.05; 2,425 +/- 175 mmHg/s) after NE pumps. In the presence of ganglionic and alpha 1-adrenergic blockades, NE (0.4 micrograms/kg/min) increased LV dP/dt by 3,656 +/- 468 mmHg/s before NE pumps and significantly less (P less than 0.01; 1,459 +/- 200 mmHg/s) after NE pumps. Confirming this, an acute challenge to NE (0.4 micrograms/kg/min) in dogs with arterial baroreceptor denervation increased LV dP/dt by 3,732 +/- 896 mmHg/s before NE pumps, and significantly less (P less than 0.05, 1,725 +/- 408 mmHg/s) after NE pumps. In addition, in cardiac denervated dogs, NE (0.4 micrograms/kg/min) increased LV dP/dt by 9,901 +/- 1,404 mmHg/s before NE pumps and significantly less (P less than 0.01, 2,690 +/- 306 mmHg/s) after NE pumps. Desensitization of heart rate responses to NE challenge was also more apparent in the absence of reflex mechanisms. Thus, neural reflex mechanisms play a major role in physiological expression of cardiac desensitization to catecholamines in conscious dogs.
J Nejima, N Uemura, D E Vatner, C J Homcy, T H Hintze, S F Vatner
Treatment of rats with the vitamin B12 analogue hydroxy-cobalamin[c-lactam] (HCCL) impairs methylmalonyl-CoA mutase function and leads to methylmalonic aciduria due to intracellular accumulation of propionyl and methylmalonyl-CoA. Since accumulation of these acyl-CoAs disrupts normal cellular regulation, the present investigation characterized metabolism in hepatocytes and liver mitochondria from rats treated subcutaneously with HCCL or saline (control) by osmotic minipump. Consistent with decreased methylmalonyl-CoA mutase activity, 14CO2 production from 1-14C-propionate (1 mM) was decreased by 76% and 82% after 2-3 wk and 5-6 wk of HCCL treatment, respectively. In contrast, after 5-6 wk of HCCL treatment, 14CO2 production from 1-14C-pyruvate (10 mM) and 1-14C-palmitate (0.8 mM) were increased by 45% and 49%, respectively. In isolated liver mitochondria, state 3 oxidation rates were unchanged or decreased, and activities of the mitochondrial enzymes, citrate synthetase, succinate dehydrogenase, carnitine palmitoyltransferase, and glutamate dehydrogenase (expressed per milligram mitochondrial protein) were unaffected by HCCL treatment. In contrast, activities of the same enzymes were significantly increased in both liver homogenate (expressed per gram liver) and isolated hepatocytes (expressed per 10(6) cells) from HCCL-treated rats. The mitochondrial protein per gram liver, calculated on the basis of the recovery of the mitochondrial enzymes, increased by 39% in 5-6 wk HCCL-treated rats. Activities of lactate dehydrogenase, catalase, cyanide-insensitive palmitoyl-CoA oxidation, and arylsulfatase A in liver were not affected by HCCL treatment. Hepatic levels of mitochondrial mRNAs were elevated up to 10-fold in HCCL-treated animals as assessed by Northern blot analysis. Thus, HCCL treatment is associated with enhanced mitochondrial oxidative capacity and an increased mitochondrial protein content per gram liver. Increased mitochondrial oxidative capacity may be a compensatory mechanism in response to the metabolic insult induced by HCCL administration.
S Krahenbuhl, D B Ray, S P Stabler, R H Allen, E P Brass
Recent studies suggest that vitamin A plays an inhibitory role with respect to "activation" of the hepatic Ito cell, a likely effector of hepatic fibrogenesis. Ito cell "activation" during fibrogenesis is characterized by a decrease in intracellular vitamin A and an increase in cellular proliferation and collagen production. To explore the hypothesis that retinoids have the capacity to diminish Ito cell activation, cultured Ito cells were exposed to retinoic acid and its effects assessed on three key features: cell proliferation, collagen protein production and mRNA abundance, and transforming growth factor beta protein production. Retinoic acid was 100-1,000X more potent than retinol with respect to inhibition of Ito cell proliferation. Interstitial collagen and transforming growth factor beta production were also reduced by 10(-6) M retinoic acid. The relative abundance of type I collagen mRNA however, was not significantly altered. By contrast, retinoic acid administration to rats caused a marked reduction in the abundance of type I collagen mRNA in both total hepatic and purified Ito cell RNA. The relative abundance of rat hepatic fibronectin or apolipoprotein E mRNA was not significantly altered. These studies demonstrate that retinoic acid can differentially modulate several key features of hepatic fibrogenesis in vitro and in vivo.
B H Davis, R T Kramer, N O Davidson
Hereditary 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant rickets (HVDRR) is an autosomal recessive disease caused by target organ resistance to the action of 1,25(OH)2D3, the active form of the hormone. The defect in target cells is heterogenous and commonly appears to be a mutation in the gene encoding the vitamin D receptor (VDR). We have studied cultured skin fibroblasts and Epstein-Barr virus transformed lymphoblasts of seven family branches of an extended kindred having eight children affected with HVDRR. We have previously shown that cells from three affected children in this group contain an "ochre" nonsense mutation coding for a premature stop codon in exon 7 within the steroid-binding domain of the VDR gene. In the current studies, we found that cells from affected children failed to bind [3H]1,25(OH)2D3 and had undetectable levels of VDR as determined by immunoblots using an anti-VDR monoclonal antibody. Measurement of VDR mRNA by hybridization to a human VDR cDNA probe showed undetectable or decreased abundance of steady-state VDR mRNA. Parents, expected to be obligate heterozygotes, showed approximately half the normal levels of [3H]1,25(OH)2D3 binding, VDR protein, and mRNA. The mutation at nucleotide 970 (counting from the mRNA CAP site) results in the conversion of GTAC to GTAA, which eliminates an Rsa I restriction enzyme site and facilitates identification of the mutation. We found that polymerase chain reaction (PCR) amplification of exons 7 and 8 from family members and subsequent Rsa I digestion allows detection of the specific genotype of the individuals. When Rsa I digests of PCR-amplified DNA are subjected to polyacrylamide gel electrophoresis, children with HVDRR exhibit a homozygous banding pattern with loss of an Rsa I site. Parents exhibit a heterozygotic DNA pattern with detection of both normal and mutant alleles. In summary, our data show that the genetic abnormality is a point mutation within the steroid-binding domain of the VDR in all seven related families with HVDRR. Analysis of restriction fragment length polymorphism at the 970 locus of PCR-amplified DNA fragments can be used to diagnose this mutation in both affected children and parents carrying the disease.
P J Malloy, Z Hochberg, D Tiosano, J W Pike, M R Hughes, D Feldman
Extracorporeal photochemotherapy is an effective treatment for cutaneous T cell lymphoma but its mode of action is uncertain. The reduction in viability of patients' photoirradiated buffy coat lymphocytes was correlated with a 35% increase in DNA single-strand breaks and marked decreases in cellular ATP and NAD levels (to 58 and 34% of control, respectively) immediately after photoirradiation. Complementary in vitro studies were conducted with normal human peripheral blood lymphocytes using a Therakos ultraviolet A (UVA) light box. UVA light was cytotoxic on its own but was potentiated by 8-methoxysporalen. 3-aminobenzamide, a poly (ADP-ribose) synthetase inhibitor, mitigated the cytotoxic effect of ultraviolet A light in the presence of 8-methoxypsoralen in lymphocytes and reduced the amount of nucleotide depletion they caused. 10 J/cm2 of UVA light in the presence of 300 ng/ml 8-methoxypsoralen increased the poly (ADP-ribose) synthetase activity of peripheral blood lymphocytes. Exposing lymphocytes to deoxycoformycin and deoxyadenosine was found to induce biochemical and physical effects similar to those of photochemotherapy. In summary, we have shown that the lymphocytotoxic effect of extracorporeal photochemotherapy for cutaneous T cell lymphoma is apparently mediated by DNA damage, subsequent poly (ADP-ribosyl)ation and adenine nucleotide depletion. It is not known how the DNA damage and resultant biochemical effects relate to the possible immunological mechanism of extracorporeal photochemotherapy; however, it is possible that its effects can be mimicked by other DNA-damaging agents.
D I Marks, S P Rockman, M A Oziemski, R M Fox
Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 184.108.40.206) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients.
T Fukao, S Yamaguchi, M Kano, T Orii, Y Fujiki, T Osumi, T Hashimoto
The metabolism of albumin and IgG was investigated in two siblings, products of a first-cousin marriage, a female aged 34 yr and a male aged 17, who had a marked reduction in their respective serum concentrations of IgG (1.3 and 3.1 mg/ml) and albumin (19 and 21 mg/ml). The metabolism of radioiodinated IgG and albumin was studied in the two patients. The total circulating and body pools of IgG were less than 28% of normal. The IgG synthetic rates were within the normal range. However, the IgG survival was short, with their respective fractional catabolic rates increased fivefold to 31% and 36% of the intravenous pool per day (normal, 6.7 +/- 2%/d). Furthermore, the patients had reduced total body pools, normal synthetic rates, and increased fractional catabolic rates for albumin. There was no proteinuria or abnormality of renal or liver function. In addition, the patients did not have circulating antibodies directed toward IgG, IgA, or albumin. Furthermore, both patients had normal fecal 51Cr-labeled albumin tests, thus excluding excessive gastrointestinal protein loss. We propose that these siblings have a previously unrecognized familial disorder characterized by reduced serum concentrations of IgG and albumin caused by a defect in endogenous catabolism, leading to a short survival of these proteins that is associated in this family with chemical diabetes and a skeletal deformity.
T A Waldmann, W D Terry
A study of cell surface phenotypes of PBL of myasthenia gravis (MG) patients showed that their T cells had a significantly higher percentage of 4B4+ T cells (the helper/inducer subset) than age- and sex-matched controls. The PBL of MG patients proliferated significantly higher than those of normal subjects (NS) in response to the purified alpha chain of the acetylcholine receptor (AChR). Anti-AChR antibody was present in sera of 88% of MG and none of the NS. The PBL B cells from MG only, when cultured with autologous T cells and stimulated with either pokeweed mitogen (69%), or AChR-alpha chain (38%), secreted antibody to AChR-alpha chain, whereas T and B cells alone secreted no antibody. T cells from PBL of MG patients were more readily cloned than T cells of NS, by limiting dilution, in the presence of recombinant IL-2 and in the absence of AChR-alpha chain. About 50% of T cell clones from MG patients, compared to none from NS, proliferated to AChR-alpha chain. This response was HLA-DR restricted. MG T cell clones did not display significant cytotoxic activity, as compared to control T cell clones. Our results indicate that in MG, 4B4+ regulatory T cells play their role in the pathogenesis of MG, not by cytotoxicity, but more likely by their ability to stimulate specific antibody production by B cells.
F Mokhtarian, M Pino, W Ofosu-Appiah, D Grob
We examined the hypothesis that impaired endothelium-dependent vasodilation in atherosclerosis is associated with decreased synthesis of nitrogen oxides by the vascular endothelium. The descending thoracic aortae of rabbits fed either normal diet, a high cholesterol diet for 2-5 wk (hypercholesterolemic, HC), or a high cholesterol diet for 6 mo (atherosclerotic, AS) were perfused in a bioassay organ chamber with physiologic buffer containing indomethacin. Despite a dramatic impairment in the vasodilator activity of endothelium-dependent relaxing factor (EDRF) released from both HC and AS aortae (assessed by bioassay), the release of nitrogen oxides (measured by chemiluminescence) from these vessels was not reduced, but markedly increased compared to NL. Thus, impaired endothelium-dependent relaxation in atherosclerosis is neither due to decreased activity of the enzyme responsible for the production of nitrogen oxides from arginine nor to arginine deficiency. Because the production of nitrogen oxides increased in response to acetylcholine in both hypercholesterolemic and atherosclerotic vessels, impairments in signal transduction are not responsible for abnormal endothelium-dependent relaxations. Impaired vasodilator activity of EDRF by cholesterol feeding may result from loss of incorporation of nitric oxide into a more potent parent compound, or accelerated degradation of EDRF.
R L Minor Jr, P R Myers, R Guerra Jr, J N Bates, D G Harrison
The interference of the recombinant HIV-1 glycoproteins gp160 and gp120 with the CD3/T cell antigen receptor (TcR)-mediated activation process has been investigated in the CD4+ diphtheria toxoid-specific human P28D T cell clone. Both glycoproteins clearly inhibit the T cell proliferation induced in an antigen-presenting cell (APC)-free system by various cross-linked monoclonal antibodies specific for the CD3 molecule or the TcR alpha chain (up to 80% inhibition). Biochemical studies further demonstrate that exposure of the T cell clone to both glycoproteins (gps) specifically inhibits the CD3/TcR phospholipase C (PLC) transduction pathway, without affecting the CD3/TcR cell surface expression. Thus, inositol phosphate production, phosphatidic acid turnover, intracellular free calcium, and intracellular pH increase induced by CD3/TcR-specific MAbs are specifically impaired in gps-treated P28D T cells. Addition of purified soluble CD4 prevents binding of gps to T cells and overcomes all observed inhibitions. Maximal inhibitions are obtained for long-term exposure of the T cell clone to gps (16 h). No early effect of gps is observed. By contrast, gp160 and gp120 fail to suppress the CD2-triggered functional and biochemical P28D T cell responses. These results demonstrate that, in addition to their postulated role in the alteration of the interaction between CD4 on T lymphocytes and MHC class II molecules on APC, soluble HIV-1 envelope glycoproteins may directly and specifically impair the CD3/TcR-mediated activation of PLC in uninfected T cells via the CD4 molecule.
D Cefai, P Debre, M Kaczorek, T Idziorek, B Autran, G Bismuth
To provide a sensitive and generally applicable method to detect clonal cells in acute lymphoblastic leukemias (ALL), we have designed a new strategy based on the polymerase chain reaction (PCR) amplification of the T cell receptor gamma delta gene rearrangements found in most T and B lineage ALLs. PCR allows rapid sequencing of variable-(diversity)-joining (V-[D]-J) junctions from tumor DNA and construction of anti-junctional oligonucleotides (AJOs) used as probes to detect clonal cells in the same patient. We have defined oligonucleotides suitable for all T cell receptor (TCR) rearrangements involving functional V gamma segments. Oligonucleotides corresponding to preferential TCR delta rearrangements in T and B lineage ALLs were also used. By analysis of the nucleotide sequence of 52 V gamma-V gamma junctions from 30 cases of B and T ALLs, we demonstrate that V-J junctional sequences are clone specific in both lineages and at all stages of differentiation examined despite the frequent presence of the recently described P nucleotides. Experiments performed with TCR gamma delta AJOs on DNA from tumor cells and polyclonal T cells show that AJOs can be used to differentiate clonal cells from polyclonal T cells, distinguish between different T cell clones, and detect residual clonal populations at 10(-4)/10(-5) dilution. AJOs were also used to detect residual disease in samples from patients in clinical and morphological complete remission. Finally, rearrangement patterns were studied by classical Southern analysis in selected cases at both presentation and subsequent relapse showing absence of clonal evolution in most cases. V-(D)-J nucleotide sequences of rearrangements with an identical pattern of rearrangement at presentation and relapse were identical in all cases analyzed. We therefore describe a new, specific, and clinically useful strategy for the detection of minor clonal populations applicable in the majority of cases of ALL.
E A Macintyre, L d'Auriol, N Duparc, G Leverger, F Galibert, F Sigaux
In two clinical trials the mouse antiidiotypic monoclonal antibody (MAb) MF11-30, which bears the internal image of human high-molecular-weight-melanoma-associated antigen (HMW-MAA) was administered by subcutaneous route without adjuvants to patients with stage IV malignant melanoma on day 0, 7, and 28. Additional injections were administered if anti-antiidiotypic antibodies were not found or their titer decreased. In the first phase I trial with 16 patients the initial dose was 0.5 mg per injection and escalated to 4 mg per injection. Neither toxicity nor allergic reactions were observed despite the development of anti-mouse Ig antibodies. Minor responses were observed in three patients. In a second clinical trial MAb MF11-30 was administered to 21 patients at a dose of 2 mg per injection, since this dose had been shown in the initial study to be effective in inducing anti-antiidiotypic antibodies. Two patients were inevaluable; in the remaining 19 patients, the average duration of treatment was 34 wk. In this trial as well, neither toxicity nor allergic reactions were observed. 17 of the 19 immunized patients increased the levels of anti-mouse Ig antibodies and 16 developed antibodies that inhibit the binding of antiidiotypic MAb MF11-30 to the immunizing anti-HMW-MAA MAb 225.28. One patient increased the level of anti-HMW-MAA antibodies. One patient achieved a complete remission with disappearance of multiple abdominal lymph nodes for a duration of 95 wk. Minor responses were observed in three patients. These results suggest that mouse antiidiotypic MAb that bear the internal image of HMW-MAA may be useful reagents to implement active specific immunotherapy in patients with melanoma.
A Mittelman, Z J Chen, T Kageshita, H Yang, M Yamada, P Baskind, N Goldberg, C Puccio, T Ahmed, Z Arlin
To investigate the hypothesis that iron absorption in man involves a carrier-mediated cellular uptake mechanism, influx velocity (Vo) of 59Fe3+ by isolated human microvillous membrane (MVM) vesicles of the upper small intestine was examined. Vo revealed saturation kinetics (Km = 315 nM; Vmax = 361 pmol Fe3+ x min-1 x mg protein-1) was temperature dependent and inhibited by pronase pretreatment of MVM. In the presence of an inwardly directed Na(+)-gradient a typical overshoot phenomenon with maximal uptake at 30-40 s was observed. The suggestion of an active, carrier-mediated uptake mechanism for iron was pursued by isolation of a 160-kD iron-binding protein from solubilized human MVM proteins. This glycoprotein was assembled as a trimer composed of 54-kD monomers. A monospecific antibody against the 54-kD subunit inhibited vesicular influx of Fe3+ into MVM by greater than 50%. Immunofluorescence and immunoblot analysis confirmed the localization of the protein in brush border plasma membranes. It was detectable in human intestinal mucosa and liver, but not in esophagus. These data indicate that the translocation of Fe3+ across human MVM represents a facilitated transport mechanism which is, at least in part, mediated by a membrane iron-binding protein.
R Teichmann, W Stremmel
Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. Sera from patients with connective tissue diseases often contain antibodies against the proteins present in these snRNPs. Antibodies against the RNA components of snRNPs, the U snRNAs, are thought to be rare. We tested 118 anti-snRNP sera for the presence of anti-snRNA antibodies and found them in 45 sera (38%). In all sera the antibodies (IgG and F(ab)2 fragments thereof) were exclusively directed against U1 snRNA. The anti-(U1) RNA antibodies were always accompanied by anti-(U1)RNP antibodies but were not found in sera which contain antibodies of the Sm serotype directed against all nucleoplasmic U snRNP particles. Like anti-RNP antibodies, anti-U1 RNA activity is confined to sera from patients with SLE or SLE overlap syndromes and is rarely found in patients with other connective tissue diseases. By analyzing binding to subfragments of U1 snRNA made in vitro, it was demonstrated that anti-(U1)RNA antibodies recognize epitopes distributed throughout the U1 RNA molecule. In most sera, however, either the second or the fourth hairpin loop is the main target of the antibody. The possible mechanisms that could lead to the production of this new type of autoantibody are discussed.
W J van Venrooij, R Hoet, J Castrop, B Hageman, I W Mattaj, L B van de Putte
The role of pancreatic B cell dysfunction in the phase preceding clinical onset of insulin-dependent and non-insulin-dependent diabetes mellitus has been much debated. In this investigation, the impact of a prolonged diabetic environment on pancreatic islet B cells transplanted syngeneically under the kidney capsule of C57BL/6 (B6) and C57BL/Ks (BKs) mice was studied. Alloxan-diabetic mice bearing a subcapsular islet graft insufficient to normalize the blood glucose level were rendered normoglycemic by a second intrasplenic islet graft after various period of hyperglycemia to examine the reversibility of hyperglycemia-induced B cell dysfunction. Using a perfusion technique of the graft-bearing, it was found that both strains of mice exhibited a diminished glucose-induced insulin secretion after 6 wk of hyperglycemia, when compared with normoglycemic mice carrying islet grafts. When normoglycemia was restituted by the splenic graft after 4 or 12 wk, there was a normalization of glucose-stimulated insulin secretion in the renal islet grafts in B6 mice, whereas insulin secretion from the grafted BKs islets remained impaired. Morphometric measurements of the islet grafts demonstrated a 50% reduction in the graft volume in diabetic BKs mice after 12 wk, compared with normoglycemic animals, whereas no such decrease was observed in B6 mice. Islet grafts removed from hyperglycemic mice of both strains exhibited diminished insulin mRNA contents, and in the BKs mice there was also a reduced glucose oxidation rate in the islet grafts in vitro. This metabolic dysfunction can only partly be explained by a reduced graft size. The present findings emphasize the genetic constitution as a decisive factor for the survival and function during a period of sustained stress on a limited B cell mass.
O Korsgren, L Jansson, S Sandler, A Andersson
The role of inositol 1,4,5-trisphosphate (1,4,5-IP3) in regulating cytosolic Ca++ by stimulating Ca++ release from intracellular organelles is well established. However, other modes of intracellular Ca++ regulation by 1,4,5-IP3 have not been determined. To determine if 1,4,5-IP3 may regulate cell cytosolic Ca++ by acting on plasma membrane bound Na(+)-Ca++ exchanger, we investigated Ca++ transport in synaptosomes using 45Ca++ as tracer. In the presence of either an inhibitor of voltage gated Na+ channels (tetrodotoxin) or the K+ ionophore (valinomycin), Ca++ uptake was significantly inhibited (P less than 0.05) by 1,4,5-IP3 in a concentration dependent manner, with half-maximal inhibition occurring at submicromolar concentrations between 10(-9) M and 10(-10) M 1,4,5-IP3. Similarly, Ca++ efflux by the exchanger was significantly inhibited 40% by 1,4,5-IP3. The inhibitory effect of 1,4,5-IP3 on the Na(+)-Ca++ exchanger was observed in the presence of Ca++ channel blockers, and in vesicles pretreated with caffeine to deplete the 1,4,5-IP3-sensitive stores of Ca++. These results suggest that during signal transduction in brain, 1,4,5-IP3 may increase cytosolic [Ca++] in part by inhibiting the Na(+)-Ca++ exchanger and thus, Ca++ efflux from cell.
C L Fraser, P Sarnacki