Messenger RNA expression and synthesis of endothelin-1 along rat nephron segments.

The kidney both produces and responds to endothelin. We examined the production and the expression of mRNA of endothelin-1 (ET-1) in tubule suspensions and microdissected nephron segments. ET-1 production was measured by RIA using an ET-1-specific antibody. We applied the reverse transcription and polymerase chain reaction (PCR) technique to detect ET-1 mRNA along the nephron segments. Stimulation of ET-1 production was observed in the presence of FCS and transforming growth factor-beta (TGF-beta) in inner medullary tubules but not in cortical or outer medullary tubule suspensions. Among dissected nephron segments, ET-1 production was observed in glomeruli and inner medullary collecting ducts (IMCD), whereas it was negligible in proximal convoluted tubules (PCT) and medullary thick ascending limbs (MAL). In addition, the PCR product of ET-1 mRNA was also higher in glomeruli and IMCD, whereas it was undetectable in PCT and MAL. Furthermore, FCS and TGF-beta increased ET-1 mRNA in microdissected glomeruli and IMCD. These data clearly demonstrated that the production sites of ET-1 are glomeruli and IMCD among the nephron segments. ET-1 is an autocrine factor in these sites.


Mucopolysaccharidosis type IVA. N-acetylgalactosamine-6-sulfate sulfatase exonic point mutations in classical Morquio and mild cases.

Mucopolysaccharidosis type IVA (MPS IVA) results from a genetic deficiency of N-acetylgalactosamine-6-sulfate (Gal-NAc6S) sulfatase. We have identified two different exonic mutations causing GalNAc6S sulfatase deficiency in two unrelated Japanese families, in one patient with classical Morquio disease, and in two brothers with a mild form of MPS IVA. The nucleotide sequence of the full-length cDNA derived from a patient with classical Morquio disease revealed a two-base deletion at nucleotide position 1343-1344 (1344-1345 or 1345-1346) that altered the reading frame (designated 1342delCA). This mutation, inherited from the proband's consanguineous parents, was revealed by TaqI restriction analysis of a cDNA fragment amplified by the polymerase chain reaction. In the proband with the mild form of the disease, a C to G transversion at nucleotide 667 predicted the substitution of Lys for Asn204 (N204K). Since a new AluI site was created by the N204K mutation, restriction analysis indicated that the affected brothers were homozygous for this mutation, as confirmed by the finding that both their parents had this lesion. Transient expression in GalNAc6S sulfatase deficient fibroblasts of these two mutant alleles showed completely deficient or markedly decreased enzyme activities, thereby indicating that these two mutations were responsible for the enzyme deficiency.


Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen.


Enhanced membrane binding of autoantibodies to cultured keratinocytes of systemic lupus erythematosus patients after ultraviolet B/ultraviolet A irradiation.

Although sunlight is known to induce skin lesions in patients with systemic lupus erythematosus (SLE) and to exacerbate systemic manifestations, the underlying mechanisms remain obscure. We report experiments that show enhanced binding of IgG autoantibodies to the cell surface membrane of ultraviolet-B (UVB) irradiated (200-1,600 J/m2) cultured SLE keratinocytes in 10 out of 12 such cell strains. The autoantibody probes showing increased binding were directed against the soluble intracellular antigens, Sm, RNP, SSA/Ro, SSB/La, whereas serum with anti-dsDNA activity did not demonstrate such binding. Control keratinocytes from several sources shared low level binding of autoantibodies after ultraviolet light exposure. In addition, 4/6 UVB-sensitive SLE strains showed increased autoantibody binding to the surface of SLE keratinocytes after UVA exposure (50-150 kJ/m2), but of lower magnitude. When UVB-sensitive nonirradiated SLE strains were exposed to autologous serum, 3/8 sera demonstrated a striking increase in IgG binding, which increased further after UVB exposure. Enhanced expression of saline-soluble intracellular antigens on the cell surface membrane of patient, but not control, keratinocytes may, in part, explain the photosensitivity of patients with SLE.


Cellular immune reactivity to recombinant OspA and flagellin from Borrelia burgdorferi in patients with Lyme borreliosis. Complexity of humoral and cellular immune responses.

Patients with Lyme borreliosis (LB) usually develop a vigorous T cell response against the causative pathogen Borrelia burgdorferi, but little is known about the antigens recognized in the cellular response. Therefore, T cell reactivities against whole bacteria, recombinant 31-kD (outer surface protein A, [OspA]), and 41-kD proteins (flagellin) from B. burgdorferi were studied in patients with LB, non-LB patients, and healthy donors. In parallel, specific antibodies were determined by Western blot analysis. Virtually all patients with LB exhibited marked cellular responses to whole B. burgdorferi, which were significantly elevated compared with the control groups in both early and late disease stages. However, analyses using the purified antigens OspA and flagellin revealed considerable heterogeneity in the cellular reactivities among individuals as well as variations during the course of infection. T cell responses to OspA were significantly increased in patients with early LB compared with both control groups whereas in late-stage disease responses only exceeded those of non-LB patients and were not different from normal donors. Cellular immune reactivities to flagellin were significantly higher only in early LB compared with both control groups. Reciprocally, several control subjects demonstrated marked cellular responses to OspA and flagellin, suggesting that reactions to these proteins may not always be related to LB. T cell reactivity did not correlate well with the presence of specific antibodies. Almost all seropositive patients in both early and late stage LB had serum antibodies against flagellin, but antibodies to OspA were detectable only in a subset of late LB sera. These data demonstrate the complexity of the humoral and the cellular immune responses to components of B. burgdorferi.


Phagocytosis of unopsonized Pseudomonas aeruginosa by murine macrophages is a two-step process requiring glucose.

Pseudomonas aeruginosa is an important pulmonary pathogen in cystic fibrosis, but the means by which it evades host defenses is understood poorly. Macrophages (M phi) are critical in protecting the lung and mucosal surfaces against infection and may need to perform their functions in the absence of opsonins before the evolution of an inflammatory response. The purpose of the present study was to define factors that regulate the capacity of macrophages to mediate nonopsonic phagocytosis. Phagocytosis of unopsonized P. aeruginosa by murine peritoneal and pulmonary alveolar M phi s was absolutely dependent upon the presence of glucose; only D-mannose could substitute. Glucose-dependent phagocytosis appears to be selective for P. aeruginosa by M phi s; ingestion of unopsonized zymosan, opsonized P. aeruginosa, EIgG, and E (IgM)C occurred in the presence or absence of glucose as did-ingestion of unopsonized P. aeruginosa by polymorphonuclear leukocytes. M phi binding and phagocytosis of unopsonized P. aeruginosa appeared to occur by a mechanism independent of complement receptor 3 and mannose receptors. Phagocytosis of P. aeruginosa killed by tobramycin or Formalin was glucose dependent, suggesting that the glucose exerted its effects on the M phi rather than the bacteria. The predilection of P. aeruginosa for lower airway disease in patients with cystic fibrosis might be explained in part by the unique dependency upon glucose for M phi phagocytosis.


Lupus anticoagulant activity of autoimmune antiphospholipid antibodies is dependent upon beta 2-glycoprotein I.

It has been reported that antiphospholipid autoantibodies do not recognize phospholipid alone, but rather the plasma protein beta 2-glycoprotein I (beta 2GPI), or a beta 2GPI-phospholipid complex. In vitro beta 2GPI binds to anionic phospholipids and inhibits the prothrombinase activity of procoagulant membranes. In light of the fact that lupus anticoagulants, a type of antiphospholipid antibody, have similar anticoagulant properties, the relationship of beta 2GPI to lupus anticoagulant activity was investigated. IgG from patients with autoimmune diseases or syphilis were tested for anticardiolipin reactivity and lupus anticoagulant activity in the presence and absence of beta 2GPI. As expected, anti-cardiolipin reactivity associated with autoimmune disease was beta 2GPI dependent. In contrast, IgG from a patient with syphilis recognized cardiolipin alone and binding was inhibited by beta 2GPI. Autoimmune antiphospholipid antibodies prolonged the dilute Russell viper venom time of normal plasma, but had no effect on beta 2GPI-depleted plasma. Antiphospholipid antibodies associated with syphilis had no anticoagulant effect. RP-1, an anti-beta 2GPI mAb, had anticoagulant effects similar to those of autoimmune antiphospholipid antibodies. These data demonstrate that antiphospholipid autoantibodies exert lupus anticoagulant activity via an interaction with beta 2GPI. These antibodies and RP-1 appear to amplify the anticoagulant effect of beta 2GPI itself.


Induction of antiphospholipid autoantibodies by immunization with beta 2 glycoprotein I (apolipoprotein H).

A subset of patients with systemic lupus erythematosus has autoantibodies to acidic phospholipids. Since lipids are poor immunogens, the mechanism responsible for the induction of these antibodies is unclear. Immunization of a normal rabbit and normal mice with purified human beta 2-glycoprotein I (apolipoprotein H) resulted in the production of high levels of two non-cross-reactive antibody populations, anti-apolipoprotein H, and antiphospholipid. The antiphospholipid antibodies had binding specificities indistinguishable from autoantibodies obtained from human and murine lupus. These findings suggest a novel mechanism for the induction of antiphospholipid autoantibodies.


Advanced glycosylation endproducts block the antiproliferative effect of nitric oxide. Role in the vascular and renal complications of diabetes mellitus.

Advanced glycosylation endproducts (AGEs) accumulate on long-lived tissue proteins such as basement membrane collagen and have been implicated in many of the long-term complications of diabetes mellitus. These products originate from glucose-derived Schiff base and Amadori products but undergo a series of complex rearrangement reactions to form ultimately protein-bound, fluorescent heterocycles. AGEs can react with and chemically inactivate nitric oxide (NO), a potent endothelial cell-derived vasodilator and antiproliferative factor. Since mesenchymal cell proliferation is an early and characteristic lesion of diabetic vasculopathy and glomerulopathy, we investigated the possibility that collagen-bound AGEs functionally inactivate the antiproliferative effect of NO. In model cell culture systems, AGEs were found to block the cytostatic effect of NO on aortic smooth muscle and renal mesangial cells. The inactivation of endothelial cell-derived NO by basement membrane AGEs may represent a common pathway in the development of the accelerated vascular and renal disease that accompany long-term diabetes mellitus.


Human bactericidal/permeability-increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria.

The bactericidal/permeability-increasing protein (BPI) of neutrophils and BPI fragments neutralize the effects of isolated Gram-negative bacterial lipopolysaccharides both in vitro and in vivo. Since endotoxin most commonly enters the host as constituents of invading Gram-negative bacteria, we raised the question: Can BPI and its bioactive fragments also protect against whole bacteria? To determine whether the bactericidal and endotoxin-neutralizing activities of BPI/fragments are expressed when Gram-negative bacteria are introduced to the complex environment of whole blood we examined the effects of added BPI and proteolytically prepared and recombinant NH2-terminal fragments on: (a) the fate of serum-resistant encapsulated Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa that survive the antibacterial actions of whole blood and (b) the ability of these bacteria to trigger cytokine release. Added BPI in nanomolar concentrations killed each of three encapsulated strains of E. coli and in closely parallel fashion inhibited tumor necrosis factor (TNF) release. Holo-BPI and its NH2-terminal fragment were equipotent toward a rough LPS chemotype K1-encapsulated strain, but the fragment was substantially more potent than holo-BPI toward two encapsulated smooth LPS chemotype strains. TNF release induced by K. pneumoniae and P. aeruginosa was also inhibited by both holo-BPI and fragment but, at the protein concentrations tested, P. aeruginosa was killed only by the fragment and K. pneumoniae was not killed by either protein. The bactericidal action of BPI/fragment toward E. coli is inhibited by C7-depleted serum, but accelerated by normal serum, indicating that BPI, acting in synergy with late complement components, enhances extracellular killing of serum-resistant bacteria. Thus, BPI and an even more potent NH2-terminal fragment may protect against Gram-negative bacteria in the host by blocking bacterial proliferation as well as endotoxin-mediated effects, not only as components of the intracellular antibacterial arsenal of the neutrophil, but also as potentially therapeutic extracellular agents.


Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-beta. Possible existence of "vascular natriuretic peptide system".

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is thus far known to be distributed mainly in the central nervous system and is considered to act as a neuropeptide, in contrast to atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which act as cardiac hormones. Recently, we and others have demonstrated that the ANP-B receptor, which is selectively activated by CNP, is localized not only in the central nervous system but in peripheral tissues, including blood vessels. This finding has made us speculate regarding the peripheral production of CNP. In the present study, cultured endothelial cells were examined for CNP production by RIA and Northern blot analysis. CNP-like immunoreactivity was detected in the conditioned media of endothelial cells. Northern blot analysis detected CNPmRNA with a size of 1.2 kb. In addition, transforming growth factor (TGF)-beta, one of the key growth factors for vascular remodeling, markedly stimulated the expression of CNPmRNA and induced a tremendous increase in CNP secretion. We could also detect CNP transcript in the bovine thoracic aorta using the reverse transcription-polymerase chain reaction method. The present study demonstrates the endothelial production of CNP and suggests that a member of the natriuretic peptide family may act as a local regulator in vascular walls. Since evidence for the pathophysiological importance of the vascular renin-angiotensin system has been accumulating and the natriuretic peptide system is known to be antagonistic to the renin-angiotensin system, the possible existence of "vascular natriuretic peptide system" may prove to be of physiological and clinical relevance.


Filamentous, helical, and tubular microstructures during cholesterol crystallization from bile. Evidence that cholesterol does not nucleate classic monohydrate plates.

Precipitation of cholesterol in gallbladder bile is believed to produce platelike cholesterol monohydrate crystals directly. We report complementary time-lapse microscopic studies of cholesterol crystallization from model bile that reveal initial assembly of filamentous cholesterol crystals covered by a monomolecular layer of lecithin. Over a few days, the filaments evolved through needle, helical, and tubular microstructures to form classical platelike cholesterol monohydrate crystals. Similar crystallization phenomena were observed in human gallbladder biles from cholesterol but not pigment stone patients. Synchrotron x-ray diffraction of the earliest filaments suggested a cholesterol monohydrate polymorph or admixture with an anhydrous cholesterol precursor. However, density gradient centrifugation of filamentous crystals revealed that their density was 1.032 g/ml, consistent with anhydrous cholesterol. Conventional x-ray diffraction of transitional crystalline forms was consistent with pure cholesterol monohydrate crystals, as were the equilibrium platelike crystals. These novel findings suggest that crystalline cholesterol in bile may not be completely mature or hydrated initially, but undergoes a series of transformations to become thermodynamically stable monohydrate plates. These observations have important implications for understanding the control of cholesterol crystallization in bile, as well as explaining putative crystal cytotoxicity during gallstone formation.


Hypertrophic gastropathy resembling Ménétrier's disease in transgenic mice overexpressing transforming growth factor alpha in the stomach.

Transforming growth factor alpha (TGF alpha) is thought to participate in the normal and pathologic processes of numerous tissues, including the gastric mucosa. To explore its role in vivo, transgenic mice were generated overexpressing TGF alpha in the stomach. TGF alpha induced dramatic structural and functional lesions of the glandular stomach that were similar to Ménétrier's disease in humans. Transgenic mice developed severe adenomatous hyperplasia that resulted in a striking nodular thickening or hypertrophy of the gastric mucosa. Secretions obtained from affected stomachs contained no detectable gastric acid, suggesting that parietal cell function had been greatly impaired. These findings demonstrate that overproduction of TGF alpha can stimulate cellular proliferation, suppress acid secretion, and perturb organogenesis of the stomach of transgenic mice. Moreover, TGF alpha may contribute to the pathogenesis of related human hypertrophic gastropathies, such as Ménétrier's disease.


Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit.

The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis.


The tissue distribution of tumor necrosis factor biosynthesis during endotoxemia.

Tumor necrosis factor (TNF) is a protein hormone implicated in the development of septic shock and other pathologic states. However, complexities inherent in detecting TNF synthesis by individual tissues have left the precise origins of this protein undefined. In addition, the possibility that localized TNF production may contribute to the pathogenesis of organ-specific diseases such as type I diabetes has not been explored in vivo. We have developed a transgenic mouse line bearing a reporter gene construct in which the TNF coding sequence and introns are replaced by a chloramphenicol acetyltransferase (CAT) coding sequence. In normal transgenic animals, CAT activity is expressed only in the thymus. When endotoxin is administered to the animals, CAT activity is also evident in kidney, heart, islets of Langerhans, spleen, lung, fallopian tubes, and uterus, but not in other organs. The biosynthesis of CAT in vivo correlated with tissue capacity to secrete TNF in vitro. Thus, TNF was secreted by all the tissues that expressed CAT, including lung, spleen, thymus, uterus/fallopian tubes, pancreatic islets, renal glomeruli, and cultured cardiac cells after exposure to endotoxin.


Airway cooling and rewarming. The second reaction sequence in exercise-induced asthma.

To determine if a relationship exists among the magnitude and rate of airway rewarming, and the severity of bronchial obstruction in thermally induced asthma, we had seven subjects perform three- to four-point stimulus response curves with isocapnic hyperventilation of frigid air with and without pretreatment with inhaled norepinephrine. The latter was employed to alter the heat supplied to the airway walls by producing vasoconstriction. 1-s forced expiratory volume (FEV1) was measured before and 5 min after the cessation of each bout of hyperpnea and before and after norepinephrine. On a separate day, the subjects repeated the above challenges while the temperatures of the airstream in the intrathoracic airways were measured. Prenorepinephrine, FEV1 progressively decreased in a stimulus response fashion as ventilation rose, while norepinephrine shifted this curve to the right. As the level of ventilation increased, the size of the temperature difference between the cooling of hyperpnea and the rewarming of recovery followed suit, and their magnitude was linearly related to the severity of bronchial narrowing. Reducing the mucosal blood supply of the airways with norepinephrine limited rewarming and attenuated the obstructive response. These data demonstrate that the airway narrowing that develops following hyperpnea and the magnitude of the thermal differences are related, and that alterations in blood supply directly affect bronchial heat flux and influence obstruction.


Intracellular ATP can regulate afferent arteriolar tone via ATP-sensitive K+ channels in the rabbit.

Studies were performed to assess whether ATP-sensitive K+ (KATP) channels on rabbit preglomerular vessels can influence afferent arteriolar (AA) tone. K+ channels with a slope conductance of 258 +/- 13 (n = 7) pS and pronounced voltage dependence were demonstrated in excised patches from vascular smooth muscle cells of microdissected preglomerular segments. Channel activity was markedly reduced by 1 mM ATP and in a dose-dependent fashion by glibenclamide (10(-9) M to 10(-6) M), a specific antagonist of KATP channels. 10(-5) M diazoxide, a K+ channel opener, activated these channels in the presence of ATP, and this effect was also blocked by glibenclamide. To determine the role of these KATP channels in the control of vascular tone, diazoxide was tested on isolated perfused AA. After preconstriction from a control diameter of 13.1 +/- 1.1 to 3.5 +/- 2.1 microns with phenylephrine (PE), addition of 10(-5) M diazoxide dilated vessels to 11.2 +/- 0.7 microns, which was not different from control. Further addition of 10(-5) M glibenclamide reconstricted the vessels to 5.8 +/- 1.5 microns (n = 5; P less than 0.03). In support of its specificity for KATP channels, glibenclamide did not reverse verapamil induced dilation in a separate series of experiments. To determine whether intracellular ATP levels can effect AA tone, studies were conducted to test the effect of the glycolytic inhibitor 2-deoxy-D-glucose. After preconstriction from 13.4 +/- 3.2 to 7.7 +/- 1.3 microns with PE, bath glucose was replaced with 6 mM 2-deoxy-D-glucose. Within 10 min, the arteriole dilated to a mean value of 11.8 +/- 1.4 microns (n = 6; NS compared to control). Subsequent addition of 10(-5) M glibenclamide significantly reconstricted the vessels to a diameter of 8.6 +/- 0.5 micron (P less than 0.04). These data demonstrate that KATP channels are present on the preglomerular vasculature and that changes in intracellular ATP can directly influence afferent arteriolar tone via these channels.


Prevention of diabetes in nonobese diabetic mice by dendritic cell transfer.

The purpose of this study was to determine the effect of dendritic cell (DC) transfers on the incidence of diabetes in female nonobese diabetic (NOD) mice. Groups of 4-wk-old NOD female mice were given a single foot pad of DCs (70-90% purity) isolated from the draining lymph nodes (LN) of the pancreas (PLN), the cervical LNs, or the axillary/inguinal LNs. In addition, other groups of NOD mice received purified spleen DCs, purified PLN T cells (the major contaminating population in DC preparations), or the injection vehicle PBS. All groups were monitored for diabetes for one year. Significant protection from diabetes was observed in NOD mice receiving greater than 1 x 10(4) PLN DCs in comparison to mice receiving other DCs populations, PLN T cells, or PBS (P less than 0.05). The pancreata of NOD mice that received PLN DCs demonstrated significantly lower levels of lymphocytic infiltration in the islets that age-sex matched nondiabetic female NOD control mice (P less than 0.05). LN cells from nondiabetic NOD mice that received PLN DC protected irradiated female recipients from the adoptive transfer of diabetes to a greater degree than LN cells from age and sex matched nondiabetic female NOD mice that did not receive PLN DC transfers at 36 d (P = 0.014) and at 1 yr (P = 0.0015) after transfer. These data suggest that the PLN DC transfers are able to modulate autoimmunity and limit diabetes expression in the NOD mouse. PLN DCs transfers may regulate autoimmunity by the induction of regulatory cells.


On the mechanism of parathyroid hormone stimulation of calcium uptake by mouse distal convoluted tubule cells.

PTH stimulates transcellular Ca2+ absorption in renal distal convoluted tubules. The effect of PTH on membrane voltage, the ionic basis of the change in voltage, and the relations between voltage and calcium entry were determined on immortalized mouse distal convoluted tubule cells. PTH (10(-8) M) significantly increased 45Ca2+ uptake from basal levels of 2.81 +/- 0.16 to 3.88 +/- 0.19 nmol min-1 mg protein-1. PTH-induced 45Ca2+ uptake was abolished by the dihydropyridine antagonist, nifedipine (10(-5) M). PTH did not affect 22Na+ uptake. Intracellular calcium activity ([Ca2+]i) was measured in cells loaded with fura-2. Control [Ca2+]i averaged 112 +/- 21 nM. PTH increased [Ca2+]i over the range of 10(-11) to 10(-7) M. Maximal stimulation to 326 +/- 31 nM was achieved at 10(-8) M PTH. Resting membrane voltage measured with the potential sensitive dye DiO6(3) averaged -71 +/- 2 mV. PTH hyperpolarized cells by 19 +/- 4 mV. The chloride-channel blocker NPPB prevented PTH-induced hyperpolarization. PTH decreased and NPPB increased intracellular chloride, measured with the fluorescent dye SPQ. Chloride permeability was estimated by measuring the rate of 125I- efflux. PTH increased 125I- efflux and this effect was blocked by NPPB. Clamping voltage with K+/valinomycin; depolarizing membrane voltage by reducing extracellular chloride; or addition of NPPB prevented PTH-induced calcium uptake. In conclusion, PTH increases chloride conductance in distal convoluted tubule cells leading to decreased intracellular chloride activity, membrane hyperpolarization, and increased calcium entry through dihydropyridine-sensitive calcium channels.


Angiotensin II receptor blockade limits glomerular injury in rats with reduced renal mass.

The effects of angiotensin II (AII) blockade were compared with the effects of angiotensin converting enzyme inhibition in rats with reduced nephron number. Rats were subjected to five-sixths renal ablation and divided into four groups with similar values for blood pressure and serum creatinine after 2 wk. Group 1 then served as untreated controls, while group 2 received the AII receptor antagonist MK954 (which has previously been designated DuP753), group 3 received the converting enzyme inhibitor enalapril, and group 4 received a combination of reserpine, hydralazine, and hydrochlorothiazide. Micropuncture and morphologic studies were performed 10 wk later. Converting enzyme inhibition, AII receptor blockade, and the combination regimen were equally effective in reversing systemic hypertension (time-averaged systolic blood pressure: group 1, 185 +/- 5 mmHg; group 2, 125 +/- 2 mmHg; group 3, 127 +/- 2 mmHg; group 4, 117 +/- 4 mmHg). Micropuncture studies showed that glomerular transcapillary pressure was reduced significantly by converting enzyme inhibition and by AII blockade but not by the combination regimen (delta P: group 1, 49 +/- 1 mmHg; group 2, 42 +/- 1 mmHg; group 3, 40 +/- 2 mmHg, group 4, 47 +/- 1 mmHg). Reduction of systemic blood pressure was associated with the development of markedly less proteinuria and segmental glomerular sclerosis in rats receiving enalapril and MK954 but not in rats receiving the combination regimen (prevalence of glomerular sclerotic lesions: group 1, 41 +/- 4%; group 2, 9 +/- 1%; group 3, 9 +/- 1%; group 4, 33 +/- 6%). These results indicate that the effects of converting enzyme inhibition on remnant glomerular function and structure depend on reduction in AII activity and are not attributable simply to normalization of systemic blood pressure.


Lower sedentary metabolic rate in women compared with men.

Since females have a greater prevalence of obesity compared with males, the question arises whether females have lower metabolic rate than males after adjusting for differences in body weight and composition. 24-h energy expenditure (24EE), basal metabolic rate (BMR), and sleeping metabolic rate (SMR) were measured in a respiratory chamber in 235 healthy, nondiabetic Caucasian subjects (114 males, 121 females). Body composition was determined by hydrodensitometry. 24EE was 124 +/- 38 kcal/d (P less than 0.002) higher in males than females after adjusting for differences in fat-free mass, fat mass, and age. Spontaneous physical activity was not significantly different between males and females. Since adjusted 24EE was 106 +/- 39 kcal/d (P less than 0.01) higher in females during the luteal phase of the menstrual cycle compared with females during the follicular phase, energy expenditure was analyzed in a subset (greater than 50 yr) to minimize the confounding effect of menstrual status. 24EE (160 +/- 66 kcal/d; P less than 0.03), BMR (116 +/- 45; P less than 0.02), and SMR (208 +/- 68 kcal/d; P less than 0.005) were higher in males compared with females of the older subset after adjusting for differences in body composition, age, and activity. In summary, sedentary 24EE is approximately 5-10% lower in females compared with males after adjusting for differences in body composition, age, and activity.


Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis.

Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P less than 0.05) in synovial fluid from RA patients (mean 25.5 +/- 8.1 ng/ml [SE]) compared to synovial fluid from osteoarthritis (OA) patients (0.92 +/- 0.08), or from patients with other arthritides (2.9 +/- 1.5). MCP-1 levels in RA sera (8.44 +/- 2.33) were significantly greater than MCP-1 in normal sera (0.16 +/- 0.06). The quantities of RA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P less than 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50 +/- 8%) as compared to either OA macrophages (5 +/- 2) or normal macrophages (1 +/- 0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18 +/- 8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine.


Anoxia-hyperoxia induces monocyte-derived interleukin-8.

Ischemia-reperfusion and hyperoxia-induced pulmonary injury are associated with the presence of activated neutrophils (PMN) and cellular injury. Although the signals orchestrating the directed migration of these PMN during the pathogenesis of these disease states remain to be fully elucidated, it appears they may be dependent upon the production of certain neutrophil activating/chemotactic factors such as C5a, leukotriene B4, platelet-activating factor, and IL-8. The production of the latter chemotaxin by mononuclear phagocytes is especially intriguing as these cells can mediate inflammatory cell migration by either directly generating IL-8, or by inducing its production from surrounding nonimmune cells. In light of these observations, we propose that ischemia-reperfusion and oxidant stress, in vivo, may be simulated by anoxia-hyperoxia induced stress in vitro, and that this stress may act as a stimulus for the production of IL-8. We now show that isolated human blood monocytes respond to such an oxygen stress with augmented production of IL-8. In initial studies, monocytes demonstrated an increase in the production of IL-8 under anoxic preconditioning. Subsequently, monocytes were cultured under one of the following conditions for 24 h: (a) room air/5% CO2; (b) 95% N2/5% CO2 for 6 h, followed by room air/5% CO2 for 18 h; (c) 95% N2/5% CO2 for 6 h, followed by 95% O2/5% CO2 for 18 h; (d) room air/5% CO2 for 6 h, followed by 95% O2/5% CO2 for 18 h; or (e) 95% O2/5% CO2. Supernatants were isolated and analyzed for IL-8 antigen by specific IL-8 ELISA, demonstrating the production of monocyte-derived IL-8: 5.9 +/- 0.9, 11.4 +/- 1.7, 21.1 +/- 2.3, 14.6 +/- 2.4, and 26.3 +/- 4.7, ng/ml by designated conditions a, b, c, d, and e listed above, respectively. This variance in IL-8 production reflects altered rates of transcription as shown by Northern blot analysis and nuclear run-off assay. Furthermore, when monocytes were concomitantly treated with LPS (100 ng/ml) under in vitro hyperoxic conditions, both IL-8 steady-state mRNA and antigenic activity were two- to threefold greater than under room air conditions. The association of anoxic preconditioning and oxygen stress with augmented production of monocyte-derived IL-8 support the potential role for ischemia-reperfusion and hyperoxia-induced IL-8 production in vivo, providing a possible mechanism for PMN migration/activation in disease states characterized by altered tissue oxygenation.


Molecular genetics of steroid 5 alpha-reductase 2 deficiency.

Two isozymes of steroid 5 alpha-reductase encoded by separate loci catalyze the conversion of testosterone to dihydrotestosterone. Inherited defects in the type 2 isozyme lead to male pseudohermaphroditism in which affected males have a normal internal urogenital tract but external genitalia resembling those of a female. The 5 alpha-reductase type 2 gene (gene symbol SRD5A2) was cloned and shown to contain five exons and four introns. The gene was localized to chromosome 2 band p23 by somatic cell hybrid mapping and chromosomal in situ hybridization. Molecular analysis of the SRD5A2 gene resulted in the identification of 18 mutations in 11 homozygotes, 6 compound heterozygotes, and 4 inferred compound heterozygotes from 23 families with 5 alpha-reductase deficiency. 6 apparent recurrent mutations were detected in 19 different ethnic backgrounds. In two patients, the catalytic efficiency of the mutant enzymes correlated with the severity of the disease. The high proportion of compound heterozygotes suggests that the carrier frequency of mutations in the 5 alpha-reductase type 2 gene may be higher than previously thought.


A fatty neuropeptide. Potential drug for noninvasive impotence treatment in a rat model.

Vasoactive intestinal peptide (VIP), a key penile neurotransmitter, induces erection after local injection in man. To augment the therapeutic potential of VIP for impotence treatment and circumvent difficulties of direct penile injections, a strategy was designed to increase peptide hydrophobicity. This was accomplished by the synthesis of a conjugate of VIP and stearic acid (stearyl-VIP). Upon penile topical application, stearyl-VIP, in contrast to native VIP, significantly increased sexual function as measured by copulatory activity and penile reflexes (erections) in testosterone-treated, castrated rats. In addition, stearyl-VIP penetrated the body in amounts severalfold greater than VIP. Pharmacokinetic studies demonstrated 10-fold higher penile concentrations of stearyl-VIP, as compared with that measured in the blood 15 min after application, with a gradual decrease thereafter. The peak of incorporation into peripheral tissues that was observed 30 min after administration was 1,000-fold less than that found in the penile tissue. Tissue extraction and chromatographic analysis revealed that stearyl-VIP remained essentially intact for greater than or equal to 15 min and was cleared after 1 h. Thus, topically administered stearyl-VIP had increased bioavailability in comparison with VIP without apparent toxicity, suggesting significant therapeutic potential.


Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.


Disorders of bile acid metabolism in cholesterol gallstone disease.

The aim of the study was to evaluate the metabolism of individual bile acids in patients with cholesterol gallstone disease. Therefore, we determined pool size and turnover of deoxycholic (DCA), cholic (CA), and chenodeoxycholic acid (CDCA) in 23 female gallstone patients classified according to their gallbladder function and in 15 healthy female controls. Gallstone patients had normal hepatic bile acid synthesis, but, depending on gallbladder function, differed with respect to turnover and size of the bile acid pools: Patients with well-emptying gallbladder (group A, n = 9) had enhanced turnover and reduced pools of CA (-46%; P less than 0.01 vs. controls) and CDCA (-24%; P less than 0.05), but normal input and size of the DCA pool. With reduced gallbladder emptying (less than 50% of volume; group B, n = 6), turnover and pools of CA, CDCA, and DCA were similar as in controls. Patients with loss of gallbladder reservoir (group C, n = 8) had increased input (+100%; P less than 0.01) and pool size of DCA (+45%; P = 0.07) caused by rapid conversion of CA to DCA, while the pools of CA (-71%; P less than 0.001 vs. controls) and CDCA (-36%; P less than 0.05) were reduced by enhanced turnover. Thus, in patients with cholesterol gallstones, the pools of primary bile acids are diminished, unless gallbladder emptying is reduced. Furthermore, in a subgroup of gallstone patients, who had completely lost gallbladder function, the CA pool is largely replaced by DCA owing to rapid transfer of CA to the DCA pool. This probably contributes to supersaturation of bile with cholesterol.


Electroneutral K+/HCO3- cotransport in cells of medullary thick ascending limb of rat kidney.

The renal medullary thick ascending limb (MTAL) of the rat absorbs bicarbonate through luminal H+ secretion and basolateral HCO3- transport into the peritubular space. To characterize HCO3- transport, intracellular pH (pHi) was monitored by use of the pH-sensitive fluorescent probe (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein in fresh suspensions of rat MTAL tubules. When cells were preincubated in HCO3-/CO2-containing solutions and then abruptly diluted into HCO3-/CO2-free media, the pHi response was an initial alkalinization due to CO2 efflux, followed by an acidification (pHi recovery). The pHi recovery required intracellular HCO3-, was inhibited by 10(-4) M diisothiocyanostilbene-2-2'-disulphonic acid (DIDS), and was not dependent on Cl- or Na+. As assessed by use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine, cell depolarization by abrupt Cl- removal from or addition of 2 mM barium into the external medium did not affect HCO3(-)-dependent pHi recovery, and the latter was not associated per se with any change in potential difference, which indicated that HCO3- transport was electroneutral. The HCO3(-)-dependent pHi recovery was inhibited by raising extracellular potassium concentration and by intracellular potassium depletion. Finally, as measured by use of a K(+)-selective extracellular electrode, a component of K+ efflux out of the cells was HCO3- dependent and DIDS sensitive. The results provide evidence for an electroneutral K+/HCO3- cotransport in rat MTAL cells.


Leukemia inhibitory factor is expressed in cartilage and synovium and can contribute to the pathogenesis of arthritis.

This study reports on leukemia inhibitory factor (LIF) in human articular connective tissues. Biologically active LIF is present in synovial fluids from patients with osteoarthritis and at higher titers in samples from patients with rheumatoid arthritis. Cultured human synoviocytes and articular chondrocytes produced biologically active LIF and synthesized and secreted LIF proteins that migrated in SDS PAGE at approximately 43 kD. This was increased after stimulation with IL-1 beta. Chondrocytes in serum-containing cultures expressed the 4.2-kb LIF mRNA. IL-1 beta, LPS, and to a lesser extent tumor necrosis factor-alpha induced LIF gene expression. LIF autoinduced its mRNA and this provides evidence for an effect of this cytokine on function of joint tissue cells. Among a series of growth factors tested, transforming growth factor (TGF beta), including the isoforms TGF-beta1, TGF-beta 2, and TGF-beta 3, platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor induced this cytokine gene but differed with respect to the duration of their effects. Cultured synoviocytes expressed the LIF gene in response to the same set of peptide regulatory factors. Analysis of signal transduction pathways showed that PMA increased LIF mRNA, whereas calcium ionophore and cAMP had no detectable effects. Cycloheximide was a potent LIF mRNA inducer and dexamethasone inhibited LIF induced by PMA or IL-1 beta. Cartilage organ cultures and synovial tissues stimulated with IL-1 expressed high levels of LIF mRNA as demonstrated by in situ hybridization. These results identify LIF as a new cytokine that is produced by joint tissue cells and is overexpressed in arthritis. The induction of this cytokine by factors that are present during joint inflammation and the effects of LIF on connective tissue cells suggest that LIF is a mediator that can contribute to the pathogenesis of arthritis.


Serum neutralizing antibody response to the vacuolating cytotoxin of Helicobacter pylori.

Approximately 50% of Helicobacter pylori isolates produce a cytotoxin in vitro that induces vacuolation of eukaryotic cells. To determine the in vivo relevance of this phenomenon, we sought to detect cytotoxin-neutralizing antibodies in sera from H. pylori-infected persons. As a group, sera from 29 H. pylori-infected patients neutralized the activity of the purified cytotoxin to a significantly greater extent than sera from 24 uninfected persons (P = 0.007). The cytotoxin neutralizing activity in sera from H. pylori-infected persons was mediated predominantly by the purified IgG fraction. Sera from H. pylori-infected persons neutralized the cytotoxins produced by multiple H. pylori strains, but failed to neutralize trimethylamine-induced cell vacuolation. Neutralization of cytotoxin activity by human or immune rabbit sera was associated with immunoblot IgG recognition of an 87-kD H. pylori protein. Similarly, neutralization of the toxin by sera was associated with IgG recognition of the purified cytotoxin in an enzyme-linked immunosorbent assay (P less than 0.0001). The presence of cytotoxin-neutralizing antibodies in sera from H. pylori-infected persons indicates that the cytotoxin is synthesized in vivo.


Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.

Complementary DNA cloning of the 130-kD pemphigus vulgaris (PV) autoantigen (PVA) has indicated that it is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules. By homology with typical cadherins, PVA has five extracellular domains (EC1 through EC5). To localize immunogenic domains and to determine whether antibodies against them might be pathogenic, we produced beta-galactosidase fusion proteins with cDNA encoding different portions of the extracellular domains of PVA (EC1-2, EC3-5, and each individual domain). Immunoblot analysis of these fusion proteins with 23 PV patients' sera demonstrated that major immunogenic regions of PVA are located on the EC1, EC2, and EC4 domains. IgG was affinity-purified from PV sera on fusion proteins representing the amino (EC1-2) and carboxy (EC3-5) terminus of the extracellular PVA, and injected into neonatal mice. PV IgG affinity-purified on the EC1-2 fusion protein caused suprabasilar acantholysis, the typical histological finding of PV, but IgG affinity-purified on the EC3-5 fusion protein or beta-galactosidase alone did not. These results indicate that at least one pathogenic epitope, which is sufficient to cause suprabasilar acantholysis in neonatal mice, is located on the amino-terminal region of PVA, an area thought to be important in cadherin homophilic adhesion.


Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck.

We find that pADEO16, a recombinant cosmid carrying the rck gene of the Salmonella typhimurium virulence plasmid, when cloned into either rough or smooth Escherichia coli and Salmonella strains, confers high level resistance to the bactericidal activity of pooled normal human serum. The rck gene encodes a 17-kD outer membrane protein that is homologous to a family of virulence-associated outer membrane proteins, including pagC and Ail. Complement depletion, C3 and C5 binding, and membrane-bound C3 cleavage products are similar in strains with and without rck. Although a large difference in C9 binding was not seen, trypsin cleaved 55.7% of bound 125I-C9 counts from rough S. typhimurium with pADEO16, whereas only 26.4% were released from S. typhimurium with K2011, containing a mutation in rck. The majority of C9 extracted from rck strain membranes sediments at a lower molecular weight than in strains without rck, suggesting less C9 polymerization. Furthermore, SDS-PAGE analysis of gradient peak fractions indicated that the slower sedimenting C9-containing complexes in rck strains did not contain polymerized C9 typical of the tubular membrane attack complex. These results indicate that complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes.


Binding of uropathogenic Escherichia coli R45 to glycolipids extracted from vaginal epithelial cells is dependent on histo-blood group secretor status.

Women with a history of recurrent Escherichia coli urinary tract infections (UTIs) are two to three times more likely to be nonsecretors of histo-blood group antigens than are women without such a history. Further, uroepithelial cells from women who are nonsecretors show enhanced adherence of uropathogenic E. coli compared with cells from secretors. To investigate the hypothesis that nonsecretors express unique receptors for uropathogenic E. coli related to their genetic background, we extracted glycosphingolipids (GSLs) from vaginal epithelial cells collected from nonsecretors and secretors and used an assay in which radiolabeled uropathogenic E. coli were bound to these GSLs separated on TLC plates. An E. coli strain (R45) expressing both P and F adhesins, which was isolated from one of these patients' UTIs, was metabolically labeled with 35S for the TLC binding assay. The radiolabeled E. coli R45 bound to two extended globo-series GSLs, sialosyl gal-globoside (SGG) and disialosyl gal-globoside (DSGG), found in the GSL extracts from nonsecretors but not from secretors. The identity of SGG in the nonsecretor GSL extracts was confirmed in radioimmunoassays using an mAb to SGG and in immunofluorescence assays with this mAb and native vaginal epithelial cells. We show that SGG and DSGG are selectively expressed by epithelial cells of nonsecretors, presumably as a result of sialylation of the gal-globoside precursor glycolipid, which in secretors is fucosylated and processed to ABH antigens. The presence of SGG and DSGG may account for the increased binding of E. coli to uroepithelial cells from nonsecretors and for their increased susceptibility to recurrent UTI.


Molecular cloning and preliminary characterization of a novel cytoplasmic antigen recognized by myasthenia gravis sera.

A cDNA clone was isolated by screening of a lambda gt11 endothelial expression library with serum from a patient with myasthenia gravis (MG). Rabbit antisera raised against the recombinant protein and human MG sera reactive with the clone immunoblotted an M(r) integral of 250,000 polypeptide (gravin) present in endothelial cells and several adherent cells. Gravin was not detected in platelets, leukocytes, U937, or human erythroleukemic (HEL) cell lines, but was expressed in HEL cells after induction with phorbol myristate acetate. Northern blot analysis showed two transcripts of approximately 6.7 and 8.4 kb in endothelial cells but not U937 or HEL cells. Indirect immunofluorescence of permeabilized cells revealed a trabecular network of gravin staining with a distinct linear component. Antibodies to gravin, were present in sera from 22:72 (31%) of MG patients. In contrast 0:50 normal sera and 1:72 sera from patients with other autoimmune diseases contained antigravin antibodies. Gravin is not likely to be a nonerythroid spectrin, talin, myosin, or actin-binding protein based on the lack of reactivity of antigravin with these polypeptides in immunoblots. The nucleotide sequence of the immunoreactive clone indicated that it encodes a highly acidic polypeptide fragment that contains the carboxyl terminus of the protein. Neither amino acid nor nucleotide sequences were present in Genbank, EMBL, or Swissprot databases as of March, 1992. These data indicate that gravin is an inducible, cell type-specific cytoplasmic protein and that auto-antibodies to gravin may be highly specific for MG.


The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization.

In addition to a nonadecapeptide homologous to the teleost melanin-concentrating hormone (MCH), the amino acid sequence predicted from a rat prepro-MCH (ppMCH) cDNA suggested that at least one (neuropeptide EI, or NEI), and possibly a second (NGE), additional neuropeptide may be encoded by this precursor. Cross-reactivity with epitopes of NEI or NGE can account for reported localization of alpha-MSH, rat CRF, and human GRF in rat dorsolateral hypothalamic neurons. We have used antisera raised against rat MCH and NEI in immunohistochemical studies at the light and electron microscopic levels, along with hybridization histochemical localization of ppMCH mRNA, to define the organization of this system. As expected, ppMCH mRNA is prominently expressed in cells in the lateral hypothalamic area and zona incerta. The MCH and NEI peptides were extensively colocalized in neurons in both of these areas. In addition, smaller cell groups in the olfactory tubercle and pontine tegmentum were also positively hybridized for ppMCH mRNA and immunostained for MCH and NEI. Fibers stained for MCH and NEI were similarly, and very broadly, distributed throughout the central nervous system in patterns that generally conformed with known projection fields of the lateral hypothalamic area and zona incerta. A differential distribution was seen in at least one region, the interanterodorsal nucleus of the thalamus, which contained a prominent terminal field stained for MCH but not NEI. At the electron microscopic level, MCH-stained perikarya displayed a prominent staining associated with the Golgi apparatus; this was not encountered in NEI-stained cells. Both peptides were distributed similarly in terminals in the lateral hypothalamic area and median eminence, with staining associated principally with dense-cored vesicles. The results suggest that ppMCH-derived peptides may serve as neurotransmitters or modulators of prominence in a surprisingly expansive projection field of incerto-hypothalamic neurons. The terminal distributions of this system seem most compatible with functional roles in generalized arousal and sensorimotor integration, processes previously implicated as being subject to modulation by the lateral hypothalamic area.


Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor.

Although the gastric tension receptor has been characterized behaviorally and electrophysiologically quite well, its location and structure remains elusive. Therefore, the vagal afferents to the rat fundus (forestomach or nonglandular stomach) were anterogradely labeled in vivo with injections of the carbocyanine dye Dil into the nodose ganglia, and the nerves and ganglia of the enteric nervous system were labeled in toto with intraperitoneal Fluorogold injection. Dissected layers and cryostat cross sections of the fundic wall were mounted in glycerin and analyzed by means of conventional and laser scanning confocal microscopy. Particularly in the longitudinal, and to a lesser extent in the circular, smooth muscle layers, Dil-labeled fibers and terminals were abundant. These processes, which originated from fibers coursing through the myenteric ganglia and connectives, entered either muscle coat and then ran parallel to the respective muscle fibers, often for several millimeters. They ran in close association with the Fluorogold-labeled network of interstitial cells of Cajal, upon which they appeared to form multiple spiny appositions or varicosities. In the myenteric plexus, two different types of afferent vagal structures were observed. Up to 300 highly arborizing endings forming dense accumulations of small puncta similar to the esophageal intraganglionic laminar endings (Rodrigo et al., '75 Acta Anat. 92:79-100) were found in the fundic wall ipsilateral to the injected nodose ganglion. They often covered small clusters of myenteric neurons or even single isolated ganglion cells (mean = 5.8 neurons) and tended to extend throughout the neuropil of the ganglia. In a second pattern, fine varicose fibers with less profuse arborizations innervated mainly the central regions of myenteric ganglia. Camera lucida analyses established that single vagal afferent fibers had separate collaterals in both a smooth muscle layer and the myenteric ganglia. Finally, Dil-labeled afferent vagal fibers were also found in the submucosa and mucosa. Control experiments in rats with supranodose vagotomy as well as rats with Dil injections directly in the distal cervical vagus ruled out the possibility of colabeling of afferent fibers of passage. In triple labeling experiments, in conjunction with Dil labeling of afferents and Fluorogold labeling of enteric neurons, the carbocyanine dye DiA was injected into the dorsal motor nucleus of the vagus to anterogradely label the efferent vagal fibers and terminals. The different distributions and morphological characteristics of the vagal afferents and efferents could be simultaneously compared. In some instances the same myenteric ganglion was apparently innervated by an afferent laminar ending and an efferent terminal.(ABSTRACT TRUNCATED AT 400 WORDS)


Innervation of the amphibian and basilar papillae in the leopard frog: reconstructions of single labeled fibers.

Amphibians have two auditory organs specialized for reception of airborne sounds: the amphibian papilla and the basilar papilla. In this report we examine the morphology of the ganglion cells and the afferent innervation of the sensory epithelium in both auditory organs of the leopard frog, Rana pipiens pipiens. Extracellular injections of either biocytin or horseradish peroxidase (HRP) were made into the VIII nerve; they labeled ganglion cells, their axons, and their terminal fibers within the papillae. Ganglion cells that projected to either the amphibian papilla or basilar papilla had cell bodies that were morphologically distinct from other labeled cells. In the amphibian papilla thick fibers terminated in the rostral portion and thin fibers terminated in the caudal portion. Labeled fibers in the rostral portion traveled short distances before making contacts with up to nine hair cells whereas labeled fibers in the caudal portion traveled longer distances and contacted no more than five hair cells. In the basilar papilla labeled fibers were thick (around 4 microns) and terminated on as many as nine hair cells. Consistent with studies from the bullfrog, Rana catesbeiana, our results suggest that the amphibian papilla of R. pipiens pipiens has a convergent innervation (i.e., multiple hair cells provide input to a single ganglion cell) and is topographically organized. However, in contrast to reports in other ranid species, a highly convergent innervation like that found in the amphibian papilla is also found in the basilar papilla.


Calcitonin gene-related peptide in afferents to the cat's cerebellar cortex: distribution and origin.

In the present study, the distribution and origin of calcitonin gene-related peptide (CGRP) were analyzed in the cat's cerebellum. Following incubation in an antibody generated against rat CGRP and processing with the peroxidase anti-peroxidase (PAP) technique, CGRP immunoreactivity (IR) is found in profiles that have morphological characteristics of both simple and complex mossy fibers. However, all mossy fibers are not CGRP-positive. Further, CGRP-IR mossy fibers have a heterogeneous distribution in the cerebellum. In the vermis, the majority of immunoreactive profiles are in lobules VII, VIII, and the dorsal folia of IX. In anterior vermal lobules, only scattered terminals, located primarily at the apex and along the shoulder of the folia, are present. Laterally, CGRP-IR mossy fibers are located in the paramedian lobule, paraflocculus, and crus II. No CGRP fibers or varicosities are observed in any of the cerebellar nuclei. However, CGRP-positive cell bodies are scattered throughout the nuclear neuropil. A double label technique revealed that CGRP-IR mossy fibers arise from neurons located in the lateral reticular nucleus, external cuneate nucleus, inferior vestibular nucleus, and basilar pons. The present findings, taken together with previous data, indicate that cerebellar afferents are chemically heterogeneous. The findings of the present study suggest that precerebellar nuclei that give rise to the mossy fibers that contain CGRP have the potential for playing a complex role in modulating circuitry in the cerebellar cortex of the cat.


The rat's postero-orbital sinus hair: II. Normal morphology and the increase in peripheral innervation with adjacent nerve section.

The morphology and innervation of the postero-orbital (PO) sinus hair has been studied in normal rats and in adult animals in which an adjacent nerve, the infraorbital nerve, was sectioned on postnatal day 0 or day 7. The normal morphology of the follicle was similar to that of mystacial sinus hairs. However, the normal innervation differed from mystacial follicles in three respects: (1) instead of a separate innervation, the deep vibrissal nerve (DVN) and dermal plexus were supplied by a common follicle and skin nerve, named here the postero-orbital cutaneous nerve, a branch of the zygomaticofacial nerve; (2) the entry of the DVN through the capsule was highly variable; in some cases fascicles entered in close proximity, but in others they were widely distributed around the capsule; and (3) two or three small nerves, called here anastomosing nerves, were found to leave the PO follicle. These arose from the DVN after it had passed through the capsule to the cavernous sinus. The anastomosing nerves passed back through the capsule and ascended on the outer surface of the follicle to join the dermal plexus. Each nerve contained 1-4 myelinated fibres and 11-35 unmyelinated fibres. Infraorbital (IO) nerve section on day 0 caused a 19% (P less than 0.001, n = 8) increase in numbers of fibers to the DVN on the lesioned side. Most of the increase was due to unmyelinated fibres with no significant change in myelinated axons. No change in axon numbers in the DVN occurred after day 7 lesions. Labelling of the mystacial pad and the PO follicle did not result in any double labelling of cells in the trigeminal ganglion, in either normal or lesioned animals, making it improbable that the increased numbers of unmyelinated axons arose from rerouting of infraorbital fibres. It is suggested that the increased innervation of the PO follicle may arise by the rescue of ganglion cells from developmentally programmed cell death.


Dual innervation of the rat vibrissa: responses of trigeminal ganglion cells projecting through deep or superficial nerves.

The rat vibrissal follicle-sinus complex is innervated by a deep vibrissal nerve (DVN) and several smaller fascicles traveling in the dermis [conus or superficial vibrissal nerves, (SVNs)]. The function of the SVNs is unknown, although it has been suggested in a comparative study that they form part of a diffuse, multivibrissal system. Anatomical and electrophysiological methods were used to test this hypothesis and to determine if DVN and SVN fibers have differing response profiles. No ganglion cells were double-labeled after retrograde tracer injections in the DVN and SVNs of single follicles. Electron microscopy showed that selective transection of the DVN caused no SVN degeneration or vice versa. Thus, the dual innervation of the vibrissa arises from separate ganglion cells that project through separate nerves. Ganglion cells with A-row vibrissa receptive fields were studied before and after cutting the DVN and/or SVNs to the responsive vibrissa in order to identify their peripheral trajectories. In this sample, 83% projected through a DVN and 17% via a SVN. SVN or DVN cells were not spontaneously active. All cells responded to single vibrissae only; none were responsive to intervibrissal hairs or skin. Latencies to electrical stimulation were similar for DVN and SVN cells. Adaptation rates and threshold measurements were also similar in the two groups: 60% of the DVN cells and 80% of the SVN cells gave slowly adapting responses to sustained vibrissal displacement; threshold displacements ranged from less than 1 degrees to greater than 15 degrees for both SVN and DVN cells. Direction sensitivity was found in all DVN and SVN slowly adapting cells, with most cells responding to movements in one or two quadrants. For SVN cells, sequential circumferential nerve sections indicated that the fiber's directional sensitivity matched the direction of the fiber's entry into the follicle. The two groups differed in their responses to pushing in or pulling on the hair shaft. All the DVN cells were responsive to both of these stimuli, while for SVN cells pushing activated only 40% and none were responsive to pulling the hair. Another difference in the two groups was that no injury discharges occurred after cutting SVNs, but were present in 44% of DVN cells. These data suggest that DVN and SVNs are similar in the majority of response properties. There is also no evidence to support the hypothesis that SVNs provide diffuse, multivibrissal inputs.


Dynamic changes in the density of radial glial fibers of the developing murine cerebral wall: a quantitative immunohistological analysis.

The density of radial glial fibers in the developing murine cerebral wall decreases dramatically during the terminal week of gestation. The present analysis characterizes these variations of fiber density quantitatively. Radial glial fibers were stained with monoclonal antibody RC2. Fibers were counted in a standard area of 2,000 microns2, passing orthogonally to the fiber long axis. At E14, fiber density is maximum and relatively uniform throughout the full width of the cerebral wall. Subsequently, the overall glial fiber density decreases and fiber density gradients emerge in the transmural span of fibers. These radial fiber density gradients are marked by sharp declines at the level of the ventricular-subventricular zone and in the zone of transition between intermediate zone (IZ) and cortical plate. In the interval E14-P0, the decrease in densities of fibers crossing the IZ is commensurate with the predicted diluting effects of tissue expansion with growth. By contrast, the decrease in fiber densities in ascent across the cortical plate beyond E14 through E17 exceeds that predicted by growth. The observations suggest that a substantial population of radial glial fibers do not span the full ventricular zone to the cerebral surface. A major contingent appears to extend only as far as the external sagittal stratum/subplate through E16. Subsequently, at a time when supragranular cortical layers are being assembled, the full set of fibers appears to extend more uniformly through the cortical strata. The late surge of fibers would be appropriate to the intercalation of groupings of neurons of the supragranular layers.


Development of serotoninlike immunoreactivity in the embryonic nervous system of the snail Lymnaea stagnalis.

In our initial effort to study the ontogeny of the gastropod nervous system, we used histological techniques to examine the post-embryonic development of cells which exhibit serotoninlike immunoreactivity in Lymnaea (Croll and Chiasson, J. Comp. Neurol. 230:122-142, '89). The present study complements that report by examining the embryonic development of these neurons. The first serotoninlike immunoreactive (SLIR) cells to be detected in the embryos are the paired C4 neurons of the cerebral ganglia. These cells are faintly visible at about 37-38% of embryonic development and have already produced axons which traverse the cerebral commissure. By about 2-3% later the axon tips reach the pedal ganglia and appose the next SLIR cells to appear, the EPe1 neurons. Over the next 30% of development four more pairs of cerebral neurons are added adjacent to the C4 neurons and over ten cells are added to each of the pedal ganglia. At about 70% of development SLIR fibers are first detected in the parietal and visceral ganglia forming the abdominal ring. Around this time the somata of the C1 neurons also first appear in the cerebral ganglia together with their prominent axons projecting to the buccal ganglia. The last 30% of development is marked by a massive addition of SLIR cells (up to 60) in each pedal ganglion. The early appearance of the first SLIR cells suggests that they may be among the first nerve cells to differentiate and that they may play central roles in the formation of the CNS. We hypothesize that most of the animal's neural circuitry is laid down during embryogenesis by a stereotypic ontogenetic program with post-embryonic neurogenesis subserving mostly compensatory and modulatory purposes.


All indoleamine-accumulating cells in the rabbit retina contain GABA.

The indoleamine-accumulating amacrine cells of the rabbit retina are wide-field and numerous. They form a dense plexus in sublamina 5 of the inner plexiform layer where they make reciprocal synapses with rod bipolar cells. To provide a quantitative test for the colocalization of serotonin (5-HT) and gamma-aminobutyric acid (GABA) in the rabbit retina, we designed two parallel double-label experiments. In the first series, the indoleamine-accumulating cells were labeled with 5,7-dihydroxytryptamine (5,7-DHT), which was subsequently visualized by photooxidation in the presence of diaminobenzidine. This was combined with autoradiography for 3H-muscimol. In the second and complementary series, 3H-5-HT uptake was combined with postembedding GABA immunocytochemistry. These two experiments provided essentially identical results: over 98% of the indoleamine-accumulating amacrine cells were double-labeled. This means that, within the limit of experimental error, all the indoleamine-accumulating amacrine cells are GABAergic. The indoleamine-accumulating amacrine cells account for 15-20% of a large diverse group of GABA amacrine cells. In addition, the rare type 3 indoleamine-accumulating cells and fine processes running in the optic fiber layer were double-labeled. If there is insufficient 5-HT to support a transmitter role in the rabbit retina, our results suggest that the indoleamine-accumulating cells may use GABA as a neurotransmitter. Thus, rod bipolar cells, in common with other bipolar cell types, receive extensive negative feedback at GABA-mediated reciprocal synapses.


Determination of liver volume from CT scans using histogram cluster analysis.

The histogram cluster analysis procedure (HICAP), which was developed by NASA for processing satellite images, classifies images into discrete clusters of pixels according to one or more arbitrary imaging variables. We incorporated this nonparametric, multivariate procedure in a semiautomatic computer algorithm for calculating total liver volume from CT scans and compared its performance with that of a human observer. Total liver volumes were calculated from CT scans in adult patients by the algorithm and by an experienced radiologist using the trackball controlled cursor at the CT console. Variability in the computer calculated volumes was determined by repeating calculations three times over the course of 3-12 months. Using HICAP in the univariate mode, we calculated total liver volumes from 28 contrast enhanced CT scans in 27 patients. Liver volumes calculated by the semiautomatic and manual methods had a median absolute difference of 3.6% (Vcomputer = 1.08 * Vmanual - 99.52 cc; r2 = 0.99). Median day-to-day variability of the computer calculated volumes was 1.9% (95% confidence interval: 1.3-2.7%). Using HICAP in a bivariate mode to illustrate its ability to incorporate two image features in one analysis, we studied an additional patient and compared total liver volume calculated from the univariate data set defined by the contrast enhanced CT scan with that calculated from the bivariate data set defined by nonenhanced and contrast enhanced CT scans. The HICAP errors were 4.1% in the univariate analysis and 0.4% in the bivariate analysis. It is concluded that this statistical clustering algorithm provides a clinically accurate, repeatable, and feasible method of in vivo liver volume determination.


Role of contrast agent perfusion and of diffusion in the NMR signal enhancement of liver lesions.

Both perfusion and diffusion contribute to NMR signal enhancement after intravenous injection of a nonspecific contrast agent. In the present study the roles of perfusion and diffusion in the contrast enhancement of induced liver lesions in the rat and the rabbit were evaluated. This was done by comparing Gd-DOTA absolute enhancement signal-to-noise versus time plots of various lesions with the findings from microangiography and histology. Four types of enhancement could be recognized. Diffusion was the major factor in the delayed enhancement seen in lesions that had large extravascular diffusion space.


Castleman disease mimicking liver tumor: CT and MR features.

We report the CT and MR features of two localized abdominal forms of Castleman disease mimicking hypervascular liver tumors due to their unusual location in the porta hepatis and the portacaval space. The MR appearance of Castleman lymph nodes is emphasized, including their characteristics on dynamic turbo-fast low angle shot (FLASH) sequences after Gd-DOTA bolus injection. Our report suggests that the CT and MR features of Castleman lymph nodes in these locations cannot enable their differentiation from other hypervascular masses such as benign liver tumors.


Direct invasion of urinary bladder from sigmoid colon cancer: CT findings.

Radiological findings in three cases of sigmoid colon carcinoma invading the urinary bladder are reported. An irregular spiculated margin or nodular indentations in region of bladder dome were seen on intravenous urography. A soft tissue mass contiguous with an irregularly thickened urinary bladder wall and a circumferentially thickened sigmoid colon wall were demonstrated on CT and sonography and led to the correct diagnosis.


Diagnosis of pneumoperitoneum: abdominal CT vs. upright chest film.

To compare the sensitivity of CT with upright chest radiography for the detection of free intraperitoneal air, we compared the results of these examinations performed on trauma patients who had introduction of intraperitoneal air from diagnostic peritoneal lavage (DPL). Thirteen patients were studied by abdominal CT within 24 h after DPL. Upright chest radiography was performed prior to abdominal CT or less than 4 h after abdominal CT. All patients demonstrated free air on abdominal CT. Only 5 of 13 (38%) patients demonstrated free air on plain radiography. The amount of free air demonstrated on CT was quantified into three groups. Upright chest radiography in the minimal group (less than three 1 mm pockets of air) was totally insensitive (0 of 2) in detecting free air. Upright chest radiography in the moderate group (greater than three 1 mm pockets, but less than 13 mm diameter collection of air) was 33% sensitive (3 of 9). Upright chest radiography in the large group (greater than 13 mm collection of air) was 100% sensitive (2 of 2). Abdominal CT is clearly superior to upright chest radiography in demonstrating free intraperitoneal air in this clinical setting.


Quantification of glucose utilization in liver metastases: parametric imaging of FDG uptake with PET.

Positron emission tomography (PET) fluorodeoxyglucose (FDG) studies are useful for identifying foci of increased FDG uptake in liver metastases, because of the high glycolytic rate of malignancies, as well as for monitoring changes in tumor glucose metabolism during treatment. We performed 15 kinetic PET FDG studies in four patients with metastatic liver disease. We produced parametric images of glucose metabolism in terms of the rate constant K (ml/min/g) for net phosphorylation of FDG. Tumor K values, estimated with nonlinear regression, correlated well with K values estimated with Patlak graphical analysis (r = 0.96), validating the assumption of low k4* values in liver metastases and supporting the use of pixel by pixel Patlak plot analysis of the data to generate parametric images. In normal liver, high levels of glucose-6-phosphatase produce much higher values of k4* than in liver metastases. Uncorrected Patlak graphical analysis underestimates K in normal liver, but this further increases the contrast between tumor and liver and facilitates both tumor detection and quantification. The technique is computationally feasible and is well suited for serial evaluations of tumor metabolism during treatment.


Chemical shift and the double-line sign in MRI of early femoral avascular necrosis.

The double-line sign (DLS) on T2-weighted images in femoral avascular necrosis (AVN) is formed by a high-intensity rim at the reactive interface inside a low-intensity margin. A hypothesis has been advanced that the sign may be seen in early AVN whether or not there is bony sclerosis. On change of direction of the frequency-encoding axis, the high-intensity area rimmed with low-intensity margin was generated on the side where chemical shift misregistration artifact was seen. Transposition of frequency-encoding and phase-encoding axes yielded the same results. The thickness of the low-density rim was almost equal to the amount of expected offset distance due to chemical shift misregistration. Therefore, chemical shift misregistration artifact is the most reasonable explanation for the DLS observed in early femoral AVN.


Abdominal CT scans in patients with blunt trauma: low yield in the absence of clinical findings.

This study undertook to determine the yield of abdominal CT scans ordered only because the patient's sensorium was decreased or general anesthesia was planned. The abdominal CT scans and hospital charts of 191 consecutive patients examined following blunt trauma were reviewed with respect to clinical indications prompting the study. In 143, some clinical or laboratory justification was in evidence. In this group, 55 patients (38.5%) were found to have trauma-related pathology at CT scan. In 48 patients, no clinical or laboratory suggestion of abdominal pathology was evident. Instead, the primary indications for ordering the CT scan were decreased sensorium (28 cases), the planning of general anesthesia for orthopedic procedures (8 cases), and a variety of non-abdominal-trauma-related reasons. In this group, only a single positive finding was identified (small pneumothorax), and in no case was the clinical course altered by findings at abdominal CT scan. Performance of abdominal CT scans without clinical or laboratory evidence of trauma, merely because of decreased patient sensorium or prophylactically prior to general anesthesia for non-abdomen-related surgery, is an extremely low yield study and should be discouraged. In the current study, no significant abdominal pathology would have been overlooked by omission of such scans. Similar findings have been reported in children. To our knowledge, this is the first such report in a largely adult population.


Comparison of skull circumference and linear measurements with CSF volume MR measurements in hydrocephalus.

In children with hydrocephalus, accurate and reproducible estimation of the presence, severity, and course of the condition is of paramount importance for both clinical and scientific purposes. In this study, 30 hydrocephalic patients were assessed with a number of commonly used methods, such as occipitofrontal skull circumference (SC) measurements, Evans ratio (ER), and bicaudate index (BCI), as well as, for comparison, another ratio of linear measurements [ventricle-skull ratio (VSR)] and MR measurements of total intracranial CSF volume. In repeated CSF volume measurements in healthy volunteers, the MR method appeared to be accurate and reproducible. This technique was simpler and easier in application, requiring less interaction than comparable MR techniques described by others. The variation coefficients were within the same range. In increased CSF volumes, our technique can be recommended; in very small CSF volumes, another technique is more adequate. Direct assessment of CSF volume as a measure of hydrocephalus was preferable over derived estimations for scientific purposes and may function as a gold standard against which to evaluate other techniques that are easier to apply clinically. In comparison, SC measurements were poor; CSF volume changes were not reflected in SC changes. VSR was preferable over ER and BCI, because it correlated more closely with CSF volume.


CT features of benign and malignant teratomas in children.

Computed tomography of 15 pediatric patients with teratomas of the chest, abdomen, and pelvis was reviewed to compare the features of benign and malignant lesions. Particular attention was paid to the frequency of characteristic CT features of this lesion, namely fat, calcification, and mural nodules. Of the 10 benign masses, 8 were predominantly cystic, usually containing less than 10% soft tissue components. One benign lesion was complex and one was nearly solid. Fat was found in 9 of 10 lesions (90%), calcification in 7 (70%), and mural nodules in 7 (70%). All five malignant teratomas were predominantly solid, containing primarily soft tissue contents. Approximately 80% of malignant tumors had CT evidence of local invasion or distant metastases. Three of five malignant lesions (60%) contained calcifications and two of five (40%) had fat. None had recognizable mural nodules. Our experience suggests that typical CT features of teratomas occur more often in benign than in malignant lesions.


Differentiation of suprasellar nonneoplastic cysts from cystic neoplasms by Gd-DTPA MRI.

Magnetic resonance imaging in 9 cases of surgically confirmed nonneoplastic intra- and/or suprasellar cysts was analyzed and compared with that from 17 cases of craniopharyngioma and 12 of cystic pituitary adenoma. Signal intensity did not help in distinguishing nonneoplastic cysts from cystic neoplasms. No postcontrast (Gd-DTPA) cyst wall enhancement was observed among the nonneoplastic cysts, although all 6 craniopharyngiomas and 12 cystic pituitary adenomas that underwent postcontrast study did enhance. Three nonneoplastic cysts were surrounded by the pituitary gland, thus mimicking wall enhancement in the postcontrast studies. Rapid enhancement in the early postcontrast-administration phase (dynamic studies) helped avoid confusion between the normal pituitary tissue and cyst wall enhancement. Magnetic resonance studies with contrast enhancement play an essential role in differentiating nonneoplastic cysts from cystic neoplasms in the sella region.


Cerebrovascular reserve capacity in ischemia due to occlusion of a major arterial trunk: studies by Xe-CT and the acetazolamide test.

Classifications based on clinical and radiographic criteria have proved to be inadequate predictors of the course of cerebral ischemia or its response to therapy. In this study the cerebrovascular reserve capacity (CRC) of 46 patients with symptomatic cerebrovascular ischemia was studied by stable xenon-enhanced CT (Xe-CT) combined with the acetazolamide test. Fifteen patients had internal carotid artery (ICA) occlusion, 10 had ICA stenosis, 10 had middle cerebral artery (MCA) occlusion, and 11 had MCA stenosis. In the patients with chronic cerebral ischemia due to occlusive lesions of the ICA and MCA, the CRC was reduced most in those with MCA occlusion, followed, in descending order, by those with ICA stenosis, MCA stenosis, and ICA occlusion. Our results indicate that measurement of the CRC elucidates cerebral hemodynamic factors that cannot be detected by angiography in patients with chronic cerebral ischemia and that Xe-CT combined with the acetazolamide test is useful for this purpose.


Volume T1-weighted gradient echo MRI in multiple sclerosis patients.

Three-dimensional (3D) volume turbo fast low angle shot (FLASH) techniques have become available which produce heavily T1-weighted images, similar to inversion recovery scans, utilizing the appropriate flip angle and inversion time. The purpose of this study was to compare the sensitivity of a rapid volume gradient echo technique [3D magnetization prepared rapid acquisition gradient echo (MP RAGE)] in identifying multiple sclerosis (MS) plaques with a conventional T2-weighted spin echo (SE) sequence. Ten patients with clinical MS were evaluated. Patients underwent a routine examination consisting of an axial T2-weighted SE sequence (2,500,22/90) and a coronal 3D MP RAGE, 10/4/10, acquired as 128 two mm partitions. In six patients, area measurements of 22 plaques were determined on both the axial T2-weighted SE examinations and the axial reformatted MP RAGE examinations. The overall number of plaques utilizing each technique was approximately the same. One hundred twenty-two plaques were visualized for the 3D MP RAGE sequence, and 128 plaques for the T2-weighted SE sequence. There were differences in detection of plaques in different regions, with plaques in gray matter better demonstrated utilizing the conventional T2-weighted SE sequence. Plaques in the corpus callosum, pons, and brachia pontis were better demonstrated utilizing 3D MP RAGE. No significant difference was found between the areas measured on the MP RAGE sequence and on the T2-weighted SE sequence. Three-dimensional MP RAGE provides a sensitive and complementary method to conventional T2-weighted SE sequences in the evaluation of patients with MS.


Gd-DTPA enhanced MRI of cervical anterior epidural venous plexus.

Seventy selected cases were reviewed retrospectively to assess the normal Gd-DTPA enhanced MR appearance of the cervical anterior epidural venous plexus. This structure can enhance quite markedly and has a somewhat variable appearance on MR imaging but we believe that it can be differentiated from pathological enhancement in the area if attention is paid to its normal anatomical characteristics.


Retrospective fusion of radiographic and MR data for localization of subdural electrodes.

Prior to epilepsy surgery, subdural electrodes are often implanted and monitored for a few days to identify the focus of abnormal electrical activity. During the implantation and subsequent brain resection, there may be uncertainty about the exact location of the electrodes with respect to features of brain anatomy such as specific gyral convolutions or lesions. In experiments with a phantom and patients, implanted electrodes were imaged with multiplanar skull radiographs (or CT scans). After retrospective registration with preimplantation MR data, the electrodes were mapped from these studies onto an MR-derived three-dimensional brain model. The resulting multimodality displays showed the relationship of the electrodes to brain anatomy. In one patient the position of each electrode with respect to a metabolic lesion was also displayed by mapping preimplantation PET data onto the same brain model. This new display of electrode positions may strengthen the interpretation of subdural electrical recordings and thereby reduce uncertainty in planning the resection of epileptic tissue.


Orbital blowout fractures as a cause of sinonasal obstructive disease.

Six cases of orbital blowout fracture with inferomedial herniation of orbital contents into the sinonasal ostiomeatal unit causing obstruction to maxillary sinus outflow are presented. Potential hazards during endoscopic sinus surgery in these cases are outlined.


Demonstration of subclavian steal by MR angiography.

We present three cases of subclavian steal syndrome demonstrated by MR angiography. By using a presaturation pulse located at the bottom or at the top of the acquisition volume we demonstrated the flow direction of vertebral arteries. We used a new investigational two-dimensional time-of-flight sequence.


Temporal correlation images derived from sequential MR scans.

In this paper a new technique is presented for measuring the local temporal changes and correlation between them on spatially aligned image sequences. A temporal correlation image is derived from a sequence of static images (frames). The method is illustrated in an application to the dynamic MRI of the rabbit kidney following bolus injection of Gd-DTPA. The similarity maps clearly separate the dynamic response of the cortex to the indicator from that of the medulla and, therefore, segment the kidney into two regions. Regions with different temporal dynamics can be displayed in different colors using a lookup table designed for that purpose.


Evaluation of high-resolution CT after tympanoplasty.

The temporal bones of 28 patients who underwent tympanoplasty were evaluated with high-resolution CT (HRCT). Seventeen patients had undergone tympanoplasty with a columella, used to form an ossicular reconstruction. The incus body was used as the columella in 4 patients and a prosthesis was used in 13 patients. No columella was used in the other 11 patients. After tympanoplasty, CT demonstrated normal appearance in 8 patients. Of the other 20 patients, 16 were diagnosed with chronic otitis media, and 4 were diagnosed with recurrent cholesteatoma on the basis of follow-up CT examinations. Seven underwent reoperation. In all 4 patients with recurrent cholesteatoma, the diagnosis was pathologically confirmed at reoperation. In 1 patient diagnosed with chronic otitis media, the pathological diagnosis was recurrent cholesteatoma. The columellae in 8 of the 17 patients could not be identified on CT because of surrounding soft tissue mass, but in the other 9 patients the condition of the ossicular reconstruction was well demonstrated. Prosthesis dislocation was apparent in 2 patients. We recommend HRCT examination in the follow-up of tympanoplasty patients.


Cardiac flow measurement by ultrafast CT: validation of continuous and pulsatile flow.

To gauge the accuracy of ultrafast CT in measuring cardiac output and myocardial perfusion in humans, measurements of continuous and pulsatile flow were made in a large asymmetrical phantom. The variation in the relationship between Hounsfield number and contrast concentration was assessed in a human thorax phantom. Radiopaque contrast medium was injected during perfusion of the phantom at a range of flow rates between 1.5 and 8 L/min. The phantom was scanned in two modes (50 and 100 ms) during continuous and pulsatile flow and with the phantom surrounded by air and by water. Flow in the tubes was calculated using indicator dilution theory, and flow in the tissue-equivalent chamber was calculated by applying first-pass distribution principles. The standard deviation of the difference between calculated and measured flow varied from 0.2 to 0.6 L/min, giving 95% limits of agreement from 0.4 to 1.2 L/min. The constant (K) relating Hounsfield unit number to iodine concentration varied widely both in different locations within the phantom and under different scan conditions (17.2-27.6 HU/mg I). Within a human thorax phantom, K varied from 14.15 to 23.18 HU/mg I and was dependent on location within the thorax phantom, the scan mode, and the cross-sectional diameter of the phantom. These data suggest that though the ultrafast CT scanner can measure continuous and pulsatile flow accurately in tubes, precise measurements of cardiac output in humans will require K to be assessed for each subject. Measurements of flow in tissue should be possible.


Performance evaluation of the positron scanner ECAT EXACT.

The Cologne Special is a prototype of the ECAT EXACT (model 921), a new generation of Siemens-CTI PET scanners. It consists of three rings of 48 BGO block detectors each, covering an axial field of view of 16.2 cm with a patient port of 56.2 cm diameter. This results in a total of 24 rings with 384 crystals each, giving 47 contiguous image planes in two-dimensional (2D) mode. Total system sensitivity is 216 kcps/microCi/ml for a 20 cm cylinder phantom in 2D. This increases to 1.5 Mcps/microCi/ml in 3D. Data are acquired in the stationary mode only (no wobble motion), resulting in a transaxial spatial resolution of better than 6 mm full width at half-maximum (FWHM) at the center, which degrades to 7.5 mm tangentially and 9.6 mm radially at a radius of 20 cm. Average axial resolution changes from 5.0 mm FWHM at the center to 8.1 mm at R = 20 cm. Count rate performance was investigated at different low energy discriminator settings and found to be linear up to 2.5 microCi/ml with a 20 cm phantom. The magnitude and distribution of scatter were evaluated for both septa-extended and septa-retracted conditions for a range of energy thresholds. Brain, heart, and whole-body studies with the new tomograph demonstrate the versatility of its applications without compromising on physical performance.


Spinal cord toxoplasma lesion in AIDS: MR findings.

Toxoplasma gondii is the cause of the most common opportunistic infection of the brain in AIDS but is extremely rare as the cause of a solitary lesion of the spinal cord. Symptoms are weakness of the lower limbs followed closely by paralysis unless diagnosed and treated early. We present such a case in an intravenous drug abuser with AIDS and emphasize that MRI is the diagnostic tool of choice and that the index of suspicion should be high in immunosuppressed patients.


Complex partial seizure-induced transient MR enhancement.

A 58-year-old man with complex partial seizures had transient symmetrical MR enhancement in the anterior mesiotemporal cortex bilaterally, correlated with the sites of seizure focus on electroencephalography. Preinfused T1-weighted and T2-weighted sequences showed no abnormality. After adequate control of seizures with medication, repeat MR was normal and the prior enhancement was no longer seen. This transient seizure-induced MR enhancement was consistent with ictal or postictal hyperemia and breakdown of the blood-brain barrier and should not be erroneously attributed to mesial temporal sclerosis, encephalitis, tumor, or infarction.


MRI and neurological complications of adult T-cell leukemia/lymphoma.

We report the assessment by MRI of a lumbosacral lymphomatous localization of adult T-cell leukemia/lymphoma (ATLL) with human T-cell lymphotropic virus-1 infection in an African patient. Brain MRI detected associated multifocal lesions of increased signal intensity in the subcortical white matter. These MRI abnormalities are compatible with reported necropsy findings in cases of ATLL with neurological complications.


Spinal cord MRI in adult polyglucosan body disease.

MRI appearance of adult polyglucosan body disease is described. A 67-year-old man presenting with a chronic progressive degenerative neurologic disorder was found to have marked atrophy of the entire cord, without signal abnormalities on long TR images. Autopsy revealed atrophy of the cord, diffuse gliosis, and numerous corpora amylacea.


CT "halo sign" in pulmonary tuberculoma.

The CT halo sign has been described as the CT finding of a low-attenuation zone surrounding a pulmonary nodule. It is an early clue to the diagnosis of invasive pulmonary aspergillosis. We describe a case of CT halo sign associated with a pulmonary tuberculoma. Therefore, we think that a diagnosis other than invasive pulmonary aspergillosis should be considered in the presence of the CT halo sign in immunocompetent patients.