Issue published November 1, 1994

I n patients with dermatomyositis (DM) the earliest lesion is microvasculopathy mediated by deposition of C5b-C9 membranolytic attack complex (MAC) on intramuscular capillaries. This leads sequentially to muscle ischemia, necrosis of muscle fibers, and muscle weakness. High-dose intravenous immunoglobulin (IVIG), which can modulate complement-dependent tissue damage in animal models, has been shown to be effective in the treatment of patients with DM. We used an in vitro C3 uptake assay to examine 55 coded sera from 13 patients with DM and 5 patients with other non-complement-mediated neuromuscular diseases, before and after treatment with IVIG or placebo. Patients with active DM had a significantly higher baseline C3 uptake compared with the others (geometric mean 12,190 vs 3,090 cpm). Post-IVIG but not post-placebo sera inhibited the C3 uptake, without depleting the complement components, by 70.6-93.4%. The maximum inhibition of C3 uptake occurred within hours after IVIG infusion, started to rebound 2 d later, and reached pretreatment levels after 30 d. The serum levels of SC5b-9 complex production were high at baseline but normalized after IVIG therapy. Repeat biopsies from muscles of improved patients showed disappearance of C3b NEO and MAC deposits from the endomysial capillaries and restoration of the capillary network. We conclude that IVIG exerts its beneficial clinical effect by intercepting the assembly and deposition of MAC on the endomysial capillaries through the formation of complexes between the infused immunoglobulins and C3b, thereby preventing the incorporation of activated C3 molecules into C5 convertase. These findings provide the first serological and in situ evidence that IVIG modulates complement attack in a human disease.

K aposi's sarcoma (KS) is the most frequent tumor of HIV-1-infected individuals (AIDS-KS). Typical features of KS are proliferating spindle-shaped cells, considered to be the tumor cells of KS, and endothelial cells forming blood vessels. Basic fibroblast growth factor (bFGF), a potent angiogenic factor, is highly expressed by KS spindle cells in vivo and after injection in nude mice it induces vascular lesions closely resembling early KS in humans. Similar lesions are induced by inoculating nude mice with cultured spindle cells from AIDS-KS lesions (AIDS-KS cells) which produce and release bFGF. Here we show that phosphorothioate antisense (AS) oligonucleotides directed against bFGF mRNA (ASbFGF) inhibit both the growth of AIDS-KS cells derived from different patients and the angiogenic activity associated with these cells, including the induction of KS-like lesions in nude mice. These effects are due to the block of the production of bFGF which is required by AIDS-KS cells to enter the cell cycle and which, after release, mediates angiogenesis. The effects of ASbFGF are specific, dose dependent, achieved at low (0.1-1 microM), nontoxic, oligomer concentrations, and are reversed by the addition of bFGF to the cells, suggesting that ASbFGF oligomers are promising drug candidates for KS therapy.

T reatment of wounds with pharmacologic doses of the BB homodimeric form of recombinant PDGF (rPDGF-BB) induces the recruitment, activation, and proliferation of mesenchymal cells, resulting in the deposition of provisional, and subsequently collagen-containing extracellular matrix. In preliminary experiments with an in vitro growth chamber model in the rat consisting of a silicone shell containing a dissected femoral vascular bundle, we found that rPDGF-BB incorporated into a rapidly dissolving collagen type I film induces the generation of a marked, but transient amount of de novo tissue around the femoral vascular bundle. In the present studies, the new tissue generated around the femoral vascular bundle was wrapped with a full thickness syngeneic skin graft to determine if functional support of the graft would lead to sustained maintenance of the underlying generated tissue and create an epithelialized soft tissue appendage. The tissue generated after a single application of rPDGF-BB was skin grafted on the 10th day, exteriorized 20 d later, and observed for an additional month. This led to the formation of soft tissue appendages which demonstrated marked neovascularization, fibroblast migration and proliferation, and increased glycosaminoglycan, fibronectin, and collagen fibril deposition, now leading to preservation of the newly generated tissue. In contrast, minimal new tissue was generated in control-treated vascular bundles or bundles treated with inactive PDGF-BB, and grafting with skin failed to sustain the underlying tissue. Thus, rPDGF-BB coupled with skin grafting induced the formation of functional large soft tissue appendages which are potentially useful clinically to fill tissue defects or to serve as a cell delivery system for transfected genes.

K eratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.

T o examine whether expression and distribution of aquaporin of collecting duct (AQP-CD) are regulated by vasopressin V2 receptor (V2R), we performed immunohistochemical studies with specific antibody against AQP-CD. Normal Wistar rats were divided into four groups and treated for 3 d; control, dehydration, vasopressin V1 receptor (V1R) antagonist (OPC-21268 120 mg/kg), V2R antagonist (OPC-31260 30 mg/kg). At time of death, urine osmolality (Uosm) in the dehydration group (1884 +/- 245 mOsm/kg) was significantly higher than that in the control (938 +/- 91). In the V2R antagonist group, Uosm was significantly decreased to 249 +/- 29, whereas V1R antagonist showed no effect on Uosm. In the control and V1R antagonist groups, immunofluorescence studies showed the AQP-CD staining of both apical membrane and subapical cytoplasm of CD cells of the cortex and the inner medulla. Dehydration increased the immunostaining of both apical membrane and subapical cytoplasm of CD cells of the inner medulla, and the degree of increase was dominant in apical membrane. In the V2R antagonist group, only faint staining of apical membrane and weak labeling of cytoplasm of CD cells of the inner medulla were observed. These changes in the localization and protein amount of AQP-CD by dehydration and V2R antagonist were quantitatively confirmed by immunogold studies and immunoblot analysis of the inner medulla. The present results indicate that the distribution and amount of AQP-CD in the CD cells are regulated by vasopressin V2 receptor.

T he cell surface metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) hydrolyzes a variety of peptide substrates and reduces cellular responses to specific peptide hormones. Because CD10/NEP modulates peptide-mediated proliferation of small cell carcinomas of the lung (SCLC) and normal fetal bronchial epithelium, we evaluated the enzyme's expression in non-small cell lung carcinomas (NSCLC). Bronchoalveolar and large cell carcinoma cell lines had low levels of CD10/NEP expression whereas squamous, adenosquamous, and adenocarcinoma cell lines had higher and more variable levels of the cell surface enzyme. Regional variations in CD10/NEP immunostaining in primary NSCLC specimens prompted us to correlate CD10/NEP expression with cell growth. In primary carcinomas of the lung, clonal NSCLC cell lines and SV40-transformed fetal airway epithelium, subsets of cells expressed primarily CD10/NEP or the proliferating cell nuclear antigen (PCNA). Cultured airway epithelial cells had the lowest levels of CD10/NEP expression when the highest percentage of cells were actively dividing; in addition, these cells grew more rapidly when cell surface CD10/NEP was inhibited. NSCLC cell lines had receptors for a variety of mitogenic peptides known to be CD10/NEP substrates, underscoring the functional significance of growth-related variability in CD10/NEP expression.

N itric oxide (NO) is an inhibitor of gastrointestinal smooth muscle. Model systems of the gut predict the NO will complex with biological thiol (SH) groups, yielding S-nitrosothiols (RS-NO), which may limit the propensity to form mutagenic nitrosamines. The inhibitory effects of NO and its biologically relevant adducts on sphincter of Oddi (SO) motility have been inferred from animal studies; however, their importance in regulating human SO is not known. The objectives of this study were to (a) provide histologic confirmation of nitric oxide synthase (NOS) in human SO; (b) characterize the pharmacology of S-nitroso-N-acetylcysteine (SNAC), an exemplary S-nitrosothiol, on SO motility in a rabbit model; and (c) study the effects of topical SNAC on SO motility in humans. Immunocytochemical and histochemical identification of NOS was performed in human SO. The pharmacologic response of SNAC was defined in isolated rabbit SO using a standard bioassay. Topical SNAC was then applied to the duodenal papilla in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) and biliary manometry. NOS was localized to nerve fibers and bundles of the SO in rabbits and humans. SNAC inhibited spontaneous motility (frequency and amplitude) as well as acetylcholine-induced elevations in SO basal pressure in the rabbit model. In patients undergoing ERCP and biliary manometry, topical SNAC inhibited SO contraction freqency, basal pressure, and duodenal motility. NOS is localized to neural elements in human SO, implicating a role for NO in regulating SO function. Supporting this concept, SNAC is an inhibitor of SO and duodenal motility when applied topically to humans during ERCP. Our data suggest a novel clinical approach using local NO donors to control gastrointestinal motility and regulate sphincteric function.

I nterleukin 12 is a heterodimeric molecule that serves as a potent co-stimulator enhancing the development of Th1 cells. As one of the classical Th1 cell-mediated responses is contact sensitivity in skin, we wondered whether IL-12 might be produced by epidermal cells and serve as a mediator of this immune response. Using a sensitive, quantitative PCR technique we demonstrate that p35 chain mRNA of IL-12 is produced constitutively by human epidermal cells, whereas p40 chain mRNA can only be detected in epidermis treated with contact allergen, but not epidermis exposed to irritants or tolerogens. Time course studies showed a dramatic induction of IL-12 p40 mRNA 4 h after in vivo allergen treatment reaching peak strength after 6 h. In cell depletion assays we show that epidermal keratinocytes are the major source of this cytokine in the epidermis. This was further supported by analysis of mRNA derived from the human keratinocyte cell line HaCat expressing IL-12 p35 and p40 mRNA upon stimulation. The presence of bioactive IL-12 in supernatants derived from allergen-stimulated epidermal cells was demonstrated by IL-12-specific bioassay. Additional evidence for the functional importance of IL-12 in primary immune reactions in skin was obtained in allogeneic proliferation assays using human haptenated epidermal cells containing Langerhans cells as APC and allogeneic CD4+ T cells as responders. Anti-IL-12 mAb inhibited the proliferation of T cells by approximately 50%. In aggregate our data demonstrate that nonlymphoid keratinocytes are capable of producing functional IL-12 and provide evidence for the functional significance of IL-12 in primary immune responses in skin.

T he ability of HIV-1 to infect macrophages is thought to be essential in AIDS pathogenesis. We tested the ability of 19 primary virus isolates to infect monocyte-derived macrophages (MDM) from different donors. Two HIV-1 isolates were able to establish a productive infection in MDM from all donors tested, whereas eight completely lacked this capacity. Next to these isolates with extreme phenotypes, 50% of the primary isolates under study displayed an intermediate phenotype. These intermediate macrophage-tropic isolates established a productive infection in MDM from some but not all donors tested. PCR analysis demonstrated that the capacity to replicate in MDM could be determined at the previously described level of virus entry. However, for intermediate macrophage-tropic isolates replication was abrogated at the level of reverse transcription. Entry of highly macrophage-tropic isolates resulted in efficient completion of the reverse transcription process, whereas entry of intermediate macrophage-tropic isolates did not. Our experiments indicate that primary HIV-1 isolates may differ in their dependency on cellular factors required for reverse transcription in MDM. Differences in susceptibility of MDM for in vitro HIV-1 infection suggest variation in the availability of these cellular factors between MDM from different individuals.

M icrobial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis.

T he glomerulus develops progressive injury with advancing age which is particularly pronounced in males and is not the result of any specific disease process. In the present studies conducted in rats, glomerular function and structure were examined in adult (8 mo), elderly (12 mo), and old (19 mo) Munich Wistar rats. Intact males and females and castrated rats of both sexes were studied to determine the role of the sex hormones in mediating age-dependent glomerular damage. Intact males developed glomerular injury and proteinuria whereas females, both intact and ovariectomized, and castrated males were protected from injury. Glomerular blood pressure did not increase with advancing age in any group and did not correlate with glomerular damage. Glomerular volume did increase with advancing age in all groups but did not correlate with glomerular damage. We found that the presence of the androgens rather than the absence of the estrogens provide the risk factor for development of age-dependent glomerular damage. Neither glomerular hypertension nor glomerular hypertrophy provide the primary mechanism by which age-dependent glomerular injury occurs in the intact male.

S hort-term culture of peripheral blood mononuclear cells (PBMC) derived from patients with human T cell lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis resulted in dominance by DR+ activated CD8+ T cells. Variations in the T cell receptor (TCR) V alpha and V beta chains in these cells were analyzed, and in all 10 patients examined, 2-3 V gene families were dominant in both TCR V alpha and V beta. In five patients we examined, cultured lymphocytes contained cytotoxic lymphocytes for p40tax (patients HAM2, 3, 7, and 8) or env protein (patient HAM4) of human T lymphotropic virus type I. In patients HAM2 and HAM8, cultured lymphocytes contained a large proportion of V beta 8+ CD8+ and/or V beta 12+ CD8+ cells. The sequence of V beta 8+ and V beta 12+ cDNA revealed that they were oligoclonal with identical or similar sequences in each patient. Elimination experiments with monoclonal antibodies for TCR V beta 8 and V beta 12 showed that they were CD8+ cytotoxic T lymphocytes (CTL) for p40tax. In addition, flow cytometry and sequencing analysis of uncultured PBMC revealed that in HAM2, V beta 8+ CTL and their precursors account for 7% and V beta 12+ CTL and their precursors account for 18% of total CD8+ cells. This indicates the presence of two markedly expanded clones in vivo. No common dominant TCR V alpha or V beta were observed among 10 HAM patients analyzed.

T o examine the role of adhesion molecules in T cell recruitment and activation during allergen-induced late asthmatic response (LAR), we evaluated the expression of lymphocyte function-associated antigen-1 alpha (LFA-1 alpha) and intercellular adhesion molecule-1 (ICAM-1) on peripheral blood T lymphocyte subsets from atopic asthmatic patients and their changes following allergen inhalation challenge. 12 atopic asthmatic patients were studied. Six patients showed only a single early response after allergen challenge, and six developed a dual response. At baseline, dual responders (DR) had a significantly higher expression of ICAM-1 on CD4+ and CD8+ T lymphocytes as compared with both single early responders (P < 0.005 and P < 0.02, respectively) and controls (P < 0.001, both comparisons). Allergen challenge was followed by a decrease of CD8+ ICAM-1+ T lymphocytes in all DR (P < 0.05) and of CD4+ ICAM-1+ T lymphocytes in four out of six DR, at the time of the LAR. At the same time, a significant rise in serum levels of the soluble form of ICAM-1 was observed in DR. These results suggest that peripheral blood immunoregulatory T lymphocytes are in a higher state of activation in DR as compared with early responders. The upregulation of ICAM-1 on these cells may be important in enhancing airway inflammation in patients with LAR.

U reteral obstruction causes impaired salt wastage and K+ secretion in the distal nephron segments, including the cortical collecting duct (CCD). Recently, we demonstrated that conductances of Na+ and K+ in the apical membrane, as well as the electrogenic Na(+)-K+ pump activity and the relative K+ conductance in the basolateral membrane of the collecting duct cell, were inhibited in the obstructed kidney after unilateral ureteral obstruction (UUO). To examine whether the increased intrarenal pressure might be causally related to these abnormalities in the CCD, the effects of unilateral renal decapsulation, a maneuver that partially blocks the increase in renal pressure, were evaluated with microelectrode techniques in isolated CCDs from UUO and sham-operated (control) rabbits 24 h after operation. Renal decapsulation had no effects on barrier voltages and conductances in the CCD from control animals. The lumen-negative transepithelial (VT) and basolateral membrane (VB) voltages as well as the transepithelial (GT) and the apical membrane (GA) conductances were decreased in the CCD from UUO animals compared with control animals. Pretreatment of renal decapsulation partially corrected the decreases in VT, VB, GT, and GA seen in the CCD from UUO animals. The changes in apical membrane voltage and GT upon addition of luminal amiloride and Ba2+, and the changes in VB upon addition of bath ouabain, were also decreased in the CCD from UUO animals compared with control animals. Pretreatment of renal decapsulation also partially corrected the above abnormalities seen in UUO animals, whereas it had no effect in control animals. The transference numbers for Cl- (tCl) and K+ (tK) in the basolateral membrane were, respectively, increased and decreased in the CCD from UUO animals compared with control animals. Pretreatment of renal decapsulation also partially corrected the changes in tCl and tK seen in UUO animals, whereas it had no effect in control animals. We conclude that, in UUO animals, renal decapsulation partially corrects the inhibition of apical Na+ and K+ conductances as well as basolateral Na(+)-K+ pump activity and relative K+ conductance seen after UUO, whereas in control animals it has no effect. The increased renal pressure may partly contribute to the defects in Na+ and K+ transport in the CCD from obstructed kidneys. Renal decapsulation has protective effects on impaired Na+ and K+ transports in the CCD after ureteral obstruction.

T he putative mannose receptor (MR), previously implicated in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle (ASM) cells, was studied to determine its properties. Specific binding of the mitogenic neoglycoprotein, mannosylated bovine serum albumin (Man-BSA) to ASM cells was saturable, with an apparent Kd = 5.0 x 10(-8) M. Cell-bound ManBSA-colloidal gold conjugate was localized by electron microscopy to clathrin-coated pits on the cell surface, and was found to undergo internalization to endosomes; this was inhibitable by weak bases and swainsonine, that also inhibited ligand-induced mitogenesis. The ASM-MR, isolated by mannose-affinity chromatography, had the same apparent molecular mass as the macrophage (Mø) MR (M(r) = 175 kD), and was immunoprecipitated by an anti-MøMR immune serum. This antiserum blocked 125I-labeled-ManBSA binding to intact ASM cells, stimulated mitogenesis, and immunolocalized the ASM-MR in cytoplasmic vesicles compatible with endosomes. A monoclonal antibody directed against the MøMR also reacted with the ASM-MR; like the polyclonal antibodies, it stimulated mitogenesis as effectively as beta-hexosaminidases. These data indicate that the ASM-MR shares a number of functional and structural properties with the MøMR and suggest that similar receptors may have different main functions in different cells.

E ndothelial adhesion molecules play an important role in the tissue recruitment of leukocytes in inflammatory conditions such as rheumatoid arthritis. We have investigated the effect of the antirheumatic drug gold sodium thiomalate on adhesion molecule protein and mRNA expression in cultured human endothelial cells. Gold sodium thiomalate inhibited cytokine (TNF, IL-1, IL-4)-stimulated expression of vascular cell adhesion molecule-1 and E-selectin but not intercellular adhesion molecule-1 on endothelial cells. Gold sodium thiomalate also suppressed TNF-stimulated increases in vascular cell adhesion molecule-1 and E-selectin mRNA levels but had no effect on intercellular adhesion molecule-1 mRNA. Thiomalate (mercaptosuccinate), but not gold thioglucose or D-penicillamine, mimics the effect of gold sodium thiomalate at equimolar concentrations. We propose that the inhibition of vascular cell adhesion molecule-1 and E-selectin expression by gold sodium thiomalate is due to its thiomalate and not its gold component. Gold sodium thiomalate has a direct effect on endothelial adhesion molecule expression, and this may contribute to its antiinflammatory activity.

G enetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.

T o determine whether the adventitia that surrounds pulmonary vessels acts as a barrier specific to nitric oxide, special lucite chambers were constructed to measure the force of contraction of rabbit pulmonary artery rings in which the endothelial or adventitial surfaces could be preferentially exposed to nitric oxide (NO), carbon monoxide (CO), or sodium nitroprusside (SNP). Delivery of NO to the endothelial and adventitial surfaces of preconstricted vessels produced markedly different concentration-response curves with maximal relaxations of 89 +/- 3 and 11 +/- 9%, respectively. In contrast, relaxations induced by both CO and SNP did not differ significantly between endothelial and adventitial exposure to these agents. Placement of a layer of pericardium onto the endothelial surface eliminated relaxation to the endothelial delivery of NO but not to CO. We conclude that the pulmonary vascular response to NO displays a striking sidedness which is not observed either with CO, another gas of similar molecular weight, or with SNP, both of which cause relaxation by stimulating guanylate cyclase. The elimination of NO but not CO relaxations with a layer of pericardium may indicate that the adventitia acts as a barrier specific to NO. This directionality of effect provides evidence for a highly localized regulation of pulmonary vascular tone by endothelial cell NO and also indicates that extravascular NO may have limited access to pulmonary vascular smooth muscle.

S timulation of endothelial cells resulted in release of arachidonic acid from phospholipids. The magnitude of this response decreased as the cells became confluent and the change coincided with a decrease in the percentage of cells in growth phases (G2+M); this was not a consequence of time in culture or a factor in the growth medium. Preconfluent cells released approximately 30% of arachidonic acid; confluent cells released only 6%. The decreasing release of arachidonic acid was demonstrated using metabolic labeling, mass measurements of arachidonic acid, and measurement of PGI2. The decrease was not due to a changing pool of arachidonic acid, and mass measurements showed no depletion of arachidonic acid. Release from each phospholipid and from each phospholipid class decreased with confluence. Conversion of confluent cells to the proliferative phenotype by mechanical wounding of the monolayer caused increased release of arachidonic acid. Potential mechanisms for these changes were investigated using assays of phospholipase activity. Phospholipase A2 activity changed in concert with the alteration in release, a consequence of changes in phosphorylation of the enzyme. The increased release of arachidonic acid from preconfluent, actively dividing cells may have important physiologic implications and may help elucidate mechanisms regulating release of arachidonic acid.

T he carbohydrate-deficient glycoprotein syndrome (CDGS) is a developmental disease associated with an abnormally high isoelectric point of serum transferrin. Carbohydrate analyses of this glycoprotein initially suggested a defect in N-linked oligosaccharide processing, although more recent studies indicate a defect in the attachment of these sugar chains to the protein. We studied both serum glycoproteins and fibroblast-derived [2-3H]mannose-labeled oligosaccharides from CDGS patients and normal controls. While there was a decrease in the glycosylation of serum glycoproteins of affected individuals, differences were not seen in either monosaccharide composition or oligosaccharide structures. The lectin-binding profiles of glycopeptides from [2-3H]-mannose-labeled fibroblasts were likewise indistinguishable. However, the incorporation of [2-3H]mannose into both glycoproteins and the dolichol-linked oligosaccharide precursor was significantly reduced. Thus, at least in some patients, CDGS is not due to a defect in processing of N-linked oligosaccharides, but rather to defective synthesis and transfer of nascent dolichol-linked oligosaccharide precursors. This abnormality could result in both a failure to glycosylate some sites on some proteins, as well as secondary abnormalities in overall glycoprotein processing and/or function.

I n stable organ systems, such as the heart and kidneys, an oxidant stress induces an increase in endogenous antioxidant systems resulting in an increased resistance of the tissue to a subsequent oxidant challenge. The development of this oxidant tolerance requires 1.5-6 d. The aim of the present study was to determine whether oxidant tolerance can be induced in the small intestinal mucosa, a labile system whose epithelium turns over every 2-3 d. Ischemia/reperfusion-induced epithelial barrier dysfunction of the small intestinal mucosa was monitored in Sprague-Dawley rats whose intestines had been exposed to an ischemic insult 1, 24, or 72 h previously. At 24 h, but not 1 or 72 h after the initial ischemic insult, the mucosa was more resistant to ischemia/reperfusion-induced barrier dysfunction. The antioxidant status of the mucosa was enhanced at 24 h, but not at 1 or 72 h after the initial ischemic insult. This adaptation appears to be specific for oxidants, since an initial ischemic insult imposed 24 h earlier also protected against H2O2-induced, but not acid- or ethanol-induced, barrier dysfunction. Further studies indicated that the increase in antioxidant status of the mucosa observed 24 h after the initial ischemic insult was a result of adaptational changes in the lamina propria, rather than the epithelium. In vitro studies with isolated epithelial cells also indicated that epithelial cells do not develop oxidant tolerance. We conclude that the development of oxidant tolerance in the small intestinal mucosa does not involve an active participation of the epithelial lining.

C lostridium difficile toxin A (Tx-A) mediates secretion and inflammation in experimental enterocolitis. Intravital video microscopy was used to define the mechanisms that underlie the inflammatory reactions elicited by direct exposure of the microvasculature to Tx-A. Leukocyte adherence and emigration, leukocyte-platelet aggregation, and extravasation of FITC-albumin were monitored in rat mesenteric venules exposed to Tx-A. Significant increases in leukocyte adherence and emigration (LAE) and albumin leakage were noted within 15-30 min of Tx-A exposure. These responses were accompanied by mast cell degranulation and the formation of platelet-leukocyte aggregates. The Tx-A-induced increases in LAE and albumin leakage were significantly attenuated by pretreatment with either monoclonal antibodies (mAbs) directed against the leukocyte adhesion glycoproteins, CD11/CD18, intercellular adhesion molecule-1, and P-selectin (but not E-selectin) or with sialyl Lewis x, a counter-receptor for P-selectin. The mast cell stabilizer, lodoxamide, an H1- (but not an H2-) receptor antagonist, and diamine oxidase (histaminase) were also effective in reducing the LAE and albumin leakage elicited by Tx-A. The platelet-leukocyte aggregation response was blunted by an mAb against P-selectin, sialyl Lewis x, and the H1-receptor antagonist. These observations indicate that Tx-A induces a leukocyte-dependent leakage of albumin from postcapillary venules. Mast cell-derived histamine appears to mediate at least part of the leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation by engaging H1-receptors on endothelial cells and platelets to increase the expression of P-selectin. The adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 also contribute to the inflammatory responses elicited by toxin A.

A cute intermittent porphyria (AIP), an autosomal dominant inborn error, results from the half-normal activity of the heme biosynthetic enzyme, hydroxymethylbilane synthase (EC 4.3.1.8). Diagnosis of AIP heterozygotes is essential to prevent acute, life-threatening neurologic attacks by avoiding various precipitating factors. Since biochemical diagnosis is problematic, the identification of hydroxymethylbilane synthase mutations has facilitated the detection of AIP heterozygotes. Molecular analyses of unrelated AIP patients revealed six exonic mutations: an initiating methionine to isoleucine substitution (M1I) in a patient with variant AIP, which precluded translation of the housekeeping, but not the erythroid-specific isozyme; four missense mutations in classical AIP patients, V93F, R116W, R201W, C247F; and a nonsense mutation W283X in a classical AIP patient, which truncated the housekeeping and erythroid-specific isozymes. Each mutation was confirmed in genomic DNA from family members. The W283X lesion was found in another unrelated AIP family. Expression of each mutation in Escherichia coli revealed that R201W, C247F, and W283X had residual activity. In vitro transcription/translation studies indicated that the M1I allele produced only the erythroid-specific enzyme, while the other mutant alleles encoded both isozymes. These mutations provide insight into the molecular pathology of classic and variant AIP and facilitate molecular diagnosis in AIP families.

T he mechanism underlying the mineralocorticoid escape phenomenon remains unknown. To assess the possible contribution of natriuretic peptides to mineralocorticoid escape, rats were injected with 5 mg deoxycorticosterone acetate for 3 d. Plasma atrial natriuretic factor (ANF) rose to twice basal levels and atrial ANF content decreased significantly by 24 h of treatment. This coincided with renal escape and with a significant increase in urinary cGMP excretion. Plasma ANF remained elevated and atrial ANF content continued to decline by 48 and 72 h while atrial ANF mRNA levels increased significantly only at 72 h. Plasma brain natriuretic peptide did not increase during escape although atrial brain natriuretic peptide mRNA levels increased significantly. Chronically administered HS-142-1 (HS), a specific antagonist of the guanylate cyclase-coupled natriuretic peptide receptors, significantly and dose-dependently impaired the escape phenomenon. The highest dose of HS completely suppressed the increase in urinary cGMP. Despite the continued suppression, partial escape was observed by the end of the observation period. HS alone influenced neither plasma nor tissue or urine parameters. These findings show that despite activation of atrial ANF, blockade of the guanylate cyclase-coupled natriuretic peptide receptors impairs the ability of the kidney to escape the Na+ retaining effect of excess mineralocorticoid in a dose-dependent fashion. Later-acting, unknown mechanisms eventually come into play to mediate the escape phenomenon through a guanylate cyclase-independent pathway. Therefore, ANF of cardiac origin appears to be a major factor initiating mineralocorticoid escape through a guanylate cyclase-dependent pathway.

E arly in human immunodeficiency virus (HIV) infection CD4+ and CD8+ T cells are qualitatively affected. Loss of responses to recall antigen precedes impaired responses to allogeneic MHC and mitogens. The selective quantitative loss of memory T cells in early infection, only partially explains the observed defects. We investigated whether functional loss of T cells is preferentially observed for memory T cells or whether both naive and memory T cell subsets are affected in the course of HIV infection. We studied the proliferative response of CD4+ T cells from HIV-infected individuals to alloantigens, to which normally both naive and memory T cells respond, by limiting dilution analysis. The decreased proliferative response to alloantigens in HIV-infected individuals was associated with a decreased precursor frequency of alloreactive cells. The frequency was decreased in both the CD45RA+ (naive) and the CD45RO+ (memory) subset of CD4+ T cells. Analysis of four individuals in the course of HIV infection revealed similar kinetics of the decline in function in both subsets. Although initially T cell defects may be accounted for by the selective quantitative loss of memory cells, in later stages of HIV infection the function of both CD45RA+ and CD45RO+ cells is affected.

U pon respiratory syncytial virus (RSV) challenge, mice previously immunized intramuscularly with inactivated whole virus express a Th2-like pattern of cytokine mRNA, while mice immunized with live virus intranasally express a Th1-like pattern. In this study, we evaluated the effects of anti-IL-4 treatment on the induction of immune responses after immunization. Mice treated with anti-IL-4 at the time of immunization with inactivated RSV had reduced clinical illness after live virus challenge, as measured by weight loss, illness score, and virus replication. This was associated with an augmented CD8+ cytotoxic T lymphocyte (CTL) activity, increased expression of IFN-gamma mRNA relative to IL-4 mRNA, and a higher titer of RSV-specific IgG2a in the anti-IL-4 treated mice before challenge. Anti-IL-4 administration at the time of challenge had no effects on illness, immunoglobulin isotype, or cytokine patterns. These results suggest that inhibition of IL-4 action at immunization can shift the selective activation of lymphocytes to a more Th1-like response. This cytokine milieu is associated with augmented CTL activity, which may be the factor responsible for rapid viral clearance and reduced illness at the time of remote RSV challenge.

R enin is produced mainly by the kidney, and cAMP is a main positive regulator of its synthesis. This study was undertaken to analyze the molecular mechanism of cAMP-mediated regulation of Ren-1C gene transcription by the proximal promoter. We first showed that the promoter region from -365 to +16 of the mouse renin gene (Ren-1C) mediated the cAMP-induced chloramphenicol acetyltransferase gene expression in embryonic kidney-derived 293 cells. Deletion analysis and heterologous promoter assay disclosed that the proximal promoter region from -75 to +16 was able to activate chloramphenicol acetyltransferase expression by cAMP, and indicated that the proximal promoter element from -75 to -47 (RP-2 element) overlapping the TATA-like region was able to confer cAMP responsiveness. Electrophoretic mobility shift assay and DNase I footprinting analysis demonstrated that novel nuclear factors in 293 cells interacted with the RP-2 element, and that cAMP increased the binding activity of these nuclear factors to the RP-2 element. Furthermore, we demonstrated that cAMP enhanced the binding of nuclear factors derived from juxtaglomerular cells, the main production site of renin in the kidney, to the RP-2 element in vivo. These results suggest that the RP-2 element plays an important role in the cAMP-mediated regulation of Ren-1C gene transcription through the proximal promoter.

W e have previously shown that treatment of endothelial cells with minimally modified LDL (MM-LDL) induces the binding of monocytes to unknown endothelial receptor molecules. We now report that a member of the GRO family of chemokines plays a role in MM-LDL-induced monocyte binding. A cDNA library made from rabbit aortic endothelial cells (RAEC) treated with MM-LDL was expression screened for molecules inducing binding of a human monocyte cell line (THP-1). A cDNA was isolated with 75% homology to GRO. GRO mRNA levels were significantly elevated after exposure of RAEC or human aortic endothelial cells (HAEC) to MM-LDL. HAEC treated with MM-LDL displayed an increase in a surface-associated protein that bound to antibody against GRO despite low levels of GRO in the medium. Antibody to GRO significantly inhibited the binding of monocytes to MM-LDL-treated RAEC and HAEC. The increase in GRO expression and monocyte binding were reduced by incubating MM-LDL-treated endothelial cells with heparin (in a method that releases heparan sulfate bound molecules from the cell surface). These results suggest that GRO related chemokines are bound to the surface of MM-LDL-treated endothelial cells and may contribute to the monocyte adhesion induced by MM-LDL.

T he results of the current study demonstrate that relaxin inhibits histamine release by mast cells. This effect is related to the peptide concentrations, and could be observed in both isolated rat serosal mast cells stimulated with compound 48/80 or calcium ionophore A 23187, and in serosal mast cells isolated from sensitized guinea pigs and challenged with the antigen. The morphological findings agree with the functional data, revealing that relaxin attenuates calcium ionophore-induced granule exocytosis by isolated rat serosal mast cells. Similar effects of relaxin have also been recognized in vivo by light microscopic and densitometric analysis of the mesenteric mast cells of rats which received the hormone intraperitoneally 20 min before local treatment of the mesentery with calcium ionophore. Moreover, evidence is provided that relaxin stimulates endogenous production of nitric oxide and attenuates the rise of intracellular Ca2+ concentration induced by calcium ionophore. The experiments with drugs capable of influencing nitric oxide production also provide indirect evidence that the inhibiting effect of relaxin on mast cell histamine release is related to an increased generation of nitric oxide. It is suggested that relaxin may have a physiological role in modulating mast cell function through the L-arginine-nitric oxide pathway.

A utoantibodies to RNA polymerases (RNAP) I, II, and III are reported to be highly specific for the diagnosis of scleroderma (systemic sclerosis, SSc). In the present study, the specificity of autoantibodies to RNAP I and III for SSc was confirmed by immunoprecipitation of 35S-labeled proteins. However, we report here the previously unrecognized production of anti-RNAP II autoantibodies by 9-14% of patients with SLE and mixed connective tissue disease/overlap syndrome. 12 out of 32 anti-RNAP II positive sera (group 1) immunoprecipitated a diffuse 220-240-kD band identified as the largest subunit of RNAP II whereas the remaining 20 (group 2) immunoprecipitated preferentially the 240-kD phosphorylated (IIo) form of the large subunit. After pulse labeling, group 1 sera immunoprecipitated only the 220-kD (IIa) RNAP II subunit, whereas the diffuse IIa/IIo band plus the 145-kD second largest RNAP II subunit (IIc) were immunoprecipitated after several hours of cold chase, suggesting that these sera recognized primarily the largest subunit of RNAP II. Group 2 sera recognized the IIc subunit after pulse labeling, and immunoprecipitated the IIc and IIo, but not the IIa, subunits after cold chase. Although it has been suggested that autoantibodies to RNAP II are usually accompanied by anti-RNAP I/III in SSc, all but one of the anti-RNAP II positive sera from SLE or mixed connective tissue disease/overlap syndrome patients, as well as most of the SSc sera, were negative for anti-RNAP I/III. Moreover, in contrast to previous reports suggesting that anti-RNAP antibodies rarely coexist with other SSc subset marker antibodies, anti-RNAP II antibodies were often accompanied by anti-Ku, anti-nRNP, or anti-topoisomerase I autoantibodies in the present study. We conclude that autoantibodies to RNAP II are not a specific marker for SSc, whereas autoantibodies to RNAP I/III are associated primarily with SSc. In addition, we have identified two distinctive patterns of RNAP II antigen recognition by autoantibodies, one of them characterized by specific recognition of the transcriptionally active (phosphorylated) form of RNAP II. The clinical significance of these different patterns remains to be determined.

R U-486 (17 beta-hydroxy-4-dimethylaminophenyl-17-alpha-propenyl estrone 4,9 diene-3-one; mifepristone) is suggested to act by binding to progesterone and glucocorticoid receptors. Based on its chemical nature, we anticipated that RU-486 may have potent antioxidant properties. We used the oxidation of LDL as our model system. RU-486 and a similar compound, onapristone, at 1-5-microM concentrations, decreased the formation of oxidized LDL. LDL isolated from plasma of subjects who were orally supplemented with RU-486 was resistant to oxidation, as compared to LDL isolated from control plasma. The antioxidant effect of RU-486 appears to reside in the dimethylaminophenyl side chain moiety. Reduction of the A-ring of the steroid molecule had no effect on its antioxidant property. Analogs of RU-486 which lack the dimethylaminophenyl group, were without antioxidant activity. Levonorgestrel, which lacks the dimethylaminophenyl group failed to inhibit the oxidation of LDL even at 100-microM levels. In contrast, ethinylestradiol and estradiol which do not possess the dimethylamino group, were able to inhibit the oxidation of LDL by virtue of their phenolic steroid "A" ring. Thus RU-486, with its long half life, high plasma concentrations, association with lipoproteins, and ability to readily enter the cell may have additional intra- and extra-cellular antioxidant effects.

H igh-dose methotrexate (HDMTX) is a component of most treatment protocols for childhood acute lymphoblastic leukemia (ALL), yet recent studies of receptor-mediated transport and saturable polyglutamylation have questioned its rationale. To investigate this in vivo, methotrexate and its active polyglutamated metabolites (MTX-PG) were measured in bone marrow blasts obtained from 101 children randomized to single-agent therapy with either HDMTX (1 g/m2 per 24 h i.v., n = 47) or low-dose MTX (LDMTX, 30 mg/m2 by mouth every 6 h x 6, n = 54), before remission induction therapy. Blast concentrations of total MTX-PGs (median 460 vs 1380 pmol/10(9) cells) and of long-chain MTX-glu4-6 were both significantly higher after HDMTX (P < 0.001). With either treatment, MTX-PGs were significantly higher in B-lineage blasts than in T-lineage blasts (LDMTX P = 0.001, HDMTX P = 0.03). In a multiple regression analysis of B-lineage ALL, blast MTX-PG was significantly related to MTX dose (or plasma MTX concentration), lymphoblast ploidy (hyperdiploid > nonhyperdiploid), and percentage S-phase. This is the first evidence that HDMTX achieves higher MTX-PG concentrations in ALL blasts in vivo, establishing a rationale for HDMTX in the treatment of childhood ALL, especially T-lineage or nonhyperdiploid B-lineage ALL, disease characteristics associated with a poor prognosis on conventional therapy.

R egulation of cytosolic Ca2+ and cytosolic Na+ is critical for lymphocyte cation homeostasis and function. To examine the influence of cytosolic Na+ on Ca2+ regulation in human peripheral blood lymphocytes, Ca2+ entry and cytosolic Ca2+ (measured with fura-2) were monitored in cells in which cytosolic Na+ was increased and/or the Na+ gradient was decreased by reduction of external Na+ concentration. Ouabain-treated cells (0.1 mM for 30 min at 37 degrees C), suspended in Na(+)-free medium, showed a 30-65% increase in Ca2+ uptake compared to cells in 140 mM Na+ medium. Enhanced Ca2+ influx was entirely dependent on ouabain pretreatment and reversal of the Na+ gradient. Na pump inhibition or Na ionophore addition and subsequent exposure to Na(+)-free medium resulted in a sustained elevation of cytosolic Ca2+. As preincubation of cells in Ca(2+)-free medium further enhanced the ouabain-dependent increase in cytosolic Ca2+, the effects of the microsomal Ca(2+)-ATPase inhibitor thapsigargin on Ca2+ influx and cytosolic Ca2+ were studied. Thapsigargin stimulated Ca2+ entry following ouabain pretreatment and reversal of the Na+ gradient; the effects of thapsigargin were retained in the presence of LaCl3, a potent inhibitor of store-dependent calcium influx pathways. These results show lymphocytes demonstrate Na+/Ca2+ exchange activity and suggest the Na+/Ca2+ exchanger modulates cytosolic Ca2+ following intracellular Ca2+ store depletion.

E ndotoxin sensitivity varies among animal species and appears to correlate with the presence of pulmonary intravascular macrophage (PIM). In rats, which lack PIM, we investigated the hypothesis that chronic cholestatic liver injury leads to induction of PIM and endotoxin sensitivity. Rats were randomized to either common bile duct ligation (BDL) or sham-surgery and studied at 1 wk (acute cholestasis), 2 wk (cholestasis, early cirrhosis), and 4 wk (cholestasis, established cirrhosis) after surgery. Intravascularly injected fluorescent latex microspheres (1 micron diameter) were taken up by large phagocytic cells in lung parenchyma of BDL rats (at 2 and 4 wk), while no uptake was observed in lungs from control rats. Electronmicroscopy revealed accumulation of large, mononuclear, macrophage-like cells containing ingested latex particles within the pulmonary capillaries. Pulmonary intravascular phagocytosis, as reflected in lung uptake of 99mTc microaggregated albumin (Microlite, mean particle diameter = 1 micron), averaged 0.7 +/- 0.1% (mean +/- SEM) of total injected dose in 13 control rats and progressively increased with time after BDL (1 wk, 1.7 +/- 0.2%; 2 wk, 10.0 +/- 3.0%; 4 wk 35.1 +/- 5.9%). Rats with biliary cirrhosis were markedly sensitive to the lethal effects of low dose endotoxin and demonstrated marked lung edema at the time of death. Furthermore, the lung uptake of intravascular 125I-lipopolysaccharide was increased five-fold in cirrhotic rats. We conclude that chronic biliary obstruction leads to the induction of pulmonary intravascular phagocytes and enhances endotoxin sensitivity in rats. Pulmonary intravascular phagocytosis in patients with advanced cirrhosis may account for their increased susceptibility to sepsis-induced adult respiratory distress syndrome.

V asoactive intestinal peptide (VIP) has potent growth-related actions that influence cell mitosis, neuronal survival, and neurodifferentiation in cell culture. VIP can also produce dramatic growth in postimplantation mouse embryos in vitro, characterized by large increases in cell number. The goal of the present study was to assess the role of VIP on early nervous system development in vivo. Pregnant mice were treated with a specific antagonist to VIP. Prenatal administration of the antagonist early in development (E9-E11) produced severe microcephaly characterized by decreased embryonic brain weight with reduced DNA and protein content. The retardation of growth was disproportionally manifested in the brain compared with the body and was prevented by co-treatment with VIP. Identical treatment with the antagonist later in gestation had no detectable effect on embryonic growth. VIP receptors, which were restricted to the central nervous system during this stage of embryonic development, were increased in the neuroepithelium of antagonist-treated embryos while the number of cells in S-phase was significantly decreased. Thus, VIP regulates brain growth in vivo and inhibition of its action provides new insight into a molecular mechanism for microcephaly.

W e produced transgenic mice which overexpress human IL-6 in the airway epithelial cells. Transgenic mice develop a mononuclear cell infiltrate adjacent to large and mid-sized airways. Immunohistochemistry reveals these cells to be predominantly CD4+ cells, MHC class II+ cells, and B220+ cells. Transgenic mice and nontransgenic mice had similar baseline respiratory system resistance (0.47 +/- 0.06 vs 0.43 +/- 0.04 cmH2O/ml per s at 9 wk of age, P = NS and 0.45 +/- 0.07 vs 0.43 +/- 0.09 cmH2O/ml per s at 17 wk of age, P = NS). Transgenic mice, however, required a significantly higher log dose of methacholine to produce a 100% increase in respiratory system resistance as compared with non-transgenic littermates (1.34 +/- 0.24 vs 0.34 +/- 0.05 mg/ml, P < or = 0.01). We conclude that the expression of human IL-6 in the airways of transgenic mice results in a CD4+, MHC class II+, B220+ lymphocytic infiltrate surrounding large and mid-sized airways that does not alter basal respiratory resistance, but does diminish airway reactivity to methacholine. These findings demonstrate an uncoupling of IL-6-induced airway lymphocytic inflammation and airway hyperresponsiveness and suggest that some forms of airway inflammation may serve to restore altered airway physiology.

W e evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.

T o elucidate the metabolism of islet amyloid polypeptide (IAPP) with respect to a possible renal elimination we investigated IAPP levels in 20 lean, nondiabetic patients with renal failure maintained on chronic hemodialysis (HD) and in 20 healthy controls. The basal levels of IAPP were significantly higher in uremic patients than in controls (15.1 +/- 3.2 vs. 3.2 +/- 0.2 pM, P < 0.001) suggesting renal excretion of IAPP. To investigate the impact of chronically elevated levels of endogenous IAPP on insulin secretion and insulin sensitivity, a frequently sampled intravenous glucose tolerance test (FSIGT) was performed in a subset of patients on hemodialysis and in age-matched healthy controls (C) and obese patients with normal (NGT) and with impaired glucose tolerance (IGT). Insulin sensitivity index (SI) was 8.7 +/- 1.5 in C (P < 0.05 vs. NGT, P < 0.01 vs. IGT), 5.4 +/- 0.9 in HD (P < 0.05 vs. IGT), 3.1 +/- 1.0 in NGT, and 2.0 +/- 0.5 in IGT. First phase insulin secretion was increased in patients on HD compared with those of several control groups. The results of this study therefore indicate a renal route of metabolism of IAPP. Increased endogenous circulating IAPP levels over a long period of time do not lead to a decrease in insulin release in patients on HD and do not cause the insulin resistance commonly seen in obesity and diabetes. Increased levels of circulating IAPP therefore are not likely to be a pathogenetic factor in the development of non-insulin-dependent diabetes mellitus (NIDDM).

P revious studies have demonstrated that the ability of beta-adrenergic receptor (beta AR) stimulation to increase cardiac contractility declines with aging. In the present study, the control mechanisms of excitation-contraction (EC) coupling, including calcium current (ICa), cytosolic Ca2+ (Cai2+) transient and contraction in response to beta AR stimulation were investigated in ventricular myocytes isolated from rat hearts of a broad age range (2, 6-8, and 24 mo). While the baseline contractile performance and the Cai2+ transient did not differ markedly among cells from hearts of all age groups, the responses of the Cai2+ transient and contraction to beta-adrenergic stimulation by norepinephrine (NE) diminished with aging: the threshold concentration and the ED50 increased in rank order with aging; the maximum responses of contraction and Cai2+ transient decreased with aging. Furthermore, the efficacy of beta AR stimulation to increase ICa was significantly reduced with aging, and the diminished responses of the contraction and Cai2+ transient amplitudes to NE were proportional to the reductions in the ICa response. These findings suggest that the observed age-associated reduction in beta AR modulation of the cardiac contraction is, in part at least, due to a deficit in modulation of Cai2+, particularly the activity of L-type calcium channels.

M acrophage-tropic, non-syncytium-inducing, HIV-1 variants predominate in the asymptomatic phase of infection and may be responsible for establishing infection in an individual exposed to the mixture of HIV-1 variants. Here, genotypical and phenotypical characteristics of virus populations, present in sexual, parenteral, or vertical donor-recipient pairs, were studied. Sequence analysis of the V3 domain confirmed the presence of a homogeneous virus population in recently infected individuals. Biological HIV-1 clones were further characterized for syncytium inducing capacity on the MT2 cell line and for macrophage tropism as defined by the appearance of proviral DNA upon inoculation of monocyte-derived macrophages. Both sexual and parenteral transmission cases revealed a selective outgrowth in the recipient of the most macrophage-tropic variant(s) present in the donor. In three out of five vertical transmission cases, more than one highly macrophage-tropic virus variant was present in the child shortly after birth, suggestive of transmission of multiple variants. In three primary infection cases, homogeneous virus populations of macrophage-tropic, non-syncytium-inducing variants were present prior to seroconversion, thus excluding humoral immunity as the selective pressure in favour of macrophage-tropic variants. These observations may have important implications for vaccine development.

C lonal expansion of T cell specificities in the synovial fluid of patients has been taken as evidence for a local stimulation of T cells. By studying the T cell receptor (TCR) repertoire of CD4+ T cells in the synovial and peripheral blood compartments of patients with early rheumatoid arthritis (RA), we have identified clonally expanded CD4+ populations. Expanded clonotypes were present in the peripheral blood and the synovial fluid but were not preferentially accumulated in the joint. Dominant single clonotypes could not be isolated from CD4+ cells of HLA-DRB1*04+ normal individuals. Clonal expansion involved several distinct clonotypes with a preference for V beta 3+, V beta 14+, and V beta 17+CD4+ T cells. A fraction of clonally related T cells expressed IL-2 receptors, indicating recent activation. The frequencies of clonally expanded V beta 17+CD4+ T cells fluctuated widely over a period of one year. Independent variations in the frequencies of two distinct clonotypes in the same patient indicated that different mechanisms, and not stimulation by a single arthritogenic antigen, were involved in clonal proliferation. These data support the concept that RA patients have a grossly imbalanced TCR repertoire. Clonal expansion may result from intrinsic defects in T cell generation and regulation. The dominance of expanded clonotypes in the periphery emphasizes the systemic nature of RA and suggests that T cell proliferation occurs outside of the joint.

W e hypothesized that normotensive sepsis affects the ability of the microcirculation to appropriately regulate microregional red blood cell (RBC) flux. An extensor digitorum longus muscle preparation for intravital study was used to compare the distribution of RBC flux and the functional hyperemic response in SHAM rats and rats made septic by cecal ligation and perforation (CLP). Using intravital microscopy, we found that sepsis was associated with a 36% reduction in perfused capillary density (from 35.3 +/- 1.5 to 22.5 +/- 1.0 capillaries/mm of test line) and a 265% increase in stopped-flow capillaries (from 0.9 +/- 0.2 to 3.3 +/- 0.4 capillaries/mm); the spatial distribution of perfused capillaries was also 72% more heterogeneous. Mean intercapillary distance (ICD) increased 30% (from 25.7 +/- 0.8 to 33.5 +/- 1.6 microns), and the proportion of capillary pairs with intercapillary distances > 33.8 microns (the 75th percentile of ICDSHAM) was greater with sepsis. Mean capillary RBC velocity increased 17% in CLP rats (391 vs 333 microns/s). Laser Doppler flowmetry was used to assess the functional hyperemic response of the extensor digitorum longus muscle before and after a period of maximal twitch contraction designed to increase oxygen demand. RBC flux was 36% lower in the CLP rats at rest. After contraction, RBC flux increased in both SHAM and CLP rats; however, the relative increase was less in the CLP group. We concluded that sepsis affects the ability of the skeletal muscle microcirculation to appropriately distribute RBC flux and to respond to increases in oxygen need.

A nti-tubular basement membrane disease (alpha TBM disease) produces T cell-mediated interstitial nephritis in SJL mice after immunization with renal tubular antigen. Initial mononuclear infiltrates appear in vivo after several weeks, with the subsequent progression to renal fibrosis and end stage renal disease over many months. We have analyzed the fine specificity of the autoreactive helper T cell repertoire in alpha TBM disease through the isolation and characterization of a panel of CD4+ Th1 clones harvested after 1-2 wk from animals immunized to produce disease. All clones capable of mediating alpha TBM disease are directed towards a 14-residue immunodominant epitope (STMSAEVPEAASEA) contained within the target antigen, 3M-1. Evaluation of the T cell receptor (TCR) V beta repertoire used by these autoreactive T cells reveals the use of several V beta genes, but with some preference for V beta 14. Sequencing across the putative CDR3 region of the TCR beta chains suggests that common amino acids at the V beta(N)D beta junction and the D beta(N)J beta junction may contribute to the specific ability of these cells to recognize the immunodominant epitope.

W e have used a murine model of organ-specific autoimmunity to characterize therapeutic modalities capable of down-regulating the cellular limb of the autoimmune response. Murine interstitial nephritis is an autoimmune disease mediated by tubular antigen-specific CD8+ nephritogenic effector T cells which are delayed-type hypersensitivity (DTH) reactive and cytotoxic to renal epithelial cells. Previous studies have demonstrated that disease can be suppressed with experimentally induced populations of T cells (Ts1 and Ts2 cells) obtained after injection of tubular antigen-coupled splenocytes into syngeneic mice. As the target of Ts2 is the CD8+ effector T cell, we have evaluated its effects on nephritogenic effector T cell clones isolated from diseased animals. Our studies demonstrate that soluble proteins expressed by Ts2 cells (TsF2) specifically abrogate the DTH, cytotoxic, and nephritogenic potential of M52 cells, although T cell receptor and IL-2 receptor expression are unchanged in these unresponsive M52 clones. TsF2-induced inhibition is dependent on new mRNA and protein synthesis. In a cytotoxic clone, M52.26, exposure to TsF2 induces expression of TGF-beta 1 which is, in turn, required for inhibition of cytotoxicity and nephritogenicity. Our studies are consistent with TGF-beta 1 behaving, at least in some T cells, as a nonspecific final effector of clone-specific suppression.

I ncreases in mesangial cell number may herald glomerular scarring, but they are not irreversible. This study sought mechanisms by which surplus glomerular mesangial cells can be cleared. A small proportion of cultured mesangial cells exhibited typical morphological features of apoptosis (programmed cell death), which was increased by growth factor deprivation or exposure to cycloheximide, stimuli known to increase apoptosis in other cell types. Apoptosis was confirmed by typical internucleosomal chromatin cleavage. In vivo, clear morphological evidence of mesangial apoptosis leading to phagocytosis by neighboring mesangial cells was obtained in self-limited mesangial proliferation induced in rats by Thy1.1 antibody, apoptosis occurring approximately 10-fold more frequently than in the healthy rat glomerulus. Indeed, changes in glomerular cell number in Thy1.1 nephritis strongly suggested that apoptosis is the major cell clearance mechanism counterbalancing cell division, thereby mediating resolution of glomerular hypercellularity in experimental mesangial proliferation.

P rotein C inhibitor (PCI) is a serpin that inhibits a number of proteases. PCI is found in urine and binds to kidney epithelial cells. To determine if kidney is a source of PCI, cDNA was produced from human kidney total RNA. Sequencing and restriction mapping showed identity between kidney and liver PCI cDNA sequences. Similar cDNAs were obtained from rhesus monkey kidney and liver RNAs. Conditioned medium from the rhesus monkey kidney cell line CCL7.1 was analyzed on immunoblots, showing a 57,000-D protein band that comigrated with human plasma PCI. Immunohistochemical staining and in situ hybridization of human kidney tissue sections showed that kidney PCI antigen and RNA were confined to tubular cells. The findings are consistent with the idea that PCI is synthesized and localized in kidney tissue where it may provide protease inhibitory activity and suggest that complexes of PCI with urokinase found in human urine may be produced locally in the kidney.

P olymorphism at the vitamin D receptor gene was examined in relation to bone mineral density (BMD) at spine, femur, and forearm in 86 monozygotic (MZ) and 39 dizygotic (DZ) adult female twins. All were white, 63 pairs (44 MZ, 19 DZ) were premenopausal, and 43 pairs (31 MZ, 12 DZ) were discordant for age at menopause or use of estrogen. Each individual of the DZ pairs and one individual of MZ pairs was genotyped for ApaI, BsmI, and TaqI polymorphism at the vitamin D receptor gene locus using Southern hybridization. Intraclass correlations for BMD in MZ and DZ twin pairs indicated that heritability accounted for over 70% of BMD. There was no relationship between genotype for any of the three polymorphisms and BMD at any skeletal site in the twin population, considered either as a total population, both with and without twins discordant for age at menopause or use of estrogen, or as a premenopausal population. In DZ twin pairs discordant for alleles for the three polymorphisms, no allele was associated with higher or lower BMD. It is concluded that in this population of healthy adult females there was no relationship between these polymorphisms at the vitamin D receptor gene locus and BMD.

T he cDNAs for two separate human 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) have been isolated and sequenced. The well-studied human placental cytosolic 17 beta-HSD (also referred to as estradiol dehydrogenase) preferentially catalyzes the reduction of estrone to estradiol-17 beta and the reduction of the C-20-ketone of progesterone to 20 alpha-dihydroprogesterone. This isoform of the enzyme has been referred to as 17 beta-HSD type 1 and localized to chromosome 17. A second 17 beta-HSD isoform (referred to as type 2) is localized in the endoplasmic reticulum of human trophoblast and is characterized by the preferential oxidation of the C-17 beta-hydroxyl group of C18- and C19-steroids and the C-20 alpha-hydroxyl group of 20 alpha-dihydroprogesterone. In this study, we determined the chromosomal localization of human 17 beta-HSD type 2, the expression of this gene in human endometrium, and the tissue distribution of the mRNA. We found that the human 17 beta-HSD type 2 gene is localized on chromosome 16, 16q24. 17 beta-HSD type 2 mRNA (approximately 1.5 kb) was identified in human endometrial tissues by Northern analysis of total RNA (10 micrograms). The highest levels of 17 beta-HSD type 2 mRNA were found in endometrial tissues obtained during the mid- to late secretory phase of the ovarian cycle (i.e., during the time of high plasma levels of progesterone). 17 beta-HSD type 2 mRNA levels were much greater in glandular epithelium than in the stromal cells isolated from secretory phase endometrium. The levels of 17 beta-HSD type 2 mRNA in secretory phase endometrium were approximately one-tenth that in villous trophoblast tissue from human placenta. We did not detect 17 beta-HSD type 1 mRNA in endometrial tissue by Northern analysis of total (10 micrograms) RNA. These findings are consistent with the view that the progestin-regulated 17 beta-HSD of the glandular epithelium of the human endometrium is primarily, if not exclusively, the product of the 17 beta-HSD type 2 gene. 17 beta-HSD type 2 mRNA was present in human placenta, liver, and small intestine; much smaller amounts, barely detectable by Northern analysis of poly(A)+ RNA, were present in prostate, kidney, pancreas, and colon, but not in heart, brain, skeletal muscle, spleen, thymus, ovary, or testis.

T ransendothelial migration of mononuclear cells is crucial in the development of allograft rejection and transplant coronary disease. Adhesion of circulating cells to endothelium is the initial step in transendothelial migration. Human aortic endothelial cell cultures were established from aortic tissue harvested at the time of organ donation for cardiac transplantation which allowed specific recipient mononuclear cell-graft endothelial interactions to be studied. Confluent untreated endothelial cells were incubated with recipient mononuclear cells for 15 min to assess adhesion. Adhesion of recipient mononuclear cells to endothelium derived from their graft was threefold higher than adhesion to nonspecific endothelium (93 +/- 20 vs. 30 +/- 11 cells/high power field, P < 0.005). Graft-specific adhesion was inhibited by preincubation of the endothelium with antibodies to class I HLA (34 +/- 16 cells/high power field, P < 0.005). Immunofluorescence performed after adhesion showed that 73 +/- 6% of both specific and nonspecific adherent cells were monocytes. The use of purified lymphocyte and monocyte preparations showed that graft-specific lymphocytes induce unrelated monocytes to become adherent. These results suggest that lymphocytes are primed in vivo to recognize endothelium derived from their graft which leads to a rapid increase in lymphocyte and monocyte adhesion. Such allo-recognition may involve endothelial class I HLA molecules.

T he effects of augmenting the nephron supply on indices of allograft injury were assessed in a rat model of "chronic rejection." Orthotopic renal allotransplantation into unine-phrectomized rats was followed by excision (allograft-alone group) or preservation of the remaining native kidney (allograft+native kidney group) such that the total kidney complement was either the allograft alone, or the allograft plus one retained native kidney. After 18 wk, values for GFR (1.85 +/- 0.3 ml/min) and kidney weights (2.3 +/- 0.2 g) in allograft-alone rats were far in excess of corresponding values in the allograft of allograft+native kidney rats (0.88 +/- 0.1 ml/min and 1.1 +/- 0.5 g, respectively). Proteinuria (35 +/- 2 mg/d) and allograft glomerulosclerosis (24 +/- 8%) also characterized allograft-alone but not allograft+native kidney rats, in whom glomerular structure (allograft glomerulosclerosis, 4 +/- 1%; native kidney glomerulosclerosis, 0%) and glomerular functional integrity (proteinuria 7 +/- 0.7 mg/d) were well preserved. Thus, the observed allograft protection derived from the presence of a retained recipient native kidney supports the hypothesis that a single renal allograft contains insufficient nephrons to prevent progressive renal injury, implicating nephron supply as a major determinant of long-term allograft outcome.

H uman villous adenomas are thought to represent premalignancies that subsequently give rise to colorectal adenocarcinomas. Currently there is no in vivo model in which to study the dedifferentiation and malignant transformation of these tumors. We establish here that human villous adenomas can be successfully engrafted into severe combined immunodeficient (scid) mice. Furthermore, these xenografts remain viable for up to 18 mo after either a subcutaneous or intraperitoneal inoculation of the human tissue. Tumors grew slowly and secreted a clear mucinous fluid. Examination of the tumors histologically at 1, 4, and 12 mo after implantation revealed that the villous polypoid structure was maintained and islands of atypical cells were observed within pockets of mucin surrounding the adenomatous tissue. No gross or histologic evidence of malignancy was detected throughout the 20-mo observation period. The human identity of the cells in the graft was confirmed by DNA in situ hybridization with a human-specific probe. We conclude that the human-scid xenograft described here represents a viable animal model with which to study the potential malignant dedifferentiation of villous adenomas over a prolonged period of time and to evaluate the possible contribution of selected oncogenic vectors on the malignant transformation of these adenomas.

A drenomedullin is a potent hypotensive peptide newly discovered in pheochromocytoma tissue by monitoring its elevating activity on platelet cAMP. We measured plasma concentration of adrenomedullin in patients with essential hypertension and chronic renal failure. As compared with normal subjects, plasma adrenomedullin was increased by 26% (P < 0.05) in hypertensives without organ damage and by 45% (P < 0.005) in those with organ damage. The increase in plasma adrenomedullin was more prominent in renal failure than in hypertension. Renal failure patients with plasma creatinine of 1.5-3, 3-6, and > 6 mg/dl had higher plasma adrenomedullin levels than healthy subjects by 78% (P < 0.05), 131% (P < 0.001), and 214% (P < 0.001), respectively. Moreover, adrenomedullin showed intimate correlations with norepinephrine, atrial natriuretic peptide, and cAMP in plasma (r = 0.625, P < 0.001; r = 0.656, P < 0.001; and r = 0.462, P < 0.001; respectively). Thus, plasma adrenomedullin is supposed to increase in association with changes in sympathetic nervous activity and body fluid volume in hypertension and renal failure. Considering its potent vasodilator effect, adrenomedullin may be involved in the defense mechanism preserving the integrity of the cardiovascular system in these disorders.

A recombinant soluble form of the alpha subunit of the human high-affinity receptor for IgE (rsFc epsilon RI alpha), one of the potent IgE-binding molecules, was tested for its ability to regulate IL-4-induced IgE synthesis by human lymphocytes. Addition of rsFc epsilon RI alpha to cultures induced a dose-dependent inhibition of the T cell-dependent and independent synthesis of IgE. The suppression of IgE synthesis was observed at the protein and the mRNA levels, and it was IgE class specific. By flow cytometry, specific binding of rsFc epsilon RI alpha was detected on surface IgE-bearing B cells as well as on U266 cells, and it was completely blocked by preincubation with IgE. rsFc epsilon RI alpha bound to the cell surface IgE could be effectively dissociated not only by a large excess of IgE, but also by an anti-rsFc epsilon RI alpha mAb that competes with IgE for the binding to rsFc epsilon RI alpha. This mAb abolished the rsFc epsilon RI alpha-mediated suppression of IgE synthesis. These data suggest that rsFc epsilon RI alpha may have a function in selectively suppressing IgE synthesis through its interaction with the membrane-bound form of IgE.