Expression cloning and characterization of the canine parietal cell gastrin receptor.

Gastrin is an important stimulant of acid secretion by gastric parietal cells and is structurally related to the peptide hormone cholecystokinin (CCK). The pharmacologic properties of the parietal cell gastrin receptor are very similar to the predominant CCK receptor in the brain, CCK-B. Neither the gastrin nor the CCK-B receptor have been cloned thus far, making it difficult to resolve whether these two receptors are distinct. We have isolated a clone encoding the canine gastrin receptor by screening a parietal cell cDNA expression library using a radioligand-binding strategy. Nucleotide sequence analysis revealed an open reading frame encoding a 453-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the beta-adrenergic family of G protein-coupled receptors. The expressed recombinant receptor shows the same binding specificity for gastrin/CCK agonists and antagonists as the canine parietal cell receptor. Gastrin-stimulated phosphatidylinositol hydrolysis and intracellular Ca2+ mobilization in COS-7 cells expressing the cloned receptor suggest second-messenger signaling through phospholipase C. Affinity labeling of the expressed receptor in COS-7 cells revealed a protein identical in size to the native parietal cell receptor. Gastrin receptor transcripts were identified by high-stringency RNA blot analysis in both parietal cells and cerebral cortex, suggesting that the gastrin and CCK-B receptors are either highly homologous or identical.


Molecular mechanisms of the partial allosteric modulatory effects of bretazenil at gamma-aminobutyric acid type A receptor.

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.


Role of a vitronectin-like molecule in embryo adhesion of the brown alga Fucus.

The rhizoid cell of the two-celled embryo of the brown alga Fucus is structurally and functionally differentiated from the thallus cell. The rhizoid cell is highly polar and transports directionally components of the cell wall to its elongating tip, which attaches the developing embryo to the substratum. Polyclonal antibodies to human vitronectin (Vn) recognize a vitronectin-like glycoprotein (Vn-F) in extracts of zygotes and two-celled embryos of Fucus, with a molecular mass (approximately 62 kDa) similar to that of human Vn. The specificity of the immunological cross-reactivity of Vn-F to rabbit polyclonal antibodies made to human Vn is demonstrated by competition experiments using pure human Vn and monospecific antibodies generated toward Vn-F. Vn-F possesses affinities for glass and heparin that are identical to those of human Vn. Immunolocalization and subcellular fractionation results demonstrate that Vn-F is localized first in the cytoplasm of the zygote, which is followed by the polar transport of Vn-F to its exclusive localization in the cell wall of the elongating rhizoid tip. Vn does not localize to the rhizoid tip under culture conditions that prevent two-celled embryos from attaching. Furthermore, an adhesion assay demonstrates that two-celled Fucus embryos do not adhere to the substratum in the presence of the Vn antibody, suggesting that the Vn-F in this brown alga not only possesses structural similarity to mammalian Vn but may also have a similar functional role in adhesion. The presence of Vn-F in brown algae suggests a high degree of evolutionary conservation of its structural and functional characteristics.


Tyrosine phosphorylation of phospholipase C-gamma 1 induced by cross-linking of the high-affinity or low-affinity Fc receptor for IgG in U937 cells.

The human monocytic cell line U937 possesses two classes of the IgG Fc receptor (Fc gamma R), a high-affinity 72-kDa Fc gamma R (Fc gamma RI) and a low-affinity 40-kDa Fc gamma R (Fc gamma RII). Cross-linking of either class of Fc gamma R in U937 cells elicits an increase in the concentration of free intracellular Ca2+. A rapid rise in the concentration of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and of several other inositol phosphates derived from Ins-1,4,5-P3 was observed after cross-linking of Fc gamma Rs in U937 cells. This result suggests that Ins-1,4,5-P3, generated by the action of phospholipase C (PLC), acts as a second messenger by which Fc gamma Rs mobilize intracellular Ca2+ in U937 cells. The mechanism by which the cross-linking of Fc gamma Rs triggers activation of PLC was studied. Cross-linking of Fc gamma RI or Fc gamma RII resulted in a rapid and transient phosphorylation of PLC-gamma 1 on tyrosine residues. It has previously been shown that phosphorylation of PLC-gamma 1 on tyrosine residues activates its enzymatic activity in cells. Prior incubation of U937 cells with a protein tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of PLC-gamma 1 and the hydrolysis of phosphatidylinositol 4,5-bisphosphate induced by the cross-linking of Fc gamma Rs. Thus, Fc gamma RI and Fc gamma RII appear to be functionally coupled to a nonreceptor tyrosine kinase that phosphorylates PLC-gamma 1 after receptor cross-linking, thereby causing activation of PLC-gamma 1.


[Late pulmonary changes following bleomycin administration in computed tomography. Nodular fibrosis mimicking a seminoma metastasis].

This is a case report of a patient with multiple metastases to the lung due to a seminoma, treated with a chemotherapeutic regimen containing bleomycin. The typical subpleural fibrosis of the lung related to bleomycin damage disappeared 6 months after the start of chemotherapy. 4 weeks later, a solitary nodule at a prior unchanged site of the lung appeared, mimicking recurrent metastasis. Explorative thoracotomy revealed a nodular fibrosis of the lung, representing a late reaction of the lung to chemotherapy damage.


[Renal tumor embolization].

We present an update on the technical aspects and clinical efficacy of the concept of capillary embolization for the treatment of renal cell carcinoma. In preoperative indications, it is shown that only this type of occlusion can safely achieve a reliable breakdown of the peritumor vasculature to meet the surgical requirements for excision of huge tumors associated with abundant retroperitoneal tributaries. In particular, in tumors infiltrating the renal vein and the vena cava, capillary embolization proved to be extremely helpful. In palliative embolization the concept achieved local and clinical efficacy comparable to nephrectomy when applied in patients free of metastatic disease. In palliative embolization of patients with metastatic disease, no change in the natural history was found. A new but still experimental concept is the combination of chemoperfusion and chemoembolization for tumor nodules in a solitary kidney and for reoccurring retroperitoneal tumors. The application of a coaxial system that features perfect maneuverability enables tumor treatment on the level of subsegmental vessels. The first short-term results seem to be promising. If definite proof of the validity of such a concept is to be obtained, however, further long-term research needs to be conducted on larger numbers of patients.


[Pneumopathy secondary to the improper use of a dust-removing spray].

We report the case of a 24 year-old woman who presented constitutional symptoms with dyspnoea on exertion accompanied by an interstitial pneumonitis in both lower lobes. The history revealed an excessive use of household dust-away cleansing spray. The diagnosis was established by bronchoalveolar lavage associated with appropriate steining. The early diagnosis is critical because of the progression to fibrosis and the risk of cancerisation.


Conditioned increase of natural killer cell activity (NKCA) in humans.

Cumulating evidence suggests that immune parameters can be modified by behavioral conditioning processes in animals. The present results suggest that this also holds true for a human immune parameter. Healthy subjects were exposed to a conditioning procedure in which a neutral sherbet sweet (conditioned stimulus) was repeatedly paired with a subcutaneous injection of 0.2 mg epinephrine (unconditioned stimulus). After epinephrine administration an increase of natural killer (NK) cell activity could be observed (unconditioned response). On the conditioning test day the conditioned group showed increased NK cell activity after reexposure of the sherbet sweet combined with saline injection. No increase was found in control groups that previously received the sherbet sweet in combination with saline (saline control) or with epinephrine in an unpaired manner (unpaired control). This study supports previous findings of conditioned modulation of immune responses and represents a model to investigate conditioning processes of a human immune function.


Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, the receptor for LPS/LBP complexes: a short review.

With the recent discovery and cloning of the lipopolysaccharide-binding protein (LBP), the "adapter-molecule" for LPS-binding to the cell surface receptor CD14 was found. The ligand-receptor pair LPS/LBP-CD14 seems to be one important element in LPS-mediated activation of monocytic cells and possibly granulocytes and B cells. Here, some of the known functions of the proteins involved, LBP and CD14, are reviewed in the context of other endotoxin recognition studies, and the outlook for ongoing and future investigations is described.


Endotoxin-neutralizing capacity of soluble CD14.

Luminol-enhanced chemiluminescence was used to determine the effect of soluble CD14 (sCD14) on the endotoxin-inducible generation of reactive oxygen species in human monocytes. It was necessary to mediate lipopolysaccharide (LPS) monocyte-activating capability by serum factors (LPS-binding proteins). sCD14 reduced LPS-inducible monocyte activation in a dose-dependent manner, even in the case of CD14- monocytes, obtained from a patient with paroxysmal nocturnal haemoglobinuria. These monocytes could be activated by opsonized LPS via other receptors. Using anti-mouse Ig-coated microbeads, it was demonstrated in FACS analysis that sCD14 mediates the binding of a mouse monoclonal anti-CD14 antibody (RoMo 1) to a complex of LPS/FITC (fluoroisothiocyanate) and a LPS-binding protein. The release of sCD14 from cultured monocytes was measured using LPS, TNF alpha (tumour necrosis factor), IL1, 4 and 6 (interleukin-1, -4 and -6) and IFN gamma (interferon-gamma) as stimulators. Addition of LPS and TNF alpha led to a dose-dependent increase in sCD14-levels in the culture supernatant, whereas IL1, IL6 and IFN gamma had no significant effect. IL4 dose-dependently depressed spontaneous sCD14 release. It is possible that elevated sCD14-serum levels in polytraumatized patients indicate a natural protective mechanism against excessive monocyte mediator production. Therefore, sCD14 may be a new therapeutic concept in endotoxic shock prevention.


Therapeutic alternatives for hormone-refractory prostatic cancer.

The currently available prognostic factors allow the identification of patients who are destined to do poorly with primary hormone therapy. Standardization of these factors is required so that they become incorporated into routine practice. For patients who relapse in osseous sites radiolabeled diphosphonates are one therapeutic alternative that can provide palliation. Future use will be directed to optimize treatment schedules, and use earlier in the course of the disease so that more durable palliation of bony metastases can be achieved. Evolving data on the use of PSA show that elevations antedate clinical relapse for most patients. In these cases, sequential changes can be used to assess therapeutic effects, which in turn can allow novel therapies to be screened more rapidly in the clinic. Criteria for the degree of change that is indicative of response must be standardized. Preliminary data suggest a 50% decline be the minimum that is used. The results with agents such as suramin, which interrupts autocrine and paracrine growth factor loops, are a therapeutic strategy that need further study. Future efforts will focus on defining which patients are best suited for specific therapeutic approaches.


[The outcome of resuscitated infants. Experience at an African university maternity unit].

The authors report a study involving 90 infants born at term or close to term and who required resuscitation at birth for per-natal fetal distress. These were 16.6 per cent early deaths (between 0 and 7 days), 11.1 per cent of infants lost to follow-up, 11.1 per cent of infants dying from various causes unrelated to their history of neonatal resuscitation and 16.6 per cent of cases considered unevaluable because of incomplete records. In a second section, they compare 40 resuscitated children (44.4 per cent of the initial sample) with 40 controls of the same age from a clinical, psychomotor and scholastic standpoint. They noted no significant difference regarding clinical and psychomotor status. Results concerning scholastic achievement, with the notable role played by socio-economic circumstances, must be interpreted with caution. At any event, results here are identical in general to those reported in other publications devoted to the same subject.


CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes.

Lymphocytes must proliferate and differentiate in response to low concentrations of a vast array of antigens. The requirements of broad specificity and sensitivity conflict because the former is met by low-affinity antigen receptors, which precludes achieving the latter with high-affinity receptors. Coligation of the membrane protein CD19 with the antigen receptor of B lymphocytes decreased the threshold for antigen receptor-dependent stimulation by two orders of magnitude. B lymphocytes proliferated when approximately 100 antigen receptors per cell, 0.03 percent of the total, were coligated with CD19. The B cell resolves its dilemma by having an accessory protein that enables activation when few antigen receptors are occupied.


Molecular cloning of the interleukin-1 beta converting enzyme.

Interleukin-1 beta (IL-1 beta) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor. A complementary DNA encoding a protease that carries out this cleavage has been cloned. Recombinant expression in COS-7 cells enabled the cells to process precursor IL-1 beta to the mature form. Sequence analysis indicated that the enzyme itself may undergo proteolytic processing. The gene encoding the protease was mapped to chromosomal band 11q23, a site frequently involved in rearrangement in human cancers.


Context dependence of hydrogen bond free energy revealed by substitutions in an RNA hairpin.

Prediction and modeling of RNA structure requires knowledge of the free energy contributions of various interactions. Many unusual hydrogen bonds were recently proposed in the structure of a GCAA hairpin determined from nuclear magnetic resonance. The contributions of these hydrogen bonds to the folding stability of the hairpin formed by rGGCGCAAGCC have now been investigated through the use of functional group substitutions. These and previous results suggest a strong context dependence for the free energy of hydrogen bond formation. The results also suggest that the phylogenetic preference for GNRA (where N = A, C, G, or U and R = A or G) tetraloops may have a functional rather than thermodynamic basis.


Cloning and characterization of inducible nitric oxide synthase from mouse macrophages.

Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.


Diacylglycerol-stimulated formation of actin nucleation sites at plasma membranes.

Diacylglycerols, which are generated during phospholipase-catalyzed hydrolysis of phospholipids, stimulated actin polymerization in the presence of highly purified plasma membranes from the cellular slime mold Dictyostelium discoideum. The increased rate of actin polymerization apparently resulted from de novo formation of actin nucleation sites rather than uncapping of existing filament ends, because the membranes lacked detectable endogenous actin. The increased actin nucleation was mediated by a peripheral membrane component other than protein kinase C, the classical target of diacylglycerol action. These results indicate that diacylglycerols increase actin nucleation at plasma membranes and suggest a mechanism whereby signal transduction pathways may control cytoskeletal assembly.


Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein.

Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions. Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane water channels.


Morphologic and quantitative changes in neurotransmitters in the lumbar spinal cord after acute or chronic mechanical compression of the cauda equina.

Changes in the neurotransmitters associated with pain transmission and regulation in the lumbar spinal cord were studied after acute or chronic mechanical compression of the cauda equina in rats. Using glyoxylic acid histofluorescence and immunohistochemical methods, it was morphologically apparent that substance P-containing nerve ending were decreased after chronic compression of the cauda equina. Somatostatin nerve terminals were reduced, and aminergic fibers and serotonin were enhanced after both acute and chronic mechanical compressions. In addition, quantitative analysis revealed that the levels of norepinephrine and serotonin remained elevated after mechanical compression of the cauda equina. It is suggested that pain in the lower back and extremities after mechanical compression of the cauda equina is controlled by these complicated changes of neurotransmitters in the lumbar spinal cord.


Metallothionein of prostatic tissues and fluids in rats and humans.

We analyzed metallothionein (MT) in rat prostates by gel filtration and radioimmunoassay. The concentration of MT in the prostate, kidney and liver of cadmium-induced rats was measured. The concentration of MT was also measured in normal prostate, benign prostatic hyperplasia, prostate cancer and the prostatic fluids from various prostatic diseases in humans. MT was detected in rat prostates by gel filtration and radioimmunoassay. The concentration of MT (micrograms/g wet tissue) was 0.3 +/- 0.1 (S.D.) in the ventral lobe, 30.4 +/- 24.0 in the lateral lobe, 5.2 +/- 0.9 in the dorsal lobe, 25.0 +/- 6.4 in the kidney and 2.0 +/- 1.5 in the liver of the rat control group. Change in MT content in CdCl2-induced organs increased quantitatively with the dose administered. The concentration of MT (micrograms/g wet tissue) in human prostate was 99.3 +/- 121.8 in the peripheral zone (PZ), 12.0 +/- 8.5 in the preprostatic region (PR), 7.3 +/- 3.1 in the central zone (CZ), 17.5 +/- 15.0 in benign hyperplastic nodules (A) and 4.2 +/- 0.5 in cancer tissue (CA). MT concentration in PZ was very high and that of CA, low (p less than 0.05). MT concentration in prostatic fluids (ng/mg protein) was 11.5 +/- 5.7 in normal patients, 3.8 +/- 2.3 in acute prostatitis, 6.5 +/- 3.7 in chronic prostatitis with pyuria, 39.6 +/- 3.9 in chronic prostatitis without pyuria and 16.9 +/- 3.0 in benign prostatic hyperplasia. We concluded that MT in the prostate is induced by heavy metals and secreted into prostatic fluid. Possibly, it is a marker of secretory function in the prostate.


Characterization of three T-lymphoid cell lines with distinct sensitivities to deoxyadenosine plus deoxycoformycin.

Three established human T-cell lines, HPB-MLT, HPB-ALL and PEER, were characterized and tested for their sensitivity to deoxyadenosine (dAdo) plus deoxycoformycin (dCoF). Phenotypic characterizations showed that all three cell lines had receptors for peanut agglutinin (PNA) and soybean agglutinin (SBA) while HPB-MLT and HPB-ALL, but not PEER, expressed the cortical thymocyte-specific marker, CD1. The majority of HPB-MLT cells (88%) expressed only CD4 but not CD8 antigen while most HPB-ALL cells (81%) co-expressed CD8 and CD4 antigens. PEER cells were negative for both CD8 and CD4. These three T cell lines showed differential sensitivity to dAdo plus dCoF in consequent tests. dAdo or dAdo plus dCoF (1 microM) had no effect on the growth, or DNA and RNA synthesis of HPB-MLT cells while the combination of dAdo and dCoF partially inhibited cellular growth and DNA and RNA synthesis of HPB-ALL cells. Further, the growth and DNA and RNA synthesis of PEER cells were strongly inhibited by the combination of dAdo and dCoF. This high sensitivity to dAdo plus dCoF reflected an immature phenotype of PEER cells despite its expression of CD3. Flow cytometric analysis of PEER cells demonstrated disappearance of the G2/M phase cells from the cell cycle after treatment with dAdo plus dCoF. Measurements of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in all three cell lines, however, did not establish correlations between purine metabolizing enzyme activities and the differential sensitivities to dAdo plus dCoF. In sum, we report here three T-cell lines of different phenotypes that displayed significantly different sensitivities to dAdo plus dCoF which may facilitate investigations on the mechanisms of ADA deficiency.


Apolipoprotein B mRNA editing: a new tier for the control of gene expression.

Two forms of apolipoprotein (apo) B are found in mammals. The shorter form is translated from an edited mRNA in which a specific cytidine base is deaminated to a uridine, creating a new stop codon. Apo B mRNA editing is mediated by a site-specific cytidine deaminase that recognizes a downstream target sequence in the RNA. The enzyme has no energy or cofactor requirements and no RNA component, and thus bears no obvious relationship to RNA processing events such as splicing or polyadenylation. While apo B mRNA editing activity may have arrived late in evolution to target dietary lipid to the liver in mammals, the discovery of the editing activity in tissues and cells that do not express apo B suggests a more widespread role in the generation of RNA and protein diversity.


The effect of bile duct ligation and bile diversion on FK506 pharmacokinetics in dogs.

Mongrel or beagle dogs were submitted to bile duct ligation, or to extraenteric biliary diversion by means of choledochoureterostomy. The kinetics of intravenously administered FK506 was not changed from control status two weeks after bile duct ligation, but the bioavailability of orally administered FK506 was nearly quadrupled. Following oral administration, the absorption of FK506 was highly variable. The results indicate that in dogs FK506 is absorbed from the intestine just as efficiently in the absence of enteric bile and in presence of exogenous bile salt supplement when compared with its absorption in presence of normal bile drainage. These findings with FK506 are different from those with cyclosporine after biliary obstruction or diversion and will have important practical as well as experimental ramifications.


Prevalence and level of antibodies to the circumsporozoite proteins of human malaria parasites, including a variant of Plasmodium vivax, in the population of two epidemiologically distinct areas in the state of Acre, Brazil.

A seroepidemiological study of the prevalence of antibodies against the repeating epitopes of circumsporozoite (CS) proteins of human malaria parasites was conducted in 2 different areas in the state of Acre, Brazil in 1987 and 1990. In 1987 antibodies against the CS protein of the VK 247 variant Plasmodium vivax as well as antibodies against the CS proteins of P. falciparum and the classic P. vivax were found at relatively high rates in the 2 areas, but significant microepidemiological differences were observed. In 1990, when large scale migration in Amazonia had ceased and control measures were applied in the study areas, the malaria endemicity decreased, as determined by the declining prevalence of anti-sporozoite antibodies against all Plasmodium species, and the small number of individuals with positive blood smears. Antibodies against sporozoites of the variant P. vivax did not cross-react with the CS proteins of the classic P. vivax, nor with antibodies against sporozoites of P. falciparum and P. malariae. Sera containing antibodies against the CS protein of P. malariae were found at a very low frequency, and only in 1987. The anti-CS protein antibody response to all Plasmodium species was age-related.


The importance of a colloid in canine pancreas preservation.

The role of hydroxyethyl starch (HES), the colloid component of the UW solution, was tested in canine pancreas preservation. Segmental pancreatic autografts were preserved for 48 hr cold storage with UW solution with HES (group 1) or UW solution without HES (group 2). After preservation, the pancreas was transplanted, and survival, serum glucose, serum amylase, intravenous glucose tolerance tests, tissue water content, and histology were compared between groups. In group 1 (with HES), 9/10 dogs were long-term survivors with one dog dying due to causes unrelated to preservation failure. In group 2 (without HES), 3/6 dogs died due to graft loss within one week posttransplant (P = 0.01). No graft failure occurred in group 1 (0/9) versus graft loss in 4/6 dogs in group 2 (P = 0.04). All animals in group 1 (with HES) showed normal serum glucose and amylase concentrations postoperatively, normal tissue water values after preservation, k values comparable to those observed after segmental autotransplantation without preservation, and relatively good histology. In group 2 (without HES), in 4/6 dogs graft failure occurred that led to the death (3 dogs) of the animals or to a diabetic state (1 dog). After 48-hr cold storage without HES, a significant increase in tissue water content, glucose and amylase levels was seen. After transplantation, hyperglycemia, hyperamylasemia, and clinical diabetes were observed in 4/6 dogs. Autopsy and histological evaluation showed evidence of thrombosis and ischemic insult. Two of 6 dogs in group 2 remained normoglycemic during follow-up with borderline k values. The results suggested that for consistently successful 48-hr preservation of the pancreas, HES is an important component of the UW solution. Although a colloid may not be essential for short-term preservation of kidney and liver, it appears to be an important factor in successful pancreas preservation.


A new dextran 40-based solution for liver preservation.

UW solution is at present the most efficient solution for preservation of livers for transplantation. We have developed an alternative solution based on dextran instead of hydroxyethyl starch and without raffinose, allopurinol, magnesium sulfate, insulin, penicillin, or dexamethasone, which all are used in UW solution. In addition, 62.5 mM potassium in UW solution is replaced with sodium. We tested this new solution for liver preservation using the isolated perfused rabbit liver. We found that livers preserved in the UW solution for 24 or 48 hr lost 11.6 +/- 2.6% and 16.8 +/- 2.0% of the prepreservation weight, respectively, as a sign of organ shrinkage (P less than 0.001). In contrast, no change in liver weight was observed after preservation in the new dextran-based solution. Similarly, no change in total tissue water of the rat liver slices was seen after preservation in the new solution. Furthermore, livers preserved for 24 hr in the UW solution or the new solution produced the same amount of bile as unpreserved livers. However, after preservation in the UW solution for 48 hr, bile production was reduced by 65% (P less than 0.05). In contrast, livers preserved for 48 hr in the new solution showed no reduction in bile production. We conclude that our new solution significantly improves long-term liver preservation, and with this modified solution, 48-hr preservation may be safe.


The clinical outcome of hepatitis C virus antibody-positive renal allograft recipients.

In order to investigate the prevalence of antibody to hepatitis C virus (anti-HCV) in renal transplant patients, the evolution of anti-HCV status, and clinical outcome in anti-HCV-positive renal allograft recipients, we tested the sera from 120 renal transplant patients for anti-HCV. Thirty-eight patients were hepatitis B surface antigen (HBsAg)-positive. Two patients were anti-delta-positive. A total of 79 patients (65.8%) had at least one serum positive for anti-HCV. Anti-HCV positivity decreased after transplantation for more than 5 years (65.5% at transplantation versus 37.9%, 78.3 +/- 13.4 months later). Among those with positive anti-HCV, the HBsAg-positive group had significantly higher incidence of chronic hepatitis (50% vs. 25.5%, P = 0.026) and liver cirrhosis (21.4% vs. 0%, P = 0.001) than HBsAg-negative group. Among the 82 HBsAg-negative patients, the prevalence of anti-HCV was significantly higher in those with chronic hepatitis than in those without (86.7% vs. 56.7%, P = 0.027). We conclude from this study: (1) anti-HCV positivity is quite prevalent in renal transplant patients; (2) coinfection of hepatitis B virus (HBV) and hepatitis C virus (HCV) may lead to aggressive liver disease and cirrhosis; HCV infection alone has a more benign clinical outcome; and (3) HCV infection is an important cause of posttransplant chronic hepatitis in HBsAg-negative patients.


Morphological and functional changes of islets of Langerhans in FK506-treated rats.

FK506, a new immunosuppressant, caused glucose intolerance in rats given daily oral doses of 1, 5, or 10 mg/kg/day for 14 days, but did not induce hyperglycemia under normal feeding. Besides the glucose intolerance, insulin secretion was impaired and insulin levels in the pancreas were lowered. Histopathologically, there was vacuolation of the Langerhans islets in the animals given 10 mg/kg. After withdrawal of the drug, these changes in pancreatic function and morphology returned to normal within 2 weeks. The findings indicate that FK506 caused dose-dependent but reversible islet toxicity in rats.


Cytomegalovirus infection of the upper gastrointestinal tract following liver transplantation--incidence, location, and severity in cyclosporine- and FK506-treated patients.

One hundred and forty randomly selected liver transplant recipients were studied before and after primary orthotopic liver transplantation for the presence or absence of CMV enteritis. Following OLTx, 65 patients were treated with cyclosporine A and 75 were treated with FK506. The two groups were similar with regard to the incidence, location, and outcome of their upper gastrointestinal CMV infection. Prior to OLTx, only one patient had evidence of enteric CMV infection. The incidence of CMV enteritis post-OLTx was 27.7% in the CsA-treated group and 20% in the FK-treated group. During the first posttransplant month, no patient in the FK-treated group developed CMV enteritis, compared with 11.5% of the patients who were treated with CsA (P less than 0.05). Gastric CMV was found in over 80% of those positive for any organ in either group. In addition to CMV infection of the upper gastrointestinal tract, clinically evident CMV disease involved more nonenteric organs in the CsA-treated group than in the FK-treated group. In the CsA-treated group, CMV-negative patients had a statistically higher 1-year survival rate (100%) than CMV-positive patients (77.8%) (P less than 0.05). In the FK-treated group, no difference in survival was observed between CMV-positive or CMV-negative cases at 1 year. Of the patients on CsA, 20% received OKT3 for persistent rejection, as compared with 13% in the FK-treated group. The patients receiving both CsA and OKT3 had a higher rate of upper gastrointestinal CMV infection than did FK-treated patients who also received OKT3 therapy (38.5% versus 20%, respectively). Based upon these data, it can be concluded that (1) patients receiving FK have a lower incidence of enteric CMV infection; (2) following OLTx, upper gastrointestinal CMV infection presents later in FK-treated patients; (3) the stomach is the most frequently involved organ in the UGIT; (4) FK-treated liver recipients have less severe enteric CMV infection than do CsA-treated patients; (5) enteric CMV is not a major cause of mortality in liver transplant recipients; and (6) in patients receiving FK, those who require OKT3 therapy do not appear to be at a greater risk for the development of CMV enteritis than those who do not.


The in vitro immunosuppressive effect of deoxymethylspergualin in man as compared with FK506 and cyclosporine.

The effect of deoxymethylspergualin (MeDSG) on in vitro human lymphocyte response was assessed in comparison with FK506 and cyclosporine. Peripheral blood mononuclear cells from normal human volunteers were used for assay of mixed lymphocyte reaction, cell mediated lympholysis, and blastogenesis by PHA, IL-2, and OKT3. MeDSG suppressed only allogeneic stimulation (MLR and CML) and IL-2-induced blastogenesis, not PHA- or OKT3-induced blastogenesis, although the other immunosuppressive agents showed some suppressive effect for all assays. A kinetic study of MLR showed that the suppressive activity did not decrease even when MeDSG was added at day 3 or day 4. The other agents, however, showed a weak suppressive effect when added at a later phase of MLR.


Langerhans cells in the lymph node: mirror section and immunoelectron microscopic studies.

Cells immunostained with antibodies against both OKT-6 and S-100 protein were observed only in superficial and hilar lymph nodes draining tissues with predominantly squamous epithelia. In contrast, in mesenteric lymph nodes and the spleen, only S-100 protein-positive, but OKT-6-negative cells were found. We suspect that the S-100 and OKT-6-positive cells might be Langerhans cells (LC) and the S-100-positive, OKT-6-negative cells, interdigitating reticulum cells (IDC). We further postulate that the LC in superficial and hilar lymph nodes might migrate from squamous epithelia, with which contact is required for the formation of Birbeck granules.


Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: the envelope of the human immunodeficiency virus (HIV-1).

Synthetic peptides and anti-peptide antibodies have been widely used as probes to map B- and T-cell epitopes on proteins. Such probes also have the potential to delineate contact sites involved generally in protein-protein interactions or in association of domains within a protein. We applied peptide/anti-peptide probes to define: (1) regions on the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41 involved in the association between these two glycoproteins; and (2) sites on gp120/gp41, essential for the association of HIV-1 with the CD4 cell receptor. Results of this examination suggested the following: (1) two segments on gp120, encompassing residues (102-126) and (425-452), contribute to the binding site for CD4 and are expected to be juxtaposed in the folded gp120 chain; (2) portions of immunodominant gp120 and gp41 epitopes, encompassing residues (303-338) and (579-611), respectively, appeared to be involved in the gp120-gp41 association, as suggested by direct binding studies and by the limited accessibility of these epitopes on HIV-1 virions: other portions of gp120 also appeared to contribute to the association between these two glycoproteins; (3) there is a partial overlap between gp41 and CD4 binding sites on gp120; (4) the fusion domain and a segment (637-666) of gp41 are not accessible to antibodies after oligomerization of gp41; and 5) the gp120-gp41 association was blocked by aurintricarboxylic acid, suggesting the possibility of developing antiviral compounds interfering with HIV-1 assembly.


Expression of authentic vaccinia virus-specific and inserted viral and cellular genes under control of an early vaccinia virus promoter is regulated post-transcriptionally in interferon-treated chick embryo fibroblasts.

The interferon sensitivity of the expression of an influenza-virus hemagglutinin (HA) gene cloned into the thymidine kinase (TK) gene of vaccinia virus was studied in chick embryo fibroblasts (CEF) and Madin-Darby bovine kidney (MDBK) cells. In CEF, the expression of the HA gene is inhibited by pretreatment of cells with homologous interferon. In MDBK cells, on the other hand, expression of the HA is not impaired by pretreatment with human interferon-alpha, and the synthesis of early vaccinia virus enzymes was also unaffected. These results indicate that the interferon sensitivity of HA gene expression is at least in part controlled by flanking regions of vaccinia virus DNA. In this report, we also address the question whether the expression of an influenza virus HA gene and the human histone H1 zero gene under control of a vaccinia virus immediate early promoter is affected in interferon-treated CEF by a post-transcriptional mechanism in the same way as the expression of the viral TK gene. In interferon-treated cells mRNA synthesis specific for all these genes was enhanced. Steady state mRNA levels 6 hr p.i. were, however, lower than the amounts expected from the rate of mRNA synthesis during the first 6 hr p.i., suggesting that part of the viral RNA was degraded. Degradation resistant mRNA accumulated in the interferon-treated cells in an amount comparable to that found in infected CEF. This RNA could be translated into viral protein in a cell-free system. Therefore the degradation of viral mRNA cannot solely be responsible for the inhibition of viral protein synthesis in interferon-treated cells.


Monoclonal IgM antibodies from cytomegalovirus-infected mice recognize the GlcNAc-containing receptor determinant of murine CMV as well as neutralizing anti-CMV IgG antibodies.

This study examines monoclonal antibodies derived from mice at different time points after infection with attenuated murine cytomegalovirus (MCMV). The antibodies obtained from mice at 3 weeks p.i. were of IgG type (designated V-antibodies) and several could neutralize the virus. Those obtained at 5 weeks p.i. were of the IgM class (designated R-antibodies), bound to uninfected (MEF, mouse embryo fibroblast) cells, and thereby blocked MCMV plaque formation. In ELISA, the IgM monoclonals (R-antibodies) bound to GalB1-3GlcNAc and GalB1-4GlcNAc, the receptor determinants for MCMV. Similarly, these GlcNAc-containing residues blocked the binding of purified IgM monoclonal antibodies (MAbs) to MEF. The R- and V-series of antibodies showed mutual binding activities; for example, IgM MAb R-2D8 bound specifically to four (V-8C4, V-1C7, V-8C7, V-9C5) of six neutralizing IgG MAbs in ELISA. The same neutralizing IgG MAbs bound to antireceptor IgM antibodies in an immunoblot assay. This suggests that the IgM monoclonals directed against the known cell surface receptor determinant are anti-idiotypic antibodies against neutralizing antiviral IgG antibodies. The neutralizing antiviral IgG MAbs bound to 60- and 66-kDa MCMV polypeptides on Western blots, suggesting that these viral polypeptides may be important in MCMV binding to this receptor. The R-series might represent anti-idiotype antibodies capable of down-regulating antiviral V-antibodies and may also represent a mechanism for the induction of IgM autoantibodies directed at cell surface glycolipids present in autoimmune CMV-associated neuropathies.


Antigenic structure of transmissible gastroenteritis virus nucleoprotein.

A group of 11 monoclonal antibodies (MAbs) raised against transmissible gastroenteritis virus (TGEV) was used to study the antigenic structure of the virus nucleoprotein (N). To identify the regions recognized by MAbs, DNA fragments derived from the N-coding region of the TGEV strain FS772/70 were cloned into pUR expression plasmids and the antigenicity of the resulting fusion proteins was analyzed by immunoblotting. A major antigenic domain was identified, covering the first 241 amino acid residues of N, within which an epitope (residues 57-117) was also found. A second antigenic domain extended from residues 175 to 360 of the nucleoprotein, within which a subsite was characterized within the region covering residues 241-349. MAb DA3 recognized a linear epitope which mapped within residues 360 and 382 at the carboxy terminus of the nucleoprotein. The binding of the majority of the MAbs (8 out of 11) to large fusions, but not to smaller fragments included in them, suggests a conformational dependence of the MAb binding sites. Our data show that the use of fusions in Western blot experiments is a useful approach to map not only linear epitopes but more complex antigenic structures found in the nucleoprotein of the TGEV.


Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells.

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.


Mechanism of interferon action: identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2 alpha protein kinase.

A molecular cDNA clone of the human RNA-dependent P1/eIF-2 alpha protein kinase was expressed in Escherichia coli. Mutant P1 proteins were examined for RNA binding activity by Northwestern blot analysis using the reovirus s1 mRNA, an activator of the kinase; the adenovirus VAI RNA, an inhibitor of kinase activation; or human immunodeficiency virus (HIV) TAR RNA as probe. Analysis of TrpE-P1 deletion mutant fusion proteins revealed that the 11-kDa N-terminal region of the P1 protein bound reovirus s1 mRNA, adenovirus VAI RNA, and HIV TAR RNA. Neither s1 RNA, VAI RNA, nor TAR RNA was bound by truncated P1 proteins which lacked the N-terminal 98 amino acids. Computer analysis revealed that the human protein P1 sequence corresponding to amino acid residues within the N-terminal RNA binding domain displays high homology (greater than 54% identity; 61 to 94% similarity) with two animal virus proteins which possess RNA binding activity (vaccinia virus E3L; rotavirus VP2) and two proteins of unknown function (murine TIK; rotavirus NS34), but which are likely RNA binding proteins.


Characterization of the endogenous reverse transcriptase reaction products of SIVagmTYO-7, a simian immunodeficiency virus isolated from an African green monkey.

The products of the endogenous reverse transcriptase reaction of SIVagmTYO-7 were characterized after the reaction conditions had been optimized. The major reaction product in the presence of actinomycin D and oligo(dT) was a DNA with a size of 300 bases. Without actinomycin D two additional reaction products with 600 or 700 bases appeared. The 300 base product was identified as the (-)strong-stop DNA, whereas the 700 base product is the (+)strong-stop DNA. The 600 base product appeared only after oligo(dT) priming. The (-)strong-stop DNA hybridized specifically with a 9 kb RNA found in virus particles and three RNA species of 1.8, 4.8 and 9 kb isolated from SIVagmTYO-7 infected cells.


Sequence analysis of human coronavirus 229E mRNAs 4 and 5: evidence for polymorphism and homology with myelin basic protein.

Human coronaviruses (HCV) are important pathogens responsible for respiratory, gastrointestinal and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the nucleotide sequence of the 5'-unique regions of mRNAs 4 and 5 were determined from cloned cDNAs. Sequence analysis of the cDNAs synthesized from mRNA 4 revealed a major difference with previously published results. However, polymerase chain reaction amplification of this region showed that the sequenced cDNAs were produced from minor RNA species, an indication of possible genetic polymorphism in this region of the viral genome. The mutated messenger RNA 4 contains two ORFs: (1) ORF4a consisting of 132 nucleotides which potentially encodes a 44-amino acid polypeptide of 4653 Da; this coding sequence is preceded by a consensus transcriptional initiation sequence, CUAAACU, similar to the ones found upstream of the N and M genes; (2) ORF4b of 249 nucleotides potentially encoding an 83-amino acid basic and leucine-rich polypeptide of 9550 Da. On the other hand, mRNA 5 contains one single ORF of 231 nucleotides which could encode a 77-amino acid basic and leucine-rich polypeptide of 9046 Da. This putative protein presents a significant degree of amino acid homology (33%) with its counterpart found in transmissible gastroenteritis coronavirus (TGEV). The proteins in the two different viruses exhibit similar molecular weights and are extremely hydrophobic. Interestingly, a sequence homology of five amino acids was found between the protein encoded by ORF4b of HCV-229E and an immunologically important region of human myelin basic protein.


Abnormalities in immune regulation precede the development of multiple myeloma.

Immunosuppression of immunoglobulin synthesis seen in patients with multiple myeloma is in part due to immunosuppressive CD5 positive B cells. In a 13 year longitudinal study of an IgA-deficient blood donor who developed multiple myeloma, the presence of immunosuppressive CD5 positive B cells and T cells preceded the diagnosis of overt multiple myeloma and the appearance of immunosuppressive monocytes. These data argue that certain immune defects may be involved in the development of myeloma and are not simply a consequence of overt malignancy.


Successful treatment of T-gamma lymphoproliferative disease with human-recombinant granulocyte colony stimulating factor.

A trial of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was attempted in a male with agranulocytosis, infection, and T-gamma lymphoproliferative disease (T-gamma-LPD). During five days of rhG-CSF (960 micrograms/day), the absolute neutrophil count (ANC) increased from 0.0 to 4.5 K/microliters. There were no changes in eosinophil or lymphocyte counts. In addition, there was no toxicity. Bone marrow cytotoxic/suppressor cells (CD57+/CD8+) were elevated (21.9%) before and decreased to 10.6% (normal less than 12%) following rhG-CSF. By contrast, there was no change in activated T cells (CD3+DR+) or T cell gene rearrangements. These findings suggest rhG-CSF can improve granulopoiesis in T-gamma-LPD, possibly by altering T-cell mediated marrow suppression.


Stretch-activated single-channel and whole cell currents in vascular smooth muscle cells.

Mechanosensitive ion channels may play a key role in transducing vascular smooth muscle (VSM) stretch into active force development. To test this hypothesis, we recorded single-channel and macroscopic currents during mechanical stimulation of enzymatically dispersed vascular smooth muscle cells. Patch pipette suction activated a nonselective cation channel that was permeable to K+, Na+, and Ca2+. Whole cell stretch was accomplished using two patch-type micropipettes attached to the cell ends with suction. Stretch elicited a sustained depolarization with a magnitude similar to that observed in pressurized arteries. Under whole cell voltage clamp, stretch activated an inward current with a reversal potential near -15 mV. In another series of experiments, whole cell stretch failed to modify the current-voltage relationship for voltage-gated calcium currents. Thus, in VSM, both single-channel and whole cell data are consistent with activation of a nonselective cation channel by stretch. This mechanism may, in part, account for pressure-induced activation of intact blood vessels.


Endothelin stimulates protein synthesis in smooth muscle cells.

The present work was carried out to assess the effect of endothelin on the relative synthesis of protein, RNA, and DNA in confluent rat aortic smooth muscle cells (SMC) derived from Wistar-Kyoto (WKY) rats maintained under serum-free medium in the presence or absence of insulin, transferrin, and selenium. Insulin stimulated protein synthesis by 42%. Endothelin (1 x 10(-7) M) rapidly induced protein synthesis by 22% (-insulin) and 30% (+insulin). Prior treatment of SMC for 4 h with endothelin resulted in 50% (-insulin) and 38% (+insulin) increase in protein synthesis. The stimulatory effect of endothelin on protein synthesis could be partially blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase C inhibitor. Atrial natriuretic factor had no effect on either the basal protein synthesis or protein synthesis stimulated by endothelin. Furthermore, endothelin stimulated RNA synthesis by twofold but had no effect on DNA synthesis in SMC derived from WKY rats. In contrast, SMC derived from spontaneously hypertensive rats showed increased DNA synthesis and cell growth after endothelin stimulation. These studies show that this hormone may play a pivotal role in the development of vascular hypertrophy in hypertension.


Gonadotropin and cAMP modulation of IGE binding protein production in ovarian granulosa cells.

Porcine granulosa cells (GC) produce insulin-like growth factor (IGF) binding protein (BP)-3 and IGFBP-2 in culture. A gonadotropin, follicle-stimulating hormone (FSH), dramatically inhibited GC production of these IGFBPs in control cultures and in cultures stimulated by insulin plus epidermal growth factor (EGF) or IGF-I plus EGF. Stimulators of adenylate cyclase (forskolin, cholera toxin) and a derivative of adenosine 3',5'-cyclic monophosphate (cAMP), 8-bromoadenosine 3',5'-cyclic monophosphate, inhibited IGFBP synthesis in a manner similar to FSH. In contrast, the antagonist of cAMP action, (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], significantly stimulated production of IGFBP-3 and IGFBP-2 compared with controls. This stimulatory effect of (R)-p-cAMPS was counteracted by cotreatment with FSH in a dose-dependent manner. Finally, treatment of GC cultures with FSH plus 3-isobutyl-1-methylxanthine resulted in a significant reduction in cellular content of mRNA coding for IGFBP-3 with no change in IGFBP-2 mRNA. In summary, agents that elevate intracellular cAMP were found to mimic the effects of FSH on IGFBP production.


Atrial natriuretic peptide synthesis in atrial tumors of transgenic mice.

Transgenic mice harboring a chimeric gene linking mouse protamine 1 5'-flanking sequence to the coding sequence of the simian virus 40 T-antigen develop spontaneous rhabdomyosarcomas of the right atria. The presence of the tumors is accompanied by dramatic elevations in plasma atrial natriuretic peptide (ANP) immunoreactivity (1,698 +/- 993 vs. 60 +/- 18 fmol/ml for controls) and hematocrit (56 +/- 8 vs. 51 +/- 2 for controls). The immunoreactive ANP (irANP) present in the tumors is similar in size to irANP found in normal mouse atria. ANP mRNA transcripts present in the tumors also appear to be very similar in overall size and 5'-termini to those produced in normal cardiac tissue. Microscopically, the tumors are composed of a disorganized array of densely packed abnormal-appearing cells. Immunocytochemistry and in situ hybridization analysis reveal considerable heterogeneity in ANP gene expression. ANP peptide and mRNA are detectable throughout the parenchyma of the tumors, but absolute levels of expression vary widely among different cells in the population. These tumors represent a potentially valuable model for the study of inappropriate ANP secretion and may provide a tissue source for the development of an ANP-producing atrial cell line.


Neutrophil accumulation in ischemic reperfused rat liver: evidence for a role for superoxide free radicals.

Oxygen-derived free radicals and leukocytes have been implicated in the pathogenesis of ischemia-reperfusion injury. This study aimed at determining, by using biochemical and histochemical techniques, whether an accumulation of neutrophils occurs in the ischemic reperfused rat liver and whether superoxide free radicals play a role in mediating this neutrophil accumulation. Hepatic ischemia was induced by occluding blood supply to the left and median lobes, and reperfusion was reinstituted by releasing the occlusion. Myeloperoxidase activity of the liver was measured with a tetramethylbenzidine-H2O2 assay after removal of glutathione (by dialysis) and in the presence of 3-aminotriazole (catalase inhibitor). A modification of Graham and Karnovsky's method was used to stain neutrophils in liver frozen sections, and the number of neutrophils was counted. Results showed that ischemia-reperfusion of the liver produced a 4.4-fold increase in myeloperoxidase activity (from 0.073 +/- 0.009 to 0.320 +/- 0.017 units/mg liver, means +/- SE), which was proportional to the number of neutrophils (3.1-fold increase from 18 +/- 7 to 57 +/- 4 cells/mm2) in the liver tissue. Pretreatment with long-acting superoxide dismutase significantly attenuated the elevated myeloperoxidase activity and the number of neutrophils. These results indicate that reperfusion after a period of ischemia induces an accumulation of neutrophils in the liver, and superoxide anion free radicals are important mediators in the mechanism of this neutrophil accumulation.


Characterization of high-conductance anion channels in rat bile duct epithelial cells.

We have utilized patch clamp recording techniques to identify a high-conductance anion channel in the plasma membrane of rat bile duct epithelial cells. Cells were isolated from the intrahepatic bile duct 2-6 wk after bile duct ligation. Channels were present in 27% (28/102) of excised patches, and, with 150 mM Cl- in bath and pipette solutions, the slope conductance of the fully open level was approximately 364 +/- 18 pS (n = 8) with current reversal = 0 +/- 1 mV. Channel characteristics were not affected by substitution of K+ for Na+ in the pipette solution; but substitution of HCO3-, gluconate, or increased NaCl caused a shift in the reversal potential toward the new equilibrium potential for Cl-. The permeability ratios were PHCO3-/PCl- = 0.51 +/- 0.03 (n = 5), Pgluconate/PCl- = 0.12 +/- 0.04 (n = 7), and PNa+/PCl- = 0.11 +/- 0.02 (n = 3). Current transitions to a subconductance level at 72% of the fully open level were present in most studies. Channel open probability was greatest near 0 mV and decreased rapidly outside of -20 to +20 mV because of voltage-dependent channel closure. The time course for current relaxation of summed single channel currents could be described by a single exponential with more rapid channel closure as the magnitude of the voltage step away from 0 mV increased. In the cell-attached configuration, the channel was rarely open (4/35, 11%) but opening could be induced by negative pipette pressure (5/14, 35%). Possible physiological roles for this channel are discussed.


Amylase release from streptolysin O permeabilized fetal pancreatic acini.

Developmental regulation of Ca(2+)-dependent protein discharge was investigated in fetal and neonatal rat pancreatic acini permeabilized with streptolysin O. When incubated at 37 degrees C in a Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/K glutamate buffer, permeabilized day 19 and 20 fetal acini demonstrated Ca(2+)-dependent release of amylase, whereas day 21 (term) fetal acini did not. Ca(2+)-dependent amylase release reappeared in day 1, 2, and 6 neonatal pancreases. ATP depletion completely inhibited Ca(2+)-stimulated amylase release from both day 20 fetal and adult acini. Ca(2+)-dependent amylase discharge from day 20 fetal acini was enhanced by the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Ca(2+)-independent GTP gamma S-stimulated amylase release was observed from adult but not from day 20 fetal acini. In contrast to its stimulatory effects in permeabilized adult acini, adenosine 3',5'-cyclic monophosphate (cAMP) alone had little effect on release from permeabilized day 20 fetal acini. Our studies indicate that the fetal pancreas is competent to undergo Ca(2+)-dependent protein secretion but that this secretion is suppressed at birth. Our studies also suggest that the fetal gland is sensitive to modulators of exocytosis active in the adult pancreas, such as GTP gamma S, TPA, and cAMP but responds differently to these agents compared with responses in adult glands.


Role of cholecystokinin in induction and maintenance of dietary protein-stimulated pancreatic growth.

The role of cholecystokinin (CCK) in induction and maintenance of pancreatic growth stimulated by a high-protein diet was investigated. Rats adapted to 5% casein diet were switched to 70% casein for 21 days. MK-329, a CCK receptor antagonist, was administered at 2.5 mg.kg-1.day-1 ip, beginning on day zero (day zero treatment) or day 7 (midcourse treatment) of feeding 70% casein and thereafter. Another group was returned to 5% casein after 7 days of feeding 70% casein. Feeding 70% casein significantly stimulated increases of 32, 87, 74, 216, and 1,450% in pancreatic DNA, RNA, wet weight, protein content, and chymotrypsin content, respectively. Midcourse treatment with MK-329 was more effective than day zero treatment, and it completely reversed increases in pancreatic weight and RNA content, partially reversed increases in protein and chymotrypsin content, and had no effect on DNA content. Return to 5% casein rapidly reversed increases in pancreatic parameters, except for DNA. The results indicate that CCK is essential for induction and maintenance of dietary protein-stimulated pancreatic hypertrophy.


Bleomycin stimulation of cytokine secretion by the human alveolar macrophage.

Bleomycin (BLM) is a very effective antineoplastic drug for many gynecologic and urinary tract carcinomas. However, its use, e.g., cumulative dosage, often is limited by the pulmonary fibrosis that it causes. The mechanism by which BLM causes fibrosis is not understood but is proposed to involve the pulmonary macrophage, a central cell in the cytokine network of the lung. To examine the direct effects of this drug on the human alveolar macrophage, we have treated human alveolar macrophages (isolated from normal subjects by bronchoalveolar lavage) with BLM in vitro and examined resultant macrophage secretory products that have importance for inflammatory and fibrotic processes. A 24-h treatment with BLM (0.5-100 mU/ml) was found to result in 1) a concentration-dependent decrease in the ability of the macrophage to produce superoxide anion in response to phorbol 12,13-dibutyrate, 2) an increase in secreted interleukin-1 beta (IL-1 beta), and 3) a decrease in intracellular levels of adenosine 3',5'-cyclic monophosphate. Kinetic studies revealed a time-dependent appearance of BLM-induced cytokines; tumor necrosis factor-alpha could be detected as early as 4 h after stimulation, followed by IL-1 beta at 8 h. The secretion of these cytokines was found to precede the release of prostaglandin E2, which became significant only at 24 h. Taken together, the present results imply that the human alveolar macrophage does not contribute to BLM-induced oxidant injury of the lung but that it may contribute to the development of BLM-induced pulmonary fibrosis.


Expression of CFTR and presence of cAMP-mediated fluid secretion in human fetal lung.

We studied the developmental expression of the cystic fibrosis (CF) gene in human lung tissue from normal and CF-affected fetuses. Two unrelated CF fetuses, both homozygous for the delta F508 deletion, were examined. Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was present in second-trimester CF lung and in first- and second-trimester normal lung as assessed by amplification of reverse transcribed total RNA with the use of the polymerase chain reaction. CFTR protein was identified by immunoprecipitation in normal second-trimester fetal lung explants. To evaluate possible functional consequences of CF in the fetus, lung tissue explants were grown in submersion organ culture. By light and electron microscopy, the CF fetal lung explants appeared normal. When explants from normal fetal lung were exposed to 8-(4-chlorophenylthio) adenosine 3',-5'cyclic monophosphate (CPT-cAMP), and 3-isobutyl-1-methylxanthine (IBMX) for 24 h, the intraluminal fluid content increased, as assessed by a 40 +/- 4% increase in cross-sectional diameter. In contrast, identically treated CF explants showed no significant change in explant diameter (3 +/- 1.6%). The transepithelial potential (psi t) across fetal lung explants was measured with microelectrodes. In normal second-trimester explants, CPT-cAMP and IBMX caused hyperpolarization of psi t (-0.93 +/- 14 mV to -4.3 +/- 1.2 mV); in contrast, CF fetal lung explants showed no significant change in psi t with CPT-cAMP and IBMX (-0.84 +/- 0.07 mV to -1.21 +/- 0.26 mV). This study confirms the presence of CFTR mRNA and protein in human fetal lung and suggests that although the CF fetal lung appears normal morphologically, there is a defect in cAMP-mediated fluid secretion in the lung of the CF fetus.


Calcium imaging of mechanically induced fluxes in tissue-cultured chick heart: role of stretch-activated ion channels.

Heart rate and contractility are sensitive to stretch. To better understand the origin of these effects, we have studied the effect of mechanical stimuli on a model system of tissue-cultured heart cells. Gently prodding cells with a pipette produced a Ca2+ influx that often led to waves of calcium-induced calcium release (CICR) spreading from the site of stimulation. Ca2+ release could also be produced by pulling on neighboring cells. The response was blocked by removing extracellular Ca2+ or by adding 20 microM Gd3+ to normal saline. The mechanical sensitivity probably arose from stretch-activated ion channels (SACs) based on several lines of evidence. Chick heart cells contain nonselective cation SACs that pass Ca2+ as well as Na+ and K+. Both the SACs and the fluorescence response are blocked by 20 microM Gd3+. Removal of Ca2+ from the extracellular medium blocked the fluorescent response. Cultures without SACs (grown in the absence of embryo extract) had no mechanically induced fluxes. These data contradict the recent claim that SAC activity is a patch-clamp artifact (C.E. Morris and R. Horn, Science Wash. DC 256: 1246-1249, 1991). The SACs had a density of approximately 1/micron 2 and were expected to pass less than 20 fA of Ca2+ current under physiological conditions. The change in intracellular concentration of Ca2+ ([Ca2+]i) resulting from activation of SACs may be too small to induce CICR unless the channels pass current into a restricted space (N. LeBlanc and J.R. Hume, Science Wash. DC 248: 372, 1990).(ABSTRACT TRUNCATED AT 250 WORDS)


A model of countercurrent shunting of oxygen in the intestinal villus.

This report describes a mathematical model of the countercurrent shunting (CCS) of O2 in the intestinal villus. The anatomic basis for the model is the close proximity of the arteriole and venule between which O2 is free to diffuse. The model divides the villus into four segments from base to tip. Steady-state equations describe the convective and diffusive fluxes of O2 in the arteriolar, capillary, and tissue compartments within each segment. Longitudinal diffusion along the length of the villus is assumed to be negligible. Simulations with the model led to the following observations: 1) CCS shifted the VO2 vs. blood flow curve down and to the right, slightly impairing VO2 at a given blood flow; 2) the base-to-tip PO2 gradient caused by the tissue O2 consumption was reduced by CCS; 3) when blood flow was reduced, the base-to-tip PO2 gradient increased until the tip PO2 fell to zero and then fell with further flow reductions; 4) lowering blood flow initially caused slight increases in shunting but further decreases in flow reduced shunting; 5) in the blood flow range in which VO2 was flow independent, increasing the O2 demand or decreasing the intervascular distance increased shunting because of the greater arteriole-to-capillary O2 concentration gradient and the decreased diffusion distance, respectively; and 6) lowering the hemoglobin's P50 to simulate fetal blood caused slight reductions in shunting and reduced VO2 at a given flow. In summary, the model confirms the potentially deleterious effects of CCS on intestinal oxygenation, and, in contrast to assertions in the literature, it shows that a base-to-tip PO2 gradient is not prima facie evidence of counter-current shunting.


Calcium currents in hearts with persistent truncus arteriosus.

We have used the neural crest model of defective heart development to characterize both L- and T-type Ca2+ currents (ICa,L and ICa,T) in ventricular myocytes from embryonic chick hearts with a severe outflow tract anomaly known as persistent truncus arteriosus (PTA). Because of smaller whole embryo weights but no significant change in the weights of ventricles with PTA, the ventricle to whole embryo weight ratios from hearts with PTA were 61% larger than normal at day 11 of incubation. There was a 51% reduction in the peak magnitude of ICa,L at a test potential of +10 mV (-1.4 vs. -0.7 microA/cm2), whereas ICa,T, a proportionately large fraction of the total Ca2+ current in the embryonic chick ventricle, was unaffected. In comparison to sham-operated controls, ICa,L was otherwise not different. Half-activation occurred at about -1 and -41 mV, whereas half-inactivation occurred at -19 and -61 mV for ICa,L and ICa,T, respectively. The time for half-recovery from inactivation were not different and were 200 and 230 ms for ICa,L and ICa,T, respectively. The time for half-decay of the currents and their responses to BAY K 8644 were also similar in both sham-operated and experimental hearts. Although the dihydropyridine receptor binding experiments suggest that the total number of L-type Ca2+ channels was not different, the results from the physiological experiments indicate that the number of functional L-type channels available for opening and/or the single-channel conductance may be reduced in hearts with PTA. Finally, our results with the neural crest model indicate that it is unlikely that the development of Ca2+ currents is influenced by the onset of cholinergic innervation in the heart.


Cardiac atrial natriuretic factor during evolution of congestive heart failure.

Congestive heart failure (CHF) is a pathophysiological condition associated with increased plasma levels of atrial natriuretic factor (ANF), a peptide hormone of cardiac origin that participates in the homeostatic control of intravascular volume and vascular tone. Atrial myocytes serve as the principal source of ANF under physiological conditions, although recent studies have demonstrated that ventricular myocardium may also synthesize ANF in models of CHF associated with ventricular hypertrophy. The current study was designed to investigate the roles of atrial and ventricular myocardium to synthesize, store, and release ANF during the evolution of tachycardia-induced CHF in the dog. The present study demonstrates a persistent elevation of plasma ANF during the evolution of CHF. In acute CHF (3 h), plasma ANF increased independent of cardiac ANF synthesis. In chronic CHF (15 and 30 days), plasma ANF is maintained by an increase in atrial synthesis and release of the peptide, without recruitment of ventricular ANF synthesis. The present study demonstrates that in acute CHF the increase in plasma ANF is regulated by release of stored peptide, and in chronic CHF the persistent elevation of plasma ANF is maintained by an increase in atrial synthesis of ANF.


Dipyridamole-induced capillary growth in normal and hypertrophic hearts.

Chronic increases in myocardial blood flow have been shown to stimulate capillary proliferation in normal growing hearts. It is unknown, however, if elevated myocardial blood flow stimulates precapillary and/or capillary growth in hearts undergoing hypertrophy. Accordingly, renal hypertension was produced in rabbits (Page, 1-kidney, 1-wrap model) in which one group of Page (n = 9) and one group of normotensive sham (n = 10) rabbits were given dipyridamole (4.0 mg/kg sc) twice daily for 2 mo. Another group of Page (n = 7) and sham (n = 12) rabbits received vehicle injections. In separate acute studies performed on conscious rabbits, this does of dipyridamole increased myocardial blood flow 35-60% over time without altering transmural distribution of flow or systemic blood pressure. Two months later, minimal coronary vascular resistance (MCVR/100 g) was calculated from perfusion during maximal coronary vasodilation in conscious animals. Histomorphometric methods were then utilized to evaluate various indexes of capillarity in perfuse-fixed hearts. Systolic pressure and left ventricle weight-to-body weight ratios were significantly higher in Page vs. sham rabbits; dipyridamole treatment did not alter these parameters within either group. Similarly, dipyridamole treatment did not significantly alter MCVR/100 g values in either normotensive or hypertensive rabbits. In contrast, dipyridamole treatment increased endomyocardial capillary length density by 33% in the hypertensive group (P less than 0.05) and 11% in the sham group (P not significant) compared with the respective vehicle-treated rabbits. In addition, intercapillary distance was significantly reduced in the endomyocardial region of both groups receiving dipyridamole injections.(ABSTRACT TRUNCATED AT 250 WORDS)


CCK-receptor antagonists attenuate suppression of sham feeding by intestinal nutrients.

To test the possibility that endogenous cholecystokinin (CCK) participates in suppression of sham feeding by intraintestinal nutrient infusions, we examined the effect of CCK-receptor antagonists on the suppression of sham feeding by intraintestinally infused oleic acid, maltose or L-phenylalanine (L-Phe). In addition, we monitored amylase activity in the intestinal lumen during some sham feeding experiments and measured plasma CCK in parallel experiments using intestinally infused animals that were not feeding. Suppression of sham feeding by oleic acid or maltose was attenuated by CCK-receptor antagonists, while suppression of sham feeding by L-Phe was not. Oleate infusion increased plasma CCK concentration and luminal amylase activity. Oleate-induced increase in luminal amylase activity was attenuated by a CCK-receptor antagonist. Intraintestinal maltose or L-Phe did not increase plasma CCK concentration or luminal amylase activity, suggesting that they did not release endocrine CCK. These results suggest 1) that endogenous CCK mediates suppression of sham feeding by oleate and maltose but not by L-Phe and 2) that CCK participating in suppression of feeding by intestinal stimuli might not be of endocrine origin.


Evaluation of radical prostatectomy specimens. A comparative analysis of sampling methods.

We evaluated 104 radical prostatectomies for clinical stage B (n = 93) and stage A (n = 11) prostate cancer. Seven (8%) stage B cases had no gross cancer. By submitting only gross stage B cancer along with standard sections of proximal and distal margins, base of seminal vesicles, and most apical section (next to distal margin), we identified 91% of capsular penetration and 96% of positive margins as compared with identification by complete microscopic examination. Although this method identified 100% capsular penetration and positive margins in stage A cases, 28% of all the cases were grossly normal. Stage A tumor was often difficult to identify because of its heterogeneous location, its gross similarity to nodular hyperplasia, and the confounding presence of post-transurethral resection scarring. In 98% of all stages B and A cases, this method identified to within 1, the Gleason sum of the totally embedded radical prostatectomy. Using this sampling method, key pathologic parameters were identified with an average of 13 blocks (range 7-36) as compared with totally embedding the prostate, using an average of 42 blocks (range 21-81). Based on our study and our understanding of stages A and B disease, we recommend that in grossly normal glands, alternate posterior sections (stage B) and alternate entire sections (stage A) be submitted. Use of this sampling method will achieve a greater uniformity in the processing of specimens and a more accurate pathologic analysis of radical prostatectomy specimens.


Nephrogenic adenofibroma. A novel kidney tumor of young people.

Nephrogenic adenofibroma is a novel kidney tumor of young people (mean age of presentation, 13 years), who present with polycythemia, hypertension, or hematuria, which resolve following nephrectomy. The typical nephrectomy specimen contains a solitary, nonencapsulated, vaguely circumscribed, irregularly shaped or spherical, firm mass with either tan, gray-white, or pale yellow coloration. Cysts are sometimes present within the tumor. The histologic appearance is distinctive and characterized by a marked proliferation of spindled mesenchymal cells resembling the classical type of congenital mesoblastic nephroma, encasing discrete nodules of embryonal epithelium similar to the hyperplastic nephrogenic rests (nephroblastomatosis) usually associated with Wilms' tumor. The mesenchymal component consists of a fascicular proliferation of tightly interlaced, uniform, benign-appearing spindled cells that immunostatin for vimentin and fibronectin, but not desmin or actin. The epithelial component consists of discrete islands of blastemal cells that are partially or fully differentiated toward tubular, tubulopapillary, or papillary structures. Psammoma bodies are plentiful. Embryonal epithelium immunostains for cytokeratin but not epithelial membrane antigen. The overall histologic appearance of the mesenchymal and epithelial components is benign, and preliminary clinical data suggest that the tumor has a benevolent course. Two cases, however, contained small, well-circumscribed papillary lesions near the renal pelvis that resembled low-grade collecting duct carcinoma. The clinical implications of the latter finding are unclear.


Primary vascular tumors of lymph nodes other than Kaposi's sarcoma. Analysis of 39 cases and delineation of two new entities.

Primary vascular tumors of lymph nodes other than Kaposi's sarcoma are very rare, as attested to by only a handful of case reports in the literature. Based on an analysis of 39 such cases, we could distinguish five major groups. Hemangiomas of capillary/cavernous, lobular capillary, and cellular types were composed of compact aggregates of blood-filled vessels, variable in size, that replaced the nodal architecture partly or almost completely; some appeared to have originated in the hilum or medulla. These hemangiomas either represented incidental findings in lymph nodes or were seen with solitary lymph node enlargement; the evolution was benign with no recurrence. A distinctive benign lesion occurring exclusively in inguinal lymph nodes, which we propose designating "angiomyomatous hamartoma," showed replacement of the nodal parenchyma by smooth muscle cells and fibrous tissue, in continuity with exuberant proliferation of muscular vessels in the hilum. Epithelioid vascular tumors, characterized by plump endothelial cells with dense eosinophilic cytoplasm and numerous vacuoles, exhibited a range of differentiation, from hemangioma with well-formed vascular channels (with or without tissue eosinophilia) to hemangioendotheliomas composed predominantly of cords and sheets of tumor cells lying in a hyaline-myxoid matrix. Epithelioid hemangioendothelioma was particularly likely to be mistaken for metastatic carcinoma, and local recurrence could occur. A variant, the spindle and epithelioid hemangioendothelioma, was characterized by the presence of an additional component of spindle cells. Another tumor we found, polymorphous hemangioendothelioma, is a previously uncharacterized borderline malignant vascular tumor exhibiting solid, primitive vascular and angiomatous patterns and relatively bland cytologic features. Lymphangiomas of lymph nodes usually showed simultaneous multifocal and extra-nodal involvement and were characterized by cystic endothelium-lined spaces filled predominantly with lymph fluid. It is important to recognize these primary vascular tumors of lymph nodes to avoid mistaking them for a variety of benign vasoproliferative lesions, Kaposi's sarcoma, angiosarcoma, and metastatic cancer.


T-cell-rich B-cell lymphomas. A clinicopathologic study of 19 cases.

T-cell-rich B-cell lymphomas (TCRBCLs) are recently described, unusual non-Hodgkin's lymphomas that have a diffuse morphology, a predominance of reactive T-cells, and a minority of neoplastic B-cells. The clinical and pathological features of 19 TCRBCLs, all of which demonstrated B-cell clonality, are presented. These lymphomas generally affected older patients by widespread disease and usually were nodal in origin. Treatment varied, but continuous complete remissions (eight patients) were achieved only in those receiving chemotherapy directed at intermediate-grade lymphomas. Although morphologically heterogeneous, all cases resembled peripheral T-cell lymphomas (PTCLs); several TCRBCLs also contained Reed-Sternberg-like cells. Flow cytometry or frozen-section immunoperoxidase failed to detect monotypic immunoglobulin (Ig) in eight of eight cases tested. In contrast, paraffin immunoperoxidase was very useful diagnostically, showing large L26 (CD20-associated) positive cells scattered singly or in small clusters among numerous small T-cells (UCHL1[CD45RO] positive) in all cases. Monotypic cytoplasmic Ig was present in 16 of 19 cases, one of which exhibited plasmacytic differentiation. Southern blot analysis demonstrated relatively faint Ig JH and/or JK bands, indicating a small monoclonal B-cell population in nine of 11 cases, one of which also showed a bcl-2 rearrangement. No T-cell receptor gene rearrangements were observed. These results showed that TCRBCLs may be easily confused with PTCLs or occasionally confused with Hodgkin's disease. TCRBCLs are probably heterogeneous biologically; some cases are of follicular center cell origin. These lymphomas respond to chemotherapy directed at intermediate-grade lymphomas, apparently have a better prognosis than PTCLs, and seem to represent morphological variants of different types of large B-cell lymphomas.


Sclerosing adenosis of the prostate gland. A lesion showing myoepithelial differentiation.

Sclerosing adenosis of the prostate is a rare lesion characterized by the proliferation of variably sized glands in a cellular stroma. We report light microscopic, immunohistochemical, and ultrastructural studies in 22 examples from 15 patients. Two cases were identified in 100 consecutive prostates embedded by a whole organ method, giving a prevalence of 2%. Antibodies directed against the following antigens were used: high-molecular-weight cytokeratin (CKH; 34 beta E12); cytokeratin (CK; AE1/AE3), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), S-100 protein, muscle-specific actin (HHF35), and vimentin (Vim). Cells within the glandular component demonstrated positive reactivity for CK, CHH, PSA, and PAP, indicating a prostatic epithelial origin. In addition, a distinct population of cells reacting for muscle-specific actin and S-100 protein was identified within this glandular element. Adequate material for ultrastructural study was available in five cases; all showed the presence of flattened cells located between the basement membrane and secretory epithelial cells, which had features typical for myoepithelial differentiation. Although the prostate gland does not normally contain myoepithelial cells, we have documented their consistent presence in this unusual lesion; we believe these cells arise by a metaplastic process from the prostatic basal cells.


Follicular neoplasms of the thyroid. Total circumferential evaluation of the fibrous capsule.

Fourteen encapsulated follicular neoplasms were extensively dissected without tangential sectioning to represent the circumference of the entire capsule on sequential histologic sections. A thorough evaluation of these sections divided the 14 cases into five benign adenomas, seven encapsulated carcinomas with only intracapsular angioinvasion, and two minimally invasive carcinomas with focal capsular invasion. Among these nine early-stage follicular carcinomas it was found that angioinvasion occurred multicentrically in at least seven and showed a geographically even distribution. Angioinvasion was found far more often than capsular invasion on the circumference of all nine early-stage carcinomas. Multiple sections produced by this extensive dissection aided the disclosure of minute but convincing findings of angioinvasion. Also, a fibrous capsule as thick as 3.8 mm at its maximum thickness as well as an irregular interface between the capsule and parenchyma were often found to be characteristic of these early-stage carcinomas. Thus, upon comparison with 38 previous cases of similarly localized follicular neoplasms, in which randomly sampled histologic sections yielded diagnoses of benign adenoma (21 cases) and encapsulated or minimally invasive carcinoma (17 cases), extensive circumferential evaluation of the capsule is considered to allow not only effective distinction of follicular neoplasm with slight invasive capability from benign adenoma but also adequate assessment of invasive foci with application of strict criteria. Despite the similarity of clinical prognosis among all the above adenomas and early-stage carcinomas, given the limited follow-up period, an extensive examination method introduced herein is practically useful and necessary for identification of malignancy in encapsulated follicular neoplasms.


Desmoplastic trichilemmoma.

Seven cases of desmoplastic trichilemmoma (DT), a recently described pseudomalignant variant of trichilemmoma, are reviewed. The tumor generally occurs in men after the fifth decade of life and presents as a small solitary nodule on the face. It is frequently misdiagnosed clinically as a basal cell carcinoma or a papilloma. Histologically DT displays a superficial lobular growth arranged about a central prominent desmoplastic stroma. At the periphery, the tumor lobules show the typical features of trichilemmoma. In contrast, at the center the cells assume a more random pattern of cords and strands traversed by the hyaline stroma, mimicking invasive carcinoma. The tumor's architectural pattern, in particular the perilobular hyaline mantle, enables DT to be differentiated from basal cell carcinoma and malignant trichilemmoma. Immunohistochemical analysis failed to demonstrate human papilloma virus (HPV), epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), and alpha-lactalbumin in tumor epithelium. Keratin was expressed by the central pseudoinvasive epithelial cords. Neither factor XIIIa nor keratin expression was found in the stromal cells, which stained only for vimentin. These findings suggest that DT is not an HPV-induced epithelial proliferation and that the stroma is not the result of degenerative changes in tumor epithelium. Instead, there appears to be a fibroblast-mediated, dendrocyte-independent, stromal reaction producing this appearance.


Distribution of cytokeratin polypeptides in syringomas. An immunohistochemical study on paraffin-embedded material.

The distribution of cytokeratin (CK) polypeptides expressed in syringomas (12 cases) was compared with that in normal eccrine sweat ducts using immunohistochemical techniques on paraffin-embedded tissue. Intradermal and intraepidermal segments of the eccrine duct showed reactivity with an antibody to CK1/5/10/11 in all cell layers, whereas CK19 expression was restricted to the luminal cell layer. CK14 was expressed in all cells of the eccrine duct except for the peripheral cells of the intraepidermal duct. Expression of CK5/6 was seen in the basal cells of the dermal duct and of the lower intraepidermal duct (sweat duct ridge) exclusively. Reactivity with an antibody to CK1 was found in the intermediate cells of the uppermost part of the eccrine dermal duct. In addition, this antibody gave a strong staining of the peripheral cells of the intraepidermal duct, leaving basal cells of the sweat duct ridge and luminal cells unstained. In syringoma, CK distribution was essentially comparable with that found in the uppermost part of the dermal duct and in the sweat duct ridge. Namely, ductal luminal cells expressed CK1/5/10/11, CK19, and variably CK14. Intermediate cells of ductal structures and solid nests were homogeneously stained by antibodies to CK1 and CK1/5/10/11, whereas CK14 was expressed heterogeneously. The basal or outermost layer of ductal structures and solid nests was reactive with antibodies to CK1/5/10/11, CK5/6, and CK14. With regard to CK expression, the results indicate that syringoma represents a tumor differentiating toward both the uppermost part of the dermal duct and the lower intraepidermal duct (sweat duct ridge) of the eccrine sweat gland.


Cystic giant solitary trichoepithelioma.

A case of a giant solitary trichoepithelioma is reported. The tumor was located on the thigh, extending from the deep dermis to the subcutaneous tissue with no epidermal contact, and showed a large central cystic cavity that measured 9 cm x 4 cm. We review the cases published under this and other names.


Reversal of streptokinase-induced bleeding with aprotinin for emergency cardiac surgery.

Two patients requiring emergency cardiac surgery following the administration of streptokinase are described. In each case aprotinin was given to counteract the haemorrhagic effects of the streptokinase.


Myelin and myelinization in the telencephalon and mesencephalon of the lizard Gallotia galloti as revealed by the immunohistochemical localization of myelin basic protein.

We have studied in the telencephalon and mesencephalon of the lizard Gallotia galloti the localization and the chronology of appearance of the immunoreactivity due to the presence of a myelin-specific protein: the Myelin Basic Protein (MBP). MBP-like immunoreactivity was present with different degrees of intensity in many nerve fibers (isolated, in tracts and in commissurae) and it was apparently more abundant in mesencephalon. During ontogeny the earliest MBP-like immunoreactivity was detected at E.36 in few tracts in mesencephalon and appeared at E.40 in telencephalon, proceeding caudo-rostrally and from the ventral (basal) to the dorsal (alar) regions. Accumulation of MBP continued after hatching. Oligodendrocyte cell bodies were not immunopositive, not even at the youngest ages studied.


Favorable clinical effects of iloprost infusion in 4 uremic patients with critical limb ischemia.

Four uremic patients with advanced peripheral arterial occlusive disease (PAOD) of lower limbs causing rest pain and ischemic-necrotic lesions were treated with a four-hour intravenous infusion of iloprost at doses of 0.75-2.5 ng/kg/min for twenty-eight days. After a week of the therapy all patients experienced disappearance of rest pain and prolonged walking distance. At the end of the trial a diabetic patient showed a complete regression of the necrotic areas of two toes while the other patients still showed ischemic-necrotic foot lesions that were well demarcated. Iloprost therapy can be effective in uremic patients with severe PAOD.


NIH conference. Insulin-like growth factors in health and disease.

The insulin-like growth factor (IGF) family of peptides, binding proteins, and receptors are ubiquitous and important for normal human growth and development. Modern techniques including specific radioimmunoassays, radioreceptor assays and recombinant DNA technology have improved our understanding of the role of IGFs in growth and development. In addition to enhancing our understanding of normal physiology, these techniques assess changes in these hormones, binding proteins, and receptors in pathologic conditions including growth retardation, acromegaly, malnutrition, diabetes, and malignancy. Further, these studies have led to improvement in the assessment of responses to certain therapies used in the treatment of these diseases and may lead to improvements in these therapies.


An experimental study of prefabricated flaps using silicone sheets, with reference to the vascular patternization process.

A study of the vascularization process in prefabricated flaps, using silicone sheets, has been undertaken in rat models. It was found that the survival rate of flaps was most stable from 2 to 3 weeks after insertion of the silicone sheet, and that this rate decreased when the silicone sheet remained in place beyond that period. The results are probably due to the decrease of delayed effect. Changes that occurred in the circulation and component blood vessels within the flap over time were studied by microangiography, and were categorized at the following four stages: at less than 1 week from the initial surgery, from 1 week but less than 2 weeks, from 2 weeks but less than 4 weeks, and 4 weeks or more. The component vessels consisted of three types, that is, transferred vessels, original vessels, and newly formed vessels, with the last type further divided into three subtypes, to clarify the timing of the vascular proliferation and dilatation for each of the component vessels.


[Neoangiogenesis and human amniotic membrane. A clinico-instrumental study in phlebostatic ulcer of the lower extremities].

The present study was made in order to determine if granulation tissue development after treatment with Human Amniotic Membrane (HAM) is related with instrumental photoplethysmographically evidenced changes of the "ulcus layer". Daily, HAM fragments were disposed along the continuity solution of external patients with opened postthrombotic ulcus cruris. Instrumental examinations were made before, a week after and two weeks after treatment, and after the complete ulcus healing. Ulcus with a slight granulous or fibrogranulous layer developed a granulation tissue within the first week of treatment. On the contrary, in continuity solutions with necrotic layer, treatment with HAM failed. After the first week of treatment, a 84% of ulcus with slight granulation and fibrinogranulation, presented some sphygmic variations respect to baseline levels, like: raising in the amplitude and dicrotism appearing. Within the first week, all of the continuity solutions showed significant sphygmograms with a regular vascularization on the layer, this period of time concurred with the complete granulation phase of the ulcus. Long-preserved HAM stimulates in a significant way the appearing of the granulation tissue, within the first week of treatment. Sphygmic raisings are directly proportional with the amount of granulated tissue evidenced on the layer of the continuity solution.


Mapping of antigenic sites to monoclonal antibodies on the primary structure of the F1-ATPase beta subunit from Escherichia coli: concealed amino-terminal region of the subunit in the F1.

To analyze relationships between the ternary and primary structures of the beta subunit of Escherichia coli F1 ATPase, we prepared two monoclonal antibodies beta 12 and beta 31 against the beta peptide. These antibodies bind to the beta subunit but do not bind to the F1 ATPase, resulting in no inhibition of the ATPase activities. Several different portions of the beta subunit peptide were prepared by constructing expression plasmids carrying the corresponding DNA segment of the beta subunit gene amplified by the polymerase chain reaction. Western blotting analysis using these peptides revealed that the antibodies bound to a peptide of 104 amino acid residues from the amino terminal end, which is outside the previously estimated catalytic domain between residues 140 and 350. These results indicated that the amino terminal portion of the maximal 104 residues is not exposed to the surface of the F1 ATPase. The binding spectrum of the antibodies to the subunit from various species including Vibrio alginolyticus and thermophilic bacterium PS3 indicated possible epitope sequences within the 104 residues. The ternary structure of the beta subunit, in terms of cleavage sites by endopeptidases, was analyzed using the antibodies. A 43-kDa peptide without binding ability to beta 12 and beta 31 appeared upon cleavage by lysyl endopeptidase. The results suggested that lysyl residues from around 70 to 100 from the amino terminus are exposed to the surface of the beta subunit.


In vitro metabolism of FK-506 in rat, rabbit, and human liver microsomes: identification of a major metabolite and of cytochrome P450 3A as the major enzymes responsible for its metabolism.

The metabolism of the immunosuppressant FK-506 was shown to be catalyzed primarily by cytochrome P450 isozymes of the P450 3A subfamily. Antibodies against rat P450 3A inhibited FK-506 metabolism by 82% in rat liver microsomes and by 35-56% in liver microsomes from humans, dexamethasone-induced rats, and erythromycin-induced rabbits. Poor species cross-reactivity of the antibodies, metabolic switching, and/or some metabolism by P450 isozymes other than P450 3A may be responsible for the incomplete inhibition observed. Besides anti-rat P450 3A, antibodies against rat P450 1A also appeared to have some inhibitory effect implicating these particular cytochrome P450 isozymes as having a minor role in FK-506 metabolism. The formation of 13-desmethyl FK-506, identified here as a major metabolite of FK-506 in all types of microsomes examined, was inhibited completely by anti-P450 3A in liver microsomes from dexamethasone-induced rats and erythromycin-induced rabbits but only partially in human and control rat liver microsomes.