Analysis and expansion of the eosinophilic esophagitis transcriptome by RNA sequencing.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder of the esophagus that is compounded by genetic predisposition and hypersensitivity to environmental antigens. Using high-density oligonucleotide expression chips, a disease-specific esophageal transcript signature was identified and was shown to be largely reversible with therapy. In an effort to expand the molecular signature of EoE, we performed RNA sequencing on esophageal biopsies from healthy controls and patients with active EoE and identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated). When clustered by raw expression levels, an abundance of immune cell-specific transcripts are highly induced in EoE but expressed at low (or undetectable) levels in healthy controls. Moreover, 66% of the gene signature identified by RNA sequencing was previously unrecognized in the EoE transcript signature by microarray-based expression profiling and included several long non-coding RNAs (lncRNA), an emerging class of transcriptional regulators. The lncRNA BRAF-activated non-protein coding RNA (BANCR) was upregulated in EoE and induced in interleukin-13 (IL-13)-treated primary esophageal epithelial cells. Repression of BANCR significantly altered the expression of IL-13-induced proinflammatory genes. Together, these data comprise new potential biomarkers of EoE and demonstrate a novel role for lncRNAs in EoE and IL-13-associated responses.

Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylases.

Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene is found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described but very few have been characterized structurally. Here we characterize 3 mutations in the URO-D gene found in patients with F-PCT, G318R, K297N, and D306Y. Expression of the D306Y mutation results in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins display reduced stability in vitro.

Longitudinal study of a mouse model of familial porphyria cutanea tarda.

Most rodent models of porphyria cutanea tarda (PCT) share in common the administration of iron and agents that induce transcription of cytochrome P450s. Dissection of changes related to porphyrin accumulation required generation of a genetic model free from exogenous precipitants. Mice heterozygous for a null Urod mutation and homozygous for null Hfe alleles spontaneously develop major increases in hepatic and urinary porphyrins several months after weaning but the high % uroporphyrin signature of PCT is established earlier, before total hepatic and urinary porphyrins rise. Total porphyrin levels eventually plateau at higher levels in females than in males. Porphyrinogens were the dominant tetrapyrroles accumulating in hepatocytes. Hepatic Urod activity is markedly reduced but total hepatic heme content does not diminish. Microsomal heme, however, is reduced and in vitro metabolism of prototype substrates showed that some but not all cytochrome P450 activities are reduced. High hepatic levels of uroporphyrinogen are also associated with increased glutathione S-transferase activity and elevated mRNA of 2 transporters, Abcc1 and Abcc4. This murine model of familial PCT affords the opportunity to study changes in porphyrinogen and porphyrin accumulation and transport in the absence of exogenous factors that alter P450 activity and transmembrane transporters.

An inherited enzymatic defect in porphyria cutanea tarda: decreased uroporphyrinogen decarboxylase activity.

Uroporphyrinogen decarboxylase activity was measured in liver and erythrocytes of normal subjects and in patients with porphyria cutanea tarda and their relatives. In patients with porphyria cutanea tarda, hepatic uroporphyrinogen decarboxylase activity was significantly reduced (mean 0.43 U/mg protein; range 0.25-0.99) as compared to normal subjects (mean 1.61 U/mg protein; range 1.27-2.42). Erythrocyte uroporphyrinogen decarboxylase was also decreased in patients with porphyria cutanea tarda. The mean erythrocyte enzymatic activity in male patients was 0.23 U/mg Hb (range 0.16-0.30) and in female patients was 0.17 U/mg Hb (range 0.15-0.18) as compared with mean values in normal subjects of 0.38 U/mg Hb (range 0.33-0.45) in men and 0.26 U/mg Hb (range 0.18-0.36) in women. With the erythrocyte assay, multiple examples of decreased uroporphyrinogen decarboxylase activity were detected in members of three families of patients with porphyria cutanea tarda. In two of these families subclinical porphyria was also recognized. The inheritance pattern was consistant with an autosomal dominant trait. The difference in erythrocyte enzymatic activity between men and women was not explained but could have been due to estrogens. This possibility was supported by the observation that men under therapy with estrogens for carcinoma of the prostate had values in the normal female range. It is proposed that porphyria cutanea tarda results from the combination of an inherited defect in uroporphyrinogen decarboxylase and an acquired factor, usually siderosis associated with alcoholic liver disease.

The role of iron in the pathogenesis of porphyria cutanea tarda. II. Inhibition of uroporphyrinogen decarboxylase.

Porphria cutanea tarda is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I and 7-carboxylporphyrins. This pattern of excretion suggest an impaired ability to decarboxylate uroporphyrinogen to the paired ability to decarboxylate uroporphyringen to the 4-carboxyl porphyrinogen, coproporphyrinogen, a reaction catalyzed by the enzyme uroporphyringen decarboxylase. Because clinical evidence has implicated iron in the pathogenesis of porphyria cutanea tarda, these experiments were designed to study the effect of iron on uroporphyrinogen decarboxylase in procine crude liver extracts. Mitochondria-free crude liver extracts were preincubated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyrinogens I and III, the substrates for the decarboxylase assay, were prepared enzymatically from (3H)porphobilinogen. The products of the decarboxylase reaction were identified and quantitated by three methods: (a) extraction into 1.5 N HCl and spectrophotometric quantitation; (b) adsorption onto talc, esterification, paper chromatographic identification, and quantitation by liquid scintillation counting; and (c) adsorption onto talc, esterification, thin-layer chromatographic identification on silica gel, and quantitation by liquid scintillation counting. The thin-layer scinllation method proved most sensitive as it was the only method which accurately identified and quantitated the 7-carboxyl porphyrin reaction product. Uroporphyrinogens I and III were decarboxylated at the same rate by porcine hepatic uroporphyrinogen decarboxylase, and the addition of iron induced marked inhibition of the decarboxylase activity. Ortholpehanthroline blocked the inhibitory effect of iron. The inhibition of uroporphyrinogen decarboxylase by ferrous ion, coupled with its previously reported inhibitory effect on uroporphyrinogen III cosynthetase, provides a possible biochemical explanation for the pattern of urinary porphyrin excretion observed in patients with porphyria cutanea tarda and the clinical association with disordered iron metabolism.

The role of iron in the pathogenesis of porphyria cutanea tarda. An in vitro model.

Porphyria cutanea tarda (PCT) is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I. Clinical evidence has implicated iron in the pathogenesis of PCT. The synthesis of the normally occurring isomer of uroporphyrin, namely uroporphyrin III, from porphobilinogen (PBG) requires two enzymes; uroporphyrinogen I synthetase and uroporphyrinogen III cosynthetase (COSYN). In the absence of COSYN only uroporphyrinogen I is formed. These experiments were designed to study the effect of iron on porphyrin biosynthesis in porcine and human crude liver extracts and to measure COSYN activity in the presence of iron.Mitochondria-free crude liver extracts were prepared in 0.25 m sucrose at pH 7.4 by centrifugation at 37,000 g. Preparations were incubated with either 0.2 mm amino-levulinic acid (ALA) or 0.1 mm PBG. The addition of ferrous ion (either from ferritin iron [4 mug/ml] and cysteine [6.7 mm] or ferrous ammonium sulfate [0.3 mm Fe] and cysteine) significantly increased the rate of uroporphyrin synthesis from either ALA or PBG. The predominant porphyrin synthesized in the presence of ferrous ion was uroporphyrin I whereas coproporphyrin III predominated in its absence. Orthophenanthroline blocked these effects of ferrous ion.To investigate the effect of ferrous ion on COSYN, crude liver extracts were incubated with ferrous ammonium sulfate (0.3 mm Fe) and cysteine (6.7 mm) and the COSYN activity of the incubates was assayed directly. In both porcine and human extracts ferrous ion caused marked inhibition of COSYN activity. Orthophenanthroline blocked the inhibitory effect.Inactivation of COSYN by heating resulted in marked enhancement of porphyrin synthesis from PBG. The sole product was uroporphyrin I.Thus, inactivation of COSYN results in accelerated synthesis of uroporphyrin I. This effect of ferrous ion provides a possible biochemical explanation for the excess production and excretion of uroporphyrin I in patients with PCT and the reversal of this defect by phlebotomy.

Biosynthesis of 5-aminolevulinic acid and heme from 4,5-dioxovalerate in the rat.

We previously demonstrated an alternate pathway for the biosynthesis of 5-aminolevulinic acid (ALA) in bovine liver mitochondria and of tetrapyrroles in suspensions of rat hepatocytes (1980. J. Biol. Chem. 255: 3742; 1981. Proc. Natl. Acad. Sci. USA. 78: 5335). This pathway involves a transamination reaction that incorporates the intact 5-carbon skeleton of 4,5-dioxovaleric acid (DOVA) into ALA. We investigated this alternate pathway in vivo by the intraperitoneal injection of DOVA into rats. Incorporation of DOVA and [5-14C]DOVA into urinary ALA and hepatic and erythroid heme was quantified and compared with the incorporation of [4-14C]ALA and [2-14C]glycine into heme. Within 3 h of injection of 175 mumol of DOVA, urinary ALA excretion increased 2.4-fold over controls. After injection of [5-14C]DOVA, 0.11% of the radioactivity was recovered as urinary ALA, which quantitatively accounted for the 2.4-fold increase in ALA excretion. After the injection 175 mumol of [5-14C]DOVA, 0.14% of the radioactivity was recovered after 3 h as hepatic heme. The injection of 1.75 mmol of [2-14C]glycine or 175 mumol of [4-14C]ALA resulted in recovery of 0.2 and 3.4%, respectively, of the radioactivity as hepatic heme after 3 h. These doses of radiolabeled DOVA, glycine, and ALA were injected into rats with phenylhydrazine-induced anemia. Recovery of radioactivity after 3 h as splenic (erythroid) heme was 0.35% for DOVA, 0.072% for glycine, and 0.25% for ALA. These studies establish that the intact 5-carbon skeleton of DOVA can be incorporated into ALA and heme in vivo.

Uroporphyrinogen decarboxylase: a splice site mutation causes the deletion of exon 6 in multiple families with porphyria cutanea tarda.

Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree.

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.

An interferon-gamma, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10- and 8-fold increase [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-15N2]arginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, high-output L-arginine/NO pathway exists in humans.

The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid.

Biosynthesis of 5-aminolevulinic acid in mammalian cells is catalyzed by aminolevulinic acid synthase in a condensation reaction utilizing glycine and succinyl X coenzyme A. An alternate pathway in mammalian cells may involve the biosynthesis of aminolevulinic acid via a transamination reaction in which L-alanine is the amino donor and 4,5-dioxovaleric acid is the acceptor. This transamination reaction, or one very similar, is employed by plants for the biosynthesis of aminolevulinic acid which is ultimately converted to chlorophyll. The effect of glyoxalase I on the diversion of dioxovaleric acid to other products was tested using both purified glyoxalase I and crude tissue homogenates. Glyoxalase I is a metalloenzyme and glutathione is a co-substrate. Purified glyoxalase I reduced the amount of aminolevulinic acid formed in the presence of dioxovaleric acid, L-alanine, glutathione, and purified L-alanine: 4,5-dioxovaleric acid aminotransferase (dioxovalerate transaminase). The conversion of dioxovaleric acid to aminolevulinic acid was inhibited by the addition of glutathione when a dialyzed bovine liver homogenate served as the source of both glyoxalase I and dioxovalerate transaminase. Removal of metals from bovine liver homogenates produced an 85% decrease in glyoxalase I activity. These 'metal-free' homogenates still affected the conversion of dioxovaleric acid to aminolevulinic acid after preincubation with MgSO4. The effect of glyoxalase I on the metabolism of dioxovaleric acid was also studied using a fluorometric enzyme assay for the quantification of dioxovaleric acid via a coupled enzyme reaction converting it to uroporphyrin. Homogenates of both liver and barley diminished the amount of dioxovaleric acid detected by the coupled assay, but this effect could be prevented by dialysis of the homogenates. Addition of glutathione to dialyzed homogenates markedly reduced the amount of uroporphyrin generated from dioxovaleric acid. Metal-free homogenates supplemented with glutathione reduced the conversion of dioxovaleric acid to uroporphyrin in the coupled assay, but preincubation with MgSO4 greatly augmented this effect. These studies point out the difficulty in evaluating dioxovaleric acid as a heme precursor using whole cell homogenates.

Mutational analysis of human uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase (URO-D), a heme biosynthetic enzyme, catalyzes the multi-step decarboxylation reaction converting uroporphyrinogen I or III to coproporphyrinogen I or III. The URO-D protein has been purified from several sources and its gene has been cloned from many organisms. In spite of this, little is known about the active site(s) of the enzyme. Inhibitor studies suggest that cysteine and histidine residues are important for enzyme activity. We employed the Kunkel method of site-directed mutagenesis to convert each of the six cysteines in human URO-D to serine and each of the three conserved histidines to asparagine. Recombinant mutant URO-D's were expressed in Escherichia coli, partially purified, and their kinetic properties compared to recombinant wild-type URO-D. All cysteine mutants retained approx. 40% wild-type enzyme activity, indicating that no single cysteine is absolutely critical for the integrity of the catalytic site. The three histidine mutants also retained significant enzyme activity and one, (H339N), displayed unique properties. The H339N mutation resulted in an enzyme with high residual activity but decarboxylation of intermediate reaction products of the I isomer series was markedly abnormal. The histidine at residue 339 is likely important in imparting isomer specificity.

Thyroid disease in hemochromatosis. Increased incidence in homozygous men.

The thyroid function of 49 patients homozygous for the hemochromatosis allele was studied by measurement of serum thyroxine and thyrotropin concentrations. Of 34 homozygous men, three were found to be hypothyroid (thyroxine, less than 3.0 micrograms/dL and thyrotropin, greater than 40 ImU/mL) and one was hyperthyroid (thyroxine, 24 micrograms/dL). All 15 homozygous women had normal thyroid function. The hypothyroid patients had elevated titers of antithyroid antibodies. Histologic examination of the thyroid at autopsy of one hypothyroid patient showed notable iron accumulation and fibrosis with modest lymphocytic infiltration. The causative importance of iron deposition in thyroid diseases associated with hemochromatosis was suggested by the reversal of the usual sex ratio of thyroid dysfunction. Men with hemochromatosis had a much greater iron load than women, and they also had a surprisingly higher incidence of thyroid disease. Iron may have caused injury to the thyroid, followed by the development of antithyroid antibodies and hypothyroidism. The frequency of thyroid disorders in men with hemochromatosis is about 80 times that of men in the general population.

Porphyria cutanea tarda associated with disinfectant misuse.

Porphyria cutanea tarda was detected in a 44-year-old janitress. The illness was probably caused by the unintentional synthesis of a polychlorinated phenol as a result of mixing commonly available household ingredients in toilet bowls and shower stalls. Although the evidence for this hypothesis is circumstantial, its likelihood and the wide-spread household use of these reagents justify calling attention to the innovative misuse of disinfectants as a potential source of toxic exposure.

Characterization and crystallization of human uroporphyrinogen decarboxylase.

The cytosolic enzyme uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO-D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 microM. URO-D containing an amino-terminal histidine tag was crystallized in space group P3(1)21 or its enantiomer P3(2)21 with unit cell dimensions a = b = 103.6 A, c = 75.2 A. There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator. Native data have been collected to 3.0 a resolution.

Screening for hemochromatosis: phenotype versus genotype.

Hereditary hemochromatosis is one of the most common inherited disorders among Caucasians of European ancestry. Malregulation of iron absorption from the duodenum eventually leads to iron overload. Although the time required to become iron loaded is variable, it is clear that most homozygotes will eventually become symptomatic. The clinical manifestations can be prevented by prophylactic phlebotomy therapy. Screening young populations is therefore a key to the prevention of disease-related morbidity. Protocols based on the phenotype of high transferrin saturation already exist. The recent identification of a candidate gene for hemochromatosis now allows for a potential genetic screen. Both the phenotypic and the genotypic methods of screening have inherent advantages and disadvantages. Iron-depletion therapy of homozygotes before the development of disease-related morbidity results in normal longevity. National initiatives for hemochromatosis screening will prevent morbidity by identifying and treating young, healthy homozygotes. Healthy, iron-depleted homozygotes should be eligible for health and life insurance at standard rates. Furthermore, healthy homozygotes would make ideal blood donors.

Fulminant amebic meningoencephalitis due to Acanthamoeba.

A 57-year-old chronically immunosuppressed woman with systemic lupus erythematosus developed fulminant meningoencephalitis due to Acanthamoeba castellanii. Amebic trophozoites were also found in the lungs, suggesting a primary pulmonary focus of infection. This case illustrates that Acanthamoeba can cause a fulminant, rapidly fatal meningoencephalitis, as well as the previously reported chronic granulomatous meningoencephalitis. X

CYP3A-inducing agents and the attenuation of uroporphyrin accumulation and excretion in a rat model of porphyria cutanea tarda.

An experimental model of porphyria cutanea tarda (PCT) can be achieved in 3 weeks by a single injection of a mixture of polychlorinated biphenyls (Aroclor 1254) into iron-loaded female Fischer 344 rats maintained continuously on delta-aminolevulinic acid-supplemented drinking water. In this model, daily treatment with 5-pregnen-3 beta-ol-20-one-16 alpha-carbonitrile (pregnenolone 16 alpha-carbonitrile) attenuated uroporphyrin and heptacarboxylporphyrin accumulation and excretion by 75%. Pregnenolone 16 alpha-carbonitrile treatment had only a minor effect on hepatic iron stores, and it had no effect on the induction of CYP1A activities by Aroclor 1254. In the absence of Aroclor 1254, pregnenolone 16 alpha-carbonitrile had no effect on the accumulation and excretion of highly carboxylated porphyrins. Attenuation of porphyrin accumulation could also be demonstrated with daily troleandomycin treatment. Troleandomycin increased CYP3A-dependent erythromycin demethylase activity, but to a lesser extent than pregnenolone 16 alpha-carbonitrile. Much of the CYP3A induced by troleandomycin was sequestered as a catalytically inactive metabolic-intermediate complex. In the absence of Aroclor 1254, troleandomycin had no effect on the accumulation and excretion of highly carboxylated porphyrins, nor did troleandomycin alter the induction of CYP1A by Aroclor 1254. The results suggest that the major attenuation of hepatic accumulation and urinary excretion of uro- and heptacarboxylporphyrins in the rat PCT model by pregnenolone 16 alpha-carbonitrile and troleandomycin is due to an enhancement of CYP3A catalytic activity.

Successful treatment of a cardiac angiosarcoma with combined modality therapy.

The patient was a 34-year-old man with a primary angiosarcoma of the heart. Initial admission was for cardiac tamponade. He was treated with preoperative chemotherapy and radiation therapy and then underwent orthotopic heart transplantation. The patient had an uneventful postoperative recovery, received two postoperative regimens of chemotherapy, and, at 33 months after transplantation, had no evidence of recurrence or metastasis. We propose that a more aggressive management of these patients with difficult conditions is warranted.

Hereditary hemochromatosis. Analysis of laboratory expression of the disease by genotype in 18 pedigrees.

Tight linkage between the hemochromatosis locus and the HLA region permits determination of genotype in members of hemochromatosis pedigrees. To determine if simple laboratory measures of iron metabolism could predict the affected genotype without the need for HLA typing, we studied seven measures of iron metabolism: serum iron concentration, total iron-binding capacity, per cent saturation of transferrin, serum ferritin concentration, deferoxamine-induced urinary iron excretion and hepatic iron concentration evaluated by both chemical and histological methods. Discriminant analysis showed a per cent saturation of transferrin above 62% to be the best simply-measured indicator of the affected genotype: homozygosity is accurately predicted in 92% of the cases. The logarithmic transform of serum ferritin concentration was only 71% accurate. Pedigree analysis estimated the frequency of the hemochromatosis gene at 0.069 +/- 0.020 with a recombination probability of 0.015 +/- 0.015 with the HLA region. This corresponds to a heterozygote frequency of 0.13 and a disease frequency of 0.005.

Comparison of stainable liver iron between symptomatic and asymptomatic hemochromatosis homozygotes and their homozygous relatives.

The authors compared the amount of hepatic parenchymal cell stainable iron in 159 hemochromatosis homozygotes. They were separated into four groups. Group 1: 59 symptomatic hemochromatosis probands with hemochromatosis (mean age 49 years) who were identified because of symptoms and signs of iron overload. Group 2: 38 asymptomatic probands with hemochromatosis (mean age 29 years) identified during population screening studies or during routine health maintenance evaluation. Group 3: 47 homozygous relatives (mean age 43 years) of Group 1 probands. Group 4: 15 homozygous relatives (mean age 30 years) of Group 2 probands. The symptomatic probands (Group 1) were 20 years older and had much more stainable hepatic iron (p less than 0.0001) than the asymptomatic probands (Group 2). The homozygous relatives (Group 3) of the symptomatic probands also were older and had much more stainable hepatic iron than the homozygous relatives (Group 4) of asymptomatic probands (p less than 0.0002). The results of this study suggest that population screening studies can result in early identification of individuals with hemochromatosis before massive hepatic iron overload occurs and before symptoms of iron overload develop.