Jonathan A. Cohn

Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis

BACKGROUND It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.

Submucosal glands are the predominant site of CFTR expression in the human bronchus

We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regulator (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non–CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with ΔF508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.

Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism

We have established Meloidogyne hapla as a tractable model plant-parasitic nematode amenable to forward and reverse genetics, and we present a complete genome sequence. At 54 Mbp, M. hapla represents not only the smallest nematode genome yet completed, but also the smallest metazoan, and defines a platform to elucidate mechanisms of parasitism by what is the largest uncontrolled group of plant pathogens worldwide. The M. hapla genome encodes significantly fewer genes than does the free-living nematode Caenorhabditis elegans (most notably through a reduction of odorant receptors and other gene families), yet it has acquired horizontally from other kingdoms numerous genes suspected to be involved in adaptations to parasitism. In some cases, amplification and tandem duplication have occurred with genes suspected of being acquired horizontally and involved in parasitism of plants. Although M. hapla and C. elegans diverged >500 million years ago, many developmental and biochemical pathways, including those for dauer formation and RNAi, are conserved. Although overall genome organization is not conserved, there are areas of microsynteny that may suggest a primary biological function in nematodes for those genes in these areas. This sequence and map represent a wealth of biological information on both the nature of nematode parasitism of plants and its evolution.

Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells

BACKGROUND Liver dysfunction is a common manifestation of cystic fibrosis (CF), a disease caused by mutations affecting the CF transmembrane conductance regulator (CFTR). The aim of this study was to examine the distribution and role of CFTR in liver. METHODS CFTR messenger RNA was detected in cryosections of human liver by in situ hybridization. CFTR immunoreactivity was detected using antibodies raised against two CFTR peptides. RESULTS The predominant site of CFTR messenger RNA and immunoreactivity in liver is the intrahepatic bile duct. CFTR is not detected in hepatocytes of normal liver or in livers exhibiting bile duct proliferation. Within bile duct cells, CFTR is localized at or near the apical plasma membrane. CONCLUSIONS The apical localization of CFTR in bile duct cells suggests a model explaining how the CFTR-associated Cl- channel contributes to normal biliary secretion. This model suggests that if CFTR expression could be promoted in intrahepatic duct cells by somatic gene therapy, this might prevent the occurrence of liver disease in CF.

Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.

Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.

Expression of the cystic fibrosis gene in adult human lung.

Critical to an understanding of the pulmonary disease in cystic fibrosis (CF) and the development of effective gene therapies is a definition of the distribution and regulation of CF gene expression in adult human lung. Previous studies have detected the product of the CF gene, the CF transmembrane conductance regulator (CFTR), in submucosal glands of human bronchi. In this report, we have characterized the distribution of CFTR RNA and protein in the distal airway and alveoli of human lungs. Samples from eight human lungs were analyzed for CFTR expression by in situ hybridization and immunocytochemistry. CFTR was detected in a subpopulation of epithelial cells at every level of the distal lung, including proximal, terminal, and respiratory bronchioles, and the alveoli. However, there was substantial variation in the level of CFTR expression between samples. In bronchioles, CFTR protein localized to the apical plasma membrane and was found primarily in a subpopulation of nonciliated cells. CFTR was expressed in the same distribution as the Clara cell marker CC10 in proximal bronchioles, however, expression was discordant in the more distal bronchioles and alveoli where CC10 was not detected. These studies suggest that epithelial cells of the distal lung may play a primary role in the pathogenesis of CF as well as expand the spectrum of target cells that should be considered in the development of gene therapies.

Determinants of HIV-1 shedding in the genital tract of women

BACKGROUND Plasma HIV-1 RNA concentration has been the best predictor for risk of heterosexual and perinatal transmission. However, direct contact with HIV-1 present locally in the genital tract might be necessary for transmission. We aimed to assess the relation between HIV-1 shedding (RNA or culturable virus) in female genital secretions and other factors that might affect HIV-1 shedding. METHODS This was a cross-sectional study within the Womens Interagency HIV Study (WIHS), a prospective longitudinal cohort study of HIV-infected women. We enrolled 311 HIV positive women from Jan 30, 1997 to July 1, 1998. We did clinical assessments, cultured HIV-1, and measured RNA in peripheral blood mononuclear cells (PBMC) and genital secretions. We compared the results with univariate and multivariate analyses. Presence of HIV-1 RNA or culturable virus in genital secretions was defined as HIV-1 shedding. FINDINGS HIV-1 RNA was present in genital secretions of 57% (152/268) of women whereas infectious virus was detected only in 6% (17/271). Genital tract HIV-1 shedding was found in 80% (130/163) of women with detectable plasma RNA and 78% (116/148) of women with positive PBMC cultures. 33% (27/83) of women with less than 500 copies/mL plasma RNA and 39% (35/90) of those with negative PBMC cultures also had genital tract shedding. INTERPRETATION Plasma RNA concentration, both qualitatively and quantitatively, was the most important factor in predicting genital HIV-1 shedding, even among women receiving potent antiretroviral therapy. However, HIV-1 shedding did occur in women with less than 500 copies/mL plasma HIV-1 RNA. This finding suggests that a separate reservoir of HIV-1 replication may exist in some women.

Partial correction of endogenous ΔF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing

Spliceosome-mediated RNA trans-splicing (SMaRT) was investigated as a means for functionally correcting endogenous ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) transcripts using in vitro human cystic fibrosis (CF) polarized airway epithelia and in vivo human CF bronchial xenografts. Recombinant adenovirus (Ad.CFTR-PTM) encoding a pre-therapeutic molecule (PTM) targeted to CFTR intron 9 corrected transepithelial cyclic AMP (cAMP)-sensitive short-circuit current (Isc) in ΔF508 homozygous epithelia to a level 16% of that observed in normal human bronchial epithelia. Molecular analyses using RT-PCR and western blotting confirmed SMaRT-mediated partial correction of endogenous ΔF508 messenger RNA (mRNA) transcripts and protein. In an in vivo model of ΔF508 CF airway epithelia, human CF bronchial xenografts infected with Ad.CFTR-PTM also demonstrated partial correction of CFTR-mediated Cl− permeability at a level 22% of that seen in non-CF xenografts. These results provide functional evidence for SMaRT-mediated repair of mutant endogenous CFTR mRNA in intact polarized CF airway epithelial models.

Correction of Aberrant Splicing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene by Antisense Oligonucleotides

The CFTR splicing mutation 3849 + 10 kb C → T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 + 10 kb C → T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2′-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose- and sequence-dependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders.

Effect of menstrual cycle on HIV-1 levels in the peripheral blood and genital tract.

ObjectiveTo assess the variation in HIV-1 over the menstrual cycle, including RNA levels in the female genital tract, plasma HIV-1-RNA levels, CD4 cell counts, and culturable virus. DesignA prospective analysis of 55 HIV-1-infected women. MethodsBlood and genital tract specimens were collected weekly over 8 weeks, spanning two complete menstrual cycles. Applying repeated-measures models that used menses as the reference level, the variation in viral RNA levels was compared in endocervical canal fluid and cells (collected by Sno-strips and cytobrush, respectively) and ectocervicovaginal lavage (CVL) fluid. Repeated-measures models were also used to assess the variation in plasma CD4 cell counts and viral load. ResultsShedding patterns differed among the three sampling methods, independent of genital tract co-infections. Genital tract HIV-1-RNA levels from CVL fluid and endocervical canal cytobrush specimens were highest during menses and lowest immediately thereafter (P = 0.001 and P = 0.04). The HIV-1-RNA level in endocervical canal fluid was highest in the week preceding menses (P = 0.003). The menstrual cycle had no effect on blood levels of RNA (P = 0.62), culturable virus (P = 0.34), or CD4 cell counts (P = 0.55). HIV-1-RNA levels were higher in endocervical canal fluid than in peripheral blood plasma during the late luteal phase (P = 0.03). ConclusionHIV-1-RNA levels vary with the menstrual cycle in the female genital tract but not the blood compartment. HIV-1-RNA levels are higher in endocervical canal fluid than in blood plasma. These findings may have important implications for sex-specific pathogenesis, heterosexual transmission, and contraceptive hormone interventions in HIV-1-infected women.