Cystic Fibrosis Gene Research Articles

Expression and maturation of Sendai virus vector-derived CFTR protein: functional and biochemical evidence using a GFP-CFTR fusion protein

Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.

Activation of CFTR-specific T Cells in Cystic Fibrosis Mice Following Gene Transfer

Gene therapy for cystic fibrosis (CF) airway disease has emerged as a potentially successful therapy, because expression of the CF gene would be expected to restore the electrophysiological function of the airway epithelium to normalcy. Although, cellular and humoral immune responses to viral gene transfer vectors have been studied extensively, there has been no evaluation of T cell-mediated responses to the therapeutic human CF gene product. Using an adenovirus vector we demonstrated that T cells against human CF gene protein are elicited in CF gene knockout (KO), heterozygote (Het), and wild-type (wt) mice. A dominant CD8 T cell epitope found in CF gene KO, Het, and wt mice was mapped to NTYLRYITV. In CF gene KO mice we also identified (to a conserved region of the CF gene CSQFSWIMPGTIKEN), a minor T cell epitope that did not show any activity in the Het or wt mice.

Integration of Inositol Phosphate Signaling Pathways via Human ITPK1

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.

United Kingdom Cystic Fibrosis Gene Therapy Consortium Multidose Trial: The Proposed Use of Computed Tomography

Interest in the role of computed tomography (CT) as a monitoring tool in cystic fibrosis (CF) research has gained momentum in the past 5 years. This is a consequence of the rapid development of therapeutic options for CF lung disease and hence the need for sensitive outcome measures that can be applied to clinical intervention trials. The following article summarizes the proposed use of CT in a forthcoming multidose gene therapy trial. Issues surrounding dose, timing of scans, scoring, and quantification of CT morphologic features are discussed. The literature which supports the use of CT as a surrogate outcome measure is also summarized.

NOVEL CFTR MISSENSE MUTATIONS IN BRAZILIAN PATIENTS WITH CONGENITAL ABSENCE OF VAS DEFERENS: COUNSELING ISSUES.

Screening for mutations in the entire Cystic Fibrosis gene (CFTR) of Brazilian infertile men with congenital absence of vas deferens, in order to prevent transmission of CFTR mutations to offspring with the use of assisted reproductive technologies. Specific polymerase chain reaction (PCR) primers were designed to each of the 27 exons and splicing sites of interest followed by single strand conformational polymorphism and Heteroduplex Analysis (SSCP-HA) in precast 12.5% polyacrylamide gels at 7 masculineC and 20 masculineC. Fragments with abnormal SSCP migration pattern were sequenced. Two novel missense mutations (S753R and G149W) were found in three patients (two brothers) together with the IVS8-5T allele in hetrozygosis. The available screenings for CF mutations do not include the atypical mutations associated to absence of vas deferens and thus, when these tests fail to find mutations, there is still a genetic risk of affected children with the help of assisted reproduction. We recommend the screening of the whole CFTR gene for these infertile couples, as part of the work-up before assisted reproduction.

Design, Development, Validation, and Use of Synthetic Nucleic Acid Controls for Diagnostic Purposes and Application to Cystic Fibrosis Testing

We have designed, tested, and validated synthetic DNA molecules that may be used as reference standard controls in the simultaneous detection of mutations in one or more genes. These controls consist of a mixture of oligonucleotides (100 to 120 bases long) each designed for the detection of one or more disease-causing mutation(s), depending on the proximity of the mutations to one another. Each control molecule is identical to 80 to 100 bases that span the targeted mutations. In addition, each oligonucleotide is tagged at the 5' and 3' ends with distinct nucleic acid sequences that allow for the design of complementary primers for polymerase chain reaction amplification. We designed the tags to amplify control molecules comprising 32 CFTR mutations, including the American College of Medical Genetics minimum carrier screening panel of 23, with one pair of primers in a single tube. We tested the performance of these controls on many platforms including the Applied Biosystems/Celera oligonucleotide ligation assay and the Tm Bioscience Tag-It platforms. All 32 mutations were detected consistently. This simple methodology allows for maximum flexibility and rapid implementation. It has not escaped our notice that the design of these molecules makes possible the production of similar controls for virtually any mutation or sequence of interest.

Liquid movement across the surface epithelium of large airways

The cystic fibrosis transmembrane conductance regulator CFTR gene is found on chromosome 7 [Kerem, B., Rommens, J.M., Buchanan, J.A., Markiewicz, D., Cox, T.K., Chakravarti, A., Buchwald, M., Tsui, L.C., 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073-1080; Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L., et al., 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073] and encodes for a 1480 amino acid protein which is present in the plasma membrane of epithelial cells [Anderson, M.P., Sheppard, D.N., Berger, H.A., Welsh, M.J., 1992. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263, L1-L14]. This protein appears to have many functions, but a unifying theme is that it acts as a protein kinase C- and cyclic AMP-regulated Cl(-) channel [Winpenny, J.P., McAlroy, H.L., Gray, M.A., Argent, B.E., 1995. Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. Am. J. Physiol. 268, C823-C828; Jia, Y., Mathews, C.J., Hanrahan, J.W., 1997. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272, 4978-4984]. In the superficial epithelium of the conducting airways, CFTR is involved in Cl(-) secretion [Boucher, R.C., 2003. Regulation of airway surface liquid volume by human airway epithelia. Pflugers Arch. 445, 495-498] and also acts as a regulator of the epithelial Na(+) channel (ENaC) and hence Na(+) absorption [Boucher, R.C., Stutts, M.J., Knowles, M.R., Cantley, L., Gatzy, J.T., 1986. Na(+) transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J. Clin. Invest. 78, 1245-1252; Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., Rossier, B.C., Boucher, R.C., 1995. CFTR as a cAMP-dependent regulator of sodium channels. Science 269, 847-850]. In this chapter, we will discuss the regulation of these two ion channels, and how they can influence liquid movement across the superficial airway epithelium.

Interesting Updates About the Cystic Fibrosis Gene

It is known that mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause congenital absence of the vas deferens

Analysis of Cystic Fibrosis Gene Mutations and Associated Haplotypes in the Croatian Population

The aim of this study was to reveal the CFTR gene mutation status in the Croatian population as well as to establish the haplotypes associated with cystic fibrosis (CF) and those associated with specific gene mutations. A total of 48 unrelated CF patients from Croatia were examined. Among 96 tested alleles, we found nine different mutations: DeltaF508, 58.33%; G542X, 3.12%; N1303K, 2.08%; R1162X; 621 + 1G --> T; G85E; Y569C; E585X; and S466X, 1.04%. Analysis of three polymorphic loci revealed 15 different haplotypes. Two of them (21-23-13 and 21-17-13) occurred with a higher frequency (40% and 24%). Both of these haplotypes also carried a CFTR gene mutation (DeltaF508 or G542X) on 27 out of 32 chromosomes. Among 12 (of all together 29) CF alleles on which no mutations were found, we detected 10 different haplotypes. Because there are still no published data on the distribution of polymorphic loci in Croatia, nor haplotypes associated with mutations in the CFTR gene, our results greatly contribute to knowledge regarding the genetic background of CF in this region.

Oral 13, An unexplained disorder of keratinization

A 6-year-old boy initially presented at the age of 1 week with perineal and perianal rash. The rash was persistent and at the age of 8 months he developed gastroenteritis. He was found to have borderline zinc deficiency. However, there was no improvement with zinc supplementation and the rash progressed to well-demarcated psoriasiform plaques involving the perianal area, penis, scrotum, upper thighs, face, axillae, right elbow and neck. He also developed palmoplantar keratoderma. Initially the hair was sparse and brittle. He developed recurrent chest and skin infections. There was a family history of malignant hyperpyrexia and mitochondrial myopathy. Skin biopsy was nonspecific, showing chronic superficial dermatitis. Analysis of skin and blood for recognized keratin and connexin disorders did not show any abnormalities. Gastroenterology investigations revealed oesophagitis, gastritis and patchy pancolitis. Routine blood tests were normal. Lactate levels, acylcarnitine profile, organic acids, tubulopathy screen, glycosaminoglycans, biotinidase, betamethylcarboxyl CoA carboxylase, holocarboxylase synthetase, prolidase and tyrosine aminotransferase levels were normal. Autoimmune screen and immunological investigations were normal. Blood tests for coeliac disease were negative. Electromyography and DNA analysis for mitochondrial myopathy were normal. Sweat studies and cystic fibrosis gene analysis were normal. DNA analysis for malignant hyperpyrexia was negative. His skin condition gradually improved on treatment with acitretin 10 mg three times a week and topical emollients. He is still troubled by recurrent infections. His gastroenteritis is well controlled by an oligoantigenic diet. We are grateful to Professor John Harper and Professor David Kelsall for their help in managing this child.

Genomic Context Vectors and Artificial Chromosomes for Cystic Fibrosis Gene Therapy

Cystic fibrosis (CF) is caused by mutations of the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP dependent chloride channel whose expression is finely tuned in space and time. Gene therapy approaches to CF lung disease have demonstrated partial efficacy and short-lived CFTR expression in the airways. Drawbacks in the use of classical gene transfer vectors include immune response to viral proteins or to unmethylated CpG motifs contained in bacterially-derived vector DNA, and shut-off of viral promoters. These limitations could be overcome by providing stable maintenance and expression of the CFTR gene inside the defective cells. This strategy makes use of large fragments of DNA of various sizes containing the CFTR transgene and its relevant regulatory regions, (genomic context vectors [GCVs], reaching ultimate complexity in the form of an artificial chromosome [AC]) as vector for the transgene. Appropriate regulation in space and time would be achieved by the presence of the endogenous promoter and other control elements, while retention in daughter cells could be ensured by the presence of sequences which guarantee episomal replication. In this review, we describe recent advances in GCVs and ACs and the technology underlying their construction. These vectors have been shown to be suitable for delivery and expression of therapeutically relevant genes, including CFTR. The major issue which now limits their routine use is delivery inefficiency. Once this issue is resolved, we will be closer to achieving the goal of regulated gene therapy for CF.

Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways

Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.

The inexhaustible mouse nose. Focus on “Olfactory epithelia exhibit progressive functional and morphological defects in CF mice”

the ability to eliminate any gene in mice and the increasing ability to identify specific genes responsible for human single-gene diseases is providing a cornucopia of biological information on the relation of genotype to phenotype. Tracing the pleiotropic effects of genes is greatly facilitated

Emerging drug treatments for cystic fibrosis

Although the gene for cystic fibrosis was discovered in 1989, a definitive cure remains elusive for this deadly orphan disease. Advances in nutritional rehabilitation, antibiotics, mucolytics and delivery of care have improved survival to a median age of 37.5 years; however, the psychosocial, personal and financial burdens of this lifelong chronic illness remain considerable. The current portfolio of investigational therapeutics is explored here and placed in a context of therapeutic target and predicted clinical benefit. Partnership between large and small pharma, the Cystic Fibrosis Foundation and academia should be fostered to accelerate therapeutic development.

Extracellular autocrine nucleotide signalling in a microenvironment: integrative physiology in a minute volume of airway surface liquid

Before the cystic fibrosis (CF) gene was cloned and the topology of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was mapped, there were multiple schools of thought and possible answers for what functional protein the CF gene encoded: dysfunctional chloride (Cl−) channel?; hyperactive sodium (Na+) channel?; and/or signal transduction effector and regulator of both Cl− and Na+ channels? The answer was a member of the ATP-binding cassette (ABC) transporter family that acted as an anion channel and as a regulator of multiple ion, acid–base and water transport proteins and cellular processes, including functioning as an inhibitor of Na+ channels. Loss of CFTR not only leads to loss of Cl− permeability and transport across epithelial cell membranes but it also to up-regulation of ENaC Na+ channels via mechanisms that remain debated, poorly defined, and likely multifactorial. Some have suggested that CFTR may be a signal transduction effector molecule itself. Its latter regulatory and signalling effector capability may be at the root of the CF genotype and lung and airways CF disease phenotype discordance observed to date in the CF condition. In an article in this issue of The Journal of Physiology, Button et al. (2007) have set the bar in both longevity and innovation in their quest to define the ion transport processes and their regulation within a tiny volume of extracellular fluid that bathes beating respiratory cilia, the so-called airway surface liquid (ASL) layer. Including this article, the UNC CF research group has published at least 11 original articles and reviews focused on aspects of the ASL and its regulation in the past several years (Lazarowski et al. 2004; Okada et al. 2004, 2006; Douillet et al. 2005; Tarran et al. 2005, 2006a,b; Matsui et al. 2006; Winters et al. 2006; Boucher, 2007; Button et al. 2007). Their power derives from the well-differentiated non-CF (‘normal’) and CF human airway epithelial cell monolayers that they grow in primary cultures to maintain cilia and a pseudo-stratified architecture. With this in vivo-like culture model, they have brought innovative methods as well as emerging mathematical modelling to these questions. Such a system is elegantly presented in an initial model figure by Button et al. (2007). To be brief, their collective work in this paper and as a whole over the past 5 years is summed by the cartoon provided in Fig. 1. It is the over-arching hypothesis of this group that secreted nucleotides and nucleosides within the ASL are key to local control of anion and cation channels and to the control of the volume of the ASL. Up-regulation of ATP secretion may be critical to the normalization of ASL depth. The ASL may be one of the most tightly regulated microenvironments in the body. With these well-differentiated non-CF and CF cultures, this paper and past papers before it showed that ASL in normal physiology is maintained at a depth of 7 μm, the approximate length of the beating respiratory cilia. In CF, however, the ASL volume is depleted to more than 50% of that depth, causing a ciliary beat and mucociliary clearance problem. When different mechanical stimuli are applied to this system (phasic motion, cyclic stress, chest percussion therapy?, exercise?), one can deepen the ASL in the normal model (not shown in Fig. 1). However and importantly for future therapeutic angles for CF, CF ASL volume is restored with these mechanical stimuli, some of which simulate normal breathing and some of which are designed to perturb the system and loosen the dehydrated and tenacious mucus. They propose and show that the accumulation of secreted ATP and its metabolities (principally adenosine) reset the normal regulation of ASL volume by rescuing Cl− and water secretion, while dampening Na+ hyperabsorption. The rescue of Cl− and anion secretion is independent of CFTR, because it remains trapped within the cell. An assumption is that water channel function remains intact in CF and follows osmotic forces within the ASL and the airway surface epithelium. Indeed, secreted ATP is also critical for regulation of cell volume within the columnar airway epithelial cells as well. Aquaporin biology is difficult to study in vitro, because expression of epithelial aquaporins is often lost in cell culture for reasons unknown. Figure 1 Cartoon depicting control of airway surface liquid via the integrative function of anion, cation and water transport and the local release of nucleotides and nucleosides with cyclic stress, phasic motion and other appropriate mechanostimuli Boucher and coworkers should be congratulated on this work and lauded for their comprehensive and innovative approach in this arena. This collective work is another example of how local signalling by purinergic ligands can never be overlooked as a critical cog in the minute-to-minute regulation of membrane, cell and tissue biology.

GREEN AND MURRAY RESPOND

We thank Ross for suggesting a potential improvement in newborn screening (NBS) for cystic fibrosis (CF) by adding a second-tier enzyme-linked immunosorbent assay for pancreatic-associated protein for those samples screening positive by immunoreactive trypsinogen (IRT).1 The addition of pancreatic-associated protein may improve the specificity of screening, perhaps bypassing some of the difficulties that we note with secondary DNA-based CF screening as it is currently practiced.2 However, the limitations of DNA-based screening by assay of only a minority of the known mutations in the CF gene can be better addressed by increasing the number of mutations analyzed. Accurate genotyping will be useful in confirming disease detection and could provide genotype-phenotype predictions of disease course and related clinical utility,3 such as for clinically less severe variants of galactosemia (i.e., the Duarte mutation) or CF (e.g., the R117H mutation). Even as protein assays progress, NBS will increasingly use DNA as a tool for the identification and treatment of children affected by CF and other disorders. Therefore, programs will be best served by improving both protein- and DNA-based screening strategies, making them more accurate, predictive, and inclusive for the diverse population of neonates. Incorporating new technologies to improve NBS and decrease health disparities remains an essential element of providing optimal public health service. Some disorders may require primary DNA-based screening. For example, a screening assay has recently been reported for severe combined immune deficiency (SCID).4,5 While we had raised questions about NBS for SCID because treatment requires bone marrow cell transplantation,2 recent evidence suggests that transplant with banked cord blood or partially matched donor hematopoietic stem cells for treatment—and cure—of SCID can be made widely available6,7 and may be cost effective.8 Such evidence reminds us that NBS should remain open to new opportunities for screening and treatment. Finally, we emphatically echo Ross’s call that NBS should help to redress, not exacerbate, health care disparities. The screening component of NBS is as close to a universal health service as exists in the United States. We must also assure that all children identified by NBS receive the care they need. The hodgepodge of eligibility criteria and funding streams that characterize health care in the United States makes this a difficult challenge, the solution to which will be found in policy, not technology.

Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors

Limited packaging capacity has hampered adeno-associated virus (AAV)-mediated gene therapy for many common genetic diseases such as cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). Trans-splicing AAV (tsAAV) vectors double AAV packaging capacity but their transduction efficiency has been too low to be useful. We have recently overcome this hurdle by rational vector design. We have shown that a pair of optimized mini-dystrophin tsAAV vectors can reach the same transduction efficiency as that of a single AAV vector after local injection in dystrophic muscle. However, global gene transfer is required to treat diseases like DMD. To test whether systemic delivery can be achieved with tsAAV vectors, we generated a set of optimized alkaline phosphatase (AP) tsAAV vectors. We delivered AAV serotype 9 pseudotyped AP tsAAV intravenously to newborn mice. Six weeks later, we observed high-level transduction in all body skeletal muscle and the heart, the tissues that are affected in DMD. We also detected efficient transduction in the lung, the primary organ affected in CF. Our results provide the first evidence of whole-body transduction with tsAAV vectors and further raise the hope of tsAAV gene therapy for DMD and CF.

Optimizing Aerosol Gene Delivery and Expression in the Ovine Lung

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

Erratum: Gene delivery to human sweat glands: a model for cystic fibrosis gene therapy

Erratum: Gene delivery to human sweat glands: a model for cystic fibrosis gene therapy

What are the useful biological and functional markers of early-stage chronic pancreatitis?

Early-stage chronic pancreatitis may be undetected as a clinical entity. However, it may carry a definite risk for subsequent secondary damage, depending on the etiology of the disease. Therefore, the most important question is whether indeed the patient in question does have early-stage chronic pancreatitis rather than oligosymptomatic advanced-stage chronic pancreatitis. This can be easily determined by appropriate imaging such as abdominal computed tomography. For early changes, endoscopic ultrasound is superior to any other technique. Endosonography may also tell about anatomical obstacles (e.g., papillary stenosis, pancreas divisum) that may be treated to prevent progression of the disease. Treatment options at this stage are endoscopic for the most part. Depending on the etiology and familiar/hereditary background of the given patient, one must look further into molecular markers. Such markers may give an estimate on the progression or dynamics of the disease in the future and include mutations in the cationic (PRSS1) and anionic (PRSS2) trypsinogen genes as well as mutations in the serine protease (SPINK1) or cystic fibrosis (CFTR) genes. Admitted ly, these are not markers of early-stage chronic pancreatitis but must be investigated if and when such pathogenesis is suspected. Further, rare forms of chronic pancreatitis, such as autoimmune pancreatitis, which can be cured by appropriate medical treatment with steroids, must be excluded. Markers for autoimmune pancreatitis are elevated serum IgG, especially IgG4, and autoantibodies to carbonic anhydrase (type II) and lactoferrin. It is noteworthy that these markers, present in almost every Asian patient with autoimmune pancreatitis, are mostly lacking in Caucasian populations of patients with autoimmune pancreatitis.