Augmentation Therapy Modulates Systemic Inflammation in Individuals with Alpha-1 Antitrypsin Deficiency and Chronic Obstructive Pulmonary Disease.

Background: Alpha-1 antitrypsin (AAT) deficiency is a genetic disorder that leads to chronic obstructive pulmonary disease (COPD) and lower circulating levels of AAT, which is a protease inhibitor with potent anti-inflammatory effects. In order to better understand the presence of systemic inflammation in AAT-deficient individuals with COPD, we investigatedthe plasma levels of C-reactive protein (CRP). Methods: AAT-deficient individuals and a matched cohort with a normal AAT genotype were recruited from the Alpha-1 Foundation DNA and Tissue Bank. AAT genotypes were determined by a combination of a Taqman-based assay. AAT and CRP levels were determined by nephelometry. Comparisons were determined by unpaired t-test and standard Pearson's correlation. Results: Our study included 40 control participants and 742 AAT-deficient participants, of which 498 received augmentation therapy. In the AAT-deficient participants, the plasma AAT was 20.2±11.6µM and 4.5±1.3µM (P<0.0001) with and without augmentation therapy, respectively, and the CRP was 0.32±0.53mg/dL and 0.69±1.97mg/dL (P=0.0169), respectively. There was a negative correlation between the percentage predicted of forced expiratory volume in 1 second and CRP in the group not receiving augmentation therapy (r=-0.2528, P<0.05), and there was no correlation in participants receiving augmentation therapy. Conclusion: Compared to healthy individuals, AAT-deficient individuals with COPD have higher levels of circulating CRP, suggesting increased systemic inflammation. However, AAT-deficient individuals receiving augmentation therapy had lower plasma CRP levels compared to those who are not.

Intradermal alpha1-antitrypsin therapy avoids fatal anaphylaxis, prevents type 1 diabetes and reverses hyperglycaemia in the NOD mouse model of the disease.

AIMS/HYPOTHESIS: Human alpha1-antitrypsin (hAAT) gene therapy prevents type 1 diabetes in a NOD mouse model of diabetes. However, repeated i.p. injections of hAAT into NOD mice leads to fatal anaphylaxis. The aim of the study was to determine if an alternative route of administration avoids anaphylaxis and allows evaluation of hAAT's potential for diabetes prevention and reversal. We also sought to determine if the addition of granulocyte colony-stimulating factor (G-CSF), augments hAAT's capacity to prevent or reverse disease in the NOD mice. METHODS: To evaluate hAAT pharmacokinetics, serum hAAT levels were monitored in NOD mice receiving a single dose (2 mg) of hAAT by i.p., s.c. or i.d. injection. For studies of type 1 diabetes prevention and reversal, mice received i.d. hAAT (2 mg/mouse/3 days) for 8 or 10 weeks or hAAT and G-CSF (i.p., 6 microg/day) for 6 weeks. Blood glucose determinations, glucose tolerance testing and insulin tolerance tests were performed. RESULTS: Both i.p. and s.c. injections resulted in fatal anaphylaxis. The i.d. injection avoided anaphylaxis and i.d. injection of hAAT into 11-week-old NOD mice prevented disease (p = 0.005, AAT vs PBS at 40 weeks of age). Treatment of diabetic NOD mice with hAAT or hAAT plus G-CSF provided long-term (at least 100 days) reversal of diabetes in 50% of treated animals. G-CSF did not enhance the reversal rates of hAAT. Glucose tolerance and insulin levels were normalised in mice with hAAT prevention and reversal. CONCLUSIONS/INTERPRETATION: Intradermal hAAT prevents and reverses disease in a NOD mouse model of type 1 diabetes without inducing anaphylaxis.

Development of predictive models for airflow obstruction in alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin deficiency is a genetic condition associated with severe, early-onset chronic obstructive pulmonary disease (COPD). However, there is significant variability in lung function impairment among persons with the protease inhibitor ZZ genotype. Early identification of persons at highest risk of developing lung disease could be beneficial in guiding monitoring and treatment decisions. Using a multicenter, family-based study sample (2002-2005) of 372 persons with the protease inhibitor ZZ genotype, the authors developed prediction models for forced expiratory volume in 1 second (FEV(1)) and the presence of severe COPD using demographic, clinical, and genetic variables. Half of the data sample was used for model development, and the other half was used for model validation. In the training sample, variables found to be predictive of both FEV(1) and severe COPD were age, sex, pack-years of smoking, bronchodilator responsiveness, chronic bronchitis symptoms, and index case status. In the validation sample, the predictive model for FEV(1) explained 50% of the variance in FEV(1), and the model for severe COPD exhibited excellent discrimination (c statistic = 0.88).

Heritability of lung function in severe alpha-1 antitrypsin deficiency.

Severe alpha-1 antitrypsin (AAT) deficiency is a proven genetic risk factor for COPD, but there is marked variation in the development of COPD among AAT deficient subjects. To investigate familial aggregation of lung function in subjects with AAT deficiency, we estimated heritability for forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) in 378 AAT deficient subjects from 167 families in the AAT Genetic Modifiers Study; all subjects were verified homozygous for the Z AAT deficiency allele. Heritability was evaluated for models that included and excluded an ascertainment correction, as well as for models that excluded, included and were stratified by a cigarette smoking covariate. In models without an ascertainment correction, and in all models without a covariate for smoking, no evidence for familial aggregation of lung function was observed. In models conditioned on the index proband with covariates for smoking, post-bronchodilator FEV1/FVC demonstrated significant heritability (0.26 +/- 0.14, p = 0.03). When we limited the analysis to subjects with a smoking history, post-bronchodilator FEV1 demonstrated significant heritability (0.47 +/- 0.21, p = 0.02). Severity rate phenotypes were also assessed as potential phenotypes for genetic modifier studies. Significant heritability was found with all age-of-onset threshold models that included smoking and ascertainment adjustments. Using the t-distribution, the heritability estimates ranged from 0.43 to 0.64, depending on the age-of-onset of FEV1 decline used for the severity rate calculation. Correction for ascertainment and consideration of gene-by-smoking interactions will be crucial for the identification of genes that may modify susceptibility for COPD in families with AAT deficiency.

Human alpha 1-antitrypsin therapy induces fatal anaphylaxis in non-obese diabetic mice.

Previous studies have shown that human alpha-1 antitrypsin (hAAT) gene delivery prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. Furthermore, hAAT protein administration prolongs acceptance of islet allografts. Therefore, we evaluated the use of purified hAAT protein therapy to prevent T1D in NOD mice. Female NOD, non-obese resistant (NOR), Balb/c and C57BL/6 mice were injected intraperitoneally with vehicle alone or vehicle containing hAAT, human albumin or mouse albumin (or mg/injection/mouse; 2x/week). Preparations of clinical-grade hAAT included API(R), Aralast, Prolastin and Zemaira. Surprisingly, hAAT administration was associated with a high rate of fatal anaphylaxis. In studies seeking T1D prevention at 4 weeks of age, 100% mice died after six injections of hAAT. When administrated at 8-10 weeks of age, most (80-100%) NOD mice died following the fourth injection of hAAT, while 0% of Balb/c and C57BL/6 mice and 10% of NOR mice died. Interestingly, repeated injections of human albumin, but not mouse albumin, also induced sudden death in NOD mice. Antibodies to hAAT were induced 2-3 weeks after hAAT administration and death was prevented by treatment with anti-platelet-activating factor along with anti-histamine. In studies of disease reversal in NOD mice, using the four pharmaceutical grade formulations of hAAT, anaphylactic deaths were observed with all hAAT preparations. The propensity for fatal anaphylaxis following antigenic administration appears to be NOD- but not hAAT-specific. The susceptibility of NOD mice to hypersensitivity provides a significant limitation for testing of hAAT. Development of strategies to avoid this unwanted response is required to use this promising therapeutic agent for T1D.

Alpha-1-antitrypsin aerosolised augmentation abrogates neutrophil elastase-induced expression of cathepsin B and matrix metalloprotease 2 in vivo and in vitro.

BACKGROUND: Neutrophil elastase (NE) activity is increased in lung diseases such as alpha(1)-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy. METHODS: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT. RESULTS: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy. CONCLUSION: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo.

Prevalence of alpha-1 antitrypsin deficiency in poorly controlled asthma--results from the ALA-ACRC low-dose theophylline trial.

In a study comparing low-dose theophylline to montelukast in poorly controlled asthmatics, 285 subjects consented to be screened for alpha-1 antitrypsin deficiency. Of the 284 for which complete data was available, 10.5% carried a deficiency gene and 2.4% were mildly deficient with an alpha-1 antitrypsin serum level of less than 20 mu M. In the non-African-American cohort, an abnormal phenotype occurred in 12% and 2.9% were mildly deficient. Baseline pulmonary function and asthma scores were not significantly different between those with normal and abnormal AAT phenotype. However those with the deficiency tended to show a greater bronchodilator response.

Pulmonary dysfunction in adults with nephropathic cystinosis.

OBJECTIVE: To characterize the pulmonary dysfunction in patients with nephropathic cystinosis after renal transplantation. DESIGN: Cross-sectional analysis of consecutive adult patients. PATIENTS: Twelve adult, nephropathic cystinosis patients and 3 adult, ocular, nonnephropathic cystinosis patients admitted to the National Institutes of Health Clinical Center. RESULTS: The 12 nephropathic cystinosis patients (age range, 21 to 40 years) showed an extraparenchymal pattern of restrictive lung disease, with inspiratory and expiratory dysfunction. Specifically, the mean FVC was 58% of predicted, the mean FEV(1) was 57% of predicted, and the mean total lung capacity was 66% of predicted, while the mean residual volume was normal. Furthermore, the mean maximal inspiratory pressure for the eight patients tested was 40% of predicted, and the mean maximal expiratory pressure was 26% of predicted. Two patients died of respiratory insufficiency. All the patients had lived at least 17 years, while lacking compliant cystine-depleting therapy with oral cysteamine. Seven patients had a conical chest, restricting excursion, and 10 of the 12 patients had evidence of the myopathy that typifies late cystinosis. In fact, the severity of pulmonary disease correlated directly with the severity of myopathy in our group of 12 patients. In contrast, the lung parenchyma was essentially normal, as gauged by chest radiographs and CT scans of the lung. The three patients with nonnephropathic cystinosis displayed entirely normal pulmonary function. CONCLUSION: The distal myopathy characteristic of nephropathic cystinosis results in an extraparenchymal pattern of restrictive lung disease in adults who have not received long-term cystine depletion. Whether or not oral cysteamine therapy can prevent this complication remains to be determined.

Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients.

The conducting airways are the primary target for gene transfer in cystic fibrosis (CF), yet the inflammation associated with CF lung disease could potentially pose a significant barrier to gene transfer vectors, such as recombinant adeno-associated virus (rAAV). In order to investigate this possibility, aliquots of bronchoalveolar lavage (BAL) fluid from eight individuals with CF were tested for their in vitro inhibitory effects on rAAV transduction, along with BAL from non-CF individuals. While the non-CF BAL fluid was not inhibitory, seven of eight CF BAL samples had significant inhibitory activity, resulting in a five- to 20-fold reduction in transduction events. Inhibition of rAAV transduction by CF BAL could be reversed by alpha-1-antitrypsin (AAT), but not by DNase. When neutrophil elastase and neutrophil alpha defensins (human neutrophil peptides, HNP) were measured in these samples, they were elevated by 500- and 10,000-fold, respectively. The levels of HNP correlated inversely with the amount of rAAV transduction. Furthermore, rAAV transduction could be blocked by purified HNP in an AAT-reversible manner at HNP concentrations within the range measured in these fluids. We conclude that products of inflammation in CF BAL fluid are inhibitory to rAAV transduction, and that these effects may be reversible by AAT.

Formation and current results of a patient-organized registry for alpha(1)-antitrypsin deficiency.

BACKGROUND: Significant challenges exist to investigating uncommon illnesses because too few patients are seen at any single clinical center to permit appropriate research studies. Recognizing this impediment to clinical research in alpha(1)-antitrypsin deficiency, the Alpha One Foundation, a patient-organized research foundation, has collaborated with clinician-scientists to organize a voluntary registry of individuals with alpha(1)-antitrypsin deficiency. PURPOSE: To facilitate clinical research in alpha(1)-antitrypsin deficiency by organizing a registry of affected individuals willing to be approached to participate in clinical studies. METHODS: Elements of the Alpha One Foundation Research Network Registry include a Medical and Scientific Advisory Committee, composed of physician-investigators and patient advocates, designated clinical resource centers at medical institutions with expertise in the management of individuals with alpha(1)-antitrypsin deficiency, and a data coordinating center with responsibility for database management and analysis. Questionnaires requesting information about demographic features, alpha(1)-antitrypsin phenotype, smoking history, and health-care utilization were distributed to prospective registrants through the following channels: mailings from the Alpha One Foundation; mailings from the clinical resource centers; and distribution by home-care and pharmaceutical companies. Information from this questionnaire formed the basis of the initial registry database. RESULTS: Between May 1997 and June 1999, 7,789 forms were distributed, and forms were returned by 712 unique registrants. Registrants have the following characteristics: mean (+/- SD) age, 49.3+/-13.2 years; women, 47.7%; white, 96.2%; PI*ZZ phenotype, 70.7%; ex-smokers, 73.3%; COPD patients, 87.2% (emphysema patients, 54.2%; chronic bronchitis patients, 33%); and self-reported liver disease, 6.4%. The mean number of physician visits reported by registrants in the preceding 12 months was 7.8+/-9.4, 59% reported currently receiving IV augmentation therapy, and 35% reported using supplemental oxygen at home. Examples of ongoing research studies using this unique database include: (1) a case-control study to evaluate occupational risk factors for obstructive lung disease in individuals with alpha(1)-antitrypsin deficiency and (2) a study to evaluate the health-care costs for affected individuals. CONCLUSIONS: A registry currently including 712 individuals with alpha(1)-antitrypsin deficiency has been organized through a collaboration between physician-investigators and a patient-organized research foundation. Use of the registry has already facilitated studies that were previously difficult because of the paucity of identifiable study subjects. The registry cohort promises to provide an important resource for future clinical and epidemiologic studies.

Molecular mechanisms of alpha1-antitrypsin null alleles.

Alpha1-antitrypsin (alpha1-AT) is the most abundant circulating inhibitor of serine proteases and therefore is essential to normal protease-anti-protease homeostasis. Inheritance of two parental alpha1-AT deficiency alleles is associated with a substantially increased risk for development of emphysema and liver disease. In very rare circumstances individuals may inherit alpha1-AT null alleles. Null alpha1-AT alleles are characterized by the total absence of serum alpha1-AT. These alleles represent the extreme end in a continuum of alleles associated with alpha1-AT deficiency. The molecular mechanisms responsible for absence of serum alpha1-AT include splicing abnormalities, deletion of alpha1-AT coding exons and premature stop codons. While these alleles comprise only a small proportion of alpha1-AT alleles associated with profound alpha1-AT deficiency, studies of their molecular mechanisms provide valuable insights into the structure, gene expression and intracellular transport of alpha1-AT.

Pulmonary function and high-resolution CT findings in patients with an inherited form of pulmonary fibrosis, Hermansky-Pudlak syndrome, due to mutations in HPS-1.

OBJECTIVE: To describe and correlate pulmonary function and high-resolution CT (HRCT) scan scores in individuals with a high risk for development of pulmonary fibrosis, ie, Hermansky-Pudlak syndrome (HPS) patients with mutations in the HPS-1 gene. DESIGN: Cross-sectional analysis of consecutive, eligible patients. PATIENTS: Thirty-eight HPS inpatients at the National Institutes of Health Clinical Center with HPS-1 mutations. RESULTS: Thirty-seven patients were Puerto Rican and exhibited the typical 16-base pair (bp) duplication in exon 15 of HPS-1. One non-Puerto Rican was homozygous for a different mutation (intervening sequence 17 -2 A-->C) previously reported in the HPS-1 gene; he died at age 35 of pulmonary insufficiency. For the 23 patients who had pulmonary symptoms, the mean age of onset was 35 years. For all 38 patients (mean age, 37 +/- 2 years), the mean FVC was 71% of predicted; the mean FEV(1), 76%; mean total lung capacity (TLC), 72%; mean vital capacity (VC), 68%; and mean diffusing capacity of the lung for carbon monoxide (DLCO), 72%. When patients were grouped according to the extent of their reduction in FVC, the other four pulmonary function parameters followed the FVC. Seventeen patients had abnormal chest radiographs, and 31 (82%) had abnormal HRCT scans of the chest, for which a scoring system of 0 (normal) to 3 (severe fibrosis) is presented. The mean +/- SEM HRCT score for 38 patients was 1.30 +/- 0.17. HRCT scores correlated inversely with FVC and DLCO. CONCLUSIONS: Mutations in the HPS-1 gene, whether or not they involve the typical 16-bp duplication seen in Puerto Rican patients, are associated with fatal pulmonary fibrosis. In affected patients, the FVC, FEV(1), TLC, VC, and DLCO fall in concert, and this functional deficit correlates with HRCT scan evidence of progression of interstitial lung disease.

Endothelial nitric oxide synthase as a potential susceptibility gene in the pathogenesis of emphysema in alpha1-antitrypsin deficiency.

A role for endothelial nitric oxide synthase (NOS3) in the susceptibility of individuals with alpha1-antitrypsin (alpha1AT) deficiency to destructive lung disease was evaluated. Six polymorphic sites were identified within the NOS3 gene (i.e., -924A/G, -788C/T, -691C/T, 774C/T, 894G/T, and 1998C/G). The genotype distribution was determined in 339 patients and 94 control individuals. Frequency of the 774T allele in severely affected individuals was 0.417 versus 0.269 in control subjects (P = 0.018), whereas the 894T allele frequency was 0.427 versus 0.280 in control subjects (P = 0.024). Patients with less severe lung disease had the 774T and 894T allele frequencies of 0.289 and 0.344, respectively, similar to frequencies in a control group (P > 0.3). No direct correlation between pulmonary function and five other NOS3 polymorphisms was observed. Thus, functional allelic variants that are in linkage disequilibrium with the 774C/T and 894G/T may be present in the specified genomic area. These data are consistent with a modulatory role for NOS3 in destructive lung disease associated with alpha1AT deficiency.

Efficient in vivo catheter-based pericardial gene transfer mediated by adenoviral vectors.

Adenoviral vectors are promising agents for a number of in vivo gene therapy applications including diseases of the heart and coronary vessels. Efficient intravascular gene transfer to specific sites has been achieved in occluded vessels, but otherwise is hampered by the effect of blood flow on localized vector uptake in the vessel wall. An alternative delivery approach to coronary arteries is the expression of diffusible gene products into the pericardial space surrounding the heart and coronary arteries. However, in vivo pericardial access is comparatively difficult and has been limited to surgical approaches. We hypothesized that efficient adenovirus-mediated gene expression in pericardial lining mesothelium could be achieved by transmyocardial vector delivery to the pericardium. To evaluate this concept, a hollow, helical-tipped penetrating catheter was used to deliver vector-containing fluid directly into the intrapericardial space. The catheter was introduced percutaneously in anesthetized mongrel dogs, advanced into the right ventricle, and the tip passed through the apical right ventricular myocardium under direct radiographic visualization until the open end of the catheter tip resided in the intrapericardial space. Adenoviral vectors expressing either nuclear-localizing beta-galactosidase, cytoplasmic luciferase, or secreted human alpha 1AT reporters (Av1nBg, Av1Lu, or Av1Aa, respectively) were instilled through the catheter into the intrapericardial space. Three days later the animals were sacrificed and reporter gene expression was evaluated in pericardium, epicardium, and multiple other tissues. In animals receiving Av1nBg, beta-galactosidase activity was evident in most of the pericardial lining endothelium, up to 100% in many areas. In animals receiving Av1Lu, luciferase reporter activity was abundant in pericardial tissues, but near-background levels were observed in other organs. In animals receiving Av1Aa, human alpha 1AT was abundant (16-29 mg/ml) in pericardial fluid, but was undetectable in serum. All animals tolerated the procedure well with no electrocardiographic changes and no clinical sequelae. These observations demonstrate highly efficient adenovirus vector delivery and gene transfer and expression in the pericardium and support the feasibility of localized gene therapy via catheter-based pericardial approaches. We suggest that the pericardial sac may serve as a sustained-release protein delivery system for the generation of desired gene products or their metabolites for diffusion into the epicardial region.

Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors.

Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-alpha promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 x 10(13) particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 micrograms/ml in SCID and over 400 micrograms/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.

Alpha 1-antitrypsin nonsense mutation associated with a retained truncated protein and reduced mRNA.

alpha 1-Antitrypsin (alpha 1AT) provides the major protection in the lung against neutrophil elastase-mediated proteolysis. Inheritance of alpha 1AT deficiency alleles is associated with an increased risk of emphysema and liver disease. alpha 1AT null alleles cause the total absence of serum alpha 1AT and represent the ultimate in a continuum of alleles associated with alpha 1AT deficiency. The molecular mechanisms responsible for absence of serum alpha 1AT include splicing abnormalities, deletion of alpha 1AT coding exons, and premature stop codons. We identified an Italian individual with asthma, emphysema, and a very low level of serum alpha 1AT. DNA sequencing demonstrated the Mprocida deficiency allele and a novel null allele, QOtrastevere (c654 G-->A, W194Z), a nonsense mutation near the intron 2 (IVS2) splice acceptor site. To determine the molecular basis of QOtrastevere and specifically to evaluate whether this nonsense mutation interfered with mRNA processing by altered splicing, we used a Chinese hamster ovary cell line permanently transfected with QOtrastevere or normal M alpha 1AT with and without IVS2. Northern blot analysis demonstrated that the normal M construct, with or without IVS2, expressed alpha 1AT mRNA of a similar size. The nonsense mutation was associated with moderately reduced alpha 1AT mRNA regardless of the presence or absence of IVS2. Reduction in alpha 1AT mRNA regardless of the opportunity for splicing supports a translational-translocation model as the cause of reduced alpha 1AT mRNA rather than the nuclear scanning model. Pulse-chase studies followed by immunoprecipitation demonstrated an endoplasmic reticulum-retained 31 kDa QOtrastevere alpha 1AT, which was rapidly degraded. Although mRNA content was moderately reduced, retention and rapid intracellular degradation of the truncated form are the major mechanisms for the absence of secreted alpha 1AT.

Inhibition of intracellular degradation increases secretion of a mutant form of alpha1-antitrypsin associated with profound deficiency.

The mutant Z form of alpha1-antitrypsin (alpha1AT) is responsible for > 95% of all individuals with alpha1AT deficiency, an important inherited cause of emphysema and liver disease. Since secreted Z alpha1AT is a functional antiprotease, we hypothesized that interrupting catabolism of retained Z alpha1AT might increase its transport out of cells, causing an increase in extracellular protease protection. Both the protein translation inhibitor cycloheximide and the specific inhibitor of proteasome function, lactacystin, prevented intracellular degradation of Z alpha1AT. Moreover, this inhibition of degradation was associated with partial restoration of Z alpha1AT vesicular transport. This effect was observed in a model system of transfected CHO cells as well as in human alveolar macrophages synthesizing Z alpha1AT. This study supports the hypothesis that altering the intracellular fate of a mutant protein may be an option in the treatment of diseases associated with misfolded but potentially functional proteins.

Genetic defects and clinical characteristics of patients with a form of oculocutaneous albinism (Hermansky-Pudlak syndrome).

BACKGROUND: Hermansky-Pudlak syndrome is characterized by oculocutaneous albinism, a storage-pool deficiency, and lysosomal accumulation of ceroid lipofuscin, which causes pulmonary fibrosis and granulomatous colitis in some cases. All identified affected patients in northwest Puerto Rico are homozygous for a 16-bp duplication in exon 15 of a recently cloned gene, HPS. We compared the clinical and laboratory characteristics of these patients with those of patients without the 16-bp duplication. METHODS: Forty-nine patients -- 27 Puerto Ricans and 22 patients from the mainland United States who were not of Puerto Rican descent -- were given a diagnosis on the basis of albinism and the absence of platelet dense bodies. We used the polymerase chain reaction to determine which patients carried the 16-bp duplication. RESULTS: Twenty-five of the Puerto Rican patients were homozygous for the 16-bp duplication, whereas none of the non-Puerto Rican patients carried this mutation. Like the patients without the duplication, the patients with the 16-bp duplication had a broad variation in pigmentation. Nine of 16 adults with the duplication, but none of the 10 without it, had a diffusing capacity for carbon monoxide that was less than 80 percent of the predicted value. High-resolution computed tomography in 12 patients with the 16-bp duplication revealed minimal fibrosis in 8, moderate fibrosis in 1, severe fibrosis in 1, and no fibrosis in 2. Computed tomography in eight patients without the duplication revealed minimal fibrosis in three and no fibrosis in the rest. Inflammatory bowel disease developed in eight patients (four in each group) between 3 and 25 years of age. CONCLUSIONS: The 16-bp duplication in exon 15 of HPS, which we found only in Puerto Rican patients, is associated with a broad range of pigmentation and an increased risk of restrictive lung disease in adults.