Research - Alpha One International Registry

Maurizio Luisetti, MD

Past Research Accomplishments

Search for genetic risk factors for COPD and bronchiectasis (CFTR gene, Serpin genes, TNF gene complex) and sarcoidosis (HLA and ACE genes)
Development of a novel method (capillary electrophoresis) to detect elastin breakdown products in urine of patients with obstructive lung disease
Development of synthetic inhibitors of human neutrophil elastase

Ongoing Research Activies

Study of a novel complement gene and TNF receptor genes in sarcoidosis
Development of a novel technique (Laser Induced Fluorescence - Capillary Electrophoresis) to improve the detection of elastin breakdown products in urine of subjects with obstructive lung disease
Funcional interactions of AAT with other human alveolar proteins

Planned Research Activities

Sib pair analysis for gene scanning in COPD
Gene expression in animal models of airspace enlargement

Eur Respir J 2003 Mar;21(3):444-9

Tumour necrosis factor family genes in a phenotype of COPD associated with
emphysema.

Ferrarotti I, Zorzetto M, Beccaria M, Gile LS, Porta R, Ambrosino N, Pignatti
PF, Cerveri I, Pozzi E, Luisetti M.

Dept of Respiratory Diseases, IRCCS San Matteo Hospital, University of Pavia,
Pavia, Italy.

Genetic factors are believed to play a role in the individual susceptibility to
chronic obstructive pulmonary disease (COPD). Tumour necrosis factor (TNF)
family genes have been widely investigated but inconsistent results may lie
either in the genetic heterogeneity of populations or in the poor phenotype
definition. A genetic study was performed using a narrower phenotype of COPD.
The authors studied 86 healthy smokers and 63 COPD subjects who were enrolled
based on irreversible airflow obstruction (forced expiratory volume in one
second/forced vital capacity <70% predicted) and a diffusing capacity for carbon
monoxide <50% predicted (moderate-to-severe COPD associated with pulmonary
emphysema). The following polymorphisms were investigated: TNF-308, the
biallelic polymorphism located in the first intron of the lymphotoxin-alpha
gene, and exon 1 and exon 6 of the TNF receptor 1 and 2 genes, respectively. No
significant deviations were found concerning the four polymorphisms studied
between the two populations. The authors confirm that the tumour necrosis factor
family genes, at least for the polymorphisms investigated, are not major genetic
risk factors for chronic obstructive pulmonary disease in Caucasians, either
defined in terms of emphysema (this study) or airflow obstruction (previous
studies). Nevertheless, the authors would like to emphasise the importance of
narrowing the phenotype in the search for genetic risk factors in chronic
obstructive pulmonary disease.

Eur Respir J 2002 Oct;20(4):1050-6

Alpha1-antitrypsin deficiency: a report from the 2nd meeting of the Alpha One
International Registry, Rapallo (Genoa, Italy), 2001.

Luisetti M, Miravitlles M, Stockley RA.

Dept of Respiratory Diseases, IRCCS San Matteo, Pavia, Italy.
m.luisetti@smatteo.pv.it

The Alpha One International Registry is a scientific foundation established to
comply with a World Health Organization recommendation to develop a
multinational registry of alpha1-antitrypsin deficiency, with the aim of
creating a common database of subjects recognised in a standardised way. A
commitment of the Alpha One International Registry members, belonging to 15
national registries, is to meet every 2 yrs in an open scientific conference to
provide a scientific and clinical update on the deficiency. The second Alpha One
International Registry meeting was held in Rapallo (Genoa, Italy) on September
27th-28th, 2001, and 26 speakers provided an exhaustive overview of all aspects
of alpha1-antitrypsin deficiency, including epidemiology, genetics,
biochemistry, associated conditions, established and novel therapeutic options,
and markers of efficacy. In the framework of a rare and often under-recognised
condition, this meeting is likely to be central to improving understanding and
increasing awareness of alpha1-antitrypsin deficiency.

Publication Types:
Congresses

Eur Respir J 2002 Jun;19(6):1128-35

Surfactant apoprotein A modulates interleukin-8 and monocyte chemotactic
peptide-1 production.

Meloni F, Alberti A, Bulgheroni A, Lupi A, Paschetto E, Marone Bianco A, Rodi G,
Fietta A, Luisetti M, Baritussio A.

Dept of Haematological, Pneumological and Cardiovascular Sciences, University of
of Pavia and Instituto di Ricovero e Cura a Carattere Scientifico Policlinico
San Matteo, Italy. federica-meloni@libero.it

Previous studies have shown that surfactant apoprotein A (SP-A) and natural or
synthetic surfactant can modulate the release of pro-inflammatory cytokines from
alveolar mononuclear phagocytes. The aim of this study was to assess whether
SP-A or Surfactant (Surf) from patients with pulmonary alveolar proteinosis
(PAP) can affect the release of two chemokines (interleukin (IL)-8 and monocyte
chemtactic peptide (MCP)-1) from human monocytes and rat lung type-II cells. In
addition IL-8 and MCP-1 levels were assessed in the brochoalveolar lavage fluid
(BALF) of seven patients with PAP and compared with those in a group of control
subjects (n=5). SP-A, tested over a wide range of concentrations, significantly
increased IL-8 and MCP-1 release from monocytes. SP-A retained its activity
after collagenase digestion, but was not active after heat treatment. The
release of IL-8 by monocytes was also stimulated by Surf. Finally, median BALF
IL-8 and MCP-1 levels in PAP patients were significantly higher than in controls
(9.50 and 9.51 pg x mL(-1) in controls versus 151.95 and 563.70 pg x mL(-1) in
PAP, respectively) and significantly correlated with SP-A concentrations in
BALF. Overall the results of this study support the view that the high content
of alveolar surfactant apoprotein A may contribute to the upregulation of
chemokine release in pulmonary alveolar proteinosis, thus contributing to airway
inflammation.

Sarcoidosis Vasc Diffuse Lung Dis 2002 Jun;19(2):83-95

HLA and sarcoidosis: new pathogenetic insights.

Martinetti M, Luisetti M, Cuccia M.

Servizio di Immunoematologia e Transfusione e Centro di Immunologia dei
Trapianti, IRCCS Policlinico S. Matteo, Pavia, Italy. m.martinetti@smatteo.pv.it

Many theories have been presented to account for the immunological and
epidemiological features of sarcoidosis; several lines of study support the
prevailing opinion that an environmental agent, possibly microbial in origin,
may cause sarcoidosis in a genetically predisposed host. Many polymorphic genes
have been suggested to contribute to this genetic susceptibility: genes encoding
angiotensin converting enzyme, vitamin D receptor, and interleukin-1, T-cell
receptor genes, Gm and Km immunoglobulin genes and, most relevant, HLA genes
(classical and non classical). There is also some evidence of an HLA-associated
protection against sarcoidosis. The main action of disease-associated HLA
molecules is to present specific antigenic peptides in such a way that the
recognizing T-lymphocytes initiate an inflammatory response with peculiar
pathological consequences. Other, so-called, non-classical HLA genes coding for
proteins involved in antigen processing and presentation, namely TAP, LMP and
DM, seem to contribute. Particular alleles of the tumor necrosis factor gene
cluster (TNFA, LTA, LTB) are known to be associated with peculiar clinical forms
of sarcoidosis. For instance, Lofgren's syndrome, which is an acute form of
pulmonary sarcoidosis with frequent spontaneous remission, is marked by the
TNFA*2, HLA-DR3 haplotype. How many HLA genes are involved is still unknown, but
it is now clear that the HLA region is strongly implicated in the development of
sarcoidosis. Probably, the future lies in isolating and sequencing the putative
peptide bound to susceptible MHC molecules which, activating reactive T-cells,
is responsible for disease initiation and/or exacerbation. However, the
investigative approach should not be confined only to genomic sequences: the
temporal and spatial expression of gene products, the post-transcriptional
modification of the protein products will be fundamental in determining the
basic functional context of developing sarcoidosis.

Publication Types:
Review
Review, Tutorial

Am J Respir Cell Mol Biol 2002 Jul;27(1):17-23

Complement receptor 1 gene polymorphisms in sarcoidosis.

Zorzetto M, Bombieri C, Ferrarotti I, Medaglia S, Agostini C, Tinelli C, Malerba
G, Carrabino N, Beretta A, Casali L, Pozzi E, Pignatti PF, Semenzato G, Cuccia
MC, Luisetti M.

Laboratorio di Biochimica e Genetica, Clinica di Malattie dell'Apparato
Respiratorio, Pavia, Italy.

Sarcoidosis is likely to result from exposure of genetically susceptible hosts
to environmental agents. Erythrocyte (E) complement receptor 1 (CR1) is a
membrane protein mediating the transport of immune complexes (ICs) to
phagocytes, and at least three polymorphisms on the CR1 gene are related to
erythrocyte surface density of CR1 molecules, in turn related to the rate of IC
clearance from circulation. We hypothesized that sarcoidosis could be associated
with increased frequency of the CR1 gene alleles coding for reduced CR1/E ratio.
We studied 91 sarcoid patients and two control groups: 94 healthy volunteers and
71 patients with chronic obstructive pulmonary disease (COPD). Three polymorphic
sites of CR1 gene, His1208Arg, intron 27 HindIII/RFLP, and Pro1827Arg, were
analyzed. The three polymorphisms were in linkage disequilibrium. The GG
genotype for the Pro1827Arg (C(5507)G) polymorphism was significantly associated
with sarcoidosis in comparison to both control groups (odds ratio [OR] = 3.13;
95% confidence interval [CI] 1.49-6.69 versus healthy control subjects, and OR=
2.82, 95% CI 1.27-6.39 versus COPD control subjects). The same genotype was
particularly associated to disease in females (OR = 7.05; 95% CI 3.10-16.61
versus healthy control subjects). These findings agree with speculations on the
role of CR1 gene as a possible susceptibility factor.

Respir Med 2002 Feb;96(2):110-4

Urinary desmosine excretion in acute exacerbations of COPD: a preliminary
report.

Fiorenza D, Viglio S, Lupi A, Baccheschi J, Tinelli C, Trisolini R, Iadarola R,
Luisetti M, Snider GL.

Laboratorio di Biochimica e Genetica della Clinica di Malattie dell'Apparato
Respiratorio, IRCCS Policlinico San Matteo, Universita degli Studi di Pavia,
Italy.

Desmosine (DES) is an elastin-derived, cross-link amino acid, which is not
metabolized; hence, its urinary levels reflect elastin breakdown. We
hypothesized that elastin degradation should increase as a result of increased
lung inflammation during an acute exacerbation of COPD and should decrease after
recovery. To test this hypothesis we measured DES in three urine samples from
nine COPD subjects during the first 5 days of an acute exacerbation and at 2
months after recovery. We also measured forced expiratory volume in 1 sec (FEV1)
to monitor the effects ofthe exacerbation on ventilatory function. The mean (SD)
FEV1 was 45 (15)% predicted during the exacerbation and 57.8 (16)% predicted 2
months later (P=0.00001).The mean (SD) DES excretion was 25.3 (9) microg g(-1)
creatinine at day 1;23.5 (9) at day 3 and 24 (9) at day 5 of the exacerbation.
The mean (SD) urinary DES excretion 60 days after discharge was 20.9 (7) microg
g(-1) creatinine (P=0.049) in comparison with the mean of the three acute-phase
values. The size of the increase in desmosine excretion during exacerbation is
small, 3.2 microg g(-1) creatinine or 16% of the recovery desmosine value. We
conclude that there is a small but statistically significant increase in lung
elastin breakdown in the body during an acute exacerbation of COPD.

Respiration 2002;69(1):81-5

A fast amplification-reverse hybridization assay kit to detect the most frequent
deficient variants in the alpha-1-antitrypsin gene.

Zorzetto M, Tamburnotti C, Maschietto B, Massi G, Battaggia C, Medaglia S,
Luisetti M.

Laboratorio di Biochimica e Genetica, Clinica di Malattie dell'Apparato
Respiratorio, IRCCS Policlinico San Matteo, Universita di Pavia, Italia.

BACKGROUND: There is worldwide growing awareness of alpha-1-antitrypsin
deficiency (AATD), a major hereditary disorder in Caucasians. The gold standard
for its laboratory diagnosis is thin-layer isoelectric focusing, which should be
performed in reference laboratories. OBJECTIVES: The aim of this study was to
check the characteristics of a commercially available amplification-reverse
hybridization assay kit in detecting at a molecular level the
alpha-1-antitrypsin (AAT) Z and S variants, i.e. the most frequent variants
associated with AATD, by comparing its performance with DNA restriction fragment
length polymorphism. METHODS: We studied samples from 36 subjects enrolled in
the Italian National Registry for Severe Alpha-1-antitrypsin Deficiency. Based
on previous plasma isoelectric focusing typing, we selected samples with the
following phenotypes: MM (9 samples), MS (9 samples), SZ (3 samples), MZ (11
samples), ZZ (3 samples), and a rare variant (1 sample). DNA was extracted by
the standard method. The presence of the AAT Z and S gene variants was
determined by the amplification-reverse hybridization test kit, following the
manufacturer's instructions, and by the restriction fragment length polymorphism
technique, according to established procedures. RESULTS: We found that the
identification of the AAT Z and S gene variants obtained by the
amplification-reverse hybridization test kit was completely in agreement with
that obtained by the restriction fragment length polymorphism technique.
CONCLUSIONS: We conclude that the test kit provides a fast, easy and unambiguous
identification of Z and S alleles. Because of its transferability to routine
laboratories, the test kit may be useful in identifying cases of severe AATD,
thus resulting in increasing awareness of this rare disorder. Copyright 2002 S.
Karger AG, Basel

Eur Respir J 2001 Oct;18(4):738

Comment on:
Eur Respir J. 2001 Apr;17(4):833.

Course and prognosis of sarcoidosis in African-Americans versus Caucasians.

Luisetti M, Beretta A, Casali L.

Publication Types:
Comment
Letter

Am J Respir Cell Mol Biol 2001 Oct;25(4):492-9

Inhibition of human neutrophil elastase by erythromycin and flurythromycin, two
macrolide antibiotics.

Gorrini M, Lupi A, Viglio S, Pamparana F, Cetta G, Iadarola P, Powers JC,
Luisetti M.

Clinica di Malattie dell'Apparato Respiratorio, Laboratorio di Biochimica e
Genetica, IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy.

Fourteen-member-ring macrolides are antibiotics with a variety of
anti-inflammatory activities, and have repeatedly been reported to reduce mucus
hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their
structure is characterized by a macrocyclic lactone ring. Because human
neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to
mucus hypersecretion, and lactones are known to be elastase inhibitors, we
hypothesized that macrolides might directly inhibit elastase. To investigate
this hypothesis we designed a series of spectrophotometric experiments using a
chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin
(FE). We determined the 1st order rate constant (k(obs)) by inhibition and
competitive substrate assays, the latter allowing us to calculate the substrate
binding constant or inhibition constant and the acylation rate constant (k(a)).
A proflavine displacement assay was used to determine the deacylation rate
constant (k(d)). Both Er and FE are good HNE inhibitors, showing a high k(a) and
a low k(d). Because the number of turnovers per inactivation of Er was congruent
with 20-fold higher than that of FE, we supposed that the lower reactivation of
HNE-FE was due to the formation of a more stable inactivated enzyme. This
hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For
Er we identified a k(d) only, whereas for FE, in addition to the k(d), an
alkylation constant (k(2)) was calculated, correlated to a fully inactivated
enzyme. From our kinetics data, we therefore conclude that Er acts as an
alternate substrate HNE inhibitor, whereas FE acts as an inactivator.

Am J Respir Crit Care Med 2000 Dec;162(6):2069-72

Short-term supplementation therapy does not affect elastin degradation in severe
alpha(1)-antitrypsin deficiency. The American-Italian AATD Study Group.

Gottlieb DJ, Luisetti M, Stone PJ, Allegra L, Cantey-Kiser JM, Grassi C, Snider
GL.

Department of Medicine, Boston University School of Medicine, MA 02118-2394,
USA. dgottleib@lung.bumc.bu.edu

We evaluated the ability of intravenous supplementation therapy with
alpha(1)-antitrypsin (AAT) to reduce the rate of urinary excretion of desmosine
(DES), a specific marker of elastin degradation, in eight men and four women
with emphysema due to severe, congenital deficiency of AAT (range 17-69 mg/dl).
Nine were former cigarette smokers, two were current smokers, and one reported
never smoking; their mean age was 54 (SD 12) yr and their mean FEV(1) was 41
(18%) of predicted. Urinary DES was measured by isotope dilution and HPLC. Prior
to the start of AAT supplementation, mean DES excretion was 13.0 (5.0) microg/g
creatinine, 73% higher than in healthy nonsmokers. During 8 wk of
supplementation therapy, mean urinary DES excretion was 13.0 (5.9) microg/g
creatinine, unchanged from the baseline period (p = 0.85 by repeated measures
ANOVA). We conclude that baseline levels of elastin degradation in emphysematous
patients with severe AAT deficiency were abnormally high and that 8 wk of AAT
supplementation therapy did not appreciably reduce the rate of elastin
degradation. These findings raise the possibilities that protective levels of
AAT in the lungs are insufficient or that elastin degradation in the lungs of
these subjects is not dependent upon neutrophil elastase at this time.

Eur Respir J 2000 Oct;16(4):768-80

Comment in:
Eur Respir J. 2001 Apr;17(4):833.

Genetic aspects in sarcoidosis.

Luisetti M, Beretta A, Casali L.

Clinica di Malattie dell'Apparato Respiratorio, IRCCS Policlinico San Matteo
Universita di Pavia, Italy.

Sarcoidosis is an immune-mediated, multiorgan, granulomatous disorder thought to
be triggered by an intricate combination of environmental and genetic factors.
Two robust lines of evidence support the hypothesis of a genetic component in
the pathogenesis of sarcoidosis: racial variation in its epidemiology and
familial clustering of cases. The relationship between epidemiology and
environmental factors affecting variations in sarcoidosis incidence/prevalence
and presentation are reviewed, as well as strategies to be pursued in the search
for susceptibility genes for the disorder. Pathogenic processes leading to
sarcoid granuloma formation and maintenance have prompted investigators
interested in the genetics of sarcoidosis to focus mainly on major
histocompatibility complex genes, and indeed a remarkable amount of data has
been accumulated during the last two decades. Whilst in contrast with some
autoimmune disorders a clear association between human leukocyte antigen (HLA)
and sarcoidosis is still a controversial issue, there is, however, a general
agreement that some HLA genes are related to phenotypic variations of the
disease. Some genetic investigators have focused on T-cell receptor genes,
immunoglobulin genes, angiotensin converting enzyme gene, chemokine genes and
others. From a review of studies performed in different racial and ethnic
groups, a reasonable suggestion arises that genetic factors are the major
determinant in the racial variations in the epidemiology of the disorder. This
assumption is, however, so far limited by lack of studies considering both
genetic and environmental factors simultaneously.

Publication Types:
Review
Review Literature

Sarcoidosis Vasc Diffuse Lung Dis 2000 Oct;17(3):288-91

Eosinophilic pleural effusion due to mesalamine. Report of a rare occurrence.

Trisolini R, Dore R, Biagi F, Luinetti O, Pochetti P, Carrabino N, Luisetti M.

Clinica di Malattie dell'Apparato Respiratorio, Universita di Pavia, Italy.

Mesalamine-induced lung toxicity has often been described. We report on a case
of a patient who underwent mesalamine treatment, though in the absence of
established criteria required for diagnosing Crohn's disease (CD) or ulcerative
colitis (UC). He developed an adverse respiratory reaction to the drug, thus
definitely proving its lung damaging capacity. The clinical presentation
included eosinophilic pleural effusion, a feature never previously described in
association with mesalamine intake.

Publication Types:
Review
Review of Reported Cases

Electrophoresis 2000 Sep;21(15):3318-26

Alpha1-antitrypsin in serum determined by capillary isoelectric focusing.

Lupi A, Viglio S, Luisetti M, Gorrini M, Coni P, Faa G, Cetta G, Iadarola P.

Dipartimento di Biochemica A. Castellani, Universita di Pavia, Italy.

A capillary isoelectric focusing (CIEF) method using bare fused-silica
capillaries filled with polyethylene oxide (PEO) and carrier ampholyte solutions
in the pH 3.5-5.0 range has been developed for the identification of
alpha1-antitrypsin (alpha1AT) phenotypes in human serum. This novel procedure
was routinely applied to the study of serum samples of five controls whose
alpha1AT phenotype was previously identified and of twelve subjects whose
alpha1AT phenotype was unknown. The results obtained allowed us to confirm or
identify the alpha1AT phenotype in all sera tested. This procedure seems
particularly suitable for identification of alpha1AT variants associated with
diseases of clinical relevance.

Eur J Hum Genet 2000 Sep;8(9):717-20

Increased frequency of CFTR gene mutations in sarcoidosis: a case/control
association study.

Bombieri C, Luisetti M, Belpinati F, Zuliani E, Beretta A, Baccheschi J, Casali
L, Pignatti PF.

Section of Biology and Genetics, DMIBG, University of Verona, 37134 Verona,
Italy. cristy@borgoroma.univr.it

A complete screening of the CFTR gene by DGGE and DNA sequencing was performed
in patients with sarcoidosis. In 8/26 cases, missense and splicing CFTR gene
mutations were found, a significant difference over controls (9/89) from the
same population (P = 0.014). The odds ratio for a person with a CFTR gene
mutation to develop the disease is 3.95 (1.18 < OR < 13.26). Seven different
CFTR gene mutations were observed: R75Q, R347P, 621 + 3 A/G, 1898 + 3 A/G,
L997F, G1069R, and a novel mutation which was detected in this study, I991V.
R75Q mutation was present in 3/26 patients, a significant increase (P = 0. 01)
in cases over controls, indicating its preferential association with
sarcoidosis. A trend towards disease progression was observed in patients with
CFTR gene mutations compared to patients without mutations. These data suggest
that CFTR gene mutations predispose to the development of sarcoidosis.

Respir Med 2000 Aug;94 Suppl C:S1-2

Introduction: the history of the Alpha One International Registry (A.I.R.).

Luisetti M.

Laboratorio di Biochimica e Genetica, Clinica di Malattie dell'Apparato
Respiratorio, Universita di Pavia, IRCCS Policlinico San Matteo, Italy.

Publication Types:
Historical Article

Eur Respir J 2000 Jul;16(1):74-80

HLA-Gm/kappam interaction in sarcoidosis. Suggestions for a complex genetic
structure.

Martinetti M, Dugoujon JM, Tinelli C, Cipriani A, Cortelazzo A, Salvaneschi L,
Casali L, Semenzato G, Cuccia M, Luisetti M.

Laboratorio HLA, Servizio di Immunoematologia e Trasfusione, IRCCS Policlinico
San Matteo and Universita degli Studi, Pavia, Italy.

The aetiology of sarcoidosis is still unknown. Environmental exposures are
believed to interact with genetic factors in determining the pattern of
sarcoidosis presentation, progression and prognosis. The frequency of
serological polymorphism of immunoglobulin G heavy chain (Gm) and kappa light
chain (kappam) markers in 107 patients with biopsy-proven sarcoidosis and in 227
controls, and their interactions with histocompatibility leukocyte antigen (HLA)
class I, II, and III markers, were studied. A "protective" effect of the Gm(3
5*) phenotype in the sarcoid group versus controls (p-value for number of
specificities tested (p(c))=0.05, odds ratio 0.15) and a reduced frequency of
Gm(3 23 5*) in patients with advanced chest radiographic stage (Chi-squared (two
degrees of freedom)(chi2(2df) 17.61, p(c)=0.0058) were observed. With reference
to epistatic interactions, the combination Gm(3 23 5*)/BfS had a "protective"
effect towards stage II (chi2(2dt) 13.86, p(c)=0.043). Finally, correspondence
analysis defined two clusters: HLA-DR4, C4BQ0, Gm(1, 3, 17 23 5*, 21, 28) and
BfF associated with stage II, and HLA-DR3, C4AQ0, kappam(1) and Gm(3 23 5*)
associated with stage I. These data further support the hypothesis that
sarcoidosis results from an interplay of environmental factors and genes, each
contributing to the susceptibility/resistance to and/or the clinical
heterogeneity of the disease. In addition, these data provide the first evidence
of an interaction between immunoglobulin G heavy chain/kappa light chain markers
and histocompatibility leukocyte antigen class III genes in a disease.

Eur Respir J 2000 Jun;15(6):1039-45

MEKC of desmosine and isodesmosine in urine of chronic destructive lung disease
patients.

Viglio S, Iadarola P, Lupi A, Trisolini R, Tinelli C, Balbi B, Grassi V,
Worlitzsch D, Doring G, Meloni F, Meyer KC, Dowson L, Hill SL, Stockley RA,
Luisetti M.

Dipartimento di Biochemical, Istituto di Ricovero e Cura a Carattere Scientifico
(IRCCS) Policlinico San matteo, Universita degli nStudi di Pavia.

Degradation of extracellular matrix components is central to many pathological
features of chronic destructive lung disorders. Desmosine and isodesmosine are
elastin-derived cross-linked amino acids whose urine levels are considered
representative of elastin breakdown. The aim of this study was to apply a novel
methodology, based on high-performance capillary electrophoresis, to the
quantification of desmosine and isodesmosine in 11 patients with stable chronic
obstructive pulmonary disease (COPD), 10 with an exacerbation of COPD, nine with
alpha1-antitrypsin deficiency, 13 with bronchiectasis, and 11 adults with cystic
fibrosis, in comparison to 24 controls. It was found that, in patients with
stable COPD, urinary desmosine levels were higher than in controls (p=0.03), but
lower than in COPD subjects with an exacerbation (p< or =0.05). The highest
desmosine levels were found in subjects with alpha1-antitrypsin deficiency,
bronchiectasis and cystic fibrosis (p<0.001 versus stable COPD). In a short-term
longitudinal study, five stable COPD patients showed a constant rate of
desmosine excretion (mean coefficient of variation <8% over three consecutive
days). In conclusion, the present method is simple and suitable for the
determination of elastin-derived cross-linked amino acid excretion in urine,
giving results similar to those obtained using other separation methods. In
addition, evidence is presented that urinary desmosine excretion is increased in
conditions characterized by airway inflammation, such as exacerbations of
chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis.
Results obtained in subjects with alphal-antitrypsin deficiency suggest that
this method might be used to evaluate the putative efficacy of replacement
therapy.

Electrophoresis 2000 Jun;21(10):1985-91

Separation of closely related peptide substrates of human proteinases by
micellar electrokinetic chromatography with anionic and nonionic surfactants.

Lupi A, Viglio S, Luisetti M, Zanaboni G, Cetta G, Iadarola P.

Laboratorio di Biochimica e Genetica Clinica di Malattie dellApparato
Respiratorio, IRCCS Policlinico San Matteo, Pavia, Italy.

In order to use micellar electrokinetic chromatography to determine the
proteolytic activity of different proteinases simultaneously present in
physiological fluids, the technique must be able to separate mixtures of
substrates with closely related structures. In an attempt to determine the best
electrophoretic conditions for resolving six p-nitroanilide peptides used as
synthetic substrates of the elastolytic enzymes (human neutrophil elastase,
cathepsin G, Pseudomonas aeruginosa elastase) most commonly involved in
pulmonary diseases, we investigated the efficiency of ionic and nonionic
surfactants in achieving the separation of this complex mixture. The results
presented here show that, of all the electrophoretic systems tested, 30 mM
sodium tetraborate, pH 9.3, containing 25 mM Brij 35 as micellar agent offered
the best performance; the separation efficiency of peptides is greater than that
obtained with other reagents and all peaks are baseline resolved and
unambiguously identifiable. Analysis of the micelle-solute interaction with the
surfactants investigated allowed better definition of the mechanism involved in
the distribution of these peptides to the micelles and identification of some
structural features that determined the magnitude of the micelle peptide complex
formation.

Chest 2000 May;117(5):1353-8

Comment in:
Chest. 2001 Jan;119(1):315-6.

Tumor necrosis factor gene complex in COPD and disseminated bronchiectasis.

Patuzzo C, Gile LS, Zorzetto M, Trabetti E, Malerba G, Pignatti PF, Luisetti M.

Istituto di Biologia e Genetica, Universita degli Studi di Verona Italy.

BACKGROUND: Tumor necrosis factor (TNF) is a potent proinflammatory cytokine
with increased levels in the sputum of COPD subjects. Two biallelic TNF gene
complex polymorphisms have been described: LtalphaNcoI, in the first intron of
the lymphotoxin alpha (previously referred to as TNF-beta) gene, and TNF-308, in
the promoter region of the TNF-alpha gene. Higher levels of TNF production are
associated with allele 1 of LtalphaNcoI (LtalphaNcoI*1) and with allele 2 of
TNF-308 (TNF-308*2). STUDY OBJECTIVES: To study the frequencies of the two TNF
gene complex polymorphisms in patients with COPD and bronchiectasis. DESIGN:
Association study. SUBJECTS AND METHODS: We studied the frequencies of these
polymorphisms in 66 subjects with COPD and in 23 subjects with disseminated
bronchiectasis and compared them to the frequencies in 98 healthy control
subjects and 45 subjects with nonobstructive pulmonary disease. Genomic DNA
samples were extracted, and TNF-alpha and LtalphaNcoI polymorphisms were
detected after polymerase chain reaction by restriction digestion. RESULTS: We
found the following frequencies: the TNF-308*2 allele was detected in 11% of
COPD individuals, 15% of bronchiectasis patients, 10% of healthy control
subjects, and 18% of subjects with nonobstructive pulmonary disease. The
LtalphaNcoI*1 allele was detected in 28% of COPD individuals, 30% of
bronchiectasis patients, 29% of healthy control subjects, and 29% of subjects
with nonobstructive pulmonary disease. We found evidence of linkage
disequilibrium between the two loci (Delta = 0.068). CONCLUSIONS: We conclude
that the TNF gene complex, at least in Caucasoid individuals and for the
considered polymorphisms, does not seem to play a major role as genetic risk
factor in COPD and bronchiectasis.

Monaldi Arch Chest Dis 1999 Oct;54(5):384-9

Lymphocyte expression of human leukocyte antigen class II molecules in patients
with chronic obstructive pulmonary disease.

Recalde H, Cuccia M, Oggionni T, Dondi E, Martinetti M, Luisetti M.

Centro Trasfusionale, Ospedale Fornaroli, Magenta, Italy.

Chronic obstructive pulmonary disease (COPD) is a multifactorial disorder,
deriving from a combination of environmental and genetic factors. Polymorphisms
of genes of the human major histocompatibility complex in COPD have been poorly
studied in the past. In a preliminary approach, it was difficult to type human
leukocyte antigen (HLA) at the protein level and it was hypothesized that there
was a reduced surface density of HLA class II molecules. The aims of this study
were to analyse, by cytofluorimetry, HLA class I and II molecule densities on
peripheral mononuclear cells of COPD patients and to investigate whether there
was a correlation with the polymorphisms of DQA and DQB promoter regions which
are supposed to be important factors involved in surface expression of HLA-DQ
molecules. The study investigated 27 male COPD patients admitted because of
disease exacerbation and 49 healthy male controls. Quantitative analysis of
fluorescence intensity of HLA class I (A, B, C) and class II (DR, DP, DQ)
molecules was performed on blood mononuclear cells by cytoron cytofluorimetry.
Polymorphisms of DQA and DQB promoters (QAP and QBP) were determined from the
DNA (PCR-SSO). The surface densities of HLA class I and HLA-DQ molecules did not
differ between the COPD patients and controls. HLA-DP molecule density seemed to
be slightly, but not significantly lower in COPD, whereas surface HLA-DR
molecules were significantly reduced (p < 0.005 vs controls). Frequencies of QAP
alleles were not different between the COPD patients and controls, but the QBP
5.12 allele was significantly more frequent in COPD than in controls (chi 2 =
10.83, p = 0.0182, RR 5.5). In conclusion, individuals with exacerbated chronic
obstructive pulmonary disease have reduced surface DR molecule expression and an
increased frequency of the QBP 5.12 allele. The possible relationship between
these two features and the possible role of cytokines in reducing human
leukocyte antigen-DR expression in exacerbated chronic obstructive pulmonary
disease is explored.

Respir Med 1999 Sep;93(9):648-54

alpha 1-antitrypsin TAQ I polymorphism and alpha 1-antichymotrypsin mutations in
patients with obstructive pulmonary disease.

Benetazzo MG, Gile LS, Bombieri C, Malerba G, Massobrio M, Pignatti PF, Luisetti
M.

Istituto di Biologia e Genetica, Universita degli Studi di Verona, Italy.

Obstructive pulmonary disease is a multifactorial condition deriving from the
interaction of environmental and genetic factors. From biochemical knowledge of
the basis of the disease, alpha 1-antitrypsin and alpha 1-antichymotrypsin are
considered two likely candidate genes. We therefore designed an association
study comprising 232 unrelated Italian individuals divided as follows: 89
individuals with obstructive lung disease (66 with COPD and 23 with disseminated
bronchiectasis) and 143 controls (45 patients with non-obstructive lung disease
and 98 healthy individuals). We screened for Taq I (G1237A) polymorphism of the
alpha 1-antitrypsin gene as well as the rare variants Bonn-1 (Pro229Ala),
Bochum-1 (Leu55Pro), Isehara-1 (Met389Val) and Isehara-2 (1258delAA), and the
common signal peptide polymorphism Thr-15Ala of the alpha 1-antichymotrypsin
gene. The frequencies of Taq I G1237A alleles were 11.7 and 10.8% in obstructed
patients and controls, respectively (P = 0.43), while those of signal peptide
Thr-15Ala alleles were 51.6 and 50.3% in obstructed patients and controls,
respectively (P = 0.42). We conclude that alpha 1-antitrypsin Taq I polymorphism
and alpha 1-antichymotrypsin Thr-15Ala mutation are not major genetic risk
factors for the development of obstructive lung disease in Italian patients. The
alpha 1-antichymotrypsin rare variants were not detected: our results do not
exclude the possibility that other alpha 1-antichymotrypsin gene mutations might
be present in Italian obstructed patients but, if so, these genetic defects must
be rare.

Respir Med 1999 Mar;93(3):169-72

A national program for detection of alpha 1-antitrypsin deficiency in Italy.
Gruppo I.D.A.

Luisetti M, Massi G, Massobrio M, Guarraci P, Menchicchi FM, Beccaria M, Balbi
B.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy. luisetti@telnetwork.it

alpha 1-antitrypsin (AAT) deficiency is an inherited condition characterized by
low serum levels of AAT and an increased risk of developing pulmonary emphysema.
The disease occurs mainly in Caucasians, but Southern Europe, including Italy,
is considered a low prevalence area. We developed a national program in Italy in
order to improve our knowledge of the epidemiology of AAT deficiency and to
establish a registry of the AAT-deficient individuals. The program had two
phases: the first lasted 36 months, during which blood from coupons mailed by
respiratory physicians from throughout the country, was isoelectrofocused by the
Central Laboratory in Rome. The second phase started in February 1996, and the
Registry was established. Up to August 1998, 151 subjects with AAT deficiency
have been identified and 64 have been enrolled in the Registry. We believe that
such a program plays a crucial role in identifying AAT deficiency in a country
such as Italy, with low prevalence and low awareness of this rare condition.

Electrophoresis 1999 Jun;20(7):1578-85

Micellar electrokinetic chromatography for analyzing active site specificity of
Pseudomonas aeruginosa elastase.

Viglio S, Zanaboni G, Lupi A, Gianelli L, Luisetti M, Casali L, Cetta G,
Iadarola P.

Dipartimento di Biochimica A. Castellani, Universita di Pavia, Italy.

The geometry of the catalytic site of Pseudomonas aeruginosa elastase was
reexamined, exploiting the specific feature of micellar electrokinetic
chromatography (MEKC), i.e., its ability to detect a decrease of intact
substrate and simultaneous formation of reaction products. We carried out a
detailed investigation using two tri- and six tetra-peptide 4-nitroanilides (NA)
differing from each other by only one or more amino acids as stable substrates.
The kinetic cleavage parameters Km and k(cat) determined by MEKC and the
catalytic efficiency Km/k(cat) values calculated allowed us to better define the
substrate specificity of this proteinase.

J Chromatogr A 1999 Jun 18;846(1-2):125-34

Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte
elastase and cathepsin G activities by micellar electrokinetic chromatography.

Viglio S, Luisetti M, Zanaboni G, Doring G, Worlitzsch D, Cetta G, Iadarola P.

Dipartimento di Biochimica A. Castellani, Universita di Pavia, Italy.

Micellar electrokinetic chromatography (MEKC) is a new method for analysing
proteolytic activities simultaneously present in incubation mixtures. Here we
demonstrate that MEKC differentiates between the enzymatic activities of
Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or
cathepsin G (Cat G) in assays using the chromogenic peptide substrates
Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl
and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at
equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products
PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the
metalloproteinase PsE with EDTA resulted in detection of NA and
Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at
equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products
Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the
PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were
separated. MEKC also allowed determination of the kinetic constants for the
interactions of PsE, Cat G and HLE with the substrates considered.

J Nat Prod 1999 May;62(5):670-3

Application of lipase-catalyzed regioselective esterification in the preparation
of digitonin derivatives.

Danieli B, Luisetti M, Steurer S, Michelitsch A, Likussar W, Riva S, Reiner J,
Schubert-Zsilavecz M.

Dipartimento di Chimica Organica e Industriale, Universita degli Studi di
Milano, Centro CNR di Studio per le Sostanze Organiche Naturali, via Venezian
21, 20133 Milano, Italy. danieli@icil64.cilea.it

The oligosaccharide chain of the monodesmosidic haemolytic saponin digitonin (1)
undergoes an efficient and regioselective acylation in organic solvent by use of
Novozym 435 (lipase B from Candida antarctica supported on acrylic resin) in the
presence of an activated ester. With vinyl acetate, acetylation occurs at C-6 OH
of glucose(II) and C-4 OH of xylose to afford the previously unreported diacetyl
derivative 2 and the monoacetyl derivatives 3 and 4. With vinyl laurate only the
monolauryl derivative 5 is formed. The structures of these acylated digitonins
have been established using modern 2D NMR techniques, which allowed complete
assignments of all proton resonances. The hemolytic activity of derivatives 2-5
is significantly reduced compared to that of digitonin.

Monaldi Arch Chest Dis 1998 Dec;53(6):614-6

Genetics of chronic obstructive pulmonary disease and disseminated
bronchiectasis.

Luisetti M, Gile LS, Bombieri C, Benetazzo MG, Pignatti PF.

Istituto di Tisiologia e Malattie Respiratoire, Universita di Pavia, IRCCS
Policlinico San Matteo, Italy.

Publication Types:
Review
Review, Tutorial

Ann N Y Acad Sci 1998 Dec 13;864:70-80

Enzymatic modification of natural compounds with pharmacological properties.

Riva S, Monti D, Luisetti M, Danieli B.

Istituto di Chimica degli Ormoni, CNR, Milano, Italy.

Glycosides of various classes of natural products are widely distributed in
nature, where they are often present esterified with aliphatic and aromatic
acids at specific OH's of their sugar moieties. Many of these compounds are
pharmacologically important molecules or possess other interesting properties.
For instance, ginsenosides (e.g., 3) are therapeutic dammarane-type
oligoglycosides isolated from the water-soluble portion of the dried roots and
leaves of Panax ginseng C.A. Meyer (Aralianceae), a plant widely used in
traditional Chinese medicine. In recent years, we have exploited the
regioselectivity of lipases and proteases in organic solvents for the synthesis
of specific esters of ginsenosides as well as the selectivity of the
beta-1,4-galactosyltransferase from bovine colostrum to obtain new glycosyl
derivatives of these compounds. The application of these two enzymatic
methodologies has also been exemplified with other natural compounds with
pharmacological properties: digitonin (5), colchicoside (6), and flavonoid
glycosides.

Publication Types:
Review
Review, Tutorial

Hum Genet 1998 Dec;103(6):718-22

Complete mutational screening of the CFTR gene in 120 patients with pulmonary
disease.

Bombieri C, Benetazzo M, Saccomani A, Belpinati F, Gile LS, Luisetti M, Pignatti
PF.

Istituto di Biologia e Genetica, Universita di Verona, Italy.
cristy@borgoroma.univr.it

In order to determine the possible role of the cystic fibrosis transmembrane
regulator (CFTR) gene in pulmonary diseases not due to cystic fibrosis, a
complete screening of the CFTR gene was performed in 120 Italian patients with
disseminated bronchiectasis of unknown cause (DBE), chronic bronchitis (CB),
pulmonary emphysema (E), lung cancer (LC), sarcoidosis (S) and other forms of
pulmonary disease. The 27 exons of the CFTR gene and their intronic flanking
regions were analyzed by denaturing gradient gel electrophoresis and automatic
sequencing. Mutations were detected in 11/23 DBE (P = 0.009), 7/25 E, 5/27 CB,
5/26 LC, 5/8 S (P = 0.013), 1/4 tuberculosis, and 1/5 pneumonia patients, and in
5/33 controls. Moreover, the IVS8-5T allele was detected in 6/25 E patients (P =
0.038). Four new mutations were identified: D651N, 2377C/T, E826K, and P1072L.
These results confirm the involvement of the CFTR gene in disseminated
bronchiectasis of unknown origin, and suggest a possible role for CFTR gene
mutations in sarcoidosis, and for the 5T allele in pulmonary emphysema.

Electrophoresis 1998 Sep;19(12):2083-9

Micellar electrokinetic chromatography: a convenient alternative to colorimetric
and high performance liquid chromatographic detection to monitor protease
activity.

Viglio S, Zanaboni G, Luisetti M, Cetta G, Guglielminetti M, Iadarola P.

Dipartimento di Biochimica A. Castellani, Universita di Pavia, Italy.

High performance capillary electrophoresis (HPCE) has been exploited as an
analytical method alternative to current procedures for the determination of
proteolytic activity of elastases from different sources. Due to some drawbacks
with capillary zone electrophoresis (CZE), the mode of operation employed for
the assay of elastolytic activity was micellar electrokinetic chromatography
(MEKC). Using a background electrolyte consisting of 35 mM sodium tetraborate,
pH 9.3, containing 65 mM SDS and 15% v/v methanol, separation of intact peptide
substrate from products of proteolytic reaction was easily achieved in a
fused-silica capillary of 50 cm effective length x 75 microm ID. This allowed us
to determine the rate of hydrolysis of substrates and to calculate the kinetic
parameters Km and k(cat) of the proteases investigated. A comparison of these
data with those obtained from high performance liquid chromatography
(HPLC)-based experiments showed that MEKC is a convenient technique for studying
protease kinetics.

J Chromatogr B Biomed Sci Appl 1998 Aug 28;714(1):87-98

Micellar electrokinetic chromatography for the determination of urinary
desmosine and isodesmosine in patients affected by chronic obstructive pulmonary
disease.

Viglio S, Zanaboni G, Luisetti M, Trisolini R, Grimm R, Cetta G, Iadarola P.

Dipartimento di Biochimica A. Castellani, Universita di Pavia, Italy.

The presence in urine of desmosine (DES) and isodesmosine (IDES), two
crosslinked amino acids unique to the elastic fiber network, can be used as a
specific indicator of degradation of mature elastin. Compared to methodologies
so far available, the capillary electrophoretic technique reported here seems to
be suitable and convenient for determining desmosines in urine of patients
affected by chronic obstructive pulmonary disease (COPD). By using 35 mM sodium
tetraborate pH 9.3 containing 65 mM SDS as the background electrolyte, the peaks
of DES and IDES could be detected in hydrolyzed urine samples from controls and
patients. Owing to the simultaneous determination of endogenous urinary
creatinine used as appropriate internal standard, the amount of these amino
acids could be accurately quantified. The results obtained were of the same
order of magnitude as the data already reported in the literature for COPD
patients. Thus micellar electrokinetic chromatography (MEKC) may be considered
as a reliable technique for studying the turnover of the elastic fiber in
clinical conditions.

Exp Lung Res 1998 May-Jun;24(3):233-51

Lung injury and degradation of extracellular matrix components by Aspergillus
fumigatus serine proteinase.

Iadarola P, Lungarella G, Martorana PA, Viglio S, Guglielminetti M, Korzus E,
Gorrini M, Cavarra E, Rossi A, Travis J, Luisetti M.

Dipartimento di Biochimica, IRCCS Policlinico San Matteo, Pavia, Italy.

Aspergillus fumigatus produces a variety of extracellular proteinases that are
believed to be virulence factors towards Aspergillus-related lung disease. Among
Aspergillus proteinases, the serine proteinase is thought to play a major
virulent role because of its widespread production. Nevertheless, evidence of
direct pulmonary injury caused by the A. fumigatus serine proteinase is still
lacking. The purpose of our work was: (1) to provide evidence for a pivotal role
of A. fumigatus serine proteinase in producing lung injury in an animal model,
and (2) to investigate the broadness of the substrate specificity of the
proteinase towards extracellular matrix components. To achieve this aim, the
proteinase from an A. fumigatus strain isolated from human airways was purified
by a four-step procedure, including cation exchange and hydrophobic interaction.
High-performance capillary electrophoresis, SDS-PAGE, determination of K(m)
towards synthetic substrates, and inhibitory studies were used to further
characterize the A. fumigatus serine proteinase. With reference to extracellular
matrix components, the A. fumigatus serine proteinase was shown to degrade human
lung elastin at a higher rate than an equimolar amount of human neutrophil
elastase. Human lung collagen, type I and type III collagens, as well as
fibronectin, were quickly digested by the A. fumigatus serine proteinase.
Finally, mice intratracheally injected with the proteinase showed a significant
degree of lower respiratory tract destruction. We conclude that the A. fumigatus
serine proteinase is capable per se of hydrolyzing the major structural barriers
of the lung.

Monaldi Arch Chest Dis 1998 Feb;53(1):105-6

Genetics of resistance and susceptibility to mycobacterial infections.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy.

Am J Physiol 1998 May;274(5 Pt 1):L737-49

In chyloptysis, SP-A affects the clearance of serum lipoproteins entering the
airways.

Alberti A, Ravenna F, Quaglino D, Luisetti M, Muraca M, Previato L, Enzi GB,
Bruni R, Baritussio A.

Institute of Internal Medicine, University of Padova, Italy.

Serum lipoproteins may enter the airways and appear in sputum (chyloptysis) when
the lymphatic circulation is impaired by inflammation, neoplasia, or an abnormal
proliferation of smooth muscle cells. While analyzing the bronchoalveolar lavage
fluid of a patient with chyloptysis, we noticed that surfactant could not be
separated from contaminating serum lipoproteins and speculated that lipoproteins
might interact with surfactant components. To clarify this point we immobilized
surfactant protein (SP) A on microtiter wells and incubated it with 125I-labeled
very low density lipoproteins (VLDLs), low-density lipoproteins, and
high-density lipoproteins. We found that SP-A binds lipoproteins. Studying in
greater detail the interaction of SP-A with VLDLs, we found that the binding is
time and concentration dependent; is inhibited by unlabeled lipoproteins,
phospholipids, and antibodies to SP-A; is increased by Ca2+; and is unaffected
by methyl alpha-D-mannopyranoside. Whole surfactant is a potent inhibitor of
binding. Furthermore, we found that SP-A increases the degradation of VLDLs by
alveolar macrophages and favors the association of VLDLs with alveolar
surfactant. We conclude that SP-A influences the disposal of serum lipoproteins
entering the airways and speculate that binding to alveolar surfactant might
represent an important step in the interaction between exogenous substances and
the lung.

Monaldi Arch Chest Dis 1997 Oct;52(5):496-7

Mapping the activities of secretory leukoprotease inhibitor.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy.

PMID: 9510674 [PubMed - indexed for MEDLINE]

Monaldi Arch Chest Dis 1997 Aug;52(4):390-1

Influence of a secondary genetic factor in cystic fibrosis genotype-phenotype
correlations.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy.

Publication Types:
Review
Review Literature

Monaldi Arch Chest Dis 1997 Jun;52(3):285-6

Multiple functions of alpha 1-antitrypsin and alpha 1-antichymotrypsin. 3.
Regulation of extracellular surfactant metabolism and amyloid fibril formation.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy.

Monaldi Arch Chest Dis 1997 Apr;52(2):176-7

Role for granulocyte/macrophage colony-stimulating factor in pulmonary
surfactant homeostasis.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratoire, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy.

Publication Types:
Review
Review, Tutorial

Monaldi Arch Chest Dis 1997 Feb;52(1):48-9

Multiple functions of alpha 1-antitrypsin and alpha 1-antichymotrypsin. 2. Role
of serpins and their target enzymes in controlling neutrophil chemotaxis.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy.

Chest 1996 Dec;110(6 Suppl):278S-283S

Bioengineering: alpha 1-proteinase inhibitor site-specific mutagenesis. The
prospect for improving the inhibitor.

Luisetti M, Travis J.

Istituto di Tisiologia e Malattie Apparato Respiratorio, Universita di Pavia,
Italy.

alpha 1-Proteinase inhibitor (alpha 1-PI) augmentation therapy has been licensed
for treatment of alpha 1-PI-deficient individuals with pulmonary emphysema. The
currently available product is purified from pooled human plasma. To obtain
larger amounts of protein free from possible unknown plasma contaminants, human
alpha 1-PI has been produced by recombinant DNA. Since wild-type alpha 1-PI is
susceptible to oxidative impairment, several alpha 1-PI variants in which the
active site oxidation-sensitive residue is replaced by inert residues have been
constructed. This article is aimed at reviewing the history, biological
efficacy, advantages, disadvantages, and concerns linked to alpha 1-PI
recombinant DNA and site-specific mutagenesis technology.

Publication Types:
Review
Review, Tutorial

Monaldi Arch Chest Dis 1996 Oct;51(5):424-5

Multiple functions of alpha 1-antitrypsin and alpha 1-antichymotrypsin. 1. Role
of lung epithelial cells in the proteinase-proteinase inhibitor balance.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico San Matteo,
Universita di Pavia, Italy.

Am J Respir Crit Care Med 1996 Sep;154(3 Pt 1):817-20

Bronchoalveolar lavage fluid composition in alveolar proteinosis. Early changes
after therapeutic lavage.

Alberti A, Luisetti M, Braschi A, Rodi G, Iotti G, Sella D, Poletti V, Benori V,
Baritussio A.

Istituto de Medicine Interna, Universita di Padova, Italy.

In patients with idiopathic alveolar proteinosis, the alveoli are filled with
materials rich in surfactant components, especially surfactant protein A (SP-A).
The anomaly could be caused by either increased secretion, decreased clearance,
or both. To clarify this point, we studied five patients who underwent
therapeutic lavage and then were ventilated mechanically for 24 h. During the
first 8 h of mechanical ventilation, a surfactant-depleted lung was lavaged at
selected intervals, and the bronchoalveolar lavage fluid was analyzed. We
observed that, after lavage, various surfactant components accumulated in the
airways with different time courses. We also observed that SP-A increased until
the second hour and then dropped rapidly, suggesting the existence of an
efficient mechanism of removal. These findings suggest that idiopathic alveolar
proteinosis might be caused by a primary defect in a slow mechanism of removal
or by the presence of factor(s) that interfere with the clearance of surfactant
and that can be removed by lavage. It seems clear, however, that an increased
secretion rate is unlikely to be the major cause of idiopathic alveolar
proteinosis.

Publication Types:
Clinical Trial
Controlled Clinical Trial

Monaldi Arch Chest Dis 1996 Aug;51(4):329-30

Role of CFTR gene in the regulation of airway mucus composition.

Luisetti M.

Istituto di Tisiologia e Malattie Respiratorie, IRCCS Policlinico S. Matteo,
Universita di Pavia, Italy.

Publication Types:
Review
Review, Tutorial

Eur Respir J 1996 Aug;9(8):1648-51

Serum type I and type III procollagen peptide levels in sarcoidosis.

Bacchella L, Tinelli C, Gile LS, Peona V, Aprile C, Gorrini M, Pasturenzi L,
Cetta G, Luisetti M.

Servizio di Medicina Nucleare, Fondazione Clinica del Lavoro, Pavia, Italy.

Type I and type III are the most abundant collagens in the lung. The aim of our
study was to compare type I and III procollagen peptides in sera of sarcoid
patients. Sixty eight patients with sarcoidosis were studied (19 with newly
recognized disease, 7 with relapsing disease, 15 with chronic disease, and 27 in
stable remission). Thirty healthy volunteers served as controls. The levels of
procollagen I and III peptides were determined by radioimmunoassay.
Angiotensin-converting enzyme (ACE) level was evaluated by means of a
colorimetric assay. In patients with newly recognized sarcoidosis, both serum
procollagen I and III peptide levels were increased with respect to controls
(p=0.0014 and p<0.00001, respectively). There was a poor correlation between
levels of procollagen I and III (r=0.26), whereas there was a closer correlation
between procollagen III and ACE (r=0.69). Procollagen I peptide level did not
identify patients in roentgenological stage III. In conclusion, in patients with
newly recognized sarcoidosis there is a significant increase in the serum level
of procollagen I peptide. However, procollagen I peptide is not a marker of
sarcoid patients with fibrosis, ie. stage III disease. Its clinical usefulness
seems to be weaker than that of procollagen III peptide.

Eur Respir J 1996 Jul;9(7):1482-6

MR889, a neutrophil elastase inhibitor, in patients with chronic obstructive
pulmonary disease: a double-blind, randomized, placebo-controlled clinical
trial.

Luisetti M, Sturani C, Sella D, Madonini E, Galavotti V, Bruno G, Peona V,
Kucich U, Dagnino G, Rosenbloom J, Starcher B, Grassi C.

Istituto di Tisiologia e Malattie Respiratorie, Universita di Pavia, IRCCS
Policlinico San Matteo, Italy.

We investigated whether MR889, a synthetic cyclic thiolic elastase inhibitor,
administered for a period of 4 weeks to chronic obstructive pulmonary disease
(COPD) patients, is well-tolerated, and whether it modifies biochemical indices
of lung destruction. The study was a double-blind, randomized,
placebo-controlled clinical trial in COPD patients. Thirty subjects were
administered MR889 orally at a dose of 500 mg b.i.d. for 4 weeks, and 30
received placebo following the same schedule. In addition to safety parameters,
MR889 efficacy was checked by a pretreatment/postreatment evaluation of levels
of plasma elastin-derived peptides and urinary desmosine. There were no
statistically significant differences between pretreatment and posttreatment
efficacy parameter levels either in the control group or in the treated group.
However, in a subset of treated patients with a short disease duration, the
level of urinary desmosine dropped significantly with respect to pretreatment
values (p = 0.004). We conclude that MR889 is safe to administer to COPD
patients for a period of at least 4 weeks. During this time, MR889 does not
modify biochemical markers of lung destruction in unselected COPD patients.
Nevertheless, a subset of treated patients with fairly short disease duration
showed a post-treatment reduction of desmosine urine levels, thus justifying the
need for further studies to prove the efficacy of MR889 in modulating indices of
lung destruction in COPD.

Publication Types:
Clinical Trial
Randomized Controlled Trial

J Nat Prod 1996 Jun;59(6):618-21

A two-step efficient chemoenzymatic synthesis of flavonoid glycoside malonates.

Riva S, Danieli B, Luisetti M.

CNR, Istituto di Chimica degli Ormoni, Milano, Italy.

A simple and high-yielding protocol for the malonylation of some flavonoid
glycosides is described. The two-step synthesis is based on the regioselective
enzymatic introduction of a benzylmalonyl group by catalysis with the lipase
from Candida antarctica, followed by Pd/C hydrogenolysis of the benzyl moiety.

Am J Hum Genet 1996 Apr;58(4):889-892

CFTR gene variant IVS8-5T in disseminated bronchiectasis.

Pignatti PF, Bombieri C, Benetazzo M, Casartelli A, Trabetti E, Gile LS,
Martinati LC, Boner AL, Luisetti M.

Publication Types:
Letter

Am J Respir Crit Care Med 1996 Feb;153(2):851-4

Polymorphism of angiotensin-converting enzyme gene in sarcoidosis.

Arbustini E, Grasso M, Leo G, Tinelli C, Fasani R, Diegoli M, Banchieri N,
Cipriani A, Gorrini M, Semenzato G, Luisetti M.

Istituto di Anatomia Patologica, Universita di Pavia, IRCCS Policlinico San
Matteo, Italy.

Sarcoidosis is the disease in which increased levels of serum
Angiotensin-converting enzyme (sACE) are most often detected. It has recently
been shown that the deletion (D) or the insertion (I) of a 250bp-DNA fragment in
the ACE gene accounts for three main ACE genotypes (i.e., II, ID, and DD) and
for 47% of total phenotypic variance in sACE level. The aim of our work was to
investigate whether or not patients with sarcoidosis have an increased incidence
of those ACE genotypes coding for highest sACE levels and to investigate whether
or not sACE level in sarcoidosis is related to ACE genotypes. We studied 61
unrelated patients with sarcoidosis (test group) and 80 unrelated healthy
control subjects (control group). The ACE I and D alleles were detected with
polymerase chain reaction on genomic DNA. In the control group we found an ACE
genotype distribution that agreed with the Hardy-Weinberg proportion. The ACE
genotype distribution was not significantly different in the test group. There
was no correlation between ACE genotype and roentgenologic stage of sarcoidosis.
Plotting the sACE level in the control group against ACE genotype, we found a
trend of increasing mean sACE value according to the order II < ID < DD. The
same trend for ACE genotype was found in the test group, in which it also
paralleled the trend of sACE values plotted against roentgenologic stage,
according to the order Stage I < Stage II < Stage III. We conclude that in
sarcoidosis the ACE genotype distribution is not altered. The trends for
increasing sACE values in sarcoidosis according to both ACE genotype and
roentgenologic stage would suggest that both mechanisms play a role in
determining sACE level.