[Multidisciplinary protocol for work resumption after occupational osteoarticular injury: case record]. / Protocollo interdisciplinare per il reinserimento lavorativo dopo infortunio osteoarticolare sul lavoro: casistica clinica.

The results obtained with a protocol aimed at work resumption after occupational osteoarticular injury and subsequent rehabilitation are presented: 159 patients (102 males and 57 females: mean age 43.7 years) were evaluated by the physiatrist and the occupational physician, providing indications based on their functional capabilities and task features. After 6 months, 105 out of 130 subjects available for telephonic follow-up (80.7%) had returned to work (8 after changing tasks, 7 part-time). The mean time for work resumption was 9.4 days for those who resumed working completely, 31.4 days for the others. After 12 months, the percentage of working patients (76 out of 93: 81.7%) was superimposable, indicating that the rehabilitation beneficial effect is maximal in the short-medium period. On the whole, the data prove the efficacy of the multidisciplinary rehabilitative approach.

[Early diagnosis of Fabry disease in children]. / Diagnosi precoce di malattia di Fabry in età pediatrica.

Fabry disease, a rare X-linked lysosomal storage disorder, is caused by deficiency of the enzyme α-galactosidase A. The incidence, ranging from one over 40 000 to one over 11 7000 worldwide is probably underestimated due to its unspecific pattern of presentation. The symptoms, including neurological, gastrointestinal, renal, ophthalmological and dermatologic manifestations, start in childhood and adolescence, cause a significant morbidity and are likely to affect the patient's quality of life. Furthermore, Anderson-Fabry disease always progress leading to a multiorgan dysfunction and life-threatening complications with end-stage renal disease, cardiomyopathy and high incidence of stroke. The estimated life in untreated patients is reduced by 15-20 years respectively in men and women. The enzyme replacement therapy, available in Europe from 2001, results in a reduction of major organs failure, morbidity and mortality. We present the case of an 8-year-old male admitted to our Division for overweight with a previous history of acroparesthesias, severe acute pain in hands and feet, abdominal pain, diarrhoea, constipation, bitemporal headache, dyshidrosis, recurrent fever, exercise intolerance and reduced quality of life. The physical examination was within normal limits. The α-galactosidase A activity was deficient in plasma and normal in peripheral leukocytes; the GLA gene showed a nucleotide substitution c.352C>T (p.Arg 118 Cys) in the eson 2 with a residual enzyme activity of the 29% suggesting the diagnosis of Fabry disease. Blood and urine chemistry, the slit-lamp examination and MRI of kidneys, heart and brain excluded any major organ involvement. The enzyme replacement therapy was then started almost three months ago using agasidase alfa at a dose of 0.2 mg/kg infused intravenously every two weeks but, unfortunately, no relief in the symptoms have been reported so far without any severe adverse reactions. This case report aims to point out the importance of an early diagnosis in order to prevent the progression of the disease, the multiorgan failure and to improve the long-term prognosis.

[Return to work after occupational osteoarticular injury: presentation of a multidisciplinary protocol and preliminary data]. / Reinserimento lavorativo dopo infortunio osteoarticolare sul lavoro: presentazione di un protocollo interdisciplinare e dati preliminari.

A protocol for work resumption after occupational osteoarticular injury and subsequent rehabilitation is presented: 97 patients (68 males and 29 females; mean age 42 years) were evaluated by the physiatrist and the occupational physician, providing indications based on their functional capabilities and task features. Up to date, 38 underwent follow-up at 6 months: 30 had returned to work (3 after changing tasks, 4 part-time). The mean time for work resumption was 15 days for the 26 subjects who resumed working completely, 1 month for the 4 who resumed partially. These data are encouraging, and highlight the importance of a multidisciplinary rehabilitative approach to facilitate return to work after occupational injuries.

Primary hyperoxaluria: report of an Italian family with clear sex conditioned penetrance.

We report the clinical and genetic study of a primary hyperoxaluria type I (PH1) family with two sisters homozygous for p.Gly170Arg who are still asymptomatic at age 29 and 35, and two brothers, also homozygous for the same mutation, who are affected since age 27 and 30. The clear sex difference observed in this family and in others reported in the literature fits well with the prevalence of males over females in the Italian registry. In the KO model of PH1, only male mice develop renal stones, suggesting that the sex difference may affect both oxalate production and stone formation. A likely mechanism is the sex-related expression of glycolate oxidase shown in experimental animals. The stable isotope method recently developed by Huidekoper and van Woerden for in vivo assessment of the endogenous oxalate production could help to clarify the issue in humans.

Down-modulation of type 1 interferon responses by receptor cross-competition for a shared Jak kinase.

In contrast to the large number of class I and II cytokine receptors, only four Janus kinase (Jak) proteins are expressed in mammalian cells, implying the shared use of these kinases by many different receptor complexes. Consequently, if receptor numbers exceed the amount of available Jak, cross-interference patterns can be expected. We have engineered two model cellular systems expressing two different exogenous Tyk2-interacting receptors. A receptor chimera was generated wherein the extracellular part of the interferon type 1 receptor (Ifnar1) component of the interferon-alpha/beta receptor is replaced by the equivalent domain of the erythropoietin receptor. Despite Tyk2 activation, erythropoietin treatment of cells expressing this erythropoietin receptor/Ifnar1 chimera did not evoke any detectable IFN-type response. However, a dose-dependent interference with signal transduction via the endogenous Ifnar complex was found for STAT1, STAT2, STAT3, Tyk2, and Jak1 activation, for gene induction, and for antiviral activity. In a similar approach, cells expressing the beta1 chain of the interleukin-12 receptor showed a reduced transcriptional response to IFN-alpha as well as reduced STAT and kinase activation. In both model systems, titration of the Tyk2 kinase away from the Ifnar1 receptor chain accounts for the observed cross-interference.

Polymorphic markers in MHC class II/III region: a study on Italian patients with myasthenia gravis.

With an Italian case series of 81 Italian patients and 130 controls, we analysed associations between myasthenia gravis (MG) and genetic polymorphisms in the MHC class II/III region. Increases in the frequency of the TNF-B*1, C4A*Q0, C4B*1, DRB1*03 supratype, which is likely part of the 8.1 ancestral haplotype, were maximal in females with early onset (EO) MG vs. controls [p<0.05, relative risk (RR)=9.9]. These patients showed neither a significantly high frequency of thymic hyperplasia, nor high levels of serum anti-acethylcholine receptor antibodies. The DRB1*03 allele was absent in patients with thymoma; however, in comparison with controls, occurrence of this marker was frequent in MG patients (p<0.005; RR=6.2), more frequent in females (p<0.005; RR=7.8) and most frequent in EOMG female patients (p<0.005; RR=15.1). Analysis of the TNF-B*1, C4A*Q0, C4B*1, DRB1*03 supratype and its recombinants showed that the MHC region between C4 and TNF might contain genes that influence susceptibility to MG in females. Polymorphic markers within the supratype, e.g. TNF-B*1 and C4A*Q0, might contribute to pathogenetically significant abnormalities in immune responses in a subset of female MG patients. The combined effect of other intervening genes cannot be excluded.

The receptor interaction region of Tyk2 contains a motif required for its nuclear localization.

Janus kinases have so far been viewed as enzymatic intermediates that couple a variety of cell surface receptors to downstream substrates with diverse effector functions. Tyk2 is a member of this family that is involved in the interferon-alpha/beta and interleukin-12 signaling pathways via its specific interaction with the IFNAR1 and the beta1 receptor subunits. Here, we have analyzed the subcellular distribution of the wild-type Tyk2 protein and of several mutants expressed in Tyk2-deficient human cells. Direct GFP-associated fluorescence and immunostaining showed a diffuse localization of Tyk2 throughout the cell, including the nuclear compartment. The nuclear localization of Tyk2 requires a nuclear localization signal-like motif rich in arginine residues that maps within the region mediating interaction with cytokine receptors. To address the question of the role of the Tyk2 nuclear pool in interferon-alpha/beta-induced biological effects, cells expressing a membrane-targeted form of Tyk2-green fluorescent protein were analyzed for their interferon-alpha responses. Our studies demonstrate that Tyk2 can reside in the nucleus independently of receptor binding and that the nuclear pool is dispensable for the transcriptional and anti-vesicular stomatitis virus responses induced by interferon-alpha.

A dual role for the kinase-like domain of the tyrosine kinase Tyk2 in interferon-alpha signaling.

Tyrosine kinases of the Janus kinase family initiate cellular responses through their association with receptors for alpha-helical cytokines. In addition to a tyrosine kinase domain, these enzymes possess a kinase-like (KL) domain, whose function remains elusive. To investigate the role of the KL domain of Tyk2 in interferon-alpha/beta signaling, we transfected a library of Tyk2 cDNAs containing random point mutations in KL into Tyk2-negative cells and selected for loss-of-function Tyk2 mutants. Four such mutants, V584D, G596V, H669P, and R856G, were identified through this screen. Like the wild-type Tyk2, the mutant proteins were able to sustain the level of IFNAR1 receptor protein. However, all four mutants were incapable of restoring high-affinity interferon-alpha binding in Tyk2-negative cells and were also catalytically impaired, even when transiently overexpressed. Interferon-alpha induced phosphorylation, and gene expression could be detected in V584D- or G596V-expressing cells, but not in H669P- or R856G-expressing cells. Furthermore, H669P and R856G proteins were constitutively highly phosphorylated. All together, our findings demonstrate that an intact KL domain is essential for the intrinsic catalytic activity of Tyk2 and for the establishment of a high-affinity interferon-alpha receptor complex.

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

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.

Exaggerated 17-hydroxyprogesterone response to short-term adrenal stimulation and evidence for CYP21B gene point mutations in true precocious puberty.

OBJECTIVE: Following the observation of two patients affected by true precocious puberty who went on to develop polycystic ovary syndrome (PCOS) and who were found to be heterozygotes (carriers) for 21-hydroxylase deficiency (21-OHD), we decided to evaluate the frequency of heterozygosity for adrenal 21-OHD in patients with true precocious puberty. STUDY DESIGN: We investigated 32 girls affected by true precocious puberty, by the single-dose ACTH stimulation test, HLA typing and the molecular analysis of the CYP21B gene encoding for the 21-OH enzyme, in order to detect gene deletions or point mutations. Twenty-eight cases were on LHRH analogue treatment and the remaining four, untreated owing to parental refusal, were investigated 0.5-1.5 years after spontaneous menarche. RESULTS: After ACTH testing, 13 out of the 32 (41%) cases displayed higher 17-hydroxyprogesterone (17-OHP) levels than normal but less than those found in patients affected by nonclassical adrenal hyperplasia (CAH); these levels were similar to those observed in obligate heterozygotes for CAH due to 21-hydroxylase deficiency (21-OHD). HLA typing showed a significantly increased frequency of the HLA alleles A28 and B14 which are peculiar to the HLA haplotypes of nonclassical CAH due to 21-OHD. Molecular analysis of the CYP21B gene showed that in four out of the 10 tested patients with an exaggerated 17-OHP response there were heterozygous point mutations of the CYP21B gene. In contrast, no CYP21B gene abnormalities were detected in the eight tested patients with normal 17-OHP. No differences were found between carriers and non-carriers of the 21-OHD with regard to age at onset of precocious puberty, clinical features, bone age acceleration and gonadal suppression induced by LH-RH analogue treatment. Two out of the four untreated patients who were investigated after menarche were found to be carriers of the 21-OHD; these girls showed signs of androgen excess, irregular menses and polycystic ovaries. CONCLUSIONS: A high frequency of heterozygosity for adrenal steroid 21-OHD was found in our patients with true precocious puberty. This adrenal defect does not seem to influence the pattern of central precocious puberty, but these patients require long-term follow-up as they might go on to develop polycystic ovary syndrome (PCOS). Whether or not heterozygosity of the 21-OHD may be related to the premature activation of the hypothalamo-pituitary-gonodal axis remains to be defined.

Male-specific transcription initiation of the C4-Slp gene in mouse liver follows activation of STAT5.

The mouse genes encoding the constitutively expressed complement component C4 and its closely related isoform C4-Slp (sex-limited protein), which is expressed only in male animals of several strains, provide a unique model to study sequence elements and trans-acting factors responsible for androgen responsiveness. Our previous studies have shown that hormonal induction of C4-Slp is mediated by a sex-specific pattern of growth hormone secretion. Promoter analyses in vitro have led to contradictory conclusions concerning the significance of C4-Slp-specific sequences in the 5' flanking region. Mutant mice carrying the H-2(aw18) haplotype, which is characterized by a large deletion in the S region covering the C4 and 21-OHase A genes, permit the direct in vivo analysis of C4-Slp transcription, unhindered by the presence of C4. Run-on analysis of transcription in liver nuclei of males and females of this strain demonstrated a 100-fold higher transcriptional activity in males, essentially determined at the transcription initiation level. The androgen dependence of transcription initiation was confirmed by run-on analysis of testosterone-treated females, where transcriptional activity started after 6 days of androgen treatment and reached male levels after 20 days. Conversely, the growth hormone-regulated activity of transcription factor STAT5 was already detected in liver nuclei after 48 hr of androgen treatment. Furthermore, we demonstrate that activated STAT5 recognizes in vitro two upstream gamma interferon-activated sequence (GAS) elements of the C4-Slp gene, centered at positions -1969 and -1831. We postulate that binding of STAT5 to these C4-Slp-specific GAS elements plays a crucial role in the chromatin remodelings that lead to transcriptional competence of the C4-Slp gene in the liver.

HLA complement gene polymorphisms in multiple sclerosis. A study on 80 Italian patients.

We studied C4A, C4B, and Bf complement gene polymorphisms in 80 Italian patients with multiple sclerosis (MS). We observed a significantly higher frequency of C4AQ0 allele in patients with the relapsing-remitting form of MS than in ethnically homogeneous controls. Restriction fragment length polymorphism analysis by Southern blotting of the C4/CYP21 gene complex showed that a structural gene deletion was present in 45% of patients with the C4AQ0 allele. Our data support the hypothesis that relapsing-remitting MS and primarily chronic progressive MS are immunogenetically distinct diseases; further, complement factor abnormalities typical of autoimmune diseases could influence the pathogenesis of MS.

Molecular analysis of CYP21 gene mutations carried on HLA-B14 positive haplotypes.

HLA-B14 positive haplotypes have increased frequencies in a group of patients with puberty disorders, IgA deficiency and cancer of the ovary. Clinical investigations demonstrated that all these patients have high values of 170H progesteron after the ACTH test which suggests an alterated function of 21 hydroxylase enzyme. In order to investigate whether these B14 positive haplotypes carry the same CYP21 mutation in the various diseases and controls, we have amplified by polymerase chain reaction (PCR) the sections of CYP21B gene which include amino acid positions 172 and 281 where typical mutations are known to occur in 21 hydroxylase deficiency. The presence or absence of the defined mutations was tested by oligonucleotide hybridization using oligonucleotides, labelled with DIG-ddUTP, designed to hybridize with the mutated or with the normal sequence. It was found that regardless of whether the subject tested was a patient or a healthy control the mutation at position 281 was found in all cases carrying HLA-B14, DR1 haplotype. Interestingly, this mutation does not seem to be in association with HLA-B14, DR7 haplotype. These findings suggest that CYP21 gene plays a role in all these differing diseases although it must be stressed that there may be alternative explanations for the observed data.

Association of Km3 allotype with increased serum levels of autoantibodies against muscle acetylcholine receptor in myasthenia gravis.

Association of immunoglobulin allotypes with myasthenia gravis was examined in a set of 84 patients presenting autoantibodies against muscle acetylcholine receptor (AChR). Km and G1m(f)/G1m(z) allotypes were determined by PCR-amplification followed by hybridization with allele-specific oligonucleotides. The Km3 kappa light chain allotype was found to be associated with significantly increased serum levels of anti-AChR autoantibodies in myasthenic patients (P = 0.014). We hypothesize that an allelic polymorphism of a regulatory (enhancer) sequence closely linked to the C kappa gene segment could account for our finding.

Adrenocorticotrophin stimulation and HLA polymorphisms suggest a high frequency of heterozygosity for steroid 21-hydroxylase deficiency in patients with Turner's syndrome and their families.

OBJECTIVE: Following the chance observation of congenital adrenal hyperplasia in a patient with Turner's syndrome we decided to evaluate the incidence of 21-hydroxylase deficiency (21-OHD) in patients with Turner's syndrome and in their relatives. SUBJECTS: Fifty-two patients with Turner's syndrome (mean age +/- SD 14.7 +/- 5.6 years) and 26 relatives were studied. MEASUREMENTS: 17-Hydroxyprogesterone (17-OHP) serum levels before and after i.m. administration of 0.25 mg of ACTH(1-24) were evaluated in patients with Turner's syndrome and relatives. In Turner patients basal testosterone and dehydroepiandrosterone concentrations were determined. The results of ACTH tests were analysed according to HLA class I and II alleles of subjects. RESULTS: The baseline 17-OHP was in the range of the classical form of 21-OHD in one Turner patient, who had severe clitoral enlargement since birth. In 11 patients the stimulated 17-OHP serum level was higher than in normal controls and similar to that found in 21-OHD heterozygous subjects. Clitoral enlargement was significantly more frequent in patients with high stimulated 17-OHP levels (P < 0.001). The frequency of heterozygous-type responses was higher in Turner subjects (1:4.6) than in the Italian population (1:47 for the classic form and 1:9.5 for the non-classic form of the disease). In our patients the frequencies of HLA antigens and haplotypes, usually associated with 21-OHD, were different compared to the controls. HLA-B8, which is negatively associated to 21-OHD, was less frequent in Turner patients than in controls and absent in those with an elevated 17-OHP level. HLA-B14, B22 and B35 were more frequent, though not significantly so, in Turner patients than in controls and even more so in the group with an elevated 17-OHP level. The same investigations performed in 26 relatives of the Turner patients showed a high frequency of carriers of 21-OHD and three subjects with the cryptic form of the disease. CONCLUSIONS: Although in the literature there are only two reports of the association of Turner's syndrome and 21-OHD, on the basis of our experience this association was more frequent, in the Italian population. Since some of the typical signs of 21-OHD (short final stature, varying degrees of virilization, menstrual irregularities, amenorrhoea, infertility) in patients with Turner's syndrome could also be attributed to the chromosomal abnormality, it is therefore more difficult to diagnose 21-OHD in Turner subjects. Adrenal function should be assessed, at least in the presence of clitoral enlargement, in patients with Turner's syndrome, particularly if their karyotype does not contain a Y chromosome. The hypothesis of the presence of cryptic Y chromosome material in these patients should also be considered.

Lack of both spinal fluid oligoclonal bands and complement 4A protein in an MS patient.

We report the case of a multiple sclerosis (MS) patient without oligoclonal bands (OCB) in three cerebrospinal fluid samples. Restriction fragment length polymorphism analysis of Class 3 genes with radiolabelled specific cDNA probes showed a homozygous deletion of the complement 4A (C4A) genes. Consequent lack of C4A protein can alter the development of humoral immune response probably preventing OCB production. Our observation may suggest a link between a rare immunogenetic pattern and the absence of OCB in MS.

Polymorphisms of HLA class III genes in allergic contact dermatitis.

HLA class III polymorphisms (BF, C4A, C4B) were studied in 55 patients of different age and sex suffering from allergic contact dermatitis, with sensitization to different substances. In the overall group of patients no significant correlation between the disease and HLA markers was found. BF F allele was present in 34% and BS S in 64% of patients suffering from allergic contact dermatitis to nickel only versus 16.45% (relative risk, RR = 2.61) and 80.76% (RR = 0.42), respectively, of the control population. The BF FB subtype frequency was 23.91% versus 7.57% in the control samples (RR = 3.88). We thus hypothesize that this polymorphic serum protein might be involved in the pathogenesis of allergic contact dermatitis to nickel.

[HLA polymorphism in the susceptibility or resistance to dilated cardiomyopathy]. / Il polimorfismo HLA nella suscettibilità o resistenza alla cardiomiopatia dilatativa.

Much of the surrounding studies on the association between HLA and diseases reflects a new insight into the key role of HLA molecules in the generation and regulation of the immune response. HLA molecules, on the surface of antigen presenting cells, bind foreign peptides. This HLA-antigen complex is then recognized by T lymphocytes and triggers the alloresponse against the peptide. Since many diseases associated with peculiar HLA antigens are thought to be autoimmune, the idea that certain Major Histocompatibility Complex (MHC) molecules could form complexes with self-peptides in anomalous ways, leading to an autoimmune reaction, is particularly attractive. Recent advances in molecular technology, x-ray crystallography and DNA studies have allowed the determination of the three-dimensional structure of some HLA class I and II molecules and also the amino acid sequences involved in binding of antigen fragments. This new information has prompted a search for differences, at the amino acid level, between HLA alleles previously shown to be positively or negatively associated with a pathology. Our own experience on the immunogenetic aspect of dilated cardiomyopathy (DCM) allowed us to assess some predisposing (HLA-DR4, DR5, C4A4) and protective (HLA-DR3) factors for DCM. Clinical heterogeneity also seems to imply a peculiar genetic background. The actual research is addressed to the study of the antigen binding site sequences and to the consideration of other new loci such as those entrapped within the HLA class III subregion (HSP70) and those lying within the class II region (PSF).

Sarcoidosis and major histocompatibility complex genes with special emphasis on BF F subtypes.

Frequencies for HLA class I and II histoglobulins and C4A, C4B, BF complement proteins were performed for 59 sarcoidosis patients. The DR5 allele was present in 55.9% of patients as compared to 31.5% of controls. We noticed that its increase was more relevant in males and in those with a poor prognosis. BF F allele was significantly over-represented in patients (29.09% vs. 19.15% of control), especially in women. Special emphasis was given to BF F subtyping, to define an association between a particular BF F subtype and patient's sex or disease outcome.