Recommendations for pre-symptomatic genetic testing for hereditary transthyretin amyloidosis in the era of effective therapy: a multicenter Italian consensus.

Hereditary transthyretin amyloidosis (ATTRv, v for variant) is a late-onset, autosomal dominant disease caused by progressive extracellular deposition of transthyretin amyloid fibrils, leading to organ damage and death. For other late-onset fatal diseases, as Huntington's disease, protocols for pre-symptomatic genetic testing (PST) are available since decades. For ATTRv, limited experience has been reported to date, mostly gathered before the availability of approved therapies. We aimed at developing recommendations for a safe and feasible PST protocol in ATTRv in the era of emerging treatments, taking also into account Italian patients' characteristics and healthcare system rules. After an initial survey on ongoing approaches to PST for ATTRv in Italy, two roundtable meetings were attended by 24 experts from 16 Italian centers involved in the diagnosis and care of this disease. Minimal requirements for PST offer and potential critical issues were highlighted. By November 2019, 457 families affected by ATTRv with 209 molecularly confirmed pre-symptomatic carriers were counted. The median age at PST was 41.3 years of age, regardless of the specific mutation. Half of the Italian centers had a multidisciplinary team, including a neurologist, an internist, a cardiologist, a medical geneticist and a psychologist, although in most cases not all the specialists were available in the same center. A variable number of visits was performed at each site. Experts agreed that PST should be offered only in the context of genetic counselling to at risk individuals aged 18 or older. Advertised commercial options for DNA testing should be avoided. The protocol should consist of several steps, including a preliminary clinical examination, a pre-test information session, an interval time, the genetic test and a post-test session with the disclosure of the test results, in the context of an experienced multidisciplinary team. Recommendations for best timing were also defined. Protocols for PST in the context of ATTRv can be refined to offer at risk individuals the best chance for early diagnosis and timely treatment start, while respecting autonomous decisions and promoting safe psychological adjustment to the genetic result.

An accurate and reliable real time SNP genotyping assay for the HLA-G +3142 bp C>G polymorphism.

Human leukocyte antigen (HLA)-G is a non classical HLA class I antigen with immuno-modulatory functions. The HLA-G gene is characterized by a +3142C>G variant in the 3' untranslated region which is suggested to control protein production and to be associated with pathological conditions. DNAs form 221 randomly selected healthy subjects were genotyped for HLA-G +3142C>G polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (BaeGI), real-time PCR and sequencing. The 19% of the PCR-RFLP heterozygous samples were genotyped as 3142GG by real-time PCR and sequencing. This disagreement is caused by digestion efficiency in PCR-RFLP. This real-time PCR method will guarantee an accurate genotyping for future research and clinical purposes, where large cohorts should be tested.

A functional role for soluble HLA-G antigens in immune modulation mediated by mesenchymal stromal cells.

BACKGROUND: It has been suggested that soluble factors produced by bone marrow (BM) mesenchymal stromal cells (MSC) play a fundamental role in mediating immune modulation. HLA-G antigens (Ag) are major histocompatibility complex (MHC) class Ib molecules characterized by a limited polymorphism and a splicing mechanism that regulates the production of membrane-bound and soluble isoforms. Interleukin-10 (IL-10) cytokine is one of the main up-modulators of soluble HLA-G Ag (sHLA-G) production by CD14+ peripheral blood monocyte cells and increased IL-10 levels are reported to be associated with MSC immune modulation. METHODS: We investigated, by specific enzyme-linked immunosorbent assay (ELISA), the possible role of sHLA-G molecules in the inhibition of the peripheral blood mononuclear cell (PBMC) response to phytohemagglutinin (PHA) mediated by MSC from different sources. RESULTS: There was a significant correlation between the presence of increased levels of sHLA-G and IL-10 in the MSC/PBMC/PHA culture supernatants and lymphoproliferative inhibition. Neutralizing experiments performed with monoclonal Ab directed against HLA-G and IL-10 molecules confirmed the inhibitory ability of sHLA-G Ag. Furthermore, exogenous IL-10 induced sHLA-G molecule secretion by MSC alone in a polymorphic way, while a longitudinal analysis confirmed the loss of MSC inhibitory functions in relation to in vitro MSC aging. DISCUSSION: Overall the results obtained suggest a functional role for sHLA-G molecules in inhibiting the PBMC response mediated by MSC. Moreover, the ability of IL-10 to induce sHLA-G Ag production by MSC alone could be proposed as a marker of MSC functional ability.

HLA-G genotype and HLA-G expression in systemic lupus erythematosus: HLA-G as a putative susceptibility gene in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease mainly mediated by the deposit of immune complexes and defects in T lymphocytes and antigen-presenting cells along with a high production of T-helper 2 cytokines. A tolerance-inducible function of nonclassical class Ib human leukocyte antigen (HLA)-G molecule in innate and adaptive cellular responses has been reported, suggesting a role in inflammatory diseases. A 14 bp sequence insertion/deletion polymorphism (rs16375) in the 3'-untranslated region of the HLA-G gene has been associated to the stability of HLA-G messenger RNA. The insertion of the 14 bp sequence seems to be associated with lower levels of soluble HLA-G (sHLA-G). The aim of this study was to evaluate the possible association of the presence of the 14 bp sequence (+14 bp) with SLE. We have HLA-G genotyped 200 SLE patients and 451 healthy control subjects (HS; Italian) and analyzed the plasma levels of sHLA-G and interleukin-10 (IL-10) in a subset of SLE patients and healthy subjects (Italian and Danish). A significant increase of the +14 bp HLA-G allele was detected in the Italian SLE patients compared with HS [P = 0.003, OR 1.44 (95% CI 1.13-1.82)]. A significant increased frequency of HLA-G +14/+14 bp and a decreased frequency of HLA-G -14/-14 bp were observed in SLE patients. There median concentration of sHLA-G was significantly lower in the plasma of SLE patients compared with that in the plasma of healthy controls (P < 0.0001). Furthermore, the results confirmed higher concentrations of IL-10-positive plasma in SLE patients. These results support a potential role for HLA-G in the susceptibility of SLE.

CSF levels of soluble HLA-G and Fas molecules are inversely associated to MRI evidence of disease activity in patients with relapsing-remitting multiple sclerosis.

Cerebrospinal fluid (CSF) concentrations of soluble human leukocyte antigen class I (HLA-I) (sHLA-I), HLA-G (sHLA-G) and anti-apoptotic Fas (sFas) molecules were measured by enzyme linked immunosorbent assay technique in 65 relapsing-remitting (RR) MS patients classified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. Sixty-four patients with other inflammatory neurological disorders (OIND) and 64 subjects with noninflammatory neurological disorders (NIND) served as controls. CSF concentrations were higher in RRMS and in OIND than in NIND patients for sHLA-I (P < 0.02), greater in RRMS than in OIND and in NIND for sHLA-G (P < 0.001 and P < 0.01, respectively) and lower in RRMS than in OIND and in NIND for sFas (P < 0.001 and P < 0.02, respectively). An increase in CSF levels was identified in MRI active RRMS for sHLA-I (P < 0.01) and in MRI stable RRMS for sHLA-G (P < 0.01), whereas CSF values of sFas were decreased in RRMS without Gd-enhancing lesions (P < 0.02). In MS patients with no evidence of MRI disease activity, a trend towards an inverse correlation was found between CSF concentrations of sHLA-G and sHLA-I and between CSF levels of sHLA-G and sFas. Our results indicate that enhanced CSF levels of sHLA-I antigens most likely represent an indirect manifestation of intrathecal immune activation taking place in neuroinflammation. Conversely, reciprocal fluctuations in CSF sHLA-G and sFas levels observed when MRI disease activity resolved suggest that sHLA-G could play an immunomodulatory role in MS through Fas/FasL-mediated mechanisms.

Soluble HLA-G molecules in follicular fluid: a tool for oocyte selection in IVF?

Currently, different approaches are used to select oocytes for in vitro fertilization (IVF) procedures, but they do not assure a significant association with the pregnancy outcome. Since several studies have proposed the expression of HLA-G antigens in early embryos to be a possible marker of elevated implantation rate, we have investigated the presence of soluble HLA-G molecules in 50 follicular fluids (FFs). The results have shown soluble HLA-G antigens (sHLA-G) in 19/50 (38%) FFs. Furthermore, we have related the presence of sHLA-G molecules in FFs to detection of the soluble antigens in culture supernatants of the corresponding fertilized oocyte, evidencing a significant relationship (p=1.3 x 10(-6); Fisher exact p-test). Specific ELISA and Western blot approaches identified both HLA-G5 and soluble HLA-G1 molecules in FFs while immunocytochemical analysis indicated polymorphonuclear-like and granulosa cells as responsible for production of sHLA-G1 and HLA-G5 molecules. In contrast, only sHLA-G1 antigens were detected in culture supernatants of fertilized oocytes. Overall, these results suggest a role for sHLA-G molecules in the ovulatory process and propose the FFs analysis for sHLA-G molecule presence as a useful tool for oocyte selection in IVF.

Intrathecal synthesis of soluble HLA-G and HLA-I molecules are reciprocally associated to clinical and MRI activity in patients with multiple sclerosis.

The aim of this study was to provide further insight into the effective contribution of classical soluble HLA-A, B and C class Ia (sHLA-I) and non-classical soluble HLA-G class Ib (sHLA-G) molecules in immune dysregulation occurring in multiple sclerosis (MS). We evaluated by enzyme-linked immunosorbent assay (ELISA) technique intrathecal synthesis and cerebrospinal fluid (CSF) and serum levels of sHLA-I and sHLA-G in 69 relapsing-remitting (RR), 21 secondary progressive (SP) and 13 primary progressive (PP) MS patients stratified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also tested, as neurological controls, 91 patients with other inflammatory neurological disorders (OIND) and 92 with non-inflammatory neurological disorders (NIND). Eighty-two healthy volunteers served as further controls for sHLA-I and sHLA-G determinations. An intrathecal production of sHLA-I and sHLA-G detected by specific indexes was significantly more frequent in MS patients than in controls (P<0.01). An intrathecal synthesis of sHLA-I was prevalent in clinically (P<0.02) and MRI active (P<0.001) MS, whereas a CSF-restricted release of sHLA-G predominated in clinically (P<0.01) and MRI stable (P<0.001) MS. sHLA-I levels were low in the serum of clinically active (P<0.001) and high in the CSF of MRI active (P<0.01) MS. Conversely, sHLA-G concentrations were decreased in the serum of clinically stable MS (P<0.01) and increased in the CSF of MRI inactive MS (P<0.001). The trends towards a negative correlation observed between CSF and serum concentrations and intrathecal synthesis of sHLA-I and sHLA-G in patients without evidence of clinical and MRI activity confirmed that intrathecal production and fluctuations in CSF and serum concentrations of sHLA-I and sHLA-G were reciprocal in MS. Our results suggest that, in MS, a balance between classical sHLA-I and non-classical sHLA-G products modulating both MRI and clinical disease activity in opposite directions may exist.

Embryonic soluble HLA-G as a marker of developmental potential in embryos.

BACKGROUND: In human reproduction, embryo implantation is complex and poorly understood. At present, no single markers are used in routine treatment to assay biochemical functions of the human embryo. Soluble human leukocyte antigen-G (sHLA-G) could be considered a possible marker of embryo developmental potential. It is localized primarily on the extravillous trophoblast, making this antigen a potential mediator of immune interaction at the maternal-fetal interface during gestation. METHODS: Soluble-HLA-G levels were evaluated by an enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibody MEM-G9. It was evaluated in 318 media of single embryo cultures. We correlated the presence of sHLA-G with embryo morphology and the pregnancy obtained in that treatment cycle. RESULTS: No correlation was found between embryo morphology and sHLA-G levels. Pregnancy was observed only when the medium of at least one transferred embryo contained sHLA-G. In 26 out of 66 patients, none of the obtained embryos showed any detectable sHLA-G molecules and no pregnancy occurred. CONCLUSIONS: From our results, we propose sHLA-G as a potential marker of embryo development: the sHLA-G ELISA can be a useful biochemical assay in addition to embryo morphology in embryo selection for transfer in IVF treatment if there are other embryos with the same morphology.

Allogeneic gene-modified tumour cells in metastatic kidney cancer. Report II.

In a limited study, comprising only ten patients, we have previously reported that allogeneic irradiated RCC-cell-line cells, engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells were used to vaccinate MRCC patients in progression of disease and also receiving IL-2 immunotherapy. The cells, admixed to autologous TC, were administered subcutaneously. We now report an extended study on thirty patients and one hundred thirty-one controls. Patients received 4-20 injections (mean 10 +/- 4), containing an average of 92 x 10(6) +/- 45 x 10(6) ACHN-IL-2 transfected cells (a minimum of 25 x 10(6), and a maximum of 200 x 10(6)). Autologous TC, admixed to allogeneic, were also administered by 4-16 s.c. injections (mean 7 +/- 3), i.e. a total of 12 x 10(6)-160 x 10(6) cells. Vaccination was administered during 73-1451 (307 +/- 316) days, and the follow-up continued for 1122 +/- 1240 days (106-5137). Throughout this period, the patients continued receiving the previously set immunotherapy treatment. No adverse side effects related to the treatment were noticed. One complete and four partial tumour responses were observed, as well as nine cases of stable disease. Thirteen patients died in the treated group (43%) and 63 (44%) in the control group. Responding patients resumed progression in 4-11 months and died 18 and 36 months after beginning the vaccine therapy. The Gehan Wilcoxon's test showed a significantly (P < 0.01) better survival in the vaccinated patients compared to that of the controls. Thus, we confirm, in an increased number of patients and an extensive follow-up, that our vaccination protocol is safe, devoid of adverse side effects, and promising.

Allogeneic gene-modified tumour cells in metastatic kidney cancer. Preliminary report.

An allogeneic irradiated RCC cell line, engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells, was used to vaccinate ten MRCC patients in progression of disease in spite of IL-2 immunotherapy. The cells were administered subcutaneously and/or intra-tumourally. Sixty-four MRCC patients in progressive disease, not treated by vaccination but receiving similar IL-2 immunotherapy, were considered as the control group. Patients received 4-16 injections (mean 9 +/- 4), containing an average of 10.6 x 10(7) +/- 7.7 x 10(7) ACHN-IL-2-transfected cells (a minimum of 4 x 10(7), and a maximum of 31 x 10(7)). Four patients also received intra-tumour injections. Vaccination was administered during 30-418 days, and the follow-up continued for 649 +/- 353 days (190-1342). Throughout this period, the patients continued receiving the previously set immunotherapy treatment. No adverse side effects related to the treatment were observed. One complete and one partial tumour response were observed, as well as two stable and one no-relapse disease. All but one patient died. Responding patients resumed progression in 4-11 months and died 18 and 36 months after beginning the vaccine therapy. In spite of the small number of treated patients, Wilcoxon's test showed a significant (P < 0.05) improvement of the survival in the vaccinated group compared to that of the control. The described vaccination protocol seems safe, devoid of adverse side effects and promising. It warrants further investigation.

Increased proliferation rate of lymphoid cells transfected with the P2X(7) ATP receptor.

Human leukocytes can express the P2X(7) purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X(7) cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serum-starved P2X(7) transfectants is abolished by the P2X(7) receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X(7)-transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.

Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages.

Extracellular ATP (ATPe) is known to cause release of processed IL-1 beta from LPS-treated macrophages and microglial cells. IL-1 beta release is fast and thought to be associated with cell death. We have reinvestigated this process to identify 1) the purinergic receptor involved; 2) the relationship to cell death; and 3) pharmacologic agonists or antagonists able to modulate IL-1 beta release. Our data confirm that ATPe is a powerful stimulus for IL-1 beta release from LPS-treated human macrophages; however, we also show that IL-1 beta release is not necessarily associated with cell death, as it occurs at lower ATP concentrations and much earlier than leakage of cytoplasmic markers. The selective purinergic P2Z receptor agonist benzoylbenzoyl ATP was at least one order of magnitude more powerful than ATP, but also had a strong cytotoxic effect. 2-Methylthio-ATP was equipotent as ATPe at the optimal concentration of 1 mM, but markedly inhibitory at higher concentrations. The irreversible P2Z blocker-oxidized ATP completely inhibited ATPe-induced IL-1 beta release. IL-1 beta release also was inhibited by increasing the K+ concentration of the incubation medium. These data suggest that ATPe triggers IL-1 beta via the purinergic P2Z receptor recently shown to be expressed by human macrophages and identified as a new member of the P2X family (P2X7), and provide pharmacologic tools for the modulation of IL-1 beta release in vitro and, possibly, in vivo.

An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes.

We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation.

Increased sensitivity of DMD lymphoblastoid cell to low doses of X-irradiation.

Several cell membrane abnormalities affecting various cell populations have been reported in Duchenne muscular dystrophy (DMD) by different investigators. In peripheral blood lymphocytes intrinsic cellular membrane defect evidentiated by impairment of capping capacities has been repeatedly obtained, suggesting that DMD product could act in such cellular phenotype at the cytoskeletal compartment. It has been previously reported that lymphoid cells are characterized by high radiosensitivity. On the assumption that DMD phenotypes could increase this susceptibility, we have compared the radiosensitivity of normal and DMD lymphoblastoid cell lines (LCLs) to small doses (0-2Gy) of x-irradiation. The results obtained suggest an increased sensitivity of DMD cells without Ca++ uptake or apoptotic phenomena, associated with an effect upon cell cycle length.

Responses to extracellular ATP of lymphoblastoid cell lines from Duchenne muscular dystrophy patients.

We have observed a striking difference in the response to extracellular ATP in lymphoblastoid cell lines established from Duchenne muscular dystrophy patients and normal subjects. Duchenne muscular dystrophy cells stimulated by extracellular ATP underwent a large increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization, while normal cell lines were little or not at all responsive. These changes in intracellular ion homeostasis were due to activation of an ATP-gated membrane channel permeable to Na+ and Ca2+, with little or no contribution of Ca2+ release from intracellular stores. The channel was selectively activated by ATP, since other purine/pyrimidine nucleotides were ineffective, and it was inhibited by pretreatment with oxidized ATP, a compound previously reported to irreversibly inhibit P2 purinergic receptors. In the presence of extracellular ATP, lymphoblastoid cells established from Duchenne muscular dystrophy patients, but not from healthy controls, underwent rounding and swelling and eventually lysed. The results of this study suggest that lymphoblastoid cells isolated from Duchenne muscular dystrophy patients are eminently sensitive to stimulation by extracellular ATP.

Fibroblasts increase adhesion to neutrophils after stimulation with phorbol ester and cytokines.

In our research there was spontaneous adhesion between resting fibroblasts and neutrophils in vitro which could be increased by stimulating either the coculture of cells or each cell type separately with various stimulants. Interferon-gamma, interleukin-1, and interleukin-6 significantly increased adhesion; however, the highest adhesive response was obtained when cocultures were treated with phorbol myristate acetate (PMA). As PMA-stimulated fibroblasts show the highest adhesion to resting neutrophils, it was suggested that adhesion was primarily due to an effect on fibroblasts. Without Mg2+ PMA did not stimulate fibroblast adhesion, whereas in the absence of Ca2+ the response was only partially reduced. Spontaneous adhesion was independent of both neutrophil integrins and fibroblast ICAM-1, whereas cytokine-stimulated adhesion was blocked by mAbs against ICAM-1; PMA-stimulated adhesion was not affected by mAbs anti-ICAM-1, but was partially inhibited by mAbs anti-beta 2 integrins. These results suggested the presence of mechanisms able to modulate the adhesive fibroblast-neutrophil interaction in inflammatory and wound healing processes.

Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains.

The immunosuppressive action of phosphatidylserine has been studied in mitogen-activated human peripheral blood mononuclear cells. The addition of phospholipid (10-60 nmol/10(6) cells) causes a dose-dependent inhibition of DNA synthesis induced by PHA, anti-CD3 mAb, allogeneic lymphocytes and tetradecanoylphorbol acetate plus ionomycin. In contrast, the interleukin-2-dependent DNA synthesis is less affected. Flow cytometric analysis and binding of radioiodinated interleukin-2 show that the phospholipid prevents the expression of interleukin-2 and transferrin receptors. Removal of monocytes by adherence does not change the action of phosphatidylserine. Furthermore, the phospholipid is equally effective in preparations depleted of CD4+ or CD8+ lymphocytes. Phosphatidylinositol partly reproduces the action of phosphatidylserine. Phosphatidic acid, phosphatidylglycerol and phosphatidylcholine are inactive. Also unsaturated phosphatidylserine analogues inhibit DNA synthesis whereas saturated phosphatidylserines do not. The data suggest that phosphatidylserine mainly affect the steps of T cell activation preceding the production of interleukin-2 and the expression of its receptor. The phosphorylserine headgroup and the unsaturated acyl chains contribute to this effect.

HLA B51 antigen associated with neutrophil hyper-reactivity.

HLA B51 specificity is strongly associated with Behcet's disease (BD) (for references see Baricordi et al., 1986), a multisystem vasculitis of unknown aetiology, for which an immunological pathogenesis has been proposed (O'Duffy et al., 1983; O'Duffy et al., 1990). Neutrophil abnormalities observed in BD patients even during clinical remission suggest prominent involvement of these phagocytic cells in the pathogenesis of the disease (Niwa et al., 1982). In order to clarify how HLA B51 antigen might confer susceptibility to BD, we have investigated neutrophil function in 13 B51-positive and 13 B51-negative healthy subjects. Lymphocyte spontaneous proliferation and circulating immune complexes were also evaluated. Whereas neutrophils from B51-positive subjects showed an increase in the chemotactic response toward casein (P = 0.003) and LPS (P = 0.033) and also in the PMA-induced superoxide production (P = 0.008) no evidence of enhanced lymphocyte activation emerged. These results suggest that the HLA region can exert a regulatory control on PMN functions.

Stable hyperzinchemic condition in patients with Behçet's syndrome. Lack of correlation with in vitro cellular immune response capacity.

We have observed a significant increase of zinc concentration in plasma and erythrocytes in a group of Italian Behcet patients, evidencing a rare hyperzinchemic condition associated with the disease. The increased zinc level observed does not show any relation to the presence of therapy or with the impairment of the blastogenic response to HSV1, a specific cellular defect in Behcet disease, confirmed in the present study. The possible biological meaning of these observations is briefly discussed.

Impairment of capping in lymphoblastoid cell lines of Duchenne patients indicates an intrinsic cellular defect.

Duchenne muscular dystrophy (DMD) is a lethal sex-linked degenerative disorder of the muscle in man. Generalized cell membrane abnormalities seem to be involved in the pathogenesis of the disease; in particular, the impairment of lymphocyte capping capacities has been repeatedly confirmed. To clarify whether capping impairment is a consequence of factors related to the activity of the disease or an expression of an intrinsic cellular defect, we have investigated the capping capacities of DMD EBV-transformed cell lines. The results indicate a significant impairment of capping capacity in cultured cell lines, providing evidence for an intrinsic cell deficiency in DMD.