Patrizia Luppi

How immune mechanisms are affected by pregnancy

Pregnancy requires physiologic adaptations in all maternal systems, including the immune system. This process is complex and includes modifications at different levels and compartments of the maternal immune system. Although many of these changes are only partially explored and understood, recent investigations have showed that during pregnancy maternal circulating immune cells undergo modifications in cell counts, phenotypes, functions and ability to produce soluble factors, such as cytokines. The ultimate goal is to establish and maintain a successful pregnancy, which involves a state of selective immune tolerance, immunosuppression and immunomodulation in the presence of a strong anti-microbial immunity. The mammalian immune system has evolved to coexist with these needs by down-regulating potentially dangerous T-cell-mediated immune responses, while activating certain components of the innate immune system, such as monocytes and neutrophils. This unique dysregulation between different components of the immune system plays a central role in the maternal adaptation to pregnancy.

Revised Guidelines for Neonatal Screening Programmes for Primary Congenital Hypothyroidism

Since the first guidelines for neonatal screening for congenital hypothyroidism (CH) were issued by ESPE in 1993 [1], there have been considerable advances in our understanding of CH and our appreciation of the various geographical and logistic difficulties involved. Therefore, an updating of the guidelines is overdue. Experience from countries where screening began in the late 1970s and early 1980s has indicated that treatment should be started no later than the first 2 weeks of life using a ‘high’ dosage regime of L-thyroxine (10–15 Ìg/kg/day). It has also been shown that the quality of long-term outcome is closely related to the quality of follow-up. In Eastern Europe, screening programmes for CH have either been started or will start soon in almost all countries, and although many programmes are operating satisfactorily, it is important to standardise screening procedures and management of suspected cases as much as possible in order to optimise outcome. A degree of uniformity throughout Europe would not only facilitate early detection and treatment of individual patients but give insight into the economic and epidemiological aspects of the screening programmes as well as the epidemiological aspect of CH.

Molecular Basis of Evolutionary Loss of the α1,3-Galactosyltransferase Gene in Higher Primates

Galactose-α1,3-galactose (αGal) epitopes, the synthesis of which requires the enzyme product of α1,3-galactosyltransferase (α1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The αGal-negative species as well as mice with deletion of the α1,3GT gene produce abundant anti-αGal antibodies. The evolutionary loss of αGal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the α1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of αGal expression, may have implications in medical research.

Preeclampsia Activates Circulating Immune Cells with Engagement of the NF‐κB Pathway

Compelling evidence implicates peripheral immune activation in the pathophysiology of preeclampsia. Polymorphonuclear neutrophils appear to be the cells most strongly affected, with changes in expression of surface markers and release of granule enzymes. Here, we investigated activation in additional leukocyte populations among women with preeclampsia.

Human proinsulin C-peptide reduces high glucose-induced proliferation and NF-κB activation in vascular smooth muscle cells

Excessive proliferation of vascular smooth muscle cells (VSMCs) is one of the primary lesions in atherosclerosis development during diabetes. High glucose triggers VSMC proliferation and initiates activation of the transcription factor nuclear factor (NF)-kappaB. Recently, clinical studies have demonstrated that replacement therapy with C-peptide, a cleavage product of insulin, to type 1 diabetic (T1D) patients is beneficial on a variety of diabetes-associated vascular complications. However, the mechanisms underlying the beneficial activity of C-peptide on the vasculature in conditions of hyperglycemia are largely unknown. The effects of C-peptide on the proliferation of human umbilical artery smooth muscle cell (UASMC) and aortic smooth muscle cell (AoSMC) lines cultured under high glucose for 48 h were tested. To gain insights on potential intracellular signaling pathways affected by C-peptide, we analyzed NF-kappaB activation in VSMCs since this pathway represents a key mechanism for the accelerated vascular disease observed in diabetes. High glucose conditions (25 mmol/L) stimulated NF-kappaB-dependent VSMC proliferation since the addition of two NF-kappaB-specific inhibitors, BAY11-7082 and PDTC, prevented proliferation. C-peptide at the physiological concentrations of 0.5 and 1 nmol/L decreased high glucose-induced proliferation of VSMCs that was accompanied by decreased phosphorylation of IkappaB and reduced NF-kappaB nuclear translocation. These results suggest that in conditions of hyperglycemia C-peptide reduces proliferation of VSMCs and NF-kappaB nuclear translocation. In patients with T1D, physiological C-peptide levels may exert beneficial effects on the vasculature that, under high glucose conditions, is subject to progressive dysfunction.

Idiopathic Dilated Cardiomyopathy: A Superantigen-Driven Autoimmune Disease

BACKGROUND Many cases of idiopathic dilated cardiomyopathy (IDC) result from an inflammatory myocarditis. The specific immunological mechanisms are not yet defined. Various autoimmune diseases are associated with superantigen-triggered immune responses, resulting in massive T-cell activation and tissue damage. We studied 3 cases in a search for evidence that such a phenomenon is also implicated in IDC. METHODS AND RESULTS Myocardial, lymph node, and thymic tissue samples were obtained from IDC patients who were undergoing heart transplantation. Infiltrating immune-cell phenotypes and gene expression of T-cell receptor (TCR) alpha- and beta-chain variable (Valpha and Vbeta) regions were analyzed by immunostaining and polymerase chain reaction. Similar technical approaches were used to assay the tissues for the presence of coxsackievirus B (CVB). In all the specimens analyzed, an overexpression of the TCR Vbeta3, Vbeta7, and Vbeta13.1 gene families was detected among the infiltrating T cells. These tissues were also found to be CVB3-positive. In vitro exposure of peripheral blood mononuclear cells to lysates of cells infected with CVB3 was capable of stimulating expansion of the same TCR Vbeta families. The TCR Valpha repertoire was never found to be skewed. CONCLUSIONS A superantigen-mediated immune response is involved in human heart disease. CVB3 may directly or indirectly trigger this response, suggesting a possible mechanistic link between CVB infection and myocarditis development progressing to IDC.

Retroviral superantigens and type 1 diabetes mellitus

Matters Arising diagnosed for less than three weeks), and four healthy Retroviral Superantigens and Type 1 adult controls. All subjects were of Caucasoid extrac-Diabetes Mellitus tion. RT-PCRs, with the Conrad et al. (1997) protocol and reagents, generated a band of the expected size (489 bp) for IDDMK 1,2 22 with U3-R-poly(A) primer from In 1994, Conrad et al. reported initial results that rekin-all samples, but in both the presence and absence of dled the debate about the involvement of an infectious RT. The U3-R-poly(A) primer also generated a 489 bp agent in type 1 diabetes. They analyzed the T cell (anti-band from genomic DNA samples. The RT-PCR product gen) receptor repertoire of T lymphocytes that had in-hybridized with the U3-R probe, as reported by Conrad vaded the pancreatic islets of two recently deceased, et al. (1997). However, DNase treatment of the RNA acutely diabetic patients. Antigen receptors carrying samples prevented generation of the PCR product and the V␤7 variable region were shown to be strikingly en-its detection by Southern blot. We could not achieve riched in the T cells, evoking the involvement of a super-specificity for RNA, by extending 5Ј the U3-R-poly(A) antigen (SAG). Recently, Conrad et al. (1997) (see also primer from four to 11Ts and/or by subtracting eight of Benoist and Mathis, 1997) described the isolation of a the 3Ј nucleotides, whereas the Pittsburgh and London hitherto unknown endogenous HERV-K10-like retroviral laboratories (see below) detected viral RNAs with modi-genome, IDDMK 1,2 22, related to mouse mammary tumor fied primers, although these were also not disease spe-virus, whose env gene encoded a SAG. The SAG stimu-cific. It should be noted, however, that the various modi-lated V␤7-bearing T cells, but not those that expressed fied primers were not identical. other V␤ genes. Transcripts derived from, or at least Conrad et al. (1997) added RT and PCR reagents to related to, the IDDMK 1,2 22 genome were detectable the same reaction tube. When we added the PCR re-by RT-PCR in the plasma of 10/10 newly diagnosed type 1 agents after the RT reactions, the yield of the U3-R-diabetes patients, but not from 10 age-matched, nondia-poly(A) product significantly increased, but again it was betic control individuals. In order to detect IDDMK 1,2 22 not specific for RNA. The increased yield was most likely RNA, Conrad et al. (1997) performed RT-PCR on total due to separation of Pwo, a DNA polymerase with 3Ј–5Ј …

Genetic background and environment contribute synergistically to the onset of autoimmune diseases.

Autoimmune diseases result from the breakdown of “self” tolerance. Environmental factors appear to be responsible for triggering this errant immune response, directed against self-tissue determinants, only when a susceptible genetic background is present in an individual. Autoimmune diseases, normally characterized by their association with certain HLA alleles, also share other features: the presence of autoantibodies, autoreactive T lymphocytes, and an intermittent clinical course of exacerbations and remissions. In cases of organ-specific diseases, as well as in cases of multi-system autoimmune diseases, viruses are increasingly implicated as such environmental triggers. Current molecular biology techniques have permitted a fine dissection of the genetic background of susceptible individuals and have enabled a more complete characterization of the immunocompetent cells involved in this autoaggression. Molecular approaches will soon allow us to pinpoint the characteristics of the environmental stimuli, so that protective strategies could be formulated to spare susceptible individuals from their ill effects.

Restricted TCR Vβ gene expression and enterovirus infection in Type I diabetes: a pilot study

Aims/hypothesis. High frequencies of T-cell receptor (TCR) Vβ7+ T cells were detected among the lymphocytes isolated from pancreatic islets of children at the onset of Type I (insulin-dependent) diabetes mellitus. We assessed whether a preferential expression of certain TCR Vβ gene families could also be detected among the peripheral blood mononuclear cells from diabetic patients. Methods. T-cell receptor repertoires were evaluated by using a semi-quantitative RT-PCR-based technique and confirmed by FACS analysis in peripheral blood mononuclear cells from diabetic patients before, at and after onset of the disease. These patients were also tested for exposure to enteroviruses by RT-PCR and by measuring titres of enterovirus-specific antibodies of the IgA, IgG, and IgM classes. Results. T-cell receptor Vβ7 gene family values were higher in recently-diagnosed diabetic patients (10.5 % ± 3.7) than in age-matched non-diabetic control subjects (5.1 % ± 1.6) (p < 0.001). In a time-course analysis of people who developed diabetes during clinical monitoring (i. e., converters), T-cell receptor Vβ7 gene expression showed values consistently above 10 % (p < 0.0005). Long-standing diabetic patients showed lower percentage of Vβ7 expression compared to values measured at disease onset. In the longitudinal study of the converters, multiple acute enterovirus infections were also detected. These infections appeared to be temporally related to increased percentage of Vβ7 gene transcripts. Conclusion/interpretation. The deviation in the T-cell receptor Vβ repertoire among circulating T cells from diabetic patients seems to re-emphasize the importance of enterovirus infections in accelerating the progression of Type I diabetes. [Diabetologia (2000) 43: 1484–1497]

Increased Expression of Monocyte CD11b (Mac-1) in Overweight Recent-Onset Type 1 Diabetic Children.

AIM Compelling evidence implicates inflammation in the pathogenesis of type 1 diabetes mellitus (T1DM) and associated vascular complications. Obesity is also characterized by low-grade systemic inflammation. In this study, we characterized the inflammatory response in diabetes by analyzing the expression of a panel of activation markers on the surface of peripheral blood monocytes in recently-diagnosed T1DM patients. The potential effects of glycemic control and body mass index (BMI) on monocyte phenotype were also investigated. METHODS Using flow cytometry, we analyzed the expression of CD11b, CD49d, CD54, CD62L and CD64 antigens on monocytes in a cohort of 51 T1DM patients (</= 2 months after diagnosis). To test whether phenotype change in monocytes was associated with abnormal cellular function, we studied the adhesive capacity of monocytes to human umbilical vein endothelial cells (HUVEC). RESULTS We found that circulating monocytes from T1DM patients tested at the clinical onset of the disease (i.e. within 1 week of diagnosis) had higher CD11b expression compared to patients analyzed 2 months after diagnosis (p = 0.02). The highest CD11b levels were detected in patients with HbA1c < 8% (p = 0.04 vs. patients with HbA1c < 8%). In T1DM children analyzed 2 months after diagnosis, we found that those who were overweight (BMI >/= 85th percentile) had higher levels of monocyte activation than those who were not (BMI </= 85th percentile) (p = 0.03). CD11b and HbA1c were significantly correlated (correlation coefficient 0.329, p = 0.02). Finally, monocytes from T1DM patients showed higher adhesion to HUVEC compared to controls. CONCLUSIONS Circulating immune cells from T1DM patients display many aspects of a proinflammatory state, as indicated by primed or activated monocytes. Obesity is an important factor in monocyte activation during diabetes.