Biomedical subjects

Roberta Bulla

Publications and source records attributed to Roberta Bulla.

10 recordsLinked to original sources

PubMed2026

The role of SYNE1/2 variants as a potential predisposition factor for the onset of endometriosis.

Endometriosis (EM) is a chronic, inflammatory gynaecological disorder defined by the presence of endometrial-like tissue outside the uterine cavity, most frequently affecting the ovaries, peritoneum, and uterosacral ligaments. Despite its prevalence and the significant impact on life quality, EM is often underdiagnosed, with an average delay of about nine years, particularly affecting adolescents and young women. The complex aetiology involves genetic, environmental, and immune factors, with whole-exome sequencing (WES) emerging as a potential tool for identifying relevant genetic variants. Research indicates that innate immune dysfunction, mechanotransduction, and epithelial-to-mesenchymal transition promote endometrial cell migration and lesion formation, processes regulated by nuclear envelope integrity and cytoskeletal dynamics. The LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, specifically Nesprin-1 and Nesprin-2, encoded by SYNE1 and SYNE2, is crucial for these processes. Genome-wide studies have linked SYNE genes to EM risk, showing downregulation in affected patients, and rare variants in these genes have been identified, though their functional implications are still unclear. To this purpose, WES was performed on 204 EM patients to identify rare (MAF <0.1%), damaging variants in SYNE1/2. Primary endometriotic cells (EMCs) were isolated from ovarian lesions of variant carriers (n=4) and wild-type (WT) non-carrier controls (n=4). Functional characterization included somatic WES, RT-qPCR, Western blot, confocal immunofluorescence, and Transwell migration assays. WES identified 11 rare, likely damaging SYNE1/2 variants in 12 patients. Immunofluorescence revealed a distinct protein mislocalization, WT EMCs displayed physiological Nesprin-2 confinement at the nuclear envelope, whereas variant carriers exhibited a diffuse cytoplasmic distribution polarized along actin stress fibres. We demonstrated that SYNE1/2 mutated EMCs had a markedly higher migratory capacity compared to WT controls. Here, in vitro experiments demonstrated, for the first time, the involvement of Nesprin-2 in endometrial cell migration, supporting a mechanistic link between nuclear-cytoskeletal disruption and the invasive phenotype of endometriotic cells (EMCs). These findings provide new insights into EM pathogenesis and highlight SYNE2 as a promising molecular marker for improved diagnosis and disease management.

Humans↗

PubMed2006

EMILIN1 represents a major stromal element determining human trophoblast invasion of the uterine wall.

The detection of EMILIN1, a connective tissue glycoprotein associated with elastic fibers, at the level of the ectoplacental cone and trophoblast giant cells of developing mouse embryos (Braghetta et al., 2002) favored the idea of a structural as well as a functional role for this protein in the process of placentation. During the establishment of human placenta, a highly migratory subpopulation of extravillous trophoblasts (EVT), originating from anchoring chorionic villi, penetrate and invade the uterine wall. In this study we show that EMILIN1, produced by decidual stromal and smooth muscle uterine cells, is expressed in the stroma and in some instances as a gradient of increasing concentration in the perivascular region of modified vessels. This distribution pattern is consistent with the haptotactic directional migration observed in in vitro functional studies of freshly isolated EVT and of the immortalized HTR-8/SVneo cell line of trophoblasts. Function-blocking monoclonal antibodies against alpha4-integrin chain and against EMILIN1 as well as the use of EMILIN1-specific short interfering RNA confirmed that trophoblasts interact with EMILIN1 and/or its functional gC1q1 domain via alpha4beta1 integrin. Finally, membrane type I-matrix metalloproteinase (MT1-MMP) and MMP-2 were upregulated in co-cultures of trophoblast cells and stromal cells, suggesting a contributing role in the haptotactic process towards EMILIN1.

Cell Adhesion↗

PubMed2006

The complement system in the pathophysiology of pregnancy.

A fully active complement system deriving from the maternal circulation as well as from local production by various cell source is present in the placenta. The role of this system at the placental level, as in any other tissue in the body, is to protect both the fetus and the mother against infectious and other toxic agents. As fetal tissues are semi-allogeneic and alloantibodies commonly develop in the mother, the placenta is potentially subject to complement-mediated immune attack at the feto-maternal interface with the potential risk of fetal loss. Uncontrolled complement activation is prevented in successful pregnancy by the three regulatory proteins DAF, MCP and CD59 positioned on the surface of trophoblasts. The critical role played by these complement regulators is supported by the embryonic lethality observed in mice deficient in the complement regulator Crry. Excessive complement activation in the placenta places the fetus at risk for growth restriction or death. The role played by the complement system in the fetal damage induced by anti-phospholipid antibodies in a mouse model will be examined.

Animals↗

PubMed2005

Thrombus formation induced by antibodies to beta2-glycoprotein I is complement dependent and requires a priming factor.

We monitored the number of intravascular platelet-leukocyte aggregates (PLAs) and thrombotic occlusions (TOs) by intravascular microscopy in the mesentery of rats receiving antiphospholipid (aPL) immunoglobulin G (IgG) purified from the sera of patients with antiphospholipid syndrome. aPL IgG had no procoagulant effect, but it caused rapid endothelial deposition of fibrinogen, followed by PLA and TO in rats receiving an intraperitoneal injection of bacterial lipopolysaccharide 3 hours before IgG infusion. Anti-beta2-glycoprotein I-depleted aPL IgG failed to induce PLAs and TOs. C3 and C9 colocalized with aPL IgG on the mesenteric vessels. The number of PLAs and TOs was markedly reduced in C6-deficient rats and in animals treated with anti-C5 miniantibody, suggesting the contribution of the terminal complement (C) complex to the aPL antibody-mediated intravascular thrombosis. In conclusion, our data indicate that antibodies to beta2-glycoprotein I trigger coagulation subsequent to a priming proinflammatory factor and that the terminal C complex is the main mediator of the coagulation process.

Animals↗

PubMed2005

VE-cadherin is a critical molecule for trophoblast-endothelial cell interaction in decidual spiral arteries.

Fetal cytotrophoblasts colonize the decidual spiral arteries during pregnancy and partially replace the endothelium by an as yet unknown mechanism. To clarify this issue, we cocultured trophoblast cells (TCs) and decidual endothelial cells (DECs) isolated from first trimester placentae and found by electron microscopic analysis that TCs adhered to DECs and migrated through the interendothelial junctions within 24 h. Since extravillous TCs were shown by FACS analysis to express vascular-endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM)-1, we investigated the role of these junctional molecules in TC adhesion to DECs and transendothelial migration of cytotrophoblasts. Both VE-cadherin and PECAM-1 were present at the contact sites between TCs and DECs in decidual sections. TC adhesion and migration were markedly inhibited by mAbs to VE-cadherin and marginally by mAb to PECAM-1. Increased expression of VE-cadherin was observed at the contact areas between TCs and DECs, whereas PECAM-1 was found to be redistributed from intercellular junctions. The induction of apoptosis of DECs by TCs, as the mechanism responsible for their replacement, was ruled out by the negative staining with TUNEL of DECs cocultured with TCs and the absence of DNA fragmentation. In conclusion, VE-cadherin is involved in transendothelial migration of TCs, and replacement of DECs by TCs is not the result of apoptosis.

Antigens, CD↗

PubMed2005

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59.

Expression of the complement-regulatory proteins (CRP) CD46, CD55 and CD59 represents a strategy used by tumor cells to evade complement-dependent cell cytoxicity stimulated by monoclonal antibodies. We have isolated two single-chain variable fragments (scFv) to CD55 and CD59 from a human phage-display library and from these scFv we have produced two miniantibodies (MB), MB-55 (against CD55) and MB-59 (against CD59), containing the human hinge-CH2-CH3 domains of IgG1. The specificity of the two MB for the corresponding CRP was assessed by ELISA using purified CD46, CD55 and CD59. MB-55 and MB-59 neutralized the inhibitory activity of CD55 and CD59, respectively, restoring the complement-mediated lysis of sheep and guinea pig erythrocytes that was otherwise inhibited by the two CRP. FACS analysis revealed binding of MB-55 and MB-59 to the lymphoma cell line Karpas 422. The two MB induced a two-fold increase in the complement-dependent killing of these cells stimulated by Rituximab, a chimeric anti-CD20 monoclonal antibody. Transfection of HEK293T cells with vectors encoding MB-55 or MB-59 markedly reduced the expression of CD55 and CD59. We conclude that the human antibodies MB-55 and MB-59 may represent a therapeutic tool to increase the complement-dependent killing activity of Rituximab in the treatment of non-Hodgkin's lymphoma.

Antibodies, Monoclonal↗

PubMed2005

The complement system at the fetomaternal interface.

The placenta has a unique structural organization that allows fetal cells expressing paternal alloantigens to establish a peaceful cohabitation with the maternal immune system. The fetal cells are continuously exposed to the humoral and cellular components of the maternal immune system present in the maternal blood that circulates in the intervillous space and in the decidual vessels. This review deals with the role played by the complement system at the placental level both in physiological and pathological conditions of pregnancies. Complement components found in the placental tissue derive to a large extent from blood circulating in placental vessels. However, some complement components may also be produced locally by macrophages and other cell types. Deposition of complement components at tissue level is usually found in association with inflammatory diseases. This is not the case in placentae in which deposits of complement components can also be documented in physiological conditions not resulting in fetal damage. Protection of the semiallogenic human conceptus against maternal complement activation products is achieved by surface expression of complement regulators that act at different steps of the complement sequence. These complement regulators are localized in a strategic position on the surface of villous trophoblast protecting the fetus from the damage that may derive from uncontrolled complement activation. However, pathological conditions of pregnancies may lead to deposition of a higher amount of complement activation products that may exceed the protection of local complement regulators.

Animals↗

PubMed2004

Platelet-activating factor and kinin-dependent vascular leakage as a novel functional activity of the soluble terminal complement complex.

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.

Animals↗

PubMed2003

Placental trophoblast and endothelial cells as target of maternal immune response.

Pregnancy is a unique physiologic condition that guarantees the survival of the semiallogenic embryo during the long period of gestation. The placenta plays a key role in the maintenance of local tolerance and allows the mother to accept the embryo until completion of pregnancy. The complex process of tolerance accompanying the survival of the foetus is controlled at the embryo-maternal interface by factors deriving from decidualized endometrium and from the trophoblast itself. Trophoblasts develop various strategies to evade the damaging attack by the maternal immune response including expression of non-classical MHC class I antigens and of complement regulatory proteins. Also, cytokines released at the feto-maternal interface play an important role in regulating embryo survival controlling not only the maternal immune response but also angiogenesis and vascular remodelling. The delicate equilibrium established between the mother and the foetus can be compromised in pathological condition of pregnancy as a result of humoral and/or cellular response of the mother against trophoblast antigens leading to spontaneous miscarriage. Cytotoxic cells and antibodies to trophoblast and endothelial cells are frequently found in patients with recurrent spontaneous abortion. This review article focuses on the delicate equilibrium established at the feto-maternal interface during pregnancy examining the various strategies devised by the embryo to evade the maternal immune attack, and the pathological conditions in which this equilibrium is compromised leading to serious complications of pregnancy.

Complement Activation↗

PubMed2002

Cytolytically inactive terminal complement complex causes transendothelial migration of polymorphonuclear leukocytes in vitro and in vivo.

Intravital microscopy was used to monitor leukocyte traffic across rat mesenteric postcapillary venules induced by the inactive terminal complement (C) complex (iTCC) topically applied to ileal mesentery. Leukocytes started rolling within 15 minutes from the administration of iTCC, and by 1 hour they adhered almost completely to the endothelium emigrating from the vessels in the next 3 hours. C5a caused a similar, though less marked, effect, whereas boiled iTCC was inactive, excluding the contribution of contaminating lipopolysaccharide. The complex stimulated the migration of polymorphonuclear neutrophils (PMNs) across endothelial cells (ECs) in a transwell system after a 4-hour incubation of ECs with iTCC added to the lower chamber of the transwell, whereas a 30-minute incubation was sufficient for C5a and interleukin (IL)-8 to induce the passage of PMNs. C5a was not responsible for the effect of iTCC because this complex had no chemotactic activity and contained too small an amount of C5a to account for the transendothelial migration of PMNs. Similarly, the effect of iTCC was not mediated by IL-8 released by stimulated ECs because anti-IL-8 failed to inhibit the migration of PMNs induced by the complex. Unlike tumor necrosis factor-alpha, iTCC did not cause the redistribution of platelet-endothelial cell adhesion molecule-1 (PECAM-1), and PMN mobilization was partially blocked by anti-PECAM-1 antibodies.

Animals↗