Prenatal screening service for fetal RHD genotyping to guide prophylaxis: the two-year experience of the Friuli Venezia Giulia region in Italy.

BACKGROUND: Fetal RHD genotyping of cell-free fetal DNA (cff-DNA) from RhD-negative pregnant women can be used to guide anti-D prophylaxis: the knowledge of fetal RhD type can direct and restrict the use of prenatal anti-D immunoglobulin exclusively to RhD-negative women carrying a RhD-positive fetus. Since November 2019 in the region of Friuli Venezia Giulia (Italy) a prenatal screening service has been offered to RhD-negative women at 22-24 weeks of gestation. MATERIALS AND METHODS: The cff-DNA is extracted from a simple peripheral maternal blood sample to analyze the fetal RHD gene: the results are interpreted as RHD-positive fetus, RHD-negative fetus, or Inconclusive. The service is shared with all regional hospitals and tests are provided free of charge by the National Health System. RESULTS: Overall, 142 RhD-negative pregnant women were recruited in nearly 2 years. Fetal RHD genotyping was negative in 53 pregnancies and positive in 89 pregnancies. Thus, unnecessary treatment of pregnant women and exposure to a scarce plasma-derived medicinal product was avoided, by the use of a single blood sample, in 37.8% of cases, representing 100% of the RhD-negative women carrying a RhD-negative fetus in our cohort. DISCUSSION: The first Italian region-wide screening service for fetal RHD genotyping has been implemented for 2 years, despite the COVID-19 pandemic, in order to obtain the predicted fetal RhD phenotype before the 28th week of gestation, during which prenatal prophylaxis is usually administered. Giving prenatal anti-D immunoglobulin exclusively to RhD-negative women carrying a RhD-positive fetus reduces the overall use of anti-D immunoglobulin, which is becoming an ever more limited resource. The high sensitivity of the procedure provides evidence that the implementation of a diagnostic test in a reference laboratory guarantees the quality of the results, the concordance of reports and the sustainability of costs, representing an excellent guide to targeted use of prophylaxis.

Innovations in the field of fungal biofilms: looking for new targets and new chemical compounds.

Biofilms pose a serious problem for public health. Penetration of pharmacological agents within a biofilm is hampered by the morphological structure of such microbial communities. A biofilm infection therefore entails adverse outcomes both in the field of cost management and patient prognosis. The problem is further complicated if the drugs available to combat a biofilm-related fungal infection versus a bacterial one are compared: in the case of a fungal infection, the drugs available are less efficacious than antibiotics used to counteract a bacterial infection. Furthermore, even the fairly recent introduction of antifungals, such as echinocandins, start presenting some limits of usage, such as ineffectiveness in treating some fungal populations and increased resistance. It therefore becomes imperative to search for innovative molecules in order to combat this condition. The discovery of new molecules and/or new targets can make a difference. This paper illustrates the main innovative molecules that are coming to light in the field of infection by fungal biofilms.

Bloodstream infection with Oligella ureolytica: a case report and review of the literature.

Oligella ureolytica is an emerging bacteria rarely implicated as a human pathogen. It is mostly recovered from urinary and respiratory tract specimens as a commensal organism, but very seldom from bloodstream infections. It is rarely reported in the literature, probably due to misidentification of the organism or uncertainty of its pathogenicity.

Mast cells, basophils and B cell connection network.

It has been proven that both resting and activated mast cells (MCs) and basophils are able to induce a significant increase in proliferation and survival of naïve and activated B cells, and their differentiation into antibody-producing cells. The immunological context in which this regulation occurs is of particular interest and the idea that these innate cells induce antibody class switching and production is increasingly gaining ground. This direct role of MCs and basophils in acquired immunity requires cell to cell contact as well as soluble factors and exosomes. Here, we review our current understanding of the interaction between B cells and MCs or basophils as well as the evidence supporting B lymphocyte-MC/basophil crosstalk in pathological settings. Furthermore, we underline the obscure aspects of this interaction that could serve as important starting points for future research in the field of MC and basophil biology in the peculiar context of the connection between innate and adaptive immunity.

Mast cells enhance proliferation of B lymphocytes and drive their differentiation toward IgA-secreting plasma cells.

The evidence of a tight spatial interaction between mast cells (MCs) and B lymphocytes in secondary lymphoid organs, along with the data regarding the abundance of MCs in several B-cell lymphoproliferative disorders prompted us to investigate whether MCs could affect the proliferation and differentiation of B cells. To this aim, we performed coculture assays using mouse splenic B cells and bone marrow-derived MCs. Both nonsensitized and activated MCs proved able to induce a significant inhibition of cell death and an increase in proliferation of naive B cells. Such proliferation was further enhanced in activated B cells. This effect relied on cell-cell contact and MC-derived interleukin-6 (IL-6). Activated MCs could regulate CD40 surface expression on unstimulated B cells and the interaction between CD40 with CD40 ligand (CD40L) on MCs, together with MC-derived cytokines, was involved in the differentiation of B cells into CD138(+) plasma cells and in selective immunoglobulin A (IgA) secretion. These data were corroborated by in vivo evidence of infiltrating MCs in close contact with IgA-expressing plasma cells within inflamed tissues. In conclusion, we reported here a novel role for MCs in sustaining B-cell expansion and driving the development of IgA-oriented humoral immune responses.

CD4+CD25+ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction.

T regulatory (Treg) cells play a role in the suppression of immune responses, thus serving to induce tolerance and control autoimmunity. Here, we explored whether Treg cells influence the immediate hypersensitivity response of mast cells (MCs). Treg cells directly inhibited the FcvarepsilonRI-dependent MC degranulation through cell-cell contact involving OX40-OX40L interactions between Treg cells and MCs, respectively. When activated in the presence of Treg cells, MCs showed increased cyclic adenosine monophosphate (cAMP) concentrations and reduced Ca(2+) influx, independently of phospholipase C (PLC)-gamma2 or Ca(2+) release from intracellular stores. Antagonism of cAMP in MCs reversed the inhibitory effects of Treg cells, restoring normal Ca(2+) responses and degranulation. Importantly, the in vivo depletion or inactivation of Treg cells caused enhancement of the anaphylactic response. The demonstrated crosstalk between Treg cells and MCs defines a previously unrecognized mechanism controlling MC degranulation. Loss of this interaction may contribute to the severity of allergic responses.

APE/Ref-1 makes fine-tuning of CD40-induced B cell proliferation.

Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches.

TRAF2 and p38 are involved in B cells CD40-mediated APE/Ref-1 nuclear translocation: a novel pathway in B cell activation.

The interaction between CD40 and its ligand CD40L plays a key role in the regulation of B cell proliferation, activation, isotype switching and the humoral memory response. APE/Ref-1 plays a key role in transcriptional responses during CD40-mediated B cell activation. It is demonstrated that CD40 signaling is mediated principally through TRAF adapter proteins. Different TRAFs exhibit specific biological functions and the role of individual TRAFs in the activation of different CD40-dependent signaling pathways has not yet been defined. To better understand the role of these factors in CD40-mediated B cell activation and how they contribute to APE/Ref-1 activity, we investigated the TRAF molecules and the downstream protein kinases directly activated in the pathways triggered by CD40. Here we show that TRAF2 is involved in CD40-mediated induction of APE/Ref-1 nuclear translocation and that the two proteins physically interact in vitro and in vivo. Moreover, treatment with the p38 inhibitor, SB203580 or site directed mutagenesis of the serine 54 (Ser(54)) in the MAP kinase consensus site present in APE/Ref-1 blocks its nuclear translocation caused by CD40-mediated B cell activation and reveals a potential role of p38 in this pathway. These data together uncovers a new signaling pathway regulating APE/Ref-1 nuclear translocation involving CD40-crosslinking, TRAF2 and p38.

Cooperative activity of Ref-1/APE and ERp57 in reductive activation of transcription factors.

ERp57, a protein disulfide isomerase localized mainly in the endoplasmic reticulum, has also been found in lesser amounts in the cytosol and nucleus, where its function is still not characterized. We report here that ERp57 displays affinity for Ref-1, a protein involved in DNA repair as well as in the reduction and activation of transcription factors. Immunoprecipitation experiments showed that Ref-1 and ERp57 also interact in vivo in at least three types of cultured human cells, namely HepG2, M14, and Raji. Oxidative stress increased the amount of nuclear Ref-1 associated with ERp57. Moreover, ERp57 reduced by the thioredoxin-reductase/thioredoxin system stimulated the binding of AP-1 to its consensus sequence on DNA, and HeLa cells stably transfected and overexpressing ERp57 were protected against hydrogen peroxide-induced cell killing. Accordingly, ERp57 appears to cooperate with Ref-1 in the regulation of gene expression mediated by redox-sensitive transcription factors and in the adaptive response of the cell to oxidative insult.

CD40 stimulation induces Pax5/BSAP and EBF activation through a APE/Ref-1-dependent redox mechanism.

CD40 is a member of the growing tumor necrosis factor receptor family that has been shown to play important roles in T cell-mediated B lymphocyte activation. Ligation of B cell CD40 by CD154, mainly expressed on activated T cells, stimulates B cell proliferation, differentiation, isotype switching, up-regulation of surface molecules contributing to antigen presentation, development of the germinal center, and the humoral memory response. In this study we demonstrate that the redox factor APE/Ref-1 acts as a key signaling intermediate in response to CD40-mediated B cell activation. The transcription factors Pax5a or BSAP (B cell lineage-specific activator protein) and EBF (early B cell factor) are constitutively expressed in spleen B cells and CD40 cross-linking induces increases in Pax5a and EBF binding activity compared with nonstimulated B cells. We show that upon CD40 antibody-mediated cross-linking, APE/Ref-1 translocates from the cytoplasm to the nucleus of activated B cells, where it modulates the DNA binding activity of both Pax5a and EBF. Moreover, we show that the repression of APE/Ref-1 protein production is able to block CD40-mediated Pax5a activation. We also provide evidence that APE/Ref-1 can modulate the cooperative activation of the blk promoter operated by Pax5a and EBF and that APE/Ref-1 might directly regulate EBF functional activity. Finally, we show that the interaction between Pax5a and EBF enhances EBF binding activity to its consensus sequence, suggesting that Pax5a can physically interact with EBF and modulate its DNA binding activity.