Radiotherapy, PARP Inhibition, and Immune-Checkpoint Blockade: A Triad to Overcome the Double-Edged Effects of Each Single Player.

Radiotherapy and, more recently, PARP inhibitors (PARPis) and immune-checkpoint inhibitors represent effective tools in cancer therapy. Radiotherapy exerts its effects not only by damaging DNA and inducing tumor cell death, but also stimulating anti-tumor immune responses. PARPis are known to exert their therapeutic effects by inhibiting DNA repair, and they may be used in combination with radiotherapy. Both radiotherapy and PARPis modulate inflammatory signals and stimulate type I IFN (IFN-I)-dependent immune activation. However, they can also support the development of an immunosuppressive tumor environment and upregulate PD-L1 expression on tumor cells. When provided as monotherapy, immune-checkpoint inhibitors (mainly antibodies to CTLA-4 and the PD-1/PD-L1 axis) result particularly effective only in immunogenic tumors. Combinations of immunotherapy with therapies that favor priming of the immune response to tumor-associated antigens are, therefore, suitable strategies. The widely explored association of radiotherapy and immunotherapy has confirmed this benefit for several cancers. Association with PARPis has also been investigated in clinical trials. Immunotherapy counteracts the immunosuppressive effects of radiotherapy and/or PARPis and synergies with their immunological effects, promoting and unleashing immune responses toward primary and metastatic lesions (abscopal effect). Here, we discuss the beneficial and counterproductive effects of each therapy and how they can synergize to overcome single-therapy limitations.

Severe Acute Respiratory Syndrome Coronavirus-2 Infection and Autoimmunity 1 Year Later: The Era of Vaccines.

Impressive efforts have been made by researchers worldwide in the development of target vaccines against the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and in improving the management of immunomodulating agents. Currently, different vaccine formulations, such as viral vector, mRNA, and protein-based, almost all directed toward the spike protein that includes the domain for receptor binding, have been approved. Although data are not conclusive, patients affected by autoimmune rheumatic diseases (ARDs) seem to have a slightly higher disease prevalence, risk of hospitalization, and death from coronavirus disease-2019 (COVID-19) than the general population. Therefore, ARD patients, under immunosuppressive agents, have been included among the priority target groups for vaccine administration. However, specific cautions are needed to optimize vaccine safety and effectiveness in these patients, such as modification in some of the ongoing immunosuppressive therapies and the preferential use of mRNA other than vector-based vaccines. Immunomodulating agents can be a therapeutic opportunity for the management of COVID-19 patients; however, their clinical impact depends on how they are handled. To place in therapy immunomodulating agents in the correct window of opportunity throughout the identification of surrogate markers of disease progression and host immune response is mandatory to optimize patient's outcome.

p53 Activation Effect in the Balance of T Regulatory and Effector Cell Subsets in Patients With Thyroid Cancer and Autoimmunity.

Carcinomas evade the host immune system by negatively modulating CD4+ and CD8+ T effector lymphocytes through forkhead box protein 3 (FOXP3) positive T regulatory cells' increased activity. Furthermore, interaction of the programmed cell death 1 (PD1) molecule and its ligand programmed cell death ligand 1 (PDL1) inhibits the antitumor activity of PD1+ T lymphocytes. Immunotherapy has become a powerful strategy for tailored cancer patients' treatment both in adult and pediatric patients aiming to generate potent antitumor responses. Nevertheless, immunotherapies can generate autoimmune responses. This study aimed to investigate the potential effect of the transformation-related protein 53 (p53) reactivation by a peptide-based inhibitor of the MDM2/MDM4 heterodimer (Pep3) on the immune response in a solid cancer, i.e., thyroid carcinoma frequently presenting with thyroid autoimmunity. In peripheral blood mononuclear cell of thyroid cancer patients, Pep3 treatment alters percentages of CD8+ and CD4+ T regulatory and CD8+ and CD4+ T effector cells and favors an anticancer immune response. Of note that reduced frequencies of activated CD8+ and CD4+ T effector cells do not support autoimmunity progression. In evaluating PD1 expression under p53 activation, a significant decrease of activated CD4+PD1+ cells was detected in thyroid cancer patients, suggesting a defective regulation in the initial activation stage, therefore generating a protective condition toward autoimmune progression.

Inhibition of Phosphodiesterase-4 in Psoriatic Arthritis and Inflammatory Bowel Diseases.

Phosphodiesterases (PDEs) are a heterogeneous superfamily of enzymes which catalyze the degradation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Among PDEs, PDE4 is the most widely studied and characterized isoenzyme. PDE4 blocking can lead to increased levels of intracellular cAMP, which results in down-regulation of inflammatory responses by reducing the expression of tumor necrosis factor (TNF), interleukin (IL)-23, IL-17, interferon-γ, while increasing regulatory cytokines, such as IL-10. Therefore, PDE4 has been explored as a therapeutic target for the treatment of different chronic inflammatory conditions such as psoriatic arthritis (PsA) and inflammatory bowel disease (IBD). PsA shares clinical, genetic, and pathogenic features with IBD such as ulcerative colitis (UC) and Crohn's disease (CD), and enteropathic spondyloarthritis (eSpA) represent a frequent clinical evidence of the overlap between gut and joint diseases. Current therapeutic options in PsA patients and underlying UC are limited to synthetic immunosuppressants and anti-TNF. Apremilast is an oral PDE4 inhibitor approved for the treatment of active PsA patients with inadequate response to synthetic immunosuppressants. The efficacy and a good safety profile observed in randomized clinical trials with apremilast in PsA patients have been confirmed by few studies in a real-life scenario. In addition, apremilast led to significant improvement in clinical and endoscopic features in UC patients in a phase II RCT. By now there are no available data regarding its role in eSpA patients. In view of the above, the use of apremilast in eSpA patients is a route that deserves to be deepened.

ADP-ribosylation in evasion, promotion and exacerbation of immune responses.

ADP-ribosylation is the addition of one or more (up to some hundreds) ADP-ribose moieties to acceptor proteins. This evolutionary ancient post-translational modification (PTM) is involved in fundamental processes including DNA repair, inflammation, cell death, differentiation and proliferation, among others. ADP-ribosylation is catalysed by two major families of enzymes: the cholera toxin-like ADP-ribosyltransferases (ARTCs) and the diphtheria toxin-like ADP-ribosyltransferases (ARTDs, also known as PARPs). ARTCs sense and use extracellular NAD, which may represent a danger signal, whereas ARTDs are present in the cell nucleus and/or cytoplasm. ARTCs mono-ADP-ribosylate their substrates, whereas ARTDs, according to the specific family member, are able to mono- or poly-ADP-ribosylate target proteins or are devoid of enzymatic activity. Both mono- and poly-ADP-ribosylation are dynamic processes, as specific hydrolases are able to remove single or polymeric ADP moieties. This dynamic equilibrium between addition and degradation provides plasticity for fast adaptation, a feature being particularly relevant to immune cell functions. ADP-ribosylation regulates differentiation and functions of myeloid, T and B cells. It also regulates the expression of cytokines and chemokines, production of antibodies, isotype switch and the expression of several immune mediators. Alterations in these processes involve ADP-ribosylation in virtually any acute and chronic inflammatory/immune-mediated disease. Besides, pathogens developed mechanisms to contrast the action of ADP-ribosylating enzymes by using their own hydrolases and/or to exploit this PTM to sustain their virulence. In the present review, we summarize and discuss recent findings on the role of ADP-ribosylation in immunobiology, immune evasion/subversion by pathogens and immune-mediated diseases.

IgM on the surface of T cells: a novel biomarker of pediatric-onset systemic lupus erythematosus.

BACKGROUND: Children with systemic lupus erythematosus (SLE) frequently have kidney involvement. Lupus nephritis sometimes presents alone, without systemic SLE features, representing the so-called full-house nephropathy (FHN). Distinguishing patients with SLE or FHN has therapeutic and prognostic implications. METHODS: In this retrospective observational study, we determined the presence of IgM on the surface of T cells (T cell IgM) by flow cytometry and characterized its ability in distinguishing SLE and FHN patients in a large pediatric cohort (n = 84). Fifty-seven patients with SLE (≥ 4 SLICC criteria at disease onset or during the follow-up) and 27 patients with FHN (3 or less SLICC criteria) were enrolled. RESULTS: Elevated T cell IgM levels were found in 24/25 SLE patients in active phase of disease and in 29/45 SLE patients in remission. In contrast, among FHN patients, only 1/9 presented this characteristic in active phase of disease and 0/20 in remission. Compared with standardized SLICC laboratory parameters, i.e., autoantibody titers and hypocomplementemia, T cell IgM positivity showed an extremely high sensitivity and specificity for the diagnosis of SLE, with the highest area under the curve (0.97, p < 0.001) by receiver operating characteristic analysis, similar to ANA (0.96, p < 0.001) and anti-dsDNA (0.90, p < 0.001) autoantibodies. CONCLUSIONS: Altogether, our data indicate that T cell IgM intensity may be a useful tool to correctly classify patients with lupus nephritis as SLE or FHN since disease onset.

Cytokine Release Syndrome in COVID-19 Patients, A New Scenario for an Old Concern: The Fragile Balance between Infections and Autoimmunity.

On 7 January 2020, researchers isolated and sequenced in China from patients with severe pneumonitis a novel coronavirus, then called SARS-CoV-2, which rapidly spread worldwide, becoming a global health emergency. Typical manifestations consist of flu-like symptoms such as fever, cough, fatigue, and dyspnea. However, in about 20% of patients, the infection progresses to severe interstitial pneumonia and can induce an uncontrolled host-immune response, leading to a life-threatening condition called cytokine release syndrome (CRS). CRS represents an emergency scenario of a frequent challenge, which is the complex and interwoven link between infections and autoimmunity. Indeed, treatment of CRS involves the use of both antivirals to control the underlying infection and immunosuppressive agents to dampen the aberrant pro-inflammatory response of the host. Several trials, evaluating the safety and effectiveness of immunosuppressants commonly used in rheumatic diseases, are ongoing in patients with COVID-19 and CRS, some of which are achieving promising results. However, such a use should follow a multidisciplinary approach, be accompanied by close monitoring, be tailored to patient's clinical and serological features, and be initiated at the right time to reach the best results. Autoimmune patients receiving immunosuppressants could be prone to SARS-CoV-2 infections; however, suspension of the ongoing therapy is contraindicated to avoid disease flares and a consequent increase in the infection risk.

Atypical IgM on T cells predict relapse and steroid dependence in idiopathic nephrotic syndrome.

The clinical heterogeneity of idiopathic nephrotic syndrome in childhood may reflect different mechanisms of disease that are as yet unclear. Here, we evaluated the association between an atypical presence of IgM on the surface of T cells (T-cell IgM) and the response to steroid therapy in a total of 153 pediatric patients with idiopathic nephrotic syndrome in different phases of disease. At disease onset, T-cell IgM median levels were significantly elevated and predictive of risk of relapse in 47 patients. They were also significantly increased comparing 58 steroid-dependent to 8 infrequently relapsing and 14 frequently relapsing patients, especially during relapse, whereas they were within the normal range in 7 genetic steroid-resistant patients. T-cell IgM in vivo was not affected by the amount of total circulating IgM, nor by concomitant acute infections or oral immunosuppression. However, it was affected by rituximab treatment in 21 steroid-dependent patients. By in vitro experiments, elevated T-cell IgM was not influenced by total circulating IgM levels or by the presence of other circulating factors, and there was no distinctive antigen-specificity or atypical IgM polymerization. Rather, we found that increased T-cell IgM correlates with reduced IgM sialylation, which influences T-cell response to steroid inhibition and T-cell production of podocyte-damaging factors. Thus, the atypical presence of IgM on the surface of T cells may predispose a subset of steroid-sensitive pediatric patients with idiopathic nephrotic syndrome to a poor response to steroid therapy since disease onset.

Lack of Gut Secretory Immunoglobulin A in Memory B-Cell Dysfunction-Associated Disorders: A Possible Gut-Spleen Axis.

Background: B-1a B cells and gut secretory IgA (SIgA) are absent in asplenic mice. Human immunoglobulin M (IgM) memory B cells, which are functionally equivalent to mouse B-1a B cells, are reduced after splenectomy. Objective: To demonstrate whether IgM memory B cells are necessary for generating IgA-secreting plasma cells in the human gut. Methods: We studied intestinal SIgA in two disorders sharing the IgM memory B cell defect, namely asplenia, and common variable immune deficiency (CVID). Results: Splenectomy was associated with reduced circulating IgM memory B cells and disappearance of intestinal IgA-secreting plasma cells. CVID patients with reduced circulating IgM memory B cells had a reduced frequency of gut IgA+ plasma cells and a disrupted film of SIgA on epithelial cells. Toll-like receptor 9 (TLR9) and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) induced IgM memory B cell differentiation into IgA+ plasma cells in vitro. In the human gut, TACI-expressing IgM memory B cells were localized under the epithelial cell layer where the TACI ligand a proliferation inducing ligand (APRIL) was extremely abundant. Conclusions: Circulating IgM memory B cell depletion was associated with a defect of intestinal IgA-secreting plasma cells in asplenia and CVID. The observation that IgM memory B cells have a distinctive role in mucosal protection suggests the existence of a functional gut-spleen axis.

Immune-Modulating Perspectives for Low Frequency Electromagnetic Fields in Innate Immunity.

In recent years, the effects of electromagnetic fields (EMFs) on the immune system have received a considerable interest, not only to investigate possible negative health impact but also to explore the possibility to favorably modulate immune responses. To generate beneficial responses, the immune system should eradicate pathogens while "respecting" the organism and tolerating irrelevant antigens. According to the current view, damage-associated molecules released by infected or injured cells, or secreted by innate immune cells generate danger signals activating an immune response. These signals are also relevant to the subsequent activation of homeostatic mechanisms that control the immune response in pro- or anti-inflammatory reactions, a feature that allows modulation by therapeutic treatments. In the present review, we describe and discuss the effects of extremely low frequency (ELF)-EMF and pulsed EMF on cell signals and factors relevant to the activation of danger signals and innate immunity cells. By discussing the EMF modulating effects on cell functions, we envisage the use of EMF as a therapeutic agent to regulate immune responses associated with wound healing.

TCD4pos lymphocytosis in rheumatoid and psoriatic arthritis patients following TNFα blocking agents.

BACKGROUND: Lymphocyte expansion and true lymphocytosis are commonly observed in the everyday clinical practice. The meaning of such phenomenon is often poorly understood so that discrimination between benign and malignant lymphocytosis remains difficult to establish. This is mainly true when lymphocytosis rises in patients affected by immune-mediated chronic inflammatory diseases under immunosuppressive treatment, conditions potentially associated with lymphomagenesis. In this brief report the development of mild T CD4pos lymphocytosis in a group of patients with chronic arthritis under anti-TNF-α treatment is described. METHODS: Two hundred eight rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients have been evaluated longitudinally for at least 1-year before and 2-years after anti-TNF-α therapy introduction for the possible appearance of a lymphocyte expansion. In patients who developed lymphocyte expansion, T, B and NK cells were analysed. RESULTS: Twenty-five out of 208 (12%) subjects developed a mild T CD4pos lymphocytosis, during anti-TNF-α therapy, which reverted after its interruption. Higher lymphocyte count, more frequent use of steroids and shorter disease duration, before biological therapy start, have emerged as risk factors for lymphocytosis development. CONCLUSIONS: This is the first longitudinal cohort study evaluating the onset of lymphocytosis in RA and PsA patients under anti-TNF-α treatment and its possible clinical relevance. A mild T CD4pos lymphocytosis has been observed in 12% of RA and PsA patients probably related to anti-TNF-α treatment as previously reported by anecdotal cases. Patients with higher baseline lymphocyte count, use of steroids and shorter disease duration before the introduction of biologic therapy, seem to be prone to develop this laboratory reversible abnormality.

Altered B cell homeostasis and Toll-like receptor 9-driven response in patients affected by autoimmune polyglandular syndrome Type 1: Altered B cell phenotype and dysregulation of the B cell function in APECED patients.

APECED is a T-cell mediated disease with increased frequencies of CD8+ effector and reduction of FoxP3+ T regulatory cells. Antibodies against affected organs and neutralizing to cytokines are found in the peripheral blood. The contribution of B cells to multiorgan autoimmunity in Aire-/- mice was reported opening perspectives on the utility of anti-B cell therapy. We aimed to analyse the B cell phenotype of APECED patients compared to age-matched controls. FACS analysis was conducted on PBMC in basal conditions and following CpG stimulation. Total B and switched memory (SM) B cells were reduced while IgM memory were increased in patients. In those having more than 15 years from the first clinical manifestation the defect included also mature and transitional B cells; total memory B cells were increased, while SM were unaffected. In patients with shorter disease duration, total B cells were unaltered while SM and IgM memory behaved as in the total group. A defective B cell proliferation was detected after 4day-stimulation. In conclusion APECED patients show, in addition to a significant alteration of the B cell phenotype, a dysregulation of the B cell function involving peripheral innate immune mechanisms particularly those with longer disease duration.

Expression of PD-1 Molecule on Regulatory T Lymphocytes in Patients with Insulin-Dependent Diabetes Mellitus.

Type 1 diabetes is caused by autoreactive T cells that destroy pancreatic beta cells. Animal models suggested that a CD4⁺CD25⁺ population has a regulatory function capable of preventing activation and effector functions of autoreactive T cells. However, the role of CD4⁺CD25high T cells in autoimmunity and their molecular mechanisms remain the subject of investigation. We therefore evaluated T regulatory cell frequencies and their PD-1 expression in the peripheral blood of long-standing diabetics under basal conditions and after CD3/CD28 stimulation. Under basal conditions, the percentages of T regulatory cells were significantly higher while that of T effector cells were significantly lower in patients than in controls. The ratio of regulatory to effector T cells was higher in patients than that in controls, suggesting that T regulatory cells were functional in patients. Percentages of total PD-1⁺, PD-1low and PD-1high expressing T regulatory cells did not change in patients and in controls. After stimulation, a defect in T regulatory cell proliferation was observed in diabetics and the percentages of total PD-1⁺, PD-1low and PD-1high expressing cells were lower in patients. Our data suggest a defective activation of T regulatory cells in long-standing diabetics due to a lower expression of PD-1 on their surface.

Inhibition of B-cell proliferation and antibody production by mesenchymal stromal cells is mediated by T cells.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4(+) and CD8(+) cells playing a role. Moreover, cell-cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell-cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired.

Effects of GSM-modulated 900 MHz radiofrequency electromagnetic fields on the hematopoietic potential of mouse bone marrow cells.

Studies describing the influence of radiofrequency electromagnetic fields on bone marrow cells (BMC) often lack functional data. We examined the effects of in vivo exposure to a Global System for Mobile Communications (GSM) modulated 900 MHz RF fields on BMC using two transplantation models. X-irradiated syngeneic mice were injected with BMC from either RF-field-exposed, sham-exposed or cage control mice. Twelve weeks after transplantation, no differences in thymocyte number, frequency of subpopulations and cell proliferation were found in mice receiving BMC from either group. Also, in the spleen cell number, percentages of B/T cells, B/T-cell proliferation, and interferon γ (IFN-γ) production were similar in all groups. In parallel, a mixture of BMC from congenic sham- and RF-exposed mice were co-transplanted into lymphopenic Rag2 deficient mice. BMC from RF-exposed and sham-exposed mice displayed no advantage or disadvantage when competing for the replenishment of lymphatic organs with mature lymphocytes in Rag2 deficient mice. This model revealed that BMC from sham-exposed and RF-exposed mice were less efficient than BMC from cage control mice in repopulating the thymus, an effect likely due to restraint stress. In conclusion, our results showed no effects of in vivo exposure to GSM-modulated RF-fields on the ability of bone marrow (BM) precursors to long-term reconstitute peripheral T and B cell compartments.

P-glycoprotein and drug resistance in systemic autoimmune diseases.

Autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are chronic inflammatory disorders of unknown etiology characterized by a wide range of abnormalities of the immune system that may compromise the function of several organs, such as kidney, heart, joints, brain and skin. Corticosteroids (CCS), synthetic and biologic immunosuppressive agents have demonstrated the capacity to improve the course of autoimmune diseases. However, a significant number of patients do not respond or develop resistance to these therapies over time. P-glycoprotein (P-gp) is a transmembrane protein that pumps several drugs out of the cell, including CCS and immunosuppressants; thus, its over-expression or hyper-function has been proposed as a possible mechanism of drug resistance in patients with autoimmune disorders. Recently, different authors have demonstrated that P-gp inhibitors, such as cyclosporine A (CsA) and its analogue Tacrolimus, are able to reduce P-gp expression and or function in SLE, RA and PsA patients. These observations suggest that P-gp antagonists could be adopted to revert drug resistance and improve disease outcome. The complex inter-relationship among drug resistance, P-gp expression and autoimmunity still remains elusive.

Beyond DNA repair, the immunological role of PARP-1 and its siblings.

ADP-ribosylation is the addition of one or more (up to some hundreds) ADP-ribose moieties to acceptor proteins. There are two major families of enzymes that catalyse this reaction: extracellular ADP-ribosyl-transferases (ARTs), which are bound to the cell membrane by a glycosylphosphatidylinositol anchor or are secreted, and poly(ADP-ribose)-polymerases (PARPs), which are present in the cell nucleus and/or cytoplasm. Recent findings revealed a wide immunological role for ADP-ribosylating enzymes. ARTs, by sensing extracellular NAD concentration, can act as danger detectors. PARP-1, the prototypical representative of the PARP family, known to protect cells from genomic instability, is involved in the development of inflammatory responses and several forms of cell death. PARP-1 also plays a role in adaptive immunity by modulating the ability of dendritic cells to stimulate T cells or by directly affecting the differentiation and functions of T and B cells. Both PARP-1 and PARP-14 (CoaSt6) knockout mice were described to display reduced T helper type 2 cell differentiation and allergic responses. Our recent findings showed that PARP-1 is involved in the differentiation of Foxp3+ regulatory T (Treg) cells, suggesting a role for PARP-1 in tolerance induction. Also ARTs regulate Treg cell homeostasis by promoting Treg cell apoptosis during inflammatory responses. PARP inhibitors ameliorate immune-mediated diseases in several experimental models, including rheumatoid arthritis, colitis, experimental autoimmune encephalomyelitis and allergy. Together these findings show that ADP-ribosylating enzymes, in particular PARP-1, play a pivotal role in the regulation of immune responses and may represent a good target for new therapeutic approaches in immune-mediated diseases.

Folic acid and methionine in the prevention of teratogen-induced congenital defects in mice.

INTRODUCTION: Periconceptional supplementation with multivitamins containing folic acid reduces the risk of congenital malformations. We have previously investigated the effect on the murine development of a multiple retinoic acid competitive antagonist, Bristol-Myers-Squibb 189453, showing that treated fetuses were affected with heart defects, thymus aplasia or hypoplasia, and severe anomalies of the central nervous system. Hereby, we analyzed the effects of nutritive therapy involving folic acid and methionine on teratogen-induced congenital defects in mice. MATERIALS AND METHODS: A total of 132 outbred CD1 litters were studied. Pregnant mice were divided into four experimental groups, and an oral supplementation of H(2)O or folic acid, or methionine, or folic acid+methionine was administered from 0.5 days postcoitum until the end of pregnancy. At 7.5 days postcoitum, mice from all these groups were administered Bristol-Myers-Squibb 189453 to induce the teratogenic effect. At the end of pregnancy, fetuses were dissected and tissues were analyzed by histology and flow cytometric assays. RESULTS: Folic acid reduces congenital heart diseases from 81.3% to 64.8%, neural tube defects from 20.3% to 3.7%, and thymus abnormalities from 98.4% to 27.8%, restoring a normal number of differentiated thymus cells. Methionine is less effective in contrasting congenital heart diseases and neural tube defects, and induces thymus cell proliferation but not differentiation. Folic acid+methionine weakly reduce congenital heart diseases and neural tube defects, but consistently reduce the incidence of fetuses affected with thymus pathologies from 98.4% to 67.7%. CONCLUSIONS: Our results suggest that folic acid and methionine periconceptional supplementations may influence the incidence of congenital defects and may probably induce negative selection of embryos presenting developmental anomalies.

CpG drives human transitional B cells to terminal differentiation and production of natural antibodies.

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.

Effects of GSM-modulated radiofrequency electromagnetic fields on mouse bone marrow cells.

We examined the effects of in vivo exposure to a GSM-modulated 900 MHz RF field on the ability of bone marrow cells to differentiate, colonize lymphatic organs, and rescue lethally X-irradiated mice from death. X-irradiated mice were injected with medium alone or containing bone marrow cells from either RF-field-exposed (SAR 2 W/kg, 2 h/day, 5 days/ week, 4 weeks) or sham-exposed or cage control donor mice. Whereas all mice injected with medium alone died, mice that received bone marrow cells survived. Three and 6 weeks after bone marrow cell transplantation, no differences in thymus cellularity and in the frequencies of differentiating cell subpopulations (identified by CD4/CD8 expression) were observed among the three transplanted groups. Mitogen-induced thymocyte proliferation yielded comparable levels in all transplanted groups. As to the spleen, no effects of the RF-field exposure on cell number, percentages of B and T (CD4 and CD8) cells, B- and T-cell proliferation, and IFN-gamma production were found in transplanted mice. In conclusion, our results show no effect of in vivo exposure to GSM-modulated RF fields on the ability of bone marrow precursor cells to home and colonize lymphoid organs and differentiate in phenotypically and functionally mature T and B lymphocytes.