Behçet disease (BD) and BD-like clinical phenotypes: NF-κB pathway in mucosal ulcerating diseases.

Behçet's disease (BD) is a heterogeneous multi-organ disorder in search of a unified pathophysiological theory and classification. The disease frequently has overlapping features resembling other disease clusters, such as vasculitides, spondyloarthritides and thrombophilias with similar genetic risk variants, namely HLA-B*51, ERAP1, IL-10, IL-23R. Many of the BD manifestations, such as unprovoked recurrent episodes of inflammation and increased expression of IL-1, IL-6 and TNFα, overlap with those of the hereditary monogenic autoinflammatory syndromes, positioning BD at the crossroads between autoimmune and autoinflammatory syndromes. BD-like disease associates with various inborn errors of immunity, including familial Mediterranean fever, conditions related to dysregulated NF-κB activation (eg TNFAIP3, NFKB1, OTULIN, RELA, IKBKG) and either constitutional trisomy 8 or acquired trisomy 8 in myelodysplastic syndromes. We review here the recent advances in the immunopathology of BD, BD-like diseases and the NF-κB pathway suggesting new elements in the elusive BD etiopathogenesis.

Leukocyte Nucleus Reveals a Linear Order of Chromosomes Separated in Two Parental Genomes That Favors the Process of Gene Activation.

Analysis of trisomy 8 cells and the chromosome-specific fluorescence in situ hybridization (FISH) signals on the ring-shaped nucleus of a neutrophil reveal that homologue chromosomes orient in diametrical opposition to each other. This positioning results in a separation of the two haploid sets of parental chromosomes organized as two exclusive groups. These two groups impart the nucleus a symmetry that fortifies immune protection by accelerating chemotaxis. The ring form of the nucleus is a legacy of the orientation of chromosomes as a rosette during metaphase and telophase stages. A dual control maintains this spatial order: (1) chromosomes are tethered to the centriole all through the cell cycle, and (2) during their circular orientation in telophase the chromosomes bind to each other with lamins, which reorganize the nuclear membrane of the daughter nuclei, generating an additional anchorage. Here, chromosomes serve as temporary packets to assure proper distribution of the nuclear DNA during mitosis. The remainder time of the cell cycle the chromosomes are chained together across the telomeres, allowing a continuous sequence of genes of the two genomes, maternal and paternal, thus facilitating easy reading of the gene sequence. Exceptions to these orders are either physiological and temporary, or pathological and disease causing.

Evidence of increased humoral endocrine organ-specific autoimmunity in severe and classic X-chromosome aneuploidies in comparison with 46,XY control subjects.

OBJECTIVE: In literature, the importance of X-linked gene dosage as a contributing factor for autoimmune diseases is generally assumed. However, little information is available on the frequency of humoral endocrine organ-specific autoimmunity in X-chromosome aneuploidies. In our preliminary study, we investigated the endocrine organ-specific humoral autoimmunity relative to four different organ-specific autoimmune diseases in a group of adult 47,XXY KS patients and in adults 46,XY control males (type 1 diabetes, T1DM; Addison's disease, AD; Hashimoto thyroiditis, HT; autoimmune chronic atrophic gastritis, AG). The aim of the present study is to evaluate the frequency of autoantibodies (Abs) specific for T1DM, AD, HT, and AG in rarer higher grade X-chromosome aneuploidies (HGA) and in 47,XXY children. DESIGN AND METHODS: Samples from 192 Caucasian patients with an X-chromosome aneuploidy (176 patients (55 children, 121 adults) with 47,XXY karyotype (KS patients) and 16 HGA patients (eight children, eight adults)) recruited from Sapienza, University of Rome (2007-2017) were tested for Abs specific for T1DM (insulin-Abs, GAD-Abs, IA-2-Abs, Znt8-Abs), HT (TPO-Abs), AD (21-OH-Abs), and AG (APC-Abs). The results were compared to those found in 213 46,XY control subjects (96 children, 117 adults). RESULTS: Altogether humoral organ-specific immunoreactivity was found in 13% of KS and HGA patients, with a significantly higher frequency than in the controls (p=.008). Almost 19% of HGA patients were positive for at least one of the organ-specific Abs investigated compared to 12.5% of KS patients. The frequency of the overall immunoreactivity was higher in KS children than in KS adults. The frequency of diabetes-specific Abs was significantly higher in the patient cohort than in controls (p=.005). Thyroid- and gastric-specific autoimmunity was also found in KS and HGA patients, while adrenal-specific immunoreactivity was rare. CONCLUSIONS: These results suggest for the first time that the risk of endocrine organ-specific humoral autoimmunity progressively increases with the severity of X-chromosome polisomy. The screening for diabetes-, thyroid-, and gastric-specific autoimmunity should be considered in clinical practice for identifying rare and classic X-chromosome aneuploid patients at risk of developing organ-specific autoimmune diseases.

The role of cytokines in first trimester pregnancy losses with fetal chromosomal anomaly.

OBJECTIVES: The contribution of local inflammation to the pathophysiology of abnormal choromosomally miscarriages remains unclear The objective of this study was to investigate the inflammatory response at the maternofetal interface of women presenting with first trimester miscarriage with abnormal choromosomally MATERIAL AND METHODS: Level of TNF-α , IL-6 ve IL-17 were asseyed using immunohistochemistry technique at decidual and placental bed biopsy samples from 23 women with elective termination of pregnancy 21 euploid and 18 aneuploid missed miscarriages. Immunostainig for TNF-α, IL-6 ve IL-17 has been evaluated semi-quantitatively by 'quickscore' method. RESULTS: We found that the intensity of TNF-α staining was high in the miscarriage group, and this has been found in previous studies. Unlike some previous studies, the intensity of IL-6 staining was higher in the miscarriage groups only in decidual glandular epithelium. The intensity of IL-6 staining was found to be higher in the miscarriage group with chromosome anomaly than in the miscarriage group without chromosome anomaly. There was no significant difference in IL-17 levels between any of the groups. CONCLUSIONS: Cytokines are considered to play an important role in the maintenance of pregnancy but the exact mechanism between them and the mutual regulation relationship were not been fully understood, which need our further study.

Development of plasma cell myeloma in a B-cell chronic lymphocytic leukemia patient with chromosome 12 trisomy.

BACKGROUND: Cancer development results from the progressive accumulation of genomic abnormalities that culminate in the neoplastic phenotype. Cytogenetic alterations, mutations and rearrangements may be considered as molecular legacy which trace the clonal history of the disease. Concomitant tumors are reported and they may derive from a common or divergent founder clone. B-cell chronic lymphocytic leukemia (B-CLL) and plasma cell myeloma (PCM) are both mature B-cell neoplasms, and their concomitancy, albeit rare, is documented. CASE PRESENTATION: Here, we described a patient with prior B-CLL with secondary development of PCM. Cytogenetic and multi parametric flow cytometry analyses were performed. The B-CLL population presented chromosome 12 trisomy, unlikely the arisen PCM population. CONCLUSION: The close follow up of B-CLL patients is important for early intervention in case of development of other malignancy, such as myeloma. Our observation suggests these two diseases may have arisen from different clones. We understand that the investigation of clonal origin may provide important information regarding therapeutic decisions, and should be considered in concomitant neoplasm.

Molecular Diagnostic Challenges and Complex Management of Consecutive Twin Pregnancies in a Family with CD40 Ligand Deficiency.

X-linked hyper-IgM syndrome (XHIGM) is a primary immunodeficiency disorder (PID) caused by mutation in the gene encoding the CD40 ligand (CD40L) expressed on activated T cells. Prenatal genotyping in carriers with twin pregnancies is more challenging than in women with singleton pregnancies. In addition, women with twin pregnancies may decide on selective termination for which the risk of loss of the healthy foetus may exceed 7%. We report here on a family affected by XHIGM. Diagnosis of the disease was made in a male patient as late as 33 years of age. After family screening, the sister of the proband conceived male twins in two consecutive pregnancies. In the first pregnancy, one of the male foetuses was hemizygous for the c.521A>G (Q174R) mutation in the CD40L gene. In the second pregnancy, ultrasound scan showed one foetus to have exencephaly and karyotyping revealed this foetus to have trisomy 18. Several options were discussed, but the parents decided on selective termination in both pregnancies. The interventions were successful in both cases, and the mother now has two healthy sons. This report demonstrates the way in which advanced technologies in molecular medicine and obstetric interventions may assist families with decisions about possible selective termination in case of life-threatening molecular or chromosomal disorders. Diagnosis of CD40L deficiency at the age of 33 years in the proband was striking and indicated that PIDs are still neglected as disease entities in the evaluation of patients with recurrent severe infectious diseases.

Immunodeficiency in a child with partial trisomy 6p.

AIM: We present a mentally retarded boy with partial trisomy of the short arm of chromosome 6 as a result of an interstitial tandem duplication of 6p12.2-p21.31 and immunodeficiency. Patients with mental retardation because of a chromosomal disorder or eponymous syndrome often experience recurrent respiratory tract infections as a result of their associated anatomical or neurological abnormalities. However, associated immune defects may also significantly contribute to their susceptibility to infections. Timely recognition and appropriate treatment of their immunodeficiency will greatly improve quality of life in these patients. CONCLUSION: Immunodeficiency may be the direct cause of recurrent respiratory tract infections in patients with mental retardation because of a chromosomal disorder or eponymous syndrome, even in the face of feeding difficulties and multiple episodes of aspiration, as is illustrated in this boy with partial trisomy 6p.

T-cell immune responses to Wilms tumor 1 protein in myelodysplasia responsive to immunosuppressive therapy.

Clinical observations and laboratory evidence link bone marrow failure in myelodysplastic syndrome (MDS) to a T cell-mediated immune process that is responsive to immunosuppressive treatment (IST) in some patients. Previously, we showed that trisomy 8 MDS patients had clonally expanded CD8(+) T-cell populations that recognized aneuploid hematopoietic progenitor cells (HPC). Furthermore, microarray analyses showed that Wilms tumor 1 (WT1) gene was overexpressed by trisomy 8 hematopoietic progenitor (CD34(+)) cells compared with CD34(+) cells from healthy donors. Here, we show that WT1 mRNA expression is up-regulated in the bone marrow mononuclear cells of MDS patients with trisomy 8 relative to healthy controls and non-trisomy 8 MDS; WT1 protein levels were also significantly elevated. In addition, using a combination of physical and functional assays to detect the presence and reactivity of specific T cells, respectively, we demonstrate that IST-responsive MDS patients exhibit significant CD4(+) and CD8(+) T-cell responses directed against WT1. Finally, WT1-specific CD8(+) T cells were present within expanded T-cell receptor Vß subfamilies and inhibited hematopoiesis when added to autologous patient bone marrow cells in culture. Thus, our results suggest that WT1 is one of the antigens that triggers T cell-mediated myelosuppression in MDS.

Generation of a panel of antibodies against proteins encoded on human chromosome 21.

BACKGROUND: Down syndrome (DS) is caused by trisomy of all or part of chromosome 21. To further understanding of DS we are working with a mouse model, the Tc1 mouse, which carries most of human chromosome 21 in addition to the normal mouse chromosome complement. This mouse is a model for human DS and recapitulates many of the features of the human syndrome such as specific heart defects, and cerebellar neuronal loss. The Tc1 mouse is mosaic for the human chromosome such that not all cells in the model carry it. Thus to help our investigations we aimed to develop a method to identify cells that carry human chromosome 21 in the Tc1 mouse. To this end, we have generated a panel of antibodies raised against proteins encoded by genes on human chromosome 21 that are known to be expressed in the adult brain of Tc1 mice RESULTS: We attempted to generate human specific antibodies against proteins encoded by human chromosome 21. We selected proteins that are expressed in the adult brain of Tc1 mice and contain regions of moderate/low homology with the mouse ortholog. We produced antibodies to seven human chromosome 21 encoded proteins. Of these, we successfully generated three antibodies that preferentially recognise human compared with mouse SOD1 and RRP1 proteins on western blots. However, these antibodies did not specifically label cells which carry a freely segregating copy of Hsa21 in the brains of our Tc1 mouse model of DS. CONCLUSIONS: Although we have successfully isolated new antibodies to SOD1 and RRP1 for use on western blots, in our hands these antibodies have not been successfully used for immunohistochemistry studies. These antibodies are freely available to other researchers. Our data high-light the technical difficulty of producing species-specific antibodies for both western blotting and immunohistochemistry.

Behçet disease associated with myelodysplastic syndrome.

There have been 22 reported cases of Behçet disease associated with myelodysplastic syndrome. The majority of cases belong to incomplete types of Behçet disease and the refractory anemia subtype of myelodysplastic syndrome. We describe a case of a 49-year-old woman with Behçet disease who developed myelodysplastic syndrome with abnormal karyotype-trisomy 8. This change was not due to immunosuppressive agents because her Behçet disease was not treated with these drugs before the onset of myelodysplastic syndrome. This is the first report of a case of Behçet disease with pathologic evidence associated with the chronic myelomonocytic leukemia subtype of myelodysplastic syndrome. After reviewing the past case studies, we suggest that patients with myelodysplastic syndrome and trisomy 8 might be prone to have Behçet disease. Furthermore, more intestinal ulcers but with less eye lesions and arthritis have been noted in patients of Behçet disease with myelodysplastic syndrome than in those without myelodysplastic syndrome.

Characterization of trisomic natural killer cell abnormalities in a patient with constitutional trisomy 8 mosaicism.

Malignancies found in children and adults with constitutional trisomy 8 mosaicism (CT8M) could be in part the consequence of dysfunction of trisomic immune cells. An adult patient exhibiting trisomy in the entire natural killer (NK) cell population has made possible the characterization of trisomy 8-positive NK cells. The study showed normal cytotoxic activity but predominance of an immunosenescent phenotype (CD56(dim)CD94/NKG2(bright)) characterized by a weak response to IL-2, increased upregulation of CD95/Fas, and impaired TNF-alpha production. As these defects may contribute to the escape and expansion of neoplastic cells, the authors hypothesize that cancer predisposition in CT8M may be partly a result of altered immunosurveillance.

Prenatally diagnosable differences in the cellular immunity of fetuses with Down's and Edwards' syndrome.

INTRODUCTION: Lymphocyte subpopulations are identified by the uniform CD classification (Cluster of Differentiation) and can be accurately differentiated with monoclonal antibodies using the method of flow cytometry. With the aid of cordocentesis it is possible to perform studies on the status and development of cellular immunity as early as in the second trimester of pregnancy. OBJECTIVE: To compare lymphocyte subpopulations present in fetuses with chromosomal abnormalities (Down's syndrome (DS), Edwards' syndrome (ES)) and fetuses with normal karyotype. STUDY DESIGN: Prospective observational study. METHODS: We examined a total of 61 pregnant women with an average age of 31.5 years (20- 46 years). RESULTS: In fetuses with DS we found a significant decrease in B lymphocytes (CD19),a decrease in the subpopulations of multi-reactive B-cells (CD5(+)CD19(+), B-CLL),and a decrease in the index of CD4/CD8 and class II HLA-DR. In contrast, the representation of NK cells expressing /CD3-CD (16 + 56)+/ was greatly increased. In ES we found a decrease in T lymphocytes (CD3), a decrease in T-helper lymphocytes (monocytes CD4), a decreased index of CD4/CD8 and a greater representation of NK cells /CD3-CD (16 + 56)+/. CONCLUSION: We determined the normal values of lymphocyte subpopulations in physiological fetuses. We demonstrated that the immunological defect of the affected fetuses is already present antenatally, and can be reliably diagnosed in the second trimester of pregnancy.

Mice triallelic for the Ig heavy chain locus: implications for VHDJH recombination.

V(H)DJ(H) recombination has been extensively studied in mice carrying an Ig heavy chain rearranged transgene. In most models, inhibition of endogenous Ig rearrangement occurs, consistently with the feedback model of IgH recombination. Nonetheless, an incomplete IgH allelic exclusion is a recurrent observation in these animals. Furthermore, transgene expression in ontogeny is likely to start before somatic recombination, thus limiting the use of Ig-transgenic mice to access the dynamics of V(H)DJ(H) recombination. As an alternative approach, we challenged the regulation of somatic recombination with the introduction of an extra IgH locus in germline configuration. This was achieved by reconstitution of RAG2(-/-) mice with fetal liver cells trisomic for chromosome 12 (Ts12). We found that all three alleles can recombine and that the ratio of Ig allotype-expressing B cells follows the allotypic ratio in trisomic cells. Although these cells are able to rearrange the three alleles, the levels of Ig phenotypic allelic exclusion are not altered when compared with euploid cells. Likewise, we find that most VDJ rearrangements of the silenced allele are unable to encode a functional mu-chain, indicating that the majority of these cells are also genetically excluded. These results provide additional support for the feedback model of allelic exclusion.

Evidence for an interferon-related inflammatory reaction in the trisomy 16 mouse brain leading to caspase-1-mediated neuronal apoptosis.

The trisomy of human chromosome 21 (Down syndrome) is the leading genetic cause of learning difficulties in children, and predisposes this population to the early onset of the neurodegeneration of Alzheimer's disease. Down syndrome is associated with increased interferon (IFN) sensitivity resulting in unexpectedly high levels of IFN inducible gene products including Fas, complement factor C3, and neuronal HLA I which could result in a damaging inflammatory reaction in the brain. Consistent with this possibility, we report here that the trisomy 16 mouse fetus has significantly increased whole brain IFN-gamma and Fas receptor immunoreactivity and that cultured whole brain trisomy 16 mouse neurons have increased basal levels of caspase 1 activity and altered homeostasis of intracellular calcium and pH. The trisomic neurons also showed a heightened sensitivity to the increase in both Fas receptor levels and caspase 1 activity we observed when IFN-gamma was added to the neuron culture media. Because of the autoregulatory nature of IFN activity, and the IFN inducing capability of caspase-1-activated cytokine activity, our data argue in favor of the possibility of an interferon-mediated, self-perpetuating, inflammatory response in the trisomy brain that could subserve the loss of neuron viability seen in this trisomy 16 mouse model for Down syndrome.

Common variable immunodeficiency with mosaic trisomy 8: report of one case.

This case study reported a 17-year-old female of common variable immunodeficiency (CVID) associated with bronchiectasis, pernicious anemia and mosaic trisomy 8. Clinically this patient presented with recurrent sinopulmonary infections, intractable diarrhea, macrocytic anemia, and primary amenorrhea. Immunological tests showed pan-hypogammaglobulinemia and a decrease of peripheral blood B cells (4%) and CD4+ cells (25%). Lymphoproliferative responses to mitogen (PHA) and specific antigen (BCG) were profoundly impaired in the patient in comparison to those in control. Production of interleukin 4 (IL-4) and gamma interferon (IFN-gamma) in the in vitro lymphoproliferation was also profoundly depressed. Pernicious anemia demonstrated by larger MCV (112.9 fl) and hyper-segmental granulocytes on peripheral blood smear responded to parental administration of vitamin B12. Interestingly, she had a mosaic trisomy 8 in peripheral blood mononuclear cells but normal 46XX karyotype in the bone marrow cells. To our knowledge, this is the first case of CVID associated with mosaic trisomy 8 reported in the literature. As the case exemplifies, CVID should be considered when the physicians evaluate the patient presenting with recurrent sinopulmonary infections, diarrhea, malnutrition, and pernicious anemia. It requires further study to explore whether the genes in the chromosome 8 are linked to CVID.

Neutrophil and monocyte beta 2-integrin expression in trisomic fetuses.

Flow cytometry was used to measure neutrophil and monocyte beta 2-integrin expression in fetuses with trisomy 18 (n = 7) and trisomy 21 (n = 7) at 20-25 weeks' gestation. The values were compared with those of 112 chromosomally normal fetuses. There were no significant differences in beta 2-integrin expression between normal and aneuploid fetuses. These findings demonstrate that in trisomies 21 and 18, alteration in beta 2-integrin expression is unlikely to contribute to the pathogenesis of immunological deficiencies that have been observed in these aneuploidies both prenatally and postnatally.

Abnormal immunological development in fetuses with trisomy 18.

Flow cytometry was used to enumerate the lymphocyte subpopulations in fetal blood obtained by cordocentesis from eight trisomy 18 fetuses at 20-36 weeks' gestation. Compared with values in chromosomally normal fetuses, in trisomy 18 the mean T- and natural killer (NK) cell counts were significantly lower (t = -7.63, P < 0.001 and t = -3.58, P < 0.01, respectively); the mean B-cell count was not significantly different (t = -1.32). These findings demonstrate that in trisomy 18 there is abnormal intrauterine development of the immune system.