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Undiagnosed monogenic diseases represent a challenging group of human conditions highly suspicious to have a genetic origin, but without conclusive evidences about it. We identified two brothers born prematurely from a non-consanguineous healthy couple, with a neonatal-onset, chronic disease characterized by severe skin and bone inflammatory manifestations and a fatal outcome in infancy. We conducted DNA and mRNA analyses in the patients' healthy relatives to identify the genetic cause of the patients' disease. DNA analyses were performed by both Sanger and next-generation sequencing, which identified two novel heterozygous IL1RN variants: the intronic c.318 + 2T>G variant in the father and a ≈2,600-bp intragenic deletion in the mother. IL1RN mRNA production was markedly decreased in both progenitors when compared with healthy subjects. The mRNA sequencing performed in each parent identified two novel, truncated IL1RN transcripts. Additional experiments revealed a perfect intrafamilial phenotype-genotype segregation following an autosomal recessive inheritance pattern. The evidences shown here supported for the presence of two novel loss-of-function (LoF) IL1RN pathogenic variants in the analyzed family. Biallelic LoF variants at the IL1RN gene cause the deficiency of interleukin-1 receptor antagonist (DIRA), a monogenic autoinflammatory disease with marked similarities with the patients described here. Despite the non-availability of the patients' samples representing the main limitation of this study, the collected evidences strongly suggest that the patients described here suffered from a lethal form of DIRA likely due to a compound heterozygous genotype at IL1RN , thus providing a reliable genetic diagnosis based on the integration of old medical information with currently obtained genetic data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Urbaneja, Bonet, Solis-Moruno, Mensa-Vilaro, de Landazuri, Tormo, Lara, Plaza, Fabregat, Yagüe, Casals and Arostegui.)

Background: The vacuoles, E1-enzyme, X linked, autoinflammatory and somatic (VEXAS) syndrome is an adult-onset autoinflammatory disease (AID) due to postzygotic UBA1 variants., Objectives: To investigate the presence of VEXAS syndrome among patients with adult-onset undiagnosed AID. Additional studies evaluated the mosaicism distribution and the circulating cytokines., Methods: Gene analyses were performed by both Sanger and amplicon-based deep sequencing. Patients' data were collected from their medical charts. Cytokines were quantified by Luminex., Results: Genetic analyses of enrolled patients (n=42) identified 30 patients carrying UBA1 pathogenic variants, with frequencies compatible for postzygotic variants. All patients were male individuals who presented with a late-onset disease (mean 67.5 years; median 67.0 years) characterised by cutaneous lesions (90%), fever (66.7%), pulmonary manifestations (66.7%) and arthritis (53.3%). Macrocytic anaemia and increased erythrocyte sedimentation rate and ferritin were the most relevant analytical abnormalities. Glucocorticoids ameliorated the inflammatory manifestations, but most patients became glucocorticoid-dependent. Positive responses were obtained when targeting the haematopoietic component of the disease with either decitabine or allogeneic haematopoietic stem cell transplantation. Additional analyses detected the UBA1 variants in both haematopoietic and non-haematopoietic tissues. Finally, analysis of circulating cytokines did not identify inflammatory mediators of the disease., Conclusion: Thirty patients with adult-onset AID were definitively diagnosed with VEXAS syndrome through genetic analyses. Despite minor interindividual differences, their main characteristics were in concordance with previous reports. We detected for the first time the UBA1 mosaicism in non-haematopoietic tissue, which questions the previous concept of myeloid-restricted mosaicism and may have conceptual consequences for the disease mechanisms., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Pathogenic RIPK1 variants have been described as the cause of two different inborn errors of immunity. Biallelic loss-of-function variants cause the recessively inherited RIPK1 deficiency, while monoallelic variants impairing the caspase-8-mediated RIPK1 cleavage provoke a novel autoinflammatory disease (AID) called cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome. The aim of this study was to characterize the pathogenicity of two novel RIPK1 variants located at the cleavage site of caspase-8 detected in patients with dominantly-inherited, early-onset undefined AID. RIPK1 genotyping was performed by Sanger and next-generation sequencing. Clinical and analytical data were collected from medical charts, and in silico and in vitro assays were performed to evaluate the functional consequences. Genetic analyses identified two novel heterozygous RIPK1 variants at the caspase-8 cleavage site (p.Leu321Arg and p.Asp324Gly), which displayed a perfect intrafamilial phenotype-genotype segregation following a dominant inheritance pattern. Structural analyses suggested that these variants disrupt the normal RIPK1 structure, probably making it less accessible to and/or less cleavable by caspase-8. In vitro experiments confirmed that the p.Leu321Arg and p.Asp324Gly RIPK1 variants were resistant to caspase-8-mediated cleavage and induced a constitutive activation of necroptotic pathway in a similar manner that previously characterized RIPK1 variants causing CRIA syndrome. All these results strongly supported the pathogenicity of the two novel RIPK1 variants and the diagnosis of CRIA syndrome in all enrolled patients. Moreover, the evidences here collected expand the phenotypic and genetic diversity of this recently described AID, and provide interesting data about effectiveness of treatments that may benefit future patients., (© 2022. The Author(s).)

Objective: Autoinflammatory diseases are inherited disorders of innate immunity that usually start during childhood. However, several recent reports have described an increasing number of patients with autoinflammatory disease starting in adulthood. This study was undertaken to investigate the underlying cause of a case of late-onset uncharacterized autoinflammatory disease., Methods: Genetics studies were performed using Sanger sequencing and next-generation sequencing (NGS) methods. In silico, in vitro, and ex vivo analyses were performed to determine the functional consequences of the detected variant., Results: We studied a 57-year-old woman who at the age of 47 years began to have recurrent episodes of fever, myalgias, arthralgias, diffuse abdominal pain, diarrhea, adenopathies, and systemic inflammation, which were relatively well controlled with anti-interleukin-1 (anti-IL-1) drugs. NGS analyses did not detect germline variants in any of the known autoinflammatory disease-associated genes, but they identified the p.Ser171Phe NLRC4 variant in unfractionated blood, with an allele fraction (2-4%) compatible with gene mosaicism. Structural modeling analyses suggested that this missense variant might favor the open, active conformation of the NLRC4 protein, and in vitro and ex vivo analyses confirmed its propensity to oligomerize and activate the NLRC4 inflammasome, with subsequent overproduction of IL-18., Conclusion: Our findings indicate that the postzygotic p.Ser171Phe NLRC4 variant is a plausible cause of the disease in the enrolled patient. Functional and structural studies clearly support, for the first time, its gain-of-function behavior, consistent with previously reported NLRC4 pathogenic variants. These novel findings should be considered in the diagnostic evaluation of patients with adult-onset uncharacterized autoinflammatory disease., (© 2021 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Identification of the novel HLA-B*27 null allele officially designated HLA-B*27:225N., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The influence of polymer composition on the sensitivity towards nitrite (NO 2 - ) and nitric oxide (NO) of a series of 19 polymeric hydrogel films has been studied. The polymers, based on the hydrophilic monomer 2-hydroxyethylmethacrylate (HEMA), are able to encapsulate the colorimetric indicator 1,2-diaminoanthraquinone (DAQ) and to respond to NO 2 - and NO by visual changes. In the case of nitrite, the calculated limits of detection (LOD) for two of the polymeric sensors (10 μM) are very close to the sensitivity estimated for free DAQ in solution (LOD 5 μM), but with the advantage of a solid supported sensor with the format of a disposable test-strip made with affordable starting chemicals. The results are interpreted taking into account the nature and proportions of monomers and cross-linkers used for the synthesis of polymers. Key factors for obtaining sensitive materials are the hydrophilic character of the film along with the utilization of low levels of cross-linker and the use of an acidic monomer, like acrylic acid, as a building block.

Two new photoactive materials compatible with environmentally friendly solvents (water and methanol) have been synthesized and characterized. They are comprised of a porous matrix of polystyrene and divinylbenzene with bound Rose Bengal and additional pendant groups added to increase the hydrophilicity (ethylenediamine and γ-gluconolactone). The new polymers are efficient photocatalysts capable of generating singlet oxygen after irradiation with visible light. Photochemical oxygenations of 9,10-anthracenedipropionic acid and 2-furoic acid have been carried out. The measured conversions indicate that the new supported photosensitizers are more effective than the parent hydrophobic polymer.

Nitric oxide (NO) is involved in many biological processes. Aromatic ortho-diamine derivatives are commonly used in the fluorescence imaging of NO in living cells. ortho-diamino (o-diamino) compounds are believed to react with NO in an oxygenated medium leading to the formation of a triazole derivative. One such o-diamino compound, 1,2-diaminoanthraquinone (DAA), is a nontoxic probe for the detection of NO in living tissues and cells. The formation of the DAA triazole derivative (DAA-TZ) upon reaction of DAA with NO/O(2) within cells has not been demonstrated previously. The aim of this study was to confirm that DAA-TZ is the species formed intracellularly when DAA reacts with NO in the presence of oxygen. The chemical synthesis and characterisation of DAA-TZ was performed together with intracellular studies of DAA and DAA-TZ. Raw 264.7 macrophages were loaded with the DAA or DAA-TZ under conditions of no-stimulation or stimulation with interferon-γ and lipopolysaccharide to produce NO. Confocal microscopy was used to image the DAA-loaded macrophage cells. Analysis of the emission spectra allowed precise discrimination of the fluorescence of each species in the macrophage cells, and confirmed the identity of DAA-TZ as the intracellular reaction product between DAA and NO in the presence of oxygen., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The CD2, CD3, and CD5 antigens are down-modulated from the cell surface of peripheral blood mononuclear cells after a 24-hr incubation with specific monoclonal antibodies (mAb). Here we show that active (phorbol myristate acetate, phorbol dibutyrate acetate, and mezerein) but not inactive (4 beta-phorbol) tumor-promoting agents inhibit the mAb-induced modulation of CD2 and CD5, but not CD3, without concomitant changes in the surface distribution of these antigens (such as capping). This inhibitory effect is not protein synthesis dependent and is reversed by protein kinase C inhibitors (staurosporine and H-7). The use of cytoskeleton-disrupting agents shows the existence of different cytoskeletal interactions driving the mAb-induced modulation of CD2 and CD5 with respect to CD3. Treatment with cytochalasin D (an agent that inhibits microfilament polymerization) but not colchicine (an agent that inhibits microtubule polymerization) reproduced the effect of TPA on the mAb-induced modulation of CD2, CD3, and CD5. Our results indicate that the mAb-induced modulation of CD2 and CD5 is dependent on microfilament (namely actin) polymerization and PKC activation, while the modulation of CD3 is not.

PPL.1, a Jurkat cell line variant deficient in CDw50 surface expression, has been selected by fluorescence-activated cell sorting and expanded in cell culture. We have studied the expression of several leukocyte surface markers (CD2, CD3, CD4, CD8, CD7, CD26, CD25, CD14, CD18, kCD20, CD43, CD45, CD45R, CD71 and HLA class I and II) and we find no differences in their expression between PPL.1 and its parental Jurkat cell line. Immunoprecipitation analysis of metabolically labelled PPL.1 cells ([35S]-cysteine plus [35S]-methionine) fails to detect the presence of a preformed cytoplasmic pool of CDw50 molecules. The deficient CDw50 expression on PPL.1 cells is stable after several weeks of continuous culture and even after exposure of cells to several lymphocyte activating agents (PGE2, PHA, Con A, calcium ionophore A23187 and human recombinant IFN-gamma). No karyotype changes responsible for such phenotype deficiency are found. PPL.1 cells are as efficient as wild-type Jurkat or K562 cells, when used as targets in cytotoxicity assays with fresh or PHA-stimulated peripheral blood lymphocytes. No blocking effects of CDw50-specific mAb are observed in such assay. These results are consistent with the fact that CDw50 is not involved in alloreactive T-cell-specific cytotoxicity. They also suggest that this antigen is implicated only on a very specialized type of cell-cell interactions.

The expression and role of interleukin-2/interleukin-2 receptor (IL-2/IL-2R) system in the pokeweed mitogen (PWM)-induced T cell mitogenesis was studied. In the absence of monocytes (Mo), both soluble and Sepharose-bound PWM fail to induce T cell mitogenesis even when exogenous IL-2 or IL-1 or IL-1 + IL-2 or IL-4 are also present. In the presence of Mo, PWM stimulation of T lymphocytes (highly depleted of B lymphocytes) induces as much IL-2 mRNA as phytohemagglutinin (PHA), but results in higher and persistent IL-2 levels in culture supernatants despite the concomitant T cell mitogenesis, suggesting that PWM-activated T cells do not utilize the IL-2 they produce. Confirming this notion, Mo-dependent PWM-preactivated T cells, as compared to PHA-preactivated ones: (a) failed to consume exogenous IL-2 and their mitogenic response did not increase upon exposure to exogenous IL-2; (b) exhibited very low numbers of high-affinity IL-2R; and (c) showed lower expression of IL-2R p55 and undetectable expression of IL-2R p75 on their surface. Moreover, the PWM-induced T cell mitogenesis was not inhibited by anti-IL-2 or CD25 antibodies and only partially (50%-60%) inhibited by cyclosporin A, while these treatments abrogated the PHA-induced one. PWM-activated T cells, as compared to the PHA-activated ones, exhibited as high (p55) or even higher (p75) mRNA expression of both IL-2R p55 and p75 subunits. The possibility that PWM interferes with IL-2R subunits once expressed on the T cell surface was excluded. Thus, intracellular PWM-related events are likely to impair IL-2R expression post-transcriptionally. Possible explanations for this effect and its relation with the capacity of PWM to induce T cell-dependent B cell differentiation are discussed.

The p53 gene is a tumor suppressor gene located on chromosome 17p. Deletions of this chromosome and point mutations of p53 have been implicated in the development of colonic neoplasms. We have analyzed the loss of heterozygosity of the human p53 tumor suppressor gene in 40 cases of colorectal carcinoma using two restriction fragment length polymorphisms detected by BglII and AccII restriction enzymes. p53 gene product expression was studied immunohistochemically in 64 colorectal carcinomas, 18 adenomas, and 40 normal colonic mucosae using an anti-human p53 monoclonal antibody (Pab 1801) and the avidin-biotin-peroxidase complex technique. Twelve of the 40 patients (30%) were polymorphic for the p53 gene. In ten of these informative patients (83%), the tumor samples showed the loss of one allele when compared with normal colorectal samples of the same patient. One of the homozygous patients showed a loss of both p53 alleles. p53 immunostaining was observed in 43 of 64 carcinomas (67%) but only in two adenomas (11%). These two positive adenomas showed areas of carcinoma in situ. The normal mucosa was always negative. No relation could be found between p53 immunostaining and the degree of differentiation, the extension of the tumor, or the Ki-67 proliferative index. Mucinous carcinomas and right-side carcinomas were less p53 immunoreactive (25% and 52%, respectively) than the usual adenocarcinomas (73%) and distal tumors (72%). These findings suggest that p53 may be a target of chromosome 17 deletions and that this gene may play a role in the malignant transformation of adenomas. BglII and AccII restriction fragment length polymorphism analysis of the p53 gene may be a useful and direct technique to detect allelic loss of this gene in tumors.