Neutrophil Disorders Research Articles

Association of busulfan exposure and outcomes after HCT for patients with an inborn error of immunity

AbstractAllogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment strategy for patients with inborn errors of immunities (IEIs). The objective of this study was to assess the optimal busulfan exposure before allogeneic HCT for patients with an IEI who received an IV busulfan–based conditioning regimen. Patients from 17 international centers were included. The main outcome of interest was event-free survival (EFS). Patients were categorized into 4 IEI subgroups: combined immunodeficiency (CID), severe combined immunodeficiency (SCID), neutrophil disorders, and hemophagocytic lymphohistiocytosis (HLH)–related disorders. Busulfan exposure was calculated by individual centers (area under the curve [AUC]CENTER) and re-estimated using a nonlinear mixed–effects model (NONMEM; exposure defined as AUCNONMEM). Overall, 562 patients were included: 173 (30.8%) with CID, 154 (27.4%) with SCID, 101 (18.0%) with HLH-related disorders, and 134 (23.8%) with neutrophil disorders. The median busulfan AUCNONMEM was 69.0 mg × h/L and correlated poorly with the AUCCENTER (r2 = 0.54). In patients with SCID, HLH-related, and neutrophil disorders with a busulfan AUCNONMEM of 70 to 90 mg × h/L, 2-year EFS was superior to <70 mg × h/L, and >90 mg ×h/L. Full donor chimerism increased with higher busulfan AUCNONMEM, plateauing at 90 mg × h/L. For patients with CID, the optimal AUCNONMEM for donor chimerism was found to be >70 mg × h/L. Improved EFS and higher donor chimerism may be achieved by targeting a cumulative busulfan AUCNONMEM of 80 mg × h/L (range, 70-90). Our study stresses the importance of uniformly using a validated population pharmacokinetic model to estimate AUCNONMEM.

Functional neutrophil disorders: Chronic granulomatous disease and beyond.

Since their description by Metchnikoff in 1905, phagocytes have been increasingly recognized to be the entities that traffic to sites of infection and inflammation, engulf and kill infecting organisms, and clear out apoptotic debris all the while making antigens available and accessible to the lymphoid organs for future use. Therefore, phagocytes provide the gateway and the first check in host protection and immune response. Disorders in killing and chemotaxis lead not only to infection susceptibility, but also to autoimmunity. We aim to describe chronic granulomatous disease and the leukocyte adhesion deficiencies as well as myeloperoxidase deficiency and G6PD deficiency as paradigms of critical pathways.

Clinical and functional spectrum of RAC2-related immunodeficiency

AbstractMutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

Morphological changes in hematopoietic parameters in COVID-19 and their prognostic significance

The types of morphological changes in the complete blood count, along with other laboratory parameters, can help establish the severity of the condition and provide the prognostic value in determining the survival of a patient with COVID-19. Peripheral blood smear (PBS) examination is a routine and simple procedure that can be performed at any medical institution. However, data on morphological changes in PBS during COVID-19 are still insufficient. Therefore, it is relevant to identify the features and patterns of these changes, taking into account the severity of the clinical presentation of COVID-19, and establish their prognostic significance. The aim. To review the literature on the presence of morphological changes in hematopoietic parameters in the case of SARS-CoV-2 infection with respect to the disease course.Results and Conclusions. Morphological changes in at least one blood cell lineage were observed in all cases of SARS-CoV-2 infection. The most common are nuclear and cytoplasmic disorders of neutrophils, such as hypogranulation and hypo/hypersegmentation of neutrophil nuclei, often with toxogenic stippling. Typical changes in lymphocyte morphology include monocytoid, lymphoplasmacytoid, granular, and atypical cells with nucleoli. Prognostically unfavorable morphological signs in the PBS include giant neutrophils and their toxic granulation, plasmacytization of lymphocytes, and a dynamic decrease in granular and abnormal lymphocytes and monocytes. A low count of lymphocytes and their subpopulations is associated with a severe clinical presentation and an unfavorable outcome of COVID-19 disease, which suggests the feasibility of their immunophenotypic evaluation and monitoring. The detection and serial monitoring of inflammatory monocytes using flow cytometry may be valuable in predicting the course and choosing adequate therapy for SARS-CoV-2 infection. Additionally, an increase in the red cell distribution width was also shown to have prognostic importance.

Zeldzame neutrofiele dermatose met belangrijke hematologische implicaties

Rare neutrophilic dermatosis with significant hematologic implications A patient was seen in the dermatology department because of a solitary lesion on the right forearm. This lesion had an annular aspect with a raised, bright erythematous, inflammatory border of confluent papules with an oedematous aspect. Previously, similar lesions had developed, regressing spontaneously after a few weeks. Histological examination showed a marked oedema and a dens dermal predominant neutrophilic infiltrate with leucocytoclasia without vasculitis. Based on the clinical picture, the chronic recurrent course and the histology, the diagnosis of chronic recurrent annular neutrophilic dermatosis (CRAND) was made. Since CRAND belongs to the neutrophilic skin disorders, a comprehensive blood test was performed, showing a monoclonal B cell population. In consultation with a fellow haematologist, additional CT scans of the thorax and abdomen were performed. These showed neither adenopathies nor hepatosplenomegaly, upon which the diagnosis of monoclonal B cell lymphocytosis was made. This condition increases the risk of developing chronic lymphatic leukaemia. This case report thus shows a rare neutrophilic dermatosis that differs from Sweet syndrome given the absence of fever, systemic symptoms or neutrophilia in the blood. This case is specifically relevant because of the additional finding of a monoclonal B cell lymphocytosis, increasing the risk of developing chronic lymphatic leukaemia. This demonstrates the importance of further additional investigation to exclude possible (pre)malignancies when the diagnosis of CRAND is made.

Neutrophil extracellular traps formation in the lesional skin of various types of pyoderma gangrenosum.

Pyoderma gangrenosum (PG) is a chronic neutrophilic disorder characterized by recurrent painful ulcers. Aseptic inflammation by neutrophils plays an essential role, and neutrophil extracellular traps (NETs) formation can contribute to the pathogenesis of PG. Seventy-five patients were diagnosed as having PG in our department, among which 58 ulcerative, 4 bullous, 3 pustular and 10 vegetative type. We examined the 20 skin biopsy specimens (11 ulcerative, 3 bullous, 2 pustular and 4 vegetative type), and local NETs formation in various types of PG was compared among each type. The biopsied specimens were double labelled for myeloperoxidase, citrullinated histone H3. Immunofluorescent images indicated that the histopathologic location and depth of NETs formation in PG varied by the clinical subtypes. In ulcerative PG, NETs formation was observed in the upper to deep dermis. In bullous PG, NETs formation was mainly observed in the epidermis. Pustular type showed NETs formation in the epidermis near the pustules, and in vegetative type, showed NETs formation mainly in the upper dermis. These results indicate that NETting neutrophils play an important role in the pathogenesis of various forms of PG, although the location and depth of NETs formation in the skin lesion of PG differ depending on each type. Further studies are necessary to examine what factors identify different clinical features of PG.

Bronchoalveolar Lavage Cytology in Severe Equine Asthma: Cytocentrifugated versus Sediment Smear Preparations.

Equine asthma is a common respiratory disease that may affect horses of any age. The diagnosis of severe equine asthma (SEA) (historically referred as recurrent airway obstruction or RAO) is based mainly on the history of the animal and clinical signs, which are further supported by the cytological examination of the bronchoalveolar lavage (BAL). This can also be helpful in monitoring the inflammation of the lower airways in response to environmental management and medication. The cytocentrifugated preparation is usually considered the method of choice for BAL cytological interpretation. The aim of this study was to compare the results in terms of differential cell counts (DCC) in BAL cytology performed on sedimented smears and cytocentrifugated preparations. To carry this out, 48 BAL samples were collected from six horses with SEA that were subjected to a process of exacerbation of the disease by environmental stimuli, which was later followed by the appropriate treatment. Each collected BAL fluid was equally divided into duplicate portions: one processed by cytocentrifugation and one by sediment smear from simple centrifugation. Cytologic examination of all BAL by both methods showed poor concordance in DCC, although it was still able to allow diagnostic recognition of severe lung neutrophilic disorders. These results suggest that sediment smear preparation, although remaining a useful method in general equine practice associated with clinical assessments in the diagnosis of SEA under conditions where there is no possibility of using a cytocentrifuge, cannot be considered a comparable alternative.

Microglia-Mediated Neuroimmune Response Regulates Cardiac Remodeling After Myocardial Infarction.

Background Sympathetic hyperactivity contributes to pathological remodeling after myocardial infarction (MI). However, the mechanisms underlying the increase in sympathetic activity remain unknown. Microglia are the predominant immune cells in the central nervous system and can regulate sympathetic neuron activity through neuroimmune response in the hypothalamic paraventricular nucleus. The present study aimed to investigate whether microglia-mediated neuroimmune response can regulate sympathetic activity and cardiac remodeling after MI. Methods and Results PLX3397 (pexidartinib) was used to deplete central microglia via intragastric injection or intracerebroventricular injection. After that, MI was induced by ligation of the left anterior descending coronary artery. Our study showed that MI resulted in the activation of microglia in the paraventricular nucleus. Microglia depletion, which was induced by PLX3397 treatment via intragastric injection or intracerebroventricular injection, improved cardiac function, reduced infarction size, and attenuated cardiomyocyte apoptosis, fibrosis, pathological electrical remodeling, and myocardial inflammation after MI. Mechanistically, these protective effects were associated with an attenuated neuroimmune response in the paraventricular nucleus, which contributed to the decrease of sympathetic activity and attenuation of sympathetic remodeling in the heart. However, intragastric injection with PLX3397 obviously depleted macrophages and induced neutrophil and T-lymphocyte disorders in the heart, blood, and spleen. Conclusions Microglia depletion in the central nervous system attenuates pathological cardiac remodeling after MI by inhibiting neuroimmune response and sympathetic activity. Intragastric administration of PLX3397 leads to serious deleterious effects in peripheral immune cells, especially macrophages, which should be a cause for concern in animal experiments and clinical practice.

Improved Outcome Following Busulfan-Based Conditioning in Children with Functional Neutrophil Disorders Undergoing Hematopoietic Stem Cell Transplant from HLA-Matched Donors.

Hematopoietic stem-cell transplantation (HSCT) is the only curative treatment for chronic granulomatous disease (CGD) and leukocyte-adhesion deficiency (LAD), but both diseases have high rates of graft failure in transplant and patients with these diseases are often referred to HSCT with significant comorbidity. The intensity of the conditioning regimen should be balanced between the need to ensure durable engraftment and to minimize toxicity when transplanting young children with infections and organ damage. We report on 26 children transplanted at our institution with CGD and LAD over 24years. We found a higher incidence of graft failure in patients receiving treosulfan based conditioning for their first transplant. There was no effect of conditioning regimen on overall survival, as all 8 patients that proceeded to a second busulfan-based HSCT were salvaged. We recommend giving patients with CGD and LAD fully myeloablative conditioning with either a busulfan-based regimen or the combination of treosulfan, fludarabine, and thiotepa.

The paradox of autoimmunity and autoinflammation in inherited neutrophil disorders - in search of common patterns.

Congenital defects of neutrophil number or function are associated with a severe infectious phenotype that may require intensive medical attention and interventions to be controlled. While the infectious complications in inherited neutrophil disorders are easily understood much less clear and explained are autoimmune and autoinflammatory phenomena. We survey the clinical burden of autoimmunity/autoinflammation in this setting, search for common patterns, discuss potential mechanisms and emerging treatments.

PIODERMA GANGRENOSUM ABOUT A CASE

Pyoderma gangrenosum is an inflammatory skin disease characterized by the formation of deep, painful ulcers that may heal with prominent scarring. This condition is classified as a neutrophilic disorder, which means it involves an exaggerated response from immune cells called neutrophils. Next, we will describe a clinical case of pyoderma gangrenosum that illustrates the typical characteristics of this disease. For its diagnosis, it was important to perform a biopsy of the lesions and their treatment. It is important to keep in mind that the disease can affect not only the physical health of the patient, but also her mental and emotional health.

Hypoxia Enhances Oxidative Stress in Neutrophils from ZZ Alpha-1 Antitrypsin Deficiency Patients.

Alpha-1 antitrypsin deficiency (AATD) is a neutrophilic inflammatory disorder that may result in local hypoxia, reactive oxygen and nitrogen species (ROS/RNS) production, and increased damage in adjacent tissues. This study aims to determine the impact of hypoxia on neutrophil oxidative stress profile in AATD patients. Neutrophils were isolated from AATD patients and control volunteers and exposed to hypoxia (1% O2 for 4 h), ROS/RNS, mitochondrial parameters, and non-enzymatic antioxidant defenses measured by flow cytometry. The expression of enzymatic antioxidant defenses was determined by qRT-PCR. Our results indicate that ZZ-AATD neutrophils produce higher amounts of hydrogen peroxide, peroxynitrite, and nitric oxide and decreased levels of the antioxidant enzymes catalase, superoxide dismutase, and glutathione reductase. Likewise, our results show a decrease in mitochondrial membrane potential, indicating that this organelle could be involved in the production of the reactive species observed. No decrease in glutathione and thiol levels were observed. The accumulation of substances with high oxidative capacity would explain the greater oxidative damage observed in proteins and lipids. In conclusion, our results indicate that, compared to MM control individuals, ZZ-AATD neutrophils show increased ROS/RNS production under hypoxic conditions opening a new rationale for using antioxidant therapies to treat the disease.

A viewpoint on the genetic determinants of generalised pustular psoriasis.

Generalised pustular psoriasis (GPP) is a rare and severe neutrophilic skin disorder, manifesting with acute episodes of pustulation and systemic upset. The discovery of recessive IL36RN mutations associated with GPP has transformed our understanding of disease drivers, paving the way for the development of targeted anti-IL36 therapeutics. In the light of these remarkable successes, this viewpoint reviews the significance of IL36RN mutations in GPP, their functional impact and their correlation with clinical phenotypes. It then covers the discovery of further genetic determinants (recessive MPO mutations) and risk factors (AP1S3 and CARD14 low-frequency variants) for the disease. It discusses the growing evidence for genetic complexity in GPP and concludes by outlining collaborative strategies that may be adopted to overcome the challenges ahead.

Use of Negative Pressure Wound Therapy to Successfully Treat Postoperative Pyoderma Gangrenosum Following Laparoscopic Surgery: A Case Report

Introduction. PG is a rare neutrophilic skin disorder with a variable clinical presentation, and diagnosis is often delayed. PPG typically presents as a necrotic ulcerative lesion with surrounding erythema and is often mistaken for a surgical site infection. Delayed diagnosis can lead to extensive morbidity and a prolonged healing time. Case Report. A 75-year-old male presented to the emergency department 2 weeks following laparoscopic appendectomy for acute appendicitis. He had fever and a suspected surgical site infection. Antibiotic treatment was initiated, and debridement was performed. Eventually, the diagnosis of PPG was made. Immunosuppressive therapy combined with NPWT was initiated. Complete wound healing was achieved 2 months after admission. Conclusions. Surgeons should have a high index of suspicion of underlying skin disorders when presumed postoperative wound infections do not respond to aggressive antibiotic and surgical treatment. Early multidisciplinary consultation should be considered. In the case of PPG, the authors of the present report advise early initiation of systemic immunosuppressive therapy combined with atraumatic wound care. NPWT should be considered as appropriate. Surgical debridement may be indicated in the setting of extensive necrosis or uncontrollable superinfection.

Assessment of the Frequency of Variants of Undetermined Significance (VUS) in Adult Patients with Chronic Idiopathic Neutropenia Studied for Myeloid Gene Mutations By Next Generation Sequencing

Background: Chronic Idiopathic Neutropenia (CIN) is a neutrophil disorder characterized by the persistent and unexplained reduction in the number of peripheral blood (PB) neutrophils. The diagnosis of CIN is based on exclusion criteria, namely the absence of clinical/laboratory evidence of any underlying disease or use of drugs that might be associated with neutropenia, absence of history of exposure to irradiation or use of chemical compounds, negative antineutrophil antibodies, and normal bone marrow (BM) morphology and karyotype. We have previously performed next generation sequencing (NGS) analysis of genes recurrently mutated in myeloid malignancies in a cohort of CIN patients and we found that 11.35% of the patients display clonal hematopoiesis in term of presence of pathogenic/likely pathogenic variants with VAF higher than 2%. Variants of undetermined significance (VUS) were ruled out from the analysis and their potential significance was not assessed. Aims: In the current study we have assessed the presence, frequency and potential clinical significance of VUS detected in our cohort of CIN patients previously studied for clonal hematopoiesis. Methods: Genomic DNA was extracted from CIN patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted NGS on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies (Tsaknakis G et al. Blood. 2021;138:1249-1257). Results: NGS analysis of myeloid genes has been performed in 139 CIN patients (106 female subjects and 33 male subjects, aged 21 to 89 years with a median age of 59 years). Re-analysis of patients' NGS data and assessment of VUS revealed that 35 out of 139 patients (25.18%) carried one or more VUS (a total of 39 VUS were detected, all with an allelic frequency of 50% and all missense variants). Genetic variants were evaluated according to the ACMG guidelines and classified as VUS. The majority of VUSs were observed in ATM (n=7) and SETBP1 (n=6) genes followed by TET2, NOTCH1 (4 occurrences respectively) and PLCG2 (n=3). Two different patients shared the same variant in TET2 gene whereas two siblings shared the same variant in SETBP1 gene. Pathogenicity score calculation based on the combined evidence from multiple other in-silico predictors identified 3 variants in 3 different genes as having the highest probability to be pathogenic. Namely these were PLCG2K456E,CXCR4R235C and SF3B1T56I. PLCG2 variants have been implicated in familial cold autoinflammatory syndrome 3 (PLCG2 associated antibody deficiency and immune dysregulation, PLAID) and in autoinflammation, antibody deficiency, and immune dysregulation syndrome (APLAID) whereas CXCR4 variants are known to be associated with Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. SF3B1 mutations have not yet been shown to be involved in hereditary disease. Conclusions: Our study of VUS assessment in a cohort of 139 well characterized adult CIN patients identified 39 VUS, all heterozygous, in a total of 35 patients. Further steps include paired analysis with constitutional reference tissue to determine the germline nature of the mutations. Moreover, these variants should be functionally tested and integrated with functional results and clinical phenotypes to more accurately interpret their potential contribution to disease onset. This study of VUS analysis in CIN patients is anticipated to contribute to the better understanding of the pathogenetic causes of this rare disease.

Brocq's geometric phagedena over penis with keloidal scars.

Pyoderma gangrenosum is a rare neutrophilic inflammatory skin disorder commonly seen over lower limbs. Involvement of penile area is rare. We report this rare case of occurrence of ulcerative type of pyoderma gangrenosum over penis with pustular type elsewhere over the body, healing with keloids in an immunocompetent young man with no systemic associations.

Intestinal inflammation alters the antigen-specific immune response to a skin commensal.

SUMMARYResident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Characterization of Eosinophilic Bronchiectasis: A European Multicohort Study.

Rationale: Bronchiectasis is classically considered a neutrophilic disorder, but eosinophilic subtypes have recently been described. Objectives: To use multiple datasets available through the European Multicentre Bronchiectasis Audit and Research Collaboration to characterize eosinophilic bronchiectasis as a clinical entity focusing on the impact of eosinophils on bronchiectasis exacerbations. Methods: Patients were included from five countries to examine the relationships between blood eosinophil counts and clinical phenotypes after excluding coexisting asthma. 16S rRNA sequencing was used to examine relationships between eosinophil counts and the sputum microbiome. A post hoc analysis of the PROMIS (Inhaled Promixin in the Treatment of Non-Cystic Fibrosis Bronchiectasis) phase 2 trial was used to examine the impact of blood eosinophil counts on exacerbations in patients with Pseudomonas aeruginosa infection. Measurements and Main Results: A relationship between sputum and blood eosinophil counts was demonstrated in two cohorts. In analysis of 1,007 patients from five countries, 22.6% of patients had blood eosinophil counts of ⩾300 cells/μl. Counts of <100 cells/μl were associated with higher bronchiectasis severity and increased mortality. There was no clear relationship with exacerbations. Blood eosinophil counts of ⩾300 cells/μl were associated with both Streptococcus- and Pseudomonas-dominated microbiome profiles. To investigate the relationship of eosinophil counts with exacerbations after controlling for the confounding effects of infection, 144 patients were studied in a clinical trial after treatment with antipseudomonal antibiotics. Compared with patients with blood eosinophil counts of <100 cells/μl (reference), elevated eosinophil counts of 100-299 cells/μl (hazard ratio, 2.38; 95% confidence interval, 1.33-4.25; P = 0.003) and ⩾300 cells/μl (hazard ratio, 3.99; 95% confidence interval, 2.20-7.85; P < 0.0001) were associated with shorter time to exacerbation. Conclusions: Eosinophilic bronchiectasis affects approximately 20% of patients. After accounting for infection status, raised blood eosinophil counts are associated with shortened time to exacerbation.

Treatment Options and Goals for Patients with Generalized Pustular Psoriasis.

Generalized pustular psoriasis (GPP) is a rare, severe neutrophilic skin disorder characterized by sudden widespread eruption of superficial sterile pustules with or without systemic inflammation. GPP flares can be life-threatening if untreated due to potential severe complications such as cardiovascular failure and serious infections. Currently, there are no GPP-specific therapies approved in the USA or Europe. Retinoids, cyclosporine, and methotrexate are the most commonly used non-biologic therapies for GPP. The evidence that supports the currently available treatment options is mainly based on case reports and small, open-label, single-arm studies. However, recent advances in our understanding of the pathogenic mechanisms of GPP and the identification of gene mutations linked to the disease have paved the way for the development of specific targeted therapies that selectively suppress the autoinflammatory and autoimmune mechanisms induced during GPP flares. Several biologic agents that target key cytokines involved in the activation of inflammatory pathways, such as tumor necrosis factor-α blockers and interleukin (IL)-17, IL-23, and IL-12 inhibitors, have emerged as potential treatments for GPP, with several being approved in Japan. The evidence supporting the efficacy of these agents is mainly derived from small, uncontrolled trials. A notable recent advance is the discovery of IL36RN mutations and the central role of IL-36 receptor ligands in the pathogenesis of GPP, which has defined key therapeutic targets for the disease. Biologic agents that target the IL-36 pathway have demonstrated promising efficacy in patients with GPP, marking the beginning of a new era of targeted therapy for GPP.Graphical abstract

Increased Frequency of Mutations in the Gene Responsible for Familial Mediterranean Fever (MEFV) in a Cohort of Patients with Chronic Idiopathic Neutropenia

▪Introduction-Aim: Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by the prolonged and unexplained reduction in the number of peripheral blood (PB) absolute neutrophil counts (ANC). The underlying pathogenesis in CIN implicates the production of proinflammatory cytokines by activated lymphocytes and monocytes that induce excessive apoptotic death of the bone marrow (BM) granulocytic progenitor cells. Clonal hematopoiesis identified by next generation sequencing (NGS) of myeloid genes is found in 11% of CIN patients conferring an increased risk for MDS/AML transformation whereas the non-clonal patients display usually a benign course. The basis for the immune cell activation and proinflammatory cytokine production in CIN remains obscure. Based on previously reported data showing increased frequency of mutations of the MEFV gene encoding pyrin in patients with idiopathic inflammatory conditions other than typical Familial Mediterranean Fever (FMF), we sought to investigate the common MEFV mutations in a cohort of well characterized CIN patients.Patients-Methods: We have studied 50 patients fulfilling the previously reported diagnostic criteria of CIN (median ANC 1.5x10 9/L, range 0.2-1.7 x10 9/L), 44 females and 6 males with a median age of 56 years (range 25-87 years) and a long-follow-up (median 132 months, range 8-336 months) in the Department of Hematology of the University Hospital of Heraklion, Crete, Greece. Nonisotopic RNase cleavage assay (NIRCA) analysis was used as first screening method to detect MEFV exons 10 and 2 mutations in DNA extracted from PB or BM samples from CIN patients, confirmed by direct NGS analysis. These sequences contain the main disease-related mutations and polymorphisms.Results: Genetics alterations of MEFV were detected in 22 out of 50 CIN patients (44%). Pathogenic mutations (variants associated with typical or “atypical” FMF phenotype in Greek population) were identified in 10/50 CIN patients (20%). The 20% frequency of MEFV mutations in exon 10 and/or exon 2 in CIN patients is significantly higher compared to the carrier rate of common MEFV mutations in the healthy Greek population (0.7%) according to our previously reported data (P<0.0001, Fisher's exact test). NGS analysis confirmed the mutational pattern of NIRCA and specifically showed: (a) one patient with heterozygous I720M (ATC>ATG; Ile>Met), two patients with heterozygous A744S (GCC>TCC; Ala>Ser) and one with homozygosity, one patient with heterozygous M694V (ATG>GTG; Met>Val), one with heterozygous K695R (AAG>AGG; Lys>Arg) and one with heterozygous M680I (ATG>ATC; Met>Ile), all in exon 10, and (b) four patients with homozygous R202Q mutation in exon 2 (one patient with homozygous A744S co-mutation in exon 10) and two patients with R202Q heterozygosity combined with heterozygosity of I720M and A744S of exons 10, respectively. None of the patients displayed any symptoms/signs of FMF or other systemic inflammatory disease. No statistically significant differences were identified between MEFV mutated and non-mutated CIN patients in the severity of neutropenia or in lymphocyte, monocyte, hemoglobin and platelet counts. A significant difference was identified between the two patient groups in serum IgG (1440±264 vs 1133±245 mg/dl; P = 0.0023, Mann-Whitney test) but not IgA or IgM levels.Discussion: This study reports for the first time that 20% of unselected, consecutive patients with CIN carry mutations of the MEFV gene without clinical manifestations of FMF. Whether these patients represent atypical cases of FMF or the identified MEFV genetic alterations have a pathogenetic/modifying effect in the inflammatory responses associated with CIN is an open/novel field of research. As a first step we are currently investigating the neutrophil autophagic status, IL-1β production and the neutrophil extracellular trap (NET) formation in CIN patients with mutations in MEFV to clarify their potential effect in the immune deregulation known to characterize CIN. DisclosuresNo relevant conflicts of interest to declare.