Dysfunctional mitochondria, disrupted levels of reactive oxygen species, and autophagy in B cells from common variable immunodeficiency patients.

Introduction: Common Variable Immunodeficiency (CVID) patients are characterized by hypogammaglobulinemia and poor response to vaccination due to deficient generation of memory and antibody-secreting B cells. B lymphocytes are essential for the development of humoral immune responses, and mitochondrial function, hreactive oxygen species (ROS) production and autophagy are crucial for determining B-cell fate. However, the role of those basic cell functions in the differentiation of human B cells remains poorly investigated. Methods: We used flow cytometry to evaluate mitochondrial function, ROS production and autophagy processes in human naïve and memory B-cell subpopulations in unstimulated and stimulated PBMCs cultures. We aimed to determine whether any alterations in these processes could impact B-cell fate and contribute to the lack of B-cell differentiation observed in CVID patients. Results: We described that naïve CD19+CD27- and memory CD19+CD27+ B cells subpopulations from healthy controls differ in terms of their dependence on these processes for their homeostasis, and demonstrated that different stimuli exert a preferential cell type dependent effect. The evaluation of mitochondrial function, ROS production and autophagy in naïve and memory B cells from CVID patients disclosed subpopulation specific alterations. Dysfunctional mitochondria and autophagy were more prominent in unstimulated CVID CD19+CD27- and CD19+CD27+ B cells than in their healthy counterparts. Although naïve CD19+CD27- B cells from CVID patients had higher basal ROS levels than controls, their ROS increase after stimulation was lower, suggesting a disruption in ROS homeostasis. On the other hand, memory CD19+CD27+ B cells from CVID patients had both lower ROS basal levels and a diminished ROS production after stimulation with anti-B cell receptor (BCR) and IL-21. Conclusion: The failure in ROS cell signalling could impair CVID naïve B cell activation and differentiation to memory B cells. Decreased levels of ROS in CVID memory CD19+CD27+ B cells, which negatively correlate with their in vitro cell death and autophagy, could be detrimental and lead to their previously demonstrated premature death. The final consequence would be the failure to generate a functional B cell compartment in CVID patients.

Hyperinflammatory Immune Response in COVID-19: Host Genetic Factors in Pyrin Inflammasome and Immunity to Virus in a Spanish Population from Majorca Island.

The hyperinflammatory response caused by SARS-CoV-2 infection contributes to its severity, and many critically ill patients show features of cytokine storm (CS) syndrome. We investigated, by next-generation sequencing, 24 causative genes of primary immunodeficiencies whose defect predisposes to CS. We studied two cohorts with extreme phenotypes of SARS-CoV-2 infection: critical/severe hyperinflammatory patients (H-P) and asymptomatic patients (AM-risk-P) with a high risk (older age) to severe COVID-19. To explore inborn errors of the immunity, we investigated the presence of pathogenic or rare variants, and to identify COVID-19 severity-associated markers, we compared the allele frequencies of common genetic polymorphisms between our two cohorts. We found: 1 H-P carries the likely pathogenic variant c.887-2 A>C in the IRF7 gene and 5 H-P carries variants in the MEFV gene, whose role in the pathogenicity of the familial Mediterranean fever (FMF) disease is controversial. The common polymorphism analysis showed three potential risk biomarkers for developing the hyperinflammatory response: the homozygous haplotype rs1231123A/A-rs1231122A/A in MEFV gene, the IFNAR2 p.Phe8Ser variant, and the CARMIL2 p.Val181Met variant. The combined analysis showed an increased risk of developing severe COVID-19 in patients that had at least one of our genetic risk markers (odds ratio (OR) = 6.2 (95% CI) (2.430-16.20)).

Elevated complement C3 and increased CD8 and type 1 helper lymphocyte T populations in patients with post-COVID-19 condition.

BACKGROUND: Biological markers associated to post-COVID-19 condition (PCC) have not been clearly identified. METHODS: Eighty-two patients attending our post-COVID-19 outpatient clinic were recruited and classified as fully recovered (40.2%) or presenting with PCC (59.8%). Clinical and radiological data, laboratory markers, cytokines, and lymphocyte populations were analyzed. RESULTS: Median number of days after hospitalization was 78.5 [p25-p75: 60-93] days. PCC was significantly more frequent in women, in patients with a previously critical COVID-19, and in those with two or more comorbidities. No differences were found in lymphocyte counts, ferritin, C-reactive protein, D-dimer or sCD25, IL-1ß, IL-1Ra, IL-6, CXCL8, IL-17A, IL-18, IL-22, IFN-γ, TNF-α, and IL-10 cytokines levels. PCC patients showed significantly higher levels of complement factor C3 than fully recovered patients: median C3 128 mg/dL [p25-p75:107-135] vs 111 mg/dL [p25-p75: 100-125] (p =.005), respectively. In the flow cytometry assessment of peripheral blood lymphocyte subpopulations, PCC patients showed significantly increased CD8 populations compared to fully recovered patients: median CD8: 529 [p25-p75: 384-683] vs 370/mm3 [p25-p75:280-523], p =.007. When type 1, 2, 17/22, and 17.1 helper and follicular T lymphocyte subpopulations were analyzed, the frequency of Th1 was significantly higher in PCC patients compared to fully recovered patients (30% vs 38.5%, p =.028). CONCLUSION: Patients with a post-COVID-19 condition showed significantly increased immunological parameters of inflammation (complement factor C3 and CD8 and Th1 T lymphocyte populations) compared to fully recovered patients. These parameters could be used as biological markers of this condition.

Clinical manifestations and approach to the management of patients with common variable immunodeficiency and liver disease.

Purpose: The clinical spectrum of common variable immunodeficiency (CVID) includes predisposition to infections, autoimmune/inflammatory complications and malignancy. Liver disease is developed by a proportion of patients with CVID, but limited evidence is available about its prevalence, pathogenesis and prognostic outcome. This lack of evidence leads to the absence of guidelines in clinical practice. In this study, we aimed at defining the characteristics, course and management of this CVID complication in Spain. Methods: Spanish reference centers were invited to complete a cross-sectional survey. Thirty-eight patients with CVID-related liver disease from different hospitals were evaluated by a retrospective clinical course review. Results: In this cohort, abnormal liver function and thrombocytopenia were found in most of the patients (95% and 79% respectively), in keeping with the higher incidence of abnormal liver imaging and splenomegaly. The most common histological findings included nodular regenerative hyperplasia (NRH) and lymphocytic infiltration, which have been associated with portal hypertension (PHTN) leading to a poorer prognosis. Autoimmune/inflammatory complications occurred in 82% of the CVID patients that developed liver disease and 52% of the patients treated with immunomodulators showed a reduction in the liver function tests' abnormalities during treatment. Among the experts that conducted the survey, there was 80% or more consensus that the workup of CVID-related liver disease requires liver profile, abdominal ultrasound and transient elastography. The majority agreed that liver biopsy should be essential for diagnosis. There was 94% consensus that endoscopic studies should be performed in the presence of PHTN. However, there was 89% consensus that there is insufficient evidence on the management of these patients. Conclusion: Liver disease varies in severity and may contribute substantially to morbidity and mortality in patients with CVID. Hence the importance of close follow-up and screening of this CVID complication to prompt early targeted intervention. Further research is needed to evaluate the pathophysiology of liver disease in patients with CVID to identify personalized treatment options. This study emphasizes the urgent need to develop international guidelines for the diagnosis and management of this CVID complication.

Hyperinflammatory State and Low T1 Adaptive Immune Response in Severe and Critical Acute COVID-19 Patients.

Background: A better understanding of COVID-19 immunopathology is needed to identify the most vulnerable patients and improve treatment options. Objective: We aimed to identify immune system cell populations, cytokines, and inflammatory markers related to severity in COVID-19. Methods: 139 hospitalized patients with COVID-19-58 mild/moderate and 81 severe/critical-and 74 recovered patients were included in a prospective longitudinal study. Clinical data and blood samples were obtained on admission for laboratory markers, cytokines, and lymphocyte subsets study. In the recovered patients, lymphocyte subsets were analyzed 8-12 weeks after discharge. Results: A National Early Warning Score 2 >2 (OR:41.4; CI:10.38-167.0), ferritin >583 pg/mL (OR:16.3; CI: 3.88-69.9), neutrophil/lymphocyte ratio >3 (OR: 3.5; CI: 1.08-12.0), sIL-2rα (sCD25) >512 pg/mL (OR: 3.3; CI: 1.48-7.9), IL-1Ra >94 pg/mL (OR: 3.2; IC: 1.4-7.3), and IL-18 >125 pg/mL (OR: 2.4; CI: 1.1-5.0) were associated with severe/critical COVID-19 in the multivariate models used. Lower absolute values of CD3, CD4, CD8, and CD19 lymphocytes together with higher frequencies of NK cells, a CD4 and CD8 activated (CD38+HLA-DR+) memory T cell and effector memory CD45RA+ (EMRA) phenotype, and lower T regulatory cell frequencies were found in severe/critical patients relative to mild/moderate and recovered COVID-19 patients. A significant reduction in Th1, Tfh1, and Tc1 with higher Th2, Tfh2, Tc2, and plasma cell frequencies was found in the most severe cases. Conclusion: A characteristic hyperinflammatory state with significantly elevated neutrophil/lymphocyte ratio and ferritin, IL-1Ra, sIL-2rα, and IL-18 levels together with a "low T1 lymphocyte signature" was found in severe/critical COVID-19 patients.

Prostate Cancer Survival by Risk and Other Prognostic Factors in Mallorca, Spain.

Studies about the survival of patients with prostate cancer by stage or risk of progression are scarce. The aims of this study were (1) to determine the cause-specific survival by risk in prostate cancer patients in Mallorca diagnosed in the period 2006-2011; (2) to identify the factors that explain and predict the likelihood of survival and the risk of dying from this type of cancer; and (3) to determine the distribution of prostate cancer by risk in the patients in Mallorca diagnosed in the period 2006-2011. Incident prostate cancer cases diagnosed between 2006 and 2011 were identified through the Mallorca Cancer Registry. We collected age; date and method of diagnosis; date of follow-up or death; T, N, M and stage according to the TNM 7th edition; Gleason score; prostate-specific antigen (PSA); histology according to the International Classification of Diseases for Oncology (ICD-O) 3rd edition, comorbidities and treatments. We calculated risk in four categories: low, medium, high and very high. The end point of follow-up was 31 December 2014. Multiple imputation (MI) was performed to estimate cases with unknown risk. We identified 2921 cases. Five years after diagnosis, survival after MI was 89% globally, and was 100% for low-risk cases, 96% for medium risk, 93% for high risk and 69% for very-high-risk cases. Cases with histology other than adenocarcinoma, with high (and especially very high) risk, as well as with systemic, mixed and observation/unspecified treatments had worse prognoses.

Predictive Immunological, Virological, and Routine Laboratory Markers for Critical COVID-19 on Admission.

BACKGROUND: Early identification of COVID-19 patients at risk of critical illness is a challenging endeavor for clinicians. We aimed to establish immunological, virological, and routine laboratory markers, which, in combination with clinical information, may allow identifying such patients. METHODS: Blood tests to measure neutrophil/lymphocyte ratio (NLR) and levels of ferritin, CRP, D-dimer, complement components (C3 and C4), cytokines, and lymphocyte subsets, as well as SARS-Cov-2 RT-PCR tests, were performed in COVID-19-confirmed cases within 48 hours of admission. RT-PCR cycle threshold (Ct) values from oropharyngeal or nasopharyngeal swabs were determined on the day of admission. Symptom severity was categorized as mild (grade 1), severe (grade 2), or critical (grade 3). RESULTS: Of 120 patients who were included, 49 had mild, 32 severe, and 39 critical COVID-19. Levels of ferritin >370 ng/mL (OR 16.4, 95% CI 5.3-50.8), D-dimer >440 ng/mL (OR 5.45, 95% CI 2.36-12.61), CRP >7.65 mg/dL (OR 11.54, 95% CI 4.3-30.8), NLR >3.77 (OR 13.4, 95% CI 4.3-41.1), IL-6 >142.5 pg/mL (OR 8.76, 95% CI 3.56-21.54), IL-10 >10.8 pg/mL (OR 16.45, 95% CI 5.32-50.81), sIL-2rα (sCD25) >804.5 pg/mL (OR 14.06, 95% CI 4.56-43.28), IL-1Ra >88.4 pg/mL (OR 4.54, 95% CI 2.03-10.17), and IL-18 >144 pg/mL (OR 17.85, 95% CI 6.54-48.78) were associated with critical COVID-19 in the univariate age-adjusted analysis. This association was confirmed in the multivariate age-adjusted analysis only for ferritin, CRP, NLR, IL-10, sIL-2rα, and IL-18. T, B, and NK cells were significantly decreased in critical patients. SARS-CoV-2 was not detected in blood except in 3 patients who had indeterminate results. RT-PCR Ct values from oropharyngeal or nasopharyngeal swabs on admission were not related to symptom severity. CONCLUSION: Ferritin, D-dimer, CRP, NLR, cytokine (IL-18 and IL-10), and cytokine receptor (IL-6, IL1-Ra, and sCD25) test results combined with clinical data can contribute to the early identification of critical COVID-19 patients.

Association between vitamin D status and allergen sensitization in pediatric subjects in the Balearic Islands.

INTRODUCTION: Vitamin D has known effects on the immune system, and its deficiency has been associated with allergen sensitization. MATERIAL AND METHODS: A retrospective study was performed to evaluate the association between 25(OH)vitamin D (25(OH)D) and specific IgE for the most frequent allergens in our area in children and adolescents. All subjects under 15 years of age with a determination of Phadiatop® or Phadiatop Infant® and close serum 25(OH)D determination were included, from 2012 to 2019. From this sample, demographic and analytical variables were collected: specific IgE to Dermatophagoides pteronyssinus; dog and cat dander; grass, olive, and pellitory pollens; egg white; and cow milk; as well as complete blood count analysis and immunoglobulins. RESULTS: A total of 749 subjects were recruited. Clusters according to deficiency, insufficiency, or sufficiency of 25(OH)D showed an association with age, Phadiatop® , D. pteronyssinus, cat and dog dander specific IgE, and a higher frequency of positive allergen sensitization (P < .05). Logistic regression, compared with vitamin D levels of greater than 30 ng/mL after age adjustment, showed that deficient 25(OH)D was associated with D. pteronyssinus (OR 1.90; 95% CI, 1.25-2.90) and dog dander (OR, 2.10; 1.10-4.02); whereas insufficient 25(OH)D was associated with cat dander (OR, 2.46; 1.15-5.28) and also with D. pteronyssinus (OR, 1.55; 1.06-2.29) (P < .05). No associations were found between 25(OH) D levels and other analytical parameters. CONCLUSION: Vitamin D status is associated with sensitization to D. pteronyssinus, and cat and dog dander in children and adolescents, and also with a higher number of positive specific IgE.

IL-21 and anti-CD40 restore Bcl-2 family protein imbalance in vitro in low-survival CD27+ B cells from CVID patients.

Common variable immunodeficiency (CVID) is characterized by an abnormal B cell differentiation to memory and antibody-secreting B cells. The defective functionality of CVID patients' B cells could be the consequence of alterations in apoptosis regulation. We studied the balance of Bcl-2 family anti-/pro-apoptotic proteins to identify molecular mechanisms that could underlie B cell survival defects in CVID. We used flow cytometry to investigate Bcl-2, Bcl-XL, Bax, and Bim expression in B cells ex vivo and after anti-CD40 or anti-BCR activation with or without IL-21, besides to spontaneous and stimulation-induced Caspase-3 activation and viable/apoptotic B cell subpopulations. We found increased basal levels of Bax and Bim in CVID B cells that correlated with low viability and high Caspase-3 activation only in CD27+ B cells, particularly in a subgroup of apoptosis-prone CVID (AP-CVID) patients with low peripheral B cell counts and high autoimmunity prevalence (mostly cytopenias). We detected a broad B cell defect in CVID regarding Bcl-2 and Bcl-XL induction, irrespective of the stimulus used. Therefore, peripheral CVID memory B cells are prompted to die from apoptosis due to a constitutive Bcl-2 family protein imbalance and defective protection from activation-induced apoptosis. Interestingly, anti-CD40 and IL-21 induced normal and even higher levels of Bcl-XL, respectively, in CD27+ B cells from AP-CVID, which was accompanied by cell viability increase. Thus low-survival memory B cells from AP-CVID can overcome their cell death regulation defects through pro-survival signals provided by T cells. In conclusion, we identify apoptosis regulation defects as disease-contributing factors in CVID. B cell counts and case history of cytopenias might be useful to predict positive responses to therapeutic approaches targeting T-dependent signaling pathways.

Relapsing-Remitting Multiple Sclerosis Is Characterized by a T Follicular Cell Pro-Inflammatory Shift, Reverted by Dimethyl Fumarate Treatment.

Multiple sclerosis (MS) is considered a T cell-mediated autoimmune disease, although several evidences also demonstrate a B cell involvement in its etiology. Follicular T helper (Tfh) cells, a CXCR5-expressing CD4+ T cell subpopulation, are essential in the regulation of B cell differentiation and maintenance of humoral immunity. Alterations in circulating (c)Tfh distribution and/or function have been associated with autoimmune diseases including MS. Dimethyl fumarate (DMF) is a recently approved first-line treatment for relapsing-remitting MS (RRMS) patients whose mechanism of action is not completely understood. The aim of our study was to compare cTfh subpopulations between RRMS patients and healthy subjects and evaluate the impact of DMF treatment on these subpopulations, relating them to changes in B cells and humoral response. We analyzed, by flow cytometry, the distribution of cTfh1 (CXCR3+CCR6-), cTfh2 (CXCR3-CCR6-), cTfh17 (CXCR3-CCR6+), and the recently described cTfh17.1 (CXCR3+CCR6+) subpopulations of CD4+ Tfh (CD45RA-CXCR5+) cells in a cohort of 29 untreated RRMS compared to healthy subjects. CD4+ non-follicular T helper (Th) cells (CD45RA-CXCR5-) were also studied. We also evaluated the effect of DMF treatment on these subpopulations after 6 and 12 months treatment. Untreated RRMS patients presented higher percentages of cTfh17.1 cells and lower percentages of cTfh2 cells consistent with a pro-inflammatory bias compared to healthy subjects. DMF treatment induced a progressive increase in cTfh2 cells, accompanied by a decrease in cTfh1 and the pathogenic cTfh17.1 cells. A similar decrease of non-follicular Th1 and Th17.1 cells in addition to an increase in the anti-inflammatory Th2 subpopulation were also detected upon DMF treatment, accompanied by an increase in naïve B cells and a decrease in switched memory B cells and serum levels of IgA, IgG2, and IgG3. Interestingly, this effect was not observed in three patients in whom DMF had to be discontinued due to an absence of clinical response. Our results demonstrate a possibly pathogenic cTfh pro-inflammatory profile in RRMS patients, defined by high cTfh17.1 and low cTfh2 subpopulations that is reverted by DMF treatment. Monitoring cTfh subsets during treatment may become a biological marker of DMF effectiveness.

Surveillance study on the tolerability and safety of Flebogamma® DIF (10% and 5% intravenous immunoglobulin) in adult and pediatric patients.

Direct comparisons of tolerability and safety of concentrated intravenous immunoglobulin (IVIG) versus less concentrated products are scarce. In this postauthorization, prospective, observational, multicenter study, a systematic comparison of 10% and 5% concentrations of Flebogamma® DIF IVIG was performed in both adult and pediatric patients treated with the studied IVIG products according to the approved indications under routine conditions. Dose of product administered, adverse events (AEs), physical assessments, laboratory tests, and concomitant therapy were analyzed. Patient recruitment in the 10% and 5% product groups was, respectively, 34 (32 analyzed, 13 of them children, receiving 130 IVIG infusions) and 35 (34 analyzed, receiving 135 IVIG infusions). Twenty-four infusions (18.5%; 95% CI: 11.8, 25.1) with the 10% product and 3 (2.2%; 95% CI: -0.3, 4.7) with the 5% product were associated with potentially treatment-related AEs (P < 0.0001). Nine patients (28.1%) infused with the 10% product and 3 (8.8%) infused with the 5% product presented, respectively, 33 and 8 treatment-related AEs (of which 7 and 6, respectively, were serious AEs, experienced by only three hypersensitive patients). The profile of AEs occurring with the infusion of 10% and 5% products were comparable. The most frequent treatment-related AEs were headache (n = 17, 3 patients; 15 episodes, 1 patient) and pyrexia (n = 6, 4 patients). In conclusion, no unpredictable risk was detected for both Flebogamma DIF 10% and 5% concentrations, which were therefore deemed as safe and well-tolerated IVIG in the studied population. The frequency of infusions associated with treatment-related AEs was lower with the 5% concentration.

B Cell-Activating Factor. An Orchestrator of Lymphoid Follicles in Severe Chronic Obstructive Pulmonary Disease.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) have increased pulmonary lymphoid follicle (LF) counts. B cell-activating factor of tumor necrosis factor family (BAFF) regulates B cells in health, but its role in COPD pathogenesis is unclear. OBJECTIVES: To determine whether BAFF expression in pulmonary LFs correlates with COPD severity, LF size or number, and/or readouts of B-cell function in LFs. METHODS: We correlated BAFF immunostaining in LFs in lung explants or biopsies from nonsmoking control subjects (NSC), smokers without COPD (SC), and patients with COPD with the number and size of LFs, and LF B-cell apoptosis, activation, and proliferation. We analyzed serum BAFF levels and BAFF expression in B cells in blood and bronchoalveolar lavage samples from the same subject groups. We assessed whether: (1) cigarette smoke extract (CSE) increases B-cell BAFF expression and (2) recombinant BAFF (rBAFF) rescues B cells from CSE-induced apoptosis by inhibiting activation of nuclear factor-κB (NF-κB). MEASUREMENTS AND MAIN RESULTS: Patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD had increased numbers and larger pulmonary LFs than patients with GOLD stages I-II COPD and SC. We identified two main types of pulmonary LFs: (1) type A, the predominant type in GOLD stages I-II COPD and SC, characterized by abundant apoptotic but few BAFF-positive cells (mostly B cells); and (2) type B, the main type in GOLD stage IV COPD, characterized by abundant BAFF-positive cells but few apoptotic cells (mostly B cells). BAFF levels were also higher in blood and bronchoalveolar lavage B cells in patients with COPD versus NSC and SC. Surprisingly, rBAFF blocked CSE-induced B-cell apoptosis by inhibiting CSE-induced NF-κB activation. CONCLUSIONS: Our data support the hypothesis that B-cell BAFF expression creates a self-perpetuating loop contributing to COPD progression by promoting pulmonary B-cell survival and LF expansion.

B cells from common variable immunodeficiency patients fail to differentiate to antibody secreting cells in response to TLR9 ligand (CpG-ODN) or anti-CD40+IL21.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.