A Critical Review on the Standardization and Quality Assessment of Nonfunctional Laboratory Tests Frequently Used to Identify Inborn Errors of Immunity.

Inborn errors of immunity (IEI), which were previously termed primary immunodeficiency diseases, represent a large and growing heterogeneous group of diseases that are mostly monogenic. In addition to increased susceptibility to infections, other clinical phenotypes have recently been associated with IEI, such as autoimmune disorders, severe allergies, autoinflammatory disorders, benign lymphoproliferative diseases, and malignant manifestations. The IUIS 2019 classification comprises 430 distinct defects that, although rare individually, represent a group affecting a significant number of patients, with an overall prevalence of 1:1,200-2,000 in the general population. Early IEI diagnosis is critical for appropriate therapy and genetic counseling, however, this process is deeply dependent on accurate laboratory tests. Despite the striking importance of laboratory data for clinical immunologists, several IEI-relevant immunoassays still lack standardization, including standardized protocols, reference materials, and external quality assessment programs. Moreover, well-established reference values mostly remain to be determined, especially for early ages, when the most severe conditions manifest and diagnosis is critical for patient survival. In this article, we intend to approach the issue of standardization and quality control of the nonfunctional diagnostic tests used for IEI, focusing on those frequently utilized in clinical practice. Herein, we will focus on discussing the issues of nonfunctional immunoassays (flow cytometry, enzyme-linked immunosorbent assays, and turbidimetry/nephelometry, among others), as defined by the pure quantification of proteins or cell subsets without cell activation or cell culture-based methods.

Contribution of Complement System pathways to the killing of Leptospira spp.

The Complement System (CS) plays an important role in the immune response against leptospirosis and can be activated by the Alternative and Lectin Pathways (Innate Immunity) and by the Classical Pathway (Acquired Immunity). Here we analyzed a broad range of nonpathogenic and pathogenic Leptospira strains considering their interaction with each CS pathway. We determined bacterial survival rate and CS protein deposition in the presence of purified proteins, specific component depleted sera and NHS treated with the chelating agents EDTA (inhibits all three activation pathways) or EGTA (inhibits the Classical and Lectin Pathways). We suggest that the Lectin and the Alternative Pathways have an important role to eliminate saprophytic leptospires since i) approximately 50% survival of both saprophytic strains was observed in the presence of MBL-deficient serum; ii) approximately 50% survival of Leptospira biflexa Patoc I was observed in the presence of NHS - EGTA and iii) C1q-depleted serum caused significant bacterial lysis. In all serovars investigated the deposition of C5-C9 proteins on saprophytic Leptospira strains was more pronounced when compared to pathogenic species confirming previous studies in the literature. No difference on C3 deposition was observed between nonpathogenic and pathogenic strains. In conclusion, Leptospira strains interact to different degrees with CS proteins, especially those necessary to form MAC, indicating that some strains and specific ligands could favor the binding of certain CS proteins.

Primary Immunodeficiencies in a Mesoregion of São Paulo, Brazil: Epidemiologic, Clinical, and Geospatial Approach.

Background: Primary immunodeficiencies (PIDs) are rare genetic disorders leading to immunologic abnormalities that can affect different organs and systems. We determined the epidemiology, clinical, and geospatial characteristics of PID disorders among patients diagnosed over a 5 year period in a reference hospital covering a mesoregion in São Paulo, Brazil. Methods: A retrospective analysis of 39 patients with recognizable PIDs according to the criteria of the European Society of Primary Immunodeficiencies were enrolled. Thirty-four patients came from outpatient immunodeficiency clinics and five patients from active search. Demographic, clinical, and immunologic data were collected, and maps were constructed using a geographic information system. Results: The ratio of females to males was 1.4:1, and 48.7% of patients were younger than 17 years of age. The mean age at the onset of symptoms in children was 2.0 years [standard error of the mean (SEM), 1.7 years] and the diagnosis lag was 5.1 years (SEM, 3.1 years); the mean age at diagnosis in adults was 16.3 years (SEM, 11.8 years) and the lag was 10.8 years (SEM, 10.9 years). Antibody deficiency and common variable immunodeficiencies were the most common categories and phenotypes, respectively. The need for intravenous antibiotics and respiratory tract infections were the most prevalent warning signs, with an overall mortality rate of 15.3%. Autoimmune diseases were diagnosed in 56.4% and visceral leishmaniasis in 5.1% of patients. In the active search, 29 patients were investigated and 17.2% were diagnosed; early diagnosis, the involvement of multidisciplinary professionals, and dissemination of knowledge achieved milestone benefits. The distribution of PID networks in Brazil shows great asymmetry between regions and at a regional level; it was shown that the patients lived mainly in Presidente Prudente municipality. Conclusions: The implementation of an immunodeficiency outpatient clinic in a referral hospital covering a mesoregion with a large population has led to the generation of policies and practices to improve the diagnosis, quality of life, and care of patients with PIDs and their families. Furthermore, the search for hospitalized patients with warning signs for PIDs showed great benefits. Inequality in the distribution of PID network centers in Brazil was demonstrated.

Disruption of an antimycobacterial circuit between dendritic and helper T cells in human SPPL2a deficiency.

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a-/- mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells.

Th17 pathway in recent-onset autoimmune diabetes.

AIMS: Evaluate the participation of IL-17 pathway in T1D pathogenesis. T helper 17 cells are potent, highly inflammatory cells that produce interleukin 17A (IL-17A), considered a mediator of various immune disorders. However, their role in Type 1 diabetes (T1D) pathogenesis in humans is not totally elucidated. METHODS: The expression of IL-17 Receptor A (IL-17RA) in peripheral T lymphocytes and IL-17A serum levels in recent-onset patients with T1D were compared with healthy controls. IL-17A gene variants were evaluated in a greater cohort. RESULTS: Patients with recent-onset T1D (less than 6 months of diagnosis) exhibited lower expression of IL-17RA in CD3+ T (% of cells = 31.3% × 43.6%; p = .041) and CD4+ T cells (11.1% × 25.2%; p = .0019) and lower number of IL-17RA in CD4+ T cells (MFI = 1.16 × 4.56; p = .03) than controls. IL-17RA expression in CD8+ T cells and IL-17A serum levels were similar in both groups. The coding regions and boundary intron sequences of IL17A were sequenced. Seventeen allelic variants, including three novel variants in exon 3 (3'UTR n) were identified, but no one was associated with T1D susceptibility, as well as the resulting haplotypes and diplotypes. The expression of IL-17RA was not correlated with metabolic variables (glucose and HbA1c levels) or pancreatic autoantibodies titers. CONCLUSIONS: The lower expression of IL-17RA in CD3+ and CD4+ T cells suggests a reduced effect of IL-17A in immune response of recent-onset T1D patients, at least at peripheral tissues. IL-17A allelic variants were not related with T1D susceptibility.

Binding of human complement C1 sterase inhibitor to Leptospira spp.

Leptospirosis is an important zoonosis of global importance caused by bacteria Leptospira spp. Pathogenic Leptospira is resistant to Complement System killing while non-pathogenic Leptospira is rapidly killed by exposure to normal human serum (NHS). Pathogenic Leptospira interact with Complement Regulators such as Factor H, C4b binding protein and Vitronectin avoiding Complement activation and killing by Alternative and Classical Pathways. One important regulator is C1-inhibitor (C1INH) that interacts with C1s or MASPs controlling the cleavage of C4 and C2 molecules, thereby inhibiting the activation of the Classical and Lectin Pathways. In this study, we demonstrate that attenuated, saprophytic and pathogenic Leptospira interact with C1INH that maintain its regulatory capacity of interaction with C1s preventing the activation of Complement system. Although the interaction with C1INH is not crucial for pathogenic Leptospira survival, it seems to be important for the survival of attenuated and saprophytic Leptospira in normal human serum.

Chemokine, cytokine and type I interferon production induced by Toll-like receptor activation in common variable immune deficiency.

Common variable immunodeficiency (CVID) is the most common symptomatic primary antibody deficiency and is associated with recurrent infections and chronic inflammatory diseases. We evaluated the ability of Toll-like receptor (TLR) ligands to induce secretion of chemokines, cytokines and type I interferons by peripheral blood mononuclear cells (PBMCs) from CVID patients. High levels of CXCL10, CCL2, CXCL9, CCL5, CXCL8, and IL-6 were detected in sera of CVID patients compared with healthy controls. Increased chemokine levels were observed in unstimulated PBMCs, but after stimulation with TLR2 and TLR4 agonists, equivalent chemokine and pro-inflammatory cytokine secretion, as in healthy controls, was observed, whereas TLR4 agonist induced a decreased secretion of CCL2 and CXCL8 and increased secretion of TNF. Decreased IFN-α secretion induced by TLR7/TLR8 activation was observed in CVID, which was recovered with TLR9 signaling. Our findings revealed that TLR9 activation has an adjuvant effect on the altered type I response in CVID.

Impaired CD8(+) T cell responses upon Toll-like receptor activation in common variable immunodeficiency.

BACKGROUND: Infections caused by bacteria or viruses are frequent in common variable immunodeficiency (CVID) patients due to antibody deficiencies, which may be associated with altered T cell function. CVID patients are frequently in contact with pathogen-associated molecular patterns (PAMPs), leading to the activation of innate immunity through Toll-like receptors (TLR) affecting T cell activation. We evaluated the effect of TLR activation on T cells in CVID patients undergoing intravenous immunoglobulin (IVIg) replacement using synthetic ligands. METHODS: Expression of exhaustion, activation and maturation markers on T cells from peripheral blood as well as regulatory T cells and follicular T cells in peripheral blood mononuclear cells (PBMCs) from CVID and healthy individuals were evaluated by flow cytometry. PBMCs cultured with TLR agonists were assessed for intracellular IFN-γ, TNF, IL-10, IL-17a or IL-22 secretion as monofunctional or polyfunctional T cells (simultaneous cytokine secretion) by flow cytometry. RESULTS: We found increased expression of the exhaustion marker PD-1 on effector memory CD4(+) T cells (CD45RA(-)CCR7(-)) in the peripheral blood and increased expression of CD38 in terminally differentiated CD8(+) T cells (CD45RA(+)CCR7(-)). Furthermore, a decreased frequency of naïve regulatory T cells (CD45RA(+)Foxp3(low)), but not of activated regulatory T cells (CD45RA(-)Foxp3(high)) was detected in CVID patients with splenomegaly, the non-infectious manifestation in this CVID cohort (43.7 %). Moreover, the frequency of peripheral blood follicular helper T cells (CD3(+)CD4(+)CXCR5(+)PD-1(+)ICOS(+)) was similar between the CVID and control groups. Upon in vitro TLR3 activation, a decreased frequency of CD8(+) T cells secreting IFN-γ, IL-17a or IL-22 was detected in the CVID group compared to the control group. However, a TLR7/TLR8 agonist and staphylococcal enterotoxin B induced an increased Th22/Tc22 (IL-22(+), IFN-γ(-), IL-17a(-)) response in CVID patients. Both TLR2 and TLR7/8/CL097 activation induced an increased response of CD4(+) T cells secreting three cytokines (IL-17a, IL-22 and TNF)in CVID patients, whereas CD8(+) T cells were unresponsive to these stimuli. CONCLUSION: The data show that despite the unresponsive profile of CD8(+) T cells to TLR activation, CD4(+) T cells and Tc22/Th22 cells are responsive, suggesting that activation of innate immunity by TLRs could be a strategy to stimulate CD4(+) T cells in CVID.

Primary immunodeficiency diseases in different age groups: a report on 1,008 cases from a single Brazilian reference center.

Primary immunodeficiencies (PIDs) represent a large group of diseases that affect all age groups. Although PIDs have been recognized as rare diseases, there is epidemiological evidence suggesting that their real prevalence has been underestimated. We performed an evaluation of a series of 1,008 infants, children, adolescents and adults with well-defined PIDs from a single Brazilian center, regarding age at diagnosis, gender and PID category according to the International Union of Immunological Societies classification. Antibody deficiencies were the most common category in the whole series (61 %) for all age groups, with the exception of <2-year-old patients (only 15 %). In the >30-year-old group, antibody deficiencies comprised 84 % of the diagnoses, mostly consisting of common variable immunodeficiency, IgA deficiency and IgM deficiency. Combined immunodeficiencies represented the most frequent category in <2-years-old patients. Most congenital defects of phagocytes were identified in patients <5 -years of age, as were the diseases of immune dysregulation, with the exception of APECED. DiGeorge syndrome and ataxia-telangiectasia were the most frequent entities in the category of well-defined syndromes, which were mostly identified in patients <10-years of age. Males represented three-quarters and two-thirds of <2 -years-old and 2-5-years -old patients, respectively, whereas females predominated among the >30-year-old patients. Our data indicated that some PIDs were only detected at early ages, likely because affected patients do not survive long. In addition, our data pointed out that different strategies should be used to search for PIDs in infants and young children as compared to older patients.

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with neurocryptococcosis.

BACKGROUND: Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis. METHODS: Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of 3H-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05. RESULTS: The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis. CONCLUSION: Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

Hereditary autoinflammatory syndromes: a Brazilian multicenter study.

OBJECTIVE: To evaluate the prevalence of genetic defects in clinically suspected autoinflammatory syndromes (AIS) in a Brazilian multicenter study. METHODS: The study included 102 patients with a clinical diagnosis of Cryopyrin Associated Periodic Syndromes (CAPS), TNF Receptor Associated Periodic Syndrome (TRAPS), Familial Mediterranean Fever (FMF), Mevalonate Kinase Deficiency (MKD) and Pediatric Granulomatous Arthritis (PGA). One of the five AIS-related genes (NLRP3, TNFRSF1A, MEFV, MVK and NOD2) was evaluated in each patient by direct DNA sequencing, based on the most probable clinical suspect. RESULTS: Clinical diagnoses of the 102 patients were: CAPS (n = 28), TRAPS (n = 31), FMF (n = 17), MKD (n = 17) and PGA (n = 9). Of them, 27/102 (26 %) had a confirmed genetic diagnosis: 6/28 (21 %) CAPS patients, 7/31 (23 %) TRAPS, 3/17 (18 %) FMF, 3/17 (18 %) MKD and 8/9 (89 %) PGA. CONCLUSION: We have found that approximately one third of the Brazilian patients with a clinical suspicion of AIS have a confirmed genetic diagnosis.

Chronic mucocutaneous candidiasis and systemic lupus erythematosus: a new variant of chronic mucocutaneous candidiasis?

Chronic mucocutaneous candidiasis (CMC) is characterized by susceptibility to Candida infection of skin, nails, and mucous membranes. Autoimmune endocrinopathies are common in CMC patients, but there are no reports of the involvement of systemic autoimmune disorders. We present here the first case of this kind of association in a patient with an autosomal dominant variant of CMC. The individual had had this disorder since childhood and systemic lupus erythematosus with secondary antiphospholipid syndrome, as well as renal, articular and hepatic manifestations without thymoma.

IRF8 mutations and human dendritic-cell immunodeficiency.

BACKGROUND: The genetic analysis of human primary immunodeficiencies has defined the contribution of specific cell populations and molecular pathways in the host defense against infection. Disseminated infection caused by bacille Calmette-Guérin (BCG) vaccines is an early manifestation of primary immunodeficiencies, such as severe combined immunodeficiency. In many affected persons, the cause of disseminated BCG disease is unexplained. METHODS: We evaluated an infant presenting with features of severe immunodeficiency, including early-onset disseminated BCG disease, who required hematopoietic stem-cell transplantation. We also studied two otherwise healthy subjects with a history of disseminated but curable BCG disease in childhood. We characterized the monocyte and dendritic-cell compartments in these three subjects and sequenced candidate genes in which mutations could plausibly confer susceptibility to BCG disease. RESULTS: We detected two distinct disease-causing mutations affecting interferon regulatory factor 8 (IRF8). Both K108E and T80A mutations impair IRF8 transcriptional activity by disrupting the interaction between IRF8 and DNA. The K108E variant was associated with an autosomal recessive severe immunodeficiency with a complete lack of circulating monocytes and dendritic cells. The T80A variant was associated with an autosomal dominant, milder immunodeficiency and a selective depletion of CD11c+CD1c+ circulating dendritic cells. CONCLUSIONS: These findings define a class of human primary immunodeficiencies that affect the differentiation of mononuclear phagocytes. They also show that human IRF8 is critical for the development of monocytes and dendritic cells and for antimycobacterial immunity. (Funded by the Medical Research Council and others.).

Retinoic acid inhibits dendritic cell differentiation driven by interleukin-4.

All-trans-retinoic acid (atRA) appears to affect Th1-Th2 differentiation and its effects on immune responses might also be mediated by dendritic cell (DC). Nonetheless, studies have been showing contradictory results since was observed either induction or inhibition of DC differentiation. Our aim was to investigate atRA action on human monocyte derived DC differentiation. For this purpose we tested pharmacological and physiological doses of atRA with or without cytokines. Cell phenotypes were analyzed by flow cytometry and function was investigated by phagocytosis and respiratory burst. DC, positive control group, was differentiated with GM-CSF and IL-4 and maturated with TNF-alpha. We demonstrated that atRA effects depend on the dose used as pharmacological doses inhibited expression of all phenotypic markers tested while a physiological dose caused cell differentiation. However, atRA combined or not with cytokines did not promote DC differentiation. In fact, atRA was detrimental on IL-4 property as a DC inductor.

Esophageal cancer associated with chronic mucocutaneous candidiasis. Could chronic candidiasis lead to esophageal cancer?

Chronic mucocutaneous candidiasis (CMC) is a rare disease associated with immunodeficiency and characterized by persistent and refractory infections of the skin, appendages and mucous membranes caused by members of the genus Candida. Several different disorders are classified under this common denominator, including chronic and recurrent mucocutaneous infections due to Candida spp., which are sometimes linked to autoimmune endocrinopathies. These fungal infections are usually confined to the mucocutaneous surface, with little propensity for systemic disease or septicemia. We describe a patient with CMC who had an esophageal candidiasis refractory to treatment for decades and who developed an epidermoid esophageal cancer. No risk factors such as familiar susceptibility, smoking, alcohol drinking, or living in an endemic area were verified. This case report suggests the participation of nitrosamine compounds produced by chronic Candida infections as a risk factor for esophageal cancer in a patient with autosomal-dominant chronic mucocutaneous candidiasis.

Evaluation of dendritic cell-tumor cell hybrid vaccine storage.

Clinical trials using dendritic cells (DCs) to treat cancer patients have generated promising results in recent years. However, even simple aspects of this therapy are still not well understood, including the storage and distribution of manufactured vaccines. These processes are essential and must be elucidated in order to reduce costs. We evaluated the effects of different storage conditions on vaccine functionality using mixed lymphocyte reaction (MLR). Vaccine storage at 4°C for up to 72 h had no significant effect on vaccine activity. Shipping to distant places is possible, if vaccines are kept at 4°C and used up to 3 days after manufacture date.

Paracoccidioides brasiliensis disseminated disease in a patient with inherited deficiency in the beta1 subunit of the interleukin (IL)-12/IL-23 receptor.

BACKGROUND: Paracoccidioides brasiliensis is a facultative intracellular dimorphic fungus that causes paracoccidioidomycosis (PCM), the most important deep mycosis in Latin America. Only a small percentage of individuals infected by P. brasiliensis develop clinical PCM, possibly in part because of genetically determined interindividual variability of host immunity. However, no primary immunodeficiency has ever been associated with PCM. METHODS: We describe the first patient, to our knowledge, with PCM and a well-defined primary immunodeficiency in the beta 1 subunit of the interleukin (IL)-12/IL-23 receptor, a disorder previously shown to be specifically associated with impaired interferon (IFN)-gamma production, mycobacteriosis, and salmonellosis. RESULTS: Our patient had a childhood history of bacille Calmette-Guérin disease and nontyphoid salmonellosis and, at the age of 20 years, presented to our clinic with a disseminated (acute) form of PCM. He responded well to antifungal treatment and is now doing well at 24 years of age. CONCLUSIONS: This unique observation supports previous studies of PCM suggesting that IL-12, IL-23, and IFN-gamma play an important role in protective immunity to P. brasiliensis. Tuberculosis and PCM are thus not only related clinically and pathologically, but also by their immunological pathogenesis. Our study further expands the spectrum of clinical manifestations of inherited defects of the IL-12/IL-23-IFN-gamma axis. Patients with unexplained deep fungal infections, such as PCM, should be tested for defects in the IL-12/IL-23-IFN- gamma axis.

Low penetrance, broad resistance, and favorable outcome of interleukin 12 receptor beta1 deficiency: medical and immunological implications.

The clinical phenotype of interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency and the function of human IL-12 in host defense remain largely unknown, due to the small number of patients reported. We now report 41 patients with complete IL-12Rbeta1 deficiency from 17 countries. The only opportunistic infections observed, in 34 patients, were of childhood onset and caused by weakly virulent Salmonella or Mycobacteria (Bacille Calmette-Guérin -BCG- and environmental Mycobacteria). Three patients had clinical tuberculosis, one of whom also had salmonellosis. Unlike salmonellosis, mycobacterial infections did not recur. BCG inoculation and BCG disease were both effective against subsequent environmental mycobacteriosis, but not against salmonellosis. Excluding the probands, seven of the 12 affected siblings have remained free of case-definition opportunistic infection. Finally, only five deaths occurred in childhood, and the remaining 36 patients are alive and well. Thus, a diagnosis of IL-12Rbeta1 deficiency should be considered in children with opportunistic mycobacteriosis or salmonellosis; healthy siblings of probands and selected cases of tuberculosis should also be investigated. The overall prognosis is good due to broad resistance to infection and the low penetrance and favorable outcome of infections. Unexpectedly, human IL-12 is redundant in protective immunity against most microorganisms other than Mycobacteria and Salmonella. Moreover, IL-12 is redundant for primary immunity to Mycobacteria and Salmonella in many individuals and for secondary immunity to Mycobacteria but not to Salmonella in most.