Possibilities for the analysis of peripheral blood B cell subpopulations in a routine immunological laboratory.

B cells play a vital role in the defence of the body against infectious agents. Apart from their ability to present antigen to T cells, B cells are mainly producers of antibodies. These play a crucial role in the effective elimination of infection and are also involved in the regulation of the immune response. The analysis of peripheral blood B cell subpopulations that makes it possible to monitor the development of B cells to the stage of antibody producing plasmablasts provides a valuable laboratory parameter which is important for both the study of the pathogenesis and diagnosis of some diseases. Laboratory analysis of B cell subpopulations is now a routinely available laboratory option thanks to the development of multicolour flow cytometry. This article summarizes the core knowledge which is currently applied to the analysis of B cell subpopulations in immunological laboratories.

When to initiate immunoglobulin replacement therapy (IGRT) in antibody deficiency: a practical approach.

Primary antibody deficiencies (PAD) constitute the majority of all primary immunodeficiency diseases (PID) and immunoglobulin replacement forms the mainstay of therapy for many patients in this category. Secondary antibody deficiencies (SAD) represent a larger and expanding number of patients resulting from the use of a wide range of immunosuppressive therapies, in particular those targeting B cells, and may also result from renal or gastrointestinal immunoglobulin losses. While there are clear similarities between primary and secondary antibody deficiencies, there are also significant differences. This review describes a practical approach to the clinical, laboratory and radiological assessment of patients with antibody deficiency, focusing on the factors that determine whether or not immunoglobulin replacement should be used. The decision to treat is more straightforward when defined diagnostic criteria for some of the major PADs, such as common variable immunodeficiency disorders (CVID) or X-linked agammaglobulinaemia (XLA), are fulfilled or, indeed, when there is a very low level of immunoglobulin production in association with an increased frequency of severe or recurrent infections in SAD. However, the presentation of many patients is less clear-cut and represents a considerable challenge in terms of the decision whether or not to treat and the best way in which to assess the outcome of therapy. This decision is important, not least to improve individual quality of life and reduce the morbidity and mortality associated with recurrent infections but also to avoid inappropriate exposure to blood products and to ensure that immunoglobulin, a costly and limited resource, is used to maximal benefit.

Hereditary angio-oedema with C1 inhibitor deficiency: Characteristics and diagnostic delay of Czech patients from one centre

BACKGROUND: Hereditary angio-oedema (HAE) is manifested by repeated episodes of localised subcutaneous or sub-mucosal oedema. Symptoms are extremely variable in frequency, localisation, and severity. Atypical or mild clinical symptoms of the disease may lead to erroneous diagnosis, causing diagnostic delay. The goal of this study was to assess how diagnostic delay has changed over 33 years at a single referral centre. METHODS: We analysed diagnostic delay and first symptoms of HAE in patients who were diagnosed at an immunology department between 1980 and 2013. Patient's records were analysed. RESULTS: The median diagnostic delay in 77 HAE type 1 and 2 patients was seven (range, 0-42) years. The difference observed in diagnostic delay between probands (18 [0-42] years) and others (1 [0-37] year) was significant (p < 0.001). Our data show a significant negative correlation between the length of diagnostic delay and the year of diagnosis in our group of patients (p = 0.024). The median age of first symptoms among all HAE patients (N = 64) was 17 (1-40) years. The first symptoms of HAE in 64 patients were analysed. Twenty-six patients had abdominal, seventeen peripheral, five facial, two urogenital, and three had laryngeal oedema as the first manifestation of the disease. The last death that was attributed to HAE was in 1977. CONCLUSIONS: Our observations demonstrate improved awareness of HAE among physicians, as documented by the significant decrease in diagnostic delay. It is believed that earlier treatment will improve patient quality of life and life expectancy

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Hereditary angio-oedema with C1 inhibitor deficiency: Characteristics and diagnostic delay of Czech patients from one centre.

BACKGROUND: Hereditary angio-oedema (HAE) is manifested by repeated episodes of localised subcutaneous or sub-mucosal oedema. Symptoms are extremely variable in frequency, localisation, and severity. Atypical or mild clinical symptoms of the disease may lead to erroneous diagnosis, causing diagnostic delay. The goal of this study was to assess how diagnostic delay has changed over 33 years at a single referral centre. METHODS: We analysed diagnostic delay and first symptoms of HAE in patients who were diagnosed at an immunology department between 1980 and 2013. Patient's records were analysed. RESULTS: The median diagnostic delay in 77 HAE type 1 and 2 patients was seven (range, 0-42) years. The difference observed in diagnostic delay between probands (18 [0-42] years) and others (1 [0-37] year) was significant (p<0.001). Our data show a significant negative correlation between the length of diagnostic delay and the year of diagnosis in our group of patients (p=0.024). The median age of first symptoms among all HAE patients (N=64) was 17 (1-40) years. The first symptoms of HAE in 64 patients were analysed. Twenty-six patients had abdominal, seventeen peripheral, five facial, two urogenital, and three had laryngeal oedema as the first manifestation of the disease. The last death that was attributed to HAE was in 1977. CONCLUSIONS: Our observations demonstrate improved awareness of HAE among physicians, as documented by the significant decrease in diagnostic delay. It is believed that earlier treatment will improve patient quality of life and life expectancy.

[Recurrent meningitis and inherited complement deficiency]. / Recidivující meningitidy a vrozený deficit komplementového systému.

Complement deficiency represents 5% of primary immunodeficiencies worldwide. A total of seven patients with deficiencies of the classical complement pathway were reported in the Czech Republic by the end of 2015. Typical manifestations of complement deficiency are recurrent meningitis, other bacterial infections, autoimmunity and kidney disease.Two case reports are presented of patients with molecularly confirmed C7 (compound heterozygote, c.663_644del in exon 6 and c.2350+2T:>C in intron 16) and C8 (homozygous c.1282C>T in exon 9) deficiency. The first patient had four attacks of meningococcal meningitis and an episode of pneumonia of unknown aetiology in childhood. The second had six attacks of meningitis. He also suffered from recurrent infections (otitis media, tonsillitis, chronic mucopurulent rhinitis and subsequent pansinusitis complicated by nasal polyposis) since childhood. No autoimmune disease was documented in either patient. They both received meningococcal and pneumococcal vaccines. Antibiotic prophylaxis was used only in the second patient, leading to a decline in the number of ENT infections.Complement deficiency should be suspected in patients with recurrent meningococcal infections, especially if combined with other infections caused by encapsulated bacteria or autoimmunity diseases. Prophylaxis with conjugate polysaccharide vaccines is recommended and antibiotic prophylaxis should be considered in individual cases.

Spectrum and management of complement immunodeficiencies (excluding hereditary angioedema) across Europe.

INTRODUCTION: Complement immunodeficiencies (excluding hereditary angioedema and mannose binding lectin deficiency) are rare. Published literature consists largely of case reports and small series. We collated data from 18 cities across Europe to provide an overview of primarily homozygous, rather than partial genotypes and their impact and management. METHODS: Patients were recruited through the ESID registry. Clinical and laboratory information was collected onto standardized forms and analyzed using SPSS software. RESULTS: Seventy-seven patients aged 1 to 68 years were identified. 44 % presented in their first decade of life. 29 % had C2 deficiency, defects in 11 other complement factors were found. 50 (65 %) had serious invasive infections. 61 % of Neisseria meningitidis infections occurred in patients with terminal pathway defects, while 74 % of Streptococcus pneumoniae infections occurred in patients with classical pathway defects (p < 0.001). Physicians in the UK were more likely to prescribe antibiotic prophylaxis than colleagues on the Continent for patients with classical pathway defects. After diagnosis, 16 % of patients suffered serious bacterial infections. Age of the patient and use of prophylactic antibiotics were not associated with subsequent infection risk. Inflammatory/autoimmune diseases were not seen in patients with terminal pathway, but in one third of patients classical and alternative pathway defects. CONCLUSION: The clinical phenotypes of specific complement immunodeficiencies vary considerably both in terms of the predominant bacterial pathogen, and the risk and type of auto-inflammatory disease. Appreciation of these phenotypic differences should help both immunologists and other specialists in their diagnosis and management of these rare and complex patients.

Low ficolin-2 levels in common variable immunodeficiency patients with bronchiectasis.

Common variable immunodeficiency (CVID) encompasses a heterogeneous group of antibody deficiencies characterized by susceptibility to recurrent infections and sequelae, including bronchiectasis. We investigated the relevance of the lectin complement pathway in CVID patients by analysing ficolin-2 and ficolin-3 serum levels and genotyping single nucleotide polymorphisms (SNPs) in the FCN2 and FCN3 genes. Our results show that ficolin-2 levels in CVID patients are significantly lower (P < 0.0001) than in controls. The lowest ficolin-2 levels are found in CVID patients with bronchiectasis (P = 0.0004) and autoimmunity (P = 0.04). Although serum levels of ficolin-3 were similar in CVID patients and controls, CVID patients with bronchiectasis again showed lower levels when compared to controls (P = 0.0001). Analysis of single nucleotide polymorphisms in the FCN2 gene confirmed known influences on ficolin-2 serum levels, but did not support a genetic basis for the observed ficolin-2 deficiency in CVID. We found that CVID patients with bronchiectasis have very low levels of ficolin-2. The reason for the deficiency of ficolin-2 in CVID and any possible causal relationship is currently unknown. However, as bronchiectasis is a very important factor for morbidity and mortality in CVID, ficolin-2 could also serve as biomarker for monitoring disease complications such as bronchiectasis.

Outcomes of splenectomy in patients with common variable immunodeficiency (CVID): a survey of 45 patients.

Splenectomy has been used in patients with common variable immunodeficiency disorders (CVID), mainly in the context of refractory autoimmune cytopenia and suspected lymphoma, but there are understandable concerns about the potential of compounding an existing immunodeficiency. With increasing use of rituximab as an alternative treatment for refractory autoimmune cytopenia, the role of splenectomy in CVID needs to be re-examined. This retrospective study provides the largest cohesive data set to date describing the outcome of splenectomy in 45 CVID patients in the past 40 years. Splenectomy proved to be an effective long-term treatment in 75% of CVID patients with autoimmune cytopenia, even in some cases when rituximab had failed. Splenectomy does not worsen mortality in CVID and adequate immunoglobulin replacement therapy appears to play a protective role in overwhelming post-splenectomy infections. Future trials comparing the effectiveness and safety of rituximab and splenectomy are needed to provide clearer guidance on the second-line management of autoimmune cytopenia in CVID.

Chronic immune activation in common variable immunodeficiency (CVID) is associated with elevated serum levels of soluble CD14 and CD25 but not endotoxaemia.

Common variable immunodeficiency (CVID), the most frequent symptomatic immunoglobulin primary immunodeficiency, is associated with chronic T cell activation and reduced frequency of CD4(+) T cells. The underlying cause of immune activation in CVID is unknown. Microbial translocation indicated by elevated serum levels of lipopolysaccharide and soluble CD14 (sCD14) has been linked previously to systemic immune activation in human immunodeficiency virus/acquired immune deficiency syndrome (HIV-1/AIDS), alcoholic cirrhosis and other conditions. To address the mechanisms of chronic immune activation in CVID, we performed a detailed analysis of immune cell populations and serum levels of sCD14, soluble CD25 (sCD25), lipopolysaccharide and markers of liver function in 35 patients with CVID, 53 patients with selective immunoglobulin (Ig)A deficiency (IgAD) and 63 control healthy subjects. In CVID subjects, the concentration of serum sCD14 was increased significantly and correlated with the level of sCD25, C-reactive protein and the extent of T cell activation. Importantly, no increase in serum lipopolysaccharide concentration was observed in patients with CVID or IgAD. Collectively, the data presented suggest that chronic T cell activation in CVID is associated with elevated levels of sCD14 and sCD25, but not with systemic endotoxaemia, and suggest involvement of lipopolysaccharide-independent mechanisms of induction of sCD14 production.

Sequence variants of the TNFRSF13B gene in Czech CVID and IgAD patients in the context of other populations.

Mutations in the TNFRSF13B gene, encoding TACI, have been found in common variable immunodeficiency (CVID) and selective IgA deficient (IgAD) patients, but only the association with CVID seems to be significant. In this study, Czech CVID, IgAD and primary hypo/dysgammaglobulinemic (HG/DG) patients were screened for all TNFRSF13B sequence variants. The TNFRSF13B gene was mutated in 4/70 CVID patients (5.7%), 9/161 IgAD patients (5.6%), 1/17 HG/DG patient (5.9%) and none of 195 controls. Eight different mutations were detected, including the most frequent p.C104R and p.A181E mutations as well as 1 novel missense mutation, p.R189K. A significant association of TNFRSF13B gene mutations was observed in both CVID (p=0.01) and IgAD (p=0.002) Czech patients. However, when combined with all published data, only the association with CVID remained significant compared with the controls (9.9% vs. 3.2%, p<10(-6)), while statistical significance disappeared for IgAD (5.7% vs. 3.2%, p=0.145). The silent mutation p.P97P was shown to be associated significantly with CVID compared with the controls in both Czech patients (allele frequency 4.3% vs. 0.2%, p=0.01) and in connection with the published data (5.1% vs. 1.8%, p=0.003). The relevance of some TNFRSF13B gene variants remains unclear and needs to be elucidated in future studies.

No evidence for linkage between the hereditary angiooedema clinical phenotype and the BDKR1, BDKR2, ACE or MBL2 gene.

Hereditary angiooedema (HAE) is a life-threatening disease with poor clinical phenotype correlation with its causal mutation in the C1 inhibitor (SERPING1) gene. It is characterized by substantial symptom variability even in affected members of the same family. Therefore, it is likely that genetic factors outside the SERPING1 gene have an influence on disease manifestation. In this study, functional polymorphisms in genes with a possible disease-modifying effect, B1 and B2 bradykinin receptors (BDKR1, BDKR2), angiotensin-converting enzyme (ACE) and mannose-binding lectin (MBL2), were analysed in 36 unrelated HAE patients. The same analysis was carried out in 69 HAE patients regardless of their familial relationship. No significant influence of the studied polymorphisms in the BDKR1, BDKR2, ACE and MBL2 genes on overall disease severity, localization and severity of particular attacks, frequency of oedema episodes or age of disease onset was detected in either group of patients. Other genetic and/or environmental factors should be considered to be responsible for HAE clinical variability in Caucasians.

[A simple method for the detection of CD154 (CD40L) on peripheral blood lymphocytes]. / Jednoduchá metoda k prukazu CD154 (CD40L) na lymfocytech v periferní krvi.

OBJECTIVE: CD154 (also called CD40L) is a transmembrane glycoprotein predominantly expressed on the surface membrane of activated CD4+ T cells. Its receptor CD40 is present on all B cells, but also on other cells. The interaction CD154-CD40 is necessary for the optimal development of the adaptive immune response and also has consequences for the modulation of the inflammatory response. A defect in the expression of CD154 is pathognomonic of congenital immunodeficiency called X-linked Hyper-IgM syndrome (XHIGM). To detect the abnormality of CD154 is essential for making the diagnosis of XHIGM. MATERIAL AND METHODS: We worked out a microtest for the detection of CD154 expression on in vitro activated CD4+ T cells in whole blood and compared it with that on isolated cells from peripheral blood. Heparinized peripheral blood was activated with phorbol 12-myristate 13-acetate and ionomycin for 4 hours, labeled with monoclonal antibodies and analyzed by flow cytometry. Considering that the CD4 marker on the plasma membrane surface decreases during the activation, CD4+ T cells are mostly recognized as CD5+/CD8- cells. Their activation is monitored based on the expression of CD69. Three-colour immunofluorescence staining was used for simultaneous detection of CD154. RESULTS: Ten blood donors were tested. As little as 0.5 ml of heparinized whole blood is needed to complete the test. Optimal time for activation and detection of CD154 on T lymphocytes is 4 hours. We found that the number of CD4 molecules on the surface of T cells decreases during the activation. The expression of CD154 in our whole blood microtest is fully comparable with that in the test on isolated leukocytes. CONCLUSION: The presented microtest for the detection of CD154 on activated lymphocytes in whole blood is fast and blood saving, since as little as 0.5 ml of blood is needed to complete it. It can be recommended as the initial test for suspected hyper-IgM syndrome in children. We demonstrate that this screening method can help to detect also carriers of XHIGM.

NOD2 polymorphisms in clinical phenotypes of common variable immunodeficiency disorders.

Common variable immunodeficiency disorders (CVIDs) are a heterogeneous group of diseases characterized by hypogammaglobulinaemia and consequent susceptibility to infection. CVID patients commonly develop a variety of additional manifestations for which the causative factors are not fully understood. Two such manifestations are granulomatous disease and enteropathy. Because the ability to predict complications would aid clinical management, we continue to search for possible disease modifier genes. NOD2 acts a microbial sensor and is involved in proinflammatory signalling. Particular mutations of the NOD2 gene are associated with Crohn's disease including gly908arg, leu1007finsc and arg702trp polymorphisms. We hypothesized that NOD2 polymorphisms may be a disease modifier gene towards an enteropathic or granulomatous phenotype within CVIDs. Sequence-specific primers returned genotypes for 285 CVID patients from centres across the United Kingdom and Europe. We present the frequencies of the different phenotypes of patients within our international cohort. Arg702trp polymorphisms were significantly less frequent than wild-type (WT) (P = 0·038) among international CVID patients with splenomegaly. Gly908arg polymorphisms were more prevalent than WT in UK patients with autoimmune disorders (P = 0·049) or enteropathy (P = 0·049). NOD2 polymorphisms were not more prevalent than WT in CVID patients with clinical phenotypes of granulomata. UK allele frequencies of 0·014, 0·056 and 0·026 were found for gly908arg, arg702trp and leu1007finsc NOD2 polymorphisms, respectively. These do not differ significantly from UK immunocompetent controls confirming, as expected, that in addition these NOD2 polymorphisms do not confer susceptibility to CVIDs per se.

Association of FcRn expression with lung abnormalities and IVIG catabolism in patients with common variable immunodeficiency.

The neonatal Fc receptor (FcRn) acts as a key regulator of IgG homeostasis and is an important sensor of luminal infection. We analyzed the influence of FcRn expression on disease phenotype and the catabolism of therapeutically administered intravenous immunoglobulins (IVIG) in 28 patients with common variable immunodeficiency (CVID). Patients with generalized bronchiectasis and fibrosis had lower levels of FCRN mRNA compared to patients without these complications (P=0.027 and P=0.041, respectively). Moreover, FCRN mRNA levels correlated negatively with the extent of bronchiectasis and the rate of IgG decline after infusion of IVIG (P=0.027 and P=0.045, respectively). No relationship of FCRN expression with age at disease onset, age at diagnosis, diagnostic delay, IgG levels or frequency of infections before or during replacement immunoglobulin treatment, the presence of lung functional abnormalities, chronic diarrhea, granulomas, lymphadenopathy, splenomegaly or autoimmune phenomena was observed. Our results showed that FcRn might play a role in the development of lung structural abnormalities and in the catabolism of IVIG in patients with CVID.

No association of FCRN promoter VNTR polymorphism with the rate of maternal-fetal IgG transfer.

The neonatal Fc receptor (FcRn) plays a critical role in maternal-fetal IgG transfer. Recently, a functionally active promoter polymorphism in the FCRN gene, represented by variable number of tandem repeats (VNTR), has been described. We analysed 103 single fetal samples and 103 paired maternal and fetal samples collected from umbilical cord blood of full-term neonates born from the 38th to the 41st week of pregnancy and detected no significant influence of maternal FCRN VNTR genotype on maternal IgG levels or of fetal FCRN VNTR genotype on fetal IgG levels or the fetal/maternal IgG ratio.

[Common variable immunodeficiency in pregnancy (set of case reports)]. / Bezný variabilní imunodeficit v tehotenství (soubor kazuistik).

Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency, which is characterized by impaired antibody responses. It's manifestation includes mainly severe and recurrent bacterial infections affecting predominantly upper and lower respiratory tract. Because of the improved standard of hypogammaglobulinemic patients many affected females become pregnant. When a woman treated for CVID gets pregnant the adequate treatment is necessary not only to protect patient from infections, but also to allow suffitient transfer of IgG through the placenta to supply the fetus and consequently the newborn. Regular periodic replacement therapy with intravenous immunoglobulins (IVIG) is applied in pregnant women as well as in other hypogammaglobulinemic patients. Regimen of IVIG administration must be modified in order to reach satisfactory IgG levels in the newborns' blood. Here we present a set of case reports of five pregnant women with CVID treated by immunoglobulins during pregnancy. In all cases labor was induced in term after the last IVIG infusion. The mode of delivery depended on the obstetric indication. All pregnancies resulted in healthy newborns.

Matrix metalloproteinase-9 and matrix metalloproteinase-2 as biomarkers of various courses in multiple sclerosis.

BACKGROUND: Matrix metalloproteinases are notable contributors to neuroinflammation and blood-brain barrier disruption in multiple sclerosis (MS). OBJECTIVE: The goal of this study was to determine the serum levels of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-2 (MMP-2), and their tissue inhibitors (TIMP-1) and (TIMP-2), and to investigate their possible relations to type, disability, and severity of MS. MATERIALS AND METHODS: Eighty-seven patients with definite MS according to the McDonald criteria and 50 healthy controls were enrolled in the study. Their clinical status was evaluated with the Expanded Disability Status Scale. Serum levels were analyzed by enzyme-linked immunoassay. RESULTS: A significant elevation in MMP-9 serum levels and in the MMP-9/TIMP-1 ratio was found in the whole MS group (P<0.001), in the relapsing-remitting MS (RRMS) (P<0.001), and secondary-progressive MS (SPMS) (P<0.001) groups when compared with the controls. A significant elevation in MMP-2 serum levels and in the MMP-2/TIMP-2 ratio was observed in the primary progressive (P<0.001) and the SPMS (P<0.002) groups when compared with the RRMS group, and this increase was also associated with the disability (P<0.001) and severity (P<0.05) of the disease. CONCLUSION: We confirmed that metalloproteinases are useful biological markers in MS, providing information about the clinical type, disability, and severity of the disease.

Reduced memory B cells in patients with hyper IgE syndrome.

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

Mannose-binding lectin gene polymorphic variants predispose to the development of bronchopulmonary complications but have no influence on other clinical and laboratory symptoms or signs of common variable immunodeficiency.

Mannose-binding lectin (MBL), activating protein of the lectin pathway of the complement system, is an important component of the non-specific immune response. MBL2 gene polymorphisms, both in the coding and promoter regions, lead to low or deficient serum MBL levels. Low serum MBL levels were shown to be associated with serious infectious complications, mainly in patients in whom other non-specific immune system barriers were disturbed (granulocytopenia, cystic fibrosis). We have analysed two promoter (-550 and -221) and three exon (codons 52, 54 and 57) MBL2 polymorphisms in a total of 94 patients with common variable immunodeficiency (CVID) from two immunodeficiency centres. Low-producing genotypes were associated with the presence of bronchiectasis (P = 0.009), lung fibrosis (P = 0.037) and also with respiratory insufficiency (P = 0.029). We could not demonstrate any association of MBL deficiency with age at onset of clinical symptoms, age at diagnosis, the number of pneumonias before diagnosis or serum immunoglobulin (Ig)G, IgA and IgM levels before initiation of Ig treatment. No association with emphysema development was observed, such as with lung function test abnormalities. No effect of MBL2 genotypes on the presence of diarrhoea, granuloma formation, lymphadenopathy, splenomegaly, frequency of respiratory tract infection or the number of antibiotic courses of the patients was observed. Our study suggests that low MBL-producing genotypes predispose to bronchiectasis formation, and also fibrosis and respiratory insufficiency development, but have no effect on other complications in CVID patients.