Pneumococcal vaccination responses in adults with subnormal IgG subclass concentrations.

BACKGROUND: We sought to compare Pneumovax®23 responses in adults with subnormal IgG subclass concentrations. We studied adults with normal total IgG, frequent/severe respiratory infection, and subnormal IgG1, IgG3, or IgG1 + IgG3 before and after Pneumovax®23. We defined response as serotype-specific IgG > 1.3 µg/mL and aggregate response as IgG > 1.3 µg/mL for ≥70% of all serotypes tested. We compared patients with and without serotype-specific responses and performed logistic regression on aggregate responses using: age; male sex; body mass index; autoimmune condition(s); atopy; other allergies; subnormal IgGSc immunophenotypes; IgA; and IgM. RESULTS: There were 59 patients (mean age 44 ± 13 (SD) years; 83.1% women). Median days between pre- and post-Pneumovax®23 testing was 33 (range 19-158). The median post-vaccination summated concentration of serotype-specific IgG was higher in patients with subnormal IgG1 than subnormal IgG3 (responders and non-responders). All subnormal IgG1 + IgG3 non-responders responded to serotypes 8, 9 and 26, unlike other non-responders. Subnormal IgG3 responders had lower responses to serotypes 1, 4, 12, 23, 26, and 51. Subnormal IgG3 non-responders had higher responses to serotypes 1, 3, 8, 9, 12, 14, 19, 51, and 56. Response rates decreased with increasing age. Aggregate responders were: subnormal IgG1, 54%; IgG3, 46%; and IgG1 + IgG3, 46%. Regression on aggregate response revealed lower response with male sex (odds ratio 0.09 [95% CI 0.01, 0.77]) and atopy (0.17 [0.03, 0.83]). CONCLUSIONS: Serotype-specific IgG responses to Pneumovax®23 were greater in patients with subnormal IgG1 than subnormal IgG3. Male sex and atopy were associated with lower aggregate responses.

Measurement of Typhim Vi IgG as a Diagnostic Tool to Determine Anti-polysaccharide Antibody Production Deficiency in Children.

Background: The assessment of specific polysaccharide antibody production plays a pivotal role in the diagnosis of humoral primary immunodeficiencies (PID). The response to 23-valent pneumococcal vaccine (PPV) remains the gold standard for the diagnosis of polysaccharide antibodies. However, in Spain, the interpretation of pure polysaccharide 23-valent immunization is hampered by the high endemicity of pneumococcal disease and the generalization of the 13-valent adjuvant pneumococcal vaccination. Specific Typhim Vi vaccination (TV) immunoglobulin G IgG response to immunization is useful in adult PID, but there is no data regarding children. Objectives: To evaluate the clinical utility of TV IgG production as a diagnostic tool to determine anti-polysaccharide antibody production deficiency in children, when the response to PPV is unclear and isolated determination of serotypes is unfeasible. Methods: We conducted a single-institution prospective observational study on 61 children with recurrent infections. Baseline specific antibodies against PPV and TV were evaluated. In 28 children (46%), the response to the production of antibodies confirmed a clinical suspicion of humoral PID, and they were therefore immunized with 23-valent pneumococcal vaccine and Typhim Vi. Both specific antibody responses were measured by ELISA (The Binding Site Group Ltd, Birmingham, UK) using previously published cut-offs. Results: Seventy percent of the 61 children displayed baseline PPV IgG > 27 mg/L, whereas only 8% showed TV IgG > 28 U/mL (p < 0.0001). Twenty-one of 28 children (75%) achieved a 3-fold increase in post-vaccination TV IgG levels, whereas only 3% achieved a 4-fold increase in PPV IgG post vaccination, mainly due to high baseline PPV IgG titers. When we classified children according to their response to TV as responders or non-responders and compared this with the well-known clinical warning signs of the Jeffrey Modell Foundation. The proportions of children with history of pneumonia and the need for intravenous antibiotics were significantly higher in TV IgG non-responders than in TV IgG responders (p = 0.02 and p = 0.01, respectively). Conclusion: Response to TV can be considered an ancillary diagnostic tool to determine polysaccharide antibodies in children, particularly when isolated determination of pneumococcal serotypes is not feasible. TV provides a useful asset for clinicians in the era of conjugate PPV vaccination, with clinical relevance. Further research is warranted for validation.

The total IgM, IgA and IgG antibody responses to pneumococcal polysaccharide vaccination (Pneumovax®23) in a healthy adult population and patients diagnosed with primary immunodeficiencies.

BACKGROUND: Interpretation of the responses to the pneumococcal polysaccharide vaccine (Pneumovax®23, PPV) has proven challenging. In addition, there are few studies documenting the longevity of these responses. METHODS: The age-specific PPV IgM, IgA, IgG and IgG2 concentrations were determined pre, 4-6 weeks and 6 years post-vaccination in the serum of Prevnar®-naïve adults using VaccZyme™ pneumococcal capsular polysaccharide ELISAs. RESULTS: The median pre-vaccination concentrations were; PPV IgM 53 U/mL (5-95% CI: 16-169 U/mL), IgA 23 U/mL (6-103 U/mL), IgG 41 mg/L (10-184 U/mL) and IgG2 18 mg/L (3-95 U/mL). 4-6 weeks post-vaccination there was a median 6-fold (5-95% CI: 2-24) increase in PPV IgM (median 315 U/mL (5-95% CI: 60-1133 U/mL), 18-fold (4-74) increase in IgA (369 U/mL (78-1802 U/mL)), 9-fold (2-19) increase in IgG (375 mg/L (77-1238 mg/L)) and 8-fold (1-20) increase in IgG2 (141 mg/L (25-573 mg/L)). This was significant for all isotypes in all age ranges (p < 0.0001). Six years post-vaccination median PPV concentrations were; IgM 54 U/mL (17-128), IgA 85 U/mL (19-279), IgG 148 mg/L (30-997) and IgG2 57 mg/L (9-437). The median concentrations for all ages 6 years post-vaccination were significantly elevated compared to the pre-vaccination titres for PPV IgA, IgG and IgG2 isotypes only. The PPV IgM and IgA responses were influenced by age. At 6 years post vaccination, in individuals with normal PPV IgG, 34 individuals had PPV IgM and/or IgA concentrations below the lower limit of the healthy adult ranges. We also used the healthy adult reference ranges developed in this study to assess a cohort of primary immunodeficiency (PID) patients. CONCLUSION: These ranges will help to provide a framework for assessment and definition of normal response to PPV, which will facilitate clinical interpretation of a deficient polysaccharide response in those suspected of antibody deficiency.

Quantification of human C1 esterase inhibitor protein using an automated turbidimetric immunoassay.

BACKGROUND: Impaired levels or function of C1 inhibitor (C1-INH) results in angioedema due to increased bradykinin. It is important to distinguish between angioedema related to C1-INH deficiency and that caused by other mechanisms, as treatment options are different. In hereditary (HAE) and acquired (AAE) angioedema, C1-INH concentration is measured to aid patient diagnosis. Here, we describe an automated turbidimetric assay to measure C1-INH concentration on the Optilite® analyzer. METHODS: Linearity, precision, and interference were established over a range of C1-INH concentrations. The 95th percentile reference interval was generated from 120 healthy adult donors. To compare the Optilite C1-INH assay with a predicate assay used in a clinical laboratory, samples sent for C1-INH investigation were used. The predicate results were provided to allow comparison. RESULTS: The Optilite C1-INH assay was linear across the measuring range at the standard sample dilution. Intra and interassay variability was <6%. The 95th percentile adult reference interval for the assay was 0.21-0.38 g/L. There was a strong correlation between the Optilite concentrations and those generated with the predicate assay (R2  = 0.94, P < 0.0001, slope y = 0.83x). All patients with Type I HAE (n = 24) and AAE (n = 3) tested had concentrations below the measuring range in both assays, while all patients with unspecified angioedema (UAE), not diagnosed with HAE or AAE had values within the reference range. CONCLUSION: The Optilite assay allows the automated and precise quantification of C1-INH concentrations in patient samples. It could therefore be used as a tool to aid the investigation of patients with angioedema.

Cerebrospinal fluid immunoglobulin light chain ratios predict disease progression in multiple sclerosis.

OBJECTIVE: To determine whether the ratio of cerebrospinal fluid (CSF) immunoglobulin kappa to lambda light chains at time of multiple sclerosis (MS) diagnosis predicts disease progression and whether this was intrinsic to CSF plasmablasts. METHODS: CSF and peripheral blood were obtained from patients undergoing elective diagnostic lumbar puncture and included clinically isolated syndrome (CIS) (n=43), relapsing remitting MS (RRMS; n=50), primary progressive MS (PPMS; n=20) and other neurological disease controls, both inflammatory (ONID; n=23) and non-inflammatory (OND; n=114). CSF samples were assayed for free and immunoglobulin-associated light chains and on B cells and plasmablasts. Clinical follow-up data were collected during a 5-year follow-up period where available. RESULTS: There was an increased median CSF κ:λ free light chain (FLC) in all MS groups (CIS: 18.2, 95% CI 6.8 to 30.3; RRMS: 4.4, 95% CI 2.7 to 11.4; PPMS: 12.0, 95% CI 3.6 to 37.1) but not controls (OND: 1.61, 95% CI 1.4 to 1.9; ONID: 1.7, 95% CI 1.3 to 2.2; p<0.001). This ratio predicted Expanded Disability Status Scores (EDSS) progression at 5 years, with a lower median EDSS in the group with high (>10) CSF κ:λ FLC (0.0, 95% CI 0 to 2.5 vs 2.5, 95% CI 0 to 4, high vs low; p=0.049). CSF κ:λ FLC correlated with CSF IgG1 κ:λ (r=0.776; p<0.0001) and was intrinsic to CSF plasmablasts (r=0.65; p=0.026). CONCLUSIONS: These data demonstrate that CSF immunoglobulin κ:λ ratios, determined at the time of diagnostic lumbar puncture, predict MS disease progression and may therefore be useful prognostic markers for early therapeutic stratification.

Measurement and interpretation of Salmonella typhi Vi IgG antibodies for the assessment of adaptive immunity.

Response to polysaccharide vaccination can be an invaluable tool for assessing functionality of the adaptive immune system. Measurement of antibodies raised in response to Pneumovax®23 is the current gold standard test, but there are significant challenges and constraints in both the measurement and interpretation of the response. An alternative polysaccharide vaccine approach (Salmonella typhi Vi capsule (ViCPS)) has been suggested. In the present article, we review current evidence for the measurement of ViCPS antibodies in the diagnosis of primary and secondary antibody deficiencies. In particular, we review emerging data suggesting their interpretation in combination with the response to Pneumovax®23 and comment upon the utility of these vaccines to assess humoral immune responses while receiving immunoglobulin replacement therapy (IGRT).

The Clinical Utility of Measuring IgG Subclass Immunoglobulins During Immunological Investigation for Suspected Primary Antibody Deficiencies.

Measurement of IgG subclass concentrations is a standard laboratory test run as part of a panel to investigate the suspicion of antibody deficiency. The assessment is clinically important when total IgG is within the normal age-specific reference range. The measurement is useful for diagnosis of IgG subclass deficiency, to aid the diagnosis of specific antibody deficiency, as a supporting test for the diagnosis of common variable immunodeficiency, as well as for risk stratification of patients with low IgA. The measurement of IgG subclasses may also help determine a revaccination strategy for patients and support patient management. In certain circumstances, the measurement of IgG subclasses may be used to monitor a patient's humoral immune system. In this review, we discuss the utility of measuring IgG subclass concentrations.

The response to Typhi Vi vaccination is compromised in individuals with primary immunodeficiency.

Measurement of an individuals ability to respond to polysaccharide antigens is a crucial test to determine adaptive immunity. Currently the response to Pneumovax® is utilized but with the success of Prevnar®, measurement of the response to Pneumovax may be challenging. The aim of the study was to assess the response to Typhi Vi vaccination in both children and adult control groups and patients with primary immunodeficiency (PID). In the control groups, >95% of the individuals had pre Typhi Vi vaccination concentrations <100 U/mL and there was significant increase in concentration post Typhi Vi vaccination (p<0.0001) with>94% achieving ≥3 fold increase in concentration (FI). The response to Typhi Vi vaccination was significantly lower in both children (p = 0.006) and adult (p = 0.002) PID groups when compared to their control groups. 11% and 55% of the children and adult PID groups respectively did not obtain a response >3FI. There were no significant differences between the responses obtained in the children and adult PID groups. When all individuals with PID were separated into those with either hypogammaglobulinemia (HYPO) or common variable immunodeficiency (CVID), both groups had a significantly lower median FI than the control group (19, 95%CI 5-56 vs 59, 95%CI 7-237; p = 0.01 and 1, 95%CI 1-56 vs 32, 95%CI 5-136; p = 0.005). Further, a >3FI differentiated the antibody responses between both the CVID and HYPO groups and their control groups (AUC: 0.83, 95%CI: 0.65-1.00, p = 0.005 and 0.81, 95% CI: 0.65-0.97, p = 0.01). The data suggests that measurement of the response to Typhi Vi vaccination could represent a complementary assay for the assessment of the response to a polysaccharide vaccine.

Concentrations of Pneumococcal IgA and IgM are compromised in some individuals with antibody deficiencies.

The response to pneumococcal vaccination is assessed by measurement of antigen specific IgG only and is compromised in a number of antibody deficiencies. We measured the concentrations of Pneumococcal IgA and IgM in individuals with both normal and abnormal pneumococcal capsular polysaccharide (PCP) IgG concentrations. A higher number of individuals had abnormal pre-vaccination IgA and IgM concentrations below the lower limit of the normal range compared to the control group. Post vaccination a lower number of individuals had IgA and IgM concentrations below the upper limit of the normal range compared to the control group. Non responders had a higher percentage of individuals with a prior history of infection. In addition, individuals with a history of prior infection had lower pre- and post-vaccination concentrations of PCP IgG, IgA, and IgM. Post-vaccination IgA and IgM concentrations identified four groups of responses which correlated with prior history of infection. A higher percentage of individuals with abnormal PCP IgA and IgM concentrations had a history of prior infection compared to the percentage of individuals with normal concentrations. In individuals with an antibody deficiency, measurement of Pneumococcal IgA and IgM correlates with the number of individuals with prior history of infection.

Purification and characterisation of anti-pneumococcal capsular polysaccharide IgG immunoglobulins.

OBJECTIVES: The production of reference materials for quantifying pneumococcal antibody concentrations relies upon large scale vaccination. An alternative simple, reproducible protocol has been developed for the affinity purification of 23 serotype anti-pneumococcal capsular polysaccharide (PCP) IgG immunoglobulins. DESIGN & METHODS: The purification protocol utilised IgG fractionation, capsular polysaccharide (CPS) adsorption, and affinity chromatography using Pneumovax®-Sepharose. Purification efficiency and method reproducibility were assessed by comparison of 4 batches of anti-PCP IgG. Immunoglobulin composition was determined using nephelometry and functionality was evaluated using VaccZyme™ ELISAs. RESULTS: Anti-PCP IgG preparations were ≥95% pure by SDS-PAGE analysis with no contaminating IgA or IgM immunoglobulins or IgG antigen specific antibodies towards haemophilus influenzae b, diphtheria toxoid or tetanus toxoid. The predominant IgG subclass in the preparation was IgG2. CONCLUSIONS: This novel purification procedure produced highly specific anti-PCP IgG preparations that compared well to both Lot 89SF and 007sp international serum standards and could be used as an alternative method for the production of reference materials.

Concentration of anti-pneumococcal capsular polysaccharide IgM, IgG and IgA specific antibodies in adult blood donors.

OBJECTIVES: Anti-pneumococcal capsular polysaccharide (PCP) IgM, IgG and IgA ELISAs have been developed to aid assessment of the adaptive immune system. The relationship between the concentrations of PCP IgM, IgG, and IgA was investigated. DESIGN AND METHODS: The concentrations of PCP IgM, IgG, and IgA were measured in sera obtained from 231 adult blood donors. RESULTS: Concentrations of each isotype were not normally distributed. The median concentration for PCP IgM was 54 U/mL (range 37-75 U/mL), IgG 40 mg/L (range 26-79 mg/L) and IgA 21 U/mL (range 13-44 U/mL). The median PCP IgM titres decreased with age and were significantly lower in patients aged 81-90 years compared to those aged 18-80 years. By contrast, there was a significantly higher median serum PCP IgG titre in the 61-90 years group compared to those aged 18-60 years and a significantly higher median serum PCP IgA titre in the 51-90 years group compared to those aged 18-50 years. The correlation between PCP IgG and IgA was more significant than between IgM and IgA and between IgM and IgG. Correlation of PCP IgA and IgM concentrations identified four phenotypes: high PCP IgM and IgA; high PCP IgM only; high PCP IgA only; and low PCP IgM and IgA. A significant number of individuals with a PCP IgG concentration >50 mg/L had low PCP IgA and IgM concentrations. CONCLUSION: The additional measurement of PCP IgA and PCP IgM, alongside PCP IgG, in individuals investigated for a compromised immune system may provide a more detailed antibody profile.

Significant, quantifiable differences exist between IgG subclass standards WHO67/97 and ERM-DA470k and can result in different interpretation of results.

OBJECTIVES: Accurate measurement of IgG subclass (IgGSc) levels are essential to aid in the diagnosis of disease states such as primary immunodeficiencies. However, there is no single standardisation of nephelometric and turbidimetric assays for these analytes and two reference materials have been utilised. We expand on previous reports and present data from a multi-site analysis that both identifies and quantitatively defines the differences in calibration resulting from the use of different reference materials. DESIGN AND METHODS: IgGSc antibodies in the serum specimens and reference materials were measured according to the manufacturers' instructions using commercially available IgGSc assays or components. RESULTS: Data from four independent sites showed that in spite of the different commercial suppliers of IgGSc assays calibrating to different reference materials, ERM-DA470k and WHO67 /97, the resulting calibrations were comparable for IgG1 and IgG2. However, for IgG3 and IgG4 the calibrations were significantly different. The use of assay specific normal ranges should compensate for these calibration differences, however, the two manufacturers' assays can give differing clinical classifications. The agreement between the different manufacturers' IgGSc assays was between 85.1% and 95.8% for all IgGSc assays, the discordance of sample classification for IgG1 and IgG2 assays was approximately 12% and 15% respectively, whilst that for IgG3 and IgG4 was 4% and 13% respectively. CONCLUSION: We discuss the similarities and differences between assays that utilise the different reference materials.

Anti-gene padlocks eliminate Escherichia coli based on their genotype.

OBJECTIVES: Several therapeutic strategies that target nucleic acids exist; however, most approaches target messenger RNA, rather than genomic DNA. We describe a novel oligonucleotide-based strategy, called anti-gene padlocks (AGPs), which eliminate Escherichia coli based on their genotype. METHODS: The strategy employs an oligonucleotide with a double hairpin structure where both strands of the AGP are complementary to both strands of a target gene. We tested AGPs for in vitro binding and inhibition of DNA polymerization. AGPs were electroporated into bacterial cells with and without gene targets along with an ampicillin resistance plasmid, and cell survival was measured. RESULTS: In vitro, AGPs bound the DNA target in a sequence-dependent fashion and inhibited DNA synthesis. When transformed into bacterial cells containing 10, 20 or 30 bp lacZ or 20 bp proA DNA targets in their genomes, AGPs selectively killed or otherwise inhibited growth of these cells, while those lacking the target demonstrated little, if any, toxicity. A single transformation resulted in approximately 30% to 40% loss of target-bearing cells. Structure-function experiments were performed to define essential AGP requirements. CONCLUSIONS: These results suggest that AGPs may be a useful tool to eliminate specific cell populations.

A novel functionally deficient MYH variant in individuals with colorectal adenomatous polyposis.

Inherited biallelic mutations in the base excision repair gene MYH confer susceptibility to colorectal adenomas and carcinoma. Approximately 85% of Caucasians with MYH mutations carry the (c.494A>G) p.Y165C and (c.1145G>A) p.G382D variants. Only a few other clearly pathogenic mutations have been identified, and mutation analyses tend to focus on the two founder mutations rather than the whole coding region of the gene. We sequenced the entire coding region of MYH in a population-based series of 24 Finnish APC-mutation negative polyposis patients in order to identify novel pathogenic MYH variants. A population-based series of 1,042 Finnish colorectal cancer patients and 85 cancer-free controls were available for further evaluation. A functional cleavage assay was designed to evaluate consequences of possible novel variants to protein function. Three novel MYH variants, (c.270C>T) p.Y90Y, (c.1376C>A) p.A459D, and (c.1389G>C) p.T469T, were observed. p.A459D variant in exon 14 was identified in two patients from the polyposis series, once in homozygosity and once in compound heterozygosity with p.Y165C. In the population-based series of 1,042 colorectal cancer patients, the p.A459D mutation was identified once, in homozygosity (allele frequency 0.1%). No p.A459D mutations were identified in the control individuals. In vitro cleavage assay showed significantly reduced repair activity in p.A459D cells. Interestingly, another variant in the same codon has previously been described in a British study, supporting a key role for the codon 459 in MYH function. We therefore suggest that screening of mutations in MYH exon 14 should be added to the molecular analysis at-risk individuals.

Cells with pathogenic biallelic mutations in the human MUTYH gene are defective in DNA damage binding and repair.

Inherited biallelic mutations in the human MUTYH gene are responsible for the recessive syndrome--adenomatous colorectal polyposis (MUTYH associated polyposis, MAP)--which significantly increases the risk of colorectal cancer (CRC). Defective MUTYH activity causes G:C to T:A transversions in tumour APC and other genes thereby altering genomic integrity. We report that of the four established cell lines, derived from patients with the MAP phenotype and containing biallelic MUTYH mutations, three contain altered expressions of MUTYH protein (MUTYH Y165C(-/-), MUTYH 1103delC/G382D and MUTYH Y165C/G382D but not MUTYH G382D(-/-)), but that all four cell lines have wild type levels of MUTYH mRNA. Mutant MUTYH proteins in these four cell lines possess significantly lowered binding and cleavage activities with heteroduplex oligonucleotides containing A.8-oxoG and 8-oxoA.G mispairs. Transfection of mitochondrial or nuclear MUTYH cDNAs partially correct altered MUTYH expression and activity in these defective cell lines. Finally, we surprisingly find that defective MUTYH may not alter cell survival after hydrogen peroxide and menadione treatments. The Y165C and 1103delC mutations significantly reduce MUTYH protein stability and thus repair activity, whereas the G382D mutation produces dysfunctional protein only suggesting different functional molecular mechanisms by which the MAP phenotype may contribute to the development of CRC.

The life and death of DNA-PK.

Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous end-joining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PK(cs)), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK(-/-) mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR.