Evaluating patient immunocompetence through antibody response to pneumococcal polysaccharide vaccine using a newly developed 23 serotype multiplexed assay.

Assessing T-cell independent antibody response to polysaccharide vaccines is crucial for diagnosing humoral immune deficiencies. However, immunocompetence criteria based on S. pneumoniae vaccination remain unclear. We evaluated IgG antibody vaccine response in healthy individuals to establish interpretive criteria. Pre- and 4-week post-vaccination sera were collected from 79 adults. Antibody concentrations to PNEUMOVAX 23 serotypes were measured using a multiplexed platform. Immunocompetence was determined by fold increase in post-vaccination response, percentage of serotypes achieving 4- or 2-fold antibody ratio, and post-vaccination concentration ≥ 1.3 µg/mL. Immunogenicity varied widely across the 23 serotypes (26.6% to 94.9% for ≥4-fold increase, 51.9% to 98.7% for ≥2-fold increase). Immunocompetence based on historic criteria of ≥4-fold increase in antibody ratio to ≥70% of serotypes was low (72.2%), but increased to 98.7% with criteria of at least a 2-fold increase and/or post-vaccination concentration ≥ 1.3 µg/mL. Current criteria for assessing immunocompetence may be overly stringent and require updating.

Specimen type validation and establishment of normal cytokine reference intervals in cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a critical body fluid to examine in attempts to discover potential biomarkers for neuroinflammatory and other disorders of the central nervous system (CNS). Serum and/or plasma cytokine levels have been associated with a variety of inflammatory conditions, and some have been shown to be actionable therapeutic targets. Less is known, however, about cytokine levels in CSF. Serum and plasma cytokine testing is widely available in clinical and research laboratories, but cytokine testing in CSF is extremely limited and if performed, accompanied by a disclaimer that it is an unvalidated specimen type. In this study, we validate CSF as a suitable specimen type and determine normal reference intervals for multiple cytokines as well as a soluble cytokine receptor. CSF was validated as a specimen type for testing using a laboratory developed multiplexed cytokine assay previously validated to measure 13 cytokines/markers in serum and plasma. Performance parameters including specimen dilution, specimen interference, linearity and precision were examined. Reference intervals were established using 197 normal and control CSF specimens by non-parametric quantile-based methods. CSF cytokine analysis demonstrated within and between run precision of <10% and < 20% CV, respectively and linearity of ±15% for all analytes throughout the analytical measurement range of the assay. Reference intervals for the 13 cytokines/markers were established from 197 normal and control CSF specimens (78 Male; mean 44.8 y ± 21.7 SD, 119 Female; mean 42.8 y ± 20.3 SD). Cytokine concentrations in CSF from normal donors and controls were less than the lower limit of quantitation of our assay for 6 of the 13 measured cytokines/markers. The chemokine IL8 demonstrated the highest concentration of all analytes measured. CSF demonstrated acceptable performance as a specimen type in our multiplexed cytokine assay. By validating CSF as a specimen type and establishing normal reference intervals for cytokine concentrations in CSF, their potential as biomarkers for infectious, autoimmune and other inflammatory CNS disorders can be more appropriately investigated.

Neurologic Manifestations of Common Variable Immunodeficiency: Impact on Quality of Life.

BACKGROUND AND OBJECTIVES: Common variable immunodeficiency is a systemic disease and not solely a disease of humoral immunity. Neurologic symptoms associated with common variable immunodeficiency are underrecognized and warrant further study. This work aimed to characterize the neurologic symptoms reported by people living with common variable immunodeficiency. METHODS: We conducted a single academic medical center study of neurologic symptoms reported by adults previously diagnosed with common variable immunodeficiency. We used a survey of common neurologic symptoms to determine the prevalence of these symptoms in a population with common variable immunodeficiency and further assessed these patient-reported symptoms with validated questionnaires and compared symptom burden with other neurologic conditions. RESULTS: A volunteer sample of adults (aged 18 years or older) previously diagnosed with common variable immunodeficiency at the University of Utah Clinical Immunology/Immune Deficiency Clinic who were able to read and comprehend English and willing and able to answer survey-based questions were recruited. Of 148 eligible participants identified, 80 responded and 78 completed the surveys. The mean age of respondents was 51.3 years (range 20-78 years); 73.1% female and 94.8% White. Patients with common variable immunodeficiency reported many common neurologic symptoms (mean 14.6, SD 5.9, range 1-25), with sleep issues, fatigue, and headache reported by more than 85%. Validated questionnaires addressing specific neurologic symptoms supported these results. T-scores on Neuro QoL questionnaires for sleep (mean 56.4, SD 10.4) and fatigue (mean 54.1, SD 11) were higher, indicating more dysfunction, than in the reference clinical population (p < 0.005). The Neuro QoL questionnaire for cognitive function showed a lower T-score (mean 44.8, SD 11.1) than that in the reference general population (p < 0.005), indicating worse function in this domain. DISCUSSION: Among survey respondents, there is a marked burden of neurologic symptoms. Given the impact of neurologic symptoms on health-related quality-of-life measures, clinicians should screen patients with common variable immunodeficiency for the presence of these symptoms and offer referral to neurologists and/or symptomatic treatment when indicated. Frequently prescribed neurologic medications may also affect the immune system, and neurologists should consider screening patients for immune deficiency before prescribing them.

Plasma cytokine levels reveal deficiencies in IL-8 and gamma interferon in Long-COVID.

Up to half of individuals who contract SARS-CoV-2 develop symptoms of long-COVID approximately three months after initial infection. These symptoms are highly variable, and the mechanisms inducing them are yet to be understood. We compared plasma cytokine levels from individuals with long-COVID to healthy individuals and found that those with long-COVID had 100% reductions in circulating levels of interferon gamma (IFNγ) and interleukin-8 (IL-8). Additionally, we found significant reductions in levels of IL-6, IL-2, IL-17, IL-13, and IL-4 in individuals with long-COVID. We propose immune exhaustion as the driver of long-COVID, with the complete absence of IFNγ and IL-8 preventing the lungs and other organs from healing after acute infection, and reducing the ability to fight off subsequent infections, both contributing to the myriad of symptoms suffered by those with long-COVID.

An enzyme-linked immunospot assay to evaluate Pneumovax response when on intravenous immunoglobulin.

BACKGROUND: Testing for common variable immunodeficiency (CVID) requires evaluation of specific antibody responses to vaccines. Current practice of evaluating pneumococcal serotype-specific immunoglobulin (Ig)G levels after Pneumovax (P23) has several limitations and is not accurate for patients already on immunoglobulin replacement. In contrast, the enzyme-linked immunospot (ELISPOT) assay can be interpreted in patients on immunoglobulin replacement as ex vivo measurement of specific antibody-secreting cells (ASCs). OBJECTIVE: To optimize and test an ELISPOT assay to evaluate vaccination response to P23 and compare with P23 serotype-specific IgG for patients on intravenous immunoglobulin (IVIG). METHODS: We prospectively enrolled a total of 15 adults: normal controls (n = 8), patients with CVID on IVIG replacement (n = 2), patients on IVIG replacement for recurrent infections who did not meet diagnostic criteria for CVID, considered IgG deficiency (n = 2), and patients without immunodeficiency on high-dose IVIG for other diagnosis (n = 3). We measured P23 serotype-specific IgG before and 4 weeks after P23 and ELISPOT ASCs before and 1 week after P23 (with B-cell subpopulation analysis by flow cytometry in patients on IVIG). RESULTS: Normal controls had a vaccination response by P23 serotype-specific IgG, whereas patients on IVIG did not. Except for true patients with CVID on IVIG, a P23 ELISPOT ASC response was found in normal controls (highest) and most patients on IVIG for recurrent infections or other diagnosis. CONCLUSION: Our pilot study suggests that an optimized ELISPOT protocol has utility to evaluate the P23-specific antibody response after vaccination. Our ELISPOT assay seemed reliable for patients on IVIG and may help differentiate true patients with CVID from those with a less stringent diagnosis while on IVIG.

Cytokine Deficiencies in Patients with Long-COVID.

Up to half of individuals who contract SARS-CoV-2 develop symptoms of long-COVID approximately three months after initial infection. These symptoms are highly variable, and the mechanisms inducing them are yet to be understood. We compared plasma cytokine levels from individuals with long-COVID to healthy individuals and found that those with long-COVID had 100% reductions in circulating levels of Interferon Gamma (IFNγ) and Interleukin-8 (IL-8). Additionally, we found significant reductions in levels of IL-6, IL-2, IL-17, IL-13, and IL-4 in individuals with long-COVID. We propose immune exhaustion as the driver of long-COVID, with the complete absence of IFNγ and IL-8preventing the lungs and other organs from healing after acute infection, and reducing the ability to fight off subsequent infections, both contributing to the myriad of symptoms suffered by those with long-COVID.

Spinal fluid IgG antibodies from patients with demyelinating diseases bind multiple sclerosis-associated bacteria.

A panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases (DD). The anti-microbial ELISA assays follow on prior human brain tissue RNA sequencing studies that established multiple sclerosis (MS) microbial candidates. Lysates included in the ELISA panel were derived from Akkermansia muciniphila, Atopobium vaginae, Bacteroides fragilis, Lactobacillus paracasei, Odoribacter splanchnicus, Pseudomonas aeruginosa, Cutibacterium (Propionibacterium) acnes, Fusobacterium necrophorum, Porphyromonas gingivalis, and Streptococcus mutans. CSF responses from patients with demyelinating diseases (DD, N = 14) were compared to those with other neurological diseases (OND, N = 8) and controls (N = 13). Commercial positive and negative control CSF specimens were run with each assay. ELISA index values were derived for each specimen against each of the 10 bacterial lysates. CSF reactivity was significantly higher in the DD group compared to the controls against Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium. Four of the 11 tested DD group subjects had elevated antibody indexes against at least one of the 10 bacterial species, suggesting intrathecal antibody production. This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients. KEY MESSAGES: A panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases, including multiple sclerosis and acute disseminated encephalomyelitis. CSF reactivity was significantly higher in the demyelination group compared to the controls against the bacteria Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium. Several of the demyelination subjects had elevated antibody indexes against at least one of the 10 antigens, suggesting at least limited intrathecal production of anti-bacterial antibodies. This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients.

Clinical significance of measuring serum cytokine levels as inflammatory biomarkers in adult and pediatric COVID-19 cases: A review.

Coronavirus disease 2019 (COVID-19) is a rapidly evolving infectious/inflammatory disorder which has turned into a global pandemic. With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as its etiologic agent, severe COVID-19 cases usually develop uncontrolled inflammatory responses and cytokine storm-like syndromes. Measuring serum levels of pro-inflammatory cytokines (e.g., IL-6 and others) as inflammatory biomarkers may have several potential applications in the management of COVID-19, including risk assessment, monitoring of disease progression, determination of prognosis, selection of therapy and prediction of response to treatment.This is especially true for pediatric patients with COVID-19 associated Kawasaki-like disease and similar syndromes. In this report, we review the current knowledge of COVID-19 associated cytokines, their roles in host immune and inflammatory responses, the clinical significance and utility of cytokine immunoassays in adult and pediatric COVID-19 patients, as well as the challenges and pitfalls in implementation and interpretation of cytokine immunoassays. Given that cytokines are implicated in different immunological disorders and diseases, it is challenging to interpret the multiplex cytokine data for COVID-19 patients. Also, it should be taken into consideration that biological and technical variables may affect the commutability of cytokine immunoassays and enhance complexity of cytokine immunoassay interpretation. It is recommended that the same method, platform and laboratory should be used when monitoring differences in cytokine levels between groups of individuals or for the same individual over time. It may be important to correlate cytokine profiling data with the SARS-CoV-2 nucleic acid amplification testing and imaging observations to make an accurate interpretation of the inflammatory status and disease progression in COVID-19 patients.

Role of Host Immune and Inflammatory Responses in COVID-19 Cases with Underlying Primary Immunodeficiency: A Review.

Coronavirus disease 2019 (COVID-19) has spread rapidly and become a pandemic. Caused by a novel human coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe COVID-19 is characterized by cytokine storm syndromes due to innate immune activation. Primary immunodeficiency (PID) cases represent a special patient population whose impaired immune system might make them susceptible to severe infections, posing a higher risk to COVID-19, but this could also lead to suppressed inflammatory responses and cytokine storm. It remains an open question as to whether the impaired immune system constitutes a predisposing or protective factor for PID patients when facing SARS-CoV-2 infection. After literature review, it was found that, similar to other patient populations with different comorbidities, PID patients may be susceptible to SARS-CoV-2 infection. Their varied immune status, however, may lead to different disease severity and outcomes after SARS-CoV-2 infection. PID patients with deficiency in antiviral innate immune signaling [eg, Toll-like receptor (TLR)3, TLR7, or interferon regulatory factor 7 (IRF7)] or interferon signaling (IFNAR2) may be linked to severe COVID-19. Because of its anti-infection, anti-inflammatory, and immunomodulatory effects, routine intravenous immunoglobulin therapy may provide some protective effects to the PID patients.

Frontline Science: Cxxc5 expression alters cell cycle and myeloid differentiation of mouse hematopoietic stem and progenitor cells.

CXXC5 is a member of the CXXC-type zinc finger epigenetic regulators. Various hematopoietic and nonhematopoietic roles have been assigned to CXXC5. In the present study, the role of Cxxc5 in myelopoiesis was studied using overexpression and short hairpin RNA-mediated knockdown in mouse early stem and progenitor cells defined as Lineage- Sca-1+ c-Kit+ (LSK) cells. Knockdown of Cxxc5 in mouse progenitor cells reduced monocyte and increased granulocyte development in ex vivo culture systems. In addition, ex vivo differentiation and proliferation experiments demonstrated that the expression of Cxxc5 affects the cell cycle in stem/progenitor cells and myeloid cells. Flow cytometry-based analyses revealed that down-regulation of Cxxc5 leads to an increase in the percentage of cells in the S phase, whereas overexpression results in a decrease in the percentage of cells in the S phase. Progenitor cells proliferate more after Cxxc5 knockdown, and RNA sequencing of LSK cells, and single-cell RNA sequencing of differentiating myeloid cells showed up-regulation of genes involved in the regulation of cell cycle after Cxxc5 knockdown. These results provide novel insights into the physiologic function of Cxxc5 during hematopoiesis, and demonstrate for the first time that it plays a role in monocyte development.

Fitness genes of group A streptococci in necrotizing fasciitis and myositis.

Necrotizing fasciitis and myositis caused by group A streptococci (GAS) are among the most fulminating infections, with a mortality rate of 20% to 30%. Although numerous regimens have been utilized in attempts to control these devastating infections, such as combinations of various antimicrobial agents and intravenous immunoglobulin (IVIG) as well as hyperbaric oxygen therapy, none have been the complete answer. Zhu and colleagues have utilized a transposon-directed insertion-site sequencing (TraDIS) protocol to identify 126 genes of M1 and 116 genes of M28 strains of GAS required for myositis, of which 25% encode transporters, which could be used as possible targets for future therapeutic protocols.

Specific Immunoglobulin (Ig) G Reference Intervals for Common Food, Insect, and Mold Allergens.

BACKGROUND: The clinical utility of serum IgG measurement in the diagnosis of allergy and food-induced hypersensitivity has been largely discredited. Recent studies, however, have shown that specific IgG can inhibit IgE mediated allergies, and may play a role in allergen specific desensitization. Accurate reference intervals for IgG specific allergens have not been widely established and are needed for better interpretation of serum antibody concentrations. In this study we established 64 IgG reference intervals for 48 common food allergens, 5 venoms, and 11 molds. DESIGN: Specific IgG concentrations were determined employing an automated fluorescent enzyme immunoassay on serum samples from 130 normal adults (65 males and 65 females), age range 18-69 y, mean 37.3 y. RESULTS: The lower reference interval limit for all allergens tested (n=64) was <2 mcg/mL. The median upper reference interval value for all 64 allergens was 12.9 mcg/mL, with Tuna (f40) having the lowest upper interval limit at 3.8 mcg/mL, and the mold Setomelanomma rostrate (m8) demonstrating the highest upper interval limit at 131 mcg/L. CONCLUSIONS: The considerable variation observed among the upper reference interval limits emphasizes the need for the establishment of allergen specific ranges for IgG. These newly established ranges should be a useful aid for clinicians in the interpretation of laboratory serum IgG results.

Detection of Acetylcholine Receptor Blocking Antibodies by Flow Cytometry.

OBJECTIVES: Detection of acetylcholine receptor (AChR) blocking antibodies through the use of a radiolabel has become standard procedure in most laboratories. Known drawbacks associated with radioassay, including cost of radioisotopes, hazards to laboratory professionals, and manufacture and disposal of radioactive materials, have prompted investigation into replacement assays. We describe here a high-throughput immunofluorescent flow cytometric assay designed for the detection of AChR blocking antibodies. METHODS: In total, 323 serum samples were tested on both the AChR blocking radioassay and the new immunofluorescent flow cytometric assay. RESULTS: Analysis of the results revealed a 96.9% concordance between the two assay methods. CONCLUSIONS: Our results indicate that a new immunofluorescent flow cytometric AChR blocking antibody assay is not only feasible but clinically comparable in both sensitivity (91%) and specificity (99%) compared with radioassay.

Fertility, pregnancies and outcomes reported by females with common variable immune deficiency and hypogammaglobulinemia: results from an internet-based survey.

BACKGROUND: Issues of fertility and pregnancy place an extra burden on females with primary immunodeficiencies. Patients lack reliable information and providers lack guidelines to counsel patients on these anxiety-provoking matters. OBJECTIVE: To collate concerns and experiences related to fertility and pregnancy from females with humoral immune deficiencies. METHODS: We conducted an internet-based survey of female patients who self-identified as having a diagnosis of primary humoral immune deficiency. RESULTS: Responses from 490 women with common variable immune deficiency and 100 with hypogammaglobulinemia were evaluated. The reported fertility measure (% of women who had had a birth) was statistically significantly lower as compared to the general US population (70 % vs. 85 %, p < 0.0001) whereas the rates of spontaneous pregnancy loss were comparable. This group reported a total of 966 pregnancies; 72 % resulted in a live birth. A majority of the pregnancies progressed with no incident and with continuation of their IgG replacement therapy; 23 % reported an increase in IgG dosing during pregnancy. Only 15 % of those reporting a first pregnancy indicated that they had been diagnosed with immune deficiency prior to their first pregnancy; these women expressed concern regarding the effect of immune deficiency on their fertility, pregnancy and decision to have children. CONCLUSION: With inherent limitations of self-reported responses to surveys, females with humoral immune deficiencies reported relatively good rates of fertility and pregnancies ending in live births. Results of the survey will serve as peer support for patients and inform counseling guidelines for providers.

Detection of acetylcholine receptor modulating antibodies by flow cytometry.

OBJECTIVES: To determine the clinical utility and performance characteristics of a laboratory-adapted flow cytometric method for the detection of acetylcholine receptor (AChR) modulating antibodies in myasthenia gravis (MG). METHODS: Serum samples from 120 healthy donors and 100 patients with suspected MG were assessed for the ability to reduce surface AChR concentrations (antigenic modulation) in RD (TE671) or DB40 human muscle cell lines by flow cytometry. Reference ranges were established by receiver operating characteristic curve analysis, and results were then compared with those of the current radioimmunoassay (RIA). RESULTS: Flow cytometric results from the RD cell line had an interpretive threshold of 46% modulation or greater and correlated best (98% sensitivity, 99% specificity) with those of the current RIA. CONCLUSIONS: The new flow cytometric method using the RD cell platform provided higher quality clinical results, a more robust and efficient assay format, a significant cost savings, and less environmental burden.

Blood pressure, salivary cortisol, and inflammatory cytokine outcomes in senior female cancer survivors enrolled in a tai chi chih randomized controlled trial.

PURPOSE: Older cancer survivors are a vulnerable population due to an increased risk for chronic diseases (e.g., cardiovascular disease) compounded with treatment late-effects and declines in physical functioning. Therefore, interventions that reduce chronic disease risk factors (i.e., blood pressure, chronic inflammation, and cortisol) are important in this population. Tai chi chih (TCC) is a mind-body exercise associated with reductions in chronic disease risk factors, but has not been examined with older cancer survivors. In a feasibility randomized controlled trial of TCC, we examined secondary outcomes of blood pressure, salivary cortisol, and inflammatory cytokines (interleukin (IL)-6, IL-12, tumor necrosis factor-α, IL-10, IL-4) due to their implications in chronic diseases. METHODS: Sixty-three senior female cancer survivors (M age = 67 years, SD = 7.15) with physical functioning limitations (SF-12 physical functioning ≤80 or role-physical ≤72) were randomized to 12-weeks (60-min, three times a week) of TCC or Health Education control (HEC) classes. Resting blood pressure, 1-day salivary cortisol samples, and fasting plasma samples for cytokine multiplex assays were collected at baseline and 1-week post-intervention. RESULTS: Controlling for baseline values, the TCC group had significantly lower systolic blood pressure (SBP, p = 0.002) and cortisol area-under-curve (AUC, p = 0.02) at post-intervention than the HEC group. There was no intervention effect on inflammatory cytokines (p's > 0.05). CONCLUSIONS: This TCC feasibility trial was associated with significant reductions in SBP and cortisol AUC in senior female cancer survivors. Larger, definitive trials are needed to confirm these findings. IMPLICATIONS FOR CANCER SURVIVORS: Senior survivors' have an increased risk for chronic diseases; however, TCC interventions may help reduce associated risk factors.

Identification of patients with RAG mutations previously diagnosed with common variable immunodeficiency disorders.

PURPOSE: Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients. METHODS: Two different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients. RESULTS: Two young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection. CONCLUSION: Astute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.