Long-Term Humoral Immune Response against SARS-CoV-2 after Natural Infection and Subsequent Vaccination According to WHO International Binding Antibody Units (BAU/mL).

The new WHO reference standard allows for the definition of serum antibodies against various SARS-CoV-2 antigens in terms of binding antibody units (BAU/mL) and thus to compare the results of different ELISA systems. In this study, the concentration of antibodies (ABs) against both the S- and the N-protein of SARS-CoV-2 as well as serum neutralization activity were evaluated in three patients after a mild course of COVID-19. Serum samples were collected frequently during a period of over one year. Furthermore, in two individuals, the effects of an additional vaccination with a mRNA vaccine containing the S1-RBD sequence on these antibodies were examined. After natural infection, the antibodies (IgA, IgG) against the S1-protein remained elevated above the established cut-off to positivity (S-IgA 60 BAU/mL and S-IgG 50 BAU/mL, respectively) for over a year in all patients, while this was not the case for ABs against the N-protein (cut-off N-IgG 40 BAU/mL, N-IgA 256 BAU/mL). Sera from all patients retained the ability to neutralize SARS-CoV-2 for more than a year. Vaccination resulted in a rapid boost of antibodies to S1-protein but, as expected, not to the N-protein. Most likely, the wide use of the WHO reference preparation will be very useful in determining the individual immune status of patients after an infection with SARS-CoV-2 or after vaccination.

Progranulin signaling in sepsis, community-acquired bacterial pneumonia and COVID-19: a comparative, observational study.

BACKGROUND: Progranulin is a widely expressed pleiotropic growth factor with a central regulatory effect during the early immune response in sepsis. Progranulin signaling has not been systematically studied and compared between sepsis, community-acquired pneumonia (CAP), COVID-19 pneumonia and a sterile systemic inflammatory response (SIRS). We delineated molecular networks of progranulin signaling by next-generation sequencing (NGS), determined progranulin plasma concentrations and quantified the diagnostic performance of progranulin to differentiate between the above-mentioned disorders using the established biomarkers procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) for comparison. METHODS: The diagnostic performance of progranulin was operationalized by calculating AUC and ROC statistics for progranulin and established biomarkers in 241 patients with sepsis, 182 patients with SIRS, 53 patients with CAP, 22 patients with COVID-19 pneumonia and 53 healthy volunteers. miRNAs and mRNAs in blood cells from sepsis patients (n = 7) were characterized by NGS and validated by RT-qPCR in an independent cohort (n = 39) to identify canonical gene networks associated with upregulated progranulin at sepsis onset. RESULTS: Plasma concentrations of progranulin (ELISA) in patients with sepsis were 57.5 (42.8-84.9, Q25-Q75) ng/ml and significantly higher than in CAP (38.0, 33.5-41.0 ng/ml, p < 0.001), SIRS (29.0, 25.0-35.0 ng/ml, p < 0.001) and the healthy state (28.7, 25.5-31.7 ng/ml, p < 0.001). Patients with COVID-19 had significantly higher progranulin concentrations than patients with CAP (67.6, 56.6-96.0 vs. 38.0, 33.5-41.0 ng/ml, p < 0.001). The diagnostic performance of progranulin for the differentiation between sepsis vs. SIRS (n = 423) was comparable to that of procalcitonin. AUC was 0.90 (95% CI = 0.87-0.93) for progranulin and 0.92 (CI = 0.88-0.96, p = 0.323) for procalcitonin. Progranulin showed high discriminative power to differentiate bacterial CAP from COVID-19 (sensitivity 0.91, specificity 0.94, AUC 0.91 (CI = 0.8-1.0) and performed significantly better than PCT, IL-6 and CRP. NGS and partial RT-qPCR confirmation revealed a transcriptomic network of immune cells with upregulated progranulin and sortilin transcripts as well as toll-like-receptor 4 and tumor-protein 53, regulated by miR-16 and others. CONCLUSIONS: Progranulin signaling is elevated during the early antimicrobial response in sepsis and differs significantly between sepsis, CAP, COVID-19 and SIRS. This suggests that progranulin may serve as a novel indicator for the differentiation between these disorders. TRIAL REGISTRATION: Clinicaltrials.gov registration number NCT03280576 Registered November 19, 2015.

Persisting Neutralizing Activity to SARS-CoV-2 over Months in Sera of COVID-19 Patients.

The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein, as well as the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted COVID-19 patients, needs further longitudinal investigation. Several new serological methods to examine these parameters were developed, validated and applied in three patients of a family which underwent an ambulatory course of COVID-19 for six months. The virus load had almost completely disappeared after about four weeks. Serum IgA levels to the S1-RBD-protein and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau at approximately six weeks, and stayed elevated over the observation period. Virus-neutralizing activity reached a peak about 4 weeks after disease onset but dropped slowly. The longitudinal associations of virus neutralization and the serological immune response suggest immunity in patients even after a mild clinical course of COVID-19.

Relative leptin deficiency in children with severe early-onset obesity (SEOO) - results of the Early-onset Obesity and Leptin - German-Polish Study (EOL-GPS).

Background Severe early-onset obesity (SEOO) in children is a common feature of monogenic obesity. Gene defects of the leptin-melanocortin pathway can be analysed biochemically and genetically. The aim of this study was to search for children with leptin deficiency or biologically inactive leptin in a cohort of children with SEOO and to study associations between leptin parameters and anthropometric data. Methods The cohort included n = 50 children with SEOO (22 boys) who were recruited at one of four study centres (Germany: Ulm; Poland: Katowice, Szczecin, Rzeszow) between October 2015 and October 2017. Weight (kg) and height (m) were measured, Tanner stage was obtained and a fasting serum blood sample was taken. Serum levels of total leptin (LEP, ng/mL), biologically active leptin (bioLEP, ng/mL) and soluble leptin receptor (sLEPR, ng/mL) were measured. The body mass index (BMI [kg/m2]), BMI z-score (World Health Organization [WHO]), quotient of bioLEP/LEP and leptin-standard deviation score (LEP-SDS) (Tanner stage, BMI and sex-adjusted) were calculated. Results We did not find any child with leptin deficiency or biologically inactive leptin in our cohort. The serum LEP and bioLEP levels were strongly correlated with age (r = 0.50, p < 0.05) and BMI (r = 0.70; p < 0.0001). Girls had higher LEP and bioLEP levels (49.7 ± 35.9 vs. 37.1 ± 25.5 ng/mL, p > 0.05) as well as lower LEP-SDS than boys (-1.77 ± 2.61 vs. -1.40 ± 2.60, p > 0.05). sLEPR levels were negatively correlated with BMI values (r = -0.44; p < 0.05), LEP (r = -0.39; p < 0.05) and bioLEP levels (r = -0.37; p < 0.05). Interestingly, there was a strong inverse relationship between LEP-SDS and BMI (r = -0.72, p < 0.001). Conclusions In this cohort with SEOO, we identified no new cases of children with leptin deficiency or bioinactive leptin. A strong negative correlation between the LEP-SDS and BMI values could be interpreted as relative leptin deficiency in children with SEOO. In case this hypothesis can be confirmed, these children would benefit from a substitution therapy with methionyl human leptin (metreleptin™).

Measurement of immunofunctional leptin to detect and monitor patients with functional leptin deficiency.

CONTEXT AND AIMS: Functional leptin deficiency is characterized by high levels of circulating immunoreactive leptin (irLep), but a reduced bioactivity of the hormone due to defective receptor binding. As a result of the fact that affected patients can be successfully treated with metreleptin, it was aimed to develop and validate a diagnostic tool to detect functional leptin deficiency. METHODS: An immunoassay capable of recognizing the functionally relevant receptor-binding complex with leptin was developed (bioLep). The analytical quality of bioLep was validated and compared to a conventional assay for immune-reactive leptin (irLep). Its clinical relevance was evaluated in a cohort of lean and obese children and adults as well as in children diagnosed with functional leptin deficiency and their parents. RESULTS: In the clinical cohort, a bioLep/irLep ratio of 1.07 (range: 0.80-1.41) was observed. Serum of patients with non-functional leptin due to homozygous amino acid exchanges (D100Y or N103K) revealed high irLep but non-detectable bioLep levels. Upon treatment of these patients with metreleptin, irLep levels decreased, whereas levels of bioLep increased continuously. In patient relatives with heterozygous amino acid exchanges, a bioLep/irLep ratio of 0.52 (range: 0.48-0.55) being distinct from normal was observed. CONCLUSIONS: The new bioLep assay is able to diagnose impaired leptin bioactivity in severely obese patients with a homozygous gene defect and in heterozygous carriers of such mutations. The assay serves as a diagnostic tool to monitor leptin bioactivity during treatment of these patients.

The biometric measurement of alcohol consumption.

BACKGROUND: Proper ascertainment of the history of alcohol consumption by an individual is an important component of medical diagnosis of disease and influences the implementation of appropriate treatment strategies that include prescription of medication, as well as intervention for the negative physical and social consequences of hazardous/harmful levels of alcohol consumption. Biological (biometric) diagnostic tests that provide information on current and past quantity and frequency of alcohol consumption by an individual, prior to onset of organ damage, continue to be sought. METHODS: Platelet monoamine oxidase B (MAO-B) protein was quantitated in 2 populations of subjects who had histories of different levels of alcohol consumption. Levels were assayed by immunoblotting or by ELISA. The development and evaluation of the new ELISA-based measure of platelet MAO-B protein levels is described. RESULTS: One subject population constituted a nontreatment-seeking, cross-sectional subject sample, and the other population was a longitudinally followed, hospitalized group of subjects. An algorithm combining measures of platelet MAO-B protein with the plasma levels of carbohydrate-deficient transferrin (CDT) and with liver enzymes (aspartate aminotransferase or γ-glutamyltransferase [GGT]) can detect hazardous/harmful alcohol use (HHAU) with the highest sensitivity and specificity in the cross-sectional nontreatment-seeking population. In the treatment-seeking population, low MAO-B protein levels at admission are associated with heavy drinking prior to admission, and these protein levels increase over a period of abstinence from alcohol. CONCLUSIONS: The platelet MAO-B protein measurement is particularly effective for male alcohol consumers. The combined use of MAO-B protein measures together with measures of CDT and GGT does, however, improve the diagnostic utility of both markers for ascertaining HHAU in women. Furthermore, measurement of changes in platelet MAO-B protein levels during treatment for alcohol dependence may help monitor the success of the treatment program.

Detection of myelin autoantibodies: evaluation of an assay system for diagnosis of multiple sclerosis in differentiation from other central nervous system diseases.

BACKGROUND: Multiple sclerosis (MS) is a frequent and often severe autoimmune disease of the central nervous system. We describe a newly developed enzyme-linked immunosorbent assay (ELISA)-based test system for the assessment of neuronal autoantibodies in serum and cerebrospinal fluid (CSF). This tool could help define autoimmune status and thus be a potential means of therapeutic surveillance. METHODS: We used an assay system (ELISA, E100, Mediagnost) based on purified bovine antigens [myelin-oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipid protein (PLP) and alpha-B-crystalline (CRY)] antibodies for the measurement of specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies. Assay characteristics and preliminary validation were conducted by measurement of serum and CSF samples from 41 MS patients and 128 patients with other neurological diseases (OND). RESULTS: We measured the inter- (17.8/10.1%) and intra-assay variability (5.5/6.7%); linearity (1:250- 1:16,000), and specificity of IgG and IgM. We demonstrate that by the results of this test system MS patients can be differentiated from patients with OND. CONCLUSIONS: The ELISA kit we evaluated is suitable for the measurement of neuronal autoantibodies. The initial validation demonstrates its potential use in the differential diagnosis of central neuronal system diseases.

Alcoholism, craving, and hormones: the role of leptin, ghrelin, prolactin, and the pro-opiomelanocortin system in modulating ethanol intake.

Evidence is growing that appetite regulating peptides such as leptin and ghrelin, but also other hormones including prolactin are altered in alcoholism. The brain pro-opiomelanocortin (POMC) system which has important mediating roles in alcohol intake also has important functions in prolactin regulation and energy homeostasis. Furthermore, it has been demonstrated to be functionally integrated with leptin regulation. The satiety factor leptin seems to be counteracted by the gut-derived peptide ghrelin which increases hunger and food intake. Consequently, the POMC system may have a role in integrating regulation of alcohol effects and these seemingly disparate regulatory systems. The goal of this mini-review is to discuss the results of some recent investigations of the potential interactions of these systems with acute and chronic alcohol responses.

Gender differences for ghrelin levels in alcohol-dependent patients and differences between alcoholics and healthy controls.

BACKGROUND: Ghrelin is a 28-amino acid gut-brain peptide, mainly secreted by the gastric mucosa. Its effects are linked to energy homeostasis and particularly seem to increase hunger and food intake. In recent years, studies suggested that appetite-regulating peptides, such as ghrelin play a relevant role in alcoholism. Since data published to date on the potential role of ghrelin as state and/or trait marker in alcoholism and the association with craving are controversial, we aimed at further elucidating these aspects. PATIENTS AND METHODS: One-hundred nine alcohol-dependent abstinent patients after withdrawal (27 f, 82 m), (ICD 10 F 10.25) and 45 healthy volunteers (12 f, 33 m) were included. Laboratory testing (Ghrelin RIA 90, Mediagnost Inc., Germany) was performed and several craving scales [Obsessive Compulsive Drinking Scale, Alcohol Urge Questionnaire and Visual Analogue Scale (VAS)] were applied at the beginning and at the end of the 3-week rehabilitation program. RESULTS: (1) Ghrelin levels are significantly higher in female alcohol-dependent patients as compared to controls, not, however, in men alcoholics. (2) In several statistical subanalyses, an association of craving and ghrelin was found. The results, however, remain heterogeneous. CONCLUSION: The data suggest gender-dependent ghrelin levels in alcohol-dependent patients. We therefore conclude, that it might be useful to perform statistical analyses gender-specific. With regard to a potential correlation of ghrelin and craving the results seem to depend on gender, duration of the abstinence period and the instrument used.

No significant effect of acute moderate alcohol intake on leptin levels in healthy male volunteers.

As, for ethical reasons, it is difficult to investigate by an experiment the effect of acute intoxication on leptin levels in alcoholics, we tested the hypothesis of lowered levels as an effect of acute ethanol intake in healthy volunteers. The subjects comprised (1) 17 healthy male participants, recruited via newspaper advertisements [age 29+/-3.75 years, body mass index (BMI) 24.3+/-3.5, leptin at baseline 3.3+/-3.1 ng/ml]; (2) for comparison, leptin levels of 16 male alcoholic patients at day 1 of withdrawal were used. They were characterized as follows: (mean, median, standard deviation and range) age in years (41.1, 40.5, 10.2, 24, 57), BMI (23.3, 21.7, 5.4, 16.6, 37.5), 1,032 g of ethanol (median) consumed within the last 7 days, leptin levels 2.3 mg/ml. A placebo-controlled double-blind trial was performed. Leptin levels of blood samples were taken at baseline (t(1)), before ethanol intake (t(2)), when blood alcohol had reached its maximum (t(3)) and the morning after (t(4)). The oral dose of ethanol administered was 0.6 g/kg ethanol. (1) VOLUNTEERS: (a) the ethanol and placebo group exhibited leptin levels corresponding closely with levels measured at baseline (t(1)) (rs=0.85, p<0.0001) and follow-up (t(4)) (rs=0.768, p<0.0001). (b) Leptin levels for the placebo and the alcohol-consuming (verum) group did not differ significantly at baseline, after ethanol intake or on the morning after [Mann-Whitney U-test (p=0.669, p=1.0 and p=0.887, respectively)]. (2) Leptin levels in relation to BMI did not significantly differ at any measurement time in either group. (3) Leptin levels/BMI of the healthy volunteers at t(1) and t(4) were not significantly different from those of 16 alcoholics. The data do not support the hypothesis of a significant effect of acute moderate alcohol intake on leptin levels in healthy volunteers.

Leptin levels of alcohol abstainers and detoxification patients are not different.

AIMS: Leptin is a cytokine-type peptide hormone, recently implicated as a putative state marker of alcohol use and in craving. Our goal was to evaluate the potential of leptin as a state and trait marker and to rule out the role of current alcohol intoxication on leptin levels. METHODS: Eighteen alcohol withdrawal patients (16 males, 2 females) whose blood contained 202 mg/dl (median) of ethanol at hospitalization, who had a median age of 43.5 years and had consumed 1075 g of ethanol (median) in the last 7 days were included in the study. Leptin was determined in samples at day 1 (when still intoxicated) and day 7 of withdrawal. Expected leptin levels were calculated with a formula. For comparison, 27 blood samples of 18 abstinent persons, matched for gender, age and body mass index were used. Furthermore, mean cell volume, gamma-glutamyl transferase (GGT), blood glucose, cholesterol, triglycerides and body composition (bioimpedance device) were determined. For statistical analysis, SPSS 11 was used. RESULTS: Expected leptin levels were 1.71 ng/ml (median), leptin measured at day 1 was 2.65 ng/ml (median) and 2.85 ng/ml on day 7 for the alcohol withdrawal patients and 2.2 ng/ml (median) for the abstainers. These concentrations were not significantly different. Significant correlations were found between leptin day 1 and expected leptin levels, percentage fat body mass, cigarettes smoked per day, GGT and blood alcohol concentration. CONCLUSIONS: Our preliminary data do not support the hypothesis of leptin as a state or trait marker and suggest only a minor influence of acute intoxication on leptin levels in alcohol detoxification patients.