Objectives
To expand the knowledge about the immunological consequences of cladribine (CLAD), a pulsed immune reconstitution therapy approved for active multiple sclerosis (MS), beyond the known ...short‐term effects on peripheral immune cell subsets.
Methods
In this study, we characterized depletion and restitution kinetics as well as cytokine profiles of peripheral immune cell subsets in 18 patients with MS following treatment with oral CLAD. The methods involved blood collection prior to CLAD and every three months over a period of 24 months, and extensive characterization of various immune cells subsets by multiparametric flow cytometry.
Results
We found a selectivity of CLAD towards central memory T cells and memory B cells and detected a hyper‐repopulation of maturing B cells. Counts of classical (−65%) and various nonclassical TH17 cells (−84% to −87%) were markedly reduced 24 months after treatment start, and were comparable with depletion rates of class‐switched memory B‐cell phenotypes (−87% to −95%). The nadir of TH cells was more pronounced in the second treatment year. We observed a proportional surge of CD20 T‐cell subsets and an expansion of regulatory T, B and NK cells. Natural killer T cells (NKT) were only depleted in year two and did not recover.
Interpretation
Peripheral immune cell profiling revealed more differentiated insights into the immunological effects of CLAD. While some immune cell subsets expanded, we also observed additive depleting effects after the second treatment course. Further studies are required to elucidate whether these changes are paramount for the consistent and prolonged disease‐modifying effect of CLAD.
Objective
Repeated intravenous administration of anti‐CD20 depleting monoclonal antibodies 6 months apart is among the highly effective treatment options in multiple sclerosis (MS). Here, we aimed to ...investigate peripheral immune cell subset depletion kinetics following either rituximab (RTX) or ocrelizumab (OCR) infusions in people with MS (pwMS).
Methods
We studied pwMS treated de‐novo with either RTX (n = 7) or OCR (n = 8). The examinations were scheduled before the initiation of anti‐CD20 therapy and every 12 weeks for up to 15 months. Immunophenotyping of immune cell subsets in peripheral blood was performed by multiparametric fluorescence cytometry.
Results
A significant, persistent decrease of CD19+ B cells was observed already with the first anti‐CD20 infusion (p < 0.0001). A significant proportional reduction of memory B cells within the B‐cell pool was achieved only after two treatment cycles (p = 0.005). We observed a proportional increase of immature (p = 0.04) and naive B cells (p = 0.004), again only after the second treatment cycle. As for the peripheral T‐cell pool, we observed a continuous proportional increase of memory T helper (TH) cells/central memory TH cells (p = 0.02/p = 0.008), while the number of regulatory T cells (Treg) decreased (p = 0.007). The percentage of B‐cell dependent TH17.1 central memory cells dropped after the second treatment cycle (p = 0.02). No significant differences in the depletion kinetics between RTX and OCR were found.
Interpretation
Peripheral immune cell profiling revealed more differentiated insights into the prompt and delayed immunological effects of repeated intravenous anti‐CD20 treatment. The observation of proportional changes of some pathogenetically relevant immune cell subsets only after two infusion cycles deserves further attention.
Objective
To compare plasma levels of amino acids and clinical chemistry parameters in healthy infants at 1 and 4 months of age and to establish corresponding reference limits.
Methods
Data of three ...multicenter studies assessing the safety of new infant formulas were used. During these studies infants of both age-groups were either breast-fed or received formulas of low or high protein content. All samples were analyzed centrally in the same accredited laboratory.
Results
Plasma was collected from 521 infants in total, 157 boys and 135 girls aged 1 month and 121 boys and 108 girls aged 4 months. At the age of 1 month, 62 infants had received exclusively breast milk, 198 exclusively formula, and 27 both; in the 4-months age group corresponding numbers were 49, 158 and 18, respectively; for 9 infants, diet was unknown. Concentrations of most amino acids and clinical chemistry parameters differed significantly between both ages. Regardless of age, most plasma amino acid levels were comparable or lower in breast-fed than in formula-fed infants whereas at 1 month of age most clinical chemistry parameters were higher. While in breast-fed infants the plasma urea concentration decreased over 4 months of age, it increased in formula-fed infants. There were significant differences between infants fed a low and high protein formula. At both ages, high protein formulas resulted in significantly higher threonine, 2-aminobutyrate, and urea concentrations.
Conclusions
For clinical use, age- and diet specific reference limits in infants are warranted.
Underuse of laboratory testing has been previously investigated in preselected populations, such as documented malpractice claims. However, these numbers might not reflect real-life situations.
To ...evaluate the underuse and misuse of laboratory follow-up testing in a real-life hospital patient population with microcytic anemia, using laboratory results ordered during routine patient care.
From all patients in whom a microcytic anemia was detected during routine diagnostics in 2018, all available laboratory data were collected and screened for appropriateness of diagnostic workup of iron deficiency and thalassemia. Subgroup analysis was performed for patient groups with mean corpuscular volume values 75 to 79 μm3 (group 1), 65 to 74 μm3 (group 2), and <65 μm3 (group 3).
A total of 2244 patients with microcytic anemia were identified. Follow-up testing for iron deficiency was not performed in 761 cases (34%). For inconclusive ferritin levels due to elevated C-reactive protein results (n = 336), reticulocyte hemoglobin content or soluble transferrin receptor levels were missing in 86 cases (26%). In patients with suspected thalassemia (n = 127), follow-up testing for hemoglobin variants was not performed in 70 cases (55%). Subgroup analysis showed that the frequency of underuse of iron status as well as thalassemia/hemoglobinopathy testing decreased from group 1 to group 3. When considering relevant preexisting anemia diagnoses, laboratory tests were underused in 904 cases (40.3%).
Because 40% (n = 904) of the patients with microcytic anemia were potentially not followed up correctly, laboratory specialists are advised to act by implementing demand management strategies in collaboration with clinicians to overcome underuse of laboratory tests and to improve patient safety.
Background
Novel biomarkers representing different pathobiological pathways and their role in patients with acute myocardial infarction (AMI) were studied.
Methods
We retrospectively analysed serum ...levels of soluble suppression of tumorigenicity (sST2), growth‐differentiation factor‐15 (GDF‐15), soluble urokinase plasminogen activator receptor (suPAR), heart‐type fatty acid‐binding protein (H‐FABP) and plasma fetuin A in blood of patients with AMI (STEMI, n = 61; NSTEMI, n = 57) compared to controls with excluded coronary artery disease (n = 76). Furthermore, detailed correlation analysis was performed.
Results
Compared with controls, in patients with STEMI and NSTEMI higher levels expressed as median of sST2 in pg/mL (STEMI: 13210·9, NSTEMI: 11989·1, control: 5248; P < 0·001), GDF‐15 in pg/mL (STEMI: 818·8, NSTEMI 677·5, control 548·6; P < 0·001), suPAR in pg/mL (STEMI: 3461·1, NSTEMI: 3466·7, control: 2463·6; P < 0·001), H‐FABP in ng/mL (STEMI: 5·8, NSTEMI: 5·4, control: 0·0; P < 0·001) and lower plasma fetuin A levels in μg/mL (STEMI: 95, NSTEMI: 54, control: 116·6; P < 0·001) were detected. Correlation analysis found clinical and biochemical parameters such as ejection fraction, length of hospital stay, creatine kinase, NT‐proBNP and hs Troponin T levels as well as inflammatory markers (CRP, leucocytes) to be significantly correlated with novel biomarkers.
Conclusion
Plasma levels of novel biomarkers were significantly elevated (sST2, GDF‐15, H‐FABP, suPAR) or inversely downregulated (fetuin A) in patients with AMI compared to a control group with excluded coronary artery disease. Significant correlations with various clinical parameters and standard biochemical markers were found.
C-X-C chemokine ligand 13 (CXCL13) is frequently elevated in cerebrospinal fluid (CSF) in a variety of inflammatory central nervous system (CNS) diseases, has been detected in meningeal B cell ...aggregates in brain tissues of multiple sclerosis patients, and proposedly recruits B cells into the inflamed CNS. Besides B cells also follicular helper T (Tfh) cells express the cognate receptor C-X-C chemokine receptor type 5 (CXCR5) and follow CXCL13 gradients in lymphoid tissues. These highly specialized B cell helper T cells are indispensable for B cell responses to infection and vaccination and involved in autoimmune diseases. Phenotypically and functionally related circulating CXCR5+CD4 T cells occur in blood. Their co-recruitment to the inflamed CSF is feasible but unresolved.
We approached this question with a retrospective study including data of all patients between 2017 and 2019 of whom immune phenotyping data of CXCR5 expression and CSF CXCL13 concentrations were available. Discharge diagnoses and CSF laboratory parameters were retrieved from records. Patients were categorized as pyogenic/aseptic meningoencephalitis (ME, n = 29), neuroimmunological diseases (NIMM, n = 22), and non-inflammatory neurological diseases (NIND, n = 6). ANOVA models and Spearman's Rank-Order correlation were used for group comparisons and associations of CXCL13 levels with immune phenotyping data.
In fact, intrathecal CXCL13 elevations strongly correlated with CXCR5+CD4 T cell frequencies in the total cohort (p < 0.0001, r = 0.59), and ME (p = 0.003, r = 0.54) and NIMM (p = 0.043, r = 0.44) patients. Moreover, the ratio of CSF-to-peripheral blood (CSF/PB) frequencies of CXCR5+CD4 T cells strongly correlated with CXCL13 levels both in the total cohort (p = 0.001, r = 0.45) and ME subgroup (p = 0.005, r = 0.50), indicating selective accumulation. ME, NIMM and NIND groups differed with regard to CSF cell counts, albumin quotient, intrathecal IgG, CXCL13 elevations and CXCR5+CD4 T cells, which were higher in inflammatory subgroups.
The observed link between intrathecal CXCL13 elevations and CXCR5+CD4 T cell frequencies does not prove but suggests recruitment of possible professional B cell helpers to the inflamed CSF. This highlights CSF CXCR5+CD4 T cells a key target and potential missing link to the poorly understood phenomenon of intrathecal B cell and antibody responses with relevance for infection control, chronic inflammation and CNS autoimmunity.
The chemokine CXCL13 is an intensively investigated biomarker in Lyme neuroborreliosis (LNB). Its role in other neuroinfections is increasingly recognized but less clear.
To determine the ...significance of CXCL13 in established central nervous system (CNS) infections other than LNB by matching cerebrospinal fluid (CSF) CXCL13 elevations with severity of the disease course.
We investigated 26 patients with bacterial (n = 10) and viral (n = 16; tick-borne encephalitis, n = 6; varicella zoster infection, n = 10) neuroinfections of whom CSF CXCL13 levels were available twice, from lumbar punctures (LP) performed at admission and follow-up. As outcome classification, we dichotomized disease courses into "uncomplicated" (meningitis, monoradiculitis) and "complicated" (signs of CNS parenchymal involvement such as encephalitis, myelitis, abscesses, or vasculitis). CXCL13 elevations above 250 pg/ml were classified as highly elevated.
Eight of 26 patients (31%) with both bacterial (n = 4) and viral (n = 4) neuroinfections had a complicated disease course. All of them but only 3/18 patients (17%) with an uncomplicated disease course had CSF CXCL13 elevations > 250 pg/ml at the follow-up LP (p < 0.001). At admission, 4/8 patients (50%) with a complicated disease course and 3/18 patients (17%) with an uncomplicated disease course showed CXCL13 elevations > 250 pg/ml. All four patients with a complicated disease course but only one with an uncomplicated disease course had sustained CXCL13 elevations at follow-up. Patient groups did not differ with regard to age, time since symptom onset, LP intervals, type of infections, and anti-pathogen treatments.
Our study revealed pronounced CXCL13 elevations in CSF of patients with severe disease courses of bacterial and viral neuroinfections. This observation indicates a role of CXCL13 in the CNS immune defense and points at an additional diagnostic value as biomarker for unresolved immune processes leading to or associated with complications.
Elevation of the chemokine CXCL13 in CSF frequently occurs during active and acute CNS inflammatory processes and presumably is associated with B cell-related immune activation. Elevation levels, ...however, vary a lot and "leaking" of CXCL13 from blood across dysfunctional brain barriers is a possible source. The aim was to clarify the relation between CXCL13 concentrations in CSF, CXCL13 concentrations in serum and blood-CSF barrier (BCSFB) function for a correct interpretation of the intrathecal origin of CXCL13.
We retrospectively analyzed CXCL13 of banked CSF/serum samples (n = 69) selected from patient records and categorized the CSF CXCL13 elevations as CXCL13 negative (< 30 pg/ml), low (30-100 pg/ml), medium (101-250 pg/ml), or high (> 250 pg/ml). CXCL13 concentrations in CSF and serum and the corresponding CSF/serum CXCL13 quotients (Qcxcl13) were compared to CSF/serum albumin quotients (QAlb) as a measure for BCSFB function. The CXCL13 negative category included two subgroups with normal and dysfunctional BCSFB.
Serum CXCL13 concentrations were similar across categories with median levels around 100 pg/ml but differed between individuals (29 to > 505 pg/ml). Despite clear evidence in serum, CXCL13 was detectable only at trace amounts (medians 3.5 and 7.5 pg/ml) in CSF of the two CXCL13 negative subgroups irrespective of a normal or pathological QAlb. Moreover, we found no association between CSF and serum CXCL13 levels or between QAlb and CSF CXCL13 levels in any of the CSF CXCL13-delineated categories. CXCL13 apparently does not "leak" from blood into CSF. This implies an intrathecal origin also for low CSF CXCL13 levels and a caveat for analyzing the Qcxcl13, because higher serum than CSF concentrations arithmetically depress the Qcxcl13 resulting in misleadingly low CSF/serum quotients.
We demonstrated that CXCL13 does not cross from blood into CSF, not even during severe BCSFB dysfunction. CSF CXCL13 elevations therefore most likely always are CNS-derived, which highlights their relevance as indicator of inflammatory CNS processes. We recommend data should not be corrected for BCSFB permeability (QAlb) and not to calculate CSF/serum quotients for CXCL13 as these may introduce error.
Background:
Anti-CD20 therapies induce pronounced B-cell depletion and blunt humoral responses to vaccines. Recovery kinetics of anti-CD20 therapy-mediated cellular and humoral effects in people with ...multiple sclerosis (pwMS) are poorly defined.
Objective:
To investigate the duration of the anti-CD20 treatment-induced effects on humoral responses to COVID-19 vaccines.
Methods:
This retrospective observational study included pwMS who had discontinued anti-CD20 therapy for ⩾12 months and remained without immunomodulation. We retrieved demographics and laboratory parameters including B-cell counts and immunoglobulin (IgG, IgM, IgA) levels prior to anti-CD20 commencement (baseline) and longitudinally after anti-CD20 treatment discontinuation from electronic medical records. Humoral responses to SARS-CoV-2 vaccines were compared with a population of 11 pwMS with ongoing anti-CD20 medication (control cohort).
Results:
A total of 24 pwMS had discontinued anti-CD20 therapy for a median of 34 months (range: 16–38 months). Antibody responses to COVID-19 vaccines were available in 17 (71%). Most individuals (n = 15, 88%) elicited a measurable antibody response mean: 774 BAU/ml (±SD 1283 BAU/ml) to SARS-CoV-2 immunization on average 22 months (range: 10–30 months) from the last anti-CD20 infusion, which was higher compared with the population with ongoing anti-CD20 therapy (n = 11, mean: 12.36 ± SD 11.94 BAU/ml; p < 0.00001). Significantly increased antibody levels compared with the control cohort were found among pwMS who were vaccinated >18 months after treatment discontinuation (19–24 months: n = 2, p = 0.013; 25–36 months: n = 9; p < 0.001). The interindividual kinetics for B-cell reconstitution were heterogeneous and mean B-cell counts approached normal ranges 18 months after treatment discontinuation. There was no correlation of B-cell repopulation and vaccine responses. Mean total IgG, IgM, and IgA levels remained within the reference range.
Conclusion:
Anti-CD20-induced inhibition of humoral responses to COVID-19 vaccines is transient and antibody production was more pronounced >18 months after anti-CD20 treatment discontinuation. The immunological effect on B-cell counts appears to wane by the same time.
Cladribine (CLAD) is a deoxyadenosine analogue prodrug which is given in multiple sclerosis (MS) as two short oral treatment courses 12 months apart. Reconstitution of adaptive immune function ...following selective immune cell depletion is the presumed mode of action. In this exploratory study, we investigated the impact of CLAD tablets on immune cell surface molecules for adhesion (CAMs) and costimulation (CoSs) in people with MS (pwMS). We studied 18 pwMS who started treatment with CLAD and 10 healthy controls (HCs). Peripheral blood mononuclear cells were collected at baseline and every 3 months throughout a 24-month period. We analysed ICAM-1, LFA-1, CD28, HLADR, CD154, CD44, VLA-4 (CD49d/CD29), PSGL-1 and PD-1 with regard to their expression on B and T cells (T helper (Th) and cytotoxic T cells (cT)) and surface density (mean fluorescence intensity, MFI) by flow cytometry. The targeted analysis of CAM and CoS on the surface of immune cells in pwMS revealed a higher percentage of ICAM-1 (B cells, Th, cT), LFA-1 (B cells, cT), HLADR (B cells, cT), CD28 (cT) and CD154 (Th). In pwMS, we found lower frequencies of Th and cT cells expressing PSGL-1 and B cells for the inhibitory signal PD-1, whereas the surface expression of LFA-1 on cT and of HLADR on B cells was denser. Twenty-four months after the first CLAD cycle, the frequencies of B cells expressing CD44, CD29 and CD49d were lower compared with the baseline, together with decreased densities of ICAM-1, CD44 and HLADR. The rate of CD154 expressing Th cells dropped at 12 months. For cT, no changes were seen for frequency or density. Immune reconstitution by oral CLAD was associated with modification of the pro-migratory and -inflammatory surface patterns of CAMs and CoSs in immune cell subsets. This observation pertains primarily to B cells, which are key cells underlying MS pathogenesis.
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