Association of Clinical Relapses With Disease Outcomes in Multiple Sclerosis Patients Older Than 50 Years.

BACKGROUND AND OBJECTIVES: Relapse and MRI activity usually decline with aging but are replaced by progression independent of relapse activity (PIRA) in patients with multiple sclerosis (PwMS). However, several older PwMS continue to experience clinical relapses, and the impact on their disease remains undetermined. We aimed to determine the impact of an index relapse on disease outcomes in patients older than 50 years and to identify risk factors of disadvantageous outcomes. METHODS: We performed a secondary analysis from 3 prospective cohorts in Germany. We evaluated all PwMS 50 years and older with a relapse ≤60 days before a baseline visit and at least 18 months of follow-up compared with a control cohort of PwMS without a relapse. Patients were stratified according to age ("50-54" vs "55-59" vs "60+") or disease outcomes ("stable" vs "active" vs "progressive," according to the Lublin criteria). We analyzed relapses, MRI activity, relapse-associated worsening, and PIRA. Regression analysis was performed to evaluate the association of specific baseline risk factors and treatment regimen changes with disease outcomes at month 18. RESULTS: A total of 681 patients were included in the "relapse cohort" (50+: 361; 55+: 220; 60+: 100). The "control cohort" comprised 232 patients (50+: 117; 55+: 71; 60+: 44). Baseline epidemiologic parameters were balanced among cohorts and subgroups. We observed increased abundance of inflammatory activity and relapse-independent disability progression in the "relapse" vs "control" cohort. In the "relapse" cohort, we identified 273 patients as "stable" (59.7%), 114 patients as "active" (24.9%), and 70 patients as "progressive" (15.3%) during follow-up. Cardiovascular risk factors (CVRFs) and older age at baseline were identified as risk factors of progressive, whereas disease-modifying treatment (DMT) administration at baseline favored stable disease. DMT during follow-up was associated with stable over active, but not over progressive disease. DISCUSSION: A relapse-suggesting underlying active disease-in PwMS older than 50 years was associated with continued disease activity and increased risk of PIRA. Presence of CVRF and absence of DMT at baseline appeared as risk factors of disadvantageous disease courses. An escalation of DMT switch was associated with stable over active but not progressive disease.

Immune Response to Seasonal Influenza Vaccination in Multiple Sclerosis Patients Receiving Cladribine.

Cladribine has been approved for the treatment of multiple sclerosis (MS) and its administration results in a long-lasting depletion of lymphocytes. As lymphopenia is known to hamper immune responses to vaccination, we evaluated the immunogenicity of the influenza vaccine in patients undergoing cladribine treatment at different stages vs. controls. The antibody response in 90 cladribine-treated MS patients was prospectively compared with 10 control subjects receiving platform immunotherapy (NCT05019248). Serum samples were collected before and six months after vaccination. Response to vaccination was determined by the hemagglutination-inhibition test. Postvaccination seroprotection rates against influenza A were comparable in cladribine-treated patients and controls (H1N1: 94.4% vs. 100%; H3N2: 92.2% vs. 90.0%). Influenza B response was lower in the cladribine cohort (61.1% vs. 80%). The increase in geometric mean titers was lower in the cladribine group vs. controls (H1N1: +98.5 vs. +188.1; H3N2: +225.3 vs. +300.0; influenza B: +40.0 vs. +78.4); however, titers increased in both groups for all strains. Seroprotection was achieved irrespective of vaccination timing and lymphocyte subset counts at the time of vaccination in the cladribine cohort. To conclude, cladribine-treated MS patients can mount an adequate immune response to influenza independently of treatment duration and time interval to the last cladribine administration.

Immunological consequences of cladribine treatment in multiple sclerosis: A real-world study.

BACKGROUND: Cladribine is a synthetic deoxyadenosine analogue approved for the treatment of highly active relapsing multiple sclerosis (RMS). Cladribine is considered to be a semi-selective immune-reconstitution therapy (IRT) that induces long-term remission following short course of treatment. Here, we evaluated the effect of cladribine on immune cell reduction and reconstitution during the first two years of treatment. METHODS: We analyzed our longitudinal, prospective, real-world cohort of 80 cladribine-treated RMS patients from two tertiary centers in Germany. Laboratory testing was conducted monthly and included evaluation of cellular as well as soluble parameters. Laboratory outcomes were correlated with infectious adverse events (AEs) and clinical or paraclinical disease activity. RESULTS: Selective alterations in immune cell populations occurred following cladribine treatment, with the most marked effects observed in year two of treatment. Specifically, a rapid reduction in CD56+ natural killer cells (nadir: month 1 (year 1) and 14 (year 2); -37 and -41% from baseline) was followed by a greater reduction in CD19+ B cells (nadir: month 2 and 14; -81 and -82%); a moderate effect on CD4+ (nadir: month 3 and 15; -48 and -61%) and CD8+ T cells (nadir: month 5 and 18; -40 and -48%). Despite the marked effect on B cells, immunoglobulin levels were unaffected. There was no or minimal effect on thrombocytes and innate immune cells. Clinical and paraclinical disease activity was unrelated to the observed immune alterations. Lymphopenia was the most commonly observed AE (86.3% of patients; grade III-IV lymphopenia: 38.8%). The cumulative incidence of infections was 55% with cladribine treatment, which were mostly mild or moderate. In total, 19 herpes infections developed in 8 (10%) cladribine-treated patients; all cases were dermatomal and 94.7% of the herpetic infections occurred during a period of lymphopenia. CONCLUSIONS: The immunophenotyping data obtained in our real-world setting are comparable to those demonstrated in pivotal clinical trials and provide further evidence that cladribine may represent a form of IRT. However, regarding the side-effect profile of cladribine, severe lymphopenia (exceeding grade II CTCAE) was more frequent, which may have prompted the development of herpes infections. Of note, lymphocyte dynamics did not correlate with clinical and paraclinical measures of disease activity in the two-year follow-up period.

Cognitive Impairment in Adult Patients with 5q-Associated Spinal Muscular Atrophy.

In previous studies, a below-average, average, or above-average intelligence quotient (IQ) in children with SMA was detected but, aside from a severe physical disability, the cognitive performance of adult SMA patients has not yet been evaluated. The intelligence test used in this study, the Wechsler Adult Intelligence Scale, fourth edition (WAIS-IV), was used to measure major intelligence components of adult SMA patients. The WAIS-IV determines four index scores representing verbal comprehension, perceptual reasoning, working memory, and processing speed. Due to time-dependent demands on motor function, the processing speed index score was excluded. IQ index scores of 33 adult SMA patients did not differ from IQ index scores of the normal population. In SMA type-3 patients, the index scores for verbal comprehension, perceptual reasoning, and working memory did not differ from the normal population but showed a trend of IQ scores towards lower points. Patients with SMA type 2 had lower IQ index scores for working memory (90.33 ± 12.95; p = 0.012) and perceptual reasoning (90.73 ± 12.58; p = 0.013) than the normal population. This study provided further evidence that SMA is a multi-systemic disease and may refute the widespread hypothesis that SMA patients might improve their cognitive skills to compensate for their physical impairment.

Differences in Cell Cycle Status Underlie Transcriptional Heterogeneity in the HSC Compartment.

Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftment potential in comparison with RA-CFP-bright HSCs. Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA in vitro. Single-cell RNA sequencing (RNA-seq) of >1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs.

The Anaphylatoxin C3a Receptor Expression on Human M2 Macrophages Is Down-Regulated by Stimulating the Histamine H4 Receptor and the IL-4 Receptor.

The anaphylatoxin C3a triggers inflammation by binding to its specific G-protein-coupled C3a receptor (C3aR). Since the number of C3aR, which is expressed on the cell surface, affects the response to C3a, we investigated the expression levels of C3aR on human M2 macrophages in allergic situations where high levels of the Th2 cytokine IL-4 and histamine are present in a local microenvironment. The histamine H1 receptor (H1R), H2R and the H4R mRNA expressions were induced or up-regulated during the differentiation process of M2 macrophages. The presence of histamine or agonists targeting the H1R, H2R and, in particular, the H4R during in vitro differentiation from monocytes to macrophages modified the M2 phenotype by regulating the macrophage differentiation marker CD68 and CD163 expressions. In -addition, the C3aR expression was also down-regulated by -ST-1006 during this process. Histamine and ST-1006 down-regulated the expression of C3aR with different time kinetics on fully differentiated M2 macrophages. By analysing C3a-induced IL-6 mRNA expression, we observed a diminished response to C3a in ST-1006-treated M2 macrophages when compared to un-treated cells. Expression of C3 was not affected by histamine, whereas IL-4 strongly down-regulated C3aR and C3 expressions. Our data suggests that down-regulation of C3aR expression by mediators present in allergic situations such as IL-4 or histamine has an anti-inflammatory impact by reducing the sensitivity to C3a-induced down-stream signaling, thereby contributing to the regulation of local inflammatory responses in the skin.

DNA Methylation Levels of the ELMO Gene Promoter CpG Islands in Human Glioblastomas.

Complete surgical resection of glioblastoma is difficult due to the invasive nature of this primary brain tumor, for which the molecular mechanisms behind remain poorly understood. The three human ELMO genes play key roles in cellular motility, and have been linked to metastasis and poor prognosis in other cancer types. The aim of this study was to investigate methylation levels of the ELMO genes and their correlation to clinical characteristics and outcome in patients diagnosed with glioblastoma. To measure DNA methylation levels we designed pyrosequencing assays targeting the promoter CpG island of each the ELMO genes. These were applied to diagnostic tumor specimens from a well-characterized cohort of 121 patients who received standard treatment consisting of surgery, radiation therapy, plus concomitant and adjuvant chemotherapy. The promoter methylation levels of ELMO1 and ELMO2 were generally low, whereas ELMO3 methylation levels were high, in the tumor biopsies. Thirteen, six, and 18 biopsies were defined as aberrantly methylated for ELMO1, ELMO2, and ELMO3, respectively. There were no significant associations between the methylation status of any of the ELMO gene promoter CpG islands and overall survival, progression-free survival, and clinical characteristics of the patients including intracranial tumor location. Therefore, the methylation status of the ELMO gene promoter CpG islands is unlikely to have prognostic value in glioblastoma.

P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts.

The ionotropic ATP-gated P2X7 receptor (P2X7R) is involved in the regulation of many physiological functions including bone metabolism. Several studies on osteoblasts from rodents and human osteoblast-like cell lines have addressed the expression and function of P2X7R on these bone-forming cells however; its role in human primary osteoblasts has not yet been reported. The aim of this study was to assess the expression of the P2X7R in bone marrow-derived stromal cells and in primary human trabecular osteoblasts and to determine the function in bone formation and cell signaling. We report that osteoblasts derived from human trabecular explants express a functional P2X7R capable of agonist-induced increase in intracellular calcium concentration and a positive permeability to fluorescent dyes. These osteoblasts are fully differentiated cells with alkaline phosphatase activity and the ability to form mineralized nodules. We show that the transcriptional regulation of osteoblastic markers can be modulated by P2X7R activity or blockade thereby influencing the differentiation, proliferation and bone matrix formation by these primary human osteoblasts. Finally, we demonstrate that the P2X7R is involved in propagation of mechanically-induced intercellular signaling in addition to the known mechanisms involving calcium signaling via P2Y2 receptors and gap junction.

Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma.

Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma.

Tumor suppressor microRNAs are downregulated in myelodysplastic syndrome with spliceosome mutations.

Spliceosome mutations are frequently observed in patients with myelodysplastic syndromes (MDS). However, it is largely unknown how these mutations contribute to the disease. MicroRNAs (miRNAs) are small noncoding RNAs, which have been implicated in most human cancers due to their role in post transcriptional gene regulation. The aim of this study was to analyze the impact of spliceosome mutations on the expression of miRNAs in a cohort of 34 MDS patients. In total, the expression of 76 miRNAs, including mirtrons and splice site overlapping miRNAs, was accurately quantified using reverse transcriptase quantitative PCR. The majority of the studied miRNAs have previously been implicated in MDS. Stably expressed miRNA genes for normalization of the data were identified using GeNorm and NormFinder algorithms. High-resolution melting assays covering all mutational hotspots within SF3B1, SRSF2, and U2AF1 (U2AF35) were developed, and all detected mutations were confirmed by Sanger sequencing. Overall, canonical miRNAs were downregulated in spliceosome mutated samples compared to wild-type (P = 0.002), and samples from spliceosome mutated patients clustered together in hierarchical cluster analyses. Among the most downregulated miRNAs were several tumor-suppressor miRNAs, including several let-7 family members, miR-423, and miR-103a. Finally, we observed that the predicted targets of the most downregulated miRNAs were involved in apoptosis, hematopoiesis, and acute myeloid leukemia among other cancer- and metabolic pathways. Our data indicate that spliceosome mutations may play an important role in MDS pathophysiology by affecting the expression of tumor suppressor miRNA genes involved in the development and progression of MDS.

Hypermethylation of the VTRNA1-3 Promoter is Associated with Poor Outcome in Lower Risk Myelodysplastic Syndrome Patients.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders. MDS is frequently associated with deletions on chromosome 5q as well as aberrant DNA methylation patterns including hypermethylation of key tumor suppressors. We have previously shown that hypermethylation and silencing of the non-coding RNA VTRNA2-1 are correlated with poor outcomes in acute myeloid leukemia patients. In this study, we find that VTRNA1-2 and VTRNA1-3, both located on chromosome 5q, can be regulated and silenced by promoter DNA methylation, and that the hypomethylating agent 5-aza-2-deoxycytidine causes reactivation these genes. In normal hematopoiesis, we find that vault RNAs (vtRNAs) show differential methylation between various hematopoietic cell populations, indicating that allele-specific methylation events may occur during hematopoiesis. In addition, we show that VTRNA1-3 promoter hypermethylation is frequent in lower risk MDS patients and is associated with a decreased overall survival.

Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction.

Hypermethylation of DAPK1 is an independent prognostic factor predicting survival in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Improvements in overall survival have been observed with the introduction of rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), however, prognostic markers are still needed. Methylation of the death associated protein kinase (DAPK or DAPK1) gene and TP53 mutations are likely to have prognostic value in DLBCL. We have assessed TP53 mutations and allelic DAPK1 methylation patterns in a cohort of 119 DLBCL patients uniformly treated with R-CHOP-like regimens. We found that DAPK1 promoter methylation was associated with shorter overall survival (p=0.017) and disease-specific survival (p=0.023). In multivariate analyses DAPK1 methylation remained as an independent prognostic factor predicting disease-specific survival (p=0.038). When only considering individuals heterozygous for the rs13300553 SNP monoallelic methylation of the A-allele was associated with shorter overall- and disease-specific survival (p<0.001). Patients carrying both DAPK1 methylation and a TP53 mutation had an inferior survival compared to patients carrying only one of these molecular alterations, however, this was borderline statistically significant. Allele-specific DAPK1 methylation patterns were also studied in a cohort of 67 multiple myeloma patients, and all of the methylated multiple myeloma samples heterozygous for the rs13300553 SNP were methylated on both alleles.

UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes.

Osteocytes reside as a cellular network throughout the mineralised matrix of bone and are considered the primary mechanosensors of this tissue. They sense mechanical stimulation such as fluid flow and are able to regulate osteoblast and osteoclast functions on the bone surface. Previously, we found that ATP is released load-dependently from osteocytes from the onset of mechanical stimulation. Therefore, the aim of the present study was to investigate whether and how ATP release can be evoked in osteocytes via purinergic receptor activation. ATP release was quantified by real-time determination using the luciferin-luciferase assay and the release pathway was investigated using pharmacological inhibition. The P2Y receptor profile was analysed using gene expression analysis by reverse transcription polymerase chain reaction, while functional testing was performed using measurements of intracellular calcium responses to P2 receptor agonists. These investigations demonstrated that MLO-Y4 osteocytes express functional P2Y(2), P2Y(4), P2Y(12) and P2Y(13) receptors in addition to the previously reported P2X receptors. Further, we found that osteocytes respond to nucleotides such as ATP, UTP and ADP by increasing the intracellular calcium concentration and that they release ATP dose-dependently upon stimulation with 1-10 µM UTP. In addition to this, osteocytes release large amounts of ATP upon cell rupture, which might also be a source for other nucleotides, such as UTP. These findings indicate that mechanically induced ATP signals may be propagated by P2 receptor activation and further ATP release in the osteocyte network and implicate purinergic signalling as a central signalling pathway in osteocyte mechanotransduction.

Mechanisms for the bone anabolic effect of parathyroid hormone treatment in humans.

Intermittent low-dose treatment with parathyroid hormone (PTH) analogues has become widely used in the treatment of severe osteoporosis. During normal physiological conditions, PTH stimulates both bone formation and resorption, and in patients with primary hyperparathyroidism, bone loss is frequent. However, development of the biochemical measurement of PTH in the 1980s led us to understand the regulation of PTH secretion and calcium metabolism which subsequently paved the way for the use of PTH as an anabolic treatment of osteoporosis as, when given intermittently, it has strong anabolic effects in bone. This could not have taken place without the basic understanding achieved by the biochemical measurements of PTH. The stimulatory effects of PTH on bone formation have been explained by the so-called 'anabolic window', which means that during PTH treatment, bone formation is in excess over bone resorption during the first 6-18 months. This is due to the following: (1) PTH up-regulates c-fos expression in bone cells, (2) IGF is essential for PTH's anabolic effect, (3) bone lining cells are driven to differentiate into osteoblasts, (4) mesenchymal stem cells adhesion to bone surface is enhanced, (5) PTH has a direct antiapoptotic effect on osteoblasts and (6) when PTH interferes with remodelling, the osteoblasts over-compensate, and (7) PTH also decreases sclerostin levels, thereby removing inhibition of Wnt signalling which is required for PTH's anabolic actions. Thus, the net formative effect of PTH given in intermittent treatment emerges through a complex network of pathways. In summary, the effects of PTH on bone turnover are dependent on the mode and dose of administration and studies investigating the mechanisms underlying this effect are reviewed in this article.