Monomeric α-synuclein activates the plasma membrane calcium pump.

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

Adaptor protein-3 produces synaptic vesicles that release phasic dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high-frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a distinct population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Functional and transcriptional heterogeneity within the massively expanding HLADR+CD38+ CD8 T cell population in acute febrile dengue patients.

IMPORTANCE: CD8 T cells play a crucial role in protecting against intracellular pathogens such as viruses by eliminating infected cells and releasing anti-viral cytokines such as interferon gamma (IFNγ). Consequently, there is significant interest in comprehensively characterizing CD8 T cell responses in acute dengue febrile patients. Previous studies, including our own, have demonstrated that a discrete population of CD8 T cells with HLADR+ CD38+ phenotype undergoes massive expansion during the acute febrile phase of natural dengue virus infection. Although about a third of these massively expanding HLADR+ CD38+ CD8 T cells were also CD69high when examined ex vivo, only a small fraction of them produced IFNγ upon in vitro peptide stimulation. Therefore, to better understand such functional diversity of CD8 T cells responding to dengue virus infection, it is important to know the cytokines/chemokines expressed by these peptide-stimulated HLADR+CD38+ CD8 T cells and the transcriptional profiles that distinguish the CD69+IFNγ+, CD69+IFNγ-, and CD69-IFNγ- subsets.

Time-varying brentuximab vedotin pharmacokinetics and weight-based dosing in paediatric patients despite lower exposure in those aged 2 to <6 and 6-11 years.

AIMS: We studied the pharmacokinetics and exposure-response relationships of the brentuximab vedotin (BV) antibody-drug conjugate (ADC) and unconjugated monomethyl auristatin E in haematologic malignancies. METHODS: This population pharmacokinetic analysis included data from five adult and three paediatric studies. Exposures in virtual adult and paediatric populations following BV 1.8 mg/kg (maximum 180 mg) intravenously every 3 weeks were simulated. Clinical endpoints included overall response rate, grade ≥2 peripheral neuropathy (PN) and grade ≥3 neutropenia. RESULTS: BV ADC exhibited linear pharmacokinetics, well-described by a three-compartment model, with body weight being the only significant covariate for exposure. Monomethyl auristatin E exhibited time-varying formation rate. Simulated steady-state BV ADC exposures in patients aged 12 to <18 years were similar to those of adult patients, but 23%-38% lower in patients aged 2 to <12 years. Despite lower exposure, clinical activity was observed with BV 1.8 mg/kg every 3 weeks in those aged 2 to <12 years (overall response rate: 2 to <12 years, 60%; 12 to <18 years, 43%). In adult, but not paediatric patients, increased BV ADC exposures were associated with grade ≥2 PN and grade ≥3 neutropenia occurrence. CONCLUSIONS: BV pharmacokinetics in adult and paediatric patients were consistent. BV ADC exposures were lower in patients aged 2 to <12 years vs. ≥12 years, but no apparent clinically relevant differences in efficacy, grade ≥2 PN or grade ≥3 neutropenia were observed. These data support body weight-based dosing of BV in patients irrespective of age; thus, dose adjustment in those 2 to <12 years does not appear warranted.

Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

Immune signalling by supramolecular assemblies.

Formation of supramolecular assemblies appears to be a general mechanism in immune signalling pathways. These supramolecular assemblies appear to form through a nucleated polymerization mechanism. This review examines selected immune signalling pathways that involve supramolecular assemblies, describes the concepts of protein polymerization, and discusses how those concepts of protein polymerization implicate new elegant ways for signal amplification, setting threshold and noise reduction in these pathways.

Associations of Autoimmunity, Immunodeficiency, Lymphomagenesis, and Gut Microbiota in Mice with Knockins for a Pathogenic Autoantibody.

A number of mouse strains transgenic for B-cell receptors specific for nucleic acids or other autoantigens have been generated to understand how autoreactive B cells are regulated in normal and autoimmune mice. Previous studies of nonautoimmune C57BL/6 mice heterozygous for both the IgH and IgL knockins of the polyreactive autoantibody, 564, produced high levels of autoantibodies in a largely Toll-like receptor 7-dependent manner. Herein, we describe studies of mice homozygous for the knockins that also expressed high levels of autoantibodies but, unlike the heterozygotes, exhibited a high incidence of mature B-cell lymphomas and enhanced susceptibility to bacterial infections. Microarray analyses and serological studies suggested that lymphomagenesis might be related to chronic B-cell activation promoted by IL-21. Strikingly, mice treated continuously with antibiotic-supplemented water did not develop lymphomas or abscesses and exhibited less autoimmunity. This mouse model may help us understand the reasons for enhanced susceptibility to lymphoma development exhibited by humans with a variety of autoimmune diseases, such as Sjögren syndrome, systemic lupus erythematosus, and highly active rheumatoid arthritis.

IL-21-driven neoplasms in SJL mice mimic some key features of human angioimmunoblastic T-cell lymphoma.

SJL/J mice exhibit a high incidence of mature B-cell lymphomas that require CD4(+) T cells for their development. We found that their spleens and lymph nodes contained increased numbers of germinal centers and T follicular helper (TFH) cells. Microarray analyses revealed high levels of transcripts encoding IL-21 associated with high levels of serum IL-21. We developed IL-21 receptor (IL21R)-deficient Swiss Jim Lambart (SJL) mice to determine the role of IL-21 in disease. These mice had reduced numbers of TFH cells, lower serum levels of IL-21, and few germinal center B cells, and they did not develop B-cell tumors, suggesting IL-21-dependent B-cell lymphomagenesis. We also noted a series of features common to SJL disease and human angioimmunoblastic T-cell lymphoma (AITL), a malignancy of TFH cells. Gene expression analyses of AITL showed that essentially all cases expressed elevated levels of transcripts for IL21, IL21R, and a series of genes associated with TFH cell development and function. These results identify a mouse model with features of AITL and suggest that patients with the disease might benefit from therapeutic interventions that interrupt IL-21 signaling.

A reporter mouse reveals lineage-specific and heterogeneous expression of IRF8 during lymphoid and myeloid cell differentiation.

The IFN regulatory factor family member 8 (IRF8) regulates differentiation of lymphoid and myeloid lineage cells by promoting or suppressing lineage-specific genes. How IRF8 promotes hematopoietic progenitors to commit to one lineage while preventing the development of alternative lineages is not known. In this study, we report an IRF8-EGFP fusion protein reporter mouse that revealed previously unrecognized patterns of IRF8 expression. Differentiation of hematopoietic stem cells into oligopotent progenitors is associated with progressive increases in IRF8-EGFP expression. However, significant induction of IRF8-EGFP is found in granulocyte-myeloid progenitors and the common lymphoid progenitors but not the megakaryocytic-erythroid progenitors. Surprisingly, IRF8-EGFP identifies three subsets of the seemingly homogeneous granulocyte-myeloid progenitors with an intermediate level of expression of EGFP defining bipotent progenitors that differentiation into either EGFP(hi) monocytic progenitors or EGFP(lo) granulocytic progenitors. Also surprisingly, IRF8-EGFP revealed a highly heterogeneous pre-pro-B population with a fluorescence intensity ranging from background to 4 orders above background. Interestingly, IRF8-EGFP readily distinguishes true B cell committed (EGFP(int)) from those that are noncommitted. Moreover, dendritic cell progenitors expressed extremely high levels of IRF8-EGFP. Taken together, the IRF8-EGFP reporter revealed previously unrecognized subsets with distinct developmental potentials in phenotypically well-defined oligopotent progenitors, providing new insights into the dynamic heterogeneity of developing hematopoietic progenitors.

Drug-induced immune hemolytic anemia associated with anti-carboplatin and the first example of anti-paclitaxel.

BACKGROUND: Combined chemotherapy with carboplatin and paclitaxel is first-line treatment for lung and ovarian cancer. Drug-induced antibodies to carboplatin are rare but can cause severe, even fatal, hemolysis. Paclitaxel-induced immune hemolysis has not been reported. We describe a case of immune-mediated hemolysis associated with antibodies to carboplatin and paclitaxel in a woman with ovarian cancer who had received multiple chemotherapeutic agents over 7 years, including several courses of these two drugs. She required many transfusions. During a chemotherapy infusion the patient became hypotensive, was pale, and had rigors and red urine. The nadir hematocrit was 12.4%; peak bilirubin and lactate dehydrogenase were 16.3 mg/dL and 1188 units/L, respectively. STUDY DESIGN AND METHODS: Blood samples collected within hours after chemotherapy and 2 days later were tested for antibodies to carboplatin and paclitaxel. RESULTS: The direct antiglobulin test was positive with anti-IgG (3+) and anti-C3 (2+). The plasma collected shortly after chemotherapy agglutinated carboplatin-treated red blood cells (RBCs); untreated and paclitaxel-treated RBCs both reacted at the antiglobulin test most likely due to circulating carboplatin, paclitaxel, or both drugs. Serum collected 2 days later agglutinated (titer 2) and sensitized (titer 128) carboplatin-treated RBCs; untreated and paclitaxel-treated RBCs were nonreactive. An acid eluate reacted weakly in the presence of polyethylene glycol with carboplatin-treated RBCs. The serum reacted with untreated and enzyme-treated RBCs in the presence of soluble carboplatin and paclitaxel. CONCLUSION: Anti-carboplatin and the first example of anti-paclitaxel were detected in this patient's sample.

Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions.

Innate-like B-1a cells contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. Here we show that these classic functions of B-1a cells segregate between two unique subsets defined by expression of plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). These subsets, designated B-1a.PC1(lo) and B-1a.PC1(hi), differ significantly in IgH chain utilization. Adoptively transferred PC1(lo) cells secreted significantly more circulating natural IgM and intestinal IgA than PC1(hi) cells. In contrast, PC1(hi) cells produced more IL-10 than PC1(lo) cells when stimulated with LPS and phorbol 12-myristate 13-acetate (PMA). PC1(hi) cells were also more efficient than PC1(lo) cells in regulating Th1 cell differentiation, even though both B-1a subsets were comparably active in stimulating T-cell proliferation. Furthermore, PC1(lo) cells generated antigen-specific IgM responses to pneumococcal polysaccharide antigens, whereas PC1(hi) cells do not. We found that PC1(lo) cells develop from an early wave of B-1a progenitors in fetal life, whereas PC1(hi) cells are generated from a later wave after birth. We conclude that identification of B-1a.PC1(lo) and B-1a.PC1(hi) cells extends the concept of a layered immune system with important implications for developing effective vaccines and promoting the generation of immunoregulatory B cells.

Evaluation of the mandibular arch in patients with impacted permanent lower canines.

AIM: To determine the features of the mandibular dental arch in subjects presenting with impacted permanent lower canines. METHODS: The 'impaction group' consisted of 48 Indian subjects with mandibular canine impaction (Females:Males, 1.5:1; mean age, 15.03 ± 0.49 years). The 'control group' was comprised of 96 age-, gender- and malocclusion-matched Indians who were randomly selected from subjects initially screened but who had completely erupted mandibular canines. Arch width, arch length, arch shape and space status (total tooth size, arch-length--tooth-size discrepancy) were assessed using dental models and were compared between the groups using comparative measurements and statistics. RESULTS: Statistically significant differences were demonstrated with respect to the arch length, arch shape, total tooth size and arch-length--tooth-size discrepancy (p = 0.03, 0.02, 0.04, 0.01; independent 2-sample t-tests, respectively). Crowding was more prevalent in subjects with impaction than in the controls, with the difference being statistically significant (chi-square = 13.202; degrees of freedom (df) = 4; p = 0.010). CONCLUSION: Patients with permanent mandibular canine impaction have adequately wide but shorter lower dental arch forms along with wider mandibular total tooth size and greater arch-length--tooth-size discrepancy when compared with a control sample.

Restoflex--a revolutionary change in preclinical practice for restorative dentistry and endodontics.

Preclinical exercises are very important for the dental students in order to master various dental techniques. The objective of this article is to introduce a new preclinical working model named Restoflex. It is especially designed for the students to carry out various restorative and endodontic procedures in an environment that closely simulate clinical situations. This will help them to provide a smooth transition from preclinical environment to the clinical one. It would also mean an increased confidence level and the efficiency with which the students would deal with their cases.

Permanent mandibular canine(s) impaction: expansion of our understanding.

INTRODUCTION: Eruption disturbances, tooth size and specific malocclusions are known to be genetically influenced. The clinical association between these traits may indicate common genetic controls. OBJECTIVES: A cross-sectional clinical study was designed to test the null hypothesis that the maximum mesiodistal crown diameter (MMD) of maxillary and mandibular central and lateral incisors and the prevalence of various classes of incisor relationships (Class I, II/1, II/2 and III) do not differ between the subjects with and without permanent mandibular canine(s) impaction. METHODS: Dental models of 43 subjects diagnosed with mandibular canine(s) impaction (Impaction Group - IG) were compared with those of 86 subjects of a control reference sample (Control Group - CG). Independent t-test and chi-square tests were used to determine the association between mandibular canine(s) impaction and the MMD of the incisors and the incisor relationship, respectively. The likelihood of various incisor relationships between the IG and CG were evaluated according to odds ratios. RESULTS: A fourfold increase (p < 0.0001) in the overall frequency of Class II/2 incisor relationship was observed in the IG when compared to controls. CONCLUSIONS: The null hypothesis was rejected. Subjects with mandibular canine(s) impaction appeared to be characterised with wider incisors and a remarkably high rate of Class II/2 malocclusion. This information assists the understanding of genetically controlled dental anomalies, which are likely to coexist with mandibular canine(s) impaction.

Anticancer mechanisms of ß-carbolines.

ß-Carboline nucleus is therapeutically valuable in medicinal chemistry for the treatment of varied number of diseases, most importantly cancer. The potent and wide-ranging activity of ß-carboline has established them as imperative pharmacological scaffolds especially in the cancer treatment. Numerous derivatives such as Tetrahydro ß-carbolines, metal complexed ß-carbolines, mono, di and tri substituted ß-carbolines have been reported to possess dynamic anticancer activity. These different substituted ß-carboline derivatives had shown different mechanism of action and plays important role in anticancer drug discovery and development. The review is an update of the chemistry of ß-carbolines, both synthetic and natural origin acting through various targets against cancerous cells. In addition to this, studies of multitarget molecules designed by coupling ß-carbolines along with other mechanisms for treatment of neoplasm are also summarized.

Racemose neurocysticercosis: a case series.

Neurocysticercosis (NCC) is a common parasitic condition of the central nervous system in certain parts of the world. The racemose variety of NCC is distinct from the commonly seen parenchymal form. It frequently infiltrates the basal cisterns and Sylvian fissures. Imaging plays a vital role in the diagnosis; however, as their signal intensity is similar to cerebrospinal fluid and due to the absence of enhancement in most cases, imaging diagnosis is often difficult on the conventional MRI sequences. Here, we present five cases of racemose NCC to emphasize the importance of a heavily T2-weighted sequence (Fast Imaging Employing Steady-state Acquisition) sequence in the diagnosing this entity.

Exposure-Response and Subgroup Analyses to Support Body Weight-Based Dosing of Brentuximab Vedotin in Children and Young Adults with Newly Diagnosed High-Risk Classical Hodgkin Lymphoma.

PURPOSE: To evaluate the relationships between brentuximab vedotin (BV) pharmacokinetics, age, and body weight (BW) with efficacy and safety in pediatric and young adult patients with previously untreated, high-risk classical Hodgkin lymphoma (cHL) in the phase 3 AHOD1331 study. PATIENTS AND METHODS: Overall, 296 patients (age 2-21 years) in the overall population were randomized to and received BV + chemotherapy; the pharmacokinetic subpopulation comprised 24 patients (age <13 years). Age- and/or BW-based (pharmacokinetic surrogates) subgroup analyses of efficacy and safety were conducted for the overall population. Exposure-response analyses were limited to the pharmacokinetic subpopulation. RESULTS: There were no visible trends in disease characteristics across pediatric age subgroups, while BW increased with age. Observed antibody-drug conjugate exposures in patients aged <12 years were lower than those in adults administered BV 1.8 mg/kg every 3 weeks (Q3W), as exposure increased with BW. Nevertheless, no detrimental impact on event-free survival (EFS) was seen in younger subgroups: 3-year EFS was 96.2% (2-<12-years) and 92.0% (12-<18-years), with no events observed in those aged <6 years. Neither early response nor lack of need for radiation therapy was associated with high pharmacokinetic exposure. No evidence of exposure-driven grade ≥2 or ≥3 peripheral neuropathy or grade ≥3 neutropenia was seen in exposure-safety and BW-based subgroup analyses; the incidence of these safety events was comparable across pediatric age subgroups, despite lower exposure in younger children. CONCLUSIONS: No further adjustments based on age or BW are required for the BV dose (1.8 mg/kg Q3W) approved in children.

STENOSIS: Long-term single versus dual antiplatelet therapy in patients with ischaemic stroke due to intracranial atherosclerotic disease - a randomised trial.

Rationale: Intracranial atherosclerotic disease (ICAD) is a pathological process that causes progressive stenosis and cerebral hypoperfusion, leading to stroke occurrence and recurrence around the world. The exact duration of dual antiplatelet therapy (DAPT) for ICAD is unclear in view of long-term risk of bleeding complications. Aim: The current study aims to study the efficacy and safety of long-term DAPT (up to 12 months) in patients with ICAD. Sample size: Using 80% power and an alpha error of 5 %, presuming a 10%-15% drop-out rate, a total of 2200 patients will be recruited for the study. Methodology: This is a prospective, randomised, double-blind, placebo controlled trial. Study outcomes: The primary outcomes include recurrent ischaemic stroke (IS) or transient ischaemic attack and any intracranial haemorrhage (ICH), major or minor systemic bleeding at the end of 12 months. Secondary outcomes include composite of any stroke, myocardial infarction or death at the end of 12 months. The safety outcomes include any ICH, major or minor bleeding as defined using GUSTO (Global Use of Streptokinase and tPA for occluded Coronary Arteries) classification at the end of 12 months and 1 month after completion of the drug treatment phase. Discussion: The study will provide level I evidence on the duration of DAPT among patients with IS due to ICAD of more than or equal to 50%.

RE-OPEN: Randomised trial of biosimilar TNK versus TPA during endovascular therapy for acute ischaemic stroke due to large vessel occlusions.

Rationale: Rapid and timely treatment with intravenous thrombolysis and endovascular treatment (EVT) in patients with acute ischaemic stroke (AIS) and large vessel occlusion (LVO) significantly improves patient outcomes. Bridging therapy is the current standard of care in these patients. However, an incompletely answered question is whether one thrombolytic agent is better than another during bridging therapy. Aim: The current study aims to understand if one thrombolytic agent is superior to the other during bridging therapy in the treatment of AIS and LVO. Sample size estimates: Using 80% power and an alpha error of 5 %, presuming a 10% drop out rate, a total of 372 patients will be recruited for the study. Methods and design: This study is a prospective, randomised, multicentre, open-label trial with blinded outcome analysis design. Study outcomes: The primary outcomes include proportion of patients who will be independent at 3 months (modified Rankin score (mRS) ≤2 as good outcome) and proportion of patients who achieve recanalisation modified thrombolysis in cerebral infarction grade 2b/3 at first angiography run at the end of EVT. Secondary outcomes include proportion of patients with early neurological improvement, rate of symptomatic intracerebral haemorrhage (ICH), rate of any ICH, rate of any systemic major or minor bleeding and duration of hospital stay. Safety outcomes include any intracranial bleeding or symptomatic ICH. Discussion: This trial is envisioned to confirm the theoretical advantages and increase the strength and quality of evidence for use of tenecteplase (TNK) in practice. Also, it will help to generate data on the efficacy and safety of biosimilar TNK. Trial registration number: CTRI/2022/01/039473.