Biochemical characterization of Plasmodium falciparum Sir2, a NAD+-dependent deacetylase

In Plasmodium falciparum, the causative agent of cerebral malaria, silent information regulator 2 (Sir2) has been implicated in pathogenesis through its role in var gene silencing. P. falciparum Sir2 (PfSir2) in addition to the catalytic core, has a 13 residue N-terminal and 4 residue C-terminal extension over the shorter Archaeoglobus fulgidus Sir2. In this paper, we highlight our studies aimed at understanding the kinetic mechanism of PfSir2 and the role of N- and C-terminal extensions in protein function and oligomerization. Bisubstrate kinetic analysis showed that PfSir2 exhibits a rapid equilibrium ordered sequential mechanism, with peptide binding preceding NAD(+). This study also reports on surfactin as a novel Sir2 inhibitor exhibiting competitive inhibition with respect to NAD(+) and uncompetitive inhibition with acetylated peptide. This inhibition pattern with surfactin provides further support for ordered binding of substrates. Surfactin was also found to be a potent inhibitor of intra-erythrocytic growth of P. falciparum with 50% inhibitory concentration in the low micromolar range. PfSir2, like the yeast homologs (yHst2 and Sir2p), is a trimer in solution. However, dissociation of trimer to monomers in the presence of NAD(+) is characteristic of the parasite enzyme. Oligomerization studies on N- and/or C-terminal deletion constructs of PfSir2 highlight the role of C-terminus of the protein in mediating homotrimerization. N-terminal deletion resulted in reduced catalytic efficiency although substrate affinity was not altered in the constructs. Interestingly, deletion of both the ends relaxed NAD(+) specificity.     

Placental site trophoblastic tumour: a case report

Placental site trophoblastic tumour (PSTT) is a rare form of trophoblastic disease accounting for < 2% of all gestational trophoblastic neoplasms. Most of the cases follow a normal pregnancy and a small number have a preceeding molar pregnancy or spontaneous abortion. It can occur as early as several weeks or as late as 15 years after normal delivery, molar pregnancy or abortion. Excessive intermediate trophoblastic activity is the most important diagnostic criterion of this tumour originating from non villous trophoblast. But the possibility of a PSTT should be considered when there is excessive intermediate trophoblastic activity despite the presence of chorionic villi as in the present case. This case report highlights the unusual features like rarity of the tumour (< 2%), occurrence following spontaneous abortion which happens only in a minority of cases, and presence of chorionic villi in the tumour despite the fact that the tumour is of non villous trophoblastic origin.     

Lenalidomide‐based maintenance therapy reduces TNF receptor 2 on CD4 T cells and enhances immune effector function in acute myeloid leukemia patients

A major limitation to improved outcomes in acute myelogenous leukemia (AML) is relapse resulting from leukemic cells that persist at clinical remission. Regulatory T cells (Tregs), which are increased in AML patients, can contribute to immune evasion by residual leukemic cells. Tumor necrosis factor (TNF), a pro‐inflammatory cytokine present at high levels within patients, can induce TNF receptor‐2 (TNFR2) expression on Tregs. We hypothesized that since TNFR2 is required for Treg stabilization and TNFR2+ Tregs are potent suppressors, targeting TNFR2+ Tregs may restore the effectiveness of immune‐surveillance mechanisms. In this pilot study, we report AML patients in clinical remission have substantially increased levels of TNFR2+ T cells, including TNFR2+ Tregs and impaired effector CD4 T cell function with reduced IL‐2 and IFNγ production. The immunomodulatory drug, lenalidomide, and the demethylating agent, azacitidine have been moderately successful in treating AML patients, but their combined effects on TNFR2+ T cells, including Tregs are currently unknown. Our data indicates that although treatment with lenalidomide and azacitidine increased cytokine production by effector T cells in all patients, durable clinical remissions may be observed in patients with a concomitant reduction in TNFR2+ T cells and TNFR2+ Tregs. In vitro studies further demonstrated that lenalidomide can reduce TNFR2 expression and can augment effector cytokine production by T cells, which can be further enhanced by azacitidine. These results indicate that reduction of TNFR2+ T cells in AML postremission phase may result from combined azacitidine/lenalidomide therapy and may contribute to an improved clinical outcome. Am. J. Hematol. 89:795–802, 2014. © 2014 Wiley Periodicals, Inc.     

Medical education in pediatric rheumatology—unique challenges and opportunities

Clinical Rheumatology - Pediatric rheumatology faces workforce shortages in both developed and developing regions of the world resulting in suboptimal care of children with chronic rheumatic...     

Maintenance lenalidomide in combination with 5‐azacitidine as post‐remission therapy for acute myeloid leukaemia

In this Phase 1b study, the safety and tolerability of maintenance therapy, comprising lenalidomide (0–25 mg, days 5–25) in combination with azacitidine (50–75 mg/m², days 1–5) every 28 d, was explored in 40 patients with acute myeloid leukaemia (AML) in complete remission after chemotherapy. Eligibility included AML in first complete remission (CR1) with adverse risk karyotype (n = 8), fms‐related tyrosine kinase 3‐internal tandem duplication (FLT3‐ITD) (n = 5), age ≥60 years (n = 31) or AML in second remission (CR2) (n = 14). Dose‐limiting toxicity was not reached. Common toxicities were haematological, infection, injection pain, constipation, fatigue and diarrhoea. In CR1, median relapse‐free (RFS) and overall survival (OS) was 12 and 20 months, respectively. In CR2, median RFS was 11 months, with median OS not yet reached. Among 29 patients with intermediate cytogenetic risk, RFS was 50% at 24 months. There were five patients with concomitant FLT3‐ITD and nucleophosmin (NPM1) mutation; none have relapsed and all are still alive after 17–39 months. Maintenance lenalidomide/azacitidine augmented the function of cytotoxic T lymphocytes, particularly in patients with NPM1 mutation. The lenalidomide/azacitidine maintenance combination was effective in suppressing residual DNA (cytosine‐5‐)‐methyltransferase 3 alpha (DNMT3A)‐positive disease, resulting in sustained remission in patients with concurrent NPM1 mutation. Azacitidine/lenalidomide as maintenance therapy for high‐risk AML warrants further exploration.     

Endometrial aspiration cytology in dysfunctional uterine bleeding

Aspiration cytology is a safe simple and reliable technique without any complication This can be used as a safe and reliable out patient procedure with minimum discomfort to the patient. The use of a cannula as a device for endometrial aspiration provides adequate material for cytological examination. So the present study was undertaken, to assess the ease of collection of endometrial material and to assess the relative compatibility of aspiration cytology and histopathology.