During blood stage infection, Plasmodium falciparum infected erythrocytes (IE) bind to host blood vessels. This virulence determinant enables parasites to evade spleen-dependent killing mechanisms, but paradoxically in some cases may reduce parasite fitness by killing the host. Adhesion of infected erythrocytes is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1), a family of polymorphic adhesion proteins encoded by var genes. Whereas cerebral binding and severe malaria are associated with parasites expressing DC8 and DC13 var genes, relatively little is known about the non-brain endothelial selection on severe malaria adhesive types. In this study, we selected P. falciparum-IEs on diverse endothelial cell types and demonstrate that DC8 and DC13 var genes were consistently among the major var transcripts selected on non-brain endothelial cells (lung, heart, bone marrow). To investigate the molecular basis for this avid endothelial binding activity, recombinant proteins were expressed from the predominant upregulated DC8 transcript, IT4var19. In-depth binding comparisons revealed that multiple extracellular domains from this protein bound brain and non-brain endothelial cells, and individual domains largely did not discriminate between different endothelial cell types. Additionally, we found that recombinant DC8 and DC13 CIDR1 domains exhibited a widespread endothelial binding activity and could compete for DC8-IE binding to brain endothelial cells, suggesting they may bind the same host receptor. Our findings provide new insights into the interaction of severe malaria adhesive types and host blood vessels and support the hypothesis that parasites causing severe malaria express PfEMP1 variants with a superior ability to adhere to diverse endothelial cell types, and may therefore endow these parasites with a growth and transmission advantage.

Summary Cytoadhesion of Plasmodium falciparum-infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C-EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine rich interdomain region (CIDRα1) domain from a variety of DC8 and DC13 P. falciparum erythrocyte membrane 1 (PfEMP1) proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate, and strong inhibition of the APC-EPCR interaction. Overall, there was a positive correlation between CIDRα1-EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti-EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.

Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

We report the case of a male, low birth weight, stillborn fetus of 36 weeks gestation with limb body wall complex. An interesting and rare feature noted in the propositus was the absence of the left subclavian artery and complete absence of the left upper limb. These findings seem to favor the vascular theory in the pathogenesis of this condition.

Aim: Intracranial subdural empyemas (SDEs), the majority of which are supratentorial in location, are common neurosurgical emergencies in developing countries, especially in the pediatric age group. They result in significant morbidity and mortality despite improvements in neuroimaging, surgical techniques and antibiotic therapy. In the present study, we retrospectively analyze our experience with operated cases of intracranial supratentorial SDEs in the pediatric age group. Patients and Methods: 65 pediatric patients (age ≤18 years) with supratentorial SDEs were treated in our institute between January 1988 and May 2006, and the case records analyzed with respect to clinical, radiological, bacteriological and surgical complications and outcome data. Results: There was a slight male preponderance (55%), with mean age being 9.54 ± 6.43 years (range 3 months to 18 years). Otogenic source was the most common identifiable etiology, followed by postmeningitic and rhinogenic sources. The initial surgical intervention, burr holes (44 patients; 67.7%) and craniotomy (21 patients; 32.3%), varied with individual cases and surgeon preference. Initial craniotomy was associated with lesser repeat procedures, and slightly better clinical outcome. The majority (83.3%) of patients with significant residual requiring repeat surgery were found to have undergone burr hole evacuation initially. The mortality rate in the present series was 10.8%. Follow-up was available for 41 patients (70.7%) with an average follow-up of 10.4 months. 88% of patients showed good outcomes (Glasgow Outcome Scores of 4 or 5) at the latest follow-up. Conclusion: Pediatric supratentorial SDEs, although rapidly fatal if not identified promptly, can be effectively managed with early surgical drainage (preferably craniotomy), eradication of the source, and sensitive broad-spectrum antibiotics (i.v.) with good outcomes.

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.Bioactive peptides that serve as signaling molecules in the central nervous system and periphery are inactivated or modified by a group of enzymes known as neuropeptidases. With few exceptions, these enzymes are metallopeptidases that carry out peptide bond hydrolysis with the assistance of a zinc ion cofactor. The neuropeptidases thimet oligopeptidase (TOP, 3 EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related members of the zinc metallopeptidase M3 family (1-9). They contain a thermolysin-like catalytic domain that includes a HisGlu-Xaa-Xaa-His (HEXXH) sequence motif involved in zinc ion coordination and catalysis. Both enzymes are widely distributed in mammalian tissues and are found in different subcellular locations (5, 10 -21), where they primarily metabolize small, bioactive peptides involved in a range of physiological processes. In addition, TOP has been shown to degrade peptides released by the proteasome, limiting the extent of antigen presentation by major histocompatibility complex class I molecules (22-27), and it has been associated with amyloid protein precursor processing (28).Human neurolysin and TOP share 63% sequence identity over 677 common residues, and the crystal structures of both enzymes (29,30) show that they adopt almost id...

A 6 year old girl woke up with pain and increasing swelling over the left hand and difficulty in breathing. On examination, she had swelling of the left forearm and hand, flaccid quadriparesis and was in respiratory failure requiring mechanical ventilation. Two clean puncture wounds were identified on the left thumb. A provisional diagnosis of snake bite with severe envenomation was made and she was given anti snake venom therapy. Over a period of about 4 hours her weakness progressed. She became areflexic, developed internal and external ophthalmoplegia and loss of other brain stem reflexes mimicking brain death. Mechanical ventilation was continued despite features suggestive of brain stem dysfunction. About 36 hours after ventilation she showed a flicker of movement of her fingers and gradually the power improved. She was weaned off the ventilator and extubated after 5 days. External ophthalmoplegia is an established association with cobra envenomation, but, this combination of internal and external ophthalmoplegia can mimic brain death and pose a dilemma to the caregivers regarding continuation of therapy. Case reportA 6 year old girl who was sleeping on the floor woke up at 3 AM with pain and swelling over the left hand. This was initially ignored by the parents. The pain and swelling gradually increased, and she complained of difficulty in breathing about 2 hours later. At this point she was brought to the hospital. There was no history suggestive of a snake bite. Examination revealed a drowsy child with a massively swollen left hand and forearm with shallow breathing. There were two clean puncture marks on the left thumb but no bleeding from them. There was no blistering or necrosis of the swollen limb. The local lymph nodes were enlarged. Examination of the central nervous system showed the child to be drowsy but arousable, not obeying verbal commands and was localizing pain. She had ptosis and grade 2-3/5 power in all four limbs and sluggish deep tendon reflexes with extensor plantars. Her pulse rate was 80/min, BP-100/60 mmHg SpO 2 -60% on 5 liters of oxygen by face mask and temperature 98.4°F. Endotracheal intubation was done and she was ventilated on synchronized intermittent mandatory ventilation (SIMV) mode as she had some spontaneous respiratory efforts. Polyvalent anti snake venom therapy was started and a total of 100 ml was given (Haffkine Institute, Bombay), after a provisional diagnosis of snake bite with severe envenomation was made. Over a period of about 4 hours, the weakness progressed and involved proximal muscles first and then distal muscles. She was comatose, had no motor response to painful stimuli, became areflexic and her plantar reflex was not elicitable. Her pupils

SUMMARYA developmentally normal infant presented with repeated episodes of afebrile status epilepticus following nutmeg ingestion. He had developed two episodes of afebrile status epilepticus and had received different treatments earlier, but the details of treatment were not available. On admission, he redeveloped convulsions and loading doses of phenytoin, phenobarbitone and midazolam were administered. However, seizures persisted and extrapyramidal movements, nystagmus and visual dysfunction were noted. Iatrogenic phenytoin toxicity was considered and confirmed by drug levels. His symptoms completely disappeared after discontinuation of phenytoin therapy. The initial seizures were attributed to myristicin, an active component of nutmeg, because of the temporal association. However, the subsequent seizures were due to phenytoin toxicity caused by administration of multiple loading doses. This case highlights that nutmeg, a spice, can cause serious toxic effects like status epilepticus. Furthermore, treatment of status epilepticus with phenytoin can cause iatrogenic seizures due to its narrow therapeutic range. BACKGROUND