A diversity-covering approach to immunization with Plasmodium falciparum apical membrane antigen 1 induces broader allelic recognition and growth inhibition responses in rabbits

Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a candidate malaria vaccine, is polymorphic. This polymorphism is believed to be generated predominantly under immune selection pressure and, as a result, may compromise attempts at vaccination. Alignment of 355 PfAMA1 sequences shows that around 10% of the 622 amino acid residues can vary between alleles and that linkages between polymorphic residues occur. Using this analysis, we have designed three diversity-covering (DiCo) PfAMA1 sequences that take account of these linkages and, when taken together, on average incorporate 97% of amino acid variability observed. For each of the three DiCo sequences, a synthetic gene was constructed and used to transform the methylotrophic yeast Pichia pastoris, allowing recombinant expression. All three DiCo proteins were reactive with the reduction-sensitive monoclonal antibody 4G2, suggesting the DiCo sequences had conformations similar to those of naturally occurring PfAMA1. Rabbits were immunized with FVO strain PfAMA1 or with the DiCo proteins either individually or as a mixture. Antibody titers and the ability to inhibit parasite growth in vitro were determined. Animals immunized with the DiCo mix performed similarly to animals immunized with FVO AMA1 when measured against FCR3 strain parasites but outperformed animals immunized with FVO AMA1 when assessed against other strains. The levels of growth inhibition (approximately 70%) induced by the mix of three DiCo proteins were comparable for FVO, 3D7, and HB3, suggesting that a considerable degree of diversity in AMA1 is adequately covered. This suggests that vaccines based upon the DiCo mix approach provide a broader functional immunity than immunization with a single allele.     

Gene expression profiling of ABC transporters in dermal fibroblasts of pseudoxanthoma elasticum patients identifies new candidates involved in PXE pathogenesis

Mutations in the ABCC6 gene, encoding the multidrug resistance-associated protein 6 (MRP6), cause pseudoxanthoma elasticum (PXE). This heritable disorder leads to pathological alterations in connective tissues. The implication of MRP6 deficiency in PXE is still unknown. Moreover, nothing is known about a possible compensatory expression of other ATP binding-cassette (ABC) transporter proteins in MRP6-deficient cells. We investigated the gene expression profile of 47 ABC transporters in human dermal fibroblasts of healthy controls (n=2) and PXE patients (n=4) by TaqMan low-density array. The analysis revealed the expression of 37 ABC transporter genes in dermal fibroblasts. ABCC6 gene expression was not quantifiable in fibroblasts derived from PXE patients. Seven genes (ABCA6, ABCA9, ABCA10, ABCB5, ABCC2, ABCC9 and ABCD2) were induced, whereas the gene expression of one gene (ABCA3) was decreased, comparing controls and PXE patients (with at least twofold changes). We reanalyzed the gene expression of selected ABC transporters in a larger set of dermal fibroblasts from controls and PXE patients (n=6, each). Reanalysis showed high interindividual variability between samples, but confirmed the results obtained in the array analysis. The gene expression of ABC transporter genes, as well as lineage markers of PXE, was further examined after inhibition of ABCC6 gene expression by using specific small-interfering RNA. These experiments corroborated the observed gene expression alterations, most notably in the ABCA subclass (up to fourfold, P<0.05). We therefore conclude that MRP6-deficient dermal fibroblasts exhibit a distinct gene expression profile of ABCA transporters, potentially to compensate for MRP6 deficiency. Moreover, our results point to a function for ABCC6/MRP6 in sterol transport, as sterols are preferential regulators of ABCA transporter activity and expression. Further studies are now required to uncover the role of ABCA transporters in PXE.     

Comparison of the effect of post-instruction multiple-choice and short-answer tests on delayed retention learning

Background

People forget much of what they learn, therefore students could benefit from learning strategies that yield long-lasting knowledge. Yet surprisingly, little is known about how longterm retention is most efficiently mastered. We studied the value of teacher made in class tests as learning aids and compared two types of teacher-made tests (multiple choice and short-answer tests) with a no test (control) to determine their value as aids to retention learning.

Method

The study was conducted on two separate batches of medical undergraduate students. This study compared two types of tests [multiple choice questions (MCQs) and short answer questions (SAQs)] with a no test (control) group. The investigation involved initial testing at the end of the lecture (post instruction), followed by an unannounced delayed retention test on the same material three weeks later. The unannounced delayed test comprising of MCQs and SAQs on the same material was given three weeks later to all the three groups.

Results

In batch I, the MCQ group had a higher mean delayed retention score of 10.97, followed by the SAQ group (8.42) and the control group (6.71). Analysis of variance (ANOVA) test and least significance difference (LSD) post hoc test revealed statistically significant difference between the means of the three groups. Similar results were obtained for batch II

Conclusion

Classroom testing has a positive effect on retention learning; both short-answer and multiple-choice tests being more effective than no test in promoting delayed retention learning, however, multiple-choice tests are better.     

Immunological properties of an N-terminal fragment of herpes simplex virus type 1 glycoprotein D expressed in Escherichia coli

Summary The N-terminal fragment, comprising residues –5 to 55 of herpes simplex virus type 1 glycoprotein D was expressed as a -galactosidase fusion protein inEscherichia coli. This gD-fusion protein reacts with monoclonal antibody LP14 directed against glycoprotein D of HSV. Antisera obtained after immunization of rabbits with purified gD-fusion protein react with HSV-1 gD in a Western blot and with N-terminal synthetic peptides of gD. In addition, these antisera are able to neutralize viral infectivity in vitro.     

Clinical Profile of Special Children at Asha Centre

Background

The care of special children suffering from cerebral palsy, deaf mutism, mental retardation (MR) and post encephalitic sequelae etc. is done in the armed forces at “ASHA” centre supported by Army Wives Welfare Association (AWWA).

Methods

The clinical profile and underlying etiological factors in these children were studied.

Result

Out of 30 children studied, majority were males. The commonest disability was cerebral palsy, seen in 13 (43%) cases followed by mental retardation in six (20%) and post encephalitic sequlae in four (13%) cases. Convulsions were noticed in 12 (40%) cases. The delayed speech was a significant handicap observed in 27 (90%) cases.

Conclusion

Among the etiological factors, natal causes and infections are leading factors in these children and there is an urgent need to strengthen the existing maternal and child health services in our country.Key Words: Cerebral palsy     

Effects of methylene blue-treated plasma on red cells and stored platelet concentrates

BACKGROUND: Photochemical methods can effectively inactivate extracellular viruses and bacteria found in blood components. Treatment of plasma with methylene blue (MB), a phenothiazine dye, and visible light inactivates enveloped viruses including HIV-1. The effects of MB- treated plasma on cellular components stored in vitro have not been well characterized. STUDY DESIGN AND METHODS: MB-treated plasma (83 microg MB/250 mL plasma) was added to single-donor platelets, stored AS- 1 red cells (RBCs), irradiated RBCs, and frozen-deglycerolized RBCs. In vitro platelet assays performed after 1 and 5 days of storage in MB- treated plasma included pH, pO2, pCO2, HCO3, platelet number, lactate dehydrogenase, glucose, osmotic recovery, and CD62 expression. RBC components were examined at specific intervals for leakage of potassium, plasma hemoglobin level, and percentage of hemolysis. Direct antiglobulin tests, osmotic fragilities, and RBC antigen stability tests were also performed on RBCs stored in MB-treated plasma. Components stored with autologous plasma or nontreated allogeneic plasma served as controls. RESULTS: Similar storage-induced changes in pH, glucose, and platelet numbers, as well as increases in lactate dehydrogenase, CD62 expression, and lactate were seen in single-donor platelets stored with MB-treated and control plasma. Platelet morphology scores and osmotic recoveries were not altered. Plasma hemoglobin and potassium and percentage of hemolysis increased equally in the various RBC components stored with MB-treated or nontreated plasma. Osmotic fragility and RBC antigen stability were not appreciably altered by MB-treated plasma. CONCLUSION: Plasma treated by MB photoinactivation can be used for in vitro resuspension and storage of platelets or RBCs, because of the lack of influence of MB-treated plasma on a variety of in vitro platelet and RBC assays.     

High-Level Expression of Plasmodium vivax Apical Membrane Antigen 1 (AMA-1) in Pichia pastoris: Strong Immunogenicity in Macaca mulatta Immunized with P. vivax AMA-1 and Adjuvant SBAS2

The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the Plasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Δglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Δglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Δglyc_43–487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant, Pichia-expressed PV66Δglyc_43–487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.     

Four cancer cases after esophageal atresia repair: Time to start screening the upper gastrointestinal tract

Esophageal atresia(EA) is one of the most common congenital digestive malformations and requires surgical correction early in life. Dedicated centers have reported survival rates up to 95%. The most frequent comorbidities after EA repair are dysphagia(72%) and gastroesophageal reflux(GER)(67%). Chronic GER after EA repair might lead to mucosal damage, esophageal stricturing, Barrett's esophagus and eventually esophageal adenocarcinoma. Several long-term follow-up studies found an increased risk of Barrett's esophagus and esophageal carcinoma in EA patients, both at a relatively young age. Given these findings, the recent ESPGHAN-NASPGHAN guideline recommends routine endoscopy in adults born with EA. We report a series of four EA patients who developed a carcinoma of the gastrointestinal tract: three esophageal carcinoma and one colorectal carcinoma in a colonic interposition. These cases emphasize the importance of lifelong screening of the upper gastrointestinal tract in EA patients.