A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Enhancement of metabolic rates of yeast flocculent cells through the use of polymeric additives

The influence of several polymeric additives on specific glucose uptake rate of flocs of a S. cerevisiae strain — S. cerevisiae NRRLY 265 was studied. A special continuous membrane microreactor was used to measure glucose uptake on the presence of calcium and of the tested additives — two cationic polymers — bis(polyoxyethylene-bis(amine)) 20,000 and BPA 1,000 and one anionic polymer — Magna Floc LT25.An increase on glucose uptake rate was always observed when comparing with calcium bound flocs. For bis(polyoxyethylene-bis(amine)) 20,000 the increase was only 19% but for BPA 1,000 a value of more than 50% was observed. For Magna Floc LT25 a two fold increase was measured.The determination of floc size and porosity in the presence of the additives indicated that, on the basis of these parameters, it was not possible to explain the observed glucose uptake rates. The floc porosites in additive bound flocs were similar and 10% larger than for calcium bound flocs and glucose uptake rate was larger for the largest flocs — Magna Floc LT25 bound flocs were the largest followed by BPA 1,000, bis(polyoxyethylene-bis(amine)) 20,000 and calcium bound flocs. These values disagree with what should be expected in diffusion controlled processes.The calculation of intercellular floc distance indicated that polymeric additives act on the reduction of diffusional limitations by increasing the available flux area for glucose inside the flocs. By analysing different kinds of packings, it was also observed that the packing arrangement for yeast cells in flocs is close to the cubic packing. The simulation of this arrangement for the obtained floc sizes confirmed that the 10% increase in floc porosity is sufficient to explain the increase in the available flux area.     

Preferential expression of cellular retinoic acid binding protein in a subpopulation of neural cells in the developing mouse embryo

The cellular retinoic acid binding protein is thought to be involved in the retinoic-acid-mediated signal transduction pathway. We have isolated the mouse cellular retinoic acid binding protein cDNA from an embryonal-carcinoma-derived cell line by using differential cDNA cloning strategies. In situ hybridization on sections of mouse embryos of various developmental stages indicated that the cellular retinoic acid binding protein gene, which we localized on mouse chromosome 9, is preferentially expressed in a subpopulation of neurectodermal cells. This restricted expression pattern suggests an important role for cellular retinoic acid binding protein in murine neurogenesis.     

Hemorrhage in mice produces alterations in B cell repertoires

Multiple organ system failure secondary to infection is the major cause of late deaths after trauma and hemorrhage. The production by B cells of antibodies directed against bacterial antigens is an important component of host defenses. In order to determine the effects of hemorrhage on B cell function, we examined hemorrhage-induced alterations in available (clonal precursors) and actual (plasma cells) B cell repertoires in the course of an immune response toward bacterial antigens. Hemorrhage produced greater than twofold decreases in the absolute frequency and number of clonal precursors specific for the bacterial antigens dextran, levan, and pneumococcal polysaccharide type II. After blood loss, there were decreases in absolute frequency, but not in numbers, of clonal precursors capable of producing antibodies against the nonbacterial antigens ovalbumin and mouse transferrin. Immunization with the bacterial antigen levan within 24 hr of hemorrhage resulted in approximately 50% fewer levan-specific plasma cells than that seen in normal, unhemorrhaged mice. These results demonstrate that hemorrhage produces marked alterations in B cell repertoires, which may contribute to postinjury abnormalities in host defenses.     

Two major classes of mitogen-reactive B lymphocytes defined by life span

The relative numbers of short- and long-lived mitogen-reactive B cells in the peripheral pool were evaluated by studying the decay of lipopolysaccharide (LPS)-reactive B lymphocytes in LPS nonresponder, histocompatible hosts for periods of up to 3 wk after cell transfer. The results obtained demonstrate the existence of two major classes of mitogen-reactive B cells defined by life span. The "short-lived" cell component comprises about 80 to 90% of the reactive cells and decays with a life expectancy of 18 to 24 hr. The long-lived cell component, with life expectancies of 10 to 20 days, comprises about 10 to 20% of the reactive cells and is preferentially enriched in circulating B cells. The present ratio for short- and long-lived B cells implies a highly dynamic state for the immune system which must be advantageous in the selection of available repertoires.     

Life span of B lymphocytes: the experimental basis for conflicting results

Recent claims have challenged the view that most peripheral, mature B cells are long-lived, and propose rates of peripheral decay that are compatible with bone marrow production. This disagreement can only reflect differences in the protocols and methods used to measure peripheral lymphocyte life spans. We have now assessed toxic or other nonselective effects of hydroxyurea treatment on the survival and migration of peripheral, noncycling cells, as well as possible reasons for exaggerated decays of LPS-reactive B cells transferred to LPS nonresponder hosts, the two methods leading to conclusions of short life spans. We also studied general effects on cell survival introduced by either repeated [3H]thymidine injections or the stress associated with surgery, thoracic duct cannulation in particular--methods with which the notion of long life spans had been established. The results failed to show toxic or nonselective effects of hydroxyurea treatments and artificial decays of LPS-reactive cells in adoptive hosts. In contrast, the present experiments demonstrate that both the stress associated with surgery and repeated [3H] thymidine administration profoundly deplete a pool of short-lived B cells, consequently selecting for an apparent higher proportion of long-lived cells.