Predictors of participation in parenting workshops for improving adolescent behavioral and mental health: results from the common sense parenting trial.

Engaging and retaining participants are crucial to achieving adequate implementation of parenting interventions designed to prevent problem behaviors among children and adolescents. This study examined predictors of engagement and retention in a group-based family intervention across two versions of the program: a standard version requiring only parent attendance for six sessions and an adapted version with two additional sessions that required attendance by the son or daughter. Families included a parent and an eighth grader who attended one of five high-poverty schools in an urban Pacific Northwest school district. The adapted version of the intervention had a higher rate of engagement than the standard version, a difference that was statistically significant after adjusting for other variables assessed at enrollment in the study. Higher household income and parent education, younger student age, and poorer affective quality in the parent-child relationship predicted greater likelihood of initial attendance. In the adapted version of the intervention, parents of boys were more likely to engage with the program than those of girls. The variables considered did not strongly predict retention, although retention was higher among parents of boys. Retention did not significantly differ between conditions. Asking for child attendance at workshops may have increased engagement in the intervention, while findings for other predictors of attendance point to the need for added efforts to recruit families who have less socioeconomic resources, as well as families who perceive they have less need for services.

Functional alteration of red blood cells by a megadalton protein of Plasmodium falciparum.

Proteins exported from Plasmodium falciparum parasites into red blood cells (RBCs) interact with the membrane skeleton and contribute to the pathogenesis of malaria. Specifically, exported proteins increase RBC membrane rigidity, decrease deformability, and increase adhesiveness, culminating in intravascular sequestration of infected RBCs (iRBCs). Pf332 is the largest (>1 MDa) known malaria protein exported to the RBC membrane, but its function has not previously been determined. To determine the role of Pf332 in iRBCs, we have engineered and analyzed transgenic parasites with Pf332 either deleted or truncated. Compared with RBCs infected with wild-type parasites, mutants lacking Pf332 were more rigid, were significantly less adhesive to CD36, and showed decreased expression of the major cytoadherence ligand, PfEMP1, on the iRBC surface. These abnormalities were associated with dramatic morphologic changes in Maurer clefts (MCs), which are membrane structures that transport malaria proteins to the RBC membrane. In contrast, RBCs infected with parasites expressing truncated forms of Pf332, although still hyperrigid, showed a normal adhesion profile and morphologically normal MCs. Our results suggest that Pf332 both modulates the level of increased RBC rigidity induced by P falciparum and plays a significant role in adhesion by assisting transport of PfEMP1 to the iRBC surface.

Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant.

Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)-induced Ank-1-null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in beta-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.

A Maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells.

The high mortality of Plasmodium falciparum malaria is the result of a parasite ligand, PfEMP1 (P. falciparum) erythrocyte membrane protein 1), on the surface of infected red blood cells (IRBCs), which adheres to the vascular endothelium and causes the sequestration of IRBCs in the microvasculature. PfEMP1 transport to the IRBC surface involves Maurer's clefts, which are parasite-derived membranous structures in the IRBC cytoplasm. Targeted gene disruption of a Maurer's cleft protein, SBP1 (skeleton-binding protein 1), prevented IRBC adhesion because of the loss of PfEMP1 expression on the IRBC surface. PfEMP1 was still present in Maurer's clefts, and the transport and localization of several other Maurer's cleft proteins were unchanged. Maurer's clefts were altered in appearance and were no longer found as close to the periphery of the IRBC. Complementation of mutant parasites with sbp1 led to the reappearance of PfEMP1 on the IRBC surface and the restoration of adhesion. Our results demonstrate that SBP1 is essential for the translocation of PfEMP1 onto the surface of IRBCs and is likely to play a pivotal role in the pathogenesis of P. falciparum malaria.

An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells.

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) - Isoleucine (I) - Lysine (K) - Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite's ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.

Bioinformatic prediction of the exportome of Babesia bovis and identification of novel proteins in parasite-infected red blood cells.

Babesia bovis is a pathogen of considerable economic significance to the livestock industry worldwide but the precise mechanisms by which this parasite causes disease in susceptible cattle remain poorly understood. It is clear, however, that alterations to the structure and function of red blood cells in which the parasites reside and replicate play an important role in pathogenesis and that these are secondary to the export of numerous, currently unknown and uncharacterised parasite-encoded proteins. Using a rational bioinformatic approach, we have identified a set of 362 proteins (117 of which are hypothetical) that we predict encompasses the B. bovis exportome. These exported proteins are likely to be trafficked to various cellular locations, with a subset destined for the red blood cell cytosol or the red blood cell cytoskeleton. These proteins are likely to play important roles in mediating the pathogenesis of babesiosis. We have selected three novel proteins and confirmed their predicted export and localisation within the host red blood cell by immunofluorescence using specific antibodies raised against these proteins. Complete characterisation of these novel exported parasite proteins will help elucidate their function within the host red blood cell and assist in identification of new therapeutic targets for babesiosis.

New insights into the altered adhesive and mechanical properties of red blood cells parasitized by Babesia bovis.

Sequestration of parasite-infected red blood cells (RBCs) in the microvasculature is an important pathological feature of both bovine babesiosis caused by Babesia bovis and human malaria caused by Plasmodium falciparum. Surprisingly, when compared with malaria, the cellular and molecular mechanisms that underlie this abnormal circulatory behaviour for RBCs infected with B. bovis have been relatively ignored. Here, we present some novel insights into the adhesive and mechanical changes that occur in B. bovis-infected bovine RBCs and compare them with the alterations that occur in human RBCs infected with P. falciparum. After infection with B. bovis, bovine RBCs become rigid and adhere to vascular endothelial cells under conditions of physiologically relevant flow. These alterations are accompanied by the appearance of ridge-like structures on the RBC surface that are analogous, but morphologically and biochemically different, to the knob-like structures on the surface of human RBCs infected with P. falciparum. Importantly, albeit for a limited number of parasite lines examined here, the extent of these cellular and rheological changes appear to be related to parasite virulence. Future investigations to identify the precise molecular composition of ridges and the proteins that mediate adhesion will provide important insight into the pathogenesis of both babesiosis and malaria.

The role of KAHRP domains in knob formation and cytoadherence of P falciparum-infected human erythrocytes.

Surface protrusions of Plasmodium falciparum-infected erythrocytes, called knobs, display focal aggregates of P falciparum erythrocyte membrane protein 1 (PfEMP1), the adhesion ligand binding endothelial-cell receptors. The resulting sequestration of infected erythrocytes in tissues represents an important factor in the course of fatalities in patients with malaria. The main component of knobs is the knob-associated histidine-rich protein (KAHRP), and it contributes to altered mechanical properties of parasite-infected erythrocytes. The role of KAHRP domains in these processes is still elusive. We generated stable transgenic P falciparum-infected erythrocytes expressing mutant versions of KAHRP. Using atomic force and electron microscopy we show that the C-terminal repeat region is critical for the formation of functional knobs. Elasticity of the membrane differs dramatically between cells with different KAHRP mutations. We propose that the 5' repeat region of KAHRP is important in cross-linking to the host-cell cytoskeleton and this is required for knob protrusion and efficient adhesion under physiologic flow conditions.

Misidentification of cardiac vagal pre-ganglionic neurons after injections of retrograde tracer into the pericardial space in the rat.

We evaluated whether pericardial injections of the retrograde tracers cholera toxin subunit B (CTb) or Fast Blue (FB) reliably labelled cardiac vagal pre-ganglionic neurons. Injections of CTb into the pericardial space of the rat labelled neurons in both the external and compact formations of the nucleus ambiguus. Most labelled neurons were found in the compact formation of the nucleus ambiguus, and the majority of these, and only these, expressed immunoreactivity for calcitonin gene-related peptide. This distribution of labelled neurons and their immunohistochemical properties is characteristic of oesophageal motoneurons. Examination of the oesophagus following intra-pericardial CTb applications revealed strong labelling of motor end plates within the skeletal muscle of the thoracic but not the abdominal oesophagus. When a second retrograde tracer, FB, was injected into the abdominal oesophagus, labelled somata were found adjacent to CTb-labelled neurons in the compact formation of the nucleus ambiguus. No co-localisation of tracers was found, but identical proportions of calcitonin gene-related peptide (CGRP) immunoreactivity were observed in both groups of neurons. FB injected into the pericardial space labelled intra-cardiac neurons but not brainstem neurons. We conclude that intra-pericardial, and perhaps sub-epicardial, injections of some retrograde tracers are likely to label a subset of oesophageal, as well as cardiac, vagal motor neurons in the brainstem.