Efficient control of Plasmodium yoelii infection in BALB/c and C57BL/6 mice with pre-existing Strongyloides ratti infection.

About 225 million malaria cases have been reported worldwide in 2009, and one-third of the world's population is infected with parasitic helminths. As helminths and Plasmodium are co-endemic, concurrent infections frequently occur. Helminths have been shown to modulate the host's immune response; therefore, pre-existing helminth infections may interfere with the efficient immune response to Plasmodium. To study the interaction between helminths and Plasmodium, we established a murine model of co-infection using the gastrointestinal nematode Strongyloides ratti and Plasmodium yoelii. We show that a pre-existing Strongyloides infection slightly enhanced peak parasitemia and weight loss in P. yoelii-infected BALB/c mice, while disease progression was not altered in co-infected C57BL/6 mice. The Plasmodium-induced IFN-γ production and final clearance of Plasmodium infection were not affected by S. ratti co-infection in both C57BL/6 and BALB/c mice. Interestingly, the T helper cell (Th) 2 response induced by S. ratti was significantly suppressed upon P. yoelii co-infection. This suppressed Th2 response, however, was still sufficient to allow expulsion of S. ratti parasitic adults. Taken together, we provide evidence that simultaneous presence of helminth and protist parasites does not interfere with efficient host defence in our co-infection model although changes in Th responses were observed.

Pulmonary venous obstruction and atrioventricular canal anomalies: role of cor triatriatum and double outlet right atrium.

Two-dimensional echocardiographic findings and surgical repair in three infants with atrioventricular (AV) canal and pulmonary venous obstruction are described. The AV canal was complicated by cor triatriatum in two patients and by double outlet right atrium in the other. In patients with AV canal, anatomic obstruction of pulmonary venous return should be excluded by detailed cardiac ultrasound examination.

Potentially fatal cardiac dysrhythmia and hyperkalemic periodic paralysis.

An 11-year-old boy was evaluated for mild periodic muscular weakness exacerbated on separate occasions by disopyramide phosphate and procainamide. He and his mother both had bidirectional ventricular tachydysrhythmia (BVT), short stature, microcephaly, and clinodactyly. The mother, but not the child, had lingual myotonia. The two antiarrhythmic drugs worsened the muscular weakness without benefiting the cardiac dysrhythmia. Potassium loading produced skeletal muscle weakness and transient conversion of the BVT to normal sinus rhythm. Hypokalemia aggravated the BVT without causing weakness. Acetazolamide had no effect. The patient suffered a nonfatal cardiac arrest after several days of increased carbohydrate intake. Imipramine controlled the dysrhythmia without inducing weakness. Periodic paralysis should be considered as the diagnosis in children with BVT, a potentially fatal condition.

High-dose irradiated splenic stimulator cells show no endogenous interleukin-2 production but stimulate clonally developing helper T cells to produce interleukin-2.

The effect of various physical or chemical treatments of splenic stimulator cells on their endogenous, mitogen-inducible IL-2 production and on their ability to induce IL-2 production in clonally developing helper T lymphocytes was investigated. While most methods (T cell depletion by monoclonal antibodies plus complement, glutaraldehyde fixation, heat inactivation and high-dose irradiation) effectively suppressed the endogenous IL-2 production of splenic stimulator cells, only T cell depletion and high-dose (6000-10,000 R) irradiation sustained their stimulatory capacity. High-dose irradiated stimulator cells induced high numbers of clonally developing helper T lymphocytes to secrete IL-2. Moreover, this induction was found to be antigen-specific. Hence, high-dose irradiation is a simple, rapid and reliable method for the treatment of stimulator cells, especially when large numbers of cultures are to be screened.

The T-cell receptor associated zeta-chain is required but not sufficient for CD26 (dipeptidylpeptidase IV) mediated signaling.

CD26 or dipeptidylpeptidase IV (DPP IV) is a cell surface protease involved in T-cell activation. Triggering or costimulation of T-cells via CD26 was shown to be dependent on the expression of the T-cell receptor (TCR) associated zeta-chain with at least one functional immune receptor tyrosine based activation motif (ITAM). Here we tested T-cell lines expressing chimeric proteins (hCD25-zeta) consisting of human IL-2 receptor-alpha chain derived extracellular sequences (hCD25) fused to mouse-specific zeta-chain segments, for their capacity to transfer CD26 mediated signals. Although these 'minimal receptor' expressing T-cell lines were capable of transmitting signals from other costimulatory molecules (e.g. CD2), crosslinking of CD26 did not induce IL-2 secretion. Co-cross-linking of hCD25 and CD26 molecules, however, resulted in the stimulation of the T-cells. Thus, although the zeta-chain is a prerequisite for CD26 mediated signaling events, the sole expression of zeta-protein as a signaling molecule is not sufficient for CD26 mediated triggering but permits CD26 induced costimulation in TCR negative cells.

Postoperative assessment of porcine-valved right ventricular-pulmonary artery conduits.

Insertion of extracardiac right ventricular (RV)-pulmonary artery (PA) conduits for repair of severe forms of RV-PA discontinuity has become a widely used technique. During a 10-year period, 76 patients had open heart repair with a porcine-valved RV-PA external conduit. The most common diagnoses were truncus arteriosus (15%) and pulmonary atresia with ventricular septal defect (15%). The patients were 18 days to 37 years old (median 7 years). The postoperative median follow-up period was 4 years. Four patients (8%) have been lost to follow-up. Nineteen patients (25%) died in the early postoperative period and there were 10 late deaths (13%). Calculated probability of survival was 66% at 8 years. Thirty-six patients have undergone late hemodynamic studies. A residual shunt was present in 14% of the patients; moderate to severe PA hypertension was present in 25% and significant RV-PA gradients (greater than 30 mm Hg) in 50%. The mean RV pressure for the group was 78.5 +/- 38 mm Hg. The severity of the gradients appeared to be greater in patients who had been followed longer. Ten patients have undergone reoperation because of conduit obstruction or residual shunting; 6 are alive and well. Of the surviving patients, 95% are acyanotic and without significant exercise intolerance and only 5% have cardiovascular symptoms. Thus, open heart repair using an external RV-PA conduit provides a marked improvement in the quality of life in patients who survive.

Method for recording electrical activity of the sinoatrial node and automatic atrial foci during cardiac catheterization in human subjects.

A method for recording electrical activity of the sinoatrial (S-A) node and automatic atrial foci in human subjects is described. To record S-A nodal electrograms, an electrode catheter was inserted percutaneously into the femoral vein and advanced under fluoroscopic control to the superior vena caval-right atrial junction. The distal terminal of the catheter was placed in the area of the S-A node and the proximal terminal on the free right atrial wall or in the right atrial lumen. Polarity was reversed from the conventional electrocardiographic recording; high amplification (about 100 microV/cm) and selective filters (0.1 to 20 hertz) were used. S-A nodal electrograms recorded with this method in human subjects were similar to electrograms obtained previously from the dog and rabbit and revealed negatively directed diastolic and upstroke slopes preceding the P wave of the electrocardiogram. Sinoatrial conduction time measured from the S-A nodal electrograms in 15 cases was 34.9 +/- 2.1 ms(mean +/- standard error of the mean) for a sinus cycle length of 736.4 +/- 38.6 ms. The coronary sinus electrograms in a patient with coronary sinus rhythm were recorded by the same technique except that the distal terminal of the catheter was placed at the coronary sinus ostium. A negatively directed diastolic slope preceding the P wave was consistently recorded. This method for recording electrograms of the S-A node and ectopic automatic atrial foci should prove useful in (1) assessment of both normal and abnormal S-A nodal function, (2) direct determination of conduction time from the S-A nodal pacemaker to the atrium, and (3) localization of automatic atrial foci.

Clonal specificity analysis of mitogen-activated murine T lymphoblasts.

We have determined the frequencies and specificities of MHC-reactive and MHC-restricted cytotoxic T lymphocyte precursors (CTL-p) in mitogen (ConA)-activated splenocytes of normal unprimed mice. The limiting dilution (LD) system supported the growth of one out of three Lyt2+ T cell blasts. The generated CTL-populations lysed blast cell targets specifically as determined by split well analyses. MHC-gene product expression was necessary for lysis to occur, since MHC-negative F9 teratocarcinoma cells were not lysed. The frequency determinations and split well analyses revealed: 1) equally high numbers (approximately 1/100) of CTL-p that generated specific allo-MHC or self-MHC reactive CTL populations, 2) high frequencies of CTL-p which recognized hapten (TNP) or minor H (MH)-antigens in the context of self MHC or allo-MHC determinants. The results are discussed with respect to antigen, restriction and receptor specificities of mitogen-activated unprimed T cell blasts.

Interaction of natural killer cells with Trypanosoma cruzi-infected fibroblasts.

The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues causing the pathology of Chagas' disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we demonstrate that NK cells trigger trypanocidal mechanisms in infected L929 cells that are critically dependent on inducible nitric oxide (NO) synthase (iNOS) induction which is, to a major degree, triggered by interferon (IFN)-gamma provided by NK cells. This work provides a more detailed analysis of how NK cells as a part of the innate immune system participate in the control of parasites that reside intracellularly in fibroblast-like L929 cells.

RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses.

We have reinvestigated the ability of the matrix protein (MA) (p19gag) of avian sarcoma and leukemia viruses to interact with RNA. Previous reports claimed on the one hand that MA can bind tightly and with a high degree of specificity to avian sarcoma and leukemia virus RNA in vitro and on the other that it cannot bind to RNA at all. We found that MA purified by any of several methods does bind to RNA, as measured by its ability to cause retention of radioactive RNA on nitrocellulose membranes in a filtration assay. However, this interaction is weak and lacks specificity. The interaction of MA with RNA was barely detectable by classical sedimentation analysis, and from this observation we estimate that the intrinsic MA-RNA association constant is ca. 10(3) M-1, at least 3 orders of magnitude smaller than the constant describing the interaction of the viral nucleocapsid protein (NC) (p12gag) with RNA, ca. 10(6) M-1. Separately purified phosphorylated and nonphosphorylated MA species bound RNA equally. We also found that MA can bind to DNA with an affinity similar to that for RNA. The large quantitative discrepancy between our results and earlier published reports can be traced in part to methods of data analysis.

Unchanged signaling capacity of mutant CD26/dipeptidylpeptidase IV molecules devoid of enzymatic activity.

CD26 is a proteolytic enzyme (dipeptidylpeptidase IV) that defines an alternative activation signal for human T lymphocytes. Crosslinking of CD26 via monoclonal antibodies triggers proliferation and cytotoxicity in CD26-positive T cells or provides costimulatory signals for these cells. Because there is some debate about whether the enzymatic activity plays a role in activation via CD26 we have here generated a mutant CD26 molecule devoid of enzymatic activity. After transfection into T cell receptor-positive recipient T cells, such mutant molecules were tested for their signaling capacity compared to that in the wildtype molecules. The response of transfected clones to direct stimulation with anti-CD26 antibodies and to costimulation via CD26 was variable and not solely dependent on the amount of CD26 and T cell receptor expressed on the T cells. Several mutant transfectants were more easily triggered via CD26 than cells transfected with the wildtype molecule. These data demonstrate that the enzymatic activity of CD26 is not required for its T cell activating or costimulating properties.

Cardiovascular malformations in the fetal alcohol syndrome.

Cardiovascular anomalies are a frequently reported feature of the fetal alcohol syndrome, but only rarely have such children been catheterized. This article fully described the cardiac anomaly in two infants with this syndrome. The malformations found in both cases include a ventricular septal defect, and the rather unusual finding of pulmonary artery dysplasia.

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo.

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.

Introduction of specific point mutations into RNA polymerase II by gene targeting in mouse embryonic stem cells: evidence for a DNA mismatch repair mechanism.

We have introduced two specific point mutations, located 20 base pairs apart, into the endogenous murine gene that encodes the largest subunit of RNA polymerase II (RPII215). The first mutation conferred resistance to the mushroom toxin alpha-amanitin (amar), and the second mutation generated a restriction fragment length polymorphism without altering the protein sequence. Targeted amar clones were generated at a frequency of 1 in 30 totipotent embryonic stem cells that expressed stably integrated DNA vectors after electroporation. Thirty to 40% of these clones had acquired both mutations, whereas, surprisingly, the remaining clones had acquired the specific amar point mutation but lacked the restriction fragment length polymorphism. We suggest that the latter clones were generated by independent DNA mismatch repair rather than by double crossover or gene conversion. These results demonstrate that it is possible to introduce specific point mutations into an endogenous gene in embryonic stem cells. Thus it should be possible to introduce single base substitutions into other cellular genes, including nonselectable genes, by optimizing the efficiency of gene transfer and/or the sensitivity of screening for targeted clones.