Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock

Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved β-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the β-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.     

Eosinophils in innate immunity: an evolving story

Eosinophils are innate immune leukocytes found in relatively low numbers within the blood. Terminal effector functions of eosinophils, deriving from their capacity to release their content of tissue-destructive cationic proteins, have historically been considered primary effector mechanisms against specific parasites, and are likewise implicated in tissue damage accompanying allergic responses such as asthma. However, the past decade has seen dramatic advancements in the field of eosinophil immunobiology, revealing eosinophils to also be key participants in many other facets of innate immunity, from bridging innate and adaptive immune responses to orchestrating tissue remodeling events. Here, we review the multifaceted functions of eosinophils in innate immunity that are currently known, and discuss new avenues in this evolving story.     

Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation

Sudden cardiac death caused by ventricular arrhythmias is a disastrous event, especially when it occurs in young individuals. Among the five major arrhythmogenic disorders occurring in the absence of a structural heart disease is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmias. Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation contraction coupling, the derangements in intracellular Ca(2+) handling may cause delayed afterdepolarizations (DADs), which constitute the mechanism underlying CPVT. To investigate catecholamine-induced arrhythmias in the CASQ2 mutated cells, we generated for the first time CPVT-derived induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant CASQ2 protein. The major findings were that the β-adrenergic agonist isoproterenol caused in CPVT iPSCs-CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after-contractions and diastolic [Ca(2+) ](i) rise. Electron microscopy analysis revealed that compared with control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae. In summary, our results demonstrate that the patient-specific mutated cardiomyocytes can be used to study the electrophysiological mechanisms underlying CPVT.     

Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene

MYH9 has been proposed as a major genetic risk locus for a spectrum of nondiabetic end stage kidney disease (ESKD). We use recently released sequences from the 1000 Genomes Project to identify two western African-specific missense mutations (S342G and I384M) in the neighboring APOL1 gene, and demonstrate that these are more strongly associated with ESKD than previously reported MYH9 variants. The APOL1 gene product, apolipoprotein L-1, has been studied for its roles in trypanosomal lysis, autophagic cell death, lipid metabolism, as well as vascular and other biological activities. We also show that the distribution of these newly identified APOL1 risk variants in African populations is consistent with the pattern of African ancestry ESKD risk previously attributed to MYH9. Mapping by admixture linkage disequilibrium (MALD) localized an interval on chromosome 22, in a region that includes the MYH9 gene, which was shown to contain African ancestry risk variants associated with certain forms of ESKD (Kao et al. 2008; Kopp et al. 2008). MYH9 encodes nonmuscle myosin heavy chain IIa, a major cytoskeletal nanomotor protein expressed in many cell types, including podocyte cells of the renal glomerulus. Moreover, 39 different coding region mutations in MYH9 have been identified in patients with a group of rare syndromes, collectively termed the Giant Platelet Syndromes, with clear autosomal dominant inheritance, and various clinical manifestations, sometimes also including glomerular pathology and chronic kidney disease (Kopp 2010; Sekine et al. 2010). Accordingly, MYH9 was further explored in these studies as the leading candidate gene responsible for the MALD signal. Dense mapping of MYH9 identified individual single nucleotide polymorphisms (SNPs) and sets of such SNPs grouped as haplotypes that were found to be highly associated with a large and important group of ESKD risk phenotypes, which as a consequence were designated as MYH9-associated nephropathies (Bostrom and Freedman 2010). These included HIV-associated nephropathy (HIVAN), primary nonmonogenic forms of focal segmental glomerulosclerosis, and hypertension affiliated chronic kidney disease not attributed to other etiologies (Bostrom and Freedman 2010). The MYH9 SNP and haplotype associations observed with these forms of ESKD yielded the largest odds ratios (OR) reported to date for the association of common variants with common disease risk (Winkler et al. 2010). Two specific MYH9 variants (rs5750250 of S-haplotype and rs11912763 of F-haplotype) were designated as most strongly predictive on the basis of Receiver Operating Characteristic analysis (Nelson et al. 2010). These MYH9 association studies were then also extended to earlier stage and related kidney disease phenotypes and to population groups with varying degrees of recent African ancestry admixture (Behar et al. 2010; Freedman et al. 2009a, b; Nelson et al. 2010), and led to the expectation of finding a functional African ancestry causative variant within MYH9. However, despite intensive efforts including re-sequencing of the MYH9 gene no suggested functional mutation has been identified (Nelson et al. 2010; Winkler et al. 2010). This led us to re-examine the interval surrounding MYH9 and to the detection of novel missense mutations with predicted functional effects in the neighboring APOL1 gene, which are significantly more associated with ESKD than all previously reported SNPs in MYH9.     

Chemo-immunotherapy of murine tumors using interleukin-2 (IL-2) and cyclophosphamide

Summary The antitumor effect of interleukin-2 (IL-2), alone and in combination with cyclophosphamide was assessed in mice with established sarcoma (MCA 105, H-2^b), carcinoma (M109, H-2^d) and T lymphoma (PIR-2, H-2^b). Whereas administration of IL-2 alone (5×10⁴–10×10⁴ U, i.p. twice daily, for 4–8 consecutive days) prolonged the survival of mice with the solid neoplasms, it enhanced tumor growth and decreased survival of mice with the lymphoma. In the PIR-2 lymphoma, no IL-2 receptor (TAC) could be detected, nor could we demonstrate IL-2 tumor growth stimulation in vitro. A synergistic therapeutic effect was achieved in mice with the solid tumors, but not in mice with the lymphoma, only when IL-2 was given 1–4 days after cyclophosphamide (100–200 mg/kg). Conversely, administration of IL-2 1–4 days prior to cyclophosphamide resulted, in all three tumor systems, in enhanced tumor growth and in decreased survival as compared with mice receiving cyclophosphamide alone. Similarly, treatment with IL-2 both before and after cyclophosphamide was less efficacious than a single course of IL-2 given after-wards. It is concluded that for maximal therapeutic efficacy, IL-2 should be administered following chemotherapy, and that certain tumors may respond adversely to IL-2 treatment.     

Studies on the rhizosphere microflora of yellow birch seedlings

Summary The rhizosphere microflora of yellow birch seedlings grown in a forest soil in tanks in the greenhouse has been investigated. In general it was found that the rhizosphere effect of yellow birch roots in the A horizon was lower than that observed with cropped plants; nevertheless a stimulation of microbial development was obtained. Of the micro-organisms studied, bacteria, and ammonifying and methylene-blue-reducing organisms appeared to be predominant types in the root zone. Counts were, on the whole, lowest when plants broke dormancy and began to increase in the later periods of growth. There was no appreciable difference between control and rhizosphere soils in regard to incidence of specific nutritional types of bacteria. Analysis of the rhizosphere population of roots growing in the B horizon showed a distinctly greater rhizosphere effect than that obtained in the A horizon. The data suggest that the rhizosphere effect in the latter was obscured because of its high organic-matter content and its abundant and active microflora.Contribution No. 481     

Relationship between genetic anomalies of different levels and deviation in dermatoglyphic traits

The present study is carried out to evaluate the effect of chromosomal morbidity (82% are 47XXY and in the remaining cases there is the extra X and/or Y) in the males with Klinefelter’s syndrome, based on dermatoglyphic traits and indices of diversity and asymmetry. The main objectives of the present study is to find dermatoglyphic traits and fluctuating asymmetry indices which could be “marker traits” and could indicate the degree of developmental instability of the organism. The sample of males with Klinefelter’s syndrome (N=171) was collected in the Institute of Human Genetics of Sheba Medical Center, Ramat-Gan, Israel, by Professor Bat-Miriam Katznelson during 20 years, between 1968–1988. All patients were confirmed by chromosomal examination. The finger and palm prints were collected with the aid of pads manufactured by Lamedco Inc., Knoxville, Tennessee, U.S.A. Interpretation of the prints was according to Cummins and Midlo (1961) and Penrose (1968) and included identification of patterns, ridge counts and the measurements of distances sum of and angles in the palms. 79 dermatoglyphic variables for every patient: 28 continuous traits, 9 discrete traits, 11 indices of intra-individual diversity, 15 indices of directional asymmetry and 16 indices of fluctuating asymmetry were estimated. The problem of asymmetry, fluctuating and directional and of intraindividual diversity of quantitative dermatoglyphic traits is reviewed here as well as illustrated by data obtained on a sample of healthy control group of Jews from Israel. In this first part of our paper we will discuss the data on individual dermatoglyphic traits on digits and palms. The second part will be dedicated to multivariate analysis in order to compare between Klinefelter’s syndrome and control healthy individuals based on quantitative dermatoglyphic traits and indices of diversity and asymmetry.     

Ex vivo generated regulatory T cells modulate experimental autoimmune myasthenia gravis

Naturally occurring CD4(+)CD25(+) regulatory T (Treg) cells are key players in immune tolerance and have therefore been suggested as potential therapeutic tools for autoimmune diseases. In myasthenia gravis (MG), reduced numbers or functionally impaired Treg cells have been reported. We have observed that PBL from myasthenic rats contain decreased numbers of CD4(+)CD25(high)Foxp3(+) cells as compared with PBL from healthy controls, and we have tested whether Treg cells from healthy donors can suppress experimental autoimmune MG in rats. Because the number of naturally occurring Treg cells is low, we used an approach for a large-scale ex vivo generation of functional Treg cells from CD4(+) splenocytes of healthy donor rats. Treg cells were generated ex vivo from CD4(+) cells by stimulation with anti-CD3 and anti-CD28 Abs in the presence of TGF-beta and IL-2. The obtained cells expressed high levels of CD25, CTLA-4, and Foxp3, and they were capable of suppressing in vitro proliferation of T cells from myasthenic rats in response to acetylcholine receptor, the major autoantigen in myasthenia. Administration of ex vivo-generated Treg cells to myasthenic rats inhibited the progression of experimental autoimmune MG and led to down-regulation of humoral acetylcholine receptor-specific responses, and to decreased IL-18 and IL-10 expression. The number of CD4(+)CD25(+) cells in the spleen of treated rats remained unchanged, but the subpopulation of CD4(+)CD25(+) cells expressing Foxp3 was significantly elevated. Our findings imply that Treg cells play a critical role in the control of myasthenia and could thus be considered as potential agents for the treatment of MG patients.     

An endocytic mechanism for haemoglobin-iron acquisition in Candida albicans

The fungal pathogen Candida albicans is able to utilize haemin and haemoglobin as iron sources. Haem-iron utilization is facilitated by Rbt5, an extracellular, glycosylphophatidylinositol (GPI)-anchored, haemin- and haemoglobin-binding protein. Here, we show that Rbt5 and its close homologue Rbt51 are short-lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of haemoglobin into the C.  albicans vacuole. We relied on recapitulation of the Rbt51-dependent haem-iron utilization in Saccharomyces cerevisiae to identify mutants defective in haemoglobin utilization. Homologues of representative mutants in S. cerevisiae were deleted in C. albicans and tested for haemoglobin-iron utilization and haemoglobin uptake. These mutants define a novel endocytosis-mediated haemoglobin utilization mechanism that depends on acidification of the lumen of the late secretory pathway, on a type I myosin and on the activity of the ESCRT pathway.