Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani

Background

Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown.

Methodology/Principal Findings

We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFβ and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFβ and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFβ to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFβ respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4⁺CD25⁺ (r = 0.55), CD4⁺CD25^hi (r = 0.61) as well as percentages of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFβ in comparison to corresponding CD25^- T cells.

Conclusions/Significance

Our findings shed some light on source(s) of TGFβ and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4⁺CD25⁺FoxP3⁺ Treg cells as a source of IL-10 and TGFβ during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.     

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.     

Profiles of Serum Cytokines in Acute Drug-Induced Liver Injury and Their Prognostic Significance

Drug-induced liver injury (DILI) is the most common cause of acute liver failure in the United-States. The aim of the study was to describe serum immune profiles associated with acute DILI, to investigate whether there are profiles associated with clinical features or types of DILI and/or with prognosis, and to assess temporal changes in levels. Twenty-seven immune analytes were measured in the sera of 78 DILI subjects in the Drug-Induced Liver Injury Network (DILIN) and compared with 40 healthy controls. Immune analytes (14 cytokines, 7 chemokines and 6 growth factors) were measured by BioPlex multiplex ELISA at DILI onset and after 6 months. A modeling process utilizing immune principles was used to select a final set of variables among 27 immune analytes and several additional clinical lab values for prediction of early death (within 6 months of DILI onset). Nineteen of the 27 immune analytes were differentially expressed among healthy control, DILI onset and 6-month cohorts. Disparate patterns of immune responses, especially innate and adaptive cellular (mostly TH17) immunity were evident. Low values of four immune analytes (IL-9, IL-17, PDGF-bb and RANTES) and serum albumin are predictive of early death [PPV = 88% (95% CI, 65%-100%), NPV = 97% (95% CI, 93%-100%), accuracy = 96% (95% CI, 92%-100%)].

Conclusions

Acute DILI is associated with robust and varying immune responses. High levels of expression of cytokines associated with innate immunity are associated with a poor prognosis, whereas high levels of expression of adaptive cytokines are associated with good long-term prognosis and eventual recovery. Serum immune analyte profiles at DILI onset appear to be of prognostic, and perhaps, diagnostic significance.     

The kinase homology domain of receptor guanylyl cyclase C: ATP binding and identification of an adenine nucleotide sensitive site

The role of the kinase homology domain (KHD) in receptor guanylyl cyclases is to regulate the activity of the catalytic guanylyl cyclase domain. The KHD lacks many of the amino acids required for phosphotransfer activity and, therefore, is not expected to possess kinase activity. Guanylyl cyclase activity of the receptor guanylyl cyclase C (GC-C) is modulated by ATP, and computational modeling showed that the KHD can adopt a structure similar to protein kinases, suggesting that the KHD is the site for ATP interaction. A monoclonal antibody, GCC:4D7, raised to the KHD of GC-C, fails to react with GC-C in the presence of ATP and ATP analogues that regulate GC-C catalytic activity, indicating that a conformational change occurs in the KHD on ATP binding. Mapping of the epitope of the antibody through the use of recombinant protein constructs and phage display showed that the epitope for GC-C:4D7 lies immediately C-terminal to a critical lysine residue (Lys516 in GC-C), required for ATP interaction in protein kinases. By employing a novel approach utilizing ATP-agarose affinity chromatography, we demonstrate that the intracellular domain of GC-C and the KHD bind ATP. Mutation of Lys516 to Ala abolishes ATP binding. Thus, this report is the first to show direct ATP binding to the pseudokinase domain of receptor guanylyl cyclase C, as well as to identify dramatic conformational changes that occur in this domain on ATP binding, akin to those seen in catalytically active protein kinases.     

Distribution of plasma alpha-1-B-glycoprotein phenotypes in several Mongoloid populations of East Asia

The distribution of plasma alpha 1B-glycoprotein (alpha 1B) phenotypes was determined by a simple method of two-dimensional electrophoresis followed by protein staining in a group of 1,154 individuals from 8 Mongoloid populations of East Asia. The sample comprised 581 Chinese from different localities (Singapore: 204; Taiwan: 150; Fujien and Hopeh provinces of eastern China: 146 and 81), 155 Koreans, 155 Filipinos, 152 Thais and 111 Malays. Altogether, 6 different alpha 1B phenotypes (1-1, 1-2, 2-2, 1-3, 2-3, and 1-6) were observed. The alpha 1B allele frequencies were very similar in all of the populations. The frequency of A1B*1 varied from 0.89 to 0.91 and that of A1B*2 from 0.08 to 0.10. The A1B*3 allele, reported previously only in American blacks, was observed with a frequency range of 0.003-0.01 in 3 of the Chinese populations, in Koreans and in Malays. A new alpha 1B allele (A1B*6) was observed in 2 Chinese individuals.