Human natural regulatory T cell development, suppressive function, and postthymic maturation in a humanized mouse model

CD4(+) regulatory T cells (Tregs) control adaptive immune responses and promote self-tolerance. Various humanized mouse models have been developed in efforts to reproduce and study a human immune system. However, in models that require T cell differentiation in the recipient murine thymus, only low numbers of T cells populate the peripheral immune systems. T cells are positively selected by mouse MHC and therefore do not function well in an HLA-restricted manner. In contrast, cotransplantation of human fetal thymus/liver and i.v. injection of CD34(+) cells from the same donor achieves multilineage human lymphohematopoietic reconstitution, including dendritic cells and formation of secondary lymphoid organs, in NOD/SCID mice. Strong Ag-specific immune responses and homeostatic expansion of human T cells that are dependent on peripheral human APCs occur. We now demonstrate that FOXP3(+)Helios(+) "natural" Tregs develop normally in human fetal thymic grafts and are present in peripheral blood, spleen, and lymph nodes of these humanized mice. Humanized mice exhibit normal reversal of CD45 isoform expression in association with thymic egress, postthymic "naive" to "activated" phenotypic conversion, and suppressive function. These studies demonstrate the utility of this humanized mouse model for the study of human Treg ontogeny, immunobiology and therapy.     

Proteomic analysis of cells in the early stages of herpes simplex virus type‐1 infection reveals widespread changes in the host cell proteome

During infection by herpes simplex virus type‐1 (HSV‐1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV‐1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV‐1 replication, 2‐DE and LC‐MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock‐ and HSV‐1‐infected HEp‐2 cells revealed a total of 103 protein spot changes. Of these, 63 were up‐regulated and 40 down‐regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV‐1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV‐1 infection.     

Transcriptomic response to differentiation induction

Background  

Microarrays used for gene expression studies yield large amounts of data. The processing of such data typically leads to lists of differentially-regulated genes. A common terminal data analysis step is to map pathways of potentially interrelated genes.     

Malaria prophylaxis policy for travellers from Europe to the Indian Sub Continent

Background

Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge.

Methods

The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia.

Results

Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities.

Conclusion

It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density.     

Functional classification analysis of somatically mutated genes in human breast and colorectal cancers

A recent study published by Sjoblom and colleagues [T. Sjoblom, S. Jones, L.D. Wood, D.W. Parsons, J. Lin, T.D. Barber, D. Mandelker, R.J. Leary, J. Ptak, N. Silliman, S. Szabo, P. Buckhaults, C. Farrell, P. Meeh, S.D. Markowitz, J. Willis, D. Dawson, J.K. Willson, A.F. Gazdar, J. Hartigan, L. Wu, C. Liu, G. Parmigiani, B.H. Park, K.E. Bachman, N. Papadopoulos, B. Vogelstein, K.W. Kinzler, V.E. Velculescu, The consensus coding sequences of human breast and colorectal cancers. Science 314 (2006) 268-274.] performed comprehensive sequencing of 13,023 human genes and identified mutations in genes specific to breast and colorectal tumors, providing insight into organ-specific tumor biology. Here we present a systematic analysis of the functional classifications of Sjoblom's “CAN” genes, a subset of these validated mutant genes, that identifies novel organ-specific biological themes and molecular pathways associated with disease-specific etiology. This analysis links four somatically mutated genes associated with diverse oncological types to colorectal and breast cancers through established TGF-β1-regulated interactions, revealing mechanistic differences in these cancers and providing potential diagnostic and therapeutic targets.     

Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors.