Cross-Clade Recognition of HIV-1 CAp24 by CD4+ T Cells in HIV-1-Infected Individuals in Burkina Faso and Germany

The presence of antigen-specific cellular immune responses may be an indicator of long-term asymptomatic HIV-1-disease. The detection of cellular immune responses to infection with different subtypes of HIV-1 may be hampered by genetic differences of immunodominant antigens such as the capsid protein CAp24. In Nouna, Burkina Faso, HIV-1 circulating recombinant forms CRF02_AG and CRF06_cpx are the 2 major strains detectable in HIV-1-infected individuals, while subtype B strains prevail in Europe and North America. Amino acid sequences of CAp24 were assessed in blood samples from 10 HIV-1-infected patients in Nouna, Burkina Faso. Production of interferon-gamma (IFN-γ) in peripheral blood CD4+ lymphocytes in response to recombinant HIV-1 proteins derived from clade B (including CAp24NL4-3) was measured using a modified flow-cytometry-based whole blood short term activation assay (FASTimmune, BDBiosciences). IFN-γ production following stimulation with a whole length CAp24 protein derived from clade B (CAp24NL4-3) was additionally quantified in comparison to a CAp24 protein derived from CRF02_AG (CAp24BD6-15) in 16 HIV-1-infected patients in Heidelberg, Germany. Amino acid sequence identity of CAp24 obtained from patients in Nouna ranged between 86 and 89% when compared to the clade B CAp24NL4-3 consensus sequence, between 90 and 95% when compared to the circulating recombinant form CRF06_CPX consensus sequence, and between 92 and 96% when compared to the CAp24BD6-15 consensus sequence. Significant numbers of HIV-1-specific CD4+ lymphocytes producing IFN-γ were detected in 4 of 10 HIV-1-infected patients. In 7 of 16 patients in Heidelberg, recombinant CAp24BD6-15 stimulated IFN-γ-production in CD4+ lymphocytes to a similar extent as the clade B-derived CAp24NL4-3. Thus, antigen-specific CD4+ lymphocytes from both West African and European patients infected with different strains of HIV-1 show relevant cross-clade recognition of HIV-1 CAp24 in a flow-cytometry-based whole blood short term activation assay

Disruption of Dnmt1/PCNA/UHRF1 Interactions Promotes Tumorigenesis from Human and Mice Glial Cells

Global DNA hypomethylation is a hallmark of cancer cells, but its molecular mechanisms have not been elucidated. Here, we show that the disruption of Dnmt1/PCNA/UHRF1 interactions promotes a global DNA hypomethylation in human gliomas. We then demonstrate that the Dnmt1 phosphorylations by Akt and/or PKC abrogate the interactions of Dnmt1 with PCNA and UHRF1 in cellular and acelluar studies including mass spectrometric analyses and the use of primary cultured patient-derived glioma. By using methylated DNA immunoprecipitation, methylation and CGH arrays, we show that global DNA hypomethylation is associated with genes hypomethylation, hypomethylation of DNA repeat element and chromosomal instability. Our results reveal that the disruption of Dnmt1/PCNA/UHRF1 interactions acts as an oncogenic event and that one of its signatures (i.e. the low level of mMTase activity) is a molecular biomarker associated with a poor prognosis in GBM patients. We identify the genetic and epigenetic alterations which collectively promote the acquisition of tumor/glioma traits by human astrocytes and glial progenitor cells as that promoting high proliferation and apoptosis evasion

The KCNE genes in hypertrophic cardiomyopathy: a candidate gene study

The original publication is available at http://www.jnrbm.com/content/10/1/12Includes bibliographyAbstract Background The gene family KCNE1-5, which encode modulating β-subunits of several repolarising K+-ion channels, has been associated with genetic cardiac diseases such as long QT syndrome, atrial fibrillation and Brugada syndrome. The minK peptide, encoded by KCNE1, is attached to the Z-disc of the sarcomere as well as the T-tubules of the sarcolemma. It has been suggested that minK forms part of an "electro-mechanical feed-back" which links cardiomyocyte stretching to changes in ion channel function. We examined whether mutations in KCNE genes were associated with hypertrophic cardiomyopathy (HCM), a genetic disease associated with an improper hypertrophic response. Results The coding regions of KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5 were examined, by direct DNA sequencing, in a cohort of 93 unrelated HCM probands and 188 blood donor controls. Fifteen genetic variants, four previously unknown, were identified in the HCM probands. Eight variants were non-synonymous and one was located in the 3'UTR-region of KCNE4. No disease-causing mutations were found and no significant difference in the frequency of genetic variants was found between HCM probands and controls. Two variants of likely functional significance were found in controls only. Conclusions Mutations in KCNE genes are not a common cause of HCM and polymorphisms in these genes do not seem to be associated with a propensity to develop arrhythmiaPeer Reviewe

Vectors Based on Modified Vaccinia Ankara Expressing Influenza H5N1 Hemagglutinin Induce Substantial Cross-Clade Protective Immunity

New highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine.The replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades.The highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine

A SNAP-Tagged Derivative of HIV-1—A Versatile Tool to Study Virus-Cell Interactions

Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches

GABA Maintains the Proliferation of Progenitors in the Developing Chick Ciliary Marginal Zone and Non-Pigmented Ciliary Epithelium

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABAA receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABAA receptor system. To quantify the effects on proliferation by GABAA receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABAA receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABAA receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl–transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABAA receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABAA receptors. This supported the depolarising role for the GABAA receptors. Inhibition of L-type voltage-gated Ca2+ channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABAA receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27KIP1, along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27KIP1 after inhibition of either the GABAA receptors or the L-type VGCCs suggests a link between the GABAA receptors, membrane potential, and intracellular Ca2+ in regulating the cell cycle

Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity

Breast Cancer Epigenetics: From DNA Methylation to microRNAs

Both appropriate DNA methylation and histone modifications play a crucial role in the maintenance of normal cell function and cellular identity. In cancerous cells these “epigenetic belts” become massively perturbed, leading to significant changes in expression profiles which confer advantage to the development of a malignant phenotype. DNA (cytosine-5)-methyltransferase 1 (Dnmt1), Dnmt3a and Dnmt3b are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells. Intriguingly, DNMTs were found to be overexpressed in cancerous cells, which is believed to partly explain the hypermethylation phenomenon commonly observed in tumors. However, several lines of evidence indicate that further layers of gene regulation are critical coordinators of DNMT expression, catalytic activity and target specificity. Splice variants of DNMT transcripts have been detected which seem to modulate methyltransferase activity. Also, the DNMT mRNA 3′UTR as well as the coding sequence harbors multiple binding sites for trans-acting factors guiding post-transcriptional regulation and transcript stabilization. Moreover, microRNAs targeting DNMT transcripts have recently been discovered in normal cells, yet expression of these microRNAs was found to be diminished in breast cancer tissues. In this review we summarize the current knowledge on mechanisms which potentially lead to the establishment of a DNA hypermethylome in cancer cells