Controle epigenético do gene imprinted SNRPN durante o desenvolvimento e reprogramação nuclear em equídeos

A tranferência nuclear de células somáticas (TNCS) está sendo utilizada para produzir cavalos de elite. No entanto, durante este procedimento pode ocorrer a perfuração da zona pelúcida, levando, ocasionalmente, à secção da massa celular interna, e conseqüente derivação de gêmeos monozigóticos. Além de serem relatadas alterações no processo de imprinting genômico, que conduzem ao desenvolvimento de doenças. Com a descoberta da possibilidade de reprogramar as células somáticas a um estado de pluripotência (iPSCs), estas células passaram a ser muito utilizadas em pesquisas de neurociência. Contudo, também ocorrem modificações epigenéticas durante esta reprogramação celular. Portanto, nossas hipóteses são que os gêmeos eqüinos gerados pela TNCS podem levar às irregularidades no desenvolvimento do sistema nervoso. O padrão de metilação do SNRPN nas estruturas dos fetos muares clonados, e as células iPSCs são diferentes dos padrões encontrados nos muares analisados. A expressão dos genes SNRPN, Necdin e UBE3A são maiores no cérebro, enquanto a expressão do H19 é maior nas membranas extra-embrionárias. Em nosso estudo, obtivemos duas gestações gemelares equinas derivadas da TNCS, que foram interrompidas com 40 e 60 dias de gestação, e comparados com gestações eqüinas únicas de idade similar. Diferenças no comprimento entre os embriões gêmeos foram observadas aos 40 (2.0 e 2.2 cm 10%) e aos 60 (6,5 e 8,5 cm 24%) dias de gestação. Somente o plexo coróide do quarto ventrículo apresentou-se mais desenvolvido nos fetos com maior comprimento. Ao analisarmos fetos muares clonados em diferentes idades gestacionais e compará-los com muares, nos períodos embrionário, fetal e adulto, não foi observada diferença no padrão de metilação do gene SNRPN. No entanto, na décima passagem das células iPSC o padrão de metilação alterou, em relação aos muares estudados e ao padrão observado nos fibroblastos. Ao analisarmos os fetos clonados nas diferentes idades gestacionais observou-se no cérebro menor expressão dos gene H19 e UBE3A, e maior expressão do gene SNRPN. Contudo, a expressão do gene Necdin variou entre as estruturas estudadas. Em conclusão, apesar dos gêmeos eqüinos provenientes de TNCS diferirem quanto ao tamanho, morfologicamente são iguais. Dentre as estruturas cerebrais o plexo coróide se apresentou mais desenvolvido nos fetos de maior comprimento. Os fetos muares clonados não apresentaram diferença no padrão de metilação do gene SNRPN. No entanto, as iPSCs apresentaram alteração no padrão de metilação deste gene na décima passagem. Embora os genes SNRPN, Necdin e UBE3A sejam expressos no cérebro, o SNRPN apresentou-se prevalente nessa estrutura

Identification of the nuclear localization signals within the Epstein-Barr virus EBNA-6 protein

Epstein-Barr virus nuclear antigen (EBNA)-6 is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. Previous studies have shown that EBNA-6 acts as a transcriptional regulator of viral and cellular genes; however at present, few functional domains of the 140 kDa EBNA-6 protein have been completely characterized. There are five computer-predicted nuclear localization signals (NLS), four monopartite and one bipartite, present in the EBNA-6 amino acid sequence. To identify which of these NLS are functional, fusion proteins between green fluorescent protein and deletion constructs of EBNA-6 were expressed in HeLa cells, Each of the constructs containing at least one of the NLS was targeted to the nucleus of cells whereas a construct lacking all of the NLS was cytoplasmic. Site-directed mutation of these NLS demonstrated that only three of the NLS were functional, one at the N-terminal end (aa 72-80), one in the middle (aa 412-418) and one at the C-terminal end (aa 939-945) of the EBNA-6 protein.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

Synthesis of fluorescent dendrimeric antigen efficiently internalized by human dendritic cells

A new fluorescent dendrimeric antigen (DeAn) based on a dendron with amoxicilloyl terminal groups has been synthetized. The synthesis implies a novel class of all-aliphatic polyamide dendrimer (BisAminoalkylPolyAmide Dendrimers, or BAPAD).[1] The introduction of a cystamine core allows the incorporation of this dendrons into a 1,8-naphthalimide fluorofore functionalized with a maleimide group. The fluorescence properties of this DeAn has been studied and compared with the properties of an equivalent dendron possessing amino-terminal groups. This DeAn has been used as a synthetic antigen in a biomedical assay that tests the amoxicillin sensitivity of dendritic cells (DC) from tolerant and allergic patients.

Estudo morfológico e imunohistoquímico da expressão de PCNA e p63 no carcinoma espinocelular oral em gatos

Dissertação de Mestrado Integrado em Medicina Veterinária

Rheumatoid arthritis sera react with a phage-displayed peptide selected by a monoclonal antibody to type II collagen that has homology to EBNA-1

Antibodies to type II collagen, and to Epstein Barr virus nuclear antigen-1 (EBNA-1) have been associated with rheumatoid arthritis (RA). In studies involving probing of phage-displayed random peptide libraries with an antibody to type II collagen, CII-C1, we observed that among 17 phagotopes selected 5 expressed peptides with homology with the sequence of EBNA-1. The residues in common were RLPFG. Hence we tested sera from 50 patients with RA, of whom 26 had antibodies to native type II collagen, and 43 healthy controls, for reactivity by ELISA with a phagotope selected 4 times, which expressed the peptide RRLPFGSQM. Eight RA sera (16%) but no normal sera reacted with the phagotope (p = 0.025). This reactivity could not be correlated with reactivity of RA sera with EBNA-1 by semi-quantitative western blot, with which reactivity occurred in 78% of RA patients and 81% of controls. Evidence for molecular mimicry was not found insofar as the phagotope did not inhibit reactivity of RA sera with EBNA-1 and CII-C1 was not reactive with EBNA-1. We conclude that the reactivity of the RA sera with the phagotope is most likely due to the phagotope being a mimic of an epitope of type II collagen for a proportion of RA sera.

The role of 25-hydroxyvitamin D deficiency in promoting insulin resistance and inflammation in patients with chronic kidney disease: A randomised controlled trial

BACKGROUND Approximately 50% of patients with stage 3 Chronic Kidney Disease are 25-hydroxyvitamin D insufficient, and this prevalence increases with falling glomerular filtration rate. Vitamin D is now recognised as having pleiotropic roles beyond bone and mineral homeostasis, with the vitamin D receptor and metabolising machinery identified in multiple tissues. Worryingly, recent observational data has highlighted an association between hypovitaminosis D and increased cardiovascular mortality, possibly mediated via vitamin D effects on insulin resistance and inflammation. The main hypothesis of this study is that oral Vitamin D supplementation will ameliorate insulin resistance in patients with Chronic Kidney Disease stage 3 when compared to placebo. Secondary hypotheses will test whether this is associated with decreased inflammation and bone/adipocyte-endocrine dysregulation. METHODS/DESIGN This study is a single-centre, double-blinded, randomised, placebo-controlled trial. Inclusion criteria include; estimated glomerular filtration rate 30-59 ml/min/1.73 m(2); aged >or=18 on entry to study; and serum 25-hydroxyvitamin D levels <75 nmol/L. Patients will be randomised 1:1 to receive either oral cholecalciferol 2000IU/day or placebo for 6 months. The primary outcome will be an improvement in insulin sensitivity, measured by hyperinsulinaemic euglycaemic clamp. Secondary outcome measures will include serum parathyroid hormone, cytokines (Interleukin-1beta, Interleukin-6, Tumour Necrosis Factor alpha), adiponectin (total and High Molecular Weight), osteocalcin (carboxylated and under-carboxylated), peripheral blood mononuclear cell Nuclear Factor Kappa-B p65 binding activity, brachial artery reactivity, aortic pulse wave velocity and waveform analysis, and indirect calorimetry. All outcome measures will be performed at baseline and end of study. DISCUSSION To date, no randomised controlled trial has been performed in pre-dialysis CKD patients to study the correlation between vitamin D status with supplementation, insulin resistance and markers of adverse cardiovascular risk. We remain hopeful that cholecalciferol may be a safe intervention, with health benefits beyond those related to bone-mineral homeostasis. TRIAL REGISTRATION Australian and New Zealand Clinical Trials Registry ACTRN12609000246280.

The Multifunctional PE_PGRS11 Protein from Mycobacterium tuberculosis Plays a Role in Regulating Resistance to Oxidative Stress

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/ 2-NF-kappa B signaling axis. Furthermore, PE_PGRS11 markedly diminished H2O2-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.

Characterization of RNA polymerase III transcription factor TFIIIC from the mulberry silkworm, Bombyx mori

Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori. resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts. The reconstituted transcription of tRNA genes required the presence of both components. The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa. Of these, the 51-kDa subunit could be specifically crosslinked to the B box of tRNA(1)(Gly). Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2. Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification. the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step. The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity. This is the first report on the purification and characterization of TFIIIC from insect systems.

Identification and characterization of cysteine proteinases of Trypanosoma evansi

Trypanosoma evansi is a causative agent of `surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines

Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66 kDa, 52-55 kDa and 41-43 kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280 days post infection (dpi). The protein cluster of 62-66 kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66 kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.

CINCINNATA in Antirrhinum majus directly modulates genes involved in cytokinin and auxin signaling

Mutations in the CINCINNATA (CIN) gene in Antirrhinum majus and its orthologs in Arabidopsis result in crinkly leaves as a result of excess growth towards the leaf margin. CIN homologs code for TCP (TEOSINTE-BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 AND 2) transcription factors and are expressed in a broad zone in a growing leaf distal to the proliferation zone where they accelerate cell maturation. Although a few TCP targets are known, the functional basis of CIN-mediated leaf morphogenesis remains unclear. We compared the global transcription profiles of wild-type and the cin mutant of A. majus to identify the targets of CIN. We cloned and studied the direct targets using RNA in situ hybridization, DNA-protein interaction, chromatin immunoprecipitation and reporter gene analysis. Many of the genes involved in the auxin and cytokinin signaling pathways showed altered expression in the cin mutant. Further, we showed that CIN binds to genomic regions and directly promotes the transcription of a cytokinin receptor homolog HISTIDINE KINASE 4 (AmHK4) and an IAA3/SHY2 (INDOLE-3-ACETIC ACID INDUCIBLE 3/SHORT HYPOCOTYL 2) homolog in A. majus. Our results suggest that CIN limits excess cell proliferation and maintains the flatness of the leaf surface by directly modulating the hormone pathways involved in patterning cell proliferation and differentiation during leaf growth. 10.1111/(ISSN)1469-8137

Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids

The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.

Diagnosis of invasive candidiasis by enzyme-linked immunosorbent assay using the N-terminal fragment of Candida albicans hyphal wall protein I

Background: The diagnosis of invasive candidiasis is difficult because there are no specific clinical manifestations of the disease and colonization and infection are difficult to distinguish. In the last decade, much effort has been made to develop reliable tests for rapid diagnosis of invasive candidiasis, but none of them have found widespread clinical use. Results: Antibodies against a recombinant N-terminal fragment of the Candida albicans germ tube-specific antigen hyphal wall protein 1 (Hwp1) generated in Escherichia coli were detected by both immunoblotting and ELISA tests in a group of 36 hematological or Intensive Care Unit patients with invasive candidiasis and in a group of 45 control patients at high risk for the mycosis who did not have clinical or microbiological data to document invasive candidiasis. Results were compared with an immunofluorescence test to detect antibodies to C. albicans germ tubes (CAGT). The sensitivity, specificity, positive and negative predictive values of a diagnostic test based on the detection of antibodies against the N-terminal fragment of Hwp1 by immunoblotting were 27.8 %, 95.6 %, 83.3 % and 62.3 %, respectively. Detection of antibodies to the N-terminal fragment of Hwp1 by ELISA increased the sensitivity (88.9 %) and the negative predictive value (90.2 %) but slightly decreased the specificity (82.6 %) and positive predictive values (80 %). The kinetics of antibody response to the N-terminal fragment of Hwp1 by ELISA was very similar to that observed by detecting antibodies to CAGT. Conclusion: An ELISA test to detect antibodies against a recombinant N-terminal fragment of the C. albicans germ tube cell wall antigen Hwp1 allows the diagnosis of invasive candidiasis with similar results to those obtained by detecting antibodies to CAGT but without the need of treating the sera to adsorb the antibodies against the cell wall surface of the blastospore.

Surface markers of regionalization in the vertebrate nervous system

In order to identify new molecules that might play a role in regional specification of the nervous system, we generated and characterized monoclonal antibodies (mAbs) that have positionally-restricted labeling patterns.

The FORSE-1 mAb was generated using a strategy designed to produce mAbs against neuronal cell surface antigens that might be regulated by regionally-restricted transcription factors in the developing central nervous system (CNS). FORSE-1 staining is enriched in the forebrain as compared to the rest of the CNS until E18. Between E11.5-E13.5, only certain areas of the forebrain are labeled. There is also a dorsoventrally-restricted region of labeling in the hindbrain and spinal cord. The mAb labels a large proteoglycan-like cell-surface antigen (>200 kD). The labeling pattern of FORSE-1 is conserved in various mammals and in chick.

To determine whether the FORSE-1 labeling pattern is similar to that of known transcription factors, the expression of BF-1 and Dlx-2 was compared with FORSE-1. There is a striking overlap between BF-1 and FORSE-1 in the telencephalon. In contrast, FORSE-1 and Dlx-2 have very different patterns of expression in the forebrain, suggesting that regulation by Dlx-2 alone cannot explain the distribution of FORSE-1. They do, however, share some sharp boundaries in the diencephalon. In addition, FORSE-1 identifies some previously unknown boundaries in the developing forebrain. Thus, FORSE-1 is a new cell surface marker that can be used to subdivide the embryonic forebrain into regions smaller than previously described, providing further complexity necessary for developmental patterning.

I also studied the expression of the cell surface protein CD9 in the developing and adult rat nervous system. CD9 is implicated in intercellular signaling and cell adhesion in the hematopoetic system. In the nervous system, CD9 may perform similar functions in early sympathetic ganglia, chromaffin cells, and motor neurons, all of which express the protein. The presence of CD9 on the surfaces of Schwann cells and axons at the appropriate time may allow the protein to participate in the cellular interactions involved in myelination.