Gain-of-Function Mutations in IFIH1 Cause a Spectrum of Human Disease Phenotypes Associated with Upregulated Type I Interferon Signaling

The type I interferon system is integral to human antiviral immunity. However, inappropriate stimulation or defective negative regulation of this system can lead to inflammatory disease. We sought to determine the molecular basis of genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, and of other patients with undefined neurological and immunological phenotypes also demonstrating an upregulated type I interferon response. We found that heterozygous mutations in the cytosolic double-stranded RNA receptor gene IFIH1 (MDA5) cause a spectrum of neuro-immunological features consistently associated with an enhanced interferon state. Cellular and biochemical assays indicate that these mutations confer a gain-of-function - so that mutant IFIH1 binds RNA more avidly, leading to increased baseline and ligand-induced interferon signaling. Our results demonstrate that aberrant sensing of nucleic acids can cause immune upregulation.

Effects of levamisole on experimental infections by Plasmodium berghei in mice

Levamisole (phenylimidothiazol), considered a strong immunostimulant, when administered to healthy Swiss mice did not cause a significant increase in -the weight of their thymus, liver and spleen, even though the drug was used at different times before removing such organs. High doses ofdrug used in the 4-day prophylactic scheme had no antimalarial effect. However, when given to malaria infected mice 24 hours before, at the same time, and 24 hours after the inoculation of a chloroquine-sensitive or a chloroquine-resistant strain of Plasmodium berghei small doses of the drug induced a somewhat decreased parasitemia, the dose of 1 mg/kg body weight before the inoculum being the best scheme. The mortality rates by malaria in the levamisole treated groups were also delayed although all mice finally died. The data suggest that levamisole may display a stimulant effect on the depressed immune response caused by malaria.

Analysis of PAX2 gene alterations in renal cell tumors

Dissertation for applying to a Master’s Degree in Molecular Genetics and Biomedicine submitted to the Sciences and Technology Faculty of New University of Lisbon

Hepatocyte metaplasia in experimental chagasic pancreatitis: preliminary report

Beginning the study of chronic pathologic changes in pancreas of hamsters experimentally infected with Trypanosoma cruzi Vic strain, hepatocyte metaplasia was observed in one animal from infected group. This is the first report of oncocytes in Chagas' disease, which could be due to aberrant regenerative response to pancreas inflammatory process.

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology

An efficient chronic unpredictable stress protocol to induce stress-related responses in C57BL/6 mice

Exposure to chronic stress can have broad effects on health ranging from increased predisposition for neuropsychiatric disorders to deregulation of immune responses. The chronic unpredictable stress (CUS) protocol has been widely used to study the impact of stress exposure in several animal models and consists in the random, intermittent, and unpredictable exposure to a variety of stressors during several weeks. CUS has consistently been shown to induce behavioral and immunological alterations typical of the chronic stress-response. Unfortunately C57BL/6 mice, one of the most widely used mouse strains, due to the great variety of genetically modified lines, seem to be resistant to the commonly used 4-week-long CUS protocol. The definition of an alternative CUS protocol allowing the use of C57BL/6 mice in chronic stress experiments is a need. Here, we show that by extending the CUS protocol to 8?weeks is possible to induce a chronic stress-response in C57BL/6 mice, as revealed by abrogated body weight gain, increased adrenals weight, and an overactive hypothalamic-pituitary-adrenal axis with increased levels of serum corticosterone. Moreover, we also observed stress-associated behavioral alterations, including the potentiation of anxious-like and depressive-like behaviors and a reduction of exploratory behavior, as well as subtle stress-related changes in the cell population of the thymus and of the spleen. The present protocol for C57BL/6 mice consistently triggers the spectrum of CUS-induced changes observed in rats and, thus, will be highly useful to researchers that need to use this particular mouse strain as an animal model of neuropsychiatric disorders and/or immune deregulation related to CUS.

HOXA9 as a key regulator in glioma initiation, aggressiveness and response to therapy

Tese de Doutoramento em Ciências da Saúde

Terceira contribuicäo ao conhecimento do gênero Archytas Jaennicke, 1867: (Diptera, Tachinidae)

Twelve species of the genus Archytas Jennicke, 1867, eight of which described as new are studied and figured in detail. Definitions of the species are based mainly on characters of male genitalia. The male genital characters are the most significant for separation of the species and most demonstrative of their affinities. By examining a long series of species of this genus we came to the conclusion that the presence of one pair of median marginal bristles on the third abdominal tergite seems to be characteristic of the genus. This caracter apparently so important, is not however considered fundamental. The most significant example is found in Archytas lenkoi sp. n. and Archytas vexor Curran, 1928. In A. lenkoi we can find one or two pairs or thay may, less frquently, be absent. In A. vexor these bristles are lacking. The shape of the male copulatory apparatus of Jurinia nitidiventris Curran, 1928 refered to by CURRAN in his "Revision of Archytas", is not characteristic of any species of the group and so, is not considered in this paper. To help in the identification, the species studied here are divided into groups. The analis group" includes: A. apicifer (Walker, 1894), A. californiae (Walker, 1856), A. nivalis Curran, 1928, a. giacomellii (Blanchard, 1941), A. basifulvus (Walker, 1849), A. incasanus Townsend, 1912 and A. cirphis Curran, 1927. The identification of members of these group is extremely difficult owing both to their similarity in colour pattern and to their variability. They all have black testaceous or dark brown abdomen, the last segment pale or brownish pollinose; second segment without bristles; third with a pair of strong marginals, fourth and fifth with two rows of discals on apical third. The final determination often rests upon the structure of the male copulatory apparatus. Fortunately in this group, many of the forcipes superiores and palpi genitalium are strikingly different from one another. The "zikani group" includes: A. zikani sp. n., A seabrai sp. n., A. duckei sp. n. and A. vernalis Curran, 1928. This group may be characterized as follows: forcipes interiores absent; forcipes superiores strongly chitinized an dilated at anex. Within this group, the forcipes of. A. seabrai sp. n. do not present an aberrant form. The "dissimilis group" will be studied in forthcoming papers. The limits of the genus Archyta Jaen. are not as yet sharply difined, the evaluation of the significance of each character used in the definition remaining as most difficult problem. The distinction between Archytas and other related genera is very difficult, chiefly because it is based on variable characters. In this paper we place the genera Parafabricia Towsend, 1931, Itachytas Blanchard, 1940, Archynemochaeta Blanchard, 1941, Proarchytoides Blanchard, 1941 and Archytodejeania Blanchard, 1941 in the synonymy of Archytas Jaen. The detailed examination of the characters used in their definition, proved them to be fundamentally proposed on basis of chaetotasy, these characters alone being precarious, because of the considerabel intraspecifical variation. The type of the new species are in the Oswaldo Cruz Institute collection. Rio de Janeiro, Brazil, and paratypes in the collections of the followings institutions: Departamento de Zoologia da Secretaria de Agricultura do Estado de São Paulo; Instituto de Ecologia e Experimemtação Agrícolas; Departamento de Defesa Sanitária Vegetal; Campos Seabra collection; and Barbiellini collection.

T Cells Bearing a Chimeric Antigen Receptor against Prostate-Specific Membrane Antigen Mediate Vascular Disruption and Result in Tumor Regression.

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of protumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here, we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA(+) endothelial targets in vitro, regardless of the signaling domain. T cells bearing the third-generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA(+) vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide a strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. Cancer Immunol Res; 3(1); 68-84. ©2014 AACR.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

Comprehensive Expression Analyses of Neural Cell-Type-Specific miRNAs Identify New Determinants of the Specification and Maintenance of Neuronal Phenotypes.

MicroRNAs (miRNAs) have been shown to play important roles in both brain development and the regulation of adult neural cell functions. However, a systematic analysis of brain miRNA functions has been hindered by a lack of comprehensive information regarding the distribution of miRNAs in neuronal versus glial cells. To address this issue, we performed microarray analyses of miRNA expression in the four principal cell types of the CNS (neurons, astrocytes, oligodendrocytes, and microglia) using primary cultures from postnatal d 1 rat cortex. These analyses revealed that neural miRNA expression is highly cell-type specific, with 116 of the 351 miRNAs examined being differentially expressed fivefold or more across the four cell types. We also demonstrate that individual neuron-enriched or neuron-diminished RNAs had a significant impact on the specification of neuronal phenotype: overexpression of the neuron-enriched miRNAs miR-376a and miR-434 increased the differentiation of neural stem cells into neurons, whereas the opposite effect was observed for the glia-enriched miRNAs miR-223, miR-146a, miR-19, and miR-32. In addition, glia-enriched miRNAs were shown to inhibit aberrant glial expression of neuronal proteins and phenotypes, as exemplified by miR-146a, which inhibited neuroligin 1-dependent synaptogenesis. This study identifies new nervous system functions of specific miRNAs, reveals the global extent to which the brain may use differential miRNA expression to regulate neural cell-type-specific phenotypes, and provides an important data resource that defines the compartmentalization of brain miRNAs across different cell types.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Thymoma, immunodeficiency, and herpes simplex virus infections.

Hypogammaglobulinemia develops in 3 to 6% of patients with thymoma and this association is commonly referred to as thymoma with immunodeficiency (formerly Good syndrome). Recurrent infections with encapsulated bacteria and opportunistic infections associated with disorders of both humoral and cell mediated immunity frequently occur in this rare primary, adult-onset immunodeficiency. We report a case of thymoma with immunodeficiency complicated by disseminated herpes simplex virus (HSV) infection and review five additional cases of HSV-related infections reported since 1966 in patients presenting with thymoma with immunodeficiency. Patients presented with epiglottitis, keratitis, recurrent genital herpes, ulcerative dermatitis, and acute hepatitis. Four of the six cases had a fatal outcome, two of which were directly attributable to HSV infection. Since the risk of invasive opportunistic infections is high and the presentation atypical, lymphocyte count and total serum immunoglobulin should be measured regularly in all patients presenting with thymoma with immunodeficiency.

Thymulin: biochemistry, biology and therapeutical applications

Thymulin is a pharmacologically active metallononapeptide inducing the differentiation of T cells and enhancing several functions of the various T cell subsets in normal or partially thymus-deficient recipients. Its effect on suppressor T cells is, so far, the most remarkable and should be the first to find useful clinical applications. The peptide is a natural hormone, available in synthetic form. It is not toxic and one may foresee its clinical use as one of the major immunoregulatory agents in the near future.

The pineal neurohormone melatonin and its physiologic opiatergic immunoregulatory role

The pineal gland functions as a neuroendocrine transducer that coordinate the organism response to changing environmental stimuli such as light and temperature. The main and best known pineal neurohormone is melatonin that is synthesized and released in a circadian fashion with a peak during the night darkness hours. We have recently reported that melatonin exerts important immuno regulatory functions. Here we describe the astonishing property of exogenous melatonin which is able to counteract completely the depressive effect of anxiety-restraint stress and/or of corticosterone on thymus weight, andibody production and antiviral responses. This effect seems to be mediated by antigen-activated T cells via an opiatergic mechanism.