Laura Guembe

A Peptide Inhibitor of FOXP3 Impairs Regulatory T Cell Activity and Improves Vaccine Efficacy in Mice

Immunosuppressive activity of regulatory T cells (Treg) may contribute to the progression of cancer or infectious diseases by preventing the induction of specific immune responses. Using a phage-displayed random peptide library, we identified a 15-mer synthetic peptide, P60, able to bind to forkhead/winged helix transcription factor 3 (FOXP3), a factor required for development and function of Treg. P60 enters the cells, inhibits FOXP3 nuclear translocation, and reduces its ability to suppress the transcription factors NF-κB and NFAT. In vitro, P60 inhibited murine and human-derived Treg and improved effector T cell stimulation. P60 administration to newborn mice induced a lymphoproliferative autoimmune syndrome resembling the reported pathology in scurfy mice lacking functional Foxp3. However, P60 did not cause toxic effects in adult mice and, when given to BALB/c mice immunized with the cytotoxic T cell epitope AH1 from CT26 tumor cells, it induced protection against tumor implantation. Similarly, P60 improved the antiviral efficacy of a recombinant adenovirus expressing NS3 protein from hepatitis C virus. Functional inhibition of Treg by the FOXP3-inhibitory peptide P60 constitutes a strategy to enhance antitumor and antiviral immunotherapies.

Hepatitis C virus induces the expression of CCL17 and CCL22 chemokines that attract regulatory T cells to the site of infection

BACKGROUND & AIMS The mechanisms by which Foxp3+ T regulatory cells (Treg) accumulate in HCV infected livers are not known. Here, we studied the role of chemokines CCL17 and CCL22 in this process. METHODS Chemokine mRNA levels were determined by qPCR in liver biopsies from 26 HCV chronically infected patients (CHC), 11 patients with treatment-induced sustained virological response (SVR), 16 patients with other liver diseases unrelated to HCV, and 24 normal livers. Double-immunofluorescence Foxp3/CD3 or CD11c/CCL22 was performed in liver sections. Chemokine production by monocyte-derived dendritic cells (MDDC) co-cultured with uninfected or HCV-JFH1 infected Huh7 cells was measured by qPCR and ELISA. Chemotactic activity of culture supernatants was also tested. RESULTS Foxp3+ Treg were increased in CHC livers as compared to controls. Patients with CHC showed elevated intrahepatic levels of CCL17 mRNA compared to normal livers or livers from subjects with SVR or other forms of liver disease. Intrahepatic CCL22 expression was also higher in CHC than in healthy subjects or SVR patients but similar to that observed in other liver diseases. Dendritic cells producing CCL22 could be found inside the hepatic lobule in CHC patients. Contact between MDDC and HCV-JFH1-infected Huh7 cells induced the expression of CCL17 and CCL22 in a process partially dependent on ICAM-1. Transwell experiments showed that upregulation of these chemokines enhanced Treg migration. CONCLUSIONS Contact of HCV-infected cells with dendritic cells induces the production of Treg-attracting chemokines, an effect which may favour liver accumulation of Treg in CHC. Our findings contribute to explain the mechanism by which HCV escapes the immune response and thus reveals novel therapeutic targets.

The diffuse endocrine system: from embryogenesis to carcinogenesis.

In the present review we will summarise the current knowledge about the cells comprising the Diffuse Endocrine System (DES) in mammalian organs. We will describe the morphological, histochemical and functional traits of these cells in three major systems gastrointestinal, respiratory and prostatic. We will also focus on some aspects of their ontogeny and differentiation, as well as to their relevance in carcinogenesis, especially in neuroendocrine tumors. The first chapter describes the characteristics of DES cells and some of their specific biological and biochemical traits. The second chapter deals with DES in the gastrointestinal organs, with special reference to the new data on the differentiation mechanisms that leads to the appearance of endocrine cells from an undifferentiated stem cell. The third chapter is devoted to DES of the respiratory system and some aspects of its biological role, both, during development and adulthood. Neuroendocrine hyperplasia and neuroendocrine lung tumors are also addressed. Finally, the last chapter deals with the prostatic DES, discussing its probable functional role and its relevance in hormone-resistant prostatic carcinomas.

Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy

BACKGROUND & AIMS Wilsons disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs and zinc salts, which may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. METHODS We transduced the liver of the Atp7b(-/-) WD mouse model with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific α1-antitrypsin promoter (AAV8-AAT-ATP7B). After vector administration we carried out periodic evaluation of parameters associated with copper metabolism and disease progression. The animals were sacrificed 6months after treatment to analyze copper storage and hepatic histology. RESULTS We observed a dose-dependent therapeutic effect of AAV8-AAT-ATP7B manifested by the reduction of serum transaminases and urinary copper excretion, normalization of serum holoceruloplasmin, and restoration of physiological biliary copper excretion in response to copper overload. The liver of treated animals showed normalization of copper content and absence of histological alterations. CONCLUSIONS Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.

Cellular Prion Protein Is Expressed in a Subset of Neuroendocrine Cells of the Rat Gastrointestinal Tract

Prion diseases are believed to develop from the conformational change of normal cellular prion protein (PrPc) to a pathogenic isoform (PrPsc). PrPc is present in both the central nervous system and many peripheral tissues, although protein concentration is significantly lower in non-neuronal tissues. PrPc expression is essential for internalization and replication of the infectious agent. Several works have pointed to the gastrointestinal (GI) tract as the principal site of entry of PrPsc, but how passage through the GI mucosa occurs is not yet known. Here we studied PrPc expression using Western blot, RT-PCR, and immunohistochemistry in rat GI tract. PrPc mRNA and protein were detected in corpus, antrum, duodenum, and colon. Immunoreactivity was found in scattered cells of the GI epithelium. With double immunofluorescence, these cells have been identified as neuroendocrine cells. PrPc immunostaining was found in subsets of histamine, somatostatin (Som), ghrelin, gastrin (G), and serotonin (5HT) cells in stomach. In small and large bowel, PrPc cells co-localized with subpopulations of 5HT-, Som-, G-, and peptide YY-immunolabeled cells. Our results provide evidence for a possible and important role of endocrine cells in the internalization of PrPsc from gut lumen.

Immunohistochemical Mapping of Endothelin in the Developing and Adult Mouse Lung

Endothelins (ET) are a family of regulatory peptides displaying, among other abilities, potent constrictor actions. We studied the perinatal time course expression and distribution of ET in the mouse airway epithelium. In fetal mouse, ET-immunoreactivity (IR) appeared earlier (gestational Day 18) in the epithelium of upper (bronchi and large bronchioles) than in lower airways, being scarce and mainly located in the apical cytoplasm. As the lung developed, ET-IR became gradually stronger and extended throughout the cell in both bronchi and bronchioles. ET-IR was found in most airway epithelial cells. Clara cells were positive for ET, whereas ciliated and endocrine cells were not. In adult lungs, part of the myocytes and parenchymal cells also showed ET-IR. In both developing and adult mouse lungs, the cell distribution of ET-IR in the epithelium is compatible with apical and/or basal secretion. The presence of ET in mouse airway epithelium during the perinatal period may indicate a role for ET as a growth factor in lung development and its involvement in control of lung ventilation at birth.

IMMUNOCYTOCHEMICAL MAPPING OF THE AMIDATING ENZYME PAM IN THE DEVELOPING AND ADULT MOUSE LUNG

The enzyme PAM is required for activation of many peptide hormones. In adult mouse lung, immunostaining for PAM was located in Clara cells, which constitute most of the epithelial cells of the mouse bronchial/bronchiolar tree. Immunoreactivity appeared for the first time in the epithelium on gestational Day 16, being slight and mostly restricted to the apical cytoplasm. As the lung developed, the labeling became gradually stronger and extended throughout the cell. Smooth muscle of airways and blood vessels, and some parenchymal cells, probably macrophages, also showed PAM immunoreactivity. Of the two enzymatically active domains of PAM, only PHM and not PAL immunoreactivity was found at all stages studied. The early appearance of PAM in developing mouse lung, as well as its presence in a variety of tissues, probably indicates a complex role of this enzyme in pulmonary development and function.

Characterization of PrPc-Immunoreactive Cells in Monkey (Macaca fascicularis) Gastrointestinal Tract

Abstract: The gastrointestinal tract (GIT) is one of the most likely entry sites for the pathological isoform of prions (PrPsc). To understand how PrPsc crosses the digestive mucosa, it is crucial to characterize the cells expressing normal prion protein (PrPc). By means of double immunofluorescence applied to sections of the monkey GIT, we demonstrated that, in the stomach, PrPc immunostaining occurs in subsets of histamine, somatostatin (Som), ghrelin (Ghr), gastrin (G), and serotonin (5HT) cells. In the small and large bowels, PrPc cells were found in subpopulations of cells immunolabeled for 5HT, Som, G, and peptide YY (PYY).

Comparative study of PrPc expression in rat, monkey, and cow gastrointestinal tract

Abstract: The gastrointestinal tract (GIT) appears to be the main site of entry for the pathological isoform of prions (PrPsc). To understand how the PrPsc internalization process occurs, it is important to characterize the cell types that express normal prion protein (PrPc) along the GIT. To do so, we studied the distribution of PrPc in the rat, monkey, and cow GIT. Using Western blot analysis, we found that PrPc is expressed in all digestive regions of the three species. Immunoreactivity for PrPc was found throughout the GIT in epithelial cells sharing the neuroendocrine (NE) phenotype. Immunostained cells appeared scattered throughout the epithelium of fundic and pyloric glands as well as in intestinal villi and crypts.