Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Expression of an activating mutation in the gene encoding the K(ATP) channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K(+) (K(ATP)) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of K(ATP) channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of K(ATP) channels, closed K(ATP) channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of K(ATP) channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Brabykinin B1 Receptor Antagonism Is Beneficial in Renal Ischemia-Reperfusion Injury

Previously we have demonstrated that bradykinin B1 receptor deficient mice (B1KO) were protected against renal ischemia and reperfusion injury (IRI). Here, we aimed to analyze the effect of B1 antagonism on renal IRI and to study whether B1R knockout or antagonism could modulate the renal expression of pro and anti-inflammatory molecules. To this end, mice were subjected to 45 minutes ischemia and reperfused at 4, 24, 48 and 120 hours. Wild-type mice were treated intra-peritoneally with antagonists of either B1 (R-954, 200 mg/kg) or B2 receptor (HOE140, 200 mg/kg) 30 minutes prior to ischemia. Blood samples were collected to ascertain serum creatinine level, and kidneys were harvested for gene transcript analyses by real-time PCR. Herein, B1R antagonism ( R-954) was able to decrease serum creatinine levels, whereas B2R antagonism had no effect. The protection seen under B1R deletion or antagonism was associated with an increased expression of GATA-3, IL-4 and IL-10 and a decreased T-bet and IL-1b transcription. Moreover, treatment with R-954 resulted in lower MCP-1, and higher HO-1 expression. Our results demonstrated that bradykinin B1R antagonism is beneficial in renal IRI.

Dissecting the Relation between a Nuclear Receptor and GATA: Binding Affinity Studies of Thyroid Hormone Receptor and GATA2 on TSH beta Promoter

Background: Much is known about how genes regulated by nuclear receptors (NRs) are switched on in the presence of a ligand. However, the molecular mechanism for gene down-regulation by liganded NRs remains a conundrum. The interaction between two zinc-finger transcription factors, Nuclear Receptor and GATA, was described almost a decade ago as a strategy adopted by the cell to up-or down-regulate gene expression. More recently, cell-based assays have shown that the Zn-finger region of GATA2 (GATA2-Zf) has an important role in down-regulation of the thyrotropin gene (TSH beta) by liganded thyroid hormone receptor (TR). Methodology/Principal Findings: In an effort to better understand the mechanism that drives TSH beta down-regulation by a liganded TR and GATA2, we have carried out equilibrium binding assays using fluorescence anisotropy to study the interaction of recombinant TR and GATA2-Zf with regulatory elements present in the TSH beta promoter. Surprisingly, we observed that ligand (T3) weakens TR binding to a negative regulatory element (NRE) present in the TSH beta promoter. We also show that TR may interact with GATA2-Zf in the absence of ligand, but T3 is crucial for increasing the affinity of this complex for different GATA response elements (GATA-REs). Importantly, these results indicate that TR complex formation enhances DNA binding of the TR-GATA2 in a ligand-dependent manner. Conclusions: Our findings extend previous results obtained in vivo, further improving our understanding of how liganded nuclear receptors down-regulate gene transcription, with the cooperative binding of transcription factors to DNA forming the core of this process.

Cuff-induced vascular intima thickening is influenced by titration of the Ace gene in mice

Lacchini S, Heimann AS, Evangelista FS, Cardoso L, Silva GJ, Krieger JE. Cuff-induced vascular intima thickening is influenced by titration of the Ace gene in mice. Physiol Genomics 37: 225-230, 2009. First published March 3, 2009; doi:10.1152/physiolgenomics.90288.2008.-We tested the hypothesis that small changes in angiotensin I-converting enzyme (ACE) expression can alter the vascular response to injury. Male mice containing one, two, three, and four copies of the Ace gene with no detectable vascular abnormality or changes in blood pressure were submitted to cuff-induced femoral artery injury. Femoral thickening was higher in 3- and 4-copy mice (42.4 +/- 4.3% and 45.7 +/- 6.5%, respectively) compared with 1- and 2-copy mice (8.3 +/- 1.3% and 8.5 +/- 0.9%, respectively). Femoral ACE levels from control and injured vessels were assessed in 1- and 3-copy Ace mice, which represent the extremes of the observed response. ACE vascular activity was higher in 3- vs. 1-copy Ace mice (2.4-fold, P < 0.05) in the control uninjured vessel. Upon injury, ACE activity significantly increased in both groups [2.41-fold and 2.14-fold (P < 0.05) for 1- and 3-copy groups, respectively] but reached higher levels in 3- vs. 1-copy Ace mice (P < 0.05). Pharmacological interventions were then used as a counterproof and to indirectly assess the role of angiotensin II (ANG II) on this response. Interestingly, ACE inhibition (enalapril) and ANG II AT(1) receptor blocker (losartan) reduced intima thickening in 3-copy mice to 1-copy mouse values (P < 0.05) while ANG II treatment significantly increased intima thickening in 1-copy mice to 3-copy mouse levels (P < 0.05). Together, these data indicate that small physiologically relevant changes in ACE, not associated with basal vascular abnormalities or blood pressure levels, do influence the magnitude of cuff-induced neointima thickening in mice.

EIN3-like gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Grande naine)

Ethylene signal transduction initiates with ethylene binding at receptor proteins and terminates in a transcription cascade involving the EIN3/EIL transcription factors. Here, we have isolated four cDNAs homologs of the Arabidopsis EIN3/EIN3-like gene, MA-EILs (Musa acuminata ethylene insensitive 3-like) from banana fruit. Sequence comparison with other banana EIL gene already registered in the database led us to conclude that, at this day, at least five different genes namely MA-EIL1, MA-EIL2/AB266318, MA-EIL3/AB266319, MA-EIL4/AB266320 and AB266321 exist in banana. Phylogenetic analyses included all banana EIL genes within a same cluster consisting of rice OsEILs, a monocotyledonous plant as banana. However, MA-EIL1, MA-EIL2/AB266318, MA-EIL4/AB266320 and AB266321 on one side, and MA-EIL3/AB266319 on the other side, belong to two distant subclusters. MA-EIL mRNAs were detected in all examined banana tissues but at lower level in peel than in pulp. According to tissues, MA-EIL genes were differentially regulated by ripening and ethylene in mature green fruit and wounding in old and young leaves. MA-EIL2/AB266318 was the unique ripening- and ethylene-induced gene; MA-EIL1, MA-EIL4/Ab266320 and AB266321 genes were downregulated, while MA-EIL3/AB266319 presented an unusual pattern of expression. Interestingly, a marked change was observed mainly in MA-EIL1 and MA-EIL3/Ab266319 mRNA accumulation concomitantly with changes in ethylene responsiveness of fruit. Upon wounding, the main effect was observed in MA-EIL4/AB266320 and AB266321 mRNA levels, which presented a markedly increase in both young and old leaves, respectively. Data presented in this study suggest the importance of a transcriptionally step control in the regulation of EIL genes during banana fruit ripening.

Haplotypes of vitamin D receptor modulate the circulating levels of lead in exposed subjects

Genetic factors influence whole blood lead (Pb-B) concentrations in lead exposed subjects. This study aimed at examining the combined effects (haplotype analysis) of three polymorphisms (BsmI, ApaI and FokI) in vitamin D receptor (VDR) gene on Pb-B and on the concentrations of lead in plasma (Pb-P), which is more relevant to lead toxicity, in 150 environmentally exposed subjects. Genotypes were determined by RFLP, and Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. Subjects with the bb (BsmI polymorphism) or ff (FokI polymorphism) genotypes have lower B-Pb than subjects in the other genotype groups. Subjects with the aa (ApaI polymorphism) or ff genotypes have lower P-Pb than subjects in the other genotype groups. Lower Pb-P, Pb-B, and %Pb-P/Pb-B levels were found in subjects with the haplotype combining the a, b, and f alleles for the ApaI, BsmI, and FokI polymorphisms, respectively, compared with the other haplotype groups, thus suggesting that VDR haplotypes modulate the circulating levels of lead in exposed subjects.

Allelic Variation Investigation of the Estrogen Receptor Within an Australian Multiple Sclerosis Population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII (P = 0.50) and Btg1 (P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder.

Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS

The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A valiant mRNA Tvas identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount irt situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.

Chronic ethanol exposure and withdrawal influence NMDA receptor subunit and splice variant mRNA expression in the rat cerebral cortex

Chronic ethanol exposure and subsequent withdrawal are known to change NMDA receptor activity. This study examined the effects of chronic ethanol administration and withdrawal on the expression of several NMDA receptor subunit and splice variant mRNAs in the rat cerebral cortex. Ethanol dependence was induced by ethanol vapour exposure. To delineate between seizure-induced changes in expression during withdrawal and those due to withdrawal per se, another group of naive rats was treated with pentylenetetrazol (PTZ) injection (30 mg/kg, i.p.). RNA samples from the cortices of chronically treated and withdrawing animals were compared to those from pairfed controls. Changes in NMDA receptor mRNA expression were determined using ribonuclease protection assays targetting the NR2A, -2B, -2C and NR1-pan subunits as well as the three alternatively spliced NR1 inserts (NR1-pan describes all the known NR1 splice variants generated from the 5' insert and the two 3' inserts). The ratio of NR1 mRNA incorporating the 5' insert vs, that lacking it was decreased during ethanol exposure and up to 48 h after withdrawal. NR2B mRNA expression was elevated during exposure, but returned to control levels 18 h after withdrawal. Levels of NR2A, NR2C, NR1-pan and both 3' NR1 insert mRNAs from the ethanol-treated groups did not alter compared with the pair-fed control group. No changes in the level of any NMDA receptor subunit mRNA was detected in the PTZ-treated animals. These data support the hypothesis that changes in NMDA receptor subunit composition may underlie a neuronal adaptation to the chronic ethanol-inhibition and may therefore be important in the precipitation of withdrawal hyperactivity. (C) 1999 Elsevier Science B.V. All rights reserved.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

Gene transfer and expression of a non-viral polycation-based vector in CD4(+) cells

CD4-selective targeting of an antibody-polycation-DNA complex was investigated The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine(268) (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of I-125-B-F5 to CD4(+) Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was defected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4(-) K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4(+) cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4(+) T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4(+) cells in vivo.

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer

The estrogen receptor alpha (ER alpha) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ER alpha and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ER alpha-positive compared with ER alpha-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ER alpha status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ER alpha and other steroid receptors in breast cancer.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas

Purpose: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. Methods: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. Results: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. Conclusions: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.