Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss-or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi. J. Leukoc. Biol. 86: 989-998; 2009.

Photodynamic therapy for acne vulgaris: A critical review from basics to clinical practice Part II. Understanding parameters for acne treatment with photodynamic therapy

Photodynamic therapy requires a photosensitizer, oxygen, and activating light. For acne, pilosebaceous units are ""target"" structures. Porphyrins are synthesized in vivo from 5-aminolevulinic acid (ALA), particularly in pilosebaceous units. Different photosensitizers and drug delivery methods have been reported for acne treatment. There are a variety of porphyrin precursors with different pharmacokinetic properties. Among them, ALA and methyl-ester of ALA (MAT.) are available for possible off-label treatment of acne vulgaris. In addition, various light sources, light dosimetry, drug incubation time, and pre- and posttreatment care also change efficacy and side effects. None of these variables has been optimized for acne treatment, but a number of clinical trials provide helpful guidance. In this paper, we critically analyze clinical trials, case reports, and series of cases published through 2009. (J Am Acad Dermatol 2010;63:195-211.)

Nonsurgical Periodontal Therapy Combined with Laser and Photodynamic Therapies for Periodontal Disease in Immunosuppressed Rats

Background. Periodontal disease is often associated with systemic diseases and is characterized by destruction of the tissues supporting the teeth. Patients using immunosuppressive drugs such as tacrolimus are among those who suffer from tissue destruction. Objective. We sought to evaluate the effects of laser and photodynamic therapies (PDT; nonsurgical) as an adjunct to scaling and rootplaning (SRP) in the treatment of corona-induced periodontitis in rats immunosuppressed with tacrolimus (Prograf). Materials and Methods. The animals were divided into 5 groups. Each groups had 6 rats. Group I, the control group, received only saline solution throughout the study period of 42 days and did not receive periodontal treatment; group II received saline solution and SRP; group III received tacrolimus (1 mg/kg per day) and was treated with SRP; group IV animals were treated identically to group III and then administered laser treatment; and in group V, the animals were treated identically to group III and then administered PDT. Results. Statistical analysis indicated decreased bone loss with the progression of time (P = .035). There was no difference between the bone loss associated with the types of treatment administered to groups I, II, and III (P > .9) or groups IV and V (P > .6). The analysis also indicated that immunosuppression was not a bone loss-determining factor. Conclusion. Laser and PDT therapies were effective as an adjunctive treatment to SRP in reducing bone loss caused by experimental periodontitis induced in animals being treated systemically with tacrolimus.

Development of microemulsions to topically deliver 5-aminolevulinic acid in photodynamic therapy

The aim of this study was to obtain and to characterize microemulsions containing 5-aminolevulinic acid (5-ALA) and to investigate the influence of these systems in drug skin permeation for further topical photodynamic therapy (PDT). 5-ALA was incorporated in water-in-oil (W/O), bicontinuous (Bc), and oil-in-water (O/W) microemulsions obtained by the titration of ethyl oleate and PEG-8 caprylic/capric glycerides:polyglyceryl-6 dioleate (3:1) mixtures with water. Selected systems were characterized by conductivity, viscosity, size of the droplets, and drug release. The stability of the drug in the microemulsions was also assessed. Moreover, the in vitro and in vivo skin permeation of 5-ALA was investigated using diffusion cells and confocal scanning laser microscopy (CSLM), respectively. Despite the fact that the O/W microemulsion decreased the 5-ALA diffusion coefficient and retarded the drug release, it also significantly increased the in vitro drug skin permeation when compared to other 5-ALA carriers. It was observed by CSLM that the red fluorescence of the skin increased homogeneously in the deeper skin layers when the 5-ALA microemulsion was applied in vivo, probably due to the formation of the photoactive protoporphyrin IX. The microemulsion developed carried 5-ALA to the deeper skin layers, increasing the red fluorescence of the skin and indicating the potentiality of the system for topical 5-ALA-PDT. (C) 2010 Elsevier B.V. All rights reserved.

Anxiolytic-like effects induced by acute reversible inactivation of the bed nucleus of stria terminalis

There is conflicting evidence concerning the role of the bed nucleus of the stria terminalis (BNST) in fear and anxiety-elicited behavior. Most of the studies investigating this role, however, employed irreversible lesions of this nucleus. The objective of the present study was to investigate the effects of an acute and reversible inactivation of the BNST in rats submitted to the Vogel conflict test (VCT) and contextual fear conditioning, two widely employed animal models that are responsive to prototypal anxiolytic drugs. Male Wistar rats were submitted to stereotaxic surgery to bilaterally implant cannulae into the BNST. Ten minutes before the test they received bilateral microinjections of cobalt chloride (COCl(2)) (1 mM/100 nL), a nonselective synapse blocker. COCl(2) produced anxiolytic-like effects in tests, increasing the number of punished licks in the VCT and decreasing freezing behavior and the increase in mean arterial blood pressure and heart rate of animals re-exposed to the context where they had received electrical foot shocks 24 h before. The results indicate that the BNST is engaged in behavioral responses elicited by punished stimuli and aversively conditioned contexts, reinforcing its proposed role in anxiety. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Effects of reversible inactivation of the dorsal hippocampus on the behavioral and cardiovascular responses to an aversive conditioned context

In rats, conditioned fear to context causes freezing immobility and cardiovascular changes. The dorsal hippocampus (DH) has a critical role in several memory processes, including conditioning fear to contextual information. To explore a possible involvement of the DH in contextual fear conditioning-evoked cardiovascular (mean arterial pressure and heart rate increases) and behavioral (freezing) responses, DH synaptic transmission was temporarily inhibited by bilateral microinjections of 500 nl of the nonselective synapse blocker, cobalt chloride (COCl2, 1 mmol/l), at different periods of the experimental procedure. During re-exposure to the foot shock chamber in which conditioning had taken place, bilateral DH inhibition 10 min before the conditioning session had no effect on either behavioral or cardiovascular responses. Bilateral DH inhibition immediately after the conditioning session (110 min) decreased both behavioral and cardiovascular responses during the context test. Finally, 48 h after the conditioning session, bilateral DH inhibition 10 min before re-exposure to the foot shock chamber significantly reduced cardiovascular responses but not freezing responses. These results suggest that contextual fear conditioning acquisition does not depend on the DH. This structure, however, is crucial for the consolidation of contextual fear. Moreover, although the DH appears to be less important for the behavioral (freezing) changes induced by re-exposure to the aversive conditioned context, it may play an important role on the cardiovascular responses generated by this model.

Acute reversible inactivation of the ventral medial prefrontal cortex induces antidepressant-like effects in rats

The ventral medial prefrontal cortex (vMPFC) has direct connections to subcortical, diencephalic and brainstem structures that have been closely related to depression. However, studies aimed at investigating the role of the vMPFC in the neurobiology of depression have produced contradictory results. Moreover, the precise involvement of vMPFC anatomic subdivisions, the prelimbic(PL) and the infralimbic (IL) cortices, in regulating depressive-like behavior have been poorly investigated. The forced swimming test (FST) is a widely employed animal model aimed at detecting antidepressant-like effects. Therefore, to further investigate a possible involvement of the vMFPC in depressive-like behavior, rats bilaterally implanted with cannulae aimed at the PL or IL prefrontal cortices were submitted to 15 min of forced swimming (pre-test) followed, 24 h later, by a 5-min swimming session (test), where immobility time was registered. Synaptic transmission in these regions was temporarily inhibited using local microinjection of cobalt chloride at different periods of the experimental procedure (before or after the pre-test or before the test). PL inactivation decreased immobility time independently of the time of the injection. In the IL inactivation induced a significant antidepressant-like effect when performed immediately before the pre-test or before the test, but not after the pre-test. These results suggest that activation of the vMPFC is important for the behavioral changes observed in rats submitted to the FST. They further indicate that, although both the PL and IL cortices are involved in these effects, they may play different roles. (C) 2010 Elsevier B.V. All rights reserved.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Height discordance in monozygotic females is not attributable to discordant inactivation of X-linked stature determining genes

We tested the hypothesis that X-linked genes determining stature which are subject to skewed or non-random X-inactivation can account for discordance in height in monozygotic female twins. Height discordant female monozygotic adult twins (20 pairs) were identified from the Australian Twin Registry, employing the selection criteria of proven monozygosity and a measured height discordance of at least 5 cm. Differential X-inactivation was examined in genomic DNA extracted from peripheral lymphocytes by estimating differential methylation of alleles at the polymorphic CAG triplet repeat of the Androgen receptor gene (XAR). There were 17/20 MZ pairs heterozygous at this locus and informative for analysis. Of these, 10/17 both had random X-inactivation, 5/17 showed identical X-inactivation patterns of non random inactivation and 2/17 (12%) showed discordant X-inactivation. There was no relationship between inactivation patterns and self-report chorionicity. We conclude that non-random X-inactivation does not appear to be a major contributor to intra-pair height discordance in female MZ twins.

A case of myoepithelial carcinoma displaying biallelic inactivation of the tumour suppressor gene APC in a patient with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.