Diazoxide Attenuates Autoimmune Encephalomyelitis and Modulates Lymphocyte Proliferation and Dendritic Cell Functionality

Activation of mitochondrial ATP-sensitive potassium (K_ATP) channels is postulated as an effective mechanism to confer cardio and neuroprotection, especially in situations associated to oxidative stress. Pharmacological activation of these channels inhibits glia-mediated neuroinflammation. In this way, diazoxide, an old-known mitochondrial K_ATP channel opener, has been proposed as an effective and safe treatment for different neurodegenerative diseases, demonstrating efficacy in different animal models, including the experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple Sclerosis. Although neuroprotection and modulation of glial reactivity could alone explain the positive effects of diazoxide administration in EAE mice, little is known of its effects on the immune system and the autoimmune reaction that triggers the EAE pathology. The aim of the present work was to study the effects of diazoxide in autoimmune key processes related with EAE, such as antigen presentation and lymphocyte activation and proliferation. Results show that, although diazoxide treatment inhibited in vitro and ex-vivo lymphocyte proliferation from whole splenocytes it had no effect in isolated CD4⁺ T cells. In any case, treatment had no impact in lymphocyte activation. Diazoxide can also slightly decrease CD83, CD80, CD86 and major histocompatibility complex class II expression in cultured dendritic cells, demonstrating a possible role in modulating antigen presentation. Taken together, our results indicate that diazoxide treatment attenuates autoimmune encephalomyelitis pathology without immunosuppressive effect.     

Enhanced Transdermal Peptide Delivery and Stability by Lipid Conjugation: Epidermal Permeation,Stereoselectivity and Mechanistic Insights

Purpose

Efficient delivery of therapeutic peptides to the skin will facilitate better outcomes in dermatology. The tetrapeptide AAPV, an elastase inhibitor with potential utility in the management of psoriasis was coupled to short chain lipoamino acids (Laa: C6-C10) to enhance the peptide permeation into and through human epidermis.

Methods

AAPV was conjugated to Laas by solid phase synthesis. Peptide stability, skin distribution and permeation, elastase activity and surface activity were determined.

Results

Laas increased peptide permeation into the skin. The permeation lag time and amount of peptide remaining in the skin increased with the carbon chain length of the Laa conjugate. We also demonstrated stereoselective permeation enhancement in favour of the D-diastereomer. Importantly, the elastase inhibition activity of the peptide was largely retained after coupling to the Laa conjugates, showing potential therapeutic utility. The Laa-peptide structures were shown to be surface active, suggesting that this surfactant-like activity coupled with enhanced lipophilicity may contribute to their interaction with and permeation through the lipid domains of the stratum corneum.

Conclusions

This study suggests that the Laa conjugation approach may be useful for enhancing the permeation of moderately sized peptide drugs with potential application in the treatment of skin disorders.     

alpha-MSH acetylation in the pituitary gland of the sea bream (Sparus aurata L.) in response to different backgrounds, confinement and air exposure

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl alpha-MSH or diacetyl alpha-MSH increases the bioactivity of the peptide. The aim of this study was to characterize alpha-MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The alpha-MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH2. In the pituitary gland of sea bream, des-, mono- and diacetyl alpha-MSH were identified. Then plasma alpha-MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma alpha-MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono- and diacetyl alpha-MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl alpha-MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl alpha-MSH relative to des- and diacetyl alpha-MSH was found in white-adapted fish. Levels of des- and diacetyl alpha-MSH forms were similar under all conditions. We observed that monoacetyl alpha-MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of alpha-MSH may vary from species to species.     

Ontogeny of adenohypophyseal cells in the pituitary of the American shad (Alosa sapidissima)

The distribution and ontogeny of adenohypophyseal cells have been studied in the pituitary gland of embryos, larvae, and juveniles of the clupeid American shad (Alosa sapidissima) using immunocytochemical techniques. In juvenile specimens, adenohypophysis was composed of rostral pars distalis (RPD), formed by cavities lined by prolactin (PRL), adrenocorticotropic hormone (ACTH), and gonadotropic hormone (GTH) cells; proximal pars distalis (PPD), containing growth hormone (GH), GTH, and putative thyroid stimulating hormone (TSH) cells; and pars intermedia (PI) with somatolactin (SL) and melanophore stimulating hormone (MSH) cells. At 3 days post-fertilization (3 days pre-hatching) the pituitary of embryos consisted of an oval mass of cells, close to the ventral margin of the diencephalon, divided in rostral and caudal regions. At this time PRL and ACTH cells appeared in the rostral region of the adenohypophysis, while SL cells were observed in the caudal region where MSH cells showed reactivity 1 day before hatching. At variance, GH cells showed a weak immunoreactivity in the rostral portion at hatching that increased 2 days latter. GTH cells also showed weak immunoreactivity in the rostral region of the adenohypophysis at hatching time. Two days later GTH cells were located in the rostral and central regions of the adenohypophysis. At hatching, the neurohypophysis was very small and no nerve processes were seen to penetrate the adenohypophysis tissue. After hatching, the pituitary gland elongated and in 7 days old larvae, the RPD showed a small lumen surrounded by a palisade of PRL, ACTH, and GHT cells; the PPD showed GH and GTH cells while the PI contained SL and MSH cells. The adenohypophysis and neural lobe increased in size with development and, in 42 days old larvae, they were similar to those of juvenile specimens.     

The Structure of Glycosaminoglycans and their Interactions with Proteins

Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG–protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three‐dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG–protein interactions. This review focuses on some key aspects of GAG structure–function relationships using classical examples that illustrate the specificity of GAG–protein interactions, such as growth factors, anti‐thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.     

Characterization of TMPRSS2-ERG fusion high-grade prostatic intraepithelial neoplasia and potential clinical implications

PURPOSE: More than 1,300,000 prostate needle biopsies are done annually in the United States with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer on subsequent needle biopsies in prostate-specific antigen-screened populations. In contemporary series, prostate cancer is detected in approximately 20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histologic subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in prostate cancer has also been shown to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion prostate cancer is associated with a more aggressive clinical course. EXPERIMENTAL DESIGN: In this study, we assessed a series of HGPIN lesions and paired prostate cancer for the presence of TMPRSS2-ERG gene fusion. RESULTS: Fusion-positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances, the matching cancer shared the same fusion pattern. Sixty percent of TMPRSS2-ERG fusion prostate cancer had fusion-negative HGPIN. CONCLUSIONS: Given the more aggressive nature of TMPRSS2-ERG prostate cancer, the findings of this study raise the possibility that gene fusion-positive HGPIN lesions are harbingers of more aggressive disease. To date, pathologic, molecular, and clinical variables do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion prostate cancer in subsequent biopsies.