SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-κB/STAT3 complex to its promoter in malignant lymphoid cells.

We explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with panobinostat in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis, concomitant with deacetylation of STAT3 and NF-κB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA-damage-response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA-binding activity of both NF-κB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with panobinostat enhanced the anti-growth and anti-survival effects mediated by either compound alone. Quantitative-PCR confirmed that the panobinostat in combination with SRT2183, SRT501 or resveratrol leads to greater upregulation of GADD45G than any of the single agents. Panobinostat plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-κB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-κB. In addition, the binding of NF-κB/STAT3 complexes to the GADD45G promoter is inhibited following panobinostat, SRT501 or resveratrol treatment. Moreover, the combination of panobinostat with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. These data suggest that STAT3 is a corepressor with NF-κB of the GADD45G gene and provides in vitro proof-of-concept for the combination of HDACi with SIRT1 activators as a potential new therapeutic strategy in lymphoid malignancies.

Phase I trial of a murine antibody to MUC1 in patients with metastatic cancer: evidence for the activation of humoral and cellular antitumor immunity.

BACKGROUND: BrevaRex mAb-AR20.5 is a murine anti-MUC1 monoclonal antibody generated to induce MUC1 antigen-specific immune responses through the formation of immune complexes with circulating MUC1 and/or MUC1-expressing tumor cells that may target these immune complexes (IC) to receptors on dendritic cells (DCs). PATIENTS AND METHODS: A phase I study focusing on safety and immunology evaluated 1, 2 and 4-mg doses. Seventeen patients with MUC1-positive cancers received intravenous infusions of the antibody over 30 min on weeks 1, 3, 5, 9, 13 and 17 of treatment. RESULTS: mAb-AR20.5 was well-tolerated, not associated with dose-limiting toxicity, and did not induce hypersensitivity reactions. Overall, five of 15 evaluable patients developed human anti-mouse antibodies (HAMA), five developed anti-idiotypic antibodies (Ab2) and seven developed anti-MUC1 antibodies. Immune responses were most prominent in the 2-mg dose cohort for all parameters tested, and treatment-emergent MUC1-specific T-cell responses were detected in five of 10 evaluable patients treated with mAb-AR20.5. CONCLUSIONS: The injection of a murine antibody to MUC1 induces MUC1-specific immune responses in advanced cancer patients. Anti-MUC1 antibody increases correlated with decrease or stabilization of CA15.3 levels (P=0.03). The 2-mg dose of mAb-AR20.5 showed strongest biological activity, and will be evaluated in future efficacy trials.

Nutrient intake in women with primary and secondary Sjögren's syndrome.

OBJECTIVE: Recently, it has been proposed that dietary factors may contribute to the etiology and progression of Sjögren's syndrome, and that nutritional intervention may modify the severity of pathological abnormalities. The objective of this study was to determine whether the nutrient intake of women with primary (1 degrees SS) or secondary (2 degrees SS; ie with systemic lupus erythematosus (2 degrees SS/SLE) or rheumatoid arthritis (2 degrees SS/RA) Sjögren's syndrome is significantly different than that of age- and gender-matched controls. DESIGN: Women with Sjögren's syndrome were asked to complete the 97 General Purpose Semi-Quantitative Food Frequency Questionnaire, which consists of a list of 147 separate food items that represent the major sources of multiple nutrients. Nutritional data were evaluated in terms of absolute and energy-adjusted nutrient amounts and analyzed by ANOVA. RESULTS: Our results showed: (a) greater intake of energy, glutamate, carbohydrates, lactose, phosphorus, caffeine and unsupplemental thiamin and riboflavin in 1 degrees SS, as well as supplemental calcium in 2 degrees SS/SLE, compared with controls; (b) greater nutrient intake of energy, protein, glutamate, methionine, tryptophan, carbohydrates, lactose, supplemental calcium and phosphorus, sodium, caffeine and unsupplemental calcium, riboflavin and thiamin in 2 degrees SS/RA, relative to 1 degrees SS and/or 2 degrees SS/SLE; (c) higher energy-adjusted values for supplemental calcium in 2 degrees SS/SLE, and for vitamin A and supplemental iron and zinc in 2 degrees SS/RA, compared with other groups; and (d) higher energy-adjusted intake of supplemental calcium, and a lower energy-adjusted intake of unsupplemental vitamin C, polyunsaturated fat, linoleic acid, omega-3 fatty acid, and specific other unsaturated fatty acids, in the Sjögren's syndrome group as a whole, relative to controls. CONCLUSIONS: Our findings demonstrate that nutrient intake is altered in Sjögren's syndrome.

Protective effects of prenatal choline supplementation on seizure-induced memory impairment.

Choline is an essential nutrient for rats and humans, and its availability during fetal development has long-lasting cognitive effects (Blusztajn, 1998). We investigated the effects of prenatal choline supplementation on memory deficits associated with status epilepticus. Pregnant rats received a control or choline-supplemented diet during days 11-17 of gestation. Male offspring [postnatal day 29 (P29)-32] were tested for their ability to find a platform in a water maze before and after administration of a convulsant dose of pilocarpine at P34. There were no differences between groups in water maze performance before the seizure. One week after status epilepticus (P41-P44), animals that had received the control diet prenatally had a drastically impaired performance in the water maze during the 4 d testing period, whereas prenatally choline-supplemented rats showed no impairment. Neither the seizures nor the prenatal availability of choline had any effect on hippocampal choline acetyltransferase or acetylcholinesterase activities. This study demonstrates that prenatal choline supplementation can protect rats against memory deficits induced by status epilepticus.

Androgen influence on the meibomian gland.

PURPOSE: The hypothesis in the study was that androgens control meibomian gland function, regulate the quality and/or quantity of lipids produced by this tissue, and promote the formation of the tear film's lipid layer. To test this hypothesis, a study was conducted to determine whether androgen receptor protein exists in the epithelial cell nuclei of rat meibomian glands and, in addition, whether androgen deficiency and/or treatment influences the gross morphology, neutral lipid content, and fatty acid profile of the rabbit meibomian gland, as well as the appearance of the tear film lipid layer. METHODS: Rat lids were obtained and processed for immunohistochemistry. Meibomian glands from intact, androgen- and/or placebo-treated rabbits were analyzed by histology, and glandular lipids were evaluated by gas chromatography, high-performance liquid chromatography (HPLC), and mass spectrometry. The rabbit tear film lipid layer was assessed by interferometry. RESULTS: In the current study androgen receptor protein existed within acinar epithelial cell nuclei of rat meibomian glands; androgen deficiency was associated with alterations in the lipid content of the rabbit meibomian gland; 19-nortestosterone treatment modulated the fatty acid profile in the total and neutral lipid fractions of the rabbit meibomian gland; and androgens did not appear to influence the gross morphology of meibomian tissue or to exert a demonstrable effect on the rabbit tear film lipid layer. CONCLUSIONS: The findings show that the meibomian gland is an androgen target organ and that androgens influence the lipid profile within this tissue. However, the extent to which androgens regulate the production of these lipids and whether this action may impact tear film stability remain to be determined.

Effect of androgen deficiency on the human meibomian gland and ocular surface.

The purpose of this study was to determine whether the chronic use of antiandrogen medications leads to meibomian gland dysfunction, altered lipid profiles in meibomian gland secretions, decreased tear film stability, and evaporative dry eye. Subjects taking antiandrogen therapy for prostatic indications, as well as age-related controls, were asked to complete a questionnaire that assessed dry eye symptoms and then were given a complete anterior segment examination. Moreover, meibomian gland secretions were obtained from each eye and analyzed by high-performance liquid chromatography/mass spectrometry for the relative content of cholesterol, cholesterol esters, wax esters, diglycerides, triglycerides, and specific molecular species in the diglyceride fraction. Our results demonstrate that patients taking antiandrogen treatment, compared with age-related controls, had a: 1) significant increase in the frequency of appearance of tear film debris, an abnormal tear film meniscus, irregular posterior lid margins, conjunctival tarsal injection, and orifice metaplasia of the meibomian glands; 2) significant increase in the degree of ocular surface vital dye staining; 3) significant decrease in the tear film breakup time and quality of meibomian gland secretions; and 4) significant increase in the frequency of light sensitivity, painful eyes, and blurred vision. In addition, the use of antiandrogen pharmaceuticals was associated with significant changes in the relative amounts of lipids in meibomian gland secretions. Our findings indicate that chronic androgen deficiency is associated with meibomian gland dysfunction and dry eye.

Prenatal availability of choline alters the development of acetylcholinesterase in the rat hippocampus.

Choline (Ch) supplementation during embryonic days (ED) 12-17 enhances spatial and temporal memory in adult and aged rats, whereas prenatal Ch deficiency impairs attention performance and accelerates age-related declines in temporal processing. To characterize the neurochemical and neuroanatomical mechanisms that may mediate these behavioral effects in rats, we studied the development [postnatal days (PD) 1, 3, 7, 17, 27, 35, 90, and 26 months postnatally] of acetylcholinesterase (AChE) activity in hippocampus, neocortex and striatum as a function of prenatal Ch availability. We further measured the density of AChE-positive laminae (PD27 and PD90) and interneurons (PD20) in the hippocampus as a function of prenatal Ch availability. During ED11-ED17 pregnant Sprague-Dawley rats received a Ch-deficient, control or Ch-supplemented diet (average Ch intake 0, 1.3 and 4.6 mmol/kg/day, respectively). Prenatal Ch deficiency increased hippocampal AChE activity as compared to control animals in both males and females from the 2nd to 5th week postnatally. Moreover, prenatal Ch supplementation reduced hippocampal AChE activity as compared to control animals over the same developmental period. There was no effect of prenatal Ch status on either cortical or striatal AChE activity at any age measured, and by PD90 the effect of Ch on hippocampal AChE was no longer observed. In order to localize the early changes in hippocampal AChE activity anatomically, frozen coronal brain sections (PD20, PD27, PD90) were stained histochemically for AChE. Consistent with biochemical results, the AChE staining intensity was reduced in PD27 hippocampal laminae in the Ch-supplemented group and increased in the Ch-deficient group compared to control animals. There was no effect of the diet on hippocampal AChE staining intensity on PD90. In addition, the prenatal Ch availability was found to alter the size and density of AChE-positive PD20 interneurons. These results show that prenatal Ch availability has long-term consequences on the development of the hippocampal cholinergic system.

Androgens and dry eye in Sjögren's syndrome.

Sjögren's syndrome is an extremely complex and currently incurable autoimmune disorder, which occurs primarily in females, and is associated with lacrimal gland inflammation, meibomian gland dysfunction, and severe dry eye. We hypothesize that androgen deficiency, which reportedly occurs in primary and secondary Sjögren's syndrome (e.g., systemic lupus erythematosus, rheumatoid arthritis), is a critical etiologic factor in the pathogenesis of dry eye syndromes. We further hypothesize that androgen treatment to the ocular surface will promote both lacrimal and meibomian gland function and alleviate both "aqueous-deficient" and "evaporative" dry eye. Our results demonstrate that androgens regulate both lacrimal and meibomian gland function, and suggest that topical androgen administration may serve as a safe and effective therapy for the treatment of dry eye in Sjögren's syndrome.

Imprinting of hippocampal metabolism of choline by its availability during gestation: implications for cholinergic neurotransmission.

Choline supplementation during the second half of the gestational period in rats permanently improves visuospatial memory. Choline availability during this period also alters the turnover of choline and acetylcholine in the hippocampus in 3-4 week-old animals in a complex pattern consistent with the notion that cholinergic neurotransmission is enhanced by prenatal choline supplementation.

Developmental changes in phospholipase D activity and mRNA levels in rat brain.

Phospholipase D (PLD) activity and PLD1 mRNA levels were determined in rat brain at ages ranging from embryonic day (E) 19 to postnatal day (P) 49. Basal, oleate-, and phosphatidylinositol-4, 5-bisphosphate-stimulated PLD activity increased between E19 and P24 by approximately 3-fold and remained unaltered thereafter. A similar developmental pattern of mRNA levels of PLD1 isoform was found by Northern blotting. The development of PLD correlates with synaptogenesis and myelination suggesting that the enzyme might have an important function in these processes.

Prenatal availability of choline modifies development of the hippocampal cholinergic system.

Choline supplementation during fetal development [embryonic days (E) 11-17] permanently enhances memory performance in rats. To characterize the neurochemical mechanisms that may mediate this effect, we investigated the development of indices of the cholinergic system in the hippocampus: choline acetyltransferase (ChAT), acetylcholinesterase (AChE), synthesis of acetylcholine (ACh) from choline transported by high-affinity choline uptake (HACU), and potassium-evoked ACh release. During E11-E17, Sprague-Dawley pregnant rats consumed 0 [choline-deficient (ChD)], 1.3 [control (ChC)], and 4.6 [choline-supplemented (ChS)] mmol/(kg x day) of choline, respectively. On postnatal days 17 and 27, hippocampi of the ChD animals had the highest AChE and ChAT activities, and increased synthesis of ACh from choline transported by HACU, concomitant with reductions of tissue ACh content relative to the ChC and ChS rats and an inability to sustain depolarization-evoked ACh release relative to the ChS animals. In contrast, AChE and ChAT activities, and ACh synthesized from choline transported by HACU, were lowest in ChS rats whereas depolarization-evoked ACh release was the highest. This pattern of changes suggests that the hippocampus of the ChD animals is characterized by fast ACh recycling and efficient choline reutilization for ACh synthesis, presumably to maintain adequate ACh release despite the decrease of the ACh pool, whereas in the ChS animals ACh turnover and choline recycling is slower while the evoked release of ACh is high. Together, the data show a complex adaptive response of the hippocampal cholinergic system to prenatal choline availability and provide a novel example of developmental plasticity in the nervous system governed by the supply of a single nutrient.

Dietary choline supplementation in pregnant rats increases hippocampal phospholipase D activity of the offspring.

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. The biochemical mechanism of this effect may be related to the function of choline as a precursor of phosphatidylcholine (PC), the substrate of a receptor-stimulated enzyme, phospholipase D (PLD). PLD activation initiates the sequential formation of two intracellular messengers, phosphatidic acid and 1,2-sn-diacylglycerol. We hypothesized that prenatal choline status may cause long-term modulation of PLD-catalyzed PC hydrolysis in the hippocampus, a brain region implicated in visuospatial memory functions. PLD activity was determined in hippocampal slices prelabeled with [3H]glycerol or [3H]oleic acid by measuring the PLD-catalyzed formation of [3H]phosphatidylpropanol in the presence of 1-propanol. Slices were obtained from male pups born to mothers consuming a control diet, a choline-supplemented diet, or a choline-free diet from days 11 to 17 of pregnancy. The radiolabeling of phospholipid classes was unaffected by the treatments. Prenatal choline supplementation significantly increased basal PLD activity in [3H]glycerol-labeled slices [by 46% of controls on postnatal day (P) 7 and by 36% on P21], and [3H]oleate-labeled slices (by 91% on P7), as well as glutamate-stimulated PLD activity in [3H]oleate-labeled slices (by 60% on P7). Prenatal choline deficiency failed to alter PLD activity. The actions of choline apparently required intact cells because in vitro assays of PLD activity in hippocampal homogenates, using fluorescent NBD-PC as substrate, revealed no differences between groups. The results show that prenatal choline supplementation up-regulates basal and receptor-stimulated PLD activity in the hippocampus during postnatal development.

Differences in the developmental expression of the vesicular acetylcholine transporter and choline acetyltransferase in the rat brain.

The neurotransmitter acetylcholine (ACh) is synthesized by the enzyme choline acetyltransferase (ChAT) and then transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Since the VAChT gene is located within the first intron of the ChAT gene, it is likely that expression of the two genes is coregulated. We compared the developmental expression of VAChT and ChAT mRNA and protein in rat brain. ChAT mRNA and enzyme activity increased by almost 10-fold from embryonic day 19 to adulthood, with the most pronounced increase occurring after birth. In contrast, VAChT mRNA increased by only about 2-fold from late embryonic stages to adult levels. However, VAChT protein followed the developmental pattern of ChAT activity, revealing a large excess of VAChT mRNA over VAChT protein during early stages of development. The results are suggestive of differential mechanisms of ChAT and VAChT regulation during brain development, and of possible translational control of VAChT expression.