Beata D. Przybyla

Aging alters macrophage properties in human skeletal muscle both at rest and in response to acute resistance exercise

Macrophages are involved in skeletal muscle repair through pro-inflammatory and alternative functions. We tested the hypothesis that aging alters the abundance and properties of skeletal muscle macrophages that will influence their functional response to acute resistance exercise. Total macrophages (CD 68+), as well as pro- (CD 11b+) and anti-inflammatory (CD 163+) subpopulations and associated cytokine mRNAs were quantified in vastus lateralis biopsies from young (N=17) and elderly (N=17) males pre- and 72 h post-exercise. Pre-exercise, young muscle tended to possess a greater number of macrophages, whereas elderly muscle possessed higher levels of IL-1 beta (P=0.001), IL-1 RA (P=0.003), and IL-10 (P=0.028). Post-exercise, total macrophages did not change in either group, however, the number of CD 11b+ (P=0.039) and CD 163+ (P=0.026) cells increased 55 and 29%, respectively, but only in the young. IL-1 beta (P=0.006), IL-10 (P=0.016), and AMAC-1 (P=0.044) also increased, approximately two-fold, and again only in the young. Quantitation of CD 11b+ and CD 163+ cells suggests that the majority of resident macrophages possess alternative functions, and a small subpopulation participates in the inflammatory response. Both subpopulations increased their activity post-exercise, exclusively in the young. These findings suggest that aging results in a defective regulation of muscle macrophage function, both at baseline and in response to resistance exercise, that may limit muscle hypertrophy in older adults.

Androgen Deficiency and Defective Intracrine Processing of Dehydroepiandrosterone in Salivary Glands in Sjögren’s Syndrome

Objective We hypothesized that in addition to dehydroepiandrosterone (DHEA) depletion, Sjögren’s syndrome (SS) is characterized by local androgen deficiency in salivary glands and defects in local processing of DHEA. Methods Sex steroid levels in serum and saliva were measured using enzyme immunoassays. Androgen effects on salivary gland cells were analyzed using the cysteine-rich secretory protein-3 (CRISP-3) androgen biomarker. Results Serum and salivary concentrations of androgens were low in SS. Substrate to end-product ratios and correlations suggest that in SS salivary glands DHEA is effectively converted to testosterone, but that there are defects in converting testosterone further to dihydrotestosterone (DHT). In healthy controls no such phenomenon was seen, but testosterone is effectively converted to DHT. Salivary glands contained type I 5-α-reductase, and its inhibition with dutasteride completely blocked the upregulating effect of DHEA, but not of DHT, on CRISP-3 in human salivary gland acinar cells. Conclusion DHEA and DHT upregulate CRISP-3, which is reportedly low in SS. The effect of DHEA on CRISP-3 is indirect and is inhibited by dutasteride, showing that there is intracrine processing of DHEA in salivary glands. In healthy glands, but not in SS, DHEA is effectively taken up and converted to DHT. Sex steroid concentrations in saliva in part reflect glandular uptake of DHEAsulfate and local intracrine DHEA metabolism, which seem to be defective in SS. Our study demonstrates a prominent androgen deficiency and a defect in intracrine production of active androgens in SS salivary glands, also suggesting that salivary DHT cannot be maintained at a normal level in this female-dominant autoimmune exocrinopathy.

Androgens and integrins in salivary glands in Sjogren's syndrome.

Objective. Laminin α1-chain normally induces intercalated duct progenitors to differentiate to acinar cells through integrin (INT) α1ß1 and α2ß1 receptors. Maintenance of acinar cells is impaired in Sjögren’s syndrome (SS), which is also characterized by low levels of serum and salivary androgens. We hypothesized that androgens normally support salivary gland remodeling by upregulating either laminin α1 chain or its cellular α1 or α2 INT subunit-containing receptors. Methods. Intercalated duct and acinar human salivary gland (HSG) cells and labial salivary gland (LSG) biopsies from healthy controls and patients with SS were cultured without or with sex steroids. Laminin α1 chain and INT α1 and α2 subunits were studied using quantitative reverse-transcription real-time polymerase chain reaction and INT α1 and α2 subunits using immunofluorescence staining. Results. INT α1-subunit and α2-subunit messenger RNA (mRNA) levels were increased in intercalated duct and acinar cells by DHEA and testosterone. In contrast, laminin α1-chain mRNA levels were not affected. The upregulating effect of DHEA on INT subunits was also seen at the protein level. DHEA also increased mRNA levels of both INT subunits in healthy but not SS LSG. Conclusion. Androgens increased INT α1 and α2 subunits in tubuloepithelial cells and in healthy LSG, but in SS salivary glands this androgen regulation was defective, which is likely to contribute to defective outside-in signaling, acinar atrophy, and ductal cell hyperplasia.

Co-regulation of dopamine D1 receptor and uncoupling protein-2 expression in 3-nitropropionic acid-induced neurotoxicity: neuroprotective role of L-carnitine.

This study tested the hypothesis that the expression of uncoupling proteins (UCPs) and dopamine (DA) system genes is responsive to 3-nitropropionic acid (3-NPA) neurotoxic effects and to the neuroprotective effects of the mitochondrial enhancer, L-carnitine (LC), in the rat striatum. Inactivation of mitochondrial succinate dehydrogenase (SDH) by 3-NPA results in hypoxic brain damage. Hypoxic conditions induce uncoupling protein-2 (UCP-2). An increase in UCP-2 expression may lead to a decrease in production of reactive oxygen species (ROS) associated with energy depletion. However, this adaptive response can also lead to a reduction of ATP that may further contribute to energy deficit and mitochondrial dysfunction. Here, male adult Sprague-Dawley rats (n=5/group) were injected either with saline or 3-NPA at 30 mg/kg, s.c. alone or 30 min after pre-treatment with LC (100mg/kg, i.p.). Rectal temperature was monitored before treatment and 4h following 3-NPA administration. Animals were sacrificed 4h post-treatment. Total RNA was isolated from the striatum and transcripts of UCP-2, UCP-4 and UCP-5 genes, as well as genes related to dopamine metabolism, such as DA D(1) and D(2) receptors, tyrosine hydroxylase (TH), monoamine oxidase-B (MAO-B), and vesicular monoamine transporter-2 (VMAT-2), were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). While core temperature decreased significantly in 3-NPA-treated rats, LC significantly inhibited the hypothermic effect of 3-NPA (p<0.05). 3-NPA caused a significant increase in UCP-2 and DA D(1) receptor gene expression in the striatum and both effects were attenuated by pre-treatment with LC. Since LC maintains the ATP/ADP ratio and was previously shown to be neuroprotective against 3-NPA toxicity, the modulation of UCP-2 expression by LC suggests that LC counteracts energy dissipation and thus prevents the negative effects of ATP decline on DA neurotransmission.

Blood Outgrowth Endothelial Cells Increase Tumor Growth Rates and Modify Tumor Physiology: Relevance for Therapeutic Targeting

Endothelial cell precursors from human peripheral blood have been shown to home to areas of neovascularization and may assist tumor growth by increasing or fortifying blood vessel growth. In the present study, the influence of these cells on tumor growth and physiology was investigated and the role of these cells as a therapeutic target or in determining treatment sensitivity was tested. After isolation from human blood and expansion in vitro, actively growing cells with verified endothelial phenotype (Blood Outgrowth Endothelial Cell, BOEC) were injected i.v. into tumor bearing mice for three consecutive days. The growth rate was significantly enhanced in relatively small RERF human lung tumors (i.e., less than 150 mm3) grown in immunocompromised mice by an average of 1.5-fold while it had no effect when injections were given to animals bearing larger tumors. There were no signs of toxicity or unwanted systemic effects. We also observed evidence of increased perfusion, vessel number, response to 15 Gy radiation and oxygenation in RERF tumors of animals injected with BOECs compared to control tumors. In addition, FSaII murine fibrosarcoma tumors were found to grow faster upon injection of BOECs. When FSaII tumors were subjected to a partial thermal ablation treatment using high intensity focused ultrasound (HIFU) there was consistently elevated detection of fluorescently labeled and i.v. injected endothelial precursors in the tumor when analyzed with optical imaging and/or histological preparations. Importantly, we also observed that BOECs treated with the novel anti-angiogenic peptide anginex in-vitro, show decreased proliferation and increased sensitivity to radiation. In vivo, the normal increase in FSaII tumor growth induced by injected BOECs was blunted by the addition of anginex treatment. It appears that endothelial precursors may significantly contribute to tumor vessel growth, tumor progression and/or repair of tumor damage and may improve the oxygenation and subsequent radiation response of tumors. We surmise that these cells are preferentially stimulated to divide in the tumor microenvironment, thereby inducing the significant increase in tumor growth observed and that the use of injected BOECs could be a viable approach to modulate the tumor microenvironment for therapeutic gain. Conversely, agents or approaches to block their recruitment and integration of BOECs into primary or metastatic lesions may be an effective way to restrain cancer progression before or after other treatments are applied.

Advanced Small Animal Conformal Radiation Therapy Device

We have developed a small animal conformal radiation therapy device that provides a degree of geometrical/anatomical targeting comparable to what is achievable in a commercial animal irradiator. small animal conformal radiation therapy device is capable of producing precise and accurate conformal delivery of radiation to target as well as for imaging small animals. The small animal conformal radiation therapy device uses an X-ray tube, a robotic animal position system, and a digital imager. The system is in a steel enclosure with adequate lead shielding following National Council on Radiation Protection and Measurements 49 guidelines and verified with Geiger-Mueller survey meter. The X-ray source is calibrated following AAPM TG-61 specifications and mounted at 101.6 cm from the floor, which is a primary barrier. The X-ray tube is mounted on a custom-made “gantry” and has a special collimating assembly system that allows field size between 0.5 mm and 20 cm at isocenter. Three-dimensional imaging can be performed to aid target localization using the same X-ray source at custom settings and an in-house reconstruction software. The small animal conformal radiation therapy device thus provides an excellent integrated system to promote translational research in radiation oncology in an academic laboratory. The purpose of this article is to review shielding and dosimetric measurement and highlight a few successful studies that have been performed to date with our system. In addition, an example of new data from an in vivo rat model of breast cancer is presented in which spatially fractionated radiation alone and in combination with thermal ablation was applied and the therapeutic benefit examined.

Effects of L-carnitine pretreatment in methamphetamine and 3-nitropropionic acid-induced neurotoxicity.

Abstract:  Adult, male Sprague‐Dawley rats were injected with 3‐ni‐tropropionic acid (3‐NPA) at 30 mg/kg or methamphetamine (METH) at 20 mg/kg alone or following pretreatment with L‐cartnitine (LC) at 100 mg/kg. Rectal temperature was measured before and 4 h following treatment. Animals were sacrificed at 4 h posttreatment. Monoamine neurotransmitters, dopamine (DA) and serotonin (5‐HT), and their metabolites were analyzed in the striatum using high‐performance liquid chromatography method coupled with electrochemical detection (HPLC/ED) . Transcripts of several genes related to DA metabolism were quantified using real time reverse transciption polymerase chain reaction (RT‐PCR). Core temperature decreased significantly after 3‐NPA acid and increased in METH‐treated rats (P < 0.05). Temperature change at 4 h exhibited a significant LC effect for 3‐NPA, preventing hypothermia (P < 0.05) and no effect for METH. Concentration of DA and 5‐HT, and their metabolites, 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA), increased significantly in 3‐NPA and decreased in METH‐treated rats. An increase in DOPAC/DA turnover and serotonin observed after 3‐NPA was abolished in LC‐/3‐NPA‐treated rats. In both 3‐NPA‐ and METH‐treated rats, LC prevented an increase in DA receptor D1 gene expression. It appears that carnitine effect preventing hypothermia after 3‐NPA treatments may be related not only to its mitochondriotropic actions but also to inhibitory effect on the DA and 5‐HT systems activated after the exposure to 3‐NPA. The same effect observed at the transcriptional level, at least for the DA receptor D1, may account for protection against METH toxicity.

Neurobiology and Hormonal Control of Lacrimal and Salivary Gland Function

Sjogren’s syndrome (SS) is characterized by diminished production of secretes from exocrine glands and sicca complex, which occur in an autoimmune context; 90% of patients are women, usually developing the disease when they are 40–50 years old. This characterization comprises several neuroimmunoendocrine aspects. The sympathetic, parasympathetic, vascular, acinar, and myoepithelial systems normally work in a co-ordinated fashion in two phases, resting and stimulated phases, corresponding to the secretion of resting and stimulated saliva. Stimulation of the exocrine flow with acetylcholine is coupled with a proportional local release of acinotrophic vasoactive intestinal peptide (VIP) from post-ganglionic parasympathetic nerve terminals, which helps to recover and repair the acinar cell during the “resting” (recovery) phase. Lost (dead and detached) acinar cells are replaced by remodeling based on an asymmetric division of the intercalated duct progenitor cells, which as a result are able to maintain their unipotent stemness and at the same time able to replace the lost acinar cells. This remodeling is maintained by dehydroepiandrosterone (DHEA) produced in the reticular zone of the endocrine adrenal gland in an endocrine process, but is locally in exocrine glands converted to dihydrotestosterone (DHT) in an intracrine process. SS is characterized by deranged intracrine enzymatic machinery and impaired DHEA-to-DHT conversion. This impairment leads particularly in women to acinar cell atrophy/loss and a reciprocal ductal cell hyperplasia. Men are protected because they feed the intracrine machinery also with testosterone, only one step away from DHT. Abnormally processed self antigens are released from dying cells and break the immunologic tolerance so that autoantibodies are formed against previously hidden endotopes. Immune inflammation leads to synthesis and release of tumor necrosis factor-α, interleukin-1β, and other cytokines, which impair signal transduction in acinar cells and add to the burden of the inflammatory functio laesa.

Conductive thermal ablation of 4T1 murine breast carcinoma reduces severe hypoxia in surviving tumour

Purpose: The purpose of this study was to quantify hypoxia changes in viable tumour volumes after thermal ablation of a murine breast carcinoma. Methods: Murine breast 4T1 tumours were grown in the rear leg of BALB/c mice to an average diameter of 10–12 mm. Tumours were treated with conductive interstitial thermal therapy (CITT) at a peak temperature of 80–90°C for 10 min. The animals were euthanised 72 h later, and the tumours were removed for immunohistochemical staining with pimonidazole – a marker of partial pressure of oxygen. The levels of pimonidazole staining intensity were used to quantify changes in hypoxia gradients in terms of strong, medium and weak positive pixel fractions. Results: The pimonidazole staining ratio of viable control tumour tissue to viable tissue in tumours that were ablated was 0.7 for weak staining, 2.7 for medium staining and 8.0 (p < 0.03) for strong pimonidazole staining. Conclusion: This shift of pimonidazole staining toward lower intensity pixels in the remaining tumour indicates that tumour ablation with CITT may increase radiosensitivity of the remaining tumour tissue and presents a rationale for combination therapy.

Molecular changes in bone marrow, tumor and serum after conductive ablation of murine 4T1 breast carcinoma

Thermal ablation of solid tumors using conductive interstitial thermal therapy (CITT) produces coagulative necrosis in the center of ablation. Local changes in homeostasis for surviving tumor and systemic changes in circulation and distant organs must be understood and monitored in order to prevent tumor re-growth and metastasis. The purpose of this study was to use a mouse carcinoma model to evaluate molecular changes in the bone marrow and surviving tumor after CITT treatment by quantification of transcripts associated with cancer progression and hyperthermia, serum cytokines, stress proteins and the marrow/tumor cross-talk regulator stromal-derived factor 1. Analysis of 27 genes and 22 proteins with quantitative PCR, ELISA, immunoblotting and multiplex antibody assays revealed that the gene and protein expression in tissue and serum was significantly different between ablated and control mice. The transcripts of four genes (Cxcl12, Sele, Fgf2, Lifr) were significantly higher in the bone marrow of treated mice. Tumors surviving ablation showed significantly lower levels of the Lifr and Sele transcripts. Similarly, the majority of transcripts measured in tumors decreased with treatment. Surviving tumors also contained lower levels of SDF-1α and HIF-1α proteins whereas HSP27 and HSP70 were higher. Of 16 serum chemokines, IFNγ and GM-CSF levels were lower with treatment. These results indicate that CITT ablation causes molecular changes which may slow cancer cell proliferation. However, inhibition of HSP27 may be necessary to control aggressiveness of surviving cancer stem cells. The changes in bone marrow are suggestive of possible increased recruitment of circulatory cancer cells. Therefore, the possibility of heightened bone metastasis after thermal ablation needs to be further investigated and inhibition strategies developed, if warranted.