Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine : involvement of adenosine and dopamine receptors

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge : A proof-of-concept study

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Therapeutic immunisation of rabbits with cottontail rabbit papillomavirus (CRPV) virus-like particles (VLP) induces regression of established papillomas

There is overwhelming evidence that persistent infection with high-risk human papillomaviruses (HR-HPV) is the main risk factor for invasive cancer of the cervix. Due to this global public health burden, two prophylactic HPV L1 virus-like particles (VLP) vaccines have been developed. While these vaccines have demonstrated excellent type-specific prevention of infection by the homologous vaccine types (high and low risk HPV types), no data have been reported on the therapeutic effects in people already infected with the low-risk HPV type. In this study we explored whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with CRPV VLPs. Rabbits immunised with CRPV VLPs had papillomas that were significantly smaller compared to the negative control rabbit group (P ≤ 0.05). This data demonstrates the therapeutic potential of PV VLPs in a well-understood animal model with potential important implications for human therapeutic vaccination for low-risk HPVs. © 2008 Govan et al; licensee BioMed Central Ltd.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Age-related changes in cardiac adenosine receptor expression

Adenosine is an important cardioprotective agent that works via several adenosine receptor (ADOR) subtypes to regulate cardiovascular activity. It is well established that functional responses to adenosine decline with age. What is unclear, though, is whether these changes occur at the receptor, second messenger or translational level. In this study we determined the effect of age on cardiac adenosine receptor expression using the housekeeping gene 18S rRNA versus the adenosine A2B receptor gene as internal controls. Absolute quantification showed that no age-related changes occurred in the expression of 18S rRNA or adenosine A2B receptor internal control genes. Subsequently, relative analysis of the adenosine receptor subtypes using 18S rRNA found a significant age-related reduction in the expression of the adenosine A1 receptor (5.5-fold), with no changes in the expression of the adenosine A2A, A2B and A3 receptors. When using the expression of the adenosine A2B receptor as the internal control gene, a significant down regulation of both the adenosine A1 (5.4-fold) and A2A (2.2-fold) receptors with no change in the expression of adenosine A3 receptor was found. Therefore, the high level of expression of the 18S rRNA housekeeping gene was found to mask a significant change in expression of the adenosine A2A receptor with age. Ultimately, these findings show an age-related reduction in adenosine A1 and A2A receptor expression in rat heart.

The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis

In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A(1), A(2A), A(2B) and A(3), and estrogen receptors (ER) alpha and beta. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA(2A) (>4-fold down) and consistent (>2-fold) down-regulation of ADORA(1), ADORA(3), and ER-beta, following ovariectomy. No change was found in ADORA(2B) or ER-alpha. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aims Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. Methods MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. Results Human L-MSC cultures were typically CD34−, CD45− and HLA-DR− and CD73+, CD90+, CD105+ and HLA-ABC+. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. Conclusions L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

Hepatocyte growth factor stimulates invasion across reconstituted basement membranes by a new human small intestinal cell line

Hepatocyte growth factor/scatter factor (HGF/SF) is a protein growth factor whose pleiotropic effects on epithelial cells include the stimulation of motility, mitosis and tubulogenesis. These responses are mediated by the cell surface tyrosine kinase receptor c-met. Because both the cytokine and receptor are found in the gastrointestinal tract, we have studied the effects of HGF/SF on transformed gut epithelial cells which express c-met. Here we describe the response of a new transformed human jejunal epithelioid cell line (HIE-7) to HGF/SF. Morphologically HIE-7 cells are immature. Their epithelial lineage was confirmed by reactivity with the epithelial specific antibodies AE1/AE3, Cam 5.2, Ber-EP4 and anti-EMA and is consistent with their expression of c-met mRNA and protein. In addition, electron microscopic analysis revealed the presence of primitive junctions and rudimentary microvilli, but features of polarization were absent. When grown on reconstituted basement membranes, HIE-7 cells formed closely associated multicellular cord-like structures adjacent to acellular spaces. However, the cells did not mature structurally, form lumen-like structures or express disaccharidase mRNA, even in the presence of recombinant HGF (rHGF). On the other hand, rHGF induced HIE-7 cells to scatter and stimulated their rapid migration in a modified wound assay. To determine whether the motogenic effect caused by rHGF is associated with HIE-7 cell invasiveness across reconstituted basement membranes, a Boyden chamber chemoinvasion assay was performed. rHGF stimulated a 10-fold increase in the number of HIE-7 cells that crossed the basement membrane barrier, while only stimulating a small increase in chemotaxis across a collagen IV matrix, suggesting that the cytokine activates matrix penetration by these cells. rHGF also stimulated the invasion of basement membranes by an undifferentiated rat intestinal cell line (IEC-6) and by two human colon cancer cell lines which are poorly differentiated (DLD-1 and SW 948). In contrast, two moderately well differentiated colon cancer cell lines (Caco-2 and HT-29) did not manifest an invasive response when exposed to rHGF. These results suggest that HGF/SF may play a significant role in the invasive behavior of anaplastic and poorly differentiated gut epithelial tumors.

Health education and the control of intestinal worm infections in China : a new vision

Background: The transmission of soil-transmitted helminths (STHs) is associated with poverty, poor hygiene behaviour, lack of clean water and inadequate waste disposal and sanitation. Periodic administration of benzimidazole drugs is the mainstay for global STH control but it does not prevent re-infection, and is unlikely to interrupt transmission as a stand-alone intervention. Findings: We reported recently on the development and successful testing in Hunan province, PR China, of a health education package to prevent STH infections in Han Chinese primary school students. We have recently commenced a new trial of the package in the ethnically diverse Xishuangbanna autonomous prefecture in Yunnan province and the approach is also being tested in West Africa, with further expansion into the Philippines in 2015. Conclusions: The work in China illustrates well the direct impact that health education can have in improving knowledge and awareness, and in changing hygiene behaviour. Further, it can provide insight into the public health outcomes of a multi-component integrated control program, where health education prevents re-infection and periodic drug treatment reduces prevalence and morbidity.

Exploring diarrhoea, enteral nutrition and intestinal microbial flora relationships in critically ill patients

Diarrhoea is a common complication observed in critically ill patients. Relationships between diarrhoea, enteral nutrition and aerobic intestinal microflora have been disconnectedly examined in this patient cohort. This research used a two-study, observational design to examine these associations. Higher diarrhoea incidence rates were observed when patients received enteral tube feeding, had abnormal serum blood results, received multiple medications and had aerobic microflora dysbiosis. Further, significant aerobic intestinal microflora changes were observed over time in patients who experienced diarrhoea. These results establish a platform for further work to improve the intestinal health of critically ill patients.

Study of the novel non-xanthine heterocyclic compound GU 285 as a potent non-selective adenosine receptor antagonist in the rat

The novel pyrazolo[3,4-d]pyrimidine compound GU285 (4-amino-6-alpha-carbamoylethylthio-1- phenylpyrazolo[3,4-d]pyrimidine, CAS 134896-40-5) was examined for its ability (1) to inhibit binding of adenosine (ADO) receptor ligands in rat brain membranes, (2) to antagonise functional responses to ADO agonists in rat right and left atria and coronary resistance vessels, and (3) to reduce the fall in heart rate and arterial blood pressure produced by the ADO A1 agonist N6-cyclopentyladenosine (CPA) in the intact, anaesthetized rat. GU285 competitively inhibited binding of the ADO A1 agonist [3H]-R-N6-phenylisopropyladenosine (R-PIA) yielding a Ki value of 11 (7-18) nmol.l-1 (geometric mean +/- 95% Cl). When assayed against the ADO A2A selective agonist [3H]-2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamidoadenosine, (CGS21680), a Ki of 15 (10-24) nmol.l-1 was obtained. In spontaneously beating right atria, GU285 competitively antagonized negative chronotropic effects of R-PIA with a pA2 of 8.7 +/- 0.3 and in electrically paced left atria, GU285 competitively antagonized negative inotropic effects of R-PIA with a pA2 of 9.0 +/- 0.1. In the potassium-arrested, perfused rat heart GU285 (1 mumol.l-1) antagonized only the high sensitivity, ADO A2B mediated component of the biphasic relaxation of the coronary vasculature produced by NECA. The low sensitivity component was unchanged. GU285 (1 mumol.kg-1) antagonized the negative chronotropic and hypotensive effects of the adenosine A1 agonist CPA in anaesthetized rats, producing a 10-fold rightward shift in the dose-response relationship. These data demonstrate that in the rat, GU285 is a potent, non-selective adenosine receptor antagonist that maintains its activity in vivo.

Age-related changes to adenosine in rat coronary resistance vessels

1. The vasodilator effects of adenosine receptor agonists, isoprenaline and histamine were examined in perfused heart preparations from young (4–6 weeks) and mature (12–20 weeks) rats. 2. Adenosine induced a biphasic concentration-dependent decrease in KCl (35 mM) raised coronary perfusion pressure in hearts from young and mature rats, suggesting the presence of both high- and low-affinity sites for adenosine receptors in the two age groups tested. In heart preparations from mature rats, vasodilator responses to adenosine were significantly reduced compared with responses observed in young rats. 3. Responses to 5′-N-ethylcarboxamidoadenosine (NECA) and 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) were reduced in preparations from mature rats, whereas the vasodilator actions of N6-cyclopentyladenosine (CPA) and N6-2-(4-aminophenyl)ethyladenosine (APNEA) did not change with age. 4. The results presented in this study suggest that several adenosine receptor subtypes mediate vasodilator responses in the coronary circulation of the rat and that a reduction in response to adenosine with age may be due to changes in the high-affinity receptor site.

A biphasic response to adenosine in the coronary vasculature of the K+-arrested perfused rat heart

Biphasic vasodilatory responses to adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) were observed in the coronary vasculature of K(+)-arrested perfused rat hearts. Dose-response data for both agonists were best represented by two-site models. For adenosine, two sites with negative log ED50 (pED50) values of 8.1 +/- 0.1 (mean +/- S.E.M) and 5.2 +/- 0.1 were obtained, mediating 31 +/- 2% and 69 +/- 2% of the total response. In the presence of 8-phenyltheophylline, the vasodilatory response to adenosine remained best fitted to a two-site model with pED50 values of 7.0 +/- 0.2 and 5.4 +/- 0.2. The relative contribution of each site to the total response remained unchanged. For NECA, pED50 values of 9.6 +/- 0.1 and 6.8 +/- 0.2 were obtained, representing 48 +/- 3% and 52 +/- 3% of the sites, respectively. In contrast, ATP produced a monophasic response with a pED50 value of 8.8 +/- 0.1. These results provide evidence of adenosine receptor and response heterogeneity in the in situ coronary vasculature.