Humoral and cell-mediated immune responses elicited by poly (dl-lactide) adjuvanted filarial antigen molecules

Abstract

Context: In our recent studies, Brugia malayi molecules have shown interesting immune-stimulating and immune-suppressive properties. Among these, F6 a pro-inflammatory (54–68 kDa) SDS-PAGE resolved fraction of the parasite when administered with Freund’s complete/incomplete adjuvant in animals, elicited both Th1 and Th2 type immune responses and protects the host from filarial parasite.

Objective: The present study was aimed at developing biodegradable microspheres for filarial antigenic protein molecules and to investigate the immunoadjuvanticity of microspheres (Ms)-loaded F6 molecules.

Materials and methods: Poly-lactide microspheres (DL-PLA-Ms) were prepared using double emulsification and solvent evaporation method; and studied their size, shape, antigen adsorption efficiency, in-process stability, and antigen release profiles. F6 and B. malayi adult worm (BmA: ～17 to 180 kDa) protein molecules adsorbed on the Ms were administered in a single shot into Swiss mice, subcutaneously, and investigated their immunoadjuvant effect and compared with one/two doses-schedule of plain F6/BmA.

Results: Immunization with F6/BmA-loaded DL-PLA-Ms resulted in upregulation of cellular proliferation, IFN- γ, TNF-α and NO release from host’s cells stimulated with F6/BmA or LPS/Con A, IgG, IgG1 and IgG2a levels. These responses were well comparable with the responses produced by two doses of plain BmA/F6.

Discussion and conclusion: In conclusion, a single dose of DL-PLA-Ms-F6 induced predominantly Th1 immune responses and well comparable with two doses of plain F6. This is the first ever report on potential of DL-PLA-Ms as adjuvant for filarial immunogen.     

Polymeric lamellar substrate particles for intranasal vaccination

In recent years, several strategies have been under investigation to achieve safe and effective immunisation, in terms of new antigens, adjuvants and routes of vaccination. The latter include mucosal sites such as oral, rectal, vaginal and nasal. Biodegradable microparticles produced from polymers such as poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been extensively studied for immunisation. These microparticles allow controlled release of vaccines with the aim to develop as single dose vaccines. However there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. Polymeric lamellar substrate particles (PLSP) produced by simple precipitation of PLA, form a novel polymeric system for the adsorption of antigens. This procedure avoids pH changes, exposure to organic solvents and hence allows the integrity of the antigen to be retained. The aim of this article is to discuss the factors affecting the characteristics of PLSP and adsorption of antigens onto PLSP and consider their potential as adjuvants for the nasal delivery of protein, peptide or viral vaccines.     

Cationic nanoglycolipidic particles as vector and adjuvant for the study of the immunogenicity of SIV Nef protein

The objective of this study was to test the immunogenicity of SIV Nef protein formulated in cationic nanoglycolipidic particles of 100 nm of diameter. In parallel, the adjuvant effect of these nanoglycolipidic particles was compared in similar experiments using GST-Nef in association with the commonly strongest used complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant in association with MDP or MDP alone. Our results showed that these particles do not alter the integrity of our immunogen GST-Nef, which remains stable for more than three months at 4 °C. We demonstrated that in the presence of nanoglycolipidic particles antibodies against Nef were produced since the first injection and remained stable after the third injection with high titers for long lasting periods as observed with CFA and IFA/MDP adjuvant. The analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed the preponderance of IgG1 isotypes suggesting the predominance of Th2-type immune response.     

Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses

This study investigates the efficacy of surface-modified microspheres of hepatitis B surface antigen (HBsAg) in eliciting systemic and mucosal immune responses. Positively charged poly(d,l-lactic-co-glycolic acid) microspheres were prepared by a double-emulsion solvent-evaporation method with cationic agents—stearylamine and polyethylenimine—in the external aqueous phase. Formulations were characterized for morphology, size, density, aerodynamic diameter, entrapment efficiency and in vitro drug-release profile. Immunization was performed after pulmonary administration of the formulations to female Sprague–Dawley rats and the immune response was monitored by measuring IgG levels in serum and secretory (sIgA) levels in salivary, vaginal and bronchoalveolar lavage fluids. The cell-mediated immune response was studied by measuring cytokine levels in spleen homogenates, and a cytotoxicity study was performed with Calu-3 cell line. The aerodynamic diameter of the particles was within the respirable range, with the exception of stearylamine-modified particles. Zeta potential values moved from negative (−6.76 mV) for unmodified formulations to positive (+0.515 mV) for polyethylenimine-modified particles. Compared to unmodified formulations, polyethylenimine-based formulations showed continuous release of antigen over a period of 28–42 days and increased levels of IgG in serum and sIgA in salivary, vaginal and bronchoalveolar lavage. Further, cytokine levels—interferon γ and interleukin-2—were increased in spleen homogenates. The viability of Calu-3 cells was not adversely affected by the microparticles. In summation, this study establishes that positive surface charges on poly(d,l-lactic-co-glycolic acid) particles containing HBsAg enhances both the systemic and mucosal immune response upon immunization via the respiratory route.     

Immune Modulation by Adjuvants Combined with Diphtheria Toxoid Administered Topically in BALB/c Mice After Microneedle Array Pretreatment

Purpose  In this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated. Methods  TCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers. Results  TCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DT-alum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction. Conclusions  The potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants.     

Diesel exhaust, carbon black, and silica particles display distinct Th1/Th2 modulating activity

Certain particulate air pollutants may play an important role in the increasing prevalence of respiratory allergy by stimulating T helper 2 cell (Th2)-mediated immune responses to common antigens. The study described here examined different particles, diesel exhaust particles (DEP), carbon black particles (CBP), and silica particles (SIP) for their immunomodulating capacity in both primary and secondary immune responses in female BALB/C mice. The primary response was studied after subcutaneous injection of 1 mg of particle together with 10 microgram of reporter antigen TNP-OVA (2,4,6-trinitrophenyl coupled to ovalbumin) into the hind paw. Interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) production was assessed in the popliteal lymph node (PLN) at Day 2 and Day 5 after injection by flow cytometry and ELISA. The number of IL-4-containing CD4(+) T cells increased between Day 2 and Day 5 in DEP- and CBP-exposed mice, in contrast to SIP-treated animals. IL-4 production by cultured PLN cells was also significantly increased for DEP- and CBP-treated animals. The secondary response was studied in different organs after an intranasal challenge with TNP-OVA (50 microgram), which was given 4 weeks after the initial subcutaneous injection. Five days after challenge the number of antibody-forming cells (AFCs) was assessed in peribronchial lymph nodes (PBLN), spleen, bone marrow, and PLN, and antibody levels were determined in weekly obtained blood samples. It appeared that all particles acted as adjuvant, but the different particles stimulated distinct types of immune responses to TNP-OVA. DEP-treated animals show high IgG1 and IgE levels in serum and high IgG1 and IgE-forming AFC numbers in PBLN, bone marrow, and spleen. CBP-treated animals show even higher IgG1 and IgE levels and AFC numbers, and in addition display IgG2a production. SIP-injected animals display predominantly IgG2a responses. It is concluded that DEP are able to skew the immune response toward the T helper 2 (Th2) side, whereas SIP stimulate a Th1 response and CBP have a mixed activity, stimulating both Th1 and Th2 responses in this model.     

8-Mercaptoguanosine-mediated enhancement of in vivo IgG1, IgG2 and IgG3 antibody responses to polysaccharide antigens in normal and xid mice

We have examined the effects of the immune adjuvant 8-mercaptoguanosine (8sGuo) on the in vivo antibody response to the T-cell-independent type 2 antigen, TNP-Ficoll. While 8sGuo enhanced the IgG1, IgG2 and IgG3 antibody responses, it was without effect on the IgM antibody responses. Increasing the dose of injected 8sGuo from 30 to 300 mg or the frequency or its injection led to greater enhancement in the antibody response, which varied from 20 to 100 times that of control responses. The effect of 8sGuo was relatively early acting in that it no longer enhanced anti-TNP antibody responses when given 3 days after antigen injection. Its ability to mediate an adjuvant effect on antibody responses was demonstrable even under conditions where the injected antigen by itself stimulated either no or low-level antibody responses. Thus, it enhanced the antibody response to the very weak antigen, pneumococcal polysaccharide, and restored the antibody response of nonresponder immune defective xid mice to TNP-Ficoll. These results extend the earlier observations of Goodman and coworkers by demonstrating that in vivo IgG response to type 2 polysaccharide antigens can be enhanced in normal mice and restored in xid immune-deficient mice.     

Haematological and immunological effects of repeated dose exposure of rats to integerrimine N-oxide from Senecio brasiliensis

This study is the first in the literature to focus attention on the possible immunotoxic effect of integerrimine N-oxide content in the butanolic residue (BR) of Senecio brasiliensis, a poisonous hepatotoxic plant that contains pyrrolizidine alkaloids (PAs). PAs have been reported as a pasture and food contaminant and as herbal medicine used worldwide and are responsible for poisoning events in livestock and human beings. After the plant extraction, BR extracted from Senecio brasiliensis was found to contain approximately 70% integerrimine N-oxide by elemental and spectral analyses (¹H and ¹³C NMR), which was administered to adult male Wistar Hannover rats at doses of 3, 6 and 9 mg/kg for 28 days. Body weight gain, food consumption, lymphoid organs, neutrophil analysis, humoural immune response, cellular immune response and lymphocyte analysis were evaluated. Our study showed that integerrimine N-oxide could promote an impairment in the body weight gain, interference with blood cell counts and a reducing T cell proliferative activity in rats; however, no differences in the neutrophil activities, lymphocytes phenotyping and humoural and cellular immune responses were observed. It is concluded that doses of integerrimine N-oxide here employed did not produce marked immunotoxic effects.     

Infection by mycotoxigenic fungal species and mycotoxin contamination of maize grain in Umbria, central Italy

Surveys were carried out in 2006 and 2007 in Umbria (central Italy) to evaluate the presence of mycotoxigenic fungi and mycotoxins in maize grain sampled at harvest. Fusarium spp., were the most abundant species detected in maize kernels, followed by Aspergillus species of sections Flavi and Nigri and by Penicillium spp. Among Fusarium species, F. verticillioides was the most prevalent species, as detected by PCR directly on the kernels and on the fungi isolated from the kernels, followed by F. proliferatum and F. subglutinans. Fumonisins were the predominant mycotoxins with values, on average, of 4.3 and 5.7 mg kg⁻¹, in 2006 and 2007, respectively, with a maximum of 76.3 mg kg⁻¹ in the second year. Deoxynivalenol ranged from 0.2 to 3.98 mg kg⁻¹ in 2006 (average 1.04 mg kg⁻¹) and from undetectable levels to 14 mg kg⁻¹ in 2007 (average 0.86 mg kg⁻¹). Aflatoxins, analyzed only in 2007, averaged 26.3 μg kg⁻¹, with a maximum of 820 μg kg⁻¹. Zearalenone content was always very low. Results indicate that EU legal limits for these mycotoxins were rarely exceeded with low levels across most of the examined area, suggesting that this region could be considered suitable for the production of healthy maize.     

In vitro and in vivo studies on some toxic effects of the venom from Hemiscorpious lepturus scorpion

The aim of this laboratory-based study was to investigate some of the toxic effects induced by the venom from Hemiscorpious lepturus (H. lepturus). For this aim, pharmacological, histological, biochemical methods as well as complete blood cell count were used to assess these toxic actions. In addition, in vitro haemolysis studies on human washed blood suspension and cytotoxicity on cultured fibroblasts were also undertaken. In vitro pharmacological test was made on rat isolated ileal segment. To this end, the effects of the venom on the contractile responsiveness to acetylcholine were recorded using F30 transducer and Darco chart recorder. For assessment of the haemolytic potency, varying concentrations (2, 10, 20 and 40 μg/ml) of the venom were added to 0.5 ml of 5% washed human blood and after 30 min, 2, 4, 8, 12 and 24 h of exposure, the degree of lysis (extent of redness developed in the supernatant solution after centrifugation) were measured by ELISA method. Cytotoxicity potential of the venom was assessed by trypan blue exclusion test. The venom (0.1, 1 and 10 μg/ml) was mixed with confluent fibroblast cell culture and the extent cytotoxicity was assessed microscopically. In vivo studies were conducted by a subcutaneous administration of sub-lethal dose (10 μg) of the venom and after 7 days the skin, at the site of injection, and kidney samples were stained by H & E method and examined microscopically. In addition, biochemical assessments including measurement of serum aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and amylase levels and urine analysis were made. The results showed that the venom prevented the relaxation phase of the acetylcholine-induced contractions on the isolated ileal segments and finally produced sustained spasmodic contractions. This spasmodic action was abolished by 1 μM atropine. The venom produced haemolysis of red blood cells in a concentration-dependent and duration-of-exposure manner, with 100% of haemolysis produced after 24 h following exposure to 40 μg/ml of venom. While cultured fibroblasts cells were more sensitive and disintegrated after 15 min of exposure to 1 μg/ml of the venom. Histological findings showed evidences of excessive inflammatory responses accompanied with signs of necrosis in the skin at the site of injection as well as structural damage in the nephrones. There was a significant rise in the serum enzymes. In addition, the number of the RBCs were reduced. The urine showed positive readings for proteinuria, blood and intact RBCs. The overall results suggest that the venom from H. lepturus primarily is a cytotoxic agent and has haemolytic, nephrotoxic and to some extent hepatotoxic activity.     

Toxigenicity of enniatins from Western Australian Fusarium species to brine shrimp (Artemia franciscana)

The high prevalence (14 of 24 isolates) of enniatin-producing isolates from Western Australian Fusarium species isolated from pasture legumes associated with sheep feed refusal and rat deaths, and the high toxicity of their crude extracts to brine shrimp (Artemia franciscana) from a previous study warranted further investigation of this class of mycotoxin. Crude extracts from Fusarium acuminatum, Fusarium avenaceum, Fusarium tricinctum and Fusarium sambucinum, along with enniatins A, A1, B and B1 purified from a Western Australian strain of F. acuminatum using semi-preparative HPLC, were bioassayed using brine shrimp. All Fusarium isolates produced both enniatins B and B1, except for F. tricinctum WAC 8019, and 11 of the 17 isolates produced enniatin A1. Overall, all of the F. avenaceum isolates produced high amounts of enniatins, in particular enniatin B. One isolate of F. acuminatum (WAC 5715) and of F. tricinctum (WAC 11486) also produced high amounts of both enniatins B and B1. Only F. acuminatum WAC 5715 produced enniatin A among the tested isolates. All four purified enniatins A, A1, B, B1, individually and in combination, caused brine shrimp toxicity after 6 h of exposure, implicating that this emerging class of mycotoxin as a cause of the acute toxicity to brine shrimp observed. The mixture of all four enniatins was the most toxic to brine shrimp compared to purified individual enniatins, where the relative toxicity order was B > B1 > A1 > A. Enniatin B was the individual most toxic enniatin with some bioactivity at 5 μg/mL and almost 100% brine shrimp death at 50 μg/mL after 24 h of exposure. This study is the first report to confirm the acute toxicity of enniatins A, A1, B and B1 to brine shrimp, and also highlights the need for further investigation of the potential toxicity of these cyclic hexadepsipeptides to animals and humans.     

Pharmacokinetics of a F(ab′)2 scorpion antivenom administered intramuscularly in healthy human volunteers

This paper presents the first study of F(ab′)₂ scorpion antivenom pharmacokinetics in humans after intramuscular (im) administration. The specific anti-Centruroides scorpion antivenom was used in 6 human healthy volunteers. The fabotherapeutic was administered as a 47.5 mg im bolus. Blood samples were drawn at 0, 5, 15, 30, 45, 60 , 90, 120, and 180 min, 6 h and at 1, 2, 3, 4, 10 and 21 days after antivenom administration. We measured antivenom concentrations in serum using a specific high sensitivity ELISA method for F(ab′)₂. Antivenom concentration in serum was fit to a 3 compartment model (inoculation site, plasma and extra vascular extracellular space), it was assumed that the venom may also be irreversibly removed from plasma. Calculated time course of antivenom content shows that at any time no more that 16.6 (5.3, 31.9)% (median and 95% confidence interval) of the antivenom bolus is present in plasma. The time to peak plasma [F(ab′)₂] was 45 (33, 74) h. The most significant antivenom pharmacokinetic parameters determined were: AUC_im, ∞ = 803 (605, 1463) mg · h · L^− 1; V_c = 8.8 (2.8, 23.6) L; V_ss, im = 55 (47, 64) L; MRT_im = 776(326, 1335) h; CL_t = 3.7 (0.6, 1.9) mL · min^− 1; f_im, V_ss = 0.300 (0.153, 0.466). Comparing these parameters with the ones obtained intravenously by Vázquez et al. [2], the parameters were more disperse between subjects, determined with more uncertainty in each individual subject, and the peak F(ab′)₂ in plasma occurred with considerable delay; all indicating that the IM route should not be used to administer the antivenom, with the possible exceptionof cases occurring very far from hospitals, as an extreme means to provide some protection before the IV route becomes available.     

Oral delivery of gastro-resistant microencapsulated typhoid vaccine

Oral vaccination has long been regarded as the best alternative to conventional parenteral vaccination considering practical, economical, and immunological aspects. The purpose of this study was to develop albumin–chitosan mixed matrix microsphere-filled coated capsule formulations of Typhoid Vi^® antigen and to determine whether it can induce antigen-specific mucosal and systemic immune responses on oral administration. Formulations with Typhoid Vi^® antigen were prepared and filled into hard gelatin capsules (size # 9) and enteric coated. Formulations were characterized and administered to Sprague–Dawley rats to evaluate the induction of immune response to the antigen. The results indicated that the particle size, zeta potential, swelling, and disintegration rates were optimal for the oral delivery of microencapsulated vaccines. In vivo studies displayed multifold increase of antigen-specific IgG and IgA levels 8 weeks after oral immunization. No statistically significant difference in the antigen-specific IgG and IgA levels were found between oral and parenteral injection groups 8 weeks after vaccination. On the basis of the results of the study, it can be concluded that the oral administration of Typhoid Vi antigen microspheres was successful in inducing antigen-specific systemic and mucosal immune response.     

Presynaptic action of Bothriopsis bilineata smargadina (forest viper) venom in vitro

In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1-30 μg/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 15-90 min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120 min (from 80 ± 15 to 206 ± 25 U/ml, n = 6, p < 0.05). In mouse phrenic nerve-diaphragm preparations, the venom (1, 10 and 30 μg/ml) produced marked facilitation (∼120% increase above basal) at the highest concentration followed by neuromuscular blockade; the effects at lower concentrations were considerably less marked. Venom increased the quantal content values after 15 and 30 min followed by significant inhibition at ≥90 min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22 °C instead of 37 °C delayed the onset of blockade, as did inhibition of venom PLA₂ activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving PLA₂.     

Non-ionic surfactant vesicles mediated transcutaneous immunization against hepatitis B

Transcutaneous immunization (TI) has many practical merits compared to parenteral routes of administration. In the present study, non ionic surfactant vesicular carrier, i.e. niosomes, was evaluated for topical delivery of vaccines using hepatitis B surface protein as an antigen and cholera toxin B as an adjuvant. Niosomes were characterized for size, shape, entrapment efficiency and in process antigen stability. In vitro permeation and skin deposition studies of antigen were performed using human cadaver skin. Skin penetration efficiency of niosomes was assessed by confocal laser scanning microscopy. The immune stimulating activity of these vesicles was studied by measuring the serum IgG titer, isotype ratio IgG2a/IgG1and mucosal immune responses following transcutaneous immunization in Balb/c mice and results were compared with the alum adsorbed HBsAg given intramuscularly and topically administered plain HBsAg solution. The result shows that optimal niosomal formulation could entrap 58.11 ± 0.71 of antigen with vesicle size range of 2.83 ± 0.29 μm. Serum IgG titers after three consecutive topical administrations were significantly better than single administration of hepatitis antigen with niosomal system, suggesting an effective stimulation of serum immune response; higher IgG1/IgG2a ratio revealed CTB mixed niosomes elicit both Th1 and Th2 responses. This study suggests that topical immunization with cholera toxin B is potential adjuvant for cutaneous immune responses when coadministered with the HBsAg encapsulated niosomes. Results also suggest that the investigated niosomes systems can be effective as topical delivery of vaccines.     

Correlation between blood pressure, cytokines and nitric oxide in conscious rabbits injected with Leiurus quinquestriatus quinquestriatus scorpion venom

Activation of the inflammatory response with the release and activation of pro-inflammatory cytokines is among the factors thought to be important in the pathogenesis of many deleterious inflammatory effects seen in case of scorpion envenomation. The released inflammatory mediators interact in the body with a large number of proteins and receptors; this interaction determines the eventual inflammatory effect of the venom. Thus, in the present study an attempt was made to map the time course of scorpion envenomation and correlate the effects observed on the cardiovascular and respiratory systems with the changes that could take place in the levels of selected cytokines and nitric oxide during the course of experimental envenomation. New Zealand white male conscious rabbits were prepared for blood pressure recording. Arterial blood pressure was measured from the left central ear artery while a cannula was inserted into the right central ear artery and blood samples collected at different time interval after venom injection for biochemical and hematological analyses. In general, subcutaneous injection of Leiurus quinquestriatus quinquestriatus venom caused a significant (P ± 0.05) triphasic effect on BP consisting of an initial transient reduction, followed by an increase that peaked 2 h after venom injection, and a gradual terminal hypotensive phase. The significantly high serum level of IL8, TNFα (P < 0.001) and nitric oxide (P < 0.0001) observed in the present study supports the evidence for the role of these potent vasodilators in the terminal hypotension that is usually observed in humans and animals after envenomation.     

Oral delivery of particulate prostate cancer vaccine: In vitro and in vivo evaluation

Background: Various approaches have been evaluated for generation of efficient immune response against tumor antigens. Our approach exploits usage of particulate delivery to generate immune response against prostate cancer antigens.

Purpose: The aim of this study was to evaluate the efficacy of prostate cancer vaccine derived from a murine prostate cancer cell line, TRAMP C2 in murine model via oral route using aleuria aurantia lectin as a targeting ligand for M-cells in the intestinal Peyer’s patches.

Methods: The whole cell lysate (WCL) was obtained from TRAMP C2 murine prostate cancer cell line and was formulated into particles using one step spray drying process. For in vivo studies, 4–6 week old C57BL/6 male mice were vaccinated orally biweekly for 10 weeks. Serum samples were analyzed at regular intervals to determine serum IgG levels. The mice were then challenged with live TRAMP C2 cells to determine efficacy of the vaccine.

Results: The serum IgG levels of vaccinated animals were higher compared to that of the controls. Moreover, the tumor growth was retarded significantly in the vaccinated mice compared to that of controls (p < 0.001).

Conclusions: The above findings suggest that oral particulate WCL vaccine can trigger an immune response against prostate cancer antigens.     

Antinociceptive activity of Annona diversifolia Saff. leaf extracts and palmitone as a bioactive compound

Annonas are consumed as fresh fruits, but are also widely used in folk medicine for treating pain and other ailments. Antinociceptive properties of the Annona diversifolia ethanol crude extract were tested using the pain-induced functional impairment model in rat (PIFIR) and the writhing test in mice. The ethanol extract caused a 25% recovery of limb function in rats; this response was significant and dose-dependent. Furthermore, this extract produced a similar antinociceptive response (ED₅₀ = 15.35 mg/kg) to that of the reference drug tramadol (ED₅₀ = 12.42 mg/kg) when evaluated in the writhing test in mice. Bio-guided fractionation yielded hexane and acetone active fractions from which the presence of palmitone and flavonoids was respectively detected. Palmitone produced an antinociceptive response with an ED₅₀ = 19.57 mg/kg in the writhing test. Antinociceptive responses from ethanol extract and tramadol were inhibited in the presence of either naloxone (1 mg/kg, s.c.)—an antagonist of endogenous opioids—or WAY100635 (0.8 mg/kg, s.c.)—a 5-HT_1A serotonin receptor antagonist. These results provide evidence that A. diversifolia possesses antinociceptive activity, giving support to their traditional use for treatment of spasmodic and arthritic pain. In addition, our results suggest the participation of endogenous opioids and 5-HT_1A receptors in this antinociceptive response.     

Gene expression profiles in rat lung after inhalation exposure to C60 fullerene particles

Concern over the influence of nanoparticles on human health has risen due to advances in the development of nanotechnology. We are interested in the influence of nanoparticles on the pulmonary system at a molecular level. In this study, gene expression profiling of the rat lung after whole-body inhalation exposure to C₆₀ fullerene (0.12 mg/m³; 4.1 × 10⁴ particles/cm³, 96 nm diameter) and ultrafine nickel oxide (Uf-NiO) particles (0.2 mg/m³; 9.2 × 10⁴ particles/cm³, 59 nm diameter) as a positive control were employed to gain insights into these molecular events. In response to C₆₀ fullerene exposure for 6 h a day, for 4 weeks (5 days a week), C₆₀ fullerene particles were located in alveolar epithelial cells at 3 days post-exposure and engulfed by macrophages at both 3 days and 1 month post-exposures. Gene expression profiles revealed that few genes involved in the inflammatory response, oxidative stress, apoptosis, and metalloendopeptidase activity were up-regulated at both 3 days and 1 month post-exposure. Only some genes associated with the immune system process, including major histocompatibility complex (MHC)-mediated immunity were up-regulated. These results were significantly different from those of Uf-NiO particles which induced high expression of genes associated with chemokines, oxidative stress, and matrix metalloproteinase 12 (Mmp12), suggesting that Uf-NiO particles lead to acute inflammation for the inhalation exposure period, and the damaged tissues were repaired in the post-exposure period. We suggest that C₆₀ fullerene might not have a severe pulmonary toxicity under the inhalation exposure condition.