Phytol, a Chlorophyll Component, Produces Antihyperalgesic, Anti-inflammatory, and Antiarthritic Effects: Possible NFκB Pathway Involvement and Reduced Levels of the Proinflammatory Cytokines TNF-α and IL-6.

Phytol is a diterpene constituent of chlorophyll and has been shown to have several pharmacological properties, particularly in relation to the management of painful inflammatory diseases. Arthritis is one of the most common of these inflammatory diseases, mainly affecting the synovial membrane, cartilage, and bone in joints. Proinflammatory cytokines, such as TNF-α and IL-6, and the NFκB signaling pathway play a pivotal role in arthritis. However, as the mechanisms of action of phytol and its ability to reduce the levels of these cytokines are poorly understood, we decided to investigate its pharmacological effects using a mouse model of complete Freund's adjuvant (CFA)-induced arthritis. Our results showed that phytol was able to inhibit joint swelling and hyperalgesia throughout the whole treatment period. Moreover, phytol reduced myeloperoxidase (MPO) activity and proinflammatory cytokine release in synovial fluid and decreased IL-6 production as well as the COX-2 immunocontent in the spinal cord. It also downregulated the p38MAPK and NFκB signaling pathways. Therefore, our findings demonstrated that phytol can be an innovative antiarthritic agent due to its capacity to attenuate inflammatory reactions in joints and the spinal cord, mainly through the modulation of mediators that are key to the establishment of arthritic pain.

Squalene containing solid lipid nanoparticles, a promising adjuvant system for yeast vaccines.

Potent adjuvant systems are required for subunit and single antigen based vaccines to provide sufficient immunogenicity. Furthermore, adjuvants can reduce the required number of immunisations and the antigen amount. Squalene nanoemulsions, like MF59® and AddaVax™, are potent, safe and well characterised adjuvant systems and approved for use in humans. Here, we developed squalene containing solid lipid nanoparticles, which can be sterilised by steam sterilisation and stored as freeze-dried power together with a yeast-based vaccine. Detailed size measurements using dynamic and static light scattering were applied, as the immune stimulating effect of squalene emulsions is mainly dependent on the particle size. The size range of AddaVax™ (120-170 nm) was favoured for the developed squalene containing solid lipid nanoparticles. Differential scanning calorimetry (DSC) and H NMR studies were performed to characterise the interactions of the incorporated liquid squalene with the solid hard fat matrix. A homogeneous distribution as liquid domains in the solid glyceride structure was suggested for the liquid squalene. The developed adjuvant was compared with Freund's adjuvant and a commercially available squalene nanoemulsion in a vaccine trial in the mouse model with a yeast-based vaccine directed against the infectious bursal disease virus. All squalene-based adjuvants showed excellent biocompatibility and provided immune stimulating properties comparable to Freund's adjuvant.

Contribution of platelet P2Y12 receptors to chronic Complete Freund's adjuvant-induced inflammatory pain.

Essentials The role of platelet P2Y12 receptors in the regulation of chronic inflammatory pain is unknown. Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain model was used in mice. Gene deficiency and antagonists of P2Y12 receptors attenuate hyperalgesia and local inflammation. Platelet P2Y12 receptors contribute to these effects in the chronic phase of inflammation. SUMMARY: Background P2Y12 receptor antagonists are widely used in clinical practice to inhibit platelet aggregation. P2Y12 receptors are also known to regulate different forms of pain as well as local and systemic inflammation. However, it is not known whether platelet P2Y12 receptors contribute to these effects. Objectives To explore the contribution of platelet P2Y12 receptors to chronic inflammatory pain in mice. Methods Complete Freund's adjuvant (CFA)-induced chronic inflammatory pain was induced in wild-type and P2ry12 gene-deficient (P2ry12-/- ) mice, and the potent, direct-acting and reversible P2Y12 receptor antagonists PSB-0739 and cangrelor were used. Results CFA-induced mechanical hyperalgesia was significantly decreased in P2ry12-/- mice for up to 14 days, and increased neutrophil myeloperoxidase activity and tumor necrosis factor (TNF)-α and CXCL1 (KC) levels in the hind paws were also attenuated in the acute inflammation phase. At day 14, increased interleukin (IL)-1ß, IL-6, TNF-α and KC levels were attenuated in P2ry12-/- mice. PSB-0739 and cangrelor reversed hyperalgesia in wild-type mice but had no effect in P2ry12-/- mice, and PSB-0739 was also effective when applied locally. The effects of both local and systemic PSB-0739 were prevented by A-803467, a selective NaV1.8 channel antagonist, suggesting the involvement of NaV1.8 channels in the antihyperalgesic effect. Platelet depletion by anti-mouse CD41 antibody decreased hyperalgesia and attenuated the proinflammatory cytokine response in wild-type but not in P2ry12-/- mice on day 14. Conclusions In conclusion, P2Y12 receptors regulate CFA-induced hyperalgesia and the local inflammatory response, and platelet P2Y12 receptors contribute to these effects in the chronic inflammation phase.

Denaturing action of adjuvant affects specificity of polyclonal antibodies.

Influence of the immunization procedure on the specificity of the produced antibodies towards different conformations of the antigen was investigated. It was demonstrated that intravenous immunization of a rabbit with an adjuvant-free solution of recombinant sperm-specific glyceraldehyde-3-phosphate dehydrogenase (dN-GAPDS) resulted in production of antibodies recognizing only native conformation of dN-GAPDS and exhibiting no cross-reaction with somatic isoenzyme of glyceraldehyde-3-phosphate dehydrogenase. A subcutaneous immunization with human dN-GAPDS mixed with Freund's complete adjuvant yielded antibodies recognizing both native and denatured conformation of dN-GAPDS. The oil component of the adjuvant was shown to cause inactivation and partial denaturation of dN-GAPDS, leading to exposure of the epitopes that are masked in the native protein, which resulted in production of the antibodies to the denatured antigen. These results may be of importance for biochemical research that often require polyclonal antibodies recognizing different conformations of antigens.

Particles for Local Delivery of Proteins Using Intra-Articular Route.

Designing a vehicle for local delivery of proteins using intra-articular route is an attractive option to minimize the adverse effects associated with systemic exposure and to maximize the efficacy. Slowly dissolving silylated microparticles are designed with specific size and shape that are capable of extending the retention time of a model protein (bovine serum albumin) in the murine knee joint. No cytotoxicity is observed for the reconstituted formulation when tested against synovial fibroblasts and RAW 264.7 macrophages.

Antinociceptive effects of incarvillateine, a monoterpene alkaloid from Incarvillea sinensis, and possible involvement of the adenosine system.

Incarvillea sinensis is a Bignoniaceae plant used to treat rheumatism and relieve pain in traditional Chinese medicine. As a major component of I. sinensis, incarvillateine has shown analgesic activity in mice formalin tests. Using a series of animal models, this study further evaluated the effects of incarvillateine against acute, inflammatory, and neuropathic pain. Incarvillateine (10 or 20 mg/kg, i.p.) dose-dependently attenuated acetic acid-induced writhing, but did not affect thermal threshold in the hot plate test. In a Complete Freund's Adjuvant model, incarvillateine inhibited both thermal hyperalgesia and paw edema, and increased interleukin-1ß levels. Additionally, incarvillateine attenuated mechanical allodynia induced by spared nerve injury or paclitaxel, whereas normal mechanical sensation was not affected. Incarvillateine did not affect locomotor activity and time on the rotarod at analgesic doses, and no tolerance was observed after 7 consecutive daily doses. Moreover, incarvillateine-induced antinociception was attenuated by theophylline, 1,3-dipropyl-8-cyclopentylxanthine, and 3,7-dimethyl-1-propargylxanthine, but not naloxone, indicating that the effects of incarvillateine on chronic pain were related to the adenosine system, but not opioid system. These results indicate that incarvillateine is a novel analgesic compound that is effective against inflammatory and neuropathic pain, and that its effects are associated with activation of the adenosine system.

Mother root of Aconitum carmichaelii Debeaux exerts antinociceptive effect in Complet Freund's Adjuvant-induced mice: roles of dynorpin/kappa-opioid system and transient receptor potential vanilloid type-1 ion channel.

BACKGROUND: Processed Chuanwu (PCW), the mother root of Aconitum carmichaelii Debeauxv, has been widely used as a classic Traditional Chinese Medicine for pain relieve for over two millennia clinically. However, its action on chronic inflammatory pain has not been clarified. Here, we investigated the antinociceptive effect of PCW in complete freund's adjuvant (CFA)-induced mice and its possible mechanisms associated with opioid system and TRPV1 ion channel. METHODS: Male ICR mice were intraplantarly injected with CFA. PCW (0.34, 0.68 and 1.35 g/kg) was orally given to mice once a day for 7 days. Von frey hairs and planter test were assessed to evaluate the antinociceptive effect of PCW. To investigate the participation of dynorphin/opioid system in PCW antinociception, subtype-specific opioid receptor antagonists or anti-dynorphin A antiserum were used. To eliminate other central mechanisms that contribute to PCW antinociception, hot plate (50 °C) test were performed. Further, involvements of TRPV1 in PCW antinociception were evaluated in CFA-induced TRPV1(-/-) and TRPV1(+/+) C57BL/6 male mice, and in capsaicin-induced nociception ICR naive mice pretreated with nor-BNI. Meanwhile, calcium imaging was performed in HEK293T-TRPV1 cells. Finally, rotarod, open-field tests and body temperature measurement were carried out to assess side effects of PCW. RESULTS: PCW dose-dependently attenuated mechanical and heat hypersensitivities with no tolerance, which could be partially attenuated by coadministration of k-opioid receptor antagonist nor-binaltorphimine (nor-BNI) or anti-dynorphin A (1-13) antiserum. And PCW antinociception was totally erased by pretreatment with nor-BNI in the hot plate test. In addition, PCW antinociception was decreased in TRPV1(-/-) mice compared to TRPV1(+/+) group. And PCW still manifested inhibitory effects in capsaicin-induced nociception with nor-BNI pretreatment. PCW significantly inhibited capsaicin-induced calcium influx in HEK293T-TRPV1 cells. Finally, no detectable side effects were found in naive mice treated with PCW. CONCLUSIONS: This study shows PCW's potent antinociceptive effect in inflammatory conditions without obvious side effects. This effect may result from the activation of κ-opioid receptor via dynorpin release and the inhibition of TRPV1. These findings indicate that PCW might be a potential agent for the management of chronic inflammatory pain.

Adjuvant poly(N-isopropylacrylamide) generates more efficient monoclonal antibodies against truncated recombinant histidine-rich protein2 of Plasmodium falciparum for malaria diagnosis.

Adjuvants play an important role in eliciting immune responses and subsequent generation of antibodies with high specificity. Recently, poly(N-isopropylacrylamide) (PNiPAAm), also known as a "smart" polymer, has been proposed as a potential adjuvant for making antibodies and vaccines. This material exhibits efficient delivery, protection against degradation, and preservation of antigen epitopes. In this work, we used both CFA and smart polymer to develop a highly specific murine monoclonal antibody (mAb) against recombinant truncated histidine rich protein2 (HRP2) of Plasmodium falciparum. Our results indicate that the mAbs developed using these adjuvants were able to recognize recombinant HRP2 and native PfHRP2 protein from spent medium. The mAbs generated against recombinant truncated HRP2 showed better sensitivity to the antigen and importantly mAbs generated using PNiPAAm adjuvant were in the range of 10(8)-10(9) M(-1). The mAbs generated using PNiPAAm are very efficient and sensitive in detecting HRP2. To the best of our knowledge, this is the first report of such comparison having been made between these two adjuvants and we propose that the smart polymer has huge potential as an alternative to CFA. Additionally, we discuss the utility of the mAbs generated through PNiPAAm for specific diagnosis of malaria caused by P. falciparum.

Synthetic polymer as an adjuvant in collagen-induced arthritis.

Collagen-induced arthritis (CIA), the classical animal model for experimental arthritis, resembles human rheumatoid arthritis in several aspects. However, the most widely used method of inducing CIA utilizes Freund's adjuvants, which can skew the elicited immune responses and also pose toxicity problems. This unit describes a new method of inducing CIA using a well defined stimuli-responsive synthetic polymer, poly-N-isopropylacrylamide-based adjuvant, mixed with the joint cartilage protein collagen type II (CII). PNiPAAm as an adjuvant is biodegradable and biocompatible, and does not skew immune responses. Thus, it is helpful in the development of arthritis models for studying antigen and tissue -specific autoimmune responses in an unbiased manner. This model is valuable for analyzing disease pathways, positional identification of genes regulating arthritis, validation of existing therapies, and exploring new therapeutic targets. Furthermore, this newly developed PNiPAAm adjuvant allows investigation of disease induction using specific autoantigens in several autoimmune diseases independently of toll-like receptors, as well as optimization of vaccine delivery systems for infectious diseases.

Optimizing available phosphorus in calcareous soils fertilized with diammonium phosphate and phosphoric acid using Freundlich adsorption isotherm.

In calcareous soils, phosphorus (P) retention and immobilization take place due to precipitation and adsorption. Since soil pH is considered a major soil variable affecting the P sorption, an acidic P fertilizer could result in low P adsorption compared to alkaline one. Therefore, P adsorption from DAP and phosphoric acid (PA) required to produce desired soil solution P concentration was estimated using Freundlich sorption isotherms. Two soils from Faisalabad and T. T. Singh districts were spiked with 0, 10, and 20 % CaCO3 for 15 days. Freundlich adsorption isotherms (P = aC(b/a)) were constructed, and theoretical doses of PA and DAP to develop a desired soil solution P level (i.e., 0.20 mg L(-1)) were calculated. It was observed that P adsorption in soil increased with CaCO3. Moreover, at all the levels of CaCO3, P adsorption from PA was lower compared to that from DAP in both the soils. Consequently, lesser quantity of PA was required to produce desired solution P, 0.2 mg L(-1), compared to DAP. However, extrapolating the developed relationship between soil CaCO3 contents and quantity of fertilizer to other similar textured soils needs confirmation.

Sub-unit vaccine against S. aureus-mediated infections: set-up of nano-sized polymeric adjuvant.

The aim of this work was the design of a novel adjuvanted system for vaccination against S. aureus-mediated infections: in particular, poly-lactide-co-glycolide (PLGA) nanoparticles were developed in order to efficiently load and boost a sub-unit model vaccine, namely a purified recombinant collagen binding bacterial adhesin fragment (CNA19). At first, the assessment of the actual immunogenicity of free CNA19 via subcutaneous administration was evaluated, in order to consider it as subunit antigen model. Secondly, for the development of CNA19 loaded PLGA nanoparticles, a preliminary study was focused on the production of well-formed nanoparticles by w/o/w double emulsion method exploiting ultrasonication cycles under mild conditions, then the optimization of the freeze-drying conditions and different CNA19 loading methods were considered (encapsulation, adsorption of on blank or CNA19 encapsulated nanoparticles). The set-up preparation method (process yield of about 83%) permitted to obtain CNA19 loaded nanoparticles with spherical shape, narrow size distribution (187.41 ± 51.2 nm), a slightly negative zeta-potential (-2.91 ± 0.64 mV) and to elicit satisfactory protein encapsulation efficiency (75.91 ± 4.22%) and loading capacity (8.59 ± 0.33 µg CNA19/nanoparticles mg). Then, CNA19 loaded PLGA nanoparticles were characterized by (i) an in vitro release test performed at different temperatures, namely 4°C, 25°C and 37°C, testing the antigen integrity (SDS-PAGE) and activity (ELISA); (ii) an in vitro stability study in terms of dimension and surface charge performed in a 21 days period of time. At 37°C there was evidence of a sustained release of the antigen, in active form, for almost 240 h with a burst release of about 20% in the first 2h. At 4°C stability tests and activity assays allowed to identify storage conditions useful to maintain CNA19 activity and easily NP re-suspendability with intact physical characteristics. Furthermore the evaluation of CNA19 loaded nanoparticles cytotoxicity (up to 10.652 mg PLGA/ml) by MTT assay and the study of cellular up-take assessed on human fibroblasts confirmed the feasibility to formulate a dosage form useful for vaccination against S. aureus-mediated infections.

Adjuvant properties of a biocompatible thermo-responsive polymer of N-isopropylacrylamide in autoimmunity and arthritis.

To evaluate the thermo-responsive poly(N-isopropylacrylamide) (PNiPAAm) polymer as an adjuvant, we synthesized PNiPAAm through free radical polymerization and characterized it both in vitro and in vivo. The polymer when mixed with collagen type II (CII) induced antigen-specific autoimmunity and arthritis. Mice immunized with PNiPAAm-CII developed significant levels of CII-specific IgG response comprising major IgG subclasses. Antigen-specific cellular recall response was also enhanced in these mice, while negligible level of IFN-γ was detected in splenocyte cultures, in vitro. PNiPAAm-CII-immunized arthritic mouse paws showed massive infiltration of immune cells and extensive damage to cartilage and bone. As determined by immunostaining, most of the CII protein retained its native configuration after injecting it with PNiPAAm in naive mice. Physical adsorption of CII and the high-molecular-weight form of moderately hydrophobic PNiPAAm induced a significant anti-CII antibody response. Similar to CII, mice immunized with PNiPAAm and ovalbumin (PNiPAAm-Ova) induced significant anti-ovalbumin antibody response. Comparable levels of serum IFN-γ, IL-1ß and IL-17 were observed in ovalbumin-immunized mice with complete Freund, incomplete Freund (CFA and IFA) or PNiPAAm adjuvants. However, serum IL-4 levels were significantly higher in PNiPAAm-Ova and CFA-Ova groups compared with the IFA-Ova group. Thus, we show for the first time, biocompatible and biodegradable thermo-responsive PNiPAAm can be used as an adjuvant in several immunological applications as well as in better understanding of the autoimmune responses against self-proteins.

Induction of progressive demyelinating autoimmune encephalomyelitis in common marmoset monkeys using MOG34-56 peptide in incomplete freund adjuvant.

Experimental autoimmune encephalomyelitis in the neotropical primate common marmoset (Callithrix jacchus) is a relevant autoimmune animal model of multiple sclerosis. T cells specific for peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) have a central pathogenic role in this model. The aim of this study was to assess the requirement for innate immune stimulation for activation of this core pathogenic autoimmune mechanism. Marmoset monkeys were sensitized against synthetic MOG34-56 peptide alone or in combination with the nonencephalitogenic peptide MOG74-96 formulated in incomplete Freund adjuvant, which lacks microbial components. Experimental autoimmune encephalomyelitis development was recorded by monitoring neurological signs, brain magnetic resonance imaging, and longitudinal profiling of cellular and humoral immune parameters. All monkeys developed autoimmune inflammatory/demyelinating central nervous system disease characterized by massive brain and spinal cord demyelinating white matter lesions with activated macrophages and CD3+ T cells. Immune profiling ex vivo demonstrated the activation of mainly CD3+CD4+/8+CD56+ T cells against MOG34-56. Upon ex vivo stimulation, these T cells produced more interleukin 17A compared with TH1 cytokines (e.g. interferon-gamma) and displayed peptide-specific cytolytic activity. These results indicate that the full spectrum of marmoset experimental autoimmune encephalomyelitis can be induced by sensitization against a single MOG peptide in incomplete Freund adjuvant lacking microbial compounds for innate immune activation and by eliciting antigen-specific T-cell cytolytic activity.

Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis.

BACKGROUND AND PURPOSE: The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis. EXPERIMENTAL APPROACH: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form of paracoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptide P10, either emulsified in Freund's adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at different concentrations (1 microg, 5 microg, 10 microg, 20 microg or 40 microg.50 microL(-1)). Therapeutic efficacy was assessed as fungal burden in tissues and the immune response by quantitative determination of cytokines. KEY RESULTS: Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in the lungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 microg.50 microL(-1)) was more effective than 'free' P10 emulsified in Freund's adjuvant (20 microg.50 microL(-1)), as an adjuvant to chemotherapy. After treatment for 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 microg.50 microL(-1)) were most effective. Treatment with P10 emulsified in Freund's adjuvant (20 microg.50 microL(-1)) or P10 entrapped within PLGA (1 microg.50 microL(-1)) were accompanied by high levels of interferon-gamma in lung. CONCLUSIONS AND IMPLICATIONS: Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA demonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticles dramatically reduced the peptide amount necessary to elicit a protective effect.

A novel liposome-based adjuvant CAF01 for induction of CD8(+) cytotoxic T-lymphocytes (CTL) to HIV-1 minimal CTL peptides in HLA-A*0201 transgenic mice.

BACKGROUND: Specific cellular cytotoxic immune responses (CTL) are important in combating viral diseases and a highly desirable feature in the development of targeted HIV vaccines. Adjuvants are key components in vaccines and may assist the HIV immunogens in inducing the desired CTL responses. In search for appropriate adjuvants for CD8(+) T cells it is important to measure the necessary immunological features e.g. functional cell killing/lysis in addition to immunological markers that can be monitored by simple immunological laboratory methods. METHODOLOGY/PRINCIPAL FINDINGS: We tested the ability of a novel two component adjuvant, CAF01, consisting of the immune stimulating synthetic glycolipid TDB (Trehalose-Dibehenate) incorporated into cationic DDA (Dimethyldioctadecylammonium bromide) liposomes to induce CD8(+) T-cell restricted cellular immune responses towards subdominant minimal HLA-A0201-restricted CTL epitopes from HIV-1 proteins in HLA-A*0201 transgenic HHD mice. CAF01 has an acceptable safety profile and is used in preclinical development of vaccines against HIV-1, malaria and tuberculosis. CONCLUSIONS/SIGNIFICANCE: We found that CAF01 induced cellular immune responses against HIV-1 minimal CTL epitopes in HLA-A*0201 transgenic mice to levels comparable with that of incomplete Freund's adjuvant.

Immunogenicity of novel recombinant human activated factor VII analogues on factor VII neonatally-tolerized rats.

Recombinant activated factor VII (rFVIIa; NovoSeven) has been widely used to treat bleeding in patients with haemophilia with inhibitors. To increase the intrinsic activity, analogues of rFVIIa (rFVIIa Q, rFVIIa DVQ, and rFVIIa DVQA) with altered amino acid sequence at or near the active centre have been developed. The immunogenicity of these analogues was tested in a rat immune tolerance model. Neonatal rats received rFVIIa intraperitoneally on post-natal Day 1 and were subsequently challenged with rFVIIa in Freunds Incomplete Adjuvant subcutaneously on Days 10 and 24. Rats were tested for tolerance on Day 32; the tolerant cohort and a parallel cohort of untreated control rats were challenged with rFVIIa, rFVIIa Q, rFVIIa DVQ, or rFVIIa DVQA on Days 46 and 76. Immune responses determined by enzymelinked immunosorbent assay (ELISA) on Day 84 showed no statistically significant difference between the responses in the four control cohorts. Immune responses were higher in the control than in the tolerant cohort. Compared with rFVIIa (4/16), there was no difference in the proportion of rats that broke tolerance following challenge with rFVIIa DVQ (3/16) and rFVIIa DVQA (7/16), whereas a statistically significant greater proportion broke tolerance after challenge with rFVIIa Q (11/16). Therefore, in this model rFVIIa DVQ or rFVIIa DVQA were not more immunogenic than rFVIIa.

Complement C3d conjugation to anthrax protective antigen promotes a rapid, sustained, and protective antibody response.

B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA) to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax) consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA) imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4) of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure.

Overlapping human leukocyte antigen class I/II binding peptide vaccine for the treatment of patients with stage IV melanoma: evidence of systemic immune dysfunction.

BACKGROUND: MPS160 (GRAMLGTHTMEVTV) is a glycoprotein 100-derived melanoma peptide that contains overlapping human leukemic antigen A2-, DR53-, and DQw6-restricted T-cell epitopes. In preclinical testing, MPS160 demonstrated superior immunization and antitumor activity. In this report, the authors present the results from a clinical trial that evaluated the safety and immunologic efficacy of the MPS160 vaccine in patients with metastatic melanoma. METHODS: Patients with stage IV melanoma were randomized to 1 of 3 treatment arms: 1) MPS160 in incomplete Freund adjuvant (Montanide ISA-51); 2) MPS160 in Montanide ISA-51 with 75 microg of granulocyte-macrophage-stimulating factor (GM-CSF); or 3) MPS160 in Montanide ISA-51 with 100 microg of GM-CSF. Vaccines were administered every 3 weeks until patients developed disease progression or severe toxicity. Patients were aged >or=18 years with metastatic melanoma and a good performance status. Exclusion criteria included pregnancy/nursing, brain metastases, and ongoing chemotherapy. Immunologic efficacy was ascertained by using tetramer and functional analysis of peripheral blood lymphocytes. RESULTS: None of the 28 patients exhibited objective tumor responses or severe toxicities. Four patients remained progression free for >or=100 days. Immunologic analysis was available for 21 patients. Laboratory data demonstrated 1) increased frequency of vaccine-specific, nonfunctional cytotoxic T lymphocytes in 10 patients; 2) no differences in immunization efficacy among the treatment arms; and 3) evidence of systemic cytokine/immune dysfunction. CONCLUSIONS: Clinically, the MPS160 vaccine was ineffective. Phenotypic (tetramer) evidence of immunization was ineffective functionally and most likely was caused by global immune dysfunction, as illustrated by abnormal cytokine profiles in peripheral blood. In this report, the authors discuss possible implications of the current results on future cancer vaccine studies.

Distinctive patterns of autoimmune response induced by different types of mineral oil.

Although mineral oils are generally considered nontoxic and have a long history of use in humans, the mineral oil Bayol F (incomplete Freund's adjuvant, IFA) and certain mineral oil components (squalene and n-hexadecane) induce lupus-related anti-nRNP/Sm or -Su autoantibodies in nonautoimmune mice. In the present study, we investigated whether medicinal mineral oils can induce other types of autoantibodies and whether structural features of hydrocarbons influence autoantibody specificity. Female 3-month-old BALB/c (16-45/group) mice each received an i.p. injection of pristane (C19), squalene (C30), IFA, three medicinal mineral oils (MO-F, MO-HT, MO-S), or PBS. Sera were tested for autoantibodies and immunoglobulin levels. Hydrocarbons were analyzed by gas chromatography/mass spectrometry. IFA contained mainly C15-C25 hydrocarbons, whereas MO-HT and MO-S contained C20-C40, and MO-F contained C15-C40. Pristane and n-hexadecane were found in IFA (0.17% and 0.10% w/v, respectively) and MOs (0.0026-0.027%). At 3 months, pristane and IFA induced mainly IgG2a, squalene IgG1, and MOs IgG3 and IgM in sera. Anti-cytoplasmic antibodies were common in mice treated with MO-F, as well as those treated with pristane, squalene, and IFA. Anti-ssDNA and -chromatin antibodies were higher in MO-F and MO-S than in untreated/PBS, squalene-, or IFA-treated mice, suggesting that there is variability in the induction of anti-nRNP/Sm versus -chromatin/DNA antibodies. The preferential induction of anti-chromatin/ssDNA antibodies without anti-nRNP/Sm/Su by MO-S and MO-F is consistent with the idea that different types of autoantibodies are regulated differently. Induction of autoantibodies by mineral oils considered nontoxic also may have pathogenetic implications in human autoimmune diseases.

Immunoaffinity layering enhances the sensitivity of antigen detection on nitrocellulose strips.

A simple strategy to remarkably increase the sensitivity of detection of antigens applied as dot or western blot on nitrocellulose membrane using human serum albumin as model antigen has been described. This involves subjecting the antigen bearing nitrocellulose strips to multiple incubation cycles with primary antibody and enzyme conjugated secondary antibody prior to staining for enzyme activity. The sensitivity of detection could be increased up to a thousand fold after three incubation cycles. Aggregation of human serum albumin could be detected by the multiple incubation procedure at very low protein concentration after electrophoresis and transfer onto nitrocellulose.