Cytokine Profile in Human Peripheral Blood Mononuclear Leukocytes Exposed to Immunoadjuvant and Adjuvant-Free Vaccines Against Influenza.

Background: In the last decade, adjuvant-containing vaccines, exerting different effects on the immune system, including the production of cytokines, which are one of the most important regulatory systems of the body, are introduced into practice. Objectives: An effect of the immunoadjuvant polymer-subunit and adjuvant-free vaccines against influenza on the cytokine profile of mononuclear leukocytes in 27 healthy women was studied. Methods: The study of cytokine profile in human peripheral blood mononuclear leukocytes exposed to vaccines against influenza virus was determined by flow cytometry method (Cytomix FC-500, Beckman Coulter, USA) using the Multiplex-13 test system (Bender MedSystems, Austria). Results: It was established that all the studied vaccines leaded to somewhat increased levels of Th1/Th2/Th17/Th9/Th22 cytokines in the culture fluid of peripheral blood mononuclear leukocytes (PBML), which indicates the activation of both humoral and cellular immunity. An immunoadjuvant vaccine has been shown to be superior in activating the synthesis of Th1 (IL-12, INF-g, IL-2, IL-6, IL-1ß, TNF-α) cytokines, IL-9 and IL-22, while the subunit vaccine was superior in activating the synthesis of IL-4, and split vaccine-of IL-5. Conclusions: Immunoadjuvant vaccine is superior in terms of inducing cellular immune effectors to a greater extent compared to subunit and split vaccines.

The small molecule antibody mimic SH7139 targets a family of HLA-DRs expressed by B-cell lymphomas and other solid cancers.

Selective high-affinity ligands (SHALs) belong to a novel class of small-molecule cancer therapeutics that function as targeted prodrugs. SH7139, the most advanced of the SHAL drugs designed to bind to a unique ß-subunit structural epitope located on HLA-DR10, has exhibited exceptional preclinical efficacy and safety profiles. A comparison of SH7139 and SH7129, a biotin derivative of the drug developed for use as a diagnostic, showed the incorporation of a biotin tag did not alter the SHALs ability to target or kill HLA-DR10 expressing Raji cells. The use of SH7129 in an immuno-histochemical type assay to stain peripheral blood mononuclear cells (PBMCs) obtained from individuals expressing specific HLA-DRB1 alleles has also revealed that in addition to HLA-DR10, seven other more commonly expressed HLA-DRs are targeted by the drug. Computational dockings of the SHAL's recognition ligands to a number of HLA-DR structures explain, in part, why the targeting domains of SH7129 and SH7139 bind to some HLA-DRs but not others. The results also substantiate the selectivity of SH7129 and suggest it may prove useful as a companion diagnostic for pre-screening biopsy samples to identify those patients whose tumours should respond to SH7139 therapy.

Proteinase activated receptor-2-mediated dual oxidase-2 up-regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma.

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase-activated receptor 2 (PAR-2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX-2, have been shown to be involved in airway inflammation in response to Toll-like receptor activation. However, the association between PAR-2 and DUOX-2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX-2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR-2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX-2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl-2, caspase-3). Challenge with CE led to up-regulation of DUOX-2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD-1068, a small molecule antagonist of PAR-2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX-2/ROS pathway; however, these effects were less pronounced than PAR-2 antagonism. The current study suggests that PAR-2 activation leads to up-regulation of the DUOX-2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.

[Effect of immune modulation on immunogenic and protective activity of a live plague vaccine].

AIM: Comparative evaluation of the effect of polyoxidonium and betaleukin on immunogenic and protective activity of a live plague vaccine in model animal experiments. MATERIALS AND METHODS: Plague vaccine EV, polyoxidonium, betaleukin, erythrocytic antigenic diagnosticum for determination of F1 antibodies and immune reagents for detection of lymphocytes with F1 receptors (LFR) in adhesive test developed by the authors were used. The experiments were carried out in 12 rabbits and 169 guinea pigs. RESULTS: Immune modulation accelerated the appearance and disappearance of LFR (early phase) and ensured a more rapid and intensive antibody formation (effector phase). Activation by betaleukin is more pronounced than by polyoxidonium. The more rapid and intensive was the development of early phase, the more effective was antibody response to the vaccine. Immune modulation in the experiment with guinea pigs significantly increased protective activity of the vaccine. CONCLUSION: The use of immune modulators increased immunogenic (in both early and effector phases of antigen-specific response) and protective activity of the EV vaccine. A connection between the acceleration of the first phase of antigen-specific response and general intensity of effector phase of immune response to the EV vaccine was detected. ,

Development of a specific and sensitive enzyme-linked immunosorbent assay for the quantification of imatinib.

Imatinib is an oral tyrosine kinase inhibitor used for first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring targeting trough plasma levels of about 1000 ng/mL may help to optimize imantinib's therapeutic effect. This paper reports a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for a pharmacokinetic evaluation of imatinib. Anti-imatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin and succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. Enzyme labeling of imatinib with horseradish peroxidase was similarly performed using succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. A simple ELISA for imatinib was developed using the principle of direct competition between imatinib and the enzyme marker for anti-imatinib antibody which had been adsorbed by the plastic surface of a microtiter plate. Serum imatinib concentrations lower than 40 pg/mL were reproducibly measurable using the ELISA. This ELISA was specific to imatinib and showed very slight cross-reactivity (1.2%) with a major metabolite, N-desmethyl imatinib. Using this assay, drug levels were easily measured in the blood of mice after their oral administration of imatinib at a single dose of 50 mg/kg. The specificity and sensitivity of the ELISA for imatinib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of imatinib.

Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors.

Cancer immunosurveillance relies on effector/memory tumor-infiltrating CD8(+) T cells with a T-helper cell 1 (TH1) profile. Evidence for a natural killer (NK) cell-based control of human malignancies is still largely missing. The KIT tyrosine kinase inhibitor imatinib mesylate markedly prolongs the survival of patients with gastrointestinal stromal tumors (GIST) by direct effects on tumor cells as well as by indirect immunostimulatory effects on T and NK cells. Here, we investigated the prognostic value of tumor-infiltrating lymphocytes (TIL) expressing CD3, Foxp3, or NKp46 (NCR1) in a cohort of patients with localized GIST. We found that CD3(+) TIL were highly activated in GIST and were especially enriched in areas of the tumor that conserve class I MHC expression despite imatinib mesylate treatment. High densities of CD3(+) TIL predicted progression-free survival (PFS) in multivariate analyses. Moreover, GIST were infiltrated by a homogeneous subset of cytokine-secreting CD56(bright) (NCAM1) NK cells that accumulated in tumor foci after imatinib mesylate treatment. The density of the NK infiltrate independently predicted PFS and added prognostic information to the Miettinen score, as well as to the KIT mutational status. NK and T lymphocytes preferentially distributed to distinct areas of tumor sections and probably contributed independently to GIST immunosurveillance. These findings encourage the prospective validation of immune biomarkers for optimal risk stratification of patients with GIST.

A novel KIT-deficient mouse mast cell model for the examination of human KIT-mediated activation responses.

Activation of KIT, by its ligand, stem cell factor (SCF), results in the initiation of signal transduction pathways that influence mast cell survival and proliferation. Activating mutations in KIT have thus been linked to clonal MC proliferation associated with systemic mastocytosis. SCF also modulates MC function by inducing MC chemotaxis and by potentiating antigen (Ag)/IgE-mediated MC degranulation. Thus, mutations in KIT also have the potential to affect these processes in allergic and other mast cell-related diseases. Studies to determine how native and mutated KIT may modulate MC chemotaxis and activation have, however, been limited due to the lack of availability of a suitable functional MC line lacking native KIT which would allow transduction of KIT constructs. Here we describe a novel mouse MC line which allows the study of normal and mutated KIT constructs. These cells originated from a bone marrow-derived mouse MC culture out of which a rapidly dividing mast cell sub-population spontaneously arose. Over time, these cells lost KIT expression while continuing to express functional high affinity receptors for IgE (FcεRI). As a consequence, these cells degranulated in response to Ag/IgE but did not migrate nor show any evidence of potentiation of Ag/IgE degranulation in response to SCF. Retroviral transduction of the cells with a human (hu)KIT construct resulted in surface expression of huKIT which responded to huSCF by potentiation of Ag/IgE-induced degranulation and chemotaxis. This cell line thus presents a novel system to delineate how MC function is modulated by native and mutated KIT and for the identification of novel inhibitors of these processes.

Opposite effects of mepyramine on JNJ 7777120-induced amelioration of experimentally induced asthma in mice in sensitization and provocation.

BACKGROUND: Histamine is detected in high concentrations in the airways during an allergic asthma response. In a murine model of allergic asthma, JNJ 7777120, an antagonist at the histamine H(4) receptor, reduces asthmatic symptoms, while the histamine H(1) receptor-selective antagonist mepyramine is virtually without effect. In the present study, we analyzed the effect of combined antagonism at the histamine H(1) and H(4) receptors in a murine asthma model in relation to the timing of their application, i.e. sensitization or provocation. METHODOLOGY/PRINCIPAL FINDINGS: Asthma was induced in mice by sensitization and provocation with ovalbumin. JNJ 7777120 and/or mepyramine were injected subcutaneously either during sensitization or during provocation, and typical asthma parameters were analyzed. JNJ 7777120, but not mepyramine, reduced serum concentrations of anti-OVA IgE, inflammatory infiltrations in lung tissue, and eosinophilia in bronchoalveolar-lavage (BAL)-fluids independently of the timing of application. Upon application of JNJ 7777120 plus mepyramine in combination during provocation, mepyramine inhibited the effects of JNJ 7777120. In contrast, when applied during sensitization, mepyramine enhanced the disease-ameliorating effects of JNJ 7777120. CONCLUSIONS/SIGNIFICANCE: Our study indicates that both histamine H(1) and H(4) receptors play important roles in the course of murine experimental asthma. Unexpectedly, the contribution of these receptors to the pathogenesis differs between the two phases, sensitization or provocation. Since in human asthma, repeated contact to the allergen is not only provocation but also a boost of sensitization, a combined pharmacological targeting of histamine H(1) and H(4) receptors could be taken into consideration as an option for the prevention of asthma and maybe other allergic diseases.

Tyrosine kinase inhibitors in hematological malignancies.

Recently novel treatment modalities has focused on targeted therapies. Tyrosine kinases represent a good target for cancer treatment since they are involved in transferring phosphate groups from ATP to tyrosine residues in specific substrate proteins transducing intracellular signals engaged in the many mechanisms, playing an important role in the modulation of growth factors signaling that are strongly related to carcinogenesis. Deregulation of tyrosine kinases activity was also found in hematological malignancies, particularly overexpression of tyrosine kinases was observed in chronic myeloid leukemia or acute lymphoblastic leukemia. Herein we show that tyrosine kinase inhibitors have revolutionized hematology malignancies therapy in a very short period of time and they still remain one of the most interesting anticancer compounds that could give a hope for cure and not only long-lasting complete remission. This manuscript summarizes current view on the first generation tyrosine kinase inhibititor--imatinib, second generation--dasatinib, nilotinib and bosutnib as well as new generation tyrosine kinase inhibititors--ponatinib and danusertib in hematooncology.

Immunopotentiation of live brucellosis vaccine by adjuvants.

In a series of studies in SPF and conventional guinea pigs, various adjuvants (larifan, polyoxidonium-PO, natrium thiosulphate-NT, TNF-ß and Ribi adjuvant system-RAS) were evaluated for their ability to enhance immune responses to the live brucellosis vaccine, Brucella abortus strain 82-PS (penicillin-sensitive). Combining adjuvants with S82-PS increased synthesis of antibodies against rough (R) and smooth (S) Brucella antigens. Dynamics and levels of antibodies differed dependent upon the adjuvant. Adjuvants enhanced cell-mediated responses to S82-PS, and phagocytosis by macrophages. Humoral and cellular immune responses stimulated by the adjuvants correlated with increased vaccine protection against experimental challenge. The highest protection was demonstrated by combining TNF-ß or PO with S82-PS. Our data demonstrates the potential of adjuvants to improve immunogenic properties of live brucellosis vaccines.

Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody.

Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed. Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL(-1), the calibration curve was linear from 5.0 to 40 ng mL(-1) (R(2)=0.952) with an IC(50) value of 18.2 ng mL(-1). In the extracts of 20 Chinese traditional drugs, the detection capability (CCbeta) of vardenafil was 0.08 mg g(-1), the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue. The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.

Development of a homogeneous immunoassay for the detection of zolpidem in urine.

Sleep disorders are common conditions that affect about 40 million people in the U.S every year, the most common of which is insomnia, which is characterized by difficulty falling or staying asleep. Zolpidem (Ambien) is a non-benzodiazepine prescription drug that is used to treat insomnia and is often preferred over the commonly used benzodiazepines due to a lesser side effect profile. This is because the non-benzodiazepine binding is more selective to GABA-A receptors versus the non-selective binding of benzodiazepines. With the increasing popularity of non-benzodiazepines, drug abuse and driving-while-impaired cases involving sleep-inducing drugs have risen. Therefore, a highly sensitive and rapid homogeneous immunoassay (EMIT-type assay) has been developed for the detection of zolpidem in urine. The zolpidem antibody is highly specific and does not cross-react with other newer sleep aids such as zopiclone and zaleplon. This assay has a detection limit of 5 ng/mL for zolpidem in urine. Further evaluation of this assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis of authentic urine samples demonstrated that the accuracy of the assay is greater than 90%. Because this assay is designed to measure the non-conjugated drug in urine, it resulted in simplification for gas chromatography-MS or LC-MS-MS confirmation methods that do not require urine hydrolysis before solid-phase extraction or liquid-liquid extraction.

Simultaneous evaluation of viability and Bcl-2 in small-cell lung cancer.

When designing molecular targeted therapeutic strategies against cancer, it is important to correlate protein expression and cell viability. However, such goal can be difficult if performed in separate assays, especially when only a fraction of cells has been efficiently transfected. Therefore, the aim of the present study was to establish a flow cytometry procedure to assess simultaneously Bcl-2 protein level and viability in small-cell lung cancer (SCLC) cells. Viability assessment was performed by staining cells with Annexin V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD). Intracellular detection of Bcl-2 was carried out by immunodetection with monoclonal antibodies. Regarding viability determination, the FSC/7-AAD plot identifies the same percentage of viable cells as the FSC/Annexin V-FITC plot, although with greater sensitivity. The procedures involving cells' fixation with 1% paraformaldehyde and permeabilization with digitonin, required for intracellular Bcl-2 immunostaining did not compromise the association of 7-ADD (nor Annexin V-FITC) previously incubated with SCLC cells. It was therefore possible to simultaneously assess cell viability and Bcl-2 protein in SCLC cells. A simple, sensitive, and versatile procedure was established for the first time for the simultaneous evaluation of cell viability and intracellular detection of Bcl-2 in SCLC.

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells.

The purpose of this study was to identify the effect of sildenafil citrate on IL-1beta-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1? stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1beta-induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1beta-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1?-induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.

Dormant tumor cells develop cross-resistance to apoptosis induced by CTLs or imatinib mesylate via methylation of suppressor of cytokine signaling 1.

In the BCR/ABL DA1-3b mouse model of acute myelogenous leukemia, dormant tumor cells may persist in the host in a state of equilibrium with the CD8(+) CTL-mediated immune response by actively inhibiting T cells. Dormant tumor cells also show a progressive decrease of suppressor of cytokine signaling 1 (SOCS1) gene expression and a deregulation of the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway due to methylation of the SOCS1 gene. Dormant tumor cells were more resistant to apoptosis induced by specific CTLs, but resistance decreased when SOCS1 expression was restored via demethylation or gene transfer. AG490 JAK2 inhibitor decreased the resistance of dormant tumor cells to CTLs, but MG132 proteasome inhibitor was effective only in SOCS1-transfected cells. Thus, SOCS1 regulation of the JAK/STAT pathways contributes to the resistance of tumor cells to CTL-mediated killing. Resistance of dormant tumor cells to apoptosis was also observed when induced by irradiation, cytarabine, or imatinib mesylate, but was reduced by SOCS1 gene transfer. This cross-resistance to apoptosis was induced by interleukin 3 (IL-3) overproduction by dormant tumor cells and was reversed with an anti-IL-3 antibody. Thus, tumor cells that remain dormant for long periods in the host in spite of a specific CTL immune response may deregulate their JAK/STAT pathways and develop cross-resistance to various treatments through an IL-3 autocrine loop. These data suggest possible new therapeutic targets to eradicate dormant tumor cells.

Imatinib-induced immune hepatitis: case report and literature review.

Imatinib is one of the most recent medications used for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumor (GIST). It is an orally administered protein-tyrosine kinase inhibitor, an enzyme which is produced by BCR-ABL fusion which results from translocation of 9:22 chromosome (Philadelphia chromosome). Imatinib blocks proliferation and induces apoptosis of BCR-ABL-expression in CML. Many side effects produced by imatinib have been documented but its induction of hepatotoxcity has been rarely reported. Only a few cases so far have been reported in the literature and almost all were in females. We describe another case of hepatotoxicity due to imatinib in a 17-year old female with clinical, laboratory and histopathological changes. The case described here suggests that imatinib may also induce immune hepatitis, in some patients.