Co-administration of tecovirimat and ACAM2000™ in non-human primates: Effect of tecovirimat treatment on ACAM2000 immunogenicity and efficacy versus lethal monkeypox virus challenge.

Naturally occurring smallpox has been eradicated but research stocks of variola virus (VARV), the causative agent of smallpox, still exist in secure laboratories. Clandestine stores of the virus or resurrection of VARV via synthetic biology are possible and have led to concerns that VARV could be used as a biological weapon. The US government has prepared for such an event by stockpiling smallpox vaccines and TPOXX®, SIGA Technologies' smallpox antiviral drug. While vaccination is effective as a pre-exposure prophylaxis, protection is limited when administered following exposure. Safety concerns preclude general use of the vaccine unless there is a smallpox outbreak. TPOXX is approved by the FDA for use after confirmed diagnosis of smallpox disease. Tecovirimat, the active pharmaceutical ingredient in TPOXX, targets a highly conserved orthopoxviral protein, inhibiting long-range dissemination of virus. Although indications for use of the vaccine and TPOXX do not overlap, concomitant use is possible, especially if the TPOXX indication is expanded to include post-exposure prophylaxis. It is therefore important to understand how vaccine and TPOXX may interact. In studies presented here, monkeys were vaccinated with the ACAM2000TM live attenuated smallpox vaccine and concomitantly treated with tecovirimat or placebo. Immune responses to the vaccine and protective efficacy versus a lethal monkeypox virus (MPXV) challenge were evaluated. In two studies, primary and anamnestic humoral immune responses were similar regardless of tecovirimat treatment while the third study showed reduction in vaccine elicited humoral immunity. Following lethal MPXV challenge, all (12 of 12) vaccinated/placebo treated animals survived, and 12 of 13 vaccinated/tecovirimat treated animals survived. Clinical signs of disease were elevated in tecovirimat treated animals compared to placebo treated animals. This suggests that TPOXX may affect the immunogenicity of ACAM2000 if administered concomitantly. These studies may inform on how vaccine and TPOXX are used during a smallpox outbreak.

A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for flubendiamide detection.

Flubendiamide (FD), the first commercial phthalic acid diamide that targets insect ryanodine receptor (RyRs), has played an important role in pest management. With its extensive worldwide application, a rapid and convenient method to detect its existence in the environment is necessary. In this study, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to analyse FD residue on environmental and food samples. The established icELISA showed a half maximal inhibition concentration (IC50) of 17.25 µg L-1, with a working range of 4.06-103.59 µg L-1 for FD, and showed no cross-reactivity with chlorantraniliprole, cyantraniliprole, and several FD analogues. Average FD recoveries from spinach, tap water, and soil samples were 89.3-112.3%, 93.0-102.1%, and 86.9-97.6%, respectively. Meanwhile, FD detection results of icELISA were compared with those of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The comparable results verified that icELISA was suitable for rapid detection of FD residue in environmental and agricultural samples.

A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens.

The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

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.

High throughput label-free platform for statistical bio-molecular sensing.

Sensors are crucial in many daily operations including security, environmental control, human diagnostics and patient monitoring. Screening and online monitoring require reliable and high-throughput sensing. We report on the demonstration of a high-throughput label-free sensor platform utilizing cantilever based sensors. These sensors have often been acclaimed to facilitate highly parallelized operation. Unfortunately, so far no concept has been presented which offers large datasets as well as easy liquid sample handling. We use optics and mechanics from a DVD player to handle liquid samples and to read-out cantilever deflection and resonant frequency. Also, surface roughness is measured. When combined with cantilever deflection the roughness is discovered to hold valuable additional information on specific and unspecific binding events. In a few minutes, 30 liquid samples can be analyzed in parallel, each by 24 cantilever-based sensors. The approach was used to detect the binding of streptavidin and antibodies.

Immune responses to the smallpox vaccine given in combination with ST-246, a small-molecule inhibitor of poxvirus dissemination.

The re-emerging threat of smallpox and the emerging threat of monkeypox highlight the need for effective poxvirus countermeasures. Currently approved smallpox vaccines have unacceptable safety profiles and, consequently, the general populace is no longer vaccinated, leading to an increasingly susceptible population. ST-246, a small-molecule inhibitor of poxvirus dissemination, has been demonstrated in various animal models to be safe and effective in preventing poxviral disease. This suggests that it may also be used to improve the safety of the traditional smallpox vaccine provided that it does not inhibit vaccine-induced protective immunity. In this study, we compared the immune responses elicited by the smallpox vaccine alone or in combination with ST-246 in mice. Normal lesion formation following dermal scarification with the attenuated New York City Board of Health strain (Dryvax), commonly referred to as a vaccine "take", was not inhibited although severe lesions and systemic disease due to vaccination with the virulent Western Reserve (VV-WR) strain were prevented. The vaccine given with ST-246 did not affect cellular immune responses or neutralizing antibody titers although anti-vaccinia ELISA titers were slightly reduced. Vaccination in combination with ST-246 provided equivalent short- and long-term protection against lethal intranasal challenge with VV-WR when compared to vaccine alone. These results suggest that ST-246 does not compromise protective immunity elicited by the vaccine and provide the basis for future studies examining the efficacy of ST-246 in preventing or treating adverse events due to vaccination.

Iosimenol, a new non-ionic dimeric contrast medium, does not induce immunoreactivity in the popliteal lymph node assay.

Animal studies in mice were conducted to determine the potential immunoreactivity of the new non-ionic dimeric contrast medium (CM) iosimenol using the PLNA and flow cytometric analyses. Comparative studies were performed with iodixanol. The known immune-reactive substance strepozotocin (STZ) and vehicle injections served as positive and negative controls, respectively. Our experiments did not show any immunological effect of iosimenol, concluding that the new CM iosimenol may be beneficial for use in high-risk patients.

Reversal of resistance to cytotoxic cancer therapies: DHMEQ as a chemo-sensitizing and immuno-sensitizing agent.

The development of tumor cell resistance to conventional therapeutics is a major clinical problem. There is an urgent need to develop novel therapeutics to overcome resistance and save patients from tumor recurrences. Novel therapeutics are currently being developed based on better understanding of the underlying molecular mechanisms that govern resistance and the identification of targets that control resistance. One of the major factors that controls resistance is the transcription factor nuclear factor kappaB (NF-kappaB) that has been shown to be constitutively activated in the majority of cancers and is responsible, in large part, for tumor cell survival, growth and direct activation of anti-apoptotic gene products. The development of non-toxic inhibitors of NF-kappaB activity may result in diminishing the anti-apoptotic threshold of resistant tumor cells and leading to inhibition of tumor cell growth and cell death or sensitization to the apoptotic effects of cytotoxic therapeutics. The novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), selectively prevents the translocation of NF-kappaB into the nucleus and, hence, prevents its various transcriptional functions. Thus, DHMEQ is unlike many other NF-kappaB inhibitors that target gene products of the NF-kappaB pathway and it is also unlike proteasome inhibitors that prevent the degradation of pIkappaB. DHMEQ is a small molecule shown to be non-toxic in mice and rodents and exerts direct anti-tumor effects in vitro and in vivo as well as significant chemo- and immuno-sensitizing activities in resistant tumor cells. The present review summarizes studies that have used DHMEQ as a novel anti-cancer agent.

Dissociation between sex differences in the immunological, behavioral, and physiological effects of kappa- and delta-opioids in Fischer rats.

RATIONALE: The sex of the individual can have a profound effect on sensitivity to the effects of opioids. Recently, our laboratory provided the first evidence that females may be more sensitive to the immune-altering effects of mu-opioids than males. However, it remains unknown whether kappa- and delta-opioids produce sexually dimorphic effects on immune responses. OBJECTIVE: The present study sought to determine whether kappa- and delta-opioids produce differential immunological effects in males and females using the memory-T-cell-dependent in vivo inflammatory response contact hypersensitivity (CHS). As sex differences in the magnitude of opioid effects can be outcome-specific, additional experiments were conducted to compare the immunological effects of kappa- and delta-opioids with other behavioral and physiological effects. MATERIALS AND METHODS: Contact hypersensitivity was induced in male and female Fischer rats. Prior to elicitation of CHS, animals were administered selected doses of the kappa-opioid spiradoline (0.2-20 mg/kg), delta-opioid SNC80 (1-10 mg/kg), or vehicle. The antinociceptive and diuretic effects of spiradoline were also assessed in males and females, as were the locomotor effects of SNC80. RESULTS: Spiradoline produced significantly greater enhancement of CHS in females than males, but produced comparable antinociceptive and diuretic effects in both sexes. By contrast, SNC80 did not significantly affect the course of CHS in either sex, but females were significantly more sensitive to its locomotor stimulatory effects. CONCLUSIONS: These results demonstrate that females are more sensitive than males to the CHS-altering effects of spiradoline and that sex differences in the magnitude and direction of opioid-induced sex differences are outcome dependent.

Iosimenol, a low-viscosity nonionic dimer: preclinical physicochemistry, pharmacology, and pharmacokinetics.

RATIONALE AND OBJECTIVES: Newer radiologic techniques require fast bolus injections and thus low-viscosity, high-concentration, well-tolerated contrast media (CM), especially in vulnerable patients. To this end, we designed and developed iosimenol, a novel isotonic nonionic dimer, and have conducted tests to enable its clinical evaluation. METHODS: Standard physicochemical methods were used. Effects on erythrocyte morphology and coagulation were investigated in human and rat blood. Neural tolerance was assessed by behavioral tests in rats after intracisternal injection. Immunosensitizing potential was evaluated by the skin sensitization test in guinea pigs and by the popliteal lymph node assay in rats. Pharmacokinetics and biotransformation were investigated in rats and dogs. RESULTS: Iosimenol is extremely hydrophilic, it is less viscous than any other isotonic CM, has little effect on erythrocytes and blood coagulation, and has good neural tolerance. No immunosensitizing effect was found in validated animal models. Pharmacokinetics are identical with other angio- and urographic CM. CONCLUSIONS: Iosimenol is the only CM which, although isotonic, affords, unlike current nonionic dimers, at the same iodine concentration the low viscosity of monomeric, nonionic agents, which are all hypertonic. Iosimenol's pharmacologic characteristics closely resemble those of iotrolan and iodixanol.

A quantitative enzyme-linked immunoassay for the detection of 2, 6-dichlorobenzamide (BAM), a degradation product of the herbicide dichlobenil.

2,6-Dichlorobenzamide (BAM) is the dominant degradation product in soil of the widely used herbicide dichlobenil. To detect BAM in water, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed. As an alternative to conventional coating of ELISA plates, the assay is based on direct covalent immobilisation. We achieved a surface which requires a short time for the immobilisation of ligand, is stable under dry storage, and which permits assays with a low CV. The performance of the assay was demonstrated by an inter-well CV that was generally less than 6%, a detection limit (DL(15)) of 0.02 microg/l and an IC(50) of 0.19 microg/l. Cross-reactivity was measured against nine analytes with structural homology to BAM. The highest degree of cross-reactivity (10.8%) was seen with 2,6-dichlorothiobenzamide (Chlorthiamid). Considering an EU-limit of 0.1 microg/l as the permissible maximum for the presence of pesticides in drinking water, this ELISA-procedure is suitable for large-scale screening of water samples suspected of being contaminated with BAM.

Protective effect of poly(ADP-ribose) synthetase inhibition on multiple organ failure after zymosan-induced peritonitis in the rat.

BACKGROUND AND METHODS: In the present study, we tested the hypothesis that peroxynitrite and subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) play a role in the pathogenesis of multiple organ failure induced by peritoneal injection of zymosan in the rat. Animals were randomly divided into six groups (ten rats for each group). The first group was treated with ip administration of saline solution (0.9% NaCl) and served as the sham group. The second group was treated with ip administration of zymosan (500 mg/kg suspended in saline solution). In the third and fourth groups, rats received ip administration of 3-aminobenzamide (10 mg/kg) 1 and 6 hrs after zymosan or saline administration, respectively. In the fifth and sixth groups, rats received ip administration of nicotinamide (50 mg/kg) 1 and 6 hrs after zymosan or saline administration, respectively. After zymosan or saline injection, animals were monitored for 72 hrs to evaluate systemic toxicity (conjunctivitis, ruffled fur, diarrhea, and lethargy), loss of body weight, and mortality. RESULTS: A severe inflammatory response, characterized by peritoneal exudation, high plasma and peritoneal levels of nitrate/nitrite (the breakdown products of nitric oxide), and leukocyte infiltration into peritoneal exudate, was induced by zymosan administration. This inflammatory process coincided with the damage of lung, small intestine, and liver as assessed by histologic examination and by an increase of myeloperoxidase activity, which is indicative of neutrophil infiltration. Zymosan-treated rats showed signs of systemic illness, significant loss of body weight, and high mortality rates. Peritoneal administration of zymosan in the rat also induced a significant increase in the plasma levels of peroxynitrite as measured by the oxidation of the fluorescent dihydrorhodamine 123. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the lung of zymosan-shocked rats. In vivo treatment with ip administration of 3-aminobenzamide (10 mg/kg, 1 and 6 hrs after zymosan injection) or nicotinamide (50 mg/kg, 1 and 6 hrs after zymosan injection) significantly decreased mortality, inhibited the development of peritonitis, and reduced peroxynitrite formation. In addition, PARS inhibitors were effective in preventing the development of organ failure because tissue injury and neutrophil infiltration, by myeloperoxidase evaluation, were reduced in the lung, small intestine, and liver. CONCLUSIONS: In conclusion, the major findings of our study are that peroxynitrite and the consequent PARS activation exert a role in the development of multiple organ failure and that PARS inhibition is an effective anti-inflammatory therapeutic tool.

Antibodies with high affinity for spiroperidol--II. Cross reactivity with iodobenzamide and domperidone.

Three radioligands, 3H-spiroperidol (3H-SPD), 3H-domperidone (3H-DOMP) and 125I-iodobenzamide (125I-IBZM), were used to investigate the antibody response to two haptens, aminospiroperidol (NH2SPD) and N-aminophenethylspiroperidol (NAPS). Although structurally different, these three radioligands each bind with high affinity to the D2 dopamine receptor. Antibodies with high affinity for 3H-SPD were elicited in rabbits following immunization with the hapten NH2SPD covalently linked to keyhole limpet hemocyanin (KLH). In addition, antibodies in the rabbit anti-NH2SPD antisera bound 125I-IBZM or 3H-DOMP. Rabbit anti-NH2SPD antibodies that bound 125I-IBZM or 3H-DOMP were found to have higher affinity for IBZM or DOMP, respectively, than for SPD. The binding properties of the anti-NH2SPD antibodies that bound 3H-SPD, 125I-IBZM and 3H-DOMP were characterized using a panel of competitive inhibitors and each radioligand appeared to bind to a distinct subpopulation of anti-NH2SPD antibodies. BALB/c mice were immunized with NH2SPD-KLH or NAPS-KLH. A population of antibodies that bound 3H-SPD and a population of antibodies that bound 3H-DOMP were detected. The population of antibodies that bound 3H-DOMP was found to be heteroclitic for DOMP, since DOMP was a more effective competitive inhibitor than SPD. Binding sites for 125I-IBZM were not detected in either the anti-NH2SPD or the anti-NAPS BALB/c antisera. However, two anti-NAPS monoclonal antibodies, N6-24 and N6-29, that bind 3H-SPD with high affinity (Kd = 10(-9) M), were also found to bind IBZM (Ki = 2 x 10(-7) M) and DOMP (Ki = 2 x 10(-6) M). Although anti-NH2SPD and anti-NAPS antibodies were identified that appeared to bind 3H-SPD, 3H-DOMP or 125I-IBZM with high affinity, none of the populations of polyclonal antibodies or monoclonal antibodies bound all three ligands with high affinity.