Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer.

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)-based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8(+) T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8(+) T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials.

Technologies for biological containment of GM and Non-GM crops

International Perspective The development of GM technology continues to expand into increasing numbers of crops and conferred traits. Inevitably, the focus remains on the major field crops of soybean, maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance and/or pest resistance. Although there are comparatively few GM crops that have been commercialised to date, GM versions of 172 plant species have been grown in field trials in 31 countries. European Crops with Containment Issues Of the 20 main crops in the EU there are four for which GM varieties are commercially available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in development (rye, triticale). Many of these crops have hybridisation potential with wild and weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats, sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have varieties in field trials and these too may hybridise with wild relatives in the European flora (alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm, Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice and rose). All these crops will require containment strategies to be in place if it is deemed necessary to prevent transgene movement to wild relatives and non-GM crops. Current Containment Strategies A wide variety of GM containment strategies are currently under development, with a particular focus on crops expressing pharmaceutical products. Physical containment in greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and duckweed) expressing pharmaceutical products have been adopted by some biotechnology companies. There are obvious limitations of the scale of physical containment strategies, addressed in part by the development of large underground facilities in the US and Canada. The additional resources required to grow plants underground incurs high costs that in the long term may negate any advantage of GM for commercial productioNatural genetic containment has been adopted by some companies through the selection of either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms with no wild relatives present in the local flora (safflower in the Americas). The expression of pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth and harvesting prior to and in the absence of flowering. Transgenically controlled containment strategies range in their approach and degree of development. Plastid transformation is relatively well developed but is not suited to all traits or crops and does not offer complete containment. Male sterility is well developed across a range of plants but has limitations in its application for fruit/seed bearing crops. It has been adopted in some commercial lines of oilseed rape despite not preventing escape via seed. Conditional lethality can be used to prevent flowering or seed development following the application of a chemical inducer, but requires 100% induction of the trait and sufficient application of the inducer to all plants. Equally, inducible expression of the GM trait requires equally stringent application conditions. Such a method will contain the trait but will allow the escape of a non-functioning transgene. Seed lethality (‘terminator’ technology) is the only strategy at present that prevents transgene movement via seed, but due to public opinion against the concept it has never been trialled in the field and is no longer under commercial development. Methods to control flowering and fruit development such as apomixis and cleistogamy will prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are complex and leaky. None of the genes controlling these traits have as yet been identified or characterised and therefore have not been transgenically introduced into crop species. Neither of these strategies will prevent transgene escape via seed and any feral apomicts that form are arguably more likely to become invasives. Transgene mitigation reduces the fitness of initial hybrids and so prevents stable introgression of transgenes into wild populations. However, it does not prevent initial formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents persistence of escapes and hybrids containing the transgene in an uncontrolled environment, but does not prevent transgene movement from the crop. Recoverable block of function, intein trans-splicing and transgene excision all use recombinases to modify the transgene in planta either to induce expression or to prevent it. All require optimal conditions and 100% accuracy to function and none have been tested under field conditions as yet. All will contain the GM trait but all will allow some non-native DNA to escape to wild populations or to non-GM crops. There are particular issues with GM trees and grasses as both are largely undomesticated, wind pollinated and perennial, thus providing many opportunities for hybridisation. Some species of both trees and grass are also capable of vegetative propagation without sexual reproduction. There are additional concerns regarding the weedy nature of many grass species and the long-term stability of GM traits across the life span of trees. Transgene stability and conferred sterility are difficult to trial in trees as most field trials are only conducted during the juvenile phase of tree growth. Bio-pharming of pharmaceutical and industrial compounds in plants Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigene’s avidin, β-glucuronidase, trypsin generated in GM maize; Ventria’s lactoferrin generated in GM rice). Numerous products are in clinical trials (collagen, antibodies against tooth decay and non-Hodgkin’s lymphoma from tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize; Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary supplements from Arabidopsis). The initial production platforms for plant-based pharmaceuticals were selected from conventional crops, largely because an established knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and spinach are widely used as leaves can be harvested and no flowering is required. Many of these crops can be grown in contained greenhouses. Potato is also widely used and can also be grown in contained conditions. The introduction of morphological markers may aid in the recognition and traceability of crops expressing pharmaceutical products. Plant cells or plant parts may be transformed and maintained in culture to produce recombinant products in a contained environment. Plant cells in suspension or in vitro, roots, root cells and guttation fluid from leaves may be engineered to secrete proteins that may be harvested in a continuous, non-destructive manner. Most strategies in this category remain developmental and have not been commercially adopted at present. Transient expression produces GM compounds from non-GM plants via the utilisation of bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole plants or plant parts, but do not insert them into the heritable genome. There are some limitations of scale and the field release of such crops will require the regulation of the vector. However, several companies have several transiently expressed products in clinical and pre-clinical trials from crops raised in physical containment.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

Association between handling stress in the corral and rabies antibody titers in selenium-supplemented cattle

This study determined the correlation between serum cortisol levels and rabies antibody titers in cattle primo-vaccinated against rabies and supplemented with dietary selenium (Se). Sixty Nelore male calves (10 to 12 months old) received daily and individual dietary supplementation with 0, 3.6, 5.4 and 6.4 mg Se (groups Gc, G(3.6), G(5.4) and G(6.4), respectively). The animals were vaccinated against rabies (day 0) and subjected to handling stress in the corral for 120 days. Blood sampling procedures were performed on days 0, 15, 30, 60, 90 and 120. Cortisol levels increased until day 90, but had dropped significantly by day 120 (P < 0.01). Rabies antibody titers on days 30 and 90 were similar among Se-supplemented groups; in the control group, rabies antibodies decreased significantly from day 30 to 60, and 90 to 120. Serum cortisol levels and antibody titers were not correlated in most of the groups or blood sampling days. A positive correlation among these variables was found only in G(6.4) on days 60 (R = 0.513; P = 0.05) and 120 (R = 0.644; P = 0.009). In conclusion, repeated handling in the corral stresses cattle, but without compromising rabies humoral immune response.

Monitoring Raccoon Rabies in Alabama: The Potential Effects of Habitat and Demographics

Density, morphometrics, and disease prevalence of raccoon populations were determined in 4 habitats (agriculture, riverine, managed, and forested) in central Alabama. In addition we monitored 71 collared raccoons to determine survival. Density estimates were similar in the agriculture (ag) and riverine habitats in central Alabama with 8 raccoons/km2, and lower in the forested habitat at 5 raccoons/ km2. Retention of juveniles did not appear to contribute to observed higher populations in the riverine and ag habitat. Although the riverine and ag, possibly due to supplemental resources, likely provide better habitat for raccoons, we found only body size in female raccoons to be different across habitats (P = 0.001). Human-caused mortality (either hunting or missing and presumed killed) was the main cause of mortality in several raccoon populations during fall; however, fall survival did not differ between the habitats (χ = 1.47, d.f. = 3, P = 0.69). Although rabies and distemper virus were prevalent in all habitats, they did not appear to contribute to mortality even with a high proportion of the population exhibiting positive CDV titers (ag – 44%, managed- 50%) and rabies titers (managed- 57% and riverine habitat-60%).

HPV knowledge, HPV vaccine awareness and initiation among Hispanics: The role of language, information seeking, and information scanning

Background. Among Hispanics, the HPV vaccine has the potential to eliminate disparities in cervical cancer incidence and mortality but only if optimal rates of vaccination are achieved. Media can be an important information source for increasing HPV knowledge and awareness of the vaccine. Very little is known about how media use among Hispanics affects their HPV knowledge and vaccine awareness. Even less is known about what differences exist in media use and information processing among English- and Spanish-speaking Hispanics.^ Aims. Examine the relationships between three health communication variables (media exposure, HPV-specific information scanning and seeking) and three HPV outcomes (knowledge, vaccine awareness and initiation) among English- and Spanish-speaking Hispanics.^ Methods. Cross-sectional data from a survey administered to Hispanic mothers in Dallas, Texas was used for univariate and multivariate logistic regression analyses. Sample used for analysis included 288 mothers of females aged 8-22 recruited from clinics and community events. Dependent variables of interest were HPV knowledge, HPV vaccine awareness and initiation. Independent variables were media exposure, HPV-specific information scanning and seeking. Language was tested as an effect modifier on the relationship between health communication variables and HPV outcomes.^ Results. English-speaking mothers reported more media exposure, HPV-specific information scanning and seeking than Spanish-speakers. Scanning for HPV information was associated with more HPV knowledge (OR = 4.26, 95% CI = 2.41 - 7.51), vaccine awareness (OR = 10.01, 95% CI = 5.43 - 18.47) and vaccine initiation (OR = 2.54, 95% CI = 1.09 - 5.91). Seeking HPV-specific information was associated with more knowledge (OR = 2.27, 95% CI = 1.23 - 4.16), awareness (OR = 6.60, 95% CI = 2.74 - 15.91) and initiation (OR = 4.93, 95% CI = 2.64 - 9.20). Language moderated the effect of information scanning and seeking on vaccine awareness.^ Discussion. Differences in information scanning and seeking behaviors among Hispanic subgroups have the potential to lead to disparities in vaccine awareness.^ Conclusion. Findings from this study underscore health communication differences among Hispanics and emphasize the need to target Spanish language media as well as English language media aimed at Hispanics to improve knowledge and awareness.^

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression. (c) 2006 Elsevier B.V. All rights reserved.

Characterization and optimization of bilosomes for oral vaccine delivery

Oral vaccines offer significant benefits due to the ease of administration, better patient compliance and non-invasive, needle-free administration. However, this route is marred by the harsh gastro intestinal environment which is detrimental to many vaccine formats. To address this, a range of delivery systems have been considered including bilosomes; these are bilayer vesicles constructed from non-ionic surfactants combined with the inclusion of bile salts which can stabilize the vesicles in the gastro intestinal tract by preventing membrane destabilization. The aim of this study was to investigate the effect of formulation parameters on bilosome carriers using Design of Experiments to select an appropriate formulation to assess in vivo. Bilosomes were constructed from monopalmitoylglycerol, cholesterol, dicetyl phosphate and sodium deoxycholate at different blends ratios. The optimized bilosome formulation was identified and the potential of this formulation as an oral vaccine delivery system were assessed in biodistribution and vaccine efficacy studies. Results showed that the larger bilosomes vesicles (~6 µm versus 2 µm in diameter) increased uptake within the Peyer's patches and were able to reduce median temperature differential change and promote a reduction in viral cell load in an influenza challenge study. © 2013 Informa UK, Ltd.

Comparative assessment of seller’s staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies.

Background: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller’s stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. Objective: This study was designed to assess the sensitivity and specificity of the SST in relation to the ‘gold standard’ dFAT in diagnosis of rabies in Nigeria. Methods: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. Results: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). Conclusion: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.

Rabies facts : dos and don'ts

This sheet gives facts about rabies and what you should and shouldn't do to avoid it.

Immunogenicity of a Whole-Cell Pertussis Vaccine with Low Lipopolysaccharide Content in Infants

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gamma delta-positive (gamma delta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in super-natants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gamma delta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gamma delta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gamma delta(+) cell expansions were similar with the two vaccines.

Altered Ex Vivo Expression of Caspase 8, Caspase 9, and Bcl-2 Is Associated with T-Cell Hyporeactivity in Patients with Paracoccidioidomycosis

To better understand the T-cell hyporesponsiveness of patients with paracoccidioidomycosis, we tested the hypothesis that the T cells were committed to apoptosis. We show here that T cells of patients with paracoccidioidomycosis overexpress caspase 9 and caspase 8 but express low Bcl-2 levels and that interleukin-2 was unable to revert the hyporesponsiveness. These data suggest that the T cells would in vivo be driven to a tolerant state and apoptosis.

Comparative Study of the Standard Fluorescent Antibody to Membrane Antigen (FAMA) Assay and a Flow Cytometry-Adapted FAMA Assay To Assess Immunity to Varicella-Zoster Virus

A flow cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster virus (VZV) was developed and tested in 62 serum samples, showing 90.32% accuracy obtained from a receiver operating characteristic (ROC) curve with a 0.9125 (95% confidence interval [CI], 0.829 to 1.00) area below the curve compared to the result with standard FAMA.

Clinical and Immunological Insights on Severe, Adverse Neurotropic and Viscerotropic Disease following 17D Yellow Fever Vaccination

Yellow fever (YF) vaccines (17D-204 and 17DD) are well tolerated and cause very low rates of severe adverse events (YEL-SAE), such as serious allergic reactions, neurotropic adverse diseases (YEL-AND), and viscerotropic diseases (YEL-AVD). Viral and host factors have been postulated to explain the basis of YEL-SAE. However, the mechanisms underlying the occurrence of YEL-SAE remain unknown. The present report provides a detailed immunological analysis of a 23-year-old female patient. The patient developed a suspected case of severe YEL-AVD with encephalitis, as well as with pancreatitis and myositis, following receipt of a 17D-204 YF vaccination. The patient exhibited a decreased level of expression of Fc-gamma R in monocytes (CD16, CD32, and CD64), along with increased levels of NK T cells (an increased CD3(+) CD16(+/-) CD56(+/-)/CD3(+) ratio), activated T cells (CD4(+) and CD8(+) cells), and B lymphocytes. Enhanced levels of plasmatic cytokines (interleukin-6 [IL-6], IL-17, IL-4, IL-5, and IL-10) as well as an exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within NK cells (gamma interferon positive [IFN-gamma(+)], tumor necrosis factor alpha positive [TNF-alpha(+)], and IL-4 positive [IL-4(+)]), CD8(+) T cells (IL-4(+) and IL-5(+)), and B lymphocytes (TNF-alpha(+), IL-4(+), and IL-10(+)). The analysis of CD4(+) T cells revealed a complex profile that consisted of an increased frequency of IL-12(+) and IFN-gamma(+) cells and a decreased percentage of TNF-alpha(+), IL-4(+), and IL-5+ cells. Depressed cytokine synthesis was observed in monocytes (TNF-alpha(+)) following the provision of antigenic stimuli in vitro. These results support the hypothesis that a strong adaptive response and abnormalities in the innate immune system may be involved in the establishment of YEL-AND and YEL-AVD.