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The focus of current studies was to fabricate dose flexible printlets of dapsone (DDS) for pediatric patients by selective laser sintering (SLS) 3D printing method, and evaluate its physicochemical, patient in-use stability, and pharmacokinetic attributes. Eight formulations were fabricated using Kollicoat ® IR, Eudragit ® L-100-55 and StarCap ® as excipients and evaluated for hardness, disintegration, dissolution, amorphous phase by differential scanning calorimetry and X-ray powder diffraction, in-use stability at 30 o C/75% RH for a month, and pharmacokinetic study in Sprague Dawley rats. The hardness, and disintegration of the printlets varied from 2.6±1.0 (F4) to 7.7±0.9 (F3) N and 2.0±0.4 (F2) to 7.6±0.6 (F3) sec, respectively. The drug was partially present as an amorphous form in the printlets. The drug was completely (>85%) dissolved in 20 min. No change in drug form or dissolution extent was observed after storage at in use condition. Pharmacokinetic profiles of both formulations (tablets and printlets) were almost superimposable with no statistical difference in pharmacokinetic parameters (T max , C max , and AUC 0- ¥ )between formulations (p>0.05). Values of EC 50 (half maximal effective concentration) and EC 90 (maximal concentration inducing 90% maximal response) were 0.50±0.15 and 1.32±0.26 mM, 0.41±0.06 and 1.11±0.21, and 0.42±0.13 and 1.36±0.19 mM for DDS, printlet and tablet formulations, respectively, and differences were statistically insignificant (p>0.05). In conclusion, tablet and printlet formulations are expected to be clinical similar, thus clinically interchangeable., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Improved methods are needed to prevent wildlife deaths from anthrax. Caused by Bacillus anthracis , naturally occurring outbreaks of anthrax are frequent but unpredictable. The commercially available veterinary vaccine is labeled for subcutaneous injection and is impractical for large-scale wildlife vaccination programs; therefore, oral vaccination is the most realistic method to control and prevent these outbreaks. We reported the induction of an anthrax-specific lethal toxin (LeTx) neutralizing antibody response in mice following oral vaccination with alginate microcapsules containing B. anthracis Sterne strain 34F2 spores, coated with poly-L-lysine (PLL) and vitelline protein B (VpB). We continued evaluating our novel vaccine formulation through this proof-of-concept study in white-tailed deer (WTD; Odocoileus virginianus ; n = 9). We orally vaccinated WTD via needle-free syringe with three formulations of the encapsulated vaccine: 1) PLL-VpB-coated microcapsules with 10 7-8 spores/ml ( n = 5), 2) PLL-VpB-coated microcapsules with 10 9-10 spores/ml ( n = 2), and 3) PLL-coated microcapsules with 10 9-10 spores/ml ( n = 2). Although the limited sample sizes require continued experimentation, we observed an anthrax-specific antibody response in WTD serum following oral vaccination with PLL-coated microcapsules containing 10 9 spores/ ml. Furthermore, this antibody response neutralized anthrax LeTx in vitro, suggesting that continued development of this vaccine may allow for realistic wildlife anthrax vaccination programs.

Outbreaks of anthrax, caused by the soilborne bacterium Bacillus anthracis, are a continuous threat to free-ranging livestock and wildlife in enzootic regions of the United States, sometimes causing mass mortalities. Injectable anthrax vaccines are commercially available for use in livestock, and although hand injection is not a cost- or time-effective long-term management plan for prevention in wildlife, it may provide a tool for managers to target selectively animals of high conservation or economic value. Vaccine-induced anthrax-specific antibody responses have been reported previously in white-tailed deer (Odocoileus virginianus), but the protective nature was not determined. In this study, five white-tailed deer were subcutaneously vaccinated with one dose (1 mL) of the Anthrax Spore Vaccine. Eight blood collections by jugular venipuncture were conducted over 146 d to measure the anthrax-specific antibody response in each deer's serum over time. Antibodies were first detected by ELISA and later with toxin neutralization assays to estimate in vitro protection. Average peak absorbance by ELISA occurred at 14 d postvaccination, whereas average peak in vitro protection occurred at 28 d postvaccination. Observed in vitro protection on average for white-tailed deer after this single-dose vaccination protocol lasted 42-56 d postvaccination, although three individuals still maintained lethal toxin-neutralizing serum antibody titers out to 112 d postvaccination. Vaccination responses were variable but effective to some degree in all white-tailed deer., (© Wildlife Disease Association 2024.)

To better understand the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant lineage distribution in a college campus population, we carried out viral genome surveillance over a 7-week period from January to March 2021. Among the sequences were three novel viral variants: BV-1 with a B.1.1.7/20I genetic background and an additional spike mutation Q493R, associated with a mild but longer-than-usual COVID-19 case in a college-age person, BV-2 with a T478K mutation on a 20B genetic background, and BV-3, an apparent recombinant lineage. This work highlights the potential of an undervaccinated younger population as a reservoir for the spread and generation of novel variants. This also demonstrates the value of whole genome sequencing as a routine disease surveillance tool.

Small ruminant brucellosis is caused by the Gram negative cocci-bacillus Brucella (B.) melitensis, the most virulent Brucella species for humans. In goats and sheep, middle to late-term gestation abortion, stillbirths and the delivery of weak infected offspring are the characteristic clinical signs of the disease. Vaccination with the currently available Rev. 1 vaccine is the best option to prevent and control the disease, although it is far from ideal. In this study, we investigate the safety of the B. melitensis 16MΔvjbR strain during a 15-month period beginning at vaccination of young goats, impregnation, delivery and lactation. Forty, 4 to 6 months old, healthy female crossbreed goats were randomly divided into four groups (n = 10) and immunized subcutaneously with a single vaccine dose containing 1x10 9 CFU of B. melitensis 16MΔvjbR delivered in alginate microcapsules or non-encapsulated. Controls received empty capsules or the commercially available Rev.1 vaccine. Seven months post-vaccination, when animals were sexually mature, all goats were naturally bred using brucellosis-free males, and allowed to carry pregnancies to term. Blood samples to assess the humoral immune response were collected throughout the study. At two months post-delivery, all dams and their offspring were euthanized and a necropsy was performed to collect samples for bacteriology and histology. Interestingly, none of the animals that received the vaccine candidate regardless of the formulation exhibited any clinical signs associated with vaccination nor shed the vaccine strain through saliva, vagina or the milk. Gross and histopathologic changes in all nannies and offspring were unremarkable with no evidence of tissue colonization or vertical transmission to fetuses. Altogether, these data demonstrate that vaccination with the mutant strain 16MΔvjbR is safe for use in the non-pregnant primary host., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Allison Rice-Ficht, managing partner of NanoRelease Technologies (NRT), LLC Inc., has a 95% equity interest in NRT, a company involved in vaccine delivery platforms. The terms of this arrangement have been reviewed and approved by Texas A&M Agri-Life Research and Texas A&M University in accordance with their conflict of interest policies]., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log 10 of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis., (© 2020 The Societies and John Wiley & Sons Australia, Ltd.)

An oral vaccine against anthrax ( Bacillus anthracis ) is urgently needed to prevent annual anthrax outbreaks that are causing catastrophic losses in free-ranging livestock and wildlife worldwide. The Sterne vaccine, the current injectable livestock vaccine, is a suspension of live attenuated B. anthracis Sterne strain 34F2 spores (Sterne spores) in saponin. It is not effective when administered orally and individual subcutaneous injections are not a practical method of vaccination for wildlife. In this study, we report the development of a microencapsulated oral vaccine against anthrax. Evaluating Sterne spore stability at varying pH's in vitro revealed that spore exposure to pH 2 results in spore death, confirming that protection from the gastric environment is of main concern when producing an oral vaccine. Therefore, Sterne spores were encapsulated in alginate and coated with a protein shell containing poly-L-lysine (PLL) and vitelline protein B (VpB), a non-immunogenic, proteolysis resistant protein isolated from Fasciola hepatica . Capsule exposure to pH 2 demonstrated enhanced acid gel character suggesting that alginate microcapsules provided the necessary protection for spores to survive the gastric environment. Post vaccination IgG levels in BALBc/J mouse serum samples indicated that encapsulated spores induced anti-anthrax specific responses in both the subcutaneous and the oral vaccination groups. Furthermore, the antibody responses from both vaccination routes were protective against anthrax lethal toxin in vitro, suggesting that further optimization of this vaccine formulation may result in a reliable oral vaccine that will conveniently and effectively prevent anthrax in wildlife populations., Competing Interests: Competing interestsA.C.R-.F. is a managing partner of NanoRelease Technologies LLC (NRT), a San Antonio-based company specializing in vaccine delivery platforms. The terms of this arrangement have been reviewed and approved by TXAgriLife Research and Texas A&M University in accordance with their conflict of interest policies., (© The Author(s) 2020.)

The global distribution of brucellosis and high incidence in certain areas of the world warrant the development of a safer and efficacious vaccine. For the past 10 years, we have focused our attention on the development of a safer, but still highly protective, live attenuated vaccine for human and animal use. We have demonstrated the safety and protective efficacy of the vaccine candidates 16 MΔvjbR and S19ΔvjbR against homologous and heterologous challenge in multiple immunocompetent animal models, including mice and deer. In the present study, we conducted a series of experiments to determine the safety of the vaccine candidates in interferon regulatory factor-1-knockout (IRF-1(-/-)) mice. IRF-1(-/-) mice infected with either wild-type Brucella melitensis 16 M or the vaccine strain Brucella abortus S19 succumb to the disease within the first 3 weeks of infection, which is characterized by a marked granulomatous and neutrophilic inflammatory response that principally targets the spleen and liver. In contrast, IRF-1(-/-) mice inoculated with either the 16 MΔvjbR or S19ΔvjbR vaccine do not show any clinical or major pathological changes associated with vaccination. Additionally, when 16 MΔvjbR- or S19ΔvjbR-vaccinated mice are challenged with wild-type Brucella melitensis 16M, the degree of colonization in multiple organs, along with associated pathological changes, is significantly reduced. These findings not only demonstrate the safety and protective efficacy of the vjbR mutant in an immunocompromised mouse model but also suggest the participation of lesser-known mechanisms in protective immunity against brucellosis.

Brucellosis is a zoonosis of nearly worldwide distribution. Vaccination against this pathogen is an important control strategy to prevent the disease. Currently licensed vaccine strains used in animals are unacceptable for human use due to undesirable side effects and modest protection. Substantial progress has been made during the past 10 years toward the development of improved vaccines for brucellosis. In part, this has been achieved by the identification and characterization of live attenuated mutants that are safer in the host but still can stimulate an adequate immune response. In the present study, the identification and characterization of the mucR mutant (BMEI 1364) as a vaccine candidate for brucellosis was conducted. BALB/c mice were vaccinated intraperitoneally at a dose of 10(5) CFU with the mutant to evaluate safety and protective efficacy against intraperitoneal and aerosol challenge. All animals vaccinated with the vaccine candidate demonstrated a statistically significant degree of protection against both intraperitoneal and aerosol challenge. Safety was revealed by the absence of Brucella associated pathological changes, including splenomegaly, hepatomegaly, or granulomatous disease. These results suggest that the 16MΔmucR vaccine is safe, elicits a strong protective immunity, and should be considered as a promising vaccine candidate for human use.

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.

The tremendous power of the particulate vaccine delivery system has only recently been recognized and employed strategically in vaccine design. The entrapment of antigen in particles clearly alters its acquisition and processing by antigen presenting cells and ensuing adaptive immunity. The adjuvant activity of particles has recently been described at the molecular level as engaging the Nalp3 inflammasome and complementing the activity of toll-like receptor ligands. The inclusion of antigen within erodible particles and subsequent delivery to dendritic cells (DCs), enables antigen-specific cell-mediated immunity and extended antigen presentation with protective outcomes. Particles less than 1 microm in size with amphipathic coatings efficiently deliver antigen to and activate DCs with concomitant engagement of humoral and cellular immunity. The size and dissolution rates of particles as well as surface chemistry and charge appear to be central in tuning adaptive immunity., (Copyright 2010. Published by Elsevier Ltd.)

The successful control of animal brucellosis and associated reduction in human exposure has limited the development of human brucellosis vaccines. However, the potential use of Brucella in bioterrorism or biowarfare suggests that direct intervention strategies are warranted. Although the dominant approach has explored the use of live attenuated vaccines, side effects associated with their use has prevented widespread use in humans. Development of live, attenuated Brucella vaccines that are safe for use in humans has focused on the deletion of important genes required for survival. However, the enhanced safety of deletion mutants is most often associated with reduced efficacy. For this reason recent efforts have sought to combine the optimal features of a attenuated live vaccine that is safe, free of side effects and efficacious in humans with enhanced immune stimulation through microencapsulation. The competitive advantages and innovations of this approach are: (1) use of highly attenuated, safe, gene knockout, live Brucella mutants; (2) manufacturing with unique disposable closed system technologies, and (3) oral/intranasal delivery in a novel microencapsulation-mediated controlled release formula to optimally provide the long term mucosal immunostimulation required for protective immunity. Based upon preliminary data, it is postulated that such vaccine delivery systems can be storage stable, administered orally or intranasally, and generally applicable to a number of agents.

Bison (Bison bison) and elk (Cervus elaphus nelsoni) in the Greater Yellowstone Area (GYA), USA, are infected with Brucella abortus, the causative agent of bovine brucellosis, and they serve as a wildlife reservoir for the disease. Bovine brucellosis recently has been transmitted from infected elk to cattle in Montana, Wyoming, and Idaho and has resulted in their loss of brucellosis-free status. An efficacious Brucella vaccine with a delivery system suitable for wildlife would be a valuable tool in a disease prevention and control program. We evaluated Strain 19 (S19) in a sustained release vehicle consisting of alginate microspheres containing live vaccine. In a challenge study using red deer (Cervus elaphus elaphus) as a model for elk, alginate, a naturally occurring polymer combined with a protein of Fasciola hepatica vitelline protein B was used to microencapsulate S19. Red deer were orally or subcutaneously immunized with 1.5 x 10(10) colony-forming units (CFUs) using microencapsulated S19. Humoral and cellular profiles were analyzed bimonthly throughout the study. The vaccinated red deer and nonvaccinated controls were challenged 1 yr postimmunization conjunctivally with 1 x 10(9) CFUs of B. abortus strain 2308. Red deer vaccinated with oral microencapsulated S19 had a statistically significant lower bacterial tissue load compared with controls. These data indicate for the first time that protection against Brucella-challenge can be achieved by combining a commonly used vaccine with a novel oral delivery system such as alginate-vitelline protein B microencapsulation. This system is a potential improvement for efficacious Brucella-vaccine delivery to wildlife in the GYA.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johne's disease) and a possible etiopathologic agent in human Crohn's disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5-12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric ss-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohn's disease.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. A vjbR knockout was generated in the S19 vaccine and investigated for its potential use as an improved vaccine candidate. Vaccination with a sustained-release vehicle to enhance vaccination efficacy was evaluated utilizing the live S19 DeltavjbR::Kan in encapsulated alginate microspheres containing a nonimmunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B). BALB/c mice were immunized intraperitoneally with either encapsulated or nonencapsulated S19 DeltavjbR::Kan at a dose of 1 x 10(5) CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses postvaccination indicate that the vaccine candidate was able to elicit an anti-Brucella-specific immunoglobulin G response even when the vaccine was administered in an encapsulated format. The safety was revealed by the absence of splenomegaly in mice that were inoculated with the mutant. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the nonencapsulated vaccine. These results suggest that S19 DeltavjbR::Kan is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained-release manner.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. The occurrence of the infection in humans is largely dependent on the prevalence of brucellosis in animal reservoirs, including wildlife. The current vaccine used for cattle Brucella abortus strain RB51, has proven ineffective in protecting bison (Bison bison) and elk (Cervus nelsoni) from infection and abortion. To test possible improvements in vaccine efficacy, a novel approach of immunization was examined from April 2004 to November 2006 using alginate composite microspheres containing a nonimmunogenic, eggshell-precursor protein of the parasite Fasciola hepatica (Vitelline protein B, VpB) to deliver live vaccine strain RB51. Red deer (Cervus elaphus), used as a model for elk, were vaccinated orally (PO) or subcutaneously (SC) with 1.5x10(10) viable organisms per animal. Humoral responses postvaccination (immunoglobulin G [IgG] levels), assessed at different time points, indicated that capsules containing live RB51 elicited an anti-Brucella specific IgG response. Furthermore, the encapsulated vaccine elicited a cell-mediated response that the nonencapsulated vaccinates failed to produce. Finally, red deer were challenged with B. abortus strain 19 by conjunctival exposure. Only animals that received encapsulated RB51 vaccine by either route exhibited a significant reduction in bacterial counts in their spleens. These data suggest that alginate-VpB microspheres provide a method to enhance the RB51 vaccine performance in elk.

The development of safe and efficacious immunization systems to prevent brucellosis is needed to overcome the disadvantages of the currently licensed vaccine strains that restrict their use in humans. Alginate microspheres coated with a protein of the parasite Fasciola hepatica (vitelline protein B [VpB]) and containing live Brucella melitensis attenuated mutant vjbR::Tn5 (BMEII1116) were evaluated for vaccine efficacy and immunogenicity in mice. A single immunization dose in BALB/c mice with the encapsulated vjbR mutant improved protection against wild-type B. melitensis 16M challenge compared to the nonencapsulated vaccine strain (P < 0.05). The encapsulated mutant was also shown to induce a sustained elevation of Immunoglobulin G levels. Cytokine secretion from spleen cells of mice vaccinated with the encapsulated vjbR::Tn5 revealed elevated secretion of gamma interferon and interleukin-12, but no interleukin-4, suggesting an induction of a T helper 1 response reflecting the enhanced immunity associated with microencapsulation. Together, these results suggest that microencapsulation of live attenuated organisms offers the ability to increase the efficacy of vaccine candidates.

The need to tailor release rate profiles from polymeric microspheres is a significant problem. Microsphere size, which has a significant effect on drug release rate, can potentially be varied to design a controlled drug delivery system with desired release profile. In this work the effects of microspheres mean diameter, polydispersity, and polymer degradation on drug release rate from poly(lactide-co-glycolide) (PLG) microspheres are described. Piroxicam containing PLG microspheres were fabricated at 20% loading, and at three different impeller speeds. A portion of the microspheres was then sieved giving five different size distributions. In vitro release kinetics were determined for each preparation. Based on these experimental results, a suitable mathematical theory has been developed that incorporates the effect of microsphere size distribution and polymer degradation on drug release. We show from in vitro release experiments that microsphere size has a significant effect on drug release rate. The initial release rate decreased with an increase in microsphere size. In addition, the release profile changed from first order to concave-upward (sigmoidal) as the microsphere size was increased. The mathematical model gave a good fit to the experimental release data. For highly polydisperse populations (polydispersity parameter b<3), incorporating the microsphere size distribution into the mathematical model gave a better fit to the experimental results than using the representative mean diameter. The validated mathematical model can be used to predict small-molecule drug release from PLG microsphere populations.

A robust, portable chip containing a single protein nanopore would be a significant development in the practical application of stochastic sensing technology. Here, we describe a chip in which a single alpha-hemolysin (alphaHL) pore in a planar phospholipid bilayer is sandwiched between two layers of agarose gel. These encapsulated nanopore chips remain functional after storage for weeks. The detection of the second messenger inositol 1,4,5-trisphosphate (IP3) was demonstrated with a chip containing a genetically engineered alphaHL pore as the sensor element.

Nanotechnology is finding its use as a potential technology in consumer products, defense, electronics, and medical applications by exploiting the properties of nanomaterials. Single-walled carbon nanotubes are novel forms of these nanomaterials with potential for large applications. However, the toxicity studies on this material are not explored in detail and therefore limiting its use. It has been earlier reported that single-walled carbon nanotubes induces oxidative stress and also dictates activation of specific signaling pathway in keratinocytes. The present study explores the effect of single-walled carbon nanotubes on stress genes in human BJ Foreskin cells. The results show induction of oxidative stress in BJ Foreskin cells by single-walled carbon nanotubes and increase in stress responsive genes. The genes included inducible genes like HMOX1, HMOX2, and Cyp1B1. In addition we validated increase for four genes by SWCNT, namely ATM, CCNC, DNAJB4, and GADD45A by RT-PCR. Moreover results of the altered stress related genes have been discussed and that partially explains some of the toxic responses induced by single-walled carbon nanotubes.

Despite the importance of microsphere size for controlled drug delivery, little work has been done to quantitatively predict the distribution of microspheres from manufacturing techniques. This work presents a quantitative study that describes the size distribution of poly(lactide-co-glycolide) (PLG) microspheres. A fluid mechanics based correlation for the mean microsphere diameter is formulated based on the theory of emulsification in turbulent flow under non-coalescing conditions. The correlation was constructed and validated with experimentally obtained mean microsphere diameters prepared at different stirring speeds. In addition, a Rosin Rammler distribution function was found to give an accurate representation of the microsphere distribution. The spread of the microsphere size distribution was found to decrease with stirring speed. With the validation of the mathematical correlation, it is now possible to have a good estimate of the average microsphere size prior to microsphere preparation. This is directly relevant to the pharmaceutical industry where microspheres of specified mean diameter and size distribution are desirable.

Antigen-specific CD4+ T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4+ T cells from immune cattle. Fractions obtained from merozoites separated by continuous-flow electrophoresis (CFE) that contained proteins ranging from 20 to 83 kDa were previously shown to stimulate memory CD4+ lymphocyte responses in B. bovis-immune cattle. Expression library screening with rabbit antiserum raised against an immunostimulatory CFE fraction identified a clone encoding a predicted 78 kDa protein. BLAST analysis revealed sequence identity of this B. bovis protein with Plasmodium falciparum fatty acyl coenzyme A synthetase (ACS) family members (PfACS1-PfACS11), and the protein was designated B. bovis acyl-CoA synthetase 1 (ACS1). Southern blot analysis indicated that B. bovis ACS1 is encoded by a single gene, although BLAST analysis of the preliminary B. bovis genome sequence identified two additional family members, ACS2 and ACS3. Peripheral blood lymphocytes and CD4+ T cell lines from B. bovis-immune cattle proliferated significantly against recombinant ACS1 protein, consistent with its predicted involvement in protective immunity. However, immune sera from cattle recovered from B. bovis infection did not react with ACS1, indicating that epitopes may be conformationally dependent.

Carbon nanotubes are now becoming an important material for use in day to day life because of their unique physical properties. The toxicological impact of these materials has not yet been studied in detail, thereby limiting their use. In the present study, the toxicity of single-walled carbon nanotubes (SWCNT) was assessed in human keratinocyte cells. The results show increased oxidative stress and inhibition of cell proliferation in response to treatment of keratinocytes with SWCNT particles. In addition, the signaling mechanism in keratinocytes upon exposure to SWCNT particles was investigated. Results from the study suggest that SWCNT particles activate NF-kappaB in a dose-dependent manner in human keratinocytes. Further, the mechanism of activation of NF-kappaB was due to the activation of stress-related kinases by SWCNT particles in keratinocytes. In conclusion, these studies show the mechanism of toxicity induced by SWCNT particles.

A fluid mechanics-based correlation for the average size of Bovine Serum Albumin (BSA) microspheres, prepared using a water-in-oil emulsion technique, is presented. The correlation is formulated based on the theory of turbulent dispersion and a non-dimensional Weber number. Measured average diameters of the BSA microspheres prepared in olive oil at different stirring speeds are used to construct the correlation formula. The correlation gives good agreement with experimentally measured average diameters for a wide range of Weber numbers. This correlation is particularly useful to the pharmaceutical industry for predicting the size of encapsulated microspheres used in drug delivery prior to microsphere preparation. The effect of additives on microsphere size was also explored. The average diameter of the BSA microspheres was doubled by the addition of a bioadherent vitelline protein B solution. In addition, a Rosin-Rammler statistical distribution function is shown to accurately represent the microsphere size distribution obtained at different stirring speeds.

Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20 Mycobacterium avium subsp. paratuberculosis field isolates, 1 American Type Culture Collection (ATCC) M. avium subsp. paratuberculosis isolate (ATCC 19698), and 2 M. avium subsp. avium isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among M. avium subsp. paratuberculosis isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of M. avium subsp. paratuberculosis isolates. Four M. avium subsp. paratuberculosis-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the M. avium subsp. paratuberculosis field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the M. avium subsp. avium isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of M. avium subsp. paratuberculosis and M. avium subsp. avium, many of which are based on deletions. Of the M. avium subsp. paratuberculosis-specific sequences studied, one revealed a 5,145-bp region with no homologue in the M. avium subsp. avium genome. Within this region are genes responsible for integrase-recombinase function. Three additional M. avium subsp. paratuberculosis-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.

Using monoclonal antibody (mAb) 70/52.9, generated from a Babesia bovis fraction enriched for spherical body organelles, we have identified a 135-kDa protein containing an epitope conserved in B. bovis strains from Texas, Mexico, and Australia. The protein was localized to the spherical bodies of the babesial apical complex and was designated spherical body protein 3 (SBP3), according to the established nomenclature. Immunofluorescence studies showed binding of the 70/52.9 mAb to the infected-erythrocyte membrane region but not to their uninfected counterparts, demonstrating a host-cell association shared with the previously isolated B. bovis spherical body proteins, SBP1 and SBP2. Using mAb 70/52.9, the full-length cDNA encoding SBP3 was isolated from an expression library, sequenced, and oligonucleotide primers synthesized to amplify the genomic copy by polymerase chain reaction. The genomic copy contained no introns and was identical to the cDNA sequence with each containing a single, large open reading frame encoding a protein of 1089 residues. Analysis of the SBP3 amino acid sequence revealed no significant amino acid identity to SBP1 and SBP2 and a lack of repeated epitopes, a notable feature of the other two spherical body proteins. Labeled probes derived from the coding region of SBP3 hybridized to single fragments on Southern blots containing B. bovis genomic DNA indicating a single copy gene. With the identification of this third spherical body protein, which associates with the cytoplasmic face of the infected-erythrocyte membrane, a complement of distinct B. bovis proteins have been identified that are likely to contribute to intracellular survival, growth, and development for this parasite. The encoded protein should be valuable for functional investigations and evaluation of potential targets for host immunity.

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.

CD4+ helper T cells are believed to be important for inducing protective immunity against Babesia bovis through the production of cytokines, including IFN-gamma, that will provide help to B lymphocytes for IgG production and activate macrophages to become parasiticidal. To provide maximum protection in an outbred population, an effective vaccine against B. bovis should contain antigens that would elicit an IFN-gamma response and would be recognized by cattle with diverse genetic backgrounds. To identify potentially protective "universal" T helper (Th) cell antigens, fractions of homogenized B. bovis merozoites were tested for the ability to stimulate proliferation of oligoclonal CD4+, IFN-gamma-producing T cell lines derived from four immune animals previously shown to differ in major histocompatibility complex class II expression. Homogenized B. bovis merozoites were separated by denaturing continuous flow electrophoresis (CFE) on 15, 10, and 7.5% polyacrylamide gels into fractions containing proteins ranging from <14.5 to approximately 95 kDa. Eighteen of 280 CFE fractions elicited anamnestic proliferative responses in all T cell lines tested. Nine of these cross-stimulatory fractions contained proteins of <14.5 to 24.5 kDa, and the remaining ones contained proteins with estimated molecular weights of 30, 31.5, 44.5, 49, 49.5, 54, 62, 72, and 82 kDa. Immunoblot analysis showed that four cross-stimulatory fractions contained a predicted known B. bovis antigen of similar molecular size. Previous studies had demonstrated that fractionated merozoite proteins stimulatory for CD4+ Th cell clones had apparent molecular weights similar to those present in 7 of the 18 stimulatory fractions. In the present study, two Th cell clones responded to cross-stimulatory CFE fractions, underscoring the potential to use both oligoclonal and monoclonal Th cell lines to identify commonly recognized polypeptides as potential vaccine antigens.

Despite convincing evidence that T cells are critical for both cellular and humoral immunity against haemoprotozoan parasites, the difficulty of performing meaningful experiments in cattle that would define the role of T cells in immunity to Babesia spp. has impeded research in this area. However, experiments performed ex vivo with immune T cells can reflect in-vivo events, and provide valuable insight into the nature of immunogenic proteins and the responding lymphocytes. The progress made towards identification of the immunogenic proteins and epitopes that stimulate anamnestic CD4+ type-1 (interferon-gamma-producing) T-cell responses in cattle immune to challenge with Babesia bovis or B. bigemina is the subject of the present review.

The Th1/Th2 paradigm has provided a useful framework for understanding the observed bias in immune responses that are often dominated by either cell-mediated or humoral responses, and for devising therapeutic strategies to stimulate T cell- or antibody-mediated immunity. However this paradigm is an oversimplification of a much more complex immunoregulatory network. Studies with bovine Th cell clones and immunoregulatory cytokines support this viewpoint. This paper highlights the progress that has been made in defining type 1 and type 2 responses in cattle. Evidence is presented for the presence of different subtypes of antigen-specific Th cell clones of cattle which constitute a spectrum of cell phenotypes, and for cytokine-mediated regulation of Th cell responses that differs from that observed in mice. The majority of over 60 parasite antigen-specific Th cell clones coexpress IL-4 and IFN-gamma, and polarized cytokine profiles were rarely observed. Furthermore, IL-2 and IL-10 expression was not restricted to IFN-gamma or IL-4-producing cells, respectively. This lack of coordinate regulation of "Th1" and "Th2" cytokines strengthens the emerging viewpoint that Th1 and Th2 responses, per se, do not typify the immune response to most pathogens. In addition, we provide evidence that major regulatory cytokines, IL-4, IL-10, and IL-12, do not selectively exert their negative (IL-4 and IL-10) or positive (IL-12) effects on Th1-like cells.

To improve assessment of cellular immune responses in seals, northern elephant seal (Mirounga angustirostris) interleukin 2 (IL-2) has been characterized. The gene was cloned and sequenced from a 658 base pair (bp) cDNA generated from total RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence encoded a 154 amino acid (aa) polypeptide that included a 20 aa putative signal peptide. Seal IL-2 was found to share considerable identity with published sequences. Nucleotide sequence analysis of phocine (seal) IL-2 with canine, feline, human, trichechine (manatee), bovine and murine sequences demonstrated 93, 92, 86, 82, 78 and 71% identity, respectively. Analysis of the derived amino acid sequences demonstrated 88, 89, 78, 71, 66 and 60% identity, respectively. Interleukin-2 sequence identities appear to reflect evolutionary proximity among the analyzed species, and importantly, those residues identified as critical to IL-2 biological activity and receptor binding are largely conserved. To examine the kinetics of IL-2 mRNA expression, northern elephant seal lymphocytes were stimulated with the mitogen concanavalin A (Con A), and RNA was collected at several time points thereafter. The RT-PCR demonstrated that seal IL-2 mRNA expression peaks in the first 8 hr following Con A stimulation. Lastly, genomic DNA from northern elephant seal, harbour seal (Phoca vitulina) and California sea lion (Zalophus californianus) was used as template to identify and clone genomic IL-2. Partial sequence of the genomic clones demonstrated nearly complete identity among the three species. Sequence identity indicates that probes constructed from the northern elephant seal IL-2 gene will be effective in assessing IL-2 in other pinniped species.

The objective of this study was to identify disease-related changes in lymphocyte populations within ileal mucosae of calves with cryptosporidiosis. Groups of five neonatal calves were orally infected at 3 days of age with 10(8) oocysts and maintained in enteric-pathogen-free conditions until clinical disease was established or until the animals had recovered from disease. Age-matched uninfected calves were used for comparison. Ileal mucosal lymphocytes were collected, quantitated, and phenotyped to determine whether changes in lymphocyte composition occurred in infected animals. We observed significantly larger numbers of intraepithelial CD8+ T lymphocytes in ileal mucosae from acutely infected calves compared with those from control animals. In addition, a proportion of intraepithelial CD4+ T cells from acutely infected calves coexpressed CD25, whereas there was an absence of coexpressed CD25 on CD4+ T cells from control calves. Ex vivo reverse transcriptase PCR of RNA from intraepithelial lymphocytes from control calves showed a cytokine expression pattern consisting of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), while intraepithelial lymphocytes from calves with cryptosporidiosis expressed IFN-gamma but not TNF-alpha. Together, the results indicate that changes occur in the ileal intraepithelial lymphocyte population coincidently with Cryptosporidium parvum-induced enteric disease.

The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.

Human recombinant interleukin-10 (IL-10) was previously shown to inhibit accessory cell (AC)-dependent proliferation of bovine parasite-specific T helper 1 (Th1), Th2, and Th0 cells in an IL-2-reversible manner (Brown, W.C., Woods, V.M., Chitko-McKown, C.G., Hash, S.M., and Rice-Ficht, A.C., 1994. Infect. Immun. 62, 4697-4708). The present study was therefore designed to determine whether the effect of IL-10 on T cell proliferation corresponded with downregulated expression of cytokines, or their receptors, important for T cell growth. The effects of IL-10 on cellular proliferation and expression of IL-2, IL-4, IL-2 receptor (IL-2R; p55), and IFN-gamma by Babesia bovis- or Fasciola hepatica-specific Th cell clones were simultaneously evaluated. As shown previously, IL-10 strongly inhibited proliferation of all types of Th cell clones, although this did not correspond with reduced expression of IL-2 or IL-4 mRNA or their products. In contrast, expression of IL-2R mRNA was consistently reduced in the IL-10-treated clones. These results indicate that IL-10 does not inhibit AC-dependent proliferation of bovine Th cells by downregulating T cell cytokines; rather, IL-10 may act by downregulating IL-2R p55 expression and subsequent signal transduction leading to decreased cellular proliferation. IFN-gamma production was also consistently downregulated in the presence of IL-10.

To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from < 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate antigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clones representative of five different antigenic groups. These antisera may be useful for isolating recombinant proteins that are immunogenic for Th cells of immune cattle and therefore potentially useful for vaccine development.

Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of reactivity, and proteins separated by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Studies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasite membranes as well as from the cytosolic fraction of B. bovis merozoites. The Th cell clones reactive with these different hemoparasites expressed either unrestricted or Th1 cytokine profiles, and were generally characterized by the production of high levels of IFN-gamma. A comprehensive study of T cell and macrophage responses to defined parasite antigens will help elucidate the reasons for vaccine failure or success, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.

Murine interleukin-10 (IL-10) is produced by type 2 helper (Th2) cells and selectively inhibits cytokine synthesis by type 1 helper (Th1) cells, whereas human IL-10 is produced by and inhibits proliferation and cytokine synthesis by both Th1 and Th2 subsets. This study reports that bovine IL-10 mRNA is expressed by Th0, Th1, and Th2 clones of bovine T cells specific for either Babesia bovis or Fasciola hepatica but not by two CD8+ T-cell clones. The antigen-induced proliferative responses of all three subsets of CD4+ cells were inhibited by human IL-10, and low levels (10 U/ml) of exogenous human IL-2 restored the suppressed response. However, proliferation of one Th1 clone was never inhibited but was enhanced by IL-10. Human IL-10 also inhibited the expression of gamma interferon and IL-4 mRNA in Th0 clones. In the absence of accessory cells (AC), the responses of Th clones to concanavalin A or IL-2 were not inhibited by IL-10, whereas antigen-specific responses of Th1 and Th2 cells were reduced when IL-10-pretreated macrophages were used as AC. Together, our results with bovine T cells support the concept that IL-10 primarily affects AC function and does not directly inhibit CD4+ T cells and demonstrate that the immunoregulatory effects of IL-10 are not selectively directed at Th1 populations, as they are in mice.

Babesia bovis is an economically important hemoprotozoon parasite o f cattle that is prevalent in many, tropical and subtropical regions o f the world. Effective vaccines against this tick-transmitted parasite consist o f live organisms attenuated by passage through splenectomized calves. However, the nature o f acquired resistance to challenge infection with heterologous isolates of this parasite has not been clearly defined. Unsuccessful attempts to select protective antigens have relied upon the use of antibodies to identify immunodominant proteins. In this review, Wendy Brown and Allison Rice-Ficht discuss the limitations of this approach and the rationale behind using helper T cells to select potential vaccine antigens.

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.

A cDNA encoding bovine interleukin 10 (IL10) was cloned and sequenced using total cellular RNA derived from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). A cDNA was produced with reverse transcriptase using oligo(dT) primers, and was amplified using primers chosen from consensus regions of the mouse and human IL10 genes. The nucleotide sequence derived from this cDNA shares 84, 79 and 78% homology with the human, mouse and rat cDNAs, respectively. The deduced amino-acid sequence shares an overall 77, 71 and 74% homology with the human, mouse and rat IL10 proteins, respectively. Northern blot analysis of the bovine IL10 mRNA reveals expression of a single 1.8-kb transcript, reaching maximal levels between 8 and 24 h, in ConA-stimulated peripheral T-cells, and weak expression in lipopolysaccharide-activated macrophages.

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T-dependent antibody production, and as effector cells acting directly or indirectly on intracellular parasites through the elaboration of cytokines, has prompted us to investigate the bovine cellular immune response against B. bovis antigens. T cell clones generated from four B. bovis-immune cattle by in vitro stimulation with soluble or membrane associated merozoite antigen were characterized for reactivity against various forms of antigen and different geographical isolates of B. bovis and B. bigemina. The clones were categorized into seven different groups based on differential patterns of reactivity. This panel of T cell clones and additional clones specific for either the 77 kDa merozoite apical complex associated protein (Bb-1) or the 42 kDa major merozoite protein (MSA-1) were analyzed for cytokines. Biological assays to measure IL-2/IL-4, IFN-gamma and TNF-alpha/TNF-beta and Northern blot analysis to detect mRNA encoding bovine IL2, IL-4, IFN-gamma, TNF-beta and TNF-alpha revealed the differential production of cytokines by clones with different antigen specificities. Two Bb-1-specific T cell clones produced the Th1 pattern of cytokines: IL-2, IFN-gamma, TNF-beta and TNF-alpha, but not IL-4. Clones specific for the 42 kDa protein produced undetectable levels of all cytokines, but expressed an unrestricted or Th0 pattern of cytokine mRNA: IL-2, IL-4, IFN-gamma and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Helper T cell clones from two Babesia bovis-immune cattle were characterized for use in identification of potentially protective immunogens of B. bovis merozoites. Proliferation assays with 11 CD4+ clones revealed a differential pattern of response to soluble cytosolic antigen, membrane-enriched antigen, detergent extracts of the membrane-enriched antigen, soluble culture supernatant exoantigen, and different geographical isolates of B. bovis as well as Babesia bigemina parasites. When the data were combined, the clones could be grouped according to five different patterns of response. One group recognized only the membrane-enriched fraction of New World and Australian parasites. Four remaining groups recognized antigens found in the cytosolic as well as the membrane-enriched fraction, and clones representative of each group were used to identify cytosolic antigens fractionated by anion-exchange chromatography with the use of fast-performance liquid chromatography. One clone (C97.3C3), which responded to all B. bovis isolates and to B. bigemina, recognized a single peak of activity that eluted with 0.25 M NaCl and contained protein bands of 70 and 75 kDa. The remaining clones were stimulated by a second antigenic peak that eluted between 0.35 and 0.45 M NaCl and contained protein bands of 42, 47, 56, and 84 kDa. The majority of the clones produced interferon, whereas tumor necrosis factor alpha/tumor necrosis factor beta production was less frequent. These studies provide the basis for using helper T cell clones to identify potentially protective immunogens of B. bovis and delineate a minimum of five helper T cell epitopes recognized by two immune cattle.

At least 3 structural protein precursors of the eggshell are synthesized and stockpiled in the extensive vitelline cells of the liver fluke Fasciola hepatica L. One of these, vitelline protein B, consists of a closely related family of proteins that owes its apparent electrophoretic heterogeneity to variations in the Tyr to DOPA conversion as well as to subtle variations in the primary sequence. The efficiency of the Tyr to DOPA conversion ranges from a maximum of about 90% to a minimum of 55% in the protein. Trypsin digestion in borate buffer at pH 8 was used to produce DOPA-peptides for sequencing. Notably, trypsin does not cleave Arg/Lys-DOPA sequences at borate concentrations greater than 0.15 M. Peptides with DOPA-containing sequences most frequently have flanking amino acids such as Lys, Ser, or Asp on the N-terminal side and Gly or Asp on the C-terminal side. All protein variants fall within a narrow molecular weight range (30-33 kDa), a pI range of 6.9 to 8.3, and the collective majority would appear to share a common N-terminal sequence up to residue 28. The results suggest some combination of the following: variations in post-translational hydroxylation, alternative post-transcriptional splicing and/or the existence of multiple gene copies of eggshell precursors. The latter have been shown to occur in the blood fluke Schistosoma mansoni [15].

Antibody raised against the major eggshell protein of Fasciola hepatica (vitelline protein B, vpB) is employed to isolate cDNAs from an expression library and to localize the protein in whole worms. Two cDNAs corresponding to the protein are homologous through the N-terminal and C-terminal coding regions and widely divergent internally. No repeated regions are apparent and no significant sequence homology is seen with chorion proteins of other genera although the amino acid composition closely reflects that of other chorion proteins. Microheterogeneity observed in the vpB is due to the presence of multiple coding sequences, transcripts and a gradient of post-translational modification. Relative transcription of the vpB mRNA throughout the female reproductive tract is demonstrated.

A major portion of a Babesia bovis-specific gene encoding a 77-kDa merozoite protein (Bb-1) produced during natural infection in cattle and in microaerophilous culture was subcloned into the pGEX1N expression vector. Recombinant Bb-1 protein fused to glutathione S-transferase (Bb-1-GST) was used to examine cellular immune responses in B. bovis-immune cattle. Sera from rabbits immunized with Bb-1-GST reacted with fusion protein and with the native antigen present in crude B. bovis but not with B. bigemina merozoites. Bb-1-GST but not GST induced strong proliferation of T lymphocytes from these immune cattle, and Bb-1-reactive T-cell lines which consisted of a mixed population of either CD4+ and CD8+ cells or CD4+, CD8+, and "null" (gamma delta T) cells were established by in vitro stimulation of peripheral blood mononuclear cells with the recombinant fusion protein. Three CD4+ CD8- and three CD4- CD8+ Bb-1-specific T-cell clones were identified after limiting-dilution cloning of the cell lines. The studies described here demonstrate that the 77-kDa protein of B. bovis contains T-cell epitopes capable of eliciting proliferation of two types of T cells in immune cattle, an important consideration for the design of a recombinant subunit vaccine.