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Heterogeneity in exposure to Eimeria spp. of chickens in a flock will result in differences between individual birds in oocyst output and acquired immunity, which subsequently affects transmission of the parasite in the population. The aim of this study was to quantify effects of previous infection of broilers with Eimeria acervulina on immune responses, oocyst output and transmission. A transmission experiment was carried out with pair-wise housed broilers, that differed in infection history. This "infection history" was achieved by establishment of a primary infection by inoculation of birds with 50,000 sporulated E. acervulina oocysts at day 6 of age ("primed"); the other birds did not receive a primary infection ("naïve"). The actual transmission experiment started at day 24 of age: one bird (I) was inoculated with 50,000 sporulated oocysts and was housed together with a non-inoculated contact bird (C). Oocyst excretion and parameters describing transmission, i.e. the number of infected C birds and time passed before start of excretion of C birds, were determined from day 28 to day 50 for six pairs of four different combinations of I and C birds (I-C): naïve-naïve, naïve-primed, primed-naïve and primed-primed. Immune parameters, CD4(+), CD8(+), αβTCR(+) and γδTCR(+) T cells and macrophages in duodenum, were determined in an additional 25 non-primed, non-inoculated control birds, and in the naïve-naïve and naïve-primed groups, each group consisting of 25 pairs. Although the numbers of CD4(+) T cells and γδTCR(+) T cells increased after primary infection, none of the immunological cell types provided an indication of differences in infectivity, susceptibility or transmission between birds. Oocyst output was significantly reduced in primed I and C birds. Transmission was reduced most in the primed-primed group, but nonetheless transmission occurred in all groups. This study also showed that acquired immunity significantly reduced oocyst output after inoculation and contact-infection, but not sufficiently to prevent transmission to contact-exposed birds., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The (T-cell) immune responses of two different broiler lines to a primary Eimeria acervulina infection were investigated. The lines used were a commercial fast-growing broiler line and a slow-growing type of broiler as used in organic farming. Seven-day-old broilers of both lines were infected with 5 x 10(4) oocysts of E. acervulina. The animals were weighed and a species-specific real-time PCR was used to quantify the total amount of parasites in the duodenum. In the fast-growing line, a lower parasite load was seen from day 4 onwards compared to the slow-growing line. In both lines the intestinal peak of Eimeria DNA was observed at day 5 post infection (p.i.). In the duodenum no increase in CD4(+) T-cells was found in both infected lines, but a fast increase in CD8(+) T-cells was observed in the fast-growing line. At day 3 p.i. in the slow-growing broilers an IL-18 mRNA response was observed. At day 4 p.i. strong IFN-gamma and IL-8 mRNA responses were found in both lines. No IL-4 mRNA responses were found in the duodenum. In conclusion, both lines have different growth rates and control and infected conditions. Based on the kinetics of observed phenomena a primary infection with E. acervulina in 7-day-old broilers seems to generate an early CD8alpha(+) response in fast-growing broilers compared to the slow-growing broilers. This difference in immune reaction after an E. acervulina infection could result in a different Eimeria load in the duodenum.

Effects of low-density broiler breeder diets on offspring performance and mortality were studied using 2,100 female and 210 male Cobb 500 breeders. Breeder treatments involved 4 experimental groups and a control group with normal density diets (ND, 2,600 kcal of AME/kg during rearing and 2,800 kcal of AME/kg during laying). In treatment 2, nutrient densities were decreased by 12% (LD12) and 11% (LD11) during the rearing and laying periods, respectively, whereas in treatment 3, nutrient densities were decreased by 23% (LD23) and 21% (LD21) during the rearing and laying periods, respectively. The nutrient density in these treatments was decreased through inclusion of palm kernel meal, wheat bran, wheat gluten feed, and sunflower seed meal in the diets. Treatment 4 included diets with the same nutrient densities as in treatment 2 but included oats and sugar beet pulp (LD12(OP) and LD11(OP)). In treatment 5, the same low-density diet was given to the breeders as in treatment 2 during the rearing period, but it was followed by a normal density diet during the laying period (LD12-ND). Treatments were applied from 4 to 60 wk of age. On low-density diets, offspring showed an increased 1-d-old weight. As compared with offspring of breeders that received ND, the d 38 live weight of chickens from 29-wk-old breeders fed LD11 was improved. Mortality was reduced in offspring from 60-wk-old parent stock given low-density diets. The IgM titers in 35-d-old offspring from eggs with a lower-than-average weight were reduced when 29-wk-old broiler breeders were fed low-density diets. In offspring from eggs with a higher-than-average weight from 60-wk-old parent stock given LD11 or LD21 diets, IgM titers were higher compared with ND. It was concluded that low-density broiler breeder diets can improve offspring growth rates, reduce mortality, and reduce or increase immune responses, depending on breeder age and egg weight.

T-cell responses are supposed to be the major immune reactions in broilers infected with Eimeria. The nature of such T-cell responses is influenced by the species of Eimeria involved, age of the host, amount of parasites and the preceding infection history. In young chicks the intestine is still developing in length while the lymphocyte populations in the gut develop and differentiate. In chicks infected at young age the immune response may be different in quality as compared to responses in adults. We investigated the (T-cell) immune responses of young broilers to a primary Eimeria acervulina infection in relation to the number of parasites used for infection. In our experiment we infected one-day-old broilers with a low (5 x 10(2) oocysts) and a high (5 x 10(4) oocysts) dose of E. acervulina. We used a newly developed species specific real-time PCR to quantify total amount of parasites in the duodenum as the number of oocysts in faeces may not be representative for the exposure of the gut immune system. We characterized T-cell subsets in the duodenum by means of FACS-analyses, lymphocyte proliferation assays with spleen lymphocytes and the mRNA profiles of different cytokines (TGF-beta2, -4, IFN-gamma, IL-2, -6, -8 and -18) in the duodenum by means of real-time PCR. From day 5 p.i. broilers with a high dose of E. acervulina had a significantly lower body weight than the control group. No increase in CD4(+) cells, but a strong increase in CD8(+) cells was observed at days 7 and 9 p.i. in the duodenum of broilers infected with a high dose E. acervulina. IL-8 mRNA responses were observed after infection with low and with high infection doses, but no IFN-gamma and TGF-beta mRNA responses were found in the duodenum. The specific proliferative T-cell responses to a low infectious dose were not significantly different as compared to the control group. In conclusion, based on the kinetics of observed responses a primary infection with a high dose of E. acervulina in one-day-old broilers seems to generate an immune response that shows a peak at the time of oocyst excretion, whereas the immune response to a low dose is less explicit.

DNA vaccines are capable of priming the immune system of neonates in the presence of maternal antibodies. However, it is still not clear whether the extent of priming and protection against challenge infections induced by a DNA vaccine in maternally immune newborns is better than that induced by conventional vaccines. To study this, we used the pseudorabies virus (PRV) infection model in the natural host, the pig. We compared the efficacy of a DNA vaccine with the efficacy of a conventional modified live vaccine (MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets. We measured the priming of the immune response and the degree of protection against challenge infection for all vaccine types. We vaccinated piglets with or without maternal immunity twice, at the age of 5 and 9 weeks, and we assessed protection by challenge infection with virulent PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines induced both humoral and cell-mediated immune responses in maternally immune animals. DNA vaccination seemed not to suffer from suppression by maternal immunity and resulted in similar or stronger immune responses in maternally immune piglets as compared in naïve piglets. In contrast, vaccination with conventional vaccines resulted in weaker immune responses in maternally immune piglets than in naïve piglets. Moreover, DNA vaccination provided better protection against challenge infection in maternally immune piglets than in naive piglets, whereas vaccination with conventional vaccines did not.

1. Based on earlier studies it was hypothesised that there is an immunological basis for the differences in susceptibility to malabsorption syndrome (MAS). A study was conducted to investigate base-line and MAS-induced cytokine levels in the intestine of broilers that differ in MAS susceptibility. 2. The transcription of cytokine mRNA in the intestine was quantified using a real-time polymerase chain reaction (PCR) method. At different time points after disease induction the intestines of broilers were investigated for expression of interleukin (IL)-2, IL-6, IL-8, IL-18 and interferon (IFN)-gamma. Age-matched non-MAS-induced chickens served as controls. 3. Control chickens from a MAS-resistant line had higher concentrations of mRNA for IL-2, IL-6, IL-18 and IFN-gamma in the small intestine while no difference between the lines was found for IL-8. After induction of MAS the relative amounts of IL-2, IL-6, IL-8 and IFN-gamma mRNA increased more in the intestines of the susceptible line than in the gut of the resistant line. 4. We suggest that differences in cytokine mRNA in the base-line situation and in MAS-induced conditions indicate a difference in immune response regulation in the two broiler lines. This difference in response could lead to the difference in susceptibility to MAS.

The capacity of farm animals to produce cytokines could be an important determinant of robustness and health. From research in rodents and humans it appears that the production and the balance of T helper 1 (Th1) and T helper 2 (Th2)-type cytokines influences susceptibility to autoimmune and infectious diseases. It is known that pigs show a large variation in many immune response parameters. So far the extent of individual variation in the production of Th1- and Th2-type cytokines in commercial outbred pigs has not been reported. In the current experiment we determined mRNA expression, as well as protein production of cytokines in 32 pigs from eight litters. From each litter two male and two female pigs were tested at 2, 5 and 8 weeks of age. Two Th1-type cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, and two Th2-type cytokines, IL-4 and IL-10, were measured after phytohaemagglutinin (PHA)-stimulation of blood mononuclear cells. Cytokine production and the Th1/Th2-ratio were highly variable. The variation in cytokine protein production was moderately consistent across ages, i.e. pigs that produced high levels of cytokine at 2 weeks of age tended to do so as well at 5 and 8 weeks of age. Cytokine production tended to increase with age, and gilts and boars differed in their IL-2/IL-4 ratio. Unexpectedly, age, gender and litter effects often differed for mRNA and protein production data. We hypothesize that cytokine production is a consistent trait in pigs, especially at the protein production level. Future investigations in more animals and across a wider age range are necessary.

Fasciola hepatica juveniles express immunodominant cathepsin L proteins, which are mainly found in their immature, procathepsin form. A gene encoding such a procathepsin L (FheCL3) was expressed by a baculovirus recombinant and by Saccharomyces cerevisiae. The glycosylated FheCL3 proteins obtained by both systems were used in a vaccination/challenge experiment in rats. Both antigens evoked similar antibody responses, but only the baculovirus expressed FheCL3 caused a significant protection against the number of liver flukes (52% protection, P=0.01), whereas the S. cerevisiae expressed FheCL3 did not. In a second experiment in rats, deglycosylated versions of both antigens were used, but this did not improve their efficacies.

In this study, 60 pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) from 20 different pig herds and 180 control pigs (without clinical signs of PMWS) were examined to get more insights into the frequencies of porcine circovirus 2 infections and the presence of co-infections in pigs with and without clinical signs of PMWS in the Netherlands. Porcine circovirus type 2 was detected in 100% of the pigs with clinical signs of PMWS by virus isolation and/or PCR and in 50% of the pigs from PMWS-free herds. There was an association between the levels of infectious PCV2 and/or PCV2 DNA load and the severity of clinical signs as described for PMWS. A high variation in PCV2 antibody titres was found in the clinically affected pigs, and 27% of these pigs did not mount PCV2 antibody titres higher than 1:200. A concurrent infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) was found in at least 83% of the pigs with clinical signs of PMWS and in 35% of the pigs from PMWS-free herds. Co-infections of European- and American-type PRRSV were detected only in PMWS herds and in one control herd with a history of PMWS clinical signs.

1. The aim of the experiments was to evaluate whether selected probiotic lactobacillus strains have different immunomodulating effects in layer- and meat-type strain chickens. 2. Humoral and cellular specific and non-specific immune responses were studied by experiments on cellular proliferation, entry and survival of Salmonella bacteria in gut and spleen leukocytes, immunoglobulin isotypes and specific immunoglobulin titres. 3. The effects of two different feeding regimes (short and continuous feeding) and doses for administration of lactobacilli were studied. 4. The lactobacillus strains that were evaluated showed modulating effects on the immune system of layer- and meat-type chickens. 5. In meat-type strain chickens the lactobacilli had a stimulating effect when the chickens were young (up to 3 weeks) and the dose was relatively high, whereas in layer-type chickens a lower effective dose and discontinuous administration was also effective. 6. Immunoprobiotic lactobacilli can have a positive effect on humoral and cellular immune responses in layer- and meat-type strain chickens, but the lactobacillus strain to be used, the age of the animals and effective dose of lactobacilli to be administered need to be optimised.

Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position P1 of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant (P < 0.05) reduction of fluke load compared to adjuvant controls. We conclude that the propeptide of CL3 plays an important role in inducing immunity against F. hepatica infection.

The aim of our study was to evaluate the relative importance of antibody and T cell-mediated immunity in protection against pseudorabies virus (suid herpes virus type 1) infection in pigs. We induced different levels of immune responses by using: (1) a modified live vaccine; (2) the same modified live vaccine with an oil-in-water (o/w) adjuvant; (3) an inactivated vaccine; and (4) the same inactivated vaccine with an o/w adjuvant. Subsequently, we challenged pigs with virulent pseudorabies virus (PRV). We demonstrated that best-protected pigs stood out by maintaining strong T cell-mediated immune (CMI) responses after challenge. Of the immune parameters tested, protection against virus shedding was correlated best with the magnitude of the IFN-gamma response of in vitro re-stimulated peripheral blood mononuclear cells (PBMC) with an additional role for PRV-specific IgG2 antibodies. The use of an o/w adjuvant resulted in higher antibody and CMI responses, in particular with an increased frequency of memory T helper blast cells of in vitro re-stimulated PBMC. However, this adjuvant-induced enhancement of the immune response had a limited additional effect on the efficacy of inactivated vaccines. This study suggests a major contribution of the CMI response in early protection against PRV infection and that PRV-induced IFN-gamma responses may serve as a suitable indicator for assessing the immune status of vaccinated pigs., (Copyright 2004 Elsevier B.V.)

In a case-control study, the role of porcine circovirus 2 (PCV2) and putative co-factors in the development of porcine dermatitis and nephropathy syndrome (PDNS) were investigated. Pigs with and without PDNS were examined for macroscopic lesions and histopathology. In addition, organs and tissues were collected at necropsy and examined for the presence of fibrinous deposits (immune complexes), CD8+ cells, and for the presence of bacterial and viral infections. Results from PDNS cases were compared with those of three control groups comprising pigs without clinical signs of PDNS and selected from; (1) the same compartment as PDNS cases, (2) another compartment but in the same PDNS herd, and (3) a control herd without any history of PDNS or post-weaning multisystemic wasting syndrome. Macroscopic and histopathological lesions found in PDNS cases were comparable to those previously documented for PDNS e.g. skin lesions and renal lesions representing glomerulonephritis associated with fibrinous deposits and to a lesser extent with interstitial nephritis. PCV2 was detected by PCR in 100% of the PDNS cases, mainly in lymph nodes and tonsils, and in 63% of the control pigs from PDNS free herds. Virus isolation did not reveal infectious PCV2 in all cases. In PDNS affected pigs the PCV2 serum antibody titres were consistently extremely high and the mean PCV2 antibody titre in PDNS pigs was significantly higher than the mean PCV2 antibody titres in pigs from all 3 control groups. Immunohistochemical investigation of kidneys from PDNS affected pigs revealed an increased accumulation of IgG1 + IgG2 and IgM, the complement factors C1q and C3, but also an increase of CD8+ cells. The amounts of IgA and the complement factor C5 in kidneys of PDNS pigs were only slightly increased as compared to control pigs. This study demonstrates that PCV2 infections can result in extremely high PCV2 antibody titres and that PCV2 is a candidate as primary agent in the development of PDNS. The causative physiological basis for PDNS may be the excessive levels of PCV2 antibodies.

Veterinarians specializing in pig health care responded to a questionnaire regarding their experiences with vaccinations. The goal of the questionnaire was to gain insight into a) the reasons for vaccination and b) the factors involved in the efficacy of vaccination. The results indicated that vaccinations were typically initiated because of health problems at the farm. Veterinarians worked together with farmers and other concerned parties to initiate vaccinations. Respondents predicted that the number of vaccinations would decrease substantially with optimal farm management, but would increase if farmers were allowed (under strict conditions) to vaccinate their own pigs. The results further indicated that the nature of financial compensation predicted the rate of vaccination. Veterinarians paid according to a fixed 'fee for service' system vaccinated less frequently than did veterinarians who were paid according to an ongoing farm management contract. In conclusion, veterinarians appear to be restricted in their capacity to disseminate their experience and knowledge due to the competing needs of the farming network (animals, farmers, retailers).

Oral administration of immunoprobiotic bacteria may support animal health. Species specificity of such microorganisms requires appropriate selection. An in vitro assay for the selection of immunoprobiotic lactic acid bacteria was developed in chicken. The assay allowed testing of large numbers of individual strains. Immune stimulation in vitro correlated well with the in vivo situation in two experiments and no false negative results occurred. Therefore this assay is an appropriate selection tool for immunomodulating properties of lactic acid bacteria in chicken.

Oral administration of autoantigens is a safe and convenient way to induce peripheral T-cell tolerance in autoimmune diseases like multiple sclerosis (MS). To increase the efficacy of oral tolerance induction and obviate the need for large-scale purification of human myelin proteins, we use genetically modified lactobacilli expressing myelin antigens. A panel of recombinant lactobacilli was constructed producing myelin proteins and peptides, including human and guinea pig myelin basic protein (MBP) and proteolipid protein peptide 139-151 (PLP(139-151)). In this study we examined whether these Lactobacillus recombinants are able to induce oral and intranasal tolerance in an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Lewis rats received soluble cell extracts of Lactobacillus transformants intranasally three times prior to induction of EAE. For the induction of oral tolerance, rats were fed live transformed lactobacilli for 20 days. Ten days after the first oral administration EAE was induced. Intranasal administration of extracts containing guinea pig MBP (gpMBP) or MBP(72-85) significantly inhibited EAE in Lewis rats. Extracts of control transformants did not reduce EAE. Live lactobacilli expressing guinea pig MBP(72-85) fused to the marker enzyme beta-glucuronidase (beta-gluc) were also able to significantly reduce disease when administered orally. In conclusion, these experiments provide proof of principle that lactobacilli expressing myelin antigens reduce EAE after mucosal (intranasal and oral) administration. This novel method of mucosal tolerance induction by mucosal administration of recombinant lactobacilli expressing relevant autoantigens could find applications in autoimmune disease in general, such as multiple sclerosis, rheumatoid arthritis and uveitis.

Lactobacillus strains with probiotic activity are major constituents of numerous common food products. Due to their 'generally regarded as safe'-status (GRAS-status), Lactobacillus strains can also be genetically engineered for use in oral immunotherapeutic applications, such as vaccination and T lymphocyte tolerance induction in autoimmune disease.In the current study, we demonstrate that the growth phase of orally administered individual Lactobacillus strains can differentially affect antigen-specific antibody subclasses IgG1 and IgG2a, which might reflect skewing of systemic activity of T helper cell type 2 (Th2) and T helper cell type 1 (Th1) pathways, respectively. Mice were orally fed different wild type Lactobacillus strains in log phase or stationary phase and immunized intraperitoneally with a T-cell dependent protein antigen. Sera were evaluated for the ratio of antigen-specific IgG1 and IgG2a antibodies. Stationary Lactobacillus murines and Lactobacillus casei cultures, but not two other Lactobacillus strains, evoked significantly higher IgG1/IgG2a ratios than log phase cultures, possibly relating to increased activity of the Th2-pathway. Despite normal variation in antibody responses against TNP-CGG among individual mice, a high correlation was found between the IgG1 and IgG2a responses of mice within experimental groups. This differential antibody response is likely due to growth phase-dependent differences in bacterial cell composition.Since Lactobacillus growth phase dependent skewing of antibody responses possibly reflecting T-cell pathways can inadvertently affect allergic and (auto)-immune responses, the current findings strongly caution against unidimensional views on the oral administration of individual Lactobacillus strains for probiotic or immunotherapeutic purposes, but also suggest additional possibilities for immune modulation.

To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.

An important property of the antiviral immune response is its time-dependent character. Beginning with a few antigen-specific cells upon infection, it evolves to a stage where there is an abundance of antigen-specific cells and antibodies that are needed to clear the pathogen, and ends with circulating antibodies and a population of virus-specific memory cells to protect the animal from reinfection. Short-term effects of stress on the immune system have been investigated extensively, showing that stress acutely changes many aspects of immunity. However, relatively little is known about the consequences of stress for the quality and quantity of long-term immunological memory. In the present study, we have investigated the effect of social stress, applied in mice at Days 1, 2 and 3 after inoculation with a herpes virus, on long-term antibody and memory cytokine responses to the virus. Male mice were subjected to three 5-min confrontations with an aggressive conspecific. Approximately half of the mice was wounded by bites of the aggressor during this stress procedure, and these mice were analyzed separately from nonwounded mice. It appeared that wounded mice showed suppressed protective antibody responses and impaired memory for virus-specific IL-4 and IL-10 production, whereas mice that were not wounded showed intact long-term immune responses and memory. It is concluded that the combination of wounds and the social stress of repeated confrontations is associated with impaired protective immunity as a consequence of suppressed antibody levels and impairment of some aspects of antiviral immunological memory.

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.

Mixing of unfamiliar pigs is common practice in intensive pig husbandry. Since pigs maintain a dominance hierarchy, mixing often leads to vigorous fighting. Apart from the negative impact that fighting has on welfare, there is evidence that the social stress associated with fighting suppresses immune function. In the present experiment, we investigated the impact of mixing on specific long-term immune responses and protection against challenge infection after vaccination with pseudorabies virus (PRV). Specific pathogen-free (SPF) pigs were mixed pairwise with an unfamiliar same-gender conspecific or left undisturbed with a same-gender littermate at 3 days after vaccination with PRV. Half of the pigs were females (gilts) and half were castrated males (barrows). Mixing increased agonistic behavior to the same degree in gilts and barrows. Cortisol concentrations in saliva and catecholamine excretion in urine were increased in mixed pigs, and these effects were independent of dominance status and gender. Subsequently, the effects of mixing, gender, dominance status and interactions between these factors on immune response parameters were studied. The main result was that mixed barrows showed suppressed immune responses after vaccination and increased clinical symptoms after challenge infection compared to control barrows. Mixed gilts however did not differ from control gilts. It also appeared that mixed dominants were more seriously affected than mixed subordinates were. We conclude that, in some pigs, social stress after mixing suppresses the immune response to a viral vaccine and consequently impairs protection against challenge infection.

The efficacy of edible vaccines produced in potato tubers was examined in mice. Transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation. The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B). A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum. Introduction of this gene under control of the tuber-specific patatin promoter in potato plants resulted in the production of functional, i.e. Gm1-binding, recLT-B pentamers in tubers. Selected tubers containing about 13 microg of recLT-B per gram fresh weight were used for immunisation. Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B. The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice. Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant. Naive and primed mice were fed 5 g of tubers ( approximately 65 microg of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract ( approximately 2 microg of recLT-B). In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres. In primed animals, however, oral immunisation resulted in significant anti-LT IgA antibody responses in serum and faeces. Intragastric intubation of tuber extract revealed higher responses than feeding of tubers. These results indicate clearly that functional recLT-B can be produced in potato tubers, that this recombinant protein is immunogenic and that oral administration thereof elicits both systemic and local IgA responses in parentally primed, but not naive, animals.

Different Lactobacillus strains are frequently used in consumer food products. In addition, recombinant lactobacilli which contain novel expression vectors can now be used in immunotherapeutic applications such as oral vaccination strategies and in T cell tolerance induction approaches for autoimmune disease. Both for food and clinical applications of lactobacilli, proper selection of wild type strains is crucial. For that purpose, eight different common Lactobacillus strains were analysed with respect to mucosal induction of pro- and anti-inflammatory cytokines, IgA-producing plasma cells in the gut, as well as systemic antibody responses against a parenterally administered antigen. Immunohistochemical analysis of cytokine-producing cells in the gut villi showed no significant induction of the cytokines IL-1alpha, IFN-gamma, IL-4 or IL-10 after oral administration of wild type Lactobacillus strains. In contrast, oral administration of L. reuteri and L. brevis induced expression of the proinflammatory/Th1 cytokines TNF-alpha, IL-2 and/or IL-1beta. Oral administration of these two strains and L. fermentum also significantly enhanced the IgG response against parenterally administered haptenated chicken gamma globulin (TNP-CGG). The five other strains did not show this adjuvanticity. L. reuteri induced relatively high levels of IgG2a compared to L. murines, a nonadjuving Lactobacillus strain. These findings imply that different Lactobacillus strains induce distinct mucosal cytokine profiles and possess differential intrinsic adjuvanticity. This suggests that rational Lactobacillus strain selection provides a strategy to influence cytokine expression and thereby influence immune responses.

We analysed the development of the natural immunoglobulin-secreting cell (Ig-SC) response in systemic- and mucosal-lymphoid tissues of specified pathogen free pigs between 1 and 40 weeks of age. As antigen exposure may influence the development of the Ig-SC repertoire we also compared the frequencies of Ig-SC in various lymphoid tissues of 40 weeks old specified pathogen free pigs and conventional pigs. A procedure to isolate lamina propria cells from porcine intestine was adapted for this study. The frequencies of IgM-, IgG-, and IgA-secreting (spot forming) cells were determined with a reversed enzyme linked immunospot assay, which was also adapted for detection of Ig-SC in pigs. The Ig-SC frequencies were calculated as percentage of the mononuclear leukocytes isolated from the various organs. The observations till 40 weeks of age were as follows: Splenic IgM-SC predominated at all ages and reached a plateau of 0.1-0.2% of the mononuclear leukocytes already at 4 weeks of age. The IgM-SC of mesenteric lymph node (MLN) predominated up till 12 weeks of age and reached an optimum of 0.15% reached at 4 weeks of age. The frequencies of IgG-SC of spleen and MLN had dips around 4 weeks of age and increased thereafter till 40 weeks of age (spleen 0.025%, MLN 0.05% at 40 weeks of age). The frequencies of IgA-SC were low in the spleen (< or =0.003%) and moderate in the MLN (0.01-0.02%) at all ages tested. In peripheral lymph node (PLN) and bone marrow (BM), the frequencies of IgM-SC (0.03-0.05%) were much lower than in the spleen. The IgG-SC frequencies of BM and MLN also had dips around 4 weeks of age and increased thereafter. The IgG-SC frequency of BM reached a plateau at 12 weeks of age (0.15%) and for PLN the highest frequency was observed at 40 weeks of age (0.05%). The frequencies of IgA-SC were low in BM and PLN (<0.003%). High frequencies of IgA-SC were observed in mucosa associated tissue like Peyer's patches (PP) and intestinal lamina propria (till 20% of the mononuclear leukocytes in intestinal lamina propria of 12-40 weeks of age). IgM and IgA are both important isotypes in mucosal lymphoid organs in the pig. The shift from IgM to IgAas predominant, mucosal isotype was first observed in duodenum and jejunum (12 weeks) and later in ileum (40 weeks). The influence of ageing on the frequency of Ig-SC in PP was only observed in jejunal PP. whereas in ileal PP the frequencies of Ig-SC did not vary over time. We combined our data about the frequencies of IgM-, IgG-, and IgA-SC in various organs with data obtained by others about the distribution of lymphocytes over porcine lymphoid organs at about 12 weeks of age. Based on these calculations we concluded that the small intestine, with more than 80% of all Ig-SC, is fair most the major site of Ig production in the pig. We also concluded that the small intestine is the major site of IgA and IgM production cells in the pig. Although IgA becomes predominant along the intestine, the results demonstrated that in the pig IgM is more a mucosal isotype compared with other species. With 40% of all IgG-SC the porcine BM appeared to be the major site of IgG production. Unexpected results were obtained for IgG-SC in the systemic lymphoid organs. In these organs the frequencies of IgG-SC dropped firstly from 1 to 4 weeks of age and steadily increased thereafter till 40 weeks of age. This observation is discussed in relation to the possibility that systemic IgG-SC at one week of age were passively acquired from maternal colostrum. The influence of housing/antigenic load at 40 weeks of age was mainly expressed by an increase (2-8x) of the frequency of IgG-SC in spleen, PLN, BM, and intestinal lamina propria, whereas the typical mucosal IgA-SC frequencies in the lamina propria were hardly affected.

Lactobacillus strains possess properties that make them attractive candidates as vehicles for oral administration of therapeutics. In this report we describe the construction and analysis of recombinant Lactobacillus casei applicable in oral vaccination against an infectious disease (tetanus) and in oral tolerance induction for intervention in an autoimmune disease, multiple sclerosis. Recombinant L. casei which express surface-anchored tetanus toxin fragment C (TTFC) were generated. Quantitative analysis by flow cytometry demonstrated a high level of cell wall-bound expression of TTFC and immunogenicity was demonstrated by parenteral immunization with whole cell extracts of the recombinants. A series of expression vectors was constructed to secrete human myelin basic protein (hMBP) or hMBP as a fusion protein with beta-glucuronidase from Escherichia coli. These heterologous products produced by L. casei were detected in the growth medium and parenteral immunization with this medium evoked antibodies against hMBP, confirming that secretion indeed had occurred. Based on the different localization of the heterologous proteins, lactobacilli expressing surface-anchored TTFC are ideally suited for the induction of antibody responses, whereas lactobacilli that secrete myelin proteins can be used for the induction of peripheral T-cell tolerance. In conclusion, the specific technology described here allows the construction of a wide array of safe live recombinant lactobacilli which may prove to be useful in oral intervention strategies for the prevention of infectious diseases or treatment of autoimmune diseases.

To determine the influence of ageing per se as well as of priming histories on the antibody response to influenza vaccination, haemagglutination inhibition (HI), ELISA IgG, IgA, IgM and neutralizing antibody titres were studied in 43 healthy young subjects (mean age 23 years) and 55 healthy elderly people (mean age 79 years). The HI and ELISA lgG responses to the A/Guizhou/54/89 strain (H3N2) for which both the young and the elderly had similar priming histories were equal. By contrast, the HI and IgG responses to A/Taiwan/1/86 (H1N1), where the priming histories were different, were lower in the elderly (P < 0.05). Influenza-specific IgA responses in the elderly tended to be higher for all vaccine strains. Influenza-specific postvaccination IgM titres were similar or tended to be higher in the elderly. A subgroup of elderly subjects (18%) who did not express HI activity to the A/Taiwan/1/86 (H1N1) vaccine strain, reacted in the HI assay with the closely related A/Singapore/6/86 (H1N1) strain. These elderly people, however, produced lgG antibodies which neutralized A/Taiwan/1/86 virus in vitro. It is concluded that the elderly are capable of mounting antibody responses similar to those observed in the young. Moreover, the observed age-related differences in antibody responses to H1N1 strains are probably not due to ageing of the immune system itself, but are determined by differences in priming histories.

In mice, strain dependent cytokine production profiles are induced after oral administration of Lactobacillus. Such a cytokine profile seems to determine the direction and efficacy of the humoral response. In SJL mice lactobacilli are able to enhance or inhibit the development of disease after induction of experimental autoimmune encephalomyelitis (EAE). Immuno-histochemical analysis of cytokine profiles showed that differential modulation is obtained dependent on the Lactobacillus strain applied. Serum antibody responses to i.p. immunisation with chicken gamma globulin in BALB/c mice are also modulated by oral application of Lactobacillus. Lactobacilli are now being developed as safe live antigen carriers for application in vaccine technology, but also for the excretion of autoantigens in order to induce tolerance. The findings of this study imply that by proper strain selection the direction of the response can be influenced by the induction of a specific cytokine profile.

Maternal immunity was shown to be an effector mechanism which does not include transfer of memory. 'Boosting' of maternal immunity by vaccination was not effective. Transferred maternal immunity negatively interfered with the induction of optimal protection by vaccination. Antibody formation was not observed after vaccination of maternally immune piglets. In contrast, induction of memory had occurred in animals under maternal immune suppression. Vaccination in young animals negatively interfered with or abrogated, effective maternal immune protection. There was no correlation between specific serum antibody titres in piglets and protection to PRV. Thus apart from protection provided by antibodies contributions of other soluble factors and the cellular immune compartment as represented in colostrum and/or milk were important for protection.

The two major hydrophilic regions of the hepatitis B virus surface antigen (HBsAg) have been expressed in the outer mannoprotein layer of the cell wall of "Bakers Yeast", Saccharomyces cerevisiae, by fusing them between the yeast invertase signal sequence and the yeast alpha-agglutinin carboxyterminal cell wall anchoring sequence. The fusion protein contained most of the preS sequences, including the hepatocyte receptor, and part of the S sequence including the "a" determinant, and was expressed from multiple genomic copies (MIRY) using the constitutive PCK promoter. Immunofluorescence studies showed that the fusion protein was detectable at the cell surface and was stably expressed at a relatively high level. Intraperitoneal immunization of mice revealed a very weak response against the S region, and a high response against yeast itself. It is proposed that increasing the amount of the antigen and reducing the number of native cell wall proteins, might lead to a yeast that is usable as a safe and cheap live oral vaccine.

We investigated the role of CD40-CD40 ligand (CD40L) interactions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Activated helper T cells expressing CD40L (gp39) surface protein were found in MS patient brain sections, but not in brain tissue sections of normal controls or patients with other neurological disease. CD40L-positive cells were co-localized with CD40-bearing cells in active lesions (perivascular infiltrates). Most of these CD40-bearing cells proved to be of the monocytic lineage (macrophages or microglial cells), and relatively few were B cells. To functionally evaluate CD40-CD40L interactions, EAE was elicited in mice by means of proteolipid-peptide immunization. Treatment with anti-CD40L monoclonal antibody completely prevented the development of disease. Furthermore, administration of anti-CD40L monoclonal antibody, even after disease onset, shortly before maximum disability score was reached led to dramatic disease reduction. The presence of helper T cells expressing CD40L in brain tissue of MS patients and EAE animals, together with the functional evidence provided by successful experimental prevention and therapy in an animal model, indicates that blockade of CD40-CD40L-mediated cellular interactions may be a method for interference in active MS.

Oral administration of lactobacilli evokes mucosal and systemic immune responses against epitopes associated with these organisms (Gerritse et al., 1990, 1991). The adjuvant function of different Lactobacillus species was investigated under the conditions of intraperitoneal (i.p.) injection or oral administration. After i.p. injection of trinitrophenylated chicken gamma-globulin, high DTH responses were observed with Lactobacillus casei and Lactobacillus plantarum, but low responses with Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus. In different experimental model systems L. casei and L. plantarum consistently showed significant adjuvanticity. A series of expression and expression-secretion vectors containing the strong constitutive promoter of the L. casei L-ldh gene or the regulatable promoter of the Lactobacillus amylovorus amy gene (Pouwels and Leer, 1995) was used for the intracellular, extracellular and surface-bound expression of an influenza virus antigenic determinant fused to Escherichia coli beta-glucuronidase. Intracellular expression of the fusion protein amounted to 1-2% of total soluble protein. Lactobacilli synthesizing the fusion protein intracellularly evoked an oral immune response after subcutaneous priming.

The localization and intensity of androgen receptor immunostaining was studied in the testes of 37 subfertile men with oligozoospermia and normal serum gonadotropin levels using a polyclonal antibody raised against a synthetic peptide corresponding to the first 20 N-terminal amino acid residues of the androgen receptor (AR). Furthermore, we investigated whether or not the immunoexpression of the AR in human Sertoli cells, in histologically normal testis tissue, is dependent on the stage of the spermatogenic cycle, as has been found in the rat. In the human testis, AR immunoexpression was observed in Sertoli cells, peritubular myoid cells, Leydig cells, and periarteriolar cells, but not in germinal cells. We found no evidence for a stage-dependent immunoexpression of AR in Sertoli cells. The intensity of AR immunoexpression varied substantially between biopsy specimens of different patients. There was, however, no correlation of the intensity of AR immunoexpression in either Sertoli cells or peritubular myoid cells with spermatogenic adequacy as measured by the method of Johnsen. When, in this study, the intensity of peritubular myoid cell staining was used as a standard to evaluate the intensity of Sertoli cell staining, no correlation was detected as well. Furthermore, serum gonadotropin levels were not correlated with AR immunoexpression levels in Sertoli cells and peritubular myoid cells. These results indicate that immunodetectability of the AR is not related to the condition of the spermatogenic epithelium in patients with oligozoospermia. Inappropriate expression of the AR is neither a cause nor a consequence of idiopathic infertility in the present group of patients.

Local cytokine profiles in skin biopsies from allergic and irritant patch test reactions were determined by in vivo immunohistochemistry to differentiate between these 2 clinically identical afflictions especially at the same time of final reading in diagnostic patch testing. Biopsies were taken from established allergic persons after specific allergic patch tests to epoxy resin (1%), and formaldehyde (1%) and from non-allergic individuals with irritant patch tests to sodium lauryl sulfate (10%) and formaldehyde (8%). At 72 h after application of the agents, significantly enhanced frequencies of dermal infiltrating cells, producing IL-alpha, IL-2, and IFN-gamma per 100 infiltrating cells in the dermis, were observed in allergic as well as irritant test patch reactions, as compared to normal skin. Significantly higher frequencies of IL-1alpha producing cells were observed in biopsies from epoxy resin (1%) allergen-affected and formaldehyde (8%) irritant affected skin. The allergic and irritant patch test reactions showed similar levels of expression of the Th1 cytokines IL-2 and IFN-gamma in the dermis, confirmed by probe based detection of IL-2 mRNA and IFN-gamma mRNA. In conclusion, the described similarity shows that allergens and irritants can induce the same profile of IL-1alpha, TNF-alpha, IL-2, and IFN-gamma production, resulting in the near impossibility of discriminating between allergic and irritant contact dermatitis at the time of patch test reading.

Fluorescent contact chemical allergens provoke sensitization after application on both syngeneic and allogeneic skin grafts in mice. We attempted to determine whether the functional activity in a contact sensitization response of human skin graft was affected at the level of antigen uptake and migration. After xenogeneic skin transplantation, we examined the effect of topical exposure of the graft to rhodamine B isothiocyanate (RITC). This paper describes the migration of RITC-carrying cells and human major histocompatibility complex (MHC) class II DR (HLA-DR)+ cells, from the graft to mouse draining lymph nodes. As demonstrated by immunohistochemistry, grafting resulted in a time-dependent decrease of human HLA-DR+ and CD1a+ cells, and an increase of mouse MHC class II (Ia)+ cells within the graft. Application of RITC on a 3-week-old human skin graft showed optimal migration capability compared to 6- or 9-week-old grafts. In addition, the time-dependent increase of frequencies of RITC+ and HLA-DR+ cells in the draining lymph nodes, and the time-dependent decrease of HLA-DR+ cells in the 3-week-old human skin graft, were concurrent. Supporting these data, human cytokine interleukin-1 alpha (IL-1 alpha), IL-1 beta and tumour necrosis factor-alpha (TNF-alpha), analysis in situ revealed that cytokine production by keratinocytes, a property associated with dendritic cell migration, was preserved in the human skin graft. Thus, like dendritic cells in contact sensitization in allografted skin, dendritic cells from human xenografted skin onto nude mice are capable of migration to mouse draining lymph nodes after allergen application. Induction of contact hypersensitivity is possible in a human skin graft onto nude mice model, although the use of this ex vivo model to analyze contact sensitivity is probably limited to 3 weeks after transplantation.

Distribution and regulation of androgen receptor expression during fetal and neonatal virilization of the rat fetus was assessed by immunohistochemistry. In mesonephric duct derivatives the androgen receptor expression became evident first in the efferent ductules and epididymis (on fetal day 14), subsequently in the vas deferens and finally in the seminal vesicle. Mesenchymal cells of the urogenital tubercle were positive for androgen receptors from fetal day 14 onwards. In the mesenchymal cells of the prostate anlagen, androgen receptor positive cells were found first on fetal day 16. Administration of 5alpha-dihydrotestosterone to pregnant rats from day 11 to day 20 of gestation caused a stabilization of the wolffian duct in female fetuses. The androgen receptor expression pattern became similar as found in mail fetuses, and showed an increase in density and in frequency of androgen receptor positive cells. Administration of the androgen antagonist flutamide during the same interval caused a reduction in density and frequency of androgen receptor positive cells in male fetuses. These findings indicate that androgens enhance the expression of androgen receptors in the developing rat genital tract by induction of androgen receptor positive cells, and by increasing the frequency. The developmental pattern of androgen receptor expression in the rat mesonephric duct system reflects the androgen-responsiveness of the ducts, and is consistent with induction of the androgen receptor along the ducts by testosterone reaching these structures in an exocrine fashion.

T-B cell interactions have a central role in the development of humoral immunity. The binding of a 39 kDa protein (gp39), selectively expressed on activated Th cells, to its receptor CD40, on B cells, results in the initial B cell activation. Thereafter, Th cell derived cytokines regulate the differentiation of B cells into antibody-forming cells. Most of these data are derived from in vitro experiments. This article discusses in vivo experiments dealing with T-B interactions. First, the immunohistochemical analysis of T cell activation (gp39 expression), cytokine and antibody production in murine spleens after injection of model antigens (TNP-Ficoll, TNP-KLH, and rabbit anti-IgD antibodies). Second, the in vivo role of gp39 and cytokines in these immune responses. Finally, by combining these in vivo experiments with in vitro data we present an in vivo model for T cell dependent antibody responses.

We describe the immunohistochemical detection of the human androgen receptor (AR) in routinely processed, paraffin-embedded tissue with the monoclonal antibody (MAb) F39.4. Deparaffinized sections were heated in a microwave oven for antigen retrieval. A panel of human male- and female-derived tissues was investigated. We observed a nuclear staining pattern consistent with previous results on frozen sections. Moreover, we studied the possibility of detecting AR in prolonged formalin-fixed tissue and in paraffin-embedded archival material. After prolonged fixation times or long-term storage of paraffin-embedded tissue, the staining intensity for the AR did not deteriorate. Blocking experiments with the specific synthetic peptides demonstrated the specificity of this technique. We conclude that this method is specific, allows retrospective AR studies, and offers optimally preserved morphology.

A generally applicable method was developed for the rapid and quantitative detection of both toxin- and virus neutralizing antibodies. The method was optimized for three different biological agents, i.e., Shigella toxin, influenza viruses (A/Beying, A/Taiwan and B/Yamagata) and Chikungunya virus. The in vitro micro-cytotoxicity tests developed for the detection and quantitation of neutralizing antibodies are based on the inhibition of the virus- or toxin-induced cytotoxic effect by antibodies. As a result of the cytotoxicity, infected cells are no longer attached to the solid phase and can be easily removed. Thereafter, the proteins of the remaining living cells are stained. After removing the excess dye, the remaining dye is dissolved and the absorbance values are measured. The neutralization titers are determined from the absorbance values. Since the tests are performed in wells of microtiter plates, the in vitro micro-cytotoxicity tests are less laborious and consume less reagent in comparison with classical neutralization tests.

T-B cell interactions have a central role in the development of antibody responses. Upon activation, T helper (Th) cells express the ligand for CD40, gp39, which is essential for Th cell-dependent B cell activation. The cytokines produced by activated Th cells have a regulatory role in B cell differentiation. In this study, we investigated, using immunohistochemical techniques, the in vivo time course and localization of gp39 expression and cytokine production in relation to the specific antibody production. Both the immunization with keyhole limpet hemocyanin (KLH), a thymus-dependent (TD) antigen, and trinitrophenyl (TNP)-Ficoll, a thymus-independent type 2 (TI-2) antigen, induced Th cells to express gp39. The expression of gp39 was restricted to Th cells in the outer periarteriolar lymphocyte sheaths (outer-PALS) and around the terminal arterioles (TA). Incidentally, gp39+ Th cells were found in the corona of follicles, whereas gp39+ cells were never found in the germinal centers or marginal zones of the spleen. Maximum frequencies of gp39+ cells were observed 3 and 4 d after primary and secondary immunization with KLH. After injection of TNP-Ficoll, a marked increase in gp39+ cells was observed, confirming previous observations that activated T cells are involved in TI-2 antibody responses. Analysis of the in vivo cytokine production revealed that interleukin 2 (IL-2)-, IL-4- and interferon gamma (IFN-gamma)-producing cells (IFN-gamma-PC) developed according to similar kinetics as observed for gp39+ cells. IL-2-PC and IL-4-PC were present in higher frequencies as were IFN-gamma-PC in the immune response against TNP-KLH. Double staining experiments revealed gp39+ Th cells producing IL-2, IL-4, or IFN-gamma, suggesting that these cells were involved in both the initial activation as well as the differentiation process of B cells into antibody-forming cells. Dual immunohistochemical analysis revealed gp39+ T cells and cytokine-PC in close proximity to antigen-specific, antibody-forming B cells. In conclusion, this study shows that in vivo gp39 is expressed on activated Th cells after immunization with TD and TI-2 antigens. Furthermore, the time course and compartmentalization of gp39+ expression, cytokine production and antibody formation after immunization suggest that cognate T-B cell interactions and T cell-regulated B cell differentiation occur in the outer-PALS and around the TA of the spleen.

In vitro experiments have documented the role of cytokines in the regulation of the human humoral immune response. Which cytokines are operative in vivo and in which lymphoid compartment interactions between cytokine-producing T cells and antibody-forming B cells occur is still unclear. For that reason we studied human tonsils using immunohistochemical techniques. In tissue sections from tonsils in a resting stage after recurrent tonsillitis we observed cells producing IL-1 alpha and tumour necrosis factor-alpha (TNF-alpha) which were exclusively localized in the mantle zone of the follicle and in the extrafollicular area. Furthermore, a high frequency of interferon-gamma (IFN-gamma)-producing cells was detected in the extrafollicular area, but not inside the follicles. Occasional IL-2- and IL-4-producing cells were found in the extrafollicular area. Immunohistochemical detection of antibody isotypes revealed that B cells, IgM-membrane-positive, were localized inside the follicles and mantle zones, whereas IgD-membrane-positive cells were mainly found in the mantle zones of secondary follicles. In contrast, plasma cells producing IgG1-4 and IgA1-2 were found in the extrafollicular area. No IgD and IgE antibody-forming cells were detected in tonsils, whereas IgM antibody-forming cells were detected in the extrafollicular area. The co-localization of cytokine-producing cells and antibody-forming cells in human tonsil suggests that T-B cell interactions, required for B cell differentiation and isotype switching, take place in the extrafollicular area.

The third variable domain (V3 domain) of HIV-1 gp120 is involved in virus neutralization by antibody, in determination of cell tropism, and in syncytium-inducing/non-syncytium-inducing capacity. Antibodies are highly specific tools to delineate the role of different V3 amino acid sequences in these processes, and to dissect events occurring during synthesis of gp120/160, gp120-CD4 interaction, cellular infection, and syncytium formation. We describe here an IgG1 murine monoclonal antibody (MAb), coded IIIB-V3-01, that was raised with a synthetic peptide (FVTIGKIGNMRQAHC) derived from the carboxy-terminal flank of the HIV-1 IIIB V3 domain. The binding site of this antibody was mapped to the sequence IGKIGNMRQ, using Pepscan analysis. In ELISA, this antibody binds to E. coli-derived gp120 from HIV-1 IIIB, which is denatured and not glycosylated. The antibody showed no neutralizing activity against HIV-1 IIIB, MN, SF2, or RF in a virus neutralization assay and in a syncytium formation inhibition assay. In addition, this antibody did not react with gp120 expressed on the surface of IIIB-infected MOLT-3 cells in FACS analysis. To assess whether the epitope defined by MAb IIIB-V3-01 is hidden on native gp120, reactivity of the antibody with SDS-DTT-denatured or DTT-denatured glycosylated gp120 (CHO cell produced) was tested. Both these treatments exposed the epitope for binding. From these data we conclude that the epitope defined by MAB IIIB-V3-01 is hidden on glycosylated recombinant gp120, and is not accessible on gp120 expressed on the membrane of HIV-1, IIIB-infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)