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Biomedical subjects

T G Kimman

Publications and source records attributed to T G Kimman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-PCR assay.

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5' noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

Base Sequence↗
PubMed1998

Time course of the porcine cellular and humoral immune responses in vivo against pseudorabies virus after inoculation and challenge: significance of in vitro antigenic restimulation.

We investigated the time course of porcine cellular and humoral immune responses against pseudorabies virus (PRV) after pigs were inoculated with PRV gE(-) mutant strain M141 and challenged with wild-type virus NIA-3. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples; half were used directly and half were restimulated with PRV in vitro before use in a cytolytic assay. We determined time course and extent of PRV-specific lymphoproliferative and cytolytic response. In addition, serum samples were examined for neutralizing antibodies. After inoculation, the frequency of various lymphocyte subsets in peripheral blood was determined by FACScan. One week after inoculation, T-lymphocytes proliferated abundantly and a B-lymphocyte response was observed. When PBMC were used directly without restimulation, only 15% of the PRV-infected target cells were lysed, and about 15-20% of uninfected target cells were lysed. In contrast, when PBMC were restimulated with PRV, up to 50% of the PRV-infected target cells were lysed while only 30% of the uninfected target cells were lysed. The frequency of various T-lymphocyte subsets in the circulation did not change significantly after inoculation, which indicates that the number of PRV-specific lymphocytes in circulation was very small. After challenge, the T-lymphocyte response was enhanced, but the B-lymphocyte response was not. When PBMC were used directly, only 20% of the PRV-infected and uninfected target cells were lysed after challenge. In contrast, when PBMC were restimulated with PRV, they again lysed more PRV-infected target cells than uninfected target cells. Cytolytic cells were detected for a longer period after challenge than after inoculation. Since it was only possible to clearly detect cytolysis after lymphocytes were restimulated with PRV, it may be that they do not preferentially localize in blood or that they are too few in blood to be detected without further antigenic restimulation in vitro. These lymphocytes may instead localize in other tissues, such as mucosal tissues, tonsils and draining lymph nodes. Whether such a reservoir of PRV-specific cytolytic cells is important in clearing the virus is still unknown. In this study we demonstrated PRV-specific lymphocytes in circulation after they were restimulated in vitro with PRV.

Animals↗
PubMed1998

A mouse model to study immunity against pseudorabies virus infection: significance of CD4+ and CD8+ cells in protective immunity.

In this study we firstly established a vaccination/challenge model to study pseudorabies virus infection in mice. The mouse model was used to investigate the significance of CD4+ and CD8+ cells and of IFN gamma production in protective immunity. Functional depletion of CD4+ and CD8+ and IFN gamma was obtained in vivo by intraperitoneal injection of alginate-encapsulated anti-CD4, -CD8 or -IFN gamma producing hybridoma's before and at the moment of vaccination. The observed protective immunity was correlated with underlying immunologic responses such as PRV-specific DTH reactivity, lymphoproliferation and cytotoxicity. The significance of CD4+ and CD8+ cells and of IFN gamma production was also investigated for these immunological responses by the same in vivo depletion technique. The results demonstrated that protective vaccination of mice, that could be induced by immunization with 10(7) plaque forming units of the avirulent PRV mutant NIA3 TK-, was characterized by a typical anti-viral Th1 type immune response. A clear PRV-specific, CD4-dependent DTH reactivity and a classical CD8-dependent, MHC-restricted cytotoxicity was induced after protective immunization and the humoral immune response had a bias towards PRV-specific IgG2a formation. In vivo treatment with anti-CD8 and anti-IFN gamma demonstrated that the cytotoxic response and humoral IgG2a response, respectively, were strongly reduced, whereas protection against lethal challenge was unaffected. On the other hand anti-CD4 treatment reduced the induced protection so that 30% of the mice died after lethal challenge. The results of our study demonstrated that CD4+, DTH like effector cells are a crucial effector mechanism for protective immunity against PRV.

Animals↗
PubMed1998

Silent memory induction in maternal immune young animals.

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.

Animals↗
PubMed1997

Establishment and characterization of porcine cytolytic cell lines and clones.

Although non-major-histocompatibility-complex-restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, the phenotype and functional characteristics of these cells are not well defined. To allow a detailed analysis of these subsets, we established and characterized cell lines and clones of interleukin-2-activated (IL-2) cytolytic cells. Cell lines and clones were obtained from peripheral blood mononuclear cells of minipigs of the swine-leucocyte-antigen-complex (SLA) d/d haplotype. Cells were cultured in the presence of human recombinant IL-2 and cloned by double limiting dilution in the presence of gamma-irradiated L14 cells (a retrovirus immortalized B-lymphoblastoid cell line of the haplotype SLAd/d) or gamma-irradiated autologous peripheral blood mononuclear cells as feeder cells. Cytolytic cell lines and clones were characterized for their ability to kill different target cells and for their cell surface phenotype. All obtained clones expressed CD2 and CD8 and were negative for CD4. The following three subsets of cytolytic cells were identified: Subset 1) CD3- CD5- cells that killed K562 cells (a natural killer cell susceptible target cell line), as well as the pseudorabies virus (PRV)-infected or uninfected porcine kidney cells. These cells were considered to be typical natural killer cells. Subset 2) CD3 gamma/delta + CD5- T-cells that killed K562 cells and PRV virus-infected or uninfected porcine kidney cells, infected or uninfected L14 cells, and L14 cells constitutively expressing the PRV viral glycoprotein gB or gC. These cells were considered to be gamma/delta T-cells with natural killer activity. Subset 3) CD3 alpha/beta + CD5+ T-cells that killed L14 cells, PRV-infected L14 cells, and PRV gB- and gC-transfected L14 cells. These cells were possibly induced by the L14 feeder cells, used in the in vitro culture system. None of the cytolytic effector cells killed only MHC-matched viral infected cells. In conclusion, we describe a method to isolate, clone, and culture cytolytic cells from pigs. The clones could be cultured for 5 months, which allowed appropriate phenotypic and functional characterization of the various clones. Two of the subsets, CD3 gamma/delta T- and the natural killer cell subset may be involved in antiviral immunity in this species.

Animals↗
PubMed1997

The influence of maternal immunity on the transmission of pseudorabies virus and on the effectiveness of vaccination.

The purpose of this study was to investigate whether maternal immunity could prevent transmission of pseudorabies virus (PRV) among pigs, and whether it reduced the effectiveness of a single or double vaccination with regard to the transmission of PRV. In five experiments, the transmission of PRV, expressed as the reproduction ratio R, was compared in groups of pigs with maternal immunity and in groups of pigs without maternal immunity. Transmission of PRV among unvaccinated pigs with maternal immunity (R = 0.2) was significantly lower than among pigs without maternal immunity (R = 6.3). Furthermore, maternal immunity in young pigs prevented transmission of PRV, as R was significantly below one. In once-vaccinated groups, PRV spread extensively among pigs with maternal immunity (R = 23), but did not spread extensively among pigs without maternal immunity (R = 0.6). In twice-vaccinated groups, transmission of PRV among pigs with maternal immunity (R = 0.6) did not differ significantly from the transmission of PRV among pigs without maternal immunity (R = 0.3). Thus, a single vaccination of pigs with PRV strain 783 at 10 weeks of age, when they still possessed maternal immunity, seemed not sufficient to prevent transmission of PRV. Virus transmission could be reduced, however, if maternally immune pigs were vaccinated twice at 10 and 14 weeks of age.

Animals↗
PubMed1997

Comparison of two pseudorabies virus vaccines, that differ in capacity to reduce virus excretion after a challenge infection, in their capacity of reducing transmission of pseudorabies virus.

Pseudorabies virus (PRV) vaccines are often compared for their capacity to reduce virus excretion after a challenge infection. Vaccines, used for the eradication of PRV, however, should reduce transmission of PRV among pigs. The purpose of this study was to investigate whether the amount of virus excreted after a challenge infection is an accurate measure of the capacity of a vaccine to reduce transmission of PRV among pigs. Two experiments were carried out, each using two groups of 10 pigs. The pigs in group one were intramuscularly vaccinated once with the glycoprotein E (gE)-negative vaccine X, the pigs in group two with the gE-negative strain 783. Eight weeks later, 5 pigs in each group were inoculated with wild-type PRV. A gE-ELISA was used to detect PRV infection. The transmission of PRV was estimated from the number of contact infections and expressed as the reproduction ratio R. The inoculated pigs vaccinated with vaccine X shed significantly more virus than the inoculated pigs vaccinated with strain 783. However, despite the difference in virus excretion, the transmission of PRV between the two groups did not differ. We conclude that virus excretion is not an accurate measure for determining vaccine effectiveness. However, R of vaccine X (R = 0.98) was not significantly below one, whereas R of vaccine 783 (R = 0) was significantly below one. Consequently, we cannot exclude the possibility that major outbreaks of PRV occur among pigs vaccinated with vaccine X.

Animals↗
PubMed1997

No massive spread of pseudorabies virus in vaccinated sow herds.

In this study, we quantified transmission of PRV in 99 sow herds in which the sows were vaccinated three times a year with strain 783 O/W and found that the reproduction ratio R was 0.66, which is significantly below one. This implies that massive spread of PRV cannot occur in such herds.

Animals↗
PubMed1997

Evaluation of tests for detection of antibodies to Aujeszky's disease (pseudorabies) virus glycoprotein E in the target population.

Receiver operating characteristic (ROC) curves assess the quality of tests over the entire range of test signals. We compared the ability of an ELISA to detect antibodies to Aujeszky's disease (pseudorabies) virus gE in colostrum (test A) and in a single droplet of whole blood (test B) with the results obtained in serum (gold standard) in the target population by constructing and analyzing such curves. The area under the ROC curve, which is a quantitative measure of test performance, proved to be significantly (p < 0.01) smaller in test A than in test B or the gold standard. No significant differences in the area under the ROC curve were observed between test B and the gold standard.

Animals↗
PubMed1997

Immunohistological characterization of the local cellular response directed against pseudorabies virus in pigs.

The aim of this study was to investigate the kinetics of a primary and secondary immune response against pseudorabies virus (PRV). Pigs vaccinated with strain 783 and unvaccinated pigs were challenged with wild-type PRV by either intranasal or subcutaneous infection. Non-challenged pigs were used as controls. On days 1, 3 and 7 after challenge, tissues from the site of infection, and the tonsils of intranasally and the draining lymph nodes of subcutaneously challenged pigs were sampled. Immunohistological staining was used to characterize the various cell populations at the primary site of virus replication and in the lymphoid tissue. Tissue sections were stained for the T-cell markers CD2, CD3 gamma delta, CD4 and CD8, for the B-cell markers IgM, IgA and IgG, for a macrophage marker, and for PRV antigen. After challenge, PRV was detected during a shorter period in vaccinated pigs, and was less disseminated than in unvaccinated pigs. Cellular infiltrates were detected both in the nasal mucosa and the subcutaneous tissue of both unvaccinated and vaccinated pigs. Cell infiltrates, however, appeared earlier in vaccinated than in unvaccinated pigs, indicating a difference in kinetics of the primary and secondary immune response. The appearance of T-cells preceded the appearance of B-cells, but the proportion of the various subsets did not differ between unvaccinated and vaccinated pigs. These findings suggest that the early immune response in vaccinated pigs may contribute to the rapid clearance of virus at the primary site of infection. In addition, T-cells appear to have a more important role in the clearance of PRV than B-cells.

Animals↗
PubMed1997

Poliovirus-specific immunoglobulin A in persons vaccinated with inactivated poliovirus vaccine in The Netherlands.

In The Netherlands the inactivated poliovirus vaccine (IPV) is used for protection against poliomyelitis. It is not clear if parenteral vaccination with IPV can lead to priming of the mucosal immune system. We developed and evaluated enzyme-linked immunosorbent assays for the detection of poliovirus serotype-specific immunoglobulin A (IgA) and secretory IgA antibodies. Using these assays we examined the kinetics of the IgA response in sequential serum samples from 15 poliomyelitis patients after natural infection with serotype 3 poliovirus. In 36% of the patients IgA remained present for up to 5 months postinfection. Furthermore, we examined, in an IPV-vaccinated population, the presence of IgA antibodies in sera from young children (4 to 12 years of age; n = 177), sera from older children (between 13 and 15 years of age; n = 123), sera from healthy blood donors (n = 66), and sera from naturally immune elderly persons (n = 54). The seroprevalence of IgA to all three serotypes was low in young vaccinated children (5 to 7%), and the seroprevalence of IgA types 2 and 3 was low in older vaccinated children (2 to 3%). The seroprevalence of antibodies to type 1 was significantly higher (18%) in older children than in younger children. This higher seroprevalence is most likely explained by the persistence of IgA following infection with the serotype 1 wild-type poliovirus strain during the 1978 epidemic. In healthy adults, the seroprevalence of type 1- and type 2-specific IgA was significantly higher than that in young children. These results suggest that at least part of the IgA found in the older population is induced by infections unrelated to the IPV vaccination schedule. Finally, we found that parenteral vaccination with IPV was able to boost secretory IgA responses in 74 to 87% of a naturally exposed elderly population (n = 54). While the presence of secretory IgA in IPV-vaccinated persons has been documented previously, our findings suggest that mucosal priming with live virus is necessary to obtain an IgA response after IPV booster vaccination.

Adolescent↗
PubMed1997

Evaluation of a poliovirus-binding inhibition assay as an alternative to the virus neutralization test.

An enzyme-linked immunosorbent assay (ELISA)-based poliovirus-binding inhibition (PoBI) test to detect and quantify antibodies to polioviruses was optimized and evaluated for use in population studies as an alternative to the virus neutralization test (NT) in tissue culture. The sensitivities of the inhibition ELISA compared with the NT in an inactivated poliovirus vaccine (IPV)-vaccinated population were 98.6, 97.4, and 92.1% for serotypes 1, 2, and 3, respectively. The specificities of the PoBI test, as determined with sera from nonvaccinated persons, were also high for all three serotypes (99.0, 95.8, and 100%, respectively). Antibodies to other enteroviruses did not cross-react in the serotype 1 and 3 PoBI, and only levels of cross-reactivity were found for serotype 2. We found high correlations between the PoBI and NT titers for serotypes 1 and 2 in IPV-vaccinated blood donors (0.97 and 0.95), in oral poliovirus vaccine (OPV)-vaccinated blood donors (0.91 and 0.95), and in naturally immune persons (0.90 and 0.87). The correlation coefficient for serotype 3, however, was significantly lower in OPV-vaccinated blood donors (0.73) and in naturally immune persons (0.76) than in IPV-vaccinated persons (0.94; P < 0.01). These results indicate that the antibody response to serotype 3 poliovirus in IPV recipients is different from that in OPV recipients and naturally infected persons. We conclude that the PoBI test is a suitable alternative to the NT for estimating the seroprevalence of neutralizing antibodies to poliovirus, especially in large-scale population studies.

Animals↗
PubMed1997

Pseudorabies virus infections in pigs. Role of viral proteins in virulence, pathogenesis and transmission.

This paper reviews new findings on the biological functions of pseudorabies virus (PRV) proteins. It focuses on the role of PRV proteins in the pathogenicity, immunogenicity and transmission of PRV vaccine strains in pigs. Furthermore, it evaluates potential risks that are connected with the use of PRV vector strains. Special emphasis is placed upon the spread of genetically engineered vaccine strains within pigs or between pigs.

Animals↗
PubMed1996

Comparison of the protective efficacy of recombinant pseudorabies viruses against pseudorabies and classical swine fever in pigs; influence of different promoters on gene expression and on protection.

The glycoprotein E (gE) locus in the genome of pseudorabies virus (PRV) was used as an insertion site for the expression of glycoprotein E1 of classical swine fever virus (CSFV). Transcription of E1 in the recombinants M401, M402 or M403 was regulated by the gD promoter of PRV, the immediate early gene promoter of human cytomegalovirus, or the gE promoter of PRV, respectively. Groups of four pigs were vaccinated once intramuscularly with 10(6) plaque forming units (p.f.u.) of the recombinant viruses and challenged intranasally with 100 50% lethal doses of virulent CSFV and with 10(5) p.f.u. of virulent PRV. All pigs vaccinated with M402 were fully protected against both classical swine fever and pseudorabies.

Animals↗
PubMed1996

No major outbreaks of pseudorabies virus in well-immunized sow herds.

In this study we quantified the transmission of pseudorabies virus (PRV) in well-immunized sow herds in The Netherlands. In three herds, sows were tested for antibodies to gE of PRV every time after they had been transported to another barn (survey A). In 99 other herds, sows were tested simultaneously once or twice yearly (survey B). We observed six introductions in survey A and 53 in survey B. None of these introductions resulted in extensive spread of the virus. The reproduction ratio R, which is defined as the mean number of secondary infections caused by one infectious sow, was significantly less than one. We conclude that PRV can be eliminated from sow herds by vaccination.

Animals↗
PubMed1996

Assessment of the quality of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein gE in a target population by the use of receiver operating characteristic curves.

The performance of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein E (gE) in a target population was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. These curves assess the discriminating ability of a test over the entire range of test signals. The advantages of applying the analysis to a sample of the target population (all commercial pigs in the Netherlands), as compared to using a panel of test sera, are that the estimates of sensitivity and specificity, the comparisons between tests and the choices of the cut-off values are all relevant for the target population. The results of a gE-ELISA in colostrum (test A) and in a single droplet of whole blood (test B) were compared with the results obtained with the same ELISA in serum (gold standard). The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P < 0.01) smaller with test A than test B or the gold standard, indicating that test B performed better than test A. No significant difference was observed between test B and the gold standard.

Animals↗
PubMed1996

Cell-mediated immunity to pseudorabies virus: cytolytic effector cells with characteristics of lymphokine-activated killer cells lyse virus-infected and glycoprotein gB- and gC-transfected L14 cells.

We examined cytolytic cells that lyse pseudorabies virus (PRV)-infected cells in pigs. In vitro stimulation of peripheral blood mononuclear cells from PRV-immune pigs with live PRV generated cells that lysed PRV-infected immortalized B cells. Several lines of evidence indicated a major contribution of non-major histocompatibility complex (MHC)-restricted cytolytic cells, which displayed characteristics of natural killer (NK) or lymphokine-activated killer cells: cytolysis was non-MHC-restricted, depended on CD2+CD4-CD8bright- (or CD2+CD4-CD8dull+) cells, was strongly augmented by in vitro antigenic stimulation and was not limited to virus-infected cells, i.e. the NK cell-susceptible target cell line K562 was also lysed. Cytolytic cells were also generated by in vitro antigenic stimulation with UV-inactivated PRV. Target cells transfected with and stably expressing PRV gB or gC were lysed to the same degree as PRV-infected target cells.

Animals↗