Developing 3D SEM in a broad biological context.

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.

Nephroblastoma--a 25-year review of a South African unit.

RATIONALE: To determine the outcome of patients with nephroblastoma in a South African hospital. OBJECTIVE: To determine if there is a difference in the outcome of patients with nephroblastoma comparing two treatment protocols SIOP (Société International D'Oncologie Pédiatrique Protocol) versus NWTS (National Wilms' Tumour Study Protocol). METHODS AND RESULTS: A retrospective audit of 25 years (1983-2007), of children diagnosed with nephroblastoma in Tygerberg Hospital. One hundred and seven patients were included in the study and 98 were analyzed. The average age at diagnosis was 3.8 years. Most patients (37%) presented with stage 1 of the disease, followed by patients with stage 3 (27%). Most patients were treated according to the SIOP protocol (61%). Gender and race did not influence the outcome. Patients with stage 1 and 2 of the disease had the best outcome (76% versus 43% for stages 3 and 4). The SIOP group had a better outcome than the NWTS group (p value 0.001). The two groups had an equal distribution of the stage of presentation. The tumor volumes were bigger in the NWTS group (1004cm3 compared to 613cm3). Nutritional status did not influence the outcome although more patients were underweight for age in the SIOP group. The statistical methods used were: Kaplan Meier, Gehan's Wilcoxon Test, Chi -square test and the Fisher exact test. DISCUSSION: Contrary to the other studies, patients treated according to the SIOP protocol had a statistically significant better outcome. Larger collaborative studies are needed to investigate this result in Africa.

17α-Ethinylestradiol rapidly alters transcript levels of murine coagulation genes via estrogen receptor α.

BACKGROUND: Oral estrogen use is associated with changes in plasma levels of many coagulation proteins. OBJECTIVE: To gain more insight into the underlying mechanism of estrogen-induced changes in coagulation. METHODS: Ovariectomized female mice were used to study the impact of oral 17α-ethinylestradiol (EE) on plasma coagulation, hepatic coagulation gene transcript levels, and dependence on estrogen receptor (ER) α and ERß. RESULTS: Ten days of oral EE treatment resulted in significantly reduced plasma activity levels of factor (F)VIII, FXII, combined FII/FVII/FX and antithrombin, whereas FIX activity significantly increased. Regarding hepatic transcript levels, oral EE caused significant decreases in fibrinogen-γ, FII, FV, FVII, FX, FXII, antithrombin, protein C, protein Z, protein Z inhibitor and heparin cofactor II mRNA levels, whereas FXI levels significantly increased and transcript levels of FVIII, FIX, protein S and α(2) -antiplasmin remained unaffected. All EE-induced coagulation-related changes were neutralized by coadministration of the non-specific ER antagonist ICI182780. In addition, ERα-deficient mice lacked the EE-induced changes in plasma coagulation and hepatic transcript profile, whereas ERß-deficient mice responded similarly to non-deficient littermate controls. A crucial role for the ER was further demonstrated by its rapid effects on transcription, within 2.5-5 h after EE administration, suggesting a short chain of events leading to its final effects. CONCLUSIONS: Oral EE administration has a broad impact on the mouse coagulation profile at the level of both plasma and hepatic mRNA levels. The effects on transcription are rapidly induced, mostly downregulatory, and principally mediated by ERα.

Phosphodiesterase-4 inhibition attenuates pulmonary inflammation in neonatal lung injury.

Phosphodiesterase-4 (PDE4) inhibitors may offer novel therapeutic strategies in respiratory diseases, including asthma and chronic obstructive pulmonary disease. Therefore, selective PDE4 inhibitors may also provide a therapeutic option for very pre-term infants with bronchopulmonary dysplasia (BPD). The anti-inflammatory effect of two PDE4 inhibitors was investigated in a pre-term rat model of hyperoxia-induced lung injury. Pre-term rat pups were exposed to room air, hyperoxia, or hyperoxia and one of two PDE4 inhibitors: rolipram and piclamilast. The anti-inflammatory effects of prolonged PDE4 inhibitor therapy were investigated by studying survival, histopathology, fibrin deposition, alveolar vascular leakage and differential mRNA expression (real-time RT-PCR) of key genes involved in inflammation, alveolar enlargement, coagulation and fibrinolysis. PDE4 inhibitor therapy prolonged median survival by up to 7 days and reduced alveolar fibrin deposition, lung inflammation and vascular leakage by decreasing the influx of monocytes and macrophages and protein efflux in bronchoalveolar lavage fluid. Analysis of mRNA expression of key genes involved in experimental BPD revealed a significant PDE4 inhibitor-induced improvement of genes involved in inflammation, fibrin deposition and alveolarisation. In conclusion, phosphodiesterase-4 inhibition prolongs survival by inhibiting inflammation and reducing alveolar fibrin deposition in pre-term rat pups with neonatal hyperoxic lung injury, whereby piclamilast outperformed rolipram.

A DNA vaccine coding for gB and gD of pseudorabies virus (suid herpes type 1) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines.

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.

Vaccine-induced T cell-mediated immunity plays a critical role in early protection against pseudorabies virus (suid herpes virus type 1) infection in pigs.

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.

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus.

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.

Discrimination of different subsets of cytolytic cells in pseudorabies virus-immune and naive pigs.

We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6(+) CD8(bright+) CD4(-) (CTL phenotype), CD6(+) CD8(dull+) CD4(+) (the fraction containing memory T helper cells), CD6(+) CD8(-) CD4(+) (T helper cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(+) ( gammadelta-T cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(-) (NK phenotype) and CD6(-) CD8(-) CD4(-) gammadelta-T(-) or gammadelta-T(+). Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6(+) CD8(+) and CD4(+) CD8(+) and fewer CD6(-) CD8(+) and gammadelta-T(+) CD8(+) lymphocytes than IL-2 stimulation of naive PBMC (P<0.05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6(+) CD8(+) T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6(-) CD8(+) T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.

A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection.

Glycoproteins B (gB), gC and gD of pseudorabies virus (PRV) have been implicated as important antigens in protective immunity against PRV infection. As cell-mediated immunity plays a major role in this protective immunity, we determined the significance of these glycoproteins in the actual induction of cell-mediated immunity. We vaccinated pigs with plasmid DNA constructs coding for gB, gC or gD and challenged them with the virulent NIA-3 strain of pseudorabies virus. Vaccination with plasmid DNA coding for gB induced the strongest cell-mediated immune responses including cytotoxic T cell responses, whereas plasmid DNA coding for gD induced the strongest virus neutralising antibody responses. Interestingly, vaccination with gB-DNA reduced virus excretion early after challenge infection while vaccination with gC-DNA or gD-DNA did not.This is the first study to demonstrate that DNA vaccination induces cytotoxic T cell responses in pigs and that cell-mediated immunity induced by vaccination with gB-DNA is important for the reduction of virus excretion early after challenge infection.

Cytolytic function for pseudorabies virus-stimulated porcine CD4+ CD8dull+ lymphocytes.

We previously observed that pseudorabies (PRV) virus-specific killing in vitro was mediated by CD6+ CD8+ lymphocytes. Also a high percentage of CD4+ lymphocytes, among these CD6+ CD8+ lymphocytes, was observed. The purpose of this study was, therefore, to further characterize the killing ability of PRV-stimulated CD4+ CD8+ lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from blood of PRV-immune pigs and were stimulated in vitro with PRV. After 6 days, the frequency of CD4+ CD8+ lymphocytes in peripheral blood was determined by flow cytometry analyses. Lymphocytes were separated using a magnet-activated cell sorter or a FACSVantage SE, and the cytolytic activity of the isolated populations was determined. Flow cytometry analyses demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of 26% +/- 4% CD4+ CD8dull+ lymphocytes. We further demonstrated that killing by PRV-stimulated PBMC was mediated by CD4+ CD8dull+ T lymphocytes and CD4- CD8+ T lymphocytes (classic cytolytic T lymphocytes and natural killer cells). The CD4+ CD8dull+ T lymphocytes showed major histocompatibility complex (MHC) II-restricted PRV-specific killing. The CD4- CD8+ T lymphocytes showed both PRV-specific and natural killing. The CD4+ CD8dull+ lymphocytes, which are unique in the pig, seemed to have a more heterogeneous function than was earlier demonstrated. In conclusion, we demonstrated that PRV-specific CD4+ CD8dull+ lymphocytes are able to kill PRV-infected target cells in a MHC II-restricted manner.

Effect of vaccination route and composition of DNA vaccine on the induction of protective immunity against pseudorabies infection in pigs.

Vaccination with naked DNA may be an alternative to conventional vaccines because it combines the efficacy of attenuated vaccines with the biological safety of inactivated vaccines. We recently showed that the vaccination with naked DNA coding for the immunorelevant glycoprotein D (gD) of pseudorabies virus (PRV) induced both antibody and cell-mediated immunity in pigs and provided protection against challenge infection. To determine whether the efficacy of the naked DNA vaccination against PRV could be improved, we compared three sets of variables. First, the efficacy of the naked DNA vaccine coding only for the immunorelevant gD was compared with a cocktail vaccine containing additional plasmids coding for two other immunorelevant glycoproteins, gB and gC. Second, the intramuscular route of vaccination was compared with the intradermal route. Third, the commonly used needle method of inoculation was compared with the needleless Pigjet injector method. Five groups of five pigs were vaccinated three times at 4-weeks intervals and challenged with the virulent NIA-3 strain of PRV 6 weeks after the last vaccination. Results showed that although the cocktail vaccine induced stronger cell-mediated immune responses than the vaccine containing only gD plasmid, both vaccines protected pigs equally well against challenge infection. Intradermal inoculation with a needle induced significantly stronger antibody and cell-mediated immune responses and better protection against challenge infection than intramuscular inoculation. Our data show that the route of administering DNA vaccines in pigs is important for an optimal induction of protective immunity.

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.

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.

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity.

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.