A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection.

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.

Inhibition of T-cell invasion across cultured fibroblast monolayers by phenothiazine-related calmodulin inhibitors: impairment of lymphocyte motility by trifluoperazine and chlorpromazine, and alteration of the monolayer by pimozide.

Phenothiazines inhibit the typical shape changes displayed by activated lymphocytes and thereby their migration through polycarbonate filters. The structure activity relationship of this effect is distinct from calmodulin inhibition. Our aim was to study this effect of phenothiazines on lymphocyte migration in an environment with living solid tissue cells. We assessed the effect of trifluoperazine and chlorpromazine (TFP and CP, two strong inhibitors of lymphocyte motility) and pimozide (PIM, a much weaker inhibitor of lymphocyte motility but a strong inhibitor of calmodulin) on invasion of human Molt-4 T-cells across precultured fibroblast monolayers. As expected invasion was inhibited by TFP and CP in the micromolar range that also inhibited motility. Surprisingly, PIM inhibited monolayer invasion at least as efficiently as TFP and CP (from 2.25 microM on). Preincubation of the monolayers or the lymphoid cells show that PIM exerted this novel invasion inhibiting effect on the monolayer. TFP and CP had a much weaker effect on the monolayer. Since these three compounds inhibit calmodulin in the same order, it is likely that this effect on the monolayer was caused by inhibition of a calmodulin-dependent pathway. KN-62, a specific inhibitor of calmodulin-dependent protein kinase II acted on the monolayer like PIM, whereas ML-7, a specific inhibitor of myosin regulatory light chain kinase, inhibited lymphoid cell motility like TFP and CP. In conclusion, invasion of T-cells across cellular monolayers is inhibited both by PIM and by phenothiazines like TFP and CP, but via distinct mechanisms: TFP and CP inhibit lymphocyte motility via a calmodulin independent pathway, whereas PIM impairs the monolayer's tolerance for invasion, most likely via a calmodulin and CamKII dependent pathway.

Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR.

We have identified a novel 529bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10(2)-10(4) tachyzoites and 100-3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.

DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice.

C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-gamma) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-gamma. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.

ADP-ribosylation of Rho-proteins with botulinum C3 exoenzyme inhibits invasion and shape changes of T-lymphoma cells.

C3 exoenzyme from Clostridium botulinum ADP-ribosylates the small GTP-binding protein Rho with a high specificity. The use of C3 has shown that Rho-mediated signaling is involved in the regulation of actin-dependent processes in various cell types. In order to investigate the role of Rho-proteins in lymphocyte crawling, we have analyzed the effects of C3 on a T-cell line derived from the murine BW5147 lymphoma. Pretreatment of the lymphoma cells with C3, in conditions where Rho was actually ADP-ribosylated, strongly inhibited the characteristic shape changes resulting from extension and retraction of pseudopodia. Concomitantly, invasion of the cells through a monolayer of fibroblast-like cells was also inhibited. C3-treatment did not affect the total F-actin content of the cells, as measured by flow cytometry of cells stained with phalloidin. Yet, microscopical observation revealed that the accumulations of F-actin, which were seen in the pseudopodia of untreated cells, were absent after treatment with C3. This suggests that C3 may affect actin polymerization locally. The inhibitory effect of C3 on invasion was not restricted to the murine BW5147 lymphoma cell line, as it occurred also with CCRF-CEM, a human T-cell lymphoma line. Our results demonstrate that invasion-bound motility of lymphocytes depends on a Rho-mediated signal transduction pathway.

Effects of Clostridium botulinum C2 toxin and cytochalasin D on in vitro invasiveness, motility and F-actin content of a murine T-lymphoma cell line.

In order to investigate the role of microfilaments in the crawling movements of lymphoid cells, we have analyzed the effects of botulinum C2 toxin and of cytochalasin D (cytoD) on the actin cytoskeleton and on the motility of a BW5147 T-lymphoma-derived cell line. Actin was ADP-ribosylated by C2 toxin in the living cells, and this resulted in a time and dose-dependent disappearance of F-actin, as assessed by staining with labeled phalloidin. CytoD did not affect the amount of polymerized actin, but rather changed its distribution from a diffuse peripheral network to focal accumulations on one side of the cell. Both treatments affected the motility of the lymphoma cells in two assay systems. Fourier analysis was used to quantify shape changes performed by the cells. C2 toxin as well as CytoD caused the cessation of pseudopodal protrusion. Invasion of the lymphoma cells through a monolayer of fibroblast-like cells was also inhibited by the treatments, in a dose-dependent way. C2 toxin significantly inhibited invasion at concentrations at which only part of the actin pool had been ADP-ribosylated. We conclude that partial depolymerization, as well as disorganization, of the microfilament network impairs the active cellular deformations that are involved in the crawling movements of the lymphoma cells. From previous work, there is evidence to state that the monolayer invasion assay to some extent mimics tissue infiltration by hematopoietic cells. The present study is the first to analyze the role of actin polymerization in a model system that is relevant for the migration of lymphoid cells in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Metastatic competence of BW5147 T-lymphoma cell lines is correlated with in vitro invasiveness, motility and F-actin content.

The aim of our study was to investigate whether the level of actin polymerization plays a role in the motile and tissue infiltrating behavior of malignant lymphoma cells. For a panel of cell lines derived from the murine BW5147 T-cell lymphoma, we had previously shown a correlation between experimental metastasis formation and in vitro monolayer invasion. We have analyzed the motility and the F-actin content of six nonmetastatic, noninvasive (meta-inv-) and five metastatic, invasive (meta+inv+) variants of BW5147. Fourier analysis of cell contours was used to quantify shape changes of cells. All meta+inv+ lines rapidly protruded and retracted pseudopodia, whereas only one of the six meta-inv- lines showed this type of motility. Flow cytometry of cells stained with fluorescein-labeled phalloidin showed that the motile meta+inv+ cell lines have a higher F-actin content than their nonmotile meta-inv- counterparts. The results indicate that in lymphoma cells a high level of actin polymerization is a prerequisite for the formation of pseudopodia, which in turn are necessary for infiltration of the cells into tissues, and eventually for efficient metastasis formation. A corollary of this conclusion is that regulation of actin polymerization is a possible target for intervention aimed at moderating the spread of malignant lymphoma.

Motility and invasive potency of murine T-lymphoma cells: effect of microtubule inhibitors.

ESb and BW-O-Li1 are T-lymphoma cell lines that form metastases in various organs after injection into syngeneic mice. In vitro, both cell lines invade through a fibroblastic monolayer, but ESb cells do so much slower than BW-O-Li1. By the use of Fourier analysis of cell outlines, we can relate this difference in invasiveness to a difference in cell motility: ESb cells do not perform any conspicuous shape change, whereas BW-O-Li1 cells are actively protruding and retracting large pseudopodia. However, the low-motile ESb cells become as motile and deformable as BW-O-Li1 cells when they have eventually invaded under a fibroblastic monolayer. This indicates that ESb cells do have inherent capability for shape change. Treatment of ESb cells with the microtubule disrupting agent nocodazole concomitantly increases their shape change intensity, and their invasion rate through fibroblast monolayers. On the contrary, the microtubule stabilizing drug taxol inhibits both motility and invasion of BW-O-Li1 cells. Our observations suggest that the microtubule network can repress invasion-bound motility of lymphoid cells.

Methods for computer assisted analysis of lymphoid cell shape and motility, including Fourier analysis of cell outlines.

Locomotion of lymphocytes and other leukocytes is an essential feature of the immune system, and therefore the evaluation of the locomotor behaviour of a lymphocyte population is part of its functional analysis. Paradoxically, the locomotor status of leukocytes is usually assessed on the basis of static information, by counting the number of spherical versus non-spherical cells. In this paper we describe two methods for the measurement of shape changes in microscopic images of lymphoid cells. First we computed a simple shape change factor, coined incongruence factor, based on the degree of non-overlap of the contours of the cell at the beginning and at the end of a 1 min time interval. Second we have used Fourier analysis of the cell outline: a function describing the undulations of the cell outline is broken down into sinusoidal 'waves' of increasing frequency, each with its corresponding amplitude. The amplitude values for the first ten frequencies produced a satisfactory mathematical description of lymphoid cell shapes, and the change of these amplitudes over a 1 minute time interval produced a quantitative description of the shape alterations of the cells. We have used five approaches to evaluate the shape and shape changes in the following populations of mouse lymphoma cells: a constitutively low-motile T lymphoma cell line (BW5147), a high-motile hybridoma (BW-O-Li1) either on plastic or on a precultured fibroblast-like monolayer, BW-O-Li1 cells after penetration through the monolayer, and BW-O-Li1 cells after treatment with cytochalasin B. We compare the results from direct visual evaluation of cell shape, from computer assisted assessment of sphericity and from Fourier analysis of cell shape at one moment, with the two methods for quantitative shape change analysis. All approaches revealed a clear distinction between spherical low-motile populations, and non-spherical high-motile cells. Moreover, the incongruence factor proved to be a reliable single parameter of active cell deformation. In addition, the Fourier analysis of cell outlines produced useful measures of static shape and of dynamic shape change, at any user-defined level of accuracy.

The lymphocytosis promoting action of pertussis toxin can be mimicked in vitro. Holotoxin but not the B subunit inhibits invasion of human T lymphoma cells through fibroblast monolayers.

Pertussis toxin is known to elicit lymphocytosis in whooping cough patients and experimental animals, by blocking the extravasation of lymphocytes and stimulating their release from lymphoid organs such as the thymus. The mechanisms responsible for these unique effects of PT are not fully understood. The effect of pertussis toxin (PT) on the invasive behavior of human CCRF-CEM T lymphoma cells has been investigated with the use of a monolayer invasion assay (MIA). We had previously found that invasion of murine T lymphoma cells in this model system was correlated with their ability to extravasate and form metastases after i.v. injection in syngeneic animals. We now show that human CEM cells can also penetrate through a precultured confluent monolayer of murine 10T1/2 fibroblast-like cells within a few hours. In a quantitative MIA run over 24 h, PT at concentrations above 10(-14) M inhibited invasion of the CEM cells. In addition, PT stimulated the release ('evasion') of CEM cells that had invaded under the monolayer before the toxin was added. The A subunit of PT was totally inactive, the B subunit had a small residual effect, and reconstitution of the AB complex partially restored the activity. The invasion-inhibiting activity of two different holotoxin preparations and of the subunits perfectly matched their activity in the Chinese hamster ovary cell clustering assay, which is known to depend on a functional AB complex. We suggest that inhibition of monolayer invasion by PT can be used as an in vitro model system to investigate the cellular and molecular mechanisms underlying the lymphocytosis-promoting action of the toxin. Furthermore, the method is sufficiently sensitive to be used for titration of toxin activity. Our data indicate that the ADP-ribosylating activity of the A subunit is indeed required, and that the promotion of lymphocytosis is not elicited by the binding of the B subunit alone.

Clinical efficacy of a herpes simplex subunit vaccine.

A DNA-free herpes simplex type 2 subunit vaccine was administered to 18 volunteers without past evidence of herpes simplex type 1 (HSV 1) or herpes simplex type 2 (HSV 2) infection, to 44 patients with severe recurrent genital HSV 2 infection, and to 15 patients with severe oral type 1 HSV recurrences. The vaccine elicited both humoral and cell-mediated immunity in 97% of the subjects without past HSV infections and boosted significantly the cell-mediated immunity and antibody titers in almost all the patients with recurrent HSV 1 or HSV 2. The vaccine elicited particularly the production of complement-dependent cytotoxic antibodies in 96% of the patients with recurrent HSV 2 infections. This might, at least partly, explain the clinical efficacy of the vaccine. Indeed, we observed a significant decrease (t test, p less than 0.01) in the attack rate of the recurrences and also a significant shortening of the time needed to complete healing of the lesions (t test, p less than 0.01).

Antibody and cell-mediated immunity to a DNA free herpes simplex subunit vaccine.

The immunogenicity of a DNA free herpes simplex subunit vaccine was evaluated in chimpanzees and rabbits. The results clearly demonstrate that 1 injection of 3 micrograms/kg elicited antibodies as well as cell-mediated immunity in all the animals studied. These antibodies persisted for at least 6 months. Furthermore the vaccine also protected 50% of the animals against an experimental infection and reduced the rate of latent infection in nervous sensory ganglia.

Cytomegalovirus infection and graft survival in renal graft recipients.

We have studied 85 patients who received a renal transplant for CMV infection as well as for herpes simplex (HSV), herpes zoster (HZ), measles, mumps, rubella and hepatitis B. We found no evidence of primary or secondary infections for the non herpetic viruses except for hepatitis B infection that occurred in 17 per cent of the patients. CMV infection occurred in 87 per cent of the patients while antibody rises to HZ and HSV occurred in 30 and 13 per cent of the patients, respectively. The CMV infections occurred 2 to 4 months after the transplantation (mean time 11.1 weeks) and seemed to trigger the first episode of renal rejection that occurred earlier in the CMV infected group (mean time 12.1 weeks) than in the uninfected group (mean time 18.6 weeks). This difference in time is highly significant, p less than 0.001). However these CMV injections did not decrease the longterm survival of the grafted kidneys.

Rapid detection of IgG and IgM antibodies for cytomegalovirus by the enzyme linked immunosorbent assay (ELISA).

A simple solid phase enzyme immunoassay for the detection of immunoglobulin G and M to cytomegalovirus (CMV) is described. Using this test IgM antibodies to CMV were detected in 0.7 per cent of newborns and regularly after CMV infection in transplant patients, furthermore in these latter patients IgM production was prolonged for several months. For the determination of IgG the enzyme immunoassay was more sensitive than the complement fixation test (CF) and the antibody titres were 4 to 8 fold higher. Since the ELISA test is rapid, specific and unexpensive it can become an acceptable routine diagnostic procedure.