Plasmodium: immunization with carboxyl-terminal regions of MSP-1 protects against homologous but not heterologous blood-stage parasite challenge.

A leading candidate for a vaccine targeted at the erythrocytic stages of plasmodial parasite development is the merozoite surface protein-1 (MSP-1). We have previously shown that the carboxyl-terminal region of MSP-1 derived from Plasmodium yoelii yoelii 17XL, expressed as a fusion protein with glutathione S-transferase (GST-PYC2), can immunize mice against an otherwise lethal homologous challenge infection. This protection has been shown to be predominantly mediated by antibodies. We report here on the efficacy of immunization with MSP-1 carboxyl regions when the challenge is a heterologous rodent parasite species. The course of parasitemia was not altered in mice immunized with GST-PYC2 and challenged with 10(4) heterologous Plasmodium chabaudi adami parasites, as both control and immunized mice developed infections that peaked at day 7 and then rapidly declined. Similarly, mice immunized with GST-PYC2 and challenged with 10(5) Plasmodium berghei ANKA parasites displayed virulence similar to that seen in infection control mice. The homologous region of the P. chabaudi adami MSP-1 gene was similarly expressed as a fusion protein with GST. Mice immunized with GST-PCC2 and challenged with 10(4) parasites showed significant protection against homologous P. chabaudi adami infection but no protection whatsoever against heterologous P. yoelii yoelii 17XL infection. These in vivo results correlate with the observation that sera generated by immunization with the carboxyl region of MSP-1 recognizes this protein from homologous, but not heterologous, radiolabeled parasite protein preparations.

Fc receptors are not required for antibody-mediated protection against lethal malaria challenge in a mouse model.

The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR gamma-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR gamma-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.

Strongyloides stercoralis host-adapted third-stage larvae are the target of eosinophil-associated immune-mediated killing in mice.

Host-adapted, transformed, Strongyloides stercoralis third-stage larvae (L3+) were previously found to be antigenically different from free-living, infective, third-stage larvae (L3). These antigenic differences were reproduced by transformation of free-living larvae in tissue culture medium at 37 C over 24 hr. Transformed L3 of both derivations were given as challenge infections in diffusion chambers to naive mice and mice immunized with S. stercoralis L3. Within 12 hr, the challenge infections were killed regardless of whether the L3+ were generated in vitro or in vivo. Eosinophils, previously found to be important in the immune response to S. stercoralis larvae, were recruited into the L3+ microenvironment within 12 hr of challenge infection in immune mice, which supports the previously proposed mechanisms of S. stercoralis larval killing. Thus, S. stercoralis L3+ appear to be targets of the immune response in mice instead of being involved in immune evasion.

Chronicity in Strongyloides stercoralis infections: dichotomy of the protective immune response to infective and autoinfective larvae in a mouse model.

Strongyloidiasis is an intestinal disease that can last for decades due to the occurrence of autoinfective larvae (L3a) in an infected person, which contribute to the maintenance of the population of adult worms in the intestine. The goal of the present study was to determine if L3a are susceptible to the protective immunity that targets the infective stage of the worm, the third-stage larvae (L3). Mice immunized and challenged with Strongyloides stercoralis L3 kill more than 90% of challenge larvae contained within diffusion chambers. The L3 do not remain antigenically static in mice, however, but undergo some degree of antigenic change before they are killed, becoming host-activated larvae (L3+). The L3/L3+ are killed in this model system by the combined effects of both parasite-specific IgM and eosinophils. Mice immunized with L3 were able to kill L3/L3+, but did not kill L3a, in challenge infections. Eosinophils were, however, present in diffusion chambers containing L3a, and IgM bound to the surface of L3a. We hypothesized that differential IgM recognition of soluble L3a, L3, and L3+ antigens is the reason why the immune response generated against L3 could not kill L3a. Many common antigens on L3, L3+, and L3a were recognized by serum from mice immunized with L3, as determined by immunoblotting. However, several unique L3, L3+, and L3a antigens were also recognized by immune serum, thus indicating that antigen recognition with IgM antibodies is different between the L3, L3+, and L3a stages. This difference in antigen recognition could explain why L3a are able to evade the immune response that targets L3/L3+ in chronically infected hosts.

A randomized, double-blind, crossover comparative endoscopy study on the gastroduodenal tolerability of a highly specific cyclooxygenase-2 inhibitor, flosulide, and naproxen.

BACKGROUND: Inhibition of constitutively expressed cyclooxygenase (Cox-1) is thought to play an important role in the gastrointestinal toxicity of nonsteroidal anti-inflammatory drugs (NSAID), while their therapeutic action may be due to inhibition of the enzyme Cox-2, which is specifically expressed at sites of inflammation. NSAIDs with high affinity and specifity for Cox-2 hold the promise of maintaining efficacy without the gastrointestinal side effects of conventional NSAIDs. METHODS: We assessed the gastrointestinal tolerability of flosulide (20 mg twice a day), a highly selective Cox-2 inhibitor with that of naproxen (500 mg twice a day), which has equal affinity for Cox-1 and -2 in 19 patients with osteoarthrosis in a randomized, double blind, crossover endoscopy study. Subjects were treated for 2 weeks with a 2-week washout period. Gastroduodenal damage was primarily assessed as by Lanza (grades 0-4). RESULTS: No stomach damage was seen in 13 (68%) patients after flosulide and in 5 (37%) after naproxen (P < 0.001). Lanza scores were significantly lower after flosulide (0.58) than after naproxen (1.47) (P < 0.001; odds ratio, 84.4; 95% confidence interval, 1.45-4908). Flosulide was significantly better tolerated (P < 0.005) than naproxen. CONCLUSION: These results endorse the idea that highly selective Cox-2 inhibitors may be associated with lesser gastrointestinal side effects than conventional NSAIDs.

IL-12 eliminates the Th-2 dependent protective immune response of mice to larval Strongyloides stercoralis.

The goal of the present study was to determine if immune-mediated killing of S. stercoralis L3 in mice could be modulated by shifting from a Th-2 to a Th-1 type immune response. L3 killing in immunized mice was ablated in CD4+ T cell-depleted animals, but not in CD8+ T cell-depleted or beta 2-microglobulin-deficient mice. Treatment of immunized mice with IL-4 or IL-5 neutralizing MoAb significantly reduced the protective effects of vaccination against S. stercoralis, while protective immunity was unimpaired in IFN-gamma knockout mice. Recombinant IL-12 was administered to infected mice to switch the immune response from a Th-2 to a Th-1 type response. Protective immunity was ablated in immunized mice that received IL-12 therapy. Eosinophil numbers, eosinophil peroxidase levels, and parasite-specific IgG1 levels were lowered in IL-12 treated immunized animals, and parasite-specific IgG2a levels were increased in these animals. The data indicate that eosinophils are important as mediators of larval killing, and that the establishment of Th-2 type immunity results in killing of infective S. stercoralis L3, while a shift to Th-1 type immunity abrogates protective responses.

Regression of C6 rat brain tumors by cells expressing an antisense insulin-like growth factor I receptor RNA.

We have previously shown that C6 cells expressing an antisense insulin-like growth factor I receptor (IGF-IR) RNA are no longer tumorigenic in syngeneic rats, protecting them from subsequent subcutaneous tumor challenge and causing regression of established subcutaneous tumors. In the present study, we have investigated the efficacy of this strategy on intracerebrally implanted C6 rat glioblastoma cells. We demonstrate that C6 cells expressing an antisense IGF-IR RNA implanted for 24 h in the subcutaneous tissue of the rats are able to elicit an anti-tumor response in the brain, leading to complete brain tumor regression and long-term survival of the rats. These findings suggest the possibility of therapeutic intervention in human gliomas.

Strongyloides stercoralis: eosinophil-dependent immune-mediated killing of third stage larvae in BALB/cByJ mice.

Challenge worm survival was significantly reduced when BALB/cByJ mice were vaccinated against Strongyloides stercoralis infective third stage larvae (L3) regardless of whether the challenge infections consisted of systemically migrating L3 or L3 implanted in diffusion chambers. The only cell type that increased in number in diffusion chambers in immunized mice, 1 week after booster immunizations, was the eosinophil, and maximal levels of eosinophils were coincident with parasite killing. Mice were treated with mAb to eliminate IL-5 or granulocytes to assess the role that eosinophils play in larval killing. Treated animals showed no decrease in immunity when challenge infections consisted of systemically migrating L3 administered 3 weeks after booster immunizations. Eosinophil numbers in immunized mice decreased to control levels when measured 3 weeks post-booster immunization, both in diffusion chambers and in the peripheral blood, whereas they were elevated at 1 week after booster immunizations. Direct contact between host cells and L3 was, however, still required for larval killing in immunized hosts 3 weeks after booster immunizations. Elimination of eosinophils by treatment with mAb to IL-5 or granulocytes significantly reduced protective immunity, when L3 were implanted in diffusion chambers at 1 and 3 weeks post-booster. However, as systemically migrating L3 were still killed in immunized, eosinophil-depleted animals, other cell types may play a role in larval destruction. Two human eosinophil granule products were found to be toxic for host-adapted L3+, but had no effect on infective L3, indicating that host-adapted larvae are possible targets for eosinophil-mediated destruction of third stage larvae. These findings suggest that inactivation of eosinophils by mAb treatment abolishes protective immunity to L3 contained within diffusion chambers and that small numbers of eosinophils are sufficient for immune-mediated killing of S. stercoralis L3.

Strongyloides stercoralis: role of antibody and complement in immunity to the third stage of larvae in BALB/cByJ mice.

Mice immunized against Strongyloides stercoralis L3 were shown to kill greater than 90% of challenge larvae contained within diffusion chambers. The objective of the present study was to identify the host components responsible for immunity. Serum from unprotected, control mice and protected, immune mice in doses of 25-500 microliters was transferred into naive mice at the same time and location as larval challenge. Transfer of as little as 50 microliters of immune serum was able to confer protective immunity. The serum-transferred immunity was ablated by excluding cells from the larval microenvironment or by depleting granulocytes through monoclonal antibody treatment in the recipient mice. Specific antibody isotypes were isolated using protein G and isotype-specific affinity columns. The resulting transfer experiments identified IgM as the isotype responsible for protective immunity to S. stercoralis L3. Antibody binding studies in vivo were performed and only IgM bound to the surface of infective L3 and host-derived L3 (L3+) in immune animals. Elevated levels of C3 were also found bound to the surface of L3/L3+ in immune mice. Cobra venom factor treatment of immunized mice to deplete complement completely eliminated C3 binding to the surface of L3/L3+ and ablated immunity. Therefore, IgM, complement, and granulocytes are necessary for immune elimination of S. stercoralis L3/L3+. Identification of antigens recognized by IgM may help select possible vaccine candidates.

Structural and molecular specificity of antibody responses in mice immune to third stage larvae of Onchocerca volvulus.

Immunization of mice with irradiated Onchocerca volvulus infective stage larvae (L3) has been demonstrated to confer protection against challenge infections with these larvae. Additionally, cytokine level measurements and cytokine depletion studies have shown that both IL-4 and IL-5 are important in generating a protective immune response against O. volvulus challenge infections, thus suggesting a dependency of protective immunity on IgG1, IgE and/or eosinophils. In the present study, we examined the humoral responses of immunized mice to O. volvulus L3 antigens. ELISA measurements of total serum antibody levels indicated that IgE was the only antibody isotype elevated in mice immunized with O. volvulus L3. IgM from immunized mice was the only isotype that recognized surface antigens on intact O. volvulus L3. IgG1, IgG3, IgE and IgA recognized internal parasite antigens on O. volvulus L3 frozen sections. Western blot analysis of L3 proteins showed that in serum from mice immunized with O. volvulus L3 IgG1, IgG2a/2b, IgA, and IgE, as well as IgM, recognized unique L3 proteins. Antibodies in serum from L3 immunized mice were able to detect O. volvulus adult antigens in a pattern similar to the recognition found in O. volvulus L3. Some L3 antigens were shared by adults, while other antigens were L3 specific. The ELISA, immunohistochemistry and Western blot findings thus demonstrate a complex pattern of antigen recognition of parasite antigens by antibodies found in mice immune to the L3 of O. volvulus.

Immunity to a challenge infection of Strongyloides stercoralis third-stage larvae in the jird.

Jirds support the entire life-cycle of Strongyloides stercoralis. We therefore used this host as a model to define the mechanism of the immune response to a challenge infection, as well as the parasite stage effected by the response. Jirds given a primary infection of S. stercoralis are resistant to re-infection. The use of implanted diffusion chambers containing larvae showed that the immune response killed the third-stage larvae, and this was confirmed by subcutaneous infections. The larvae of a challenge infection are killed within 48 h, a time period too short to allow for the development of L4 and adult worms. The immune response is dependent on both a serum factor and cells, suggestive of an ADCC type response.

Correlation between apoptosis, tumorigenesis, and levels of insulin-like growth factor I receptors.

We have investigated whether there is a quantitative relationship between the insulin-like growth factor I receptor (IGF-IR), the extent of apoptosis in vivo, and tumorigenesis. C6 rat glioblastoma cells were treated with increasing concentrations of antisense oligodeoxynucleotides to the IGF-IR RNA. The extent of apoptosis in vivo is correlated to the decrease in IGF-IR levels and, in turn, tumorigenesis in nude mice is correlated to the fraction of surviving cells. In syngeneic rats, a host response leads to complete inhibition of tumorigenesis. These findings establish, for the first time on a quantitative basis, the relationship between IGF-IR levels and the extent of apoptosis, as well as the relationship between the initial apoptotic event and the time of appearance of transplantable tumors.

The insulin-like growth factor I receptor protects tumor cells from apoptosis in vivo.

The role of the insulin-like growth factor I receptor (IGF-IR) in programmed cell death has been investigated in vivo in a biodiffusion chamber, where the extent of cell death could be determined quantitatively. We found that a decrease in the number of IGF-IRs causes massive apoptosis in vivo in several transplantable tumors, either from humans or rodents. Conversely, an overexpressed IGF-IR protects cells from apoptosis in vivo. We also show that the same conditions that in vitro cause only partial growth arrest with a minimum of cell death, induce in vivo almost complete cell death. We conclude that the IGF-IR activated by its ligands plays a very important protective role in programmed cell death, and that its protective action is even more striking in vivo than in vitro.

Strongyloides stercoralis: protective immunity to third-stage larvae inBALB/cByJ mice.

A murine model system was developed to study the induction and mechanism of protective immunity to L3 of Strongyloides stercoralis. L3 were implanted in BALB/cByJ mice in diffusion chambers constructed with 0.1- or 2.0-microns-pore-size membranes. Parasites survived equally well regardless or membrane type for 7 days, after which larval survival decreased in diffusion chambers constructed with 2.0-microns-pore-size membranes, which allowed host cells to enter. Survival of S. stercoralis L3 in diffusion chambers implanted in mice was assayed after immunization with live, heat-killed, and homogenized L3. Optimal immunization was achieved with 10,000 live L3, whereby immunized mice eliminated 97% of the larvae either contained within diffusion chambers or free within the tissues of the mouse by 24 hr postinfection. Sera from immunized mice had elevated levels of IgG1, IgM, and IgA parasitic-specific antibody; IgM was the only antibody isotype that recognized surface antigens of L3. Larvae were not killed in immunized mice if contact between host cells and the parasites was prevented. In the peripheral blood and diffusion chamber fluid of immunized mice, eosinophil levels were significantly higher when compared to the levels found in control mice. The rodent model developed in the present study has thus demonstrated that virtually complete immunity can be induced to the L3 of S. stercoralis and that larval killing was found to be associated with the presence of both specific antibody and eosinophils.

Double-blind, multicenter evaluation of the efficacy and tolerability of diclofenac dispersible in the treatment of acute soft-tissue injuries.

In a randomized, double-blind, parallel-group, multicenter study, the efficacy and tolerability of diclofenac dispersible were compared with placebo in the treatment of acute soft-tissue injuries. Patients seen within 48 hours of a soft-tissue injury received either diclofenac dispersible 50 mg or placebo three times daily for 3 to 7 days, with paracetamol allowed as a rescue analgesic. Of a total of 253 recruited patients. 247 patients (122 in the diclofenac dispersible group and 125 in the placebo group) were eligible for tolerability assessment and 229 patients (115 diclofenac dispersible and 114 placebo) were eligible for efficacy analysis. In general, median reductions in the intensities of pain at rest, on movement, and on local pressure (as measured on 100-mm analog chromatic continuous scales) were greater with diclofenac dispersible than with placebo after treatment (differences did not reach statistical significance). At the center that recruited the largest number of patients, the initial median levels of pain at rest and on movement were considerably lower than those at the other centers. On reanalysis of the pain data without this center, a significant difference favoring diclofenac dispersible over placebo was noted for pain on movement and on local pressure (P < or = 0.044). With respect to daily assessment of pain severity, more patients in the diclofenac dispersible group had none or mild pain while fewer had moderate or severe pain during the early posttreatment days; this treatment difference versus placebo reached statistical significance on days 3 and 4 (P < or = 0.045).(ABSTRACT TRUNCATED AT 250 WORDS)

A double-blind comparative study of soluble aspirin and diclofenac dispersible in the control of postextraction pain after removal of impacted third molars.

The analgesic efficacy and patient acceptability of soluble aspirin and diclofenac dispersible were compared in patients with postoperative pain after removal of impacted third molars. A total of 136 patients were randomly allocated to receive soluble aspirin 600 mg tds or diclofenac dispersible 50 mg tds after extraction under local anaesthesia of impacted third molars on one side of the mouth. The medication, which was both patient and operator blind, was reversed after extraction of the contralateral third molars 3 weeks later, the patients acting as their own controls in assessing postoperative pain, pain relief, and interincisal opening. Patients receiving diclofenac dispersible recorded significantly lower pain levels; pain relief was significantly greater and the patients' assessment significantly favoured diclofenac dispersible. Interincisal opening throughout the study period was significantly increased in the diclofenac dispersible group. The surgeons' postoperative assessment of extraction sites showed no significant difference between the two treatment groups in rate of healing. Two patients reported side-effects while taking soluble aspirin, and eight while taking diclofenac dispersible, two of whom discontinued treatment.

Hepatitis A antibody titres after infection and immunization: implications for passive and active immunization.

Titres of antibodies against hepatitis A virus (HAV) were determined in patients, in donors, and in volunteers after active, passive, and combined immunization. Highest titres were found in recently infected persons: in 109 IgM anti-HAV positive persons, the geometric mean titre (GMT) was 15,400 mIU/ml. The GMT in 265 anti-HAV positive blood donors was 10,700 mIU/ml. The anti-HAV seroprevalence in 19,746 donors increases with age: at the age of 40 years, 50% have antibodies. Titres after active, passive, and combined immunization were studied in three groups: 51 persons received inactivated HAV vaccine at months 0, 1, and 6. The GMT after the booster was 3,400 mIU/ml at month 7. All persons produced more than 100 mIU/ml anti-HAV. Forty-nine persons received both 5 ml immunoglobulin and three vaccinations, yielding a GMT of 1,300 mIU/ml at month 7. One person in this group produced less than 100 mIU/ml anti-HAV. Forty-nine persons received 5 ml immunoglobulin intramuscularly. At day 5 the GMT was 96 mIU/ml. The estimated minimum protective level (10 mIU/ml) was reached in 3 months. Hepatitis A vaccination may supersede the use of immunoglobulin as prophylaxis for travellers to endemic areas. Passive immunization remains necessary for protection during outbreaks. The dosage regimen for passive immunization is based on old studies using preparations with unknown anti-HAV content. Concern regarding the antibody levels in immunoglobulin preparations is justified; the prevalence of HAV antibodies in developed countries continues to fall. Our results indicate that dosage regimens should be reconsidered. Dosage should be deduced logically from the anti-HAV antibody content of the immunoglobulin preparation.