Biomedical subjects
J R Broderson
Publications and source records attributed to J R Broderson.
Chronic energy restriction versus energy cycling and mammary tumor promotion.
Chronic energy restriction significantly inhibits mammary tumor promotion in rodents. The present work studied the effect of short-term, intermittent energy restriction or energy cycling on mammary tumor promotion since this feeding paradigm mimics the dieting habits of humans. Female Sprague-Dawley rats were given 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age (5 mg ig). One week later, rats were randomly divided into three dietary groups. One group was fed ad libitum throughout the study (AL), another (ER) was fed 40% fewer calories than the AL group, and a third, energy-cycled group (EC) was fed in repeated cycles of 2 days of feeding at a level comparable to that of AL rats followed by 2 days of 40% energy restriction. At 10 weeks post-DMBA, the mammary tumor incidences in the AL and EC groups were the same, but incidence in the ER group was significantly lower. A second experiment examined serum levels of three hormones thought to play a role in mammary tumorigenesis. After 12 or 24 days on diet, ER rats had lower insulin levels compared with the other groups. Serum insulin levels in AL and EC rats were the same. After 24 days on diet, estradiol levels were significantly lower and corticosterone levels higher in the ER and EC groups compared with the AL group. Although energy cycling is a type of energy restriction that lowers overall weight gain and energy intake, it does not inhibit mammary tumor promotion as does chronic energy restriction. These data also suggest that feed efficiency and serum insulin levels correlate with susceptibility to mammary tumor promotion.
Failure of high fat diets to promote mammary cancers in spontaneously hypertensive rats.
Rat strains differ in their susceptibilities to chemically-induced mammary carcinogenesis. The present study tested the hypothesis that the spontaneously hypertensive rat (SHR) strain is resistant to mammary carcinogenesis. Resistance would imply the presence of the mammary carcinoma suppressor (MCS) gene. Therefore, we also wanted to test the ability of this gene to inhibit mammary tumor promotion in the presence of high fat diets. Female SHRs were treated with DMBA (5 mg/rat) at 50 days of age and transferred to either a 20% corn oil or 19% menhaden oil + 1% corn oil diet one week later. At 17 weeks post-DMBA none of the rats in either group developed mammary carcinomas. Multiple palpable nodules formed in the mammary gland indicating that initiation had occurred. We conclude that the SHR strain is genetically resistant to DMBA-induced mammary carcinogenesis by mechanisms involving a blockade of promotion.
Inhibitory activity against sporozoites induced by antibodies directed against nonrepetitive regions of the circumsporozoite protein of Plasmodium vivax.
In previous studies, Saimiri sciureus boliviensis monkeys have been immunized with four recombinant proteins reproducing part of the circumsporozoite (CS) protein of Plasmodium vivax sporozoites (NS1(81) V20, rPvCS-1, rPvCS-2, rPv-CS-3), or with irradiated sporozoites of P. vivax Salvador I strain. To analyze the antibody response elicited against epitopes located outside the immunodominant repeat region of the CS protein, serum samples from these animals were tested for their ability to inhibit the in vitro development of liver stages of P. vivax VK247 strain, characterized from the other strains only by a specific repeat region on the CS protein. Results indicated that there is at least one protective B-cell epitope outside the repeat region of the CS protein of P. vivax sporozoites, and that this epitope can be expressed by irradiated sporozoites, rPvCS-1 and -3, but not by rPvCS-1 or NS1(81)V20. Therefore, we designed peptides from the amino acid sequences present both in rPvCS-2 and -3, but not included in the recombinant proteins rPvCS-1 and NS1(81)V20. Anti-peptide antibodies had no activity on the development of sporozoites of P. vivax Salvador I strain, into schizonts in primary culture of Saimiri monkey hepatocytes. In addition, antisporozoite antibodies did not react with any of the peptides. These results suggest that the in vitro inhibition observed in this study could depend upon the conformation of the CS protein. This study also demonstrates that antibody response to unnatural linear epitopes can be induced by immunization with recombinant proteins.
Reinforcement of immunity in Saimiri monkeys following immunization with irradiated sporozoites of Plasmodium vivax.
To determine the duration of immunity to Plasmodium vivax following immunization, six Saimiri sciureus boliviensis monkeys were vaccinated with irradiated sporozoites of P. vivax and challenged multiple times with sporozoites. Over a period of almost four years, complete protection from repeated challenge with infective sporozoites was demonstrated in one monkey; protection in two monkeys was obtained on eight of nine occasions, in one monkey on seven of nine occasions, in one monkey on six or nine occasions, and in one monkey on four of eight occasions. Five of six monkeys were protected against infection during the last six challenges. Inoculation with blood-stage parasites at the end of the trial indicated that all animals were susceptible to infection. These results suggest that protection against sporozoite challenge may be strongly reinforced by subsequent exposure to viable sporozoites.
Immunogenicity and efficacy trials in Aotus nancymai monkeys with model compounds representing parts of a 75-kD merozoite surface antigen of Plasmodium falciparum.
We tested the ability of a recombinant DNA-encoded fragment (C7Ag) of a Plasmodium falciparum merozoite protein (p75) and of two carrier-free peptide models (28-mer and 76-mer) to stimulate boostable antibody responses in Aotus nancymai monkeys. In addition, we evaluated protection against challenge with the Uganda Palo Alto (FUP) strain of this parasite. The data indicate that C7Ag elicited a strong and boostable IgG antibody response in all the monkeys immunized. However, studies with the peptide models demonstrated that various animals produce antibodies to different portions of this structure. When the post-boost sera from monkeys immunized with C7Ag were analyzed for reactivity against two major portions of C7Ag, most of the antibody response was observed against the disulfide-bonded 76-residue region that forms a conformational immunogenic epitope. In the same sera, antibody levels against the charged helical region modeled with a 28-mer were generally low. Immunization with synthetic peptides revealed that the 76-mer stimulated an antibody response almost as strong as C7Ag, with substantial cross-reactivity against the parasite antigen. The 28-mer evoked a response that was not efficient or uniform, and showed little reactivity with the authentic parasite antigen. Aotus nancymai was shown to be susceptible to infection with the Uganda Palo Alto strain of P. falciparum; however, maximum parasitemia varied markedly in both immunized and control monkeys. Statistical analysis failed to recognize differences in maximum parasitemia between the vaccine and control groups. The variation in maximum parasitemia suggests that the FUP strain in this species of Aotus is a poor model for the detection of differences in efficacy based on maximum parasitemia. This initial study with structures based on parts of the 75-kD merozoite surface antigen of P. falciparum indicated that both the recombinant-produced protein C7 and the 76-mer synthetic peptide, when combined with a Syntex adjuvant formulation, were safe and immunogenic in A. nancymai monkeys. However, the data emphasize the problems of using animal models to evaluate the potential effects of immunogens in humans.
Renal pathology in owl monkeys vaccinated with Plasmodium falciparum asexual blood-stage synthetic peptide antigens.
Renal specimens from Aotus monkeys were studied by light microscopy and immunohistochemistry to examine pathologic changes following vaccination with synthetic peptides corresponding to the 35-kD, 55-kD, and 83-kD asexual blood stage antigens of Plasmodium falciparum. The monkeys were vaccinated and later challenged with P. falciparum. In the monkeys vaccinated with Centers for Disease Control peptides (group I), specimens from four of six postvaccinated animals had mild to severe mesangial proliferation and two had diffuse interstitial nephritis. Specimens from three monkeys vaccinated with Colombia peptides (group II) had mild to severe mesangial proliferation and one had interstitial nephritis. In the hybrid polymer-vaccinated monkeys (group III), specimens from three animals had mild to moderate mesangial proliferation and one had severe interstitial nephritis. On the other hand, the control group immunized with bovine serum albumin (group IV) showed that specimens from three animals had mild to severe mesangial proliferation and two had severe interstitial nephritis. In the nonimmunized group (group V), specimens from three animals had moderate to severe mesangial proliferation and two had severe and mild interstitial nephritis. Immunohistochemical analysis using the peroxidase-antiperoxidase method revealed mesangial deposits of P. falciparum antigens in 11 of 14 vaccinated monkeys and in five of 10 unvaccinated controls. These results show that treatment of monkeys with prospective malaria vaccines does not increase the frequency of occurrence or of the severity of renal lesions. These data thus provide a baseline for assessing the safety of synthetic malarial vaccines in the future.
Development of a polymorphic strain of Plasmodium vivax in monkeys.
A strain of Plasmodium vivax from Thailand with a polymorphic repeat unit of the circumsporozoite protein was established in Saimiri sciureus boliviensis and 3 species of Aotus monkeys. All 11 attempts to transmit infection via sporozoite inoculation, 4 times to splenectomized S. sciureus boliviensis, 2 times to splenectomized Aotus nancymai, and 5 times to intact Saimiri monkeys, were successful. Anopheles freeborni, Anopheles stephensi, Anopheles dirus, and Anopheles gambiae mosquitoes were infected by feeding on parasitemic blood from a chimpanzee and an Aotus azarae boliviensis monkey. Our results indicate that this strain may be useful in antisporozoite vaccine trials.
Susceptibility of Macaca fascicularis monkeys from Mauritius to different species of Plasmodium.
Macaca fascicularis monkeys from Mauritius were shown to be susceptible via sporozoite inoculation to 7 species of Plasmodium (P. fragile, P. coatneyi, P. gonderi, P. inui, P. cynomolgi, P. knowlesi, and P. fieldi), indigenous to macaques in southeastern Asia. Four monkeys were sequentially infected with different species of Plasmodium to determine maximum and course of parasitemia. In 2 nonsplenectomized monkeys, P. fragile developed maximum parasite counts of only 134 and 155/microliters. For Plasmodium knowlesi, a parasite that is life-threatening to rhesus monkeys, maximum parasite counts were 4,278 and 7,440/microliters. Plasmodium coatneyi developed to what must be considered as moderate levels. After animals underwent splenectomy, parasite counts of P. coatneyi were 58,280, 89,094, 4,464, and 43,524/microliters. The maximum parasite counts for P. gonderi (13,508 and 21,576/microliters) and P. fieldi (1,767 and 17,836/microliters) were lower than would be expected in M. mulatta. In 2 monkeys that developed patent parasitemia with P. inui, the maximum parasite counts (95,046 and 728,748/microliters) indicated that this parasite may be the best adapted species for development in these animals once infection is established. Finally, the reinfection of 2 monkeys with P. cynomolgi suggested that some animals may be basically more resistant than others, whether splenectomized or not, to the production of high-density parasitemia.
Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence.
Saimiri monkeys immunized with a recombinant protein containing 20 copies of the nine amino acid repeat of the Plasmodium vivax circumsporozoite (CS) protein developed high concentrations of antibodies to the repeat sequence and to sporozoites, but were not protected against challenge. After intravenous injection of an immunoglobulin G3 monoclonal antibody (NVS3) against irradiated P. vivax sporozoites, four of six monkeys were protected against sporozoite-induced malaria, and the remaining two animals took significantly longer to become parasitemic. Epitope mapping demonstrated that NVS3 recognizes only four (AGDR) of the nine amino acids within the repeat region of the P. vivax CS protein. The monkeys immunized with (DRAADGQPAG)20 did not produce antibodies to the protective epitope AGDR. Thus, determination of the fine specificity of protective immune responses may be critical to the construction of successful subunit vaccines.
Antigenic relationships among the 47 human adenoviruses determined in reference horse antisera.
Reference equine antisera to all 47 serotypes of human adenoviruses presently described have been prepared and evaluated by reciprocal neutralization and hemagglutination-inhibition tests. All tests were carried to endpoint dilutions a minimum of five times in each direction to give accurate values for homologous and heterologous antibody titers. Significant cross-reactions in the horse antisera were compared to similar data obtained from rabbit antisera. Using this analysis, major antigenic relationships exist among types 12-18-31 of subgenus A, types 7-11-14 and 34-35 of subgenus B, types 8-9-10, 10-19-37, 13-38-39, 15-22-42, 20-47, 24-32-33-46, and 29-45 of subgenus D, types 16-4 between subgenera B and E, and types 40-41 of subgenus F. Across all subgenera, types 8, 10, 13, 15, 17, 19, 26, 29, 39, 40, and 43 have antigenic moieties found most frequently in other types, averaging 12 heterologous reactions per type when summing both tests in both directions. Types 20, 30, 32, and 45 exhibit shared determinants slightly less often, with a mean of 8 heterologous reactions per type.
Plasmodium cynomolgi: immunization of a rhesus monkey with exoerythrocytic stages cultured in autologous hepatocytes.
To investigate the immune response to exoerythrocytic stages of malaria parasites, a rhesus monkey was immunized with autologous primary hepatocyte cultures infected with 7-day-old liver stage parasites of Plasmodium cynomolgi. A primary antibody response against EE stage antigens was obtained, and boosted after injection of homologous viable sporozoites. Antibodies directed against sporozoites and blood stages were also detected. The polyvalent immune response observed demonstrates the antigenicity of the liver stages and suggests their involvement in the general immune response against malaria.
Renal pathology in Saimiri monkeys during a vaccine trial using the recombinant circumsporozoite protein of Plasmodium vivax.
Renal specimens of squirrel monkeys (Saimiri sciureus boliviensis) were studied by light microscopy and immunohistochemistry to examine the pathologic changes during vaccine trials with four recombinant circumsporozoite (CS) proteins (rPvCS-1, rPvCS-2, rPvCS-3, NS1(81) V20) of Plasmodium vivax. The monkeys were vaccinated and later challenged with P. vivax sporozoites. Among the 33 posttrial biopsies, 17 had mild to moderate mesangial proliferation and nine had interstitial nephritis. Immunohistochemistry by the peroxidase-antiperoxidase (PAP) method revealed IgG deposits in only three of 24 specimens and failed to demonstrate C3 deposits and P. vivax antigens in their glomeruli. There was no relationship between the severity of nephropathy and intensity of parasitemia. The intensity of parasitemia was the same in the vaccinated and control groups. Vaccinated monkeys from the groups (rPvCS-1, rPvCs-2, rPvCS-3) had no differences in renal pathology from the unvaccinated controls, but one group vaccinated with NS1(81) V20 did not develop renal changes.
Inhibitory activity against Plasmodium vivax sporozoites induced by plasma from Saimiri monkeys immunized with circumsporozoite recombinant proteins or irradiated sporozoites.
Two Saimiri monkey vaccine trials have been conducted comparing four recombinant Plasmodium vivax circumsporozoite proteins and irradiated sporozoites. Only a small number of animals immunized with certain recombinants or irradiated sporozoites became fully protected against sporozoite challenge. Preimmunization and postimmunization plasma samples obtained from 30 monkeys on the day of challenge were tested in an in vitro assay based on sporozoite development into exoerythrocytic stages in primary cultures of Saimiri monkey hepatocytes. The percentage of inhibition was determined by comparison of the number of exoerythrocytic stages developing from sporozoites preincubated with a preimmunization and a postimmunization plasma sample of each animal. The plasma samples of the day of challenge of nearly all the immunized animals had a variable, but significant inhibitory effect, when compared with the corresponding preimmunization sample. We found no correlation between the degree of in vitro inhibition of liver stage development, and the in vivo protection against sporozoite challenge of individual animals. The variable results of the incubation of sporozoites with "normal" plasma of different animals indicates that the in vitro results were affected by plasma factors unrelated to anti-sporozoite antibodies.
Spinal dracunculiasis in an experimentally infected ferret.
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Infection of Aotus vociferans monkeys with different strains of Plasmodium falciparum.
Twenty-one splenectomized Aotus vociferans monkeys were infected with the different strains/clones of Plasmodium falciparum. Maximum parasitemia ranged from 1,302 to 1,460,000 parasites per mm3. Only the Santa Lucia strain was shown to produce gametocytes for extended periods. Gametocytes produced during the primary episode of parasitemia were highly infective to Anopheles freeborni mosquitoes. Gametocytes produced during recrudescence were not infective to mosquitoes feeding directly on the animals. This lack of mosquito infection during recrudescence periods suggests the presence of transmission-blocking immunity, which may be important in understanding the control of malaria through immunologic initiatives.
Renal pathology in owl monkeys in Plasmodium falciparum vaccine trials.
Renal specimens of 16 owl monkeys (Aotus vociferans) were studied by light microscopy and immunohistochemistry during a vaccine trial with recombinant proteins of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. Deposition of IgG, C3, and P. falciparum antigens in the mesangium was demonstrated by the peroxidase anti-peroxidase (PAP) method. A relationship between the severity of parasitemia at the time of death and the presence of nephropathy was not apparent.
Further studies on the immunization of Saimiri sciureus boliviensis with recombinant vaccines based on the circumsporozoite protein of Plasmodium vivax.
Reported are the results of a trial in squirrel monkeys of 2 Plasmodium vivax malaria vaccine candidates based on the circumsporozoite (CS) protein, namely, rPvCs-2 and rPvCS-3. Compared with an earlier recombinant P. vivax CS construct, rPvCS-1, rPvCS-2 has an additional 24 amino acids at the C-terminal, which includes the thrombospondin region of homology and a putative T cell epitope. The rPvCS-3 was generated from a chemically synthesized gene that contained an additional 54 amino acids at the amino terminus and terminates at the same carboxy-terminal amino acid as rPvCS-2. In addition, rPvCS-3 contained only 1 each of the repeat sequences DRADGQPAG and DRAAGQPAG. Both antigens were administered with alum as adjuvant. Neither formulation caused toxic side effects and both recombinant molecules induced high antibody titers. Two monkeys were protected against sporozoite challenge by immunization with rPvCS-2 antigen, while none of the rPvCS-3 immunized animals displayed any degree of protection. While there was no correlation between protection and antibody titer or the in vitro proliferation of lymphocytes in response to the antigens, this is further evidence to support the role of the repeating epitopes in generating protective immunity.