Graham F. Mitchell

Antigen-induced selective recruitment of circulating lymphocytes

Abstract Thoracic duct lymphocytes obtained from mice 1–2 days after the injection of sheep erythrocytes and injected into thymectomized, irradiated, marrow-protected syngeneic hosts were deficient in adoptively transferring immune reactivity to sheep erythrocytes but normal with respect to horse erythrocytes. Cells collected at 3 days had normal reactivity, but cells collected at 5 days allowed their hosts to produce an enhanced response to sheep erythrocytes and a somewhat depressed response to horse erythrocytes. Thoracic duct lymphocytes obtained from CBA mice 2 days after injection of CBA × C57BL)F 1 spleen cells produced splenomegaly in newborn (CBA × BALB/c)F 1 mice but not in (CBA × C57BL) F 1 recipients. When obtained 5 days after injection, they caused an increase in the splenic index of the (CBA × C57BL) F 1 recipients significantly above that given by control lymphocytes from saline-injected donors. These results are interpreted in terms of a selective recruitment from the circulation of specific antigen-sensitive cells, occurring soon after antigen administration and followed in turn by a rapid reentry of such cells into the pool in an increased proportion.

Isolation and characterization of infective and non-infective clones of Leishmania tropica

Cloning by limit dilution of an isolate of Leishmania tropica (LRC-L137) that is infective for mice resulted in 7 stable clones, only one of which was infective in BALB/c mice. Three of the non-infective clones that were examined for survival in BALB/c macrophages in vitro seemed to be killed more readily, suggesting failure to establish in macrophages as the basis for non-infectivity in vivo. Promastigotes from three non-infective clones and one infective clone were biosynthetically labelled or surface radioiodinated, and the detergent lysates were analyzed by 2-dimensional gel electrophoresis. The pattern of the radiolabelled cytoplasmic and membrane proteins of promastigotes from all L. tropica clones was similar, with minor differences. All clones as well as the uncloned population bound to the same extent to a series of lectins with galactose and N-acetylgalactosamine as specificities. They also bound in a solid-phase radioimmunoassay to 9 monoclonal antibodies raised against the uncloned L. tropica (LRC-L137). The genetic characterization of four L. tropica clones was attempted by analysis of their isolated kinetoplast DNA. The clones from two schizodemes since they possess kinetoplast DNAs which exhibit similar restriction endonuclease fingerprints and show extensive DNA sequence homology, suggesting that the four clones are closely related and that the non-infective variants may be derived from the infective presumptive parental clone L137-7-121. Further characterization of the clones of L. tropica should allow a better understanding of the genetic basis of parasite virulence in cutaneous leishmaniasis.

ISCOMs (immunostimulating complexes): The first decade

A little over a decade ago, novel immunostimulating complexes (ISCOMs) were described. This review examines the position and progress that ISCOM technology has achieved in the fields of vaccine research and medicine over this period. Much of the work on ISCOMs has remained in the area of vaccine research where there is still an urgent need for improved adjuvants to help combat important diseases such as AIDS, malaria and influenza. Currently the only widely licensed adjuvants for human use are the aluminium salts, but with the trend towards highly purified subunit vaccines, which are inherently less immunogenic than some of the older vaccines, potent adjuvants capable of promoting specific immune responses are required. ISCOMs are one such technology that offers many of these requirements and as their use in vaccines enters its second decade clinical trials are commencing that will establish whether these submicron, non‐living particles composed of saponin, cholesterol, phospholipid and in many cases protein, are useful components for a range of human vaccines.

Giardiasis In Mice: I. Prolonged infections in certain mouse strains and hypothymic (nude) mice

The natural history of Giardia muris has been studied in inbred mouse strains and hypothymic (nude) mice derived from a specific pathogen-free facility. Although giardiasis was readily established in several mouse strains, marked variation was observed in the time course of spontaneous elimination of the parasite. During a 10-week study, fecal excretion of Giardia cysts remained relatively constant in C3H/He mice, but decreased at a variable rate in other mouse strains. Resistance to reinfection was greater in strains in which the duration of primary infection was relatively short. Hypothymic (nude) mice derived from a strain showing a relatively rapid elimination of Giardia (BALB/c) maintained a stable infection with high cyst counts. Nude mice reconstituted with lymphoid cells from syngeneic thymus-intact mice showed a progressive reduction in cyst excretion and reconstitution with limited numbers of lymphoid cells from thymus-intact mice previously exposed to Giardia accelerated resolution of infection. In nude mice, giardiasis was associated with a reduction in the villus-crypt ratio of jejunal mucosa, but the degree of change was greater in nude mice reconstituted with lymphoid cells. This Giardia model involving inbred strains and nude mice permits further dissection of the function of thymus-derived cells in intestinal immune responses and induction of changes in small bowel morphology.

Analysis of Infection Characteristics and Antiparasite Immune Responses in Resistant Compared with Susceptible Hosts

Variations in susceptibility or immune responsiveness to parasites, and variations in the consequences of parasitic infection, are readily detected in natural host populations (Wakelin 1978a). Variable outcomes of parasitic infection and disease are predictable bearing in mind that genetic heterogeneity of hosts a«c/parasites is essential for evolutionary survival of either party. It has been argued that immunological events have been the key (but certainly not the only) ingredient in the evolutionary development of balanced host-parasite relationships (Sprent 1959, Burnet & White 1972, and reviewed in Mitchell 1979a,b). The animal breeder expects to exploit any high hereditability of resistance to parasites and searches for linked characteristics which are detected readily, or correlative immune responses, to be used in selection programs. Similarly, the experimental immunoparasitologist expects to exploit at least some genetically-based variations in host resistance to pinpoint immunological effector mechanisms and target parasite antigens (and to identify genetic aspects of expression oi both) which are necessary or sufficient for host protection. Of particular interest are differences in (1) disease manifestations, (2) levels and duration of primary infection, (3) responsiveness to vaccination, (4) responsive-

Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.

Responses to infection with metazoan and protozoan parasites in mice.

Publisher Summary This chapter focusses on the general elements involved in resistance to parasitic infection and the characteristics of these infections that make them likely targets of immunodiagnosis, immunoprophylaxis, and/or immunotherapy. Consideration of work using recent immunologic technology in the study of immune responses to parasitic infections is limited primarily to the mouse. The characteristic elements of these responses, such as hypergammaglobulinemia and eosinophilia, are discussed. The chapter also describes the less well-recognized immunosuppression that accompanies a number of parasitic infections. Factors such as the potential rewards of research, the biological fascination of parasites, and various new sources of research funding, ensure that the field of immunoparasitology evolves into a major area of biomedical research. Numerous model host-parasite systems are in widespread use, and this availability of systems augurs well for the rapid accumulation of knowledge in immunoparasitology. Parasites in their natural hosts are of particular fascination to immunobiologists simply because they generally infect the young in prereproductive life. Severe restraints are imposed on the parasite in terms of the life-threatening consequences of infection and, in this regard, parasites differ from disease entities characteristic of old age, such as tumors.

Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase.

The NH2-terminal amino acid sequence of the Mr 26 000 glutathione S-transferase (EC 2.5.1.18) of Schistosoma japonicum (Sj26) has been deduced by RNA and protein sequence analysis. Using this information, a bacterial plasmid has been constructed that directs the synthesis of the entire Sj26 molecule in Escherichia coli. Recombinant Sj26 exhibits glutathione S-transferase activity and can be readily purified from bacteria in a one-step procedure under non-denaturing conditions. The availability of recombinant Sj26 in essentially unlimited quantities will aid its assessment as a candidate vaccine molecule in schistosomiasis and could eventually lead to the rational design of a drug targetted on schistosome glutathione S-transferases.

Immunization against Taenia taeniaeformis in mice: Studies on the characterization of antigens from oncospheres

Abstract Mature eggs of Taenia taeniaeformis hatched readily in the presence of sodium hypochlorite and no loss in infectivity of oncospheres for mice was observed after hatching. Crude and sodium deoxycholate-solubilized antigens (termed TtO-DOC) prepared from such oncospheres stimulated high levels of protection against T. taeniaeformis infection in immunized mice similar to those described previously for oncospheres prepared by other methods. Mice immunized with TtO-DOC antigens that had been exposed to potassium metaperiodate remained significantly protected against infection. Exposure of TtO-DOC antigens to pronase and thermolysin, or to trypsin, significantly reduced the ability of these antigens to protect mice against infection. These data suggest that the antigens which immunize mice against infection include protein components. 125 I-labelled TtO-DOC antigens were immunoprecipitated with sera from mice infected with T. taeniaeformis and the immunoprecipitates analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation with sera from C3H/He mice infected for 28 days revealed a single major labelled protein antigen having a relative molecular mass ( M r ) of 31,000. Sera from 5-month infected C3H/He mice immunoprecipitated at least thirteen labelled antigens, including one at M r 31,000. Attempts to use SDS-PAGE separated proteins to immunize mice showed that oncosphere antigens exposed to the reducing conditions prior to SDS-PAGE lost their ability to protect mice against infection. It was concluded that SDS-PAGE was an unsatisfactory technique for the isolation of a host protective fraction of TtO-DOC antigens. TtO-DOC proteins were resolved by PAGE performed in the presence of sodium deoxycholate (DOC-PAGE) and mice were vaccinated with cut-outs from the gel. A fraction of the DOC-polyacrylamide gel was found to be effective in immunizing mice against infection. Thus, although the characteristics of the protein antigens in this DOC-PAGE fraction have yet to be determined, an important fractionation technique has been identified. It was shown that partial removal of DOC from oncosphere antigen preparations solubilized in 1% DOC was required for the antigen to stimulate protective immunity. These findings will facilitate further antigen characterization studies towards the development of a defined-antigen vaccine in murine cysticercosis. This is particularly so as attempts to raise anti-oncospheral monoclonal antibodies capable of passively transferring protection to mice by using crude antigen preparations to immunize donor mice have not been successful.

The development of resistance in different inbred strains of mice to infection with nematospiroides dubius.

Summary Infection by the intestinal nematode parasite Nematospiroides dubius was studied in seven different inbred mouse strains. Although there was some minor variation in the susceptibility of the different strains to a primary infection there were marked differences in their ability to develop resistance to infection following repeated exposure to infective larvae. The strains of mice which developed the best resistance also expelled adult worms arising from the previous infections. The adult worms resulting from a primary infection were slowly eliminated in two inbred strains studied whereas no loss occurred from outbred LACA mice. Although males and females of two strains, C3H/HeJand CBA/H were equally susceptible to a primary infection, the females developed better resistance than the male mice following two oral administrations of third stage larvae. Infected mice of every strain and both sexes contained high levels of IgG1, in the serum.