The role of Plasmodium knowlesi in the history of malaria research.

In recent years, a malaria infection of humans in South East Asia, originally diagnosed as a known human-infecting species, Plasmodium malariae, has been identified as a simian parasite, Plasmodium knowlesi. This species had been subject to considerable investigation in monkeys since the 1930s. With the development of continuous culture of the erythrocytic stages of the human malarial parasite, Plasmodium falciparum in 1976, the emphasis in research shifted away from knowlesi. However, its importance as a human pathogen has provoked a renewed interest in P. knowlesi, not least because it too can be maintained in continuous culture and thus provides an experimental model. In fact, this parasite species has a long history in malaria research, and the purpose of this chapter is to outline approximately the first 50 years of this history.

The major histocompatibility complex class II-linked cim locus controls the kinetics of intracellular transport of a classical class I molecule.

The dominant trans-acting major histocompatibility complex (MHC)-linked class I modifier (cim) locus, previously recognized through its ability to determine altered alloantigenicity of a rat class I molecule, RT1.A3, is shown here to influence class I intracellular transport. The MHC recombinant laboratory rat strains PVG.R1 and PVG.R8 display unusually long retention of RT1.Aa within the endoplasmic reticulum or cis-Golgi. In appropriate F1 hybrid cells heterozygous for RT1.Aa and another class I MHC allele, RT1.Ac, only the RT1.Aa protein is subject to slow transport. The cim gene product therefore shows class I allele specificity in its action, cim appears to be a polymorphic locus whose product is directly involved in the processes of class I MHC assembly and/or intracellular transport.

The role of subregions of the rat major histocompatibility complex in the rejection and passive enhancement of renal allografts.

The laboratory recombinant haplotype H-1acl of the Norway rat has been used in studies of the rejection and passive enhancement of kidney allografts. While the full H-1a haplotype provoked acute rejection, neither of the isolated subregions, H-1Aa and H-1Ba, did so. It was also found that alloantisera raised against either the H-1Aa or the H-1Ba antigens would enhance the grafts. It is suggested that both MHC subregions contain a histocompatibility locus (i) for kidney (as they do for skin) and that in the genetic combinations studied only incompatibility for both provokes a response sufficient for rejection. In other combinations, however, single region incompatibilities may be sufficient.

Immune response genes controlling responsiveness to major transplantation antigens. Specific major histocompatibility complex-linked defect for antibody responses to class I alloantigens.

We have identified two major histocompatibility complex (MHC)-linked Ir genes that control the antibody response made by rats against class I major alloantigens. We have named these genes Ir-RT1Aa and Ir-RT1Ac. These Ir genes determine responsiveness of the immunized animal in a typical codominant fashion. There is no evidence so far for trans-complementation between low-responder haplotypes. Detailed studies of Ir-RT1Aa indicate that it controls the antibody response to at least two distinct alloantigenic determinants on RT1Aa molecules. These class I molecules thus behave like hapten-carrier conjugates when the response against the carrier is under Ir gene control. Analysis of the origin of alloantibody-forming cells in tetraparental radiation chimeras indicates that Ir-RT1Aa must control the provision of effective help to B cells. In many respects therefore, the properties of Ir-RT1Aa are broadly similar to those described for Ir genes controlling antibody responses to conventional antigens. The existence of apparently conventional Ir genes controlling the antibody response to major alloantigens strongly suggest that the processing of these transmembrane molecules by host antigen-presenting cells is a prerequisite for immune induction, and that it is the MHC of the responder rather than that of the allograft to which T helper cells are restricted in alloimmune responses in vivo.

A trans-acting major histocompatibility complex-linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule.

The RT1.A locus of the rat MHC encodes the H chain of the single classical class I molecule of this species. One of the alleles of this polymorphic locus, RT1.Aa, is present in several laboratory inbred, congenic, and MHC recombinant rat strains. Studies of the RT1.Aa class I molecule from a number of these strains as a target for CTL show that its antigenicity, both as an alloantigen and a restricting element, is subject to gain and loss alterations by the action of a gene mapping in the MHC to the right of RT1.A. This locus is apparently present in two allelic forms (one possibly a null allele) corresponding to the presence or absence of a dominant transacting modifier, and has been named class I modification, or cim. The antigenic change brought about by cim is scarcely detectable serologically but highly immunogenic for CTL. Biochemical investigations show that cim affects the post-translational modification of RT1.Aa.

Generation of T cells with lytic specificity for atypical antigens. II. A novel antigen system in the rat dependent on homozygous expression of major histocompatibility complex genes of the class I-like RT1C region.

Lymphocytes from parental strain DA rats can induce potent killer cell responses to atypical antigen systems in F1 Lewis (L)/DA and DA/L recipients. Here, we describe an antigen system, H, present on homozygous parental target cells, but not on F1 cells. This antigen system is unusual in several respects: it does not involve class I RT1A gene products usually used by killer cell responses in the rat, it maps to the major histocompatibility complex (MHC) class I-like RT1C region, and it requires homozygous expression of RT1Cav1 alleles. This may be another example, this time involving the RT1C region, of an MHC gene product antigenically altered by an MHC-linked trans-activating modifier gene.

Tumorigenicity conferred to lymphoma mutant by major histocompatibility complex-encoded transporter gene.

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules requires MHC-encoded molecules of the adenosine triphosphate binding cassette (ABC) family. Defects in these proteins represent a potential risk, since they are essential links in the machinery of T cell-mediated surveillance which continuously scrutinizes peptide samples of cellular proteins. Nevertheless, transfection of the mouse lymphoma mutant RMA-S with the rat ABC gene mtp2a (homologue to mouse HAM2 and human RING11), commonly termed TAP-2 genes, led to a marked increase in tumor outgrowth potential in vivo. This occurred despite restored antigen presentation and sensitivity to cytotoxic T lymphocytes, and was found to be due to escape from natural killer (NK) cell-mediated rejection. It has previously been proposed that adequate expression of self-MHC class I is one important mechanism to avoid elimination by NK cells. Our data argue that a defect in the machinery responsible for processing and loading of peptides into MHC class I molecules is sufficient to render cells sensitive to elimination by NK cells. The latter thus appear to function as a surveillance of the peptide surveillance machinery.

Generation of T cells with lytic specificity for atypical antigens. I. A mitochondrial antigen in the rat.

F1 rats primed with normal parental strain lymphocyte populations and restimulated in culture with parental lymphoblasts generate potent cytotoxic T cell responses to unusual antigen systems. Here we describe in the Lewis (L)/DA anti-DA combination an antigen system most likely of mitochondrial origin with the following properties: it is transmitted maternally from DA strain females, inherited in an extra-chromosomal manner, restricted by class I RT1Aa major histocompatibility complex gene products, extinguished on target cells treated with chloramphenicol, and its pattern of expression in different rat strains correlates with restriction fragment-length polymorphisms of mitochondrial DNA. Sequence analysis of the rat ND1 gene indicates that the maternally transferred factor in the rat is not a homologue of the maternally transmitted factor responsible for the mitochondrial antigen in mice. In keeping with its inheritance from DA females, this antigen is present on target cells from (DA female x L male)F1 donors and all other F1 combinations derived from DA female parents, but absent from target cells from some F1 combinations (L/DA and Wistar-Furth [WF]/DA) derived from DA strain males. The presence of this antigen in other F1 combinations (Brown Norway [BN]/DA, August 2880 [AUG]/DA, and PVG/DA) indicates that this mitochondrial antigen system is shared by the DA, BN, and PVG strains, but not by the L and WF strains.

The endurance shuttle walk test: an alternative to the six-minute walk test for the assessment of ambulatory oxygen.

UK guidelines for domiciliary oxygen have suggested the six-minute walk test or shuttle walk tests as suitable functional measures for the clinical assessment of ambulatory oxygen (AO). To date, there is limited evidence that would support the use of shuttle walk tests as assessment tools for AO. The endurance shuttle walk test (ESWT) is used increasingly as an assessment tool within pulmonary rehabilitation (PR) but its potential as an investigative test for AO has not been explored. Using the same test for both PR and AO assessment is appealing since it would improve efficiency and act to standardise outcome measures in this patient population. The aim of this study was to examine the responsiveness and repeatability of the ESWT to AO and to compare the response with that of the six-minute walk test (6MWT). Twenty-three patients with chronic obstructive pulmonary disease (COPD) performed, in random order, the ESWT and the 6MWT on air and whilst breathing AO. Oxygen saturation and Borg ratings of breathlessness and perceived exertion were recorded. On a third day, eleven patients repeated the ESWT with AO in order to measure repeatability. There was a significantly greater change in the ESWT with oxygen than the change recorded from the 6MWT (66 [91] vs 6 [28] m respectively; P < .05). When repeated on a separate day, the mean difference (95% CI) between distances walked on the ESWT with AO was 0.91 (-47, 49) m. The ESWT was more responsive than the 6MWT for detecting improvements in walking endurance whilst breathing AO.

LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes.

BACKGROUND: Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. RESULTS: We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. CONCLUSIONS: Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression.

Co-evolution of rat TAP transporters and MHC class I RT1-A molecules.

The genes for rat major histocompatibility complex (MHC) class I molecules are associated either with those for the A allele of the transporter associated with antigen processing (TAP-A), which can transport peptides with basic carboxy-terminal residues, or with those for TAP-B, which cannot [1-5]. To explore whether these associations have a functional basis, we compared the sequences of 13 rat MHC class la RT1-A cDNAs from nine MHC haplotypes. Of seven TAP-A- linked RT1-A molecules, six possess strongly acidic F pockets, and these bind a high proportion of peptides with basic carboxy-terminal residues. The F pockets of TAP-B-linked molecules, by contrast, were more basic. Furthermore, we identified six positions at the 'righthand end' of the peptide-binding groove, at which a majority of TAP-B-linked molecules diverge from the consensus sequence for class la molecules whereas, at these positions, all the TAP-A-linked molecules reflect the consensus sequence. Our results suggest that the linked rat class la and TAP genes have co-evolved to maximize the supply of appropriate peptides to the presenting molecules.

Development of malaria blood-stage vaccines: learning from mosquitoes.

If current methods of vaccine development for malaria continue to result in vaccines with only relatively limited degrees of protection, what is the alternative? Here, a totally different approach to blood-stage vaccine research is suggested, focusing on malarial immunity as it develops in macaque monkeys, but using methodology already well established in mosquito research.

Effect of antibiotic growth promoters on broiler performance, intestinal growth parameters, and quantitative morphology.

The effects of addition of bacitracin methylene disalicylate (BMD) or virginiamycin (VM) to a corn-soybean meal diet on broiler performance and gastrointestinal tract (GIT) growth parameters and morphology were studied at various ages during growth and finishing. Male and female birds were killed at 1, 3, 5, or 7 wk of age for gross and histologic examination of the duodenum and ileum. Feeding either antibiotic increased BW and decreased intestinal length and weight at all times compared with control birds. However, intestinal length and weight decreases were greater in birds fed VM than BMD at 1 and 3 wk of age. The only change found in the duodenum resulting from dietary treatment was an increase in the number of villi per unit length in birds given VM but not BMD or control. In the ileum, the muscularis mucosa was thinner in birds given VM than in those fed the control diet. Chicks supplemented with VM had a smaller total villus area and shorter villus height and crypt depth in the ileum than birds fed the control diet or BMD. Physical changes in the intestine of birds given either antibiotic growth promoter, although not the same, resulted in improved performance.

Troponin of asynchronous flight muscle.

Troponin has been prepared from the asynchronous flight muscle of Lethocerus (water bug) taking special care to prevent proteolysis. The regulatory complex contained tropomyosin and troponin components. The troponin components were Tn-C (18,000 Mr), Tn-T (apparent Mr 53,000) and a heavy component, Tn-H (apparent Mr 80,000). The troponin was tightly bound to tropomyosin and could not be dissociated from it in non-denaturing conditions. A complex of Tn-T, Tn-H and tropomyosin inhibited actomyosin ATPase activity and the inhibition was relieved by Tn-C from vertebrate striated muscle in the presence of Ca2+. However, unlike vertebrate Tn-I, Tn-H by itself was not inhibitory. Monoclonal antibodies were obtained to Tn-T and Tn-H. Antibody to Tn-T was used to screen an expression library of Drosophila cDNA cloned in lambda phage. The sequence of cDNA coding for the protein was determined and hence the amino acid sequence. The Drosophila protein has a sequence similar to that of vertebrate skeletal and cardiac Tn-T. The sequence extends beyond the carboxyl end of the vertebrate sequences, and the last 40 residues are acidic. Part of the sequence of Drosophila Tn-T is homologous to the carboxyl end of the Drosophila myosin light chain MLC-2 and one anti-Tn-T antibody cross-reacted with the light chain. Lethocerus Tn-H is related to the large tropomyosins of Drosophila flight muscle, for which the amino acid sequence is known, since antibodies that recognize this component also recognize the large tropomyosins. Tn-H is easily digested by calpain, suggesting that part of the molecule has an extended configuration. Electron micrographs of negatively stained specimens showed that Lethocerus thin filaments have projections at about 39 nm intervals, which are not seen on thin filaments from vertebrate striated muscle and are probably due to the relatively large troponin complex. Decoration of the thin filaments with myosin subfragment-1 in rigor conditions appeared not to be affected by the troponin. The troponin of asynchronous flight muscle lacks the Tn-I component of vertebrate striated muscle. Tn-H occurs only in the flight muscle and may be involved in the activation of this muscle by stretch.

Elevated growth hormone responses to pyridostigmine in obsessive-compulsive disorder: evidence of cholinergic supersensitivity.

The growth hormone response to the acetylcholinesterase inhibitor pyridostigmine was measured in nine normothymic outpatients who met DSM-III-R criteria for obsessive-compulsive disorder. The responses were significantly elevated when compared to those found in a healthy comparison group (N = 9). The data suggest that cholinergic supersensitivity is present in obsessive-compulsive disorder.

Peptide binding characteristics of the non-classical class Ib MHC molecule HLA-E assessed by a recombinant random peptide approach.

BACKGROUND: Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E. RESULTS: An in vitro refolding system was used to assess binding of recombinant HLA-E to either specific peptides or a nonamer random peptide library. Peptides eluted from HLA-E molecules refolded around the nonamer library were then used to determine a binding motif for HLA-E. Hydrophobic and non-charged amino acids were found to predominate along the peptide motif, with a leucine anchor at P9, but surprisingly there was no methionine preference at P2, as suggested by previous studies. CONCLUSIONS: Compared to the results obtained with rat classical class Ia MHC molecules, RT1-A1c and RT1-Au, HLA-E appears to refold around a random peptide library to reduced but detectable levels, suggesting that this molecule's specificity is tight but probably not as exquisite as has been previously suggested. This, and a previous report that it can associate with synthetic peptides carrying a viral sequence, suggests that HLA-E, similar to its mouse counterpart (Qa-1b), could possibly bind peptides different from MHC class I leader peptides and present them to T lymphocytes.

The new recombinant haplotypes r19 and r20 in RT1, the MHC of the rat.

Two new recombinants, designated r19 and r20, have been found in RT1, the rat major histocompatibility complex. In both recombinants the A and I regions appear to be derived from one parental haplotype, and a further region, able to induce both skin graft rejection and cytotoxic lymphocytes is derived from the other. The properties of this region appear similar to the previously described RT1C and the putative genotypes of the PVG X R19 and PVG X R20 congenic lines are therefore AaIaCc and AcIcCa, respectively. Evidence suggesting that the two recombinants may not be reciprocal is discussed.

Graft rejection in a congenic panel of rats with defined immune response genes for MHC class I antigens. I. Rejection of and priming to the RT1Aa antigen.

Allograft rejection in the rat has been shown to be under stringent immune response (Ir) gene control using major histocompatibility complex recombinant animals as donors. Presentation of an isolated class I antigenic difference to high responder recipients results in rapid graft rejection, but low responders fail to reject. This striking qualitative difference is also seen in some liver grafting experiments in which the donor presents a full MHC haplotype and minor antigen mismatch to the responders. Grafts of other organs, however, do not discriminate qualitatively between high and low responders when a full haplotype mismatch exists. We have used the canonical high and low-responder animals, (PVG X PVG-RT1u)F1 and PVG to examine whether any qualitative difference in responsiveness can be detected against the a haplotype using a variety of organ grafts. We have confirmed a qualitative difference between high and low responders using PVG.R1 donors presenting an isolated class I (Aa) difference. Rapid rejection by high responders contrasted with complete failure to reject by the low responders. No difference in rejection tempo was found when a full a haplotype mismatch was introduced. This could have reflected vigorous responses to I and C region differences, because rapid rejection through these regions was demonstrated using the PVG.r1 (AaIcCc) and PVG.r8 (AaIuCu) recombinants. The feeble immunogenicity of the Aa antigen for PVG animals was revealed by priming and cross-priming experiments showing not only that r1 failed to prime for subsequent r1 graft rejection, but that the Aa antigen presented in concert with Ia and Ca also failed to prime. An unexpected result was that the Aa antigen of r1 actually suppressed responsiveness, especially when delivered by a heart graft. This suppression not only extended to subsequent r1 grafts (for example, skin rafts) but also to subsequent grafts of a tissue. The mechanism of this suppression remains unclear but preliminary experiments argue in favor of enhancement rather than active suppression.

Graft rejection in a congenic panel of rats with defined immune response genes for class I antigens. II. Quantitative aspects of Ir gene function in a full-haplotype mismatch.

Previous studies have shown that the Ir-gene-controlled rejection of rl tissues by c/u responder and non-rejection by c low responders does not extend to tissues expressing a full a haplotype mismatch. However, antibody responses and liver graft rejection are both defective in low responders, even across a full haplotype barrier. We have therefore used a titrated adoptive transfer assay to search for quantitative differences in the responsiveness of c and c/u animals to a organ grafts. We first established that a heart graft rejection could be ablated in both recipient strains with whole-body irradiation and could be restored with syngeneic cells. Titration of restorative cells revealed that 5 times as many c cells were required to restore graft rejection in c recipients as c/u cells were required in c/u recipients. Use of cells from primed donors showed that in both c/u and c animals these cells had undergone about a 5-fold increase in potency, showing that there was no failure of proliferation and differentiation in the low responder after contact with antigens. Cross-transfer experiments were done to attempt to localize the defect in low-responder animals either to a failure of low-responder antigen-presenting cells (APC) to trigger a response or a defect in the responsiveness of alloreactive cells toward the a antigens. In these experiments c cells were obtained from radiation chimeras of the c----c/u type. These cells were used to restore graft rejection in c/u irradiated recipients. Similar experiments employing c/u cells obtained from c/u----c chimeras and given to irradiated c recipients were also done. These showed that c cells from chimeras were marginally less potent than c/u cells from chimeras. In contrast when cross-transfer of c/u cells to c animals bearing a nonrejected rl heart was done, no rejection was seen even when antigen presenting cells were cotransferred. The conclusions from this series of experiments were that quantitatively small defects were present in both repertoire and antigen presentation, and that these quantitative defects in aggregate were probably sufficient to explain the documented low responsiveness of c animals to the a haplotype. The failure of high-responder c/u cells to secure rejection of rl tissues in the low-responder c environment suggests that presentation of isolated class I differences in host APCs is mandatory for rejection to occur and is highly defective in the c animal.

Major histocompatibility complex control of NK-related allogeneic lymphocyte cytotoxicity in rats. The contributions of strong and medial transplantation antigens.

Allogeneic lymphocyte cytotoxicity (ALC) describes the elimination of allogeneic lymphocytes in vivo by an NK-related activity. There is evidence that ALC is demonstrable between donor and recipient when these are incompatible at MHC gene loci alone. Since ALC is a property of T cell-deficient nude rats, the role of the MHC in this rejection process needs further study. We have determined the contribution of the MHC to ALC using congenic and recombinant rats. In our analysis we have assumed that ALC involves the recognition of classic alloantigens by clonally distributed effector cells as for other examples of transplant rejection, although this is not yet proved. Strong ALC was measured between congenic rats that differed for MHC genes only. Non-MHC incompatibility alone did not elicit ALC. In the presence of MHC incompatibility the strength of ALC generated in a recipient was dependent on non-MHC genes. The PVG background generated high ALC responses whereas ALC was not measured in the DA rat. However ALC was measured in the congenic PVG-RT1avl (DA) rat. The contributions of classic class I (RT1.A), class II (RT1.B/D), and medial transplantation (RT1.C) regions of the rat MHC were determined by comparing different recombinant donors into the same recipient strain. Single region differences alone in any of these three MHC regions did not elicit full ALC. In two sets of transfers a combination of RT1.B/D and RT1.C region incompatibility was sufficient to generate a full allogeneic response. It can be concluded that the controlling element for allogeneic lymphocyte cytotoxicity is in the RT1.B/D-RT1.C region of the rat MHC.