The types of Phasmida in the Natural History Museum, London, UK.

Type specimens of 437 Phasmida taxa have been located in the Natural History Museum, London (NHMUK, formerly BMNH), including 480 primary types of 372 taxa. Taxa with types are listed alphabetically by their specific or subspecific name, and the number of specimens, sex and locality data are given.

Distribution of parasite stages in tissues of Toxoplasma gondii infected SCID mice and human peripheral blood lymphocyte-transplanted SCID mice.

The establishment of Toxoplasma gondii infection in the tissues of SCID mice and SCID mice transplanted with human peripheral blood lymphocytes (PBL) was investigated. The presence of bradyzoites and tachyzoites was analysed in hu-PBL SCID mice using Southern blot of reverse transcriptase-polymerase chain reaction products for the expression of B1, BAG1 and SAG1 of T. gondii. BAG1 was present by week 1 in brain, lung, liver and spleen of some animals; by week 3, BAG1 was present in all animals and in all of these tissues. In contrast, SAG1 was rarely detected until week 2 (mainly in the lung and brain) and by week 3, some animals still did not have detectable SAG1 in brain, lung, liver and spleen. SAG1 expression was increased in the lungs of animals transplanted with human PBL compared to nontransplanted SCID mice. Human PBL engraftment was demonstrated, initially in uninfected mice, by the presence of human CD3+ T cells in the spleen (3.1 x 10(5) positive cells) and peritoneal cavity (3.4 x 10(5) cells) 4 weeks after transplantation. The final outcome of infection was not influenced by the presence of human PBL, with similar mortality in human PBL transplanted and nontransplanted mice. These studies provide a detailed analysis of the kinetics and distribution of both the cyst and tachyzoite stage of T. gondii. This system has been established to allow evaluation of therapies against T. gondii immunodeficient mice in the presence of human immune cells.

Effect of host genotype in determining the relative roles of natural killer cells and T cells in mediating protection against murine cytomegalovirus infection.

The influence of host genotype on the relative importance of T cell subsets and natural killer (NK) cells in controlling murine cytomegalovirus (MCMV) replication has been investigated. Genetically susceptible BALB/c and A/J, moderately resistant C57BL/10, and resistant CBA/CaH mouse strains were treated with monoclonal antibodies (MAb) to the CD4 and CD8 markers and the extent of MCMV replication in major target tissues was determined. Both mouse strain-specific and tissue-specific effects were observed. CBA/CaH and C57BL/10 mice were found not to require CD4+ or CD8+ T cells for control of MCMV replication in the spleen or liver. In contrast, in A/J mice, as well as BALB/c mice, the CD8+ T cell population was primarily responsible for the clearance of virus from these tissues. However, in all strains of mice, CD4+ T cells were required for delayed type hypersensitivity and antibody responses, and for virus clearance in the salivary glands. The dependence of mice with the BALB genetic background on CD8+ T cells for limitation of acute MCMV infection was found to be negated in the BALB.B6-Cmv1(r) congenic strain, in which an effective NK cell response has been generated through the introduction of the resistant Cmv1(r) allele from C57BL/6 mice. Depletion of NK cells in the BALB.B6-Cmv1(r) strain using anti-NK1.1 MAb restored the role of CD8+ T cells in mediating viral clearance. These analyses demonstrate that some, but not all, strains of mice use CD8+ T cells to control MCMV replication and that even when CD8+ T cell-dependence exists, this can be circumvented by an appropriate NK cell response.

Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition.

The amino acid sequence YPHFMPTNL of pp89, the ie1-encoded product of murine cytomegalovirus (MCMV; Smith strain), constitutes an immunodominant T cell epitope recognized in association with H-2Ld. Nucleotide sequencing of MCMV isolates derived from wild mice identified variation between amino acids 147-192 of pp89 in 19 of 27 isolates, including the region encompassing the CTL epitope (amino acid residues 168-176). Four groups of isolates with naturally occurring variant sequences for the CTL epitope were defined: (1) YPHFMPPNL; (2) YPHFMPPSL; (3) YPHFIPPSL; and (4) YLDFMPPNL. The remaining isolates, and the laboratory strains K181 and Vancouver, showed complete identity with the Smith strain. Polyclonal pp89 (Smith strain)-specific CTL only weakly recognized target cells infected with MCMV from most variant groups. No lysis of cells infected with isolate N1 from group 4 was detected. Analyses of cross-reactive recognition of YPHFMPTNL peptide-coated targets by CTL primed with variant MCMV isolates showed that the group 2 and 3 isolates, G4 and K6, respectively, but not the group 4 isolate N1, elicited CTL that exhibited a cross-reactive response. Furthermore, while the group 2 and 3 isolates G4 and K6 were able to prime CTL responses that displayed reactivity to homologous pp89 variant nonapeptides, the group 4 isolate N1 failed to do so. Finally, while immunization of mice with the nonapeptide YPHFMPTNL conferred significant protection against the laboratory strain K181 [correction of Kl81], no evidence of protection was observed for the group 2 and 4 variants G4 and N1, respectively. These observations raise the possibility that clinical isolates of HCMV may also differ in sequence from potential vaccine strains at immunodominant epitopes for CD8+ T cells thus reducing the efficacy of vaccination.

Viral cross-reactivity and antigenic determinants recognized by human parainfluenza virus type 1-specific cytotoxic T-cells.

To obtain information relevant to vaccination against human parainfluenza virus type 1 (hPIV-1), cytotoxic T-lymphocyte (CTL) responses to individual viral components were tested. The CD8-positive T-cell fraction was first enriched from human, adult PBL and grown for several passages in the presence of hPIV-1-infected stimulator cells. T-cell lines were then tested for CTL activity toward hPIV-1 and toward the related viruses hPIV-3 and Sendai virus (the murine parainfluenza type 1 virus). All tested cultures which responded to hPIV-1 also responded to hPIV-3 and Sendai virus, demonstrating sequence conservation between all three viruses among major antigenic determinants for CTL. Specificity for particular viral components was defined using recombinant vaccinia viruses expressing individual proteins from either mouse or human parainfluenza type 1 viruses. Strong CTL responses toward hemagglutinin-neuraminidase, phosphoprotein, and nucleoprotein (NP) were demonstrated. The testing of vaccinia constructs expressing truncated proteins then showed that there were multiple CTL determinants within NP. Several T-cell lines from one donor recognized an NP peptide (amino acids 321-336) conserved between the hPIV-1 and Sendai virus. In total, the results demonstrated that the human CTL response is directed to multiple determinants within several distinct hPIV-1 proteins.

Parainfluenza type 1 virus-infected cells are killed by both CD8+ and CD4+ cytotoxic T cell precursors.

The memory cytotoxic T lymphocyte (CTL) response to human parainfluenza type 1 virus (hPIV-1), a prominent cause of respiratory infection in young children, has been analysed for a panel of healthy adults. The CTL response to the parainfluenza viruses has not been investigated previously. Precursor CTL (CTLp) with activity against hPIV-1-infected Epstein-Barr virus (EBV)-transformed B lymphoblastoid target cells were found at a relatively high precursor frequency (approximately 1/2500-1/4700 CD8+ and CD4+ subsets respectively) in peripheral blood. Both CD4+ and CD8+ CTLp were detected by the analysis of individual microcultures set up under limiting dilution conditions from freshly isolated blood, the phenotype of the responder cell from individual wells being determined by flow cytometry. Further characterization of the CTL response demonstrated MHC restriction by the HLA-A2 glycoprotein in 3/4 HLA-A2+ donors. The presence of effective, hPIV-1-directed T cell memory may explain, in part, the protection observed in the adult population.

T-cell receptor heterogeneity among Epstein-Barr virus-stimulated T-cell populations.

The mechanism by which Epstein-Barr Virus (EBV) escapes T-cell activity during latency in immunocompetent individuals has long been debated. In order to identify potential weaknesses in the EBV-specific immune response, a study of T-cell receptors (TCR) within virus stimulated T-cell populations was performed. Membrane staining techniques and the polymerase chain reaction were used to address two questions: (1) Does EBV behave as a superantigen, thus stimulating, and possibly eliminating, T-cell subsets based on TCR V beta expression?, and (2) Are T-cells dependent on a predominant TCR V beta segment for effective cytotoxic function towards EBV? In search for superantigen effects, V beta repertoires among PBL from seropositive and seronegative individuals were compared both before and after short term in vitro exposure to EBV. In order to characterize functional TCR, the V beta usage among CD8+ EBV-specific cytotoxic T-cell clones was determined. Taken together, results illustrated the strengths rather than weaknesses of the EBV-specific T-cell immune response. T-cells did not respond to EBV in a manner typifying potent superantigen activity, nor did T-cells rely on the expression of a single V beta gene segment for efficient, EBV-specific cytotoxic function.

A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes.

Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-cell epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.

Dissection of an inflammatory process induced by CD8+ T cells.

A massive delayed type hypersensitivity (DTH) reaction occurs in the cerebrospinal fluid (CSF) of mice with lymphocytic choriomeningitis (LCM). In this article, Peter Doherty and colleagues analyze this reaction together with the population dynamics of the regional lymph node to give a comprehensive picture of the events underlying this CD8+ T-cell-mediated immunopathological disease. Their findings are of general relevance to the understanding of inflammation.

Binding of monoclonal antibodies and T cell effector function in vivo.

The capacity of adoptively transferred CD8+ effector T cells to induce meningitis in immunosuppressed, or unsuppressed, recipients infected with lymphocytic choriomeningitis virus (LCMV) may be diminished by prior incubation of the lymphocytes with IgM monoclonal antibodies (MAbs) specific for CD8 or Thy1.2. The same is true, though to a lesser extent, for the further proliferation of donor T cells in the spleens of the immunosuppressed mice. This inhibition of cell mediated immunity can be overcome, at least for the unsuppressed recipients, by increasing the numbers of cells that are transferred, even though exposure to Mab+ complement abrogates all cytotoxic T cell activity in vitro. The LCM model thus provides a quantitative system for assessing the consequences of MAb binding for T cell trafficking and effector function in vivo.

Tumor necrosis factor inhibits the development of viral meningitis or induces rapid death depending on the severity of inflammation at time of administration.

Administration of human rTNF to both male and female mice with severe, but clinically inapparent, lymphocytic choriomeningitis caused death within 2 to 3 h. The development of fatal symptoms was accompanied by a two- to threefold reduction in the number of cells present in the cerebrospinal fluid, with this effect being more apparent in female mice. Giving the same dose of rTNF earlier in the course of the disease was not fatal and reduced the level of subsequent meningitis. In addition, prior exposure to rTNF protected against the acute development of lethal disease when a second dose of the cytokine was given later. The extent of this "tolerance" to rTNF was directly related to the degree of reduction of the inflammatory process resulting from the primary exposure to the cytokine.

In vivo treatment with an appropriate anti-Thy-1.2 monoclonal antibody abrogates hybrid resistance to Thy-1.1+ virus-immune effector T cells.

The hybrid resistance (Hr) effect operates to limit the induction of T-cell-mediated inflammatory processes following transfer into recipients injected with murine lymphocytic choriomeningitis virus (LCMV). Adoptively transferred Thy-1.1+ H-2b (KbDb) LCM virus-immune T cells are inhibited in cyclophosphamide (Cy)-suppressed, virus-infected mice that are heterozygous for H-2Db (e.g. H-2k x b F1). Injection of recipient animals with monoclonal antibody (mAb) to Thy-1.2 resulted in the elimination of Thy-1.2+ H-2b x d F1 effectors on transfer to H-2b x d F1 recipients. The Hr effect was removed by mAb treatment of Thy-1.2+ F1 recipients given H-2b Thy-1.1+-immune T cells. Apparently Hr is mediated by a cell which is Thy-1+.

Contributions of host and donor T cells to the inflammatory process in murine lymphocytic choriomeningitis.

The severe inflammation characteristic of the infection of adult mice with murine lymphocytic choriomeningitis virus (LCMV) is induced earlier in unsuppressed, virus-infected recipients by the adoptive transfer of class I MHC-compatible, CD4- CD8+ LCMV-immune spleen cell populations. The time to onset of fatal LCM may also be slightly diminished, though not to the extent that would be expected from the enhanced kinetics of the extravasation of cells into cerebrospinal fluid. The development of symptoms is thus not solely related to the magnitude of the inflammatory process. The majority of the T lymphocytes in the inflammatory exudate are of host origin and have the size characteristics of resting cells, while the minority population of donor T cells show more of a lymphoblast morphology. The findings are consistent with the idea that relatively few CD8+ virus-immune effectors trigger an inflammatory process which consists largely of secondarily recruited host T cells and monocyte/macrophages.

Virus-specific memory T cells are Pgp-1+ and can be selectively activated with phorbol ester and calcium ionophore.

Memory lymphocytic choriomeningitis virus (LCMV)-immune cytotoxic T-lymphocyte precursors (CTLp) can be stimulated to proliferate and to mediate specific cytotoxic activity following incubation with phorbol myristate acetate (PMA), calcium ionophore (CaI), and interleukin 2 (IL-2). This protocol can be used to selectively induced virus-specific CTL activity under both bulk culture and limiting dilution conditions, in the absence of added antigen. There is no concurrent stimulation of alloreactive CTLp. Proliferation of the effector Lyt-2+ population in medium containing PMA and CaI requires L3T4+ cells, which can be replaced by adding IL-2, and the development of cytotoxicity is totally IL-2 dependent. The LCMV-specific memory T cells are also characterized by the expression of the Pgp-1 (Ly24) glycoprotein. The availability of this marker, together with the capacity to selectively stimulate primed CTLp in the absence of antigen, should greatly facilitate the analysis of T-cell memory in virus infections.

Immunogenetic analysis of cellular interactions governing the recruitment of T lymphocytes and monocytes in lymphocytic choriomeningitis virus-induced immunopathology.

The Lyt2+ class I major histocompatibility complex (MHC)-restricted virus-immune T cells that induce murine lymphocytic choriomeningitis (LCM) are targeted onto radiation-resistant cells in the central nervous system of virus-infected mice. The use of appropriate bone marrow radiation chimeras as LCM virus-infected, (immunosuppressed recipients for immune T-cell transfer has established that, though bone marrow-derived cells can stimulate virus-specific cytotoxic T lymphocytes (CTL) in spleen, they do not reconstitute the barrier to T-cell recruitment from blood to cerebrospinal fluid. This is true for chimeras made up to 8 months previously, even though the inflammatory monocytes and macrophages in such chimeras are all of donor bone marrow origin. Radiation-resistant cells in the spleens of these chimeras are also still able to further stimulate virus-immune CTL. There is no requirement for H-2 compatibility between virus-immune T lymphocytes and secondarily recruited monocytes, or T cells of an inappropriate specificity. The key event in LCM immunopathology may thus be localization of T cells to the antigen-presenting endothelium in brain, leading to the secretion of mediators that promote the nonspecific recruitment of monocytes and other T cells.

Lethal vaccinia infection in cyclophosphamide-suppressed mice is associated with decreased expression of Thy-1, Lyt-2 and L3T4 and diminished IL-2 production in surviving T cells.

Prior treatment of C57BL/6J mice with 300 mg/kg of cyclophosphamide (Cy) converts a subclinical infection with vaccinia virus to a lethal disease. This is accompanied by a loss of more than 80% of spleen cells and a decreased capacity, on a cell-for-cell basis, to develop virus-immune cytotoxic T lymphocytes (CTL), although the frequency of CTL precursors among surviving T cells is not greatly modified. Phenotypically, the surviving T cells express low levels of cell-surface Thy-1, Lyt-2 and L3T4 and, upon stimulation, are less able to produce IL-2 for more than 1 week following Cy treatment. The defect in capacity to generate CTL effectors both in vitro and in vivo can be corrected by providing an exogenous source of IL-2. These experiments indicate that a single dose of Cy induces changes in T cells that persist throughout the development of an immune response. Such effects are in accordance with the known property of Cy to mediate DNA damage.

Flavivirus-specific murine L3T4+ T cell clones: induction, characterization and cross-reactivity.

Murine T cell clones specific for the Kunjin (KUN), West Nile (WN) and Murray Valley encephalitis (MVE) flaviviruses were generated in vitro following priming in vivo. Clones were isolated by limiting dilution and maintained in culture with antigen stimulation and interleukin-2(IL-2). The cells were characterized as having the Thy 1+, L3T4+ and Lyt 2- phenotype by immunofluorescence. All clones proliferated strongly and secreted high levels of IL-2 and IL-3 in response to homologous antigen. Both KUN- and WN-specific clones showed extensive cross-reactivity to KUN and WN antigen, but recognized MVE to a lesser extent. In contrast, MVE-specific clones cross-reacted strongly with both KUN and WN. These data show that antigen-specific, major histocompatibility complex-restricted L3T4+ T cells are generated during flavivirus infection and are cross-reactive for viruses of the same subgroup.

Effect of dietary protein and food restriction on milk production and composition, maternal tissues and enzymes in lactating rats.

Lactating rats have been fed either a protein-restricted diet (10 vs. 20% casein in the control diet) or the control diet at 80, 60 and 40% of the voluntary intake for 7 d from d 7 of lactation. Food consumption, changes in maternal live weight, litter live weight gain and the mass of several maternal tissues were determined together with the activity of several mammary and liver enzymes, including 10 that are essential for fatty acid and complex lipid synthesis. Milk production was estimated from the litter weight gain and litter weight. Lactating rats fed the 20% protein diet ad libitum consumed three times that of nonlactating rats; their liver and kidney masses were significantly higher and their adipose mass was lower. The livers of the lactating rats were fatty, containing 118 mg lipid/g compared with 42 mg/g for the nonlactating rats. Lactating rats fed either the protein-restricted diet or the control diet at 40 and 60% of the ad libitum intake of the control diet had lower mammary, liver and kidney masses than rats consuming the control diet ad libitum. Both protein and food restriction led to lower rates of milk production than those of ad libitum-fed control rats as evidenced by the decrease in litter live weight gains. The concentrations of total lipid, total protein and lactose in milk were not affected by these dietary treatments. The concentration of alpha-lactalbumin in milk of rats fed the low protein diet was, however, lower than that in the milk of all rats receiving the control diet, irrespective of intake. Consumption of the restricted diets resulted in only small changes in specific activities (mu/mg protein) of 15 mammary enzymes. In the livers, lactation led to higher specific activities of all four soluble lipogenic enzymes examined but did not affect the particulate enzymes involved in complex lipid synthesis. The dietary restrictions resulted in lower specific activities of the soluble enzymes compared with those of the lactating rats consuming the control diet ad libitum without affecting the particulate enzymes. Total activities of these enzymes were, however, lower than those for the control rats as a result of the smaller liver mass in the rats receiving the restricted diets.

Anti-asialo GM1 eliminates both inflammatory process and cytotoxic T-cell function in the lymphocytic choriomeningitis adoptive transfer model.

The induction of severe inflammatory process and fatal neurological disease by transfer of lymphocytic choriomeningitis virus (LCMV)-immune T cells into cyclophosphamide (Cy)-suppressed LCMV-infected mice is greatly inhibited by treatment of these recipients with antibody to the asialo GM1 ganglioside (anti-ASGM1). Examination of cytotoxic activity in lymphoid tissue of the Cy-suppressed recipients at 72 hr after cell transfer revealed that anti-ASGM1 treatment prevented the development of the cytotoxic T lymphocyte (CTL) response, even though the dose of antibody used did not significantly decrease CTL generation in unsuppressed mice. Abrogation of CTL activity was also observed following antibody treatment of NK-deficient (bg/bg) Cy-suppressed recipients, indicating that the anti-ASGM1 was unlikely to be operating via removal of NK cells that are in some way involved in the development of the CTL response. The possibility that anti-ASGM1 may act directly on T cells should be considered in all protocols involving the use of this reagent in immunosuppressed mice.