Profile of the mosaic element BTMR1 in the genome of the bumble bee Bombus terrestris (Hymenoptera: Apidae).

Co-evolution involving a mariner transposon, Botmar1 and the other repeats contained in the Bombus terrestris genome was investigated. We found that the 5'-region of Botmar1 forms one of the components of a mosaic element, known as B. terrestris mosaic repeat 1 (BTMR1), which is also composed of inner segments originating from two different retrotransposons and a pseudogene corresponding to an RNA methyltransferase cDNA. The fact that BTMR1 is interspersed within chromosomes and the differences in its abundance in different species indicate that it is very probably a mobile element. Nevertheless, the absences of direct or inverted repeats at its ends and of target site duplication indicate that its mobility is not ensured by a cardinal transposable element, but putatively by a Crypton-like element.

Hypodermin A, a new inhibitor of human complement for the prevention of xenogeneic hyperacute rejection.

BACKGROUND: Hyperacute rejection (HAR) of discordant xenografts in the pig-to-human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea-pig-to-rat and in the pig-to-human combinations. METHODS: Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea-pig-to-rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC11) were transfected with HA complementary DNA (cDNA). RESULTS: The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 +/- 16 U/ml vs. 700 +/- 63 U/ml, P < 0.05). Sera from rats injected with 3.2 mg/kg of HA were less effective in lysing guinea-pig endothelial cells (12 +/- 7%) than normal rat sera (79 +/- 3%; P < 0.001). Ex vivo, guinea-pig hearts perfused by rat serum supplemented with HA survived longer than those perfused by non-treated serum (210 +/- 34 and 154 +/- 71 min, respectively; P < 0.05). In vivo, guinea-pig hearts transplanted into HA treated rats survived longer than in non-treated rats (27 +/- 5 min vs. 13 +/- 4 min; P < 0.001). In the presence of human serum, smaller amounts of C6 and C5b-9 were deposited onto HA-transfected CHO cells than onto control cells. The mHA-PAEC11 cells were significantly more resistant to lysis by human C than control PAEC11 cells. CONCLUSIONS: These data suggest that transgenic HA could be used to prevent hyperacute xenogeneic rejection.

Hypodermin a and inhibition of lymphocyte proliferation.

Infection with Hypoderma lineatum is accompanied by modulation of the host's immune system. Here, Nathalie Moiré describes some in vitro studies examining how the parasitic enzyme hypodermin A interferes with lymphocyte proliferation.

Enzymatic effect of hypodermin A, a parasite protease, on bovine lymphocyte membrane antigens.

The protease hypodermin A (HA) is produced by the parasitic warble-fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti-lymphocyte receptor antibodies tested. Anti-BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti-MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 micrograms/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)-induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

Sero-surveillance of hypodermosis in a herd under therapeutic control. Effect of a low level of infestation.

A cattle herd from the experimental farm of INRA in Nouzilly has been treated for hypodermosis since October 1990. Additionally, a regional eradication scheme has been implemented in this area since autumn 1992. Bi-monthly warble counts were performed between March and July each year on an average of 200 animals. No warble was recorded in this herd from 1991 to 1994 with the exception of two dairy cows in 1993. In autumn 1994, therapeutic control measures were stopped. Serological surveys were performed in the autumn of each year from 1991 until 1995. Anti-Hypoderma antibodies were found in 25%, 27.2%, 4.3%, 3.2% and 0% of the animals respectively. An experimental low infestation was conducted in the summer of 1994. During the spring 1994, third instars of Hypoderma bovis were collected from naturally infested animals. From a total of 13 pupae, six adults (four males and two females) emerged and were released in the herd of Nouzilly on 24 June and 4 July. In October 1994 serological investigations revealed two animals seropositive for hypodermosis. This number increased to six in January 1995. The antibody kinetics of these six animals remained parallel throughout the next 6 months: the titres increased up to April and started to fall in May to return to negative values in August. Manual examinations of the animals at weekly intervals between April and July revealed the presence of four warbled animals with one, one, two and three warbles respectively. The two other seropositive animals remained warble free. One other animal showed antibody titre fluctuations between negative and low positive values, but was warble free in the spring. In October 1995 all the animals of the herd were seronegative. The interpretation and the value of a sensitive immunodiagnosis in a large eradication programme are discussed and compared with warble counts, especially in the case of a low level of infestation.

Cross-reactive, stage-specific antigens in the Oestridae family.

The larval stages of different economically important Oestridae species were studied for their antigenicity and cross reactivity, using ELISA and immunoblotting. The immune sera of cattle from Algeria, Belgium, France and Switzerland were compared for their capacity to recognize the stage-specific antigens of their specific parasites Hypoderma bovis and H lineatum, originating from different populations. This comparison was extended to other Hypoderminae species responsible for economic losses in European animal production: H diana in roe deer and H tarandi in reindeer. The specific host for each of these parasites recognized common epitopes of hypodermin C, a collagenolytic enzyme previously well characterized in the first instar of H bovis and H lineatum. No cross reactivity was found with other Oestridae, such as Oestrus ovis or Gasterophilus intestinalis. The specificity of hypodermin C was evaluated using sera from animals harbouring other arthropods such as ticks, helminths, including Fasciola hepatica and Haemonchus contortus, or protozoa such as Anaplasma sp, and no reaction was observed. The use of hypodermin C is therefore suggested as an antigen in the immuno-survey of economically-important Hypoderminae.

Effect of the parasite enzyme, hypodermin A, on bovine lymphocyte proliferation and interleukin-2 production via the prostaglandin pathway.

The immune function of cattle infected with a primary infestation of Hypoderma lineatum is impaired during the first instar migration of the larvae. Hypodermin A (HA) is an enzyme secreted by the larvae that is implicated in immunosuppression. The response of bovine peripheral blood mononuclear cells (PBMC) to HA was examined in this study. HA blocked their proliferation in response to phytohaemagglutinin (PHA) and its effect was enhanced when cells were preincubated with HA before activation. This suggests that HA affects the lymphocyte commitment to blastogenesis during the early stages of their activation. HA also markly reduced the production of interleukin-2 (IL-2) in PHA-stimulated bovine PBMC cultures. Furthermore, indomethacin, which inhibits prostaglandin (PG) synthesis, blocked the immunosuppressive effect of HA on the PBMC proliferative response. The concentration of PGE2 in medium of PBMC or PMA-stimulated monocyte cultures was increased by incubation with HA. Thus, the HA appeared to act by reducing IL-2 production via a prostaglandin-dependent pathway.

Sequencing and gene expression of hypodermins A, B, C in larval stages of Hypoderma lineatum.

The cDNAs of hypodermins, enzymes secreted by the larvae of the parasitic fly Hypoderma lineatum, were sequenced. Four cDNA clones were isolated, one encoding hypodermin A (HA), one encoding hypodermin C (HC), and the two others encoding proteins related to hypodermin B (HB). The amino acid sequences deduced from the nucleotide sequences confirmed that these enzymes are serine proteases. HA and one of the HB proteins had potential N-linked glycosylation sites. Analysis of hypodermin protein, RNA and DNA at different larval stages indicated that protein overexpression is regulated transcriptionally for HA and HB, and by transcriptional and DNA amplification for HC.

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.

Induction of cytolytic function in resting peripheral blood CD8+/Leu-7+ T cells through IL2/p 75 IL2-receptor interaction: a study in the allogeneic human bone marrow transplantation model.

CD8+/Leu-7+ T cells which circulate in increased proportions in the blood of long-term surviving BMT patients are for the most part high-density resting lymphocytes lacking IL2R-alpha (p55) expression. We show that they can be induced by IL2 to manifest cytolytic function after 24-48 hr stimulation by using rather high concentrations of IL2 (at least 50 U/ml). This function was much more readily induced in high-density CD8+/Leu-7+ T cells than in high-density CD8+/Leu-7+ T cells and occurred in the presence of minimal cell proliferation. Other cytokines involved in primary CTL differentiation (IFN-gamma, IL4 and IL6) were without effect suggesting that CD8+/Leu-7+ T cells are, in the BMT model, in vivo preactivated CTL ready to differentiate into cytolytic effectors under the sole IL2 stimulus. TU27 Mab directed at IL2R-beta (p75) subunit almost completely prevented IL2-induced cytolytic function of CD8+ T cells while 33B3.1 Mab directed at IL2R-alpha (p55) subunit was ineffective, suggesting that the signal for this function has its origin in IL2R-beta chains constitutively expressed by these cells.

Role of interleukin 2 receptor beta chain in initiating anti-CD3 and interleukin 2-induced proliferation of human resting T cells.

We have examined the role of isolated interleukin 2 receptor (IL2R) beta chains expressed by human resting T cells in the early period of primary T cell activation induced by soluble OKT3 monoclonal antibody (mAb) and exogenous IL2. In the initial 3-day-stimulation phase, high IL2 concentrations were required, in association with soluble OKT3 mAb, to induce the formation of IL2R alpha/beta heterodimers, while later, low IL2 concentrations were sufficient to promote cell growth. When added during the initial phase, TU27 mAb directed at the IL2 binding site of IL2R beta chain substantially inhibited the appearance of functional high-affinity IL2R. Lo-Tact-1 mAb directed at the IL2 binding site of the IL2R alpha chain had only a marginal effect. Strong induction of IL2R alpha mRNA occurred within 3 days upon OKT3 and IL2 stimulation even in the presence of Lo-Tact-1 mAb, but not in the presence of TU27 mAb. OKT3 alone failed to induce significant IL2R alpha gene transcription and that induced by IL2 alone was very weak. The constitutive expression of IL2R beta mRNA was visualized in resting T cells. It remained at a rather stable level, at least during the initial stimulation period which was examined herein. Given the fact that OKT3 alone was ineffective in up-regulating IL2R beta mRNA expression and that pre-incubation of the cells with OKT3 alone did not allow them to respond to high concentrations of IL2, it is highly probable that isolated IL2R beta chains constitutively expressed by CD8+ T cells (the main reactive cells in this system) are primarily responsible for the initial interaction of IL2 with these cells. Such an interaction will result in the formation of high-affinity IL2R and in the initiation of cell proliferation provided that a CD3-derived co-signal is simultaneously delivered to the cells.

IL-2 receptors on circulating natural killer cells and T lymphocytes. Similarity in number and affinity but difference in transmission of the proliferation signal.

IL-2-binding sites expressed on purified circulating NK cells and high density T lymphocytes were enumerated in 125I-IL-2 binding assays and analyzed by autoradiography after chemical cross-linking of IL-2 to the cells. Quite similar profiles of IL-2R were exhibited by both types of cells consisting of the simultaneous expression of approximately 150 high affinity Tac+ receptors/cell (Kd congruent to 19 pM) and of approximately 540 intermediate affinity Tac- receptors/cell (Kd congruent to 800 pM) which appeared, in cross-linking experiments, to be the isolated 70-kDA protein (p70) subunit of the 55-kDa protein (p55)/p70 heterodimer. The high affinity receptors were distributed on less than 3% of the cells and could be eliminated by complement lysis with anti-p55 mAb. In these conditions, Scatchard analysis no longer revealed two classes of binding sites but only one class of binding sites with intermediate affinity. Although expressed in equal numbers on the surface of NK cells and resting T lymphocytes, the constitutive p70 chains seemed to transmit differently the proliferation signal after effective ligand interaction. Thus, NK cells proliferated strongly in the presence of only 260 pM IL-2, (40 U/ml), whereas resting T cells remained unresponsive to IL-2 concentrations able to saturate the existing p70 receptors (6.5 nM IL-2, 1000 U/ml IL-2) unless monocytes were present. The initiation of cell division seemed to involve the synthesis of p55 chains and the constitution of high affinity receptors as introducing anti-Tac antibody at the start of the cultures inhibited IL-2-induced proliferation. Tac mRNA transcripts accumulated rapidly in NK cells during a 18-h observation period whether anti-Tac antibody was present or not during IL-2 stimulation. In contrast a weak Tac mRNA induction was observed in resting T cells in the same conditions.