The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

Clathrin-lndependent endocytosis and signalling of interleukin 2 receptors IL-2R endocytosis and signalling.

Interleukin 2 receptors (IL-2R) belong to the cytokine receptor family and share subunits with other members of the family. They are essential in T cell activation and in maintaining homeostatic immune responses. These receptors do not have an intrinsic kinase activity and use multiple signalling pathways. Their endocytic pathway is different from that of classic growth factor receptors in that it does not follow the classic clathrin-coated pit and vesicle route. After uptake, one of the IL-2R chains, alpha, recycles to the plasma membrane, whereas the two other chains, beta and gamma, are targeted to late endosomes/lysosomes and degraded. This involves ubiquitination of the receptor as a sorting signal. Links between the signalling events, internalisation and intracellular sorting of these receptors are reviewed.

A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners.

p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction. Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment.

Comparison of serological tests for detection of Mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens.

Specific pathogen free hens and males were experimentally infected with Mycoplasma gallisepticum. Eggs were then collected, and a part was incubated and set for hatching. Mycoplasma cultures were performed on infected adults and antibodies to MG were analysed by use of slide agglutination (SA) test and commercial ELISA tests on adults and chicks sera and on yolks from non incubated eggs. Both ELISA tests could detect antibodies in yolks from non incubated eggs laid three weeks after infection. SA and the three ELISA tests revealed positive sera in chicks hatched from eggs laid as soon as one week after infection.

Utility of an internal control for evaluation of a Mycoplasma meleagridis PCR test.

Mycoplasma meleagridis, a turkey pathogen, can be detected by PCR directly from tracheal or genital swabs. However, up to 40% samples may contain inhibitory substances. A DNA fragment, that can be amplified with M. meleagridis primers and in the same cycling conditions, was constructed to use as an internal control (IC) to check for these inhibitors. This IC can easily be distinguished from the M. meleagridis amplicon after agarose gel electrophoresis since it is longer. Use of this IC in PCR amplifications revealed that more than 35% of turkey tracheal swabs and more than 45% of turkey cloacal swabs contained inhibitors. In most cases, dilution (1:100) of swab lysates allowed amplification of the internal control but DNA purification may be necessary to eliminate inhibitors (20% of tracheal swabs and 5% of cloacal swabs). Use of this internal control DNA allowed assessment of the efficiency of each individual reaction and ensured that the reaction was not inhibited by interfering substances.

Evaluation of polymerase chain reaction for detection of Mycoplasma meleagridis infection in turkeys.

Different methods were compared for the detection of the turkey pathogen Mycoplasma meleagridis in a turkey field flock before and after antibiotic (oxytetracycline) treatment. They included culture, serology (detection of antibodies by ELISA) and a polymerase chain reaction (PCR) based assay developed by Boyle et al. [Boyle, J.S., Good, R., Morrow, C.J., 1995. Detection of the turkey pathogens Mycoplasma meleagridis and M. iowae by amplification of genes coding for rRNA. J. Clin. Microbiol. 33, 1335-1338]. Culture and PCR assay with tracheal swab samples were much more sensitive than ELISA with serum samples. Percentages of infected birds detected by culture or PCR for samples collected prior to antibiotic treatment were almost identical but the percentage of positive samples detected after antibiotic treatment was much higher with the PCR test.

Detection of Mycoplasma synoviae in poultry environment samples by culture and polymerase chain reaction.

Successful detection of Mycoplasma synoviae (MS) by culture and PCR from samples collected in the environment of experimentally infected chickens and turkeys, or under field conditions, is described. Results showed that in the experimental infection, 10/96 and 46/96 samples of food, drinking water, feathers, droppings or dust were positive by culture and Mycoplasma-PCR. In field conditions, the number of positive results for environmental samples were respectively 7/28 and 17/28. These observations highlight the high disseminating capacities of this mycoplasma and show the usefulness of the PCR method for epidemiological studies.

Development of a blocking enzyme-linked immunosorbent assay for detection of turkey antibodies to Mycoplasma meleagridis.

A blocking enzyme-linked immunosorbent assay (B-ELISA) was developed to detect antibodies to Mycoplasma meleagridis (MM) in turkey sera. This assay was based on two mouse monoclonal antibodies recognising all MM strains tested but none of seven avian mycoplasmal species tested. Furthermore, their binding to the Tween 20 antigen was inhibited by serum from MM-infected birds. The B-ELISA test format was optimized. The cut-off was determined using a set of sera from MM-free turkeys. This B-ELISA was then compared with a commercial indirect ELISA (I-ELISA). Specificities of the two ELISA tests were not significantly different (100 or 99%, respectively). The sensitivity of B-ELISA was significantly higher than the I-ELISA when I-ELISA suspicious results were considered as negative. Testing sera from experimentally MM-infected animals showed that serum plate agglutination (SPA) test detected positive birds before both ELISA methods. Samples were collected in MM-infected commercial flocks and analyzed by SPA, ELISAs, MM-PCR or culture. Results showed that the sensitivity of the B-ELISA appeared superior to the I-ELISA. Moreover, the ability to detect maternal antibodies makes it a useful tool for eradication or control of MM infections.

Comparison of pulsed-field gel electrophoresis with random amplified polymorphic DNA for typing of Mycoplasma synoviae.

Pulsed-field gel electrophoresis (PFGE) analysis was developed and compared with random amplified polymorphic DNA (RAPD) method to type 18 Mycoplasma synoviae (MS) strains. All analysed strains were typeable by RAPD but only 89% of MS strains were typeable by PFGE because of DNA degradation. The discriminatory power of RAPD was greater than that of PFGE but the two techniques had a discriminatory index superior to 0.95, the threshold value for interpreting typing results with confidence. The in vitro, in ovo and in vivo reproducibility of both typing techniques was 100%. However, the interpretation of RAPD patterns was complicated because of inconsistent band intensity. Thus, these molecular typing techniques should be helpful for epidemiological studies of avian mycoplasma infections.

Antigen heterogeneity and epitope variable expression in Mycoplasma meleagridis isolates.

Immunoblotting was used to check the antigenic profiling of 27 Mycoplasma meleagridis strains isolated in different countries. Hyperimmune polyclonal rabbit antiserum as well as monoclonal antibodies (MAbs) raised against M. meleagridis (MM) showed antigen heterogeneity among strains. Five anti-MM MAbs were selected for lack of reaction against heterologous avian mycoplasma. Three of these five Mabs did not cross-react with 63 mycoplasma strains from six species affecting turkeys other than M. meleagridis. The five Mabs used to analyse the epitopes of 30 M. meleagridis strains indicated that some epitopes were not expressed in all strains. Moreover, other epitopes were located on proteins which differed according to number or molecular mass from strain to strain. The five Mabs therefore, recognised variable surface proteins, among which two were amphiphilic membrane proteins. Three of the selected Mabs recognised 29 or 30 of the 30 tested strains. The in vitro expression of surface epitopes in M. meleagridis ATCC 25284 was investigated by colony immunobinding and allowed demonstration of a variable antigenic system.

Efficacy of tilmicosin in the control of experimental Mycoplasma gallisepticum infection in chickens.

This study was conducted to evaluate the efficacy of 5-day, "in water" tilmicosin medication for the prevention of experimental Mycoplasma gallisepticum (MG) disease in 10-day-old specific-pathogen-free (SPF) chickens. Birds were inoculated intratracheally and into the sinus with the MG R-P10 strain. A limited dose titration of the antibiotic over the expected effective range was included, using six groups of 60 birds each: UI: uninfected untreated group; IUT: infected untreated group; IT1 to IT4: four infected treated groups, which were administered 50, 100, 200, or 300 mg/liter of tilmicosin. The birds were given tilmicosin from 8 to 13 days of age and were inoculated at 10 days of age. The birds were observed for 11 days postchallenge before terminal postmortem examination was completed including, assessment of lesions and sampling for mycoplasma culture and serology. Body-weight gains of the different groups were compared. The results showed that tilmicosin medication at dose levels of 50-300 mg/liter significantly decreased growth losses and respiratory signs due to MG infection (P < 0.05). Significant reduction in air sac and peritonitis lesions were obtained by treatment with 100, 200 or 300 mg/liter for 5 days (P < 0.05). A significant reduction in the proportion of MG-culture-positive birds was obtained at a dose level of 50 mg/liter (P < 0.05). Increasing the dose resulted in a further decrease in the number of MG shedding chickens to the extent that with the two highest doses of tilmicosin, no bird was serologically positive on day 21, compared to 46/58 positively infected untreated birds (day 21).

Dose titration study of enrofloxacin (Baytril) against respiratory colibacillosis in Muscovy ducks.

Four-week-old specific-pathogen-free Muscovy ducks were inoculated with reovirus. One week later, they were inoculated intratracheally with a O78:K80 strain of Escherichia coli. The next day, they were given enrofloxacin at different doses in the drinking water. Comparison of mortality rates, weight gain, macroscopic lesions, and E. coli re-isolations among treated and untreated birds showed that a 5-day treatment course with 12.5 or 25 ppm enrofloxacin in water for 4 hours in the morning provided good therapeutic efficacy against respiratory colibacillosis.

Experimental infection of chickens with an atypical Mycoplasma gallisepticum strain: comparison of diagnostic methods.

Fifteen chickens were inoculated with the atypical Mycoplasma gallisepticum (MG) K703 strain. On different dates post inoculation, tracheal swab samples were collected for mycoplasma culture and blood samples were analysed by slide agglutination test (SA) with commercial or homologous antigen and enzyme-linked immunosorbent assay (ELISA) with three different kits. Results showed that MG isolation rate was low on several sampling dates. The SA with commercial antigen did not yield positive results, although birds were positive when tested with homologous antigen. With commercial ELISA kits, the numbers of positive samples remained low. These results illustrate the difficulty of diagnosis of infections with such MG variant strains.

Comparison of antigenic and pathogenic properties of Mycoplasma iowae strains and development of a PCR-based detection assay.

The six reference strains of Mycoplasma iowae (I, J, K, N, Q and R) and 12 field strains, most of them isolated from turkeys, were studied with a growth-inhibition test and a dot immunobinding test with rabbit antisera to the different serovars of M iowae, 16S rDNA gene amplification by polymerase chain reaction, and pathogenicity for chicken or turkey embryos. Antigenic tests tended to be strain specific and showed that most field strains were closely related to serovars K or N. The two pairs of primers chosen in 16S rDNA guided the amplification of 332 base pairs (bp) or 892 bp fragments from all the M iowae strains tested. All the field strains tested were highly pathogenic for turkey embryos.

Efficacy of danofloxacin in the therapy of experimental mycoplasmosis in chicks.

Specific pathogen free day-old chicks were inoculated with a virulent strain of Mycoplasma gallisepticum. Birds received either danofloxacin (50 ppm), tylosin (500 ppm) or no medication in the drinking water from 24 hours after infection for three days. The effects of medication on mortality, weight gain, serology, lesions and reisolation of M gallisepticum 21 days following infection were studied. Treatment with danofloxacin and tylosin significantly decreased mortality and increased weight gain compared with infected unmedicated birds. Twenty-one days after infection, M gallisepticum was isolated from 96 per cent of unmedicated birds compared with only 6 per cent of danofloxacin-treated and 40 per cent of tylosin-treated birds, and the percentage showing positive serological tests was reduced from 100 per cent of unmedicated birds to 0 per cent of danofloxacin-treated and 29 per cent of tylosin-treated birds. In both cases, the proportion of positive birds from the danofloxacin-treated group was significantly lower than that from the tylosin-treated group. The occurrence of air sac lesions was also significantly lower in danofloxacin-treated than in tylosin-treated birds.

A new tyrosine-phosphorylated 97-kDa adaptor protein mediates interleukin-2-induced association of SHP-2 with p85-phosphatidylinositol 3-kinase in human T lymphocytes.

Interleukin (IL)-2 is a major cytokine that controls differentiation and proliferation of T lymphocytes. In this report we characterize an as yet unidentified 97-kDa protein that is a major tyrosine kinase substrate in IL-2-stimulated cells. pp97 was found to associate with the p85.p110 phosphatidylinositol 3-kinase complex, the Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2, and the adaptor molecules CrkL and Grb2. We demonstrate that these interactions are directly mediated through the SH2 domains of CrkL, p85, and SHP-2 and through the SH3 domains of Grb2. pp97 was found to mediate the IL-2-induced interaction between p85 and both a phosphorylated and a non-phosphorylated form of SHP-2. In this study we show that pp97 behaves as a docking protein and associates with at least CrkL, p85, and SHP-2 in the same multimolecular complex. We thus characterized pp97 as a new tyrosine kinase substrate in human T lymphocytes which might play a central role in the regulation of several pathways activated by IL-2.

Bcr/Abl activates transcription of the Bcl-X gene through STAT5.

Several tyrosine kinase oncogenes have been associated with myeloproliferative diseases, including Bcr/Abl, Tel/Abl, Tel/Jak2, and Tel/PDGFR. One target molecule shared by these oncogenes is known to be STAT5. We generated sublines of Ba/F3 cells in which either wild-type STAT5 or a constitutively active mutant of STAT5 (STAT5-1*6) were expressed under the control of a tetracycline-inducible promoter. These cell lines were compared with a Ba/F3 cell line in which the expression of p210(Bcr/Abl) was made inducible by a similar promoter. Before induction, all cells were dependent on interleukin 3 (IL-3) for growth and survival. Both STAT5-1*6 and Bcr/Abl enhanced viability and induced proliferation in the absence of IL-3. We found that the proviability protein Bcl-X(L), but not Bcl-2, was induced by both p210(Bcr/Abl) and STAT5-1*6. Using a Bcl-X gene promoter construct fused to a luciferase complementary DNA (cDNA), both p210(Bcr/Abl) and STAT5-1*6 were shown to induce transcription of Bcl-X. The increase in transcription of the Bcl-X promoter and the increase in Bcl-X protein, due to p210(Bcr/Abl), were blocked by expression of a dominant negative STAT5 mutant. Interestingly, however, STAT5-1*6 required the continued presence of IL-3 to cause a significant increase in Bcl-X(L) protein, whereas p210(Bcr/Abl) did not need IL-3. Studies with enzyme inhibitors suggest that the extra signal supplied by IL-3 may be supplied by the PI3K pathway. Overall, these data suggest that constitutively activated STAT5 can increase viability and proliferation of Ba/F3 cells. This may contribute to, but is not likely sufficient for, the enhanced viability associated with Bcr/Abl transformation.