Suitability of testicular tissue fluid from castrated piglets to verify sow -vaccination status and herd monitoring.

INTRODUCTION: In a proof of concept, PCV2-specific IgG-antibodies from testicular tissue fluid of seven-day-old castrated piglets were measured to verify the vaccination status of their mothers. Twelve randomly selected sows were vaccinated twice during the last third of gestation with a PCV2 vaccine, while ten controls received only adjuvant. PCV2- specific IgG-antibody titers of serum and colostrum from the sows were correlated with PCV2-specific IgG-antibody titers of serum and testicular tissue fluid of their castrated male offspring. Vaccinated sows showed significantly higher average PCV2-specific IgG-antibody titers in serum (29250 ELISA units, EU) and colostrum (65410 EU) compared to 980 EU and 2630 EU of the control group, respectively. Moreover, significantly higher average concentrations of antibodies were also measured in the serum (9362 EU vs. 247 EU) and the testicular tissue fluid (4022 EU vs. 354 EU) of piglets from vaccinated compared to piglets from adjuvant administered sows. Importantly, a strong linear correlation between PCV2-specific IgG-antibodies in the serum of the piglets and in their testicular tissue fluid was found (rs = 0.9148). PCV2-specific IgG-antibody titers of testicular tissue fluid from five randomly selected piglets allowed the determination of the vaccination status of the herd with a reliability of 98% for vaccinated and 73% for unvaccinated sows. Furthermore, using castration waste products is a very animal friendly method to replace painful and time-consuming blood samplings for herd monitoring or to verify vaccination status.

INTRODUCTION: Dans une preuve de concept, les anticorps IgG spécifiques au PCV2 provenant de liquide tissulaire testiculaire de porcelets castrés âgés de sept jours ont été mesurés pour vérifier le statut vaccinal de leur mère. Douze truies sélectionnées au hasard ont été vaccinées deux fois au cours du dernier tiers de la gestation avec un vaccin PCV2, tandis que dix témoins n'ont reçu que l'adjuvant. Les titres d'anticorps IgG spécifiques au PCV2 dans le sérum et le colostrum des truies étaient corrélés avec les titres d'anticorps IgG spécifiques au PCV2 dans le sérum et le liquide tissulaire testiculaire de leur progéniture mâle castrée. Les truies vaccinées ont montré des titres moyens d'anticorps IgG spécifiques au PCV2 significativement plus élevés dans le sérum (29250 unités ELISA, UE) et le colostrum (65410 UE) par rapport à respectivement 980 UE et 2630 UE dans le groupe témoin. De plus, des concentrations moyennes d'anticorps significativement plus élevées ont également été mesurées dans le sérum (9362 EU contre 247 EU) et le liquide tissulaire testiculaire (4022 EU contre 354 EU) de porcelets vaccinés par rapport aux porcelets de truies n'ayant reçu que l'adjuvant. Il est important de noter une forte corrélation linéaire entre les anticorps IgG spécifiques au PCV2 dans le sérum des porcelets et ceux présents dans leur liquide tissulaire testiculaire (rs = 0,9148). Les titres d'anticorps IgG spécifiques au PCV2 dans le liquide tissulaire testiculaire provenant de cinq porcelets sélectionnés au hasard ont permis de déterminer le statut vaccinal du troupeau avec une fiabilité de 98% pour les truies vaccinées et 73% pour les truies non vaccinées. De plus, l'utilisation de déchets de castration est une méthode très respectueuse des animaux pour remplacer les douloureux et longs prélèvements sanguins pour la surveillance du troupeau ou pour vérifier le statut vaccinal.

Risk factors causing postweaning multisystemic wasting syndrome (PMWS) onset in Swiss pig farms.

Postweaning multisystemic wasting syndrome (PMWS) was epizoozic between 2003 and 2008 in Switzerland. Nevertheless, infectious risk factors including porcine reproductive and respiratory syndrome virus (PRRSV) were missing at all or were seen only sporadically (enzootic pneumonia and actinobazillosis). In a case-control study, 30 farms with PMWS affected pigs were compared to 30 inconspicious farms ("matched pairs"). The case-control allocation was verified by PCV2 DNA measurements of 5 healthy weaned pigs in each control farm, 5 healthy and 5 PMWS affected weaners in each PMWS affected farm. Diseased pigs showed in average 1.8x10(8) DNA templates per ml serum significantly higher than healthy pigs from control farms with 1x10(6) DNA templates per ml serum. Virus load in healthy pigs did not differ between control- and PMWS affected farms. PMWS mainly emerged among affected pigs in the 5th to 8th week of age. In a logistic regression model risk factors were identified such as high occupancy in weaning pens (p = 0.002), large groups in gestation facilities (p = 0.03) as well as reduced birth weight < 1.3 kg (p = 0.04). We suggest these factors might have lead to chronic stress e.g. through influencing negatively social interaction in pigs or disturbances of the maturing immune system. Heavy fly and rodent infestation might not only be viewed as a vector for disease transmission, but, also as a stress factor.

[Infection related fertility disorders in Swiss pig breeding farms at the end of the postweaning multisystemic wasting syndrome (PMWS) epizooty]. / Infektionsbedingte Fruchtbarkeitsstörungen in schweizerischen Schweinezuchtbetrieben am Ende der «Postweaning Multisystemic Wasting Syndrome¼ (PMWS)-Epizootie.

Porcine Circovirus type 2 (PCV2) is able to induce reproductive failures. 286 fetuses from 113 sows of 59 farms with increased reproductive disorders which included abortions, mummies, stillborn and weak born piglets were studied six years after the beginning of the epizooty of postweaning multisystemic wasting syndrome (PMWS) in Switzerland. 14 % of the cases were bacterial infections based on histological signs of inflammation and pathogen isolation. 12 % further cases showed inflammatory reactions by histology without pathogen identification. PCV2 was identified in only 4 % of cases by immunohistochemistry (IHC). Thus, PCV2 infections are of minor importance in respect to pig reproductive failures in Switzerland. Porcine parvovirus (PPV) infections were found in 3 % of the cases and seem to occur more infrequently compared to former findings. Hitherto, Enteroviruses/Teschovirus were marginally studied in etiologically undefined cases with a prevalence of 11 %. To our knowledge this is the first identification of Enteroviruses/Teschovirus in fetal tissue from reproductive failures in Switzerland. The etiology remained unclear in more than 50 % of all cases in spite of modern diagnostic methods.

Efficacy of simultaneous vaccination with Enterisol® Ileitis and Ingelvac® CircoFLEXTM in a Swiss breeding farm.

This study explores administration of two piglet vaccines as compared to the mono- and adjuvant-application. A vaccine against the Porcine Circovirus Type 2 (PCV2) cap protein subunit and a vaccine with attenuated live culture against Lawsonia (L.) intracellularis were applied to piglets aged 23.5 days on average. 1'405 animals were divided randomly into four groups. One piglet group was immunized with both vaccines while two other groups were immunized with a combination of one vaccine and adjuvants of alternate vaccination protocol and vice versa. These piglet groups were also compared to a control group supplemented with both adjuvants only. During fattening, pigs, which were simultaneously immunized with Enterisol(®) Ileitis and Ingelvac(®) CircoFLEX(TM) vaccine, gained significantly more weight (792 g/day) when compared to piglet groups mono-vaccinated with Ingelvac® CircoFLEXTM (772 g/day) or either with Enterisol® Ileitis (774 g/day). Moreover, immunized piglet groups showed significantly higher daily weight gain when compared to adjuvants only inoculated control group (751 g/day). Additionally, during fattening the control group displayed higher mortality (6,3 %) than the three vaccinated groups (Ingelvac(®) CircoFLEX(TM) 2,5 %, Enterisol(®) Ileitis 2,3 % and the combination of both vaccines 1,1 %). These data imply that simultaneous immunization with PCV2- and L. intracellularis specific vaccines positively benefit piglet growth observed by an additive effect on growth parameters in farms harboring both pathogens. Return of investment was calculated of 2.10 on the additional Enterisol(®) Ileitis vaccination.

[Economic impact of Circovac® - vaccination in a PCV2 subclinically infected farm in Switzerland on performance parameters]. / Ökonomische Auswirkungen der CIRCOVAC® - Impfung in einem subklinisch mit PCV2 infizierten Zuchtbetrieb in der Schweiz auf die Mastleistungsparameter.

Vaccination of dams in a PCV2 subclinically infected farm 2 and 4 weeks before insemination, with a booster at 12 weeks of gestation did not influence fertility parameters of the dams. However, growth parameters of offspring of vaccinated sows improved significantly (+ 51 g/d), resulting in a shorter growing period of 9 days and a massively improved economy. Mortality of weaners and fattening pigs was not significantly influenced by dam vaccination. Nevertheless, compared to a period of 6 months before vaccination, the mortality rate declined in the weaning period by 0,3 % and in the fattening period by 5,5 %. The Return on Investment (ROI) was calculated with 1:9.5. Even, the historically low pork prices in 2011 led to a ROI of 1:7.

Vaccination of dams increases antibody titer and improves growth parameters in finisher pigs subclinically infected with porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is the obligate infectious agent in postweaning multisystemic wasting syndrome (PMWS) of pigs. To control PMWS, we vaccinated dams at 4 and 2 weeks before pregnancy and again in the 12th week of gestation with an inactivated PCV2 vaccine (Circovac). Two producer farms run under the control of Swiss Swine Health Organization were selected for the experiment. Previously, in one farm PMWS was diagnosed on pigs after weaning, whereas in the other farm, pigs wasted during the fattening period. For the experiments 113 dams were randomly vaccinated, and 111 dams were sham injected. Vaccination increased serum antibodies in dams 3- to 9-fold, accompanied by serum antibody titer increases in their offspring. In the sixth week of life, progeny from vaccinated dams had about the same IgG antibody titers as progeny of unvaccinated dams at the third day of life. In sera of vaccinated dams only low concentrations of PCV2 DNA were detected, and no progeny developed PMWS. Interestingly, at day 56 four progeny of unvaccinated dams tested positive for anti-PCV2 IgM antibodies, indicating a primary infection with PCV2. Of economic importance is the observation that progeny of vaccinated dams had a significantly higher daily weight gain in the fattening period (farm X, +51 g/day; farm Y, +30 g/day) and thus a shortened fattening period of about 6 days compared to progeny of controls. To our knowledge this is the first demonstration of subclinical circovirus infection and its effects on growth performance of fattening pigs by vaccination of dams.

Molecular evidence for chlamydial infections in the eyes of sheep.

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.

A comprehensive test system to identify suitable antibodies against p53 for immunohistochemical analysis of canine tissues.

The tumour suppressor p53 is commonly detected in tissues of companion animals by means of antibodies raised against the human protein. The following three-step procedure was devised to test the suitability of such antibodies for immunohistochemistry on canine tissues. (1) Western blot and immunohistochemical analyses on bacterially expressed recombinant canine protein to assess human-to-canine cross-reactivity. (2) Immunohistochemistry of cultured, UVB-irradiated canine keratinocytes to evaluate suitability for detection of endogenous p53. (3) Immunohistochemistry on tissue arrays to further substantiate suitability of the antibodies on a panel of normal and neoplastic human and canine tissues. Five of six antibodies cross-reacted with recombinant canine p53. Three of these (PAb122, PAb240, CM-1) also immunolabelled stabilized wild type p53 in cell cultures and elicited a consistent, characteristic labelling pattern in a subset of tumours. However, two alternative batches of polyclonal antibody CM-1 failed to detect p53 in cell cultures, while showing a characteristic labelling pattern of a completely different subset of tumours and unspecific labelling of normal tissues. The test system described is well suited to the selection of antibodies for immunohistochemical p53 detection. The results emphasize the need to include appropriate controls, especially for polyclonal antibodies.

Intensively kept pigs pre-disposed to chlamydial associated conjunctivitis.

In the present study, ocular chlamydial infections in pigs that originate from two different farming systems were investigated. In particular, the aim was to test pigs with and without clinical ocular symptoms for the presence of Chlamydiaceae and for linked infections with Acanthamoebae spp. possibly acting as vectors for Chlamydia or Chlamydia-like organisms. In a total of 181 pigs, 102 from Germany (GER), representing the intensively kept animals and 79 from Switzerland (CH), which were kept extensively, were screened for the presence of different pathogens by PCR, including a new Chlamydiaceae-specific intergenic spacer rRNA gene PCR. Additionally, results of clinical examination and cytology were compared between the symptomatic and asymptomatic pigs of the two groups. Ocular symptomatic pigs showed a high prevalence of Chlamydia suis in both groups: CH 79%, GER 90%. Only 23% asymptomatic pigs from CH, but 88% asymptomatic pigs from GER were positive for C. suis by PCR. DNA of Chlamydia-like organisms were detected in 19% CH, but only in 2% GER pigs, whereas only 4% CH and 1% GER pigs were also positive for Acanthamoebae spp. A co-infection of Acanthamoebae spp. and C. suis was present in only 3% of the CH but 28% of the GER pigs. In general, the intensively kept pigs in our study seemed to be pre-disposed to ocular chlamydial infection and associated conjunctivitis. Infections with Chlamydia-like organisms alone and in combination with Acanthamoebae played no role for clinical findings within the tested pig groups, whereas a co-infection of Acanthamoebae and C. suis was able to cause serious ocular manifestations in half of the cases of intensively kept pigs being positive for these microorganisms.

Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells.

Removal of apoptotic cells is a dynamic process coordinated by ligands on apoptotic cells, and receptors and other signaling proteins on the phagocyte. One of the fundamental challenges is to understand how different phagocyte proteins form specific and functional complexes to orchestrate the recognition/removal of apoptotic cells. One evolutionarily conserved pathway involves the proteins cell death abnormal (CED)-2/chicken tumor virus no. 10 (CT10) regulator of kinase (Crk)II, CED-5/180 kDa protein downstream of chicken tumor virus no. 10 (Crk) (Dock180), CED-12/engulfment and migration (ELMO) and MIG-2/RhoG, leading to activation of the small GTPase CED-10/Rac and cytoskeletal remodeling to promote corpse uptake. Although the role of ELMO : Dock180 in regulating Rac activation has been well defined, the function of CED-2/CrkII in this complex is less well understood. Here, using functional studies in cell lines, we observe that a direct interaction between CrkII and Dock180 is not required for efficient removal of apoptotic cells. Similarly, mutants of CED-5 lacking the CED-2 interaction motifs could rescue engulfment and migration defects in CED-5 deficient worms. Mutants of CrkII and Dock180 that could not biochemically interact could colocalize in membrane ruffles. Finally, we identify MIG-2/RhoG (which functions upstream of Dock180 : ELMO) as a possible point of crosstalk between these two signaling modules. Taken together, these data suggest that Dock180/ELMO and CrkII act as two evolutionarily conserved signaling submodules that coordinately regulate engulfment.

Prevalence of chlamydiae in semen and genital tracts of bulls, rams and bucks.

Chlamydiae infect male genital organs of ruminants. However, little is known about their prevalence. Hence, we investigated fresh and cryopreserved semen (bulls: n=304; rams: n=78; bucks: n=44) by polymerase chain reaction (PCR), as well as genital organs (bulls: n=13; rams: n=10; bucks: n=6) by immunohistochemistry (IHC) and PCR. Sera from bulls (n=104) and small ruminants (n=61) were tested by LPS and rMOMP (recombinant major outer membrane protein) ELISA and competitive ELISA (cELISA), respectively. Three PCR assays were compared in this study for detection of chlamydial DNA in semen: 16S rRNA, IGS-S (intergenic spacer 16S/23S-short), and IGS-L (intergenic spacer 16S/23S-long) PCRs. PCR sensitivity and inhibitory effects were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. In bull semen, detection limits of the 16S, IGS-S and IGS-L PCRs were 10, 10, 100 templates, respectively. However, PCR sensitivity was reduced in ram and buck semen suggesting the presence of potential PCR inhibitors. Of 304 bull semen samples, the 16S PCR revealed DNA of chlamydiae in 20 samples (6.6%), including Cp. abortus (n=2), Cp. psittaci (n=1), Chlamydia suis (n=2), and Chlamydia-like organisms (n=15). In rams, one semen sample was positive for Chlamydia-like organism. All investigated male genital organs were negative for Chlamydia. Serology revealed 47.1% (49/104) positive bulls by LPS ELISA. Of these, 30 samples were positive by rMOMP ELISA, predominantly for Cp. pecorum. In small ruminants, cELISA displayed 34.8% (16/46) and 60% (9/15) positivity for Cp. abortus in rams and bucks, respectively. There was no correlation between serology and PCR of semen. The presence of chlamydiae in semen suggests the possibility of venereal transmission, although risk may be low in Switzerland.

Chlamydia-related abortions in cattle from Graubunden, Switzerland.

In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.

Detection of chlamydiae in boar semen and genital tracts.

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.

Chlamydiales in guinea-pigs and their zoonotic potential.

The aim was to detect and characterize chlamydial infections in guinea-pigs (GP) with ocular disease, study their pathogenicity and zoonotic potential and to test for the presence of Acanthamoebae spp. in GP eyes and to investigate whether they could act as vectors for Chlamydia-like organisms. Overall 126 GP, of which 77 were symptomatic, were screened by clinical examination, cytology, gross pathology, histology, immunohistochemistry, polymerase chain reaction (PCR) and bacteriology. A new Chlamydiaceae-specific intergenic spacer rRNA gene PCR, designed to amplify this segment linking the 16S and 23S regions, was performed. DNA samples were also received from one owner including samples of his cat and rabbit. Guinea-pigs: 48 of 75 symptomatic, but only 11 of 48 asymptomatic GP were positive by PCR for Chlamydophila caviae guinea-pig inclusion conjunctivitis (GPIC) (P < 0.0001). Eighteen of 75 or 15/48, respectively, were positive for DNA from Chlamydia-like organisms. Acanthamoebae-DNA could be found in two GP, of which one was symptomatic. Owner, cat and rabbit: Samples of all three species were positive by PCR for C. caviae GPIC and the owner's one-day disposable contact lenses showed a positive PCR result for the Chlamydia-like organism Parachlamydia acanthamoebae. No Acanthamoebae-DNA could be detected. This study is the first to describe Chlamydia-like organisms in GP and to detect C. caviae GPIC in human, cat and rabbit. Therefore, C. caviae GPIC could pose a zoonotic potential. We believe that the finding of C. caviae GPIC in species other than GP is probably not unique.

CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration.

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Evidence for a conserved role for CRKII and Rac in engulfment of apoptotic cells.

Apoptosis or programmed cell death occurs in multicellular organisms throughout life. The removal of apoptotic cells by phagocytes prevents secondary necrosis and inflammation and also plays a key role in tissue remodeling and regulating immune responses. The molecular mechanisms that regulate the engulfment of apoptotic cells are just beginning to be elucidated. Recent genetic studies in the nematode Caenorhabditis elegans have implicated at least six genes in the removal of apoptotic cell corpses. The gene products of ced-2, ced-5, and ced-10 are thought to be part of a pathway that regulates the reorganization of the cytoskeleton during engulfment. The adapter proteins CrkII and Dock180 and the small GTPase Rac represent the mammalian orthologues of the ced-2, ced-5 and ced-10 gene products, respectively. It is not known whether CrkII, Dock180, or Rac proteins have any role during engulfment in mammalian cells. Here we show, using stable cell lines and transient transfections, that overexpression of wild-type CrkII or an activated form of Rac1 enhances engulfment. Mutants of CrkII failed to mediate this increased engulfment. The higher CrkII-mediated uptake was inhibited by coexpression of a dominant negative form of Rac1 but not by a dominant a negative Rho protein; this suggested that Rac functions downstream of CrkII in this process, which is consistent with genetic studies in the worm that place ced-10 (rac) downstream of ced-2 (crk) in cell corpse removal. Taken together, these data suggest that CED-2/CrkII and CED-10/Rac are part of an evolutionarily conserved pathway in engulfment of apoptotic cells.

Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells.

A central paradigm of T cell development is that CD4+8+ (DP) thymocytes differentiate into CD4+ or CD8+ T cells in response to intrathymic signals that extinguish transcription of the inappropriate coreceptor molecule. Contrary to this prevailing paradigm, we now demonstrate that signaled DP thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells. Remarkably, thymocytes that have selectively terminated CD8 transcription can be signaled by IL-7 to differentiate into CD8+ T cells by silencing CD4 transcription and reinitiating CD8 transcription, events we refer to as "coreceptor reversal." These observations significantly alter our understanding of CD8+ T cell differentiation and lead to a new perspective ("kinetic signaling") on CD4/CD8 lineage determination in the thymus. These observations also suggest a novel mechanism by which bipotential cells throughout development can determine their appropriate cell fate.

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells.

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

Functional domains of the heavy metal-responsive transcription regulator MTF-1.

Metallothioneins (MTs) constitute a class of low molecular weight, cysteine-rich, metal binding proteins which are regulated at the level of gene transcription in response to heavy metals and other adverse treatments. We have previously cloned a zinc finger factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in metallothionein promoters and shown that this factor is essential for basal and heavy metal-induced transcription. Here we report that the C-terminal part of MTF-1 downstream of the DNA binding zinc fingers harbours three different transactivation domains, namely an acidic domain, a proline-rich domain and a domain rich in serine and threonine. When fused to the heterologous DNA binding domain of the yeast factor GAL4 these activation domains function constitutively, i.e. transcription of a GAL4-driven reporter gene is not induced by heavy metals. In search of the region(s) responsible for metal induction, external and internal deletion mutations of mouse and human MTF-1 and chimeric variants thereof were tested with a reporter gene driven by a metal-responsive promoter. The N-terminal part of MTF-1 containing the zinc fingers, which are dependent on zinc for efficient DNA binding, can indeed confer a limited (3- to 4-fold) zinc-responsive transcription when fused to the heterologous activation domain of the viral VP16 protein. Another region containing the acidic and proline-rich activation domains also contributes to metal inducibility, but only in the context of intact MTF-1. This indicates that the activity of MTF-1 results from a complex interplay of different functional domains.