Barcoding quarantine nematodes and their close relatives: an update on the QBOL-project.

Identification of plant pests, in particular quarantine species, needs to be fast and accurate to enable timely plant protection measures. In addition, a false diagnosis can cause serious financial losses for trade and producers. It is now well established that genetically based diagnosis is a reliable alternative to the classical identification procedures generally based on morphological features, which usually require expert taxonomic skills. On the other hand, genetic diagnosis through the use of DNA-barcodes, i.e. stretches of DNA that contain taxon-specific information, can be performed by any skilled laboratory worker. The European Union 7th framework project QBOL aims to establish DNA-barcodes for all European quarantine organisms as well as their close relatives. The results and protocols will be disseminated in the publicly available and curated database Q-BANK. To enable genetically based identification requires knowledge of the genetic variation both within and between the species of interest as well as their close relatives. For the nematodes, several gene regions (the COI, COII, SSU, LSU and RNA polymerase subunit II) are being evaluated for their barcoding potential.

Small subunit rDNA-based phylogeny of the Tylenchida sheds light on relationships among some high-impact plant-parasitic nematodes and the evolution of plant feeding.

Cyst (Heteroderidae), root knot (Meloidogyne spp.), and lesion (Pratylenchus spp.) nematodes all belong to a single nematode order, Tylenchida. However, the relationships between and within these economically highly relevant groups, and their relatedness to other parasitic Tylenchida is unclear. We constructed a phylogeny of 116 Tylenchida taxa based on full length small subunit ribosomal DNA (small subunit [SSU] rDNA) sequences. Ancestral state reconstruction points at a gradual development of simple to more complex forms of plant parasitism. Good resolution was observed in distal clades that include cyst, root knot, and lesion nematodes, and monophyly of most families was confirmed. Our data suggest that root knot nematodes have evolved from an ancestral member of the genus Pratylenchus, but it remains unclear which species is closest to this branching point. Contrary to the notoriously polyphagous distal representatives, basal members of the genus Meloidogyne (and probably, their common ancestor) have narrow host ranges. Our analysis also shows that mitotic parthenogeny has arisen at least two times independently among root knot nematodes. In many cases resolution till species was observed, suggesting that SSU rDNA sequences have a potential for DNA barcode-based species identification with, due to the overall conserved nature of this gene, limited intra-species variation.

Cardiovascular embryology.

During the first 20 days of development, the human embryo has no cardiovascular structure. Over the next month, the heart and great vessels complete their development and look very much like they will at full gestation. This amazing process transforms isolated angiogenic cell islets into a complex, four-chambered structure. During this transformation, the single heart tube begins to beat at 23 days of development and by 30 days blood circulates through the embryo.

Living-related bowel transplantation to treat short bowel syndrome in a four-year-old child: a case report.

PURPOSE: Short bowel syndrome (SBS) is an extremely challenging clinical problem in children. Although many patients can be maintained for a period of time on total parenteral nutrition (TPN), many of these children suffer from the morbidity and mortality associated with sequential central line infections, venous thromboses, and TPN-induced liver failure. Intestinal transplantation often is the only chance for long-term survival. Unfortunately, many children die every year waiting for size-matched cadaveric intestine to become available. METHODS: After our success with living-related bowel transplantation in adults, the authors successfully transplanted 150 cm of maternal ileum into a 4-year-old 10-kg child with profound malnutrition from SBS and advanced TPN-induced liver failure. Because of the size mismatch, the abdominal cavity could not be closed primarily. The defect was covered with absorbable mesh and subsequently with skin graft. RESULTS: The patient is home with excellent bowel and liver function, off hyperalimentation, and on a regular diet. No rejection has been encountered. CONCLUSIONS: Living-related intestinal transplantation is a life-saving alternative to cadaveric intestinal transplantation in children with short bowel syndrome.

Paraneoplastic pemphigus and bronchiolitis obliterans associated with a mediastinal mass: A rare case of Castleman's disease with respiratory failure requiring lung transplantation.

Castleman's disease is an infrequent and usually benign lymphoproliferative disorder. Resection of the tumor usually is curative. The immunostimulatory nature of the tumor can, in rare instances, result in paraneoplastic manifestations. The authors present a case of a 14 year old with mucocutaneous ulcerations and progressive dyspnea that was found to have a large mediastinal mass and circulating autoantibodies that were responsible for his paraneoplastic pemphigus and bronchiolitis obliterans. In spite of aggressive immunotherapy to control the autoimmune mucocutaneous lesions, the pulmonary fibrosis was irreversible and progressed to pulmonary failure necessitating lung transplantation. J Pediatr Surg 36:E22.

Targeted delivery of anti-CTLA-4 antibody downregulates T cell function in vitro and in vivo.

CTLA-4 is a T cell surface molecule that binds to the costimulatory molecules CD80 and CD86 on antigen-presenting cells and downregulates T cell function. Therefore, we wanted to test whether antigen-specific activated T cells could be inhibited through directed CTLA-4 signaling using a bispecific antibody (BiAb) capable of simultaneously binding to CTLA-4 and a tissue-specific antigen. The BiAb was prepared by linking two separate monoclonal antibodies against CTLA-4 and the thyroid-stimulating hormone receptor (TSHR). The mouse B cell lymphoma line M12 (H2(d)) was used to induce alloreactive T cells in CBA/J mice (H2(k)); M12 cells stably transfected with the cDNA encoding murine TSHR (mM12) were used to restimulate the alloresponse in vitro. Results of assays for in vitro T cell proliferation, IL-2 production, and cytotoxicity in the presence of BiAb demonstrated that the BiAb could inhibit the T cell alloresponse when stimulated with mM12 cells but not with M12 cells. This effect was dependent on binding of TSHR-bound BiAb to CTLA-4, since the addition of soluble CTLA-4-Ig blocked the inhibitory effect. Injection of mM12 cells, along with the BiAb, not with antibodies against TSHR or CTLA-4 either separately or together, into CBA/J mice (H2(k)) downregulated alloreactive T cell responses. Our study demonstrated that the presence of CTLA-4 signaling molecules on the surface of target cells can protect those cells from immune attack by antigen-specific T cells and suggested that a similar approach could have potential therapeutic value in transplant rejection and tissue-specific autoimmune diseases.

Combinatorial search of substituted beta-cyclodextrins for phosphatase-like activity.

Thirteen per-6-akylamino-6-deoxy-beta-cyclodextrin libraries (beta-CD libraries) were generated by a solution-phase combinatorial synthesis starting from per-6-iodo-6-deoxy-beta-CD and different combinations of eleven individual amine nucleophiles. Certain libraries showed the ability to hydrolyzep-nitrophenyl phosphate in the presence of Zn2+.

Antenatal urologic intervention: current status.

The devastation caused by fetal obstructive uropathy is now well known. At the most severe end of the spectrum of obstructive uropathy not only is the developing kidney damaged but the resultant oligohydra-mnios prevents pulmonary development and causes skeletal defects. The most significant changes are noted in patients with posterior urethral values (PUV). The obvious solution to the problem is to either correct or by pass the obstruction prior to the development of permanent changes. Unfortunately, this simple concept is not easy to apply since it raises numerous ethical, legal, economic and technical problems.

Surgical implications of necrotizing fasciitis in children with chickenpox.

Varicella (chickenpox) affects approximately 90,000 children each year. Although most cases resolve, some develop necrotizing soft tissue infections secondary to group A streptococcus and staphylococcus. Delay in diagnosis is common. At the time of initial presentation, the need for surgical intervention is not always clear. The authors conducted a retrospective review of 30 patients with varicella (seen from December 1993 to June 1995) for whom there was clinical concern for necrotizing soft tissue infection. Various parameters were examined, including tachycardia, band count, temperature, and clinical symptoms, to differentiate the children who required surgery from those who did not. Of the 30, 22 underwent surgery. Eighteen had necrotizing fasciitis and required debridement, and four had abscesses that were incised and drained. Eight patients had simple cellulitis and did not require operation. Group A streptococcus was the most common organism cultured. All patients were treated with appropriate antibiotics. Twenty of the 22 surgical patients had elevated band count (> or = 5%), 21 had tachycardia, and 18 were febrile at the time of presentation (> 4 days after the onset of chickenpox). Although all patients with necrotizing fasciitis had tachycardia, this sign was a less specific indicator for surgery than was increased band count. Severe pain, erythemia, and induration was the most common signs/symptoms in the surgical patients. The survival rate for these 30 patients was 100%, and there was little long-term morbidity. The authors recommend immediate surgical intervention for children with chickenpox who present more than 2 or 3 days after the onset of the viral illness with symptoms that include fever, tachycardia, and an elevated band count in association with an erythematous, indurated, painful lesion. With this sign/symptom complex, the presumptive diagnosis must be necrotizing fasciitis until proven otherwise. If the patient has suspicious symptoms or if these symptoms are associated with tachycardia or an elevated band count, the patient warrants admission, institution of intravenous fluids, nafcillin, clindamycin, and close observation over several hours. If the symptoms progress over the next few hours or if the tachycardia persists despite rehydration and antibiotics, the patient should be taken to the operating room for exploration. The authors strongly endorse such exploration despite the risk of a negative operation, because the morbidity and mortality associated with delayed surgical treatment are potentially significant. With prompt aggressive surgical and medical treatment, a good outcome can be anticipated for these patients.

Identification and surgical management of cystic retroperitoneal lymphangioma in children.

Retroperitoneal lymphangiomas are rare, benign cystic structures that are best evaluated with computed tomography and ultrasound. Preoperative diagnosis is often difficult, in part because there is little to distinguish them from other cystic masses and because the lesion is often not considered on the differential diagnosis. Surgery may be required as both a diagnostic and therapeutic measure. The cysts may be asymptomatic for years and then present because of compression of surrounding structures or pain. The treatment is as complete surgical excision as is possible. Bowel cleansing should be done preoperatively. The long-term results are excellent when total excision or near-total excision with marsupialization is accomplished.

Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition of HLA alloantigens and utilization of HLA-A2.1 as a restriction element.

Previous studies have indicated that the frequency of murine CTL precursors (CTLp) for human class I molecules is one to two orders of magnitude lower than that for murine class I alloantigens, and that this is due to species-specific structural differences between these molecules. Transgenic mice expressing the human class I MHC Ag HLA-A2.1 were used to examine changes in the frequency of class I HLA-specific precursors after T cell differentiation in an HLA-A2.1 positive environment. The HLA-A2.1 gene product was expressed at levels comparable to those of the endogenous H-2Db molecule in thymus, bone marrow, and spleen. By limiting dilution analysis, it was observed that the frequencies of CTLp in transgenic mice responding to the human alloantigens HLA-B7 or HLA-A2.2 were comparable to or lower than those in normal C57BL/6 mice, regardless of whether the Ag was presented on human or murine cells. Thus, expression of a human class I molecule in these animals did not result in an expansion of the number of CTLp specific for other human class I Ag. In addition, the frequency of HLA-A2.1-restricted, influenza specific CTLp was substantially lower than the frequency of H-2b restricted CTLp, indicating a poor utilization of HLA-A2.1 as a restricting element. Finally, the frequencies of CTLp for HLA-A2.1 expressed on syngeneic murine tumor cells were decreased significantly. Thus, expression of HLA-A2.1 in these animals appeared to induced tolerance to this Ag. Interestingly, however, these mice were not tolerant to the HLA-A2.1 molecule expressed on human cells. This indicates that the HLA-A2.1 associated epitopes expressed on murine and human cells differ and suggests that, under these circumstances, HLA-A2.1 acts as a restricting element for human nominal Ag. These results are discussed in the context of current models of T cell repertoire development.

Species-specific structural differences in the alpha 1 + alpha 2 domains determine the frequency of murine cytotoxic T cell precursors stimulated by human and murine class I molecules.

The frequency of murine CTL precursors (CTLp) that recognize the human histocompatibility Ag HLA-A2 and HLA-B7 was measured and found to be approximately two orders of magnitude lower than the frequency of CTLp that recognize murine H-2 alloantigens. The possible contribution of other cell surface molecules to this difference in response was addressed by expression of the H-2Ld molecule on a human cell and the HLA-B7 molecule on a murine cell. It was found that both human and murine H-2Ld expressing cells elicited comparable levels of H-2Ld specific CTL. Although murine HLA-B7 positive cells stimulated a higher frequency of HLA-B7-specific CTLp than did human cells, this appeared to be largely due to stimulation of CTLp that recognized HLA-B7 in the context of H-2 molecules; consequently, it was concluded that the difference in the frequency of murine CTLp elicited by human and murine class I Ag is due to species specific structural differences in these molecules. The regions of the class I molecule that were responsible for this difference were mapped using chimeric class I molecules constructed to replace domains of the human molecule with their murine counterparts. It was found that the frequency of CTLp is controlled by structures within the alpha 1 and alpha 2 domains of the molecule. These results are discussed in the light of models for T cell recognition of class I Ag and the diversification of the T cell receptor repertoire.

The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity.

Two groups of human and murine cytotoxic T lymphocyte (CTL) clones specific for human leukocyte antigen (HLA)-A2 or -B7 can be distinguished based on their ability to kill murine transfectants expressing these molecules. The clones which do not recognize murine transfectants exhibited greatly reduced conjugate formation with these cells, indicating that the inability to lyse these cells occurs in recognition and binding. No systematic differences in inhibitory titer between the two types of CTL clones were seen with anti-CD8 (Lyt-2), anti-LFA-1, or monoclonal antibodies against HLA class I molecules. However, blocking with anti-HLA class I monoclonal antibodies suggested that different CTL clones recognized spatially separate epitopes on HLA-A2 and -B7. In addition, a correlation between the inability to recognize murine transfectants and fine specificity was seen. Eight of nine clones which did not lyse murine transfectants also failed to recognize human cells expressing HLA-A2.2 or -A2.3. In contrast only 5 of 12 clones which lysed transfectants failed to recognize the variant molecules. Analogous data were obtained with human CTL clones raised against HLA-A2.1. These findings suggest that CTL clones that do not lyse murine cells expressing appropriate antigens recognize epitopes that have been altered or lost as a consequence of expression on the murine cell surface. It is suggested that the loss of HLA-associated epitopes on the murine cell surface may be due to differences between mouse and human cells in the processing or presentation of class I-associated peptides.

HLA antigens expressed on murine cells are preferentially recognized by murine cytotoxic T cells in the context of the H-2 major histocompatibility complex.

The frequency of murine cytotoxic T lymphocytes (CTL) capable of responding to HLA antigens expressed on human or murine cells was determined by limiting dilution analysis. HLA antigens expressed on human cells stimulated CTL with a precursor frequency of about 1 in 2 X 10(5) spleen cells in primed mice, over two orders of magnitude weaker than a primary allogeneic response. There was a 10-fold increase in the frequency of precursors responding to HLA antigens when they were expressed on murine cells. It was determined that the increased frequency of responders was due to CTL that could only recognize HLA antigens on the syngeneic murine line to which they had been stimulated and that these CTL could not lyse any other HLA expressing murine cells of different H-2 haplotypes. The lytic activity of these CTL was inhibited by H-2b-specific antibodies. These results indicate that such CTL recognize HLA antigens in the context of the H-2 major histocompatibility complex. The magnitude and specificity of CTL responses to xenoantigens are discussed in the context of a model for T-cell interactions with major histocompatibility antigens.

Construction of novel class I histocompatibility antigens by interspecies exon shuffling.

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

Detection of repeated gonorrhea by a modified follow-up method.

The rate of reinfection after treatment was studied in four groups of patients. The first group of 100 patients were evaluated retrospectively after treatment with 1.0 g ampicillin twice followed by two re-examinations over a 2-week period during which time intercourse was prohibited. The second and third prospectively groups consisted of 100 and 200 patients respectively, treated with 1 g ampicillin twice and 1.4 g pivampicillin + probenecid respectively, with re-examinations at intervals of 1 week and 4 weeks after treatment. In these two groups sexual intercourse was allowed during the last 10 days of the follow-up period. The fourth study group consisted of all patients infected with gonococci having MIC greater than or equal to 0.3 micrograms/ml for ampicillin and/or benzylpenicillin, who were treated with 1 g ampicillin twice during the period of 1973-76. In the first group no positive cultures were obtained at re-examination. In groups two and three, 35 out of 300 (12%) were found to have positive cultures at re-examination. Some of these were probably treatment failures. The failure rate in group four was found to be 20%, indicating that the treatment regimen used left a narrow therapeutic margin. However, the risk of re-infection, in this urban clinic, seems to be higher than the risk of treatment failure which would have remained undetected had sexual intercourse been forbidden.

Molecular basis of bunyavirus transmission by mosquitoes: role of the middle-sized RNA segment.

In an examination of the molecular basis of oral transmission of bunyaviruses by mosquitoes., La Crosse (LAC), snowshoe hare (SSH), and LAC-SSH reassortant viruses were compared in their ability to be transmitted to laboratory mice by the natural mosquito vector of LAC virus, Aedes triseriatus. Both LAC virus and the reassortment viruses containing the middle-sized (M) segment from the LAC parent were efficiently transmitted. In contrast, SSH virus and reassortment viruses containing the M RNA from the SSH parent were inefficiently transmitted. Thus, the M RNA segment, which codes for the virion glycoproteins, may be a major determinant of oral transmission of bunyaviruses by mosquitoes.