Limited BVDV transmission and full protection against CSFV transmission in pigs experimentally infected with BVDV type 1b.

Bovine viral diarrhea virus (BVDV) in pigs may interfere with the detection and epidemiology of classical swine fever virus (CSFV). To investigate the importance of BVDV infections in pigs, first we studied the transmission dynamics of a recent BVDV field isolate. Subsequently, the protection of BVD antibodies against transmission and clinical disease of CSF virus was studied. Only limited transmission of BVDV occurred (R = 0.20), while no CSFV transmission occurred in pigs with BVDV antibodies. We concluded that BVDV transmission among pigs is possible, but seems to be limited and thus the virus should disappear from a population if no new introductions occur. Furthermore, the presence of BVD antibodies may completely prevent the transmission of CSFV and therefore could protect pigs against classical swine fever. It was also noticed that double infections with BVDV and CSFV were incorrectly diagnosed using the neutralization peroxidase linked assay (NPLA), which is the golden standard for antibody detection. This might hamper the diagnosis of CSF in herds with a high BVD prevalence.

A distinct pattern of trophic factor expression in myelin-deficient nerves of Trembler mice: implications for trophic support by Schwann cells.

Distal to a peripheral nerve transection, myelin degradation and Schwann cell (SC) proliferation are accompanied by a marked upregulation of brain-derived neurotrophic factor (BDNF) and a decrease of ciliary neurotrophic factor (CNTF) in non-neuronal cells. To investigate the role of SC differentiation in trophic factor regulation, we studied BDNF and CNTF expression in sciatic nerves from Trembler-J (Tr-J) mice. In these animals, a mutation in the pmp-22 gene causes a failure of myelination and continuous SC proliferation, but axonal continuity is preserved. In spite of the severe abnormalities in Tr-J nerves, BDNF levels remained as low as in the intact controls. Thus, the primary SC disorder in Tr-J produces a different pattern of BDNF expression from that caused by axonal breakdown due to nerve transection. Furthermore, the upregulation of BDNF mRNA triggered by transection was 70-fold in control nerves, but only 30-fold in Tr-J sciatic nerves. Because these results raised the possibility that axonal loss may influence neurotrophin expression only in SCs that have differentiated toward a myelinating phenotype, we measured BDNF mRNA after axotomy in the cervical sympathetic trunk (CST), a predominantly unmyelinated autonomic nerve. In contrast to the sciatic nerves, the BDNF mRNA level barely increased in the injured CST, supporting the idea that not all SCs are equal sources of trophic molecules. In Tr-J sciatic nerves, CNTF mRNA levels were fourfold lower than normal, implying that the downregulation of this cytokine is a sensitive indicator of a spectrum of SC perturbations that affect myelinating cells.

Effects of neurotrophins on the survival and regrowth of injured retinal neurons.

The focus of this short review is the role of certain neurotrophins and their receptors on the survival and regrowth of retinal ganglion cells (RGCs) whose axons are damaged in the optic nerve. Initial experiments in our laboratory documented patterns of RGC death after axotomy. Subsequent studies were designed to investigate the distribution of high-affinity neurotrophin receptors in neurons and glial cells of the retina and optic nerve. This information was used both in vitro and in vivo to study the effects of specific trophic molecules on the survival and regrowth of injured RGCs. During the course of experiments involving neurotrophin administration, an endogenous source of trophic support--independent of the exogenous administration of growth factors--was found within the eye. Several experiments were subsequently undertaken to define further this survival effect and determine its nature and source within the eye. Finally, anatomical techniques that help visualize fine axonal processes within the retina have provided insights into the specific effects of neurotrophins on the growth and branching of injured CNS axons.

Trophic factors.

The various neurotrophic factors influence a wide range of cell functions in the developing, mature and injured nervous system. Recent studies have provided valuable insights on the receptors that mediate these effects and on the intracellular events that follow the binding of the ligand. Although growth factors were known to be expressed by non-neuronal cells in the targets and pathways of neuronal projections, it is now clear that the neurons themselves can also be a source of these molecules. A better understanding of the mechanisms of action of trophic factors on the survival and differentiation of neurons, coupled with advances in methods for the delivery of these molecules to the nervous system have provided an impetus for exploring their use as aids to the protection and regeneration of the injured nervous system.

Non-isotopic labeling of DNA by newly developed hapten-containing platinum compounds.

A newly developed reagent was tested for its applicability in in situ hybridization and in reversed hybridization of DNA fragments generated by PCR amplification. This Platinum-complex, designated universal linkage system (ULS), equipped, for instance, with biotin or fluorescein as hapten, enables versatile nonenzymatic "one step" labeling of genomic, cloned or amplified DNA. Here we demonstrate direct in situ detection of integrated human papilloma virus (HPV) DNA in cervical carcinoma cells using DNA probes labeled with fluorescein-ULS. In cervical smears the presence of HPV or Chlamydia trachomatis was assessed by PCR. To analyze the amplified DNA, a reversed hybridization assay was developed. Immobilized probes were incubated with amplimers that were labeled post-amplification through the action of the biotinylated (BIO)-ULS complex. This novel type of nonradioactive analysis appeared to be as sensitive as its isotopic or colorimetric equivalents. This labeling procedure is simple, versatile and can be included as a universal hapten linkage system in any PCR test or in situ hybridization assay aiming at the detection and identification of DNA or RNA molecules.

Application of a new, universal DNA labeling system in the PCR mediated diagnoses of Chlamydia trachomatis and human papillomavirus type 16 infection in cervical smears.

Non-isotopic DNA labeling procedures are essential for integration of DNA diagnostics into the clinical laboratory. A newly developed reagent was tested for use in reversed hybridisation identification of DNA fragments generated by polymerase chain reaction (PCR) amplification of Chlamydia trachomatis or human papilloma virus type 16 (HPV16) DNA isolated from cervical smears. The platinum-containing chemical compound, equipped with a biotin hapten, enables versatile 'one tube' labeling of amplified DNA. A HPV16-specific probe was immobilised on a nylon strip and reverse hybridisation with the biotin labeled DNA took place. To determine the value of this new, non-isotopic label in combination with clinical material, 98 cervical smears 54 of which contained HPV16, and 51 cervical smears 26 of which contained C. trachomatis, were analysed. The novel type of non-radioactive analysis appeared to be as sensitive as its isotopic counterpart. The DNA isolation and purification method require modification only in samples of poor quality. The labeling procedure is simple, versatile and can be included as a universal linkage system in any PCR test for the detection and identification of DNA molecules.

Different forms of the neurotrophin receptor trkB mRNA predominate in rat retina and optic nerve.

The expression of TrkB mRNAs was investigated in rat retina and optic nerve. A 11.5 kb transcript that encodes full-length TRKB was found to predominate in Northern blots of retinal RNA. By in situ hybridization, this trkB expression was concentrated in the ganglion cell and inner nuclear layers. Furthermore, an antibody to the full-length TRKB immunostained retinal ganglion cells and their axons. In contrast, Northern blots of optic nerve RNA showed a prominent 9.5 kb band that encoded a form of the TRKB receptor lacking the tyrosine kinase domain. This species was also detected in both the sciatic nerve and cultured astrocytes and C6 glioma cells. These results suggest that neurons express the full-length TRKB containing the tyrosine kinase domain, while non-neuronal cells express the truncated form of the receptor. These two classes of TRKB may mediate different neurotrophic actions in the retina and optic nerve.

Sequences in E1A proteins of human adenovirus 5 required for cell transformation, repression of a transcriptional enhancer, and induction of proliferating cell nuclear antigen.

A range of deletion and other mutants in the coding region of the E1A gene of Ad5 has been assayed for transformation of baby rat kidney (BRK) cells in cooperation with ras, repression of the SV40 enhancer, and induction of proliferating cell nuclear antigen (PCNA). Transformation efficiency was drastically reduced by deletion of residues 4-25, 36-60, or 111-138 in exon 1 of the 289 residue (289R) and 243R E1A proteins. Deletion of other residues in exon 1 had little effect. With mutants in the region unique to the 289R protein, and in exon 2, the only effect on transformation seemed to be an increased tendency of mutant transformants, compared to wt, to migrate to form secondary foci. Repression assays, performed with E1A plasmids producing only the 243R protein, showed that deletion of residues 4-25 or 36-60 inhibited repression completely. Deletion of residues 128-138 reduced repression, but deletions elsewhere in exon 1 had little effect. Deletion of residues 188-204 in exon 2 reduced repression slightly, and deletion of all of exon 2 reduced it to about one-half. It is concluded that for transformation, there are two functional domains in E1A proteins, both in exon 1, both involved in binding different cellular proteins, and both probably concerned with different transforming functions. One of these domains, involving residues 4-25 and 36-60, also functions in repression, but the role of the second in repression is much less critical. All of the deletion mutants in exon 1 induced PCNA synthesis in BRK cells. This result, together with previously published work, suggests that the active site for PCNA induction either involves residues 61-69 or 82-85 in exon 1, which have not been deleted, or it does not depend on any single limited region of the E1A proteins.

Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5.

The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 E1A products were studied with E1A deletion mutants. All appeared to bind to the amino-terminal half of E1A products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of E1A products.

Improvement of a direct agglutination test for field studies of visceral leishmaniasis.

To increase the potential for the wide-scale application of our direct agglutination test for visceral leishmaniasis, modifications in the components and procedures were introduced. Supplementation with 0.056 M citrate of the suspension medium stabilized the antigen for 9 weeks at 37 degrees C. To circumvent the need for cooling systems in the field, 0.2% (wt/vol) gelatin was added to the serum diluent instead of fetal bovine serum, with reliable results. Specificity and sensitivity were improved by the incorporation of 0.1 M 2-mercaptoethanol in samples with borderline titers. The test could be performed on samples of whole blood; thus the difficulties of preparation and storage of serum, plasma, or filter paper blood are avoided. For mass screening programs, a single serum dilution of 1:6,400 could be employed, contributing to a further reduction in test expenses. Sera from different geographical areas showed equal reactivities in this direct agglutination test despite the nonhomologous Leishmania donovani antigens used.

Use of deletion and point mutants spanning the coding region of the adenovirus 5 E1A gene to define a domain that is essential for transcriptional activation.

To help in identifying functional domains within Ad5 E1A proteins, we have constructed a series of mutants that create deletions throughout these products. We have also produced several mis-sense point mutations in the unique 13 S mRNA region. These mutated E1A regions have been tested in plasmid form for their ability to activate transcription of an E3-promoted CAT gene. From the results, a major domain for transactivation has been identified. This begins between residues 138 and 147, ends between residues 188 and 204, and encompasses the unique 13 S region. This domain is sensitive to mis-sense mutations. Transactivation was unaffected by small deletions in the N-terminal half of E1A proteins between residues 4 and 138, but was destroyed when this whole region was deleted. The C-terminal 71 residues may affect transactivation, but the results with the mutant in which this region was deleted were variable. The results obtained with these mutants are discussed in relation to the transactivation obtained by J. W. Lillie et al. [(1987). Cell 50, 1091-1100] with a synthetic peptide similar to the domain described here.