Bilirubin Encephalopathy in a Domestic Shorthair Cat With Increased Osmotic Fragility and Cholangiohepatitis.

A 7-month-old female domestic shorthair cat was diagnosed with chronic regenerative hemolytic anemia characterized by increased osmotic fragility of unknown etiology. At 13 months of age, the cat was evaluated for acute collapse. The cat was icteric with severe hyperbilirubinemia but no hematocrit changes. Severe obtundation and lateral recumbency progressed to tetraparesis and loss of proprioception in all 4 limbs, and a cerebellar or brainstem lesion was suspected. Postmortem examination revealed suppurative cholangiohepatitis and acute neuronal necrosis in the nuclei of the brainstem and cerebellum, consistent with bilirubin encephalopathy. This is the first known occurrence of cholangiohepatitis and bilirubin encephalopathy in an adult cat with chronic hemolytic anemia. Although rare, bilirubin encephalopathy should be considered a possible sequela to hyperbilirubinemia in adult patients. It remains unknown whether increased osmotic fragility was related to the cholangiohepatopathy.

Meningoencephalitis associated with Carnobacterium maltaromaticum-like bacteria in stranded juvenile salmon sharks (Lamna ditropis).

Juvenile salmon sharks beach yearly along the California coast, primarily during late summer and early fall. Fresh, frozen, and formalin-fixed tissues from 19 stranded salmon sharks were collected for examination. Histopathology revealed meningitis or meningoencephalitis in 18 of 19 shark brains with intralesional bacteria observed in 6 of the affected brains. Bacterial culture of fresh or frozen brain, liver, and/or heart blood from 13 sharks yielded pure cultures characterized molecularly and/or biochemically as belonging to the genus Carnobacterium. The 16s ribosomal DNA sequence of 7 tissue isolates from 7 separate sharks was 99% homologous to C. maltaromaticum (GenBank FJ656722.1). Sequence of the large ribosomal DNA intergenic spacer region (ISR) was 97% homologous to C. maltaromaticum (AF374295.1). This is the first report of Carnobacterium infection in any shark species, and the authors posit that brain infection caused by Carnobacterium is a significant cause of morbidity and mortality in juvenile salmon sharks found stranded along the Pacific coast of California.

Neurolymphomatosis in a dog with B-cell lymphoma.

Lymphoma in the left femoral nerve of a 10-year-old English Cocker Spaniel caused complete paralysis of the affected limb. Neoplastic cells were immunopositive for CD79a and Pax5 and negative for CD3. Neoplastic cells were in multiple lymph nodes and one kidney but spared bone marrow. The clinical and histologic features in this case resemble those of the rare human condition of neurolymphomatosis.

Morbidity and mortality in Danio rerio and Pimephales promelas exposed to antilipidemic drug mixtures (fibrates and statins) during embryogenesis: Comprehensive assessment via ante and post mortem endpoints.

Antilipidemic drugs are routinely detected in effluent and surface waters downstream of wastewater treatment plants. A mixture exposure study with nine environmentally relevant antilipidemic drugs was performed with zebrafish (Danio rerio, ZF) and fathead minnow (Pimephales promelas, FHM) embryos to investigate the effects on sensitive embryologic stages. Zebrafish embryos were exposed nominally to: (a) 0.005 µM, (b) 0.05 µM, or (c) 0.5 µM of each drug in the mixture. Fathead minnow embryos were exposed nominally to: (a) 0.0005 µM, (b) 0.005 µM, or (c) 0.05 µM of each drug in the mixture. Several of the individual drug concentrations were within ranges previously found in the environment. Multiple metrics demonstrate that (a) exposure of ZF and FHM embryos to antilipidemic drugs during embryonic development results in lethal and sublethal effects, (b) ZF were more sensitive than FHM based on median lethal concentration (LC50 0.02 µM and 0.05 µM, respectively), but FHM exhibited more severe abnormal sublethal morphologies than zebrafish embryos, and (c) the sublethal effects differed between the two species. This model identified novel specific endpoints for assessing sensitive, sublethal effects of pharmaceuticals in the environment. Abnormal myofiber birefringence pattern, hemorrhage, and heart rate are not included in standard evaluations but each of these metrics demonstrated a dose-dependent response in this study. Results demonstrate risk to fish development with potential repercussions at the population level, especially if environmental concentrations increase.

Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection.

Viral infection is sometimes associated with the initiation or exacerbation of autoimmune disease, although the underlying mechanisms remain unclear. One proposed mechanism is that viral determinants that mimic host antigens trigger self-reactive T cell clones to destroy host tissue. An epitope expressed by a coat protein of herpes simplex virus-type 1 (HSV-1) KOS strain has now been shown to be recognized by autoreactive T cells that target corneal antigens in a murine model of autoimmune herpes stromal keratitis. Mutant HSV-1 viruses that lacked this epitope did not induce autoimmune disease. Thus, expression of molecular mimics can influence the development of autoimmune disease after viral infection.

Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4.

To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.

Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis.

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.

BCL2 and MYC are expressed at high levels in canine diffuse large B-cell lymphoma but are not predictive for outcome in dogs treated with CHOP chemotherapy.

Diffuse large B-cell lymphoma (DLBCL) is the most common haematopoietic malignancy in dogs. Recently, MYC and BCL2 expression levels determined with immunohistochemistry (IHC) were found to be prognostic in people with DLBCL. We hypothesized that canine DLBCL can be similarly subdivided into prognostic subtypes based on expression of MYC and BCL2. Cases of canine DLBCL treated with CHOP chemotherapy were retrospectively collected and 43 dogs had available histologic tissue and complete clinical follow-up. Median values of percent immunoreactive versus immunonegative cells were used to determine positive or negative expression status. Completion of CHOP was significantly associated with a positive outcome. Compared with human patients, our canine DLBCL patients had high IHC expression of both MYC and BCL2, and relative expression levels of one or both markers were not associated with clinical outcome.

Attenuation of herpesvirus saimiri for marmosets after successive passage in cell culture at 39 degrees C.

A variant of Herpesvirus saimiri (HVS) stably attenuated for marmosets was isolated after serial passage of wild-type HVS in cell culture at 39 degrees C. Marmosets previously inoculated with attenuated virus experienced a significant delay in the development of lymphoma when challenged with wild-type HVS.

Studies on herpes simplex virus and cancer.

Virus-induced polypeptides of cells infected by herpes simplex virus (HSV) types 1 and 2 were investigated by analysis on polyacrylamide gels and by determination of their antigenicity. Some polypeptides, VP154 and VP134, had immunological reactivity common to both virus types, while others (VP175 and VP123) were type specific. Only the glycosylated polypeptides were able to induce neutralizing antibody. The expression of viral genetic information was studied in newborn mice infected with wild-type and ts mutant viruses; some mutants had become attenuated and had lost pathogenicity for newborn mice while others had not. From induction experiments in HSV=transformed hamster cells, it appears that detection of enhanced replication of ts mutants in human cancer cells would be an indication of resident HSV genetic information. Sera obtained from cancer patients were examined for antibodies to early proteins synthesized in HSV-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. Cervical cancer patients had sera with a higher reactivity to early nonstructural polypeptides than to breast cancer patients or to matched healthy women. In contrast to the results with early polypeptides, little difference was detectable between the matched sera in their reactivity with a major capsid polypeptide, which is synthesized late in the infectious cycle.

Genetics of herpes simplex virus.

The most direct approach to elucidating the roles of herpes simplex virus (HSV) proteins in the viral replicative cycle has been to isolate temperature-sensitive, cytolysis-resistant, and drug-resistant mutants that exhibit alterations in the synthesis or activity of these proteins. The development of procedures for the introduction of temperature-sensitive mutations into physically defined regions of the viral genome and for fine mapping of these mutations has proven especially valuable. Thus, (1) hydroxylamine mutagenesis of the HSV-1 BglII I fragment (coordinates 0.312-0.415) has facilitated the genetic and functional characterization of the gene for the major viral DNA-binding protein of 130 K molecular weight; (2) the selection of a mutant conditionally able to render infected cells resistant to immune cytolysis has led to identification of an HSV gene involved in the processing of viral glycoproteins; and (3) the combined use of temperature-sensitive and drug-resistant mutants has led to a better definition of the physical limits and functional domains of the gene for HSV DNA polymerase.

Herpes simplex virus gene products involved in the induction of chromosomal aberrations.

The effect of short-term herpes simplex virus type 1 (HSV-1) infection on chromosomes of human diploid fibroblasts was examined. In addition to chromosomal breaks, gaps and pulverization, three kinds of cytogenetic damage (double minutes, polyploidy and endoreduplication) not yet reported following productive infection with HSV or other animal viruses were frequently observed. Consistent with previous studies suggesting that the expression of immediate-early and/or early viral gene products is required for the induction of chromosomal damage, was the observation that cells infected at the nonpermissive temperature with HSV-1 temperature-sensitive mutants defective in the gene for the immediate-early transcriptional regulatory protein, ICP4, and three early viral gene products--DNA polymerase (pol), the major HSV DNA-binding protein (ICP8) and an HSV-2 mutant defective in alkaline nuclease--exhibited altered patterns of chromosomal damage relative to the effects of wild-type virus on infected cells. These findings suggest a direct or indirect role for all four gene products in the induction of chromosomal damage. In cells infected with wild-type virus for 4 h or longer, HSV proved to be a more potent mitotic arresting agent than colcemid. Moreover, studies with selected mutants indicate that HSV pol specifically may be involved in mitotic arrest. Additionally, in cells infected at the non-permissive temperature with a pol mutant, the number of polyploid metaphases was reduced 4-fold relative to that seen in wild-type virus-infected cells suggesting a role for HSV pol in the amplification of cellular DNA.

A rapid procedure for the enrichment of undenaturated, antigenically active herpes simplex virus glycoproteins.

The usefulness of lentil lectin affinity chromatography for the rapid enrichment of HSV glycoproteins in an undenatured state for both research and clinical purposes was investigated. In order to compare the lentil lectin-binding characteristics and immunologic specificities of undenatured HSV-1 and HSV-2 glycoproteins, [35S]methionine-labelled extracts of virus-infected HEp-2 cells were subjected to lentil lectin affinity chromatography. Individual HSV-1 and HSV-2 glycoproteins in bound and unbound fractions were identified using monoclonal antibodies. With the exception of a portion of pgD and gD, all major viral glycoprotein species (gA, gB, gC, gD, gE and gF) and their glycosylated processive intermediates bound to lentil lectin indicating that all possess predominantly mannosyl and/or glucosyl carbohydrate moieties. Although the unbound pgD and gD species were glycosylated, no gD and only a portion of pgD bound to lentil lectin when reapplied to the column indicating that these subspecies possess alterations in factors required for efficient lectin binding. Immunoprecipitation of undenatured lectin-bound glycoproteins from infected cells using HSV-1 and HSV-2-specific rabbit and human antisera confirmed previous findings that the predominant type-specific glycoproteins of HSV-1 and HSV-2 are gC and gE/gF, respectively.

Multiple red cell transfusions and alloimmunization. Experience with 6996 antibodies detected in a total of 159,262 patients from 1985 to 1993.

This retrospective study of red cell antibodies covered the period from 1985 to 1993. A three-cell antibody screen, 22% albumin enhancement, and a polyspecific antiglobulin reagent assay were performed. From a total of 159,262 patients in the data set, 6996 antibodies were detected among the sera of 4700 patients (2.9%). Four thousand two hundred thirty-five (60.5%) alloantibodies of potential clinical significance were found. These included anti-K1 (23.0%), -E (17.6%), -D (12.4%), -Le(a) (7.3%), -C (6.3%), -Fya (5.7%), and -c (4.4%). Cold agglutinins were found in 1119 positive antibody screens, 261 had warm autoantibodies, and 554 had high-titered, low-avidity antibodies. Three hundred seven were clinically insignificant (eg, Sda and Bga). Five hundred seventeen were too weak to identify. Most patients' sera demonstrated only one antibody (69.3%), but there was a strong linear correlation between the total number of recorded red cell transfusions and the number of antibodies found (r = .976; P < .0001). There was a higher percentage of females with antibodies than the percentage of females in the total study population (59.2% versus 43.8%, P < .0001). Two hundred nine of 554 (37.7%) high-titered, low-avidity antibodies and 349 (31.2%) of 1119 of the cold agglutinins accompanied or obscured clinically significant antibodies.

Cloning and characterization of herpes simplex virus type 1 oriL: comparison of replication and protein-DNA complex formation by oriL and oriS.

The herpes simplex virus type 1 genome contains three origins of DNA replication: two copies of oriS and one copy of oriL. Although oriS has been characterized extensively, characterization of oriL has been severely limited by the inability to amplify oriL sequences in an undeleted form in Escherichia coli. We report the successful cloning of intact oriL sequences in an E. coli strain, SURE, which contains mutations in a series of genes involved in independent DNA repair pathways shown to be important in the rearrangement and deletion of DNA containing irregular structures such as palindromes. The oriL-containing clones propagated in SURE cells contained no deletions, as determined by Southern blot hybridization and DNA sequence analysis, and were replication competent in transient DNA replication assays. Deletion of 400 bp of flanking sequences decreased the replication efficiency of oriL twofold in transient assays, demonstrating a role for flanking sequences in enhancing replication efficiency. Comparison of the replication efficiencies of an 822-bp oriS-containing plasmid and an 833-bp oriL-containing plasmid demonstrated that the kinetics of replication of the two plasmids were similar but that the oriL-containing plasmid replicated 60 to 70% as efficiently as the oriS-containing plasmid at both early and late times after infection with herpes simplex virus type 1. The virus-specified origin-binding protein (OBP) and a cellular factor(s) (OF-1) have been shown in gel mobility shift experiments to bind specific sequences in oriS (C.E. Dabrowski, P. Carmillo, and P.A. Schaffer, Mol. Cell. Biol. 14:2545-2555, 1994; C.E. Dabrowski and P.A. Schaffer, J. Virol. 65:3140-3150, 1991). Although the nucleotides required for the binding of OBP to OBP binding site I in oriL and oriS are the same, a single nucleotide difference distinguishes OBP binding site III in the two origins. The nucleotides adjacent to oriS sites I and III have been shown to be important for the binding of OF-1 to oriS site I. Several nucleotide differences exist in these sequences in oriL and oriS. Despite these minor nucleotide differences, the protein-DNA complexes that formed with oriL and oriS sites I and III were indistinguishable when extracts of infected and uninfected cells were used as the source of protein. Furthermore, the results of competition analysis suggest that the proteins involved in protein-DNA complex formation with sites I and III of the two origins are likely the same.