Sinusoidal endothelial cell and hepatic stellate cell phenotype correlates with stage of fibrosis in chronic liver disease in dogs.

We evaluated the extent of hepatic fibrosis in chronic liver disease of dogs using a modification of Ishak's staging criteria for human chronic liver disease, and examined the association of stage of fibrosis with immunophenotypic markers of transdifferentiation of hepatic sinusoidal endothelial cells and hepatic stellate cells. Formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained liver biopsy specimens from 45 case dogs with chronic liver disease and 55 healthy control dogs were scored for the presence and extent of fibrosis. This stage score for fibrosis strongly correlated with upregulated von Willebrand factor (vWF) expression in lobular sinusoidal endothelial cells (Spearman correlation coefficient [SCC] = 0.57, p < 0.05). Immunoreactivity for vWF factor was identified in 68.9% of case biopsies, varying in distribution from periportal to diffuse, whereas vWF immunoreactivity was identified in only 14.5% of control specimens, and was restricted to the immediate periportal sinusoids. The majority of both case and control biopsies exhibited similar prominent lobular perisinusoidal expression of alpha-smooth muscle actin (α-SMA). A minority of specimens (17.8% of case biopsies, 1.8% of control biopsies) exhibited low perisinoidal α-SMA expression, and there was a weak negative correlation between α-SMA expression and stage of fibrosis (SCC = -0.29, p = 0.0037). These results document a method for staging the severity of fibrosis in canine liver biopsies, and show a strong association between fibrosis and increased expression of vWF in hepatic sinusoidal endothelial cells.

Defibrinated bovine plasma inhibits retroviral transcription by blocking p52 activation of the NFkappaB element in the long terminal repeat.

Bovine leukemia virus (BLV) induces a persistent but latent infection in cattle. Viral latency is invoked by a protein known as plasma blocking factor (PBF) that is found in both bovine and human plasma. We report here on pathways that mediate latency in the presence of PBF. Reporter-gene constructs driven by the promoters of 6 retroviruses were used to measure the production of chloramphenicol acetyl transferase (CAT) in cell lines cultured with or without defibrinated bovine plasma. Plasma inhibited CAT production only in constructs containing an NFkappaB-binding element proximal to the initiation site (BLV, human immunodeficiency virus, and human T-cell leukemia virus). The promoters of Bovine immunodeficiency virus, Feline immunodeficiency virus, or Feline leukemia virus were not inhibited in the presence of bovine plasma. Using gel mobility shift assays, we demonstrated that activation of viral transcription upon stimulation with phorbol esters and ionomycin was mediated through the NFkappaB element and that this was abrogated in the presence of plasma. Furthermore, analysis of individual NFkappaB proteins in nuclear extracts of mononuclear cells or Jurkat cells showed that all 5 members of the NFkappaB family were upregulated in response to stimulation, but only p52 was significantly downregulated in the presence of bovine plasma. Thus, we infer that plasma effects are mediated through interference with either p52 translocation to the nucleus or p52 synthesis.

Effects of noncytopathic type 2 bovine viral diarrhea virus on the proliferation of bone marrow progenitor cells.

The purpose of this study was to investigate the effects of isolates of noncytopathic type 2 Bovine viral diarrhea virus (ncpBVDV-2) of high and low virulence on the proliferation of bone marrow progenitor cells. Holstein calves 6 to 7 mo old and BVDV-naïve were inoculated intranasally with a BVDV isolate of high virulence (HV24515), a BVDV isolate of low virulence (LV11Q), or uninfected cell culture medium. Serial bone marrow and peripheral blood samples were collected before and after inoculation. Bone marrow mononuclear cells (BMMCs) were isolated and cultured for 5 d, and the mean number of colony-forming unit-granulocyte-macrophage (CFU-GM) colonies was determined. Tritiated (3H)-thymidine uptake by BMMCs was determined to indicate overall proliferative capacity. Virus isolation was done on concurrent samples of BMMCs and peripheral blood. Virus was isolated from BMMCs and peripheral blood buffy-coat cells as early as day 2 or 3 after inoculation. Neutropenia developed in both groups inoculated with a BVDV isolate. However, in the calves given LV11Q, neutrophil counts rebounded earlier in response to increased proliferation of BMMCs, whereas the response was delayed in calves given HV24515. Thymidine uptake was significantly increased (P = 0.0047) in BMMCs after inoculation compared with before inoculation in the calves given LV11Q but not in those given HV24515 or in the control calves. The median number of CFU-GM colonies was significantly decreased (P = 0.0164) after inoculation compared with before inoculation in the calves given HV24515, whereas there was no significant difference in the calves given LV11Q or in the control calves. The data support the hypothesis that the prolonged neutropenia observed in calves given HV24515 results at least in part from decreased proliferative capacity of bone marrow progenitor cells.

Purified bovine plasma blocking factor decreases Bovine leukemia virus p24 expression while increasing protein synthesis and transcriptional activity of peripheral blood mononuclear cells in short-term culture.

Bovine leukemia virus (BLV) induces a persistent infection in the B-cells causing polyclonal expansion of B-cells in one-third of infected cattle and lymphosarcoma in less than 5% of infected cattle. While BLV is difficult to detect in vivo, it is readily produced by cultured lymphocytes and is diminished when supplemented by bovine plasma. This phenomenon is attributed to a poorly characterized plasma blocking factor (PBF). We assessed the effects of bovine plasma on cell viability and BLV p24 expression, and the effects of purified PBF on protein synthesis and gene expression of short-term cultures of bovine lymphocytes. The addition of 25% plasma or semi-purified PBF to cultures had no significant effect on cell viability but caused significant decreases in BLV p24 production and significantly increased de novo protein synthesis. Utilizing a human microarray, the RNA messages of 83 genes involved in cell division, cell metabolism, and gene regulation were up-regulated.

Isolation of a bovine plasma fibronectin-containing complex which inhibits the expression of bovine leukemia virus p24.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects cattle worldwide. In agriculturally intensive regions, approximately 30% of dairy cows are BLV infected. Like the human T-cell leukemia virus (HTLV), there is a lengthy period of viral quiescence after initial infection with BLV. Unlike HTLV, BLV resides predominantly in B cells. Lymphoma is observed in less than 10% of BLV-infected adult cattle. Although viremia is undetectable in vivo, BLV-infected peripheral blood mononuclear cells readily become productive when cultured in vitro. Productivity is markedly diminished when cultures are supplemented with bovine plasma. This inhibitory activity of bovine plasma has been attributed to the "plasma blocking factor" (PBF). Here, we describe the purification of a PBF whose activity was resistant to heating to 65 degrees C for 10 min and was attributable to a fibronectin-containing complex of approximately 320 kDa under nonreducing conditions. By use of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry), a protein with a size of 220 kDa and a pI of 5.4 was identified as a member of the fibronectin group of molecules. Both the purified protein and the commercially available bovine fibronectin inhibited BLV production in naturally infected peripheral blood mononuclear cells, although the fibronectin was less biologically active.

Use of a commercial methylcellulose medium with and without recombinant bovine granulocyte colony-stimulating factor for culturing bovine bone marrow cells.

A commercial methylcellulose culture medium, with and without the addition of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF), was utilized for culturing bovine bone marrow cells in a colony-forming unit assay. Bone marrow mononuclear cells were isolated and cultured in a commercial methylcellulose-based medium containing several recombinant human cytokines. Cultures were prepared with and without 100 ng/mL of rbG-CSF. The size and mean number of colonies per plate from culture days 3 to 9 were compared. We concluded that bovine bone marrow colony growth was supported by this culture medium. The addition of rbG-CSF yielded larger and more numerous colonies. There were significantly more colonies on day 3 (P < 0.001), day 4 (P < 0.001), and day 5 (P = 0.03) with rbG-CSF. Both culture media had the highest colony counts on day 5.