The Role of Hypoxia in 2-Butoxyethanol–Induced Hemangiosarcoma

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore, their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow (BM), liver, and spleen of mice exposed to a single dose (4 h) or seven daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased erythropoeitin signaling and erythropoiesis in the spleen and BM, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe) demonstrated that tissue hypoxia was present in the spleen and BM. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE–induced hemangiosarcoma in mice

Early Pathogenesis of Transmucosal Feline Immunodeficiency Virus Infection

To identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-ISH and in retropharyngeal, tracheobronchial, or external iliac lymph nodes and sometimes in spleen or blood mononuclear cells by day 2, indicating that regional and distant spread of virus-infected cells occurred rapidly after mucosal exposure. By day 8, viral RNA, DNA, and culturable virus were uniformly detected in regional and distant tissues, connoting systemic infection. TSA-ISH proved more sensitive than DNA PCR in detecting early FIV-infected cells. In mucosal tissues, the earliest demonstrable FIV-bearing cells were either within or subjacent to the mucosal epithelium or were in germinal centers of regional lymph nodes. The FIV(+) cells were of either of two morphological types, large stellate or small round. Those FIV RNA(+) cells which could be colabeled for a phenotype marker, were labeled for either dendritic-cell-associated protein p55 or T-lymphocyte receptor antigen CD3. These studies indicate that FIV crosses mucous membranes within hours after exposure and rapidly traffics via dendritic and T cells to systemic lymphoid tissues, a pathway similar to that thought to occur in the initial phase of infection by the human and simian immunodeficiency viruses

Increased Serum Enzyme Levels Associated with Kupffer Cell Reduction with No Signs of Hepatic or Skeletal Muscle Injury

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is responsible for the survival and proliferation of monocytes and the differentiation of monocytes into macrophages, including Kupffer cells (KCs) in the liver. KCs play an important role in the clearance of several serum enzymes, including aspartate aminotransferase and creatine kinase, that are typically elevated as a result of liver or skeletal muscle injury. We used three distinct animal models to investigate the hypothesis that increases in the levels of serum enzymes can be the result of decreases in KCs in the apparent absence of hepatic or skeletal muscle injury. Specifically, neutralizing M-CSF activity via a novel human monoclonal antibody reduced the CD14+CD16+ monocyte population, depleted KCs, and increased aspartate aminotransferase and creatine kinase serum enzyme levels in cynomolgus macaques. In addition, the treatment of rats with clodronate liposomes depleted KCs and led to increased serum enzyme levels, again without evidence of tissue injury. Finally, in the osteopetrotic (Csf1op/Csf1op) mice lacking functional M-CSF and having reduced levels of KCs, the levels of serum enzymes are higher than in wild-type littermates. Together, these findings support a mechanism for increases in serum enzyme levels through M-CSF regulation of tissue macrophage homeostasis without concomitant histopathological changes in either the hepatic or skeletal system