Molecular localization techniques in the diagnosis and characterization of nonhuman primate infectious diseases.

Molecular localization techniques remain important diagnostic and research tools for the pathologist evaluating nonhuman primate tissues. In situ hybridization and immunohistochemistry protocols have been developed for many important pathogens of nonhuman primates, including RNA and DNA viruses, prions, and bacterial, protozoal, and fungal pathogens. Such techniques will remain critical in defining the impact and relevance of novel agents on animal health and disease. A comparative pathology perspective often provides valuable insight to the best strategy for reagent development and can also facilitate interpretation of molecular localization patterns. Such a perspective is grounded in a firm understanding of microbe-host pathobiology. This review summarizes current molecular localization protocols used in the diagnosis of selected primate infectious diseases.

Polioencephalomalacia secondary to hypernatremia in squirrel monkeys (Saimiri sciureus).

Squirrel monkeys (Saimiri spp) are one of the most consistently used New World primates in biomedical research and are increasingly being used in neuroscience research, including models of drug abuse and addiction. Spontaneous neurologic disease in the squirrel monkey is uncommonly reported but includes various infectious diseases as well as cerebral amyloidosis. Hypernatremia is an extremely serious condition of hyperosmolarity that occurs as a result of water loss, adipsia, or excess sodium intake. Neurologic effects of hypernatremia reflect the cellular dehydration produced by the shift of water from the intracellular fluid space into the hypertonic extracellular fluid space. Severe hypernatremia may result in cerebrocortical laminar necrosis (polioencephalomalacia) in human patients as well as in a number of domestic species, including pigs, poultry, and ruminants. We report the clinical, histopathologic, and immunohistochemical findings of polioencephalomalacia in 13 squirrel monkeys. Polioencephalomalacia in these animals was associated with hypernatremia that was confirmed by serum levels of sodium greater than 180 mmol/L (reference range, 134.0-154.0 mmol/L [mEq/L]). All animals had concurrent diseases or experimental manipulation that predisposed to adipsia. Immunohistochemical investigation using antibodies to neuronal nuclei (NeuN), CNPase, Iba-1, and CD31 revealed necrosis of predominantly cerebral cortical layers 3, 4, and 5 characterized by neuronal degeneration and loss, oligodendrocytic loss, microglial proliferation, and vascular reactivity. The squirrel monkey is exquisitely sensitive to hyperosmolar metabolic disruption and it is associated with laminar cortical necrosis.

Acute traumatic spinal cord injury induces glial activation in the cynomolgus macaque (Macaca fascicularis).

BACKGROUND: Traumatic spinal cord injury leads to direct myelin and axonal damage and leads to the recruitment of inflammatory cells to site of injury. Although rodent models have provided the greatest insight into the genesis of traumatic spinal cord injury (TSCI), recent studies have attempted to develop an appropriate non-human primate model. METHODS: We explored TSCI in a cynomolgus macaque model using a balloon catheter to mimic external trauma to further evaluate the underlying mechanisms of acute TSCI. RESULTS: Following 1hour of spinal cord trauma, there were focal areas of hemorrhage and necrosis at the site of trauma. Additionally, there was a marked increased expression of macrophage-related protein 8, MMP9, IBA-1, and inducible nitric oxide synthase in macrophages and microglia at the site of injury. CONCLUSIONS: This data indicate that acute TSCI in the cynomolgus macaque is an appropriate model and that the earliest immunohistochemical changes noted are within macrophage and microglia populations.

CD8+ lymphocyte depletion without SIV infection does not produce metabolic changes or pathological abnormalities in the rhesus macaque brain.

BACKGROUND: Simian immunodeficiency virus (SIV) infection and persistent CD8(+) lymphocyte depletion rapidly leads to encephalitis and neuronal injury. The objective of this study is to confirm that CD8 depletion alone does not induce brain lesions in the absence of SIV infection. METHODS: Four rhesus macaques were monitored by proton magnetic resonance spectroscopy ((1) H-MRS) before and biweekly after anti-CD8 antibody treatment for 8 weeks and compared with four SIV-infected animals. Post-mortem immunohistochemistry was performed on these eight animals and compared with six uninfected, non-CD8-depleted controls. RESULTS: CD8-depleted animals showed stable metabolite levels and revealed no neuronal injury, astrogliosis or microglial activation in contrast to SIV-infected animals. CONCLUSIONS: Alterations observed in MRS and lesions in this accelerated model of neuroAIDS result from unrestricted viral expansion in the setting of immunodeficiency rather than from CD8(+) lymphocyte depletion alone.

Ovarian pathology in rhesus macaques: a 12-year retrospective.

BACKGROUND: Ovarian pathology is an important cause of decreased fertility and reproductive capability and may impact multiple systems, particularly in aging rhesus macaques. METHODS: Retrospective histopathologic and immunohistochemical analysis of 458 female rhesus macaque necropsies over 12 years at the New England Primate Research Center in Southborough, MA. RESULTS: Degenerative and inflammatory changes in the ovaries included mineralization, infiltration by lymphocytes, macrophages and multinucleated giant cells, endometriosis, and arteriopathy. Cystic changes included follicular cysts, cystic rete, and mesonephric duct cysts with cystic rete the most common. Neoplasms included granulosa cell tumors, cystadenoma, cystadenocarcinoma, and teratoma. CONCLUSIONS: Ovarian lesions of the rhesus macaque are similar to those of cynomolgus macaques and humans. These lesions are frequently incidental findings but may impact metabolic and neurocognitive studies.

Histologic and immunohistochemical characterization of pheochromocytoma in 6 cotton-top tamarins (Saguinus oedipus).

Pheochromocytomas are uncommon neoplasms of the adrenal medulla that are most frequently reported in rats and select mouse strains. In many cases, especially those in man, pheochromocytoma is associated with familial tumor syndromes, because of inherited mutations in a variety of proto-oncogenes and tumor suppressor genes. Nonhuman primates are valuable animal models for a variety of human diseases because of their similar anatomy and physiology; however, cases of pheochromocytomas have only rarely been reported in nonhuman primates. Herein, we characterize the gross, histologic, and immunohistochemical features of pheochromocytoma in 6 cotton-top tamarins (Saguinus oedipus). Pheochromocytomas represented 6 of 114 of the total causes of death in the studied population (5.3%) and corresponded to 16% of the total number of neoplasms. The average age of affected animals was 17.9 years. On histologic examination, all cases were defined by tight bundles, nests, and cords of neoplastic chromaffin cells. All cases had concurrent myocardial degeneration and fibrosis of varying severity and chronicity. Three of the cases (50%) also had hyalinization and medial thickening of coronary arteries consistent with hypertension. On immunohistochemical examination, 6 of 6 of the cases (100%) stained positively for chromogranin A, synaptophysin, N-CAM (or CD56), and protein gene product 9.5. None of the cases stained for glial fibrillary acidic protein. Pedigree analysis revealed inter-relatedness in 4 of 6 animals, with progenitor animals also affected with pheochromocytomas. The tumors in this population illustrate comparable histologic and immunohistochemical staining patterns with cases in other laboratory animals and humans, and, therefore, may indicate common underlying genetic alterations that precipitate tumor development.

Pediatric hepatic hemangiosarcoma in a rhesus macaque (Macaca mulatta).

BACKGROUND: Pediatric hepatic angiosarcoma is a rare condition in children with poor prognosis. Microscopically this neoplasm has a particular 'Kaposi-form' arrangement. Hemangiosarcoma in non-human primates is a rare finding. METHODS: Gross and microscopic examination of a 3-year-old rhesus were performed. Immunohistochemistry was used to characterize the hepatic hemangiosarcoma. RESULTS: The gross necropsy revealed hemoabdomen and a 4 x 3 x 3 cm mass in the liver with multiple smaller masses throughout the hepatic parenchyma. Histopathology confirmed a poorly differentiated hemangiosarcoma. Other organs submitted were free of metastases. CONCLUSIONS: Hemangiosarcoma in non-human primates has been rarely reported. Diagnosis was confirmed by expression of endothelial-specific markers CD31 and vWF by immunohistochemistry. Due to the young age of this monkey and the particular solid pattern throughout the mass this neoplasm resembles pediatric hepatic angiosarcoma in humans.

Rhesus lymphocryptovirus type 1-associated B-cell nasal lymphoma in SIV-infected rhesus macaques.

Epstein-Barr virus (EBV) is a worldwide endemic gamma herpesvirus of the genus Lymphocryptovirus (LCV) that infects more than 90% of the world's population. EBV has been associated with a variety of malignancies, but it has a demonstrated role in lymphomas, especially in immunosuppressed individuals. Lymphomas of the nasal cavity, paranasal sinuses, and nasopharynx are uncommon and constitute less than 5% of all extranodal lymphomas. Sinonasal non-Hodgkin's lymphomas have been reported in patients infected with human immunodeficiency virus (HIV) at an increased frequency. Rhesus LCV (rhLCV), the rhesus viral homolog of EBV, has been cloned and is associated with B-cell lymphomas in immunosuppressed rhesus macaques. We report two cases of B-cell lymphoma within the nasal cavity from 2 simian immunodeficiency virus-infected rhesus macaques with acquired immunodeficiency syndrome. The B-cell phenotype and rhLCV association were demonstrated by immunohistochemistry and confocal microscopy. The majority of the nuclei of the neoplastic B lymphocytes were EBNA-2 positive. RhLCV type 1 sequences were verified from the neoplasms by polymerase chain reaction. Nasal lymphoma is an unusual presentation of rhLCV-associated B-cell lymphoma in immunosuppressed rhesus macaques. These tumors demonstrate comparable viral pathogenesis with EBV-induced nasal lymphomas in HIV-positive people.

Necrotizing meningoencephalitis and pneumonitis in a simian immunodeficiency virus-infected rhesus macaque due to Acanthamoeba.

Free-living amoebae of the genus Acanthamoeba can cause a fatal disease of the brain in humans called granulomatous amoebic encephalitis. We present a case of meningoencephalitis and pneumonitis in a simian immunodeficiency virus (SIV)-infected rhesus macaque caused by Acanthamoeba sp. The animal became ill 176 days after intravenous inoculation with SIVmac251 after a short history of weight loss and a sudden onset of hind limb paresis and abnormal head movements. Histopathologic examination of hematoxylin and eosin-stained tissues revealed multifocal to coalescing necrotizing neutrophilic meningoencephalitis and pneumonitis. Immunofluorescence and polymerase chain reaction were used to identify the genus of amoeba as Acanthamoeba. Immunohistochemistry of immune cell markers was used to characterize the animal's immune response to the opportunistic amoebic infection with features of both innate and adaptive cell-mediated immunity. Although not previously reported, the potential transmission to humans, either through environmental contamination or contact with an infected animal, makes this disease a threat to laboratory animal care staff and pathologists.

Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain.

Emerging data indicate that chemokine receptors on neurons and glia in the central nervous system (CNS) play a role in normal CNS development, intercellular communication, and the neuropathogenesis of AIDS. To further understand chemokine receptors in the brain and explore their potential role in HIV neuropathogenesis, particularly in pediatrics, we examined the regional and cellular distribution of CCR5 and CXCR4 in normal fetal, neonatal, and adult rhesus macaques. CCR5 and CXCR4 were detected by immunohistochemistry and immunofluorescence within the cytoplasm of subpopulations of neurons in the neocortex, hippocampus, basal nuclei, thalamus, brain stem, and cerebellum and by flow cytometry on the surface of neurons and glia. Interestingly, expression of CCR5 and CXCR4 increased significantly (p<0.05) from birth to 9 months of age. We further characterize this dynamic developmental pattern of CCR5 and CXCR4 expression in resident cells of the CNS.

Macaques with rapid disease progression and simian immunodeficiency virus encephalitis have a unique cytokine profile in peripheral lymphoid tissues.

The influence of host cytokine response on viral load, disease progression, and neurologic lesions was investigated in the simian immunodeficiency virus (SIV)-infected macaque model of AIDS. Cytokine gene expression (interleukin-1beta [IL-1beta], IL-2, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral loads were evaluated by semiquantitative reverse transcription-PCR in lymph nodes of 5 control animals and 28 animals infected with SIVmac251 at the terminal stages of AIDS. Infected animals showed higher expression of IFN-gamma, IL-6, and IL-10 mRNAs compared with controls. Levels of all cytokines were comparable between animals with rapid (survival, <200 days) or slow/normal (survival, >200 days) disease progression. However, among rapid progressors, the eight animals with SIV encephalitis had a unique cytokine profile (increased IL-2, IL-6, and IFN-gamma) that was associated with higher viral loads. These observations provide evidence that host cytokine responses may influence SIV neuropathogenesis independent of disease progression.

Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS.

The macrophage is well established as a target of HIV and simian immunodeficiency virus (SIV) infection and a major contributor to the neuropathogenesis of AIDS. However, the identification of distinct subpopulations of monocyte/macrophages that carry virus to the brain and that sustain infection within the central nervous system (CNS) has not been examined. We demonstrate that the perivascular macrophage and not the parenchymal microglia is the primary cell productively infected by SIV. We further demonstrate that although productive viral infection of the CNS occurs early, thereafter it is not easily detectable until terminal AIDS. The biology of perivascular macrophages, including their rate of turnover and replacement by peripheral blood monocytes, may explain the timing of neuroinvasion, disappearance, and reappearance of virus in the CNS, and questions the ability of the brain to function as a reservoir for productive infection by HIV/SIV.

Expression of peripherin in the brain of macaques (Macaca mulatta and Macaca fascicularis) occurs in astrocytes rather than neurones and is associated with encephalitis.

Peripherin is a member of the type III intermediate filament family, expressed in neurones of the peripheral nervous system of many species and in a discrete subpopulation of neurones of the central nervous system (CNS) during early development in rodents. Previous studies on rats have shown that peripherin immunoreactivity increased significantly in cell bodies of spinal motor neurones following axonal injury. Our study examined the expression of peripherin in the cerebrum of normal macaques (Macaca mulatta and Macaca fascicularis) and those with encephalitis of viral (simian immunodeficiency virus and simian virus 40) or autoimmune (experimental allergic encephalomyelitis) aetiology. Immunohistochemistry, immunoelectronmicroscopy, immunofluorescence and confocal microscopy were performed on tissue sections using antibodies against cell-specific markers and peripherin. Peripherin-positive cells were absent in the cerebrum of normal macaques of all ages examined, whereas animals with encephalitis had peripherin-positive cells associated with inflammatory infiltrates. Further evaluation revealed that these peripherin-positive cells were not neurones, but were predominantly astrocytes expressing glial fibrillary acidic protein. Our study suggests that peripherin is not neurone-specific in the CNS of macaques; peripherin is expressed in astrocytes of animals with encephalitis.

Early brain injury in the SIV-macaque model of AIDS.

OBJECTIVE: To specify the type and severity of cellular damage in the central nervous system soon after infection and at later stages of disease in the SIV-macaque model of AIDS. DESIGN AND METHODS: Adjacent samples of frontal cortical gray matter were taken from three groups of macaques: uninfected controls (n = 4), acute (14 days post-infection; n = 4), and chronic (mean 2 years post-infection; n = 7). In vitro high resolution magnetic resonance spectroscopy of snap frozen intact tissue and quantitative neuropathology measurements of synaptophysin, calbindin, and glial fibrillary acidic protein (GFAP) in formalin-fixed tissue were performed. RESULTS: Losses in n-acetylaspartate and calbindin (indicating neuronal injury and/or death) and decreases in synaptophysin immunoreactivity (indicating synaptodendritic injury) were detected along with increases in GFAP (indicating reactive gliosis). Cellular injury worsened progressively with increased time after infection. CONCLUSIONS: These results are the first direct evidence that neuronal injury occurs soon after infection. The exacerbation of injury with time suggests a connection between the early response of the central nervous system and dementia, which occurs late in the course of infection. This connection may have broad implications for the study of and the development of therapies for damage of the central nervous system by HIV.

Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses.

The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20(+) B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection.

Neuropathogenesis of simian immunodeficiency virus in neonatal rhesus macaques.

Neonatal human immunodeficiency virus (HIV) infection usually occurs intrapartum or postpartum and results in a higher incidence of neurological dysfunction than is seen in adults. To explore the neuropathogenesis of neonatal HIV infection, we infected neonatal macaques with simian immunodeficiency virus (SIV) and followed the course of infection focusing on early time points. Infected neonates had decreased brain growth and mild histological changes in brain that resembled those seen in pediatric AIDS, including perivascular infiltrates of mononuclear cells, mineralization of vessels in the basal ganglia, and gliosis. The perivascular lesions and gliosis were associated with the presence of occasional infected cells that required in situ hybridization with radiolabeled riboprobes for detection. Using this technique, SIV-infected cells were detected in the brain parenchyma within 7 days of infection. These findings were confirmed by nested PCR for SIVgag DNA in brain and RT-PCR for viral RNA in cerebrospinal fluid. Together, these techniques revealed SIV infection of the CNS in 12 of 13 neonates infected with SIVmac239, 3 of 3 infected with SIVmac251, and 2 of 2 infected with SIVmac239/316. The prevalence of CNS infection was indistinguishable from that of older animals infected with the same dose and stock of virus, but neonates appeared to have fewer infected cells in the CNS and detecting them required more sensitive techniques. This observation was true regardless of inoculum and despite the fact that neonates had equal or greater viral loads in the periphery compared with older animals. These data suggest that maturation-dependent host factors have a major impact on the neuropathogenesis of pediatric AIDS.

Simian immunodeficiency virus encephalitis in rhesus macaques is associated with rapid disease progression.

Central nervous system (CNS) disease is a major feature of simian immunodeficiency virus (SIV) infection of macaques. To define the spectrum of CNS lesions in SIV-infected macaques and the potential associations with viral strain and disease course, we performed a retrospective analysis of necropsies on 124 macaques with SIV-induced AIDS. Histologic evidence of CNS disease was observed in 71 (57.3%) of the 124 animals. SIV encephalitis was the most common CNS lesion occurring in 43.7% (31/71) of the animals with CNS disease and 25% of all animals. The incidence of SIVE correlated significantly with shortened survival (P=0.0207). In addition, SIVE was seen in 42.9% (15/35) of rapid progressors (animals that died within 200 days) compared to only 18% (16/89) of normal progressors (animals that lived longer than 200 days) (P=0.011). Animals with SIVE had higher viral loads in peripheral blood than those that did not, but this difference did not reach statistical significance. Similarly, while animals infected with uncloned SIVmac251 had a higher incidence of SIVE (27.5%; 14/51) than animals infected with molecularly cloned SIVmac239 and its T-cell tropic derivatives (18.5%; 10/54) this difference was not statistically significant. In this study rapid disease progression and SIVE were highly correlated making separation of viral determinants of virulence from those of neurovirulence difficult.

Chemokine receptor expression on resident and inflammatory cells in the brain of macaques with simian immunodeficiency virus encephalitis.

Although the mechanisms of human immunodeficiency virus (HIV) neuroinvasion, neuronal injury, and subsequent development of HIV-1-associated AIDS dementia complex are not fully understood, a correlation between monocyte/macrophage infiltrates in the brain and neurological disease exists. In light of the many potential roles that chemokines and chemokine receptors may play in HIV neuropathogenesis, we sought to describe their pattern of expression in the SIV-infected rhesus macaque model of HIV encephalitis. We previously demonstrated elevated expression of the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and interferon-inducible protein (IP)-10 in brain of macaque monkeys with SIV encephalitis. In this study, we demonstrate that the corresponding chemokine receptors CCR3, CCR5, CXCR3, and CXCR4 are expressed in perivascular infiltrates in these same tissues. In addition, we detected CCR3, CCR5, and CXCR4 on subpopulations of large hippocampal and neocortical pyramidal neurons and on glial cells in both normal and encephalitic brain. These findings suggest that multiple chemokines and their receptors contribute to monocyte and lymphocyte recruitment to the brain in SIV encephalitis. Furthermore, the expression of known HIV/SIV co-receptors on neurons suggests a possible mechanism whereby HIV or SIV can directly interact with these cells, disrupting their normal physiological function and contributing to the pathogenesis of AIDS dementia complex.

Toxicity of TNF alpha and platelet activating factor for human NT2N neurons: a tissue culture model for human immunodeficiency virus dementia.

A significant proportion of HIV-1 infected individuals develop a symptom complex consisting of dementia and motor deficits termed HIV Dementia (HIVD) or the AIDS Dementia complex (ADC). The pathophysiology of this neurologic complication is unclear, but neuronal injury and death may occur as a direct result of the release of cytokines from HIV-1 infected microglial cells (Everall et al, 1991). To evaluate the utility of a human neuronal cell line, NT2N, for studies of HIV-related neuronal cytotoxicity, we studied cellular viability after exposure to HIV-1 gp120, tumor necrosis factor alpha (TFN alpha), platelet activating factor (PAF), interleukin 1 beta (IL-1 beta), and interferon gamma (IFN gamma), all of which have been implicated in previous publications as having a role in HIVD (Brenneman et al, 1988; Dreyer et al, 1990; Merrill et al, 1992; Gelbard et al, 1993). Neither gp 120 nor the cytokines IL-1 beta and IFN gamma resulted in significant NT2N cell death. However, TNF alpha and PAF were highly neurotoxic in this assay. Pentoxifylline, which inhibits the effects of TNF alpha, had a significant protective effect. This system provides an excellent substrate for the evaluation of neurotoxicity and for the development of pharmacologic agents that may be useful in HIV dementia.

Hepatic disposition and biliary excretion of bilirubin and bilirubin glucuronides in intact rats. Differential processing of pigments derived from intra- and extrahepatic sources.

Mechanisms for transport of bilirubin and its conjugates in hepatocytes have not been defined. We investigated the hepatic processing of bilirubin glucuronides and their precursors, and characterized the disposition of bile pigments arising from intraversus extrahepatic sources. Tracer doses of purified radiolabeled biliverdin, bilirubin, bilirubin monoglucuronide (BMG) or diglucuronide (BDG) were administered intravenously to intact normal or jaundiced homozygous Gunn rats. Rapid sequential analysis of radiolabeled BMG and BDG in bile revealed comparable excretion patterns following biliverdin and bilirubin injection, with BDG as the major pigment. Biliary excretion of radiolabeled conjugates from injected BMG was more rapid, with BMG predominating. Excretion of injected BDG in normal rats and BMG or BDG in Gunn rats was virtually identical to that of unaltered BMG in normal rats. Model independent analysis by deconvolution provided objective comparison of the disposition of radiolabeled pigments from the different sources. These findings indicate that bilirubin glucuronides formed in the liver from endogenous (hepatic) and exogenous (extrahepatic) sources of bilirubin follow a similar excretory pathway. BMG formed endogenously is converted preferentially to BDG, whereas circulating BMG is excreted predominantly unchanged. Exogenous conjugated bilirubins are excreted more rapidly than those generated intrahepatically, by a transcellular pathway that is largely independent of the conjugation system.