Laura V. Chalifoux

Identifying the Target Cell in Primary Simian Immunodeficiency Virus (SIV) Infection: Highly Activated Memory CD4+ T Cells Are Rapidly Eliminated in Early SIV Infection In Vivo

ABSTRACT It has recently been shown that rapid and profound CD4+T-cell depletion occurs almost exclusively within the intestinal tract of simian immunodeficiency virus (SIV)-infected macaques within days of infection. Here we demonstrate (by three- and four-color flow cytometry) that this depletion is specific to a definable subset of CD4+ T cells, namely, those having both a highly and/or acutely activated (CD69+ CD38+HLA-DR+) and memory (CD45RA−Leu8−) phenotype. Moreover, we demonstrate that this subset of helper T cells is found primarily within the intestinal lamina propria. Viral tropism for this particular cell type (which has been previously suggested by various studies in vitro) could explain why profound CD4+ T-cell depletion occurs in the intestine and not in peripheral lymphoid tissues in early SIV infection. Furthermore, we demonstrate that an acute loss of this specific subset of activated memory CD4+ T cells may also be detected in peripheral blood and lymph nodes in early SIV infection. However, since this particular cell type is present in such small numbers in circulation, its loss does not significantly affect total CD4+ T cell counts. This finding suggests that SIV and, presumably, human immunodeficiency virus specifically infect, replicate in, and eliminate definable subsets of CD4+ T cells in vivo.

Induction of an AIDS virus-related tuberculosis-like disease in macaques: A model of simian immunodeficiency virus-Mycobacterium coinfection

ABSTRACT The mechanism by which human immunodeficiency virus (HIV)-Mycobacterium tuberculosis coinfection facilitates development of HIV-related tuberculosis is poorly characterized. Macaque models of simian immunodeficiency virus (SIVmac)-Mycobacterium bovis BCG coinfection were employed to explore the pathogenesis of AIDS virus-related tuberculosis. Following BCG coinfection, SIV (SIV)-infected macaques with high viral loads developed an SIV-related tuberculosis-like disease. This disease was characterized clinically by a syndrome of diarrhea, anorexia, weight loss, and altered levels of consciousness and pathologically by the presence of disseminated granulomas. In contrast, SIVmac-infected macaques with low viral loads either showed no evidence of BCG-induced disease or developed focal granulomatous lesions. Pathogenic SIV-BCG interactions appeared to play a critical role in triggering the development of this SIV-related tuberculosis-like disease. BCG coinfection enhanced the destruction of CD4+ T cells in SIVmac-infected macaques whose viral loads were high. Reciprocally, exacerbations of SIV disease led to marked suppression of BCG-specific T-cell responses, persistence of the BCG infection, and development of an SIV-related tuberculosis-like disease. Furthermore, development of this SIV-related tuberculosis-like disease was also seen in naïve macaques simultaneously inoculated with SIVmac and BCG. These results provide in vivo evidence that coinfection of AIDS virus-infected individuals with an avirulent mycobacterium can lead to development of a tuberculosis-like disease.

Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta).

CONTEXT Enterocytozoon bieneusi is the most frequent microsporidian parasite of human patients with acquired immunodeficiency syndrome and is a significant cause of diarrhea and wasting. Recently, this organism has also been recognized as a spontaneous infection of several species of captive macaques. As in humans, E bieneusi frequently causes enteropathy and cholangiohepatitis in immunodeficient simian immunodeficiency virus (SIV)-infected macaques. OBJECTIVE To examine E bieneusi as an etiologic agent of nonsuppurative proliferative serositis in immunodeficient rhesus macaques (Macaca mulatta). DESIGN Retrospective analysis of necropsy material obtained from immunodeficient SIV-infected rhesus macaques. RESULTS Examination of SIV-infected rhesus macaques (n = 225) revealed E bieneusi proliferative serositis in 7 of 16 cases of peritonitis of unknown origin. The organism could be identified by in situ hybridization and polymerase chain reaction in sections of pleura and peritoneum obtained at necropsy. Serositis was always accompanied by moderate-to-severe infection of the alimentary tract, and morphologic evidence suggested dissemination through efferent lymphatics. Colabeling experiments revealed most infected cells to be cytokeratin positive and less frequently positive for the macrophage marker CD68. Sequencing of a 607-base pair segment of the small subunit ribosomal gene revealed 100% identity to sequences obtained from rhesus macaques (Genbank accession AF023245) and human patients (Genbank accession AF024657 and L16868). CONCLUSIONS These findings indicate that E bieneusi disseminates in immunodeficient macaques and may be a cause of peritonitis in the immunocompromised host.

Nephritis and hemolytic anemia in owl monkeys (Aotus trivirgatus).

The two most common diseases of captive owl monkeys (Aotus trivirgatus) are hemolytic anemia and glomerulonephritis. The anemia is characterized by total red blood cell counts between 0.45 and 3.44 × 106/μl, hemoglobin values as low as 1.0 g/dl, and many circulating nucleated red blood cells. Centrilobular necrosis in the liver, extramedullary hematopoiesis in liver and spleen, and hemoglobin casts in kidney tubules are prominent histologic features. Hemosiderin and lipofuscin often are found in liver, spleen, kidney and lymph nodes. Microthrombi and microinfarcts sometimes are scattered throughout the brain. Glomerular lesions in Aotus have been described previously and are characterized by increased numbers of mesangial cells and matrix, glomerulosclerosis and electron dense deposits in basement membranes. Lymphocytes, plasma cells and eosinophils frequently are present in the interstitium. In the early stages the cellular infiltrate is periglomerular. The foci then grow to encompass adjacent glomeruli and tubules. Finally, large portions of the kidney are affected and connective tissue proliferates. The incidence of extramedullary hematopoiesis in the liver correlated significantly with that of interstitial nephritis (0.001 < p < 0.01) but not with glomerular lesions. The two kidney lesions, glomerulonephritis and interstitial nephritis, correlated strongly in incidence. They also were found with equal frequency in 87 monkeys with clinical evidence of anemia. This analysis indicates that there may be no common pathogenesis of the hematologic and renal abnormalities as seen in certain autoimmune diseases. However, there could be complex interactions between two or more disease mechanisms that account for the various manifestations of disease.

Antiretroviral Agents Restore Mycobacterium-Specific T-Cell Immune Responses and Facilitate Controlling a Fatal Tuberculosis-Like Disease in Macaques Coinfected with Simian Immunodeficiency Virus and Mycobacterium bovis BCG

ABSTRACT The contribution of immune reconstitution following antiretroviral treatment to the prevention or treatment of human immunodeficiency virus-related primary or reactivation tuberculosis remains unknown. Macaque models of simian immunodeficiency virus-Mycobacterium bovis BCG (SIV/BCG) coinfection were employed to determine the extent to which anti-Mycobacterium tuberculosis immunity can be restored by antiretroviral therapy. Both SIV-infected macaques with active BCG reinfection and naive animals with simultaneous SIV/BCG coinfection were evaluated. The suppression of SIV replication by antiretroviral treatment resulted in control of the active BCG infection and blocked development of the fatal SIV-related tuberculosis-like disease. The resolution of this disease coincided with the restoration of BCG purified protein derivative (PPD)-specific T-cell immune responses. In contrast, macaques similarly coinfected with SIV/BCG but not receiving antiretroviral therapy had depressed PPD-specific primary and memory T-cell immune responses and died from tuberculosis-like disease. These results provide in vivo evidence that the restoration of anti-mycobacterial immunity by antiretroviral agents can improve the clinical outcome of an AIDS virus-related tuberculosis-like disease.

Ultrastructural Morphology of Enterocytozoon bieneusi in Biliary Epithelium of Rhesus Macaques (Macaca mulatta)

Enterocytozoon bieneusi is the most common microsporidian parasite found in humans with acquired immunodeficiency syndrome. A nearly identical organism was recently recognized in rhesus macaques (Macaca mulatta). Ultrastructural examination of this microsporidian parasite in biliary epithelium of rhesus macaques reveals characteristics unique to E. bieneusi, including 1) a lack of sporophorus vesicles or pansporoblastic membranes, 2) direct contact of all stages with the host-cell cytoplasm, 3) elongated nuclei present within proliferative and sporogonial stages, 4) late thickening of the sporogonial plasmodium plasmalemma, 5) electron-lucent inclusions present throughout the life cycle, 6) precocious development of electron dense discs before plasmodial division to sporoblasts, and 7) the presence of polar tube doublets within spores and sporoblasts visualized as 5–7 coils in section.

Clinicopathologic Characterization of Canine Juvenile Cellulitis

The syndrome of canine juvenile cellulitis was observed and characterized throughout its clinical course when it occurred spontaneously in a litter of dogs. Histologically, pyogranulomatous inflammation was seen in facial skin and mandibular and superficial cervical lymph nodes of affected dogs. The predominant inflammatory cell characterized by light and electron microscopy and by immunohistochemical staining was an epithelioid macrophage. The same pyogranulomatous inflammatory process was seen in a lymph node anatomically distant from the site of apparent disease. Interestingly, a littermate with neither clinically evident dermal lesions nor lymphadenopathy had histologic evidence of a milder, but similar inflammatory process in a mandibular lymph node. The observation of canine juvenile cellulitis in clusters of dogs between 1 and 4 months of age and its apparent systemic nature suggest an infectious etiology. Bacterial, fungal, or viral agents were not isolated from affected lymph nodes. Attempts to transfer the disease by inoculation of neonatal puppies with tissue from affected dogs were also unsuccessful.

Pulmonary Cryptosporidiosis in Simian Immunodeficiency Virus—infected Rhesus Macaques

Cryptosporidiosis is a common opportunistic infection in the gastrointestinal tract of human and nonhuman primates with AIDS. Pulmonary infection associated with Cryptosporidium spp. has not been previously reported in monkeys. Two macaques experimentally infected with simian immunodeficiency virus (SIV) had lesions containing cryptosporidial organisms involving the trachea, lungs, bile ducts, pancreas, and intestine. The pulmonary sections revealed moderate to severe bronchopneumonia associated with cryptosporidiosis. Numerous 2-4 μm oval Cryptosporidium spp. organisms were present in the cytoplasm of alveolar macrophages, multinucleated giant cells, and intestinal epithelial cells. Giant cells were positive for SIV by in situ hybridization. These are the first reported cases of cryptosporidiosis with involvement of pulmonary parenchyma in SIV-infected macaques.

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. Delivery of ribosome-inactivating proteins to spleen and lymph node T cells.

The selective delivery in vivo of a T lymphocyte-specific monoclonal antibody and immunotoxin conjugates to T cells in lymph node and spleen was assessed in rhesus monkeys. A transient coating of all T lymphocytes in the lymph nodes and spleens of healthy rhesus monkeys could be achieved after infusion of unconjugated anti-T11. Because derivatized antibody is cleared more rapidly than unconjugated antibody, it was necessary to infuse a higher dose of immunotoxin than antibody alone to achieve saturation of the lymphocyte binding sites with anti-T11. When sufficient antibody-toxin conjugate was infused, toxin was readily demonstrable on lymph node and spleen T cells by 16 h after infusion. This demonstration that toxins can be successfully delivered with specificity to target T cell populations in the monkey suggests that killing of restricted cell populations in vivo should be feasible.

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. IV. Cytotoxic effect of an anti-T11-gelonin immunotoxin.

The cytotoxic effect of a lymphocyte-specific immunotoxin formed by disulfide conjugation of an anti-T11 monoclonal antibody with the ribosome-inactivating protein gelonin was assessed in vitro on peripheral blood T cells and in vivo on splenic and lymph node T cells of macaque monkeys. This immunotoxin was cytotoxic to proliferating peripheral blood T cells in vitro as measured by both direct and indirect assays. Two sequential intravenous infusions into macaque monkeys achieved plasma concentrations of immunotoxin far in excess of those shown to be cytotoxic for cultured T cells and coated all T cells in lymph nodes and spleen with intact immunotoxin for four days. However, the cytotoxic effect of the immunotoxin on T cells in vivo was considerably less than that predicted by the in vitro studies. Further experiments suggested that the state of activation of the targeted T cell population in vivo, or the appearance of anti-immunotoxin antibodies, which occurred in all infused monkeys, might attenuate immunotoxin-mediated cell killing in vivo. These studies illustrate the significant differences between the action of immunotoxin conjugates in vitro, and those seen when these conjugates are utilized as therapeutic agents in vivo.