Norbert Makori

HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages

Immature dendritic cells are among the first cells infected by retroviruses after mucosal exposure. We explored the effects of human immunodeficiency virus-1 (HIV-1) and its Tat transactivator on these primary antigen-presenting cells using DNA microarray analysis and functional assays. We found that HIV-1 infection or Tat expression induces interferon (IFN)-responsive gene expression in immature human dendritic cells without inducing maturation. Among the induced gene products are chemokines that recruit activated T cells and macrophages, the ultimate target cells for the virus. Dendritic cells in the lymph nodes of macaques infected with simian immunodeficiency virus (SIV) have elevated levels of monocyte chemoattractant protein 2 (MCP-2), demonstrating that chemokine induction also occurs during retroviral infection in vivo. These results show that HIV-1 Tat reprograms host dendritic cell gene expression to facilitate expansion of HIV-1 infection.

Pathogenesis of retinoic acid-induced ear malformations in a primate model

13-cis retinoic acid (RA) is a causative agent for human/monkey retinoic acid embryopathy (RAE), in which the most common type of malformation is microtia or anotia. In the present study, malformed ears of monkey fetuses exposed to RA during early embryogenesis were analyzed and revealed a subtype of defects., i.e., apparent duplication of the external/middle ear. A part of the posterior auricle appeared to be ectopically formed in the anterior auricular region or in the region posterior to the auricle. Additionally, there was duplication of the zygomatic arch, malleus, and incus. In order to characterize possible pathogenetic events underlying these malformations, embryos at selected stages were collected after dosing dams with RA at 5 mg/kg/day during gestational days 12-27. Cellular retinoic acid binding protein I whole-mount immunostaining showed that RA induced specific alterations in the migration of cranial neural crest cells (NCC). NCC en route to the second pharyngeal arch were bifurcated, and some of these NCC migrated abnormally into the first and/or third arches, which may underlie external ear duplication. Scanning electron microscopy and neurofilament immunostaining provided evidence that there was partial duplication of trigeminal nerve/ganglion following RA insult. The duplication of NCC neuronal derivatives in the first pharyngeal arch is consistent with duplication of NCC mesenchymal components (zygomatic arch, malleus, and incus). Therefore, RA-induced alterations in cranial NCC migration patterns are likely to be a pathogenetic event underlying ear malformations (including duplication) of RAE in monkeys.

Functional and Morphological Development of Lymphoid Tissues and Immune Regulatory and Effector Function in Rhesus Monkeys: Cytokine-Secreting Cells, Immunoglobulin-Secreting Cells, and CD5+ B-1 Cells Appear Early in Fetal Development

ABSTRACT Little is known regarding the timing of immune ontogeny and effector function in fetal humans and nonhuman primates. We studied the organization of lymphocyte and antigen-presenting cell populations in developing lymphoid tissues of rhesus monkey fetuses during the second and third trimesters (65 to 145 days of gestation; term = 165 days). Immunoglobulin-secreting and cytokine-secreting cells were detected at day 80. The thymus, spleen, lymph nodes, and intestinal mucosa were examined for cells expressing CD3, CD5, CD20, CD68, p55, and HLA-DR. In the spleens of 65-day-old fetuses (early second trimester), the overwhelming majority of total lymphocytes were CD5+ CD20+ B-1 cells. The remaining lymphocytes were CD3+ T cells. By day 80, splenic B and T cells were equal in number. Intraepithelial CD3+ CD5− T cells and lamina propria CD20+ CD5+ B cells were present in the intestines of 65-day-old fetuses. By day 80, numerous CD20+ CD5+ B cells were present in the jejunums and colons and early lymphocyte aggregate formation was evident. The spleens of 80- to 145-day-old fetuses contained immunoglobulin M (IgM)-secreting cells, while IgA-, IgG-, interleukin-6-, and gamma interferon-secreting cells were numerous in the spleens and colons. Thus, by the second trimester, the lymphoid tissues of the rhesus monkey fetus have a complete repertoire of properly organized antigen-presenting cells, T cells, and B cells.

The nonhuman primate as a model of developmental immunotoxicity

Macaques are well suited for preclinical testing of biopharmaceutics due to reproductive and developmental similarities with humans. In order to characterize ontogeny of the immune system in this model, we studied lymphocyte and antigen-presenting cell populations in developing lymphoid tissues of rhesus macaque fetuses during the second and third trimesters [gestation days (GD) 75–145, term 165 days]. Systemic lymphoid tissues (thymus, spleen and lymph nodes, and intestinal tissue) were examined for morphology and cell surface markers by immunohisto-chemistry. Lymphocytes were further characterized by flow cytometry for differentiation markers. Splenic tissue from early second trimester fetuses was populated mainly by CD20+ B cells while the thymus contained large numbers of CD3+ T cells. In the late second trimester (day 80), appro-ximately equal populations of B and T cells were present in both tissues and numerous dendritic cells (p55+) were present in the intestinal lamina propria. By the second trimester, the rhesus macaque fetal lymphoid system is well developed. Analysis of lymphoid organs from retinoic acid-treated fetuses indicated that the T-cell (thymus)-dependent compartment of the spleen white pulp in specimens with thymic aplasia showed a reduction in size and proportion of CD3+ T cells compared to controls. Our findings indicate that RA-induced thymic defects result in disrupted development of the splenic T-cell-dependent compartment.

Nonhuman primates : their role in assessing developmental effects of immunomodulatory agents

There are close physiologic similarities between humans and macaques that make them well suited for preclinical testing of biopharmaceutics. These include menstrual cycles of similar length and hormonal control, comparable cellular and endocrine processes of implantation, and similar time-tables of prenatal development. Three teratogenic agents have induced abnormal development of the macaque thymus that is a key organ in the development of the fetal immune system. Embryonic exposure to triamcinolone acetonide, a potent corticosteroid, during critical periods of thymus development caused marked hypoplasia, depletion of thymic lymphocytes, and reduction of epithelial elements. Aplasia and hypoplasia of the thymus were a distinct feature of the “retinoid syndrome” in cynomolgus macaques following exposure to 13-cis-retinoic acid (Accutane) in early pregnancy, the time of neural crest migration. Experimentally induced zinc deficiency in rhesus macaques from conception to 1-year of age caused severe alterations in immunocompetence. More recent studies have shown that the levels of IgG and IgA in cervicovaginal lavages of the rhesus macaque exhibit specific temporal patterns during the normal menstrual cycle. Taken together, theses data suggest that several macaque species are appropriate animal models for preclinical safety assessment of immunomodulatory drugs. Current teratology protocols in these models may require slight modifications to adequately assess the safety of these biologics.

High Beta-Chemokine Expression Levels in Lymphoid Tissues of Simian/Human Immunodeficiency Virus 89.6-Vaccinated Rhesus Macaques Are Associated with Uncontrolled Replication of Simian Immunodeficiency Virus Challenge Inoculum

ABSTRACT Viral suppression by noncytolytic CD8+ T cells, in addition to that by classic antiviral CD8+ cytotoxic T lymphocytes, has been described for human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. However, the role of soluble effector molecules, especially beta-chemokines, in antiviral immunity is still controversial. In an attenuated vaccine model, approximately 60% of animals immunized with simian/human immunodeficiency virus (SHIV) 89.6 and then challenged intravaginally with SIVmac239 controlled viral replication (viral RNA level in plasma, <104 copies/ml) and were considered protected (K. Abel, L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller, J. Virol. 77:3099-3118, 2003). To determine the in vivo importance of beta-chemokine secretion and CD8+-T-cell proliferation in the control of viral replication in this vaccine model, we examined the relationship between viral RNA levels in the axillary and genital lymph nodes of vaccinated, protected (n = 20) and vaccinated, unprotected (n = 11) monkeys by measuring beta-chemokine mRNA levels and protein expression, the frequency of CD8+ T cells expressing beta-chemokines, and the extent of CD8+-T-cell proliferation. Tissues from uninfected (n = 3) and unvaccinated, SIVmac239-infected (n = 9) monkeys served as controls. Axillary and genital lymph nodes from unvaccinated and vaccinated, unprotected monkeys had significantly higher beta-chemokine mRNA expression levels and increased numbers of beta-chemokine-positive cells than did vaccinated, protected animals. Furthermore, the lymph nodes of vaccinated, unprotected monkeys had significantly higher numbers of beta-chemokine+ CD8+ T cells than did vaccinated, protected monkeys. Lymph nodes from vaccinated, unprotected animals also had significantly more CD8+-T-cell proliferation and marked lymph node hyperplasia than the lymph nodes of vaccinated, protected monkeys. Thus, higher levels of virus replication were associated with increased beta-chemokine secretion and there is no evidence that beta-chemokines contributed to the SHIV89.6-mediated control of viral replication after intravaginal challenge with SIVmac239.

13‐cis‐retinoic acid alters neural crest cells expressing Krox‐20 and Pax‐2 in macaque embryos

This study investigates hindbrain and associated neural crest (NCC), otocyst, and pharyngeal arch development in monkey embryos following teratogenic exposure to 13‐cis‐retinoic acid (cRA). cRA was orally administered (5 mg/kg) to pregnant long‐tailed macaques (Macaca fascicularis) between gestational days (GD) 12 and 27. Embryos were surgically collected at desired stages during treatment, analyzed for external morphological changes, and processed for immunohistochemistry. Two transiently expressed nuclear proteins, Krox‐20 and Pax‐2, were used as markers for the target cellular and anatomical structures. Rhombomere (r) expression patterns of Pax‐2 (r4/r6) and Krox‐20 (r3/r5) were maintained after cRA treatment, but r4 and r5 were substantially reduced in size. In untreated embryos, Krox‐20 immunoreactive NCC derived from r5 migrated caudally around the developing otocyst to contribute to the third pharyngeal arch mesenchyme. In cRA‐treated embryos, a subpopulation of NCC rostral to the otocyst also showed Krox‐20 immunoreactivity, but there was a substantial reduction in Krox‐20 post‐otic NCC. Pax‐2 immunoreactive NCC migrating from r4 to the second pharyngeal arch were substantially reduced in numbers in treated embryos. Alteration in the otic anlage included delayed invagination, abnormal relationship with the adjacent hindbrain epithelium, and altered expression boundaries for Pax‐2. cRA‐associated changes in the pharyngeal arch region due to cRA included truncation of the distal portion of the first arch and reduction in the size of the second arch. These alterations in hindbrain, neural crest, otic anlage, and pharyngeal arch morphogenesis could contribute to some of the craniofacial malformations in the macaque fetus associated with exposure to cRA. Anat Rec 255:142–154, 1999.

Morphology of a Six-Legged Goat With Duplication of the Intestinal, Lower Urinary, and Genital Tracts

An adult female goat with rare malformations, which consisted of duplication of the intestinal, lower urinary, and genital tracts as well a pair of parasitic appendages, is presented.

13-cis-retinoic acid causes patterning defects in the early embryonic rostral hindbrain and abnormal development of the cerebellum in the macaque

BACKGROUND We have previously reported that exposure of embryos to 13-cis-retinoic acid (cRA) results in an abnormal phenotype of the fetal cerebellum. In this study, we analyzed early changes in the cerebellar anlagen (midbrain-hindbrain junction) as well as lesions of the fetal cerebellar vermis after a teratogenic dosing regimen of cRA in the macaque model. METHODS We examined embryo coronal sections of the midbrain-hindbrain junction immunostained for Pax-2, Engrailed-2 (En-2) and Krox-20. To characterize the cerebellum foliation and fissure formation processes, we analyzed vermal cortical cell layer development and the number and depth of the major fissures on sagittal sections of fetal vermis. We also examined Purkinje cell development in vermal sections immunostained for CD3. RESULTS Compared with controls, there was a consistent truncation of the midbrain-hindbrain region of early embryos exposed to cRA. The cRA-induced fetal vermis lesions included inhibition in its anteroposterior growth, altered folial patterning, a general loss of prominence of the fissures accompanied by a total loss of sublobular fissures, and changes in cortical cell layer development. CD3(+) Purkinje cells were abnormally dispersed deep into the molecular layer in the vermis. CONCLUSIONS Our findings indicate that the effects of cRA on the developing cerebellum involve interference with the hierarchy of complex cellular and genetic interactions that lead to the growth and subdivision of the cerebellum into smaller units. The regional vermal defects may be related to early postnatal functional deficits.

EFFECTS OF 13-CIS-RETINOIC ACID ON HINDBRAIN AND CRANIOFACIAL MORPHOGENESIS IN LONG-TAILED MACAQUES (MACACA FASCICULARIS)

Hindbrain and craniofacial development during early organogenesis was studied in normal and retinoic acid‐exposed Macaca fascicularis embryos. 13‐cis‐retinoic acid impaired hindbrain segmentation as evidenced by compression of rhombomeres 1 to 5. Immunolocalization with the Hoxb‐1 gene product along with quantitative measurements demonstrated that rhombomere 4 was particularly vulnerable to size reduction. Accompanying malformations of cranial neural crest cell migration patterns involved reduction and/or delay in pre‐ and post‐otic placode crest cell populations that contribute to the pharyngeal arches and provide the developmental framework for the craniofacial region. The first and second pharyngeal arches were partially fused and the second arch was markedly reduced in size. The otocyst was delayed in development and shifted rostrolaterally relative to the hindbrain. These combined changes in the hindbrain, neural crest, and pharyngeal arches contribute to the craniofacial malformations observed in the retinoic acid malformation syndrome manifested in the macaque fetus.