Histology and immunohistochemistry of the gut-associated lymphoid tissue of the eastern grey kangaroo, Macropus giganteus

Mesenteric lymph nodes and gut-associated lymphoid tissue (GALT) from juvenile eastern grey kangaroos were investigated. The mesenteric nodes had a similar structure to that described for eutherian mammals. They contained distinct regions of medulla and cortex, with prominent follicles and germinal centres. Gut associated lymphoid tissue consisted of areas of submucosal follicles. These varied from areas of densely packed lymphocytes with darkly staining, prominent coronas to areas with no defined follicles. The distribution of T cells in these tissues was documented by use of species-crossreactive antibodies to the surface markers CD3 and CD5; B cells were identified by antibodies to CD79b. Within the lymph nodes T cells were located mainly in the paracortex and cortex, with limited numbers observed in the follicles; B cells were located on the marginal zone of the follicles. In GALT, T cells were located in the peripheral regions of the germinal centres of secondary follicles, while B cells were abundant in primary follicles. These observations are consistent with those made in a range of other marsupials (metatherian) and eutherian mammals and are indicative of the capacity to respond to antigens entering via the mouth.     

Immunohistochemistry of the lymphoid tissues of the tammar wallaby,Macropus eugenii

The lymphoid tissues of the metatherian mammal, the adult tammar wallaby, Macropus eugenii, were investigated using immunohistochemical techniques. Five cross-reactive antibodies previously shown to recognize surface markers in marsupial tissues and five previously untested antibodies were used. The distribution of T-cells in the tissue beds of spleen, lymph node, thymus, gut-associated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT) was documented using antibodies to CD3 and CD5. Similarly, B-cells were identified in the same tissues using anti-CD79b. Antibodies to CD8, CD31, CD79a and CD68 failed to recognize cells in these tissue beds. In general the pattern of cellular distribution identified using these antibodies was similar to that observed in other marsupial and eutherian lymphoid tissues. This study provides further information on the commonality of lymphoid tissue structure in the two major groups of extant mammals, metatherians and eutherians.     

Histological Development of the Immune Tissues of a Marsupial,the Red‐Tailed Phascogale (Phascogale calura)

Red‐tailed phascogale (Phascogale calura) pouch young at birth were relatively underdeveloped in comparison with their eutherian counterparts, and the lymphoid tissues of the immune system were found to be histologically immature. The phascogale thymus rapidly developed in the first few days of pouch life and was quickly populated with lymphocytes. By the end of pouch life, involution of the thymus was underway. The bone marrow started to develop in the early stage of pouch life, although adipocytes and megakaryocytes were not observed until slightly later. The liver was hematopoietic from birth and reached histological maturity toward the end of pouch life. The lymph nodes were difficult to detect macroscopically because of their small size, but were easily identified microscopically later in pouch life, particularly in the mesentery, and these lymph nodes exhibited germinal centers by the end of pouch life. The early spleen was predominately mesenchymal, but exhibited some erythropoiesis. Follicles with well‐developed germinal centers were not observed until the latest stage of pouch life. Although intraepithelial lymphocytes were detected in the intestines early in pouch life, the discrete lymphoid aggregates and Peyer's patches characteristic of the gut‐associated lymphoid tissue (GALT) were not detected until later in pouch life. This is the first report of histological development in phascogale pouch young, as well as the first report of the thymus, bone marrow, and lymph nodes in this dasyurid species at any age. Anat Rec, 299:207–219, 2016. © 2015 Wiley Periodicals, Inc.     

Development of the thymus, spleen, lymph nodes and liver in the marsupial, Isoodon macrourus (Northern brown bandicoot, Peramelidae)

We report for the first time the development and morphological characteristics of the spleen thymus, lymph nodes and liver of the northern brown bandicoot, Isoodon macrourus. To date few marsupial species have been studied. The development and morphological characteristics of the organs shared the typical features of those few other marsupials studied as well as those of eutherians. These suggest comparative functional properties with the eutherian immune system. The thymus differentiated within the first week of pouch life and showed evidence of immunolymphopoiesis up to juvenile stages. The spleen, though seeded by lymphocytes within the first week of pouch life, was slower to mature, but differentiated and showed signs of immunocompetency by the time young left the pouch. The mature spleen displayed the same anatomical blood filtering and immunosurveillance properties as that of the eutherian spleen, with evidence of erythrocyte destruction, thrombopoietic activity, activation and differentiation of immunocompetent lymphocytes. However, the absence of sheathed capillary structures in the spleen may indicate differences in the humoral response to circulating antigens. Similarly, lymph nodes also mature by this stage with anterior nodes appearing before posterior nodes. The mature lymph nodes displayed structural features of secondary immuno-lymphoid organs consistent with production of immune responses. Finally, the liver displayed haemopoietic activity for the first four weeks of pouch life. The pattern of development in the bandicoot appears to parallel the pattern reported for other marsupials, yet the thymus matures considerably earlier than previously reported and may be of significance in the development and onset of cell-mediated immunity. Current studies to characterise cellular components, such as T/B lymphocyes and accessory cells of these organs will help to define the mechanisms of immune recognition, activation and hence outline the asis of the marsupial humoral and cellular immunity.     

Development of the thymus, spleen, lymph nodes and liver in the marsupial, Isoodon macrourus (Northern brown bandicoot, Peramelidae)

We report for the first time the development and morphological characteristics of the spleen thymus, lymph nodes and liver of the northern brown bandicoot, Isoodon macrourus. To date few marsupial species have been studied. The development and morphological characteristics of the organs shared the typical features of those few other marsupials studied as well as those of eutherians. These suggest comparative functional properties with the eutherian immune system. The thymus differentiated within the first week of pouch life and showed evidence of immunolymphopoiesis up to juvenile stages. The spleen, though seeded by lymphocytes within the first week of pouch life, was slower to mature, but differentiated and showed signs of immunocompetency by the time young left the pouch. The mature spleen displayed the same anatomical blood filtering and immunosurveillance properties as that of the eutherian spleen, with evidence of erythrocyte destruction, thrombopoietic activity, activation and differentiation of immunocompetent lymphocytes. However, the absence of sheathed capillary structures in the spleen may indicate differences in the humoral response to circulating antigens. Similarly, lymph nodes also mature by this stage with anterior nodes appearing before posterior nodes. The mature lymph nodes displayed structural features of secondary immuno-lymphoid organs consistent with production of immune responses. Finally, the liver displayed haemopoietic activity for the first four weeks of pouch life. The pattern of development in the bandicoot appears to parallel the pattern reported for other marsupials, yet the thymus matures considerably earlier than previously reported and may be of significance in the development and onset of cell-mediated immunity. Current studies to characterise cellular components, such as T/B lymphocyes and accessory cells of these organs will help to define the mechanisms of immune recognition, activation and hence outline the basis of the marsupial humoral and cellular immunity.     

Myeloid‐Lymphoid Ontogeny in the Rhesus Monkey (Macaca mulatta)

Establishment of a functional immune system has important implications for health and disease, yet questions remain regarding the mechanism, location, and timing of development of myeloid and lymphoid cell compartments. The goal of this study was to characterize the ontogeny of the myeloid‐lymphoid system in rhesus monkeys to enhance current knowledge of the developmental sequence of B‐cell (CD20, CD79), T‐cell (CD3, CD4, CD8, FoxP3), dendritic cell (CD205), and macrophage (CD68) lineages in the fetus and infant. Immunohistochemical assessments addressed the temporal and spatial expression of select phenotypic markers in the developing liver, thymus, spleen, lymph nodes, gut‐associated lymphoid tissue (GALT), and bone marrow with antibodies known to cross‐react with rhesus cells. CD3 was the earliest lymphoid marker identified in the first trimester thymus and, to a lesser extent, in the spleen. T‐cell markers were also expressed midgestation on cells of the liver, spleen, thymus, and in Peyer's patches of the small and large intestine, and where CCR5 expression was noted. A myeloid marker, CD68, was found on hepatic cells near blood islands in the late first trimester. B‐cell markers were observed mid‐second trimester in the liver, spleen, thymus, lymph nodes, bone marrow spaces, and occasionally in GALT. By the late third trimester and postnatally, secondary follicles with germinal centers were present in the thymus, spleen, and lymph nodes. These results suggest that immune ontogeny in monkeys is similar in temporal and anatomical sequence when compared to humans, providing important insights for translational studies. Anat Rec, 297:1392–1406, 2014. © 2014 Wiley Periodicals, Inc.     

The detection of mature T- and B-cells during development of the lymphoid tissues of the tammar wallaby (Macropus eugenii)

The distribution of T- and B-cells in the developing lymphoid and immunohaematopoietic tissues of the tammar wallaby were investigated using antibodies to the mature cell surface markers, CD3, CD5 and CD79b. In the thymus, CD3- and CD5-positive T-cells were first observed at day 12 postpartum whilst rare B-cells were first detected at day 23. Both T- and B-lymphocytes were first stained on day 21 postpartum in the spleen and day 24 in lymph nodes. In one sample from a 7-day-old animal, rare CD79b-positive (CD79b+) lymphocytes were observed in the gut-associated lymphoid tissues. However, CD3+ cells were not apparent until day 12 and CD5+ cells were not detected until day 74 postpartum. No lymphocytes were detected in liver or bone marrow samples and no bronchus-associated lymphoid tissues were observed. The pattern of development and the distribution of T- and B-cells in the lymphoid and immunohaematopoietic tissues were similar to those observed in eutherian mammals and in limited studies of other metatherians. However, the detection of apparently mature T- and B-cells in the thymus and gut-associated lymphoid tissues (GALT) at the same postnatal age highlights the need for a more substantial study of the development of GALT. This is, at present, limited by availability of marsupial-specific antibodies.     

Identification of dendritic cells,B cell and T cell subsets in Tasmanian devil lymphoid tissue; evidence for poor immune cell infiltration into devil facial tumors

The Tasmanian devil is under threat of extinction due to the transmissible devil facial tumor disease (DFTD). This fatal tumor is an allograft that does not induce an immune response, raising questions about the activity of Tasmanian devil immune cells. T and B cell analysis has been limited by a lack of antibodies, hence the need to produce such reagents. Amino acid sequence analysis revealed that CD4, CD8, IgM, and IgG were closely related to other marsupials. Monoclonal antibodies were produced against CD4, CD8, IgM, and IgG by generating bacterial fusion proteins. These, and commercial antibodies against CD1a and CD83, identified T cells, B cells and dendritic cells by immunohistochemistry. CD4⁺ and CD8⁺ T cells were identified in pouch young thymus, adult lymph nodes, spleen, bronchus‐ and gut‐associated lymphoid tissue. Their anatomical distribution was characteristic of mammalian lymphoid tissues with more CD4⁺ than CD8⁺ cells in lymph nodes and splenic white pulp. IgM⁺ and IgG⁺ B cells were identified in adult lymph nodes, spleen, bronchus‐associated lymphoid tissue and gut‐associated lymphoid tissue, with more IgM⁺ than IgG⁺ cells. Dendritic cells were identified in lymph node, spleen and skin. This distribution is consistent with eutherian mammals and other marsupials, indicating they have the immune cell subsets for an anti‐tumor immunity. Devil facial tumor disease tumors contained more CD8⁺ than CD4⁺ cells, but in low numbers. There were also low numbers of CD1a⁺ and MHC class II⁺ cells, but no CD83⁺ IgM⁺ or IgG⁺ B cells, consistent with poor immune cell infiltration. Anat Rec, 297:925–938, 2014. © 2014 The Authors. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology Published by Wiley Periodicals, Inc.     

Uterine remodelling during pregnancy and pseudopregnancy in the brushtail possum (Trichosurus vulpecula; Phalangeridae)

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non‐invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non‐invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.     

The immune tissues of the endangered red-tailed phascogale (Phascogale calura)

The lymphoid tissues of the red-tailed phascogale (Phascogale calura) were examined using histological and immunohistochemical techniques. The distribution of immune cells in the tissue beds was documented using antibodies to surface markers CD3 and an MHC Class II antigen (equivalent to HLA DRII). Spleen, gut-associated lymphoid tissues (GALT), lung, bronchus-associated lymphoid tissue (BALT) and liver were examined. The spleen had defined areas of red and white pulp, with follicles containing tingible-bodied macrophages. Anti-CD3 and anti-HLA DRII antibodies revealed the presence of T cells in areas of white pulp and around the peri-arterial lymphatic sheaths. GALT and BALT were detected and appeared as scattered areas of lymphocytes in the tissues beds. This is the first study to report on the lymphoid tissues of this endangered species of marsupial and the first report of the capacity of anti-human antibodies to a surface MHC molecule to react with Dasyurid cells.     

Hyperplastic Lymphoid Tissue in HIV/AIDS: An Electron Microscopic Study

The purpose of this study was to characterize the ultrastructure of lymphoid tissue from HIV/AIDS patients and to evaluate it as a reservoir and source of HIV. HIV has been demonstrated in lymph nodes and tonsils and adenoids, by immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM), to be associated with germinal center (GC) follicular dendritic cells (FDC). The presence of HIV in the larger gastrointestinal tract-associated lymphoid tissue (GALT) has been much less studied. Whether FDC themselves are productively infected by HIV in any of the lymphoid sites is controversial. Lymph nodes, tonsils, and gastrointestinal biopsies were fixed in neutral buffered glutaraldehyde and prepared for TEM. Mature HIV particles were abundant in GC of hyperplastic lymph nodes, tonsils, and the GALT. They were enmeshed within an electron-dense matrix associated with an all-encompassing branching FDC network of processes. HIV particles were seen budding from both FDC and lymphocytes. The greatest numbers of particles were seen in hyperplastic lymphoid tissue from untreated individuals and in lymph nodes co-infected with opportunistic organisms, such as Mycobacterium aviumcomplex. In addition to HIV, unidentifiable “particles” of varying sizes, possibly including other viruses, were regularly seen in association with FDC. Ultrastructural study graphically demonstrated the abundance of HIV particles associated with the complex FDC network of hyperplastic lymph nodes, tonsils, and GALT. HIV was shown to productively infect FDC, as well as lymphocytes.     

Development of lymphoid tissues of the stripe-faced dunnart (Sminthopsis macroura)

The development of the lymphoid tissues of a model marsupial, the stripe-faced dunnart, has been described from birth to weaning, a period of 2.5 months. At birth the lymphoid tissues, including the thymus, lymph nodes and mucosa-associated lymphoid tissues, were undeveloped. A thoracic thymus consisting primarily of stromal tissue was observed by day 4 after birth but by day 12, lymphocytes were observed in the thymus and some cortico-medullary differentiation was apparent. Lymph nodes were histologically mature by day 31, the earliest day investigated for this tissue. In gut tissue, lymphoid follicles were first observed by day 57 post-partum. No bronchus-associated lymphoid tissue was observed in any lung samples. The thymus, lymph nodes and gut-associated lymphoid tissues were all distinguishable before weaning (day 70) but not all were histologically mature. The sequence of development of the lymphoid tissues in the stripe-faced dunnart was similar to those observed in other marsupial species.     

A Histological and Immunohistochemical Analysis of Lymphoid Tissues of the Tasmanian Devil

Tasmanian devil lymphoid tissues (thymus, spleen, and lymph node) from seven animals, including pouch young, juvenile, and adult devils, were investigated using histological and immunohistochemical techniques. Antibodies against the conserved intracytoplasmic portion of CD3 and CD79b (T‐ and B‐cell markers, respectively) and MHC II were used to label immune cells. The thymus from the juvenile devils and the pouch young had CD3+ cells that were primarily located in the medulla of the organ. The spleen consisted of red and white pulp areas with characteristic lymphoid follicles with CD79b+ and MHC II+ cells and nonfollicular T‐cell‐dominated periarteriolar lymphoid sheaths. Peripheral lymph nodes presented three distinct regions, outer cortex and medulla (both with primarily CD79b+ and MHC II+ cells) and paracortex (mainly CD3+ cells). Tissue architecture and distribution of the immune cells were similar to that seen in eutherian mammals and other marsupials, indicating that the Tasmanian devil has all the structural elements necessary for effective adaptive immunity. Anat Rec, 292:611–620, 2009. © 2009 Wiley‐Liss, Inc.     

The roles of histology and immunohistology in the investigation of marsupial disease and normal lymphoid tissue

This review acquaints scientists with current information related to the application of histology and immunohistology to the studies of normal lymphoid tissues and specific diseases in marsupials. Histological examination of tissue is a necessary prerequisite for immunohistological examination because it establishes tissue structure and detects specific areas of disease which allow the selection of smaller areas for immunohistological examination. Information is provided on the basic techniques of histology. Immunohistology (immunohistochemistry) refers to the identification of antigenic determinants of specific substances (proteins) by the application of antibodies to histological sections. Information is provided on a technique for enzyme based, avidin-biotin enhanced immunohistology, and on antibodies that can be used to mark disease agents or marsupial tissues and cells. A summary is provided of the available information on studies of the histology and immunohistology of normal marsupial lymphoid tissue (thymus, spleen, lymph nodes and mucosa associated lymphoid tissue), and of selected marsupial diseases such as chlamydiosis and lymphosarcoma in koalas.     

An Examination of the Development and Localization of Key Immune Cells in Developing Pouch Young of the Red-Tailed Phascogale (Phascogale calura)

Cells expressing the surface markers CD3, CD4, CD79b, IgM, MHC class II, and ModoUG (nonclassical MHC class I) were detected in red-tailed phascogale tissues using immunohistochemistry, and the appearance and localization of cells observed here was consistent with previous observations in other marsupial species. CD3⁺ cells were first detected at one day postpartum (dpp) in the thymus, followed by ModoUG⁺ cells at 5–7 dpp in the thymus and lymph nodes. CD79b⁺ cells were first detected at 12–14 dpp in bone marrow, spleen, and lymph nodes. IgM⁺ cells were first detected at 12–14 dpp in thymus, bone marrow, spleen, and lymph nodes. MHC class II⁺ cells were first detected at 12–14 dpp in thymus, bone marrow, and lymph nodes. CD4⁺ cells were detected in adult thymus and spleen only. The presence of the mature immune cell populations and their localization to characteristic T and B cell zones in mature lymphoid tissues with normal histological structure indicates that red-tailed phascogales develop immunocompetence by the end of pouch life. Anat Rec, 302:1985–2002, 2019. © 2019 American Association for Anatomy     

Effects of oestrogen and exercise on caspase‐3 activity in primary and secondary lymphoid compartments in ovariectomized mice

This study investigated the effect of oestrogen exposure and exercise on caspase‐3 activity, a measure of apoptosis, in lymphocytes from the thymus, spleen, and lymph nodes in ovariectomized mice. Fifty‐nine female B6D2F₁ mice were randomized to hormone and exercise conditions. Hormone treatment consisted of implantation with oestradiol pellets (0.72 mg oestradiol) or placebo pellets (0 mg) for 21 days following bilateral ovariectomy (OVX). Exercise consisted of a single treadmill exercise bout (26 m min^?1, 6° slope, 90‐min) or sedentary condition. Mice were killed and the thymus, spleen and lymph nodes were removed for the determination of caspase‐3 expression by enzyme‐linked immunosorbent assay (ELISA), serum oestrogen levels by RIA, and tissue weights. Body weights were monitored throughout the study. In the thymus, oestrogen exposure, exercise and both treatments together were associated with higher caspase‐3 activity (P < 0.05) and lower thymus weights (P < 0.05). In contrast, oestrogen exposure and exercise treatment were not associated with greater caspase‐3 activity or change in tissue weight in secondary lymphoid tissues (spleen, lymph nodes). Oestrogen‐replaced OVX mice had a higher concentration of plasma oestradiol than placebo OVX mice (P < 0.05). Conclusion: The results suggest that oestrogen and treadmill exercise are associated with greater apoptosis, as measured by caspase‐3 activity, in the thymus but not in the spleen or lymph nodes. Clinical studies will be necessary to determine if women who take oestrogen have higher rates of apoptosis in primary lymphoid tissues and the significance of thymocyte apoptosis for maintenance of cellular immune function during the post‐menopausal years.     

Antigen receptor V-segment usage in mucosal T cells

In the accepted model of lymphocyte intestinal homing, naïve T cells recirculate via organized lymphoid tissues, whilst induced effector/memory cells home to the intestinal mucosa. In order to assess the T‐cell‐receptor repertoire in the intestine and gut‐associated lymphoid tissue (GALT), spectratyping was performed on the proximal and the distal intestine, spleen and mesenteric lymph node tissue from six PVG rats. The products were analysed with an automated sequencer and statistical analyses were performed with hierarchical cluster analysis. This demonstrated the presence of a restricted T‐cell repertoire in the small intestine compared with that in the mesenteric lymph nodes and the spleen. It also demonstrated marked differences in repertoire between individual, fully inbred rats maintained under apparently identical conditions in the same cage and fed identical diets. In addition, this work demonstrated marked differences between repertoires in the proximal and the distal intestine. Such marked differences are likely to reflect the end result of increasing divergence over time produced by relatively subtle effects of environment and antigenic load. Equally, marked differences in repertoire between small intestinal segments within individual rats indicate selective recruitment or retention of specific clones, presumably antigen‐driven.     

Lung Development of Monotremes: Evidence for the Mammalian Morphotype

The reproductive strategies and the extent of development of neonates differ markedly between the three extant mammalian groups: the Monotremata, Marsupialia, and Eutheria. Monotremes and marsupials produce highly altricial offspring whereas the neonates of eutherian mammals range from altricial to precocial. The ability of the newborn mammal to leave the environment in which it developed depends highly on the degree of maturation of the cardio‐respiratory system at the time of birth. The lung structure is thus a reflection of the metabolic capacity of neonates. The lung development in monotremes (Ornithorhynchus anatinus, Tachyglossus aculeatus), in one marsupial (Monodelphis domestica), and one altricial eutherian (Suncus murinus) species was examined. The results and additional data from the literature were integrated into a morphotype reconstruction of the lung structure of the mammalian neonate. The lung parenchyma of monotremes and marsupials was at the early terminal air sac stage at birth, with large terminal air sacs. The lung developed slowly. In contrast, altricial eutherian neonates had more advanced lungs at the late terminal air sac stage and postnatally, lung maturation proceeded rapidly. The mammalian lung is highly conserved in many respects between monotreme, marsupial, and eutherian species and the structural differences in the neonatal lungs can be explained mainly by different developmental rates. The lung structure of newborn marsupials and monotremes thus resembles the ancestral condition of the mammalian lung at birth, whereas the eutherian newborns have a more mature lung structure. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.