The pig as a model for immunology research.

The pig is an omnivorous, monogastric species with many advantages to serve as an animal model for human diseases. There are very high similarities to humans in anatomy and functions of the immune system, e g., the presence of tonsils, which are absent in rodents. The porcine immune system resembles man for more than 80% of analyzed parameters in contrast to the mouse with only about 10%. The pig can easily be bred, and there are less emotional problems to use them as experimental animals than dogs or monkeys. Indwelling cannulas in a vein or lymphatic vessel enable repetitive stress-free sampling. Meanwhile, there are many markers available to characterize immune cells. Lymphoid organs, their function, and their role in lymphocyte kinetics (proliferation and migration) are reviewed. For long-term experiments, minipigs (e.g., Göttingen minipig) are available. Pigs can be kept under gnotobiotic (germfree) conditions for some time after birth to study the effects of microbiota. The effects of probiotics can be tested on the gut immune system. The lung has been used for extracorporeal preservation and immune engineering. After genetic modifications are established, the pig is the best animal model for future xenotransplantation to reduce the problem of organ shortage for organ transplantation. Autotransplantation of particles of lymphnodes regenerates in the subcutaneous tissue. This is a model to treat secondary lymphedema patients. There are pigs with cystic fibrosis and severe combined immune deficiency available.

Histological characterization of the lingual tonsils of the one-humped camel (Camelus dromedarius).

Tonsils are located in the entrance of digestive and respiratory tracts forming Waldeyer's ring that reacts against ingested or inhaled antigens. On occasion, tonsils may be a site of entry and replication for some pathogens. The lingual tonsils are a main constituent of the Waldeyer's ring. Despite the immunological importance of the lingual tonsils, there is limited information about their structure in the one-humped camel. The lingual tonsils of 10 clinically healthy male camels (3-25 years) were collected and studied macroscopically and microscopically. Lingual tonsils were localized at the root of the tongue of camels of all ages in the form of several spherical macroscopic nodules protruding into the oropharynx. Each nodule possesses a single central crypt, covered with keratinized stratified squamous epithelium without any M cells and surrounded with an incomplete capsule. Each tonsillar crypt was lined with stratified squamous non-keratinized epithelium with lymphocytic infiltration forming patches of lymphoepithelium or reticular epithelium. Secondary lymphoid nodules extended under the apical epithelium. The interfollicular areas had diffused lymphocytes. Among these lymphocytes, high endothelial venules, macrophages, dendritic cells and plasma cells were observed. The organization of camel lingual tonsils in isolated units with separate crypts increases the surface area exposed to antigen. The present findings indicate a sustained immunological role of the lingual tonsils throughout the life of the one-humped camel.

The bone marrow is not only a primary lymphoid organ: The critical role for T lymphocyte migration and housing of long-term memory plasma cells.

In immunology and anatomy textbooks the bone marrow is described as a typical "primary lymphoid organ" producing lymphoid cells independent of antigens. The hematopoietic bone marrow is largely age-dependent organ with great anatomical and functional differences among various species. There are estimates that about 12% of all lymphoid cells in the human body are found in the bone marrow at any given time (2% in the peripheral blood). Enormous numbers of T lymphocytes migrate to the bone marrow and partly return later to the blood. Many of these lymphocytes are memory CD4+ and CD8+ T cells. A few days after immunization a wave of plasma cells and their precursors migrate to the bone marrow where they lose their migratory response to CXCL-12 and CXCL9. There is a relative enrichment of CD19+ B cells in the bone marrow outnumbering those in the blood and secondary lymphoid organs. This is not due to local production. The proliferation and migration kinetics of these lymphoid cells in the bone marrow have to be studied in more detail as this is of major clinical relevance.

The thymus is relevant in the migration of mature lymphocytes.

The thymus is a primary lymphoid organ where T lymphocyte proliferation and selection takes place. The different subsets of lymphocytes leave the thymus as recent thymic emigrants. Peripheral dendritic cells migrate to the thymus. In addition to the homing of hematopoietic progenitor cells to the thymus, there is evidence for lymphocyte entry from peripheral lymphoid tissues mainly into the medulla. The entry sites are the venules in the medullary part near to the cortex with a higher endothelium. Furthermore, there are also B lymphocytes in the thymus. The thymus is not only a primary lymphoid organ but is well integrated in lymphocyte traffic as shown in several different species.

Co-localization of lymphoid aggregates and lymphatic networks in nose- (NALT) and lacrimal duct-associated lymphoid tissue (LDALT) of mice.

BACKGROUND: The lymphatic vascular pattern in the head of mice has rarely been studied, due to problems of sectioning and immunostaining of complex bony structures. Therefore, the association of head lymphoid tissues with the lymphatics has remained unknown although the mouse is the most often used species in immunology. RESULTS: Here, we studied the association of nasal and nasolacrimal duct lymphatics with lymphoid aggregates in 14-day-old and 2-month-old mice. We performed paraffin sectioning of whole, decalcified heads, and immunostaining with the lymphatic endothelial cell-specific antibodies Lyve-1 and Podoplanin. Most parts of the nasal mucous membrane do not contain any lymphatics. Only the region of the inferior turbinates contains lymphatic networks, which are connected to those of the palatine. Nose-associated lymphoid tissue (NALT) is restricted to the basal parts of the nose, which contain lymphatics. NALT is continued occipitally and can be found at both sides along the sphenoidal sinus, again in close association with lymphatic networks. Nasal lymphatics are connected to those of the ocular region via a lymphatic network along the nasolacrimal duct (NLD). By this means, lacrimal duct-associated lymphoid tissue (LDALT) has a dense supply with lymphatics. CONCLUSIONS: NALT and LDALT play a key role in the immune system of the mouse head, where they function as primary recognition sites for antigens. Using the dense lymphatic networks along the NLD described in this study, these antigens reach lymphatics near the palatine and are further drained to lymph nodes of the head and neck region. NALT and LDALT develop in immediate vicinity of lymphatic vessels. Therefore, we suggest a causative connection of lymphatic vessels and the development of lymphoid tissues.

Isolated aggregates of lymphoid cells in the inner bronchial wall in asthma patients.

Here, we report findings in volunteers with bronchial asthma. Biopsies were obtained from the inner bronchial wall before and a short time again after segmental allergen provocation. In most of the baseline biopsies and in all evaluable biopsies after segmental allergen provocation, the follicular lymphoid tissue was detected by immunohistochemistry in the epithelium of these asthmatic patients. The basic occurrence of the tertiary lymphoid tissue in the bronchial mucosa of mild asthmatics was unexpected and may have consequences for the interpretation of pathophysiology, e.g., as a cause or consequence of bronchial asthma.

Stromal cell heterogeneity in lymphoid organs.

Although the role of stromal cells has not been clearly defined, these cells have been described as forming the extracellular matrix in all lymphoid organs. Their important role in facilitating the development of immune cells in the thymus and bone marrow has long been known. In contrast, stromal cells have been found in secondary lymphoid organs and it has been shown that they are important mediators during organogenesis. More recently, their important function in the guidance and survival of immune cells has been documented. Here, we describe the important role of stromal cells within secondary lymphoid organs and highlight the fact that the immunological function of stromal cells is site-specific and unique in each lymphoid organ.

Lymph node stromal cells strongly influence immune response suppression.

Many pathogens are initially encountered in the gut, where the decision is made to mount an immune response or induce tolerance. The mesentric lymph node (mLN) has been shown to be involved in immune response and much more in oral tolerance induction. Furthermore, using an in vivo transplantation model, we showed recently that lymph node (LN) stromal cells can affect T-cell function and influence the IgA response by supporting a site-specific environment. To elucidate the importance of LN stromal cells for tolerance induction, mLN or peripheral LN were transplanted into mice (mLNtx or pLNtx) and oral tolerance was induced via ovalbumin. A reduced delayed-type hypersensitivity (DTH) response was detected in pLNtx compared to mLNtx mice. Reduced IL-10 expression, reduced percentages of Tregs, and increased proportions of B cells were identified within the pLNtx. The increase of B cells resulted in a specific immunoglobulin production undetectable in mLNtx. Moreover, transferred IgG(+) cells of tolerized peripheral LN induced a strong reduction of the delayed-type hypersensitivity response, whereas CD4(+) cells were less efficient. Thus, stromal cells have a high impact on creating a unique environment. Furthermore, the environment of pLNtx induces a tolerogenic phenotype by B-cell accumulation and antibody production.

The bronchus-associated-lymphoid tissue (BALT) an unique lymphoid organ in man and animals.

The development structure and number of bronchus-associated lymphoid tissue (BALT) will be described in many different animals (like chicken, rabbit, mouse, rat, farm animals and particular the pig, monkey) and these data compared to healthy man and in human diseases. The term induced BALT should not be used because it is a tertiary lymphoid structure, which lacks the contact to a bronchus and does not consist of the important area (dome area) which is essential for antigen uptake of microbial stimuli, which are essential in the development of BALT. Mycoplasma seems to play a critical role as shown in pigs but there not been documented in other species like rabbits. More studies have to be performed in health and disease (e.g. in apes) to document the structural and functional basis to use BALT as an entry site for vaccination protocols.

Comparison of avascular lymph node fragment transplantation techniques to optimize lymphangiogenesis in the minipig model.

BACKGROUND: Secondary lymphoedema is a challenging pandemic. This condition may arise after oncologic resection of tumor-draining lymph nodes and/or radiation. Plastic-surgical procedures for lymphoedema comprise transplantation of vascularized lymph node flaps, which are, however, technically challenging and difficult to implement on a global level due to the scarcity of microsurgery facilities in some countries. To improve this situation, comparative research in valid animal models is needed. METHODS: A total of 33 minipigs were subjected to lymphatic resection in the hind limbs. This large animal model was used in a first phase to compare different lymph node fragmentation methods and assess lymphatic regeneration after avascular transplantation. In a second phase, several stimulants were tested for their effect on lymphatic regeneration after fragment transplantation. In a third phase, animals additionally received irradiation of the groin. In this novel animal model, autologous avascular lymph node fragment transplantation was complemented by peripheral injections of vascular endothelial growth factor-C (VEGF-C). Finally, regeneration rates were quantified in relative numbers (percentage) in the irradiated tissue. RESULTS: In the first phase, transversal lymph node fragmentation under preservation of the nodal capsule showed the best percentage of regeneration (62.5%). Peripheral intradermal administration of VEGF-C enhanced lymph node fragment regeneration (70.8%) better than injections of tetanus toxoid (41.6%) or Streptococcus suis (62.5%). Lymph node fragment regeneration also occurred in an irradiated porcine model of lymphadenectomy under VEGF-C administration (66.6%). CONCLUSIONS: The present findings provide a pre-clinical proof-of-concept for a possible simplification strategy for current operative procedures of autologous lymph node transplantation.Level of evidence : Not gradable.

Innate immunity in the human lung: pathogen recognition and lung disease.

As the human lung is exposed to a variety of microbial pathogens in the environment, a first line of defense is built up by pulmonary cells like bronchial/alveolar epithelial cells and alveolar macrophages. These cells express several pattern recognition receptors (PRRs) recognizing highly conserved microbial motifs and initiating the production of chemokines and pro- and anti-inflammatory cytokines acting as transmembrane or intracellular receptors. This might not only lead to acute but also to chronic inflammation which is discussed as an underlying mechanism in the pathogenesis of different lung diseases.

Macrophage activating lipopeptide 2 is effective in mycobacterial lung infection.

MALP-2, a synthetic lipopeptide is a Toll-like receptor 2 and -6 ligand and agonist. MALP-2 stimulates immune cells at different sites. Local stimulation in the lungs has beneficial effects in experimental pneumococci infection. The presented study investigated local effects of MALP-2 in the mycobacterial infection of lungs. MALP-2 was applied prior, simultaneously or after the pulmonary infection with Mycobacterium bovis BCG. Colony forming units were determined in the bronchoalveolar lavage fluid and lung homogenate. Numbers of Mycobacterium bovis BCG colony forming units were found to be reduced in two compartments, bronchoalveolar lavage fluid and lung homogenate after treatment with MALP-2 simultaneously to the infection for up to eight weeks. Reduction of the bacterial load in both compartments was also found up to two weeks after local treatment before and after the infection. Thus, macrophage activating lipopeptide-2 enhances the host defence in the lung in acute and long term bacterial infections.

The Hippocratic Oath and the physician's pledge and their potential role early in medical education.

This is a short view point on a possible role of physician's pledges in today's medical careers. The history of the Hippocratic Oath and the Geneva Declaration was depicted and its relevance for education in Anatomy was mooted. Anatomy is taught very early in the medical curriculum and anatomists should behave in an exemplary manner with respect to the dead bodies as well as to medical students as future colleagues.

Bronchus-associated lymphoid tissue: an entry site for antigens for successful mucosal vaccinations?

An accumulation of lymphoid cells with a typical localization of B lymphocytes preferentially in a follicle and T lymphocytes, more peripherally around high endothelial venules in the wall of bronchi, is called bronchus-associated lymphoid tissue (BALT). A further structural component is a cap-like accumulation of lymphoid cells partly bulging into the lumen of the bronchus, called the dome area. The epithelium covering the dome lacks goblet cells, is infiltrated by lymphocytes, and contains cells specialized for antigen uptake-M cells. BALT is not present in all species and age groups and can be classified as a tertiary lymphoid organ. A hypothesis is proposed for a two-step vaccination protocol: first, BALT is induced and activated, and second, an antigen-the vaccine-is applied locally. BALT is part of the integrated mucosal-associated lymphoid tissue system.

Mesenteric lymph nodes are not required for an intestinal immunoglobulin A response to oral cholera toxin.

Stimulation of the adaptive immune system in the gut is thought to be mainly initiated in the Peyer's patches as well as in the mesenteric lymph nodes (mLNs) and results in immunoglobulin A (IgA) secretion by plasma cells in the lamina propria. However, the precise role of the mLNs in the development of IgA immune responses is poorly understood. Thus, cholera toxin (CT) was administered to mLN-resected and mLN-bearing animals and the IgA response to CT in the intestine and serum was examined. Levels of CT-specific IgA antibodies and the numbers of cells producing these antibodies in the intestine were increased in mLN-resected rats. Particularly in the distal parts of the intestine, the jejunum and the ileum, IgA responses to orally administered antigens developed were stronger in the intestine after removal of the mLNs. This strongly indicates that the mLNs play a critical role in modulating the expansion of specific IgA responses. After removal of the mLNs, the lymph from the gut flows directly into the blood. It was investigated whether the spleen is involved in the initiation of an immune response to orally administered CT after removal of the mLNs. In the spleens of mLN-resected animals, proliferation was up-regulated, and germinal centres were formed in the follicles. However, CT-specific IgM(+) cells, but no IgA(+) cells, developed. Additionally, an increase of CT-specific IgM in the serum was found in mLN-resected animals. Thus, the data indicate that the spleen is involved in the immune response to CT after mLN resection.

Sex differences in a transgenic rat model of Huntington's disease: decreased 17beta-estradiol levels correlate with reduced numbers of DARPP32+ neurons in males.

Recent clinical studies have highlighted that female sex hormones represent potential neuroprotective mediators against damage caused by acute and chronic brain diseases. This evidence has been confirmed by experimental studies documenting the protective role of female sex hormones both in vitro and in vivo, although these studies did not specifically focus on Huntington's disease (HD). We therefore investigated the onset and course of HD in female and male transgenic (tg) HD (CAG(n51)) and control rats across age and focused on three aspects: (i) behavioral and physiological alterations (energy expenditure, home-cage activity, emotional disturbance and motor dysfunction), (ii) morphological markers (numbers and characteristics of striatal DARPP32(+) medium-sized spiny neurons (MSNs) and dopamine receptor autoradiography) and (iii) peripheral sex hormone levels as well as striatal estrogen receptor expression. Independent of their sex, tgHD rats exhibited increased levels of food intake, elevated home-cage activity scores and anxiolytic-like behavior, whereas only males showed an impairment of motor function. In line with the latter finding, loss and atrophy of DARPP32(+) MSNs were apparent only in male tgHD rats. This result was associated with a decreased striatal dopamine D1 receptor density and lower plasma levels of 17beta-estradiol at the age of 14 months. As DARPP32(+) MSNs expressed both alpha- and beta-estrogen receptors and showed a correlation between cell numbers and 17beta-estradiol levels, our findings suggest sex-related differences in the HD phenotype pointing to a substantial neuroprotective effect of sex hormones and opening new perspectives on the therapy of HD.

Histology of hemal nodes of the water buffalo (Bos bubalus).

Hemal nodes are independent lymphoid organs found in various mammals but are ignored by most immunologists. They seem to play a role in defense against blood-borne infections in some species. The structure of the hemal node has been described in various species but, so far, not in the water buffalo. Specimens were obtained from ten clinically healthy male animals (five calves: 2-3 months old; five bulls: 2-8 years old). Six hemal nodes were obtained from each animal from the mesenteric and perirectal region. The samples were studied by light and transmission electron microscopy. The hemal nodes are bean-shaped or spherical, with one hilus through which the hilus arteries and nerves enter the node and from which veins and lymphatics leave it. The buffalo hemal node has a thin capsule of connective tissue and a few smooth muscle cells. Trabeculae extend from the capsule partially dividing the parenchyma. Subcapsular and trabecular blood sinuses are present. The parenchyma is composed of irregular lymphoid cords rich in erythrocytes, macrophages, and plasma cells and is separated by blood sinuses of variable size engorged with blood. These blood sinuses drain into the trabecular sinuses and then into the subcapsular sinus. In calves, the size of the lymphoid cords is larger than that in adult bulls. Buffalo hemal nodes can be classified as typical hemal nodes, because they are definitely different from hemolymph nodes in other species. They may play a role in filtering the blood.

Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa.

Nosocomial infections often cause lethal pneumogenic sepsis. Information on early bacteria-host interaction in the lung is limited. In the present study, mice were sacrificed 60 min and 4 h after Pseudomonas aeruginosa (PA) infection to investigate lung morphology by using electron microscopy and light microscopy. After 1 h, bacteria were found in the alveoli partly in contact with surfactant. Alveolar macrophages were seen with up to 10 intracellular bacteria close to protrusions of alveolar epithelial type I cells and the gas/blood barrier. A rare but surprising finding was bacteria and even replicating bacteria in alveolar epithelial type II cells (AEII). No bacteria were seen in capillaries. Neither engulfment of bacteria by neutrophils nor structural damage of the pulmonary barrier was visible. After 4 h, many neutrophils were found within the capillaries, but also in the alveolar space. Thus, we hypothesize that, in early stages of infection, the uptake of PA even by single AEII can influence the course of the disease.

Age-dependent histoarchitectural changes in human lymph nodes: an underestimated process with clinical relevance?

Experimental evidence indicates that lymph nodes in humans undergo alterations during ageing. This is clinically important because of the crucial role of these organs in the immune system and their lymph reabsorption and drainage function. Although some age-related changes in lymph node histoarchitecture have been described, they are seldom taken into account in traditional depictions of lymph nodes. Recently introduced clinical procedures, such as intranodal vaccination or lymph node transplantation, have demonstrated the need for an accurate knowledge of these degenerative processes. In this study, superficial inguinal lymph nodes were obtained from 41 deceased patients between 17 and 98 years old. To minimize immunological influences, such as chronic diseases, specimens were only obtained from forensic pathology autopsies. An immunohistochemical analysis was carried out, on the basis of which lymph node degeneration was scored according to the numbers of lymphocytes and high endothelial venules, and degree of fibrosis and lipomatosis. We observed an age-dependent tendency towards the replacement of areas populated with diverse immune cells by connective tissue. Paradoxically, these changes were also detected in some of the nodes from younger age groups. In conclusion, lymph nodes can display degenerative changes that are mainly age-related and often diverge from the common description found in textbooks. These alterations should be taken into account when dealing with lymph nodes diagnostically and therapeutically in clinical practice.

Stromal cells confer lymph node-specific properties by shaping a unique microenvironment influencing local immune responses.

Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103(+) dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.