Martina Bleyer

LPS-Induced Lung Inflammation in Marmoset Monkeys – An Acute Model for Anti-Inflammatory Drug Testing

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC50). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.

Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes

Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destruction of insulin-producing beta cells. Substantial advances have been made in beta cell replacement therapies during the last decades. However, lack of eligible donor organs and the need for chronic immunosuppression to prevent rejection critically limit widespread application of these strategies. In this manuscript, we present an experimental study using a bioartificial pancreas device for the transplantation of xenogeneic islet without affecting the immune system in nonhuman primates. We could demonstrate stable graft function and adequate glucose-regulated insulin secretion without the need for immunosuppressive medication. This strategy opens up new avenues for more widespread and safe application of various cell-based therapies. Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions.

Distribution of ciliated epithelial cells in the trachea of common marmosets (Callithrix jacchus)

The distribution of ciliated cells in the tracheal epithelium of common marmosets was evaluated.

Distribution and morphology of Clara cells in common marmosets (Callithrix jacchus)

The objective of this investigation was to define the phenotype and spatial distribution of Clara cells within the respiratory tract of common marmosets and to distinguish them from other non‐ciliated cells (goblet cells, mixed type secretory cells).

Atypical cilia in the respiratory tract of common marmosets (Callithrix jacchus) with and without concurrent lung disease

ABSTRACT Ciliated cells of the respiratory epithelium play an essential role in the mechanisms of mucociliary clearance, and ultrastructural alterations of cilia are known to be associated with respiratory disease. The aim of this study was to evaluate the presence of ciliary changes in common marmosets (Callithrix jacchus) of different age groups and to compare healthy animals with animals suffering from subclinical chronic inflammatory pulmonary lesions. Therefore, samples of different sites of the tracheobronchial tree from 24 common marmosets were investigated by transmission electron microscopy. Ciliary alterations were present in all animals and were represented by compound cilia (“bulging/adhesive type”), extramatrix, extratubuli, and disorientation of the microtubular arrangement. Ciliary alterations affected less than 1% of cilia (average 0.06%–0.55%) with no statistically significant differences between age groups, sample localizations, or healthy and sick animals. The study results suggest that ciliary alterations of secondary nature are a common background finding in common marmosets with individual variability in abundance and have to be considered when interpreting ultrastructural data from respiratory studies.

Morphological and immunohistochemical characterization of spontaneous endometriosis in rhesus macaques ( Macaca mulatta )

Abstract Several cases of spontaneous endometriosis in middle-aged to old rhesus macaques (Macaca mulatta) from the breeding colony of the German Primate Center were thoroughly characterized with regards to anatomical distribution and macroscopic appearance, histological differentiation and immunohistochemical profile including somatic markers, hormonal receptors, and proliferation indices. More than half of the examined animals (five of nine) were directly related to one breeding male, supporting a strong genetic predisposition. Histologically, four different types of endometriotic lesions, depending on the degree of ectopic endometrial gland and stromal differentiation (well differentiated, purely stromal, mixed differentiation, poorly differentiated), could be constantly identified within all animals. Immunohistochemistry (IHC) of cytokeratin (CK), vimentin, smooth muscle actin (SMA), desmin, estrogen (ER), and progesterone (PR) receptors as well as of the nuclear proteins Ki67 and p53 revealed varying staining patterns in the four different types of endometriosis differentiation and compared to normal endometrium. Purely stromal, mixed, or poorly differentiated lesions, especially, showed additional cytokeratin-positive stromal cells, whereas epithelial cells of endometriosis with mixed or poor differentiation increasingly expressed mesenchymal markers (vimentin, SMA). Hormonal receptor and Ki67 expression in well-differentiated endometriotic lesions mostly reflected that of normal endometrial tissue according to the cyclic phase of the animal, while the expression gradually diminished with decreasing grade of differentiation. However, increased nuclear accumulations of p53 antigen could only be continuously detected in epithelial cells of mixed or poorly differentiated endometriosis. Altogether, these findings support the pathogenetic theory of coelomic metaplasia, since the expression profiles of somatic markers in less differentiated forms closely resembled that of mesothelial cells. Thus, the four different histological types of endometriosis might display subsequent grades of differentiation in the course of time, with poorly differentiated types representing newly formed, immature lesions and well-differentiated types being older, fully differentiated forms, rather than being the outcome of dedifferentiation processes.

A genome-wide association study reveals a locus for bilateral iridal hypopigmentation in Holstein Friesian cattle

BackgroundEye pigmentation abnormalities in cattle are often related to albinism, Chediak-Higashi or Tietz like syndrome. However, mutations only affecting pigmentation of coat color and eye have also been described. Herein 18 Holstein Friesian cattle affected by bicolored and hypopigmented irises have been investigated.ResultsAffected animals did not reveal any ophthalmological or neurological abnormalities besides the specific iris color differences. Coat color of affected cattle did not differ from controls. Histological examination revealed a reduction of melanin pigment in the iridal anterior border layer and stroma in cases as cause of iris hypopigmentation. To analyze the genetics of the iris pigmentation differences, a genome-wide association study was performed using Illumina BovineSNP50 BeadChip genotypes of the 18 cases and 172 randomly chosen control animals. A significant association on bovine chromosome 8 (BTA8) was identified at position 60,990,733 with a -log10(p) = 9.17. Analysis of genotypic and allelic dependences between cases of iridal hypopigmentation and an additional set of 316 randomly selected Holstein Friesian cattle controls showed that allele A at position 60,990,733 on BTA8 (P = 4.0e–08, odds ratio = 6.3, 95% confidence interval 3.02–13.17) significantly increased the chance of iridal hypopigmentation.ConclusionsThe clinical appearance of the iridal hypopigmentation differed from previously reported cases of pigmentation abnormalities in syndromes like Chediak-Higashi or Tietz and seems to be mainly of cosmetic character. Iridal hypopigmentation is caused by a reduced content of melanin pigment in the anterior border layer and iridal stroma. A single genomic position on BTA8 was detected to be significantly associated with iridal hypopigmentation in examined cattle. To our knowledge this is the first report about this phenotype in Holstein Friesian cattle.

Morphology and staining behavior of neutrophilic and eosinophilic granulocytes of the common marmoset (Callithrix jacchus).

Common marmosets (Callithrix jacchus) are frequently used as translational animal models for human diseases. However, a comparative study of cytological and histochemical detection methods as well as morphometric and ultrastructural characterization of neutrophils and eosinophils in this species is lacking. Blood samples of house dust mite sensitized and allergen challenged as well as lipopolysaccharide (LPS) challenged marmosets were analyzed with different cytological and histological staining methods. Furthermore, cell size and number of nuclear segments were compared between neutrophils and eosinophils. Electron microscopy was performed to characterize the ultrastructure of granulocytes. Of all applied cytological stains, three allowed differentiation of eosinophils and neutrophils and, thus, reliable quantification in blood smears: May-Grünwald-Giemsa stain, Congo Red and Naphthol AS-D Chloroacetate-Esterase. For histology, Hematoxylin-Eosin (H&E) could not demonstrate clear differences, whereas Sirius Red, Congo Red, and Naphthol AS-D Chloroacetate Esterase showed capable results for identification of eosinophils or neutrophils in lung tissue. Morphometry revealed that marmoset neutrophils have more nuclear segments and are slightly larger than eosinophils. Ultrastructurally, eosinophils presented with large homogeneous electron-dense granules without crystalloid cores, while neutrophils were characterized by heterogeneous granules of different size and density. Additionally, sombrero-like vesicles were detected in tissue eosinophils of atopic marmosets, indicative for hypersensitivity-related piecemeal degranulation. In conclusion, we provide a detailed overview of marmoset eosinophils and neutrophils, important for phenotypic characterization of marmoset models for human airway diseases.

Spontaneous meningioma in a pig-tailed macaque ( Macaca nemestrina )

Abstract We present a case of spontaneous meningioma in a female pig-tailed macaque (Macaca nemestrina) more than 24 years old. Clinically, the monkey displayed slow, weak, and insecure movements and poor vision. A tumorous mass was present at the floor of the cranial vault extending from the optic chiasm towards the foramen magnum. It compressed adjacent parts of the brain, infiltrated the sphenoidal and occipital bone, and showed transcranial expansion into the pharyngeal area. Histologically, the tumor was consistent with a meningioma displaying mostly meningothelial and some microcystic components. Since only six cases of meningiomas in nonhuman primates have been reported so far and only two of these meningiomas have been described in detail, the findings of each case should be reported to expand the knowledge base of this type of tumor. In addition, this is the first description of a meningioma in pig-tailed macaques.

TAENIA CRASSICEPS CYSTICERCOSIS IN A NILGIRI LANGUR (SEMNOPITHECUS JOHNII)

Abstract:  A captive-born adult female Nilgiri langur (Semnopithecus johnii) developed an edematous swelling of the left thigh and a firm mass around the right ankle joint. The animal also suffered from lethargy and anorexia and was euthanized because of poor general condition. Necropsy revealed that the skeletal muscle of the left thigh had been replaced by a multilocular cystic mass containing numerous sand-grain–sized whitish structures. Small cysts were also present in the lung and the myocardium. The mass of the right ankle joint was histologically consistent with a myxosarcoma. In contrast, the cystic masses from the left thigh, the lung, and the myocardium represented metacestode tissue with evidence of numerous larval cestodes consistent with cysticerci. Cysticerci showed morphological characteristics of Cysticercus longicollis, the larval form of Taenia crassiceps, which was confirmed by genetic analysis. This is the first documented case of a Taenia crassiceps cysticercosis in an Old World monkey species.