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Lectins are naturally occurring molecules which bind to specific carbohydrates of glycoconjugates. The binding specificity of lectins can therefore be used to specifically elucidate the glycosylation pattern in various tissues. While lectin histochemistry is usually carried out manually on single slides, a fully automated immunostaining system offers an easy, standardized, and high throughput system. In this study lectin histochemistry was implemented and optimized on a fully automated immunostaining system to investigate glycosylation patterns in the murine respiratory tract and the primary olfactory pathway. We tested 22 commercially available biotinylated lectins for their labelling-profiles to specifically identify morphologic structures. The results showed that lectin staining profiles using the implemented protocol on the automated system were constant and suitable for high throughput morphological studies. Further, the morphological evaluation of the stained slides revealed a complete characterization of the murine respiratory tract and primary olfactory pathway including the lectin binding profiles for the olfactory bulb, the vomeronasal organ and the nasal-associated lymphoid tissue.

Repulsive guidance molecule A (RGMa) is a potent inhibitor of axonal growth and a regulator of neuronal cell death. It is up-regulated following neuronal injury and accumulates in chronic neurodegenerative diseases. Neutralizing RGMa has the potential to promote neuroregeneration and neuroprotection. Previously we reported that a rat anti-N terminal RGMa (N-RGMa) antibody r5F9 and its humanized version h5F9 (ABT-207) promote neuroprotection and neuroregeneration in preclinical neurodegenerative disease models. However, due to its cross-reactivity to RGMc/hemojuvelin, ABT-207 causes iron accumulation in vivo, which could present a safety liability. Here we report the generation and characterization of a novel RGMa-selective anti-N-RGMa antibody elezanumab, which is currently under Phase 2 clinical evaluation in multiple disease indications. Elezanumab, a human monoclonal antibody generated by in vitro PROfusion mRNA display technology, competes with ABT-207 in binding to N-RGMa but lacks RGMc cross-reactivity with no impact on iron metabolism. It neutralizes repulsive activity of soluble RGMa in vitro and blocks membrane RGMa mediated BMP signaling. In the optic nerve crush and optic neuritis models, elezanumab promotes axonal regeneration and prevents retinal nerve fiber layer degeneration. In the spinal targeted experimental autoimmune encephalomyelitis (EAE) model, elezanumab promotes axonal regeneration and remyelination, decreases inflammatory lesion area and improves functional recovery. Finally, in the mouse cuprizone model, elezanumab reduces demyelination, which is consistent with its inhibitory effect on BMP signaling. Taken together, these preclinical data demonstrate that elezanumab has neuroregenerative and neuroprotective activities without impact on iron metabolism, thus providing a compelling rationale for its clinical development in neurodegenerative diseases.

The histopathology slide seminar “Classic Examples in Toxicologic Pathology XXVII” was held on February 21 and 22, 2020, at the Department of Pathology at the University of Veterinary Medicine in Hannover, Germany, with joint organization by the European Society of Toxicologic Pathology. The goal of this annual seminar is to present and discuss classical and actual cases of toxicologic pathology. This article summarizes the presentations given during the seminar, including images of representative lesions. Ten actual and classical cases of toxicologic pathology, mostly induced by a test article, were presented. These included small intestine pathology and transcriptomics induced by a γ-secretase modulator, liver findings in nonhuman primates induced by gene therapy, drug-induced neutropenia in dogs, device-induced growth plate lesions, polycystic lesions in CAR/PXR double knockout mice, inner ear lesions in transgenic mice, findings in Beagle dogs induced by an inhibitor of the myeloid leukemia cell differentiation protein MCL1, findings induced by a monovalent fibroblast growth factor receptor 1 antagonist, kidney lesions induced by a mammalian target of rapamycin inhibitor in combination therapy, and findings in mutation-specific drugs.

ABT-736 is a humanized monoclonal antibody generated to target a specific conformation of the amyloid-beta (Aβ) protein oligomer. Development of ABT-736 for Alzheimer’s disease was discontinued due to severe adverse effects (AEs) observed in cynomolgus monkey toxicity studies. The acute nature of AEs observed only at the highest doses suggested potential binding of ABT-736 to an abundant plasma protein. Follow-up investigations indicated polyspecificity of ABT-736, including unintended high-affinity binding to monkey and human plasma protein platelet factor 4 (PF-4), known to be involved in heparin-induced thrombocytopenia (HIT) in humans. The chronic AEs observed at the lower doses after repeat administration in monkeys were consistent with HIT pathology. Screening for a backup antibody revealed that ABT-736 possessed additional unintended binding characteristics to other, unknown factors. A subsequently implemented screening funnel focused on nonspecific binding led to the identification of h4D10, a high-affinity Aβ oligomer binding antibody that did not bind PF-4 or other unintended targets and had no AEs in vivo. This strengthened the hypothesis that ABT-736 toxicity was not Aβ target-related, but instead was the consequence of polyspecificity including PF-4 binding, which likely mediated the acute and chronic AEs and the HIT-like pathology. In conclusion, thorough screening of antibody candidates for nonspecific interactions with unrelated molecules at early stages of discovery can eliminate candidates with polyspecificity and reduce potential for toxicity caused by off-target binding.

Hepcidin was originally detected as a liver peptide with antimicrobial activity and it functions as a central regulator in the systemic iron metabolism. Consequently suppression of hepcidin leads to iron accumulation in the liver. AbbVie developed a monoclonal antibody ([mAb]; repulsive guidance molecule [RGMa/c] mAb) that downregulates hepcidin expression by influencing the RGMc/bone morphogenetic protein (BMP)/neogenin receptor complex and causes iron deposition in the liver. In a dose range finding study with RGMa/c mAb, rats were treated with different dose levels for a total of 4 weekly doses. The results of this morphometric analysis in the liver showed that iron accumulation is not homogenous between liver lobes and the left lateral lobe was the most responsive lobe in the rat. Quantitative hepcidin messenger RNA analysis showed that the left lateral lobe was the most responsive lobe showing hepcidin downregulation with increasing antibody dose. In addition, the morphometric analysis had higher sensitivity than the chemical iron extraction and quantification using a colorimetric assay. In conclusion, the Prussian blue stain in combination with semi-quantitative and quantitative morphometric analysis is the most reliable method to demonstrate iron accumulation in the liver compared to direct measurement of iron in unfixed tissue using a colorimetric assay.

Current therapies for anemia associated with high hepcidin have limited success. Humanized monoclonal antibodies targeting hemojuvelin decrease hepcidin and improve anemia in three preclinical models. The antihemojuvelin antibodies were successfully applied to a mouse and a rat model of ACD (anemia

High levels of hepcidin, the main regulator of systemic iron metabolism, lead to various diseases. Targeting hepcidin and lowering its concentration is a possible form of intervention in order to treat these diseases. High turnover rate of hepcidin is a major drawback of therapies directly targeting this peptide. We developed two monoclonal antibodies ABT-207 and h5F9-AM8 which inhibit hemojuvelin/repulsive guidance molecule C (RGMc) and downregulate hepcidin. We conducted single-application and dose response studies to understand the antibodies’ mechanism and subchronic toxicology studies to exclude safety-related concerns. Investigation was carried out at different biological levels through qPCR, Affymetrix, liquid chromatography coupled with mass spectrometry (LC-MS/MS), histopathology, serum iron, unsaturated iron binding capacity (UIBC), and drug concentration measurements. After a single application of these antibodies, hepcidin expression in liver and its serum protein levels were reduced. Serum iron increased for several weeks. The RGMc antibodies show a pronounced dose response relationship in rats with h5F9-AM8 having an IC50 (UIBC) of approximately 80-fold higher than ABT-207. When hepcidin levels were downregulated, iron deposition in the liver was visible histologically 1 week post application. These antibody-mediated iron depositions were not associated with any adverse toxicologically relevant effect at the doses and time points evaluated. Iron depositions seen after 14 weekly treatments with ABT-207 were reversible in rats and in cynomolgus monkeys. Due to their long-lasting effects and excellent safety profile, both RGMc-blocking antibodies ABT-207 and h5F9-AM8 are favorable clinical candidates for diseases characterized by high serum hepcidin levels like anemia of chronic disease. Electronic supplementary material The online version of this article (doi:10.1208/s12248-015-9770-4) contains supplementary material, which is available to authorized users.

Neonatal porcine isosporosis is known to cause serious economic losses in piglet farms by causing severe enteritis with dehydration, weight loss and reduced development in the affected animals, predominantly during the first weeks of life. In the present study, piglets experimentally infected with Isospora suis were treated with Bay Vi 9143, a symmetrical triazintrione, at different days post-infection. As shown by clinical and pathological examinations, Bay Vi 9143 is effective against the asexual and sexual stages of I. suis at all selected treatment times. However, the therapeutic use at an early stage of asexual multiplication is most effective before the onset of clinical symptoms.

Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.

3-Hydroxy-2,2-dimethyl- N -[4({[5-(dimethylamino)-1-naphthyl]sulfonyl}amino)-phenyl]propanamide (BAY 38-4766) is a novel selective nonnucleoside inhibitor of cytomegalovirus (CMV) replication with an excellent safety profile. This compound and structural analogues inhibit neither viral DNA synthesis nor viral transcription and translation. Accumulation of dense bodies and noninfectious enveloped particles coincides with inhibition of both concatemer processing and functional cleavage at intergenomic transitions, pointing to interference with viral DNA maturation and packaging of monomeric genome lengths. Resistant virus populations, including a murine CMV (MCMV) isolate with 566-fold-decreased drug sensitivity, were selected in vitro. Sequencing of the six open reading frames (ORFs) known to be essentially involved in viral DNA cleavage and packaging identified mutations in ORFs UL56, UL89, and UL104. Construction of MCMV recombinants expressing different combinations of murine homologues of mutant UL56, UL89, and UL104 and analysis of drug susceptibilities clearly demonstrated that mutant ORFs UL89 exon II (M360I) and M56 (P202A I208N) individually confer resistance to BAY 38-4766. A combination of both mutant proteins exhibited a strong synergistic effect on resistance, reconstituting the high-resistance phenotype of the in vitro mutant. These findings are consistent with genetic mapping of resistance to TCRB (2,5,6-trichloro-1-β- d -ribofuranosyl benzimidazole) (P. M. Krosky et al., J. Virol. 72:4721–4728, 1998) and provide further indirect evidence that proteins encoded by UL89 and UL56 function as two subunits of the CMV terminase. While these studies also suggest that the molecular mechanism of BAY 38-4766 is distinct from that of benzimidazole ribonucleosides, they also offer an explanation for the excellent specificity and tolerability of BAY 38-4766, since mammalian DNA does not undergo comparable maturation steps.

Aberrant craniopharyngeal structures within the neurohypophysis were analyzed in 17 rats, originating from four different colonies of Sprague-Dawley- and Wistar-derived strains, which were used for toxicity studies in five different laboratories. Males were more frequently affected than females. The incidence of these findings, which occurred spontaneously and mainly in aged rats, was very low. Predominant features included tubular or acinar glandular structures, rarely embedded in a fibrous stroma, and, as a rule, not compressing adjacent tissue. In some cases, large cysts filled with colloid-like, amorphous material and cellular debris were present. The tubular structures consisted of a rather flat epithelium, while the cystic elements were lined by a cuboidal or columnar, rarely ciliated epithelium, containing goblet cells, or by a stratified squamous epithelium. These structures reacted positively for cytokeratin. Acinar structures mimicked salivary glands of the serous or mucinous type. In a few cases, small, round or fusiform cells were present. Distribution and predominance of the various epithelial structures depended on the strain and colony of rats. Considering the ontogenic development of the pituitary gland, the morphological aspect of these lesions, their immunoreactivity and former reports on similar findings, we concluded that these rats have aberrant craniopharyngeal structures within the pars nervosa of the hypophysis, originating from remnants of the oro-pharyngeal epithelium of the craniopharyngeal duct (RATHKE's pouch). These lesions, which occurred in different strains and colonies of laboratory rats, represent heterotopias or choristomas, consisting of non-neoplastic growth disturbances. Being of a distinctly non-proliferative nature, they should not be confused with craniopharyngiomas.

To study phage-mediated gene transfer in Yersinia , the ability of Yersinia phages to transduce naturally occurring plasmids was investigated. The transduction experiments were performed with a temperate phage isolated from a pathogenic Yersinia enterocolitica strain and phage mixtures isolated from sewage. Small plasmids (4.3 and 5.8 kb) were transduced at a frequency of 10 −5 to 10 −7 /PFU. However, we could not detect the transduction of any indigenous virulence plasmid (ca. 72 kb) in pathogenic Yersinia strains. Transductants obtained by infection with the temperate phage were lysogenic and harbored the phage genome in their chromosomes.

The carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEACAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection-based infection system, we showed that binding of Opa52 involves residues 27-42, which are predicted to form beta-strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino-terminal domain. The replacement of residues 27-29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEACAM6 receptors that are bound by CEACAM1/CEACAM5-specific Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC' face of the N-terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.

SummaryThe identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.

The use of sensitive biomarkers to monitor skeletal muscle toxicity in preclinical toxicity studies is important for the risk assessment in humans during the development of a novel compound. Skeletal muscle toxicity in Sprague Dawley Rats was induced with clofibrate at different dose levels for 7 days to compare standard clinical pathology assays with novel skeletal muscle and cardiac muscle biomarkers, gene expression and histopathological changes. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) enzyme activity were compared to novel biomarkers fatty acid binding protein 3 (Fabp3), myosin light chain 3 (Myl3), muscular isoform of CK immunoreactivity (three isoforms CKBB, CKMM, CKMB), parvalbumin (Prv), skeletal troponin I (sTnI), cardiac troponin T (cTnT), cardiac troponin I (cTnI), CKMM, and myoglobin (Myo). The biomarker elevations were correlated to histopathological findings detected in several muscles and gene expression changes. Clofibrate predominantly induced skeletal muscle toxicity of type I fibers of low magnitude. Useful biomarkers for skeletal muscle toxicity were AST, Fabp3, Myl3, (CKMB) and sTnI. Measurements of CK enzyme activity by a standard clinical assay were not useful for monitoring clofibrate-induced skeletal muscle toxicity in the rat at the doses used in this study.

Spinal cord injury (SCI) is a devastating condition characterized by loss of function, secondary to damaged spinal neurons, disrupted axonal connections, and myelin loss. Spontaneous recovery is limited, and there are no approved pharmaceutical treatments to reduce ongoing damage or promote repair. Repulsive guidance molecule A (RGMa) is upregulated following injury to the central nervous system (CNS), where it is believed to induce neuronal apoptosis and inhibit axonal growth and remyelination. We evaluated elezanumab, a human anti-RGMa monoclonal antibody, in a novel, newly characterized non-human primate (NHP) hemicompression model of thoracic SCI. Systemic intravenous (IV) administration of elezanumab over 6 months was well tolerated and associated with significant improvements in locomotor function. Treatment of animals for 16 weeks with a continuous intrathecal infusion of elezanumab below the lesion was not efficacious. IV elezanumab improved microstructural integrity of extralesional tissue as reflected by higher fractional anisotropy and magnetization transfer ratios in treated vs. untreated animals. IV elezanumab also reduced SCI-induced increases in soluble RGMa in cerebrospinal fluid, and membrane bound RGMa rostral and caudal to the lesion. Anterograde tracing of the corticospinal tract (CST) from the contralesional motor cortex following 20 weeks of IV elezanumab revealed a significant increase in the density of CST fibers emerging from the ipsilesional CST into the medial/ventral gray matter. There was a significant sprouting of serotonergic (5-HT) fibers rostral to the injury and in the ventral horn of lower thoracic regions. These data demonstrate that 6 months of intermittent IV administration of elezanumab, beginning within 24 h after a thoracic SCI, promotes neuroprotection and neuroplasticity of key descending pathways involved in locomotion. These findings emphasize the mechanisms leading to improved recovery of neuromotor functions with elezanumab in acute SCI in NHPs.