A specific nanobody prevents amyloidogenesis of D76N \u3b22-microglobulin in vitro and modifies its tissue distribution in vivo

Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of \u3b22-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type \u3b22-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The \u3b22-microglobulin -binding nanobody, Nb24, more potently inhibits D76N \u3b22-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In \u3b22-microglobulin knock out mice, the D76N \u3b22-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type \u3b22-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis

The two tryptophans of β2-microglobulin have distinct roles in function and folding and might represent two independent responses to evolutionary pressure

We have recently discovered that the two tryptophans of human β2-microglobulin have distinctive roles within the structure and function of the protein. Deeply buried in the core, Trp95 is essential for folding stability, whereas Trp60, which is solvent-exposed, plays a crucial role in promoting the binding of β2-microglobulin to the heavy chain of the class I major histocompatibility complex (MHCI). We have previously shown that the thermodynamic disadvantage of having Trp60 exposed on the surface is counter-balanced by the perfect fit between it and a cavity within the MHCI heavy chain that contributes significantly to the functional stabilization of the MHCI. Therefore, based on the peculiar differences of the two tryptophans, we have analysed the evolution of β2-microglobulin with respect to these residues

Mammary carcinoma with ocular metastasis in a mare

Mammary neoplasias in mares are considered a rare event, usually documented via case reports [1,2,3] or in small case series [4], with a carcinoma prevalence rate of 0,11% [3]. An endo-ocular mass from a 24 years old nonpregnant sella italiano mare was conservatively removed and istologically and immunohistochemically diagnosed as a neuroepithelial benign tumor (adenoma of the ciliary body). A second eye mass was then surgically excised with a second histological outcome consistent with a solid carcinoma with squamous differentiation. Due to poor clinical conditions, the animal was euthanized and sent to the necropsy service of the Department of Comparative Biomedicine and Food Science. Necropsy findings showed a primary solid firm mass with a diameter of 15 cm, involving the right mammary gland, with a yellow-whitish cut surface with a central portion of necrosis. One to 6 cm in size, similar nodular masses were found in several tissues and organs including skin, lungs, lymph nodes at different sites, heart, liver, pancreas, parotid gland, intestinal wall (rectum and colon), adrenal glands, muscles and brain. Samples for histological and immunohistochemical examination were collected and routinely processed from all the grossly affected tissues. H&E sections from the primary mass showed multifocal necrosis and a primary neoplastic population of closely packed malignant epithelial cells organized in solid sheets, with foci of squamous differentiation and occasional tubule formation, admixed in abundant fibrovascular stroma and associated with a second population of benign myoepithelial cells. The latter were not detectable in most of the metastases analyzed. Selected sections of the primary tumors and metastases were then immunohistochemically stained for panCK, CK14, CK5-6, CK8-18, vimentin, p63 and calponin (avidin-biotin complex method with a BenchMark automatic immunostainer). As positive control, a dog mammary gland comprehensive of healthy skin and mammary gland was used. The antibody-panel showed: i) strong or mild positive staining in both the primary tumor and metastases for panCK (showing epithelial differentiation) and CK5-6 and CK14, consistent with a proliferation either of basal epithelial cells or myoepithelial cells; ii) occasional positivity for vimentin and p63, and positivity only in the metastases for calponin, consistent with a mild and focal myoepithelial proliferation; iii) inconsistent results for CK8-18, consistent with a low or insignificant proliferation of the luminal epithelium. The findings are compatible with an infiltrative solid carcinoma associated with hyperplasia of the myoepithelial cells. Our findings suggest that the second endo-ocular mass clinically detected was not a relapse of the adenoma of the ciliary body, but a metastasis of the undiagnosed mammary tumor. Despite the reported tendency to metastasize to multiple organs [1,2,4], this is (at the best of our knowledge) the first confirmed ocular metastasis of a mammary carcinoma in a mare

Case Report of a Mare Diagnosed with a Metastatic Mammary Carcinoma after the Excision of a Recurrent Intraocular Neuroepithelial Tumor

A 24-year-old Irish Cob mare was presented with a peripheral iris mass, which was surgically resected and diagnosed as an undifferentiated neuroepithelial tumor. A few months later, a relapse occurred with histological features characterized by a more solid appearance and squamous differentiation. Subsequently, the mare was presented with rapidly spreading multiple subcutaneous masses and, at the onset of neurological signs, was humanely euthanized and subjected to a complete post mortem examination. The necropsy confirmed the presence of numerous widespread masses in the subcutaneous tissue, several internal organs, and mammary gland. Histological and immunohistochemical (IHC) examinations were performed on all masses, allowing the diagnosis of mammary carcinoma with several visceral and subcutaneous metastases. Considering the post mortem findings, the second intraocular mass was submitted to histological and IHC re-evaluation to differentiate it from an intraocular metastasis of the mammary carcinoma. The results of the histological and IHC analyses confirmed the diagnosis of neuroepithelial tumor relapse. This is the first case of a metastatic mammary carcinoma concurrent with a recurrent intraocular neuroepithelial tumor in a mare. This case was a challenge for both clinicians and pathologists involved and highlighted the importance of post mortem and IHC evaluations

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS

The amyloidogenic variant of β2-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β2-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β2-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β2-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β2-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β2-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β2-microglobulin fibrils

Effect of tetracycline on β₂-m induced locomotory defect in transgenic <i>C. elegans</i> strains.

<p>Egg-synchronized control worms (vector), wild type β₂-m expressing worms (WT), P32G-mutated β₂-m and ΔN6-truncated β₂-m expressing nematodes (ΔN6) were placed at 20°C into fresh NMG plates seeded with tetracycline-resistant <i>E. coli</i>. At their L3/L4 larval stage, animals were fed with 50–100 µM tetracycline hydrochloride or 100 µM doxycycline (100 µl/plate). Body bends in liquid were scored after 24 hours. At least three independent assays were performed. Data are mean of number of body bends/min ± SD; **p<0.01 vs. the Vector, °°p<0.01 vs. the respective untreated group, according to one-way ANOVA (N = 60 animals for each group).</p

Genotype of <i>C. elegans</i> transgenic strains.

<p>(<b>A</b>) PCR genotyping of adult transgenic worms transfected with the empty vector (vector) or vectors for expression of wild type β₂-m (WT), P32G or 7–99 truncated form (ΔN6). The expected size of PCR products (about 360 bp) was observed. (<b>B</b>) Human β₂-m mRNA expression in different transgenic strains was normalized to worm cell division cycle 42 (cdc-42, GTP binding protein) as endogenous reference. Data are expressed as mean ± SD of three independent experiments.</p

Behavioural phenotypes of transgenic <i>C. elegans</i> strains.

<p>(<b>A</b>) Larval growth of control worms (Vector), wild type β₂-m expressing worms (WT) and nematodes expressing P32G or 7–99 truncated form of β₂-m (ΔN6). One hundred synchronized eggs were placed into fresh NMG plates seeded with OP50 as food, and the number of L1/L2, L2/L3 and L4/adult worms were scored after 24, 48 and 72 hours, respectively. Data are expressed as percentage of total worms in the plate at each time point and are given as mean of three independent experiments (N = 300). (<b>B</b>) Correlation between oligomers of β₂-m and reduction in growth rate of transgenic <i>C. elegans</i> strains. Percentage of adult worms of each transgenic strain, scored 72 after egg synchronization, was correlated to the the amount of A11-positive oligomeric assemblies detected by dot blotting. Data of both graphic axes represent mean of three independent experiments. (<b>C</b>) Kaplan-Meier survival curves of transgenic hermaphrodite adult nematodes. Animals were placed in plates seeded with OP50 starting from L4, cultured at 20°C and transferred to fresh plates for each consecutive other days. Survival rate was scored every day and expressed as percent of survival. Plots are representative of three independent experiments (N = 30). (<b>D</b>) Body bends in liquid of transgenic worms. At least three independent assays were performed (N = 100 animals for each group). Data are given as mean of number of body bends/min ± SE, *p<0.05 and **p<0.01 vs. the vector, °°p<0.01 vs. WT, according to one-way ANOVA. (<b>E</b>) Superoxide anions production in control worms (Vector), wild type β₂-m expressing worms (WT) and in nematodes expressing P32G or 7–99 truncated form of β₂-m (ΔN6). Age-synchronized worms were collected in PBS containing 1.6 ml of 1% Tween 20 and colorimetric NBT assay was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052314#s2" target="_blank">Materials and Methods</a>. Results show the fold increase in superoxide production calculated as NBT absorbance/mg of proteins (% NBT) compared to Vector; *p<0.05 vs. vehicle and ° p<0.05 vs. WT, according to one-way ANOVA. Error bars indicate SD.</p

Localization of β₂-m in transgenic <i>C. elegans</i> strains.

<p>Overlay of bright field and immunofluorescence images of head, vulva and tail of transgenic <i>C. elegans</i> strains. All animals depicted are 2 days adult worms. A specific β₂-m related signal (red, using a polyclonal anti human β₂-m antibody) was observed at the vulva muscles and anal sphincter muscle in the tail (red arrows) whereas no signal was observed in the head muscles. Scale bar, 50 µm.</p