Simultaneous testing of immunological sensitization to multiple antigens in sarcoidosis reveals an association with inorganic antigens specifically related to a fibrotic phenotype.

Organic and inorganic antigens were studied simultaneously in the same cohort of sarcoidosis patients to investigate whether correlations between clinical characteristics and immunological sensitization could reveal new phenotypes. Sensitization to antigens of mycobacteria, Propionibacterium acnes catalase and vimentin was investigated in 201 sarcoidosis and 51 obstructive sleep apnoea patients, serving as control group. Sensitization to aluminium, beryllium, silica and zirconium was also studied in 105 of the sarcoidosis patients and in 24 of the controls. A significantly higher percentage of sarcoidosis patients (27·6%) than controls (4·2%) had an immunological response to metals or silica (P = 0·014). A higher percentage of these sarcoidosis patients showed fibrosis on chest X-ray 5 years after the diagnosis (69·2 versus 30·3%, P = 0·016). No significant differences in mycobacterial or vimentin enzyme-linked immunospot (ELISPOT) assay results were observed between sarcoidosis and control patients. A significantly lower percentage of sarcoidosis patients (3·5%) than control patients (15·7%) had a positive ELISPOT for P. acnes catalase (P = 0·003). However, sarcoidosis patients sensitized to P. acnes catalase were more likely to have skin involvement, while sarcoidosis patients sensitized to mycobacterial antigens were more likely to have cardiac involvement. Our study suggests a more prominent role for inorganic triggers in sarcoidosis pathogenesis than previously thought. Immunological sensitization to inorganic antigens was associated with development of fibrotic sarcoidosis. No association was found between sensitization to bacterial antigens or vimentin and sarcoidosis in Dutch patients. However, our data suggest that trigger-related phenotypes can exist in the heterogeneous population of sarcoidosis patients.

A comprehensive biological evaluation of ceramic nanoparticles as wear debris.

Patients are exposed internally to nanoparticles (NPs) by wear mechanisms associated with total joint arthroplasty. This tissue-specific retention implies that the biological evaluation of NPs shall be integrative and niche targeting. Here, we report that ceramic zirconia and silicon nitride NPs interfere with MG63 cells' function and remarkably stimulate the secretion of TNF-α in RAW264.7 cells. However, alumina NPs significantly promote the alkaline phosphatase (ALP) activity of MG63 cells at low concentration and do not show irritation to macrophages. In this study, we prove that ceramic materials at nanoscale are bioactive to cells. These findings also suggest that a more rational paradigm for the biosafety evaluation of NPs than is currently in place is needed before their clinical applications. FROM THE CLINICAL EDITOR: In this study, the authors demonstrate that ceramic nano materials associated with normal wear-and-tear of joint prostheses at nanoscale are bioactive to cells.

Probing the limits of Q-tag bioconjugation of antibodies.

Site-selective labelling of antibodies (Abs) can circumvent problems from heterogeneity of conventional conjugation. Here, we evaluate the industrially-applied chemoenzymatic 'Q-tag' strategy based on transglutaminase-mediated (TGase) amide-bond formation in the generation of 89Zr-radiolabelled antibody conjugates. We show that, despite previously suggested high regioselectivity of TGases, in the anti-Her2 Ab Herceptin™ more precise native MS indicates only 70-80% functionalization at the target site (Q298H), in competition with modification at other sites, such as Q3H critically close to the CDR1 region.

89Zr-immuno-PET for imaging of long circulating drugs and disease targets: why, how and when to be applied?

Positron emission tomography (PET) with 89Zr-labeled monoclonal antibodies (mAbs) or other targeted vehicles (e.g., peptides, nanoparticles and cells), collectively called "89Zr-immuno-PET", can be used for better understanding of disease targets and the in vivo behavior of targeted drugs. This will become increasingly important in the development of next generation mAbs, which are characterized by high potency and/or multiple binding domains. This review provides practical information for researchers who want to implement 89Zr-immuno-PET for answering their own biological and pathological questions or for steering their own drug development program. An overview is given of the reagents, labeling protocols, quality tests and critical steps to come to high quality 89Zr-conjugates, while possibilities for further improvement are discussed. Since PET has the advantage of allowing quantitative imaging, information is provided about standardization of 89Zr quantification. Issues are summarized for consideration when starting preclinical or clinical 89Zr-immuno-PET studies, to enable at the end unequivocal interpretation of results. Finally, many appealing examples are provided of what can be learned from 89Zr-immuno-PET studies, while future directions are outlined. Most of the current examples are still on the characterization of mAbs in oncology, but the review will show that 89Zr-immuno-PET harbors potential for many kinds of targeted drugs and diseases, as well as for elucidating biological processes.

Sensitization with Cr, Ni and Zr salts and allergic type granuloma formation in the guinea pig.

Guinea pigs were sensitized to potassium dichromate, nickel sulfate and sodium zirconium lactate by three methods of immunization in Freund's complete adjuvant: Polak, split adjuvant, and maximization (modified Magnusson and Kligman). These were followed after 2 weeks by weekly intradermal injections of 25 microng of the metal salt. Delayed hypersensitivity-like reactions developed 3 to 12 weeks after initial injection. Reactivity, although strong with an increase in thickness of over 0.7 mm and/or diameter of erythema of over 8 X 8 mm, was frequently transient, the animal losing reactivity on subsequent skin test or after 2 or 3 skin tests. In two-thirds of the experiments using sodium zirconium lactate, delayed hypersensitivity-like reactions at 24 hr developed into nodular lesions which reached peak intensity at 8 days and histologically contained histiocytes with an epithelioid cell appearance and giant cells. In some experiments, sodium zirconium lactate-sensitive animals showed cross reactivity with potassium dichromate, but not with nickel sulfate.

Mitogenic effects of beryllium and zirconium salts on mouse splenocytes in vitro.

The beryllium (Be) and zirconium (Zr) salts, BeSO4 and Zr(SO4)2, each exerted a concentration-dependent stimulation of mouse spleen cell proliferation as measured by an increase in [3H]thymidine incorporation into lymphocyte DNA, although the maximal response induced by Zr(SO4)4 (4-5 fold at 100-200 microM) was greater than that by BeSO4 (2-3 fold at 1-5 microM). Preincubation of splenocytes with low concentrations of BeSO4 (less than 1 microM) or a broad range of Zr(SO4)2 concentrations (2-100 microM) was also found to assist subsequent lectin (concanavalin A; ConA)-mediated lymphocyte proliferation. The results indicate that at defined concentrations Be and Zr salts can both act as lymphocyte mitogens and augment the functional responsiveness of immune cells, which may help explain the characteristic induction of delayed hypersensitivity and production of immunological granulomas by these metals in vivo.

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116.

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 ((89)Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of (89)Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of (89)Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg (89)Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent (89)Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of (89)Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of (89)Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, (89)Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.

Site-specifically 89Zr-labeled monoclonal antibodies for ImmunoPET.

UNLABELLED: Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled (89)Zr-Df-thio-trastuzumab conjugates were investigated. METHODS: The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N-hydroxysuccinimidyl iodoacetate, or N-hydroxysuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with (89)Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model. RESULTS: The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with (89)Zr in yields exceeding 75%. (89)Zr-Df-Ac-thio-trastuzumab and (89)Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20-25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1-7.1. CONCLUSIONS: The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with (89)Zr. The site-specifically (89)Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.

Bronchopulmonary cellular response to aluminum and zirconium salts.

The bronchopulmonary cellular immunological response to repeated intratracheal inoculation of aluminum chlorhydrate, sodium zirconium lactate, and zirconium aluminum glycine was examined in rabbits. Results of a dose-response experiment using 0.1, 1.0, and 10.0-mg intratracheal inoculations of each metallic salt demonstrated significant bronchopulmonary histopathology in the 10.0-mg dose-response groups only. Acute lesions were histologically characterized by an inflammatory response centered around respiratory bronchioles. Although epithelioid cell formation was evident in 10.0 mg of aluminum salt (aluminum chlorhydrate and zirconium aluminum glycine) -injected animals, no well-defined granulomas characterized by an orderly arrangement of epithelioid cells, lymphocytes, and giant cells were evident in any of the experimental groups employed. All three metallic salts induced "activated" bronchopulmonary macrophages as determined by an in vitro phagocytic assay. This activation was likely nonimmunological since no measurable differences were observed in metallic salt-induced delayed skin reactivity or migration inhibition factor production between inoculated and uninoculated rabbits. The above observations suggest that aluminum and zirconium salts administered in comparatively high dosage via the respiratory tract route can induce respiratory bronchiolitis and activation of alveolar macrophages in the absence of demonstrable delayed hypersensitivity.