The changing microRNA landscape by color and cloudiness:a cautionary tale for nipple aspirate fluid biomarker analysis

Purpose: Investigation of nipple aspirate fluid (NAF)-based microRNAs (miRNAs) as a potential screening tool for women at increased risk of developing breast cancer is the scope of our research. While aiming to identify discriminating NAF-miRNAs between women with different mammographic densities, we were confronted with an unexpected confounder: NAF sample appearance. Here we report and alert for the impact of NAF color and cloudiness on miRNA assessment. Methods: Seven classes of NAF colors coupled with cloudiness appearance were established. Using 173 NAF samples from 154 healthy women (19 samples were bilaterally collected), the expression of 14 target and 2 candidate endogenous control (EC) miRNAs was investigated using Taqman Advanced miRNA assays to identify significant differential expression patterns between color-cloudiness classes. Inter- and intra-individual variation of miRNA expression was analyzed using the coefficient of variation (CV). Results: We found that between the seven NAF classes, fold change miRNA expression differences ranged between 2.4 and 19.6 depending on the interrogated miRNA. Clear NAF samples exhibited higher miRNA expression levels compared to cloudy NAF samples with fold change differences ranging between 1.1 and 6.2. Inter-individual and intra-individual miRNA expression was fairly stable (CV &lt; 15 %), but nevertheless impacted by NAF sample appearance. Within NAF classes, inter-individual variation was largest for green samples (CV 6-15 %) and smallest for bloody samples (CV 2-6 %). Conclusions: Our data indicate that NAF color and cloudiness influence miRNA expression and should, therefore, be systematically registered using an objective color classification system. Given that sample appearance is an inherent feature of NAF, these variables should be statistically controlled for in multivariate data analyses. This cautionary note and recommendations could be of value beyond the field of NAF-miRNAs, given that variability in sample color and cloudiness is likewise observed in liquid biopsies such as urine, cerebrospinal fluid and sputum, and could thereby influence the levels of miRNAs and other biomarkers.</p

Resolution of psoriasis upon blockade of IL-15 biological activity in a xenograft mouse model

Psoriasis is a chronic inflammatory disease of the skin characterized by epidermal hyperplasia, dermal angiogenesis, infiltration of activated T cells, and increased cytokine levels. One of these cytokines, IL-15, triggers inflammatory cell recruitment, angiogenesis, and production of other inflammatory cytokines, including IFN-γ, TNF-α, and IL-17, which are all upregulated in psoriatic lesions. To investigate the role of IL-15 in psoriasis, we generated mAb’s using human immunoglobulin-transgenic mice. One of the IL-15–specific antibodies we generated, 146B7, did not compete with IL-15 for binding to its receptor but potently interfered with the assembly of the IL-15 receptor α, β, γ complex. This antibody effectively blocked IL-15–induced T cell proliferation and monocyte TNF-α release in vitro. In a human psoriasis xenograft model, antibody 146B7 reduced the severity of psoriasis, as measured by epidermal thickness, grade of parakeratosis, and numbers of inflammatory cells and cycling keratinocytes. These results obtained with this IL-15–specific mAb support an important role for IL-15 in the pathogenesis of psoriasis

Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20(+) B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab ')2 fragments, as well as C1q-binding-deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed "accessory CDC," remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20(+) cells: OFA-F(ab ')(2) fragments were able to induce CDC in the CD20(+)BCR(+) cell population, but not in the CD20(+)BCR(-) population. Importantly, OFA-F(ab ')(2) fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenstrom macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic

Promoting Fc:Fc interactions alleviates CDC sensitivity to target and mCRP expression.

<p>(A) Cell lines were opsonized with saturating amounts of wild-type or mutated RTX and analyzed by CDC assay. Left panel: cell lines with increasing CD20:mCRP ratio (left to right); right panel: cell lines with decreasing CD20:mCRP ratio (left to right). (B) In vivo analysis of subcutaneous tumor growth in a severe combined immunodeficiency (SCID) xenograft model. SCID mice were injected with luciferase-expressing Raji cells. Eight days after tumor cell injection, mice were randomized at an average tumor volume of 85 mm³ per group and treated with 50 μg IgG1 antibody. Tumor volumes were monitored over time and depicted as average tumor volume ± standard error of the mean (SEM) (left panel), and as Kaplan-Meier curves with time to progression cutoff set at a tumor volume of >700 mm³ (right panel).</p

Biophysical characterization of antibody hexamerization in solution.

<p>(A) Native MS analysis of solutions of IgG1-005 and mutant antibodies at 2 μM. IgG1-005-RGY (RGY), a previously described triple mutant, is used as a positive control. The charge envelope of the IgG monomers appears around <i>m/z</i> 5,500 (~ 147 kD Mw) and that of the IgG hexamers around <i>m/z</i> 12,500 (~890 kD Mw). The signals of the hexameric species are displayed at a 100-fold or 10-fold (RGY only) amplification. (B–C) Dynamic light scattering analysis of 7D8 and mutant solutions formulated in PBS pH 7.4. Three independent experiments are shown. (B) Linear regression of diffusion coefficient versus Ab concentration. (C) Hydrodynamic radii (Rhs) of mutants measured by dynamic light scattering (DLS) were divided by Rh of wild-type 7D8 to correct for viscosity without masking the contribution of Fc:Fc self-association. (D) HP-SEC analysis of 100 mg/mL 7D8 and mutant antibody solutions formulated in PBS, incubated for three months at 5°C. Multimer detection limit of 1% is indicated by a dashed line.</p

Efficient lysis of CLL patient tumor cells.

<p>Peripheral B cells isolated from the blood of six CLL patients were opsonized with wild-type or mutant, 7D8, RTX or control (IgG1-b12) antibody and tested in CDC assays. The CLL cells of patient 4 were highly refractory to CDC due to their very low CD20 expression.</p

E345 and E430 limit Fc:Fc interactions.

<p>(A) Left: overview of the IgG1 antibody hexamer observed in the IgG1-b12 crystal structure (PDB access code 1HZH [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002344#pbio.1002344.ref020" target="_blank">20</a>]). Right: zoomed-in view of two neighboring Fc domains with enhancing amino acid positions highlighted. (B) Summary of CDC inhibition data. The surface view is rotated 90° relative to panel A to show the Fc:Fc interface. Daudi cells were opsonized with mutants of anti-CD38 mAb IgG1-005 (1.0 μg/mL) and assessed for CDC. Light and dark grey colors indicate the unmodified amino acids of the two heavy chain regions composing a single Fc domain. All mutants assessed were ranked by efficiency per amino acid position, and the resulting median lysis efficacy is indicated by coloring per position; orange and red colors indicate positions with reduced lysis. Blue: >60% lysis; orange: 30%–60% lysis; red: <30% lysis. (C) Summary of CDC enhancement data. Wien 133 cells were opsonized with mutants of anti-CD38 mAb IgG1-005 (1.0 μg/mL) and assessed for CDC. Light and dark grey colors indicate unmodified amino acids as described under (B). The maximal lysis of the most efficient mutant per amino acid position is displayed with green indicating positions for which CDC was enhanced. Blue: <20% lysis; light green: 20%–40% lysis; dark green: >40% lysis. (D) Dose response in CDC of Wien 133 cells by IgG1-005 and selected mutants. A representative of 3 replicate experiments is shown. (E) Dose response in CDC of Ramos cells induced by RTX and selected mutants. A representative experiment of 3 replicates is shown. (F) Structural view zoomed to residue E345. Amino acids facing E345 at the Fc:Fc interface are indicated in blue and named with apostrophe. (G) Structural view zoomed to residue E430, forming a salt bridge with K338 at the intramolecular C_H2–C_H3 domain interface.</p