Safety and efficacy of a single intra-articular injection of a novel enhanced protein solution (JTA-004) compared to hylan G-F 20 in symptomatic knee osteoarthritis: a randomized, double-blind, controlled phase II/III study.

BACKGROUND: New minimally invasive treatments are vital to delay joint replacement surgery in patients with knee osteoarthritis. This study was designed to select the most effective among three formulations of an enhanced protein solution containing clonidine, hyaluronic acid, and human plasma (JTA-004), and compare the safety and efficacy of intra-articular administration of the selected formulation with a reference treatment (hyaluronic acid) in symptomatic knee osteoarthritis patients. METHODS: In this two-stage, double-blind, phase II/III study conducted in 12 Belgian centers, 50-79-year-old patients with primary knee osteoarthritis were randomized (1:1:1:1) to receive one dose of one of three JTA-004 formulations (differing in clonidine concentration [50 or 100 µg/ml] and volume [2 or 4 ml]) or the reference treatment (hylan G-F 20). Patients were evaluated using Western Ontario McMaster Universities (WOMAC®) Scores and the Short-Form health survey up to 6 months post-injection (Month 6). Drug consumption and safety were evaluated. RESULTS: Among 164 treated patients, 147 completed the study. The JTA-004 formulation containing 200 µg clonidine and 20 mg hyaluronic acid in 2 ml (JTA-200/2) was selected based on interim results at Month 6. The difference in adjusted mean change in WOMAC Pain Subscale Score from baseline (JTA-200/2 minus reference group) at Month 6 was - 9.49 mm; statistical superiority of JTA-200/2 over the reference was not demonstrated. No statistically significant differences in adjusted mean changes from baseline between JTA-200/2 and reference groups were observed for Pain, Physical Function and Stiffness Subscales WOMAC Scores, Total WOMAC Score, and Well-being Score at any timepoint, although JTA-200/2 induced larger improvements in WOMAC Scores than the reference. Statistically significantly larger improvements in WOMAC Pain Subscale Scores for JTA-004 versus the reference were observed in post-hoc analyses on pooled data from all JTA-004 formulations at Month 6 (p = 0.030) and Month 3 (p = 0.014). All JTA-004 formulations had clinically acceptable safety profiles. CONCLUSIONS: This study provided preliminary evidence of the safety of intra-articular injection of JTA-004 in knee osteoarthritis patients. Phase III randomized controlled trials with larger sample sizes are needed to evaluate the efficacy of JTA-004 in knee osteoarthritis. TRIAL REGISTRATION: Clinicaltrials.gov/identifier NCT02740231; clinicaltrialsregister.eu/identifier 2015-002117-30. Retrospectively registered 13/4/2016.

Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, ß1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane.

Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation.

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by > 90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.

Validation of housekeeping gene and impact on normalized gene expression in clear cell renal cell carcinoma: critical reassessment of YBX3/ZONAB/CSDA expression.

BACKGROUND: YBX3/ZONAB/CSDA is an epithelial-specific transcription factor acting in the density-based switch between proliferation and differentiation. Our laboratory reported overexpression of YBX3 in clear cell renal cell arcinoma (ccRCC), as part of a wide study of YBX3 regulation in vitro and in vivo. The preliminary data was limited to 5 cases, of which only 3 could be compared to paired normal tissue, and beta-Actin was used as sole reference to normalize gene expression. We thus decided to re-evaluate YBX3 expression by real-time-PCR in a larger panel of ccRCC samples, and their paired healthy tissue, with special attention on experimental biases such as inter-individual variations, primer specificity, and reference gene for normalization. RESULTS: Gene expression was measured by RT-qPCR in 16 ccRCC samples, each compared to corresponding healthy tissue to minimize inter-individual variations. Eight potential housekeeping genes were evaluated for expression level and stability among the 16-paired samples. Among tested housekeeping genes, PPIA and RPS13, especially in combination, proved best suitable to normalize gene expression in ccRCC tissues as compared to classical reference genes such as beta-Actin, GAPDH, 18S or B2M. Using this pair as reference, YBX3 expression level among a collection of 16 ccRCC tumors was not significantly increased as compared to normal adjacent tissues. However, stratification according to Fuhrman grade disclosed higher YBX3 expression levels in low-grade tumors and lower in high-grade tumors. Immunoperoxidase confirmed homogeneous nuclear staining for YBX3 in low-grade but revealed nuclear heterogeneity in high-grade tumors. CONCLUSIONS: This paper underlines that special attention to reference gene products in the design of real-time PCR analysis of tumoral tissue is crucial to avoid misleading conclusions. Furthermore, we found that global YBX3/ZONAB/CSDA mRNA expression level may be considered within a "signature" of RCC grading.

An ex vivo culture system to study thyroid development.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the "angio-follicular" units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood. This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo. Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR. In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.