Mesenchymal stromal/stem cells promote intestinal epithelium regeneration after chemotherapy-induced damage.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for leukemia and a range of non-malignant disorders. The success of the therapy is hampered by occurrence of acute graft-versus-host disease (aGvHD); an inflammatory response damaging recipient organs, with gut, liver, and skin being the most susceptible. Intestinal GvHD injury is often a life-threatening complication in patients unresponsive to steroid treatment. Allogeneic mesenchymal stromal/stem cell (MSC) infusions are a promising potential treatment for steroid-resistant aGvHD. Data from our institution and others demonstrate rescue of approximately 40-50% of aGvHD patients with MSCs in Phase I, II studies and minor side effects. Although promising, better understanding of MSC mode of action and patient response to MSC-based therapy is essential to improve this lifesaving treatment. METHODS: Single cell human small intestine organoids were embedded in Matrigel, grown for 5 days and treated with busulfan for 48 h. Organoids damaged by treatment with busulfan or control organoids were co-cultured with 5000, 10,000, and 50,000 MSCs for 24 h, 48 h or 7 days and the analyses such as surface area determination, proliferation and apoptosis assessment, RNA sequencing and proteomics were performed. RESULTS: Here, we developed a 3D co-culture model of human small intestinal organoids and MSCs, which allows to study the regenerative effects of MSCs on intestinal epithelium in a more physiologically relevant setting than existing in vitro systems. Using this model we mimicked chemotherapy-mediated damage of the intestinal epithelium. The treatment with busulfan, the chemotherapeutic commonly used as conditioning regiment before the HSCT, affected pathways regulating epithelial to mesenchymal transition, proliferation, and apoptosis in small intestinal organoids, as shown by transcriptomic and proteomic analysis. The co-culture of busulfan-treated intestinal organoids with MSCs reversed the effects of busulfan on the transcriptome and proteome of intestinal epithelium, which we also confirmed by functional evaluation of proliferation and apoptosis. CONCLUSIONS: Collectively, we demonstrate that our in vitro co-culture system is a new valuable tool to facilitate the investigation of the molecular mechanisms behind the therapeutic effects of MSCs on damaged intestinal epithelium. This could benefit further optimization of the use of MSCs in HSCT patients.

In situ Synthesis of Supramolecular Polymers: Finding the Right Conditions when Combining Covalent and Non-Covalent Synthesis.

The combination of covalent and non-covalent synthesis is omnipresent in nature and potentially enables access to new materials. Yet, the fundamental principles that govern such a synthesis are barely understood. Here, we demonstrate how even simple reaction mixtures behave surprisingly complex when covalent reactions are coupled to self-assembly processes. Specifically, we study the reaction behavior of a system in which the in situ formation of discotic benzene-1,3,5-tricarboxamide (BTA) monomers is linked to an intertwined non-covalent reaction network including self-assembly into helical BTA polymers. This system shows an unexpected phase-separation behavior in which an interplay of reactant/product concentrations, side-products and solvent purity determines the system composition. We envision that these insights can bring us one step closer to how to design the synthesis of systems in a combined covalent/non-covalent fashion.

T cell-derived interferon-γ programs stem cell death in immune-mediated intestinal damage.

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.

Characterizing early contrast uptake of ductal carcinoma in situ with high temporal resolution dynamic contrast-enhanced MRI of the breast: a pilot study.

Improvements in the reliable diagnosis of preinvasive ductal carcinoma in situ (DCIS) by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) are needed. In this study, we present a new characterization of early contrast kinetics of DCIS using high temporal resolution (HiT) DCE-MRI and compare it with other breast lesions and normal parenchyma. Forty patients with mammographic calcifications suspicious for DCIS were selected for HiT imaging using T(1)-weighted DCE-MRI with ∼7 s temporal resolution for 90 s post-contrast injection. Pixel-based and whole-lesion kinetic curves were fit to an empirical mathematical model (EMM) and several secondary kinetic parameters derived. Using the EMM parameterized and fitted concentration time curve for subsequent analysis allowed for calculation of kinetic parameters that were less susceptible to fluctuations due to noise. The parameters' initial area under the curve (iAUC) and contrast concentration at 1 min (C(1 min)) provided the highest diagnostic accuracy in the task of distinguishing pathologically proven DCIS from normal tissue. There was a trend for DCIS lesions with solid architectural pattern to exhibit a negative slope at 1 min (i.e. increased washout rate) compared to those with a cribriform pattern (p < 0.04). This pilot study demonstrates the feasibility of quantitative analysis of early contrast kinetics at high temporal resolution and points to the potential for such an analysis to improve the characterization of DCIS.

Autologous conditioned serum (Orthokine) is an effective treatment for knee osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is prevalent and difficult to treat. Autologous conditioned serum (ACS), marketed under the trade name Orthokine, is a novel, injectable antiarthritic derived from the patient's own blood. The present study is the first time ACS has undergone a controlled clinical trial. METHOD: We investigated 376 patients with knee OA in a prospective, randomized, patient- and observer-blinded, placebo-controlled trial using an intention-to-treat analysis (ITT). The clinical effects of ACS were compared to hyaluronan (HA) and saline (placebo) as assessed by patient-administered outcome instruments (Western Ontario and McMaster Universities osteoarthritis index, global patient assessment, visual analog scale, Short-Form 8) after 7, 13 and 26 weeks. After 104 weeks an observer-blinded follow-up was carried out. Frequency and severity of adverse events were used as safety parameters. RESULTS: In all treatment groups, intra-articular injections produced a reduction in symptoms as well as an improvement in quality of life. However, the effects of ACS were significantly superior to those of HA and saline for all outcome measures and time points, and improvements were clinically relevant; there were no differences between the effects of HA and saline. The frequency of adverse events was comparable in the ACS and saline groups, but higher in the HA group. CONCLUSION: The data demonstrate that ACS injection considerably improves clinical signs and symptoms of OA. It remains to be determined whether ACS is disease-modifying, chondroprotective, or chondroregenerative.

Detection of in situ mammary cancer in a transgenic mouse model: in vitro and in vivo MRI studies demonstrate histopathologic correlation.

Improving the prevention and detection of preinvasive ductal carcinoma in situ (DCIS) is expected to lower both morbidity and mortality from breast cancer. Transgenic mouse models can be used as a 'test bed' to develop new imaging methods and to evaluate the efficacy of candidate preventive therapies. We hypothesized that despite its microscopic size, early murine mammary cancer, including DCIS, might be accurately detected by MRI. C3(1) SV40 TAg female mice (n=23) between 10 and 18 weeks of age were selected for study. Eleven mice were subjected to in vitro imaging using a T(2)-weighted spin echo sequence and 12 mice were selected for in vivo imaging using a T(1)-weighted gradient echo, a T(2)-weighted spin echo and high spectral and spatial resolution imaging sequences. The imaged glands were carefully dissected, formalin fixed and paraffin embedded, and then H&E stained sections were obtained. The ratio of image-detected versus histologically detected cancers was obtained by reviewing the MR images and H&E sections independently and using histology as the gold standard. MR images were able to detect 12/12 intramammary lymph nodes, 1/1 relatively large (approximately 5 mm) tumor, 17/18 small (approximately 1 mm) tumors and 13/16 ducts distended with DCIS greater than 300 microm. Significantly, there were no false positives--i.e., image detection always corresponded to a histologically detectable cancer in this model. These results indicate that MR imaging can reliably detect both preinvasive in situ and early invasive mammary cancers in mice with high sensitivity. This technology is an important step toward the more effective use of non-invasive imaging in pre-clinical studies of breast cancer prevention, detection and treatment.

Tobramycin and gentamycin elution analysis between two in situ polymerizable orthopedic composites.

This research analyzed Tobramycin and Gentamycin elution characteristics for two antibiotic-impregnated bone composites: PMMA-based Simplex P and the novel, hybrid, bioactive, CORTOSS. Experimental results were correlated with composite hydrophilicity and antibiotic phase partitioning behaviors. The phase partitioning experiment was conducted to understand antibiotic solubility in aqueous environments. By comparing experimental results with calculated data, antibiotic release behavior was predicted. Total Tobramycin elution percentages from CORTOSS and Simplex P were 12.5 and 6.4%, respectively. Total Gentamycin elution percentages from CORTOSS and Simplex P were 6.95 and 10.17%, respectively. Phase partitioning data indicate 100% of Tobramycin remains in aqueous phases, being extremely hydrophilic. This is supported by its calculated theoretical value (log P = - 7.32). Results suggest that Tobramycin elution can be attributed to composite hydrophilicity as well as its high degree of hydrophilicity. Fifteen percent of Gentamycin distributes in hydrophobic phases (log P = - 4.22). Despite a lower Gentamycin hydrophilicity, its release was affected by its complexation with polar salts in the leaching buffer, thereby increasing its elution potential, making it appreciably water soluble. CORTOSS is more hydrophilic; therefore the migration of aqueous liquids into the polymer network of CORTOSS facilitates greater antibiotic elution compared with hydrophobic Simplex P.

A fast and efficient determination of amines and preservatives in cough and cold liquid and suspension formulations using a single isocratic ion-pairing high performance [correction of power] liquid chromatography method.

A single, highly selective ion-pairing reverse phase-high power liquid chromatography (RP-HPLC) method has been developed for the determination of amines and preservatives in a wide range of Tylenol((R)) liquid and suspension liquid products. As with many OTC products, the challenge is to quantitatively extract the analytes from difficult matrices and specifically analyze them in the presence of various excipients and flavors. Historically, separate analytical methods were used for each class of analytes (acids, bases and neutral compounds). In this method a mobile phase consisting of a buffered ion-pairing agent with acetonitrile, methanol and tetrahydrofuran was used to separate the charged amines from neutral and acidic compounds on a Phenomenex LUNA C8(2) 75 x 4.6 mm i.d. analytical column with a 3-microm particle size. The analytes include acids (benzoic acid), bases (pseudoephedrine, chlorpheniramine, dextromethorphan, doxylamine and diphenhydramine) and a neutral compound (butylparaben). The effects of pH, the chain length of the ion-pairing reagent, ionic strength and organic modifiers on the separation are discussed. The method is linear from 15 to 150% of the target amounts. The optimized method proves to be specific, robust and accurate for the analysis of the compounds.