Background Post-transplantation cyclophosphamide (PTCy) reduces severe graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Additionally, the gut microbiota taxa and diversity have been associated with occurrence of GVHD and mortality in HCT patients. Methods To understand better the impact of PTCy on the gut microbiota and the importance of the gut microbiota for PTCy's mechanisms of GVHD prevention, we used our T-cell-replete MHC-haploidentical murine HCT model (B6C3F1→B6D2F1). PTCy 25mg/kg/day or vehicle (PBS) was given on days +3/+4. Results PTCy treatment was associated with increased frequencies of bacterial taxa known to be protective against GVHD, such as ruminococcacae, clostridiales, lachnospiraceae, and blautia. This change coincided with increased levels of plasma butyrate, a metabolite known to protect against GVHD; butyrate levels positively correlated with percentages of regulatory T cells (Tregs) at day +21 in mice treated with or without PTCy. Many of the bacterial taxa modulations induced by PTCy were not affected by prophylactic treatment with levofloxacin. PTCy also led to faster numerical recovery of bacteria over time. To assess the mechanistic importance of PTCy modulation of the gut microbiota, we performed fecal microbiota transplants (FMT), in which mice did not receive post-HCT treatment themselves, but rather pre-HCT received FMT via oral gavage using stool from mice treated with PTCy or PBS. Results showed that mice receiving stool from PTCy-treated mice had significantly better weights at early post-HCT time points and a trend towards better overall survival. We repeated these experiments with germ-free (GF) recipient mice reconstituted with FMT via oral and rectal gavage using stool from donors transplanted under specific pathogen-free conditions; GF mice that received PTCy-modified microbiota also had significantly higher weights in the first few weeks after HCT. Nevertheless, PTCy maintained its efficacy in preventing GVHD in GF recipients that were not FMT reconstituted. Moreover, neither levofloxacin treatment from days 0 to +14 nor aggressive depletion of the gut microbiota via continual treatment using oral broad-spectrum antibiotic cocktail (imipenem, vancomycin, neomycin) starting 2 weeks prior to HCT worsened clinical or histopathologic GVHD in PTCy-treated mice. Conclusion PTCy modulates the microbiome towards taxa protective against GVHD, which may contribute to Treg recovery via increased butyrate levels. Even so, antibiotic treatment does not hinder GVHD prevention by PTCy, and consequently concerns regarding antibiotic exposure perhaps may be of less concern for affecting post-HCT outcomes in PTCy-treated recipients. Overall, PTCy's modulation of the microbiota contributes to GVHD prevention, but is not necessary for the mechanisms by which PTCy prevents severe GVHD.

BackgroundAlthough systemic 2H2O labeling is an attractive clinically relevant strategy for enhancing tumor imaging given relative ease of administration, gradual, slow ramp-up of labeling is preferable to avoid side effects, such as nausea and dizziness. Slow labeling periods are more easily tolerated but are suboptimal in the setting of cancer given the need to promptly diagnose and stage patients to initiate treatment. Hence, we sought to deploy low systemic concentrations of 2H2O in total body water (TBW) with short durations and low magnetic field strength to test clinically relevant experimental parameters conducive to clinical translation.MethodsHT-29 cells and KPC cells were used to establish subcutaneous and orthotopic murine tumor models, respectively. Systemic 2H2O administration was performed and TBW enrichment was confirmed via urine testing. All mice were imaged using the 7T MRI with a dual tuned (1H/2H) leg or body coil after 1, 3, or 7 days of systemic 2H2O labeling to 2 or 4% TBW (v/v).ResultsThe concentrations of 2H2O in urine samples closely reflected the target 2H2O enrichment level specified in the systemic labeling protocol. In all tested experimental schemas, there were no statistically significant differences in tumor-muscle contrast-to-noise ratio (CNR) and signal ratio among different labeling durations (p > 0.05), despite a significantly higher CNR at 4% compared with 2% (p = 0.02).ConclusionReasonable and stable imaging contrast between tumor and healthy tissue during the systemic labeling was achieved in this pre-clinical study, which offer preliminary indications of potential clinical translation of this imaging approach in cancer.

In vivo deuterated water ( 2 H 2 O) labeling leads to deuterium ( 2 H) incorporation into biomolecules of proliferating cells and provides the basis for its use in cell kinetics research. We hypothesized that rapidly proliferating cancer cells would become preferentially labeled with 2 H and, therefore, could be visualized by deuterium magnetic resonance imaging (dMRI) following a brief period of in vivo systemic 2 H 2 O administration. We initiated systemic 2 H 2 O administration in two xenograft mouse models harboring either human colorectal, HT-29, or pancreatic, MiaPaCa-2, tumors and 2 H 2 O level of ~ 8% in total body water (TBW). Three schemas of 2 H 2 O administration were tested: 1) starting at tumor seeding and continuing for 7 days of in vivo growth with imaging on day 7, 2) starting at tumor seeding and continuing for 14 days of in vivo growth with imaging on day 14, and 3) initiation of labeling following a week of in vivo tumor growth and continuing until imaging was performed on day 14. Deuterium chemical shift imaging of the tumor bearing limb and contralateral control was performed on either day 7 of 14 after tumor seeding, as described. After 14 days of in vivo tumor growth and 7 days of systemic labeling with 2 H 2 O, a clear deuterium contrast was demonstrated between the xenografts and normal tissue. Labeling in the second week after tumor implantation afforded the highest contrast between neoplastic and healthy tissue in both models. Systemic labeling with 2 H 2 O can be used to create imaging contrast between tumor and healthy issue, providing a non-radioactive method for in vivo cancer imaging.

Bronchiolitis obliterans syndrome (BOS) is a severe manifestation of chronic graft-versus-host disease (cGVHD) following hematopoietic cell transplantation (HCT). Montelukast interrupts cysteinyl leukotriene (CysLT) activity and may diminish the activation and homing of cells to bronchioles and subsequent fibrosis. We performed a prospective phase II trial to test whether montelukast altered lung decline for patients with BOS after HCT. In this single-arm, open-label, multi-institutional study, the primary endpoints were stability or improvement (<15% decline) in forced expiratory volume in 1 second (FEV1) and a <1-point decline in the slope of FEV1 after 6 months of treatment. Secondary endpoints included symptom and functional responses and immune correlates investigating the role of leukotrienes in BOS progression. The study enrolled 25 patients with moderate to severe lung disease after 3 months of stable cGVHD therapy. Montelukast was well tolerated, and no patient required escalation of BOS-directed therapy. At the primary endpoint, all 23 evaluable patients met the criteria for treatment success using FEV1% predicted, and all but 1 patient had stable or improved FEV1 slope. In those with a >5% improvement in FEV1, clinically meaningful improvements were seen in the Lee scores of breathing, energy, and mood. Improvements in the Human Activity Profile and 6-minute-walk test were observed in those with a <5% decline in FEV1. Overall survival was 87% at 2 years. Immune correlates showed elevated leukotriene receptor levels on blood eosinophils and monocytes versus healthy controls, elevated urine leukotrienes in 45% of the cohort, and CysLT receptors in bronchoalveolar lavage subsets and a predominance of Th2 cells, all pretreatment. These data suggest that montelukast may safely halt the progression of BOS after HCT, and that leukotrienes may play a role in the biology of BOS.

Graft-versus-host disease (GvHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (HSCT). Definitive diagnosis of GvHD is invasive and biopsies of involved tissues pose a high risk of bleeding and infection. Our previous studies in a chronic GvHD mouse model demonstrated that alloreactive CD4 + T cells are distributed to target organs ahead of overt symptoms, meanwhile CD4 + T cell activation is tied to increased glycolysis. Thus, we hypothesized that metabolic imaging of glycolysis would allow non-invasive detection of insipient GvHD in target organs infiltrated by glycolytic effector memory CD4 + T cells. We metabolically characterized CD4 + T cell subsets on day 14 post-transplant before the onset of chronic GvHD in a pre-clinical mouse model and performed 13 C hyperpolarized magnetic resonance imaging (MRI) to quantify glycolytic activity in the liver of mice over the course of the disease. Intracellular metabolic screening and ex vivo metabolic profiling of CD4 + T cell subsets at day 14 confirmed that activated CD4 + T cells were highly glycolytic. Concurrently, hyperpolarized 13 C-pyruvate MRI of the liver showed high conversion of pyruvate to lactate, indicative of increased glycolytic activity, that distinguished allogeneic from syngeneic HSCT recipients prior to the development of overt chronic GvHD. Furthermore, single cell sequencing of T cells in patients undergoing allogeneic HSCT indicated that similar metabolic changes may play a role in acute GvHD, providing a rationale for testing this imaging approach in the clinical post-HSCT setting. Our imaging approach is amenable to clinical translation and may allow early, non-invasive diagnosis of GvHD.

Water is an essential component of many biochemical reactions. Deuterated water (D2O) has been used to study cell kinetics, protein synthesis, and metabolism. We hypothesized that rapidly proliferating cancer cells would become preferentially labeled with deuterium due to high metabolic activity, thus allowing imaging of biosynthetically labeled metabolites within tumors in vivo . We initiated systemic D2O labeling in two established tumor xenograft models, HT-29 and MiaPaCa-2 and imaged mice by deuterium magnetic resonance spectroscopic imaging (dMRSI). After 14 days of tumor growth and 7 days of in vivo labeling, a clear contrast was demonstrated between the xenograft and the contralateral control limb in both models. The origin of the contrast was traced to an aliphatic peak at 1.8 ppm, which was identified by ex vivo NMR analysis to originate from cholesterol and cholesterol esters. Cholesterol is important for tumor cell proliferation, signaling, and malignant transformation, while current methods to monitor cholesterol synthesis and accumulation are limited. This deuterated water labeling-imaging approach could complement current cancer imaging techniques, allowing not only imaging of uptake but also synthesis of cholesterol to elucidate effects on tumor cholesterol metabolism in vivo . ### Competing Interest Statement NPB, DEF, NM, HM, MCK, and REG are inventors on a patent application related to this work, PCT/US2017/058886. Additionally, JRB, SK, HM, MCK are inventors on patent application pertaining to the signal-to-noise reduction algorithm, PCT/US2018/018217.

Introduction Chronic graft-versus host disease (cGvHD) is a major complication of allogeneic hematopoietic stem cell transplantation. Standard treatment mainly consists of corticosteroids but is limited by a high percentage of steroid-refractory cases. Alloreactive T cells are a major driver of the disease, infiltrating target organs such as the liver. However, global ablation of T cells interferes with the graft-versus-leukemia effect and bears the risk of increased infections. An increased use of glycolysis has been shown to drive T cell activation and its inhibition can blunt T cell effector functions. Dimethyl fumarate (DMF), an immunomodulatory drug already approved for the treatment of multiple sclerosis and psoriasis, has been shown to inhibit the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme. Objective We hypothesized that metabolic targeting of T cells could specifically inhibit activated T cells while preserving other T cell populations in cGvHD. Methods Chronic GvHD was induced using the B10.D2 into BALB/c minor-mismatch mouse model. Daily oral treatment with 100 mg/kg DMF or vehicle was initiated on the day of the transplant and continued for a 42-day period. Weight and cGvHD severity were scored twice weekly and liver and spleen tissue was analyzed via flow cytometry on day 42. Results DMF treatment reduced weight loss and cGvHD severity over a 42-day period. In addition, DMF decreased the frequency of activated CD4/CD8 T cells in the liver while simultaneously increasing the frequency of naive T cells. Importantly, the frequency of regulatory T cells was not affected by the treatment. Conclusion DMF has already been shown to be safe and effective in autoimmune diseases. Our studies indicate that DMF could have therapeutic potential in the setting of cGvHD as a selective inhibitor of activated, alloreactive T cells.

A rapid and selective method for the quantitation of neurotransmitters, l-Glutamic acid (GA) and γ-Aminobutyric acid (GABA), was developed and validated using gas chromatography-tandem mass spectrometry (GC-MS/MS). The novel method utilized a rapid online hot GC inlet gas phase sample derivatization and fast GC low thermal mass technology. The method calibration was linear from 0.5 to 100μg/mL, with limits of detections of 100ng/mL and 250ng/mL for GA and GABA, respectively. The method was used to investigate the effects of deletion of organic anion transporter 1 (Oat1) or Oat3 on murine CNS levels of GA and GABA at 3 and 18 mo of age, as compared to age matched wild-type (WT) animals. Whole brain concentrations of GA were comparable between WT, Oat1-/-, and Oat3-/- 18 mo at both 3 and 18 mo of age. Similarly, whole brain concentrations of GABA were not significantly altered in either knockout mouse strain at 3 or 18 mo of age, as compared to WT. These results indicate that the developed GC-MS/MS method provides sufficient sensitivity and selectivity for the quantitation of these neurotransmitters in mouse brain tissue. Furthermore, these results suggest that loss of Oat1 or Oat3 function in isolation does not result in significant alterations in brain tissue levels of GA or GABA.

Deuterated water (2H2O) is a label commonly used for safe quantitative measurement of deuterium enrichment into DNA of proliferating cells. More recently, it has been used for labeling proteins and other biomolecules. Our in vitro - in vivo research reports important stable isotopic labeling enrichment differences into the DNA nucleosides and their isotopologues (e.g. deoxyadenosine (dA) M + 1, dA M + 2, dA M + 3), as well as tumor cell proliferation effects for various forms of commercially available stable heavy water (2H2O, H218O, and 2H218O). Using an in vitro mouse thymus tumor cell line, we determined that H218O provides superior DNA labeling enrichment quantitation, as measured by GC-positive chemical ionization (PCI)-MS/MS. In addition, at higher but physiologically relevant doses, both 2H218O and 2H2O down modulated mouse thymus tumor cell proliferation, whereas H218O water had no observable effects on cell proliferation. The in vivo labeling studies, where normal mouse bone marrow cells (i.e. high turnover) were evaluated post labeling, demonstrated DNA enrichments concordant with measurements from the in vitro studies. Our research also reports a headspace-GC-NCI-MS method, which rapidly and quantitatively measures stable heavy water levels in total body water.

Cardiac ischemia associated with acute coronary syndrome and myocardial infarction is a leading cause of mortality and morbidity in the world. A rapid detection of the ischemic events is critically important for achieving timely diagnosis, treatment and improving the patient's survival and functional recovery. This minireview provides an overview on the current biomarker research for detection of acute cardiac ischemia. We primarily focus on inosine and hypoxanthine, two by-products of ATP catabolism. Based on our published findings of elevated plasma concentrations of inosine/hypoxanthine in animal laboratory and clinical settings, since 2006 we have originally proposed that these two purine molecules can be used as rapid and sensitive biomarkers for acute cardiac ischemia at its very early onset (within 15 min), hours prior to the release of heart tissue necrosis biomarkers such as cardiac troponins. We further developed a chemiluminescence technology, one of the most affordable and sensitive analytical techniques, and we were able to reproducibly quantify and differentiate total hypoxanthine concentrations in the plasma samples from healthy individuals versus patients suffering from ischemic heart disease. Additional rigorous clinical studies are needed to validate the plasma inosine/hypoxanthine concentrations, in conjunction with other current cardiac biomarkers, for a better revelation of their diagnostic potentials for early detection of acute cardiac ischemia.

The use of bone marrow-derived mesenchymal stromal cells (BMSC) in the treatment of alloimmune and autoimmune conditions has generated much interest, yet an understanding of the therapeutic mechanism remains elusive. We therefore explored immune modulation by a clinical-grade BMSC product in a model of human-into-mouse xenogeneic graft-versus-host disease (x-GVHD) mediated by human CD4+ Th1 cells. BMSC reversed established, lethal x-GVHD through marked inhibition of Th1 cell effector function. Gene marking studies indicated BMSC engraftment was limited to the lung; furthermore, there was no increase in regulatory T cells, thereby suggesting a paracrine mechanism of BMSC action. BMSC recipients had increased serum CD73 expressing exosomes that promoted adenosine accumulation ex vivo. Importantly, immune modulation mediated by BMSC was fully abrogated by pharmacologic therapy with an adenosine A2A receptor antagonist. To investigate the potential clinical relevance of these mechanistic findings, patient serum samples collected pre- and post-BMSC treatment were studied for exosome content: CD73 expressing exosomes promoting adenosine accumulation were detected in post-BMSC samples. In conclusion, BMSC effectively modulate experimental GVHD through a paracrine mechanism that promotes adenosine-based immune suppression. Stem Cells 2015;33:1200–1212 Stem Cells 2015;33:1200–1212

A rapid and sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g., dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multiparameter fluorescence activated cell sorting. Cell lysis, DNA extraction, and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid online hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25-20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25-20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20 000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and nonrelapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g., T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), arthritis, inflammatory bowel disease, and multiple sclerosis.

A rapid and simple chemiluminescence method was developed for detection of inosine and hypoxanthine in human plasma. The method utilized a microplate luminometer with direct injectors to automatically dispense reagents during sample analysis. Enzymatic conversions of inosine to hypoxanthine, followed by hypoxanthine to xanthine to uric acid, generated superoxide anion radicals as a useful metabolic by-product. The free radicals react with Pholasin(®) , a sensitive photoprotein used for chemiluminescence detection, to produce measurable blue-green light. The use of Pholasin(®) and a chemiluminescence signal enhancer, Adjuvant-K™, eliminated the need for plasma clean-up steps prior to analysis. The method used 20 μL of heparinized plasma, with complete analysis of total hypoxanthine levels (inosine is metabolized to hypoxanthine using purine nucleoside phosphorylase) in approximately 3.7 min. The rapid chemiluminescence method demonstrated the capability of differentiating total hypoxanthine levels between healthy individuals, and patients presenting with non-traumatic chest pain and potential acute cardiac ischemia. The results support the potential use of chemiluminescence methodology as a diagnostic tool to rapidly screen for elevated levels of inosine and hypoxanthine in human plasma, potential biomarkers of acute cardiac ischemia.

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C18 column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.