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Optimizing multiplexed imaging experimental design through tissue spatial segregation estimation [article]

Pierre Bost, Daniel Schulz, Stefanie Engler, Clive Wasserfall, Bernd Bodenmiller
2021 bioRxiv   pre-print
Recent advances in multiplexed imaging methods allow simultaneous detection of dozens of proteins or RNAs enabling deep spatial characterization of both healthy and tumor tissues. Parameters for design of optimal sequencing-based experiments have been established, but such parameters are lacking for multiplex imaging studies. Here, using a spatial transcriptomic atlas of healthy and tumor human tissues, we developed a new statistical framework that determines the number of fields of view
more » ... ry to accurately identify all cell types that are part of the tissue. Using this strategy on imaging mass cytometry data, we identified a measurement of tissue spatial segregation that enables optimal experimental design and that is technology invariant. This strategy will enable significantly improved design of multiplexed imaging studies.
doi:10.1101/2021.11.28.470262 fatcat:rtsmliiylndh3k3hlen57n3ou4

Suppression of islet homeostasis protein thwarts diabetes mellitus progression

Seh-Hoon Oh, Marda L Jorgensen, Clive H Wasserfall, Altin Gjymishka, Bryon E Petersen
2017 Laboratory Investigation  
During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report
more » ... t in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.
doi:10.1038/labinvest.2017.15 pmid:28218739 fatcat:ql34rnhzjvauhg3wv4n2l6j4fq

Autologous umbilical cord blood infusion for type 1 diabetes

Michael J. Haller, Hilla-Lee Viener, Clive Wasserfall, Todd Brusko, Mark A. Atkinson, Desmond A. Schatz
2008 Experimental Hematology  
Objective. The physical, emotional, and economic costs of type 1 diabetes (T1D) mandate continued efforts to develop effective strategies to prevent or reverse the disease. Herein, we describe the scientific and therapeutic rationale underlying efforts utilizing umbilical cord blood (UCB) as a therapy for ameliorating the progression of this autoimmune disease. Materials and Methods. We recently embarked on a pilot study to document the safety and potential efficacy of autologous UCB infusion
more » ... subjects with T1D. Under this protocol, patients recently diagnosed with the disease and for whom autologous cord blood is stored, undergo infusion. Studies are performed before infusion and every 3 to 6 months postinfusion for immunologic and metabolic assessment. To date, 15 autologous infusions have been performed. Results. Preliminary observations suggest that autologous cord blood transfusion is safe and provides some slowing of the loss of endogenous insulin production in children with T1D. Mechanistic studies demonstrate that umbilical cord blood contains highly functional populations of regulatory T cells (Treg) and that increased Treg populations may be found in the peripheral blood of subjects more than 6 months after cord blood infusion. We provide the rationale for cord blood-based therapies, a summary of our initial protocol, and plans for future studies designed to explore the potential of cord blood-derived regulatory T cells to treat T1D. Conclusions. Prolonged follow-up and additional mechanistic efforts are urgently needed to determine if umbilical cord blood-derived stem cells can be used as part of safe and effective therapies for T1D. Ó
doi:10.1016/j.exphem.2008.01.009 pmid:18358588 pmcid:PMC2444031 fatcat:samj6fpgnbagxcmy7bimpuxjmq

Untargeted Metabolomic Analysis In Naturally Occurring Canine Diabetes Mellitus Identifies Similarities To Human Type 1 Diabetes [article]

Allison L. O'Kell, Timothy J. Garrett, Clive Wasserfall, Mark A. Atkinson
2017 bioRxiv   pre-print
While predominant as a disease entity, knowledge voids exist regarding the pathogenesis of canine diabetes. To test the hypothesis that diabetic dogs have similar metabolomic perturbations to humans with type 1 diabetes (T1D), we analyzed serum metabolomic profiles of breed- and body weight-matched, diabetic (n=6) and healthy (n=6) dogs by liquid chromatography-mass spectrometry (LC-MS) profiling. We report distinct clustering of diabetic and control groups based on heat map analysis of known
more » ... d unknown metabolites. Random forest classification identified 5/6 dogs per group correctly with overall out of bag error rate=16.7%. Diabetic dogs demonstrated significant upregulation of glycolysis/gluconeogenesis intermediates (e.g., glucose/fructose, C6H12O6, keto-hexose, deoxy-hexose, (P<0.01)), with significant downregulation of tryptophan metabolism metabolites (e.g., picolinic acid, indoxyl sulfate, anthranilate, (P<0.01)). Multiple amino acids (AA), AA metabolites, and bile acids were also significantly lower in diabetic versus healthy dogs (P<0.05) with the exception of the branched chain AA valine, which was elevated in diabetic animals (P<0.05). Metabolomic profiles in diabetic versus healthy dogs shared similarities with those reported in human T1D (e.g., alterations in glycolysis/gluconeogensis metabolites, bile acids, and elevated branched chain AA). Further studies are warranted to evaluate the utility of canine diabetes to provide novel mechanistic insights to the human disorder.
doi:10.1101/139113 fatcat:74s7zg75dfcwzaqsa52xgqtdye

Freeze-thaw increases adeno-associated virus transduction of cells

Sifeng Chen, Clive Wasserfall, Matthias H. Kapturczak, Mark Atkinson, Anupam Agarwal
2006 American Journal of Physiology - Cell Physiology  
combination of gene and cell-based therapies may provide significant advantages over existing treatments in terms of their effectiveness. However, long-term efficient gene delivery has been difficult to achieve in many cell types, including endothelial cells. We developed a freeze-thaw technique which significantly increases the transduction efficiency of recombinant adeno-associated virus vectors in human aortic endothelial cells (23-fold) and in human renal proximal tubular epithelial cells
more » ... 28-fold) in comparison to current methods for transduction. Freeze-thaw resulted in a transient but significant increase in cell surface area by 1,174 Ϯ 69.8 M 2 per cell. Reduction of cryogenic medium volume and repeated freeze-thaw further increased transduction efficiency by 2.8-and 2.4-fold, respectively. Trypsinization, dimethylsulfoxide, and cold temperatures, which are also involved in cell preservation, had no significant impact on transduction efficiency. Increased transduction was also observed in mesenchymal stem cells (42-fold) by the freeze-thaw method. The potential mechanism of this novel technique likely involves an increase in the net permeable area of biological membranes caused by water crystallization. These findings provide a new approach for gene delivery in various cell types, particularly in those resistant to transduction by conventional methods.
doi:10.1152/ajpcell.00582.2005 pmid:16510845 fatcat:iazhum3yk5gxjdpvuv6xfg5ye4

The influence of type 1 diabetes on pancreatic weight

Martha L. Campbell-Thompson, John S. Kaddis, Clive Wasserfall, Michael J. Haller, Alberto Pugliese, Desmond A. Schatz, Jonathan J. Shuster, Mark A. Atkinson
2015 Diabetologia  
Aims/hypothesis Previous studies of pancreases obtained at autopsy or by radiography note reduced pancreas weight (PW) and size, respectively, in type 1 diabetes; this finding is widely considered to be the result of chronic insulinopenia. This literature is, however, limited with respect to the influence of age, sex, anthropometric factors and disease duration on these observations. Moreover, data are sparse for young children, a group of particular interest for type 1 diabetes. We
more » ... that the pancreas-to-body weight ratio would normalise confounding inter-subject factors, thereby permitting better characterisation of PW in type 1 diabetes. Methods Transplant-grade pancreases were recovered from 216 organ donors with type 1 diabetes (n=90), type 2 diabetes (n=40) and no diabetes (n= 86). Whole-organ and
doi:10.1007/s00125-015-3752-z pmid:26358584 pmcid:PMC4670792 fatcat:5z5mriuwtrcirof5o7lwg6kv54

T regulatory cell function in idiopathic minimal lesion nephrotic syndrome

Carlos Araya, Leila Diaz, Clive Wasserfall, Mark Atkinson, Wei Mu, Richard Johnson, Eduardo Garin
2009 Pediatric nephrology (Berlin, West)  
The purpose of this study was to test the hypothesis that, in idiopathic minimal lesion nephrotic syndrome (IMLNS), the T regulatory (T reg) cell suppressor mechanism is deficient, thereby enhancing cytokine release by T effector cells. Twenty-one patients with IMLNS, eight healthy controls and two patients with nephrotic syndrome and membranoproliferative glomerulonephritis were studied. The percentage of T reg cells was similar in the healthy controls and in patients with IMLNS in relapse or
more » ... n remission. Thymidine incorporation in autologous T effector cells, as well as expression of the regulatory cytokine interleukin (IL)-10, was significantly reduced in patients in relapse when compared with patients in remission and healthy subjects. IL-2 expression was also reduced in patients in relapse but did not achieve statistical significance. In a different set of experiments, T cells, from subjects with IMLNS in remission, when stimulated with antiCD3-antiCD28 antibodies, secreted increased levels of cytokines. No such increase in cytokines was observed when cells from healthy controls were stimulated with same mitogen. The impaired T reg cell function observed in these patients may have pathogenic and therapeutic implications, because it could explain the persistence of the proposed pathogenic cytokines observed in the patients with IMLNS.
doi:10.1007/s00467-009-1214-x pmid:19495805 pmcid:PMC2785122 fatcat:7h42hzdhsbhwvfstgmyzlu3hc4

71. Alpha 1 Antitrypsin (AAT) Gene Therapy for the Prevention of Type 1 Diabetes

Sihong Song, Yuanqing Lu, Mei Tang, Bin Zhang, Clive Wasserfall, Martha Campbell-Thompson, Mark Atkinson
2006 Molecular Therapy  
TEM Intravascular Interaction between Adenovirus and Platelets Promotes Adenovirus Sequestration, Degradation and Toxicity Sihong Song, Yuanqing Lu, Mei Tang, Bin Zhang, Clive Wasserfall, Martha Campbell-Thompson  ... 
doi:10.1016/j.ymthe.2006.08.088 fatcat:zyukmv4novhjzhzptleaa5lon4

Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation

Todd M. Brusko, Clive H. Wasserfall, Maigan A. Hulme, Roniel Cabrera, Desmond Schatz, Mark A. Atkinson, Derya Unutmaz
2009 PLoS ONE  
Citation: Brusko TM, Wasserfall CH, Hulme MA, Cabrera R, Schatz D, et al. (2009) Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation.  ... 
doi:10.1371/journal.pone.0007980 pmid:19956753 pmcid:PMC2775921 fatcat:2wkmrwm6bvd75gpyt5wbob7wfy

BCG Vaccine-Induced Neuroprotection in a Mouse Model of Parkinson's Disease

Jing Yong, Goran Lacan, Hoa Dang, Terry Hsieh, Blake Middleton, Clive Wasserfall, Jide Tian, William P. Melega, Daniel L. Kaufman, Jacques Zimmer
2011 PLoS ONE  
There is a growing interest in using vaccination with CNS antigens to induce autoreactive T cell responses that home to damaged areas in the CNS and ameliorate neurodegenerative disease. Neuroprotective vaccine studies have focused on administering oligodendrocyte antigens or CopaxoneH in complete Freund's adjuvant (CFA). Theoretical considerations, however, suggest that vaccination with a neuronal antigen may induce more robust neuroprotective immune responses. We assessed the neuroprotective
more » ... otential of vaccines containing tyrosine hydroxylase (a neuronal protein involved in dopamine synthesis) or CopaxoneH in CFA in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Surprisingly, we observed that the main beneficial factor in these vaccines was the CFA. Since the major immunogenic component in CFA is Mycobacterium tuberculosis, which closely related to the bacille Calmette-Guérin (BCG) that is used in human vaccines, we tested BCG vaccination in the MPTP mouse model. We observed that BCG vaccination partially preserved markers of striatal dopamine system integrity and prevented an increase in activated microglia in the substantia nigra of MPTP-treated mice. These results support a new neuroprotective vaccine paradigm in which general (nonself-reactive) immune stimulation in the periphery can limit potentially deleterious microglial responses to a neuronal insult and exert a neurorestorative effect in the CNS. Accordingly, BCG vaccination may provide a new strategy to augment current treatments for a wide range of neuropathological conditions.
doi:10.1371/journal.pone.0016610 pmid:21304945 pmcid:PMC3031604 fatcat:rrvwl3fqurganmk27bls624gvq

Influence of Fecal Sample Storage on Bacterial Community Diversity

Luiz F.W. Roesch, George Casella, Olli Simell, Jeffrey Krischer, Clive H. Wasserfall, Desmond Schatz, Mark A. Atkinson, Josef Neu, Eric W. Triplett
2009 Open Microbiology Journal  
Previous studies have identified a correlation, either positive or negative, between specific stool bacteria strains and certain autoimmune diseases. These conflicting data may relate to sample collection. The aim of this work was to evaluate the influence of the collection parameters of time and temperature on bacterial community composition. Samples were taken from healthy children and immediately divided in 5 sub-samples. One sample was frozen immediately at -80°C, while the other aliquots
more » ... re frozen 12, 24, 48, and 72h later DNA extracted from each sample was used to amplify the 16S rRNA with barcoded primers. The amplified products were pooled and partial 16S rRNA sequences were obtained by pyrosequencing. Person-to-person variability in community diversity was high. A list of those taxa that comprise at least 1% of the community was made for each individual. None of these were present in high numbers in all individuals. The Bacteroides were present in the highest abundance in three of four subjects. A total of 23,701 16S rRNA sequences were obtained with an average of 1,185 reads per sample with an average length of 200 bases. Although pyrosequencing of amplified 16S rRNA identified changes in community composition over time (~10%), little diversity change was observed at 12 hours (3.06%) with gradual changes occurring after 24 (8.61%), 48 (9.72%), and 72 h (10.14%), post collection. © Roesch et al.; Licensee Bentham Open. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
doi:10.2174/1874285800903010040 pmid:19440250 pmcid:PMC2681173 fatcat:hlklioh67nftldzofkpucw4qgm

Impact of Resistant Starch on Body Fat Patterning and Central Appetite Regulation

Po-Wah So, Wei-Sheng Yu, Yu-Ting Kuo, Clive Wasserfall, Anthony P. Goldstone, Jimmy D. Bell, Gary Frost, Daniel Tomé
2007 PLoS ONE  
Background. Adipose tissue patterning has a major influence on the risk of developing chronic disease. Environmental influences on both body fat patterning and appetite regulation are not fully understood. This study was performed to investigate the impact of resistant starch (RS) on adipose tissue deposition and central regulation of appetite in mice. Methodology and Principle Findings. Forty mice were randomised to a diet supplemented with either the high resistant starch (HRS), or the
more » ... digestible starch (LRS). Using 1 H magnetic resonance (MR) methods, whole body adiposity, intrahepatocellular lipids (IHCL) and intramyocellular lipids (IMCL) were measured. Manganese-enhanced MRI (MEMRI) was used to investigate neuronal activity in hypothalamic regions involved in appetite control when fed ad libitum. At the end of the interventional period, adipocytes were isolated from epididymal adipose tissue and fasting plasma collected for hormonal and adipokine measurement. Mice on the HRS and LRS diet had similar body weights although total body adiposity, subcutaneous and visceral fat, IHCL, plasma leptin, plasma adiponectin plasma insulin/glucose ratios was significantly greater in the latter group. Adipocytes isolated from the LRS group were significantly larger and had lower insulin-stimulated glucose uptake. MEMRI data obtained from the ventromedial and paraventricular hypothalamic nuclei suggests a satiating effect of the HRS diet despite a lower energy intake. Conclusion and Significance. Dietary RS significantly impacts on adipose tissue patterning, adipocyte morphology and metabolism, glucose and insulin metabolism, as well as affecting appetite regulation, supported by changes in neuronal activity in hypothalamic appetite regulation centres which are suggestive of satiation.
doi:10.1371/journal.pone.0001309 pmid:18074032 pmcid:PMC2111051 fatcat:3p52eddwvranvpk475tld5ssti

Serum levels of soluble CD25 as a marker for hepatocellular carcinoma

2012 Oncology Letters  
In a previous study, we showed that the level of soluble CD25 (sCD25) was elevated in a small series of patients with hepatocellular carcinoma (HCC). In the present study, we determined the capacity of serum levels of sCD25 to detect the presence and the early stage of HCC using a larger cohort of HCC patients and evaluated the correlation between sCD25 level and tumor burden. Serum levels of sCD25 were quantified using ELISA in patients with HCC (n=145), controls with advanced fibrosis (n=61)
more » ... nd healthy control subjects (n=30). The levels of sCD25 in patients with HCC (median, 6,955 pg/ml) were significantly higher than those in cirrhosis-only patients (4,310 pg/ml; P<0.0001). At a cut-off value of 2,180 pg/ml, sCD25 had a sensitivity of 92.3% and a specificity of 37.7% in detecting HCC presence [area under the curve (AUC) of 0.685; P<0.0001]. By comparison, α-fetoprotein (AFP) had a sensitivity of 53.8% and a specificity of 86.8% at a cut-off value of 32.8 ng/ml (AUC=0.755; P<0.0001) for HCC presence detection. For early HCC, the sensitivity of sCD25 was 89.6% and its specificity was 39.3% (AUC=0.630; P<0.0001) at a cut-off value of 2,859 pg/ml, while AFP had a sensitivity of 41.7% and a specificity of 82.6% at a cut-off value of 20.6 ng/ml (AUC=0.630; P=0.0257). We also found a significant positive correlation between serum levels of sCD25 and tumor stage. In the present study study, sCD25 was more effective than AFP at detecting the presence and early stages of HCC. This immune factor may hold promise as a novel predictive marker of HCC presence and may be useful in distinguishing early HCC from advanced cirrhosis, currently areas of global unmet need.
doi:10.3892/ol.2012.826 pmid:23205111 pmcid:PMC3506698 fatcat:232s43un7zfvbdurvf55ipi3km

Insulitis and β-Cell Mass in the Natural History of Type 1 Diabetes

Martha Campbell-Thompson, Ann Fu, John S. Kaddis, Clive Wasserfall, Desmond A. Schatz, Alberto Pugliese, Mark A. Atkinson
2015 Diabetes  
Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and b-cell and a-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb + , n = 18) and without islet autoantibodies (AAb 2 , n = 61). Insulitis was observed in
more » ... our of four donors (100%) with type 1 diabetes duration of £1 year and two AAb + donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = 20.58, P = 0.01) but not with age at disease onset. Residual b-cells were observed in all type 1 diabetes donors with insulitis, while b-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin + islets compared with 2% of insulin 2 islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45 + , CD3 + , CD4 + , CD8 + , and CD20 + cell numbers within the insulitis (r = 0.53-0.73, P = 0.004-0.04), but not CD68 + or CD11c + cells. The presence of b-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal.
doi:10.2337/db15-0779 pmid:26581594 pmcid:PMC4764143 fatcat:jonxxqxudvf3zblrci76k4x6pe

Glucose transporter-2 (GLUT2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice

Brant R. Burkhardt, Mathew J. Parker, Y. Clare Zhang, Sihong Song, Clive H. Wasserfall, Mark A. Atkinson
2005 FEBS Letters  
Insulin production afforded by hepatic gene therapy (HGT) retains promise as a potential treatment for type 1 diabetes, but successful approaches have been limited. We employed a novel and previously untested promoter for this purpose, glucose transporter-2 (GLUT2) to drive insulin production via delivery by recombinant adeno-associated virus (rAAV). In vitro, the GLUT2 promoter was capable of robust glucose-responsive expression in transduced HepG2 human hepatoma cells. Therefore, rAAV
more » ... ts were designed to express the furin-cleavable human preproinsulin B10 gene, under the control of the murine GLUT2 promoter and packaged for delivery with rAAV expressing the type 5 capsid. Streptozotocin-induced diabetic mice were subjected to hepatic portal vein injection immediately followed by implantation of a sustained-release insulin pellet to allow time for transgenic expression. All mice injected with the rAAV5-GLUT2-fHPIB10 virus remained euglycemic for up to 35 days post-injection, with 50% euglycemic after 77 days post-injection. In contrast, mock-injected mice became hyperglycemic within 15 days post-injection following dissolution of the insulin pellet. Serum levels of both human insulin and C-peptide further confirmed successful transgenic delivery by the rAAV5-GLUT2-fHPIB10 virus. These findings indicate that the GLUT2 promoter may be a potential candidate for regulating transgenic insulin production for hepatic insulin gene therapy in the treatment of type I diabetes.
doi:10.1016/j.febslet.2005.09.060 pmid:16223491 fatcat:wx7iqbtlxzgfboughhumcuysum
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