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1.
Lasers Surg Med ; 56(3): 239-248, 2024 03.
Article in English | MEDLINE | ID: mdl-38311811

ABSTRACT

BACKGROUND: Systemically delivered hedgehog inhibitors including vismodegib and sonidegib are widely used to treat basal cell carcinomas (BCCs). Ablative fractional laser (AFL)-assisted topical delivery of vismodegib has been demonstrated in preclinical studies. The aim of this explorative clinical study was to evaluate intratumoral vismodegib concentrations and effect on hedgehog pathway gene expression following AFL-assisted topical vismodegib delivery to BCCs. METHODS: In an open-label clinical trial, 16 nodular BCCs (in n = 9 patients) received one application of CO2 -AFL (40 mJ/microbeam, 10% density) followed by topical vismodegib emulsion. After 3-4 days, vismodegib concentrations in tumor biopsies (n = 15) and plasma were analyzed and compared with samples from patients receiving oral treatment (n = 3). GLI1, GLI2, PTCH1, and PTCH2 expression was determined by quantitative polymerase chain reaction (n = 7) and GLI1 additionally by in situ hybridization (n = 3). RESULTS: Following AFL-assisted topical administration, vismodegib was detected in 14/15 BCCs and reached a median concentration of 6.2 µmol/L, which compared to concentrations in BCC tissue from patients receiving oral vismodegib (9.5 µmol/L, n = 3, p = 0.8588). Topical vismodegib reduced intratumoral GLI1 expression by 51%, GLI2 by 55%, PTCH1 and PTCH2 each by 73% (p ≤ 0.0304) regardless of vismodegib concentrations (p ≥ 0.3164). In situ hybridization demonstrated that GLI1 expression was restricted to tumor tissue and downregulated in response to vismodegib exposure. CONCLUSION: A single AFL-assisted topical application of vismodegib resulted in clinically relevant intratumoral drug concentrations and significant reductions in hedgehog pathway gene expressions.


Subject(s)
Anilides , Antineoplastic Agents , Carcinoma, Basal Cell , Lasers, Gas , Pyridines , Skin Neoplasms , Humans , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Hedgehog Proteins/therapeutic use , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein GLI1/therapeutic use , Carcinoma, Basal Cell/drug therapy , Carcinoma, Basal Cell/genetics , Carcinoma, Basal Cell/pathology , Antineoplastic Agents/adverse effects , Gene Expression
2.
Nat Commun ; 14(1): 7099, 2023 11 04.
Article in English | MEDLINE | ID: mdl-37925520

ABSTRACT

Inhibition of Janus kinase (JAK) family enzymes is a popular strategy for treating inflammatory and autoimmune skin diseases. In the clinic, small molecule JAK inhibitors show distinct efficacy and safety profiles, likely reflecting variable selectivity for JAK subtypes. Absolute JAK subtype selectivity has not yet been achieved. Here, we rationally design small interfering RNAs (siRNAs) that offer sequence-specific gene silencing of JAK1, narrowing the spectrum of action on JAK-dependent cytokine signaling to maintain efficacy and improve safety. Our fully chemically modified siRNA supports efficient silencing of JAK1 expression in human skin explant and modulation of JAK1-dependent inflammatory signaling. A single injection into mouse skin enables five weeks of duration of effect. In a mouse model of vitiligo, local administration of the JAK1 siRNA significantly reduces skin infiltration of autoreactive CD8+ T cells and prevents epidermal depigmentation. This work establishes a path toward siRNA treatments as a new class of therapeutic modality for inflammatory and autoimmune skin diseases.


Subject(s)
Janus Kinase Inhibitors , Vitiligo , Mice , Animals , Humans , RNA, Small Interfering/genetics , CD8-Positive T-Lymphocytes/metabolism , Autoimmunity/genetics , Vitiligo/drug therapy , Vitiligo/genetics , Janus Kinase 1/genetics , Janus Kinase 1/metabolism , RNA, Double-Stranded
3.
J Immunother Cancer ; 9(10)2021 10.
Article in English | MEDLINE | ID: mdl-34615703

ABSTRACT

BACKGROUND: Targeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC) in vitro and in vivo in monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice. METHODS: The murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studied in vitro on human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studied in vivo as monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects. In vivo depletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC. RESULTS: MSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6, CCL20, CXCL10, and stimulator of interferon genes (STING)-related genes) in vitro as determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activation in vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy. CONCLUSIONS: These in vitro and in vivo data on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies.


Subject(s)
B7-H1 Antigen/antagonists & inhibitors , Immunoconjugates/therapeutic use , Thorium/therapeutic use , Animals , Gene Expression Profiling , Immunoconjugates/pharmacology , Immunotherapy , Mice , Thorium/pharmacology , Transfection , Xenograft Model Antitumor Assays
4.
Mol Ther Nucleic Acids ; 11: 441-454, 2018 Jun 01.
Article in English | MEDLINE | ID: mdl-29858079

ABSTRACT

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 µM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

5.
Diabetes ; 55(10): 2705-12, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17003334

ABSTRACT

Pancreatic beta-cell growth and survival and insulin production are stimulated by growth hormone and prolactin through activation of the transcription factor signal transducer and activator of transcription (STAT)5. To assess the role of STAT5 activity in beta-cells in vivo, we generated transgenic mice that expressed a dominant-negative mutant of STAT5a (DNSTAT5) or constitutive active mutant of STAT5b (CASTAT5) under control of the rat insulin 1 promoter (RIP). When subjected to a high-fat diet, RIP-DNSTAT5 mice showed higher body weight, increased plasma glucose levels, and impairment of glucose tolerance, whereas RIP-CASTAT5 mice were more glucose tolerant and less hyperleptinemic than wild-type mice. Although the pancreatic insulin content and relative beta-cell area were increased in high-fat diet-fed RIP-DNSTAT5 mice compared with wild-type or RIP-CASTAT5 mice, RIP-DNSTAT5 mice showed reduced beta-cell proliferation at 6 months of age. The inhibitory effect of high-fat diet or leptin on insulin secretion was diminished in isolated islets from RIP-DNSTAT5 mice compared with wild-type islets. Upon multiple low-dose streptozotocin treatment, RIP-DNSTAT5 mice exhibited higher plasma glucose levels, lower plasma insulin levels, and lower pancreatic insulin content than wild-type mice, whereas RIP-CASTAT5 mice maintained higher levels of plasma insulin. In conclusion, our results indicate that STAT5 activity in beta-cells influences the susceptibility to experimentally induced type 1 and type 2 diabetes.


Subject(s)
Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 1/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Insulin-Secreting Cells/physiology , STAT5 Transcription Factor/physiology , Animals , Blood Glucose/metabolism , Cell Proliferation/drug effects , Dietary Fats/administration & dosage , Glucose Tolerance Test , Insulin/blood , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Leptin/blood , Mice , Mice, Transgenic , STAT5 Transcription Factor/genetics
6.
Endocrinology ; 147(12): 5752-9, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16973727

ABSTRACT

Trefoil factors (TFFs) 1, 2, and 3 are expressed in mucosal epithelia. TFFs are particular abundant in the intestine in which they play a crucial role in maintenance and restitution of the epithelium. Because pancreas developmentally arises from the primitive foregut, we explored the expression of TFFs in the pancreas in man and rat. Immunocytochemical staining of adult human pancreas showed abundant TFF3 immunoreactivity in pancreatic islets and some duct cells, whereas weak TFF1 and no TFF2 staining were detected. In the islets TFF3 localized to most insulin and some glucagon and pancreatic polypeptide-producing cells. TFF3 immunoreactivity was colocalized with insulin and glucagon in distinct cell clusters in human fetal pancreas at wk 14 and in the newborn rat pancreas. In isolated human and rat islets, TFF3 and TFF1 mRNA was identified by RT-PCR, and TFF3 protein was detected in human pancreas and islets by ELISA. Exposure of neonatal rat islets or insulinoma cells to GH, a known beta-cell growth factor, resulted in markedly increased TFF3 but decreased TFF1 mRNA levels. The effect of GH on TFF3 expression was confirmed by Western blot. Culture of neonatal rat islets in the presence of TFF3 resulted in attachment and migration of the islet cells, but no effects on proliferation, insulin secretion or cytokine-induced apoptosis were seen. These data demonstrate expression of TFFs in the endocrine pancreas, but their possible functions remain unknown.


Subject(s)
Growth Hormone/metabolism , Islets of Langerhans/metabolism , Peptides/metabolism , Adult , Animals , Animals, Newborn , Apoptosis/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation , Humans , Insulin/metabolism , Insulin Secretion , Insulinoma/metabolism , Islets of Langerhans/embryology , Pancreatic Neoplasms/metabolism , Peptides/pharmacology , Rats , Tissue Distribution , Trefoil Factor-2 , Trefoil Factor-3 , Tumor Cells, Cultured
7.
Inflamm Bowel Dis ; 22(9): 2078-97, 2016 09.
Article in English | MEDLINE | ID: mdl-27542128

ABSTRACT

BACKGROUND: Beneficial roles for glucagon-like peptide 1 (GLP-1)/GLP-1R signaling have recently been described in diseases, where low-grade inflammation is a common phenomenon. We investigated the effects of GLP-1 in Brunner's glands and duodenum with abundant expression of GLP-1 receptors, as well as GLP-1 effect on colonic inflammation. METHODS: RNA from Brunner's glands of GLP-1R knockout and wild-type mice were subjected to full transcriptome profiling. Array results were validated by quantitative reverse transcription polymerase chain reaction in wild-type mice and compared with samples from inflammatory bowel disease (IBD) patients and controls. In addition, we performed a detailed investigation of the effects of exogenous liraglutide dosing in a T-cell driven adoptive transfer (AdTr) colitis mouse model. RESULTS: Analyses of the Brunner's gland transcriptomes of GLP-1R knockout and wild-type mice identified 722 differentially expressed genes. Upregulated transcripts after GLP-1 dosing included IL-33, chemokine ligand 20 (CCL20), and mucin 5b. Biopsies from IBD patients and controls, as well as data from the AdTr model, showed deregulated expression of GLP-1R, CCL20, and IL-33 in colon. Circulating levels of GLP-1 were found to be increased in mice with colitis. Finally, the colonic cytokine levels and disease scores of the AdTr model indicated reduced levels of colonic inflammation in liraglutide-dosed animals. CONCLUSIONS: We demonstrate that IL-33, GLP-1R, and CCL20 are deregulated in human IBD, and that prophylactic treatment with 0.6 mg/kg liraglutide improves disease in AdTr colitis. In addition, GLP-1 receptor agonists upregulate IL-33, mucin 5b, and CCL20 in murine Brunner's glands. Taken together, our data indicate that GLP-1 receptor agonists affect gut homeostasis in both proximal and distal parts of the gut.


Subject(s)
Brunner Glands/metabolism , Colitis/pathology , Colon/metabolism , Inflammatory Bowel Diseases/pathology , Liraglutide/pharmacology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Chemokine CCL20/metabolism , Colitis/drug therapy , Female , Gene Expression , Glucagon-Like Peptide-1 Receptor/agonists , Glucagon-Like Peptide-1 Receptor/genetics , Humans , Inflammation/pathology , Interleukin-33/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Mucin-5B/metabolism , RNA, Messenger/analysis , Young Adult
8.
Gene ; 299(1-2): 219-25, 2002 Oct 16.
Article in English | MEDLINE | ID: mdl-12459269

ABSTRACT

Insulin receptor (IR)-deficient mice develop severe diabetes mellitus, diabetic ketoacidosis (DKA) and liver steatosis and die within 1 week after birth. We examined in this work whether the metabolic phenotype of IR(-/-) mutants could be improved by transgenic complementation with IR selectively in the liver. We first generated transgenic mice expressing a human DNA complementary to RNA encoding a truncated constitutively activated form of IR (IRdelta) under the control of liver-specific phenylalanine hydroxylase (PAH) gene promoter. These mice presented more pronounced fasting hypoglycemia and showed slightly improved glucose tolerance as compared to controls. The transgenic mice were crossed with IR(+/-) mutants to generate IR(-/-) mice carrying the PAH-IRDelta transgene. Although such mutants developed glycosuria, DKA was delayed by more than 1 week and survival was prolonged to 8-20 days in approximately 10% of mice. In these partially rescued pups, serum glucose and triglyceride levels were lowered, hepatic glycogen stores were reconstituted and liver steatosis was absent as compared with pups which developed strong DKA and died earlier. Thus, lack of insulin action in the liver is responsible in large part for the metabolic disorders seen in IR(+/-) mice. This study should stimulate interest in therapeutic strategies aimed at improving hepatic function in diabetes.


Subject(s)
Liver/metabolism , Receptor, Insulin/genetics , Animals , Blood Glucose/metabolism , Female , Gene Expression Regulation , Genetic Complementation Test , Genotype , Glucose Tolerance Test , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Mutation , Phenylalanine Hydroxylase/genetics , Receptor, Insulin/metabolism , Recombinant Fusion Proteins/genetics , Survival Analysis
9.
EMBO Rep ; 3(4): 323-8, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11943762

ABSTRACT

Non-insulin-dependent diabetes mellitus (NIDDM) is a complex heterogeneous polygenic disease characterized mainly by insulin resistance and pancreatic beta-cell dysfunction. In recent years, several genetically engineered mouse models have been developed for the study of the pathophysiological consequences of defined alterations in a single gene or in a set of candidate diabetogenes. These represent new tools that are providing invaluable insights into NIDDM pathogenesis. In this review, we highlight the lessons emerging from the study of some of the transgenic or knockout mice in which the expression of key actors in insulin signaling, action or secretion has been manipulated. In addition to contributing to our knowledge of the specific roles of individual genes in the control of glucose homeostasis, these studies have made it possible to address several crucial issues in NIDDM that have remained controversial or unanswered for a number of years.


Subject(s)
Diabetes Mellitus, Type 2/metabolism , Glucose/metabolism , Insulin/physiology , Muscle Proteins , Signal Transduction/physiology , Animals , Animals, Genetically Modified , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/physiopathology , Glucose Transporter Type 4 , Insulin/genetics , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Mice , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Phosphoproteins/metabolism , Potassium Channels/metabolism
10.
Transgenic Res ; 13(5): 463-73, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15587270

ABSTRACT

We report here knock-in of diphteria toxin A chain (dta) gene at the Ins2 locus, using the strategy previously employed to insert lacZ under control of the Ins2 promoter. Mutant Ins2(dta/+), Ins2(dta/lacZ) or Ins2(lacZ/+) mouse pups were generated by breeding and analyzed to study the effects of toxigenetic beta-cell ablation on islet development and to localize the extrapancreatic Ins2 expression site in the brain. Ins2(dta/+) and Ins2(dta/lacZ) pups developed a severe diabetic ketoacidosis and died rapidly. Histological analysis of their pancreas revealed that beta-cells completely disappeared in their islets as evidenced by loss of lacZ activity or insulin immunonostaining. beta-cell ablation did not alter the size of other islet cell populations which were normal at birth, although the glucagon-cell population was reduced by 85% at embryonic day E12.5. In the brain, comparative analysis of lacZ expression in Ins2(lacZ/+) and Ins2(dta/laZ) mice identified the choroid plexus (CP) as a major Ins2 expression site. This finding was confirmed by RT-PCR analysis of insulin transcripts in RNAs prepared from microdissected wild-type CP. Transcripts for other key beta-cell markers, with the notable exception of Pdx-1, were also found in CP RNAs. These results must revive interest in studies focused on extrapancreatic insulin gene expression.


Subject(s)
Choroid Plexus/metabolism , Diphtheria Toxin/genetics , Gene Expression Regulation, Developmental , Islets of Langerhans/metabolism , Pancreas/metabolism , Peptide Fragments/genetics , Animals , Choroid Plexus/pathology , Diabetic Ketoacidosis/metabolism , Diabetic Ketoacidosis/pathology , Diphtheria Toxin/metabolism , Genetic Vectors/genetics , Insulin/metabolism , Islets of Langerhans/pathology , Lac Operon/genetics , Mice , Mice, Transgenic , Pancreas/pathology , Peptide Fragments/metabolism , Promoter Regions, Genetic
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