36 results on '"K. Nocka"'
Search Results
2. 722 Type 2 helper T-cell (Th2) effects of crisaborole versus 3 topical corticosteroids (TCSs) with a range of potencies: An ex vivo human skin model study
- Author
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G. Berstein, K. Nocka, Jessica Neil, William C. Ports, Bonnie Vlahos, Marc B. Brown, S.R. Feldman, Chuanbo Zang, and A.V. Caserta
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Range (biology) ,Chemistry ,Model study ,T cell ,Crisaborole ,Human skin ,Cell Biology ,Dermatology ,Pharmacology ,Biochemistry ,medicine.anatomical_structure ,medicine ,Molecular Biology ,Ex vivo - Published
- 2019
3. Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen
- Author
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V C, Broudy, N L, Lin, G V, Priestley, K, Nocka, and N S, Wolf
- Subjects
Anemia, Hemolytic ,Stem Cell Factor ,Immunology ,Antibodies, Monoclonal ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Biochemistry ,Phenylhydrazines ,Rats ,Colony-Forming Units Assay ,Mice, Inbred C57BL ,Mice ,Proto-Oncogene Proteins c-kit ,Phenotype ,Bone Marrow ,Cell Movement ,Mice, Inbred DBA ,Immunoglobulin G ,Radiation Chimera ,Animals ,Erythropoiesis ,Female ,Spleen - Abstract
The phenotypes of mice that harbor a defect in the genes encoding either stem cell factor (SCF) or its receptor, c-kit, indicate that this ligand/receptor pair is necessary for maintenance of normal hematopoiesis in the adult. Our objective was to determine whether SCF, like erythropoietin, is necessary for acute erythroid expansion during recovery from hemolytic anemia. Monoclonal antibody ACK2, which recognizes the murine c-kit receptor, was used to selectively block the hematopoietic growth-promoting effects of SCF. Mice were treated with phenylhydrazine on day 0 and day 1 to induce hemolytic anemia and also received no antibody, control IgG, or ACK2 on day 0. The mice were killed on day 3 and the hematocrit (Hct), reticulocyte count, and numbers of erythroid and myeloid hematopoietic progenitor cells (colony- forming unit-erythroid [CFU-E], burst-forming unit [BFU]-E, and CFU- granulocyte-macrophage [GM]) were quantitated in the femoral marrow and spleen using hematopoietic colony-forming assays. Induction of hemolytic anemia with phenylhydrazine resulted in a drop in the Hct from approximately 50% to 30%, and an approximate 8- to 10-fold increase in the reticulocyte count. The numbers of CFU-E increased modestly in the femur, and approximately 25- to 50-fold in the spleen, in comparison with normal mice. BFU-E and CFU-GM values did not increase in the femur but expanded 6- to 10-fold in the spleen, in comparison with normal mice. This confirms that much of the erythroid expansion in response to hemolytic anemia occurs in the murine spleen. Neutralizing quantities of the ACK2 antibody reduced femoral CFU-E, BFU- E, and CFU-GM content to less than half that found in phenylhydrazine- treated control mice and nearly totally ablated splenic hematopoiesis. These results suggest that c-kit receptor function may be required for optimal response to acute erythropoietic demand and that erythropoiesis in the splenic microenvironment is more dependent on SCF/c-kit receptor interaction than is erythropoiesis in the marrow microenvironment. Because expansion of late erythropoiesis in the spleen was preferentially blocked, we tested the hypothesis that homing of more primitive hematopoietic cells to the spleen was dependent on c-kit receptor function. Lethally irradiated mice were injected with marrow cells obtained from mice that had received phenylhydrazine plus control IgG or with marrow cells obtained from mice that had received phenylhydrazine plus ACK2. In parallel experiments, normal murine marrow cells were treated in vitro with control IgG or with ACK2 and were injected into lethally irradiated mice. The fraction of BFU-E and CFU-GM retrieved from the marrow and spleen of the recipient mice 4 hours later was reduced by approximately 75% when progenitor cells had been exposed to ACK2, in comparison with control IgG. These data suggest that interaction of SCF with the c-kit receptor affects the homing behavior of hematopoietic progenitor cells in the adult animal.
- Published
- 1996
4. Targeted disruption of guanosine diphosphate-dissociation inhibitor for Rho-related proteins, GDID4: normal hematopoietic differentiation but subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation
- Author
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J C, Guillemot, B A, Kruskal, C N, Adra, S, Zhu, J L, Ko, P, Burch, K, Nocka, K, Seetoo, E, Simons, and B, Lim
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Histamine Release ,Minor Histocompatibility Antigens ,Mice ,Phagocytosis ,rho Guanine Nucleotide Dissociation Inhibitor beta ,GTP-Binding Proteins ,Superoxides ,Animals ,rho-Specific Guanine Nucleotide Dissociation Inhibitors ,Mast Cells ,Calcimycin ,Cells, Cultured ,Guanine Nucleotide Dissociation Inhibitors ,Respiratory Burst ,Stem Cell Factor ,Ionophores ,Macrophages ,Stem Cells ,Proteins ,Cell Differentiation ,Fibroblasts ,Immunoglobulin E ,Embryo, Mammalian ,Hematopoietic Stem Cells ,Hematopoiesis ,Gene Targeting ,Calcium - Abstract
The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.
- Published
- 1996
5. Gonadal expression of c-kit encoded at the W locus of the mouse
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Peter Besmer, Katia Manova, K. Nocka, and Rosemary F. Bachvarova
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Male ,medicine.medical_specialty ,Gonad ,Gene Expression ,Mice, Inbred Strains ,In situ hybridization ,Testicle ,Biology ,Andrology ,Mice ,Internal medicine ,Proto-Oncogene Proteins ,Gene expression ,Testis ,medicine ,Animals ,Genitalia ,Microscopy, Immunoelectron ,Molecular Biology ,Gametogenesis ,Embryogenesis ,Ovary ,Embryo ,Protein-Tyrosine Kinases ,Oocyte ,Blotting, Northern ,Spermatozoa ,Proto-Oncogene Proteins c-kit ,Endocrinology ,medicine.anatomical_structure ,Oocytes ,Female ,Developmental Biology - Abstract
Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting ( W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries, c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.
- Published
- 1990
6. The mouse W/c-kit locus
- Author
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A, Bernstein, B, Chabot, P, Dubreuil, A, Reith, K, Nocka, S, Majumder, P, Ray, and P, Besmer
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ErbB Receptors ,Mice ,Proto-Oncogene Proteins c-kit ,Hematopoietic System ,Proto-Oncogene Proteins ,Animals ,Chromosome Mapping ,Protein-Tyrosine Kinases ,Chromosomes ,Mice, Mutant Strains - Abstract
The mature cells in the haemopoietic system arise as the result of the extensive developmental and proliferative capacity of pluripotential stem cells. In order to understand the molecular basis for these developmental processes, it will be necessary to identify and characterize the cellular genes that control early steps in haemopoiesis. Mutations at the mouse W locus on chromosome 5 lead to pleiotropic developmental defects, including sterility, coat colour abnormalities, severe macrocytic anaemia and mast cell deficiency. The defects in all these lineages are cell autonomous and intrinsic, suggesting that the W locus encodes a gene product required directly for cellular differentiation. In an attempt to understand this classical mouse developmental mutation, we have demonstrated that the c-kit proto-oncogene, which encodes a transmembrane receptor tyrosine kinase, is very closely linked to W. Several further observations are consistent with the idea that W and c-kit are allelic: first, c-kit is expressed in those cell populations affected by W mutations; second, the expression of c-kit transcripts can be affected by mutations at the W locus; third, the tyrosine kinase activity associated with the protein encoded by c-kit is functionally impaired in mast cells derived from mutant W/Wv mice; and fourth, rearrangements within the c-kit gene have been reported in two W mutant alleles. These observations suggest that the dominant phenotype associated with W mutations results from loss-of-function alterations that affect the receptor tyrosine kinase encoded by c-kit. The demonstration that the W locus encodes a transmembrane growth factor receptor provides a molecular basis for understanding the intrinsic haemopoietic defect in W mutant mice and the role that this cellular proto-oncogene plays in haemopoiesis and other developmental processes.
- Published
- 1990
7. Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice
- Author
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S Majumder, Benoit Chabot, Prabir Ray, M Cervone, K Nocka, Peter Besmer, and Alan Bernstein
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Heterozygote ,Mutant ,Melanoma, Experimental ,Biology ,Gene product ,Embryonic and Fetal Development ,Mice ,Cell Movement ,Proto-Oncogene Proteins ,Proto-Oncogenes ,Gene expression ,Tumor Cells, Cultured ,Genetics ,Animals ,Anemia, Macrocytic ,Mast Cells ,RNA, Messenger ,Tyrosine ,Protein kinase A ,Alleles ,Kinase ,Protein-Tyrosine Kinases ,Hematopoietic Stem Cells ,Molecular biology ,Mice, Mutant Strains ,Transmembrane protein ,Hematopoiesis ,Proto-Oncogene Proteins c-kit ,Haematopoiesis ,Liver ,Neural Crest ,Organ Specificity ,Melanocytes ,Genes, Lethal ,Pigmentation Disorders ,Developmental Biology - Abstract
The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.
- Published
- 1989
8. Phosphodiesterase-4 Inhibitors Increase Pigment Cell Proliferation and Melanization in Cultured Melanocytes and within a 3-Dimensional Skin Equivalent Model.
- Author
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Goldstein NB, Berk ZBK, Tomb LC, Hu J, Hoaglin LG, Roop DR, Adiri R, Zhuang Y, Canosa JM, Sanders P, Norris DA, Nocka K, Cha A, and Birlea SA
- Abstract
Vitiligo is a common chronic autoimmune disease characterized by white macules and patches of the skin, having a negative impact on patients' life and without any definitive cure at present. Identification of new compounds to reverse depigmentation is therefore a pressing need for this disease. The pharmacologic compounds phosphodiesterase-4 inhibitors (PDE4is) are small molecules with immunomodulatory properties used for treatment of inflammatory dermatoses. PDE4is have shown repigmentation effects in patients with vitiligo, in some case reports. We characterized the proliferative and melanogenic potential of 2 known PDE4is-crisaborole and roflumilast-and of a more recently designed compound, PF-07038124. We used 2 in vitro model systems-the primary human melanocyte culture and a 3-dimensional cocultured skin model (MelanoDerm)-with an exploratory testing platform composed of complementary assays (spectrophotometry, melanin and proliferation assays, immunostaining, Fontana-Masson staining, RT-qPCR, western blot, and whole-transcriptome RNA sequencing). We identified that treatment with PDE4is was associated with increased melanocyte proliferation and melanization in both in vitro models and with increase in the melanogenic genes and proteins expression in cultured melanocytes. These effects were found to be enhanced by addition of α-melanocyte-stimulating hormone. Our findings support the further evaluation of PDE4is with or without α-melanocyte-stimulating hormone agonists in vitiligo trials., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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9. Crisaborole reverses dysregulation of the mild to moderate atopic dermatitis proteome toward nonlesional and normal skin.
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Kim M, Del Duca E, Cheng J, Carroll B, Facheris P, Estrada Y, Cha A, Werth J, Bissonnette R, Nocka K, Zang C, Pavel AB, and Guttman-Yassky E
- Subjects
- Adult, Humans, Boron Compounds pharmacology, Boron Compounds therapeutic use, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Bridged Bicyclo Compounds, Heterocyclic therapeutic use, Ointments therapeutic use, Proteome, Proteomics, Dermatitis, Atopic drug therapy, Dermatitis, Atopic pathology
- Abstract
Background: Safe and effective long-term topical treatments for atopic dermatitis (AD) remain limited., Objective: In this phase 2a, single-center, intrapatient, and vehicle-controlled study, we examine the mechanism of action of crisaborole 2% ointment, a topical nonsteroidal PDE4 (phosphodiesterase-4) inhibitor, in a proteomic analysis of 40 adults with mild to moderate AD and 20 healthy subjects., Methods: Within the AD cohort, 2 target lesions were randomized in an intrapatient (1:1) manner to double-blind crisaborole/vehicle applied twice daily for 14 days. Punch biopsy specimens were collected for biomarker analysis at baseline from all participants, then from AD patients only at day 8 (optional) and day 15., Results: Compared to the vehicle, crisaborole significantly reversed dysregulation of the overall lesional proteome and of key markers and pathways (eg, Th2, Th17/Th22, and T-cell activation) associated with AD pathogenesis toward both nonlesional and normal skin. Significant clinical correlations were observed with markers associated with nociception and Th2, Th17, and neutrophilic activation., Limitations: Study limitations include predominance of white patients in the cohort, relatively short treatment time, and regimented administration of crisaborole., Conclusion: Our results demonstrate crisaborole-induced normalization of the AD proteome toward a nonlesional molecular phenotype and further support topical PDE4 inhibition in the treatment of mild to moderate AD., Competing Interests: Conflicts of interest Dr Bissonnette is an Advisory Board Member, Consultant, Speaker and/or Investigator for and received honoraria and/or grants from AbbVie, Almirall, Amgen, AnaptysBio, Arcutis, Bausch Health, Boehringer-Ingelheim, Bristol-Myers Squibb, Dermavant, Eli Lilly, Escalier, Janssen, Kyowa Kirin, LEO Pharma, Nimbus, Pfizer, Regeneron, Sienna, and UCB. He is also an employee and shareholder of Innovaderm Research. Dr Guttman-Yassky has served as a consultant for AbbVie, Amgen, Allergan, Asana Bioscience, Celgene, Concert, Dermira, DS Biopharma, Escalier, Galderma, Glenmark, Kyowa Kirin, LEO Pharmaceuticals, Lilly, Mitsubishi Tanabe, Novartis, Pfizer, Regeneron, Sanofi, and Union Therapeutics; a member of advisory boards of Allergan, Asana Bioscience, Celgene, DBV, Dermavant, Dermira, Escalier, Galderma, Glenmark, Kyowa Kirin, LEO Pharma, Lilly, Novartis, Pfizer, Regeneron, and Sanofi; and a recipient of research grants from AbbVie, AnaptysBio, AntibioTx, Asana Bioscience, Boehringer-Ingelheim, Celgene, DBV, Dermavant, DS Biopharma, Galderma, Glenmark, Innovaderm, Janssen Biotech, Kiniska Pharma, LEO Pharmaceuticals, Lilly, Medimmune, Sienna Biopharmaceuticals, Novan, Novartis, Ralexar, Regeneron, Pfizer, UCB, and Union Therapeutics. Dr Pavel is an employee of the Icahn School of Medicine at Mount Sinai and conducts research sponsored by Pfizer. Dr Werth is a full-time employee of and shareholder in Pfizer, Inc. Kim, Duca, Cheng, Carroll, Facheris, Estrada, Cha, Nocka, and Zhang have no conflicts of interest to declare., (Copyright © 2023 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)
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- 2023
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10. Paying for PrEP: A qualitative study of cost factors that impact pre-exposure prophylaxis uptake in the US.
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Sosnowy C, Predmore Z, Dean LT, Raifman J, Chu C, Galipeau D, Nocka K, Napoleon S, and Chan P
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- Male, Humans, United States, Homosexuality, Male, Bisexuality, Pre-Exposure Prophylaxis, Sexual and Gender Minorities, HIV Infections prevention & control, HIV Infections drug therapy
- Abstract
Background: Concerns about the actual and perceived costs of pre-exposure prophylaxis (PrEP) continue to be a major barrier to uptake among gay, bisexual and men who have sex with men (GBMSM) in the United States., Methods: We conducted semi-structured interviews with 25 GBMSM who presented for routine health care at a STD clinic in the northeastern United States. The cohort included GBMSM who were or were not currently taking PrEP and represented varied health care coverage and financial resources. We used a structured coding scheme to analyze transcripts and identify themes relevant to cost factors., Results: Participants shared their perspectives about PrEP and their experiences with accessing and paying for PrEP. Our findings suggest that health care coverage or financial assistance were essential to PrEP access but were not easily accessible to all people and did not always cover all costs. Therefore, paying for PrEP had to be balanced with other life expenses. Participants had multiple sources for information about PrEP cost and assistance from clinic and pharmacy staff helped reduce burden and resolve difficulties., Conclusion: Addressing gaps in health care coverage, providing financial support, and improving the enrollment process in a financial assistance program may improve PrEP uptake.
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- 2022
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11. Discovery and multi-parametric optimization of a high-affinity antibody against interleukin-25 with neutralizing activity in a mouse model of skin inflammation.
- Author
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Bone R, Fennell BJ, Tam A, Sheldon R, Nocka K, Varghese S, Chang CS, Hawerkamp HC, Yeow A, Saunders SP, Hams E, Walsh PT, Cunningham O, and Fallon PG
- Abstract
Background: Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo ., Methods: In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody's therapeutic potential for targeting IL25 in inflammation., Results: A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule., Conclusions: The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
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- 2022
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12. A database of US state policies to mitigate COVID-19 and its economic consequences.
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Skinner A, Flannery K, Nocka K, Bor J, Dean LT, Jay J, Lipson SK, Cole MB, Benfer EA, Scheckman R, Raderman W, Jones DK, and Raifman J
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- Humans, Masks, Pandemics prevention & control, Policy, Public Health, United States epidemiology, COVID-19 epidemiology, COVID-19 prevention & control
- Abstract
Background: Since COVID-19 first appeared in the United States (US) in January 2020, US states have pursued a wide range of policies to mitigate the spread of the virus and its economic ramifications. Without unified federal guidance, states have been the front lines of the policy response., Main Text: We created the COVID-19 US State Policy (CUSP) database ( https://statepolicies.com/ ) to document the dates and components of economic relief and public health measures issued at the state level in response to the COVID-19 pandemic. Documented interventions included school and business closures, face mask mandates, directives on vaccine eligibility, eviction moratoria, and expanded unemployment insurance benefits. By providing continually updated information, CUSP was designed to inform rapid-response, policy-relevant research in the context of the COVID-19 pandemic and has been widely used to investigate the impact of state policies on population health and health equity. This paper introduces the CUSP database and highlights how it is already informing the COVID-19 pandemic response in the US., Conclusion: CUSP is the most comprehensive publicly available policy database of health, social, and economic policies in response to the COVID-19 pandemic in the US. CUSP documents widespread variation in state policy decisions and implementation dates across the US and serves as a freely available and valuable resource to policymakers and researchers., (© 2022. The Author(s).)
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- 2022
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13. Primary Care for Transgender Adolescents and Young Adults in Rhode Island: An Analysis of the all Payers Claims Database.
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Nocka K, Montgomery MC, Progovac A, Guss CE, Chan PA, and Raifman J
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- Adolescent, Humans, Primary Health Care, Rhode Island, United States, Young Adult, Gender Dysphoria, Pre-Exposure Prophylaxis, Transgender Persons
- Abstract
Purpose: Structural stigma has shaped disparities across several domains of health for transgender relative to cisgender (nontransgender) adolescents in the United States. Research on transgender health has largely overlooked the role of preventive care, especially for adolescents., Methods: We used ICD-9 and ICD-10 codes to identify transgender adolescents in the Rhode Island All Payers Claims Database (APCD) from 2011 to 2017 based on a diagnosis for gender identity disorder (GID). We evaluated differences in the use of preventive care services between transgender and cisgender adolescents. We compared the frequency of sexually transmitted infection and HIV screening and the percentage prescribed pre-exposure prophylaxis among transgender and cisgender adolescents using t-tests and chi-square tests. We used logistic regression to evaluate the association between attending regular physical exams and receiving preventive health services., Results: There was no significant difference in the proportion of transgender and cisgender adolescents who received regular influenza vaccinations, physical exams, and HPV vaccinations. Transgender adolescents were significantly more likely to receive regular cholesterol and BMI screenings compared to cisgender adolescents. While there was a significant positive association between having regular physical exams and receiving most preventive screenings in the cisgender population, in the transgender population, regular physical exams were only significantly positively associated with STI screening., Conclusions: Transgender adolescents accessing the healthcare system received similar, if not greater, levels of preventive health services compared to their cisgender peers. Because regular physical exams were not associated with receiving most preventive services among transgender adolescents, these services may be delivered outside of primary care settings., (Copyright © 2020 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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14. Evaluating statewide HIV preexposure prophylaxis implementation using All-Payer Claims Data.
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Raifman J, Nocka K, Galárraga O, Wilson IB, Crowley C, Tao J, Napoleon S, Marak T, and Chan PA
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- Adult, Female, Humans, Insurance Claim Review, Male, Pre-Exposure Prophylaxis methods, Rhode Island, Anti-HIV Agents administration & dosage, Emtricitabine administration & dosage, HIV Infections prevention & control, Pre-Exposure Prophylaxis statistics & numerical data, Tenofovir administration & dosage
- Abstract
Purpose: Preexposure prophylaxis (PrEP) in the form of daily emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) is effective for preventing HIV infection. Implementation has been limited by an inability to systematically evaluate uptake and use. All-Payer Claims Databases (APCDs) provide an opportunity to evaluate population-level PrEP implementation., Methods: We used 2012-2017 data from Rhode Island's APCD and developed an algorithm to identify individuals prescribed FTC/TDF for PrEP. We describe PrEP implementation by patient demographics and provider type and mapped PrEP implementation across ZIP codes. We compared APCD data to electronic medical record data and comprehensive pharmaceutical claims data (AIDSVu.org)., Results: The Rhode Island APCD represented approximately 87% of the state's population. PrEP use increased 31-fold from 2012 to 2017. Users were predominantly privately insured, male, and concentrated in Providence County (76.6%). Infectious diseases providers had 3.2 times the odds of being a PrEP prescriber compared to primary care providers. Compared to other pharmaceutical and electronic medical record data, the APCD underestimated the number of PrEP users in Rhode Island but improved in capturing users over time., Conclusions: APCDs are a useful data source for characterizing PrEP use across a state. There is a need to increase PrEP prescribing among primary care providers, especially in areas with underserved populations., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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15. Modulation of the IL-33/IL-13 Axis in Obesity by IL-13Rα2.
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Duffen J, Zhang M, Masek-Hammerman K, Nunez A, Brennan A, Jones JEC, Morin J, Nocka K, and Kasaian M
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- Adipose Tissue immunology, Adipose Tissue metabolism, Animals, Humans, Interleukin-13 immunology, Interleukin-13 Receptor alpha2 Subunit immunology, Interleukin-33 immunology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Interleukin-13 metabolism, Interleukin-13 Receptor alpha2 Subunit metabolism, Interleukin-33 metabolism, Obesity immunology, Obesity metabolism
- Abstract
In obesity, IL-13 overcomes insulin resistance by promoting anti-inflammatory macrophage differentiation in adipose tissue. Endogenous IL-13 levels can be modulated by the IL-13 decoy receptor, IL-13Rα2, which inactivates and depletes the cytokine. In this study, we show that IL-13Rα2 is markedly elevated in adipose tissues of obese mice. Mice deficient in IL-13Rα2 had high expression of IL-13 response markers in adipose tissue, consistent with increased IL-13 activity at baseline. Moreover, exposure to the type 2 cytokine-inducing alarmin, IL-33, enhanced serum and tissue IL-13 concentrations and elevated tissue eosinophils, macrophages, and type 2 innate lymphoid cells. IL-33 also reduced body weight, fat mass, and fasting blood glucose levels. Strikingly, however, the IL-33-induced protection was greater in IL-13Rα2-deficient mice compared with wild-type littermates, and these changes were largely attenuated in mice lacking IL-13. Although IL-33 administration improved the metabolic profile in the context of a high fat diet, it also resulted in diarrhea and perianal irritation, which was enhanced in the IL-13Rα2-deficient mice. Weight loss in this group was associated with reduced food intake, which was likely related to the gastrointestinal effects. These findings outline both potentially advantageous and deleterious effects of a type 2-skewed immune response under conditions of metabolic stress, and identify IL-13Rα2 as a critical checkpoint in adipose tissues that limits the protective effects of the IL-33/IL-13 axis in obesity., (Copyright © 2018 by The American Association of Immunologists, Inc.)
- Published
- 2018
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16. Alternative splicing of interleukin-33 and type 2 inflammation in asthma.
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Gordon ED, Simpson LJ, Rios CL, Ringel L, Lachowicz-Scroggins ME, Peters MC, Wesolowska-Andersen A, Gonzalez JR, MacLeod HJ, Christian LS, Yuan S, Barry L, Woodruff PG, Ansel KM, Nocka K, Seibold MA, and Fahy JV
- Subjects
- Adult, Asthma metabolism, Basophils metabolism, Cell Line, Epithelial Cells metabolism, Female, Gene Expression Profiling methods, Humans, Inflammation metabolism, Interleukin-1 Receptor-Like 1 Protein genetics, Interleukin-1 Receptor-Like 1 Protein metabolism, Interleukin-33 metabolism, Male, Mast Cells metabolism, Middle Aged, Respiratory Mucosa metabolism, Respiratory Mucosa pathology, Sputum cytology, Sputum metabolism, Young Adult, Alternative Splicing, Asthma genetics, Inflammation genetics, Interleukin-33 genetics
- Abstract
Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an "alarmin" during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms.
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- 2016
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17. Developing predictive assays: the phenotypic screening "rule of 3".
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Vincent F, Loria P, Pregel M, Stanton R, Kitching L, Nocka K, Doyonnas R, Steppan C, Gilbert A, Schroeter T, and Peakman MC
- Subjects
- Endpoint Determination, Humans, Phenotype, Biological Assay methods, Drug Evaluation, Preclinical methods
- Abstract
Phenotypic drug discovery approaches can positively affect the translation of preclinical findings to patients. However, not all phenotypic assays are created equal. A critical question then follows: What are the characteristics of the optimal assays? We analyze this question and propose three specific criteria related to the disease relevance of the assay-system, stimulus, and end point-to help design the most predictive phenotypic assays., (Copyright © 2015, American Association for the Advancement of Science.)
- Published
- 2015
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18. Low sputum MMP-9/TIMP ratio is associated with airway narrowing in smokers with asthma.
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Chaudhuri R, McSharry C, Brady J, Grierson C, Messow CM, Spears M, Miele G, Nocka K, MacNee W, Connell M, Murchison JT, Sproule M, Hilmi OJ, Miller DK, and Thomson NC
- Subjects
- Adult, Bronchography methods, Female, Forced Expiratory Volume, Humans, Male, Middle Aged, Tomography, X-Ray Computed, Asthma physiopathology, Bronchi pathology, Matrix Metalloproteinase 9 analysis, Smoking adverse effects, Sputum chemistry, Tissue Inhibitor of Metalloproteinase-1 analysis, Tissue Inhibitor of Metalloproteinase-2 analysis
- Abstract
Asthmatic smokers have poor symptom control and accelerated decline in lung function. A reduced ratio of matrix metalloproteinase (MMP)-9/tissue inhibitors of metalloproteinases (TIMPs) in nonsmokers with asthma has been implicated in airway remodelling. We tested the hypothesis that sputum MMP-9 activity/TIMPs ratios are reduced in smokers compared with never-smokers with asthma and are associated with reduced lung function and altered computed tomography (CT) measures of airway wall dimensions. Lung function, airway dimensions by CT, and induced sputum concentrations (and activity) of MMP-9 and TIMP-1 and -2 were measured in 81 asthmatics and 43 healthy subjects (smokers and never-smokers). Respiratory epithelial MMP9 and TIMP mRNA was quantified in 31 severe asthmatics and 32 healthy controls. Sputum MMP-9 activity/TIMP-1 and TIMP-2 ratios, and nasal epithelial MMP9/TIMP1 and MMP9/TIMP2 expression ratios were reduced in smokers with asthma compared with never-smokers with asthma. Low sputum ratios in asthmatic smokers were associated with reduced post-bronchodilator forced expiratory volume in 1 s (FEV1), FEV1/forced vital capacity ratio and segmental airway lumen area. The association of a low sputum MMP-9 activity/TIMP-1 ratio with persistent airflow obstruction and reduced CT airway lumen area in smokers with asthma may indicate that an imbalance of MMP-9 and TIMPs contributes to structural changes to the airways in this group., (©ERS 2014.)
- Published
- 2014
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19. Arachidonic acid metabolites and enzyme transcripts in asthma are altered by cigarette smoking.
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Thomson NC, Chaudhuri R, Spears M, Messow CM, Jelinsky S, Miele G, Nocka K, Takahashi E, Hilmi OJ, Shepherd MC, Miller DK, and McSharry C
- Subjects
- Adult, Anti-Asthmatic Agents pharmacology, Anti-Asthmatic Agents therapeutic use, Asthma diagnosis, Asthma drug therapy, Cross-Sectional Studies, Female, Gene Expression, Humans, Leukocytes metabolism, Leukotriene E4 blood, Leukotriene E4 metabolism, Leukotriene E4 urine, Male, Middle Aged, Prostaglandins blood, Prostaglandins urine, RNA, Messenger genetics, Respiratory Function Tests, Respiratory Mucosa metabolism, Risk Factors, Sputum metabolism, Surveys and Questionnaires, Arachidonic Acid metabolism, Asthma genetics, Asthma metabolism, Smoking, Transcription, Genetic
- Abstract
Background: Arachidonic acid metabolites are implicated in the pathogenesis of asthma although only limited information is available on the impact of current smoking history on these metabolites. The aim of the study was to examine the effect of smoking status on urinary, sputum, and plasma eicosanoid concentrations and relevant enzyme transcripts in asthma., Methods: In 108 smokers and never smokers with asthma and 45 healthy controls [smokers and never smokers], we measured urinary tetranor prostaglandin (PG)D2 (PGDM) and leukotriene (LT)E4 , induced sputum fluid LTB4 , LTE4 , PGD2 , and PGE2 , plasma secretory phospholipase A2 (sPLA2 ), and 11β prostaglandin F2α (11βPGF2α ), and, in a subgroup with severe asthma, airway leukocyte and epithelial cell mRNA expression levels of arachidonic acid metabolic enzymes., Results: Smokers with asthma had higher urinary LTE4 ; 83 (59, 130) vs 59 (40, 90) pg/mg creatinine, P = 0.008, and PGDM; 60 (35, 100) vs 41 (28, 59) ng/mg creatinine, P = 0.012 concentrations, respectively, and lower sputum PGE2 concentrations 80 (46, 157) vs 192 (91, 301) pg/ml, P = 0.001 than never smokers with asthma. Sputum LTB4 (P = 0.013), and plasma 11βPGF2α (P = 0.032), concentrations, respectively, were increased in smokers with asthma compared with healthy smokers. Asthma-specific and smoking-related increases (>1.5-fold expression) in arachidonate 15-lipoxygenase and gamma-glutamyltransferase transcripts were demonstrated., Conclusions: Several arachidonic acid metabolites and enzyme transcripts involving both lipoxygenase and cyclooxygenase pathways are increased in smokers with asthma and differ from never smokers with asthma. Possibly targeting specific lipoxygenase and cyclooxygenase pathways that are activated by asthma and cigarette smoking may optimize therapeutic responses., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
- Published
- 2014
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20. Sputum matrix metalloproteinase-9 is associated with the degree of emphysema on computed tomography in COPD.
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Chaudhuri R, McSharry C, Spears M, Brady J, Grierson C, Messow CM, Miele G, Nocka K, MacNee W, Connell M, Murchison JT, Sproule M, Hilmi O, Miller DK, and Thomson NC
- Abstract
Background: Matrix-metalloproteinase (MMP)-9 has been implicated in the pathogenesis of COPD, although its link to disease severity is unclear. The purpose of the study was to examine the relationship between disease severity assessed by lung function and computed tomography (CT) and sputum MMP-9 expression, concentration and activity in patients with COPD., Findings: In 53 COPD subjects, smokers and ex-smokers; 46 healthy controls, smokers and never smokers, we measured sputum MMP-9 concentrations (ELISA) and enzyme activity (FRET), sputum MMP-9 mRNA expression, spirometry, diffusing capacity for carbon monoxide (DLco) and CT assessment of emphysema (% low attenuation areas below-950 Hounsfield units). Sputum MMP-9 concentrations and mRNA expression in COPD subjects were significantly greater than in healthy never-smokers (p = 0.007 and p = 0.001 respectively) and similar to those in healthy smokers. Disease severity when assessed by the extent of emphysema measured by CT, but not by spirometry or DLco values, was directly associated with sputum MMP-9 concentrations [r = 0.442 (0.171, 0.634), p = 0.020], and MMP-9 activity [r = 0.447 (0.219, 0.643), p = 0.010]. In moderate to severe COPD, increased MMP-9 mRNA expression levels were associated with reduced post-bronchodilator FEV1 [r = -0.530 (-0.686, -0.327), p < 0.001], FEV1/FVC ratio [r = -0.551 (-0.701, -0.354), p < 0.001] and reduced DLco [r = -0.399 (-539, -0.102), p = 0.048]., Conclusions: Sputum MMP-9 concentrations in COPD are directly associated with the extent of emphysema measured by CT and MMP-9 expression levels are inversely associated with DLco. These findings support a role for MMP-9 in the pathogenesis of COPD.
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- 2013
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21. Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit.
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Fish PV, Filippakopoulos P, Bish G, Brennan PE, Bunnage ME, Cook AS, Federov O, Gerstenberger BS, Jones H, Knapp S, Marsden B, Nocka K, Owen DR, Philpott M, Picaud S, Primiano MJ, Ralph MJ, Sciammetta N, and Trzupek JD
- Subjects
- Cell Cycle Proteins, Crystallography, X-Ray, Humans, Models, Molecular, Molecular Probes chemical synthesis, Molecular Structure, Nuclear Proteins antagonists & inhibitors, Protein Binding, Quinazolinones chemical synthesis, Structure-Activity Relationship, Sulfonamides chemical synthesis, Sulfonamides chemistry, Molecular Probes pharmacology, Nuclear Proteins metabolism, Quinazolinones pharmacology, Sulfonamides pharmacology, Transcription Factors antagonists & inhibitors
- Abstract
The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogated and interpreted by an acetyllysine-specific protein-protein interaction with bromodomain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a small molecule is feasible, and this may represent an opportunity for disease intervention through the recently disclosed antiproliferative and anti-inflammatory properties of such inhibitors. Herein, we describe the discovery and structure-activity relationship (SAR) of a novel, small-molecule chemical probe for BET family inhibition that was identified through the application of structure-based fragment assessment and optimization techniques. This has yielded a potent, selective compound with cell-based activity (PFI-1) that may further add to the understanding of BET family function within the bromodomains.
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- 2012
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22. Sputum matrix metalloproteinase-12 in patients with chronic obstructive pulmonary disease and asthma: relationship to disease severity.
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Chaudhuri R, McSharry C, Brady J, Donnelly I, Grierson C, McGuinness S, Jolly L, Weir CJ, Messow CM, Spears M, Miele G, Nocka K, Crowther D, Thompson J, Brannigan M, Lafferty J, Sproule M, Macnee W, Connell M, Murchison JT, Shepherd MC, Feuerstein G, Miller DK, and Thomson NC
- Subjects
- Adult, Aged, Asthma complications, Asthma diagnosis, Cross-Sectional Studies, Disease Progression, Emphysema diagnosis, Emphysema enzymology, Female, Fluorescence Resonance Energy Transfer, Follow-Up Studies, Humans, Male, Matrix Metalloproteinase 12 genetics, Matrix Metalloproteinase 12 immunology, Middle Aged, Pulmonary Disease, Chronic Obstructive complications, Pulmonary Disease, Chronic Obstructive diagnosis, Respiratory Function Tests, Severity of Illness Index, Tomography, X-Ray Computed, Asthma immunology, Asthma physiopathology, Matrix Metalloproteinase 12 metabolism, Pulmonary Disease, Chronic Obstructive immunology, Pulmonary Disease, Chronic Obstructive physiopathology, Sputum enzymology
- Abstract
Background: Matrix metalloproteinase (MMP)-12 has been implicated in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and asthma. The influence of disease severity on sputum MMP-12 concentrations and activity is not known., Objectives: We sought to examine the relationship between disease severity assessed by means of lung function and computed tomography (CT) and induced sputum MMP-12 concentrations and activity in patients with asthma and COPD., Methods: In 208 subjects (109 asthmatic patients, smokers and never smokers, mild, moderate, and severe; 53 patients with COPD, smokers and exsmokers, mild, moderate, and severe; and 46 healthy control subjects, smokers and never smokers), we measured induced sputum MMP-12 concentrations (ELISA) and enzyme activity (fluorescence resonance energy transfer), sputum cell MMP12 mRNA expression (quantitative PCR [qPCR]), diffusing capacity for carbon monoxide (Dlco), and CT assessment of emphysema (percentage of low-attenuation areas at less -950 Hounsfield units)., Results: Sputum MMP-12 concentrations are greater in patients with COPD and smokers with asthma than in healthy nonsmokers (P = .003 and P = .035, respectively) but similar to those seen in healthy smokers. In patients with COPD, disease severity, when measured by means of CT-assessed emphysema, but not by means of spirometry or Dlco values, is directly associated with sputum MMP-12 concentrations and activity. In the asthma groups there is no significant association between disease severity and sputum MMP-12 concentrations or activity., Conclusions: Sputum MMP-12 concentrations and activity in patients with COPD are directly associated with the extent of emphysema measured by means of CT. This finding supports a role for MMP-12 in the pathogenesis of COPD and might suggest that blocking MMP-12 activity in patients with COPD could prevent the further development of emphysema., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)
- Published
- 2012
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23. Acidic mammalian chitinase is not a critical target for allergic airway disease.
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Fitz LJ, DeClercq C, Brooks J, Kuang W, Bates B, Demers D, Winkler A, Nocka K, Jiao A, Greco RM, Mason LE, Fleming M, Quazi A, Wright J, Goldman S, Hubeau C, and Williams CM
- Subjects
- Allergens immunology, Animals, Asthma genetics, Asthma immunology, Bronchoalveolar Lavage Fluid immunology, Chitinases deficiency, Chitinases genetics, Chitinases immunology, Female, Humans, Hypersensitivity genetics, Hypersensitivity immunology, Inflammation enzymology, Inflammation genetics, Inflammation immunology, Interleukin-13 immunology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Neutrophils immunology, Th1 Cells immunology, Th2 Cells immunology, Asthma enzymology, Chitinases antagonists & inhibitors, Hypersensitivity enzymology
- Abstract
The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.
- Published
- 2012
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24. A fluorescent assay suitable for inhibitor screening and vanin tissue quantification.
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Ruan BH, Cole DC, Wu P, Quazi A, Page K, Wright JF, Huang N, Stock JR, Nocka K, Aulabaugh A, Krykbaev R, Fitz LJ, Wolfman NM, and Fleming ML
- Subjects
- Amidohydrolases antagonists & inhibitors, Amidohydrolases genetics, Amino Acid Sequence, Animals, Cell Line, Enzyme Inhibitors pharmacology, Fluorescent Dyes chemistry, GPI-Linked Proteins, High-Throughput Screening Assays, Humans, Kidney enzymology, Mice, Molecular Sequence Data, Pantothenic Acid chemistry, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins genetics, Recombinant Proteins metabolism, Spectrophotometry, Ultraviolet, Amidohydrolases metabolism, Chromatography, High Pressure Liquid methods, Enzyme Inhibitors chemistry, Mass Spectrometry methods
- Abstract
Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently labeled pantothenate derivative. The assay has been used for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from high-throughput screening (HTS), and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in kidney. We demonstrated that the purified recombinant vanin-1 consisting of the extracellular portion without the glycosylphosphatidylinositol (GPI) linker was highly active with an apparent K(m) of 28 microM for pantothenate-7-amino-4-methylcoumarin (pantothenate-AMC), which was converted to pantothenic acid and AMC based on liquid chromatography-mass spectrometry (LC-MS) analysis. The assay also performed well in a 384-well microplate format under initial rate conditions (10% conversion) with a signal-to-background ratio (S/B) of 7 and a Z factor of 0.75. Preliminary screening of a library of 1280 pharmaceutically active compounds identified inhibitors with novel chemical scaffolds. This assay will be a powerful tool for target validation and drug lead identification and characterization., (Copyright 2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
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25. Human alveolar macrophage gene responses to Mycobacterium tuberculosis strains H37Ra and H37Rv.
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Silver RF, Walrath J, Lee H, Jacobson BA, Horton H, Bowman MR, Nocka K, and Sypek JP
- Subjects
- Bronchoalveolar Lavage Fluid cytology, Bronchoalveolar Lavage Fluid microbiology, Down-Regulation genetics, Granulocyte-Macrophage Colony-Stimulating Factor biosynthesis, Humans, Interleukin-23 biosynthesis, Intracellular Space microbiology, Monocytes metabolism, Monocytes microbiology, Mycobacterium tuberculosis classification, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, Tuberculosis genetics, Tuberculosis microbiology, Up-Regulation genetics, Gene Expression Regulation, Macrophages, Alveolar metabolism, Macrophages, Alveolar microbiology, Mycobacterium tuberculosis physiology
- Abstract
H37Rv and H37Ra have been widely used as models of virulent and avirulent strains, respectively, of Mycobacterium tuberculosis. Since the sequencing of H37Rv, microarrays have been used to investigate gene expression of M. tuberculosis strains under various conditions, and to compare gene expression of specific isolates of the organism. Because differences in the virulence of these organisms could also be manifest via their differential induction of host genes, we used Affymetrix Human Genome Arrays U133A and U133B to evaluate human alveolar macrophage (AM) responses to infection with H37Rv and H37Ra. H37Rv altered expression of far more genes than did H37Ra. Moreover, the genes induced by H37Rv to a greater extent than by H37Ra were predominantly associated with the development of effective immunity. H37Rv markedly increased expression of IL-23 p19, whereas neither organism significantly induced IL-12 p35 expression. Quantitative PCR confirmed that H37Rv induced significantly more AM p19 expression than did H37Ra. After low-level infection of both AM and peripheral blood monocytes (MN) with H37Rv, neither cell type produced IL-12 (by ELISA). In contrast, AM displayed significant IL-23 production in response to H37Rv, whereas MN did not. Our findings thus suggest an important role for IL-23 in human host responses to pulmonary infection with M. tuberculosis, and are consistent with epidemiologic and genetic studies that imply that H37Rv may not have unusual capacity to cause human disease.
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- 2009
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26. Cutting edge: Interleukin 4-dependent mast cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling.
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Jiang Y, Kanaoka Y, Feng C, Nocka K, Rao S, and Boyce JA
- Subjects
- Animals, Cell Proliferation drug effects, Cells, Cultured, Glutathione Transferase deficiency, Glutathione Transferase genetics, Glutathione Transferase metabolism, Humans, Mast Cells drug effects, Mice, Mice, Knockout, Signal Transduction drug effects, Autocrine Communication drug effects, Cysteine metabolism, Interleukin-4 pharmacology, Leukotrienes metabolism, Mast Cells cytology, Mast Cells metabolism, Paracrine Communication drug effects
- Abstract
Reactive mastocytosis (RM) in epithelial surfaces is a consistent Th2-associated feature of allergic disease. RM fails to develop in mice lacking leukotriene (LT) C4 synthase (LTC4S), which is required for cysteinyl leukotriene (cys-LT) production. We now report that IL-4, which induces LTC4S expression by mast cells (MCs), requires cys-LTs, the cys-LT type 1 receptor (CysLT1), and Gi proteins to promote MC proliferation. LTD4 (10-1000 nM) enhanced proliferation of human MCs in a CysLT1-dependent, pertussis toxin-sensitive manner. LTD4-induced phosphorylation of ERK required transactivation of c-kit. IL-4-driven comitogenesis was likewise sensitive to pertussis toxin or a CysLT1-selective antagonist and was attenuated by treatment with leukotriene synthesis inhibitors. Mouse MCs lacking LTC4S or CysLT1 showed substantially diminished IL-4-induced comitogenesis. Thus, IL-4 induces proliferation in part by inducing LTC4S and cys-LT generation, which causes CysLT1 to transactivate c-kit in RM.
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- 2006
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27. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam.
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Lynch BA, Lambeng N, Nocka K, Kensel-Hammes P, Bajjalieh SM, Matagne A, and Fuks B
- Subjects
- Animals, Binding Sites, Brain cytology, Brain metabolism, Fibroblasts, Gene Deletion, Humans, Inhibitory Concentration 50, Intracellular Membranes metabolism, Levetiracetam, Membrane Glycoproteins chemistry, Membrane Glycoproteins genetics, Mice, Mice, Knockout, Molecular Weight, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins genetics, Photoaffinity Labels, Piracetam analogs & derivatives, Precipitin Tests, Protein Binding, Rats, Seizures, Synaptic Vesicles metabolism, Anticonvulsants metabolism, Membrane Glycoproteins metabolism, Nerve Tissue Proteins metabolism, Piracetam metabolism
- Abstract
Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of approximately 90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.
- Published
- 2004
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28. Targeted disruption of guanosine diphosphate-dissociation inhibitor for Rho-related proteins, GDID4: normal hematopoietic differentiation but subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation.
- Author
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Guillemot JC, Kruskal BA, Adra CN, Zhu S, Ko JL, Burch P, Nocka K, Seetoo K, Simons E, and Lim B
- Subjects
- Animals, Calcimycin pharmacology, Calcium metabolism, Cell Differentiation, Cells, Cultured, Embryo, Mammalian cytology, Fibroblasts metabolism, GTP-Binding Proteins metabolism, GTP-Binding Proteins physiology, Hematopoiesis, Histamine Release drug effects, Immunoglobulin E pharmacology, Ionophores pharmacology, Mast Cells cytology, Mice, Minor Histocompatibility Antigens, Phagocytosis, Proteins physiology, Stem Cell Factor pharmacology, Stem Cells cytology, rho Guanine Nucleotide Dissociation Inhibitor beta, rho-Specific Guanine Nucleotide Dissociation Inhibitors, Gene Targeting, Guanine Nucleotide Dissociation Inhibitors, Hematopoietic Stem Cells cytology, Macrophages metabolism, Proteins genetics, Respiratory Burst, Stem Cells metabolism, Superoxides metabolism
- Abstract
The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.
- Published
- 1996
29. Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen.
- Author
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Broudy VC, Lin NL, Priestley GV, Nocka K, and Wolf NS
- Subjects
- Anemia, Hemolytic chemically induced, Anemia, Hemolytic pathology, Animals, Antibodies, Monoclonal pharmacology, Bone Marrow pathology, Cell Movement physiology, Colony-Forming Units Assay, Erythropoiesis physiology, Female, Hematopoietic Stem Cells metabolism, Immunoglobulin G pharmacology, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Phenotype, Phenylhydrazines toxicity, Proto-Oncogene Proteins c-kit drug effects, Radiation Chimera, Rats, Stem Cell Factor antagonists & inhibitors, Hematopoietic Stem Cells pathology, Proto-Oncogene Proteins c-kit physiology, Spleen pathology, Stem Cell Factor physiology
- Abstract
The phenotypes of mice that harbor a defect in the genes encoding either stem cell factor (SCF) or its receptor, c-kit, indicate that this ligand/receptor pair is necessary for maintenance of normal hematopoiesis in the adult. Our objective was to determine whether SCF, like erythropoietin, is necessary for acute erythroid expansion during recovery from hemolytic anemia. Monoclonal antibody ACK2, which recognizes the murine c-kit receptor, was used to selectively block the hematopoietic growth-promoting effects of SCF. Mice were treated with phenylhydrazine on day 0 and day 1 to induce hemolytic anemia and also received no antibody, control IgG, or ACK2 on day 0. The mice were killed on day 3 and the hematocrit (Hct), reticulocyte count, and numbers of erythroid and myeloid hematopoietic progenitor cells (colony-forming unit-erythroid [CFU-E], burst-forming unit [BFU]-E, and CFU-granulocyte-macrophage [GM]) were quantitated in the femoral marrow and spleen using hematopoietic colony-forming assays. Induction of hemolytic anemia with phenylhydrazine resulted in a drop in the Hct from approximately 50% to 30%, and an approximate 8- to 10-fold increase in the reticulocyte count. The numbers of CFU-E increased modestly in the femur, and approximately 25- to 50-fold in the spleen, in comparison with normal mice. BFU-E and CFU-GM values did not increase in the femur but expanded 6- to 10-fold in the spleen, in comparison with normal mice. This confirms that much of the erythroid expansion in response to hemolytic anemia occurs in the murine spleen. Neutralizing quantities of the ACK2 antibody reduced femoral CFU-E, BFU-E, and CFU-GM content to less than half that found in phenylhydrazine-treated control mice and nearly totally ablated splenic hematopoiesis. These results suggest that c-kit receptor function may be required for optimal response to acute erythropoietic demand and that erythropoiesis in the splenic microenvironment is more dependent on SCF/c-kit receptor interaction than is erythropoiesis in the marrow microenvironment. Because expansion of late erythropoiesis in the spleen was preferentially blocked, we tested the hypothesis that homing of more primitive hematopoietic cells to the spleen was dependent on c-kit receptor function. Lethally irradiated mice were injected with marrow cells obtained from mice that had received phenylhydrazine plus control IgG or with marrow cells obtained from mice that had received phenylhydrazine plus ACK2. In parallel experiments, normal murine marrow cells were treated in vitro with control IgG or with ACK2 and were injected into lethally irradiated mice. The fraction of BFU-E and CFU-GM retrieved from the marrow and spleen of the recipient mice 4 hours later was reduced by approximately 75% when progenitor cells had been exposed to ACK2, in comparison with control IgG. These data suggest that interaction of SCF with the c-kit receptor affects the homing behavior of hematopoietic progenitor cells in the adult animal.
- Published
- 1996
30. Gonadal expression of c-kit encoded at the W locus of the mouse.
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Manova K, Nocka K, Besmer P, and Bachvarova RF
- Subjects
- Animals, Blotting, Northern, Female, Male, Mice, Mice, Inbred Strains, Microscopy, Immunoelectron, Oocytes physiology, Ovary physiology, Proto-Oncogene Proteins c-kit, Spermatozoa physiology, Testis physiology, Gene Expression genetics, Genitalia physiology, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins genetics
- Abstract
Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.
- Published
- 1990
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31. The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus.
- Author
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Huang E, Nocka K, Beier DR, Chu TY, Buck J, Lahm HW, Wellner D, Leder P, and Besmer P
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Line, Cloning, Molecular, DNA genetics, DNA isolation & purification, Genes, Hematopoietic Cell Growth Factors isolation & purification, Hematopoietic Cell Growth Factors metabolism, Ligands, Mice, Mice, Inbred BALB C, Mice, Inbred Strains, Molecular Sequence Data, Molecular Weight, Proto-Oncogene Proteins c-kit, Transcription, Genetic, Chromosome Mapping, Hematopoietic Cell Growth Factors genetics, Mutation, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins metabolism
- Abstract
Mutations at the steel locus (Sl) of the mouse affect the same cellular targets as mutations at the white spotting locus (W), which is allelic with the c-kit proto-oncogene. We show that KL, a hematopoietic growth factor obtained from conditioned medium of BALB/c 3T3 fibroblasts that stimulates the proliferation of mast cells and early erythroid progenitors, specifically binds to the c-kit receptor. The predicted amino acid sequence of isolated KL-specific cDNA clones suggests that KL is synthesized as an integral transmembrane protein. Linkage analysis maps the KL gene to the Sl locus on mouse chromosome 10, and KL sequences are deleted in the genome of the Sl mouse. These results indicate that the Sl locus encodes the ligand of the c-kit receptor, KL.
- Published
- 1990
- Full Text
- View/download PDF
32. Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors.
- Author
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Nocka K, Buck J, Levi E, and Besmer P
- Subjects
- Alleles, Animals, Cell Division drug effects, Cell Line, Cells, Cultured, Chromatography, Gel, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Fibroblast Growth Factors isolation & purification, Fibroblast Growth Factors metabolism, Hematopoietic Stem Cells drug effects, Isoelectric Focusing, Ligands, Mast Cells drug effects, Mice, Mice, Inbred BALB C, Mice, Inbred Strains, Proto-Oncogene Proteins c-kit, Fibroblast Growth Factors pharmacology, Hematopoietic Stem Cells cytology, Mast Cells cytology, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins metabolism
- Abstract
The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis and hematopoiesis during development and in adult life. Cellular targets of W mutations in hematopoiesis include distinct cell populations in the erythroid and mast cell lineages as well as stem cells. In the absence of interleukin-3 (IL-3) mast cells derived from normal mice but not from W mutant mice can be maintained by co-culture with 3T3 fibroblasts. Based on the defective proliferative response of W mast cells in the 3T3 fibroblast co-culture system it had been proposed that fibroblasts produce the c-kit ligand. We have used a mast cell proliferation assay to purify a 30 kd protein, designated KL, from conditioned medium of Balb/3T3 fibroblasts to apparent homogeneity. KL stimulates the proliferation of normal bone marrow derived mast cells but not mast cells from W mice, although both normal and mutant mast cells respond similarly to IL-3. Connective tissue-type mast cells derived from the peritoneal cavity of normal mice were found to express a high level of c-kit protein on their surface and to proliferate in response to KL. The effect of KL on erythroid progenitor cells was investigated as well. In combination with erythropoietin, KL was found to stimulate early erythroid progenitors (BFU-E) from fetal liver and spleen cells but not from bone marrow cells of adult mice and from fetal liver cells of W/W mice.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1990
- Full Text
- View/download PDF
33. Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W.
- Author
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Nocka K, Tan JC, Chiu E, Chu TY, Ray P, Traktman P, and Besmer P
- Subjects
- Alleles, Amino Acid Sequence, Animals, Base Sequence, Cells, Cultured, Chromosome Mapping, Fibroblasts physiology, Mast Cells physiology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Molecular Sequence Data, Phenotype, Pigmentation genetics, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins biosynthesis, Proto-Oncogene Proteins c-kit, RNA, Messenger biosynthesis, Mutation, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins genetics
- Abstract
The proto-oncogene c-kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white-spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 are severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. Wv and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co-culture assay. We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c-kit coding sequence (W37 E----K at position 582; Wv T----M position 660 and W41 V----M position 831), which affect the c-kit associated tyrosine kinase to varying degrees. The c-kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c-kitW37 protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c-kit protein. A 125 kd c-kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1990
- Full Text
- View/download PDF
34. The dominant W42 spotting phenotype results from a missense mutation in the c-kit receptor kinase.
- Author
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Tan JC, Nocka K, Ray P, Traktman P, and Besmer P
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cells, Cultured, DNA genetics, Gene Expression, Homozygote, Liver analysis, Liver cytology, Liver embryology, Mast Cells metabolism, Mice, Molecular Sequence Data, Polymerase Chain Reaction, Proto-Oncogene Proteins c-kit, RNA analysis, Receptors, Cell Surface genetics, Signal Transduction, Mutation, Phenotype, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins genetics
- Abstract
The murine white spotting locus (W) is allelic with the proto-oncogene c-kit, which encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and migration of primordial germ cells and melanoblasts during development to varying degrees of severity. The W42 mutation has a particularly severe effect in both the homozygous and the heterozygous states. The molecular basis of the W42 mutation was determined. The c-kit protein products in homozygous mutant mast cells were expressed normally but displayed a defective tyrosine kinase activity in vitro. Nucleotide sequence analysis of mutant complementary DNAs revealed a missense mutation that replaces aspartic acid with asparagine at position 790 in the c-kit protein product. Aspartic acid-790 is a conserved residue in all protein kinases. These results provide an explanation for the dominant nature of the W42 mutation and provide insight into the mechanism of c-kit-mediated signal transduction.
- Published
- 1990
- Full Text
- View/download PDF
35. The mouse W/c-kit locus.
- Author
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Bernstein A, Chabot B, Dubreuil P, Reith A, Nocka K, Majumder S, Ray P, and Besmer P
- Subjects
- Animals, Chromosome Mapping, ErbB Receptors genetics, Hematopoietic System cytology, Hematopoietic System metabolism, Hematopoietic System ultrastructure, Mice, Mice, Mutant Strains, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-kit, Chromosomes ultrastructure, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins genetics
- Abstract
The mature cells in the haemopoietic system arise as the result of the extensive developmental and proliferative capacity of pluripotential stem cells. In order to understand the molecular basis for these developmental processes, it will be necessary to identify and characterize the cellular genes that control early steps in haemopoiesis. Mutations at the mouse W locus on chromosome 5 lead to pleiotropic developmental defects, including sterility, coat colour abnormalities, severe macrocytic anaemia and mast cell deficiency. The defects in all these lineages are cell autonomous and intrinsic, suggesting that the W locus encodes a gene product required directly for cellular differentiation. In an attempt to understand this classical mouse developmental mutation, we have demonstrated that the c-kit proto-oncogene, which encodes a transmembrane receptor tyrosine kinase, is very closely linked to W. Several further observations are consistent with the idea that W and c-kit are allelic: first, c-kit is expressed in those cell populations affected by W mutations; second, the expression of c-kit transcripts can be affected by mutations at the W locus; third, the tyrosine kinase activity associated with the protein encoded by c-kit is functionally impaired in mast cells derived from mutant W/Wv mice; and fourth, rearrangements within the c-kit gene have been reported in two W mutant alleles. These observations suggest that the dominant phenotype associated with W mutations results from loss-of-function alterations that affect the receptor tyrosine kinase encoded by c-kit. The demonstration that the W locus encodes a transmembrane growth factor receptor provides a molecular basis for understanding the intrinsic haemopoietic defect in W mutant mice and the role that this cellular proto-oncogene plays in haemopoiesis and other developmental processes.
- Published
- 1990
36. Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice.
- Author
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Nocka K, Majumder S, Chabot B, Ray P, Cervone M, Bernstein A, and Besmer P
- Subjects
- Alleles, Anemia, Macrocytic enzymology, Animals, Cell Movement, Embryonic and Fetal Development, Genes, Lethal, Hematopoiesis, Hematopoietic Stem Cells enzymology, Heterozygote, Liver embryology, Liver enzymology, Mast Cells enzymology, Melanocytes enzymology, Melanoma, Experimental, Mice, Mice, Mutant Strains embryology, Mice, Mutant Strains metabolism, Neural Crest pathology, Organ Specificity, Pigmentation Disorders embryology, Pigmentation Disorders enzymology, Protein-Tyrosine Kinases deficiency, Proto-Oncogene Proteins biosynthesis, Proto-Oncogene Proteins c-kit, RNA, Messenger analysis, Tumor Cells, Cultured enzymology, Anemia, Macrocytic genetics, Mice, Mutant Strains genetics, Pigmentation Disorders genetics, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins genetics, Proto-Oncogenes
- Abstract
The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.
- Published
- 1989
- Full Text
- View/download PDF
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