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We demonstrate a gradient refractive index (GRIN) microendoscope with an outer diameter of â¼1.2 mm and a length of â¼186 mm that can fit into a stereotactic surgical cannula. Two photon imaging at an excitation wavelength of 900â nm showed a field of view of â¼180 microns and a lateral and axial resolution of 0.86 microns and 9.6 microns respectively. The microendoscope was tested by imaging autofluorescence and second harmonic generation (SHG) in label-free human brain tissue. Furthermore, preliminary image analysis indicates that image classification models can predict if an image is from the subthalamic nucleus or the surrounding tissue using conventional, bench-top two-photon autofluorescence.
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Fragile X Syndrome (FXS) is a neurodevelopmental disorder and the most common monogenic cause of intellectual disability, autism spectrum disorders (ASDs) and anxiety disorders. Loss of fragile x mental retardation protein (FMRP) results in disruptions of synaptic development during a critical period (CP) of circuit formation in the basolateral amygdala (BLA). However, it is unknown how these alterations impact microcircuit development and function. Using a combination of electrophysiologic and behavioral approaches in both male (Fmr1-/y) and female (Fmr1-/-) mice, we demonstrate that principal neurons (PNs) in the Fmr1KO BLA exhibit hyperexcitability during a sensitive period in amygdala development. This hyperexcitability contributes to increased excitatory gain in fear-learning circuits. Further, synaptic plasticity is enhanced in the BLA of Fmr1KO mice. Behavioral correlation demonstrates that fear-learning emerges precociously in the Fmr1KO mouse. Early life THIP intervention ameliorates fear-learning in Fmr1KO mice. These results suggest that CP plasticity in the amygdala of the Fmr1KO mouse may be shifted to earlier developmental timepoints.SIGNIFICANCE STATEMENTIn these studies we identify early developmental alterations in principal neurons in the FXS BLA. We show that as early as P14, excitability and feed-forward excitation, and synaptic plasticity is enhanced in Fmr1KO lateral amygdala. This correlates with precocious emergence of fear-learning in the Fmr1KO mouse. Early life THIP intervention restores CP plasticity in WT mice and ameliorates fear-learning in the Fmr1KO mouse.
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Fragile X syndrome (FXS) is a neurodevelopmental disorder (NDD) characterized by intellectual disability, autism spectrum disorders (ASDs), and anxiety disorders. The disruption in the function of the FMR1 gene results in a range of alterations in cellular and synaptic function. Previous studies have identified dynamic alterations in inhibitory neurotransmission in early postnatal development in the amygdala of the mouse model of FXS. However, little is known about how these changes alter microcircuit development and plasticity in the lateral amygdala (LA). Using whole-cell patch clamp electrophysiology, we demonstrate that principal neurons (PNs) in the LA exhibit hyperexcitability with a concomitant increase in the synaptic strength of excitatory synapses in the BLA. Further, reduced feed-forward inhibition appears to enhance synaptic plasticity in the FXS amygdala. These results demonstrate that plasticity is enhanced in the amygdala of the juvenile Fmr1 knock-out (KO) mouse and that E/I imbalance may underpin anxiety disorders commonly seen in FXS and ASDs.
Assuntos
Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Tonsila do Cerebelo/metabolismo , Animais , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Camundongos , Camundongos Knockout , Sinapses/metabolismo , Transmissão SinápticaRESUMO
Fragile X Syndrome (FXS) is a neurodevelopmental disorder instigated by the absence of a key translation regulating protein, Fragile X Mental Retardation Protein (FMRP). The loss of FMRP in the CNS leads to abnormal synaptic development, disruption of critical periods of plasticity, and an overall deficiency in proper sensory circuit coding leading to hyperexcitable sensory networks. However, little is known about how this hyperexcitable environment affects inhibitory synaptic plasticity. Here, we show that in vivo layer 2/3 of the primary somatosensory cortex of the Fmr1 KO mouse exhibits basal hyperexcitability and an increase in neuronal firing rate suppression during whisker activation. This aligns with our in vitro data that indicate an increase in GABAergic spontaneous activity, a faulty mGluR-mediated inhibitory input and impaired inhibitory plasticity processes. Specifically, we find that mGluR activation sensitivity is overall diminished in the Fmr1 KO mouse leading to both a decreased spontaneous inhibitory postsynaptic input to principal cells and a disrupted form of inhibitory long-term depression (I-LTD). These data suggest an adaptive mechanism that acts to homeostatically counterbalance the cortical hyperexcitability observed in FXS.
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Síndrome do Cromossomo X Frágil/fisiopatologia , Homeostase/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Córtex Somatossensorial/fisiopatologia , Animais , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Potenciais Pós-Sinápticos Inibidores/fisiologia , Camundongos , Camundongos KnockoutRESUMO
AIM: To study whether DPD epitope-specific glutamate decarboxylase autoantibodies are found more frequently in children with milder forms of Type 1 diabetes. METHODS: We prospectively evaluated 75 children with new-onset autoimmune Type 1 diabetes, in whom we collected demographic, anthropometric and clinical data and measured islet autoantibodies. Glutamate decarboxylase 65 autoantibody-positive samples were analysed for epitope specificities using recombinant Fab against the DPD-defined epitope of glutamate decarboxylase 65. RESULTS: After adjustment for age, positive DPD epitope recognition was significantly associated with higher C-peptide levels at onset (P = 0.02, r2 =0.21, n = 35), and high DPD recognition in the highest quartile tended to be associated with HbA1c ≤ 53 mmol/mol (7%) at the last follow-up [mean (sd) follow-up 1.3 (0.4) years; P = 0.07; for the model, P = 0.044, n = 30)]. Age- and sex-adjusted BMI percentile was significantly correlated with recognition of the DPD-defined epitope (P < 0.03, r2 =0.14, n = 34), but this correlation was driven by the older age group (age ≥ 10 years; P = 0.016, r2 =0.27, n = 21) and was not significant in younger children (P = 0.93, n = 13). There were no independent associations with sex, race/ethnicity, diabetic ketoacidosis, HbA1c , HLA DR3-DQ2/DR4-DQ8 or autoantibody number. CONCLUSIONS: Our findings suggest that recognition of the DPD-defined glutamate decarboxylase 65 autoantibody epitope at Type 1 diabetes onset is directly associated with ß-cell function, BMI and age, which supports the hypothesis that immunological factors contribute to the clinical heterogeneity of Type 1 diabetes. Larger studies relating epitope-specific glutamate decarboxylase 65 autoantibody to clinical phenotype in children with Type 1 diabetes are warranted.
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Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/sangue , Glutamato Descarboxilase/imunologia , Adolescente , Especificidade de Anticorpos , Autoanticorpos/química , Peptídeo C/sangue , Proteínas de Transporte de Cátions/imunologia , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Feminino , Glutamato Descarboxilase/química , Humanos , Lactente , Masculino , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Transportador 8 de ZincoRESUMO
Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder of the esophagus that is compounded by genetic predisposition and hypersensitivity to environmental antigens. Using high-density oligonucleotide expression chips, a disease-specific esophageal transcript signature was identified and was shown to be largely reversible with therapy. In an effort to expand the molecular signature of EoE, we performed RNA sequencing on esophageal biopsies from healthy controls and patients with active EoE and identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated). When clustered by raw expression levels, an abundance of immune cell-specific transcripts are highly induced in EoE but expressed at low (or undetectable) levels in healthy controls. Moreover, 66% of the gene signature identified by RNA sequencing was previously unrecognized in the EoE transcript signature by microarray-based expression profiling and included several long non-coding RNAs (lncRNA), an emerging class of transcriptional regulators. The lncRNA BRAF-activated non-protein coding RNA (BANCR) was upregulated in EoE and induced in interleukin-13 (IL-13)-treated primary esophageal epithelial cells. Repression of BANCR significantly altered the expression of IL-13-induced proinflammatory genes. Together, these data comprise new potential biomarkers of EoE and demonstrate a novel role for lncRNAs in EoE and IL-13-associated responses.
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Esofagite Eosinofílica/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Linhagem Celular , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Interleucina-13/farmacologia , Interferência de RNA , RNA não Traduzido/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
AIMS: Diabetes mellitus results from an absolute or relative deficiency of insulin-producing pancreatic ß-cells. The turnover rate of adult human ß-cells remains unknown. We employed two techniques to examine adult human islet ß-cell turnover and longevity in vivo. METHODS: Subjects enrolled in National Institutes of Health clinical trials received thymidine analogs [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8 d to 4 yr prior to death. Archival autopsy samples from 10 patients (aged 17-74 yr) were employed to assess ß-cell turnover by scoring nuclear analog labeling within insulin-staining cells. Human adult ß-cell longevity was determined by estimating the cells' genomic DNA integration of atmospheric (14)C. DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15-yr-old donor, and purified ß-cell DNA was obtained from two donors (ages 48 and 80 yr). (14)C levels were then determined using accelerator mass spectrometry. Cellular "birth date" was determined by comparing the subject's DNA (14)C content relative to a well-established (14)C atmospheric prevalence curve. RESULTS: In the two subjects less than 20 yr of age, 1-2% of the ß-cell nuclei costained for BrdU/IdU. No ß-cell nuclei costained in the eight patients more than 30 yr old. Consistent with the BrdU/IdU turnover data, ß-cell DNA (14)C content indicated that the "birth date" of cells occurred within the subject's first 30 yr of life. CONCLUSIONS: Under typical circumstances, human ß-cells and their cellular precursors are established by young adulthood.
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Envelhecimento/fisiologia , Bromodesoxiuridina/farmacocinética , Proliferação de Células , Células Secretoras de Insulina/fisiologia , Datação Radiométrica , Adolescente , Adulto , Idoso , Envelhecimento/metabolismo , Feminino , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Datação Radiométrica/métodos , Coloração e Rotulagem/métodos , Timidina/análogos & derivados , Timidina/farmacocinética , Doadores de Tecidos , Adulto JovemRESUMO
Beta-cell regeneration represents a major goal of therapy for diabetes. Unravelling the origin of beta cells during pancreatic regeneration could help restore a functional beta-cell mass in diabetes patients. This scientific question has represented a longstanding interest still intensively investigated today. This review focuses on pioneering observations and subsequent theories made 100 years ago and describes how technical innovation helped resolve some, but not all, of the controversies generated by these early investigators. At the end of the 19th century, complete pancreatectomy demonstrated the crucial physiological role of the pancreas and its link with diabetes. Pancreatic injury models, including pancreatectomy and ductal ligation, allowed investigators to describe islet function and to assess the regenerative capacity of the pancreas. Three main theories were proposed to explain the origins of newly formed islets: (i) transdifferentiation of acinar cells into islets, (ii) islet neogenesis, a process reminiscent of islet formation during embryonic development, and (iii) replication of preexisting islet cells. Despite considerable technical innovation in the last 50 years, the origin of new adult beta cells remains highly controversial and the same three theories are still debated today.
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Diferenciação Celular/fisiologia , Ilhotas Pancreáticas/fisiologia , Modelos Biológicos , Pâncreas/fisiologia , Regeneração/fisiologia , Proliferação de Células , Transdiferenciação Celular/fisiologia , Humanos , Ilhotas Pancreáticas/citologia , Pâncreas/anatomia & histologia , Pâncreas/citologia , PancreatectomiaRESUMO
Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene is found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described but very few have been characterized structurally. Here we characterize 3 mutations in the URO-D gene found in patients with F-PCT, G318R, K297N, and D306Y. Expression of the D306Y mutation results in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins display reduced stability in vitro.
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Uroporfirinogênio Descarboxilase/metabolismo , Adulto , Idoso , Domínio Catalítico , Cristalografia por Raios X , Feminino , Genótipo , Heterozigoto , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Porfiria Cutânea Tardia/etiologia , Porfiria Cutânea Tardia/genética , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Uroporfirinogênio Descarboxilase/química , Uroporfirinogênio Descarboxilase/genéticaRESUMO
Most rodent models of porphyria cutanea tarda (PCT) share in common the administration of iron and agents that induce transcription of cytochrome P450s. Dissection of changes related to porphyrin accumulation required generation of a genetic model free from exogenous precipitants. Mice heterozygous for a null Urod mutation and homozygous for null Hfe alleles spontaneously develop major increases in hepatic and urinary porphyrins several months after weaning but the high % uroporphyrin signature of PCT is established earlier, before total hepatic and urinary porphyrins rise. Total porphyrin levels eventually plateau at higher levels in females than in males. Porphyrinogens were the dominant tetrapyrroles accumulating in hepatocytes. Hepatic Urod activity is markedly reduced but total hepatic heme content does not diminish. Microsomal heme, however, is reduced and in vitro metabolism of prototype substrates showed that some but not all cytochrome P450 activities are reduced. High hepatic levels of uroporphyrinogen are also associated with increased glutathione S-transferase activity and elevated mRNA of 2 transporters, Abcc1 and Abcc4. This murine model of familial PCT affords the opportunity to study changes in porphyrinogen and porphyrin accumulation and transport in the absence of exogenous factors that alter P450 activity and transmembrane transporters.
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Porfiria Cutânea Tardia/genética , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Feminino , Genótipo , Glutationa Transferase/metabolismo , Heme/análise , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Fígado/metabolismo , Estudos Longitudinais , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Porfiria Cutânea Tardia/patologia , Porfirinas/sangue , Porfirinas/urina , Uroporfirinogênio Descarboxilase/deficiência , Uroporfirinogênio Descarboxilase/genética , Uroporfirinogênio Descarboxilase/metabolismo , Uroporfirinogênios/metabolismoRESUMO
AIMS/HYPOTHESIS: We recently demonstrated that humans with hereditary haemochromatosis have decreased insulin secretory capacity with a compensatory increase in insulin sensitivity. We therefore determined how these measures change after correction of tissue iron overload. SUBJECTS AND METHODS: Five non-diabetic subjects who had been studied previously at the time of initial diagnosis by means of the OGTT and frequently sampled intravenous glucose tolerance tests (FSIVGTT) underwent phlebotomy to normalise their serum ferritin. After normalisation of ferritin they were studied again (33+/-4 months after the initial studies) by OGTT and FSIVGTT. RESULTS: Normalisation of tissue iron stores resulted in an average 1.8-fold increase in the integrated area under the insulin curve during OGTT (p<0.0001), but no significant change in the area under the glucose curve (10% decrease, p=0.32). After phlebotomy, there was a 2.2-fold increase in insulin secretory capacity as determined by FSIVGTT (acute insulin response to glucose [AIRg], p<0.02) but a concomitant 70% fall in insulin sensitivity (Si, p<0.05). The disposition index (AIRgxSi) was unchanged (5% increase, p=0.90). BMI and fasting glucose were unchanged. At the time of diagnosis of haemochromatosis, four of the subjects had IGT. After normalisation of ferritin, two achieved NGT and two remained with IGT, despite 2.5- and 3.7-fold increases in insulin secretory capacity. CONCLUSIONS/INTERPRETATION: Insulin secretory capacity improves after normalisation of iron stores in subjects with hereditary haemochromatosis. Glucose tolerance status improves incompletely because of decreased insulin sensitivity after phlebotomy. We conclude that tissue iron levels are an important determinant of insulin secretion and insulin action.
Assuntos
Hemocromatose/genética , Hemocromatose/terapia , Insulina/metabolismo , Sobrecarga de Ferro/terapia , Flebotomia , Glicemia/metabolismo , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Secreção de Insulina , Estudos RetrospectivosRESUMO
AIMS/HYPOTHESIS: The prevalence and mechanisms of diabetes in hereditary haemochromatosis are not known. We therefore measured glucose tolerance, insulin secretory capacity and insulin sensitivity in adults with haemochromatosis. SUBJECTS AND METHODS: Subjects recruited from referrals to a haemochromatosis clinic underwent OGTT and frequently sampled IVGTT. A chart review of former clinic patients was also performed. RESULTS: The prevalence of diabetes (23%) and IGT (30%) was increased in haemochromatosis compared with matched control subjects (0% diabetes and 14% IGT). Subjects with haemochromatosis and diabetes were overweight (14%) or obese (86%). The prevalence of diabetes, as determined by chart review of fasting glucose values, in subjects who had haemochromatosis and were in the 40-79 years age range was 26%. Overall, patients with haemochromatosis and control subjects had similar values for acute insulin response to glucose and insulin sensitivity. However, patients with haemochromatosis and IGT had a 68% decrease in acute insulin response to glucose (p<0.02) compared with those with NGT. They were not insulin-resistant, exhibiting instead a 62% increase in insulin sensitivity (NS). Haemochromatosis subjects with diabetes exhibited further declines in acute insulin response to glucose, insulin resistance, or both. CONCLUSIONS/INTERPRETATION: Diabetes and IGT are common in haemochromatosis, justifying screening for diabetes and therapeutic phlebotomy. The major abnormality associated with IGT is decreased insulin secretory capacity. Diabetes is usually associated with obesity and concomitant insulin resistance.
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Intolerância à Glucose/epidemiologia , Hemocromatose/epidemiologia , Insulina/metabolismo , Adulto , Idoso , Glicemia/análise , Complicações do Diabetes/sangue , Complicações do Diabetes/epidemiologia , Diabetes Mellitus/sangue , Diabetes Mellitus/epidemiologia , Feminino , Intolerância à Glucose/complicações , Hemocromatose/sangue , Hemocromatose/complicações , Homeostase , Humanos , Resistência à Insulina , Secreção de Insulina , Masculino , Pessoa de Meia-Idade , PrevalênciaRESUMO
Our aim was to examine the relationship between mouthrinse matrix metalloproteinases (MMPs) and whole albumin levels (AL) relative to oral mucositis (OM) in allogeneic stem cell transplant (alloSCT) patients. Mouthrinse vertebrate collagenase levels are positively correlated with connective tissue destruction (CTD) in periodontitis and may also be involved in CTD associated with OM. Increases in salivary AL have been noted prior to OM onset and may serve as a predictive tool for OM and as a positive control in this study. A total of 23 alloSCT patients were visited eight times over 4 weeks following the transplant. OM was scored via a previously validated examiner-based ordinal system. Mouthrinse samples were collected and analyzed for MMP-1, 8, 13 (members of the vertebrate collagenase group) and AL. No significant correlation was found for MMP levels relative to OM scores. AL were positively and significantly associated with OM scores (P<0.001). MMP levels may not be an important factor in OM development and severity; however, mouthrinse AL may serve as a more objective measure of OM development and severity.
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Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Metaloproteinases da Matriz/análise , Saliva/enzimologia , Albumina Sérica/análise , Índice de Gravidade de Doença , Estomatite/diagnóstico , Adulto , Colagenases/análise , Tecido Conjuntivo/patologia , Diagnóstico Bucal , Humanos , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 13 da Matriz , Metaloproteinase 8 da Matriz/análise , Pessoa de Meia-Idade , Boca/enzimologia , Boca/patologia , Mucosa Bucal , Estomatite/etiologia , Transplante HomólogoRESUMO
Transfusion therapy for inherited anemias and acquired refractory anemias both improves the quality of life and prolongs survival. A consequence of chronic transfusion therapy is secondary iron overload, which adversely affects the function of the heart, the liver and other organs. This session will review the use of iron chelating agents in the management of transfusion-induced secondary iron overload. In Section I Dr. John Porter describes techniques for the administration of deferoxamine that exploit the pharmacokinetic properties of the drug and minimize potential toxic side effects. The experience with chelation therapy in patients with thalassemia and sickle cell disease will be reviewed and guidelines will be suggested for chelation therapy of chronically transfused adults with refractory anemias. In Section II Dr. Nancy Olivieri examines the clinical consequences of transfusion-induced secondary iron overload and suggests criteria useful in determining the optimal timing of the initiation of chelation therapy. Finally, Dr. Olivieri discusses the clinical trials evaluating orally administered iron chelators.
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Quelantes de Ferro/uso terapêutico , Sobrecarga de Ferro/tratamento farmacológico , Anemia Falciforme/complicações , Ensaios Clínicos como Assunto , Humanos , Quelantes de Ferro/farmacocinética , Quelantes de Ferro/toxicidade , Sobrecarga de Ferro/etiologia , Guias de Prática Clínica como Assunto , Talassemia/complicações , Reação TransfusionalRESUMO
This paper examines the current state of the blood supply in the US and focuses on the potential for augmenting blood availability by attention to the iron status of donors. Increasing demands are being made upon the national blood supply as rates of blood donation are declining, in part because of the loss of blood donors as a result of enhanced screening and testing procedures. Iron-related means of expanding the blood supply include the use of blood from individuals undergoing therapeutic phlebotomy for hereditary hemochromatosis and enhancing the retention and commitment of women of childbearing age as donors by using iron supplementation to prevent iron deficiency. In Section I, Dr. Klein discuss the circumstances responsible for a decline in the population of eligible donors, including public attitudes toward donation, factors influencing the retention of donors by blood centers, and the effects of increased screening and testing to maintain the safety of the blood supply. In Section II, Drs. Kushner and Ajioka focus on the consequences of the decision by the US Food and Drug Administration (FDA) to develop recommendations to permit blood centers to collect blood from patients with hereditary hemochromatosis and to distribute this blood obtained without disease labeling if all other screening and testing procedures are passed. After summarizing the pathophysiology of hereditary hemochromatosis, the use by blood centers of blood obtained from heterozygotes and homozygotes for hereditary hemochromatosis is considered. In Section III, Dr. Brittenham reviews the use of low dose, short-term carbonyl iron supplementation for women donors of childbearing age. Replacing the iron lost at donation can help prevent iron deficiency in women of childbearing age and, by decreasing deferral, enhance the retention and commitment of women who give blood regularly. He emphasizes the use by blood centers of iron-related means to enhance recruitment and retention of blood donors.
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Bancos de Sangue/normas , Doadores de Sangue/provisão & distribuição , Anemia Ferropriva/tratamento farmacológico , Feminino , Hemocromatose/sangue , Hemocromatose/diagnóstico , Hemocromatose/tratamento farmacológico , Humanos , Ferro/administração & dosagem , Ferro/sangue , Masculino , Opinião Pública , Estados UnidosRESUMO
Functional consequences of 12 mutations-10 missense, 1 splicing defect, and 1 frameshift mutation-were characterized in the uroporphyrinogen decarboxylase (URO-D) gene found in Utah pedigrees with familial porphyria cutanea tarda (F-PCT). All but one mutation altered a restriction site in the URO-D gene, permitting identification of affected relatives using a combination of polymerase chain reaction and restriction enzyme digestion. In a bacterial expression system, 3 of the missense mutants were found in inclusion bodies, but 7 were expressed as soluble proteins. Enzymatic activity of soluble, recombinant mutant URO-D genes ranged from 29% to 94% of normal. URO-D mRNA levels in Epstein-Barr-virus transformed cells derived from patients were normal (with the exception of the frameshift mutation) even though protein levels were lower than normal, suggesting that missense mutations generally cause unstable URO-Ds in vivo. The crystal structures of 3 mutant URO-Ds were solved, and the structural consequences of the mutations were defined. All missense mutations reported here and by others were mapped to the crystal structure of URO-D, and structural effects were predicted. These studies define structural and functional consequences of URO-D mutations occurring in patients with F-PCT.
Assuntos
Mutação , Porfiria Cutânea Tardia/genética , Uroporfirinogênio Descarboxilase/genética , Linhagem Celular Transformada , Cristalização , Mutação da Fase de Leitura , Expressão Gênica , Herpesvirus Humano 4 , Humanos , Linfócitos/química , Modelos Moleculares , Estrutura Molecular , Mutação de Sentido Incorreto , Linhagem , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Uroporfirinogênio Descarboxilase/química , Uroporfirinogênio Descarboxilase/metabolismo , UtahRESUMO
OBJECTIVE: To report two cases of seizures following administration of levofoxacin and ciprofloxacin. CASE SUMMARY: A 75-year-old white woman admitted to the hospital was prescribed levofloxacin for ischemic toes. After receiving three doses of oral levofloxacin, the patient experienced a seizure. One month later, the patient was rechallenged with ciprofloxacin and again experienced a seizure. The patient was hypomagnesemic and had elevated serum creatinine at the time of both seizures, and was hyponatremic during the second seizure. A 74-year-old white woman admitted to the hospital was prescribed levofloxacin for bacterial pneumonia. After five doses, the patient experienced a seizure. The woman had no electrolyte imbalances at the time of the seizure and no history of a seizure disorder. DISCUSSION: Quinolone antibiotics vary in their ability to induce seizures, with trovafloxacin having the greatest potential and levofloxacin possibly having the least potential. Neither patient had a history of a previous seizure disorder. Electrolyte imbalances are common with previous reports of fluoroquinolone-induced seizures. CONCLUSIONS: Although levofloxacin monotherapy has not been implicated in inducing seizures, it appears to be the causative agent in the second case. In the first case, the quinolones may have been a necessary, but not sufficient, cause in a patient with electrolyte abnormalities. Risk factors for fluoroquinolone-induced seizures may Include seizure history, electrolyte imbalances,dose unadjusted for renal insufficiency, and concomitant treatment with agents that lower the seizure threshold.
Assuntos
Anti-Infecciosos/efeitos adversos , Ciprofloxacina/efeitos adversos , Levofloxacino , Ofloxacino/efeitos adversos , Convulsões/induzido quimicamente , Idoso , Feminino , Humanos , Magnésio/sangue , Sódio/sangueRESUMO
2-(4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy)propionic acid (XK469) is among the most highly and broadly active antitumor agents to have been evaluated in our laboratories and is currently scheduled to enter clinical trials in 2001. The mechanism or mechanisms of action of XK469 remain to be elaborated. Accordingly, an effort was initiated to establish a pharmacophore hypothesis to delineate the requirements of the active site, via a comprehensive program of synthesis of analogues of XK469 and evaluation of the effects of structural modification(s) on solid tumor activity. The strategy formulated chose to dissect the two-dimensional parent structure into three regions-I, ring A of quinoxaline; II, the hydroquinone connector linkage; and III, the lactic acid moiety-to determine the resultant in vitro and in vivo effects of chemical alterations in each region. Neither the A-ring unsubstituted nor the B-ring 3-chloro-regioisomer of XK469 showed antitumor activity. The modulating antitumor effect(s) of substituents of differing electronegativities, located at the several sites comprising the A-ring of region I, were next ascertained. Thus, a halogen substituent, located at the 7-position of a 2-(4-[(2-quinoxalinyl)oxy]phenoxy)propionic acid, generated the most highly and broadly active antitumor agents. A methyl, methoxy, or an azido substituent at this site generated a much less active structure, whereas 5-, 6-, 8-chloro-, 6-, 7-nitro, and 7-amino derivatives all proved to be essentially inactive. When the connector linkage (region II) of 1 was changed from that of a hydroquinone to either a resorcinol or a catechol derivative, all antitumor activity was lost. Of the carboxylic acid derivatives of XK469 (region III), i.e., CONH2, CONHCH3, CON(CH3)2, CONHOH, CONHNH2, CN, or CN4H (tetrazole), only the monomethyl- and N,N-dimethylamides proved to be active.
Assuntos
Antineoplásicos/síntese química , Quinoxalinas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Quinoxalinas/química , Quinoxalinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase (URO-D) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene (HFE) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D. URO-D(+/-) mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D(+/-) mice were injected with iron-dextran and given drinking water containing delta-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption (HFE(-/-)) to URO-D(+/-) mice, generating mice with the URO-D(+/-)/HFE(-/-) genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D(+/-) mice. The URO-D(+/-) mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.
Assuntos
Modelos Animais de Doenças , Hemocromatose/genética , Porfiria Cutânea Tardia/genética , Uroporfirinogênio Descarboxilase/genética , Ácido Aminolevulínico/farmacologia , Animais , Clonagem Molecular , Coproporfirinogênios/química , Coproporfirinogênios/metabolismo , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Marcação de Genes , Genótipo , Humanos , Ferro/análise , Complexo Ferro-Dextran/administração & dosagem , Complexo Ferro-Dextran/farmacologia , Fígado/química , Fígado/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Porfiria Cutânea Tardia/induzido quimicamente , Porfiria Cutânea Tardia/enzimologia , Porfiria Cutânea Tardia/metabolismo , Porfirinas/análise , Porfirinas/urina , Células-Tronco/metabolismo , Uroporfirinogênio Descarboxilase/análise , Uroporfirinogênio Descarboxilase/antagonistas & inibidores , Uroporfirinogênio Descarboxilase/metabolismo , Uroporfirinogênios/química , Uroporfirinogênios/metabolismoRESUMO
Biosynthesis of heme is important for both prokaryotes and eukaryotes. The enzymes for this multistep process are distributed between the cytosol and mitochondria in eukaryotes. In humans there are inherited and acquired disorders characterized by over synthesis of one or more enzymes or absence of an enzyme. This overview discusses each enzyme in the heme biosynthesis pathway.