RESUMO
In this study, liver biopsy sections fixed in formalin and embedded in paraffin (FFPE) from patients with alcoholic hepatitis (AH) were used. The results showed that the expression of the SYK protein was up regulated by RNA-seq and real time PCR analyses in the alcoholic hepatitis patients compared to controls. The results were supported by using the IHC fluorescent antibody staining intensity morphometric quantitation. Morphometric quantification of fluorescent intensity measurement showed a two fold increase in SYK protein in the cytoplasm of the cells forming MDBs compared to surrounding normal hepatocytes. The expression of AKT1 was also analyzed. AKT1 is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. The AKT protein was also increased in hepatocyte balloon cells forming MDBs. This observation demonstrates the role of SYK and its subsequent effect on the internal signaling pathways such as PI3K/AKT as well as p70S6K, as a potential multifunctional target in protein quality control mechanisms of hepatocytes when ER stress is activated.
Assuntos
Citoplasma/metabolismo , Fígado/metabolismo , Corpos de Mallory/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , Transdução de Sinais , Quinase Syk/biossíntese , Biópsia , Citoplasma/genética , Hepatite Alcoólica/genética , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/patologia , Hepatócitos/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Fígado/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Quinase Syk/genéticaRESUMO
Several research strategies have been used to study the pathogenesis of alcoholic hepatitis (AH). These strategies have shown that various signaling pathways are the target of alcohol in liver cells. However, few have provided specific mechanisms associated with Mallory-Denk Bodies (MDBs) formed in Balloon cells in AH. The formation of MDBs in these hepatocytes is an indication that the mechanisms of protein quality control have failed. The MDB is the result of aggregation and accumulation of proteins in the cytoplasm of balloon degenerated liver cells. To understand the mechanisms that failed to degrade and remove proteins in the hepatocyte from patients suffering from alcoholic hepatitis, we investigated the pathways that showed significant up regulation in the AH liver biopsies compared to normal control livers (Liu et al., 2015). Analysis of genomic profiles of AH liver biopsies and control livers by RNA-seq revealed different pathways that were up regulated significantly. In this study, the focus was on Tec kinase signaling pathways and the genes that significantly interrupt this pathway. Quantitative PCR and immunofluorescence staining results, indicated that several genes and proteins are significantly over expressed in the livers of AH patients that affect the Tec kinase signaling to PI3K which leads to activation of Akt and its downstream effectors.
Assuntos
Biomarcadores/metabolismo , Hepatite Alcoólica/patologia , Hepatócitos/patologia , Fígado/patologia , Corpos de Mallory/patologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Hepatite Alcoólica/metabolismo , Hepatócitos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fígado/metabolismo , Corpos de Mallory/metabolismo , Proteínas Tirosina Quinases/genéticaRESUMO
There are many homeostatic mechanisms for coping with stress conditions in cells, including autophagy. In many studies autophagy, as an intracellular pathway which degrades misfolded and damaged protein, and Mallory-Denk Body (MDB) formation have been shown to be protective mechanisms against stress such as alcoholic hepatitis. Alcohol has a significant role in alteration of lipid homeostasis, sterol regulatory element-binding proteins (SREBPs) and peroxidase proliferator-activated receptors through AMP-activated protein kinase (AMPK)-dependent mechanism. AMPK is one of the kinases that regulate autophagy through the dephosphorylation of ATG1. Activation of ATG1 (ULK kinases family) activates ATG6. These two activated proteins relocate to the site of initial autophagosome and activate the other downstream components of autophagocytosis. Many other proteins regulate autophagocytosis at the gene level. CHOP (C/EBP homologous protein) is one of the most important parts of stress-inducible transcription that encodes a ubiquitous transcription factor. In this report we measure the upregulation of the gene that are involved in autophagocytosis in liver biopsies of alcoholic hepatitis and NASH. Electron microscopy was used to document the presence of autophagosomes in the liver cells. Expression of AMPK1, ATG1, ATG6 and CHOP in ASH were significantly (p value<0.05) upregulated in comparison to control. Electron microscopy findings of ASH confirmed the presence of autophagosomes, one of which contained a MDB, heretofore undescribed. Significant upregulations of AMPK-1, ATG-1, ATG-6, and CHOP, and uptrending of ATG-4, ATG-5, ATG-9, ATR, and ATM in ASH compared to normal control livers indicate active autophagocytosis in alcoholic hepatitis.
Assuntos
Autofagia , Hepatite Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Regulação para Cima , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Estudos de Casos e Controles , Hepatite Alcoólica/enzimologia , Humanos , Hepatopatia Gordurosa não Alcoólica/enzimologia , Fagossomos/metabolismo , Fagossomos/ultraestruturaRESUMO
Background: Economic and health care restraints strongly impact on drug prescription for chronic diseases. We aimed to identify potential factors for prescription behavior in chronic disease. Multiple sclerosis was chosen as a model disease due to its chronic character, incidence, and high socioeconomic impact. Methods: Germany was used as a model country as the health-care system is devoid of economic and drug availability restraints. German statutory health insurance data were analyzed retrospectively. The impact of number of university hospitals and neurologists as well as the gross domestic product (GDP) as potential factors on prescriptions of platform and high-efficacy disease-modifying therapies (DMTs) was analyzed. Results: Prescription of platform DMTs increased over time in almost all federal states with varying degree of increase. Univariate regression analysis showed that the prescription volume of platform DMTs positively correlated with the number of university hospitals and neurologists, as well as the GDP per federal state. Stepwise forward regression analysis including all potential factors indicated a statistically significant model for platform DMT (R2 = 0.55; 95%-CI [0.28, 0.82]; p=0.001) revealing GDP as the main contributor. This was confirmed in the independent analysis. Conclusion: This study illustrates that even without overt drug prescription inequity, access to medication is not evenly distributed and depends on economic strength and regional medical care density.
Assuntos
Esclerose Múltipla , Fatores Socioeconômicos , Humanos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/economia , Alemanha/epidemiologia , Estudos Retrospectivos , Acessibilidade aos Serviços de Saúde , Prescrições de Medicamentos/estatística & dados numéricos , Prescrições de Medicamentos/economia , Feminino , MasculinoRESUMO
Ocrelizumab is a B cell-depleting drug widely used in relapsing-remitting multiple sclerosis (RRMS) and primary-progressive MS. In RRMS, it is becoming increasingly apparent that accumulation of disability not only manifests as relapse-associated worsening (RAW) but also as progression independent of relapse activity (PIRA) throughout the disease course. This study's objective was to investigate the role of PIRA in RRMS patients treated with ocrelizumab. We performed a single-center, retrospective, cross-sectional study of clinical data acquired at a German tertiary multiple sclerosis referral center from 2018 to 2022. All patients with RRMS treated with ocrelizumab for at least six months and complete datasets were analyzed. Confirmed disability accumulation (CDA) was defined as a ≥ 12-week confirmed increase from the previous expanded disability status scale (EDSS) score of ≥ 1.0 if the previous EDSS was ≤ 5.5 or a ≥ 0.5-point increase if the previous EDSS was > 5.5. PIRA was defined as CDA without relapse since the last EDSS measurement and at least for the preceding 12 weeks. RAW was defined as CDA in an interval of EDSS measurements with ≥ 1 relapses. Cox proportional hazard models were used to analyze the probability of developing PIRA depending on various factors, including disease duration, previous disease-modifying treatments (DMTs), and optical coherence tomography-assessed retinal degeneration parameters. 97 patients were included in the analysis. Mean follow-up time was 29 months (range 6 to 51 months). 23.5% developed CDA under ocrelizumab therapy (n = 23). Of those, the majority developed PIRA (87.0% of CDA, n = 20) rather than RAW (13.0% of CDA, n = 3). An exploratory investigation using Cox proportional hazards ratios revealed two possible factors associated with an increased probability of developing PIRA: a shorter disease duration prior to ocrelizumab (p = 0.02) and a lower number of previous DMTs prior to ocrelizumab (p = 0.04). Our data show that in ocrelizumab-treated RRMS patients, the main driver of disability accumulation is PIRA rather than RAW. Furthermore, these real-world data show remarkable consistency with data from phase 3 randomized controlled trials of ocrelizumab in RRMS, which may increase confidence in translating results from tightly controlled RCTs into the real-world clinical setting.
Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Estudos Transversais , Estudos Retrospectivos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Doença CrônicaAssuntos
Fígado Gorduroso Alcoólico/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Retículo Endoplasmático/metabolismo , Fígado Gorduroso Alcoólico/fisiopatologia , Humanos , Fígado/citologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Streptozocin-induced diabetes in rats causes changes in urinary bladder function and increases the responsiveness of isolated bladder strip preparations to contractile agents and field stimulation. We monitored the role of extracellular glucose in the contractile responsiveness of bladder body strips from control, 2-month diabetic, and sucrose-drinking rats to the muscarinic agonist bethanechol. Consumption of sucrose and induction of diabetes caused increases in bladder mass compared with that of controls. In the presence of normal glucose levels (5.6 mmol/L), bladder strips from diabetic rats responded to bethanechol with significantly larger responses than those from control or sucrose-drinking rats. Removal of glucose from the bathing medium caused time-dependent decreases in contractile response of bladder strips from all groups; there were no differences in the percent decrease in response between the three groups. The presence of insulin (100 mU/mL) had no effects on contractile responsiveness or the rate of decline of response. Following return of glucose to the medium, there were progressive increases in contractile responsiveness in all groups, which returned to original contractile values within 60 minutes and were unaffected by insulin. Pyruvate (9.1 mmol/L) was able to substitute for glucose in maintaining the contractile responses. Increasing the glucose concentration of the medium to 30 mmol/L had no effects on contractile responses. Unstimulated bladder adenosine triphosphate (ATP) and creatine phosphate concentrations were similar in control, diabetic, and sucrose-drinking rats. In conclusion, changes in glucose utilization and high-energy phosphate levels cannot explain the increased contractile responsiveness of bladder body strips from diabetic rats to contractile agents.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Bexiga Urinária/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Betanecol , Compostos de Betanecol/farmacologia , Peso Corporal , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Experimental/fisiopatologia , Espaço Extracelular/metabolismo , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Tamanho do Órgão , Fosfocreatina/metabolismo , Piruvatos/farmacologia , Ácido Pirúvico , Ratos , Ratos Sprague-Dawley , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiopatologiaRESUMO
F wave latency has been shown to be a valuable method in evaluation of a variety of neurologic disorders. We measured F wave values in 50 healthy individuals in Shiraz. Maximum normal F wave latency for median nerve was 25.7 ms for women and 28.5 ms for men with stimulation at the wrist. It was 23 ms for women and 25 ms for men with stimulation at the elbow. Maximum normal F wave latency for ulnar nerve was 26.45 ms for women and 28.9 ms for men with stimulation at the wrist. It was 23.1 ms for women and 25.3 ms for men with stimulation at the elbow. Maximum normal difference in F wave latency between right and left upper extremities with stimulation at the wrist for total group was 2.2 ms for median nerve and 2.4 ms for ulnar nerve. Maximum normal difference in F wave latency between median and ulnar nerve in an extremity with stimulation at the wrist for total group was 2.7 ms. There was statistically significant difference in F wave latency between women and men.
Assuntos
Nervo Mediano/fisiologia , Tempo de Reação/fisiologia , Nervo Ulnar/fisiologia , Adolescente , Adulto , Cotovelo/inervação , Estimulação Elétrica , Feminino , Humanos , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Punho/inervaçãoRESUMO
This study examined contractile responses of the in vitro rabbit whole-bladder preparation to field stimulation, bethanechol and KCl in Tyrode's solution and minimum essential medium (MEM). We found frequency-dependent increases in intravesical pressure in bladders incubated in Tyrode's solution and MEM. However, bladders incubated in MEM consistently responded with greater increases in intravesical pressure compared to those incubated in Tyrode's solution. Similarly, there were frequency-dependent increases in the rate of pressure generation in bladders incubated in Tyrode's solution and MEM, and bladders incubated in MEM responded with greater rates of pressure generation than those incubated in Tyrode's solution at frequencies of 1, 2 and 4 Hz. In addition, there were significant increases in intravesical pressure generated in response to administration of 500 mmol/l bethanechol and 186.4 mmol/l KCl in bladders incubated in MEM compared to Tyrode's solution but no changes in the rate of pressure generation. The influence of L-methionine, one of the constituents of MEM, on the responses of whole bladders to nerve stimulation was also investigated. L-methionine at concentrations of 0.1 and 1.0 mmol/l had no effects on the increase in intravesical pressure or rate of pressure generation following nerve stimulation. It is speculated that the increases in contractile responsiveness of bladders incubated in MEM are related to the combination of amino acids, vitamins and other constituents of the cell culture medium.
Assuntos
Contração Muscular/fisiologia , Músculo Liso/fisiologia , Bexiga Urinária/fisiologia , Animais , Betanecol , Compostos de Betanecol/administração & dosagem , Estimulação Elétrica , Soluções Isotônicas , Masculino , Músculo Liso/inervação , Coelhos , Bexiga Urinária/inervaçãoRESUMO
While it is well established clinically that urinary tract infection in the presence of outflow obstruction may be associated with difficulty in eradicating bacteria, it is not clear whether this is secondary to the presence of residual urine volume or other local effects of the obstruction such as attenuation of the intrinsic antibacterial defense mechanisms of the mucosal surface. Experiments in our laboratory and others over the past several years have demonstrated that the primary antibacterial defense mechanism of the bladder is the antiadherence effect of the bladder surface mucin layer. Additional studies have shown that heparin can duplicate this antiadherence activity of bladder mucin. The present report demonstrates that one hour of overdistension or ischemia and one week of partial outlet obstruction cause a functional defect in the intrinsic antiadherence effect of the bladder mucosa as evidenced by increased bacterial adherence. This defect can be reversed by heparin exposure prior to bacterial challenge. These results indicate that partial outlet obstruction and its potential sequelae such as overdistension and, particularly, mucosal ischemia, have dramatic adverse effects on the intrinsic antiadherence defense mechanism of the bladder. These effects can be reversed by intravesical exposure to an exogenous anionic polyelectrolyte (heparin).