Brigatinib, a newly discovered AXL inhibitor, suppresses AXL-mediated acquired resistance to osimertinib in EGFR-mutated non-small cell lung cancer.

In addition to the classical resistance mechanisms, receptor tyrosine-protein kinase AXL is a main mechanism of resistance to third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) osimertinib in EGFR-mutated non-small cell lung cancer (NSCLC). Developing an effective AXL inhibitor is important to sensitize osimertinib in clinical application. In this study we assessed the efficacy of brigatinib, a second-generation of anaplastic lymphoma kinase (ALK)-TKI, as a novel AXL inhibitor, in overcoming acquired resistance to osimertinib induced by AXL activation. We established an AXL-overexpression NSCLC cell line and conducted high-throughput screening of a small molecule chemical library containing 510 anti-tumor drugs. We found that brigatinib potently inhibited AXL expression, and that brigatinib (0.5 µM) significantly enhanced the anti-tumor efficacy of osimertinib (1 µM) in AXL-mediated osimertinib-resistant NSCLC cell lines in vitro. We demonstrated that brigatinib had a potential ability to bind AXL kinase protein and further inhibit its downstream pathways in NSCLC cell lines. Furthermore, we revealed that brigatinib might decrease AXL expression through increasing K48-linked ubiquitination of AXL and promoting AXL degradation in HCC827OR cells and PC-9OR cells. In AXL-high expression osimertinib-resistant PC-9OR and HCC827OR cells derived xenograft mouse models, administration of osimertinib (10 mg·kg-1·d-1) alone for 3 weeks had no effect, and administration of brigatinib (25 mg·kg-1·d-1) alone caused a minor inhibition on the tumor growth; whereas combination of osimertinib and brigatinib caused marked tumor shrinkages. We concluded that brigatinib may be a promising clinical strategy for enhancing osimertinib efficacy in AXL-mediated osimertinib-resistant NSCLC patients.

Heterogeneity of immune control in chronic hepatitis B virus infection: Clinical implications on immunity with interferon-α treatment and retreatment.

Hepatitis B virus (HBV) infection is a global public health issue. Interferon-α (IFN-α) treatment has been used to treat hepatitis B for over 20 years, but fewer than 5% of Asians receiving IFN-α treatment achieve functional cure. Thus, IFN-α retreatment has been introduced to enhance antiviral function. In recent years, immune-related studies have found that the complex interactions between immune cells and cytokines could modulate immune response networks, in-cluding both innate and adaptive immunity, triggering immune responses that control HBV replication. However, heterogeneity of the immune system to control HBV infection, particularly HBV-specific CD8+ T cell heterogeneity, has consequ-ential effects on T cell-based immunotherapy for treating HBV infection. Altogether, the host's genetic variants, negative-feedback regulators and HBV components affecting the immune system's ability to control HBV. In this study, we reviewed the literature on potential immune mechanisms affecting the immune control of HBV and the clinical effects of IFN-α treatment and retreatment.

Role of transcribed ultraconserved regions in gastric cancer and therapeutic perspectives.

Gastric cancer (GC) is the fourth leading cause of cancer-related death. The occurrence and development of GC is a complex process involving multiple biological mechanisms. Although traditional regulation modulates molecular functions related to the occurrence and development of GC, the comprehensive mechanisms remain unclear. Ultraconserved region (UCR) refers to a genome sequence that is completely conserved in the homologous regions of the human, rat and mouse genomes, with 100% identity, without any insertions or deletions, and often located in fragile sites and tumour-related genes. The transcribed UCR (T-UCR) is transcribed from the UCR and is a new type of long noncoding RNA. Recent studies have found that the expression level of T-UCRs changes during the occurrence and development of GC, revealing a new mechanism underlying GC. Therefore, this article aims to review the relevant research on T-UCRs in GC, as well as the function of T-UCRs and their regulatory role in the occurrence and development of GC, to provide new strategies for GC diagnosis and treatment.

Diagnostic possibility of the combination of exhaled nitric oxide and blood eosinophil count for eosinophilic asthma.

BACKGROUND: Tests to identify reversible airflow limitation are important in asthma diagnosis, but they are time-consuming and it may be difficult for patients to cooperate. We aimed to evaluate whether the combination of fractional exhaled nitric oxide (FeNO) and blood eosinophil (B-Eos) can be used to distinguish some asthma patients who could avoid objective tests. METHODS: We conducted a retrospective cohort study on 7463 suspected asthma cases between January 2014 and December 2019 in Chongqing, China, and identified 2349 patients with complete FeNO, B-Eos count, and spirometry data. Asthma was diagnosed by clinicians by the criteria of recurrent respiratory symptoms and a positive bronchial-provocation or bronchodilation test (BPT, BPD). We evaluated the diagnostic accuracy of FeNO or B-Eos alone or both in combination for asthma using receiver operating characteristic (ROC) curve analysis. RESULTS: In this study, 824 patients were diagnosed with asthma. When FeNO and B-Eos counts were used in combination, the area under the ROC curve (AUC) for diagnosing asthma increased slightly (0.768 vs. 0.745 [FeNO] or 0.728 [B-Eos]; both P < 0.001). The odds ratio for having asthma increased progressively with a gradual increase in FeNO or B-Eos count (both P < 0.001; assessed using the Cochran-Armitage trend test). Further analysis of in-series combinations of different threshold values for these biomarkers indicated that moderately elevated biomarker levels (FeNO > 40 ppb and B-Eos > 300 cells/µl) support a diagnosis of asthma because diagnostic specificity was > 95% and the positive likelihood ratio (PLR) was > 10. This conclusion was verified when selecting the 2017-2019 data as the internal validation dataset. CONCLUSION: FeNO or B-Eos count alone is insufficient to accurately diagnose asthma. Patients with moderately elevated biomarkers (FeNO > 40 ppb and B-Eos > 300 cells/µl) could be diagnosed with asthma and avoid objective tests when such tests are not feasible.

New therapeutic options for persistent low-level viremia in patients with chronic hepatitis B virus infection: Increase of entecavir dosage.

Chronic hepatitis B virus (HBV) infection (CHB) is a public health concern worldwide. Current therapies utilizing nucleos(t)ide analogs (NA) have not resulted in a complete cure for CHB. Furthermore, patients on long-term NA treatment often develop low-level viremia (LLV). Persistent LLV, in addition to causing the progression of liver disease or hepatocellular carcinoma, may shed light on the current plight of NA therapy. Here, we review the literature on LLV, NA treatment, and various doses of entecavir to find a strategy for improving the efficacy of this antiviral agent. For LLV patients, three therapeutic options are available, switching to another antiviral monotherapy, interferon-α switching therapy, and continuing monotherapy. In real-world clinical practice, entecavir overdose has been used in antiviral therapy for CHB patients with NA refractory and persistent LLV, which encouraged us to conduct further in-depth literature survey on dosage and duration related entecavir studies. The studies of pharmacodynamics and pharmacokinetics show that entecavir has the maximal selected index for safety, and has great potential in inhibiting HBV replication, in all of the NAs. In the particular section of the drug approval package published by the United States Food and Drug Administration, entecavir doses 2.5-20 mg/d do not increase adverse events, and entecavir doses higher than 1.0 mg/d might improve the antiviral efficacy. The literature survey led us to two suggestions: (1) Increasing entecavir dose to 1.0 mg/d for the treatment of NA naïve patients with HBV DNA >2 × 106 IU/mL is feasible and would provide better prognosis; and (2) Further research is needed to assess the long-term toxic effects of higher entecavir doses (2.5 and 5.0 mg/d), which may prove beneficial in treating patients with prior NA treatment, partial virological response, or LLV state.

TWIST1 transcriptionally regulates glycolytic genes to promote the Warburg metabolism in pancreatic cancer.

Reprogrammed glucose metabolism is essential for tumor initiation and development, especially for pancreatic ductal adenocarcinoma (PDAC). Most cancer cells rely on aerobic glycolysis, a phenomenon termed "the Warburg effect", to support uncontrolled proliferation and evade apoptosis. However, the direct regulators of the Warburg effect remain areas of active investigation. In this study, we found that the highly conserved transcription factor, TWIST1, is a crucial regulator of aerobic glycolysis in PDAC. Genetic silencing of TWIST1 significantly inhibited the glycolytic phenotypes of PDAC cells as revealed by reduced glucose uptake, lactate production, and extracellular acidification rate, which can be restored by re-expression of siRNA-resistant TWIST1. Moreover, tamoxifen-inducible expression of TWIST1 promoted the Warburg metabolism of PDAC cells. Mechanistically, by luciferase reporter assay and chromatin immunoprecipitation experiment, we showed that TWIST1 can directly increase the expression of several glycolytic genes, including SLC2A1, HK2, ENO1, and PKM2. Of note, the transcriptional regulation by TWIST1 was not dependent on HIF1α or c-Myc. In The Cancer Genome Atlas and Gene Expression Omnibus accession GSE15471, we confirmed that TWIST1 was closely associated with the glycolysis pathway. Collectively, our findings indicate that TWIST1 is likely to act as important regulator of the Warburg effect in PDAC.

Efficacy of interferon for chronic hepatitis B in patients with nucleoside and nucleotide combination therapy failure.

BACKGROUND AND AIM: In China, inappropriate therapies with nucleos(t)ide analogues (NA) have induced hepatitis B virus resistance, combination therapy with nucleoside and nucleotide (ComTNsNt) failure, or multi-drug resistant mutations. However, the efficacy of combination therapy with entecavir plus tenofovir for ComTNsNt failure is limited. In the current study, the regimens of interferon-α (IFN-α) therapy, switching from NAs to IFN-α, and subsequent re-treatment with IFN-α were applied to treat ComTNsNt failure. We further evaluated the efficacy of this therapy. METHODS: Eleven patients with ComTNsNt failure were enrolled in this study. Nine subjects (9/11) received IFN-α switching therapy. Combination therapy with IFN-α and ComTNsNt was administered in the first 4 weeks. Then, ComTNsNt was discontinued at the end of Week 4, and IFN-α monotherapy was continued for 6 months. Two (2/11) patients discontinued ComTNsNt without receiving IFN-α treatment. All 11 patients received the first re-treatment of IFN-α when they experienced hepatitis relapses after the withdrawal of IFN-α or ComTNsNt. Six (6/11) patients received a second re-treatment of IFN-α. Follow up was conducted after IFN-α therapy in all 11 patients. RESULTS: Two patients (2/9) receiving IFN-α switching therapy experienced alanine aminotransferase (ALT) flare. In contrast, the two patients without IFN-α switching therapy experienced ALT flare. Multiple re-treatments with IFN-α resulted in a sustained response. CONCLUSIONS: Interferon-α switching therapy and IFN-α re-treatment might be applied for treatment of ComTNsNt failure. IFN-α switching therapy resulted in safe ComTNsNt cessation, and IFN-α re-treatment induced a sustained response of IFN-α in all patients. This IFN-α treatment is an optional treatment for ComTNsNt failure.

Efficacy and safety of tenofovir in a kidney transplant patient with chronic hepatitis B and nucleos(t)ide multidrug resistance: a case report.

INTRODUCTION: Five nucleos(t)ide analogs are used to treat chronic hepatitis B. Ideal nucleos(t)ide analog therapy in chronic hepatitis B patients with kidney transplantation must ensure virological suppression and minimize renal injury. However, resistance to nucleos(t)ide analogs frequently results in virological breakthrough, hepatitis flare, and complicated deterioration of the transplanted kidney. Inappropriate rescue therapy for drug resistance may subsequently cause hepatitis B virus multidrug resistance. Currently, tenofovir is used to treat chronic hepatitis B patients with kidney transplantation. In the field, we first reported combination therapy with tenofovir plus entecavir in a kidney transplant chronic hepatitis B patient with nucleos(t)ide analog multidrug resistance. CASE PRESENTATION: A 50-year-old Chinese man with chronic hepatitis B and kidney transplantation received nucleos(t)ide analog therapy with sequential monotherapy and combination therapy. Virological parameters, hepatic enzymology and renal function were monitored. Drug-resistance mutations were detected by sequence analysis. Our patient received sequential nucleos(t)ide analog monotherapy and inappropriate combination therapy during 132 months, which caused multidrug resistance and renal functional injury. Entecavir plus adefovir was administered in month 106, resulting in decreased hepatitis B virus load, normal hepatic function, and stabilized creatinine clearance. As a result of rebounded viral load and significantly declining creatinine clearance, tenofovir plus entecavir was administered in month 133. After eight weeks, undetectable hepatitis B virus DNA, normal hepatic function and improved creatinine clearance were present. Compared with combination therapy with adefovir plus entecavir, tenofovir plus entecavir showed a potent antiviral effect for multidrug resistance and minimized renal injury. CONCLUSIONS: In chronic hepatitis B patients with kidney transplantation, sequential monotherapy with antiviral agents with low barriers to resistance should be avoided, and initial therapy with entecavir is a better option. Combination therapy with tenofovir plus entecavir in this setting with multidrug resistance is safe and effective.

Effects of costimulation on intrahepatic immunopathogenesis in patients with chronic HBV infection.

OBJECTIVE: Chronic HBV infection can lead to "immune tolerance" in asymptomatic carriers (ACs), "immune injury" in active chronic hepatitis (ACH) patients or "immune abnormality" in cirrhosis (Cir) and hepatocellular carcinoma (HCC) patients. Previous investigations reported that chronic hepatitis presented abnormal expression of costimulatory molecules. We investigated the costimulation profile in the liver of ACs and patients with ACH, Cir and HCC. METHODS: Patients with ACH, Cir and HCC, ACs and normal controls were recruited into the present study. The costimulation profiles and cytokines in the liver of patients were investigated by Western blotting, immunohistochemistry and real-time quantitative PCR. Correlations between serum alanime aminotransferase (ALT) levels, necroinflammation scores, cytokines and costimulatory proteins were assessed. RESULTS: The ACs presented decreased inflammatory and increased inhibitory costimulation, which was negatively correlated with inflammatory costimulatory proteins and ALT, whereas the ACH patients exhibited increased inflammatory costimulation and decreased inhibitory costimulation, which was correlated with increased ALT. The Cir patients showed both increased inhibitory and inflammatory costimulation. The HCC patients exhibited both decreased inhibitory and inflammatory costimulation. CONCLUSION: Costimulation participates in intrahepatic immune responses, and plays important roles in immune tolerance, immune injury and immune abnormalities in patients with chronic HBV infection.

Effects of resuscitation with crystalloid fluids on cardiac function in patients with severe sepsis.

BACKGROUND: The use of hypertonic crystalloid solutions, including sodium chloride and bicarbonate, for treating severe sepsis has been much debated in previous investigations. We have investigated the effects of three crystalloid solutions on fluid resuscitation in severe sepsis patients with hypotension. METHODS: Ninety-four severe sepsis patients with hypotension were randomly assigned to three groups. The patients received the following injections within 15 min at initial treatment: Ns group (n = 32), 5 ml/kg normal saline; Hs group (n = 30), with 5 ml/kg 3.5% sodium chloride; and Sb group (n = 32), 5 ml/kg 5% sodium bicarbonate. Cardiac output (CO), systolic blood pressure, mean arterial pressure (MAP), body temperature, heart rate, respiratory rate and blood gases were measured. RESULTS: There were no differences among the three groups in CO, MAP, heart rate or respiratory rate during the 120 min trial or the 8 hour follow-up, and no significant differences in observed mortality rate after 28 days. However, improvement of MAP and CO started earlier in the Sb group than in the Ns and Hs groups. Sodium bicarbonate increased the base excess but did not alter blood pH, lactic acid or [HCO3]- values; and neither 3.5% hypertonic saline nor 5% sodium bicarbonate altered the Na+, K+, Ca2+ or Cl- levels. CONCLUSION: All three crystalloid solutions may be used for initial volume loading in severe sepsis, and sodium bicarbonate confers a limited benefit on humans with severe sepsis. TRIAL REGISTRATION: ISRCTN36748319.

Early-phase endotoxic shock-induced myocardial injury increases iNOS and selectin expression in macaque primate.

BACKGROUND: Our previous study has established a macaque model with early-phase endotoxic shock. The present study further investigated myocardial and blood vessel injury in Macaques by examining the subsequent expression of ACP, selectins, iNOS, and cTnI in response to LPS treatment. METHODS: In an experiment with anaesthetised, instrumental macaques, eleven animals were randomised into: an En group (n=6), receiving a dose of 2.8 mg kg(-1) lipopolysaccharides (LPS) by i.v.; and a Co group (n=5), injected with normal saline of 1 ml kg(-1). Cytochemistry of acid phosphatase (ACPase) in heart was performed by electron microscope at 120 min following endotoxin injection. Three immunochemical stains, namely, L-selectin, P-selectin and iNOS protein in heart, were studied. In addition, cardiac troponin I (cTnI), L-selectin and P-selectin in plasma were detected. RESULTS: In the early phase of endotoxic shock, LPS caused myocardial lysosome damage. The data of immunochemical staining showed the thrombus formation in vessels and the increase of iNOS, L-Selectin and P-Selectin expression in heart, but LPS challenge did not change L-selectin, P-selectin and cTnI in plasma. CONCLUSION: The increase of iNOS, L-selectin and P-selectin protein expression following endotoxin administration may have caused vessel injury and myocardial damage in macaques.

[Effects of nitric oxide synthases on kidney in early phase of endotoxic shock in macaque].

OBJECTIVE: To study the pathogenesis of kidney injury in early phase of endotoxic shock. METHODS: Eleven macaques were anesthetized and randomly divided into two groups: an experimental group (n=6), receiving a dose of 2.8 mg/kg lipopolysaccharides (LPS) by intravenous injection, and a control group (n=5), injected with normal saline of 1 ml/kg. The animals were sacrificed 120 minutes following endotoxin injection. With immunohistochemical technique endothelium nitric oxide synthases (eNOS) and inducible nitric oxide synthases (iNOS) protein of the renal tissue were determined. The ultrastructure was studied with electron microscope. Acid phosphatase (ACP) was also assayed. RESULTS: One hundred and twenty minutes after LPS challenge, damages to the glomerulus and renal tubules were found, and lysosomes were increased in the renal cells. iNOS was positively expressed in vascular endothelium of glomerulus and epithelium of renal tubules in the experimental group, but not in the control group. eNOS was positively expressed in vascular endothelium of glomerulus; and peri-vascular region in both groups. CONCLUSION: The present findings indicate that NO contributed to the pathogenesis of renal injury in the early phase of endotoxic shock in primates.

Effect of fluid resuscitation on ryanodine receptor in macaques with endotoxic shock.

OBJECTIVE: Our recent study demonstrated that sodium bicarbonate improved cardiac function in macaque models with early-phase endotoxic shock. In the present study, we investigated further the ryanodine receptor/calcium release-channel (RyR) and calcium pump after fluid resuscitation of macaques with early-phase endotoxic shock. METHODS: Twenty-four anaesthetised macaques were assigned to four groups. Nineteen animals were given an intravenous dose of 2.8 mgkg(-1) lipopolysaccharide (LPS). Sixty minutes after the LPS challenge, the animals were given (i) 5 mLkg(-1) normal saline (Ns group, n = 6), (ii) 5 mLkg(-1) of 5% sodium bicarbonate (Sb group, n = 6) or (iii) 5 mLkg(-1) of 3.5% hypertonic sodium chloride (Hs group, n = 7). The control group (Co group, n = 5) received 1 mLkg(-1) normal saline and then with 5 mLkg(-1) normal saline 60 min later. RESULTS: Endotoxin produced a reduction of the density of RyR but did not alter the affinity of RyR. Compared with normal saline, sodium bicarbonate or hypertonic saline induced a restoration of density of RyR but did not influence the affinity of RyR and the calcium pump. CONCLUSION: Up-regulation of RyR performance in myocardium following administration of sodium bicarbonate contributes to the improvement of cardiac function in macaques in the early phase of endotoxic shock.

[Clinical characteristics and ultrastructural features of livers in children with Wilson disease manifested mainly as hepatic injuries].

OBJECTIVES: To study the feasibility and possibility to diagnose Wilson disease with electronmicroscopical examination of liver biopsies. METHODS: Clinical analysis, histological observation and ultrastructural examination were performed on 15 children with Wilson disease. RESULTS: All 15 subjects had symptoms of hepatic disorders, such as jaundice. Morphological signs of hepatocyte injury in three phase, namely steatosis, mitochondrion changes and cholestasis in bile canaliculi of the early phase, nucleus injury, dilation of endoplasmic reticulum, increase of lysosomes and appearance of residual bodies of the second phase, and massive autophagy and cirrhosis of the late phase were shown. A few inflammatory cells in the liver specimens were observed. Accumulation of copper in lysosomes and autophagosomes was found by energy-dispersion X-ray. CONCLUSION: The diagnostic signs for Wilson disease are autophagosomes in hepatocytes, cirrhosis accompanied with a few of inflammatory cells. A certain diagnosis of the disease depends on the finding of copper accumulation in hepatocytes.

Endotoxic shock model with fluid resuscitation in Macaca mulatta.

These studies established a macaque model of early-phase endotoxic shock, and investigated the resuscitation effects of three different solutions. Twenty-four macaques were assigned to four groups. Nineteen animals were given an intravenous dose of 2.8 mg/kg lipopolysaccharide (LPS). At 60 min after LPS challenge, the animals were given (i) 5 mL/kg normal saline (Ns group, n=6), (ii) 5% of 5 mL/kg sodium bicarbonate (Sb group, n=6), (iii) hypertonic 3.5% sodium chloride of 5 mL/kg (Hs group, n=7). The control group (Co group, n=5) was first injected with 1 mL/kg Ns and with 5 mL/kg Ns 60 min later. Haemodynamic parameters and blood gases were measured during the experiment, and myocardial morphology was examined on termination of the experiment. Administration of LPS caused hypotension and decreases of the left ventricular work index (LVWI). In the Sb group, mean arterial pressure, cardiac index, systemic vascular resistance index, LVWI and right ventricular work index were significantly higher than those of the Ns group. Pathological changes of myocardium were identified in all of the LPS groups. The studies suggest that macaques are suitable models for studying endotoxic shock and potential fluid therapies.

[Changes in selectin in early stage of lung injury induced by endotoxic shock in macaque].

OBJECTIVE: To investigate the change in selectin and its effect on lung injury induced by endotoxic [lipopolysaccharide (LPS)] shock in macaque. METHODS: Eleven macaques were randomly divided into two groups: control group (n=5) and LPS group (n=6). The animals of the control group received injection of 1 ml/kg normal saline, and the animals of the LPS group received a dose of 2.8 mg/kg LPS intravenously. The plasma contents of P-selectin and L-selectin were assayed before LPS challenge, 60 and 120 minutes after LPS challenge. Ultrastructure of lung tissue and immunohistochemical assay of P-selectin and L-selectin in the lung were observed. RESULTS: Administration of LPS did not changed P-selectin level in plasma, but decreased the L-selectin level at 120 minutes after LPS challenge in both groups (all P<0.05). By immunohistochemical staining, P-selectin and L-selectin were identified on endothelial cells of alveolar wall of LPS animals, whereas no positive staining of P-selectin and L-selectin was showed in control animals. Damages to alveolar type I and II cells, slight transudation of red blood corpuscles, and damage to the basement membrane were observed with electron microscopy in the endotoxin challenged macaques. No pathological changes were observed in the control group. CONCLUSION: Administration of LPS induces expression of P-selectin and L-selectin in alveolar wall and causes alveolar damages in early-phase of endotoxic shock. In the meantime, the L-selectin and P-selectin in plasma do not change. The selectins play an important role in the pathogenesis of lung injury in the early-phase of endotoxic shock.

Changes of inflammation-associated cytokine expressions during early phase of experimental endotoxic shock in macaques.

AIM: To study changes of inflammation-associated cytokine expressions during early phase of endotoxic shock in macaque. METHODS: Experiments were performed in Macaque mulatta treated with LPS 2.8 mg/kg in shock model group or with normal saline in control group. Blood samples were collected before, or 60 min, or 120 min after LPS injection, respectively. Liver and spleen tissues were obtained at 120 min after LPS injection. The plasma levels of TNF-alpha, IL-1beta, IL-10 and IL-12P40 were determined by double-antibody sandwich ELISA with antibodies against human cytokines. The mRNA levels of TNF-alpha, IL-1beta, and IL-18 in peripheral blood mononuclear cells (PBMCs), liver and spleen were examined by real-time fluorescence semi-quantitative RT-PCR with the primers based on human genes. RESULTS: Mean systemic arterial pressure (MAP), systemic vascular resistance index (SVRI) and left ventricular work index (LVWI) of macaques were significant declined in shock model group on average 60 min after LPS injection. The plasma levels of TNF-alpha and IL-10 were significantly increased 60 min after LPS injection and then decreased. The plasma levels of IL-1beta and IL-12P40 were significantly increased at 120 min after LPS injection. The mRNA levels of TNF-alpha and IL-1beta were significantly increased 60 min after LPS stimulation in PBMCs and 120 min after LPS stimulation in livers. The mRNA level of IL-18 was significantly increased 120 min after LPS stimulation in PBMCs and livers. But in spleen, only TNF-alpha mRNA level in LPS group was significantly higher 120 min after LPS stimulation, compared with that in control group. CONCLUSION: An endotoxic shock model of Macaque mulatta was successfully established. Both antibodies for ELISA and PCR primers based on human cytokine assays were successfully applied to detect macaque cytokines. In the model, inflammatory cytokines, such as TNF-alpha, IL-1beta, IL-12 and IL-18 as well as anti-inflammation cytokine IL-10, were released at very early phase of endotoxic shock within 120 min after LPS injection. PBMCs and liver cells might be the important sources of these cytokines.

[Changes of blood-cerebrospinal fluid barrier in rabbits with diabetic ketoacidosis].

OBJECTIVE: To explore the mechanism of the changes of blood-cerebrospinal fluid barrier in rabbits with diabetic ketoacidosis (DKA). METHODS: The New Zealand rabbits were injected with 150 mg/kg streptozotion and alloxan monohydrate each (model group, n=6) intravenously, or equal volume of normal saline (control group, n=6). After 72 hours, blood sugar and uric ketone were detected. All of animals were injected with Evans blue (EB). After 6 hours, arterial blood gases were measured and animals were killed. Absorbency of EB of brain tissue was detected. All brains of animals were examined with light and electron microscopy. Marker of blood-cerebrospinal fluid barrier, cytochemical stains of alkaline phosphatase (ALPase) was operated by ultrastructure. The inducible nitric oxide synthase (iNOS) in brain tissue was detected by immunohistochemical method. RESULTS: The models of DKA were established after 72 hours of injecting streptozotion and alloxan monohydrate. Absorbency of EB of model group rabbits was slightly increased, but had no significant difference compared with controls (P>0.05). The brain edema, damages of vessel endothelium and necrosis of neuron were observed through histological and ultrastructure examination in model group. ALPase activity of model group was evidently decreased in brain blood vessel endothelium in comparison with controls. Compared to controls, the iNOS activity of model group was increased and its positive cells were aggregated on blood vessel of brain membrane. CONCLUSION: By streptozotion and alloxan monohydrate inducing DKA models, NO could induce blood-cerebrospinal fluid barrier damages and result in brain edema.