Release Of Toxic Cytokines Research Articles

A novel tumor-bearing humanized mouse model for evaluating the efficacy and predictive safety of bispecific T cell engager

Abstract Bispecific T cell engager (BiTE) is one of the most promising therapeutic approaches for treatment of various malignancies. Humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs) have currently become the preclinical platform for evaluating the effectiveness of various cancer immunotherapies including BiTE. However, the wide application of this humanized animal model is limited by a strong xenoreaction of human T cells against the mouse tissues. In this study, we used humanized murine major histocompatibility class I and class II double knock-out NSG mice (referred to DKO) to evaluate the antitumor effect of BiTE. The DKO mice were implanted with human pre-B acute lymphoblastic leukemia cell line (Nalm6). Five days later, the mice were reconstituted with PBMCs. Following CD19xCD3 BiTE treatment whole-body tumor burden was quantified by a in vivo imaging system. Our data demonstrated that the mice developed consistent clinical signs of leukemia-related body weight loss and hind-limb paralysis within 4 weeks. With serial dose of CD19xCD3 BiTE treatment, the tumor cell growing was significantly suppressed. A lower incidence of hind-limb paralysis with prolonged survival were observed in BiTE-treated mice compared with controls. In addition, after treatment with CD19xCD3 BiTE, the humanized mice exhibited elevated serum levels of human IFN-γ, TNF-a, IL-6, IL-10, IL-2, and IL-4. The cytokine production was dose-dependent and associated with the increase of CD19xCD3 BiTE effectiveness. This tumor-bearing humanized model provides a promising preclinical platform for evaluating the efficacy, toxic cytokine release, and dosing requirements of any T cell-based immunotherapies for cancer treatment.

Trametinib, a novel MEK kinase inhibitor, suppresses lipopolysaccharide-induced tumor necrosis factor (TNF)-α production and endotoxin shock

Lipopolysaccharide (LPS), one of the most prominent pathogen-associated molecular patterns (PAMPs), activates macrophages, causing release of toxic cytokines (i.e. tumor necrosis factor (TNF)-α) that may provoke inflammation and endotoxin shock. Here, we tested the potential role of trametinib, a novel and highly potent MAPK/ERK kinase (MEK) inhibitor, against LPS-induced TNF-α response in monocytes, and analyzed the underlying mechanisms. We showed that trametinib, at nM concentrations, dramatically inhibited LPS-induced TNF-α mRNA expression and protein secretion in transformed (RAW 264.7 cells) and primary murine macrophages. In ex-vivo cultured human peripheral blood mononuclear cells (PBMCs), this MEK inhibitor similarly suppressed TNF-α production by LPS. For the mechanism study, we found that trametinib blocked LPS-induced MEK-ERK activation in above monocytes, which accounted for the defective TNF-α response. Macrophages or PBMCs treated with a traditional MEK inhibitor PD98059 or infected with MEK1/2-shRNA lentivirus exhibited a similar defect as trametinib, and nullified the activity of trametinib. On the other hand, introducing a constitutively-active (CA) ERK1 restored TNF-α production by LPS in the presence of trametinib. In vivo, mice administrated with trametinib produced low levels of TNF-α after LPS stimulation, and these mice were protected from LPS-induced endotoxin shock. Together, these results show that trametinib inhibits LPS-induced TNF-α expression and endotoxin shock probably through blocking MEK-ERK signaling.

Vanishing Bile Duct Syndrome: A Rare Cause of Cholestasis in Hodgkinʼs Lymphoma

Case: A 46 year-old Hispanic male presented with a 3-day history of jaundice, poor appetite, and abdominal pain. Past medical history was unremarkable, and the patient was not taking medications, illicit drugs, or alcohol. He denied B symptoms. Physical exam was unremarkable except for jaundice; no stigmata of chronic liver disease. Investigations showed total bilirubin of 14.2 mg/dL (direct 9mg/dL), GGT 362, AP 581, AST 120, ALT 248, albumin 4.1g/dL. Serology was negative for HIV, hepatitis A, B and C. CT showed normal liver without intra or extra-hepatic duct dilatation, and multiple large para-aortic and peri-hepatic lymph nodes (LN). Biopsy of a LN showed mixed cellularity Hodgkin lymphoma (HL), stage I/II. Bilirubin continued to rise to 27.2 mg/dL, and despite two cycles of chemotherapy with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), bilirubin increased to 41 g/dL, which lead to ABVD cessation and initiation of radiotherapy (RT) to the abdomen. An ERCP was normal. A liver biopsy showed marked canalicular and focal hepatocytic cholestasis, minimal inflammation, and ductopenia; no malignant cells, steatosis or iron deposition were seen, and a diagnosis of vanishing bile duct dyndrome (VBDS) as the cause of cholestasis was established. Ursodeoxycholic acid was started, RT was completed, and ABVD was restarted when bilirrubin decreased. Treatment was completed and a followup CT showed a decrease of LN size, and a PET scan was negative. Currently,18 months after diagnosis, the patient remains well and in remission. His bilirubin has normalized, and LFT have improved to near normal. Discussion: Cholestasis as the presenting symptom of HL is very uncommon (<4%). It can be caused by lymphomatous infiltration of the liver, extrahepatic biliary obstruction by lymphadenopathy, concurrent viral infections, or drug toxicities. Rarely, it can be due to a paraneoplastic phenomenon, which can be divided into VBDS and pure intrahepatic cholestasis without ductopenia. The pathophysiology of VBDS is unknown, but is thought to be caused by release of toxic cytokines from lymphoma cells, leading to destruction of bile ducts without significant inflammation. Diagnosed by exclusion and supported by histology in the absence of liver involvement by HL. VBDS is rare, and usually fatal. Only 18 cases of VBDS have been reported since its description in 1993. The overall mortality is 65%, liver failure being the leading cause, followed by sepsis and recurrence of HL. However, 30% of cases have normal LFT without recurrence at 24 months. There is no difference in outcome across sex, age, type of HL, B-symptoms, or chemotherapy or steroids, but those who are stage I/II at presentation and receive radiotherapy appear to have a better outcome.

Microglial activation is not prevented by tacrolimus but dopamine neuron damage is reduced in a rat model of Parkinson's disease progression

A progressive model of Parkinson's disease has been recently developed in the rat where a unilateral excitotoxic injection into the globus pallidus leads to a gradual loss of dopaminergic neurons in the ipsilateral substantia nigra over a period of at least 6 weeks. In this model microglial activation is observed in the ipsilateral substantia nigra 3 weeks after the lesion and could contribute to neuronal death at this time. The immunosuppressant drug tacrolimus (FK506) reduces dopamine cell death at 3 weeks following a globus pallidus lesion, but not thereafter. Tacrolimus-mediated neuroprotection could result from suppression of microglial activation but the microglial activation at three weeks post-lesion was not much reduced. Microglial activation was observed even in the apparent absence of neuronal death, prompting the suggestion that tacrolimus may prevent, or at least delay, the release of toxic cytokines from activated microglia. By 6 weeks after the GP lesion, even this mechanism fails to protect the dopamine cells from damage.

Helicobacter pylori and the pathogenesis of gastroduodenal disease: Implications for the management of peptic ulcer disease.

The discovery of Helicobacter pylori has changed our understanding of the pathophysiology of peptic ulcer disease. An estimated one billion people harbour the organism worldwide but the highest prevalence is found in developing countries with up to 80% of people infected. The most favoured modes of transmission are faeco-oral and oral-oral. The mechanisms of H pylori-induced gastroduodenal disease include the provocation of local inflammatory reaction with the release of toxic cytokines, elevation of gastrin concentration and cytotoxic epithelial injury from the activity of urease and other enzymes produced by the bacterium. However, a large proportion of infected persons have no disease or are asymptomatic thereby suggesting that there may be other factors apart from H pylori infection necessary for ulcer formation. The simple finger prick test, a variant of serology and the newly developed ELISA-based Faecal Antigen Test hold the ace for large-scale epidemiological studies. The eradication of H pylori is now a very important goal of treatment of gastric and duodenal ulcers. Most H pylori eradication regimens combine anti-secretory agent, usually a proton pump inhibitor or H2-receptor antagonist and two antibiotics (usually, Clarithromycin and Amoxycillin or Metronidazole). Emergence of antibiotic resistance is worrisome but a quadruple therapy that incorporates bismuth may be used if the triple therapy fails.

Lipopolysaccharide Signals Activation of Tumor Necrosis Factor Biosynthesis Through the Ras/Raf-1/MEK/MAPK Pathway

Lipopolysaccharide (LPS) is known to activate macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One of the most important and best studied of these cytokines is tumor necrosis factor (TNF). Details of the signaling pathway leading to TNF biosynthesis remain unclear. The pathway is branched in the sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP kinase homologs become phosphorylated in LPS-stimulated cells, suggesting their possible involvement in signal transduction. We sought to test this hypothesis. Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensitive cells. Transfection studies, in which dominant inhibitors of ras and raf-1 were used to block signaling to the level of MAP kinase, were carried out in order to judge whether the TNF gene transcription and TNF mRNA translation are modulated through this pathway. In RAW 264.7 mouse macrophages, both ERK2 and MEK1 activity are induced by LPS treatment. In the same cell line, dominant negative inhibitors of ras and raf-1 block LPS-induced activation of the TNF promoter, as well as derepression of the translational blockade normally imposed by the TNF 3'-untranslated region. A constitutively active form of raf-1 (raf-BXB) was found to augment, but not replace, the LPS signal. In LPS-insensitive cells (RAW 264.7 x NIH 3T3 fusion hybrid cells and primary macrophages derived from C3H/HeJ mice), ERK2 activity was found to be refractory to induction by LPS. The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to the level of the TNF gene and mRNA. raf and raf-1 lie upstream from (or actually represent) the physical branchpoints of the transcriptional and translation activation signals generated by LPS. The lesions that prevent LPS signaling in macrophages from C3H/HeJ mice, or in RAW 264.7 x NIH 3T3 fusion hybrid cells, occupy a proximal position in the signaling pathway.