PD-1-induced encephalopathy: a report of 2 cases on neurological toxicities with immune checkpoint inhibitors.

Background: Immune-related adverse effects (irAEs) often occur during immune checkpoint inhibitor (ICI) therapy. In the nervous system, the incidence of irAEs ranges from 0.1-12%, with 80% occurring within the first 4 months of ICI application. For complications of the nervous system, adequate diagnosis is made by signs, symptoms, imaging and cerebrospinal fluid. If severe irAEs occur, ICIs should be discontinued and patients should be treated with high-dose glucocorticoids, immunoglobulins, or immunosorbent therapy with systemic support. Patients who develop severe neurologic irAEs have a poorer prognosis. Case Description: In this article, we report 2 cases of encephalopathy induced by anti-programmed cell death protein 1 (PD-1) monoclonal antibodies at the initial diagnoses. Our findings may help clinicians to differentiate between encephalopathy caused by immunotherapy and other neurological disorders. Case 1 was a 24-year-old male patient who had undergone PD-1 immunotherapy to treat olfactory neuroblastoma. After the 6th course of therapy, he began to develop persistent epilepsy, which decreased significantly after high doses of glucocorticoid and immunosorbent therapy were administered. Based on his medical history and laboratory examination results, PD-1-induced encephalopathy was the most likely diagnosis. Case 2 was a 67-year-old female patient who had been treated with PD-1/programmed death ligand-1 therapy for lung adenocarcinoma. She began to have headaches after 1 cycle of treatment, and her cognitive function gradually decreased with the continuation of immunotherapy. Conclusions: These case reports show the difficulty in distinguishing PD-1-induced encephalopathy from other neurological disorders, especially paraneoplastic neurological syndromes. If not treated properly, patients' lives may be endangered. Thus, early identification and early treatment are very important.

Rotational vertebral artery occlusion in a patient with basilar invagination.

Basilar invagination (BI) is a congenital or acquired craniovertebral junction (CVJ) anomaly with odontoid process superiorly migrating into the foramen magnum. Compression of neural structures is the most relevant complication of BI. However BI is also a rare cause of ischemic stroke. In this case we reported a 30-year-old female with BI who developed recurrent ischemic stroke in posterior circulation. Before the onset of ischemic stroke, she didn't present neck pain or clinical signs of lower cranial nerve dysfunction, brainstem compression or transient ischemic attack. At first she suffered from sudden onset of left-sided hemidysesthesia. Magnetic resonance imaging from a local hospital revealed an acute infarction in the right thalamus. Cerebral MR angiography was unremarkable at that time. The tip of the odontoid process had protruded into the foramen magnum and could be observed at the level of the lower medulla, but unfortunately it was ignored by the clinicians and the radiologists. She was given antiplatelet therapy and the sensory disturbance disappeared gradually. However she experienced a recurrence in the pontine and midbrain region 2 months later. At this time she was transferred to our hospital, and reconstructed computed tomography of cervical spine demonstrated basilar invagination, atlanto-axial dislocation, and atlanto-occipital assimilation. Computed tomographic angiography (CTA) revealed a dominant right vertebral artery (VA) and a redundant loop in its third segment. Dynamic cerebral angiogram demonstrated that the patient had a Bow Hunter's type phenomenon, with dynamic occlusion of the right dominant VA during contralateral head turn. This case highlighted the necessary of hemodynamic evaluation in asymptomatic basilar invagination.

Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis.

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

Epstein-Barr Virus Induces Lymphangiogenesis and Lympth Node Metastasis via Upregulation of VEGF-C in Nasopharyngeal Carcinoma.

Lymphatic metastasis is a common clinical symptom in nasopharyngeal carcinoma (NPC), the most common Epstein-Barr virus (EBV)-associated head and neck malignancy. However, the effect of EBV on NPC lymph node (LN) metastasis is still unclear. In this study, we demonstrated that EBV infection is strongly associated with advanced clinical N stage and lymphangiogenesis of NPC. We found that NPC cells infected with EBV promote LN metastasis by inducing cancer-associated lymphangiogenesis, whereas these changes were abolished upon clearance of EBV genomes. Mechanistically, EBV-induced VEGF-C contributed to lymphangiogenesis and LN metastasis, and PHLPP1, a target of miR-BART15, partially contributed to AKT/HIF1a hyperactivity and subsequent VEGF-C transcriptional activation. In addition, administration of anti-VEGF-C antibody or HIF1α inhibitors attenuated the lymphangiogenesis and LN metastasis induced by EBV. Finally, we verified the clinical significance of this prometastatic EBV/VEGF-C axis by determining the expression of PHLPP1, AKT, HIF1a, and VEGF-C in NPC specimens with and without EBV. These results uncover a reasonable mechanism for the EBV-modulated LN metastasis microenvironment in NPC, indicating that EBV is a potential therapeutic target for NPC with lymphatic metastasis. IMPLICATIONS: This research demonstrates that EBV induces lymphangiogenesis in NPC by regulating PHLPP1/p-AKT/HIF1a/VEGF-C, providing a new therapeutic target for NPC with lymphatic metastasis.

[Effect of exosomes derived from human Epstein-Barr virus-positive nasopharyngeal carcinoma cells on lymphangiogenesis and lymph node metastasis].

OBJECTIVE: To investigate the effect of exosomes derived from Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) cells on lymphangiogenesis and lymph node metastasis of NPC. METHODS: Exosomes from NP69 cells and EBV-positive HK1 (HK1-EBV) cells were obtained by ultracentrifugation and identified by Western blotting and nanoparticle tracking analysis. Dio dye phagocytosis test was performed to observe exosome uptake by lymphatic endothelial cells. Lymphatic endothelial cells were treated with exosomes from nasopharyngeal epithelium (NP69), HK1-EBV, and C666-1 cells or exosome-free supernatant of HK1-EBV and C666-1 cells, and tube formation and migration of the cells were observed. In a nude mouse model of popliteal lymph node metastasis of NPC, the effects of normal saline, NP69 cell-derived exosomes, HK1-EBV cell-derived exosomes, exosome-free supernatant of HK1-EBV cells, and HK1-EBV exosome-free supernatant protein on lymphangiogenesis and lymph node metastasis of the tumor were observed. RESULTS: The exosomes obtained by ultracentrifugation contained abundant exosome-specific proteins and showed a normal size range. The exosomes from NPC cells and NP69 cells could be taken up by lymphatic endothelial cells. Compared with the blank control and exosomes form NP69 cells, exosomes derived from HK1-EBV and C666-1 cells significantly promoted tube formation and migration of lymphatic endothelial cells (P < 0.05), and the exosomes and exosome-free supernatant of HK1-EBV and C666-1 cell produced similar effects (P > 0.05). In the tumor-bearing nude mice, exosomes derived from HK1-EBV cells significantly promoted metastasis of NPC cells and local lymphangiogenesis compared with the blank control, NP69 cell-derived exosomes and exosome-free supernatant of HK1-EBV cells (P < 0.05). CONCLUSIONS: Exosomes from EBV-positive NPC cells can significantly promote lymphangiogenesis and lymph node metastasis of NPC.

[Effect of Heat-Clearing and Detoxifying Health Function of Lonicera japonica in Rats Based on Metabonomics].

Objective: To study the metabonomics for heat-clearing and detoxifying health function of Lonicera japonica in rats. Methods: Male Wistar rats were treated with decoction of Lonicera japonica through intragastric for 5 d. GC-MS was used to detect the changes in rats serum metabolites. Mass spectrometry analysis,PCA and other technologies were used to analyze the differences among their metabolites,and analyzed the endogenous product. Results: There was a decline trend in citric acid cycle intermediates of Lonicera japonica treated group. On the contrary,amino acids and fatty acid intermediate product showed significant elevation,which revealed that the pathway of tricarboxylic acid cycle has been inhibited,and consistented with heat-clearing and detoxifying effect of Lonicera japonica. Conclusion: The metabonomics method by detecting the low-molecular-weight compounds can evaluate the heat-clearing and detoxifying health function of Lonicera japonica.

In vivo and in vitro evidence of the sex-dependent pharmacokinetics and disposition of G004, a potential hypoglycemic agent, in rats.

G004 is a novel sulfonylurea hypoglycemic drug which aimed at reducing macro- and micro-vascular complications as well as controlling glucose excursion in type 2 diabetes mellitus. The pharmacokinetics of G004 in rats was sex- and dose-dependent over the dose range of 1-10 mg kg(-1). The mean AUC values in the female rats were fivefold higher than those in males. Drug blood and tissue levels in female rats were higher than the most counterparts of males. Compared with male rats, G004 was eliminated slowly from female rats in the bile, urine and feces. Consistent with the in vivo observations, marked sex-related difference of the metabolizing activity between the male and female liver microsomes (RLM) was observed. The intrinsic clearance (V max/K m) of G004 was 3.1-fold larger in the RLM from male than female rats. Seventeen oxidative metabolites were identified in rat liver microsomes. The amount of three metabolites of G004 showed relatively sex-related difference in RLM incubations. CYP2C11 was demonstrated mainly contributing to the sex-related differences in the pharmacokinetics and disposition of G004 in rats.

A sensitive LC-MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring.

Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC-MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 µL glacial acetic acid to 200 µL aliquot of freshly collected plasma samples before storage at -80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC-MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets.

Interference by metabolites and the corresponding troubleshooting during the LC-MS/MS bioanalysis of G004, a bromine-containing hypoglycemic agent.

The quantitative determination of drugs in bio-samples may be interfered by the drug-related metabolites during the high-throughput LC-MS/MS analysis. When quantifying bromine or chlorine containing compounds, the 8¹Br/³7Cl isotopic forms of their mono-hydroxylated metabolites after in-source dehydration could produce ions which are isobaric with the precursor ions of the parent compounds at the 79Br/³5Cl isotopic form. In this report, we described the identification of an interfering hydroxylated metabolite of G004, a novel bromine-containing hypoglycemic agent, during LC-MS/MS analysis of plasma samples. Several different MRM transitions were tested and evaluated to minimize the metabolite influence on the quantification of G004. Furthermore, the standard addition method using incurred samples was used to evaluate the matrix effect caused by the interfering metabolite. The lower limit of quantitation of the established method was 0.2 ng/ml, which was 10 times lower than the existing one. The method was successfully applied to investigate the single-dosing pharmacokinetic profile of G004 in beagle dogs. The above results indicated that when quantifying chlorine or bromine containing compounds, evaluation of the interference from mono-hydroxylation or dehydrogenation metabolites should be undertaken, and if such metabolites existed, their impact on quantification of the parent compounds could be eliminated by the proper selection of the MRM transitions.

[Nummular headache: 21 new cases and therapeutic results].

OBJECTIVE: To evaluate the clinical feature of nummular headache and the efficiency of treatment in China. METHOD: The data of 21 NH patients treated from February 2006 to February 2008, were analyzed. RESULTS: They were 9 men, 12 women, aged 37 +/- 12 (18 - 63) years. Headache history ranged from 7 days to 30 years. 13 patients reported head pain confined to a circular area of 0.6 to 5 centimeter, and the other 8 patients had pain in an elliptical area of 1 - 2 centimeter x 1.5 - 3 centimeter. The symptomatic area was located in the parietal (n = 7), occipital(n = 8), temporal (n = 6), or border regions between temporal and parietal (n = 1). The right side was affected in 10 patients and the left side in the 9 patients, the middle of occipital and parietal areas were affected in 1 patient, respectively. Seven patients had mild pain, 8 patients had moderate pain and 6 patients had severe pain. The characteristics of headache included stabbing pain (n = 6), sharp pain (n = 3), pulsating apin (n = 3), grinding pain (n = 1), exploding pain (n = 5), included light-dull apin (n = 3). Three patients were treated by local nerve block with lidocaine plus dexamethasone phosphate/acetate and one of them got some 30 hours of prominent pain relief, no relief was obtained for other two patients. One patient obtained pain remission by acupuncture after amitriptyline, paroxetine had been tried ineffectively. One patient, woman, 38 years old, was treated by carbamazepine (tegretol) and amitriptyline for 3 months and pain relief was obtained gradually and the head pain disappeared finally. 8 patients were treated effectively by amitriptyline combining indomethacium, ibuprofen or carbamazepine. One patient was treated effectively by nimodipine. 2 patients were treated ineffectively by amitriptyline combining indomethacium. 4 patients were not treated. 3 patients were lost to follow-up. CONCLUSION: NH is not very rare and some of NH patients may be treated effectively by acupuncture or amitriptyline combining indomethacium, ibuprofen or carbamazepine.