Corrigendum: Advanced lung cancer inflammation index predicts survival outcomes of hepatocellular carcinoma patients receiving immunotherapy.

[This corrects the article DOI: 10.3389/fonc.2023.997314.].

Advanced lung cancer inflammation index predicts survival outcomes of hepatocellular carcinoma patients receiving immunotherapy.

Objective: We evaluate the predictive significance of the Advanced Lung Cancer Inflammation Index (ALI) in patients with advanced hepatocellular carcinoma (HCC) following therapy with immune checkpoint drugs. Methods: In 2018-2020, 98 patients with advanced hepatocellular carcinoma who were treated with immune checkpoint inhibitors at our hospital were compiled. Using the receiver operating characteristic (ROC) curve, the appropriate cut-off point for ALI was determined. Kaplan-Meier analysis, the Cox proportional hazards model, and Nomogram plots highlighted the relationship between ALI and overall survival (OS). The model was validated using calibration plots, receiver operating characteristic curves (ROC), and decision curve analysis (DCA), which was performed on 52 patient sets by external validation. Results: The AUC for ALI was 0.663. The best cutoff value was 36.5, with a median overall survival (OS) of 473 days for patients with ALI≤ 36.5 and 611 days for those with ALI > 36.5. Univariate analysis revealed that the presence or absence of local treatment, alpha-fetoprotein (AFP), and ALI were prognostic factors; LASSO regression analysis identified four candidate variables. Multifactorial COX analysis revealed that high ALI was an independent prognostic factor for overall survival in both groups (HR = 0.411; 95% CI: 0.244-0.651; P<0.001). In addition, the Nomogram model that included ALI was able to predict the success of immunotherapy in patients with advanced liver cancer more accurately. Conclusion: ALI is a novel prognostic marker in immunotherapy-treated patients with advanced hepatocellular cancer.

The value of the Advanced Lung Cancer Inflammation Index (ALI) in assessing the prognosis of patients with hepatocellular carcinoma treated with camrelizumab: a retrospective cohort study.

Background: The Advanced Lung Cancer Inflammation Index (ALI) is considered a useful prognostic biomarker for clinical outcome in patients with malignancy. However, the prognostic value of ALI in patients with advanced hepatocellular carcinoma (HCC) is unclear. In this study we assessed the prognostic value of the ALI in patients with HCC treated with camrelizumab. Methods: This retrospective study analyzed patients with advanced hepatocellular carcinoma treated with the ICI, camrelizumab alone or in combination at Henan Cancer Hospital from January 2017 to January 2020. Sixty-five patients were finally screened for at least 2 years of follow-up according to the inclusion criteria, with no significant differences in patient baseline data. The receiver operating characteristic (ROC) curve was used to determine the optimal cut-off point for the ALI which was compared to other clinical indicators for predicting survival. A Kaplan-Meier analysis and Cox proportional analysis were conducted to examine the association between the ALI and patient prognosis. Results: The median overall survival (OS) for the overall group of patients was 383 days, the area under the curve for ALI was 0.815 and the optimal cut-off value for predicting OS was 34.65. The median OS for patients with an ALI score ≤34.65 was 336 days and that for patients with an ALI score >34.65 was 524 days. The univariate analysis showed that the Eastern Cooperative Oncology Group (ECOG) score, aspartate aminotransferase (AST) level, and the ALI score predicted OS. The multivariate analysis showed that the ALI score was an independent prognostic factor of OS in patients with advanced HCC who had been treated with immunotherapy [hazard ratio (HR) =0.285, 95% confidence interval (CI): 0.097-0.833, P=0.022]. A nomogram that included ALI performed well relative to the prediction of prognosis after immunotherapy for patients with advanced liver cancer. Conclusions: The ALI may be a new prognostic marker in patients with advanced HCC undergoing immunotherapy.

Phase 1 multicenter, dose-expansion study of ARX788 as monotherapy in HER2-positive advanced gastric and gastroesophageal junction adenocarcinoma.

ARX788 is an anti-human epidermal growth factor receptor 2 (HER2) antibody-drug conjugate with AS269 as cytotoxic payload. In this phase 1 multicenter dose-expansion clinical trial, patients with HER2-positive advanced gastric/gastroesophageal junction adenocarcinoma failing to respond to prior trastuzumab-based standard treatment were enrolled. Between July 15th, 2019, and March 14th, 2022, 30 participants were enrolled. Twenty-eight (93.3%) patients experienced at least one drug-related adverse event (AE) and 13.3% experienced grade 3 ARX788-related AEs. The confirmed objective response rate is 37.9% (95% confidence interval [CI]: 20.7%-57.7%) and the disease control rate is 55.2% (95% CI: 35.7%-73.6%). With a median follow up of 10 months, the median progression-free survival and overall survival are 4.1 (95% CI: 1.4-6.4) and 10.7 months (95% CI: 4.8-not reached), respectively. The median duration of response is 8.4 (95% CI: 2.1-18.9) months. ARX788 is well tolerated and has promising anti-tumor activity in patients with HER2-positive advanced gastric adenocarcinoma (ChinaDrugTrials.org.cn: CTR20190639).

Novel insertions in the mitochondrial maxicircle of Trypanosoma musculi, a mouse trypanosome.

Trypanosoma musculi is a, globally distributed, mouse-specific haemoflagellate, of the family Trypanosomatidae, which shares similar characteristics in morphology with Trypanosoma lewisi. The kinetoplast (mitochondrial) DNA of Trypanosomatidae flagellates is comprised of catenated maxicircles and minicircles. However, genetic information on the T. musculi kinetoplast remains largely unknown. In this study, the T. musculi maxicircle genome was completely assembled, with PacBio and Illumina sequencing, and the size was confirmed at 34 606 bp. It consisted of 2 distinct parts: the coding region and the divergent regions (DRs, DRI and II). In comparison with other trypanosome maxicircles (Trypanosoma brucei, Trypanosoma cruzi and T. lewisi), the T. musculi maxicircle has a syntenic distribution of genes and shares 73.9, 78.0 and 92.7% sequence identity, respectively, over the whole coding region. Moreover, novel insertions in MURF2 (630 bp) and in ND5 (1278 bp) were found, respectively, which are homologous to minicircles. These findings support an evolutionary scenario similar to the one proposed for insertions in Trypanosoma cruzi, the pathogen of American trypanosomiasis. These novel insertions, together with a deletion (281 bp) in ND4, question the role of Complex I in T. musculi. A detailed analysis of DRII indicated that it contains numerous repeat motifs and palindromes, the latter of which are highly conservative and contain A5C elements. The comprehensively annotated kinetoplast maxicircle of T. musculi reveals a high degree of similarity between this parasite and the maxicircle of T. lewisi and suggests that the DRII could be a valuable marker for distinguishing these evolutionarily related species.

A new species of mammalian trypanosome, Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis.

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27-37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.

Down-Regulation of miR-7 in Gastric Cancer Is Associated With Elevated LDH-A Expression and Chemoresistance to Cisplatin.

MicroRNAs (miRNAs) are dysregulated in the context of many cancer types, making them potentially ideal diagnostic or therapeutic targets in patients in which they are aberrantly expressed. In the present study, we found miR-7 to be downregulated in gastric cancer (GC), and we further determined its expression to be closely linked to GC sensitivity to the chemotherapeutic compound cisplatin. This effect appears to be at least partially attributable to the regulation of LDH-A, which is a miR-7 target gene and expression of LDH-A is negatively correlated with miR-7 expression in primary GC tumor samples. When upregulated, we also determined that miR-7 was able to inhibit the proliferation, colony formation, and glycolysis of GC cells owing to its regulation of LDH-A. Moreover, overexpression of miR-7 render cells more sensitive to cisplatin. Our results thus provide novel evidence that miR-7 is a key mediator of GC growth and chemosensitivity through its regulation of LDH-A, thus potentially highlighting this pathway as a therapeutic target for treating affected patients.

Novel organization of mitochondrial minicircles and guide RNAs in the zoonotic pathogen Trypanosoma lewisi.

Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.

MiR-200b Inhibits Tumor Growth and Chemoresistance via Targeting p70S6K1 in Lung Cancer.

Downregulation of microRNA-200b (miR-200b) has been identified in a range of cancers, yet the specific mechanisms whereby it influences lung cancer growth require further exploration. We determined that lung cancer patient tumor samples exhibit decreased miR-200b expression, and we further found this miRNA to inhibit tumor growth via interfering with ERK1/2 and AKT signaling, targeting p70S6K1 to suppress HIF-1α expression. This miRNA further rendered H1299 cells more sensitive to cisplatin while impairing their proliferative and invasive potential through its ability to target and inhibit the activity of p70S6K1. These results were further confirmed in a murine xenograft model in which miR-200b also inhibited the growth of tumor and suppressed p70S6K1, p-AKT, p-ERK1/2, and HIF-1α expression. These findings clearly demonstrate a role for miR-200b in suppressing lung cancer development, making it a potentially relevant target for future diagnostic and therapeutic interventions.

Apigenin Inhibits IL-6 Transcription and Suppresses Esophageal Carcinogenesis.

Esophagus cancer is the seventh cause of cancer-related deaths globally. In this study, we analyzed interleukin 6 (IL-6) gene expression in human esophagus cancer patients and showed that IL-6 mRNA levels are significantly higher in tumor tissues and negatively correlated with overall survival, suggesting that IL-6 is a potential therapeutic target for esophagus cancer. We further demonstrated that apigenin, a nature flavone product of green plants, inhibited IL-6 transcription and gene expression in human esophagus cancer Eca-109 and Kyse-30 cells. Apigenin significantly and dose-dependently inhibited cell proliferation and promoted apoptosis while stimulating the cleaved PARP (poly ADP-ribose polymerase) (C-PARP) and caspase-8 expression. It suppressed VEGF (Vascular endothelial growth Factor) expression and tumor-induced angiogenesis. Pretreatment of cells with IL-6 could completely reverse apigenin-induced cellular changes. Finally, using a preclinical nude mice model subcutaneously xenografted with Eca-109 cells, we demonstrated the in vivo antitumor activity and mechanisms of apigenin. Taken together, this study revealed for the first time that apigenin is a new IL-6 transcription inhibitor and that inhibiting IL-6 transcription is one of the mechanisms by which apigenin exhibits its anticancer effects. The potential clinical applications of apigenin in treating esophagus cancer warrant further investigations.

Effects of obatoclax combined with gemcitabine on the biological activity of pancreatic cancer cells under hypoxic conditions.

The present study aimed to investigate the effects of obatoclax (OBX) combined with gemcitabine (GEM) treatment on the proliferation, migration, invasion and epithelial­mesenchymal transition (EMT) related proteins of pancreatic cancer cell line BxPC­3 under hypoxic conditions. Protein expression levels of hypoxia­inducible factor 1α (HIF­1α) in BxPC­3 pancreatic cancer cells under normoxic and hypoxic conditions were detected by western blotting. Cells were divided into four groups: Normoxia group, hypoxia group, OBX group and OBX + GEM group. The proliferation activity of BxPC­3 cells was detected by Cell Counting kit­8. The migratory and invasive abilities of BxPC­3 cells were detected by the scratch test and Matrigel assay, respectively. The protein expression levels of vimentin, E­cadherin and p53 in BxPC­3 cells were also detected by western blotting. HIF­1α expression under hypoxic conditions was significantly increased compared with expression under normoxic conditions. Under hypoxic conditions, OBX treatment reduced cell activity, decreased cell migration and invasion, promoted the expression of E­cadherin and p53. In the OBX + GEM group, BxPC­3 cell activity decreased significantly, cell migration and invasion decreased significantly, the expression of vimentin was significantly reduced and the expression of E­cadherin and p53 further increased. In conclusion, the present results demonstrated that under hypoxic conditions, OBX combined with a small dose of GEM may be able to inhibit the growth, migration and invasion of pancreatic cancer cells, possibly via inhibition of EMT process. These results may provide a promising strategy for pancreatic cancer therapy.

ECRG2 enhances the anti-cancer effects of cisplatin in cisplatin-resistant esophageal cancer cells via upregulation of p53 and downregulation of PCNA.

AIM: To explore the anti-tumor effects of esophageal cancer-related gene 2 (ECRG2) in combination with cisplatin (DDP) in DDP-resistant esophageal cancer cells (EC9706/DDP). METHODS: A drug-resistant cell model was established, with EC9706/DDP cells being treated with ECRG2 and/or DDP. Cell viability was examined by MTT assay. The rate of cell apoptosis was determined by flow cytometry. The mRNA expression levels of proliferating cell nuclear antigen (PCNA), metallothionein (MT), and p53 were determined by RT-PCR and PCNA, while MT and p53 protein expression levels were determined by western blotting. RESULTS: The anti-proliferative effect of ECRG2 in combination with DDP was superior when compared to ECRG2 or DDP alone. The inhibition rate for the combination reached its peak (51.33%) at 96 h. The early apoptotic rates of the control, ECRG2 alone, DDP alone, and ECRG2 plus DDP groups were 5.71% ± 0.27%, 12.68% ± 0.61%, 14.15% ± 0.87%, and 27.96% ± 0.36%, respectively. Although all treatment groups were significantly different from the control group (P < 0.05), the combination treatment of ECRG2 plus DDP performed significantly better when compared to either ECRG2 or DDP alone (P < 0.05). The combination of ECRG2 and DDP significantly upregulated p53 mRNA and protein levels and downregulated PCNA mRNA and protein levels compared to ECRG2 or DDP alone (P < 0.05). However, no changes were seen in the expression of MT mRNA or protein. CONCLUSION: ECRG2 in combination with DDP can inhibit viability and induce apoptosis in esophageal cancer DDP-resistant cells, possibly via upregulation of p53 expression and downregulation of PCNA expression. These findings suggest that the combination of ECRG2 and DDP may be a promising strategy for the clinical treatment of esophageal cancers that are resistant to DDP.

The HepaRG cell line, a superior in vitro model to L-02, HepG2 and hiHeps cell lines for assessing drug-induced liver injury.

Drug-induced liver injury (DILI) is a leading cause of discontinuation of new drug approval or withdrawal of marketed medicine based on safety due to organ vulnerability. The aim of this research is to investigate the potential abilities of four different in vitro cell models (L-02, HepG2, HepaRG, and hiHeps cell lines) in assessing marketed drugs labeled with apparently different types of liver injury. A total of 17 drugs with versatile pharmacological profiles were chosen, of which, 14 drugs are recognized as DILI agents and 3 drugs are DILI irrelevant. Preliminary cellular screening assays indicated that the HepaRG cell line had an advantage over other cell lines in predicting drugs associated with DILI in vitro as it had the highest Youden's index (71.4%). A multi-parametric screening assay showed that oxidative stress, mitochondrial damage, and disorders of neutral lipid metabolism were changed notably in the HepaRG cell line after DILI-related drugs exposure, accounting for its high sensitivity in comparison with other three cell lines. In addition, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) all correlated with the cytotoxic effects of diclofenac sodium (p < 0.05), buspirone hydrochloride (p < 0.01), and danazol (p < 0.01) in the HepaRG cell line. We conclude that the HepaRG cell line is a superior in vitro cell model to other three cell lines for evaluating drugs with DILI potential.

[Effects of Germanium Concentrations on Germanium Accumulation and Biotransformation of Polysaccarified Germanium in Cordyceps militaris].

OBJECTIVE: To study the effects of Germanium (Ge) concentration on Ge accumulation and biotransformation of polysaccarified Ge (PG) in Cordyceps militaris. METHODS: Solid and liquid culture were used in this study. RESULTS: In the solid culture conditions, when the Ge concentration of medium was 200 mg/L, the sporophore biomass of Cordyceps militaris was the maximum; and when Ge concentration was 300 mg/L,the amount of biotransformation of PG in sporophore was the highest; and when the Ge concentration is 250 mg/L, conversion rate of organic germanium (OG) in sporophore reached the highest value. In the liquid culture conditions, when the Ge concentration was 250 mg/L, the mycelium biomass of Cordyceps militaris was the maximum; and when Ge concentration was 150 mg/L, the amount of organic conversion of PG in mycelium was the most; and conversion rate of OG in mycelium was the highest in media with the Ge concentration of 200 mg/L. This study showed the germanium concentrations in 150 - 300 mg/L was more suitable for Ge accumulation and biotransformation of PG in Cordyceps militaris. In general, the biotransformation capacity to germanium of sporophore was stronger than that of mycelium of Cordyceps militaris. CONCLUSION: Germanium can significantly affect Ge accumulation and biotransformation of PG in Cordyceps militaris (P < 0.05) at different concentration. This result has practical value for Ge enriched cultivation of fruiting body in Cordyceps militaris.

[Study on monosaccharide composition of intracellular polysacchride and contents of cordycepin and cordyceps polysacchride produced by Cordyceps militaris induced by blue light].

OBJECTIVE: To study effects of blue light irradiation on monosaccharide composition of intracellular polysacchride and contents of cordycepin and cordyceps polysacchride of mycelium and sporocarp in Cordyceps militaris. METHODS: The monosaccharide composition of intracellular polysacchride of mycelium and sporocarp in Cordyceps militaris as materials were determined by gas chromatography after 144 h blue light irradiation. The contents of cordycepin and cordyceps polysacchride of mycelium and sporocarp in Cordyceps militaris were detected at different blue light irradiation periods. At the same time, the growth of mycelium and sporocarp in Cordyceps militaris were observed during blue light irradiation. RESULTS: Mycelium polysaccharide in Cordyceps militaris was a kind of heteropolysaccharide containing four kinds of monosaccharide and fruiting body polysaccharide was a kind of heteropolysaccharide containing five kinds of monosaccharide. Whether blue light irradiation or dark culture, the content changes of cordyceps polysacchride in two groups showed similar patterns in the test of mycelium polysaccharides. The content changes of cordyceps polysacchride in two groups were basically the same in the detection of sporocarp polysacchride. Cordycepin content in the two set of experiments of blue light irradiation all showed a clear upward trend in the detection of mycelium and sporocarp in Cordyceps militaris. CONCLUSION: The blue light irradiation has certain effect on the species and quantity of monosaccharide in intracellular polysaccharide. The content increase of cordycepin and cordyceps polysacchride in Cordyceps militaris are promoted by blue light irradiation. Blue light can help the morphogenesis and promote the differentiation and growth of sporocarp in Cordyceps militaris. This study is the first report about the effect of blue light on the type and quantity of the monosaccharide composition in polysaccharide of Cordyceps militaris, which will lay the foundation for further study on the metabolism of active substance in Cordyceps militaris by blue light irradiation.

A safety study of inactivated Enterovirus 71 vaccine.

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot and mouth disease (HFMD) in childhood. Nowadays, HFMD or EV71 infections have already become an important public health issue throughout the world. Vaccination may be the most effective measure to control the transmission of the virus. Therefore, to pave EV71 vaccine into human clinical trial, in the present study a comprehensive preclinical safety assessment of inactivated EV71 vaccine including single- and repeat-dose toxicity studies were conducted in rats and cynomolgus monkeys. No abnormal findings were observed in rats following single intramuscular administration with EV71 vaccine (640 U). The results also showed no obvious systemic toxicities from four repetitive intramuscular injections, with a 14-d interval, of two dosages of EV71 vaccine in the two animal species. Antinuclear antibody response was not detected after the repeated administrations. Histopathological examination demonstrated the minimal to severe inflammatory changes in muscle tissues of the injection sites in EV71 vaccine-injected animals and most of findings have been improved over time. Furthermore, test article could induce highly EV71-specfic neutralizing antibody response in both animal species. Taken together, these data suggested a favorable safety profile for inactivated EV71 vaccine and supported this product to enter human phase I clinical trial.

Expression of Bmi-1 gene in esophageal carcinoma cell EC9706 and its effect on cell cycle, apoptosis and migration.

BACKGROUND AND OBJECTIVE: Previous studies have shown that Bmi-1 is overexpressed in a variety of tumors, suggesting that Bmi-1 plays an important role in tumorigenesis. In this study, we investigated the effect of Bim-1 siRNA on cell proliferation, cell cycle, cell apoptosis and migration of human esophageal carcinoma EC9706 cells, and explored its potential mechanisms. METHODS: Bmi-1 small interfering RNA (siRNA) was transferred into EC9706 cells. Then, cell proliferation was measured using cell counting kit-8 (CCK-8), cell cycle and cell apoptosis were analyzed by flow cytometry, cell migration ability was detected using Boyden chamber assay, and the mRNA and protein expression levels of Bmi-1, p16, Bcl-2, Bax, and MMP-2 were determined using real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. RESULTS: Bmi-1 siRNA treatment significantly inhibited the expression of Bmi-1 at both mRNA and protein levels in EC9706 cells. Cell proliferation rate decreased dramatically in the Bmi-1 siRNA treated group than in the untreated group and in the scrambled siRNA treated group (both P < 0.001). In Bmi-1 treated group, the percentage of cells at G(0)/G(1) stage was 71.93%, which was higher than that in the untreated group (47.36%) or scramble siRNA treated group (48.47%) (both P < 0.001). Early cell apoptosis rate also increased significantly in the Bmi-1 siRNA treated group (both 17.32%) than in the untreated group (2.61%) and in the scramble siRNA treated group (2.73%) (both P < 0.001). Further experiment suggested that downregulation of Bmi-1 led to less cell migration. In EC9706 cells transfected by Bmi-1 siRNA, the expression levels of p16 and Bax increased, while the expression level of Bcl-2 decreased. CONCLUSIONS: Bmi-1 downregulation in esophageal carcinoma cells inhibits cell proliferation, cell cycle, and cell migration, while increases cell apoptosis. These results suggest that Bmi-1 is a potential molecular target of treating esophageal cancer.

Clozapine-induced myocarditis: role of catecholamines in a murine model.

Clozapine, an atypical antipsychotic, is very effective in the treatment of resistant schizophrenia. However, cardiotoxicity of clozapine, particularly in young patients, has raised concerns about its safety. Increased catecholamines have been postulated to trigger an inflammatory response resulting in myocarditis, dilated cardiomyopathy, and death, although this has not yet been thoroughly studied. Here, we used the mouse to study whether clozapine administration could cause adverse myocarditis associated with an increase in catecholamines. Male Balb/C mice, age ~6 weeks, were administered 5, 10 or 25 mg/kg clozapine daily for 7 and 14 days; one group was administered 25 mg/kg clozapine plus 2 mg/kg propranolol for 14 days. Saline-treated mice served as controls. Heart sections were stained with hematoxylin and eosin for histopathological examination. Plasma catecholamines were measured with HPLC. Myocardial TNF-alpha concentrations were determined by ELISA. Histopathology of clozapine-treated mice showed a significant dose-related increase in myocardial inflammation that correlated with plasma catecholamine levels and release of TNF-alpha. Propranolol significantly attenuated these effects. A hypercatecholaminergic state induced by clozapine could explain the occurrence of myocarditis in some patients. Our data suggest that a beta-adrenergic blocking agent may be effective in reducing the incidence and severity of clozapine-induced myocarditis.

Successful implantation of intravenously administered stem cells correlates with severity of inflammation in murine myocarditis.

The present study was designed to determine whether cardiac inflammation is important for the successful homing of stem cells to the heart after intravenous injection in a murine myocarditis model. Male Bagg albino/c mice were infected with encephalomyocarditis virus (EMCV) to produce myocarditis. Subgroups of mice received single injections by tail vein of embryonic stem cells (ESCs) transfected with green fluorescent protein (GFP) as a marker at days 3, 14, or 60 after infection; other subgroups without stem cell injections were killed at each of these time points to assess the degree of inflammation present. The surviving mice were killed at day 90 after virus infection and hemodynamics, gross pathology, histology, and inflammatory cytokine production in the hearts were measured. Our results indicate that myocardial inflammation was most severe and cytokine production highest at day 14 after EMCV inoculation, and in particular, was strongly positive for interleukin 6. Mice receiving intravenous ESC injections on day 14 after EMCV inoculation showed the largest number of GFP-positive cells at the time of death and the greatest functional improvement compared to uninfected controls without inflammation. We conclude that factors released from myocardium during inflammation are important for enhancing the homing, migration, and implantation of systemically infused stem cells.

Propranolol ameliorates and epinephrine exacerbates progression of acute and chronic viral myocarditis.

Recent studies point to important interactions between proinflammatory cytokines and neurohumoral mediators in heart failure. Here we investigate the influence of the beta-adrenergic system on cytokines and neurohumoral factors and the sequelae of viral myocarditis. In an experimental model with virus-infected BALB/c mice, we studied the acute and chronic effects of epinephrine and propranolol on myocardial morphology, cytokine gene expression, and survival. BALB/c mice were inoculated with the encephalomyocarditis virus (EMCV) or sham inoculated with saline and followed for 30 days. Epinephrine increased the severity of inflammatory cell infiltration and myocardial necrosis induced by EMCV. Gene expression of TNF-alpha, IL-6, and IL-10 was markedly enhanced by epinephrine in EMCV-inoculated mice. Survival rate after 30 days was reduced to 40% in epinephrine-treated EMCV-inoculated mice compared with 70% in untreated EMCV-inoculated mice (P < 0.05). Treatment with the beta-blocker propranolol significantly decreased mortality, myocardial necrosis, and infiltration of inflammatory cells in EMCV-inoculated mice. Propranolol also suppressed gene expression of TNF-alpha, IL-6, and IL-10. A single dose of epinephrine 120 days after EMCV inoculation caused sudden death in 70% of infected mice; propranolol significantly reduced incidence of death to 33%. These results indicate that acute and chronic stages of viral myocarditis are modulated by the beta-adrenergic system and its interactions with proinflammatory cytokines.