Progress of engineered bacteria for tumour therapy.

Finding novel therapeutic modalities, improving drug delivery efficiency and targeting, and reducing the immune escape of tumor cells are currently hot topics in the field of tumor therapy. Bacterial therapeutics have proven highly effective in preventing tumor spread and recurrence, used alone or in combination with traditional therapies. In recent years, a growing number of researchers have significantly improved the targeting and penetration of bacteria by using genetic engineering technology, which has received widespread attention in the field of tumor therapy. In this paper, we provide an overview and assessment of the advancements made in the field of tumor therapy using genetically engineered bacteria. We cover three major aspects: the development of engineered bacteria, their integration with other therapeutic techniques, and the current state of clinical trials. Lastly, we discuss the limitations and challenges that are currently being faced in the utilization of engineered bacteria for tumor therapy.

Targeted paclitaxel prodrug nanoassemblies to improve therapeutic effects for liver cancer.

Prodrug nanoassemblies fabricated by anticancer drug conjugates exhibited more advantages in controlled drug release and bioavailability and favorable antitumor efficacy. In this paper, lactobionic acid (LA) was connected with polyethylene glycol through amido linkages, and paclitaxel was joined with polyethylene glycol by means of ester bonds to form the prodrug copolymer LA-PEG-PTX. Then, LA-PEG-PTX was automatically assembled into LA-PEG-PTX nanoparticles (LPP NPs) by dialysis. The LPP NPs had a relatively uniform size of approximately 200 nm, a negative potential (-13.68 mV), and a spherical shape under TEM. The drug loading of LPP NPs was 3.91%, which was measured by HPLC. The in vitro release profile of LPP NPs exhibited a sustained release feature. The results of the pharmacokinetic test in rats showed that LPP NPs had higher T1/2 and AUC values than the control group (free PTX) and a prolonged in vivo circulation time, thus increasing the bioavailability of PTX. Remarkably, the LPP NPs were absorbed into HepG2 cells after galactose-directed internalization and enhanced cytotoxicity. Consequently, LPP NPs displayed notable antitumor activity in Kunming mice with H22 hepatocellular carcinoma. Collectively, these findings suggested that paclitaxel prodrug-based self-assembled nanoparticles were a promising alternative for improving the bioavailability and antitumor effect of PTX.

Effects of centrifugation prior to pneumatic tube system transport on routine biochemical and immunological tests of susceptibility to hemolysis.

BACKGROUND: Pneumatic tube system (PTS) may be associated with preanalytical hemolysis. The objective of this study was to evaluate the effects of PTS on biochemical and immunological tests susceptible to hemolysis and try to find ways to reduce the result bias caused by PTS. METHODS: Laboratory parameters were compared between PTS without centrifuging group, PTS after centrifuging group, PTS with serum group, and hand-delivered (HD) group. Studies were performed to access the influence of different PTS transport frequencies on laboratory assays. RESULTS: PTS transportation resulted in obviously increase in LDH (lactate dehydrogenase) and NSE (neuron-specific enolase) results (LDH: Bias = 17.95%, 95% confidence interval (CI) = -3.13-39.02; p < 0.001; NSE: Bias = 64.26%, 95% CI = -21.29-149.82; p < 0.001; respectively). After pre-centrifugation, no statistical difference was observed in LDH results (Bias = 2.83%, 95% CI = -13.00-18.65; p = 0.737). However, the bias of NSE still reach 19.16% (95% CI = -41.78-80.11), which exceeded the clinical acceptable range (p = 0.017). Both LDH(p = 0.931) and NSE(p > 0.999) show no statistical difference between PTS with serum group and HD group (LDH: Bias = -1.60%, 95% CI = -6.00-2.81; NSE: Bias = -3.68%, 95% CI = -11.35-3.99). CONCLUSION: PTS can lead to falsely increased LDH and NSE test results. Only loading the centrifuged upper serum in new tubes during PTS transport can eliminate the results bias of NSE.

Glutamatergic lateral hypothalamus promotes defensive behaviors.

The glutamatergic lateral hypothalamus (LH) has been implicated in a variety of behaviors, such as evasion and feeding, while its role in defensive behaviors and relevant neurocircuits remains unclear. Here, we demonstrated that the glutamatergic LH is a critical structure regulating defensive behaviors. Trimethylthiazole (TMT), the odor of mice predator, significantly increased c-Fos expression in the LH. Using fiber photometry technology, we found that TMT exposure increased the activity of LH glutamatergic neurons. Selective activation of LH glutamatergic neurons with optogenetics and chemogenetics promoted a series of defense-related behaviors, including fleeing, avoidance, and hiding, while selective inhibition of LH glutamatergic neurons suppressed the avoidance provoked by TMT. Activation of both the glutamatergic LH terminals in the hypothalamic paraventricular nucleus (PVN) and the glutamatergic projection from the basolateral amygdala (BLA) to the LH elicited defensive behaviors. Finally, by combining the viral-mediated retrograde tracing with anterograde activation, we found that PVN-projecting glutamatergic neurons in the LH were activated by BLA glutamatergic inputs. Taken together, our results illustrate that the glutamatergic LH is a pivotal relay of defensive behaviors and possibly promotes these behaviors through the BLA→LH→PVN pathway.

Liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

BACKGROUND: Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed. AIM: To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method. METHODS: A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay. RESULTS: Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P < 0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with marked-severe inflammation were significantly higher than those with mild-moderate inflammation (P < 0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R 2 = 0.673, P < 0.01) or plasma DNA (R 2 = 0.597, P < 0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild-moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively. CONCLUSION: The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

Preliminary Research on Syndrome Types of Chinese Medicine in Children with Primary Nephrotic Syndrome.

OBJECTIVE: To provide an objective reference for the syndrome types of Chinese medicine (CM) associated with pediatric primary nephrotic syndrome (PNS). METHODS: A cross-sectional study was performed. Data on clinical symptoms, CM syndrome types, biochemical indices, and medications used were collected from 98 children with PNS. Then, the correlation between CM syndromes and biochemical indices, as well as medications used, was analyzed. RESULTS: The four most common symptoms in children with PNS were brown urine, red tongue, excessive sweating, and swelling of the face and limbs. The syndromes of qi deficiency of Fei (Lung) and Shen (Kidney) (FSQD) and yin deficiency of Gan (Liver) and Shen (GSYD) were the most common main CM syndrome types. FSQD syndrome score correlated significantly with the total cholesterol level, urine protein/creatinine ratio, and urine IgG and albumin levels (P<0.01 or P<0.05). The use of maintenance glucocorticoids combined with immunosuppressive agents correlated with FSQD syndrome, and the use of maintenance glucocorticoids alone correlated with GSYD syndrome (P<0.05). CONCLUSION: Two of the most common CM syndrome types were FSQD and GSYD syndromes. FSQD syndrome may be caused by some factors related to lipid levels, protein loss, and the use of immunosuppressive agents. The use of maintenance glucocorticoids may cause GSYD syndrome.

Chitosan-modified cholesterol-free liposomes for improving the oral bioavailability of progesterone.

Based on the structurally similar properties of progesterone and cholesterol, chitosan-coated cholesterol-free liposomes (CS-Lipo/Prog) were formulated. CS-Lipo/Prog are spherical and uniform in size (662.1±19.3nm) with positive potential (28.19±1.97mV). The average drug entrapment efficiency (EE) is approximately 80%. The in vitro release profile of CS-Lipo/Prog shows sustained release. The in vitro stability evaluation demonstrated that CS-Lipo/Prog can efficiently shield Prog from degradation in the gastrointestinal tract. CS-Lipo/Prog showed a longer MRT and higher AUC0-infinite after oral administration to mice than in the control group (progesterone-free). The relative bioavailability of CS-Lipo/Prog was higher than that of progesterone soft capsules (QINING®) and Lipo/Prog. Collectively, these findings suggest that cholesterol-free chitosan-coated liposomes are a promising alternative for improving the oral bioavailability of progesterone.

In vivo synergistic antitumor effect and safety of siRNA and lonidamine dual-loaded hierarchical targeted nanoparticles.

Based on development of nano-delivery system, co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) has exerted a promising advantage in cancer therapy. In this work, the superiority of synergistic therapy and safety of the hierarchical targeted co-delivery system loaded with siRNA and lonidamine (LND) were evaluated. The in vivo tumor accumulation ability and cancer growth inhibition effect of the polymer-blend nanocarriers were evaluated by a H22 subcutaneous sarcoma model. Moreover, hematoxylin and eosin (H&E) staining and transferase-mediated dUTP nick end-labeling (TUNEL) staining of tumor sections from each group were compared to assess the therapeutic efficacy. The dual-loaded nanocarriers had better tumor accumulation ability, remarkably inhibited growth of solid tumor in a synergistic manner, even significantly decreased hepatotoxicity of LND, and had good in vivo biocompatibility whereas LND alone showed serious hepatotoxicity. We believed that the dual-loaded hierarchical targeted delivery system with high effectiveness and biocompatibility would provide a promising approach for cancer combination therapy.

High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay.

BACKGROUND: Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment. METHODS: A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals. RESULTS: The average TRACP level of the high-UA individuals (1.47 ± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p < 0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24). CONCLUSIONS: High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.

Biocompatible polymeric nanocomplexes as an intracellular stimuli-sensitive prodrug for type-2 diabetes combination therapy.

Combination therapy is usually considered as a promising strategy owing to its advantages such as reduced doses, minimized side effects and improved therapeutic efficiency in a variety of diseases including diabetes. Here we synthesized a new highly intracellular stimuli-sensitive chitosan-graft-metformin (CS-MET) prodrug by imine reaction between oxidative chitosan and metformin for type 2 diabetes (T2D) therapy. Hypothetically, CS-MET functions dually as an anti-diabetes prodrug as well as a gene delivery vector without superfluous materials. CS-MET formed nanocomplexes with therapeutic gene through electrostatic interactions and entered cells by Organic Cation Transporter (OCT)-independent endocytosis. The incorporation of metformin into chitosan has been found to increase endosomal escape via the proton sponge effect. When vector carrying a short-hairpin RNA (shRNA) silencing sterol regulatory element-binding protein (SREBP), a major transcription factor involved in de novo lipogenisis, it reduced the SREBP mRNA and proteins efficiently. Furthermore, by intraperitoneal injection, CS-MET/shSREBP nanocomplexes effectively knocked down SREBP in livers of western-type diet (WD)-induced obese C57BL/6J mice, markedly reversed insulin resistance and alleviated the fatty liver phenotype without obvious toxic effects. Thus we were able to show that the intracellular stimuli-sensitive CS-MET prodrug renders a potential platform to increase the anti-diabetes activity with synergistic enhancement of gene therapy.

Mitochondria apoptosis pathway synergistically activated by hierarchical targeted nanoparticles co-delivering siRNA and lonidamine.

The mitochondria-mediated apoptosis pathway is an effective option for cancer therapy due to the presence of cell-suicide weapons in mitochondria. However, anti-apoptotic proteins that are over-expressed in the mitochondria of many malignant tumors, such as Bcl-2 protein, could allow the cancer cells to evade apoptosis, greatly reducing the efficacy of this type of chemotherapy. Here, we constructed a hierarchical targeted delivery system that can deliver siRNA and chemotherapeutic agents sequentially to tumor cells and mitochondria. In detail, the copolymer TPP-CP-LND (TCPL) was synthesized by the mitochondria-targeting ligand triphenylphosphine (TPP) and therapeutic drug lonidamine (LND) conjugated to the polyethyleneimine in chitosan-graft-PEI (CP), and then complexed with siRNA. Followed, the complexes were coated with poly(acrylic acid)-polyethylene glycol-folic acid (PPF) copolymer to form a hierarchical targeted co-delivery system (TCPL/siRNA/PPF NPs). The TCPL/siRNA/PPF NPs had a neutral surface charge, were stable in plasma and exhibited pH-responsive shell separation. Remarkably, the TCPL/siRNA/PPF NPs simultaneously released siBcl-2 into the cytoplasm and delivered LND to mitochondria in the same cancer cell after FA-directed internalization, and even synergistically activated mitochondria apoptosis pathway. This work demonstrated the potential of RNA-interference and mitochondria-targeted chemotherapeutics to collaboratively stimulate the mitochondria apoptosis pathway for cancer therapy.

A novel potential biocompatible hyperbranched polyspermine for efficient lung cancer gene therapy.

The clinical successful application of gene therapy critically depends upon the development of non-toxic and efficient delivery system. Although polycationic non-viral vectors hold great promise in nanomedicine, the exploring of application in clinics still remains a big challenge. To develop a non-toxic and efficient non-viral gene delivery system, two kinds of endogenous substance, citric acid (CA) and spermine (SPE), were used to prepare a new low charge density hyperbranched polyspermine (HPSPE) by one-pot polymerization. The biocompatibility evaluated by hemolytic activity and red blood cell (RBC) aggregation indicated that HPSPE was highly biocompatible without causing hemolysis and RBC aggregation compared with PEI as well as SPE. The MTS assay also demonstrated that the cell viability of HPSPE was above 90% even at 200 µg/mL at different time (24 and 72 h), which much higher than PEI 25K. Besides, HPSPE showed high transfection efficiency without any toxic effect after aerosol delivery to the mice. Moreover, aerosol delivery of HPSPE/Akt1 shRNA significantly reduced tumor size and numbers and efficiently suppressed lung tumorigenesis ultimately in K-ras(LA1) lung cancer model mice. These results suggest that low charge density as well as endogenous substance skeleton endow HPSPE with great potential for toxicity-free and efficient gene therapy.

[cagA,vacA and iceA genotypes of Helicobacter pylori isolated from children in Shanghai].

OBJECTIVE: To investigate cagA, vacA and iceA genotypes of Helicobacter pylori (H. pylori) isolated from children suffering from gastric and duodenal diseases in Shanghai and to explore a possible genotype-phenotype correlation. METHODS: From May 2007 to January 2008, 59 children were confirmed with Hp infection by gastroscopy. Biopsied specimens were taken from the gastric antrum. cagA, vacA and iceA genes were determined by PCR. The histological changes in the gastric mucosa were evaluated. The levels of IFN-gamma and IL-4 in the gastric mucosa were measured using ELISA. RESULTS: cagA, vacAs1/m1, vacAs1/m2, iceA1 and iceA2 were found in 65%, 19%, 40%, 63% and 19% of H. pylori strains, respectively. Both iceA1 and iceA2 were detected in 9% of strains. There were no statistical differences in the distribution of various genotypes between the children with chronic gastritis and peptic ulcer. No association was observed between the genotypes and the degree of inflammation of gastric mucosa. There were no significant differences in levels of IFN-gamma and IL-4 in the gastric mucosa infected by different genotypes of H. pylori strains. CONCLUSIONS: cagA/vacAs1/m2/iceA1 may be the commonest genotype combination of H.pylori in children from Shanghai. That there was no association between H.pylori genotypes and clinical variables suggests the potential role of host and environment factors in the development of clinical diseases at a later life.

[Mitochondrial DNA mutation analysis in patients with mitochondrial myopathy].

OBJECTIVE: To examine mitochondrial DNA mutations in mitochondrial myopathy. METHODS: Three suspected cases of mitochondrial myopathy were examined by HE staining, histochemical staining methods and electron microscopy. The mutations in all 22 tRNA genes of mitochondrial genome were screened by polymerase chain reaction-single strand conformation polymorphism and DNA sequencing. RESULTS: The three cases were diagnosed as mitochondrial myopathy. The examinations revealed that patient 1 had a homoplasmic A1627G mutation in tRNA-Val gene, and patient 2 had a heteroplasmic A1627G/A mutation in tRNA-Val gene, and patient 3 had two mutationsuone was homoplasmic T5554C mutation in tRNA-Trp gene, the other was heteroplasmic A10412C/A mutation in tRNA-Arg gene. CONCLUSION: tRNA genes mutations of mtDNA might be one of the etiologies of mitochondrial myopathy.

[Evaluation of the serum endothelin-1 level and sleep architecture change in patients with obstructive sleep apnea hypopnea syndrome accompanied hypertension].

OBJECTIVE: To explore the role of endothelin-1 (ET-1) in the pathogenesis of hypertension in obstructive sleep apnea hypopnea syndrome (OSAHS). METHODS: The levels of serum ET-1 in 30 OSAHS patients accompanied by hypertension, 30 normotensive OSAHS patients and 30 healthy controls were measured by ET-1 enzyme immunoassay kit. Meanwhile the correlation about the concentration of ET-1 in OSAHS patients with the clinic, polysomnography (PSG) parameters was analyzed. RESULTS: OSAHS patients with or without hypertension compared with snoring group and normal people, the sleep structure was significantly disturbed. The time percentages of awake and stage I sleep were increased, while stage II sleep decreased significantly in OSAHS patients than those in snoring group (P < 0.01, respectively). There were no significantly difference about the sleep structure in the two OSAHS groups. The levels of serum ET-1 (mean +/- s) were significantly higher in OSAHS patients accompanied by hypertension and normotensive OSAHS patients(42.5 +/- 8.4) ng/L and (38.6 +/- 4.7) ng/L respectively than those in the healthy controls(33.1 +/- 5.4) ng/L (P < 0.01, respectively). In the two OSAHS groups, the levels of serum ET-1 were significantly higher in OSAHS patients accompanied by hypertension than those in the normotensive OSAHS patients (P < 0.05). There were positive correlations between the concentration of ET-1 and the apnea hypopnea index (AHI) in all the 60 OSAHS patients with and without hypertension (r = 0.334, P < 0.01). There were negative correlations between the concentration of ET-1 and the lowest oxygen desaturation in all the 60 OSAHS patients with and without hypertension (r = -0.230, P < 0.05). CONCLUSION: These results indicate that the sleep disordered breathing and hypoxia may contribute to the elevation of ET-1 in the OSAHS patients and OSAHS patients accompanied by hypertension. ET-1 may play an important role in the pathogenesis of OSAHS-induced hypertension.