MGP+ and IDO1+ tumor-associated macrophages facilitate immunoresistance in breast cancer revealed by single-cell RNA sequencing.

Immunotherapy is widely applied for the treatment of breast cancer, but to which some patients respond poorly or develop resistance. Therefore, the mechanism needs to be further studied. Transcriptomic data of 31 breast cancer patients treated with anti-programmed death receptor 1 (PD-1) was downloaded from the VIB-KULeuven Center for Cancer Biology to analyze the changes in myeloid cells in tumor tissues before and after immunotherapy. And 24 cell populations that may be immune-related were further identified. Representative cell populations were also screened and validated through cellular and animal experiments to evaluate the relevant molecular expression and pathways of tumor-associated macrophages (TAMs) in the tumor microenvironment. The results demonstrated that MGP+ TAMs and IDO1+ TAMs influenced the efficacy of immunotherapy in breast cancer patients. After anti-PD-1 treatment, Increased numbers of MGP+ TAMs and IDO1+ TAMs in breast cancer patients upregulated pro-tumorigenic factors associated with resistance to immunosuppressive therapy. This study provides new biomarkers for immunotherapy to predict therapeutic responses and overcome potential resistance to immunotherapy. It is an important complement to the immunosuppression caused by TAMs after immunotherapy for breast cancer.

Utilizing a Dual Human Transporter MDCKII-MDR1-BCRP Cell Line to Assess Efflux at the Blood Brain Barrier.

To facilitate the design of drugs readily able to cross the blood brain barrier (BBB), a Madin-Darby canine kidney (MDCK) cell line was established that over expresses both P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), the main human efflux transporters of the BBB. Proteomics analyses indicate BCRP is expressed at a higher level than Pgp in this cell line. This cell line shows good activity for both transporters [BCRP substrate dantrolene efflux ratio (ER) 16.3 ± 0.9, Pgp substrate quinidine ER 27.5 ± 1.2], and use of selective transporter inhibitors enables an assessment of the relative contributions to overall ERs. The MDCKII-MDR1-BCRP ER negatively correlates with rat unbound brain/unbound plasma ratio, Kpuu Highly brain penetrant compounds with rat Kpuu ≥ 0.3 show ERs ≤ 2 in the MDCKII-MDR1-BCRP assay while compounds predominantly excluded from the brain, Kpuu ≤ 0.05, demonstrate ERs ≥ 20. A subset of compounds with MDCKII-MDR1-BCRP ER < 2 and rat Kpuu < 0.3 were shown to be substrates of rat Pgp using a rat transfected cell line, MDCKII-rMdr1a. These compounds also showed ERs > 2 in the human National Institutes of Health (NIH) MDCKI-MDR1 (high Pgp expression) cell line, which suggests that they are weak human Pgp substrates. Characterization of 37 drugs targeting the central nervous system in the MDCKII-MDR1-BCRP efflux assay show 36 have ERs < 2. In drug discovery, use of the MDCKII-MDR1-BCRP in parallel with the NIH MDCKI-MDR1 cell line is useful for identification of compounds with high brain penetration. SIGNIFICANCE STATEMENT: A single cell line that includes both the major human efflux transporters of the blood brain barrier (MDCKII-MDR1-BCRP) has been established facilitating the rapid identification of efflux substrates and enabling the design of brain penetrant molecules. Efflux ratios using this cell line demonstrate a clear relationship with brain penetration as defined by rat brain Kpuu.

Regional neuroinflammation induced by peripheral infection contributes to fatigue-like symptoms: a [18F]DPA-714 positron emission tomography study in rats.

Introduction: A series of symptoms, including fever, widespread pain, fatigue, and even ageusia, have frequently been reported in the context of various infections, such as COVID-19. Although the pathogenic mechanisms underlying an infection causing fever and pain have been well established, the mechanisms of fatigue induced by infection in specific brain regions remain unclear. Methods: To elucidate whether and how the peripheral infection cause fatigue via regional neuroinflammation, we performed a brain-wide investigation of neuroinflammation in a peripheral pseudoinfection rat model using [18F]DPA-714 positron emission tomography (PET) imaging analysis, in which the polyriboinosinic: polyribocytidylic acid (poly I:C) was intraperitoneally injected. Results: Transient fever lasting for several hours and subsequent suppression of spontaneous activity lasting a few days were induced by poly I:C treatment. Significant increase in plasma interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α were observed at 2 and 4 h following poly I:C treatment. PET imaging analysis revealed that the brain uptake of [18F]DPA-714 was significantly increased in several brain regions one day after poly I:C treatment, such as the dorsal raphe (DR), parvicellular part of red nucleus (RPC), A5 and A7 noradrenergic nucleus, compared with the control group. The accumulation of [18F]DPA-714 in the DR, RPC and A5 was positively correlated with subsequent fatigue-like behavior, and that in the A7 tended to positively correlate with fever. Discussion: These findings suggest that peripheral infection may trigger regional neuroinflammation, which may cause specific symptoms such as fatigue. A similar mechanism might be involved in COVID-19.

Cerebral Small Vessel Disease: Neuroimaging Features, Biochemical Markers, Influencing Factors, Pathological Mechanism and Treatment.

Cerebral small vessel disease (CSVD) is the most common chronic vascular disease involving the whole brain. Great progress has been made in clinical imaging, pathological mechanism, and treatment of CSVD, but many problems remain. Clarifying the current research dilemmas and future development direction of CSVD can provide new ideas for both basic and clinical research. In this review, the risk factors, biological markers, pathological mechanisms, and the treatment of CSVD will be systematically illustrated to provide the current research status of CSVD. The future development direction of CSVD will be elucidated by summarizing the research difficulties.

Study on the Correlation Between NF-κB and Central Fatigue.

In recent years, the World Health Organization (WHO) has included fatigue as a major risk factor for human life and health. The incidence rate of fatigue is high. In Europe and America, nearly 1/3 of the population is suffering from fatigue. Due to the acceleration of modern people's life rhythm and the increase of work pressure, more and more attention has been paid to central fatigue. The activation of NF-κB is related to central fatigue, which has been paid little attention by previous studies. At the same time, previous studies have mostly focused on the immune regulation function of NF-κB, while the NF-κB pathway plays an equally important role in regulating nerve function. NF-κB can participate in the occurrence and development of central fatigue by mediating immune inflammatory response, regulating central excitability and inhibitory transmitters, regulating synaptic plasticity and regulating central nervous system (CNS) functional genes. In addition to neuroprotective effects, NF-κB also has nerve damage effects, which is also closely related to the occurrence and development of central fatigue. In this review, we focus on the relationship between NF-κB pathway and central fatigue and further explore the biological mechanism of central fatigue. At the same time, the clinical application and potential of typical NF-κB inhibitors in the treatment of fatigue were analyzed to provide reference for the clinical treatment of central fatigue.

Alleviation of the chronic stress response attributed to the antioxidant and anti-inflammatory effects of electrolyzed hydrogen water.

Reactive oxygen species (ROS) are highly reactive and directly attack surrounding biomolecules to deteriorate cellular and tissue functions. Meanwhile, ROS also serve as signaling mediators to upregulate pro-inflammatory cytokine expression via activation of the nuclear factor kappa B signaling pathway, and the increased pro-inflammatory cytokines trigger respiratory burst of inflammatory cells that further accelerates ROS production in the inflamed tissue. Such crosstalk between ROS and inflammatory responses leads to a chain reaction of negativity, and cause progression of several chronic pathologies. Since molecular hydrogen is known to preferentially remove cytotoxic hydroxyl radicals and peroxynitrites, and to prevent cell and tissue damage, we here examined whether electrolyzed hydrogen water (EHW) enriched with molecular hydrogen and reactive hydrogen storing platinum nanoparticles dissolved from an electrode could alleviate oxidative stress and inflammation induced by continuous stress challenges. Five-day continuous stress loading to rats elevated reactive oxygen metabolites-derived compounds (d-ROMs), interleukin (IL)-1ß, and adrenocorticotropic hormone (ACTH) levels and decreased the biological antioxidant potential (BAP) level. Drinking EHW during 5-day continuous stress loading significantly alleviated all of these changes. The results suggest that EHW could suppress stress-response-associated oxidative stress and IL-1ß level elevation in vivo, and that drinking of EHW is effective for controlling stress responses via its antioxidant potential.

Homeostatic disturbance of thermoregulatory functions in rats with chronic fatigue.

Chronic fatigue syndrome (CFS) is characterized by long-lasting fatigue, and a range of symptoms, and is involved in homeostasis disruption. CFS patients frequently complain of low grade fever or chill even under normal body temperature indicating that thermosensory or thermoregulatory functions might be disturbed in CFS. However, little is known about the detailed mechanisms. To elucidate whether and how thermoregulatory function was altered during the development of chronic fatigue, we investigated temporal changes in body temperature with advance of fatigue accumulation in a chronic fatigue rat model using a wireless transponder. Our findings demonstrated that the body temperature was adaptively increased in response to fatigue loading in the early phase, but unable to retain in the late phase. The tail heat dissipation was often observed and the frequency of tail heat dissipation gradually increased initially, then decreased. In the late phase of fatigue loading, the body temperature for the tail heat dissipation phase decreased to a value lower than that for the non-dissipation phase. These results suggest that adaptive changes in thermoregulatory function occurred with fatigue progression, but this system might be disrupted by long-lasting fatigue, which may underlie the mechanism of fatigue chronification.

Modified Si-Ni-San Decoction Ameliorates Central Fatigue by Improving Mitochondrial Biogenesis in the Rat Hippocampus.

The traditional Chinese medicine (TCM) decoction Si-Ni-San (SNS) has been utilised for millennia to improve physiological coordination of the functions of the liver and spleen, which are regarded as the main pathological organs of central fatigue in TCM. This study evaluates the effect of a modified SNS (MSNS) formula on central fatigue in rats and explores molecular changes associated with hippocampal mitochondrial biogenesis. Central fatigue was induced through a 21-day sleep deprivation protocol. We assessed MSNS's effects on behaviour, blood and liver biomarkers, and mitochondrial ultrastructure. We found that MSNS could reverse various signs of central fatigue such as its effects on hippocampal gene and protein expression levels of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and nuclear respiratory factor 1 (NRF1). We also observed evidence of MSNS decreasing central fatigue, such as decreasing creatine kinase activity, decreasing levels of malondialdehyde and blood urea nitrogen, increasing lactate dehydrogenase and superoxide dismutase activities, increasing mitochondrial DNA copy number, and reversing mitochondrial ultrastructure changes. These findings suggest that MSNS can ameliorate central fatigue and that its molecular mechanism involves mitochondrial biogenesis enhancement mediated by hippocampal SIRT1, PGC-1α, and NRF1.

An Investigation into the Prediction of the Plasma Concentration-Time Profile and Its Interindividual Variability for a Range of Flavin-Containing Monooxygenase Substrates Using a Physiologically Based Pharmacokinetic Modeling Approach.

Our recent paper demonstrated the ability to predict in vivo clearance of flavin-containing monooxygenase (FMO) drug substrates using in vitro human hepatocyte and human liver microsomal intrinsic clearance with standard scaling approaches. In this paper, we apply a physiologically based pharmacokinetic (PBPK) modeling and simulation approach (M&S) to predict the clearance, area under the curve (AUC), and Cmax values together with the plasma profile of a range of drugs from the original study. The human physiologic parameters for FMO, such as enzyme abundance in liver, kidney, and gut, were derived from in vitro data and clinical pharmacogenetics studies. The drugs investigated include itopride, benzydamine, tozasertib, tamoxifen, moclobemide, imipramine, clozapine, ranitidine, and olanzapine. The fraction metabolized by FMO for these drugs ranged from 21% to 96%. The developed PBPK models were verified with data from multiple clinical studies. An attempt was made to estimate the scaling factor for recombinant FMO (rFMO) using a parameter estimation approach and automated sensitivity analysis within the PBPK platform. Simulated oral clearance using in vitro hepatocyte data and associated extrahepatic FMO data predicts the observed in vivo plasma concentration profile reasonably well and predicts the AUC for all of the FMO substrates within 2-fold of the observed clinical data; seven of the nine compounds fell within 2-fold when human liver microsomal data were used. rFMO overpredicted the AUC by approximately 2.5-fold for three of the nine compounds. Applying a calculated intersystem extrapolation scalar or tissue-specific scalar for the rFMO data resulted in better prediction of clinical data. The PBPK M&S results from this study demonstrate that human hepatocytes and human liver microsomes can be used along with our standard scaling approaches to predict human in vivo pharmacokinetic parameters for FMO substrates.

An Investigation into the Prediction of in Vivo Clearance for a Range of Flavin-containing Monooxygenase Substrates.

Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CLint) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CLint u) for each compound. This was compared with observed unbound intrinsic clearance (observed CLint u) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CLint u using hepatocytes (R2 = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained (R2 = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates.

[Peptide bond scission of staphylococcal enterotoxin C2 and related factors].

OBJECTIVE: To investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions. METHODS: The purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE. RESULT: The cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2). CONCLUSION: The recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.

Expression and bioactivity analysis of Staphylococcal enterotoxin M and N.

Staphylococcal enterotoxins (SEs) are powerful superantigens that stimulate non-specific T-cell proliferation produced by Staphylococcus aureus and draw considerable attention as ideal drugs for cancer therapy. The filtrate of S. aureus culture has been used as ampul named Staphylococcal enterotoxin C injection in clinic for 10 years in China and proved to be effective. The superantigen SEC claimed to be the only active component without certifiable evidences. For further investigations of the active components of this injection and establishment of foundations for the development of novel anti-cancer drugs, in this research we extracted total DNA from S. aureus (FRI 1230), cloned, expressed and purified recombinant proteins of Staphylococcal enterotoxin M and N (rSEM and rSEN). The MTT assay of the purified rSEM and rSEN demonstrated that their abilities of stimulating T cells and inhibiting the proliferation of K562-ADM cells and B16 cells were equivalent to that of purified SEC2 in vitro. These findings suggested that SEC was not the only active component of Staphylococcal enterotoxin C injection and the effective procedure of expression and purification may be useful for mass productions of these therapeutically important proteins.

[Expression and bioactivity analysis of staphylococcal enterotoxin C2].

AIM: To clone the gene of staphylococcal enterotoxin C2 and express it in the form of a soluble fusion protein in E. coli. Then the activation of SEC2 on mice lymphocyte and its lethal effects on tumor cells were studied. METHODS: Staphylococcus aureus SEC2 gene was cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEC2 was used to transform E. coli BL21, where the GST-SEC2 fusion protein was expressed efficiently. The rSEC2 protein was purified with Glutathione Sepharose 4B affinity column and digested with thrombin. The in vitro culture system was utilized to observe the activation of the SEC2 on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte. RESULTS: The proper gene of SEC2 was cloned and purified rSEC2 was obtained. The MTT results indicated that rSEC2 have strong ability to stimulate mice lymphocyte to proliferate with a dose-dependent manner. With the proliferation of mice splenic lymphocyte, rSEC2 has a strong lethal effect on tumor cells B16, K562 and K562-AD. CONCLUSION: In this study, the gene of SEC2 was cloned and the rSEC2 protein was obtained, which had strong lethal effect on tumor cells B16, K562 and K562-AD.