Osteoarthritis (OA) causes chronic pain that significantly impairs quality of life, with current treatments often proving insufficient and accompanied by adverse effects. Recent research has identified the dorsal root ganglion (DRG) and its resident macrophages as crucial mediators of chronic OA pain through neuroinflammation driven by macrophage polarization. We present a novel injectable thermo-sensitive hydrogel system, KAF@PLEL, designed to deliver an anti-inflammatory peptide (KAF) specifically to the DRG. This biodegradable hydrogel enables sustained KAF release, promoting the reprogramming of DRG macrophages from pro-inflammatory to anti-inflammatory phenotypes. Through comprehensive in vitro and in vivo studies, we evaluated the hydrogel's biocompatibility, effects on macrophage polarization, and therapeutic efficacy in chronic OA pain management. The system demonstrated significant capabilities in preserving macrophage mitochondrial function, suppressing neuroinflammation, alleviating chronic OA pain, reducing cartilage degradation, and improving motor function in OA rat models. The sustained-release properties of KAF@PLEL enabled prolonged therapeutic effects while minimizing systemic exposure and side effects. These findings suggest that KAF@PLEL represents a promising therapeutic approach for improving outcomes in OA patients through targeted, sustained treatment.

Objective:To investigate immunotherapy effects and mechanism of BCG and recombinant BCG(rBCG)with c-di-AMP as adjuvant on melanoma in mice model.Methods:Melanoma mice model was established by B16F10 cell subcutaneous injec-tion in groin,and treated with 1×106 CFU of BCG and rBCG by adjacent injection of subcutaneous tumor for 3 times,respectively.Survival of melanotic mice,tumor growth and metastasis were observed.Tumor tissues of mice were isolated to prepare cell suspen-sion,and proportion of immune cells were detected by flow cytometry.Transcriptional levels of immune-related genes in tumor tissues were detected by qRT-PCR.Results:Both BCG and rBCG immunotherapy could significantly inhibit growth in melanoma mice and prolong survival time of mice.rBCG showed better inhibition on metastasis than BCG.Both strains significantly reduced proportion of M2-type macrophages and myeloid-derived suppressor cell associated with tumor growth and metastasis.Both two strains promoted infiltration of lymphocytes in tumor tissues,and rBCG significantly increased proportion of B cells in tumor.BCG immunotherapy upregulated transcription levels of metastasis-related cytokines,while rBCG therapy had no effects on transcriptions of these genes.Conclusion:Both BCG and rBCG have immunotherapeutic effects on melanotic mice,and rBCG with c-di-AMP as adjuvant shows better inhibition on tumor metastasis than BCG,which mechanism was related to regulation of immune response in tumor tissues.

Objective:To investigate immunotherapy effects and mechanism of BCG and recombinant BCG(rBCG)with c-di-AMP as adjuvant on melanoma in mice model.Methods:Melanoma mice model was established by B16F10 cell subcutaneous injec-tion in groin,and treated with 1×106 CFU of BCG and rBCG by adjacent injection of subcutaneous tumor for 3 times,respectively.Survival of melanotic mice,tumor growth and metastasis were observed.Tumor tissues of mice were isolated to prepare cell suspen-sion,and proportion of immune cells were detected by flow cytometry.Transcriptional levels of immune-related genes in tumor tissues were detected by qRT-PCR.Results:Both BCG and rBCG immunotherapy could significantly inhibit growth in melanoma mice and prolong survival time of mice.rBCG showed better inhibition on metastasis than BCG.Both strains significantly reduced proportion of M2-type macrophages and myeloid-derived suppressor cell associated with tumor growth and metastasis.Both two strains promoted infiltration of lymphocytes in tumor tissues,and rBCG significantly increased proportion of B cells in tumor.BCG immunotherapy upregulated transcription levels of metastasis-related cytokines,while rBCG therapy had no effects on transcriptions of these genes.Conclusion:Both BCG and rBCG have immunotherapeutic effects on melanotic mice,and rBCG with c-di-AMP as adjuvant shows better inhibition on tumor metastasis than BCG,which mechanism was related to regulation of immune response in tumor tissues.

We investigated the prevalence and genetic variation of porcine pseudorabies virus(PRV)in Hunan Province.A total of 18 861 serum samples and 1 725 suspected PRV-infected tissue sam-ples were collected from Hunan Province from 2021 to 2022.ELISA and qPCR assays were used to detect PRV-gE antibodies in serum samples and the PRV-gE nucleic acids in tissue samples.The positive rates of samples in different years and seasons were analyzed correspondingly.Moreover,the gC gene sequences of 15 PRV-positive samples were amplified,sequenced,and analyzed.The results showed that 2 004 serum and 56 tissue samples tested positive,with an average positive rate of 10.74％and 3.25％,respectively.The sequence analysis showed that the nucleotide and ami-no acid sequence identities of the gC gene among 15 PRV-positive samples were 98.4％-100.0％and 98.8％-100.0％,respectively.Further genetic evolution analysis showed that 13 PRV strains belonged to the same branch with the PRV variants,while two strains were clustered into the same branch with the classical PRV strains.The above results indicate that PRV remains widely prevalent in different regions of Hunan Province,and the variant strain is the dominant epi-demic subtype,which provided a scientific basis for exploring the epidemiological characteristics of PRV and the development of vaccines against PR in Hunan Province.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) has been recently identified as a neurotrophic factor, but its role in hepatic fibrosis is unknown. Here, we found that MANF was upregulated in the fibrotic liver tissues of the patients with chronic liver diseases and of mice treated with CCl₄. MANF deficiency in either hepatocytes or hepatic mono-macrophages, particularly in hepatic mono-macrophages, clearly exacerbated hepatic fibrosis. Myeloid-specific MANF knockout increased the population of hepatic Ly6C^high macrophages and promoted HSCs activation. Furthermore, MANF-sufficient macrophages (from WT mice) transfusion ameliorated CCl₄-induced hepatic fibrosis in myeloid cells-specific MANF knockout (MKO) mice. Mechanistically, MANF interacted with S100A8 to competitively block S100A8/A9 heterodimer formation and inhibited S100A8/A9-mediated TLR4-NF-κB signal activation. Pharmacologically, systemic administration of recombinant human MANF significantly alleviated CCl₄-induced hepatic fibrosis in both WT and hepatocytes-specific MANF knockout (HKO) mice. This study reveals a mechanism by which MANF targets S100A8/A9-TLR4 as a "brake" on the upstream of NF-κB pathway, which exerts an impact on macrophage differentiation and shed light on hepatic fibrosis treatment.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting both upper and lower motor neurons (MNs) with large unmet medical needs. Multiple pathological mechanisms are considered to contribute to the progression of ALS, including neuronal oxidative stress and mitochondrial dysfunction. Honokiol (HNK) has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke, Alzheimer's disease and Parkinson's disease. Here we found that honokiol also exhibited protective effects in ALS disease models both in vitro and in vivo. Honokiol improved the viability of NSC-34 motor neuron-like cells that expressed the mutant G93A SOD1 proteins (SOD1-G93A cells for short). Mechanistical studies revealed that honokiol alleviated cellular oxidative stress by enhancing glutathione (GSH) synthesis and activating the nuclear factor erythroid 2-related factor 2 (NRF2)-antioxidant response element (ARE) pathway. Also, honokiol improved both mitochondrial function and morphology via fine-tuning mitochondrial dynamics in SOD1-G93A cells. Importantly, honokiol extended the lifespan of the SOD1-G93A transgenic mice and improved the motor function. The improvement of antioxidant capacity and mitochondrial function was further confirmed in the spinal cord and gastrocnemius muscle in mice. Overall, honokiol showed promising preclinical potential as a multiple target drug for ALS treatment.

Objective:To analyze the detection strategy and basic detection situation of markers of infectious diseases transmitted by transfusion in blood testing laboratories of blood stations in China.Methods:Based on the data of practice comparison working party of Blood Stations in Mainland of China from 2017 to 2021, the data on the testing strategies and the basic detection information of the markers for the transmission of infectious diseases through transfusion in the member laboratories of the practice comparison working party of Blood Stations in Mainland of China from 2017 to 2021 were collected, and the situation of the selection for testing markers, testing strategy and the testing method and other relevant aspects were sorted out and analyzed by charts.Results:The selection of the testing markers was consistent, but HTLV testing item was added in some member laboratories. The detection strategy of using two ELISA reagents and one nucleic acid testing (NAT) reagent simultaneously was adopted in 47 member blood stations; 3) NAT method was dominated by mini pool-NAT in member laboratories. The number of members adopting mini-pools of 8 (MP8)-NAT decreased from 17 in 2017 to 14 in 2021, while the number of members adopting mini-pools of 6 (MP6)-NAT increased from 13 in 2017 to 22 in 2021; Roche NAT system accounted for the largest proportion.Conclusions:In order to ensure blood safety and avoid missing detection, the blood stations still adopt the detection strategy of using two ELISA reagents and one nucleic acid testing (NAT) reagent simultaneously; Meanwhile, in order to increase the NAT positive rate, the proportion of mini pool-NAT mainly decreased year by year despite its dominating role, while the proportion of individual donation-NAT increased year by year; NAT method is transiting from mini-pools of 8 (MP8) to mini-pools of 6 (MP6); The proportion of imported NAT system used in NAT laboratory is relatively large.

OBJECTIVE：To establish the content determin ation method of ferulic acid ，verbascoside，ligustilide and astragaloside in Shengyu decoction lyophilized powder. METHODS ：HPLC method was adopted to determine 4 components in 3 batches of lyophilized powder. The determination of ferulic acid ，verbascoside and ligustilide was performed on Inertsil ODS-SP C 18 column with mobile phase consisted of methanol- 0.1％ phosphoric acid （gradient elution ）at the flow rate of 1.0 mL/min；detector was diode array detector ；detection wavelength was set at 330 nm；column temperature was 30 ℃，the sample size was 10 μL. The determination of astragaloside was performed on Kromasil C 18 column with mobile phase consisted of acetonitrile-water （32∶68，V/ V）；detector was evaporative light scattering detector ；the drift tube temperature wa s 100 ℃，the carrier gas （air）flow rate was 2.5 L/min at the flow rate of 1.0 mL/min；column temperature was 30 ℃，the sample size was 10 μL. RESULTS：The linear ranges of ferulic acid ，verbascoside，ligustilide and astragaloside were 0.050 15-10.03 μg（r＝0.999 8），0.067 80-13.56 μg（r＝ 0.999 9），0.057 30-11.46 μg（r＝0.999 5），1.128-11.28 μg（r＝0.999 3），respectively. The detection limits were 2.12×10－4，1.30× 10－3，8.02×10－4，1.09×10－3 μg，respectively. The limit of quantification were 7.43×10－4，3.87×10－3，2.34×10－3，3.36×10－3 μg， respectively. RSDs of precision ，stability（12 h）and reproducibility tests were all lower than 2％（n＝6）. Average recovery rates were 99.6％（RSD＝0.83％，n＝6），100.9％（RSD＝1.07％，n＝6），98.8％（RSD＝0.84％，n＝6）and 101.3％（RSD＝0.99％， n＝6），respectively. The contents of ferulic acid ，verbascoside，ligustilide and astragaloside in 3 batches of samples were 1.225-1.248， 0.413-0.424， 0.325-0.332， 0.394-0.404 mg/g， respectively （RSDs among batches were lower than 1.5％ ）. CONCLUSIONS：Established method is stable ，reproducible，rapid and accurate for the content determination of ferulic acid ， verbascoside， ligustilide and astragaloside in Shengyu

In recent years, 16S rRNA amplicon sequencing technology has been widely used to study human gut microbiota and to detect unknown pathogens in clinical samples. However, its resolution to bacterial population can only reach the relative abundance of genus level, and different factors affect the final bacterial profile, such as sample concentrations, PCR cycle numbers and amplification primers. In order to solve these problems, we developed a quantitative 16S rRNA amplicon sequencing method by combining random tag and internal marker method. The new methods improved the accuracy of human gut microbiota, reduced the impact of experimental operation on the results, and improved the comparability between sequencing and other molecular biological methods.
Bacteria/genetics* ; Gastrointestinal Microbiome/genetics* ; High-Throughput Nucleotide Sequencing ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics*

Objective:To investigate the clinicopathological characteristics of the T cell lymphomas with CD20 expression, and to better understand this rare entity.Methods:Two-hundred cases of T-cell lymphoma diagnosed in the Department of Pathology of the Affiliated Hospital of Qingdao University from November 2016 to February 2020 were examined, and 5 cases of CD20-positive T-cell lymphomas were identified and included. Combined with clinical data and review of the literature, the clinicopathological characteristics of the disease were analyzed.Results:The five patients were all male, and had an average age of 56 years (range, 47 to 64 years). There were 2 cases of monomorphic epitheliotropic intestinal T-cell lymphoma, 2 cases of mycosis fungoides (1 case was plaque stage and the other was tumor stage) and 1 case of indolent T-cell lymphoproliferative disorder of the gastrointestinal tract. Immunohistochemistry showed that all 5 cases expressed multiple T cell markers (CD3/CD4/CD5/CD7/CD8) and only one of B cell markers (CD20). Three of the 5 cases were negative for CD20 at the first diagnosis, while CD20 was diffusely positive on the second biopsy from the recurrence or progression of the disease, without expression of CD79a or PAX5. Epstein-Barr encoding region (EBER) in situ hybridization was negative in all 5 cases. T-cell receptor gene analysis showed monoclonal rearrangement of β or/and δ chains;Ig rearrangements were all polyclonal. None of the five patients were treated with rituximab, and 4 patients survived with disease and 1 patient survived without disease at the end of follow-up. Among them, the patient with mycosis fungoides at the cancerous stage has progressed rapidly and had poor quality of life.Conclusions:CD20-positive T-cell lymphoma is extremely rare. Its prognosis is closely related to the type of T-cell lymphoma, clinical stage and initial therapeutic effect. However, the expression of CD20 indicates the recurrence or progression of the disease, and the prognosis is relatively poor. When CD3 expression is absent in T-cell lymphoma, it is easy to be misdiagnosed as B-cell lymphoma. The combination of multiple immunohistochemical antibodies and molecular detection can improve the accuracy of diagnosis.