OBJECTIVES: To characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues. METHODS: Leukoreduction filters containing residues from 400 mL whole blood units (n=6) were collected from a blood center. Filters were back-flushed with normal saline, and the eluate was concentrated to obtain leukoreduction filter residues. Leukocytes in the residues were counted by dual-fluorescence staining. DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation. Both cryopreserved and fresh unstimulated DNT cells derived from the residues were subjected to in vitro culture. Following culture, cells were assessed for expansion fold, viability, immunophenotype, differentiation status, and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry, with comparisons made to DNT cells derived from whole blood. RESULTS: The leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was (41.9±14.7)%. Compared to whole blood, the DNT cell starting material obtained from filter residues showed no significant difference in total T-cell content (P>0.05). However, the viability and purity of the resulting DNT cell starting materials were significantly lower (both P<0.05). After 17 days of culture, DNT cells from filter residues and whole blood showed no significant differences in expansion fold, immunophenotype, differentiation status, or cytotoxicity toward target cells (all P>0.05). However, the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells [(86.0±4.2)% vs. (92.2±1.2)%, P<0.05]. After thawing (post 3 or 15 days of cryopreservation) and 17 days of culture, DNT cell starting materials from residues showed comparable immunophenotype, expansion fold, and differentiation status to their non-cryopreserved counterparts from the same source (all P>0.05). However, the viability of DNT cells cryopreserved for 3 days [92.4% (91.8%, 92.8%)] and the cytotoxicity against target cells of those cryopreserved for 15 days [91.3% (89.4%, 95.1%)] were significantly higher than those of non-cryopreserved DNT cells [87.8% (82.0%, 89.0%) and 70.9% (67.3%, 80.2%), respectively] (P<0.05). CONCLUSIONS DNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion, purity, differentiation, and biological potency. Furthermore, their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells. These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf, allogeneic DNT cell therapeutics.

Objective:To analyze the latent prevalence of hepatitis B and type 2 diabetes and their correlation through an observational study.Methods:This study used a case-control design. The cases with diabetes were recruited through the diabetes management system and village doctors, while the controls without diabetes were screened from volunteers recruited by village health clinics. Capillary blood samples were collected from the study participants for the measurement of real-time blood glucose level, and venous blood samples were taken from them for the detections of HBV serological markers. Firth logistic regression model was used to fit the relationship between HBsAg positive status and diabetes status.Results:The study included 1 218 diabetes patients, 62 patients with impaired fasting glucose and 491 cases without diabetes. In the cases without diagnosis of diabetes, 11.15% had impaired fasting blood glucose and 4.43% had diabetes. Among those who reported no or unknown diagnosis of hepatitis B, 1.73% were positive for HBsAg, while 18.80% were positive for both HBV core antibody and surface antibody, indicating latent infection of hepatitis B virus. In the non-diabetes group, 0.81% reported hepatitis B history, and in the diabetes group, 2.76% reported hepatitis B history. After adjustment, the HBsAg positive rate was higher in the diabetes group ( OR=2.90, 95% CI: 1.21-6.91). Conclusions:Both diabetes and hepatitis B exhibited a high degree of latent prevalence. The HBsAg positive rate was significantly higher in those with diabetes than in those without diabetes, indicating a potential correlation. These findings highlighted the importance of strengthened screening and management of comorbidities.

Objective:Exploration of the immunogenicity and persistence of three different immunization regimens of hepatitis B vaccines in diabetic patients.Methods:Participants with diabetes and non-diabetic individuals were recruited from study sites and assigned to different vaccination regimens: the diabetic group (①D60Yeast0-1: received 60 μg Saccharomyces cerevisiae-derived recombinant HBV vaccine on a 0-1-month schedule; ②D20Yeast0-1-6: received 20 μg Saccharomyces cerevisiae-derived recombinant HBV vaccine on a 0-1-6-month schedule; ③D20CHO0-1-6: received 20 μg Chinese hamster ovary (CHO) cell-derived recombinant HBV vaccine on a 0-1-6-month schedule) and the non-diabetic group (ND20Yeast0-1-6: non-diabetic individuals received 20 μg Saccharomyces cerevisiae-derived recombinant HBV vaccine on a 0-1-6-month schedule). Venous blood samples were collected at 1,12, and 48 months post-full vaccination to measure anti-HBs levels. Differences in immunogenicity between diabetic and non-diabetic groups, as well as among diabetic subgroups, were analyzed.Results:This study enrolled a total of 564 subjects. In the D20CHO0-1-6 group, the seroconversion rate decreased from 90.72% (95% CI: 84.84%-96.60%) at 1 month to 74.23% (95% CI: 65.37%-83.08%) at 48 months, and the antibody geometric mean concentration (GMC) decreased from 676.08 (95% CI: 389.05- 1 148.20) mIU/ml at 1 month to 33.11 (95% CI: 23.44-46.77) mIU/ml at 48 months. In the D20Yeast0-1-6 group, the seroconversion rate declined from 93.81% (95% CI: 89.29%-98.32%) at 1 month to 63.72% (95% CI: 54.71%-72.72%) at 48 months, with antibody GMC dropping from 630.96 (95% CI: 407.40-954.99) mIU/ml to 25.70 (95% CI: 17.78-38.02) mIU/ml over the same period. For the D60Yeast0-1 group, seroconversion rate fell from 82.03% (95% CI: 75.29%-88.77%) to 56.25% (95% CI: 47.54%-64.96%), and antibody GMC decreased from 81.28 (95% CI: 51.29-128.82) mIU/ml to 15.49 (95% CI: 11.75-20.89) mIU/ml between 1 and 48 months. The ND20Yeast0-1-6 group (non-diabetic control) exhibited a higher initial seroconversion rate of 97.56% (95% CI: 94.80%- 100.00%) at 1 month, but it still declined to 76.42% (95% CI: 68.82%-84.03%) at 48 months, with antibody GMC decreasing from 1 318.30 (95% CI: 912.01- 1 905.50) mIU/ml to 34.67 (95% CI: 25.12-47.86) mIU/ml. Multivariate analysis on factors influencing the GMC of antibodies revealed statistically significant differences in antibody GMC between the D20Yeast0-1-6 group and ND20Yeast0-1-6 group at 12 months (a OR=0.73, 95% CI: 0.58-0.93) and 48 months (a OR=0.79, 95% CI: 0.63-0.99) post-vaccination (all P<0.05). As for the diabetic population, when compared with the D20Yeast0-1-6 group, the D60Yeast0-1 group also showed statistically significant differences in antibody GMC at 12 months (a OR=0.57, 95% CI: 0.44-0.74) and 48 months (a OR=0.60, 95% CI: 0.47-0.76)(all P<0.05). Conclusions:The seroconversion rate and antibody GMC gradually decreased over time (1, 12, and 48 months) in the four groups. Diabetic patients showed poor immunogenicity and persistence to hepatitis B vaccines. The immunogenicity and persistence of hepatitis B vaccination in diabetic patients were associated with vaccine type, antigen dose, and vaccination regimen. The CHO cell-recombinant hepatitis B vaccine demonstrated better performance in terms of immunogenicity and persistence among the diabetic population.

BACKGROUND: Severe stroke has high rates of mortality and morbidity. This study aimed to investigate the clinical course, causes of worsening, and outcomes of severe ischemic stroke. METHODS: This prospective, multicenter cohort study enrolled adult patients admitted ≤30 days after ischemic stroke from nine hospitals in China between September 2017 and December 2019. Severe stroke was defined as a score of ≥15 on the National Institutes of Health Stroke Scale (NIHSS). Clinical worsening was defined as an increase of 4 in the NIHSS score from baseline. Unfavorable functional outcome was defined as a modified Rankin scale score ≥3 at 3 months and 1 year after stroke onset, respectively. We performed Logistic regression to explore baseline features and reperfusion therapies associated with clinical worsening and functional outcomes. RESULTS: Among 4201 patients enrolled, 854 patients (20.33%) had severe stroke on admission. Of 3347 patients without severe stroke on admission, 142 (4.24%) patients developed severe stroke in hospital. Of 854 patients with severe stroke on admission, 33.95% (290/854) experienced clinical worsening (median time from stroke onset: 43 h, Q1-Q3: 20-88 h), with brain edema (54.83% [159/290]) as the leading cause; 24.59% (210/854) of these patients died by 30 days, and 81.47% (677/831) and 78.44% (633/807) had unfavorable functional outcomes at 3 months and 1 year respectively. Reperfusion reduced the risk of worsening (adjusted odds ratio [OR]: 0.24, 95% confidence interval [CI]: 0.12-0.49, P  <0.01), 30-day death (adjusted OR: 0.22, 95% CI: 0.11-0.41, P  <0.01), and unfavorable functional outcomes at 3 months (adjusted OR: 0.24, 95% CI: 0.08-0.68, P <0.01) and 1 year (adjusted OR: 0.17, 95% CI: 0.06-0.50, P  <0.01). CONCLUSIONS: Approximately one-fifth of patients with ischemic stroke had severe neurological deficits on admission. Clinical worsening mainly occurred in the first 3 to 4 days after stroke onset, with brain edema as the leading cause of worsening. Reperfusion reduced the risk of clinical worsening and improved functional outcomes. REGISTRATION ClinicalTrials.gov , NCT03222024.
Humans ; Male ; Female ; Prospective Studies ; Ischemic Stroke/mortality* ; Aged ; Middle Aged ; Aged, 80 and over ; Stroke ; Brain Ischemia

Objective:To identify core genes with co-expression trends in the suprachiasmatic nucleus (SCN) and lateral habenula (LHb) through miRNA sequencing, and to explore the molecular mechanisms underlying the onset and progress of sleep disturbances in depression.Methods:A rat model of depression with sleep disturbances was established. Behavioral assessments were conducted using the pentobarbital-induced sleep test, sucrose water consumption test, and open field test. Differentially expressed genes (DEGs) in the SCN and LHb of model rats were analyzed using biological replicates with the limma package and DESeq2 software. Gene ontology (GO), KEGG, Reactome pathway-gene set enrichment analysis (GSEA) were performed on the DEGs.Co-expression networks and protein-protein interaction (PPI) networks were constructed using R and Cytoscape software for further screening of genes with potential regulatory roles.Results:After modeling, the sleep latency in the model group ((679.88±350.05) s) was longer than that in the normal group ((316.25±87.35) s) ( t=-2.851, P=0.022), while the sleep duration in the model group ((33.13±8.41) min) was shorter than that in the normal group ((69.72±25.11) min) ( t=3.907, P=0.004). Additionally, the model group exhibited lower sucrose water consumption, horizontal movement counts, and vertical movement counts compared to the normal group (all P<0.05). Differential expression gene analysis showed, in the SCN, 379 genes were upregulated and 132 genes were downregulated, with the top five most significantly differentially expressed genes being Sec61g, Cox6b1, Ephx2, Thrsp, and Pak2. In the LHb, 1 657 genes were upregulated and 1 737 were downregulated, with the top five most significantly differentially expressed genes being Mrpl32, Mrpl20, Mrpl27, Celsr2, and Uba52. Biological pathways enriched in the SCN were mainly associated with the positive regulation of responses to external and biological stimuli. In contrast, pathways enriched in the LHb were related to neurotransmitter level regulation, developmental growth, neurotransmitter transport, and processes such as learning and memory. Co-expression network analysis identified 864 interactions involving 100 nodes in the SCN and 524 interactions involving 99 nodes in the LHb. Conclusion:The biological functions and signaling pathways of the suprachiasmatic nucleus and lateral habenula are different in the occurrence and development of depression with sleep disturbance, and the significantly differentially expressed genes may play an important role.

Objective:To identify core genes with co-expression trends in the suprachiasmatic nucleus (SCN) and lateral habenula (LHb) through miRNA sequencing, and to explore the molecular mechanisms underlying the onset and progress of sleep disturbances in depression.Methods:A rat model of depression with sleep disturbances was established. Behavioral assessments were conducted using the pentobarbital-induced sleep test, sucrose water consumption test, and open field test. Differentially expressed genes (DEGs) in the SCN and LHb of model rats were analyzed using biological replicates with the limma package and DESeq2 software. Gene ontology (GO), KEGG, Reactome pathway-gene set enrichment analysis (GSEA) were performed on the DEGs.Co-expression networks and protein-protein interaction (PPI) networks were constructed using R and Cytoscape software for further screening of genes with potential regulatory roles.Results:After modeling, the sleep latency in the model group ((679.88±350.05) s) was longer than that in the normal group ((316.25±87.35) s) ( t=-2.851, P=0.022), while the sleep duration in the model group ((33.13±8.41) min) was shorter than that in the normal group ((69.72±25.11) min) ( t=3.907, P=0.004). Additionally, the model group exhibited lower sucrose water consumption, horizontal movement counts, and vertical movement counts compared to the normal group (all P<0.05). Differential expression gene analysis showed, in the SCN, 379 genes were upregulated and 132 genes were downregulated, with the top five most significantly differentially expressed genes being Sec61g, Cox6b1, Ephx2, Thrsp, and Pak2. In the LHb, 1 657 genes were upregulated and 1 737 were downregulated, with the top five most significantly differentially expressed genes being Mrpl32, Mrpl20, Mrpl27, Celsr2, and Uba52. Biological pathways enriched in the SCN were mainly associated with the positive regulation of responses to external and biological stimuli. In contrast, pathways enriched in the LHb were related to neurotransmitter level regulation, developmental growth, neurotransmitter transport, and processes such as learning and memory. Co-expression network analysis identified 864 interactions involving 100 nodes in the SCN and 524 interactions involving 99 nodes in the LHb. Conclusion:The biological functions and signaling pathways of the suprachiasmatic nucleus and lateral habenula are different in the occurrence and development of depression with sleep disturbance, and the significantly differentially expressed genes may play an important role.

Objective:To analyze the latent prevalence of hepatitis B and type 2 diabetes and their correlation through an observational study.Methods:This study used a case-control design. The cases with diabetes were recruited through the diabetes management system and village doctors, while the controls without diabetes were screened from volunteers recruited by village health clinics. Capillary blood samples were collected from the study participants for the measurement of real-time blood glucose level, and venous blood samples were taken from them for the detections of HBV serological markers. Firth logistic regression model was used to fit the relationship between HBsAg positive status and diabetes status.Results:The study included 1 218 diabetes patients, 62 patients with impaired fasting glucose and 491 cases without diabetes. In the cases without diagnosis of diabetes, 11.15% had impaired fasting blood glucose and 4.43% had diabetes. Among those who reported no or unknown diagnosis of hepatitis B, 1.73% were positive for HBsAg, while 18.80% were positive for both HBV core antibody and surface antibody, indicating latent infection of hepatitis B virus. In the non-diabetes group, 0.81% reported hepatitis B history, and in the diabetes group, 2.76% reported hepatitis B history. After adjustment, the HBsAg positive rate was higher in the diabetes group ( OR=2.90, 95% CI: 1.21-6.91). Conclusions:Both diabetes and hepatitis B exhibited a high degree of latent prevalence. The HBsAg positive rate was significantly higher in those with diabetes than in those without diabetes, indicating a potential correlation. These findings highlighted the importance of strengthened screening and management of comorbidities.

Objective:Exploration of the immunogenicity and persistence of three different immunization regimens of hepatitis B vaccines in diabetic patients.Methods:Participants with diabetes and non-diabetic individuals were recruited from study sites and assigned to different vaccination regimens: the diabetic group (①D60Yeast0-1: received 60 μg Saccharomyces cerevisiae-derived recombinant HBV vaccine on a 0-1-month schedule; ②D20Yeast0-1-6: received 20 μg Saccharomyces cerevisiae-derived recombinant HBV vaccine on a 0-1-6-month schedule; ③D20CHO0-1-6: received 20 μg Chinese hamster ovary (CHO) cell-derived recombinant HBV vaccine on a 0-1-6-month schedule) and the non-diabetic group (ND20Yeast0-1-6: non-diabetic individuals received 20 μg Saccharomyces cerevisiae-derived recombinant HBV vaccine on a 0-1-6-month schedule). Venous blood samples were collected at 1,12, and 48 months post-full vaccination to measure anti-HBs levels. Differences in immunogenicity between diabetic and non-diabetic groups, as well as among diabetic subgroups, were analyzed.Results:This study enrolled a total of 564 subjects. In the D20CHO0-1-6 group, the seroconversion rate decreased from 90.72% (95% CI: 84.84%-96.60%) at 1 month to 74.23% (95% CI: 65.37%-83.08%) at 48 months, and the antibody geometric mean concentration (GMC) decreased from 676.08 (95% CI: 389.05- 1 148.20) mIU/ml at 1 month to 33.11 (95% CI: 23.44-46.77) mIU/ml at 48 months. In the D20Yeast0-1-6 group, the seroconversion rate declined from 93.81% (95% CI: 89.29%-98.32%) at 1 month to 63.72% (95% CI: 54.71%-72.72%) at 48 months, with antibody GMC dropping from 630.96 (95% CI: 407.40-954.99) mIU/ml to 25.70 (95% CI: 17.78-38.02) mIU/ml over the same period. For the D60Yeast0-1 group, seroconversion rate fell from 82.03% (95% CI: 75.29%-88.77%) to 56.25% (95% CI: 47.54%-64.96%), and antibody GMC decreased from 81.28 (95% CI: 51.29-128.82) mIU/ml to 15.49 (95% CI: 11.75-20.89) mIU/ml between 1 and 48 months. The ND20Yeast0-1-6 group (non-diabetic control) exhibited a higher initial seroconversion rate of 97.56% (95% CI: 94.80%- 100.00%) at 1 month, but it still declined to 76.42% (95% CI: 68.82%-84.03%) at 48 months, with antibody GMC decreasing from 1 318.30 (95% CI: 912.01- 1 905.50) mIU/ml to 34.67 (95% CI: 25.12-47.86) mIU/ml. Multivariate analysis on factors influencing the GMC of antibodies revealed statistically significant differences in antibody GMC between the D20Yeast0-1-6 group and ND20Yeast0-1-6 group at 12 months (a OR=0.73, 95% CI: 0.58-0.93) and 48 months (a OR=0.79, 95% CI: 0.63-0.99) post-vaccination (all P<0.05). As for the diabetic population, when compared with the D20Yeast0-1-6 group, the D60Yeast0-1 group also showed statistically significant differences in antibody GMC at 12 months (a OR=0.57, 95% CI: 0.44-0.74) and 48 months (a OR=0.60, 95% CI: 0.47-0.76)(all P<0.05). Conclusions:The seroconversion rate and antibody GMC gradually decreased over time (1, 12, and 48 months) in the four groups. Diabetic patients showed poor immunogenicity and persistence to hepatitis B vaccines. The immunogenicity and persistence of hepatitis B vaccination in diabetic patients were associated with vaccine type, antigen dose, and vaccination regimen. The CHO cell-recombinant hepatitis B vaccine demonstrated better performance in terms of immunogenicity and persistence among the diabetic population.

Objective:To investigate the interactive effect of mismatch repair (MMR) protein status and adverse clinicopathological features on prognosis of stage Ⅰ-Ⅲ colon cancer.Methods:The retrospective cohort study was conducted. The clinicopathological data of 1 650 patients with colon cancer of stage Ⅰ-Ⅲ who were admitted to 7 hospitals in China from January 2016 to December 2017 were collected. There were 963 males and 687 females, aged 62(53,71)years. Patients were classified as 230 cases of MMR deficiency (dMMR) and 1 420 cases of MMR proficiency (pMMR) based on their MMR protein status. Observation indicators: (1) comparison of clinicopathological characteristics between patients of different MMR protein status; (2) analysis of factors affecting the survival outcomes of patients of dMMR; (3) analysis of factors affecting the survival outcomes of patients of pMMR; (4) interaction analysis of MMR and adverse clinicopathological features on survival outcomes. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the independent t test. Measurement data with skewed distribution were represented as M( Q1, Q3), and comparison between groups was conducted using the Mann-Whitney U test. Count data were described as absolute numbers, and comparison between groups was conducted using the chi-square test or Fisher exact probability. Comparison of ordinal data was conducted using the Mann-Whitney U test. The random forest interpolation method was used for missing values in data interpolation. Univariate analysis was conducted using the COX proportional risk regression model, and multivariate analysis was conducted using the COX stepwise regression with forward method. The coefficient of multiplication interaction effect was obtained using the interaction term coefficient of COX proportional risk regression model. Evaluation of additive interaction effects was conducted using the relative excess risk due to interaction ( RERI). Results:(1) Comparison of clinicopathological characteristics between patients of different MMR protein status. There were significant differences in age, T staging, the number of lymph node harvest, the number of lymph node harvest <12, high grade tumor between patients of dMMR and pMMR ( P<0.05). (2) Analysis of factors affecting the survival outcomes of patients of dMMR. Results of multivariate analysis showed that T staging, N staging, the number of lymph node harvest <12 were independent factors affecting the disease-free survival (DFS) of colon cancer patients of dMMR ( hazard ratio=3.548, 2.589, 6.702, 95% confidence interval as 1.460-8.620, 1.064-6.301, 1.886-23.813, P<0.05). Age and N staging were independent factors affecting the overall survival (OS) of colon cancer patients of dMMR ( hazard ratio=1.073, 10.684, 95% confidence interval as 1.021-1.126, 2.311-49.404, P<0.05). (3) Analysis of factors affecting the survival outcomes of patients of pMMR. Results of multivariate analysis showed that age, T staging, N staging, vascular tumor thrombus were independent factors affecting the DFS of colon cancer patients of pMMR ( hazard ratio=1.018, 2.214, 2.598, 1.549, 95% confidence interval as 1.006-1.030, 1.618-3.030, 1.921-3.513, 1.118-2.147, P<0.05). Age, T staging, N staging, high grade tumor were independent factors affecting the OS of colon cancer patients of pMMR ( hazard ratio=1.036, 2.080, 2.591, 1.615, 95% confidence interval as 1.020-1.052, 1.407-3.075, 1.791-3.748, 1.114-2.341, P<0.05). (4) Interaction analysis of MMR and adverse clinicopathological features on survival outcomes. Results of interaction analysis showed that the multiplication interaction effect between the number of lymph node harvest <12 and MMR protein status was significant on DFS of colon cancer patients ( hazard ratio=3.923, 95% confidence interval as 1.057-14.555, P<0.05). The additive interaction effects between age and MMR protein status, between high grade tumor and MMR protein status were significant on OS of colon cancer patients ( RERI=-0.033, -1.304, 95% confidence interval as -0.049 to -0.018, -2.462 to -0.146). Conclusions:There is an interaction between the MMR protein status and the adverse clinicopathological features (the number of lymph node harvest <12, high grade tumor) on prognosis of colon cancer patients of stage Ⅰ-Ⅲ. In patients of dMMR, the number of lymph node harvest <12 has a stronger predictive effect on poor prognosis. In patients of pMMR, the high grade tumor has a stronger predictive effect on poor prognosis.