In Vitro and In Vivo Antigen Presentation and Diagnosis Development of Recombinant Overlapping Peptides Corresponding to Mtb ESAT-6/CFP-10.

Cellular immunity in Mycobacteria tuberculosis (Mtb) infection is important for the pathogenesis and final clearance of intracellular Mtb infection. In addition, it is valuable for the diagnosis of tuberculosis. In this pioneering work, we tested in vitro and in vivo antigen presentation and diagnostic application of a recombinant overlapping peptide-protein derived from two Mtb RD1 antigens ESAT-6 and CFP-10 (ROP-TB). The overlapping peptide sequence of ROP-TB is cleaved by the cathepsin S enzyme and covers the entire length of the two proteins. ROP-TB can be expressed and purified from E. coli. Once taken in by antigen-presenting cells, ROP-TB can be cleaved into a peptide pool by cathepsin S within the cells. We found that in dendritic cells, ROP-TB can be processed in 6 hours of co-culture, while the ESAT-6/CFP-10 fusion protein remained in the endosomal compartment. In Mtb-infected mice, ROP-TB stimulated stronger specific T cell responses than pooled synthetic peptides derived from ESAT-6 and CFP-10. With regard to the presentation of in vivo antigens, in a guinea pig model infected with Mtb, ROP-TB induced delayed type hypersensitivity (DTH) responses comparable to those of the tuberculin purified protein derivative (PPD) and ESAT-6/CFP-10 fusion protein. In Mycobacterium bovis (Bovine TB)-infected cattle, ROP-TB elicited DTH responses. Finally, in Mtb infected patients, ROP-TB stimulated cellular immune responses in majority of patients (16/18) of different HLA phenotypes while a single peptide derived from the same proteins did not elicit the immune responses in all patients. In summary, in vitro and in vivo data suggest that ROP-TB stimulates a strong cellular immune response irrespective of HLA phenotypes and is therefore suitable for use in vitro and in vivo diagnostics.

Effects of psychological nursing care on anxiety and depression in perioperative patients with lung cancer: A systematic review and meta-analysis.

BACKGROUND: This study aimed to investigate the effects of psychological nursing care (PNC) on anxiety relief in perioperative lung cancer (LC) patients. METHODS: We searched the Cochrane Library, PubMed, Embase, CNKI, CBM, and Wangfang electronic databases from inception to May 1, 2022. Eligible randomized controlled trials (RCTs) investigating the effects and safety of PNC on anxiety relief in perioperative LC patients. Anxiety was the primary outcome measure. The secondary outcomes were depression, length of hospital stay, and the occurrence of adverse events. RESULTS: Six eligible RCTs with 494 patients were included in this study. Compared with routine nursing care, PNC showed better outcomes in terms of anxiety relief (mean difference [MD] = -13.24; random 95% confidence interval (CI), -18.28 to -8.20; P<.001), depression decrease (MD = -11.84; random 95% CI, -18.67 to -5.01; P < .001), and length of hospital stay (MD = -2.6; fixed 95% CI, -3.13 to -2.07; P < .001). No data on adverse events were pooled because only 1 trial reported this outcome. CONCLUSIONS: This study showed that PNC may benefit more than routine nursing care for patients with LC in anxiety, depression, and length of hospital stay. High-quality RCTs are needed to validate the current findings in the future.

HPV-16 E7-Specific Cellular Immune Response in Women With Cervical Intraepithelial Lesion Contributes to Viral Clearance: A Cross-Sectional and Longitudinal Clinical Study.

High-risk human papillomavirus (HPV) infection is the cause of almost all cervical cancers. HPV16 is one of the main risk subtypes. Although screening programs have greatly reduced the prevalence of cervical cancer in developed countries, current diagnostic tests cannot predict if mild lesions may progress into invasive lesions or not. In the current cross-sectional and longitudinal clinical study, we found that the HPV16 E7-specific T cell response in peripheral blood mononuclear cells of HPV16-infected patients is related to HPV16 clearance. It contributes to protecting the squamous interaepithelial lesion (SIL) from further malignant development. Of the HPV16 infected women enrolled (n = 131), 42 had neither intraepithelial lesion nor malignancy (NILM), 33 had low-grade SIL, 39 had high-grade SIL, and 17 had cervical cancer. Only one of 17 (5.9%) cancer patients had a positive HPV16 E7-specific T cell response, dramatically lower than the groups of precancer patients. After one year of follow-up, most women (28/33, 84.8%) with persistent HPV infection did not exhibit a HPV16 E7-specific T cell response. Furthermore, 3 malignantly progressed women, one progressed to high-grade SIL and two progressed to low-grade SIL, were negative to the HPV16 E7-specific T cell response. None of the patients with a positive HPV16 E7-specific T cell response progressed to further deterioration. Our observation suggests that HPV16 E7-specific T cell immunity is significant in viral clearance and contributes in protection against progression to malignancy.

Evaluation of monoclonal antibody-based direct, rapid immunohistochemical test for rabies diagnosis.

Rabies is a major public health problem in developing countries in Asia and Africa. Although a number of laboratory diagnoses can be used for rabies control, the WHO and OIE recommended gold standard for rabies diagnosis is the direct fluorescent antibody test (FAT). However, FAT is not widely used in developing countries because of deficient financial sources to procure fluorescent microscope. Recently the direct rapid immunohistochemical test (dRIT) has been developed and has a worldwide promising application, particularly in developing countries, since its result can be read by inexpensive light microscopy, in addition to be consistent with that of FAT. However, no commercial conjugated antibody is available to meet the laboratory demand. We describe here the production of a monoclonal antibody (MAb) against rabies virus (RABV) N protein and its use as a biotinylated conjugate in a dRIT. Tested against a batch of 107 brain specimens representing a wide phylogenetic diversity of RABV collected from different animal species with multiple geographical origins in China, results showed that the dRIT had 100% specificity (95% CI 0.93-1.00) and 96.49% sensitivity (95% CI 0.88-1.00) as compared with the gold standard FAT. It therefore provides a simple, economical alternative to FAT, particularly for use in rabies diagnosis in developing countries.

Protective cellular immunity generated by cross-presenting recombinant overlapping peptide proteins.

Priming of naive CD8+ and CD4+ T cells by dendritic cells (DCs) requires effective antigen presentation on both MHC class I and II molecules. We have developed a novel technology to use recombinant overlapping peptides (ROP) that stimulate both CD8+ and CD4+ T cell immune responses. The single chain protein of a ROP is made up of overlapping peptides linked by the target sequence (LRMK) for cathepsin S, a protease found in the endosomes of DCs. We designed synthetic genes encoding ROPs derived from ovalbumin (OVA), tuberculosis protein (CFP10-ESAT6), human papilloma virus (HPV) protein (E7) and survivin, a protein commonly over-expressed in tumour cells. An epitope from ROP-OVA was cross-presented and detected by a CD8+ T cell receptor-like antibody (TCR like Ab). Human DCs pulsed with ROP-survivin activated CD8+ T cells. CD4-low PBMCs from HIV and TB co-infected patients recognized ROP-CFP10-ESAT6 compared to a soluble form of the antigen. Immunization of mice with ROP-survivin or ROP-HPV-E7 generated specific cellular immune responses and protected mice from inoculation with melanoma B16 cells expressing survivin or HPV-E7 proteins. Together these data provide evidence to support ROP as a central component of a new platform for therapeutic vaccines and diagnostics.

[The protective effect of interleukin-1 receptor antagonist on postischemic reperfused myocardium and its possible mechanism].

OBJECTIVE: To observe the dynamic changes of plasma inflammatory cytokine interleukin-1 beta (IL-1 beta) in patients with acute myocardiac infarction (AMI) before and after recanalization of the infarct related artery (IRA) and to observe the effect of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the postischemic reperfused myocardium in experimental rabbits. METHODS: (1) ELISA was used to measure the plasma IL-1 beta of 22 AMI patients, 20 males and 2 females, aged 64 +/- 12, before emergency percutaneous coronary intervention (PCI), and 12 hours and 24 hours after-intervention, and measure the plasma IL-1 beta of 8 healthy controls, 6 males and 2 females, aged 56 +/- 9. (2) Forty rabbits underwent ligation of the left circumflex branch of coronary artery (LCX) for 50 minutes and reperfusion for 4 hours after the ligatures were untied. The rabbits were randomly divided into 4 groups of 10 rabbits to be injected into the left ventricle immediately before the reperfusion with rhIL-1ra 10 mg/kg (group A), 20 mg/kg (group B), or 40 mg/kg (group C), and normal saline (control group) respectively. After reperfusion of 4 hours, the LCX was re-ligated. Evans blue was injected into the left ventricle. 15% KCl was injected intravenously to kill the rabbits. Their hearts were taken out to weigh the non-ischemic, ischemic, and necrotic cardiac muscles so as to calculate the infarct size. The myoperoxidase (MPO) activity was measured by colorimetry. Sections of myocardium were made. The number of apoptotic cardiomyocytes was evaluated by TUNEL method. The apoptotic rate of cardiomyocyte was measured by annexin V method. The DNA expression of myocardium was detected by DNA laddering method. The expressions of Bcl-2 and Bax apoptosis genes were assessed. RESULTS: (1) The average plasma IL-1 beta level of the 22 AMI patients before emergency PCI was significantly higher than that of the controls (28 pg/ml +/- 9 pg/ml vs. 20 pg/ml +/- 11 pg/ml, P < 0.05), and became the highest 12 hours after the intervention (86 pg/ml +/- 14 pg/ml), and the high level lasted to 24 hours after emergency PCI. (2) In the ischemia-reperfusion rabbit model, the infarct size was 47% +/- 7% in the group A, 34% +/- 8% in the group B, 31% +/- 6% in the group C, and 61% +/- 11% in the control group (P < 0.05, 0.01, and 0.01 respectively). The activity of myocardial MPO was 16.6 +/- 3.6 min(-1).g.w.w(-1) in the group A, 10.9 min(-1).g.w.w(-1) +/- 1.9 min(-1).g.w.w(-1) in the group B, 7.8 min(-1).g.w.w(-1) +/- 2.2 min(-1).g.w.w(-1) in the group C, and 20.5 min(-1).g.w.w(-1) +/- 4.5 min(-1).g.w.w(-1) in the control group (P < 0.05, 0.01, and 0.01 respectively). The cardiomyocyte apoptosis evaluated by TUNEL was 38.3 n/HP +/- 7.4 n/HP in the group A, 25.6 n/HP +/- 6.8 n/HP in the group B, 12.2 n/HP +/- 3.3 n/HP in the group C, and 44.4 n/HP +/- 9.5 n/HP in the control group (P < 0.05, and P < 0.01 respectively in comparison between the group B and the control group and between the group C and the control group). The apoptotic rate by annexin V method was 11.6% +/- 2.7% in the group A; 7.7% +/- 2.4% in the group B, 4.7% +/- 1.4% in the group C, and 15.6% +/- 3.5% in the control group (P < 0.05, 0.01, and 0.01 respectively). DNA electrophoresis showed scaling ladder pattern only in the control group. The fluorescent density of the apoptosis gene Bax in myocardium was 24.9 +/- 8.2 in the group A; 15.5 +/- 3.4 in the group B, 10.6 +/- 2.5 in the group C, and 33.3 +/- 9.4 in the control group (P = 0.0298, 0091, and 0052 respectively) and no significant difference in the expression of Bcl-2 was shown among the 4 groups. Myocardial MPO was correlated with cardiomyocyte apoptosis (r = 0.86 by TUNEL, P < 0.01; r = 0.75 by Annexin V method, P < 0.05). CONCLUSION: Inflammatory cytokine IL-1 beta is involved in myocardial ischemia-reperfusion injury. With potential therapeutic value in prevention and treatment of ischemia-reperfusion injury to myocardium, rhIL-1ra may reduce myocardial ischemia-reperfusion injury by suppression of cardiomyocytes apoptosis mediated by IL-als; 0.86 by TUNEL, P < 0.01; r = 0.75 by Annexin V method, P < 0.05). CONCLUSION: Inflammatory cytokine IL-1 beta is involved in myocardial ischemia-reperfusion injury. With potential therapeutic value in prevention and treatment of ischemia-reperfusion injury to myocardium, rhIL-1ra may reduce myocardial ischemia-reperfusion injury by suppression of cardiomyocytes apoptosis mediated by IL-1.