AIM: To investigate the inhibitory effects of cholecystokinin octapeptide(CCK-8) on nuclear factor-?B(NF-?B) activities stimulated by lipopolysaccharide(LPS) by using forskolin,the activator of adenylate cyclase,and PKA inhibitor H-89 in rat pulmonary interstitial macrophages(PIMs).METHODS: PIMs were isolated and purified.EMDA was applied to detect NF-?B activities and Western blotting was used to analyze the I?B-? protein level in rat PIMs.RESULTS: The NF-?B activity was not detected in normal control rat PIMs.The NF-?B activity in LPS-treated rat PIMs was obviously higher than that in control group(P0.05).The NF-?B activity in CCK+LPS group and LPS+Fsk group were obviously lower than that in LPS group(P

The morphological and molecular identifications of Cyclospora-like oocysts of dogs were underwent in the present study, in which the morphological characteristics of the Cyclospora-like oocysts firstly found in the stool samples of dogs, such as shape, size, acid-fast staining,sporulation and auto-fluorescene, were observed. According to the published sequence of the rDNA gene of Cyclospora in GenBank, 3 primers were designed and were used to amplify part of the 18S rDNA gene of dog-associated Cyclospora-like organism by nested PCR.The amplicons were purified and cloned into vector pMD19T. Then, the positive clones screened were sequenced and subjected to sequence homology and phylogenetic analysis. It was found that the morphological charactertistics of the Cyclospora-like oocysts in dogs were similar to that of the human Cyclospoa oocysts and the size of-the amplified fragment of 18S rDNA was proved to be 715 bp, that was identical to that of the target fragment. Based on the results of sequence homology and phylogenetic analysis, the dog-associated Cyclospora-like organism was identified as the Cyclospora species.

AIM: To investigate the binding characteristics of cholecystokinin receptors in rat pulmonary interstitial macrophages (PIMs). METHODS: The PIMs were isolated from rat lung tissues, purified using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. The PIM membrane was obtained by supercentrifuge. Receptors for CCK in PIMs were examined using [~3H] labeled CCK-8S as ligand. The specificity of [~3H]-CCK-8S binding to PIMs membrane and the subtypes of CCK receptors were determined by competitive inhibition experiments with CCK-8S, CCK-A and CCK-B receptors selective antagonists (CR1409 and CR2945). The effects of time and incubation temperature on the specific binding were also observed. RESULTS: The specific binding of [~3H]-CCK-8S was not detected in normal rat PIMs, but was detected in the rat administrated with LPS for 48 h. The capacity of ligand-receptor binding was dependent on the incubation temperature and time. Scatchard analysis of the saturation curves suggested that the presence of CCK receptors with high affinity [Kd=(0.68?0.28)mmol/L] and low binding capacity [Bmax=(32.50?2.70) pmol?g~(-1) protein] in PIMs. By means of competitive inhibition studies, the specific binding of [~3H]-CCK-8S to rat PIMs was inhibited by unlabelled CCK-8S [IC_(50)=(3.20?1.13) nmol/L], CCK-AR specific antagonist CR 1 409 [IC_(50)=(0.19?0.06)?mol/L] and CCK-BR specific antagonist CR 2945[IC_(50)=(2.30?0.80)nmol/L]. CONCLUSION: These results suggest the presence of two subtypes of CCK-AR and CCK-BR and provide a structural basis for CCK to play a pivotal role in PIMs.

Objective To investigate the relationship between peripheral inflammatory cytokines and anxiety symptoms in patients with the first?episode generalized anxiety disorder. Methods 48 patients diagnosed with the first?episode generalized anxiety disorder according to ICD?10 criteria and 48 healthy sub?jects were recruited. Peripheral levels of IL?1, IL?2, IL?4, IL?5, IL?6, IL?8, IL?10, IL?12p70, GM?CSF and IFN?γ of both groups were evaluated by enzyme?linked immunosorbent assay ( ELISA) ,and CRP was evalua?ted by immunoturbidimetric method. Generalized Anxiety Disorder Scale( GAD?7) and State?Trait Anxiety Inventory ( STAI ) were used to assess the levels of overall anxiety, state anxiety and trait anxiety. Results The levels of CRP ( ( 1. 19 ± 0. 80 ) mg/L vs ( 0. 68 ± 0. 70 ) mg/L, t=3. 31 ) , IL?1α( ( 70. 34 ± 3.60)pg/ml vs (16.94±3.42)pg/ml, t=74.50),IL?2((7.25±3.42)pg/ml vs (4.95±2.31)pg/ml, t=3.85), IL?4((102.02±73.14)pg/ml vs (75.55±32.78)pg/ml, t=2.29),IL?6((12.55±2.37)pg/ml vs (2.71±1.35) pg/ml, t=14.79),IL?8((44.64±16.21)pg/ml vs (35.69±11.70)pg/ml, t=3.10),IL?12((18.16±24.17) pg/ml vs (10.82±4.72)pg/ml, t=2.06),IFN?γ((23.32±15.52)pg/ml vs (16.48±6.80)pg/ml, t=2.79), GM?CSF((19.07±11.12)pg/ml vs (13.40±8.54)pg/ml, t=2.80) in patients with the first?episode general?ized anxiety disorder were significantly higher than normal controls(P<0.05) . Both SAI and TAI had signifi? cantly positive correlation with the levels of IL?1α, IL?2, IL?6, IL?8, IL?12, IFN?γ and GM?CSF ( r=0.24?0.76, P<0.05) . Conclusion The levels of some peripheral inflammatory cytokines in patients with the first?episode generalized anxiety disorder are significantly increased,and they have positive correlation with gener?al anxiety,state anxiety and trait anxiety,which may suggest some immune system defects in the patients.

To establish a highly sensitive and specific method to detect the presence of Cryptosporidium homini, the RT-PCR-ELISA assay was tried, in which the primer with a biotin-labeled probe was designed to amplify fragment containing the highly variable region by multiple alignment between p23 gene of C.hominis and other Cryptosporidium spp. The RT-PCR was used to amplify the target fragment, and the amplified product was used to hybridize with the probe primer. The hybridized product was then captured on micro-plate wells coated with streptavidin and reacted with anti-digoxin antibody labeled with horse-radish peroxidase. This method of testing was then used for the detection of C.hominis in 22 clinical specimens and compared with the conventional methods of testing. It was demonstrated that the RT-PCR--ELISA for the detection of C.hominis was proved to be quite sensitive and specific. Its sensitivity was 100 times higher than that of the general PCR. From the result of clinic detection, the detection rate of RT-PCR-ELISA assay attained to 86%(19/22), while those of RT-PCR, sucrose floating method and anti-acid staining were 27%, 27% and 50% respectively. This result indicates that the RT-PCR-ELISA assay is more sensitive to detect C.hominis than the other three methods of testing.

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.

BackgroundCognitive impairment， a core clinical feature of schizophrenia， is considered to be associated with the aberrant functional connectivity in patients with schizophrenia， whereas previous studies on the characteristics of cognitive impairment in patients with chronic schizophrenia and its correlation with interhemispheric voxel-mirrored homotopic connectivity （VMHC） are somewhat inadequate. ObjectiveTo investigate the characteristics of cognitive impairment in patients with chronic schizophrenia who are clinically stable on their antipsychotic medication， and to explore its correlation with resting-state interhemispheric VMHC， so as to provide theoretical basis for the identification of neurobiological mechanism possibly responsible for cognitive impairment in chronic schizophrenia. MethodsA total of 15 patients with chronic schizophrenia who met the criteria of the International Classification of Diseases， tenth edition （ICD-10） and hospitalized in Suzhou Guangji Hospital from January 2021 to December 2022 were included. Another 15 healthy community-dwelling individuals were concurrently recruited. All participants were requested to complete the Positive and Negative Symptom Scale （PANSS）， Repeatable Battery for the Assessment of Neuropsychological Status （RBANS） and resting-state functional Magnetic Resonance Imaging （rs-fMRI） scanning to evaluate their mental symptoms， cognitive function and interhemispheric functional connectivity. The rs-fMRI data were analyzed with VMHC method. Then Pearson correlation analysis was performed to examine the correlation between VMHC values of regions of interest and scores of RBANS and PANSS within patient group. ResultsPatient group obtained lower scores than control group based on RBANS immediate memory， visuospatial/constructional， language， attention and total score， with statistically significant differences （t=-2.853， -2.107， -5.576， -7.108， -5.354， P<0.05 or 0.01）. The VMHC values of left superior occipital gyrus （t=-5.188， P<0.05） and right cuneus （t=-5.188， P<0.05） in patient group were lower than those in control group， with statistical difference. Correlation analysis denoted that the VMHC values of left superior occipital gyrus （r=0.612， P=0.015） and right cuneus （r=0.612， P=0.015） were positively correlated with visuospatial/constructional index score in RBANS. ConclusionThe resting-state VMHC is abnormal in left superior occipital gyrus and right cuneus of patients with chronic schizophrenia， and yields a correlation with the visuospatial/constructional performance of patients. ［Funded by Suzhou Science and Technology Development Plan Project （number， SKJYD2021131； SKJY2021143）］

Objective:To investigate the level of serum vascular endothelial growth factor (VEGF) and its correlation with clinical symptoms in patients with first-episode drug-naive schizophrenia patients of different genders.Methods:From January 2016 to October 2019, a total of 81 first-episode drug-naive schizophrenia patients(patient group, 41 male, 40 female) and 64 healthy controls (control group, 40 male, 24 female) were included in this study.The serum level of VEGF was detected with flow cytometric bear array (CBA). Positive and negative symptom scale (PANSS) was used to evaluate the relevant clinical symptoms of patients.SPSS 22.0 software was used for statistical analysis.Independent sample t-test and nonparametric test were used for comparison between groups.The relationship between VEGF and clinical variables was analyzed by Pearson correlation analysis and Spearman correlation analysis. Results:The level of serum VEGF in the patient group was significantly lower than that in the control group(148.08(75.89, 208.61)pg/mL, 179.94(99.14, 318.41)pg/mL, Z=-2.20, P=0.028). The total PANSS score((82.71±17.30), (73.45±16.36), t=2.473, P=0.016)and cognitive score((7.88±3.36), (6.23±2.81), t=2.402, P=0.019) in male patients were higher than those in female patients.There was a negative correlation between VEGF level and PANSS negative symptom score in the patient group( r=-0.228, P=0.041), as well as significant negtive correlation between VEGF level and cognitive score in male patients( r=-0.425, P=0.007). Conclusion:The level of serum VEGF is reduced in first-episode patients with schizophrenia, which influences their negative symptom. Moreover, the decline in serum VEGF level is implicated in cognitive impairments in male patients with first-episode schizophrenia.

Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide (mHSP/P) vaccine in combination with a programmed death ligand 1 (PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and en-zyme-linked immunosorbent assay (Elisa) was used to quantitatively identify the expression of heat shock proteins (HSP70, HSP90, Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide (Grp94/P) sarcoma vac-cines were isolated using chromatography and were identified by Western blot (WB). Flow cytometry was used to determine their cy-totoxic effects. The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes (HSP70, HSP90, Grp94). We successfully isolated sarco-ma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cy-totoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation (P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor (P<0.05). Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune eva-sion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.