Objective To investigate the feasibility of IgG4 as a biomarker of the activity and outcome of phospholipase A2 receptor (PLA2R)-associated membranous nephropathy (PLA2R-MN). Methods Serum and urine samples were collected from 56 patients with PLA2R-MN,13 patients with secondary membranous nephropathy (SMN),and 10 patients with primary IgA nephropathy (IgAN) when kidney biopsy was performed in the Department of Nephrology,Hangzhou Hospital of Traditional Chinese Medicine from April 2017 to January 2018.Sandwich enzyme-linked immunosorbent assay was employed to measure the serum and urinary IgG4 levels. Results The PLA2R-MN group had higher median serum IgG4/IgG ratio than the SMN group (P=0.009) and the IgAN group (P<0.001) and higher median urinary IgG4/creatinine ratio than the SMN group (P=0.008).In the patients with PLA2R-MN,the median serum IgG4/IgG ratio and urinary IgG4/creatinine ratio were significantly higher in the renal insufficiency group than in the normal renal function group (P=0.049,P=0.015).Moreover,the median serum IgG4/IgG ratio was higher in those with a serum albumin level<30 g/L than in those with a serum albumin level ≥30 g/L (P=0.005).Fifty-three patients with PLA2R-MN were followed up for at least 1 year,and the serum IgG4/IgG ratios of the patients in remission were lower than those of the patients without remission (P=0.005).The median serum IgG4/IgG ratio of 23 patients in remission decreased from 5.82% (4.54%,10.20%)(at initial enrollment) to 2.91% (2.11%,5.37%)(after 1-year follow up) in remission patients (P<0.001).The receiver operating characteristic curve showed that the patients with a serum IgG4/IgG ratio<10.24% had a higher possibility of remission (P=0.005). Conclusion Serum and urinary IgG4 levels may be an indicator of the activity in PLA2R-MN patients and thus may be a predictive biomarker of the outcomes.
Biomarkers ; Creatinine ; Glomerulonephritis, Membranous/pathology* ; Humans ; Immunoglobulin G ; Receptors, Phospholipase A2 ; Serum Albumin

BACKGROUND: The aim of study is to investigate the initial functional changes of muscle in rats induced to have myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). The authors investigated the functional changes of muscle evaluated by mechanomyography (MMG) and the expression of acetylcholine receptors (AChRs). METHODS: After the institutional approval, 39 male Lewis rats were randomly allocated into study. 26 animals were immunized to induce EAMG by Torpedo AChR (T-AChR) emulsified with complete Freund's adjuvant (CFA) and phosphate buffer saline (PBS)/bovine serum albumin (BSA) 0.01% at the base of tail, and received booster immunizations twice by T-AChR with incomplete Freund's adjuvant (IFA) and PBS/BSA 0.01% at all different site on the upper back. 13 animals were sham immunized as control group by the same method of EAMG except T-AChR. Clinical EAMG scores were examined. Anti T-AChR and anti rat-AChR (R-AChR) antibodies (Ab) were compared by using (125)I-alpha-bungarotoxin ((125)I-alpha-BuTx) radioimmunoassay. Under the anesthesia, neuromuscular functions were monitored by MMG using single twitch (ST) and TOF. AChRs were quantitated using (125)I-alpha-BuTx. RESULTS: Overall weight gain and final body mass, muscle force (ST), specific muscle force of ST, TOF fade ratio and AChRs were reduced in EAMG score 3 compared to control (P < 0.0001). Anti T-AChR Ab and anti R-AChR Ab were increased in score 3 EAMG (P < 0.0001). CONCLUSIONS: EAMG score 3 rats showed characteristic neuromuscular functions as depressed initial ST and its specific force, initial TOF fade and increased anti AChR Abs. Those above characteristics had significant correlations with the clinical EAMG scores. AChRs were significantly down-regulated according to their functional characteristics and clinical EAMG scores.
Acetylcholine* ; Anesthesia ; Animals ; Antibodies ; Freund's Adjuvant ; Humans ; Immunization, Secondary ; Male ; Myasthenia Gravis* ; Myasthenia Gravis, Autoimmune, Experimental ; Radioimmunoassay ; Rats* ; Receptors, Cholinergic* ; Serum Albumin ; Tail ; Torpedo ; Weight Gain

PURPOSE: To determine the optimum culture conditions by investigating isolated rat hepatocytes cultured in medium containing different glucose concentrations. METHODS: Hepatocytes were isolated from rats using a two-step perfusion technique and divided into the following two groups cultured in medium containing different glucose concentrations: (1) low-glucose group and (2) high-glucose group. Total cell count and viability of cultured rat hepatocytes and liver function parameters (i.e., concentrations of albumin, ammonia, and urea in the culture medium) were measured. The morphology of cultured rat hepatocytes was examined by staining with hematoxylin and eosin, and albumin receptor expression was confirmed by immunofluorescence. RESULTS: Total cell count and viability showed smaller increases in the low-glucose group than the high-glucose group, although the difference was not statistically significant (P = 0.112 and P = 0.147, respectively). The levels of albumin (P = 0.943), ammonia (P = 0.744), and urea (P = 0.709) were not significantly different between the two groups. In both groups, the function of cultured hepatocytes decreased significantly over time. The morphology of hepatocytes was well maintained in both groups at 3 days. On day 7, the cytoplasm was transformed into a spindle shape. On day 10, these changes were exaggerated, and were more prominent in the high-glucose group. CONCLUSION: Morphological assessment indicated that low-glucose culture medium is better than high-glucose culture medium for culturing of hepatocytes, although there was not significantly different in functional assessment. The cultured hepatocytes with low-glucose culture medium could be maintained for 7 days.
Ammonia ; Animals ; Cell Count ; Cell Transplantation ; Cytoplasm ; Eosine Yellowish-(YS) ; Fluorescent Antibody Technique ; Glucose* ; Hematoxylin ; Hepatocytes* ; Liver ; Perfusion ; Rats* ; Receptors, Albumin ; Urea

BACKGROUND: To identify the optimum culture conditions by investigating isolated rat hepatocytes cultured in medium containing different growth factors. METHODS: Hepatocytes were isolated from rats using a two-step perfusion technique and divided into the following four groups cultured in medium containing different growth factors: control, epidermal growth factor (EGF), insulin, and EGF+insulin. The viability of the cultured rat hepatocytes and liver function parameters, including albumin, ammonia, and urea in the culture medium, were measured. Hepatocyte morphology was examined by staining with hematoxylin and eosin, and albumin receptor expression was confirmed by immunofluorescence. RESULTS: Slightly higher viability was observed in the growth factor groups than in the control group, although without significance (P=0.073). The levels of albumin (P=0.001), ammonia (P<0.001), and urea (P=0.041) differed significantly among the four groups. The functional parameters in the growth factor groups, particularly the EGF+insulin group, were significantly superior to those in the control group. The morphology of the hepatocytes in all growth factor groups was well maintained at 10 days. However, the control group showed deterioration in cell morphology by day 7. CONCLUSIONS: Morphological and functional assessment indicated that the presence of growth factors, particularly EGF+insulin, provided culture conditions superior to those of non-supplemented medium.
Ammonia ; Animals ; Eosine Yellowish-(YS) ; Epidermal Growth Factor ; Fluorescent Antibody Technique ; Hematoxylin ; Hepatocytes* ; Insulin ; Intercellular Signaling Peptides and Proteins ; Liver ; Perfusion ; Rats* ; Receptors, Albumin ; Urea

BACKGROUNDTo investigate the relationship between interleukin-6 (IL-6) levels in serum and HBV replication and the role of IL-6 in liver damage in chronic hepatitis B patients.

METHODSDetected IL-6 levels and polymerized human serum albumin receptor (PHSA-R) in serum of different type of HBV markers positive patients and of different degree of chronic hepatitis B patients.

RESULTSThe results showed that the serum IL-6 levels were increased significantly in PHSA-R positive patients than in PHSA-R negative patients in chronic hepatitis B (P<0.01). The serum IL-6 levels were correlated with the levels of PHSA-R (r=0.694, P<0.01), and the degree of symptoms.

CONCLUSIONSThis study suggested that the IL-6 levels in serum of hepatitis B patients correlated HBV replication and the degree of liver damage, serum IL-6 levels may be used as a value indicating of HBV replication, the degree of symptoms and the effect of treatment.

Adult ; Aged ; Hepatitis B virus ; physiology ; Hepatitis B, Chronic ; blood ; Humans ; Interleukin-6 ; blood ; Middle Aged ; Receptors, Albumin ; blood ; Virus Replication

OBJECTIVE: To evaluate the value of pharmacogenetic testing for improving the efficacy and safety of treatment with cyclosporine, tacrolimus, and cyclophosphamide (CTX) for PLA2R-related membranous nephropathy and for determing individualized and precise treatment plans for the patients. METHODS: A total of 63 patients with PLA2R-related membranous nephropathy hospitalized in the Department of Nephrology at our hospital from January, 2019 to October, 2021 were enrolled in this study. Thirty-three of the patients underwent pharmacogenetic testing before taking the immunosuppressive drugs selected based on the results of genetic screening for sensitive targets, and the other 30 patients were empirically given immunosuppressive drugs according to the guidelines (control group). The clinical efficacy and adverse effects of the immunosuppressive drugs were analyzed for all the patients. The two groups of patients were compared for demographic and biochemical parameters including 24-h urine protein, serum albumin, renal function, and serum anti-phospholipase A2 receptor antibody both before and at 3 months after the beginning of the treatment. RESULTS: Among the 33 patients undergoing pharmacogenetic testing, 51.5% showed a GG genotype for cyclosporine, and 61.6% had an AG genotype for tacrolimus; for CTX, 51.5% of the patients showed a homozygous deletion and 63.6% had an AA genotype. After treatment for 3 months, serum anti-phospholipase A2 receptor antibody, 24-h urine protein, and serum albumin levels were significantly improved in pharmacogenetic testing group as compared with the control group (P < 0.05). CONCLUSION Individualized and precise administration of immunosuppressive drugs based on pharmacogenetic testing better controls proteinuria and serum antiphospholipase A2 receptor antibodies and increases serum albumin level in patients with PLA2R-related membranous nephropathy.
Humans ; Autoantibodies ; Cyclosporine/therapeutic use* ; Glomerulonephritis, Membranous/diagnosis* ; Homozygote ; Immunosuppressive Agents/therapeutic use* ; Pharmacogenomic Testing ; Receptors, Phospholipase A2 ; Sequence Deletion ; Serum Albumin ; Tacrolimus/therapeutic use*

Bovine serum albumin nanoparticles(BSANP) were prepared by desolvation method. The activated folic acid (N-hydroxysuccinimide ester of folic acid) was conjuated to the surface of BSANP via the amino groups. Then the folate-conjugated BSANPs (folate-BSANP) were purified with Sephadex G-50 column and completely separated from unreacted folic acid. After chymotryptic hydrolysis, the extent of folate conjugation on the BSANP was determined by quantitative ultraviolet(UV) spectrophotometric analysis. It was found that the spectrum of trypsin digest of folate-conjugate BSANP is basically identical with that of folate, thus indicating folate is successfully expressed on the surface of BSANP. The folate-BSANP was averagely 66 nm in diameter and was spherical in shape. Folate-conjugated BSANP was achieved, which represents a potential new drug carrier for tumor cell-selective targeting.
Animals ; Antineoplastic Agents ; administration & dosage ; Carrier Proteins ; chemistry ; Cattle ; Drug Delivery Systems ; methods ; Folate Receptors, GPI-Anchored ; Folic Acid ; chemistry ; Immunotoxins ; Microspheres ; Nanotechnology ; Receptors, Cell Surface ; chemistry ; Serum Albumin, Bovine ; chemistry

OBJECTIVETo investigate the accumulation of advanced glycation end products (AGE) and the inflammatory response of skin and wound in diabetic patients, and to analyze their relationship in vitro.

METHODSHistological staining and immunohistochemical staining was respectively performed on skin and wound tissue specimens collected from 10 patients with Type II diabetes mellitus (diabetes group) and 12 non-diabetic patients with skin injury (control group) to observe the arrangement of collagen and the distribution of inflammatory cells, and to determine the expression levels of AGE and its receptor (RAGE). Malondialdehyde (MDA) levels in skin and wound tissue homogenates were assayed by enzyme-linked immunosorbent assay. In vitro, human neutrophils were isolated and treated with RPMI-1640 culture medium or that containing AGE-human serum albumin in the concentration of 0.315, 0.625, 1.250 mg/mL, and they were identified as normal control (NC) group, low concentration (L) group, moderate concentration (M) group, and high concentration (H) group. Cell viability in each group was determined by MTT colorimetric assay, and the reactive oxygen species (ROS) in cell was measured with 2', 7'-dichlorofluorescein-diacetate. Data were processed with t test.

RESULTSCompared with those of skin in control group, collagens of skin tissues in diabetes group atrophied and disorderly arranged. Inflammatory cells in wounds in diabetes group were dispersed, in which collagens arranged loosely and irregularly, as compared with those of wounds in control group. Expression levels of AGE and RAGE of skin in diabetes group were higher than those in control group. In diabetes and control groups, especially in diabetes group, the numbers of RAGE-positive cells in wound tissue were more than those in skin tissue. Large amount of inflammatory cells with positive expression of RAGE were observed in diabetes group. MDA level of skin and wound tissue in diabetes group was respectively (6.3 ± 1.0), (7.1 ± 2.4) nmol per milligram protein, which were obviously higher than those in control group [(2.9 ± 1.0), (3.6 ± 1.4) nmol per milligram protein, with t value respectively 8.017, 4.349, P < 0.05 or P < 0.01]. Cell viability and ROS levels in neutrophils were increased in L, M, and H groups [(59 ± 8)%, (77 ± 5)%, (67 ± 6)% and 1.67 ± 0.14, 2.13 ± 0.17, 3.48 ± 0.48] as compared with those in NC group [(34 ± 5)% and 0.58 ± 0.06, with t value respectively 7.195, 14.890, 11.130 and 20.195, 24.905, 16.864, P < 0.05 or P < 0.01].

CONCLUSIONSAbnormal oxidative stress in diabetic skin leads to an atypical origin of wound repair. AGE-RAGE effect is a critical mediator for oxidative stress in diabetic wound tissue during wound healing.

Aged ; Case-Control Studies ; Diabetes Mellitus, Type 2 ; metabolism ; pathology ; Female ; Glycation End Products, Advanced ; metabolism ; Humans ; Male ; Middle Aged ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Serum Albumin ; metabolism ; Serum Albumin, Human ; Skin ; metabolism ; pathology ; Wound Healing

OBJECTIVETo observe and compare the effects of Hanfangji Compound and IFN-gamma on expressions of transthyretin (TTR) , inter-alpha inhibitor H1 (ITIH1) and serpin peptidase inhibitor clade F member 2 (SERPINF2) of hepatic stellate cells (HSC-T6).

METHODHanfangji Compound and IFN-gammaof different concentrations were used in hepatic stellate cell-T6 (HSC-T6) for 48 h. Flow cytometer was used to detect the effects of Hanfangji Compound and IFN-gamma on HSC proliferation. RT-PCR method was adopted to detect mRNA expressions of TFR, ITIH1 and SERPINF2. TTR, ITIH1 and SERPINF2 secretions were detected by ELISA. The protein localizations of TTR, ITIH1 and SERPINF2 were examined by immune fluorescence. The protein expression of TfR and ITIHI were determined by Western blot.

RESULTAfter Hanfangji Compound and IFN-gamma were adopted in HSC-T6, compared with the control group, the cell proliferation was inhibited obviously (P < 0. 05) , protein expressions of TTR, ITIH1 and SERPINF2 and mRNA expression increased significantly, with certain correlation with concentrations of Hanfangji Compound. The 2. 5 g L-I Hanfangji Compound group was superior to the IFN-gamma group (P <0. 05).

CONCLUSIONHanfangji Compound can inhibit HSC proliferation, upregulated TTR, ITIH1 and SERPINF2 proteins and mRNA expression, which may be one of mechanisms of anti-hepatic fibrosis of Hanfangji Compound.

Alpha-Globulins ; genetics ; metabolism ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Receptors, Albumin ; genetics ; metabolism ; alpha-2-Antiplasmin ; genetics ; metabolism

OBJECTIVETo investigate the effects of advanced glycosylation end products (AGEs) modified bovine serum albumin (AGE-BSA) on the expression of reactive oxygen species (ROS) and monocyte chemoattractant protein-1 (MCP-1) in human renal mesangial cells (HRMCs).

METHODSHRMCs were cultured in vitro with medium containing different doses of AGE-BSA or BSA (50,100, 200, 400 mg/L) for 48 hours, or with AGE-BSA (200 mg/L) for different times (12, 24, 48, 72 h). Immunocytochemistry assay was used to estimate the protein level of RAGE. The ROS in cells were measured by flow cytometry and the mRNA expression of MCP-1 were analyzed by semi-quantiative reverse transcription-polymerase chain reaction (RT-PCR) after treatment with AGE-BSA or BSA.

RESULTSThe protein level of RAGE was upregulated in the HRMCs with AGE-BSA. The expression of ROS and MCP-1 significantly enhanced by incubation of AGE-BSA in a time- and dose-dependent manner. The effects of AGE-BSA-induced up-regulation of ROS and MCP-1 level was significantly blocked by neutralizing antibodies to RAGE, while the expression of ROS and MCP-1 stood nearly unchanged after cultured with huamn IgG.

CONCLUSIONThe expression of ROS and MCP-1 in HRMCs is induced by AGE-BSA through RAGE, which may have potential effects in the pathgenic mechanism of diabetic nephropathy.

Cells, Cultured ; Chemokine CCL2 ; metabolism ; Glycation End Products, Advanced ; pharmacology ; Humans ; Mesangial Cells ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Reactive Oxygen Species ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Serum Albumin, Bovine ; pharmacology