[Clinical characteristics of 11 patients with Vibrio vulnificus infection and the establishment of a rapid diagnosis procedure for this disease].

Objective: To analyze the clinical characteristics of patients with Vibrio vulnificus infection, share diagnosis and treatment experience, and establish a rapid diagnosis procedure for this disease. Methods: This study was a retrospective case series study. From January 2009 to November 2022, 11 patients with Vibrio vulnificus infection who met the inclusion criteria were admitted to the Department of Burns and Wound Repair of Guangdong Provincial People's Hospital Affiliated to Southern Medical University. The gender, age, time of onset of illness, time of admission, time of diagnosis, route of infection, underlying diseases, affected limbs, clinical manifestations and signs on admission, white blood cell count, hemoglobin, platelet count, C-reactive protein (CRP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, procalcitonin, albumin, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and blood sodium levels on admission, culture results and metagenomic next-generation sequencing (mNGS) results of pathogenic bacteria and the Vibrio vulnificus drug susceptibility test results during hospitalization, treatment methods, length of hospital stay, and outcomes of all patients were recorded. Comparative analysis was conducted on the admission time and diagnosis time of patients with and without a history of exposure to seawater/marine products, as well as the fatality ratio and amputation of limbs/digits ratio of patients with and without early adequate antibiotic treatment. For the survived patients with hand involvement, the hand function was assessed using Brunnstrom staging at the last follow-up. Based on patients' clinical characteristics and treatment conditions, a rapid diagnosis procedure for Vibrio vulnificus infection was established. Results: There were 7 males and 4 females among the patients, aged (56±17) years. Most of the patients developed symptoms in summer and autumn. The admission time was 3.00 (1.00, 4.00) d after the onset of illness, and the diagnosis time was 4.00 (2.00, 8.00) d after the onset of illness. There were 7 and 4 patients with and without a history of contact with seawater/marine products, respectively, and the admission time of these two types of patients was similar (P>0.05). The diagnosis time of patients with a history of contact with seawater/marine products was 2.00 (2.00, 5.00) d after the onset of illness, which was significantly shorter than 9.00 (4.25, 13.00) d after the onset of illness for patients without a history of contact with seawater/marine products (Z=-2.01, P<0.05). Totally 10 patients had underlying diseases. The affected limbs were right-hand in 8 cases, left-hand in 1 case, and lower limb in 2 cases. On admission, a total of 9 patients had fever; 11 patients had pain at the infected site, and redness and swelling of the affected limb, and 9 patients each had ecchymosis/necrosis and blisters/blood blisters; 6 patients suffered from shock, and 2 patients developed multiple organ dysfunction syndrome. On admission, there were 8 patients with abnormal white blood cell count, hemoglobin, and albumin levels, 10 patients with abnormal CRP, procalcitonin, and NT-proBNP levels, 5 patients with abnormal creatinine and blood sodium levels, and fewer patients with abnormal platelet count, ALT, and AST levels. During hospitalization, 4 of the 11 wound tissue/exudation samples had positive pathogenic bacterial culture results, and the result reporting time was 5.00 (5.00, 5.00) d; 4 of the 9 blood specimens had positive pathogenic bacterial culture results, and the result reporting time was 3.50 (1.25, 5.00) d; the mNGS results of 7 wound tissue/exudation or blood samples were all positive, and the result reporting time was 1.00 (1.00, 2.00) d. The three strains of Vibrio vulnificus detected were sensitive to 10 commonly used clinical antibiotics, including ciprofloxacin, levofloxacin, and amikacin, etc. A total of 10 patients received surgical treatment, 4 of whom had amputation of limbs/digits; all patients received anti-infection treatment. The length of hospital stay of 11 patients was (26±11) d, of whom 9 patients were cured and 2 patients died. Compared with that of the 6 patients who did not receive early adequate antibiotic treatment, the 5 patients who received early adequate antibiotic treatment had no significant changes in the fatality ratio or amputation of limbs/digits ratio (P>0.05). In 3 months to 2 years after surgery, the hand function of 8 patients was assessed, with results showing 4 cases of disabled hands, 2 cases of incompletely disabled hands, and 2 cases of recovered hands. When a patient had clinical symptoms of limb redness and swelling and a history of contact with seawater/marine products or a pre-examination triage RiCH score of Vibrio vulnificus sepsis ≥1, the etiological testing should be initiated immediately to quickly diagnose Vibrio vulnificus infection. Conclusions: Vibrio vulnificus infection occurs most frequently in summer and autumn, with clinical manifestations and laboratory test results showing obvious infection characteristics, and may be accompanied by damage to multiple organ functions. Both the fatality and disability ratios are high and have a great impact on the function of the affected limbs. Early diagnosis is difficult and treatment is easily delayed, but mNGS could facilitate rapid detection. For patients with red and swollen limbs accompanied by a history of contact with seawater/marine products or with a pre-examination triage RiCH score of Vibrio vulnificus sepsis ≥1, the etiological testing should be initiated immediately to quickly diagnose Vibrio vulnificus infection.

Bone-filling mesh container versus percutaneous kyphoplasty in treating Kümmell's disease.

Kümmell's disease (eponymous name for osteonecrosis and collapse of a vertebral body due to ischemia and non-union of anterior vertebral body wedge fractures after major trauma) cannot heal spontaneously. Bone-filling mesh container (BFMC) can significantly relieve pain, help the correction of kyphosis, and may prevent cement leakage. This pilot study may provide the basis for the design of future studies. PURPOSE: To compare the effectiveness and safety of BFMC and percutaneous kyphoplasty (PKP) for treatment of Kümmell's disease. METHODS: From August 2016 to May 2018, 40 patients with Kümmell's disease were admitted to Guizhou Provincial People's Hospital. Among them, 20 patients (20 vertebral bodies) received PKP (PKP group) and the other 20 received BFMC (BFMC group). Operation time, Visual Analogue Scale (VAS), Oswestry Disability Index (ODI), Cobb's angle changes, and related complications were recorded. RESULTS: All patients underwent operations successfully. VAS scores and ODI of both groups at each postoperative time point were lower than preoperatively, with statistically significant difference (p < 0.05). Postoperative Cobb's angle of both groups postoperatively was lower than preoperatively (p < 0.05). Cement leakage occurred in eight vertebrae (8/20) in the PKP group and in one vertebra (1/20) in the BFMC group. No complications such as pulmonary embolism, paraplegia, or perioperative death occurred during operation in both groups. Adjacent vertebral refractures occurred in five patients (5/20) in the PKP group and in four patients (4/20) in the BFMC group, with no significant difference in the incidence rate of refractures in both groups but the material is too small to verify statistically. CONCLUSIONS: Both PKP and BFMC technologies can significantly relieve pain and help the correction of kyphosis while treating Kümmell's disease. Moreover, the BMFC may prevent cement leakage.

MiR-127-3p targets KIF3B to inhibit the development of oral squamous cell carcinoma.

OBJECTIVE: Recently, increased microRNAs have been shown to play an important role in the pathogenesis and progression of human cancers, including oral squamous cell carcinoma (OSCC). In this study, we focused on the function of microRNA-127-3p (miR-127-3p) associated with OSCC carcinogenesis. PATIENTS AND METHODS: MiR-127-3p and KIF3B expressions were observed via quantitative Real-time polymerase chain reaction (qRT-PCR) or Western blot in OSCC. The functions of mR-127-3p and KIF3B were investigated through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assays. And luciferase reporter assay was performed to confirm the relationship between mR-127-3p and KIF3B. RESULTS: First, down-regulation of miR-127-3p was identified in OSCC, which was associated with malignant clinicopathological features and poor prognosis in OSCC patients. Functionally, overexpression of miR-127-3p led to inhibition of cell proliferation and metastasis in OSCC. Further, KIF3B was confirmed to be a direct target of miR-127-3p. Moreover, upregulation of KIF3B was also observed in OSCC, which promoted tumorigenesis of OSCC. In particular, the upregulation of KIF3B partially attenuated the inhibitory effect of miR-127-3p on the development of OSCC. CONCLUSIONS: MiR-127-3p targeted KIF3B to inhibit the development of OSCC through suppressing cell proliferation, migration and invasion.

[Relationship between C-C chemokine receptor type 2 and P38 mitogen-activated protein kinase signaling pathway in the spinal cord of rats with bone cancer pain].

Objective: To investigate the relationship between C-C chemokine receptor type 2(CCR2) and P38 mitogen-activated protein kinase (P38MAPK) signaling pathway in the spinal cord of rats and further clarify the mechanism of bone cancer pain (BCP). Methods: A total of 92 healthy female SD rats, of which 60 were subjected to behavioral tests using a ciliary mechanical stimulation needle. SD rats were randomly divided into six groups: sham operation group (group S), bone cancer pain group (group B), sham operation + DMSO solvent group (group SD), bone cancer pain + DMSO solvent group (group BD), sham operation + RS102895 CCR2 inhibitor group (group SR), bone cancer pain + RS102895 CCR2 inhibitor group (group BR), and Von Frey was used in the behavioral test. Another 32 SD rats were randomly divided into the following 8 groups (n=4): sham operation group (group S), bone cancer pain 5 d group (group B5), bone cancer pain 9 d group (group B9), bone cancer pain 14 d group (group B14), bone cancer pain + DMSO solvent group (group BD), bone cancer pain + RS102895 CCR2 inhibitor 0.5 h group (group BR0.5 h), bone cancer pain + RS102895 CCR2 inhibitor 4 h group (group BR4 h), bone cancer pain + RS102895 CCR2 inhibitor 12 h group (group BR12 h). Western blot was used to detect the expression of P38, p-P38 and CCR2 in spinal cord of rats. Results: At day 5, 7, 9, 14, 21 post-injection, mechanical withdrawal thresholds of group S were(30.9±1.5), (31.9±1.2), (32.0±1.1), (31.6±1.5), (32.2±1.4)g respectively, the mechanical withdrawal thresholds of group B were( 26.4±0.7), (24.4±0.8), (21.4±0.8), (13.5±0.4), (9.9±0.2)g respectively, the mechanical withdrawal thresholds in group B decreased obviously versus group S, and the differences were statistically significant(t=-13.177, -16.660, -23.778, -35.574, -48.401, all P<0.01). At day 9 post-injection, the mechanical withdrawal thresholds in SD, BD, SR and BR groups were (32.4±1.7), (19.4±1.1), (32.1±1.3), (26.3±1.0) g respectively, the difference was statistically significant (F=224.681, P<0.01), and the mechanical withdrawal thresholds in group BD decreased obviously versus group SD, while the mechanical withdrawal thresholds in group BR increased obviously versus group BD. The expression levels of p-P38 in spinal cord of group S, group B5, group B9 and group B14 were(0.08±0.03), (0.20±0.05), (0.40±0.17), (0.65±0.14)respectively, the expression levels of CCR2 were(0.08±0.04), (0.18±0.05), (0.30±0.09), (0.58±0.07)respectively, the difference was statistically significant(F=19.123, 40.746, all P<0.01), and the expression of p-P38 and CCR2 in group B9 were showed a significant up-regulation versus group S. The expression levels of p-P38 in spinal cord of group BD, group BR0.5 h, group BR4 h and group BR12 h were (0.57±0.06), (0.17±0.11), (0.03±0.01), (0.25±0.11)respectively, and the difference was statistically significant(F=29.582, P<0.01). The expression of p-P38 in group BR0.5 h, BR4 h, BR12 h showed a significant down-regulation versus group BD. Conclusion: CCR2 in the spinal cord may be involved in the development of bone cancer pain by activating P38MAPK signaling pathway in rats.

Jujube leaf green tea extracts inhibits hepatocellular carcinoma cells by activating AMPK.

Here we evaluated the anti-hepatocellular carcinoma activity by the Jujube leaf green tea extracts (JLGTE). We showed that JLGTE exerted anti-proliferative, cytotoxic and pro-apoptotic activities against HepG2 and primary human hepatocellular carcinoma cells. It was however non-cytotoxic to the normal hepatocytes. JLGTE activated AMP-activated protein kinase (AMPK) signaling, which was required for its cytotoxicity against hepatocellular carcinoma cells. Silence of AMPKα1, via targeted short hairpin RNAs or CRISPR-Cas9 genome editing, inhibited JLGTE-induced AMPK activation and HepG2 cell apoptosis. Further, in-activation of AMPK by a dominant negative AMPKα1 (T172A) also alleviated JLGTE's cytotoxicity against HepG2 cells. On the other hand, forced-activation of AMPK by introduction of a constitutively-active AMPKα1 (T172D) mimicked JLGTE's actions and led to HepG2 cell apoptosis. These results suggest that JLGTE inhibits human hepatocellular carcinoma cells possibly via activating AMPK.

Presence of diabetic complications in type 1 diabetic patients correlates with low expression of mononuclear cell AGE-receptor-1 and elevated serum AGE.

BACKGROUND: Receptors for advanced glycation endproducts (AGE-R) mediate AGE turnover, but can also trigger inflammatory genes that promote diabetic tissue injury and diabetic complications (DC). High AGE levels and reduced AGE-R sites in kidneys of NOD mice prone to type 1 diabetes (T1D) and to renal disease (RD) suggested that impaired AGE-R function may contribute to RD in these mice. MATERIALS AND METHODS: In this study, after confirming reduced AGE-R1 expression in NOD mouse peritoneal macrophages, we tested for differences in AGE-R1, -R2, and -R3 gene expression in 54 human subjects by RT-PCR and Western analysis. Fresh peripheral blood mononuclear cells (PBMN) were isolated from 36 persons: 18 T1D patients with severe RD (DC); 11 age-and DM-duration matched patients without DC (n-DC); and 7 normal volunteers (NL). EBV-transformed lymphoblasts were obtained from an additional 18 subjects (12 T1D patients, 6 with and 6 without DC, and 6 nondiabetics). RESULTS: AGE-R1 mRNA and protein of PBMN from n-DC patients were enhanced (p < .05 versus NL) in proportion to serum AGE levels (sAGE) (p < .005 versus NL). In contrast, PBMN from DC patients exhibited no up-regulation of AGE-R1 mRNA or protein, despite higher sAGE levels (p < .005 versus NL). A similar unresponsiveness in AGE-R1 gene expression was observed in EBV-transformed lymphoblasts from DC patients versus NL (p < .01), but not in n-DC (p = NS). AGE-R2 and -R3 mRNA and protein levels were enhanced in both T1D groups (DC > n-DC) (n-DC AGE-R3, p < .05, DC AGE-R3, p < .05) compared to NL. AGE-R2 mRNA levels correlated with sAGE levels (r = .61, p < .05), and with creatinine clearance (r = -.63, p < .05). No differences were noted in AGE-R2 and -R3 mRNA expression in cultured cells. CONCLUSIONS: The consistent pattern of elevated serum AGE and low expression of AGE-R1 gene in macrophages from T1D mice (NOD), fresh PBMN and EBV-transformed cells from T1D patients with advanced DC suggests ineffective regulation of R1-mediated AGE turnover, possibly of genetic basis.

Differential expression of renal AGE-receptor genes in NOD mice: possible role in nonobese diabetic renal disease.

BACKGROUND: Nonobese diabetic mice (NOD) are prone to glomerular pathology, which is accelerated with the onset of diabetes. Advanced glycation end product (AGE) interactions with AGE-receptors (AGE-Rs) in kidneys can contribute to glomerular injury and diabetic nephropathy (DN). The significant elevation in kidney AGE deposits noted in prediabetic NOD mice suggested that delayed AGE turnover in this model may contribute to its propensity toward DN. METHODS: To explore whether excess tissue AGE was linked to altered AGE-R status in the kidney, mRNA/protein expression, and of several AGE-Rs [AGE-R1, AGE-R2, AGE-R3, scavenger receptor II (ScR-II), and receptor for AGE (RAGE)], was determined in renal cortex and in mesangial cells (MCs) isolated from ND-, D-NOD, and ILE mice (N = 20 per group). Ligand binding, receptor site number, and affinity were determined in MCs from the same mouse groups. RESULTS: Prediabetic NOD kidney AGE-R1 mRNA and protein level were threefold lower than that of ILE mice (P < 0.01), while AGE-R3 mRNA was enhanced by twofold (P < 0.05) and AGE-R2, RAGE, and ScR-II mRNA remained close to normal (ILE). The onset of diabetes in NOD mice, while enhancing AGE-R1 mRNA expression by approximately twofold, failed to raise it above the normal (ILE) level, despite increases in tissue, and serum AGE. The latter was associated with higher elevation in AGE-R3 (sixfold, P < 0.05), RAGE (twofold, P = NS), and ScR-II mRNA (2. 8-fold, P = NS) above control. MCs from prediabetic NOD mice showed a threefold lower level of AGE-R1 mRNA (P < 0.02 vs. ILE) and AGE-R1-protein, and AGE-binding activity (<40% of control ILE). In contrast, AGE-R3 mRNA was enhanced (twofold), while AGE-R2 showed no change. Cultured ND-NOD MCs displayed only one fourth of the AGE-binding sites/cell present on ILE MCs (1.6 x 10(6) vs. 6.6 x 10(6), P < 0.05), which after the onset of diabetes rose to the normal range (7.0 x 10(6)/cell), but failed to exceed it. CONCLUSIONS: Reduced AGE-R1 gene expression in this strain may contribute to delayed AGE removal from and early AGE deposition in kidney tissues. This may act as a trigger for those AGE-R genes involved in growth-promoting changes, leading to DN in this strain.

Programmed cell death and aerenchyma formation in roots.

Lysigenous aerenchyma contributes to the ability of plants to tolerate low-oxygen soil environments, by providing an internal aeration system for the transfer of oxygen from the shoot. However, aerenchyma formation requires the death of cells in the root cortex. In maize, hypoxia stimulates ethylene production, which in turn activates a signal transduction pathway involving phosphoinositides and Ca2+. Death occurs in a predictable pattern, is regulated by a hormone (ethylene) and provides an example of programmed cell death.

Nature and severity of the glomerular response to nephron reduction is strain-dependent in mice.

Nephron reduction is an important factor in the development of glomerulosclerosis. In a study of the oligosyndactyly (Os) mutation that causes a congenital 50% reduction in nephron number, we previously found that ROP Os/+ mice developed glomerulosclerosis whereas C57B1/6J Os/+ mice did not. We concluded that the predisposition to glomerulosclerosis depended largely on the genetic background, the ROP being sclerosis-prone whereas the C57 strain was sclerosis-resistant. In the current experiments we asked whether the intensity of the sclerotic response to nephron reduction in the ROP strain was related to the time at which it occurred, ie, a pre- or post-natal event. We also determined whether the absence of lesions in C57 Os/+ mice was caused by a higher threshold for the induction of a sclerotic response in C57 mice. We further examined the relationship between glomerular hypertrophy and sclerosis. C57 +/+, C57 Os/+, ROP +/+, and ROP Os/+ mice were uninephrectomized (NX) at age 10 weeks and followed for 8 weeks. We found no sclerotic changes in NX C57 +/+ and C57 Os/+ mice, despite a 75% reduction in nephron number in the latter. In contrast, both NX ROP +/+ and NX ROP Os/+ mice had glomerulosclerosis, which was more severe in the NX ROP Os/+ mice. Examination of extracellular matrix synthesis and degradation at the mRNA level revealed that synthesis exceeded degradation in ROP Os/+ mice. The lesions in NX ROP +/+ were less severe than in sham-operated ROP/Os mice, suggesting that the timing of nephron reduction affected the amplitude of the sclerotic response in this strain. Following NX, an increase in glomerular volume was found in C57 +/+, ROP +/+, and ROP Os/+ mice. However, NX did not lead to a further increase in glomerular volume in C57 Os/+ mice. We make three conclusions: 1) sclerosis was more severe in the ROP strain when nephron reduction occurred in utero; 2) the absence of glomerulosclerosis in C57 mice was not related to a higher threshold for a sclerosis response in this strain; and 3) whereas glomerular size continued to increase as nephron number decreased in ROP mice, it reached a plateau in C57 mice.

Orally absorbed reactive glycation products (glycotoxins): an environmental risk factor in diabetic nephropathy.

Endogenous advanced glycation endproducts (AGEs) include chemically crosslinking species (glycotoxins) that contribute to the vascular and renal complications of diabetes mellitus (DM). Renal excretion of the catabolic products of endogenous AGEs is impaired in patients with diabetic or nondiabetic kidney disease (KD). The aim of this study was to examine the oral absorption and renal clearance kinetics of food AGEs in DM with KD and whether circulating diet-derived AGEs contain active glycotoxins. Thirty-eight diabetics (DM) with or without KD and five healthy subjects (NL) received a single meal of egg white (56 g protein), cooked with (AGE-diet) or without fructose (100 g) (CL-diet). Serum and urine samples, collected for 48 hr, were monitored for AGE immunoreactivity by ELISA and for AGE-specific crosslinking reactivity, based on complex formation with 125I-labeled fibronectin. The AGE-diet, but not the CL-diet, produced distinct elevations in serum AGE levels in direct proportion to amount ingested (r = 0.8, P < 0.05): the area under the curve for serum ( approximately 10% of ingested AGE) correlated directly with severity of KD; renal excretion of dietary AGE, although normally incomplete (only approximately 30% of amount absorbed), in DM it correlated inversely with degree of albuminuria, and directly with creatinine clearance (r = 0.8, P < 0.05), reduced to <5% in DM with renal failure. Post-AGE-meal serum exhibited increased AGE-crosslinking activity (two times above baseline serum AGE, three times above negative control), which was inhibited by aminoguanidine. In conclusion, (i) the renal excretion of orally absorbed AGEs is markedly suppressed in diabetic nephropathy patients, (ii) daily influx of dietary AGEs includes glycotoxins that may constitute an added chronic risk for renal-vascular injury in DM, and (iii) dietary restriction of AGE food intake may greatly reduce the burden of AGEs in diabetic patients and possibly improve prognosis.

Transduction of an Ethylene Signal Is Required for Cell Death and Lysis in the Root Cortex of Maize during Aerenchyma Formation Induced by Hypoxia.

Ethylene has been implicated in signaling cell death in the lysigenous formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays) subjected to hypoxia. Various antagonists that are known to modify particular steps in signal transduction in other plant systems were applied at low concentrations to normoxic and hypoxic roots of maize, and the effect on cell death (aerenchyma formation) and the increase in cellulase activity that precedes the appearance of cell degeneration were measured. Both cellulase activity and cell death were inhibited in hypoxic roots in the presence of antagonists of inositol phospholipids, Ca2+- calmodulin, and protein kinases. By contrast, there was a parallel promotion of cellulase activity and cell death in hypoxic and normoxic roots by contact with reagents that activate G-proteins, increase cytosolic Ca2+, or inhibit protein phosphatases. Most of these reagents had no effect on ethylene biosynthesis and did not arrest root extension. These results indicate that the transduction of an ethylene signal leading to an increase in intracellular Ca2+ is necessary for cell death and the resulting aerenchyma development in roots of maize subjected to hypoxia.

Increased placental antifibrinolytic potential and fibrin deposits in pregnancy-induced hypertension and preeclampsia.

Preeclampsia is characterized by maternal hypercoagulable state and intravascular coagulation, microthromboses in several organs, and impairment of uteroplacental circulation. Excessive fibrin deposition occurs in the placenta, suggesting that disorders of placental coagulation and fibrinolysis physiologic systems may have a role in hemostasis activation. Term placentas were collected from 17 hypertensive/preeclamptic women and from 17 healthy pregnant women, and processed for both histologic and hemostasis studies. Placental fibrinoid deposition was visualized by cresyl-violet staining and quantified by histomorphometric analysis. The content in hemostasis factors was measured on extracts from homogenized placentas treated by a nonionic detergent. The percentage of villi with fibrinoid deposits was higher in the diseased placentas than in controls: 13.2 +/- 11.2 versus 6.75 +/- 2.7% (p < 0.001) for the total amount of deposits; 4.8 +/- 6.7 versus 1.5 +/- 1.0% (p = 0.04) for perivillous fibrinoid deposits, which are considered as histologic markers of intraplacental fibrin. The content in type 2 plasminogen activator inhibitor (PAI-2) antigen was higher in the diseased placentas than in controls: 124 +/- 8 versus 104 +/- 6 ng/mg placental protein (p = 0.046); there was a negative correlation between PAI-2 antigen and thrombomodulin activity (r = -0.57, p = 0.02) in the diseased placentas. No significant differences were found between the two groups for placental procoagulant tissue factor and anticoagulant thrombomodulin activities, and for the content in plasminogen activators and PAI-1 antigens. Placental antifibrinolytic potential is increased in pregnancy-induced hypertension and preeclampsia. This change, and the association of the highest PAI-2 placental concentrations with the lowest concentrations of thrombomodulin, may contribute to the prethrombotic state and to the excessive placental perivillous fibrin deposition observed in these situations.

Thrombin stimulation of renal cells.

Numerous glomerular and vascular nephritides are associated with fibrin formation and deposition within the kidney. Thrombin, which induces fibrin formation, also exerts numerous cellular effects on both circulating cells and intrinsic glomerular cells through the activation of the functional thrombin receptor. By immunohistochemistry and in situ hybridization, we demonstrated the constitutive expression of the functional thrombin receptor in the normal human kidney. Both glomerular epithelial and mesangial cells in culture also express the functional thrombin receptor, as shown by the binding of a specific monoclonal antibody against this receptor and Northern blot analysis. Thrombin has a potent mitogenic effect on cultured glomerular cells, suggesting that it could be at least, in part, one of the mediators that induces glomerular cell proliferation in glomerular diseases. Furthermore, we demonstrated that thrombin upregulates the synthesis of plasminogen activators and their type 1 inhibitor in these cells. Thrombin induces protein kinase C activation and an increase in intracellular calcium in these cells. Finally, we demonstrated that the functional thrombin receptor is internalized after addition of thrombin and thrombin receptor-activating peptide (homologous internalization) and also after phorbol myristate acetate addition (heterologous internalization).

Relationships between mesangial cell proliferation and types I and IV collagen mRNA levels in vitro.

Changes in the composition of the mesangial extracellular matrix (ECM) and cell turnover are present in glomerular disease. To determine if ECM changes play a role in perpetuating mesangial cell dysfunction, we examined a line of mouse mesangial cells cultured on films or gels of several ECM components and also on methyl cellulose, an inert substrate that prevents attachment. Cells on films of fibronectin or type IV or I collagen had persistently high growth rates and high levels of alpha 1-I and alpha 1-IV collagen mRNAs. In contrast, on gels of type IV or I collagen or matrigel, the growth rate was low. The alpha 1-IV collagen mRNA levels were low on type IV collagen gel or matrigel, whereas the alpha 1-I collagen mRNA levels remained high. In contrast, the alpha 1-I collagen mRNA levels were low on type I collagen gel, and the alpha 1-IV collagen mRNA levels were high. Cells on methyl cellulose formed floating aggregates, did not proliferate, and had a 5- to 10-fold decrease in both alpha 1-I and alpha 1-IV collagen mRNA levels. These phenotypic changes were largely reversible. Finally, when matrigel was layered over cells on fibronectin films, alpha 1-IV collagen mRNA levels decreased, but alpha 1-I collagen mRNA levels and proliferation remained high. Thus proliferation and alpha 1-I and alpha 1-IV collagen mRNA levels in mesangial cells were independently regulated and depended on attachment and the nature of the adjacent matrix.

Collagen and collagenase mRNAs in normal and sclerotic glomeruli: predictors of progression and response to therapy.

Progressive glomerulosclerosis is associated with decreasing kidney function, eventuating in end-stage renal failure. There are multiple components of the extracellular matrix, and the exact composition in various renal diseases is not known. Thus, we examined some of the major components of the extracellular matrix (ECM) in murine and human glomerular diseases. We studied matrix synthesis and degradation at the level of gene expression and ECM composition in the intact glomerulus. To determine whether the composition of sclerosis was similar among diseases, we examined a normal mouse strain and compared it with strains which spontaneously developed glomerulosclerosis. The baseline levels of matrix components varied between different mouse strains, and this level correlated with their propensity to develop glomerulosclerosis. In addition, when glomerulosclerosis was induced, the baseline ECM mRNA level predicted the subsequent outcome. We studied mice transgenic for bovine growth hormone, since they develop progressive glomerulosclerosis. Treatment with heparin substantially decreased the lesions without changes in type IV collagen mRNAs. However, there was an up-regulation of both the mRNA and enzyme activity for the 92 kD matrix metalloproteinase. In contrast, when these mice were treated with either angiotensin converting enzyme inhibitors or angiotensin II (Ang II) receptor antagonists, the glomerulosclerosis was accentuated histologically and the ECM synthetic and degradative mRNAs were elevated. These data suggest that the mRNA levels reflect response to therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin and phorbol ester induce internalization of thrombin receptor of human mesangial cells through different pathways.

Thrombin is a potent activator of human mesangial cells probably by activation of its functional receptor. Northern blot analysis demonstrates the presence of mRNA encoding the functional thrombin receptor in mesangial cells, and surface expression of thrombin receptor antigen has been confirmed by immunocytochemistry. Using 125I-labeled ATAP2, a monoclonal antibody against the functional thrombin receptor, we found that thrombin and thrombin receptor agonist peptide (TRAP) induce homologous internalization of thrombin receptor in a dose-dependent manner. Redistribution of thrombin receptor from the cell surface to vesicular structures in the cytoplasm has been followed by immunocytochemistry. Additionally, a dose-dependent loss of cell surface thrombin receptor is induced by phorbol 12-myristate 13-acetate (PMA), suggesting that thrombin receptor undergoes heterologous internalization in response to PMA. The time course of thrombin-induced receptor internalization is different from that observed with TRAP and PMA. Protein kinase C inhibitors, staurosporine and GF 109 203 X, do not affect thrombin receptor internalization induced by thrombin and TRAP but block receptor internalization stimulated by PMA. These data suggest that heterologous thrombin receptor internalization induced by PMA is mediated by protein kinase C. However, activation of protein kinase C is not responsible for homologous thrombin receptor internalization caused by thrombin and TRAP.

Overexpression of transforming growth factor-beta 1 mRNA is associated with up-regulation of glomerular tenascin and laminin gene expression in nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop immune-mediated insulin-dependent diabetes mellitus and nephropathy, providing an opportunity to study the early molecular events in a model of diabetic glomerulosclerosis. The expression of several genes coding for growth factors and extracellular matrix was examined in microdissected glomeruli, by the use of reverse transcription-competitive polymerase chain reaction, in diabetic NOD mice (mean duration of diabetes, 28.5 +/- 7 days) and age-matched nondiabetic NOD mice with normal glucose tolerance. The levels of mRNA coding for transforming growth factor-beta 1, tenascin, and laminin B1 increased 1.9-, 2.0-, and 1.7-fold, respectively, whereas platelet-derived growth factor (PDGF)-B, alpha 1(IV) collagen, 72-kd collagenase, alpha-smooth muscle actin, and beta-actin mRNA remained stable in the diabetic mice. The kidney advanced glycosylation end-products levels increased 2.1-fold in the diabetic mice, and the diabetic glomeruli showed an accumulation of tenascin and laminin but not of type IV collagen by immunofluorescence microscopy. There was no increase in cell number per glomerulus after the onset of diabetes, a finding consistent with stable PDGF-B and alpha-smooth muscle actin mRNA levels. These findings provide evidence that increased glomerular transforming growth factor-beta 1, but not PDGF-B, mRNA is associated with the up-regulation of tenascin and laminin expression after advanced glycosylation endproduct accumulation, early after the onset of diabetes.