Apoptosis of Renal Cortical Cells in the Hemolytic-Uremic Syndrome: In Vivo and In Vitro Studies

ABSTRACT This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing Escherichia coli. Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with E. coli O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and E. coli O157:H7 extracts were examined for apoptotic changes. Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains. Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group. Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells. DNA fragmentation increased when cells were prestimulated with tumor necrosis factor alpha. Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation. The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury.

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Thrombotic Thrombocytopenic Purpura and Sporadic Hemolytic-Uremic Syndrome Plasmas Induce Apoptosis in Restricted Lineages of Human Microvascular Endothelial Cells

Blood ◽

10.1182/blood.v89.4.1224 ◽

1997 ◽

Vol 89 (4) ◽

pp. 1224-1234 ◽

Cited By ~ 120

Author(s):

Debashis Mitra ◽

Eric A. Jaffe ◽

Babette Weksler ◽

Katherine A. Hajjar ◽

Carl Soderland ◽

...

Keyword(s):

Endothelial Cells ◽

Hemolytic Uremic Syndrome ◽

Thrombotic Thrombocytopenic Purpura ◽

Apoptotic Cell ◽

Umbilical Vein ◽

Thrombocytopenic Purpura ◽

Related Disorder ◽

Uremic Syndrome ◽

Vessel Coronary Artery

Abstract Thrombotic thrombocytopenic purpura (TTP) and sporadic hemolytic-uremic syndrome (HUS) are thrombotic microangiopathies that occur in the absence of an inflammatory response. Ultrastructural features of tissues involved in TTP/sporadic HUS suggest an apoptotic process. Consistent with these findings, we observed that TTP plasmas induce apoptosis in primary human endothelial cells (EC) of dermal microvascular but not umbilical vein origin (Laurence et al, Blood 87:3245, 1996). We now document the ability of plasmas from both TTP and sporadic HUS patients, but not from a patient with childhood/diarrhea-associated HUS, to induce apoptosis and expression of the apoptosis-associated molecule Fas (CD95) in restricted lineages of microvascular EC. EC of small vessel dermal, renal, and cerebral origin were susceptible to induction of Fas and an apoptotic cell death. In contrast, microvascular EC of pulmonary and hepatic origin, as well as EC of a large vessel, coronary artery, were resistant to both processes. This dichotomy parallels the in vivo pathology of TTP/sporadic HUS, with notable sparing of the pulmonary and hepatic microvasculature. Apoptotic EC also had some features of a procoagulant phenotype, including depressed production of prostaglandin I2 (prostacyclin). These phenomena support the pathophysiologic significance of microvascular EC apoptosis in TTP, extend it to a related disorder (sporadic HUS), and suggest consideration of apoptosis inhibitors in the experimental therapeutics of these syndromes.

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Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome

Blood ◽

10.1182/blood.v74.3.978.bloodjournal743978 ◽

1989 ◽

Vol 74 (3) ◽

pp. 978-983 ◽

Cited By ~ 57

Author(s):

PM Mannucci ◽

R Lombardi ◽

A Lattuada ◽

P Ruggenenti ◽

GL Vigano ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Thrombotic Thrombocytopenic Purpura ◽

Von Willebrand Factor ◽

Relative Increase ◽

Thrombocytopenic Purpura ◽

Uremic Syndrome ◽

Von Willebrand ◽

Willebrand Factor

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.

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Differential Tumor Necrosis Factor Alpha Expression and Release from Peritoneal Mouse Macrophages In Vitro in Response to Proliferating Gram-Positive versus Gram-Negative Bacteria

Infection and Immunity ◽

10.1128/iai.68.8.4422-4429.2000 ◽

2000 ◽

Vol 68 (8) ◽

pp. 4422-4429 ◽

Cited By ~ 18

Author(s):

Wei Cui ◽

David C. Morrison ◽

Richard Silverstein

Keyword(s):

Tumor Necrosis ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

E Coli ◽

Tnf Α ◽

Mouse Macrophages ◽

Factor Alpha ◽

Necrosis Factor

ABSTRACT Viable Escherichia coli and Staphylococcus aureus bacteria elicited markedly different in vitro tumor necrosis factor alpha (TNF-α) responses when placed in coculture with peritoneal murine macrophages. These include quantitative differences in TNF-α mRNA expression and corresponding protein product secretion as well as kinetic differences in the profiles of the TNF-α responses. Further, lipopolysaccharide (from E. coli) is a major contributing factor to these differences, as revealed by comparative experiments with endotoxin-responsive (C3Heb/FeJ) and endotoxin-hyporesponsive (C3H/HeJ) macrophages. Nevertheless, the eventual overall magnitude of the TNF-α secretion of macrophages in response to S. aureus was at least equivalent to that observed with E. coli, while appearing at time periods hours later than the E. coli-elicited TNF-α response. Both the magnitude and kinetic profile of the TNF-α responses were found to be relatively independent of the rate of bacterial proliferation, at least to the extent that similar results were observed with both viable and paraformaldehyde-killed microbes. Nevertheless, S. aureus treated in culture with the carbapenem antibiotic imipenem manifests markedly altered profiles of TNF-α response, with the appearance of an early TNF-α peak not seen with viable organisms, a finding strikingly similar to that recently reported by our laboratory from in vivo studies (R. Silverstein, J. G. Wood, Q. Xue, M. Norimatsu, D. L. Horn, and D. C. Morrison, Infect. Immun. 68:2301–2308, 2000). In contrast, imipenem treatment of E. coli-cocultured macrophages does not significantly alter the observed TNF-α response either in vitro or in vivo. In conclusion, our data support the concept that the host inflammatory response of cultured mouse macrophages in response to viable gram-positive versus gram-negative microbes exhibits distinctive characteristics and that these distinctions are, under some conditions, altered on subsequent bacterial killing, depending on the mode of killing. Of potential importance, these distinctive in vitro TNF-α profiles faithfully reflect circulating levels of TNF-α in infected mice. These results suggest that coculture of peritoneal macrophages with viable versus antibiotic-killed bacteria and subsequent assessment of cytokine response (TNF-α) may be of value in clarifying, and ultimately controlling, related host inflammatory responses in septic patients.

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The Fibrinolytic System in the Hemolytic Uremic Syndrome: In Vivo and In Vitro Studies

Pediatric Research ◽

10.1203/00006450-199408000-00019 ◽

1994 ◽

Vol 36 (2) ◽

pp. 257-264 ◽

Cited By ~ 27

Author(s):

Nicole C A J Van De Kar ◽

Victor W M Van Hinsbergh ◽

Emile J P Brommer ◽

Leo A H Monnens

Keyword(s):

Hemolytic Uremic Syndrome ◽

In Vitro Studies ◽

Fibrinolytic System ◽

Uremic Syndrome

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Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome

Blood ◽

10.1182/blood.v74.3.978.978 ◽

1989 ◽

Vol 74 (3) ◽

pp. 978-983 ◽

Cited By ~ 5

Author(s):

PM Mannucci ◽

R Lombardi ◽

A Lattuada ◽

P Ruggenenti ◽

GL Vigano ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Thrombotic Thrombocytopenic Purpura ◽

Von Willebrand Factor ◽

Relative Increase ◽

Thrombocytopenic Purpura ◽

Uremic Syndrome ◽

Von Willebrand ◽

Willebrand Factor

Abstract To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.

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Differential Efficacy of Caspase Inhibitors on Apoptosis Markers during Sepsis in Rats and Implication for Fractional Inhibition Requirements for Therapeutics

Journal of Experimental Medicine ◽

10.1084/jem.20031791 ◽

2004 ◽

Vol 199 (2) ◽

pp. 199-207 ◽

Cited By ~ 41

Author(s):

Nathalie Méthot ◽

JingQi Huang ◽

Nathalie Coulombe ◽

John P. Vaillancourt ◽

Dita Rasper ◽

...

Keyword(s):

Cell Death ◽

Dna Fragmentation ◽

Caspase 3 ◽

Apoptotic Cell ◽

Mitochondrial Pathway ◽

Caspase Inhibitors ◽

Caspase Inhibition ◽

Caspase Activated Dnase

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

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Development of DNA Vaccines against Hemolytic-Uremic Syndrome in a Murine Model

Infection and Immunity ◽

10.1128/iai.71.7.3971-3978.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3971-3978 ◽

Cited By ~ 20

Author(s):

Alejandra V. E. Capozzo ◽

Virginia Pistone Creydt ◽

Graciela Dran ◽

Gabriela Fernández ◽

Sonia Gómez ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Humoral Response ◽

Host Cells ◽

Newborn Mice ◽

B Subunit ◽

Adult Mice ◽

Uremic Syndrome ◽

A Subunit

ABSTRACT Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2ΔA). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.

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Activation of the Nlrp3 Inflammasome Contributes to Shiga Toxin-Induced Hemolytic Uremic Syndrome in a Mouse Model

Frontiers in Immunology ◽

10.3389/fimmu.2020.619096 ◽

2021 ◽

Vol 11 ◽

Author(s):

Liqiong Song ◽

Yuchun Xiao ◽

Xianping Li ◽

Yuanming Huang ◽

Guangxun Meng ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Nlrp3 Inflammasome ◽

Gene Knockout ◽

Peritoneal Macrophages ◽

Inflammasome Activation ◽

Nlrp3 Inflammasome Activation ◽

Uremic Syndrome ◽

Biochemical Indices

ObjectiveTo explore the role of the Nlrp3 inflammasome activation in the development of hemolytic uremic syndrome (HUS) induced by Stx2 and evaluate the efficacy of small molecule Nlrp3 inhibitors in preventing the HUS.MethodsPeritoneal macrophages (PMs) isolated from wild-type (WT) C57BL/6J mice and gene knockout mice (Nlrc4-/-, Aim2-/-, and Nlrp3-/-) were treated with Stx2 in vitro and their IL-1β releases were measured. WT mice and Nlrp3-/- mice were also treated with Stx2 in vivo by injection, and the biochemical indices (serum IL-1β, creatinine [CRE] and blood urea nitrogen [BUN]), renal injury, and animal survival were compared. To evaluate the effect of the Nlrp3 inhibitors in preventing HUS, WT mice were pretreated with different Nlrp3 inhibitors (MCC950, CY-09, Oridonin) before Stx2 treatment, and their biochemical indices and survival were compared with the WT mice without inhibitor pretreatment.ResultsWhen PMs were stimulated by Stx2 in vitro, IL-1β release in Nlrp3-/- PMs was significantly lower compared to the other PMs. The Nlrp3-/- mice treated by Stx2 in vivo, showed lower levels of the biochemical indices, alleviated renal injuries, and increased survival rate. When the WT mice were pretreated with the Nlrp3 inhibitors, both the biochemical indices and survival were significantly improved compared to those without inhibitor pretreatment, with Oridonin being most potent.ConclusionNlrp3 inflammasome activation plays a vital role in the HUS development when mice are challenged by Stx2, and Oridonin is effective in preventing HUS.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

Current Drug Delivery ◽

10.2174/1567201818666201214125237 ◽

2020 ◽

Vol 18 ◽

Author(s):

Zirui Zhang ◽

Shangcong Han ◽

Panpan Liu ◽

Xu Yang ◽

Jing Han ◽

...

Keyword(s):

Wound Healing ◽

Mrna Expression ◽

Tissue Injury ◽

Inflammatory Cells ◽

Pcr Analysis ◽

Protective Effects ◽

Deep Tissue ◽

Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

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