A randomized phase 2 trial of oral vitamin A for graft-versus-host disease in children and young adults.

ABSTRACT: Vitamin A plays a key role in the maintenance of gastrointestinal homeostasis and promotes a tolerogenic phenotype in tissue resident macrophages. We conducted a prospective randomized double-blinded placebo-controlled clinical trial in which 80 recipients of hematopoietic stem cell transplantation (HSCT) were randomized 1:1 to receive pretransplant high-dose vitamin A or placebo. A single oral dose of vitamin A of 4000 IU/kg, maximum 250 000 IU was given before conditioning. The primary end point was incidence of acute graft-versus-host disease (GVHD) at day +100. In an intent-to-treat analysis, incidence of acute GVHD was 12.5% in the vitamin A arm and 20% in the placebo arm (P = .5). Incidence of acute gastrointestinal (GI) GVHD was 2.5% in the vitamin A arm (P = .09) and 12.5% in the placebo arm at day +180. Incidence of chronic GVHD was 5% in the vitamin A arm and 15% in the placebo arm (P = .02) at 1 year. In an "as treated" analysis, cumulative incidence of acute GI GVHD at day +180 was 0% and 12.5% in recipients of vitamin A and placebo, respectively (P = .02), and cumulative incidence of chronic GVHD was 2.7% and 15% in recipients of vitamin A and placebo, respectively (P = .01). The only possibly attributable toxicity was asymptomatic grade 3 hyperbilirubinemia in 1 recipient of vitamin A at day +30, which self-resolved. Absolute CCR9+ CD8+ effector memory T cells, reflecting gut T-cell trafficking, were lower in the vitamin A arm at day +30 after HSCT (P = .01). Levels of serum amyloid A-1, a vitamin A transport protein with proinflammatory effects, were lower in the vitamin A arm. The vitamin A arm had lower interleukin-6 (IL-6), IL-8, and suppressor of tumorigenicity 2 levels and likely a more favorable gut microbiome and short chain fatty acids. Pre-HSCT oral vitamin A is inexpensive, has low toxicity, and reduces GVHD. This trial was registered at www.ClinicalTrials.gov as NCT03202849.

Obesity Alters cytokine signaling and gut microbiome in septic mice.

Sepsis is a leading cause of mortality. Plasma cytokine levels may identify those at increased risk of mortality from sepsis. Our aim was to understand how obesity alters cytokine levels during early sepsis and its correlation with survival. Six-week-old C57BL/6 male mice were randomized to control (non-obese) or high fat diet (obese) for 5-7 weeks. Sepsis was induced by cecal ligation and perforation (CLP). Cytokine levels were measured from cheek bleeds 8 h after CLP, and mice were monitored for survival. Other cohorts were sacrificed 1 h after CLP for plasma and tissue. Septic obese mice had higher survival. At 8 h after sepsis, obese mice had higher adiponectin, leptin, and resistin but lower TNFα and IL-6 compared to non-obese mice. When stratified by 24-h survival, adipokines were not significantly different in obese and non-obese mice. TNFα and IL-6 were higher in non-obese, compared to obese, mice that died within 24 h of sepsis. Diet and to sepsis significantly impacted the cecal microbiome. IL-6 is a prognostic biomarker during early sepsis in non-obese and obese mice. A plausible mechanism for the survival difference in non-obese and obese mice may be the difference in gut microbiome and its evolution during sepsis.

Intestinal permeability in patients undergoing stem cell transplantation correlates with systemic acute phase responses and dysbiosis.

Intestinal permeability may correlate with adverse outcomes during hematopoietic stem cell transplantation (HSCT), but longitudinal quantification with traditional oral mannitol and lactulose is not feasible in HSCT recipients because of mucositis and diarrhea. A modified lactulose:rhamnose (LR) assay is validated in children with environmental enteritis. Our study objective was to quantify peri-HSCT intestinal permeability changes using the modified LR assay. The LR assay was administered before transplant, at day +7 and +30 to 80 pediatric and young adult patients who received allogeneic HSCT. Lactulose and rhamnose were detected using urine mass spectrometry and expressed as an L:R ratio. Metagenomic shotgun sequencing of stool for microbiome analyses and enzyme-linked immunosorbent assay analyses of plasma lipopolysaccharide binding protein (LBP), ST2, REG3α, claudin1, occludin, and intestinal alkaline phosphatase were performed at the same timepoints. L:R ratios were increased at day +7 but returned to baseline at day +30 in most patients (P = .014). Conditioning regimen intensity did not affect the trajectory of L:R (P = .39). Baseline L:R ratios did not vary with diagnosis. L:R correlated with LBP levels (r2 = 0.208; P = .0014). High L:R ratios were associated with lower microbiome diversity (P = .035), loss of anaerobic organisms (P = .020), and higher plasma LBP (P = .0014). No adverse gastrointestinal effects occurred because of LR. Intestinal permeability as measured through L:R ratios after allogeneic HSCT correlates with intestinal dysbiosis and elevated plasma LBP. The LR assay is well-tolerated and may identify transplant recipients who are more likely to experience adverse outcomes.

IEC-intrinsic IL-1R signaling holds dual roles in regulating intestinal homeostasis and inflammation.

Intestinal epithelial cells (IECs) constitute a critical first line of defense against microbes. While IECs are known to respond to various microbial signals, the precise upstream cues regulating diverse IEC responses are not clear. Here, we discover a dual role for IEC-intrinsic interleukin-1 receptor (IL-1R) signaling in regulating intestinal homeostasis and inflammation. Absence of IL-1R in epithelial cells abrogates a homeostatic antimicrobial program including production of antimicrobial peptides (AMPs). Mice deficient for IEC-intrinsic IL-1R are unable to clear Citrobacter rodentium (C. rodentium) but are protected from DSS-induced colitis. Mechanistically, IL-1R signaling enhances IL-22R-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in IECs leading to elevated production of AMPs. IL-1R signaling in IECs also directly induces expression of chemokines as well as genes involved in the production of reactive oxygen species. Our findings establish a protective role for IEC-intrinsic IL-1R signaling in combating infections but a detrimental role during colitis induced by epithelial damage.

Patient-Reported Outcomes Correlate With Microbial Community Composition Independent of Mucosal Inflammation in Pediatric Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel diseases (IBDs) involve an aberrant host response to intestinal microbiota causing mucosal inflammation and gastrointestinal symptoms. Patient-reported outcomes (PROs) are increasingly important in clinical care and research. Our aim was to examine associations between PROs and fecal microbiota in patients 0 to 22 years of age with IBD. METHODS: A longitudinal, prospective, single-center study tested for associations between microbial community composition via shotgun metagenomics and PROs including stool frequency and rectal bleeding in ulcerative colitis (UC) and abdominal pain and stool frequency in Crohn's disease (CD). Mucosal inflammation was assessed with fecal calprotectin. A negative binomial mixed-effects model including clinical characteristics and fecal calprotectin tested for differentially abundant species and metabolic pathways by PROs. RESULTS: In 70 CD patients with 244 stool samples, abdominal pain correlated with increased relative abundance of Haemophilus and reduced Clostridium spp. There were no differences relative to calprotectin level. In 23 UC patients with 76 samples, both rectal bleeding and increased stool frequency correlated with increased Klebsiella and reduced Bacteroides spp. Conversely, UC patients with lower calprotectin had reduced Klebsiella. Both UC and CD patients with active symptoms exhibited less longitudinal microbial community stability. No differences in metabolic pathways were observed in CD. Increased sulfoglycolysis and ornithine biosynthesis correlated with symptomatic UC. CONCLUSIONS: Microbial community composition correlated with PROs in both CD and UC. Metabolic pathways differed relative to PROs in UC, but not CD. Data suggest that microbiota may contribute to patient symptoms in IBD, in addition to effects of mucosal inflammation.

Commensal segmented filamentous bacteria-derived retinoic acid primes host defense to intestinal infection.

Interactions between the microbiota and mammalian host are essential for defense against infection, but the microbial-derived cues that mediate this relationship remain unclear. Here, we find that intestinal epithelial cell (IEC)-associated commensal bacteria, segmented filamentous bacteria (SFB), promote early protection against the pathogen Citrobacter rodentium, independent of CD4+ T cells. SFB induced histone modifications in IECs at sites enriched for retinoic acid receptor motifs, suggesting that SFB may enhance defense through retinoic acid (RA). Consistent with this, inhibiting RA signaling suppressed SFB-induced protection. Intestinal RA levels were elevated in SFB mice, despite the inhibition of mammalian RA production, indicating that SFB directly modulate RA. Interestingly, RA was produced by intestinal bacteria, and the loss of bacterial-intrinsic aldehyde dehydrogenase activity decreased the RA levels and increased infection. These data reveal RA as an unexpected microbiota-derived metabolite that primes innate defense and suggests that pre- and probiotic approaches to elevate RA could prevent or combat infections.

Reducing acute kidney injury in pediatric oncology patients: An improvement project targeting nephrotoxic medications.

BACKGROUND: Nephrotoxic medication exposure and associated acute kidney injury (AKI) occur commonly in hospitalized children. At Cincinnati Children's Hospital Medical Center, there is an initiative to increase awareness of nephrotoxic medication exposure and decrease rates of associated AKI. The oncology service utilized these data in a quality improvement project to drive reductions in AKI rates. METHODS: Three interventions were implemented targeted at decreasing the incidence of nephrotoxic exposure, as well as protecting against the conversion of exposures to AKI episodes. Cefepime replaced piperacillin-tazobactam for febrile neutropenia, vancomycin stewardship limited empiric courses to 72 hours, and nephroprotection for intravenous contrast administration was standardized for defined high-risk patients. RESULTS: The study cohort comprised 42 520 noncritically ill patient days admitted to the oncology service at Cincinnati Children's Hospital Medical Center. A total of 273 unique patients were exposed to combination nephrotoxic medications, leading to 111 AKI episodes. The rate of nephrotoxic medication exposure within the oncology service decreased by 49% from 16.08 to 8.17 per 1000 patient days. Episodes of AKI associated with nephrotoxic medication exposure decreased by 45% from 3.48 to 1.92 per 1000 patient days. CONCLUSION: Interventions to decrease AKI took a three-pronged approach. Collectively, this approach was proven successful with significant reductions in both rates of nephrotoxic medication exposure and associated AKI among hospitalized oncology patients.

Microbial Shifts and Shorter Time to Bowel Resection Surgery Associated with C. difficile in Pediatric Crohn's Disease.

BACKGROUND: Clostridioides difficile infection and colonization are common in pediatric Crohn's disease (CD). Our aims were to test the relationship between C. difficile positivity and bowel resection surgery and to characterize microbial shifts associated with C. difficile carriage and surgery. METHODS: A retrospective single-center study of 75 pediatric CD patients tested for association between C. difficile carriage and bowel resection surgery. A prospective single-center study of 70 CD patients utilized C. difficile testing and shotgun metagenomic sequencing of fecal samples to define microbiota variation stratified by C. difficile carriage or history of surgery. RESULTS: The rate of bowel resection surgery increased from 21% in those without C. difficile to 67% in those with (P = 0.003). From a Kaplan-Meier survival model, the hazard ratio for time to first surgery was 4.4 (95% CI, 1.2-16.2; P = 0.00) in patients with positive C. difficile testing in the first year after diagnosis. Multivariable logistic regression analysis confirmed this association (odds ratio 16.2; 95% CI, 2.2-120; P = 0.006). Larger differences in microbial abundance and metabolic pathways were observed in patients with prior surgery than in those with C. difficile carriage. Depletion of Alistipes and Ruminococcus species and reduction in methionine biosynthesis were noted in patients with both C. difficile carriage and past surgery. CONCLUSIONS: A positive C. difficile test during the first year after diagnosis is associated with decreased time to first bowel resection surgery in pediatric Crohn's disease. Depletion of beneficial commensals and methionine biosynthesis in patients with C. difficile carriage may contribute to increased risk for surgery.

Reduction in Nephrotoxic Antimicrobial Exposure Decreases Associated Acute Kidney Injury in Pediatric Hematopoietic Stem Cell Transplant Patients.

Exposure to nephrotoxic medications is a common risk factor for acute kidney injury (AKI) in pediatric stem cell transplantation (SCT). We hypothesized that reducing nephrotoxic antimicrobial exposure for SCT patients would be associated with lower nephrotoxin-associated AKI (NTMx-AKI) rates and no increase in infection treatment failures. We conducted a prospective cohort analysis of all inpatient SCT patients at Cincinnati Children's Hospital Medical Center between January 2014 and December 2017. In January 2016, first line fever coverage was changed from piperacillin-tazobactam to cefepime, acknowledging that the change resulted in a loss of enterococcal coverage, and the duration of antimicrobial exposures was limited, specifically including vancomycin. We collected data using prospective NTMx-AKI and antimicrobial utilization monitoring platforms within the electronic health record. AKI days and severity were extracted for patients exposed to 3+ nephrotoxins, 3+ days of IV aminoglycosides, or 3+ days of IV vancomycin. AKI was identified using KDIGO serum creatinine criteria. We assessed rates of nephrotoxin exposure and NTMx-AKI in all SCT inpatients for 2 years pre- and post-intervention. Data were grouped and analyzed by calendar month, normalized to a denominator of 1000 patient-days. Statistical process control methods were used to monitor adherence to the intervention and identify changes in mean rate of nephrotoxin exposure and NTMx-AKI. Infection rates, alternate antimicrobial usage rates, and the fraction of repeat positive cultures were used to identify treatment failures. PTZ usage decreased from 196 to 33 days/1000 patient days, cefepime usage increased from 62 to 290 days/1000 patient days, and vancomycin usage decreased from 62 to 41 days/1000 patient days. High nephrotoxin exposure decreased by 33% (143 to 96 days/1000 patient days), and NTMx-AKI decreased by 74% (24 to 6 days/1000 patient days). Rates of all KDIGO stages of NTMx-AKI decreased ≥50% after the intervention. Stage 3, the most severe, decreased by >80%. The fraction of repeat positive cultures remained stable between the two eras at .1 (standard deviation 0.21) and .07 (standard deviation 0.17), respectively. There were no increases in infection rates, alternate antimicrobial usage rates, or treatment failures. Reduction of nephrotoxic antimicrobial exposure can decrease the amount and severity of NTMx-AKI in SCT patients without an increase in treatment failures.

A Pilot Study of Human Milk to Reduce Intestinal Inflammation After Bone Marrow Transplant.

OBJECTIVE: Human milk administration in the early peritransplant period would lower intestinal inflammation after bone marrow transplant (BMT). MATERIALS AND METHODS: Children 0-5 years undergoing BMT received either a ready-to-feed human milk preparation designed for these children (Prolacta Bioscience, Duarte, CA) or standard formula. Babies breastfeeding at the time of BMT were also enrolled on the human milk arm. Human milk was administered from day -3 until day +14 after BMT. Metagenomic shotgun sequencing and metabolomics of stool, plasma cytokines, and regenerating islet-derived 3α (REG3α) levels were measured at enrollment and day +14. Human leukocyte antigen-DR isotype (HLA-DR), CD38, and CD69 expression on T cells were evaluated at day +21. RESULTS: Forty-six children were enrolled, 32 received human milk (donor milk n = 23, breastfeeding babies n = 9), and 14 were controls who received standard feeds supervised by a BMT dietician. Twenty-four patients received at least 60% of goal human milk and were evaluable. Plasma interleukin (IL)-8 (p = 0.04), IL-10 (p = 0.02), and REG3α (p = 0.03) were decreased in the human milk cohort. Peripheral blood CD69+ CD8+ T cells were higher in controls (p = 0.01). Species abundance of Adenovirus (p = 0.00034), Escherichia coli (p = 0.0017), Cryptosporidium parvum (p = 0.0006), Dialister invisus (p = 0.01), and Pseudomonas aeruginosa (p = 0.05) from stool was higher in controls. Stool alanine, tyrosine, methionine, and the ratio of fecal alanine to choline and phosphocholine were higher in controls (p < 0.05). No difference was observed in stool propionate and butyrate levels as measures of short-chain fatty acids between the two cohorts. CONCLUSIONS: Administration of human milk resulted in decreased markers of intestinal inflammation and could be a valuable adjunct for patients after BMT.

Antibiotic Exposure and Reduced Short Chain Fatty Acid Production after Hematopoietic Stem Cell Transplant.

Human studies have shown loss of diversity of the gut microbiome following hematopoietic stem cell transplantation (HSCT) in association with significant gut injury caused by the preparative regimen. Prolonged antibiotic use worsens loss of microbiome diversity and increases risk of complications such as graft-versus-host disease (GVHD). Our data support the hypothesis that loss of intestinal commensals that produce short-chain fatty acids (SCFAs) may increase dysbiosis. Here, we report an extensive longitudinal examination of changes in the luminal SCFAs in children undergoing allogeneic HSCT, and the relationship of those changes to the microbiota and antibiotic exposure. We found significant and progressive alterations in butyrate, and in additional SCFAs in stool in the first 14 days after transplant, a finding not observed in published mouse studies. SCFA levels were lower in children receiving antibiotics with activity against anaerobic organisms. Moreover, day 14 post-HSCT butyrate and propionate levels are lower in children who went on to develop GVHD, although our disease population was small. These data provide insight into the mechanism of prior observations that loss of diversity and increased antibiotic use are associated with GVHD following HSCT. Our findings offer potential modifiable targets to reduce risk of GVHD and improve survival after HSCT.

Thermoneutral housing exacerbates nonalcoholic fatty liver disease in mice and allows for sex-independent disease modeling.

Nonalcoholic fatty liver disease (NAFLD), a common prelude to cirrhosis and hepatocellular carcinoma, is the most common chronic liver disease worldwide. Defining the molecular mechanisms underlying the pathogenesis of NAFLD has been hampered by a lack of animal models that closely recapitulate the severe end of the disease spectrum in humans, including bridging hepatic fibrosis. Here we demonstrate that a novel experimental model employing thermoneutral housing, as opposed to standard housing, resulted in lower stress-driven production of corticosterone, augmented mouse proinflammatory immune responses and markedly exacerbated high-fat diet (HFD)-induced NAFLD pathogenesis. Disease exacerbation at thermoneutrality was conserved across multiple mouse strains and was associated with augmented intestinal permeability, an altered microbiome and activation of inflammatory pathways that are associated with the disease in humans. Depletion of Gram-negative microbiota, hematopoietic cell deletion of Toll-like receptor 4 (TLR4) and inactivation of the IL-17 axis resulted in altered immune responsiveness and protection from thermoneutral-housing-driven NAFLD amplification. Finally, female mice, typically resistant to HFD-induced obesity and NAFLD, develop full disease characteristics at thermoneutrality. Thus, thermoneutral housing provides a sex-independent model of exacerbated NAFLD in mice and represents a novel approach for interrogation of the cellular and molecular mechanisms underlying disease pathogenesis.

MAPK-activated protein kinase 2 contributes to Clostridium difficile-associated inflammation.

Clostridium difficile infection (CDI) results in toxin-induced epithelial injury and marked intestinal inflammation. Fecal markers of intestinal inflammation correlate with CDI disease severity, but regulation of the inflammatory response is poorly understood. Previous studies demonstrated that C. difficile toxin TcdA activates p38 kinase in tissue culture cells and mouse ilium, resulting in interleukin-8 (IL-8) release. Here, we investigated the role of phosphorylated mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2 kinase, pMK2), a key mediator of p38-dependent inflammation, in CDI. Exposure of cultured intestinal epithelial cells to the C. difficile toxins TcdA and TcdB resulted in p38-dependent MK2 activation. Toxin-induced IL-8 and GROα release required MK2 activity. We found that p38 and MK2 are activated in response to other actin-disrupting agents, suggesting that toxin-induced cytoskeleton disruption is the trigger for kinase-dependent cytokine response. Phosphorylated MK2 was detected in the intestines of C. difficile-infected hamsters and mice, demonstrating for the first time that the pathway is activated in infected animals. Furthermore, we found that elevated pMK2 correlated with the presence of toxigenic C. difficile among 100 patient stool samples submitted for C. difficile testing. In conclusion, we find that MK2 kinase is activated by TcdA and TcdB and regulates the expression of proinflammatory cytokines. Activation of p38-MK2 in infected animals and humans suggests that this pathway is a key driver of intestinal inflammation in patients with CDI.

Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessing-independent mechanism.

Clostridium difficile is the most common cause of antibiotic-associated nosocomial infection in the United States. C. difficile secretes two homologous toxins, TcdA and TcdB, which are responsible for the symptoms of C. difficile associated disease. The mechanism of toxin action includes an autoprocessing event where a cysteine protease domain (CPD) releases a glucosyltransferase domain (GTD) into the cytosol. The GTD acts to modify and inactivate Rho-family GTPases. The presumed importance of autoprocessing in toxicity, and the apparent specificity of the CPD active site make it, potentially, an attractive target for small molecule drug discovery. In the course of exploring this potential, we have discovered that both wild-type TcdB and TcdB mutants with impaired autoprocessing or glucosyltransferase activities are able to induce rapid, necrotic cell death in HeLa and Caco-2 epithelial cell lines. The concentrations required to induce this phenotype correlate with pathology in a porcine colonic explant model of epithelial damage. We conclude that autoprocessing and GTD release is not required for epithelial cell necrosis and that targeting the autoprocessing activity of TcdB for the development of novel therapeutics will not prevent the colonic tissue damage that occurs in C. difficile - associated disease.

The MAP kinase-activated protein kinase 2 (MK2) contributes to the Shiga toxin-induced inflammatory response.

Infection with Shiga toxin (STx)-producing bacteria can progress to a toxemic, extraintestinal injury cascade known as haemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Mounting evidence suggests that STx activates stress response pathways in susceptible cells and has implicated the p38 mitogen-activated protein kinase (MAPK) pathway. More importantly, some of the pathology associated with HUS is believed to be a result of a STx-induced inflammatory response. From a siRNA screen of the human kinome adapted to a high-throughput format, we found that knock-down of the MAPK-activated protein kinase 2 (MK2), a downstream target of the p38 MAPK, protected against Shiga toxicity. Further characterization of the in vitro role of MK2 revealed that STx activates the p38-MK2 stress response pathway in both p38- and MK2-dependent manners in two distinct cell lines. MK2 activation was specific to damage to the ribosome by an enzymatically active toxin and did not result from translational inhibition per se. Genetic and chemical inhibition of MK2 significantly decreased the inflammatory response to STx. These findings suggest that MK2 inhibition might play a valuable role in decreasing the immuopathological component of STx-mediated disease.

Contribution of the HEDJ/ERdj3 cysteine-rich domain to substrate interactions.

Cytoplasmic type I DnaJ/Hsp40 chaperones contain a Cys-rich domain consisting of four CXXCXG motifs that are in a reduced state and coordinate zinc, stabilizing the intervening sequence in a loop structure. However, the Cys-rich region of the endoplasmic reticulum localized HEDJ (ERdj3/ERj3p), is considerably different in sequence and arrangement. Unlike the typical type I molecule, the HEDJ CXC, and CXXC motifs were demonstrated in this study to be predominantly oxidized in intramolecular disulfide bonds. In the native state, HEDJ bound to immobilized, denatured thyroglobulin. Unlike its binding partner GRP78, redox conditions affected the interaction of HEDJ with substrate. Substitution of the Cys-rich domain cysteine residues with serine diminished or abolished HEDJ binding in the in vitro assay. These findings suggest that the Cys-rich region of HEDJ and its oxidation state are important in maintaining the substrate interaction domain in a binding-competent conformation.

A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis.

Inhibition of protein synthesis is a common mechanism by which bacterial and plant toxins injure human cells. Examples of toxins that inhibit protein synthesis include shiga toxins of Escherichia coli, diphtheria toxin, Pseudomonas exotoxin A and the plant toxin ricin. In order to facilitate studies on toxin pathogenesis and to enable screening for inhibitors of toxin action, a quantitative and highly sensitive assay for the action of these toxins on mammalian cells was developed. The cDNA encoding destabilized luciferase was cloned into an adenoviral expression plasmid and a high-titre viral stock was prepared. Following transduction of Vero cells, luciferase expression was found to be linear with respect to viral multiplicity of infection. Luciferase expression by as few as 10 cells was readily detected. Treatment of transduced cells with either cycloheximide or shiga toxin resulted in a decrease in luciferase activity, with a half-life ranging from 1 to 2 h. Inhibition of luciferase expression was evident at toxin concentrations as low as 1 pg ml(-1). The assay was adapted for use in 24-, 96- and 384-well plates, enabling rapid processing of large numbers of samples. Using this approach, susceptibility of Vero, Hep2, Chang, A549, COS-1 and HeLa cells to three different toxins was determined. These results demonstrate that the luciferase-based assay is applicable to the study of numerous cell types, is quantitative, highly sensitive and reproducible. These features will facilitate studies on pathophysiology of toxin-mediated diseases and allow high-throughput screening for inhibitors of cytotoxicity.