Remodeling of the Fetal Collecting Duct Epithelium

Congenital urinary tract obstruction induces changes to the renal collecting duct epithelium, including alteration and depletion of intercalated cells. To study the effects of obstruction on the ontogeny of intercalated cell development, we examined normal and obstructed human fetal and postnatal kidneys. In the normal human fetal kidney, intercalated cells originated in the medullary collecting duct at 8 weeks gestation and remained most abundant in the inner medulla throughout gestation. In the cortex, intercalated cells were rare at 18 and 26 weeks gestation and observed at low abundance at 36 weeks gestation. Although early intercalated cells exhibit an immature phenotype, Type A intercalated cells predominated in the inner and outer medullae at 26 and 36 weeks gestation with other intercalated cell subtypes observed rarely. Postnatally, the collecting duct epithelium underwent a remodeling whereby intercalated cells become abundant in the cortex yet absent from the inner medulla. In 18-week obstructed kidneys with mild to moderate injury, the intercalated cells became more abundant and differentiated than the equivalent age-matched normal kidney. In contrast, more severely injured ducts of the late obstructed kidney exhibited a significant reduction in intercalated cells. These studies characterize the normal ontogeny of human intercalated cell development and suggest that obstruction induces premature remodeling and differentiation of the fetal collecting duct epithelium.Congenital urinary tract obstruction is an important cause of morbidity and mortality in affected fetuses.^1,2,3,4,5 Children born with less severe obstruction are at risk of developing chronic kidney disease and its inherent complications, including progression to end-stage renal disease and the need for dialysis and transplantation.⁶ In utero kidney obstruction results in altered glomerular development, cortical glomerular cystogenesis, tubular atrophy, apoptosis of tubular cells, and expansion and fibrosis of the renal interstitium.^7,8,9 In the fetal monkey kidney, obstruction causes significant alteration to the collecting duct (CD) epithelium, including mesenchymal transition and loss of ICs.¹⁰Unlike other nephron segments, the CD is derived exclusively from the ureteric bud. During fetal kidney development, this ureteric bud branches with the earliest differentiation of the CD epithelium occurring in the initial branches of the ureteric bud of the medulla.¹¹ The mature and differentiated CD epithelium comprises two unique cells types with principal cells responsible for vasopressin-regulated water reabsorption via aquaporin-2, and intercalated cells (ICs) regulating acid-base homeostasis.¹² ICs can further be subdivided into type A, type B, and non-A non-B cells, which are identifiable by their expression of transporter proteins (Figure 1) including vacuolar H+-ATPase (vATPase; apical in type A and non-A non-B and basolateral in type B ICs), the bicarbonate transporters pendrin (in type B and non-A non-B cells) and anion exchanger 1 (basolateral in type A), and the ammonium transporter Rhesus blood group C glycoprotein (RhCG; in type A and non-A non-B ICs).^{13,14,15,16,17} In addition to pH regulation, ICs exhibit phenotypic plasticity in vitro allowing them to differentiate into other IC subtypes and into principal cells.^18,19 This putative pluripotency raises the possibility that these subtypes may be responsible for CD epithelial renewal or the adaptive response to injury.Open in a separate windowFigure 1Classification of ICs. ICs and their functionally specific subtypes are identifiable by their expression of transport proteins, including the hydrogen ion transporter vATPase, ammonium transporter RhCG, and the chloride/bicarbonate exchangers pendrin and anion exchanger 1 (AE1).Little is known about the normal development and ontogeny of ICs in the human fetal kidney. In mice, ICs are first observed in the developing medullary CD and connecting tubule, yet are not seen in the cortical CD throughout prenatal development.^20,21 In the immediate postnatal period, the IC distribution shifts from the inner medullary CD to the cortical CD, similar to the adult distribution across several species.^{14,22,23,24,25}The full extent of the effects of obstruction on CD epithelial remodeling and on postnatal CD physiology is unknown. The purpose of the current study was to define the normal ontogeny of human IC development, and to characterize the changes in IC abundance and distribution after fetal urinary tract obstruction.     

Effects of Shiga Toxin Type 2 on a Bioengineered Three-Dimensional Model of Human Renal Tissue

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy, following infections by Shiga toxin-producing Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood renal failure worldwide. There are currently no specific therapies for STEC-associated HUS, and the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully representative animal models and an inability to monitor disease progression on a molecular or cellular level in humans at early stages. Three-dimensional (3D) tissue models have been shown to be more in vivo-like in their phenotype and physiology than 2D cultures for numerous disease models, including cancer and polycystic kidney disease. It is unknown whether exposure of a 3D renal tissue model to Stx will yield a more in vivo-like response than 2D cell culture. In this study, we characterized Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine interleukin-8 compared to the 2D cell cultures. This study represents the first application of 3D tissues for the study of Stx-mediated kidney injury.     

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A₁ peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A₂ peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A₂ sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens representing the major etiological agents of hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease characterized by hemolytic anemia, thrombocytopenia, and renal failure (19). The infection correlates with ingestion of contaminated meat or vegetables but is also transmitted by water or even person-to-person contact (8, 14, 44). Sporadic or massive outbreaks have been reported in several developed countries but, in Argentina, HUS is endemic and represents a serious public health problem with high morbidity and mortality rates (29, 40). Production of verocytotoxin or Shiga-like toxin (Stx) is the basis of EHEC pathogenesis (18, 20). The toxin is formed by a single A subunit, which possesses N-glycosidase activity to the 28S rRNA and promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells (9, 28). Although two major types (Stx1 and Stx2) and several subtypes have been described, Stx2 and Stx2c are the most frequently found toxins in severe HUS cases among EHEC-infected subjects (12, 41). The degree of antigenic cross-reactivity between Stx2 and Stx1 is low, and several authors have reported that the two toxins do not provide heterologous protection, particularly concerning the B subunits (45, 47). On the other hand, Stx2c and Stx2d variants are readily neutralized by antibodies against Stx2 (27).Despite the magnitude of the social and economic impacts caused by EHEC infections, no licensed vaccine or effective therapy is presently available for human use. So far, attempts to develop vaccine formulations against EHEC-associated sequelae have relied mainly on induction of serum anti-Stx antibody responses. Several approaches have been pursed to generate immunogenic anti-Stx vaccine formulations and include the use of live attenuated bacterial strains (2, 32), protein-conjugated polysaccharides (21), purified B subunit (33, 48), B-subunit-derived synthetic peptides (15), and mutated Stx1 and Stx2 nontoxic derivatives (5, 6, 13, 16, 37, 39, 42, 45).In a previous report we described anti-Stx2 DNA vaccines encoding either the B subunit or a fusion protein between the B subunit and the first N-terminal amino acid of the A₁ subunit (8). The DNA vaccine encoding the hybrid protein elicited Stx-specific immune responses in mice and partial protection to Stx2 challenge (1, 33). Recent data have indicated that epitopes leading to generation of Stx-neutralizing antibodies are present on both the B as well as the A subunit (34, 45, 46). In addition, further evidence indicates that the A₂ subunit contains some of the most immunogenic epitopes of the Stx2 toxin (4). Thus, in line with such evidence, we attempted the construction of a new DNA vaccine encoding the last 32 amino acids from the A₂ subunit, in addition to the complete B subunit of Stx2, as separated polypeptides which could enhance the immunogenicity and protective effects of the vaccine formulation. In the present report, we describe the generation of a new DNA vaccine encoding both Stx2 A2 and B subunits as an approach to elicit protective antibody responses to Stx2. The results obtained demonstrate that immunization with this vaccine formulation results in systemic antibody responses to Stx2 A and B subunits and toxin neutralization activity both in vitro and in vivo.     

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.     

Identification and Characterization of a Newly Isolated Shiga Toxin 2-Converting Phage from Shiga Toxin-Producing Escherichia coli

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A. D. O’Brien, J. W. Newlands, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal, Science 226:694–696, 1984). In this study, we isolated two Stx2-converting phages, designated Stx2Φ-I and Stx2Φ-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Φ-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Φ-II was distinct from them. The sizes of the plaques of Stx2Φ-I and Stx2Φ-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Φ-I and Stx2Φ-II were not identical; rather, Stx2Φ-II DNA was approximately 3 kb larger than Stx2Φ-I DNA. Furthermore, Stx2Φ-I and Stx2Φ-II showed different phage immunity, with Stx2Φ-I and 933W belonging to the same group. Infection of C600 by Stx2Φ-I or 933W was affected by environmental osmolarity differently from that by Stx2Φ-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Φ-I and 933W was greatly decreased compared with that of Stx2Φ-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Φ-I and 933W for the fadL mutant decreased to less than 10⁻⁷ compared with that for C600 whereas the efficiency of Stx2Φ-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Φ-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Φ-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His₆-tagged FadL and LamB proteins. Based on the data, we concluded that FadL acts as the receptor for Stx2Φ-I and Stx2Φ-II whereas LamB acts as the receptor only for Stx2Φ-II.     

Anaerobic Bacteria on the Mucosal Epithelium of the Murine Large Bowel

Anaerobic bacteria can be detected at population levels of 10(11) organisms per g of cecum or colon in adult mice from four different colonies widely spaced in the United States. Most of these microorganisms are oxygen-intolerant fusiform-shaped bacteria. At least one type of these tapered, rod-shaped bacteria can be seen in layers in the epithelial mucin in frozen-section histological preparations of the large bowels of mice. In addition, such microorganisms can be seen within 0.5 mum of the epithelium in ultrathin sections of colon or cecum examined in an electron microscope. These fusiform-shaped bacteria predominate in the mucin layers. However, spiral-shaped microorganisms can be found as well near the mucosal epithelia in ultrathin sections of colon. Also, such organisms can be seen in negatively-stained preparations of washings of the colonic mucosal epithelia examined in an electron microscope. At least three types of spiral-shaped organisms, including both spiral-shaped bacteria and spirochetes, can be found in preparations from mice from three of the four colonies. Such spiral-shaped microorganisms can be detected at population levels as great as 10(9) organisms per g of cecum or colon in anaerobic cultures of the large bowels of mice from all four colonies. One anaerobic spiral bacterium was isolated in pure culture. This particular organism was found by immunofluorescence to be intermingled with the fusiform-shaped bacteria in the mucin on the mucosal epithelium in the mouse large bowel.     

Retinoid Levels Influence Enterohemorrhagic Escherichia coli Infection and Shiga Toxin 2 Susceptibility in Mice

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that produces Shiga toxin (Stx) and causes hemorrhagic colitis. Under some circumstances, Stx produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome. Although retinoids like vitamin A (VA) and retinoic acid (RA) are beneficial to gut integrity and the immune system, the effect of VA supplementation on gastrointestinal infections of different etiologies has been controversial. Thus, the aim of this work was to study the influence of different VA status on the outcome of an EHEC intestinal infection in mice. We report that VA deficiency worsened the intestinal damage during EHEC infection but simultaneously improved survival. Since death is associated mainly with Stx toxicity, Stx was intravenously inoculated to analyze whether retinoid levels affect Stx susceptibility. Interestingly, while VA-deficient (VA-D) mice were resistant to a lethal dose of Stx2, RA-supplemented mice were more susceptible to it. Given that peripheral blood polymorphonuclear cells (PMNs) are known to potentiate Stx2 toxicity, we studied the influence of retinoid levels on the absolute number and function of PMNs. We found that VA-D mice had decreased PMN numbers and a diminished capacity to produce reactive oxygen species, while RA supplementation had the opposite effect. These results are in line with the well-known function of retinoids in maintaining the homeostasis of the gut but support the idea that they have a proinflammatory effect by acting, in part, on the PMN population.     

An Orally Applicable Shiga Toxin Neutralizer Functions in the Intestine To Inhibit the Intracellular Transport of the Toxin

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which causes gastrointestinal diseases and sometimes fatal systemic complications. Recently, we developed an oral Stx2 inhibitor known as Ac-PPP-tet that exhibits remarkable therapeutic potency in an STEC infection model. However, the precise mechanism underlying the in vivo therapeutic effects of Ac-PPP-tet is unknown. Here, we found that Ac-PPP-tet completely inhibited fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. Interestingly, Ac-PPP-tet accumulated in the ileal epithelial cells only through its formation of a complex with Stx2. The formation of Ac-PPP-tet-Stx2 complexes in cultured epithelial cells blocked the intracellular transport of Stx2 from the Golgi apparatus to the endoplasmic reticulum, a process that is essential for Stx2 cytotoxicity. Thus, Ac-PPP-tet is the first Stx neutralizer that functions in the intestine by altering the intracellular transport of Stx2 in epithelial cells.Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) in humans causes gastrointestinal diseases that are often followed by potentially fatal systemic complications such as acute encephalopathy and hemolytic-uremic syndrome (12, 22, 25, 26). Stx is produced in the gut, traverses the epithelium, and passes into the circulation. Circulating Stx then causes vascular damage in specific target tissues such as the brain and the kidney, resulting in systemic complications. For this reason, development of a neutralizer that specifically binds to and inhibits Stx in the gut and/or in the circulation would be a promising therapeutic approach.Stx is classified into two subgroups, Stx1 and Stx2. Stx2 is more closely related to the severity of STEC infections than Stx1 (6, 23, 31, 33). Stx consists of a catalytic A subunit and a pentameric B subunit. The former has 28S rRNA N-glycosidase activity and inhibits eukaryotic protein synthesis, while the latter is responsible for binding to the functional cell surface receptor Gb3 [Galα(1-4)-Galβ(1-4)-Glcβ1-ceramide] (11, 17, 25). The crystal structure of Stx reveals the presence of three distinctive binding sites (i.e., sites 1, 2, and 3) on each B subunit monomer for the trisaccharide moiety of Gb3 (7, 16). Highly selective and potent binding of Stx to Gb3 is attributed mainly to the multivalent interaction between the B subunit pentamer and the trisaccharide. This so-called clustering effect has formed the basis for the development of several synthetic Stx neutralizers that contain the trisaccharide in multiple configurations (3, 5, 14, 18, 19, 36). These neutralizers can strongly bind to Stx and inhibit its cytotoxic activity. Some are also effective in STEC infection models (18, 19, 36). However, the clinical application of these neutralizers has been substantially hampered by the synthetic complexity of the trisaccharide moiety.We have recently screened a library of novel tetravalent peptides that exert a clustering effect and have identified four peptide motifs that are superior to trisaccharide in binding Stx (20). Tetravalent forms of these peptides bind with high affinity to one trisaccharide-binding site (site 3) of Stx2 and effectively inhibit Stx2 cytotoxicity. This is particularly true of the neutralizer designated PPP-tet, which contains four Pro-Pro-Pro-Arg-Arg-Arg-Arg motifs. PPP-tet protects mice from a fatal dose of E. coli O157:H7, even when the peptide is orally administered after the establishment of infection (20). Moreover, the addition of acetyl groups to all the amino termini of PPP-tet (yielding Ac-PPP-tet) makes this compound resistant to proteolysis and markedly enhances its protective activity against STEC infection, indicating that Ac-PPP-tet holds promise as a therapeutic agent for STEC infections.After binding to Gb3, Stx is first transported to the Golgi apparatus in a retrograde manner and then transported to the endoplasmic reticulum (ER). On the other hand, the Stx catalytic A subunit is released into the cytoplasm, where it inhibits protein synthesis (27, 29). The retrograde transport of Stx is known to be essential for Stx cytotoxicity (2, 27, 28). In Vero cells, one of the cell types most sensitive to Stx, PPP-tet prevents Stx2 cytotoxicity by inducing the aberrant transport of Stx from the Golgi apparatus to an acidic compartment rather than to the ER, leading to the degradation of Stx (20). An advantage of PPP-tet is its ability to partially permeate cells, which allows it to inhibit the cytotoxicity of Stx2 already incorporated into cells (20). Nevertheless, the precise mechanism by which PPP-tet and Ac-PPP-tet function in vivo, as well as the identities of the organs or cells targeted by these compounds, is unknown.To understand how orally administered Ac-PPP-tet functions in vivo, we investigated the effect of Ac-PPP-tet on fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. The rabbit ileal loop system is highly valid for evaluating the toxicity of Stx2 produced in the intestine after infection. We also examined the localization of the tetravalent peptide and Stx2 in the intact rabbit ileum, cultured ileal specimens, and Caco-2 intestinal epithelial cells. Our results reveal that Ac-PPP-tet functions as a potent Stx2 neutralizer in the intestine by altering the intracellular transport of Stx2 in epithelial cells.     

Toxicity of Shiga Toxin 1 in the Central Nervous System of Rabbits

The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.     

Suppressive and Enhancing Effect of T-2 Toxin on Murine Lymphocyte Activation and Interleukin 2 Production

T-2 toxin, a fungal metabolite shown previously to exert potent immunosuppressive effects, was examined for its effects on activation and interleukin 2 (IL 2) production by murine and rat splenocytes. Splenocytes (1 × 10⁶ cells/well) were incubated with 1 μg Concanavalin A (Con A) for 48h at which time cellular protein and DNA synthesis by these cells were ascertained using radiolabeled precursors. IL 2 synthesis was determined from the cell supernatant using the IL 2-requiring cell line CTLL. Spleen cells from mice treated for 4 consecutive days with 2 mg/kg toxin exhibited a 50% reduction in in vitro Con A activation but the supernatant IL 2 levels from these cells was 4-fold higher than cells from control mice. In vitro exposure of Con A-activated normal spleen cells to various toxin doses for 48h resulted in diminished protein and DNA synthesis at 0.4 ng toxin with maximum inhibition at 1 ng (50% inhibition (TC1D₅₀) - 0.5 ng). Enhanced synthesis of both products was observed at lower toxin concentrations. IL 2 production by these cells followed a similar toxin dose response. Rat splenocytes were slightly more resistant and CTLL cells were slightly more sensitive to T-2 toxin than mouse splenocytes. These results indicate the variable effects a cytotoxic agent can have on lymphoid cells and that dosage is an important parameter for these effects.     

Immunohistochemical Study of Cytokeratin Distribution in the Collecting Duct of the Human Kidney

In order to assess the expression of different cytokeratins in the collecting duct cells (CDCs) of the human kidney, three consecutive sections were stained with periodic acid-Schiff, CAM 5.2, and AE-1 (CAM 5.2 recognizes cytokeratins #19,18,8 and AE 1 #19,16,15,14,10 of Moll's catalog.), respectively. By comparing these sections, it was found that most CDCs in the inner medulla were both CAM 5.2 and AE 1 positive, whereas in the outer medulla and cortex, 77% of the CDCs were both CAM 5.2 and AE 1 positive, 15% CAM 5.2 positive and AE 1 negative, 8% both CAM 5.2 and AE-1-negative, and 0.4% CAM 5.2 negative and AE 1 positive. Recent studies have shown that most CDCs express low-molecular weight cytokeratins #7,8,18 and 19(17,18,19,20). Of these cytokeratins, CAM 5.2 recognizes cytokeratins #8,18,19 and AE-1 recognizes cytokeratin #19. Therefore, most CDCs belong to one of the following three major types; 1. Those positive for cytokeratins #8,18 and 19 (CAM 5.2 and AE 1 positive), 2. Those positive for cytokeratins #8 and 18 and negative for #19 (CAM 5.2-positive and AE 1 -negative) and 3. Those negative for cytokeratins #8,18 and 19 (CAM 5.2 and AE 1 negative). A few CAM 5.2 negative and AE 1 positive cells were thought to express high molecular weight cytokeratins. The significance of these various cytokeratin expressions is discussed. Acta Pathol Jpn 41: 516 520, 1991.     

Monoclonal Antibody 11E10, Which Neutralizes Shiga Toxin Type 2 (Stx2), Recognizes Three Regions on the Stx2 A Subunit,Blocks the Enzymatic Action of the Toxin In Vitro,and Alters the Overall Cellular Distribution of the Toxin

Monoclonal antibody (MAb) 11E10 recognizes the Shiga toxin type 2 (Stx2) A₁ subunit. The binding of 11E10 to Stx2 neutralizes both the cytotoxic and lethal activities of Stx2, but the MAb does not bind to or neutralize Stx1 despite the 61% identity and 75% similarity in the amino acids of the A₁ fragments. In this study, we sought to identify the segment or segments on Stx2 that constitute the 11E10 epitope and to determine how recognition of that region by 11E10 leads to inactivation of the toxin. Toward those objectives, we generated a set of chimeric Stx1/Stx2 molecules and then evaluated the capacity of 11E10 to recognize those hybrid toxins by Western blot analyses and to neutralize them in Vero cell cytotoxicity assays. We also compared the amino acid sequences and crystal structures of Stx1 and Stx2 for stretches of dissimilarity that might predict a binding epitope on Stx2 for 11E10. Through these assessments, we concluded that the 11E10 epitope is comprised of three noncontiguous regions surrounding the Stx2 active site. To determine how 11E10 neutralizes Stx2, we examined the capacity of 11E10/Stx2 complexes to target ribosomes. We found that the binding of 11E10 to Stx2 prevented the toxin from inhibiting protein synthesis in an in vitro assay but also altered the overall cellular distribution of Stx2 in Vero cells. We propose that the binding of MAb 11E10 to Stx2 neutralizes the effects of the toxin by preventing the toxin from reaching and/or inactivating the ribosomes.Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) strains cause approximately 110,000 cases of infection and over 90 deaths each year in the United States according to the Centers for Disease Control and Prevention (16). Infections with STEC can lead to diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). HUS occurs in about 6 to 15% of individuals after infection with E. coli O157:H7 (15)—but less frequently with other STEC strains (5)—and is characterized by hemolytic anemia, thrombotic thrombocytopenia, and renal failure. The development of this sequela is linked to the expression of Stxs by the bacteria (18).The Stx family comprises two serogroups, Stx/Stx1 and Stx2, and polyclonal antisera raised against either Stx1 or Stx2 do not cross-neutralize the other toxin (29, 30). Stx is produced by Shigella dysenteriae type 1 and differs by only 1 amino acid from the Stx1 made by the prototypic STEC O157:H7 strain, EDL933. A single isolate of STEC can express Stx1 (or one of its variants), Stx2 (or one of its variants), or both toxins. Variants of each toxin type are defined by either a biological or immunological difference from the prototypical toxin (31). Stx1 variants include Stx1c and Stx1d, while the variants of Stx2 are Stx2c, Stx2d, Stx2d-activatable (Stx2d_act), Stx2e, and Stx2f (reviewed in reference 18).Stxs are complex holotoxins with a stoichiometry of five identical binding (B) subunits and a single active (A) domain. These AB₅ molecules are potent cytotoxins with an N-glycosidase activity that stops protein synthesis by inactivation of the 60S ribosome (6); this activity eventually leads to eukaryotic cell death. The ∼32-kDa A subunit contains the enzymatic activity of the toxin with the active site glutamic acid residue at position 167. The A subunit is asymmetrically cleaved by trypsin or furin into an enzymatically active ∼28-kDa A₁ fragment and an ∼4-kDa A₂ peptide. The A₂ peptide remains linked to the large enzymatic domain through a disulfide bond and is encircled by the five identical B subunits of ∼7.7 kDa. The B subunits of the Stxs typically bind to the eukaryotic glycolipid receptor globotriaosylceramide (Gb₃), also known as CD77. The mature A and B subunits of Stx1 and Stx2 are approximately 68 and 73% similar at the amino acid level. The crystal structures of Stx and Stx2 have been resolved, and the two structures are remarkably similar (7, 8). Nevertheless, there are some features of these three-dimensional models that differ (summarized in reference 8).Currently, there are no Food and Drug Administration-approved therapies in the United States to treat STEC infections. However, our research group is one of several that investigate passive immunization strategies to neutralize the Stxs associated with STEC infections (3, 4, 10, 13, 19, 20). Our passive immunization strategy is based on murine monoclonal antibodies (MAbs) developed in this laboratory that specifically bind to and neutralize Stx/Stx1 or Stx2 (21, 28). The MAb 11E10 was generated by immunization of BALB/c mice with Stx2 turned into a toxoid (“toxoided”) by treatment with formaldehyde (21). MAb 11E10 specifically recognizes the A₁ fragment of Stx2 and neutralizes Stx2 for Vero cells and mice but does not bind to or neutralize Stx/Stx1 (21). The murine MAb 11E10 was modified to contain a human constant region to reduce the potential for an antibody recipient to generate an antimouse antibody response (4). This human-mouse chimeric antibody, called cαStx2, successfully underwent phase I clinical testing (3). In this report, we define the epitope on the A subunit of Stx2 recognized by the murine MAb 11E10 (and, therefore, also by cαStx2) and present evidence that the MAb blocks the enzymatic action of the toxin in vitro and also alters toxin trafficking in Vero cells.