CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Nov. 1996, p. 701–705 1071-412X/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 3, No. 6
Lipopolysaccharide-Specific Antibodies in Plasma and Stools of Children with Shigella-Associated Leukemoid Reaction and Hemolytic-Uremic Syndrome TASNIM AZIM,* FIRDAUSI QADRI, SHARMEEN AHMED, M. SAFIULLAH SARKER, RAMESH C. HALDER, JENA HAMADANI, AKHTARUZZAMAN CHOWDHURY, M. A. WAHED, M. ABDUS SALAM, AND M. JOHN ALBERT International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka 1000, Bangladesh Received 16 May 1996/Returned for modification 3 July 1996/Accepted 1 August 1996
Antibody responses to the lipopolysaccharide (LPS) of shigellae were compared between children with uncomplicated and complicated Shigella dysenteriae 1 infection. One hundred fifteen children between 12 and 60 months of age with S. dysenteriae 1 infection were studied. Of these children, 42 had complications (leukemoid reaction and/or hemolytic-uremic syndrome [complicated shigellosis]) and 73 had no complications (uncomplicated shigellosis). Antibodies to the LPS of S. dysenteriae 1 and Shigella flexneri Y were measured in plasma and stools, as were total immunoglobulin A (IgA) and IgG concentrations in plasma and the total IgA concentration in stool, on enrollment and 3 to 5 days later. In the plasma, the concentrations of homologous (IgG) and heterologous (IgA) LPS antibodies on enrollment were higher in children with complicated shigellosis than in those with uncomplicated shigellosis. In stool, the concentrations on enrollment were similar between the two groups of children. There was a rise in antibody concentrations in the plasma (homologous and heterologous) and stool (homologous) between the day of enrollment and 3 to 5 days later in children with uncomplicated shigellosis but not in those with complicated shigellosis. These findings suggest that systemic stimulation is more marked in children with complications, so that a subsequent rise in plasma antibody concentrations does not occur in these children. In contrast, the lack of a rise in stool antibody concentrations in children with complicated shigellosis is suggestive of a lower-level mucosal response. Because the duration of diarrhea before enrollment influenced the homologous antibody concentrations, children were further divided into three subgroups (short [3 to 5 days], medium [6 to 9 days], and long [>9 days] diarrhea durations before enrollment). Comparisons of homologous antibody concentrations between the two groups of children following such subdivisions showed that in children with complicated shigellosis, antibody concentrations were higher in the plasma of children in the short diarrhea duration subgroup but lower in the stool of children in the medium diarrhea duration subgroup. No differences in antibody concentrations were observed in children in the other diarrhea duration subgroups. Thus, complications in shigellosis are associated with an early and strong systemic stimulation without a concomitant stimulation of the mucosal antibody response. children (11). The cause(s) of these complications is not fully understood, but Shiga toxin and Shiga-like toxin II have been implicated because they are both potent cytotoxic agents for vascular endothelial cells (14, 17). In addition, LPS acts synergistically with Shiga toxin in damaging endothelial cells (13). The role of antigen-specific antibodies in the development of HUS and leukemoid reaction is not clear. In enterohemorrhagic E. coli-associated HUS, high levels of antibodies to the LPS are detected in serum (3). However, in more severe cases of HUS, the levels of anti-LPS IgG antibodies are lower in serum (9), and it has been hypothesized that the lower levels of antibodies may be due to their consumption by the large amount of endotoxin released into the circulation. In Shigellaassociated HUS, high levels of endotoxin are present in blood (15), but there is no information about antibody responses in complications associated with shigellosis. In the present study we compared the LPS antibody responses in the plasma and stools of children with complicated shigellosis with those in children with uncomplicated shigellosis.
The humoral immune response in both the systemic (5, 10, 16) and mucosal (6, 10, 16, 21) compartments has been well documented in uncomplicated shigellosis. The role of these antibodies in protection is unclear, but there is evidence to suggest that the non-immunoglobulin M (non-IgM) fraction to the lipopolysaccharide (LPS) may be protective (7). Children living in areas where Shigella infection is endemic, such as Bangladesh, are most at risk of acquiring Shigella infections. Also, in children below 5 years of age, infection with Shigella dysenteriae 1 can lead to a life-threatening complication, hemolytic-uremic syndrome (HUS) (consisting of a triad of hemolytic anemia, thrombocytopenia, and acute renal failure) (18). The leukemoid reaction (characterized by an increase in the blood leukocyte count of $40,000/ml, granulocytosis, and an increase in the number of immature granulocytes) (4), although not serious in itself, may accompany or precede HUS, so that its presence indicates poor diagnosis (4). S. dysenteriae 1 is not the only enteropathogen associated with the development of HUS; enterohemorrhagic Escherichia coli O157:H7 has a similar association with HUS in young
MATERIALS AND METHODS Study population. Children between 12 and 60 months of age attending the Clinical Research and Service Centre of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), located in Dhaka, were studied. Initial enrollment was done on the basis of a history of dysentery (visible blood in stool, with or without fever). Stools were examined microscopically and were
* Corresponding author. Mailing address: Laboratory Sciences Division, ICDDR,B, GPO Box 128, Dhaka 1000, Bangladesh. Phone: 880 2 871751 to 880 2 871760. Fax: 880 2 883116 and 880 2 886050. Electronic mail address: tasnim%
[email protected]. 701
702
AZIM ET AL.
CLIN. DIAGN. LAB. IMMUNOL. TABLE 1. Clinical characteristics of children on enrollment No. (%) withc
Study group (children with shigellosis)
Age (mo)a
No. (%) male
Wt for age (% of NCHSb median)a
Duration of diarrhea (days)a
Stool frequency (per 24 h)a
.20 WBCs/HPF in stool
.20 RBCs/HPF in stool
Uncomplicated Complicated
31.8 6 13.0 27.8 6 12.6
39 (53.4) 25 (59.5)
66.8 6 11.6 70.0 6 11.3
7.7 6 5.0 7.7 6 3.7
25.2 6 16.6 22.0 6 14.5
65 (94.2) 27 (65.9)
47 (67.1) 11 (26.8)
NSe
NS
NS
NS
NS
,0.001
,0.001
Pd
Expressed as mean 6 SD. b NCHS, National Center for Health Statistics. c WBCs, leukocytes; HPF, high-power microscopic field; RBCs, erythrocytes. d The Mann-Whitney U test (for continuous variables) and the chi-square statistic (for categorical variables) were used to compare children with uncomplicated and complicated shigellosis. e NS, not significant. a
cultured for enteric bacterial pathogens (24), and only children with cultureconfirmed S. dysenteriae 1 infection were finally included in the study. All children were clinically evaluated by a daily physical examination and determination of vital signs. Laboratory investigations included determination of hematocrit, total and differential leukocyte counts, platelet counts, and numbers of fragmented erythrocytes. Serum electrolyte and creatinine concentrations were measured when required. All children with shigellosis were treated with amdinocillin pivoxil or ciprofloxacin. Some children received additional antibiotics for concomitant infections such as respiratory tract infections, middle ear infections, or septicemia. Venous blood (5 ml) was collected on enrollment and 3 to 5 days after enrollment from all children except those children who had received blood transfusions or who were transferred to another hospital in the interim period and was placed in sterile Vacutainer tubes (Becton-Dickinson, Rutherford, N.J.). The study was approved by the Ethical Review Committee of ICDDR,B. Collection of plasma. Fresh blood was separated on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), in which plasma collected as a clear supernatant above the band of mononuclear cells at the interface. Plasma was collected and filtered through a 0.22-mm-pore-size filter (Sartorius, Goettingen, Germany), and aliquots were frozen at 2208C until use. Preparation of stool extracts. Stools were mixed 1:1 (for liquid stools) or 1:4 (for solid stools) (weight/volume with phosphate-buffered saline [pH 7.2]) containing soybean protein inhibitor (1 mg/ml; Sigma Chemical Co., St. Louis, Mo.), phenylmethylsulfonyl fluoride (1 mg/ml; Sigma), and 0.05% Tween 20 (Sigma). The mixture was vortexed and centrifuged at 20,000 3 g, and the supernatant was filtered through a 0.45-mm-pore-size filter (Sartorius). Aliquots were frozen at 2208C until use. Determination of total immunoglobulins. The total IgA level in stool was measured by a standard enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Nunc, Roskilde, Denmark) were coated with rabbit anti-human IgA (Dakopatts, Glostrup, Denmark) at 1:2,000 in carbonate buffer. The wells were blocked with 1% bovine serum albumin (BSA) in carbonate buffer. Doubling dilutions of purified human IgA (Dakopatts) were used as a standard with a starting concentration of 1 mg/ml. Diluted stool extract samples (in PBS-Tween containing 1% BSA) were added in tripling dilutions. All samples were tested in duplicate. Goat anti-human IgA conjugated to horseradish peroxidase (HRP) (Sigma) at 1:1,000 in PBS-Tween containing 1% BSA was then added to the plate. The substrate, ortho-phenylenediamine dihydrochloride (OPD; Sigma) in 0.1 M citrate buffer–30% hydrogen peroxide (Sigma), was added, and the reaction was stopped with 1 M sulfuric acid after color development. The optical density was measured in a spectrophotometer (Titertek Multiskan Plus) with a 492-nm filter. Concentrations were calculated by interpolation from the standard curve and were expressed as milligrams per milliliter of stool. Total IgA and IgG levels in plasma were measured by turbidimetry in a discrete analyzer (COBAS-BIO; Roche) with monospecific antisera (Dakopatts). Results were expressed as milligrams per milliliter of plasma. Assays for antibodies to the LPS. The specific titers of the antibodies to the LPS were measured by ELISA as described before (21). Briefly, microtiter plates (Nunc) were coated with the LPSs (22) of S. dysenteriae 1 and S. flexneri Y at 10 mg/ml in carbonate buffer. The LPS of S. flexneri was used to assess the heterologous antibody response to antigens of other shigellae or cross-reacting LPS. After blocking with 1% BSA in carbonate buffer, diluted plasma or stool samples (volume/volume in PBS-Tween with 0.1% BSA) were added to duplicate wells with threefold serial dilutions. Goat anti-human IgA (Sigma) or IgG (Jackson Immunoresearch Laboratories Inc., West Grove, Pa.) conjugated to HRP at 1:1,000 each in PBS-Tween containing 0.1% BSA was then added. The substrate OPD was used for color development, and the optical density was measured at 492 nm as before. End point titers were determined as a reciprocal of the interpolated dilution of the sample giving an optical density 0.2 unit above the background. Calculations were carried out by using a computer-based program (Multi; DataTree Inc.,
Watthams, Mass.). Results were expressed as the specific titer per milligram of IgA or IgG in plasma and as the specific titer per milligram of IgA in stool. Statistical analyses. The Mann-Whitney U test was used to compare two groups of continuous, nonparametric data. Comparisons between proportions were done by using the chi-square statistic. Differences for paired samples between the study periods were assessed by using the Wilcoxon matched pairs signed-rank test (for continuous variables) and the McNemar test (for categorical variables). Differences were considered significant when P was #0.05. Multiple regression analyses were performed to evaluate the effects of clinical parameters on antibody concentrations. Data analyses were carried out by using the Statistical Package for Social Sciences (version 6.0 for Windows; SPSS Inc., Chicago, Ill.).
RESULTS Study groups. One hundred fifteen children with S. dysenteriae 1 infection were enrolled in the study. Of these, 42 had complications including leukemoid reaction (n 5 25) and HUS (n 5 17) (complicated shigellosis), while the others (n 5 73) had no such complications (uncomplicated shigellosis). The clinical characteristics of the children on enrollment are presented in Table 1. The children in the two study groups were comparable for all parameters except for inflammatory cells in stool. The numbers of leukocytes and erythrocytes in stools per high-powered microscopic field (determined with a 340 objective of a light microscope; BH-2 Olympus) were lower in children with complicated shigellosis than in those with uncomplicated shigellosis (P , 0.001 for both types of cells). Antibodies in plasma on enrollment. Total IgG concentrations in plasma were lower in children with complicated shigellosis (median, 4.9 mg/ml; standard deviation [SD], 3.4 mg/ml; n 5 36) than in those with uncomplicated shigellosis (median, 7.7 mg/ml; SD 3.9 mg/ml; n 5 58) (P , 0.001). Total IgA concentrations were similar in the two groups of children (for children with uncomplicated shigellosis, median, 1.2 mg/ml; SD 5 0.4 mg/ml; n 5 52; for children with complicated shigellosis, median, 1.0 mg/ml; SD, 0.4 mg/ml; n 5 26). Comparisons of homologous and heterologous LPS antibody concentrations between the two groups of children (Fig. 1) indicated that children with complicated shigellosis had higher concentrations of anti-LPS IgG to S. dysenteriae 1 (P 5 0.021) and anti-LPS IgA to S. flexneri (P 5 0.001). Plasma LPS antibody concentrations on the day of enrollment and 3 to 5 days later. Comparisons of the LPS antibody concentrations between the day of enrollment and 3 to 5 days later were carried out for the two groups of children for whom plasma samples from both study periods were available. In children with uncomplicated shigellosis, homologous LPS (IgA and IgG) (Fig. 2A and B, respectively) and heterologous LPS (IgA) (Fig. 2C) antibody concentrations increased from the day of enrollment to 3 to 5 days later (P 5 0.005, 0.001, and
VOL. 3, 1996
FIG. 1. Concentrations of homologous and heterologous LPS IgA and IgG antibodies in the plasma of children with uncomplicated and complicated shigellosis on the day of enrollment. Values are medians and SDs. Comparisons were done between children with uncomplicated (h) and complicated (u) shigellosis by using the Mann-Whitney U test. P values are provided only when differences were significant. *, P 5 0.021; **, P 5 0.001.
0.015, respectively). In children with complicated shigellosis, there were no differences in homologous (Fig. 2A and B) or heterologous (Fig. 2C and D) antibody concentrations between the two study periods. Antibodies in stool on enrollment. Total stool IgA concentrations were lower in children with complicated shigellosis (median, 0.6 mg/ml; SD, 0.5 mg/ml; n 5 40) than in those with uncomplicated shigellosis (median, 1.1 mg/ml; SD, 0.8 mg/ml; n 5 66) (P , 0.001). In children with uncomplicated shigellosis, concentrations of anti-LPS IgA to S. dysenteriae 1 (median, 238.8 titer per mg of IgA; SD, 888.3 titer per mg of IgA; n 5 57) and to S. flexneri (median, 264.4 titer per mg of IgA; SD, 568.9 titer per mg of IgA; n 5 62) were similar to those in
ANTIBODIES IN COMPLICATED SHIGELLOSIS
703
children with complicated shigellosis (for S. dysenteriae 1, median, 190.7 titer per mg of IgA; SD, 385 titer per mg of IgA; n 5 36; for S. flexneri, median, 210.4 titer per mg of IgA; SD, 380.9 titer per mg of IgA; n 5 36). Stool LPS antibody concentrations on the day of enrollment and 3 to 5 days later. Comparisons of LPS antibody concentrations in stools between the day of enrollment and 3 to 5 days later were carried out for children with uncomplicated and complicated shigellosis for whom samples from both study periods were available. In children with uncomplicated shigellosis, the concentrations of homologous antibodies (Fig. 3A) increased from the day of enrollment to 3 to 5 days later (P 5 0.003), while the concentrations of heterologous antibodies (Fig. 3B) were similar. In children with complicated shigellosis, there were no differences in antibody concentrations (homologous and heterologous) (Fig. 3A and B, respectively) between the two study periods. Effect of clinical parameters on antibody concentrations. Multiple regression analyses showed that there was no effect of nutritional status (as a percentage of the median weight for age of the National Center for Health Statistics), sex, stool frequency on enrollment, and concomitant infections on the homologous antibody response. However, the homologous antibody response was influenced by the duration of diarrhea before enrollment, for which reason the children were further divided into short (3 to 5 days), medium (6 to 9 days), and long (.9 days) diarrhea duration subgroups on the basis of the observed kinetics of LPS antibody responses in adults (20). Comparisons of homologous antibody concentrations between the two groups of children in each diarrhea duration subgroup were then carried out. Such comparisons indicated that the antibody concentrations (IgA and IgG) in plasma (Fig. 4A and
FIG. 2. Concentrations of LPS antibodies in the plasma of children with uncomplicated and complicated shigellosis on the day of enrollment (h) and 3 to 5 days later (u) for children for whom samples were available at both study periods. (A) LPS IgA antibody to S. dysenteriae 1; (B) LPS IgG antibody to S. dysenteriae 1; (C) LPS IgA antibody to S. flexneri Y; (D) LPS IgG antibody to S. flexneri Y. Values are medians and SDs. Comparisons were done between the two study periods by using the Wilcoxon matched pairs signed-rank test. P values are provided only when differences were significant. *, P 5 0.005; **, P 5 0.001; ***, P 5 0.015.
704
AZIM ET AL.
CLIN. DIAGN. LAB. IMMUNOL.
ellosis, which is in contrast to the time of the systemic humoral response. This is further emphasized by the lack of an increase in fecal antibody concentrations from the day of enrollment to 3 to 5 days later. This shift in response from a predominantly mucosal response to a predominantly systemic one is similar to that observed for interleukin 6 and tumor necrosis factor alpha in children with complicated shigellosis (1, 8) and may reflect the systemic nature of these complications. The mechanism of the earlier stimulation of the systemic immune system is not understood. Systemic stimulation may occur directly by bacterial antigens such as LPS, because high levels of circulating endotoxin are found in patients with Shigella-associated HUS (15), or by circulating cytokines, because children with complicated shigellosis have high levels of cytokines in the blood (8). However, whether these antibodies have a role in protection or merely reflect increased stimulation of the immune system is not clear. High levels of anti-LPS antibodies have been reported for enterohemorrhagic E. coli-associated HUS (3), and in patients with more severe cases of HUS, the antibody levels are lower (9), possibly because of their consumption by high concentrations of circulating endotoxin (9). In the present study, not enough samples were available to compare antibody concentrations in patients with more severe HUS versus the concentrations in those with less severe
FIG. 3. Concentrations in stool of homologous (A) and heterologous (B) LPS antibodies in children with uncomplicated and complicated shigellosis on the day of enrollment (h) and 3 to 5 days later (u) for children for whom samples were available at both study periods. Values are medians and SDs. Comparisons were done between the two study periods by using the Wilcoxon matched pairs signed-rank test. The P value is provided only when differences were significant. *, P 5 0.003.
B, respectively) were higher in children with complicated shigellosis than in those with uncomplicated shigellosis in the short diarrhea duration subgroup only. Antibody concentrations were lower in the stools (Fig. 4C) of children with complicated shigellosis than in the stools of those with uncomplicated shigellosis in the medium diarrhea duration subgroup only. DISCUSSION The role of antibodies to the LPS of Shigella spp. in systemic complications associated with shigellosis, either as markers of activation of the immune response or for protection, has not been defined. In the present study the LPS antibody response in plasma was found to be higher on enrollment in children with complicated shigellosis than in those with uncomplicated shigellosis. In addition, antibody concentrations in plasma were higher only in those children with complicated shigellosis who had had a short diarrhea duration before enrollment, suggesting that there is a stronger and earlier stimulation of the systemic immune system in these children than in children with uncomplicated shigellosis. The lack of a rise in antibody concentrations in plasma between the day of enrollment and 3 to 5 days later in children with complicated shigellosis reflects the already elevated antibody concentrations at enrollment. In stool, differences in LPS antibody concentrations between children at enrollment were observed only when children were divided into diarrhea duration subgroups. The observation that concentrations were lower in children with complicated shigellosis than in those with uncomplicated shigellosis in the medium diarrhea duration subgroup only, suggests that the local humoral response is slower in children with complicated shig-
FIG. 4. Homologous LPS IgA (A) and IgG (B) concentrations in plasma and IgA concentrations in stools (C) from children with uncomplicated and complicated shigellosis on enrollment in the following diarrhea duration subgroups: 3 to 5 days (short), 6 to 9 days (medium), and .9 days (long). Values are medians and SDs. Comparisons were done between children with uncomplicated (h) and complicated (u) shigellosis by using the Mann-Whitney U test. P values are provided only when differences were significant. *, P 5 0.044; **, P 5 0.018; ***, P 5 0.014.
VOL. 3, 1996
ANTIBODIES IN COMPLICATED SHIGELLOSIS
HUS. However, we did not find any difference between children with leukemoid reaction and HUS. In the stools of children with complicated shigellosis, it is not only LPS antibodies that are lowered but also total IgA; leukocyte numbers; erythrocyte numbers, which is an indicator of protein loss from the gut (2), and hence of the extent of intestinal inflammation; and cytokines (1). This suggests that intestinal inflammatory responses are different in children with complicated shigellosis. It is unlikely that the extent of intestinal inflammation is lower in children with complications because colonic studies of patients with diarrhea-associated HUS have shown variable but often extensive colitis (23). Recently, cytokine mRNA-positive cells were found to be higher in number than the corresponding protein-synthesizing cells in rectal biopsy specimens from adults with shigellosis (19). The cause for this inhibition of protein translation is not clear and does not appear to be mediated by Shiga toxin (19). It is possible that this phenomenon is enhanced in children with complicated shigellosis. In summary, there is a strong and early stimulation of the systemic antibody response in children with Shigella-associated leukemoid reaction and/or HUS which may reflect the systemic nature of these complications. There is no evidence from the present study to suggest a role for these antibodies in protection from the development of complications. ACKNOWLEDGMENTS This research was supported by the U.S. Agency for International Development under grant DPE-5986-A-1009-00 and ICDDR,B. ICDDR,B is supported by the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, China, Denmark, Germany, Japan, The Netherlands, Norway, Republic of Korea, Saudi Arabia, Sri Lanka, Sweden, Switzerland, Thailand, the United Kingdom, and the United States; international organizations including the Arab Gulf Fund, Asian Development Bank, European Union, the United Nations Children’s Fund, the United Nations Development Programme, the United Nations Population Fund, and the World Health Organization; private foundations including Aga Khan Foundation, Child Health Foundation, Ford Foundation, Population Council, Rockefeller Foundation, and the Sasakawa Foundation; and private organizations including American Express Bank, Bayer AG, CARE, Family Health International, Helen Keller International, the Johns Hopkins University, Macro International, New England Medical Center, Procter & Gamble, RAND Corporation, SANDOZ, Swiss Red Cross, the University of Alabama at Birmingham, the University of Iowa, and others. We thank Manzurul Haque and Khairun Nessa for secretarial assistance. REFERENCES 1. Azim, T., R. C. Halder, M. S. Sarker, S. Ahmed, J. Hamadani, A. Chowdhury, F. Qadri, M. A. Salam, R. B. Sack, and M. J. Albert. 1995. Cytokines in the stools of children with complicated shigellosis. Clin. Diagn. Lab. Immunol. 2:492–495. 2. Bennish, M. L., M. A. Salam, and M. A. Wahed. 1993. Enteric protein loss during shigellosis. Am. J. Gastroenterol. 88:53–54. 3. Bitzan, M., E. Moebius, K. Ludwig, D. E. Mu ¨ller-Wiefel, J. Heesemann, and H. Karch. 1991. High incidence of serum antibodies to Escherichia coli O157 lipopolysaccharide in children with hemolytic-uremic syndrome. J. Paediatr. 119:380–385.
705
4. Butler, T., M. R. Islam, and P. K. Bardhan. 1984. The leukemoid reaction in shigellosis. Am. J. Dis. Child. 138:162–165. 5. Ca ´ceres, A., and L. J. Mata. 1974. Serologic response of patients with Shiga dysentery. J. Infect. Dis. 129:439–443. 6. Cleary, T. G., D. K. Winsor, D. Reich, G. Ruiz-Palacios, and J. J. Calva. 1989. Human milk immunoglobulin A antibodies to Shigella virulence determinants. Infect. Immun. 57:1673–1679. 7. Cohen, D., M. S. Green, C. Block, R. Slepon, and I. Ofek. 1991. Prospective study of the association between serum antibodies to lipopolysaccharide O antigen and the attack rate of shigellosis. J. Clin. Microbiol. 29:386–389. 8. De Silva, D. G. H., L. N. Mendis, N. Sheron, G. J. M. Alexander, D. C. A. Candy, H. Chart, and B. Rowe. 1993. Concentrations of interleukin 6 and tumour necrosis factor in serum and stools of children with Shigella dysenteriae 1 infection. Gut 34:194–198. 9. Heyderman, R. S., H. M. Fitzpatrick, and G. R. Barclay. 1994. Haemolyticuraemic syndrome. Lancet 343:1042. 10. Islam, D., B. Wretlind, M. Ryd, A. A. Lindberg, and B. Christensson. 1995. Immunoglobulin subclass distribution and dynamics of Shigella-specific antibody responses in serum and stool samples in shigellosis. Infect. Immun. 63:2054–2061. 11. Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and H. Lior. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151:775–782. 12. Li, A., A. Karnell, P. T. Huan, P. D. Cam, N. B. Minh, L. N. Tram, N. P. Quy, D. D. Trach, K. Karlsson, G. Lindberg, and A. A. Lindberg. 1993. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in adult Vietnamese volunteers. Vaccine. 11:180–189. 13. Louise, C. B., and T. G. Obrig. 1992. Shiga toxin-associated hemolyticuremic syndrome: combined cytotoxic effects of Shiga toxin and lipopolysaccharide (endotoxin) on human vascular endothelial cells in vitro. Infect. Immun. 60:1536–1543. 14. Louise, D. B., and T. G. Obrig. 1995. Specific interaction of Escherichia coli O159:H7-derived Shiga-like toxin II with human renal endothelial cells. J. Infect. Dis. 172:1394–1401. 15. Marchant, A., M. L. Bennish, M. A. Salam, W. A. Khan, A. M. Khan, M. J. Streulens, J. Griffiths, and G. T. Keusch. 1991. Endotoxemia during shigellosis: relationship in hemolytic-uremic syndrome (HUS) and leukemoid reaction, abstr. 11, p. 100. In Program and abstracts of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 16. Oberhelman, R. A., D. J. Kopecko, E. Salazar-Lindo, E. Gotuzzo, J. M. Buysse, M. M. Venkatesan, A. Yi, C. Fernandez-Prada, M. Guzman, R. Leon-Barua, and R. B. Sack. 1991. Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides. Infect. Immun. 59:2341–2350. 17. Obrig, T., P. J. Del Vecchio, J. E. Brown, T. P. Moran, B. M. Rowland, T. K. Judge, and S. W. Rothman. 1988. Direct cytotoxic action of Shiga toxin on human vascular endothelial cells. Infect. Immun. 56:2373–2378. 18. Rahaman, M. M., A. K. M. J. Alam, M. R. Islam, W. B. Greenough III, and J. Lindenbaum. 1975. Shiga bacillus dysentery associated with marked leukocytosis and erythrocyte fragmentation. Johns Hopkins Med. J. 136:65–70. 19. Raqib, R., Å. Ljungdahl, A. A. Lindberg, B. Wretlind, U. Andersson, and J. Andersson. 1996. Dissociation between cytokine mRNA expression and protein production in shigellosis. Eur. J. Immunol. 26:1130–1138. 20. Raqib, R., S. Tzipori, M. Islam, and A. A. Lindberg. 1993. Immune response to Shigella dysenteriae 1 and Shigella flexneri lipopolysaccharide and polysaccharide antigens in Bangladeshi patients with shigellosis. Serodiagn. Immunother. Infect. Dis. 1:37–45. 21. Schultsz, C., F. Qadri, S. A. Hossain, F. Ahmed, and I. Ciznar. 1992. Shigellaspecific IgA in saliva of children with bacillary dysentery. FEMS Microbiol. Immunol. 89:65–72. 22. Westphal, O., and K. Jann. 1965. Bacterial LPS: extraction with phenolwater and further applications of the procedures. Methods Carbohydr. Chem. 5:83–91. 23. Whitington, P. F., A. L. Friedman, and R. W. Chesney. 1979. Gastrointestinal disease in the hemolytic-uremic syndrome. Gastroenterology 76:728–733. 24. World Health Organization. 1987. Manual for laboratory investigations of acute enteric infections, p. 9–20. World Health Organization, Geneva.
