- Email: [email protected]
Effect of human recombinant interferon-γ on Salmonella typhimurium invasion in cultured human intestinal cells
Immunology, Microbiology, and InflammatoryDisorders A1063
April 1998 immunity is vital in outbreak investigation. Specific antibody responses to Cryptosporidium parvum infection may be detected by enzyme linked immunosorbent assay (ELISA) or Western blot (WB). We compared anticryptosporidial seroprevaience using ELISA and WB in comparable individuals resident within and outside a contaminated water supply area during a waterborne cryptosporidiosis outbreak. Methods. Serum samples were collected anonymously from 26 individuals not identified as infected who were resident within a contaminated water supply area during a waterborne outbreak of cryptosporidiosis in South England; 47 individuals residing outside this water supply area; 5 individuals convalescing from confirmed cryptosporidiosis, and 90 UK blood donors. An ELISA was developed for the detection of total, IgG and IgM anticryptosporidial antibodies in the serum samples collected. Seropositivity was defined as an optical density (OD) value greater than the mean OD value for the blood donor samples + 2SDs. The serum samples were further characterised and compared for their reactivity to three low molecular weight C.parvum antigenic determinants; 17, 14, and 6kDa in size; by WB. Sera reactive to 2 or more of these determinants was taken as positive. Results. Groups ELISA +ve (%) W. blot +ve (%)
Method. Caco-2 cell monolayers were grown to confluence on glass coverslips in 24 well plates. 2x105 C. parvum oocysts suspended in DMEM alone or containing a concentration of 0, 0.1, 1, and 10~tM p-bromophenacyl bromide (pBPB), an irreversible inhibitor of PL A 2 and PL C, were added to each well. After 2h incubation wells were washed and replaced with complete media. Following a further 48h incubation and washing, monolayers were fixed with methanol and infection quantified by immunofluorescence assay (IFA). Two controls were included (1) wells pre-treated with 10urn pBPB in DMEM for 30min and washed prior to oocyst inoculation and (2) wells inoculated with heat inactivated oocysts. Infection was confirmed by transmission and scanning electron microscopy. IFA was performed using a rabbit polyclonal anti-C parvum antibody raised against sporozoites specific for intracellular parasitic forms. Parasitic stages were counted in 20 microscopic fields per coverslip using a Seescan TM image analyser. Results. Electron microscopy confirmed the development of asexual stages at 48h. Coincubation of C. parvum and pBPB significantly reduced infection in a dose dependent fashion (ANOVA p<0.001). No difference in infection was noted between control wells pre-treated with pBPB and untreated wells. Infection was not detected in monolayersinoculated with heat inactivated oocysts.
Positive controls (n = 5) 5 (100) 5 (100) Blood donors (n = 90) 2 (2.2) Within supply area (n = 26) 8 (30.8)# 13 (50.0)* Outside supply area (n = 47) 3 (6.3) 3/36 (8.3) (chi-square test: # •2 = 7.8, p < 0.05; * X2 = 9.0, p < 0.005 vs. outside supply area) Conclusion. We have demonstrated a 3-4 fold increase in anti-cryptosporidial seropositivity amongst exposed individuals during a waterbome outbreak of cryptosporidiosis than unexposed controls by both ELISA and WB infection. These results imply a high rate of occult C.parvum infection amongst these individuals during the epidemic. • G4352 ENTERIC VIRAL INFECTION IN PATIENTS WITH THE ACQUIRED IMMUNODEFICIENCY SYNDROME: A CAUSE OF DIARRHEA? RCG Pollok 1, P Thomas2, BG Gazzard 3. Digestive Disease Research Centre, St Bartholomew's & The Royal London School of Medicine 1, St Mark's Hosp2, The Chelsea & Westminster Hosp, London, UK3. Introduction. The role of non-cytomegalovirus (CMV) enteric viral infection in causing diarrhoea in patients with human immunodeficiency virus (HIV) is poorly understood. The aim of the study was to investigate the role of these infections in causing acute and chronic diarrhoea and their association with other gastrointestinal pathogens. Methods. In a prospective study, 862 stool specimens from 377 HIV infected patients presenting with diarrhoea were studied for evidence of noncytomegalovirus enteric viral infection. Patients with diarrhoea underwent examination of stool, small and large bowel biopsies for bacterial, protozoal, and viral pathogens. The case records of patients in whom enteric virus was identified were studied for the relationship of enteric virus to their diarrhoeal symptomatology. Results. Sixty one (15.9%) HIV positive individuals were positive for coronavirus (n=13, 22%), rotavirus (n--ll, 18%), adenovirus (n=30, 50%), and small round structured viruses (n=5, 8%), or dual infection (n=2, 3%). 33/52 (63%) cases available for analysis had acute diarrhoea, and 9/52 (36%) had chronic diarrhoea. 23/52 (44%) cases had a concurrent gut pathogen. Acute diarrhoea was significantly more common in those patients with enteric viral infection alone than in those with a concurrent gut pathogen in whom chronic diarrhoea was more common (p=0.001). Twenty one cases of adenovirus colitis were identified by histological examination. Following stratification for the presence of concurrent gut pathogens, the presence of adenovirns colitis was significantly more likely to be associated with chronic diarrhoea (18/21 cases) than adenovirus isolated from stool alone (7/23 cases) (p=0.002). A significantly higher association of cytomegalovirus colitis with adenovirus colitis was also noted (p=0.004). Conclusion. Enteric viral infection is strongly associated with acute diarrhoea in patients with HIV. Light microscopic examination of large bowel biopsies can identify adenovirus colitis which is significantly associated with chronic diarrhoea, and in addition may facilitate gastrointestinal co-infection with CMV. • G4353 THE ROLE OF CRYPTOSPORIDIUM PARVUM-DERIVED PHOSPHOLIPASE IN MEDIATING ENTERIC HOST CELL INVASION IN VITRO. RCG Pollok, MJG Farthing. Digestive Disease Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, London, UK. Introduction. The mechanisms of host cell invasion by C. parvum are poorly understood. In the related pathogen, Toxoplasma gondii, parasite derived phospholipase (PL) is crucial to host cell penetration. We hypothesised that PL plays a similar role in host cell invasion by C. parvum. Using a human intestinal epithelial cell line we developed an in vitro assay of C. parvum infection to determine the role of parasite derived PL in host cell penetration.
o
o.1
t
Io
Discussion. This study suggests a role for parasite derived PL in C. parvum infection. We speculate that PL modifies the host cell membrane, allowing penetration and formation of the parasitophornus vacuole. • G4354 EFFECT
OF
HUMAN
RECOMBINANT
INTERFERONff
ON
SALMONELLA TYPHIMURIUM INVASION IN CULTURED HUMAN INTESTINAL CELLS. JF Poschet, RCG. Pollok & PD Fairclough, DDRC, St. Bartholomew's & The Royal London School of Medicine & Dentistry, London E1 2AD. Introduction: Throughout the world Salmonella infections are a major health problem, leading to gastro-enteritis and typhoid fever. Following oral ingestion Salmonellae adhere to intestinal cell surfaces and penetrate the epithelium. Upon entering enterocytes they are relatively protected from host defences and antibiotic therapy. Human recombinant interferon-'/(INF-'/) has previously been shown to prevent Toxoplasma gondii and Rotavirus infection in IEC-6, and Caco-2 cells respectively. We therefore studied the effect of INF-'/on S. typhimurium invasion of Caco-2 cells. Methods: Caco-2 cell monolayers were grown in Transwells (0.4 pm pore size) to 14 days post confluency, reaching a monolayer resistance (MR) of<300D./cmL 72 hours prior to the experiment 0, 0.01, 0.1, 1, 10, 100 and 1000 U/ml !NF-'/was added to the apical and basolateral side of monolayers. After 72 hrs INF-'/was removed and S. typhimurium (SR11) at an inoculum of approximately 107 bacteria/well were added to the apical side. After 2.5 hrs external bacteria was removed and remaining external bacteria were killed with 100 pg/ml gentamicin. Invasion was quantified by counting viable intracellular bacteria. Internalised bacteria were released, serially diluted, cultured on LB-agar plates and counted the following day. Results: 72 hrs pre-incubation with 1000 U/ml INF-'/ reduced MR by 16-+3%. All other concentrations did not have an effect compared to controls. Compared to negative controls, addition of SR11 reduced MR at all INF-'/ concentrations by >85%, including positive controls. However, invasion of Caco-2 monolayers by SR11 was greatly inhibited by INF-'/ concentrations of 10 U/ml and above, compared to controls (no INF-7). All experiments were carded out in triplicates. Invasion
Control 100 _+33%
1.0 U/ml 95 _+44%
10 U/ml 23 _+18%
100 U/ml 21 _+3%
1000 U/ml 18 _+8%
Conclusion: We have shown that INF-'/ at concentrations equal or above 10 U/ml inhibit S. typhimurium invasion by up to 80%. INF-'/ epithelial effector mechanisms are poorly understood. We are currently studying these mechanisms and the role of related cytokines on S. typhimurium invasion of Caco-2 cells.
April 1998 immunity is vital in outbreak investigation. Specific antibody responses to Cryptosporidium parvum infection may be detected by enzyme linked immunosorbent assay (ELISA) or Western blot (WB). We compared anticryptosporidial seroprevaience using ELISA and WB in comparable individuals resident within and outside a contaminated water supply area during a waterborne cryptosporidiosis outbreak. Methods. Serum samples were collected anonymously from 26 individuals not identified as infected who were resident within a contaminated water supply area during a waterborne outbreak of cryptosporidiosis in South England; 47 individuals residing outside this water supply area; 5 individuals convalescing from confirmed cryptosporidiosis, and 90 UK blood donors. An ELISA was developed for the detection of total, IgG and IgM anticryptosporidial antibodies in the serum samples collected. Seropositivity was defined as an optical density (OD) value greater than the mean OD value for the blood donor samples + 2SDs. The serum samples were further characterised and compared for their reactivity to three low molecular weight C.parvum antigenic determinants; 17, 14, and 6kDa in size; by WB. Sera reactive to 2 or more of these determinants was taken as positive. Results. Groups ELISA +ve (%) W. blot +ve (%)
Method. Caco-2 cell monolayers were grown to confluence on glass coverslips in 24 well plates. 2x105 C. parvum oocysts suspended in DMEM alone or containing a concentration of 0, 0.1, 1, and 10~tM p-bromophenacyl bromide (pBPB), an irreversible inhibitor of PL A 2 and PL C, were added to each well. After 2h incubation wells were washed and replaced with complete media. Following a further 48h incubation and washing, monolayers were fixed with methanol and infection quantified by immunofluorescence assay (IFA). Two controls were included (1) wells pre-treated with 10urn pBPB in DMEM for 30min and washed prior to oocyst inoculation and (2) wells inoculated with heat inactivated oocysts. Infection was confirmed by transmission and scanning electron microscopy. IFA was performed using a rabbit polyclonal anti-C parvum antibody raised against sporozoites specific for intracellular parasitic forms. Parasitic stages were counted in 20 microscopic fields per coverslip using a Seescan TM image analyser. Results. Electron microscopy confirmed the development of asexual stages at 48h. Coincubation of C. parvum and pBPB significantly reduced infection in a dose dependent fashion (ANOVA p<0.001). No difference in infection was noted between control wells pre-treated with pBPB and untreated wells. Infection was not detected in monolayersinoculated with heat inactivated oocysts.
Positive controls (n = 5) 5 (100) 5 (100) Blood donors (n = 90) 2 (2.2) Within supply area (n = 26) 8 (30.8)# 13 (50.0)* Outside supply area (n = 47) 3 (6.3) 3/36 (8.3) (chi-square test: # •2 = 7.8, p < 0.05; * X2 = 9.0, p < 0.005 vs. outside supply area) Conclusion. We have demonstrated a 3-4 fold increase in anti-cryptosporidial seropositivity amongst exposed individuals during a waterbome outbreak of cryptosporidiosis than unexposed controls by both ELISA and WB infection. These results imply a high rate of occult C.parvum infection amongst these individuals during the epidemic. • G4352 ENTERIC VIRAL INFECTION IN PATIENTS WITH THE ACQUIRED IMMUNODEFICIENCY SYNDROME: A CAUSE OF DIARRHEA? RCG Pollok 1, P Thomas2, BG Gazzard 3. Digestive Disease Research Centre, St Bartholomew's & The Royal London School of Medicine 1, St Mark's Hosp2, The Chelsea & Westminster Hosp, London, UK3. Introduction. The role of non-cytomegalovirus (CMV) enteric viral infection in causing diarrhoea in patients with human immunodeficiency virus (HIV) is poorly understood. The aim of the study was to investigate the role of these infections in causing acute and chronic diarrhoea and their association with other gastrointestinal pathogens. Methods. In a prospective study, 862 stool specimens from 377 HIV infected patients presenting with diarrhoea were studied for evidence of noncytomegalovirus enteric viral infection. Patients with diarrhoea underwent examination of stool, small and large bowel biopsies for bacterial, protozoal, and viral pathogens. The case records of patients in whom enteric virus was identified were studied for the relationship of enteric virus to their diarrhoeal symptomatology. Results. Sixty one (15.9%) HIV positive individuals were positive for coronavirus (n=13, 22%), rotavirus (n--ll, 18%), adenovirus (n=30, 50%), and small round structured viruses (n=5, 8%), or dual infection (n=2, 3%). 33/52 (63%) cases available for analysis had acute diarrhoea, and 9/52 (36%) had chronic diarrhoea. 23/52 (44%) cases had a concurrent gut pathogen. Acute diarrhoea was significantly more common in those patients with enteric viral infection alone than in those with a concurrent gut pathogen in whom chronic diarrhoea was more common (p=0.001). Twenty one cases of adenovirus colitis were identified by histological examination. Following stratification for the presence of concurrent gut pathogens, the presence of adenovirns colitis was significantly more likely to be associated with chronic diarrhoea (18/21 cases) than adenovirus isolated from stool alone (7/23 cases) (p=0.002). A significantly higher association of cytomegalovirus colitis with adenovirus colitis was also noted (p=0.004). Conclusion. Enteric viral infection is strongly associated with acute diarrhoea in patients with HIV. Light microscopic examination of large bowel biopsies can identify adenovirus colitis which is significantly associated with chronic diarrhoea, and in addition may facilitate gastrointestinal co-infection with CMV. • G4353 THE ROLE OF CRYPTOSPORIDIUM PARVUM-DERIVED PHOSPHOLIPASE IN MEDIATING ENTERIC HOST CELL INVASION IN VITRO. RCG Pollok, MJG Farthing. Digestive Disease Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, London, UK. Introduction. The mechanisms of host cell invasion by C. parvum are poorly understood. In the related pathogen, Toxoplasma gondii, parasite derived phospholipase (PL) is crucial to host cell penetration. We hypothesised that PL plays a similar role in host cell invasion by C. parvum. Using a human intestinal epithelial cell line we developed an in vitro assay of C. parvum infection to determine the role of parasite derived PL in host cell penetration.
o
o.1
t
Io
Discussion. This study suggests a role for parasite derived PL in C. parvum infection. We speculate that PL modifies the host cell membrane, allowing penetration and formation of the parasitophornus vacuole. • G4354 EFFECT
OF
HUMAN
RECOMBINANT
INTERFERONff
ON
SALMONELLA TYPHIMURIUM INVASION IN CULTURED HUMAN INTESTINAL CELLS. JF Poschet, RCG. Pollok & PD Fairclough, DDRC, St. Bartholomew's & The Royal London School of Medicine & Dentistry, London E1 2AD. Introduction: Throughout the world Salmonella infections are a major health problem, leading to gastro-enteritis and typhoid fever. Following oral ingestion Salmonellae adhere to intestinal cell surfaces and penetrate the epithelium. Upon entering enterocytes they are relatively protected from host defences and antibiotic therapy. Human recombinant interferon-'/(INF-'/) has previously been shown to prevent Toxoplasma gondii and Rotavirus infection in IEC-6, and Caco-2 cells respectively. We therefore studied the effect of INF-'/on S. typhimurium invasion of Caco-2 cells. Methods: Caco-2 cell monolayers were grown in Transwells (0.4 pm pore size) to 14 days post confluency, reaching a monolayer resistance (MR) of<300D./cmL 72 hours prior to the experiment 0, 0.01, 0.1, 1, 10, 100 and 1000 U/ml !NF-'/was added to the apical and basolateral side of monolayers. After 72 hrs INF-'/was removed and S. typhimurium (SR11) at an inoculum of approximately 107 bacteria/well were added to the apical side. After 2.5 hrs external bacteria was removed and remaining external bacteria were killed with 100 pg/ml gentamicin. Invasion was quantified by counting viable intracellular bacteria. Internalised bacteria were released, serially diluted, cultured on LB-agar plates and counted the following day. Results: 72 hrs pre-incubation with 1000 U/ml INF-'/ reduced MR by 16-+3%. All other concentrations did not have an effect compared to controls. Compared to negative controls, addition of SR11 reduced MR at all INF-'/ concentrations by >85%, including positive controls. However, invasion of Caco-2 monolayers by SR11 was greatly inhibited by INF-'/ concentrations of 10 U/ml and above, compared to controls (no INF-7). All experiments were carded out in triplicates. Invasion
Control 100 _+33%
1.0 U/ml 95 _+44%
10 U/ml 23 _+18%
100 U/ml 21 _+3%
1000 U/ml 18 _+8%
Conclusion: We have shown that INF-'/ at concentrations equal or above 10 U/ml inhibit S. typhimurium invasion by up to 80%. INF-'/ epithelial effector mechanisms are poorly understood. We are currently studying these mechanisms and the role of related cytokines on S. typhimurium invasion of Caco-2 cells.