The targeting of immunoliposomes to tumour cells (A431) and the effects of encapsulated methotrexate.

Immunoliposomes have been prepared from lipid mixtures of dipalmitoylphosphatidylcholine, wheat germ phosphatidylinositol and a reactive lipid (the m-maleimidobenzoyl-N-hydroxysuccinimide derivative of dipalmitoylphosphatidylethanolamine) which was conjugated to the N-succinimidyl-S-acetylthioacetate (SATA) derivative of a monoclonal antibody (H17E2) raised to human placental alkaline phosphatase (PLAP). The immunoliposomes were prepared by the extrusion technique (VETs) and by reverse phase evaporation (REVs) and were found to effectively target to immobilised PLAP and to PLAP or PLAP-like enzyme on the surface of a tumour cell line (A431) using an ELISA and autoradiography. The extent of binding to immobilised PLAP was a function of immunoliposomal lipid concentration, the weight-average number of antibody molecules per liposome (Pw) and the liposome size. The effectiveness of methotrexate-loaded immunoliposomes in inhibiting the proliferation of A431 cells was investigated relative to equivalent levels of the free drug. The immunoliposomes prepared by the extrusion technique (VETs) inhibited growth of A431 cells but had no effect on the growth of a normal human fibroblastic cell line. Immunoliposomes prepared by reverse phase evaporation (REVs) were less effective in inhibiting A431 cell proliferation. The immunoliposomes probably enter the tumour cells largely by receptor-mediated endocytosis although other mechanisms of uptake cannot be excluded.

Sulphasalazine treatment and the colorectal mucosa-associated flora in ulcerative colitis.

AIM: To study the influence of sulphasalazine treatment on the mucosa-associated bacterial flora of rectal biopsy tissue specimens in patients with ulcerative colitis. PATIENTS: Twenty-four patients had newly diagnosed active ulcerative colitis; 20 patients had acute relapse of ulcerative colitis (10 not taking maintenance sulphasalazine); (40 patients had quiescent ulcerative colitis; 21 not taking maintenance sulphasalazine). The influence of 3 weeks of sulphasalazine treatment on the mucosa-associated flora was studied in the patients presenting with active disease. RESULTS: Comparison of patients according to sulphasalazine usage revealed few differences in the mucosal flora. In patients with quiescent ulcerative colitis, Escherichia coli was found at lower counts in patients taking maintenance sulphasalazine; however, this effect was not evident in patients with active disease. Inconsistent changes in other facultatives were seen between the two active disease groups, particularly for a miscellaneous group of unidentified Gram-positive rods. Three patients, all receiving sulphasalazine, were colonized with Clostridium difficile, but this did not appear to influence their disease. CONCLUSION: Sulphasalazine treatment in ulcerative colitis causes only minor disturbance to the populations of bacteria colonizing the colorectal mucosa.

Visual versus mechanised leucocyte differential counts: costing and evaluation of traditional and Hemalog D methods.

Visual differential counts were examined for efficiency, cost effectiveness, and staff acceptability within our laboratory. A comparison with the Hemalog D system was attempted. The advantages and disadvantages of each system are enumerated and discussed in the context of a large general hospital.

Antigenic conservation of the ureases of spiral- and helical-shaped bacteria colonising the stomachs of man and animals.

A monoclonal antibody, CP11, has been produced which is directed against the ureas of Campylobacter pylori. This antibody has been used to look for antigenic cross-reactivity, in other ureolytic and non-ureolytic campylobacters, by immunohistological techniques. It has also been used to investigate the helical-shaped organisms found in the stomach of the human, monkey and cat (CS1) and the ileum of the rat (ST1). Interestingly the antibody cross-reacted with the gastric helical organisms from the human, monkey and cat but not with the rat helical organism. No cross-reactivity was observed with C. mustelae or the other ureolytic campylobacters, C. nitrofigilis and the urease positive thermophilic campylobacters. These results are discussed in relation to the phylotaxonomy of these organisms.

The urinary bacterial flora in patients with three types of urinary tract diversion.

There is known to be a high risk of malignancy at uretero-colic anastomoses and it has been postulated that bacteria catalyse both the local formation and the activation of carcinogens such as N-nitrosamines. If this theory is correct, then the risk of malignancy should be greatest when the prevalence of mixed urinary tract infection is greatest. Bacterial culture was performed on samples of urine obtained from 56 patients with three forms of urinary tract diversion: ureterosigmoidostomy, isolated ileal and colon conduits. As expected, the rectal urine of all patients with a ureterosigmoidostomy had a rich bacterial flora. However, 11 of the 15 patients with ileal conduits (73%) had significant mixed growths of bacteria in the loop urine, whereas only six out of 16 colon loop urines (37.5%) were infected, all being monobacterial infections. The implications of these bacteriological findings for carcinogenesis associated with urinary diversion are discussed.

Isolation of a gastric campylobacter-like organism from the stomach of four rhesus monkeys, and identification as Campylobacter pylori.

Campylobacter-like organisms, isolated from the gastric antrum of Rhesus monkeys, were compared with Campylobacter jejuni and C. pylori. They were similar to C. pylori by light microscopy, in ultrastructural morphology, in enzymic, fatty-acid-methyl-ester, and protein-profile analysis, and in antigenic reactivity with rabbit antisera to C. jejuni and C. pylori and with C. pylori-specific monoclonal antibody. Because this natural infection of the Rhesus monkey is associated with chronic gastritis, resembling the disease in humans colonised with C. pylori, we recommend the animal as a model for the investigation of human gastritis.

Experimental campylobacter infection and diarrhoea in immunodeficient mice.

The responses of previously untested immunodeficient mouse strains to campylobacter infection are described. Three strains of adult immunodeficient mice (SCID-Beige, C.B-17-SCID-Beige and RAG-2) were inoculated intragastrically with Campylobacter jejuni NCTC 11168. All mice became heavily colonised, but none developed clinical signs of disease. Immunocompetent BALB/c mice inoculated similarly had much lower colonisation levels. The co-administration of iron dextran had no effect on colonisation levels nor the development of clinical signs of disease. In contrast, C.B-17-SCID-Beige mice, when inoculated with one of a series of 10 clinical isolates of C. jejuni, were more heavily colonised for extended periods (up to 5 months) and approximately 10-20% of the mice became ill with diarrhoea. C. jejuni was detected in mouse faeces throughout at levels of 10(7)-10(9) cfu/g. All mice killed whilst ill with diarrhoea displayed histopathological lesions typical of human campylobacteriosis. Severe pathology was limited to the large intestine and was suggestive of an acute, bacteria-induced inflammation. Although blood was detected in the diarrhoeal stools, no evidence of mucosal epithelial cell invasion was found by immunohistology. No pathology was detected in tissue sections from any of the animals that had not developed signs of disease following C. jejuni inoculation. These immunodeficient mouse strains are readily, and heavily, colonised as adults by C. jejuni. The diarrhoea, although sporadic, was reproducibly produced, and could provide the basis for pathogenicity studies.

The microbial flora of the rectal mucosa and faeces of patients with Crohn's disease before and during antimicrobial chemotherapy.

The faecal flora and mucosa-associated flora (MAF) of rectal biopsy material from 12 patients with active Crohn's disease were studied before and during treatment with a combination of metronidazole and cotrimoxazole given orally for at least 2 weeks. The total faecal flora was greater than the MAF although the proportions of bacterial groups were similar. The changes observed during treatment were: obligate anaerobes such as Bacteroides spp. decreased in faeces (p less than 0.05) and in MAF (p less than 0.02); the total count of facultative bacteria increased in the faeces (p less than 0.002) but not in the MAF. Streptococci, predominantly enterococci, increased significantly in faeces (p less than 0.001) and in MAF (p less than 0.02) such that they became predominant components of these florae. Facultative gram-negative bacilli were unaltered in faeces but significantly reduced in the MAF (p less than 0.05). Sporing clostridia were infrequently isolated from the MAF but were significantly reduced in the faeces (p less than 0.01). During the treatment period, eight of the 12 patients showed clinical improvement, but this could not be related to the site or extent of disease or to specific changes in faecal flora or MAF. This combination of antibacterial agents causes profound alterations to the bacterial flora of mucosa and faeces and these changes may help to define the role of bacteria in the pathogenesis of Crohn's disease.

The rectal mucosa-associated microflora in patients with ulcerative colitis.

The rectal mucosa-associated flora (MAF) of patients with ulcerative colitis has been studied in 25 patients with newly diagnosed disease, 20 with relapse of existing disease, and 44 who were in remission. Patients with active disease were re-examined twice during treatment. The MAF was simpler and less dense than the microflora of faeces. Obligate anaerobes usually predominated in the MAF although the ratio of obligate anaerobes to facultative species was lower than that found in faeces. Viable counts of the total flora and of its constituent genera varied considerably between patients. Counts of the total flora, of obligate anaerobes (including bifidobacteria, eubacteria and clostridia), and facultative organisms and micro-aerobes (enterobacteria and lactobacilli) were reduced in patients with active disease compared with those with inactive disease; corresponding carriage rates were also lower. Counts and carriage rates increased during treatment and approached those found in quiescent disease. The alterations in the MAF were especially marked in patients experiencing their first attack of ulcerative colitis. The relationship between these alterations and the aetiology and pathogenesis of this disease remains unclear.

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines.

Serogroup C meningococcal conjugate vaccines generally use diphtheria or tetanus toxoids as the protein carriers. The use of alternative carrier proteins may allow multivalent conjugate vaccines to be formulated into a single injection and circumvent potential problems of immune suppression in primed individuals. Bordetella pertussis fimbriae were assessed as carrier proteins for Neisseria meningitidis serogroup C polysaccharide. Fimbriae were conjugated to the polysaccharide using modifications of published methods and characterised by size exclusion chromatography; co-elution of protein and polysaccharide moieties confirmed conjugation. The conjugates elicited boostable IgG responses to fimbriae and serogroup C polysaccharide in mice, and IgG:IgM ratios indicated that the responses were thymus-dependent. High bactericidal antibody titres against a serogroup C strain of N. meningitidis were also observed. In a mouse infection model, the conjugate vaccine protected against lethal infection with N. meningitidis. Therefore, B. pertussis fimbriae are effective carrier proteins for meningococcal serogroup C polysaccharide and could produce a vaccine to protect against meningococcal disease and to augment protection against pertussis.

Product development horizons--a view from industry.

Against the background of what appears to be an increasing incidence of many allergic disorders, the production of safe, effective 'hypoallergenic' diets on a commercial scale has become of increasing importance. Combining knowledge of protein biochemistry and immunology with clinical science has provided not only a greater understanding of what is required for effective treatment of allergic disorders, but is also facilitating a unique insight into the use of dietary management in allergy prevention. Infants who have become sensitised to intact proteins either in utero or during early feeding usually require a diet with significantly reduced allergenicity for a period of time. One of the most effective methods of allergen avoidance has traditionally been achieved by subjecting dairy proteins to extensive hydrolysis until virtually no antigenic epitopes remain recognisable to the infant's immune system (Sampson, Bernhisel-Broadbent, Yang et al., 1991). This 'hypoallergenic' protein base can then be used together with other non-sensitizing ingredients, to create a formula feed which will meet the essential nutritional requirements of infants during the specified period for recovery--usually the first 12 months of life or beyond.

Flotation removal of As(V) onto goethite.

Arsenic oxyanions, considered as priority pollutants, were removed from dilute aqueous solutions by sorption onto synthetic goethite, a typical inorganic adsorbent. Flotation was subsequently applied as an effective solid/liquid separation method. The combined process produced a foam concentrate, containing the arsenic-loaded goethite particles. The dispersed-air flotation technique was used for the generation of fine gas bubbles. The main parameters affecting the process were studied and promising results, in terms of arsenic removal and of goethite separation, were obtained.

Novel in vitro functional assays for the determination of anthrax toxin components.

The characterisation and evaluation of the UK licensed human anthrax vaccine depends on several in vivo tests that determine its safety and potency. Assays for the determination of functionally active and/or immunoreactive toxin components and S-layer proteins have been developed and applied to the characterisation of anthrax vaccine. These technologies may support production of consistent and effective vaccines, and may ultimately reduce the requirements for in vivo testing.

Swine dysentery: protection of pigs by oral and parenteral immunisation with attenuated Treponema hyodysenteriae.

An attenuated strain of Treponema hyodysenteriae was used to immunise 18 pigs in three experiments. Live attenuated spirochaetes were dosed orally and injected intra-peritoneally, and killed spirochaetes were injected intramuscularly with adjuvant. The vaccinated pigs, which developed high serum agglutination titres against T hyodysenteriae, and 18 unvaccinated litter-mates were repeatedly challenged with virulent T hyodysenteriae. Nine vaccinated pigs and 16 control pigs developed typical swine dysentery.

A comparison study of different methods used in the detection of Giardia lamblia.

OBJECTIVE: The purpose of this study was to compare results obtained using a single fecal specimen for O&P examination, direct immunofluorescent assay (DFA), and three immunodiagnostic techniques. DESIGN: Sixty-eight human fecal specimens were collected and examined by each method. The O&P and the DFA were used as the reference method. SETTING: The study was performed at the research laboratory in the Medical Technology Department at The University of Southern Mississippi. PATIENTS OR OTHER PARTICIPANTS: The fecal specimens were collected from individuals with a suspected Giardia lamblia infection. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The amount of agreement and disagreement between methods. 1. The sensitivity and specificity of each method. 2. The working time and cost per specimen for each method. RESULTS: There was complete agreement among methods on 52 specimens (21 positive, 31 negative). Eight specimens were positive by all immunologic methods, but negative by O&P. The remaining eight specimens (12%) demonstrated discrepancies among methods. Sensitivity and specificity of each assay ranged from 91% to 100% and 89% to 100%, respectively. The cost per specimen ranged from $11.62 for the DFA method to $32.54 for the O&P method. The average cost per specimen for ELISA and EIA averaged $26.86. CONCLUSION: The study supported findings of other investigators who concluded that immunologic methods have the greater sensitivity. The immunologic methods were more efficient, quicker, and economical than the conventional O&P method.

Diagnosis of swine dysentery: spirochaetes which may be confused with Treponema hyodysenteriae.

Spirochaetes, designated PWS, microscopically resembling Treponema hyodysenteriae, were isolated from the colon contents of pigs with post-weaning scours from three herds, which on clinical and epidemiological grounds were thought to be free from swine dysentery. One of the isolates was fed to pigs experimentally but no evidence of disease was noted. Their cultural characteristics differed from those of T hyodysenteriae but they were similar to those of a non-pathogenic spirachaete (4/71) which had been isolated previously from the laboratory's dysentery-free herd. Smears prepared from cultures of the PWS spirochaete, 4/71 and two virulent and one avirulent strain of T hyodysenteriae were all positive to a fluorescent antibody test which was in use as an aid to the diagnosis of swine dysentery in the United Kingdom.

Study of infectious intestinal disease in England: rates in the community, presenting to general practice, and reported to national surveillance. The Infectious Intestinal Disease Study Executive.

OBJECTIVE: To establish the incidence and aetiology of infectious intestinal disease in the community and presenting to general practitioners. Comparison with incidence and aetiology of cases reaching national laboratory based surveillance. DESIGN: Population based community cohort incidence study, general practice based incidence studies, and case linkage to national laboratory surveillance. SETTING: 70 general practices throughout England. PARTICIPANTS: 459 975 patients served by the practices. Community surveillance of 9776 randomly selected patients. MAIN OUTCOME MEASURES: Incidence of infectious intestinal disease in community and reported to general practice. RESULTS: 781 cases were identified in the community cohort, giving an incidence of 19.4/100 person years (95% confidence interval 18.1 to 20.8). 8770 cases presented to general practice (3.3/100 person years (2.94 to 3.75)). One case was reported to national surveillance for every 1.4 laboratory identifications, 6.2 stools sent for laboratory investigation, 23 cases presenting to general practice, and 136 community cases. The ratio of cases in the community to cases reaching national surveillance was lower for bacterial pathogens (salmonella 3.2:1, campylobacter 7.6:1) than for viruses (rotavirus 35:1, small round structured viruses 1562:1). There were many cases for which no organism was identified. CONCLUSIONS: Infectious intestinal disease occurs in 1 in 5 people each year, of whom 1 in 6 presents to a general practitioner. The proportion of cases not recorded by national laboratory surveillance is large and varies widely by microorganism. Ways of supplementing the national laboratory surveillance system for infectious intestinal diseases should be considered.

Evaluation of mercury exposure reduction through a fish consumption advisory program for Anishinaabe tribal members in Northern Wisconsin, Michigan, and Minnesota.

The Great Lakes Indian Fish and Wildlife Commission has an extensive program to inform Anishinaabe tribal members from northern Wisconsin, Michigan, and Minnesota who harvest and consume walleye about the health risks of consuming these fish, and to encourage harvest and consumption practices that reduce exposure to MeHg. We report here the results of a probabilistic analysis of exposure to methyl mercury (MeHg) among tribal members who consume walleye. The model predicts that the potential for greatest exposures to MeHg occur among women of child-bearing age and children who consume large walleye from lakes that contain heavily contaminated (MeHg concentration >0.5 mg/kg) fish. The analysis allows GLIFWC to evaluate, focus, and fine-tune its initiatives to protect the health of tribal members in ways that result in exposure and risk reduction for tribal harvesters, women of child-bearing age, and children, while maintaining important tribal lifeways, which include the harvest and consumption of walleye.