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F D Gillin

Publications and source records attributed to F D Gillin.

At least 73 records · Page 4Linked to original sources
PubMed1986

Human milk kills Giardia lamblia by generating toxic lipolytic products.

This study supports our previous hypotheses that normal human milk kills Giardia lamblia trophozoites in vitro and that this killing is due to the release of free fatty acids (FFAs) from milk triglycerides by action of the bile salt-stimulated lipase (BSL) of human milk. Heat-stable killing of G. lamblia was generated when normal human milk was preincubated with sodium cholate, which activates BSL. Moreover, both the skim-milk (containing BSL) and cream (containing mainly triglycerides) fractions were required to kill G. lamblia. We measured the toxicity of FFA and other products of lipolysis to G. lamblia. cis-Unsaturated FFAs (LD50 less than 12 microM), three of four monoglycerides, and four of five lysophosphatidylcholines were toxic to the parasites (LD50 less than 100 microM). In contrast, the parasites were not harmed by the corresponding diglycerides, phosphatidylcholines, triolein, or glycerol. Thus, products of lipid hydrolysis in the normal digestive tract are toxic to G. lamblia. We also demonstrated that albumin and conjugated bile salts, which bind FFA, partially protected trophozoites from killing by oleic acid.

Bile Acids and Salts↗
PubMed1986

Biliary lipids support serum-free growth of Giardia lamblia.

Giardia lamblia has been grown in vitro only in media containing serum or serum fractions. How this pathogen can grow in the human small intestinal lumen without serum is not known. We found that samples of human hepatic or gall bladder bile maintained G. lamblia survival for 24 to 48 h in medium without serum but did not support growth. By contrast, an artificial biliary lipid dispersion containing six bile salts, phosphatidylcholine (PC), and cholesterol, in the ratios characteristic of human bile, supported parasite growth in medium without serum or serum fractions. To define the requirements, we showed that 1-palmitoyl-2-linoleoyl-PC or 1-palmitoyl-2-oleoyl-PC (which predominate in human bile) satisfied the requirement for PC. Moreover, either glycocholate or glycodeoxycholate could be substituted for the bile salt mixture. The finding that biliary lipids can support serum-free growth of G. lamblia may help explain why this parasite colonizes the upper small intestine.

Animals↗
PubMed1985

Tolerance of axenically cultured Entamoeba histolytica and Giardia lamblia to a variety of antimicrobial agents.

Twelve antimicrobial agents were tested for use in axenic cultures of Entamoeba histolytica and Giardia lamblia. Ten did not inhibit parasite growth at concentrations of at least two-fold above the effective antibacterial or antifungal levels. We have found the combination of piperacillin (500 micrograms/ml) plus amikacin (125 micrograms/ml) to be useful in axenization of a new strain of E. histolytica, eradication of multiply drug-resistant contaminants in cultures and prevention of bacterial growth in experiments with non-sterile components.

Amikacin↗
PubMed1985

Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals.

The DNA banding pattern of 11 human and four animal isolates (two beaver, one cat, and one guinea pig) of Giardia were compared by using two related techniques. Patterns were compared after endonuclease restriction of DNA followed by agarose gel electrophoresis and ethidium bromide staining and after Southern blot analysis using recombinant plasmids containing Giardia DNA as probes. Two major groups could be distinguished with ethidium bromide staining of eight isolates. Southern blot analysis, however, could distinguish nine different patterns among the 15 isolates studied. One common banding pattern was seen in six isolates (two animal and four human); the remainder of the isolates were unique, with the exception of two identical isolates from sisters. Three isolates (one from a beaver and two from humans) were markedly different from Giardia with the common banding pattern, whereas the other six unique isolates varied moderately. Beavers and other mammals do not seem to possess their own species of Giardia. This methodology introduces a way of distinguishing one species of Giardia isolate from another and promises to be helpful in epidemiological investigations.

Animals↗
PubMed1985

Interactions of Giardia lamblia with human intestinal mucus: enhancement of trophozoite attachment to glass.

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 micrograms/ml. Attachment increased with mucus concentrations from 1 to 1000 micrograms/ml but declined toward the unstimulated level at concentrations above 1000 micrograms/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.

Adhesiveness↗
PubMed1985

Cholate-dependent killing of Giardia lamblia by human milk.

We showed previously that nonimmune human milk (NHM) kills Giardia lamblia trophozoites in vitro and presented evidence that killing requires the bile salt-stimulated lipase of milk. Since this enzyme is activated by bile salts, killing should be dependent on the presence of bile salts. We now show that killing by fresh NHM or NHM stored at -70 degrees C is totally dependent on sodium cholate (a bile salt). With less than 0.4 mM cholate, no parasites were killed, whereas with 1 mM cholate, greater than 99.7% were killed by 5% NHM in 30 min. Moreover, killing activity was completely heat labile. The G. lamblia-killing activity of human milk was greatly altered by storage at -10 or -20 degrees C. In less than 23 days, the 50% lethal dose decreased, cholate dependence was lost, and killing activity became heat stable. In contrast, the activity of milk stored at -70 degrees C remained unchanged. Milk lipase activity, like killing activity, became cholate independent during storage at -10 or -20 degrees C. On the basis of these results, we hypothesize that killing of G. lamblia by fresh NHM or NHM stored at -70 degrees C depends on bile salt-stimulated lipase, which must be activated by bile salts. In contrast, NHM stored at -20 degrees C accumulated free fatty acids which kill G. lamblia. In support of this thesis, milk stored at -10 degrees C had a concentration of 18.7 mM free fatty acids compared with only 1.1 mM in an identical sample stored at -70 degrees C.

Cholic Acid↗
PubMed1984

Thiol groups on the surface of anaerobic parasitic protozoa.

Evidence is presented that Giardia lamblia and Entamoeba histolytica, phylogenetically unrelated aerotolerant anaerobes, have crucial thiol groups on or easily accessible to their external surface. Both parasites were killed by three structurally unrelated thiol-blocking reagents which penetrate intact cells poorly or not at all. The parasites were protected from p-chloromercuribenzenesulfonic acid (10-100 microM) by cysteine or by reduced glutathione. Killing was arrested with identical kinetics by addition of either cysteine (which quickly penetrates the cells) or bovine serum albumin (which does not penetrate intact cells) at various times after p-chloromercuribenzenesulfonic acid, indicating that the reactive site may be on the outer surface of the cell. Proteins lacking cysteine did not protect. Sensitivity of three other protozoa to p-chloromercuribenzenesulfonic acid was also tested. Trichomonas vaginalis (anaerobic) was at least as sensitive as E. histolytica and G. lamblia, while Crithidia fasciculata and Paramecium tetraurelia (both aerobic) were less sensitive. Thiol groups on the G. lamblia surface were demonstrated directly by fluorescence-activated cell sorter analysis of trophozoites which had been modified with a thiol-specific hapten, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylenediamine and reacted with fluorescent antibody to this hapten.

Anaerobiosis↗
PubMed1984

Inhibition of growth of Giardia lamblia by difluoromethylornithine, a specific inhibitor of polyamine biosynthesis.

Difluoromethylornithine (DFMO) is a specific and irreversible inhibitor of ornithine decarboxylase, an enzyme which catalyzes the first step in the biosynthetic pathway of the polyamines. We tested the effect of DFMO on the growth of Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis. Growth of G. lamblia was inhibited by DFMO at concentrations of greater than or equal to 1.25 mM. Culture doubling time increased with increasing DFMO concentration. Growth inhibition was reversed if spermidine was added within 53 h of addition of DFMO; no growth was observed if spermidine was added later, indicating eventual parasite death. The growth of E. histolytica and T. vaginalis, two unrelated mucosal-dwelling parasites of humans, was not inhibited by 20 mM DFMO. These studies indicate that polyamine biosynthesis from ornithine is required for growth of G. lamblia.

Animals↗
PubMed1983

Human milk kills parasitic intestinal protozoa.

Giardia lamblia, a common pathogenic intestinal parasite of humans, was rapidly killed by exposure to normal human milk in vitro. The killing did not depend on secretory immunoglobulin A. Entamoeba histolytica, the dysentery amoeba, was also killed by normal human milk. Giardia-cidal activity cochromatographed with an unusual lipase that is present in the milk of humans but not of lower mammals. Human milk may play a protective role in infants exposed to this parasite.

Entamoeba histolytica↗
PubMed1983

Killing of Giardia lamblia trophozoites by normal human milk.

The clinical course of giardiasis is variable, and serum antibodies do not appear to be protective. We propose that natural factors either produced by intestinal tissue, transported into the intestine, or ingested (ie, by breast-fed babies) might promote resistance to this disease. Human milk is very rich in secretory IgA (S-IgA) antibodies, as well as nonspecific antibacterial factors (eg, lactoferrin, lysozyme). Previous studies showed that Giardia lamblia trophozoites were killed by nonimmune human milk (NHM) in a time- and concentration-dependent manner. Removal of greater than 99% of the S-IgA from NHM did not decrease its Giardia-cidal activity. Thus, the killing was not antibody dependent. This is the first demonstration of nonimmune antiparasitic defenses in human milk. The present studies show that in the presence of NHM, trophozoites lost motility, swelled, and lysed. The Giardia-cidal activity (GCA) may be specific to human milk, since unheated cow's and goat's milk were virtually devoid of activity. Much, but not all, of the GCA was lost when NHM was heated or reacted with diisopropylfluorophosphate (DIFP), a specific esterase inhibitor. Activity of the major human milk lipase (BSL, bile salt-stimulated lipase, a fatty acid esterase) was lost after heat or DIFP treatment and was absent from cow's or goat's milk. The parasites were also killed by pure BSL. These studies suggest that BSL may be a heat-labile Giardia-cidal component of NHM.

Animals↗
PubMed1983

Excretory-secretory products of Giardia lamblia.

The surface of Giardia lamblia strain WB was radioiodinated with either 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (IODOGEN) or lactoperoxidase, and the labeled membrane components as well as the released excretory-secretory (E-S) products were identified. The surface of G. lamblia was easily labeled, and G. lamblia excretory-secretory (E-S) products were identified in the medium. Over 70% of the label on the cell surface was released over 24 hr. The major E-S product released was polydisperse (m.w. 225 to 94,000), protease VI and periodate-sensitive, chloroform-methanol insoluble, and failed to adhere to a series of carbohydrate-binding lectins or to diethylaminoethyl (DEAE) cellulose. Hydrophobicity was suggested by adherence to phenyl-Sepharose. The major E-S product of another G. lamblia isolate, Portland-1 (P-1), was antigenically different. A previous study showed that strain P-1 lacked a major antigenic component of strain WB. In the present study, this material was identified as the major secretory product of WB by crossed immunoelectrophoresis.

Animals↗
PubMed1982

Effects of oxygen tension and reducing agents on sensitivity of Giardia lamblia to metronidazole in vitro.

The effects of oxygen tension and reducing agents upon the sensitivity of Giardia lamblia to metronidazole in vitro were determined using two different assays. First, the parasites were exposed to drug under high or low oxygen tension in liquid medium with or without fresh cysteine and ascorbic acid. Survival was determined by colony assay. Second, the effect of thiol reducing agents was measured directly by colony assay in semi-solid media (low pO2 conditions). Trophozoites were strikingly (greater than 100-fold) less sensitive to metronidazole under the aerobic condition of the first assay. In contrast, the reducing agents had much lesser effects (2- to 5-fold) depending upon the assay. Finally, G. lamblia isolated from a patient treated with metronidazole unsuccessfully four times showed similar metronidazole sensitivity to a standard strain under both aerobic and reduced O2 tension conditions.

Animals↗
PubMed1982

Bruceantin, a potent amoebicide from a plant, Brucea antidysenterica.

Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytica in vitro (IC50 [the concentration of drug which decreased the number of colonies to half that of controls] = 0.018 microgram/ml). Six related quassinoids of 17 tested were also amoebicidal. No relationship between quassinoid structure and amoebicidal activity was apparent.

Amebicides↗
PubMed1982

Antigenic analysis of Giardia lamblia from Afghanistan, Puerto Rico, Ecuador, and Oregon.

The trophozoite antigens of Giardia lamblia to which host humoral and cellular immune responses are directed have not been identified. Therefore, we initiated studies to characterize these antigens in strains of G. lamblia from Afghanistan, Oregon, Ecuador, and Puerto Rico. By polyacrylamide gel electrophoresis, the electrophoretic mobility patterns of proteins of the four strains were similar; molecular weights of protein bands ranged between 12,000 and 140,000. The antigens which reacted with rabbit and anti-G. lamblia antisera by immunoelectrophoresis were also similar for the four strains. However, comparison by crossed immunoelectrophoresis showed the Oregon strain, which has been the longest in culture, lacked a set of anodic antigens and the single neutral antigen which were present in the other three strains. In addition, other minor antigen differences between the strains were detected by this technique. When we employed trophozoites from each strain as antigen in an enzyme-linked immunosorbent assay against 10 human antisera of various liters, we also detected some differences between the strains. Although polyacrylamide gel electrophoresis and immunoelectrophoresis revealed gross similarity among G. lamblia from widely differing geographic locations, subtle differences detected by crossed electrophoresis and enzyme-linked immunosorbent assay suggest the existence of potentially important antigenic differences among these strains.

Afghanistan↗
PubMed1982

Attachment of the flagellate Giardia lamblia: role of reducing agents, serum, temperature, and ionic composition.

The flagellated protozoan Giardia lamblia has been grown only in highly complex media under reduced oxygen tension. Therefore, the organic and physiological requirements for in vitro attachment and short-term (12-h) survival of this organism were determined. In defined maintenance media, a thiol reducing agent (e.g., cysteine) was absolutely required for attachment and survival of this aerotolerant anaerobe. The crude bovine serum Cohn III fraction greatly stimulated attachment and survival. Attachment was decreased at a reduced temperature (24 degrees C as compared with 35.5 degrees C) and absent at 12 degrees C or below. Attachment and survival were strongly dependent upon pH and ionic strength, with optima at pH 6.85 to 7.0 and 200 to 300 mosmol/kg. Sodium chloride was better tolerated than KC1. Reduction of Ca2+ and Mg2+ to below 10(-8) M did not significantly affect attachment.

Animals↗
PubMed1982

Chronic giardiasis: studies on drug sensitivity, toxin production, and host immune response.

UNLABELLED: We investigated a patient (W.B.) with chronic symptomatic Giardiasis despite seven separate courses of either quinacrine or metronidazole who was cured by combined quinacrine and metronidazole. After isolating Giardia lamblia from W.B., we cultured the trophozoites to make the following observations. In vitro drug testing showed that (a) W.B.'s organisms were not more drug resistant than three other isolates and that (b) W.B.'s organisms were more sensitive to combined quinacrine and metronidazole than to either drug alone. Isolates of Giardia lamblia from W.B. and 3 other patients did not produce detectable enterotoxin in four different assays. W.B. had normal levels of circulating immunoglobulins, detectable intestinal immunoglobulin A, circulating immunoglobulin G anti-Giardia lamblia antibodies, and lymphocyte responsiveness to solubilized giardia lamblia. However, monocytes-macrophages from W.B. exhibited reduced killing for Giardia lamblia compared with normal subjects. CONCLUSIONS: (a) The chronicity of our patient's infection was not due to the organism having unique properties of drug resistance. (b) Combined quinacrine and metronidazole, which cured our patient's chronic giardiasis, should be tried in patients in whom infection persists after single drug therapy. (c) The diarrhea in our patient was probably caused by a mechanism other than Giardia lamblia-induced secretion by currently recognized enterotoxins. (d) Reduced cellular cytotoxicity for Giardia lamblia may have contributed to the persistence of our patient's infection and should be suspected in other patients with chronic giardiasis.

Adult↗