Biomedical subjects
Publications and source records attributed to D S Reiner.
A technique for the insertion of a central venous access device in the patient with thrombocytopenia is described. Using the Seldinger technique, a wire is placed into the internal jugular vein. A catheter tunneled from the anterior part of the chest is inserted through a peel-away sheath into the central venous system. The incision is then closed.
To study the interactions between the parasitic protozoan Giardia lamblia and its environment, we have cloned the gene that encodes the two major surface-labeled trophozoite protein species. Sequence analysis of this gene reveals a single open reading frame specifying a hydrophilic, cysteine-rich (11.8%) protein of 72.5-kDa molecular mass with an amino-terminal signal peptide and a postulated hydrophobic membrane-spanning anchor region near the carboxyl terminus. Most of the cysteine residues (58 of 84) are in the motif Cys-Xaa-Xaa-Cys, which is dispersed 29 times throughout the sequence. Antibodies against the recombinant protein react with the entire surface of live trophozoites, including flagella and adhesive disc. These antibodies inhibit trophozoite attachment, prevent growth, and immunoprecipitate the major approximately 66- and 85-kDa proteins from surface-labeled live trophozoites. The recombinant Escherichia coli also expresses polypeptides of approximately 66- and 85-kDa molecular mass, which are not fusion proteins. This suggests that the processing and/or conformational changes that lead to production of these two peptide species in E. coli reflect those that occur in Giardia. The abundance of cysteine residues suggests that the native proteins on the parasite surface may contain numerous disulfide bonds, which would promote resistance to intestinal fluid proteases and to the detergent activity of bile salts and would help to explain the survival of Giardia in the human small intestine.
Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.
Surgeons have long recognized that a proportion of hospitalized trauma patients present with a history of a previous admission for trauma, termed by the authors as "trauma recidivism." The incidence of trauma recidivism was addressed by a review of 150 consecutive admissions to a level I Trauma Center. This study identifies this subset of trauma patients, establishes their magnitude, and analyzes mechanisms of injury and hospital courses. The implications for those who care for trauma patients is discussed.
Large numbers (10(4) to greater than 10(5)/ml) of Type I water-resistant Giardia lamblia cysts were produced in vitro under conditions that are characteristic of the human intestinal lumen. We define Type I cyst morphology as oval shaped, smooth, and refractile, with cyst wall, axostyle, and median body visible in relief by Normarski differential interference contrast optics. Human and porcine bile induced higher levels of encystation than bovine bile at the alkaline pH (7.8) which occurs in the human lower small intestine. High-pressure liquid chromatography analysis showed that the porcine bile had a preponderance of hyocholate, rather than cholate, while bovine bile had less chenodeoxycholate and more deoxycholate than human bile. Lactic acid, a major product of bacterial metabolism in the human colon, further stimulated encystation. Growth of the preencystation culture without bile also increased subsequent encystation. More than 90% of Type I cysts produced with porcine bile plus lactic acid were viable as indicated by the uptake and retention of fluorescein diacetate and exclusion of propidium iodide. Biological activity of in vitro-derived water-resistant cysts was demonstrated by the observation that 1 to 9.5% excysted in vitro. The percentage of excystation was greatly decreased following encystation at pH 7.0 or by omission of bile or lactic acid. This is the first quantitative in vitro demonstration of the complete life cycle of G. lamblia from humans.
We induced Giardia lamblia trophozoites to encyst in vitro by exposure to conditions which are specific to the human small intestinal milieu. We now show that encystation entails the appearance of two new groups of antigens detected in Western blots by rabbit antiserum against cysts which had been purified from human feces. A heterodisperse group of lower-molecular-mass antigens (approximately 21 to 39 kilodaltons) was expressed relatively early (less than 19 h) in encystation. In contrast, discrete bands at approximately 66, 78, 92, and 103 kilodaltons only appeared after 24 h of incubation under conditions which lead to production of large numbers of water-resistant cysts. We also describe for the first time the appearance of prominent cytoplasmic vesicles, which were the earliest morphologic change in encysting trophozoites observable by light microscopy. Early in encystation, cyst wall antigens were concentrated in these vesicles, as shown by immunocytochemistry, suggesting that the vesicles function in export of cyst wall constituents.
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Bile salts and fatty acids stimulated differentiation of cultured Giardia lamblia trophozoites into water-resistant cysts at the slightly alkaline pH of the small intestinal lumen. Maximum encystation occurred at pH 7.8. Thus, specific small-intestinal factors may influence encystation in vivo as well as in vitro.
The cyst form of Giardia lamblia is responsible for transmission of giardiasis, a common waterborne intestinal disease. In these studies, encystation of Giardia lamblia in vitro was demonstrated by morphologic, immunologic, and biochemical criteria. In the suckling mouse model, the jejunum was shown to be a major site of encystation of the parasite. Small intestinal factors were therefore tested as stimuli of encystation. An antiserum that reacted with cysts, but not with cultured trophozoites was raised in rabbits and used as a sensitive probe for differentiation in vitro. Cultured trophozoites that were exposed to bile salts showed a more than 20-fold increase in the number of oval, refractile cells that reacted strongly with anticyst antibodies, and in the expression of major cyst antigens. Exposure to primary bile salts resulted in higher levels of encystation than exposure to secondary bile salts. These studies will aid in understanding the differentiation of an important protozoan pathogen.
One of the major problems in studying Giardia cysts is separating this form of the parasite from faecal debris and bacteria. We approached this problem by passing filtered stool suspensions over a column of Sephadex G-50 followed by low speed centrifugations. The Sephadex retained most of the faecal debris and the centrifugation reduced the bacterial flora more than 10(5)-fold to less than 3 X 10(4)/ml. Cyst recovery was 61% (+/- 28%). This new method of separating G. lamblia from faecal material is simple, rapid, and effective.
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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.
Comatose blunt trauma patients undergo diagnostic peritoneal lavage to aid in the management of severe injuries. In deference to routine performance of lavage, patients felt to be a risk for abdominal injury were prospectively studied by using selective criteria for lavage. One hundred consecutive comatose blunt trauma patients were studied; five underwent urgent celiotomy for refractory hypotension. Five criteria for lavage were: history of postinjury hypotension, abdominal physical findings suggestive of underlying abdominal injuries, evidence of fracture of the bones of the trunk and/or femur, blunt trauma of unknown etiology, and operative general anesthesia required for nonabdominal injuries. Fifty-five patients satisfied at least one of the above criteria and underwent peritoneal lavage. Forty patients not satisfying the criteria were observed without peritoneal lavage, as it was felt they had a decreased probability of serious abdominal injury. One of these patients had a minor liver laceration found at the time of autopsy following his death from severe head injury. None of the other patients had evidence of intra-abdominal injury by autopsy or subsequent clinical course. Utilizing the selective criteria, none of the 100 consecutive blunt trauma patients had major delay in abdominal diagnosis or missed significant abdominal pathology defined by operation, clinical course, or autopsy. Routine peritoneal lavage in all comatose blunt trauma patients may subject the patient to unnecessary risk, waste valuable time, increase the cost of care, and alter subsequent diagnostic procedures. Based on this small study, it appears that comatose blunt trauma victims not fulfilling the criteria may be effectively evaluated without the use of peritoneal lavage.(ABSTRACT TRUNCATED AT 250 WORDS)
Controversy persists regarding the use of antibiotics in association with t tube thoracostomy for trauma patients. We conducted a prospective randomized study of patients requiring tube thoracostomy for pneumo- and/or hemothorax complicating blunt or penetrating thoracic trauma in an attempt to assess the efficacy of prophylactic antibiotic therapy. Fifty-eight patients were included in the study. The control group (Group I) included 28 patients who received no antibiotic therapy: the experimental group (Group II) included 30 patients who received cefoxitin (1.0 gm IV q 6 h) commencing before tube thoracostomy and terminating 12 hours after its removal. The incidence of infectious complications (pneumonia and/or empyema) was recorded. Among the patients not receiving antibiotics, eight of 28 (29%) developed infectious chest complications. Of the patients receiving antibiotics, there was one infectious complication (3%). This difference is statistically significant (p = 0.0227). Cultures demonstrated significant conversion from negative to positive both within each group and between groups. The organism most commonly recovered was S. aureus. Our findings strongly suggest that patients requiring tube thoracostomy for trauma, whether blunt or penetrating, should receive the benefit of systemic prophylactic antibiotic therapy.
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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.
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.
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.