Antigenic variation of a cysteine-rich protein in Giardia lamblia.

The WB isolate of Giardia lamblia expresses a cysteine-rich 170-kD surface antigen (CRP170) that undergoes antigenic variation. An (6E7), cytotoxic for isolates expressing CRP170, was used in another study to select antigenic variants from clones of the WB isolate of Giardia. CRP170 was replaced by surface-labeled bands ranging in size from approximately 50 to 170 kD. In this study, mAb 6E7 was used to isolate a 1-kb portion of the CRP170 gene (M2-1) from a lambda gt 11 expression library. The M2-1 clone hybridized to a 5.4-kb transcript from isolates expressing CRP170 but did not hybridize to RNA from antigenic variants. Evidence was found for frequent rearrangements at the CRP170 gene locus. DNA sequencing of the M2-1 clone revealed the presence of long tandem repeats. The putative amino acid sequence of M2-1 reveals a 12% cysteine content, and CRP170 is readily labeled in vivo with cysteine.

Identification of the nucleoli of Giardia lamblia with TEM and CFM.

Giardia species are flagellated parasites of vertebrates and belong to the diplomonads, most of which have two nuclei. These organisms were classified among the earliest branching eukaryotes on the basis of small subunit rDNA sequences and their lack of many canonical organelles. However, some of these organelles have subsequently been identified in rudimentary form, such as Golgi that become apparent during encystation. One of the "missing" organelles has been the nucleolus, the site of rRNA synthesis, since it was not identified in earlier ultrastructural studies. In the current study, we visualized in-vitro-grown Giardia lamblia trophozoites by transmission electron microscopy and laser scanning confocal microscopy. We found that each of the two nuclei contains a single small and deeply stained granular nucleolus, thus demonstrating that Giardia does indeed have nucleoli.

The cysteine-rich protein gene family of Giardia lamblia: loss of the CRP170 gene in an antigenic variant.

Giardia lamblia trophozoites demonstrate variable expression of a repertoire of cysteine-rich surface antigens in vitro and in vivo. The size of the repertoire has been estimated at 20 to 184, and specific variants can be detected after approximately 12 generations of in vitro growth for the WB isolate. In earlier studies, we cloned a portion of the gene for a 170-kDa surface antigen (CRP170) and demonstrated by DNA sequencing that it was cysteine rich (12%) and contained 2.6 copies of a tandemly repeated 195-bp pair sequence. The clone hybridized to multiple bands on a Southern blot of G. lamblia DNA in a pattern that was variable among the cloned lines but did not correlate with expression of CRP170. We have now cloned a nearly full length cDNA as well as genomic clones for CRP170 from the WBA6 cloned isolate. In addition, we have isolated a cDNA clone from the WB1269 line (expressing CRP72), an antigenic variant which was derived from WBA6. Sequence analysis of the CRP170 and CRP72 genes revealed marked C-terminal amino acid homology, suggesting a conserved functional role such as membrane anchoring. The CRP170 repeat oligonucleotide hybridized to a stairstep of bands approximately 6 kb in size on HindIII-digested WBA6 DNA representing the expressed copy(ies) of CRP170. In contrast, there was no hybridization to a fragment of similar size in WB1269, suggesting that WB1269 trophozoites have lost the expressed copy of the CRP170 gene.

Telomeric location of Giardia rDNA genes.

Giardia lamblia telomeres have been isolated from a library enriched for repaired chromosome ends by (i) screening with a Plasmodium falciparum telomere and (ii) differential hybridization with Bal 31-digested and total G. lamblia DNA. Analysis of three clones isolated by this strategy has identified multiple tandem repeats of the 5-mer TAGGG. An oligonucleotide containing these repeats recognizes Bal 31-sensitive bands in Southern hybridizations and detects all G. lamblia chromosomes in pulsed-field gel electrophoresis separations. An abrupt transition from the G. lamblia rDNA sequence to telomeric repeats has been found in all three clones. In two of the clones the transition occurs at the same site, near the beginning of the large subunit rDNA sequence. In the third clone the transition occurs at a site in the intergenic spacer sequence between the rDNA genes. Hybridization of an rDNA probe to a pulsed-field separation of G. lamblia chromosomes indicates that rDNA genes are present on several chromosomes but vary in location from isolate to isolate. These results suggest that rRNA genes are clustered at telomeric locations in G. lamblia and that these clusters are mobile.

Rhinocerebral mucormycosis. Therapy with amphotericin B lipid complex.

Rhinocerebral mucormycosis with intracranial involvement has a high mortality. The standard therapy consists of aggressive surgical débridement accompanied by high doses of amphotericin B deoxycholate. Even with this therapy, the mortality rate has been 48% in the series reported since 1980. We treated a 60-year-old diabetic woman with rhinocerebral mucormycosis involving the cavernous sinus whose infection responded to medical therapy with amphotericin B lipid complex. To our knowledge, this is the only well-documented medical cure of a patient with rhinocerebral mucormycosis and intracranial involvement.

Phaeohyphomycosis caused by the fungal genera Bipolaris and Exserohilum. A report of 9 cases and review of the literature.

We have reported 7 new cases of Bipolaris infection and 2 of Exserohilum infection, which demonstrate the capability of these 2 genera to cause invasive as well as "allergic" disease. As noted previously, it is likely that all of the cases of "Helminthosporium" and Drechslera infections reported in the literature were caused by Bipolaris or Exserohilum. Infections due to these 2 genera are probably more common than previously recognized. They should be included in the differential diagnosis of central nervous system and disseminated fungal disease, sinusitis, keratitis, peritonitis associated with continuous ambulatory peritoneal dialysis, and allergic bronchopulmonary disease. These various entities have distinct histopathologic characteristics. With disseminated disease in the immunocompromised patient, the most frequent findings are acute inflammation with prominent vascular invasion, thrombosis, and infarction. In contrast, granulomatous inflammation and leukocytoclastic vasculitis are seen in meningoencephalitis caused by these fungi. The histologic features of allergic bronchopulmonary disease and sinusitis are similar. A chronic inflammatory infiltrate of lymphocytes, plasma cells and eosinophils within edematous granulation tissue is found in addition to squamous metaplasia and thickening of the basement membrane. Infections caused by Bipolaris/Exserohilum and Aspergillus show many clinical and pathologic similarities despite the lack of taxonomic relationship between these fungi. Both cause disseminated disease in immunocompromised patients that is characterized by tissue necrosis and vascular invasion. Both cause central nervous system disease, osteomyelitis, and sinusitis and are associated with allergic bronchopulmonary disease. Sinusitis, the most common form of disease caused by Bipolaris and Exserohilum, occurs in otherwise healthy patients with nasal polyposis and allergic rhinitis. Although pathologic evidence of bone invasion may not be found, there frequently is radiographic evidence of invasive disease. Most patients who are treated initially with surgical debridement and amphotericin B have apparently been cured. However, longer follow-up will be necessary in these patients. Amphotericin B appears to be the treatment of choice for invasive infections caused by Bipolaris/Exserohilum species. Ketoconazole and other imidazole derivatives may also be effective in certain of the disease entities caused by these black moulds; however, their role has yet to be defined.(ABSTRACT TRUNCATED AT 400 WORDS)

A group of Giardia lamblia variant-specific surface protein (VSP) genes with nearly identical 5' regions.

The surfaces of Giardia lamblia trophozoites contain one of a set of variant-specific surface proteins. The genes encoding these proteins are highly conserved at the 3' terminus, but frequently demonstrate little similarity in the remainder of the coding region. This report describes a family of vsp genes highly similar to a repeat-containing vsp gene (vspC5) at the 5' coding and flanking regions, but which diverge abruptly from vspC5 in the first repeat and do not themselves contain full copies of the repeat. This observation suggests the possibility that recombination among different vsp genes may have played a role in development of the vsp gene repertoire.

Structural basis of karyotype heterogeneity in Giardia lamblia.

The molecular karyotype of a series of Giardia lamblia isolates representing the two major genotypes (Groups 1 and 3) was generated by assigning 13 genetic markers to chromosomes separated by pulsed-field gel electrophoresis. The co-localization identified five linked groups of genetic markers in Group 1 isolates. For each of the five linkage groups, there were up to four size variants that hybridized with the same genetic markers. Long range physical maps of the regions flanking the low copy number genetic markers indicated that these size variants were homologous chromosomes. The linkage groups were similar in Group 1 and 3 isolates. The core of each chromosome was stable while the subtelomeres were variable. The location of the ribosomal DNA repeats was variable among the different isolates and they were found in the subtelomeric regions of any of the five linkage groups. The data suggest a functional ploidy of at least four. Hypervariable subtelomeric regions of homologous chromosomes provide the structural basis of the chromosome size heterogeneity that is characteristic of G. lamblia.

Giardia lamblia trophozoites contain multiple alleles of a variant-specific surface protein gene with 105-base pair tandem repeats.

Giardia lamblia trophozoites undergo antigenic variation of a variant-specific surface protein (VSP). All VSPs that have been reported have had high cysteine contents, including numerous copies of a CXXC motif. The first vsp gene described (vspA6; from the cloned line, WBA6), contained 21 copies of a 195 base pair tandem repeat, but other reported VSPs have not contained repeats. In this report, we describe the vsp gene from WBC5, a cloned line derived from WBA6. The vspC5 gene contains short 5' and 3' regions flanking 26 copies of a 105-bp tandem repeat, which comprises 93% of the coding region. In addition to the copy containing 26 repeats, the genome contains other copies of the vspC5 with fewer copies of the repeat. The sequences flanking the repeats are identical, and all copies map to the same location on chromosomal Band 5, suggesting that multiple alleles of the vspC5 gene are present.

Pharmacokinetics and relative bioavailability of clofazimine in relation to food, orange juice and antacid.

BACKGROUND: Clofazimine is potentially useful for the treatment of disease due to multidrug resistant Mycobacterium tuberculosis, as well as leprosy and certain chronic skin diseases. Its pharmacokinetics have been incompletely characterized. This study was conducted to explore issues relating to bioavailability in the presence of food, orange juice, and antacid. METHODS: A 5 drug regimen consisting of clofazimine, cycloserine, ethionamide, para-aminosalicyclic acid, and pyridoxime was administered to healthy subjects four times using a four period cross-over design with two weeks washout between treatments. Subjects also received orange juice, a high fat meal, aluminum/magnesium antacid, or only water in random order with the drug regimen. The pharmacokinetics of clofazimine were assessed using individual- and population-based methods and relative bioavailability compared to fasting administration was determined. RESULTS: Clofazimine exhibited a sometimes prolonged and variable lag-time and considerable variability in plasma concentrations. From the population analysis (one-compartment model), the mean oral clearance was 76.7 l/h (CV=74.2%) and mean apparent volume of distribution was 1470 l (CV=36.3%). The first-order absorption rate constant ranged from 0.716 to 1.33 h(-1) (pooled CV=61.7%). Residual (proportional) error was 49.1%. Estimates of bioavailability compared to fasting administration were 145% (90% CI, 107-183%) for administration with high fat food, 82.0% (63.2-101%) for administration with orange juice, and 78.5% (55.1-102%) for administration with antacid. CONCLUSION: Administration of clofazimine with a high fat meal provides the greatest bioavailability, however, bioavailability is associated with high inter- and intra-subject variability. Both orange juice and aluminum-magnesium antacid produced a reduction in mean bioavailability of clofazimine.

The Giardia lamblia genome.

Giardia lamblia is a protozoan parasite of humans and other mammals that is thought to be one of the most primitive extant eukaryotic organisms. Although distinctly eukaryotic, it is notable for its lack of mitochondria, nucleoli, and perixosomes. It has been suggested that Giardia spp. are pre-mitochondriate organisms, but the identification of genes in G. lamblia thought to be of mitochondrial origin has generated controversy regarding that designation. Giardi lamblia trophozoites have two nuclei that are identical in all ways that have been studied. They are polyploid with at least four, and perhaps eight or more, copies of each of five chromosomes per organism and have an estimated genome complexity of 1.2x10(7)bp of DNA, and GC content of 46%. There is evidence for recombination at the telomeres of some of the chromosomes, and multiple size variants of single chromosomes have been identified within cloned isolates. However, the internal regions of the chromosomes demonstrate no evidence of recombination. For example, there is no evidence for control of vsp gene expression by DNA recombination, and no evidence for rapid mutation in the vsp genes. Single pass sequences of approximately 9% of the G. lamblia genome have already been obtained. An ongoing genome project plans to obtain approximately 95% of the genome by a random approach, as well as a complete physical map using a bacterial artificial chromosome library. The results will facilitate a better understanding of the biology of Giardia spp. as well as their phylogenetic relationship to other primitive organisms.

Molecular comparison of Giardia lamblia isolates.

Giardia lamblia (also Giardia duodenalis, Giardia intestinalis) isolates have been variably divided into two or three genotypes by different investigators. We have compared the triose phosphate isomerase sequences of the three genotypes (Groups 1, 2, and 3) described by Nash and shown that Groups 1 and 2 are similar, while Group 3 is markedly different from Groups 1 and 2, indicating that Group 1/2 and Group 3 correspond to the two major genotypes identified by other investigators. We have also analysed three Chinese isolates and showed that two fit into Group 3, while the third contained a mixture of Groups 1 and 3 isolates. These results confirm the relatedness of G. lamblia isolates from throughout the world, and established the feasibility of using DNA amplification and sequence analysis for detecting mixed isolates.

Haemophilus influenzae: an important cause of maternal and neonatal infections.

Although Haemophilus influenzae is recognized as a major pathogen of infants, its role in maternal and neonatal infections is not as well appreciated. We analyzed the records of all mothers and neonates infected with H influenzae over a 10-year period. Twenty-eight mother/neonate sets were identified in which at least one had documented infection with H influenzae. Of the 18 mothers with documented infection, 13 had chorioamnionitis, endometritis, or both, and two of these mothers were bacteremic with H influenzae. Of the 23 infected neonates, 15 presented with early sepsis and/or pneumonia and nine had conjunctivitis. During the period of the study, only group B streptococci and Escherichia coli were more common as causes of early neonatal bacteremia. Under the conditions of this retrospective study, maternal infection predicted neonatal infection. However, prospective studies in which asymptomatic patients are cultured will be required to determine how well maternal colonization/infection with H influenzae predicts neonatal infection.

The Giardia genome project database.

The Giardia genome project database provides an online resource for Giardia lamblia (WB strain, clone C6) genome sequence information. The database includes edited single-pass reads, the results of BLASTX searches, and details of progress towards sequencing the entire 12 million-bp Giardia genome. Pre-sorted BLASTX results can be retrieved based on keyword searches and BLAST searches of the high throughput Giardia data can be initiated from the web site or through NCBI. Descriptions of the genomic DNA libraries, project protocols and summary statistics are also available. Although the Giardia genome project is ongoing, new sequences are made available on a bi-monthly basis to ensure that researchers have access to information that may assist them in the search for genes and their biological function. The current URL of the Giardia genome project database is www.mbl.edu/Giardia.

Prolonged effect of nadolol on heart rate in hyperthyroidism.

There is extensive experience in the treatment of hyperthyroidism with beta-blockade. Because of the short half-life of propranolol the drug must be taken in divided doses. The effect of a single daily dose of a long-acting beta-blocking drug, nadolol, was investigated in 7 hyperthyroid patients. A satisfactory and prolonged reduction in heart rate was observed during continuous monitoring over a 24-hour period. Nadolol, therefore, is a possible alternative to propranolol in the treatment of hyperthyroidism with an advantage in the poorly-compliant patient.

Pharmacokinetics of cycloserine under fasting conditions and with high-fat meal, orange juice, and antacids.

STUDY OBJECTIVES: To determine the effect of a high-fat meal, orange juice, and antacids on absorption of a single oral dose of cycloserine and to estimate its population pharmacokinetic parameters. DESIGN: Randomized, four-period, crossover study. SETTING: Clinical research center. PATIENTS: Twelve healthy volunteers. INTERVENTIONS: Subjects received single doses of cycloserine 500 mg after a 12-hour fast (reference), with a high-fat meal, with orange juice, and with antacids. They also received clofazimine 200 mg, ethionamide 500 mg, and p-aminosalicylic acid granules 6000 mg. MEASUREMENTS AND MAIN RESULTS: Plasma samples were collected for 48 hours and assayed by validated high-performance capillary electrophoresis assay. Concentration-time data were analyzed with noncompartmental, one-compartment, and population methods. The maximum concentration (Cmax) of cycloserine was decreased (p=0.02) by the high-fat meal. No other statistically significant differences were observed for Cmax and area under the curve from time zero to infinity across the four treatments. The high-fat meal significantly (p<0.0001) delayed time to maximum concentration by 4.7 times compared with that of the reference (1.1 hr). CONCLUSION: The pharmacokinetics of cycloserine were minimally affected by orange juice and antacids, whereas the high-fat meal delayed absorption. Administering cycloserine without a high-fat meal avoids potential alterations in the pattern of absorption.

Technical note: an evaluation of a Diamentor based system for estimation of spot film dose-area product values.

In the audit of barium studies it is not common to assess the contribution of spot films to the total dose. This study investigates the accuracy with which the number of spot films and associated dose-area product (DAP) may be estimated. The study was undertaken for barium enemas, the swallow part of barium swallow studies, and the abdominal part of barium meal studies on X-ray sets with film-screen radiography, digital fluorography and 100 mm camera fluorography. DAP readings from a Diamentor were input to a computer at a rate of three per second and the difference in successive DAP values calculated. These data were used to assess the number and total DAP from spot films. For Diamentor based systems which sample the DAP values at three times per second the threshold algorithm works well for film-screen radiography of barium meals and enemas. However, the estimated number of spot films is generally in error. For the Diamentor M2 it is unlikely that the sample rate can be increased to a high enough value to provide sufficient accuracy for all barium studies obtained using all spot film modalities, although use of the modified threshold algorithm has the potential to provide some increase in accuracy.

Clinical and metabolic features of overdosage with Amesec.

A 23-year-old woman ingested 2g. amylobarbitone, 10.4g. aminophylline and 2g. ephedrine. She was deeply unconscious, hypothermic, and went on to have supraventricular and ventricular dysrhythmias, convulsions and haematemesis. During the last convulsion she aspirated vomitus and died. The peak plasma concentration of amylobarbitone was 19mg. per l. and those of ephedrine and theophylline were 13 times higher than accepted therapeutic levels. During the course of the poisoning marked hypokalaemia (1.8mmol./l.) and hyperinsulinaemia (greater than 240mU./l.) were found in conjunction with mild hyperglycaemia (9.6mmol./l.) and elevation of free fatty acid levels (1860mumol./l.). The mechanism of these changes is discussed.