Cancer-homing toxins.

Cancer-homing toxins are a group of man-made cytotoxic molecules targeting cancer cells. In the past decade they have demonstrated potential as cancer therapeutics. These molecules contain a toxin, natural or usually derivatized, connected to a cancer-homing module, such as a monoclonal antibody or growth factor or their derivatives. Various cancer-homing toxins have been designed and tested in cell-lines, animal-models and clinical trials. We review some of these data and discuss ways to better design cancer-homing toxins in the light of advances in cancer genomics, antibody-engineering techniques and computational algorithms.

Antenatal seroprevalence of herpes simplex virus type 2 (HSV-2) in Canadian women: HSV-2 prevalence increases throughout the reproductive years.

BACKGROUND: This study sought to provide the first population estimates of herpes simplex type 2 (HSV-2) seroprevalence in Canada. GOAL: To measure the antenatal seroprevalence of HSV-2 antibodies in reproductive age women. STUDY DESIGN: An anonymous unlinked seroprevalence study used stored sera collected from pregnant women in British Columbia during 1999. Randomized sampling within age strata selected a total of 1215 subjects, ages 15 to 44 years. Serologic testing used the Gull Meridian Test. Overall prevalence was directly standardized to the 1999 Canadian female population. RESULTS: The age-adjusted prevalence for HSV-2 was 17.3% (95% CI, 15.2-19.4). Prevalence ranged from 7.1% (ages, 15-19 years) to 28.1% (ages, 40-44 years), with the largest increases after the age of 24 years. CONCLUSIONS: The HSV-2 seroprevalence among pregnant women in British Columbia is similar to that in the United States and other countries. Seroprevalence continues to rise through the later reproductive years. This observation may relate to continued transmission, an age cohort effect, or both.

Helicobacter pylori in familial clusters based on antibody profile.

Studies have shown a high prevalence of Helicobacter pylori infection in close communities and that intrafamilial spread during early childhood may be a route of transmission. A total of 72 household members from 21 families were enrolled in this study. Sera from individuals showed 50/72 (69.4%) seropositive for IgG against H. pylori by ELISA. Western blots showed diversity in the protein profiles with molecular masses ranging from approximately 8 to 130 kDa. Cohen's kappa statistical analysis of the blot patterns showed that nine families demonstrated similar profiles (100%), while 4 other families showed varying similarities (17-50%). The results support the hypothesis of intrafamilial transmission of H. pylori. Furthermore, serological studies can be used as an effective approach to determine the familial status in relation to H. pylori infection.

Bile duct injuries during laparoscopic cholecystectomy: a collective experience of four teaching hospitals and results of repair.

BACKGROUND: Laparoscopic cholecystectomy has been performed in Singapore since 1990 and, up until the end of 1997, a total of 4445 procedures had been performed in the four major teaching hospitals. Although bile duct injuries were thought to have increased following the introduction of laparoscopic cholecystectomy, there have been no reviews done on the incidence of these injuries in the Singapore context. METHODS: The present retrospective review aimed to audit the rate of bile duct injuries in the four major teaching hospitals in Singapore and to document the results of management of these injuries. RESULTS: Of the 4445 procedures performed, there were 19 (0.43%) cases of bile duct injuries. These involved the common hepatic duct (n = 8), common bile duct (n = 10), and the right hepatic duct (n = 1). The underlying gall bladder pathology included non-inflamed gall bladders (n = 10), acute cholecystitis (n = 4), Mirrizzi's syndrome (n = 3) and mucocele of the gall bladder (n = 2). Transection of the duct accounted for the majority of the injuries. Eleven bile duct injuries were identified at the time of operation. These were primarily repaired over a T tube (n = 4) or by a bilio-enteric bypass (n = 7). The remainder were diagnosed at a median of 7 days (range: 1-556 days) after surgery with a presentation of jaundice or pain. These were repaired by bilio-enteric anastomosis (n = 7) and closure over a T tube (n = 1). Three patients developed strictures subsequently, two following bilio-enteric repair after delayed diagnosis and one following immediate primary repair over a T tube. One patient developed intrahepatic stones and required a left lateral segmentectomy. CONCLUSIONS: The experience of a 0.43% bile duct injury rate is comparable to the best results from most large series in the West. Inflammation at Calot's triangle is an important associated factor for injury. Early recognition and prompt repair affords good results, and hepaticojejunostomy is recommended as the repair of choice.

Unchanged characteristics of Helicobacter pylori during its morphological conversion.

Helicobacter pylori strains RH 54 and NCTC 11637 were grown in brain-heart infusion broth up to 56 days, and the coccoid form was obtained during prolonged incubation. Two morphological types of coccoids were observed, one of which was electron-dense and had an intact cellular membrane and flagella, indicating that it was likely to be viable. The other coccoid form was sphaeroblast-like and weakly stained, showing features of degeneration. Catalase activity was positive for aged cultures even up to 160 days. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that most of the protein bands appeared to be similar in both the spiral and coccoid forms. In addition, Lewis blood group antigens were detected in cultures of up to 8 weeks. Furthermore, two sets of primers for the vacA and cagA genes were used in polymerase chain reaction, and these two important genes remained conserved in both the spiral and coccoid forms. The present study shows that the coccoid form of H. pylori retained many important characteristics present in the spiral form despite the morphological conversion, and thus supports the notion that some of the coccoid forms of H. pylori are likely to be viable.

Predominance of a single strain of Helicobacter pylori in gastric antrum.

BACKGROUND: Diversity of DNA among H. pylori strains isolated from different patients can serve as a useful marker for differentiating strains. DNA profiles of H. pylori obtained from sequential gastric biopsies were identical in most patients indicating that a given strain can persist from months to years. Patients colonized with more than two strains isolated mainly from different anatomical sites have been reported. This work examined whether the gastric antrum of patients with dyspepsia is colonized by single or multiple strains of H. pylori as well as the in vitro competition of different strains of H. pylori. MATERIALS AND METHODS: Two antral biopsy specimens from each of the 124 patients were cultured for H. pylori. DNA fingerprinting of H. pylori isolates was performed using random amplified polymorphic DNA (RAPD) analysis. To elucidate the possible interaction among H. pylori isolates, bacterial populations of two H. pylori strains cogrown in broth medium over 21 days were enumerated and DNA fingerprinting was compared. RESULTS: A total of 58 patients showed the presence of H. pylori in both antral specimens, while five patients had H. pylori in only one of the two samples. These 58 patients were shown to harbor a single strain of H. pylori as analyzed by RAPD fingerprinting. In vitro studies of bacterial interaction of two different strains of H. pylori showed growth competition resulting in the predominance of a single strain. CONCLUSIONS: The results support the concept that a single strain predominates in the gastric antrum site of patients studied.

Severe gastritis in guinea-pigs infected with Helicobacter pylori.

An appropriate animal model is essential to study Helicobacter pylori infection. The aim of this study was to investigate if H. pylori can colonise the guinea-pig stomach and whether the infection causes gastritis and a serological response similar to that observed in man. Guinea-pigs were infected either with fresh H. pylori isolates from human gastric biopsies or with a guinea-pig passaged strain. When the animals were killed, 3 and 7 weeks after inoculation, samples were taken for culture, histopathology and serology. H. pylori was cultured from 22 of 29 challenged animals. All culture-positive animals exhibited a specific immune response against H. pylori antigens in Western blotting and gastritis in histopathological examination. Antibody titres in enzyme immunoassay were elevated among animals challenged with H. pylori. The inflammatory response was graded as severe in most animals and consisted of both polymorphonuclear leucocytes and lymphocytes. Erosion of the gastric epithelium was found in infected animals. These results suggest that the guinea-pig is suitable for studying H. pylori-associated diseases. Moreover, guinea-pigs are probably more similar to man than any other small laboratory animal as regards gastric anatomy and physiology.

Characterization of clinical isolates of Helicobacter pylori in Singapore.

Of the 69 Helicobacter pylori isolates analysed, 31 strains (45%) showed resistance to metronidazole, one strain (1.4%) was resistant to amoxicillin while two strains (2.9%) were resistant to clarithromycin. It was found that metronidazole resistance rates increased in Singapore from 20% to 62% between late 1995 and early 1997. By biotyping using API ZYM, a total of 80% (55/69) strains were characterized as biotype II, while the remaining 20% (14/69) strains belonged to biotype III. Interestingly, 71% (10/14) of biotype III were resistant to metronidazole compared with 38% (21/55) of biotype II. DNA profiles generated by random amplified polymorphic DNA from 69 isolates showed highly diversified DNA fingerprints allowing effective discrimination among strains. Of the 60 H. pylori isolates from peptic ulcer patients, it was found that cagA and vacA occurred in 80% (48/60) and 82% (49/60) isolates, respectively.

Improving the success of culturing Helicobacter pylori from gastric biopsies.

Factors influencing the successful isolation of Helicobacter pylori from human gastric biopsies were studied. Within 24 h, each of the gastric biopsies was inoculated onto chocolate blood agar media and incubated for up to 2 weeks. Among 63 (70%) culture positive cases in 90 patients, 58 (64%) cases were culture positive for both specimens, while five (6%) cases were culture positive in only one biopsy. Of the 63 positive cultures, 51 H. pylori strains (81%) grew on both media with and without antibiotics. Eight strains (13%) grew only on medium without antibiotics, while four isolates (6%) were obtained only from medium with antibiotics. These results support the previous histological observation of patchy colonization of H. pylori in the stomach. The success rate for culture of H. pylori from gastric biopsies increased when two biopsies were taken and inoculated on chocolate blood agar media with and without antibiotics.

Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium DNA from dried blood spots.

The Plasmodium parasite possesses two extrachromosomal genomes; the mitochondrial genetic element and the extrachromosomal plastid-like DNA. The latter has only been fully described for one culture strain of P. falciparum. In this study, a rapid procedure for amplifying plastid DNA from dried blood spots of blood infected with different malaria species was developed. PCR amplification of a 595 bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene was achieved using primers derived from the P. falciparum sequence. The PCR product was observed in all Plasmodium species examined. Sequence analysis of amplified products homologous to an LSU-rRNA fragment of the plastid-like extrachromosomal circle revealed extensive conservation between Plasmodium species including P. falciparum, P. vivax, P. malariae and P. berghei.

Electrohydraulic lithotripsy: an effective and economical modality of endoscopic ureteric lithotripsy.

BACKGROUND: Electrohydraulic lithotripsy (EHL) has been available for endoscopic treatment of urinary calculi since 1960, but the large probe size and concerns regarding safety had previously restricted its use to the treatment of bladder calculi. However, recent refinements have made it particularly suitable for the treatment of ureteric calculi. METHODS: The authors report their initial experience using EHL in conjunction with mini-ureteroscopy in the treatment of 94 ureteric calculi in 89 patients. The size of the calculi ranged from 3 to 19 mm in diameter, with a mean of 8.2 mm. The mean operating time was 29 min, ranging from 10 to 120 min. RESULTS: A complete fragmentation rate of 91.5% of the calculi was achieved. There were no major complications and a low incidence of minor complications: haematuria (2.2%), urinary tract infection (3.4%) and postoperative ureteric colic (2.2%). There were four cases of minor ureteric perforations (4.5%); all were successfully treated using conservative measures. CONCLUSIONS: It is concluded that EHL is a safe and effective method of treating ureteric calculi.

Nucleotide sequence of ToxPK1 gene from Toxoplasma gondii.

We report here for the first time a complete nucleotide sequence (6.8 kb) of a protein kinase gene (ToxPK1) from the obligate intracellular protozoan parasite of man, Toxoplasma gondii. This gene comprising putatively of 9 exons and 8 introns forms the Toxoplasma gene with the largest number and size of introns reported so far. The predicted protein with 508 amino acids contains the 15 invariant residues as well as the characteristic motifs specific to protein serine/threonine kinases. Homology-based computational comparisons suggested that TOXPK1 belongs to or closely resembles the SNF1 subfamily of protein-serine/threonine kinases. Based on the functions of SNF1 homologs in other organisms and our RT-PCR results, it is likely that TOXPK1 may be transiently expressed to up-regulate glycogen biosynthesis during the development of tachyzoites into bradyzoites.

Identification of two protein serine/threonine kinase genes and molecular cloning of a SNF1 type protein kinase gene from Toxoplasma gondii.

We report for the first time the identification of two protein kinase genes, in Toxoplasma gondii. Based on the conserved amino acid sequence motifs of catalytic subdomains VI(b) and IX of known protein serine/threonine kinases, degenerate oligonucleotides were synthesized and used in PCR to produce 196 and 373 bp DNAs. They encoded stretches of amino acid sequences characteristic of protein serine/threonine kinases. Dot and Southern blot analysis confirmed that these PCR products were of Toxoplasma origin. Screening of a genomic library of the organism with the 196 bp PCR product as a probe yielded 3 ToxPK1 g clones. Nucleotide sequence of two of these clones, revealed that the protein encoded, TOXPK1 resembled other carbon catabolite derepressing regulatory protein kinases. Therefore, we suggest that TOXPK1 could play a role in the interconversion of active and passive life-cycle stages of this parasite. RT-PCR studies on Toxoplasma tachyzoites' total RNA suggested that ToxPK1 gene is developmentally regulated. The 373 bp PCR product, however encoded a polypeptide that resembled the catalytic subunit of other cAMP-dependent protein kinases. Hence, this protein (TOXPK2) was considered as a product of another gene, ToxPK2.