Effect of treating Schistosoma haematobium infection on Plasmodium falciparum-specific antibody responses.

BACKGROUND: The overlapping geographical and socio-economic distribution of malaria and helminth infection has led to several studies investigating the immunological and pathological interactions of these parasites. This study focuses on the effect of treating schistosome infections on natural human immune responses directed against plasmodia merozoite surface proteins MSP-1 (DPKMWR, MSP1(19)), and MSP-2 (CH150 and Dd2) which are potential vaccine candidates as well as crude malaria (schizont) and schistosome (whole worm homogenate) proteins. METHODS: IgG1 and IgG3 antibody responses directed against Schistosoma haematobium crude adult worm antigen (WWH) and Plasmodium falciparum antigens (merozoite surface proteins 1/2 and schizont extract), were measured by enzyme linked immunosorbent assay (ELISA) in 117 Zimbabweans (6-18 years old) exposed to S. haematobium and P. falciparum infection. These responses were measured before and after anti-helminth treatment with praziquantel to determine the effects of treatment on anti-plasmodial/schistosome responses. RESULTS: There were no significant associations between antibody responses (IgG1/IgG3) directed against P. falciparum and schistosomes before treatment. Six weeks after schistosome treatment there were significant changes in levels of IgG1 directed against schistosome crude antigens, plasmodia crude antigens, MSP-1(19), MSP-2 (Dd2), and in IgG3 directed against MSP-1(19). However, only changes in anti-schistosome IgG1 were attributable to the anti-helminth treatment. CONCLUSION: There was no association between anti-P. falciparum and S. haematobium antibody responses in this population and anti-helminth treatment affected only anti-schistosome responses and not responses against plasmodia crude antigens or MSP-1 and -2 vaccine candidates.

A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae.

The Coronaviridae family, comprising the Coronavirus and Torovirus genera, is part of the Nidovirales order that also includes two other families, Arteriviridae and Roniviridae. Based on genetic and serological relationships, groups 1, 2 and 3 were previously recognized in the Coronavirus genus. In this report we present results of comparative sequence analysis of the spike (S), envelope (E), membrane (M), and nucleoprotein (N) structural proteins, and the two most conserved replicase domains, putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL), aimed at a revision of the Coronaviridae taxonomy. The results of pairwise comparisons involving structural and replicase proteins of the Coronavirus genus were consistent and produced percentages of sequence identities that were distributed in discontinuous clusters. Inter-group pairwise scores formed a single cluster in the lowest percentile. No homologs of the N and E proteins have been found outside coronaviruses, and the only (very) distant homologs of S and M proteins were identified in toroviruses. Intragroup sequence conservation was higher, although for some pairs, especially those from the most diverse group 1, scores were close or even overlapped with those from the intergroup comparisons. Phylogenetic analysis of six proteins using a neighbor-joining algorithm confirmed three coronavirus groups. Comparative sequence analysis of RdRp and HEL domains were extended to include arterivirus and ronivirus homologs. The pairwise scores between sequences of the genera Coronavirus and Torovirus (22-25% and 21-25%) were found to be very close to or overlapped with the value ranges (12 to 22% and 17 to 25%) obtained for interfamily pairwise comparisons, but were much smaller than values derived from pairwise comparisons within the Coronavirus genus (63-71% and 59-67%). Phylogenetic analysis confirmed toroviruses and coronaviruses to be separated by a large distance that is comparable to those between established nidovirus families. Based on comparison of these scores with those derived from analysis of separate ranks of several multi-genera virus families, like the Picornaviridae, a revision of the Coronaviridae taxonomy is proposed. We suggest the Coronavirus and Torovirus genera to be re-defined as two subfamilies within the Coronavirdae or two families within Nidovirales, and the current three informal coronavirus groups to be converted into three genera within the Coronaviridae.

Aspirin effects on endometrial cancer cell growth.

OBJECTIVE: To find whether aspirin (acetylsalicylic acid, ASA) inhibits the growth of endometrial cancer cells in vitro in a way similar to that in colorectal cancer cells and to investigate the mechanisms by which aspirin might lead to growth inhibition. METHODS: Ishikawa human endometrial tumor cells were grown in the presence of ASA (1-5 mM) for 96 hours. Controls were treated with vehicle (absolute ethanol). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Analysis of cell-cycle distribution and bcl-2 expression was assessed by flow cytometry. RESULTS: Acetylsalicylic acid induced a dose-dependent inhibition of Ishikawa cells in vitro. The percentage of growth inhibition was 21-88% at concentrations of 1-5 mM. It also induced apoptosis and reduced bcl-2 expression in Ishikawa cells in a dose-dependent manner. Control cells and cells treated with the lowest concentration of ASA exhibited 2% apoptosis and more than 60% of the population expressed bcl-2. Apoptosis levels increased as levels of ASA increased from 2 to 5 mM (7-58%) with a concommitant decrease in bcl-2 expression from 46% at 2 mM to 2% at 5 mM. Acetylsalicylic acid concentrations of 3 mM or greater induced a shift from the resting phase (G0/G1) to S phase of the cell cycle. CONCLUSION: Acetylsalicylic acid inhibited Ishikawa cell growth in vitro in a dose-dependent manner. Apoptosis is one of the mechanisms involved in the response, which can be mediated in part by downregulation of bcl-2.

Innovation and discovery: the application of nucleic acid-based technology to avian virus detection and characterization.

Polymerase chain reaction (PCR)-based approaches to the detection, differentiation and characterization of avian pathogens continue to be developed and refined. The PCRs, or reverse transcriptase-PCRs, may be general, designed to detect all or most variants of a pathogen, or to be serotype, genotype or pathotype specific. Progress is being made with respect to making nucleic acid approaches more suitable for use in diagnostic laboratories. Robotic workstations are now available for extraction of nucleic acid from many samples in a short time, for routine diagnosis. Following general PCR, the DNA products are commonly analyzed by restriction endonuclease mapping (restriction fragment length polymorphism), using a small number of restriction endonucleases, based on a large body of sequence data. Increasingly, however, nucleotide sequencing is being used to analyze the DNA product, in part due to the expanding use of non-radioactive sequencing methods that are safe and enable high throughout. In this review, I highlight some recent developments with many avian viruses: Newcastle disease virus; circoviruses in canary and pigeon; infectious bursal disease virus (Gumboro disease virus); avian adenoviruses, including Angara disease/infectious hydropericardium virus, haemorrhagic enteritis virus of turkeys, and egg drop syndrome virus; avian herpesviruses, including infectious laryngotracheitis virus, duck plague virus, psittacine herpesvirus (Pacheco's parrot disease virus), Marek's disease virus and herpesvirus of turkeys; avian leukosis virus (associated with lymphoid leukosis or myeloid leukosis, and egg transmission); avian pneumoviruses (turkey rhinotracheitis virus); avian coronaviruses, including infectious bronchitis virus, turkey coronavirus and pheasant coronavirus; astrovirus, in the context of poult enteritis and mortality syndrome, and avian nephritis virus; and avian encephalomyelitis virus, a picornavirus related to hepatitis A virus.

cis-acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging.

The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5' terminus was replicated; the 5' untranslated region (UTR) comprises 528 nt. Region I of the 3' UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3' UTR, was replicated. Thus, the 5'-terminal 544 nt and 3'-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5' end of region II of the 3' UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.

Distal external iliac lymph nodes in early cervical cancer.

OBJECTIVE: To evaluate the utility of the deep circumflex iliac vein as a landmark for the caudal limit of external iliac lymphadenectomy for early cervical cancer. METHODS: During dissection of the distal (caudal), anterior (ventral) aspect of the external iliac vessels in conjunction with radical hysterectomy for carcinoma of the cervix, a careful search was made for the deep circumflex iliac vein. Lymph nodes immediately above this vein were sent as adjacent and lymph nodes caudal to the vein were sent as distal. The distance from the vein to the femoral canal was measured. RESULTS: Seventy-one women were studied over 40 months. Fifty-five had squamous cell carcinoma, 15 had adenocarcinoma, and one had adenosquamous carcinoma. The mean distance from the deep circumflex iliac vein to the femoral canal was 16 mm. Sixty-three patients had dissections distal to identified deep circumflex iliac veins and 49 (77%) of these had distal lymph nodes removed. The median number of pelvic lymph nodes removed was 24 (range nine to 68), and the median number of distal lymph nodes removed was 1.0 from each side. Lymph node metastases were found in 13 women (18%). One subject with multiple macroscopically positive nodes had a single positive distal lymph node. Thus, a small number of distal lymph nodes were found in most women with early invasive cervical cancer, and 8% of those with positive nodes had involvement of this nodal group. CONCLUSION: The deep circumflex iliac vein was an appropriate landmark for the caudal limit of external iliac lymphadenectomy.

Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus.

Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.

Lateral microscopic extension of squamous cell carcinoma of the vulva.

PURPOSE: The aim of this study was to measure the radial occult microscopic spread of tumor in patients with invasive squamous cell carcinoma of the vulva. MATERIALS AND METHODS: In the operating room the gross tumor border was marked. The pathologist took a radial section in each quadrant and measured the distance of occult lateral spread of the tumor. RESULTS: From 7/01/93 to 6/30/96, 24 tumors from 21 patients were studied. The mean maximum tumor diameter was 3. 2 cm (0.5-7.0) and the mean depth of invasion was 9.1 mm (1.1-28.0). The gross and microscopic extent correlated in 20 tumors. Maximum lateral microscopic extent of the other 4 tumors was 3.5, 5 (to the margin), 10, and 16 mm. These 4 tumors were ulcerative and infiltrative and arose from or involved mucosa. CONCLUSION: The gross and microscopic periphery of most invasive squamous vulvar cancers are approximately the same. Ulcerative tumors with an infiltrative pattern of invasion which involve mucosal epithelium may be more likely to extend beyond what is grossly apparent. Measurement of the tumor-free margin should be included in future studies.

Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus.

The complete RNA genome of avian encephalomyelitis virus (AEV) has been molecularly cloned and sequenced. This revealed AEV to be a member of the Picornaviridae and consequently it is the first avian picornavirus for which the genome has been sequenced. Excluding the poly(A) tail the genome comprises 7032 nucleotides, which is shorter than that of any mammalian picornavirus sequenced to date. An open reading frame commencing at nucleotide 495 and terminating at position 6896 (6402 nucleotides) potentially encodes a polyprotein of 2134 amino acids. The polyprotein sequence has 39% overall amino acid identity with hepatitis A virus (HAV; genus Hepatovirus), compared to 19 to 21% for viruses from the other five picornavirus genera. Eleven cleavage products were predicted. The highest identity (49%) with HAV was in the P1 region, encoding the capsid proteins. The 5' and 3' untranslated regions (UTRs) comprise 494 and 136 nucleotides, respectively. The 5' UTR is the shortest of any picornavirus sequenced to date and, unlike HAV, it does not contain a long polypyrimidine tract.

Adenocarcinoma of the Endometrium: An Institutional Review.

BACKGROUND: Many oncologists regard endometrial cancer as a relatively benign and easily treatable gynecologic tumor. Inadequate care can result in poor outcomes. METHODS: The authors review the epidemiology and pathology of the disease, and they compare disease characteristics and outcomes of FIGO staging with their own 11-year experience at a tertiary referral center. RESULTS: Patients referred to tertiary referral centers tend to present with more advanced stages of disease than those reported by FIGO, although the profile of histologic types is similar. CONCLUSIONS: Prevention and early detection of endometrial cancer can minimize the impact of this disease. Complete staging and tumor removal including extrafascial hysterectomy with bilateral salpingo-oophorectomy, pelvic lymphadenectomy, and selective paraaortic lymphadenectomy are the cornerstones of surgical therapy.

Does ligation of the hypogastric artery at the time of radical hysterectomy and lymphadenectomy decrease blood loss? Results of a prospective randomized trial.

A prospective, randomized study of patients undergoing radical hysterectomy for gynecologic malignancies was undertaken from 10/95 to 11/96 to determine if ligation of the hypogastric arteries at the time of radical hysterectomy decreases blood loss. Patients were randomized to either ligation of the hypogastric artery (Group 1) or no ligation (Group 2) prior to a standard Piver type III radical hysterectomy. Surgeries were performed by Board certified gynecologic oncologists with gynecologic oncology fellows and/or OB/GYN residents. Patients were analyzed for demographic characteristics and intraoperative and postoperative parameters. Statistical analysis was performed with independent samples t-test, Mann-Whitney rank sum test, Chi square and Fisher exact test. Twenty-one patients were randomized to group 1 and 22 to group 2. Groups were similar with respect to demographics and preoperative parameters except for age. There were no differences among the groups with respect to intraoperative and postoperative parameters. The mean estimated blood loss for group 1 was 600 ml and 550 ml for group 2 (P = NS). Hypogastric artery ligation (HAL) at the time of radical hysterectomy for gynecologic malignancy does not reduce blood loss.

Does IBV change slowly despite the capacity of the spike protein to vary greatly?

We have sequenced that part of the spike protein (S) gene which encodes the aminoterminal and most variable quarter (hypervariable region, HVR) of the S1 subunit of 28 isolates of the 793/B (also known as CR88 and 4/91) serotype of infectious bronchitis virus (IBV) and the whole of S1 for nine of them. The isolates were from France and Britain between the years 1985 (first isolation) and 1996. The maximum nucleotide and amino acid differences between the first isolate and the others were 4.1% and 7.6%, respectively, for the whole of S1 and 7.1% and 14.6%, respectively, in the HVR. Analysis within clearly recognisable subgroups suggested that even in the HVR the nucleotide mutation rate was only 0.3 to 0.6% per year. However, there was no evidence that mutations had become fixed in a progressive manner; this serotype did not appear to be evolving. Strains isolated several years apart could be more similar than those isolated in a given year. It is likely that the amino acid changes are largely at positions where amino acid differences are tolerated rather than as a consequence of immune pressure. Reasons for this conclusion are discussed.

Hormonal modulation of Ishikawa cells during three-dimensional growth in vitro.

OBJECTIVES: The Ishikawa endometrial cancer cell line is hormonally responsive, expressing estrogen and progesterone receptors (ER, PR) when grown in traditional monolayer culture. The purpose of this paper is to demonstrate a three-dimensional spheroid culture system for cancer cells. We used this system to determine the response of the Ishikawa cell line to estradiol-17 beta (E), tamoxifen (T), megestrol acetate (MA), and progesterone (P). METHODS: Ishikawa cells were incubated in polyurethane culture bags using phenol red-free media containing ethanol (0.1%, controls), E (1 mumol, or 1 nmol), T (1 mumol, or 10 nmol), MA (1 mumol, or 10 nmol), or P (1 mumol). Cellular morphology was assessed by hematoxylin and eosin staining, and expression of estrogen and progesterone receptors was determined immunohistochemically using an immunoperoxidase technique. RESULTS: Cells in control cultures demonstrated minimal organization and lacked hormone receptors. In contrast, cells exposed to either E or T displayed significant glandular formation, with multicellular, microvilli-rich, columnar epithelia exhibiting polarized nuclear arrangements. Within 4 weeks, E- and T-treated cultures showed upregulated nuclear staining for PR, with little ER present. Cells treated with MA or P showed less glandular organization but expressed ER with PR downregulation. CONCLUSIONS: These data support the use of this novel three-dimensional culture system to study the modulation of tumor cell biologic activity in response to hormonal agents. Future applications of this model include examining in vitro responsiveness of cancer cell lines to additional biologic agents and chemotherapeutic regimens.

Body mass predicts the survival of patients with new International Federation of Gynecology and Obstetrics Stage IB1 and IB2 cervical carcinoma treated with radical hysterectomy.

BACKGROUND: The authors evaluated the impact of body mass on survival and morbidity of patients with new International Federation of Gynecology and Obstetrics (FIGO) Stage IB1 and IB2 cervical carcinoma managed with radical hysterectomy. METHODS: Two hundred twenty-nine patients with Stage IB1 or IB2 cervical carcinoma treated with radical hysterectomy were studied in a multivariate logistic regression analysis. The body mass index (BMI) and the ponderal index (PI) were used as measures of body mass and were analyzed as predictors of recurrence, survival, and complications in light of the new staging system. RESULTS: Twenty-seven of 229 patients died of recurrent disease. A low BMI or a high PI were predictive of poor survival. Tumor greatest dimension, lymph node involvement, BMI, and PI were all independent predictors of survival (P=0.0006). The only independent predictor of complications was para-aortic lymph node dissection (P=0.0026). CONCLUSIONS: Cervical carcinoma patients with a low body mass, as indicated by a low BMI or a high PI, were found to have poor survival after undergoing radical hysterectomy. Additional predictors of poor survival included lymph node metastases and increased tumor size. BMI and PI are more important predictors of survival than the new FIGO Stages IB1 and IB2. Body mass is not predictive of complications.

The ectodomains but not the transmembrane domains of the fusion proteins of subtypes A and B avian pneumovirus are conserved to a similar extent as those of human respiratory syncytial virus.

The fusion glycoprotein (F(B)) gene of five strains of the B subtype of avian pneumovirus (APV; turkey rhinotracheitis virus) has been sequenced. The length of the F(B) protein was 538 amino acids, identical to that of the F protein of subtype A virus, with which it had 74% and 83% overall nucleotide and deduced amino acid identities, respectively. The F(B) and F(A) ectodomains had 90% amino acid identity, very similar to the 91% identity between the ectodomains of the F proteins of subtype A and B human respiratory syncytial virus (HRSV). As with HRSV, the F2 polypeptide was less conserved (83% identity) than F1 (94%). In contrast to the ectodomain, the transmembrane and cytoplasmic domains of the two APV subtypes were much less conserved (30% and 48% identity, respectively) than those of HRSV (92% and 87%, respectively). Comparisons within all the genera of the Paramyxoviridae (Pneumovirus, Morbillivirus, Paramyxovirus and Rubullavirus) show that low amino acid identity between F protein transmembrane domains is a feature of different species of virus rather than of strain differences. This may indicate that the two subtypes of APV have evolved in different geographical regions and/or different avian species. This is the first report of an F gene sequence from a subtype B APV.

A pilot study utilizing intraoperative lymphoscintigraphy for identification of the sentinel lymph nodes in vulvar cancer.

OBJECTIVE: To identify sentinel lymph nodes using intraoperative lymphoscintigraphy. METHODS: Technetium-99-labeled sulfur colloid was injected at the site of primary vulvar carcinoma. An intraoperative gamma counter was used to identify one or more sentinel lymph nodes. RESULTS: Ten patients underwent bilateral inguinal and femoral lymphadenectomy. The clinical stages are as follows: T1 in 6, T2 in 2, and T3 in 2. A total of four groins (3 patients) were positive for metastases. In one patient only the sentinel node was positive for disease. In a second patient, two unilateral nodes were positive for disease and both were identified with the gamma counter as sentinel nodes. In the third patient, a single sentinel node was positive for malignancy in each groin. Multiple nonsentinel lymph nodes were positive in each groin in this patient. In no case was the sentinel node negative when other nonsentinel nodes were positive. CONCLUSION: Intraoperative lymphoscintigraphy quantitatively identifies one or more sentinel lymph nodes. Since sentinel lymph nodes can be localized transcutaneously, this technique may be useful for selective lymphadenectomy. Larger patient accrual is necessary to verify this technique.

Management of gynecologic oncology patients with a preoperative deep vein thrombosis.

Our experience with gynecologic oncology patients presenting preoperatively with a deep vein femoral thrombosis is reported. Over a 3-year period data were collected on all patients at the University of South Florida (USF) requiring surgery for a known or suspected gynecologic cancer and having a concomitant active femoral venous thrombosis. Twelve such patients were managed. Management was divided among three options: heparinization, preoperative inferior vena cava (IVC) filter, and intraoperative IVC ligation. For two patients a filter could not be placed preoperatively due to tumor compression of the IVC. Both underwent IVC ligation intraoperatively. One of the two died intraoperatively, possibly related to pulmonary embolism. One of eight with a preoperative IVC filter had obvious clot propagation postoperatively, managed with heparin. One of two managed with heparin only had severe bleeding and heparin-associated thrombocytopenia (HAT) preoperatively. Based on our experience and a review of the literature, we recommend therapeutic heparinization and a preoperatively placed IVC filter for most preoperative gynecologic oncology patients with femoral deep venous thrombosis.

The clinical significance of calculated versus measured creatinine clearance in patients with gynecologic malignancy.

Measurement of creatinine clearance remains in convenient because of the need for 24-hour urine collection. Our study compared creatinine clearance calculated from serum creatinine (CC alpha) to measured creatinine clearance (MC alpha) using an unsupervised 8-hour urine collection at home. Forty-two women admitted to our gynecologic oncology service participated in this study. Twenty-six of these patients had cancer originating from the ovaries, 8 from the uterus, 7 from the cervix, and 1 from the fallopian tube. There were adequate data for 61 collections. MC alpha using unsupervised 8-hour urine collection at home correlated well with CC alpha. This calculated value may be sufficient to be used as a guide in evaluating renal function in patients with gynecologic malignancy. However, if it becomes necessary to measure creatinine clearance, our data suggest that an 8-hour collection may be used.