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Unlabelled: POPE (Phylogeny, Ortholog and Paralog Extractor) provides an integrated platform for automatic ortholog identification. Intermediate steps can be visualized, modified and analyzed in order to assess and improve the underlying quality of orthology and paralogy assignments., Availability: POPE is available for download from the website: http://www.well.ox.ac.uk/~tota/pope.

The steroid and xenobiotic receptor SXR (human pregnane X receptor) is a nuclear receptor that plays a key role in the body's detoxification response by regulating genes involved in drug metabolism and transport. SXR ligands include a wide range of compounds, which induce transcription of SXR target genes via activation of a heterodimeric transcription factor consisting of SXR and the related nuclear receptor retinoid X receptor (RXR). We investigated the effect of RXR-selective ligands, rexinoids, on SXR/RXR activity. In agreement with previous reports, we found that rexinoids are weak activators of SXR, but we also found that they can antagonize SXR activation by the potent SXR agonist rifampicin. This antagonism included suppression of rifampicin-induced expression of SXR target genes, as well as reduced binding of SXR/RXR to SXR response elements both in vivo and in vitro. Interestingly, two rexinoids, bexarotene (LGD1069/Targretin) and LG100268, caused a rapid and sustained decrease in the protein levels of both SXR and RXR. The decrease in SXR level was due to an enhanced rate of protein degradation and was dependent on calpain activity, as opposed to rexinoid-induced RXR degradation, which is mediated via the proteasome. Thus, we have demonstrated a novel, rexinoid-modulated mechanism regulating SXR protein stability, which may explain why rexinoids are only weak activators of SXR/RXR-mediated transcription, despite reports that they bind to SXR with high affinity. We suggest that the ability of rexinoids to induce degradation of both SXR and RXR, in combination with competition for binding to SXR, can also explain why rexinoids antagonize the activation of SXR by drugs like rifampicin.

Background: Genome wide association (GWA) studies are now being widely undertaken aiming to find the link between genetic variations and common diseases. Ideally, a well-powered GWA study will involve the measurement of hundreds of thousands of single nucleotide polymorphisms (SNPs) in thousands of individuals. The sheer volume of data generated by these experiments creates very high analytical demands. There are a number of important steps during the analysis of such data, many of which may present severe bottlenecks. The data need to be imported and reviewed to perform initial quality control (QC) before proceeding to association testing. Evaluation of results may involve further statistical analysis, such as permutation testing, or further QC of associated markers, for example, reviewing raw genotyping intensities. Finally significant associations need to be prioritised using functional and biological interpretation methods, browsing available biological annotation, pathway information and patterns of linkage disequilibrium (LD)., Results: We have developed an interactive and user-friendly graphical application to be used in all steps in GWA projects from initial data QC and analysis to biological evaluation and validation of results. The program is implemented in Java and can be used on all platforms., Conclusion: Very large data sets (e.g. 500 k markers and 5000 samples) can be quality assessed, rapidly analysed and integrated with genomic sequence information. Candidate SNPs can be selected and functionally evaluated.

Transcriptional activation by nuclear hormone receptors is well characterized, but their cooperation with other signaling pathways to activate transcription remains poorly understood. Tumor necrosis factor alpha (TNFalpha) and all-trans retinoic acid (RA) induce monocytic differentiation of acute promyelocytic leukemia (APL) cells in a synergistic manner. We used the promoter of DIF2, a gene involved in monocytic differentiation, to model the mechanism underlying the cooperative induction of target genes by RA and TNFalpha. We show a functional RA response element in the DIF2 promoter, which is constitutively bound by PML/RARalpha in APL cells. RA stimulates release of corepressors and recruitment of chromatin modifying proteins and additional transcription factors to the promoter, but these changes cause only a modest induction of DIF2 mRNA. Co-stimulation with RA plus TNFalpha facilitates binding of NF-kappaB to the promoter, which is crucial for full induction of transcription. Furthermore, RA plus TNFalpha greatly enhanced the level of RNA Pol II phosphorylation on the DIF2 promoter, via synergistic recruitment of TFIIH. We propose that RA mediates remodeling of chromatin to facilitate binding of transcription factors, which cooperate to enhance Pol II phosphorylation, providing a mechanism whereby nuclear receptors interact with other signaling pathways on the level of transcription.

Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional "peak" in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important virulence factor on the surface of infected erythrocytes. Naturally acquired antibodies to PfEMP1 expressed by parasites causing severe malaria are suggested to be protective and of major interest for the development of a vaccine against severe disease. In this study, the PfEMP1 expressed by a parasite clone displaying a multiadhesive phenotype associated with severe malaria was well recognized by sera of malaria semi-immune children. The efficiency of the Duffy binding-like 1 alpha (DBL1 alpha) domain of this PfEMP1 was therefore, alone or in combination with two additional DBL1 alpha domains, evaluated as a potential vaccine candidate using both a rodent model and a primate model. Antibodies against the DBL1 alpha domain were generated by immunization with recombinant DBL1 alpha-Semliki Forest virus particles and recombinant protein and analyzed in vitro. The immunized animals were challenged in vivo with various parasite strains or clones. Immunization with the PfEMP1-DBL1 alpha domain abolished the PfEMP1-dependent sequestration of the homologous strain in immunized rats and substantially inhibited parasite adhesion in immunized monkeys. Protection against sequestration of heterologous parasite strains was also confirmed by direct or indirect challenge in the rat model. These results strongly support the use of the DBL1 alpha domain in the development of a vaccine targeting severe malaria.

Severe human malaria is attributable to an excessive sequestration of Plasmodium falciparum-infected and uninfected erythrocytes in vital organs. Strains of P. falciparum that form rosettes and employ heparan sulfate as a host receptor are associated with development of severe forms of malaria. Heparin, which is similar to heparan sulfate in that it is composed of the same building blocks, was previously used in the treatment of severe malaria, but it was discontinued due to the occurrence of serious side effects such as intracranial bleedings. Here we report to have depolymerized heparin by periodate treatment to generate novel glycans (dGAG) that lack anticoagulant-activity. The dGAGs disrupt rosettes, inhibit merozoite invasion of erythrocytes and endothelial binding of P. falciparum-infected erythrocytes in vitro, and reduce sequestration in in vivo models of severe malaria. An intravenous injection of dGAGs blocks up to 80% of infected erythrocytes from binding in the micro-vasculature of the rat and releases already sequestered parasites into circulation. P. falciparum-infected human erythrocytes that sequester in the non-human primate Macaca fascicularis were similarly found to be released in to the circulation upon a single injection of 500 mug of dGAG. We suggest dGAGs to be promising candidates for adjunct therapy in severe malaria.

The interactions of Plasmodium falciparum infected erythrocytes parasitized red blood cells (pRBC) with endothelial receptors and erythrocytes are mediated by multiple Duffy-binding like (DBL) and cysteine-rich interdomain region (CIDR) domains harboured in the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The success of a subunit vaccine based on PfEMP1 depends on its ability to elicit cross-reactive responses to a substantial number of PfEMP1 variants. We have here evaluated serological PfEMP1 cross-reactivity by immunizing rats with phylogenetically diverse recombinant NTS-DBL-1alpha/x fusion domains from the 3D7 genome parasite emulsified in Montanide ISA 720. Cross-reactivity was elicited to these diverse DBL-1alpha/x domains as measured by ELISA and by immunoblotting. Employing a novel in vivo model of human infected erythrocyte sequestration, immunized animals were challenged with the FCR3S1.2 clone and cross-protection in terms of reduction in lung sequestration amounting to approximately 50% was demonstrated. Our results suggest that immunization with phylogenetically distant DBL-1alpha/x variants, can elicit partial cross-protection to challenge with the parasites harbouring a distant variant. These observations have implications for the design of multi-component vaccines against P. falciparum malaria.

Radiotherapy is one of the mainstays of glioblastoma (GBM) treatment. This study aims to investigate and characterise differences in protein expression patterns in brain tumour tissue following radiotherapy, in order to gain a more detailed understanding of the biological effects. Rat BT4C glioma cells were implanted into the brain of two groups of 12 BDIX-rats. One group received radiotherapy (12 Gy single fraction). Protein expression in normal and tumour brain tissue, collected at four different time points after irradiation, were analysed using surface enhanced laser desorption/ionisation - time of flight - mass spectrometry (SELDI-TOF-MS). Mass spectrometric data were analysed by principal component analysis (PCA) and partial least squares (PLS). Using these multivariate projection methods we detected differences between tumours and normal tissue, radiation treatment-induced changes and temporal effects. 77 peaks whose intensity significantly changed after radiotherapy were discovered. The prompt changes in the protein expression following irradiation might help elucidate biological events induced by radiation. The combination of SELDI-TOF-MS with PCA and PLS seems to be well suited for studying these changes. In a further perspective these findings may prove to be useful in the development of new GBM treatment approaches.

A new approach is presented for predicting ligand binding to proteins using hierarchical partial-least-squares regression to latent structures (Hi-PLS). Models were based on information from the 2002 release of the PDBbind database containing (after in-house refinement) high-resolution X-ray crystallography and binding affinity (Kd or Ki) data for 612 protein-ligand complexes. The complexes were characterized by four different descriptor blocks: three-dimensional (3D) structural descriptors of the proteins, protein-ligand interactions according to the Validate scoring function, binding site surface areas, and ligand 2D and 3D descriptors. These descriptor blocks were used in Hi-PLS models, generated using both linear and nonlinear terms, to relate the characterizations to pKd/i. The results show that each of the four descriptor blocks contributed to the model, and the predictions of pKd/i of the internal test set gave a root-mean-square error of prediction (RMSEP) of 1.65. The data were further divided according to the structural classification of the proteins, and Hi-PLS models were constructed for the resulting subclasses. The models for the four subclasses differed considerably in terms of both their ability to predict pKd/i (with RMSEPs ranging from 0.8 to 1.56) and the descriptor block that had the strongest influence. The models were validated with an external test set of 174 complexes from the 2003 release of the PDBbind database. The overall results show that the presented Hi-PLS methodology could facilitate the difficult task of predicting binding affinity.

The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with 99mtechnetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria.

Background: All eukaryotic organisms need to distinguish each of their chromosomes. A few protein complexes have been described that recognise entire, specific chromosomes, for instance dosage compensation complexes and the recently discovered autosome-specific Painting of Fourth (POF) protein in Drosophila. However, no sequences have been found that are chromosome-specific and distributed over the entire length of the respective chromosome. Here, we present a new, unbiased, exhaustive computational method that was used to probe three Drosophila genomes for chromosome-specific sequences., Results: By combining genome annotations and cytological data with multivariate statistics related to three Drosophila genomes we found sequence signatures that distinguish Muller's F-elements (chromosome 4 in D. melanogaster) from all other chromosomes in Drosophila that are not attributable to differences in nucleotide composition, simple sequence repeats or repeated elements. Based on these signatures we identified complex motifs that are strongly overrepresented in the F-elements and found indications that the D. melanogaster motif may be involved in POF-binding to the F-element. In addition, the X-chromosomes of D. melanogaster and D. yakuba can be distinguished from the other chromosomes, albeit to a lesser extent. Surprisingly, the conservation of the F-element sequence signatures extends not only between species separated by approximately 55 Myr, but also linearly along the sequenced part of the F-elements., Conclusion: Our results suggest that chromosome-distinguishing features are not exclusive to the sex chromosomes, but are also present on at least one autosome (the F-element) in Drosophila.

GOLDsurfer is a java-based analysis and graphics program for three-dimensional plotting of linkage disequilibrium (LD). Simultaneous presentation of LD measures, including recombination rate estimates and disease association statistics, helps to clarify LD patterns and facilitates interpretations based on multiple indices of local genetic data.

Retinoids are vitamin A derivatives, which cause growth inhibition, differentiation and/or apoptosis in various cell types, including some breast cancer cells. In general, estrogen receptor (ER)-positive cells are retinoic acid (RA) sensitive, whereas ER-negative cells are resistant. In this report, we show that ER-negative MDA-MB-231 cells are strongly growth inhibited by retinoids in combination with a PKC inhibitor. While neither RA nor GF109203X (GF) has a significant growth inhibitory effect in these cells, RA+GF potently suppress proliferation. We found that RA+GF induce apoptosis, as shown by an increase in fragmented DNA, Annexin-V-positive cells and caspase-3 activation. Apoptosis was also induced by GF in combination with two synthetic retinoids. Expression of phosphorylated as well as total PKC was decreased by GF and this was potentiated by RA. In addition, treatment with GF caused a strong and sustained activation of ERK1/2 and p38-MAPK, as well as a weaker activation of JNK. Importantly, inhibition of ERK but not p38 or JNK suppressed apoptosis induced by RA+GF, indicating that activation of ERK is specifically required. In support of this novel finding, the ability of other PKC inhibitors to cause apoptosis in combination with RA correlates with ability to cause sustained activation of ERK.

The ability of retinoids to inhibit breast cancer cell growth correlates with estrogen receptor (ER) alpha status, as shown by the antiproliferative effects of retinoids in ERalpha-positive breast cancer cells and their use as chemopreventive agents in premenopausal women. The discovery of ERbeta, also present in breast cancer cells, has added a new level of complexity to this malignancy. To determine the retinoid response in ERbeta-expressing breast cancer cells, we used retroviral transduction of ERbeta in ER-negative MDA-MB-231 cells. Western blot and immunofluorescence confirmed expression and nuclear localization of ERbeta, whereas functionality was shown using an estrogen response element-containing reporter. A significant retinoic acid (RA)-mediated growth inhibition was observed in the transduced ERbeta-positive cells as shown by proliferation assays. Addition of estradiol, tamoxifen, or ICI 182,780 had no effect on cell growth and did not alter RA sensitivity. We observed that retinoids altered ERbeta-mediated transcriptional activity from an estrogen response element, which was confirmed by decreased expression of the pS2 gene, and from an activator protein response element. Conversely, the expression of ERbeta altered RA receptor (RAR) beta expression, resulting in greater induction of RARbeta gene expression on RA treatment, without altered expression of RARalpha. Our data provide evidence of transcriptional crosstalk between ERbeta and RAR in ERbeta-positive breast cancer cells that are growth inhibited by RA., (Copyright 2004 American Association for Cancer Research)

A family of parasite antigens known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is believed to play an important role in the binding of infected erythrocytes to host receptors in the micro-vasculature. Available data advocates the existence of a subset of very adhesive (rosetting, auto-agglutinating) and antigenic PfEMP1s implicated as virulence factors. Serum antibodies that disrupt rosettes are rarely found in children with severe malaria but are frequent in those with mild disease suggesting that they may be protective. Here we have developed a Semliki forest virus (SFV) vaccine construct with a recombinant gene (mini-var gene) encoding a mini-PfEMP1 (DBL1alpha-TM-ATS) obtained from a particularly antigenic and rosetting parasite (FCR3S1.2). The mini-PfEMP1 is presented to the host mimicking the location of the native molecule at the infected erythrocyte surface. Antibodies generated by a regimen of priming with SFV RNA particles and boosting with a recombinant protein recognize the infected erythrocyte surface (immuno-fluorescence/rosette-disruption) and prevent the sequestration of P. falciparum-infected erythrocytes in an in vivo model of severe malaria. The data prove the involvement of DBL1alpha in the adhesion of infected- and uninfected erythrocytes and the role of rosette-disruptive antibodies in preventing these cellular interactions. The work supports the use of DBL1alpha in a vaccine again severe malaria.

The Carlsson research group has developed a series of compounds capable of stabilising the dopamine system without inducing the deleterious hypodopaminergia that encumbers the currently used antipsychotic drugs. In the present study one of these dopaminergic stabilisers, ACR16, was tested in a mouse model for cognitive deficits of schizophrenia and autism. Since we believe that hypoglutamatergia is a key element in both schizophrenia and autism we used mice rendered hypoglutamatergic by treatment with the N-methyl-D-aspartate (NMDA) antagonist MK-801. MK-801 causes both hyperactivity and a behavioural primitivization. ACR16 attenuated the MK-801-induced hyperactivity and, in addition, caused a marked improvement of behavioural quality with a movement pattern approaching that of control animals. Since we believe that the impoverishment of the behavioural repertoire caused by MK-801 may correspond to the cognitive deficits seen in schizophrenia and autism, these results suggest that ACR16 may improve cognitive status in these disorders.

The objective to this retrospective study of 341 cases of primary carcinoma of vagina (PCV) diagnosed between 1956 and 1996 was to find whether epidemiological, clinical, and histopathological variables were related to the age at diagnosis of patients with PCV. The univariate statistical analysis showed that younger age at diagnosis significantly correlated with a history of cervical dysplasia, hysterectomy, gynecological infections, and tumors located in the upper part of the vagina, whereas older age at diagnosis significantly correlated with late menarche and exophytically growing tumors. In the multivariate regression analysis, the remaining independent predictors were a history of cervical dysplasia and age at menarche. Further, parity >/=4 as well as nulliparity, smoking, and unstable marital status were more common among patients with PCV than among those in the general Swedish female population. This study indicates that the etiology of vaginal carcinoma may be age related. In young patients, the disease seems to be etiologically related to cervical neoplasia and thus human papillomavirus (HPV) dependent. However, in the most common age group, the older patients, there might be another (probably non-HPV-related) etiology associated with hormonal factors and trauma to the vagina.

We have analyzed the abundance of mRNAs expressed from 11 nuclear genes in leaves of a free-growing aspen (Populus tremula) tree throughout the growing season. We used multivariate statistics to determine the influence of environmental factors (i.e. the weather before sampling) and developmental responses to seasonal changes at the mRNA level for each of these genes. The gene encoding a germin-like protein was only expressed early in the season, whereas the other tested genes were expressed throughout the season and showed mRNA variations on a day-to-day basis. For six of the genes, reliable models were found that described the mRNA level as a function of weather, but the leaf age was also important for all genes except one encoding an early light-inducible protein (which appeared to be regulated purely by environmental factors under these conditions). The results confirmed the importance of several environmental factors previously shown to regulate the genes, but we also detected a number of less obvious factors (such as the variation in weather parameters and the weather of the previous day) that correlated with the mRNA levels of individual genes. The study shows the power of multivariate statistical methods in analyzing gene regulation under field conditions.

Transcriptional cross-talk exists between the estrogen receptor (ERalpha) and retinoic acid receptor (RAR) pathways in human breast cancer cells. We have previously shown that re-expression of ERalpha in ER-negative cells stimulates the transcriptional and growth inhibitory effects of all-trans-retinoic acid (tRA) by a mechanism that is independent of the ER ligands estradiol and tamoxifen. In this study, we generated cell lines stably expressing ERalpha-deletion mutants to elucidate the mechanism whereby ERalpha modulates RAR transcriptional activity. Using RT-PCR and RNAse protection assays, we observed that expression of ERalpha suppresses basal expression of the RA-responsive gene RARbeta2, while allowing it to be strongly induced by tRA. Repression of basal RARbeta2 transcription was confirmed by transient expression of the reporter plasmid betaRE-tk-CAT, containing the RARbeta2 promoter. In the ERalpha-negative cells, on the other hand, transcription was only weakly induced by RA. We further determined that this effect of ERalpha on RARbeta induction required the N-terminal AF-1-containing region, including the DNA-binding domain, but was independent of the C-terminal ligand-binding domain. Consistent with these results, the ER agonist estradiol and the AF-2 antagonist 4-hydroxytamoxifen had no significant effect on betaRARE activity. Conversely, the full ER antagonist ICI 182,780, which blocks ERalpha AF-1 activity, was able to completely relieve repression of basal betaRARE activity. The effect of ERalpha is specific for RAR-mediated transcription and does not occur on promoters containing typical response elements for the Vitamin D or thyroid hormone receptors. Moreover, the cross-talk between ERalpha and RAR does not seem to be mediated by sequestration of a number of common co-regulators, suggesting a novel mechanism whereby the N-terminal region of ERalpha modulates the transcriptional activity of RAR.

All-trans-retinoic acid and 9-cis-retinoic acid have been reported to have inhibitory effects on pancreatic adenocarcinoma cells and we have shown that this is partly due to induction of apoptosis. In this study, the mechanisms whereby 9-cis-retinoic acid induces apoptosis in these cells were investigated. An involvement of the Bcl-2 family of proteins was shown, such that 9-cis-retinoic acid causes a decrease in the Bcl-2/Bax ratio. Overexpression of Bcl-2 also resulted in inhibition of apoptosis induced by 9-cis-retinoic acid. Furthermore, two broad-range caspase inhibitors blocked DNA fragmentation induced by 9-cis-retinoic acid, but had no effect on viability defined by mitochondrial activity. Using synthetic retinoids, which bind selectively to specific retinoic acid receptor subtypes, we further established that activation of retinoic acid receptor-gamma is essential for induction of apoptosis. Only pan-retinoic acid receptor and retinoic acid receptor-gamma selective agonists reduced viability and a cell line expressing very low levels of retinoic acid receptor-gamma is resistant to the effects of 9-cis-retinoic acid. A retinoic acid receptor-beta/gamma selective antagonist also suppressed the cytotoxic effects of 9-cis-retinoic acid in a dose-dependent manner. This study provides important insight into the mechanisms involved in suppression of pancreatic tumour cell growth by retinoids. Our results encourage further work evaluating the clinical use of receptor subtype selective retinoids in pancreatic carcinoma.

Introduction: Retinoids, which are derivatives of vitamin A, are important factors involved in the control of biologic functions such as cell growth and differentiation, development, and carcinogenesis. We have shown previously that the naturally occurring retinoids all-trans-retinoic acid (ATRA) and 9-cisretinoic acid (9cRA) induce growth inhibition followed by apoptosis in pancreatic adenocarcinoma cells in vitro., Aim: To evaluate the efficacy of retinoids in combination with the chemotherapeutic drugs gemcitabine and cisplatin., Methodology: In vitro growth inhibition and induction of apoptosis by different combinations of retinoids and cytotoxic drugs were studied by using the T3M-4 and BxPc-3 cell lines. For in vivo studies, T3M-4 cells were injected subcutaneously in nude mice., Results: Pre-treatment of pancreatic adenocarcinoma cells with ATRA or 9cRA before the addition of the drugs resulted in significant reduction in cell number compared with treatment with the drugs alone. Pre-treatment with 9cRA followed by gemcitabine or cisplatin alone also resulted in a strong increase in the percentage of cells undergoing programmed cell death, or apoptosis. Furthermore, there was an indication that the combination of ATRA and gemcitabine caused increased apoptosis in vivo., Conclusion: Our results clearly suggest the need for additional studies exploring the potential role of the combination of retinoids and gemcitabine in the management of pancreatic cancer.

The sequestration of Plasmodium falciparum-infected erythrocytes (pRBC) away from the peripheral circulation is a property of all field isolates. Here we have examined the pRBC of 111 fresh clinical isolates from children with malaria for a number of adhesive features in order to study their possible coexpression and association with severity of disease. A large number of adhesion assays were performed studying rosetting, giant rosetting, and binding to CD36, intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, thrombospondin, heparin, blood group A, and immunoglobulins. Suspension assays were performed at the actual parasitemia of the isolate, while all the static adhesion assays were carried out at an equal adjusted parasitemia. The ability to bind to multiple receptors, as well as the ability to form rosettes and giant rosettes, was found to be more frequent among isolates from children with severe versus mild malaria (P = 0.0015). Rosettes and giant rosettes were more frequent for children with severe malaria, and the cell aggregates were larger and tighter, than for those with mild disease (P = 0.0023). Binding of immunoglobulins (97% of isolates) and of heparin (81% of isolates) to infected erythrocytes was common, and binding to heparin and blood group A was associated with severity of disease (P = 0.011 and P = 0.031, respectively). These results support the idea that isolates that bind to multiple receptors are involved in the causation of severe malaria and that several receptor-ligand interactions work synergistically in bringing about severe disease.

Background: The purpose of this retrospective study is to evaluate the longterm prognosis for cervical stump cancer compared to matched controls with cancer in an intact uterus., Methods: From 1959 to 1987, 145 patients were treated for an infiltrating carcinoma of the cervical stump at Radiumhemmet representing 2.2% of all cervical cancers. Three control cases to each case were selected from the cohort of cervical carcinoma cases - matched to year of treatment, stage, histology and age (plus, minus 2 years). Actuarial survival was calculated for cases and controls. Survival differences were analyzed with the Kaplan-Meier technique. The age distribution for cases ranged between 36 and 84 years with a mean age of 60.6 years. The mean age for the control series is 9 years of age (range 35-86 years). Among the cases 87.6% were squamous cell carcinoma and 12.4% were adenocarcinomas. Treatment of carcinoma of the uterine stump at Radiumhemmet followed the same modality as was practised for ordinary cervical cancer cases i.e. two brachyradium applications with 3 weeks interval followed by external irradiation. The dose of irradiation from the intracavitary application given to the stump cancers was lower than that given to comparable cases of the common cervical cases., Results: No evidence was found of poorer longterm prognosis for radiologically treated squamous cell carcinoma of the uterine stump compared to that of the ordinary cervical carcinomas. Stump cancers of the adenocarcinoma type had a worse prognosis than adenocarcinomas in an intact uterus (p<0.07) and also compared with stump cancers of the squamous epithelial type (p=0.05). The complication rate was higher for the stump cancer cases compared with that for cervical cancers in intact uterus. The mean time interval from subtotal hysterectomy to the stump cancer diagnosis was 17.6 years with a range from 1 to 46 years., Conclusions: Recent discussions argue for a better sexual function after subtotal hysterectomy. Our study gave no convincing argument in terms of poorer prognosis for radiologically treated carcinoma of the uterine stump compared to that of the total cervical cancer series. It is thus necessary to weigh the possible gains with subtotal hysterectomy against the relatively low risk to fall victim of a stump cancer. Complications following surgery, as well as possible physiologic and sexual functions of the cervix, should be taken into account.

Objectives: To investigate and analyze the process from first recognition of symptom(s) to final diagnosis at operation in patients with ovarian epithelial cancer., Method: The medical records of 160 women with ovarian cancer were studied and traced back to the doctor first consulted, in order to obtain information on patient- and doctor-related delay., Results: Symptom duration was 12 weeks in patients with serous tumors and 7 weeks in patients with other histopathological classes; 56% were diagnosed within 4 weeks. Women in stages I and II were diagnosed faster than those in stages III and IV; 4% were diagnosed within 3 days because of an emergency operation; 30% were not diagnosed within 8 weeks., Conclusion: Diagnosis of ovarian cancer is difficult due to the vagueness of the symptoms which mislead both patients and doctors. Methods to enable earlier diagnosis must be sought.

A collaborative group of cancer registries and hospitals carried out a case-control study of tumours of the bladder in women who had previously been treated for ovarian cancer. A total of 63 cases of bladder tumours were identified, and 188 controls were selected matching for age, year of ovarian cancer diagnosis and survival time. Full details of the treatment for ovarian cancer were sought for both cases and for controls. The risk of bladder tumours was increased for patients who had been treated by radiotherapy alone (1.9; 95% confidence interval, 0.77-4.9), by chemotherapy alone (3.2; 0.97-10), and by chemotherapy and radiotherapy (5.2; 1.6-16), when comparison was made with patients treated only by surgery. Patients treated by chemotherapy were separated into 2 groups according to whether they had received cyclophosphamide. Among those who had, there was a clear increase in risk (approximately 4-fold) regardless of whether or not they had also received radiotherapy. For those who received only other drugs, risk was increased substantially among patients who had also been treated by radiation, as compared with patients treated by surgery alone, and those who had received radiotherapy only. Both melphalan and thiotepa were implicated as potential bladder carcinogens on the basis of these results. The estimated risk of bladder tumours due to cyclophosphamide was more than twice the risk following radiation to the bladder, and it appeared substantially earlier. For both agents, the risk continued to increase more than 10 years after treatment began.

Twenty-eight patients with a diagnosis of 'extramammary Paget's disease of the vulva' were referred to the Radiumhemmet, Karolinska Sjukhuset, Stockholm, during the period 1975-1990. A clinical and histopathologic retrospective review was undertaken. Six patients had associated malignancies (21.4%). The disease was considered primary invasive in three cases, whereas three patients later developed an invasive cancer. Surgery-local resection, hemivulvectomy or vulvectomy-was performed in 24 cases. Twelve patients, in which surgery was supposed to be radical with respect to free margins, had a significantly longer recurrence-free survival than 12 patients in which the surgical margins were dubious.

Objective: To investigate and analyze symptoms in patients with different types of ovarian cancer., Method: Records of 160 women with ovarian cancer were studied in detail following the patients from first consultation to operation and diagnosis at Sodersjukhuset, Stockholm., Result: No specific group of symptoms could be linked to type or stage of ovarian cancer. Gastrointestinal symptoms were more common in patients with class 1C tumors. Only 21% complained of gynecological symptoms. Women with class 1C cancer had significantly more advanced disease than those with 2C-5C cancer as 77% had a stage III-IV tumor compared with 40% of class 2C-5C patients., Conclusion: Diagnosis of ovarian cancer is difficult due to the multitude of symptoms often appearing late in the disease. The majority of women did not experience symptoms in the genital organs. Methods to encourage early consultation should be investigated.

The earlier optimistic predictions that invasive carcinoma of the uterine cervix could be totally eradicated by means of Pap screening have failed. Experiences from different countries give evidence, however, that a considerable reduction of incidence and mortality can be gained with this type of secondary prevention. Improved knowledge of the epidemiology of carcinoma of the uterine cervix and of its natural history could be anticipated to give a better basis for the planning of preventive measures. Maintenance of a high laboratory standard, a good technique for taking of smears, and improved communications between laboratories and the doctor or nurse taking the smear and the doctor or clinic performing the treatment and an adequate reaction to the report from the laboratory with adequate treatment of the precancerous stages is supposed to improve the effect of the screening programmes.

We have studied 15 women who were previously treated by irradiation for pelvic malignancies and who all developed symptoms of arterial occlusive disease of the iliac arteries and the distal aorta. Risk factors were evaluated in a case control study in which each patient was compared with three individually matched controls who were treated during the same period for the same type of cancer and who did not develop arterial occlusions. We conclude that smoking habits as well as treatment by irradiation are strong predictors of the development of atherosclerotic occlusive disease. Side effects of irradiation occur in the arteries more often in patients who have also radiation reactions in other organs--for example, the bowel and bladder. Irradiation acts in combination with other atherogenic factors and this should be kept in mind when radiation treatment is planned for patients with a high risk of developing atherosclerosis.

Twenty-eight cases of endometrial stromal sarcoma treated from 1936 to 1981 at Radiumhemmet were reviewed. Histopathologically, 16 were classified as high-grade stromal sarcomas and 12 were of a low grade. A significant relation between mitotic count and relative survival within 10 years of the diagnosis was found (p less than 0.05) showing a more favorable outcome for patients with low counts compared with patients with higher counts. Primary treatment included surgery and radiotherapy in 21 cases. Two advanced cases were only irradiated and five cases were treated with surgery alone. Adjuvant radiotherapy seems to be of benefit in high-grade tumors, demonstrated in a 5-year survival rate of 73%, which exceeds most reported results. Five out of seven women dying of low-grade tumors were diagnosed in an advanced stage.

Of 788 women treated with ionizing irradiation for benign bleeding disorders (metropathia) 308 (39%) died of cardiovascular disease. In a control group, consisting of 1219 women with metropathia who were not irradiated, 257 (21%) cardiovascular deaths occurred. Although the risk of cardiovascular death in the two studied cohorts compared with national death rates was only 0.92 (lower limit 0.82; upper limit 1.03) and 0.88 (lower limit 0.78; upper limit 0.99) respectively, it was found that women irradiated before the menopause (age 50 years) run a higher risk of cardiovascular death than the controls in the same age group and those irradiated after the menopause.

An international collaborative group of cancer registries and hospitals identified 114 cases of leukemia following ovarian cancer. We investigated the possible etiologic role of chemotherapy, radiotherapy, and other factors, using a case-control study design, with three controls matched to each case of leukemia. Chemotherapy alone was associated with a relative risk of 12 (95 percent confidence interval, 4.4 to 32), as compared with surgery alone, and patients treated with both chemotherapy and radiotherapy had a relative risk of 10 (95 percent confidence interval, 3.4 to 28). Radiotherapy alone did not produce a significant increase in risk as compared with surgery alone. The risk of leukemia was greatest four or five years after chemotherapy began, and the risk was elevated for at least eight years after the cessation of chemotherapy. The drugs cyclophosphamide, chlorambucil, melphalan, thiotepa, and treosulfan were independently associated with significantly increased risks of leukemia, as was the combination of doxorubicin hydrochloride and cisplatin. Chlorambucil and melphalan were the most leukemogenic drugs, followed by thiotepa; cyclophosphamide and treosulfan were the weakest leukemogens, and the effect per gram was substantially lower at high doses than at lower doses. The extent to which the relative risks of leukemia are offset by differences in chemotherapeutic effectiveness is not known.

One hundred and forty-three patients with uterine leiomyosarcomas treated at Radiumhemmet from 1936 through 1981 were reviewed. Clinical and histopathological findings were analysed by bivariate and multivariate analyses to assign prognostic factors. Mitotic count, menopausal status and stage showed to be significant predictors of survival. The series was divided into two periods of time and a significantly higher survival rate in the second period was noted for patients with stage I disease. Thirty-seven patients with tumors showing less than 10 mitotic figures per 10 high power fields had a 5-year survival rate of 65% compared with 17% for 106 patients with 10 or more mitotic figures per 10 high power field. No significant predictor of a bad outcome for these low mitotic count tumors was found.

A cancer registry cohort of 16,704 cases of invasive carcinoma of the uterine cervix and 56,116 cases of in situ carcinomas of the uterine cervix was followed up and second new primary cancers were recorded. The invasive carcinomas contributed 127,118 woman-years at risk and the in situ carcinomas contributed 453,362 woman-years at risk. The main treatment for the invasive carcinomas had been radiotherapy and for the in situ carcinomas, conization and other types of surgical intervention. 767 new primaries occurred after treatment of invasive carcinoma of the uterine cervix, compared with 644.5 expected. O/E is 1.19. After the in situ carcinomas, 1,421 malignant tumors were observed, vs. 1,188.0 expected (O/E 1.19). If, however, cases of invasive carcinoma of the uterine cervix after in situ carcinomas are excluded, the ratio observed/expected is 1.10. For some sites the increased observed/expected ratios were found after both invasive and in situ carcinomas, which speaks for some common carcinogenic effect other than irradiation (for instance, in bronchus and trachea, pharynx, nose, sinus and larynx, but also in rectum, urinary bladder, other female genital organs, pancreas, lymphosarcoma, as well as acute and non-lymphatic leukemia). A lower risk than expected--after both in situ and invasive carcinoma of the uterine cervix--is observed for breast cancer, cancer of the corpus uteri and for multiple myeloma. However, analyses based on time since treatment provide evidence of a carcinogenic effect of irradiation, especially in intensively irradiated organs such as bladder, rectum, corpus uteri and ovary, and also for acute and non-lymphatic leukemia.

One hundred and seven cases of malignant tumours occurred among 788 women irradiated 1912 to 1977 for metropathia. One hundred and seventy-three women out of 1,219 referred for the same diagnosis and not irradiated developed a malignant tumour. The tumours were diagnosed between 1958 and 1982. The relative risk of malignant tumours among the irradiated women was 1.22 and among the non-irradiated 1.09 compared to cancer registry data. A statistically non-significant increase of the relative risk was found in the irradiated patients for tumours of the rectum (1.58), colon (1.46), and the nervous system (1.67). A decreased overall relative risk was seen for cancer of the breast (0.92) after irradiation, but women treated at the age of 50 or more had an increased risk (2.08). The relative cancer risk of the heavily irradiated sites was not increased during the first 20 years after irradiation, but a statistically significant increase of the risk was seen after 30 years.

During the five-year period 1964-68 96 733 births were registered in the 28 hospitals equipped with maternity facilities in the Uppsala hospital region. Of these babies, 1 636 were born in 818 twin deliveries. Data on gestational age, sex, weight and length at birth, birth order, hospital type, congenital malformations and perinatal mortality are analysed. Altogether 17.3 per 1 000 of the children born during this period were born in multiple births. The perinatal mortality for the twin babies was 64 per 1 000 born, with the mortality higher in the less specialized hospitals than the others. Twin no. 1 suffered perinatal death in 67 cases per 1 000 and twin no. 2 in 60 cases per 1 000. For twins of primiparae the losses were 92 per 1 000 children and for twins born to multiparae 51 per 1 000. Altogether 72 per 1 000 male twins died perinatally compared to 52 per 1 000 female twins. The most heavy losses occurred among the low-weight premature twins and in these cases both twins often suffered perinatal death.

A new sampling method for uterine cervical cancer detection is described. In this method, sampling of cytologic material is done by using a pulse wash instrument. Liquid jets with a diameter of 0.2 mm at a speed of 20 m/s create a successful rinsing effect of cervical epithelial cells due to the high kinetic energy produced. Because cells are suspended in the flushing liquid it is possible to collect material for additional cytochemical, immunocytochemical, and microbiologic diagnostic techniques in addition to a conventional smear technique. Compared to a conventional Papanicolaou smear technique performed in 75 women at two cervical atypia clinics at the Karolinska Hospital, the pulse wash technique is suggested to result in a more representative cellular sample, thus offering a method to decrease false negative diagnoses in uterine cervical cancer detection.

Over the period 1923--72 a total of 305 patients with granulosa-cell, theca-cell tumor and mixed tumor were seen at Radiumhemmet. The commonest symptom was abnormal uterine bleeding. Twenty-three per cent of married women were nulliparous. The menarche seems to have occurred earlier in this group than in the general population. Eight of the granulosa-cell tumor group gave birth to 12 children after treatment for the tumor. Nineteen women with granulosa-cell tumor and 4 with thecoma had received radiotherapy earlier in life for benign lesions. The risk of endometrial cancer was approximately 10 times greater for the women with granulosa- or theca-cell tumor than for the general population. Two hundred and fifty-two patients were given both surgical treatment and radiotherapy. Fifty-three received only surgery (37 granulosa-cell and 16 theca-cell tumors). None of the thecoma patients but 21 per cent of those with granulosa-cell tumor died from their disease. The 5-year survival for the latter group, all stages, was 85 per cent.

Development of in-situ or invasive carcinoma of the uterine cervix was studied among 930,127 women reported to a Swedish Central Screening Register through a record linkage between that Register and the National Cancer Registry. The relative protection against cervical cancer after one negative smear, calculated on incidence level prior to screening, is 14.5 for the first year and drops to 3.4 five years after the screening. The relative protection after two or more negative smears is considerably higher for the first three years but for the years following drops to the same level as after one negative smear.

To evaluate oral contraceptive (OC) use as a possible cause of the changed ratio between adenocarcinoma and squamous cell carcinoma of the uterine cervix a case-control study was performed. The OC use among 23 women with adenocarcinoma of the uterine cervix was compared with that of a matched group of 46 women with squamous cell carcinoma. No differences in percentage of OC use, duration of such use or period of OC use in relation to diagnosis could be demonstrated between the two groups compared.

The efficacy of a new sampling technique performed for early detection of cervical carcinoma is compared with Pap smears with the swab-and-wooden spatula technique in 312 women. In this new method, sampling of cytologic material is achieved by using a pulse wash instrument described in a previous article. Cells are rinsed and detached by liquid jets of 0.2 mm in diameter which are produced by a spray nozzle connected with a pressure hose to a high pressure pump. The liquid molecules pass through the spray nozzle at a speed of 20 m/s thus creating a successful rinsing effect on cervical epithelium due to high kinetic energy. Rinsed cells are mixed with the small amount of the flushing liquid. The suspension of cells and liquid accumulated in the speculum is then transported to a small container by a suction pump. The results of this work suggest that the pulse wash technique gives a more representative cell sample than the Pap smear sampling technique, thus offering a simple method to decrease false negative diagnoses in the detection of carcinoma of the uterine cervix. Samples by the new technique give an abundance of cells for slide preparation for cytodiagnostic techniques as well as for additional cytochemical, immunocytochemical and microbiologic diagnostic techniques.

Papanicolaou screening for cancer of the uterine cervix was introduced in Sweden in the late 1950's. Screening programmes covering the age groups 30-49 years were organized in various countries between 1965 and 1973. The approximate number of smears rose from 100 000 in 1960 to one million in 1970, in a female population of four million. Almost 60 000 cases of in situ carcinoma and 17 100 invasive carcinomas of the uterine cervix were registered in Sweden between 1958 and 1980. The age-standardized incidence of invasive carcinoma fell in this period by about 40%. Within the screened cohorts and age groups, the incidence was reduced by two-thirds and there was a parallel fall in mortality from the disease. At least part of these reductions seemed to be explained by the intensity of screening.

Seventy-four patients with carcinoma in situ (CIS) of the vulva were followed over a 10-year period. The mean age was 52.8 years (range 21 to 90 years). Pruritus was the most common symptom. Forty per cent of the women were asymptomatic. Twenty-three per cent had a history of carcinoma in situ of the uterine cervix. Single lesions of CIS were found in the perineal area in 22 per cent, in the labia major and minor in 23 per cent, and in the clitoris in 7 per cent. Forty-two per cent of the patients had CIS in two or more of those areas. Seven of 74 patients (10%) had previously been irradiated for pelvic malignant tumours. Three women developed invasive squamous cell carcinoma of the vulva, two of them were previously irradiated. All 74 patients were treated surgically in different ways. Out of 38 women with histologically free margins of surgery, 6 had recurrence of dysplasia or CIS.