Conserved peptide sequences bind to actin and enolase on the surface of Plasmodium berghei ookinetes.

The description of Plasmodium ookinete surface proteins and their participation in the complex process of mosquito midgut invasion is still incomplete. In this study, using phage display, a consensus peptide sequence (PWWP) was identified in phages that bound to the Plasmodium berghei ookinete surface and, in selected phages, bound to actin and enolase in overlay assays with ookinete protein extracts. Actin was localized on the surface of fresh live ookinetes by immunofluorescence and electron microscopy using specific antibodies. The overall results indicated that enolase and actin can be located on the surface of ookinetes, and suggest that they could participate in Plasmodium invasion of the mosquito midgut.

The chaperone micronemal protein Hsp70-1 from Plasmodium berghei ookinetes is shed during gliding on solid surface sustrata.

Plasmodium the causative agent of malaria is a member of the phylum Apicomplexa, where all invasive forms have a substrate-dependent motility called gliding, key to malaria transmission. Gliding allows parasite host-cell recognition, binding, cell entry and trespassing the cytoplasm. In this process Plasmodium releases molecules from micronemes and the cell surface that are deposited on trails left behind on the substratum as the parasite progresses. Previously we identified the heat shock protein 70-1 (HSP 70-1) on the surface and micronemes of P. berghei ookinetes, the parasite form that invades the mosquito midgut. To investigate if this protein is shed of from the parasite during invasion, we searched HSP 70-1 in gliding trails deposited on a solid surface by P. berghei ookinetes.

Prostaglandin E2 modulates the expression of antimicrobial peptides in the fat body and midgut of Anopheles albimanus.

Prostaglandins (PGs) participate in the regulation of vertebrate and in at least six insect orders' immune responses. We identified PGE2 in midgut, fat body, Malpighian tubules, and ovarioles of Anopheles albimanus (Aa) mosquitoes. Our data indicate that PGE2 synthesis in cultured midguts responds to the presence of two bacterial species, Micrococcus luteus and Klebsiella pneumoniae. The production of mRNA coding for antimicrobial peptides Aa-Attacin, Aa-Cecropin, and Aa-Gambicin was observed in cultured fat bodies and midguts. The production of these messengers was reduced in the presence of dexamethasone, and this effect was reversed by arachidonic acid. Adding PGE2 to cultures resulted in increased Aa-cecropin mRNA and decreased Aa-attacin and Aa-gambicin mRNAs.

[Symptomatic hepatic arterioportal shunt resolved through conservative treatment]. / Fístula arterioportal sintomática resuelta mediante tratamiento conservador.

Arterioportal shunt in the liver is a rare vascular disorder that may be due to congenital vascular malformation (hereditary hemorrhagic telangiectasia), trauma, iatrogenic causes (after a hepatic biopsy) or neoplasm. Initial treatment consists of transcatheter arterial embolization with different kinds of materials. We present the case of a 64-year-old woman with signs of portal hypertension and severe diarrhea. Doppler ultrasonography, computed tomography and angiography revealed arterioportal fistulae between the hepatic artery and right portal vein. Transcatheter arterial embolization with n-butyl-2-cyanoacrylate surgical glue (Glubran) was successfully performed. After 2 years of follow-up, the patient remains asymptomatic. Transcatheter arterial embolization with Glubran should be considered as a therapeutic option in arterioportal shunts and could be a definitive therapy.

IL-11 directly stimulates murine and human erythroid burst formation in semisolid cultures.

The effect of interleukin-11 (IL-11) on cultures of bone marrow cells was investigated. We found that IL-11 increased, in a dose-dependent manner, the number of bursts in the presence of Epo in murine or human cells cultures. This effect was also observed in cultures of murine cells established in serum-free conditions as well as in cultures of CD34+ enriched human cells in serum-containing (but not serum-free) cultures. A linear relationship between the number of bursts and the plated cell number was observed with murine bone marrow cells or non-adherent mononuclear cells (NA-MNC) human bone marrow cells. Moreover, the effect of IL-11 was not abrogated when either anti-stem cell factor-receptor (anti-SCF-R), anti-IL-3, or anti-granulocyte/macrophage colony-stimulating factor (GM-CSF), three cytokines known to greatly synergize with IL-11, was added to cultures. These results lead us to conclude that IL-11 directly stimulates the proliferation of murine and human burst-forming units-erythroid (BFU-E).

Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom.

A novel peptide, scorpine, was isolated from the venom of the scorpion Pandinus imperator, with anti-bacterial activity and a potent inhibitory effect on the ookinete (ED(50) 0.7 microM) and gamete (ED(50) 10 microM) stages of Plasmodium berghei development. It has 75 amino acids, three disulfide bridges with a molecular mass of 8350 Da. Scorpine has a unique amino acid sequence, similar only to some cecropins in its N-terminal segment and to some defensins in its C-terminal region. Its gene was cloned from a cDNA library.

Two novel mutations in EGF-like domains of human factor IX dramatically impair intracellular processing and secretion.

We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.

The P-selectin cytoplasmic domain directs the cellular storage of a recombinant chimeric factor IX.

Hemophilia B was recognized as a good candidate for gene therapy. Several strategies have been attempted and gave promising results in hemophilic animals but failed to achieve corrective levels in humans. To overcome this inconvenience we aimed to generate intracellular pools of factor (F)IX in cells that are implicated in the hemostatic response, e.g. endothelial cells and platelets. Upon stimulation, these cells release their granule content, which in this case would result in an increase in local FIX concentration, and could locally produce an effective hemostasis. In an attempt to produce an intracellular pool of releasable coagulation FIX, the cytoplasmic domain of the P-selectin (pselCT) molecule was fused to the carboxy-terminal extremity of the human FIX protein. The properties of this chimeric molecule (FIX-pselCT) were studied in AtT20, a cell line which possesses storage granules. As previously shown for transmembrane molecules but not for a soluble protein such as FIX, the pselCT fragment induces the storage of FIX-pselCT. The coagulant activity of FIX-pselCT was not affected by the addition of the pselCT tail. The treatment of AtT20 cells with different inhibitors revealed that FIX-pselCT was not submitted to intracellular degradation and that the half-life of the chimeric molecule was at least two times longer than that of FIX-WT. An immunoelectron microscopic analysis demonstrated a specific localization of FIX-pselCT within the ACTH-containing granules. Cell stimulation using Phorbol Myristrate Acetate (PMA), ionophore A-23187 or 8-Br-cAMP induced efficient release of an active FIX-pselCT. These data demonstrate that the addition of the cytoplasmic domain of P-selectin to FIX modifies the cellular fate of the FIX molecule by directing the recombinant protein toward regulated-secretory granules without altering its coagulant activity.

A factor VIII minigene comprising the truncated intron I of factor IX highly improves the in vitro production of factor VIII.

The biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.

The three in-frame ATG, clustered in the translation initiation sequence of human factor IX gene, are required for an optimal protein production.

Three in-frame potential methionine codons have been identified in human factor IX gene and are clustered at amino acids -46, -41 and -39. In view of initiating a gene therapy approach, human factor IX production has been evaluated after modifications of these first three in-frame translation start sites. To characterize the most efficient translation initiation context, five factor IX cDNA expression vectors directed by CMV promoter-enhancer were generated. These vectors contained different starting site combinations including one, two or three ATG. A quantitative analysis of factor IX production in stably transfected CHO cells and in a rabbit reticulocyte lysate cell free system revealed the ability of all single site to generate fully active factor IX. However, the factor IX production level increased with the ATG number and the wild type (WT) cDNA bearing the 3 ATG induced the highest protein production. A truncated intron I of factor IX, previously suggested of having an expression-augmenting activity, was also placed in the WT factor IX cDNA. In stably transfected CHO cells, a 8-fold increase in protein production was measured. These results show that at least in vitro, the presence of the three ATG seems to be crucial for a maximal factor IX production. The data also suggest that both the three ATG and the truncated intron I are required for an optimal factor IX production in a perspective of a human gene therapy of haemophilia.

Expression of coagulation factor IX in a haematopoietic cell line.

We have developed a gene therapy project for haemophilia B which aims to express factor IX (FIX) in haematopoietic lineage. Haematopoietic stem cells and subsequent megakaryocyte-derived cells represent the target cells of this approach. Our speculation is that platelets can deliver the coagulation factor at the site of injury, and subsequently correct the haemostasis defect. In order to direct FIX expression in cells from the megakaryocytic lineage, we designed a FIX cassette where the FIX cDNA was placed under the control of the tissue-specific glycoprotein IIb (GPIIb) promoter. In stably transfected HEL cells, FIX production was higher when driven by the GPIIb promoter compared to the CMV promoter. Using a cassette containing both the GPIIb promoter and a truncated FIX intron 1, FIX synthesis was dramatically increased in HEL cells. Northern blot analysis demonstrated an increase in FIX mRNA amounts, which paralleled with an increase of FIX antigen in the culture supernatants. Using a one-stage clotting assay and an activation by FXIa and FVIIa/TF, the HEL-derived recombinant FIX was shown to be a biologically active protein. This recombinant protein exhibited a 60-kDa molecular mass and was more heterogeneous than plasma immunopurified FIX (Mononine). The molecular mass difference could be partly explained by a different glycosylation pattern. The GPIIb promoter appears therefore to be a very attractive sequence to specifically direct FIX production in the megakaryocytic compartment of hematopoietic cells. These data also demonstrate that hematopoietic cells may represent potential target cells in an approach to gene therapy of haemophilia B.

The environment, remote sensing, and malaria control.

Results of studies in California, Mexico and Belize demonstrate the value of remote sensing technology for studying vector-borne diseases. These studies have also shown that it is necessary to fully define the environmental factors associated with the presence of vectors and disease transmission, and to be able to detect these environmental factors with image data. These studies, and other published reports, are demonstrating many potential uses of remotely sensed data in managing and targeting vector and disease control measures.

Twice a cesarean, always a cesarean?

The cesarean delivery rate has quadrupled during the past two decades, resulting in considerable attention focused on alternatives to cesarean birth. One option, vaginal birth after one previous cesarean, has come to be recognized as an acceptable alternative to routine elective repeat cesarean delivery. The purpose of this report was to evaluate whether women with two previous cesareans can safely undergo a trial of labor. Between July 1, 1982 and June 30, 1986, data were collected prospectively on all women with previous cesareans. Those with a known classical incision or a medical or obstetric contraindication to a trial of labor were excluded from an attempted vaginal delivery. During this period, 67,784 patients were delivered, of whom 6250 (9.2%) had had a previous cesarean. Of the 6250 previous-cesarean patients, 1088 (17.4%) had had two previous cesareans; of these, 501 (46%) underwent a trial of labor and 346 (69%) delivered vaginally. Whereas the overall rate of uterine dehiscence was 3%, the rate in those women who attempted a vaginal delivery was 1.8%, versus 4.6% in those who did not. Overall, oxytocin was used in 284 (57%) and was associated with a dehiscence rate of 2.1%, versus 1.4% in the no-oxytocin group. Successful vaginal delivery was related significantly to the use of oxytocin and to a previous vaginal delivery. Trial of labor in patients with two previous cesareans appears to be a reasonable consideration.

Acquired transmission-blocking immunity to Plasmodium vivax in a population of southern coastal Mexico.

Naturally acquired transmission-blocking immunity to Plasmodium vivax was studied in three groups of patients from the southern coast of Mexico: primary cases (Group A, 61% of the study population), secondary cases with the prior infection seven or more months earlier (Group B, 23%), and secondary cases with the previous malaria experience within six months of the present study (Group C, 16%). Anopheles albimanus mosquitoes were fed with patients' infected blood cells in the presence of autologous or control serum, with or without heat-inactivation. Patients from all three groups had transmission-blocking immunity, although the quality and quantity of this blocking activity was significantly higher in the two secondary patient groups (B and C). Only primary malaria cases produced transmission-enhancing activity (23% of the cases), which was dependent on heat-labile serum components. The levels of patient group transmission-blocking immunity and mosquito infectivity were used to calculate the probabilities of a mosquito becoming infective after taking a blood meal from a P. vivax-infected patient from any one of the three groups. This probability was 0.025, with Group A patients providing the major source of these infections (92% risk from Group A and 4% risk for Groups B and C).

Effect of semiannual treatments of ivermectin on the prevalence and intensity of Onchocerca volvulus skin infection, ocular lesions, and infectivity of Simulium ochraceum populations in southern Mexico.

The effect of semiannual ivermectin treatment along with nodulectomy on filarial transmission levels were estimated during the three dry seasons of 1991-1993 in a hyperendemic village in southern Mexico. Parasitologic and ophthalmologic examinations were carried out every six months until five drug treatments were completed. Ivermectin mass treatment with a coverage of approximately 80% had a significant impact (P < 0.05) on the prevalence of skin infection and the mean microfilarial skin density (CMFL), which were reduced 38% and 89%, respectively. A gradual and significant (P < 0.05) decrease in the mean microfilariae number in the anterior chamber of the eye and in corneal opacities was also observed as the CMFL was reduced. After three treatments, these were reduced 84% and 69%, respectively. However, after two years of continuous intervention, no significant differences (P > 0.05) were observed in either the daily mean infective biting density and the daily mean transmission potential. This was probably due to the remaining microfilarial load provided by the untreated resident population and migrant groups. On the whole, our results confirm both the efficacy of ivermectin to alleviate the clinical manifestations of the disease and its minimal impact on Onchocerca volvulus transmission, and indicate the need both to achieve higher levels of drug coverage and to incorporate other measures to stop transmission until a macrofilaricide drug is found.

Serologic and genetic characterization of Plasmodium vivax from whole blood-impregnated filter paper discs.

The presence in the New World of a variant strain of Plasmodium vivax (VK247) containing a unique circumsporozoite (CS) repeat domain was determined by the detection of antibodies to the variant CS protein and by genetic analysis of the CS gene from field isolates. Whole blood specimens were collected on filter paper from patients infected with P. vivax in Mexico and Peru. Plasmodium vivax DNA was eluted from filter paper samples and the CS gene was amplified by the polymerase chain reaction (PCR) and analyzed for the presence of VK247 or VK210 DNA by oligoprobe hybridization. Sera eluted from a companion filter paper sample were screened for antibodies reactive with the predominant and variant repeat peptides by enzyme-linked immunosorbent assays (ELISA) and with sporozoites by the immunofluorescent antibody (IFA) test. All 24 patients were positive by PCR and oligoprobe hybridization for either VK210 (16 of 24), VK247 (3 of 24), or both (5 of 24). Mixed infections were common (5 of 7) in Peru, but were not observed in the Mexican isolates (0 of 17). All three VK247 infections from Mexico occurred in residents of the foothills above Tapachula (P = 0.02). Of patients with smear-positive P. vivax infection, 42% (10 of 24) had detectable antibodies eluted from dried blood dots that were reactive with the CS protein by IFA or ELISA. These findings establish the widespread distribution of the P. vivax variant CS protein in the New World and indicate that dried blood filter paper samples represent a valuable source of material for the serologic and molecular analysis of plasmodial infections.

Assessment of a remote sensing-based model for predicting malaria transmission risk in villages of Chiapas, Mexico.

A blind test of two remote sensing-based models for predicting adult populations of Anopheles albimanus in villages, an indicator of malaria transmission risk, was conducted in southern Chiapas, Mexico. One model was developed using a discriminant analysis approach, while the other was based on regression analysis. The models were developed in 1992 for an area around Tapachula, Chiapas, using Landsat Thematic Mapper (TM) satellite data and geographic information system functions. Using two remotely sensed landscape elements, the discriminant model was able to successfully distinguish between villages with high and low An. albimanus abundance with an overall accuracy of 90%. To test the predictive capability of the models, multitemporal TM data were used to generate a landscape map of the Huixtla area, northwest of Tapachula, where the models were used to predict risk for 40 villages. The resulting predictions were not disclosed until the end of the test. Independently, An. albimanus abundance data were collected in the 40 randomly selected villages for which the predictions had been made. These data were subsequently used to assess the models' accuracies. The discriminant model accurately predicted 79% of the high-abundance villages and 50% of the low-abundance villages, for an overall accuracy of 70%. The regression model correctly identified seven of the 10 villages with the highest mosquito abundance. This test demonstrated that remote sensing-based models generated for one area can be used successfully in another, comparable area.

Antigenic characterization of Plasmodium vivax with monoclonal antibodies.

Monoclonal antibodies were produced against Plasmodium vivax obtained from patients living in southeastern Mexico, where P. vivax malaria is endemic. Nine hybridomas specific for this parasite were obtained. By an indirect immunofluorescence assay, seven antibodies were found to react with epitopes present in the cytoplasm of the infected erythrocyte and two with the parasite itself. By immunoblotting, five monoclonal antibodies reacted with a 17-kD protein band, three with an 85-kD band, and two with one of 45 kD. By immunogold electron microscopy, two antibodies that reacted with the cytoplasm of infected erythrocytes by immunofluorescence also labeled cytoplasmic clefts, and one, in addition, recognized caveola-vesicle complexes and the parasite matrix. These results demonstrate the value of monoclonal antibodies in identifying P. vivax antigens and disclosing their subcellular distribution.

Antibody response to Plasmodium vivax antigens in human malaria.

The present study investigated the serum antibody response to parasite antigens involved in human Plasmodium vivax malaria. Analysis was performed by protein immunoblotting of a pool of P. vivax preparations obtained from blood of patients from Tapachula, Chiapas (southeastern Mexico), and sera from local malarious patients. Patient sera recognized 19 P. vivax antigens with molecular sizes between 17 and 170 kD. The most frequently recognized antigens were proteins of 19, 31, 43, 72, 93, and 97 kD. These proteins could be useful in diagnostic methods and possibly relevant in vaccine design.

Different prevalences of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico.

The geographic distribution of Plasmodium vivax circumsporozoite protein phenotypes from patient blood used to infect colonized Anopheles albimanus and An. pseudopunctipennis was investigated in southern Mexico. Parasite phenotype types were determined in blood samples by a polymerase chain reaction and oligoprobe hybridization or by immunofluorescent assay of sporozoites. The proportion of infected mosquitoes and the number of oocysts per mosquito confirmed previous in vitro observations indicating that An. albimanus is more susceptible to VK210 and that An. pseudopunctipennis is more susceptible to VK247. All patients living on the coast were infected with VK210 and most patients living above 170 meters above sea level had VK247. Both phenotypes infected patients from intermediate altitudes. These results concur with the distribution of the anophelines, indicating that An. albimanus is the main vector of the phenotype VK210, but that An. pseudopunctipennis transmits both phenotypes. These conditions have direct implications on parasite transmission rates and malaria epidemiology in Mexico.