The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection.

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

Caracterização da anemia em cadelas com piometra

A piometra é uma condição mórbida caracterizada pela inflamação do útero com acúmulo de exsudatos, resultante de ações hormonais e geralmente associada à presença de bactérias no lúmen uterino. A anemia é a alteração hematológica mais frequentemente observada em cadelas com piometra e está associada à cronicidade da doença, diminuição da eritropoiese, devido ao efeito toxêmico na medula óssea, diminuição da disponibilidade de ferro ou perda de sangue para o útero. Adicionalmente, o efeito das toxinas bacterianas e os radicais livres gerados pelo metabolismo oxidativo dos neutrófilos podem resultar na modificação da estrutura antigênica da membrana do eritrócito, permitindo a ligação de imunoglobulinas em sua superfície e acelerando a destruição eritrocitária. Essa hipótese pode ser comprovada pela detecção de imunocomplexos na superfície eritrocitária de cadelas com piometra. O diagnóstico de piometra foi estabelecido em 33 cadelas atendidas no Serviço de Obstetrícia/Ginecologia do Hospital Veterinário da Universidade de São Paulo com base na anamnese, exame físico e exames subsidiários (ultrassonografia, hemograma e concentrações séricas de ureia e creatinina). As amostras sanguíneas foram coletadas em dois momentos. A primeira anterior a ovariosalpingohisterectomia (OSH) e a segunda, sete a dez dias após a OSH. A quantificação de hemácias com deposição de imunocomplexos IgG e IgM foi realizada utilizando-se anticorpos anti-IgG e anti-IgM (Bethyl®Laboratories) conjugadas a fluoresceína de isotiocianato (FITC), e a leitura realizada com citômetro de fluxo (FACS Calibur; Becton, Dickinson and Company© 2007 BD), sendo os resultados expressos em percentual de hemácias marcadas. Foram utilizados o Teste de Shapiro-Wilk para a avaliação da distribuição de dados e a comparação entre os grupos controle, pré e pós-OSH foi realizada valendo-se do Teste t ou Teste t pareado e Correlação de Pearson, e do Teste U de Mann-Whitney e Correlação de Spearman, para as variáveis com distribuição normal e não-normal, respectivamente. O valor de alfa estipulado foi de 0,05. Analisando os valores hematológicos de cada um dos cães incluídos no estudo, observa-se que 19 (57,6%) apresentavam anemia normocítica normocrômica não regenerativa no momento pré-OSH e cinco (15,2%) no momento pós-OSH. Em cães do grupo controle foram observadas 0,14 - 0,77% (0,43±0,18%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (0,68±0,29%) para anticorpos anti-IgM FITC. Já nos cães com piometra, foram encontradas 0,14 - 4,19% (0,96±0,86%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (1,37±1,71%) com anticorpos anti-IgM FITC, antecedendo a OSH. No momento pós-OSH observou-se 0,18 - 16,2% (2,77±3,67%) de hemácias marcadas para anticorpos anti-IgG FITC e 0,15 - 19,8% (4,01±4,46%) para anticorpos anti-IgM FITC. O percentual de hemácias marcadas com anticorpos anti-IgG FITC diferiu entre os grupos controle e piometra, pré-OSH (p<0,001) e pós-OSH (p<0,001). Em relação a anticorpos anti-IgM FITC, não foram observadas diferenças entre os grupos controle e pré-OSH (p=0,09), porém, após a OSH houve aumento na marcação de hemácias, quando comparado ao grupo controle (p<0,001). Apenas alguns animais apresentaram mais de 5% de hemácias marcadas, e isto ocorreu, principalmente, no momento pós-OSH. Entretanto, não resultou no agravamento da anemia, indicando que a piometra em cadelas está associada à deposição de imunoglobulinas G ou M na superfície das hemácias, sem, no entanto, promover hemólise ou agravamento da anemia

A pore-forming haemolysin from the hookworm, Ancylostoma caninum

Hookworms feed on blood, but the mechanism by which they lyse ingested erythrocytes is unknown. Here we show that Ancylostoma caninum, the common dog hookworm, expresses a detergent soluble, haemolytic factor. Activity was identified in both adult and larval stages, was heat-stable and unaffected by the addition of protease inhibitors, metal ions, chelators and reducing agents. Trypsin ablated lysis indicating that the haemolysin is a protein. A closely migrating doublet of hookworm proteins with apparent molecular weights of 60-65 kDa bound to the erythrocyte membrane after lysis of cells using both unlabeled and biotinylated detergent-solubilised hookworm extracts. In addition, separation of detergent-soluble parasite extracts using strong cation-exchange chromatography, resulted in purification of 60-65 kDa proteins with trypsin-sensitive haemolytic activity. Erythrocytes lysed with particulate, buffer-insoluble worm extracts were observed using scanning electron microscopy and appeared as red cell ghosts with approximately 100 nm diameter pores formed in the cell membranes. Red blood cell ghosts remained visible indicating that lysis was likely caused by pore formation and followed by osmotic disruption of the cell. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria

Individuals living in regions where malaria is endemic develop an acquired immunity to malaria which enables them to remain asymptomatic while still carrying parasites. Field studies indicate that cumulative exposure to a variety of diverse Plasmodium parasites is required for the transition from symptomatic to asymptomatic malaria. This study used a simulation model of the within-host dynamics of P. falciparum to investigate the development of acquired clinical immunity under different transmission conditions and levels of parasite diversity. Antibodies developed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), a clonally variant molecule, were assumed to be a key human immunological response to P. falciparum infection, along with responses to clonally conserved but polymorphic antigens. The time to the development of clinical immunity was found to be proportional to parasite diversity and inversely proportional to transmission intensity. The effect of early termination of symptomatic infections by chemotherapy was investigated and found not to inhibit the host's ability to develop acquired immunity. However, the time required to achieve this state was approximately double that compared to when no treatment was administered. This study demonstrates that an immune response primarily targeted against PfEMP1 has the ability to reduce clinical symptoms of infections irrespective of whether treatment is administered, supporting its role in the development of acquired clinical immunity. The results also illustrate a novel use for simulation models of P. falciparum infections, investigation of the influence of intervention strategies on the development of naturally acquired clinical immunity.

Detection sensitivity and quantitation of Plasmodium falciparum var gene transcripts by real-time RT-PCR in comparison with conventional RT-PCR

Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples.

Physical linkage to drug resistance genes results in conservation of var genes among west pacific Plasmodium falciparum isolates

The multicopy var gene family encoding the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 is highly diverse, with little overlap between different P. falciparum isolates. We report 5 var genes (varS1-varS5) that are shared at relatively high frequency among 63 genetically diverse P. falciparum isolates collected from 5 islands in the West Pacific region. The varS1, varS2, and varS3 genes were localized to the internal region on chromosome 4, similar to 200 kb from pfdhfr-ts, whereas varS4 and varS5 were mapped to an internal region of chromosome 7, within 100 kb of pfcrt. The presence of varS2 and varS3 were significantly correlated with the pyrimethamine-resistant pfdhfr genotype, whereas varS4 was strongly correlated with the chloroquine-resistant pfcrt genotype. Thus, the conservation of these var genes is the result of their physical linkage with drug-resistant genes in combination with the antimalarial drug pressure in the region.

Efeito do exercício físico na estabilidade de membrana de eritrócitos

CHAPTER II - This study evaluated the effects of two different types of acute aerobic exercise on the osmotic stability of human erythrocyte membrane and on different hematological and biochemical variables that are associated with this membrane property. The study population consisted of 20 healthy and active men. Participants performed single sessions of two types of exercise. The first session consisted of 60 min of moderate-intensity continuous exercise (MICE). The second session, executed a week later, consisted of high-intensity interval exercise (HIIE) until exhaustion. The osmotic stability of the erythrocyte membrane was represented by the inverse of the salt concentration (1/H50) at the midpoint of the sigmoidal curve of dependence between the absorbance of hemoglobin and the NaCl concentration. The values of 1/H50 changed from 2.29 ± 0.1 to 2.33 ± 0.09 after MICE and from 2.30 ± 0.08 to 2.23 ± 0.12 after HIIE. In MICE has occurred an increase in the mean corpuscular volume, probably due to in vivo lysis of older erythrocytes, with preservation of cells that were larger and more resistant to in vitro lysis. The study showed that a single bout of acute exercise affected the erythrocyte osmotic stability, which increased after MICE and decreased after HIIE.

Studies on the transport of lanthanides ions through the membrane of human erythrocyte in vitro by ICP-MS

A method was developed for the determination of lanthanum in the cytoplasm of human erythrocytes after they were incubated in lanthanum nitrate or citrate solutions. The lanthanum concentration in the cytoplasm of incubated erythrocytes is much higher than that in normal erythrocytes. It is suggested lanthanum can transport through the membrane of erythrocyte in vitro. Solutions containing chelator are unsuitable to be washing buffer in the investigation.

Common variants in TMPRSS6 are associated with iron status and erythrocyte volume

We report a genome-wide association study to iron status. We identify an association of SNPs in TPMRSS6 to serum iron (rs855791, combined P = 1.5 x 10(-20)), transferrin saturation (combined P = 2.2 x 10(-23)) and erythrocyte mean cell volume (MCV, combined P = 1.1 x 10(-10)). We also find suggestive evidence of association with blood hemoglobin levels (combined P = 5.3 x 10(-7)). These findings demonstrate the involvement of TMPRSS6 in control of iron homeostasis and in normal erythropoiesis.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

The interaction of aldolase with the cytoplasmic domain of human erythrocyte band 3 inhibited by lanthanum ions

The effect of lanthanum ions on the activity of the cytoplasmic domain of human erythrocyte band 3 (CDB3), which was measured according to the inhibition to aldolase, was studied. In the presence of low concentration of lanthanum ions, the function of CDB3 to inhibit aldolase activity decreased significantly. It indicated that lanthanum ions in the erythrocyte would change the conformation of CDB3 and influence the control on aldolase activity.

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.

In vitro and in vivo evaluation of a primaquine prodrug without red blood cell membrane destabilization property

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)