Monkey Erythrocytes Research Articles

Genetic characterisation of the erythrocyte-binding protein ($$\hbox {Pk}{\upbeta }\hbox {II}$$) of Plasmodium knowlesi isolates from Malaysia

Plasmodium knowlesi contributes to the majority of human malaria incidences in Malaysia. Its uncontrollable passage among the natural monkey hosts can potentially lead to zoonotic outbreaks. The merozoite of this parasite invades host erythrocytes through interaction between its erythrocyte-binding proteins (EBPs) and their respective receptor on the erythrocytes. The region II of P. knowlesi EBP, P. knowlesi beta ( $$\hbox {Pk}{\upbeta }\hbox {II}$$ ) protein is found to be mediating merozoite invasion into monkey erythrocytes by interacting with sialic acid receptors. Hence, the objective of this study was to investigate the genetic diversity, natural selection and haplotype grouping of $$\hbox {Pk}{\upbeta }\hbox {II}$$ of P. knowlesi isolates in Malaysia. Polymerase chain reaction amplifications of $$\hbox {Pk}{\upbeta }\hbox {II}$$ were performed on archived blood samples from Malaysia and 64 $$\hbox {Pk}{\upbeta }\hbox {II}$$ sequences were obtained. Sequence analysis revealed length polymorphism, and its amino acids at critical residues indicate the ability of $$\hbox {Pk}{\upbeta }\hbox {II}$$ to mediate P. knowlesi invasion into monkey erythrocytes. Low genetic diversity ( $${\uppi } = 0.007$$ ) was observed in the $$\hbox {Pk}{\upbeta }\hbox {II}$$ of Malaysia Borneo compared to Peninsular Malaysia ( $${\uppi } = 0.015$$ ). The $$\hbox {Pk}{\upbeta }\hbox {II}$$ was found to be under strong purifying selection to retain infectivity in monkeys and it plays a limited role in the zoonotic potential of P. knowlesi. Its haplotypes could be clustered into Peninsular Malaysia and Malaysia Borneo groups, indicating the existence of two distinct P. knowlesi parasites in Malaysia as reported in an earlier study.

Transcriptome profiling of Plasmodium vivax in Saimiri monkeys identifies potential ligands for invasion

Unlike the case in Asia and Latin America, Plasmodium vivax infections are rare in sub-Saharan Africa due to the absence of the Duffy blood group antigen (Duffy antigen), the only known erythrocyte receptor for the P. vivax merozoite invasion ligand, Duffy binding protein 1 (DBP1). However, P. vivax infections have been documented in Duffy-negative individuals throughout Africa, suggesting that P. vivax may use ligands other than DBP1 to invade Duffy-negative erythrocytes through other receptors. To identify potential P. vivax ligands, we compared parasite gene expression in Saimiri and Aotus monkey erythrocytes infected with P. vivax Salvador I (Sal I). DBP1 binds Aotus but does not bind to Saimiri erythrocytes; thus, P. vivax Sal I must invade Saimiri erythrocytes independent of DBP1. Comparing RNA sequencing (RNAseq) data for late-stage infections in Saimiri and Aotus erythrocytes when invasion ligands are expressed, we identified genes that belong to tryptophan-rich antigen and merozoite surface protein 3 (MSP3) families that were more abundantly expressed in Saimiri infections compared with Aotus infections. These genes may encode potential ligands responsible for P. vivax infections of Duffy-negative Africans.

Human erythrocyte antigens in Brazilian Capuchin monkeys (Sapajus sp.).

Investigation of erythrocyte antigens in New World monkeys, especially in the Brazilian ones, is scant and incomplete. Determining the presence of 29 erythrocyte antigens from 11 human blood group systems (ABO, H, Rh, Kell, Duffy, Kidd, Lewis, P, MNS, Lutheran and Diego) on erythrocytes in nine Capuchin monkeys (Sapajus sp.). A majority (20 of 29) of human erythrocyte antigens were not found in this monkey genus. Erythrocyte phenotyping was very similar within this animal group, as five Capuchin monkeys differed from the other four in the ABO system only. The erythrocyte phenotype for this group of animals is less diversified than in humans. Some monkey erythrocyte antigens were similar in frequency, whereas others were different from those observed in human ethnicities.

The Duffy binding protein (PkDBP\u03b1II) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes

BackgroundThe zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated.MethodsThe PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes.ResultsAnti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes.ConclusionThis study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

Plasmodium knowlesi Skeleton-Binding Protein 1 Localizes to the 'Sinton and Mulligan' Stipplings in the Cytoplasm of Monkey and Human Erythrocytes.

The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as ‘Sinton and Mulligan’ stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum.

Role of Plasmodium vivax Duffy-binding protein 1 in invasion of Duffy-null Africans

The ability of the malaria parasite Plasmodium vivax to invade erythrocytes is dependent on the expression of the Duffy blood group antigen on erythrocytes. Consequently, Africans who are null for the Duffy antigen are not susceptible to P. vivax infections. Recently, P. vivax infections in Duffy-null Africans have been documented, raising the possibility that P. vivax, a virulent pathogen in other parts of the world, may expand malarial disease in Africa. P. vivax binds the Duffy blood group antigen through its Duffy-binding protein 1 (DBP1). To determine if mutations in DBP1 resulted in the ability of P. vivax to bind Duffy-null erythrocytes, we analyzed P. vivax parasites obtained from two Duffy-null individuals living in Ethiopia where Duffy-null and -positive Africans live side-by-side. We determined that, although the DBP1s from these parasites contained unique sequences, they failed to bind Duffy-null erythrocytes, indicating that mutations in DBP1 did not account for the ability of P. vivax to infect Duffy-null Africans. However, an unusual DNA expansion of DBP1 (three and eight copies) in the two Duffy-null P. vivax infections suggests that an expansion of DBP1 may have been selected to allow low-affinity binding to another receptor on Duffy-null erythrocytes. Indeed, we show that Salvador (Sal) I P. vivax infects Squirrel monkeys independently of DBP1 binding to Squirrel monkey erythrocytes. We conclude that P. vivax Sal I and perhaps P. vivax in Duffy-null patients may have adapted to use new ligand-receptor pairs for invasion.

Abstract 4271: Lack of CD47 membrane mobility contributes to the poor erythrocyte binding of SIRPαFc, a novel CD47-blocking cancer immunotherapeutic

Abstract CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal that suppresses phagocytosis. There is strong evidence that many liquid and solid tumors exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of CD47 using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells. We have previously reported data demonstrating that human SIRPαFc binds strongly to tumor cells but very poorly to human red blood cells (RBCs), despite abundant surface expression of CD47 on RBCs and strong reactivity with CD47-specific antibodies. Here we expand upon these early findings and assess inter-species differences in RBC binding. Our results, based on a panel of 43 human donors, clearly show that SIRPαFc binds very poorly to human RBCs regardless of gender, ABO blood group or Rh antigen status. Consistent with this finding, SIRPαFc was unable to induce agglutination of RBCs in vitro, although hemagglutination was triggered by CD47-blocking antibodies. Curiously, although the binding affinity of human SIRPαFc to cynomolgus macaque CD47 is approximately 10-fold lower than the binding to the human target, it binds strongly to cyno RBCs. This indicates that affinity alone does not predict the ability to bind erythrocytes. Instead, we hypothesized that the mobility of CD47 in the RBC membrane is a key determinant of SIRPαFc binding and thus compared the detergent solubilization profile of CD47 in human and cyno RBCs. CD47 was observed to segregate largely into the detergent-soluble fraction in monkey erythrocytes but was localized primarily to the insoluble pellet fraction in human RBCs, suggesting greater membrane mobility in cyno compared to human RBCs. This finding is consistent with a model in which CD47 mobility is required to form high affinity clusters with SIRPαFc, and indeed we have previously observed that pre-clustering CD47 with a non-blocking antibody converts human RBCs into strong SIRPαFc binders. Finally, the consequences of cyno RBC binding were assessed in vivo. Significant depletion of RBCs was evident following intravenous infusions of SIRPαFc in cyno monkeys. We speculate that similar anemia is likely to occur in humans treated with CD47-blocking antibodies that bind to human erythrocytes, but not with a low RBC-binding SIRPαFc therapeutic. In conclusion, human SIRPαFc binds very poorly to human RBCs but is highly reactive with cyno erythrocytes. This unusual pattern of species cross-reactivity may result from species-specific differences in CD47 membrane mobility and is consistent with a model in which SIRPαFc binding requires mobile CD47 to form high affinity clusters. We predict that anemia, which occurs in monkeys following SIRPαFc administration, is not likely to occur in human patients where significant RBC binding is absent. Citation Format: Penka S. Petrova, Karen Dodge, Tanya Prasolava, Vien Chai, Xinli Pang, Robert A. Uger. Lack of CD47 membrane mobility contributes to the poor erythrocyte binding of SIRPαFc, a novel CD47-blocking cancer immunotherapeutic. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4271. doi:10.1158/1538-7445.AM2015-4271

A comparative histological study on the blood film of young and adult, male and female Monkeys (Chlorocebus pygerythrus)

Twenty Vervets monkeys were used to study the effect of sexes and ages on morphohistological and hematological pictures. The study revealed that age and sex affect blood values. Vervet monkeys registered a significant dominancy of hematological parameters in adult males. Erythrocytes of vervet monkeys were of macrocytic type, howell-Jolly bodies were observed. Rouleaux formation, Anisocytosis, Aggregation, and Agglutination were not observed. A significant predominancy of neutrophils in females and lymphocytes in males was recorded. The neutrophil was well defined, large with ring-shaped highly segmented nucleus. Band neutrophils were not observed in peripheral blood. Eosinophils recorded high values. Circulating leukocytes acted as a transient phase in the functional development of tissue leukocytes. Lymphocytes were the most common cells among leukocytes. Three sizes of lymphocytes and two of monocytes were registered. The value of monocytes percent was higher in males. The herein study declared that blood platelets showing different sizes, shapes and affinities for basic stains and according to these functional affinity, light resting and dark stimulating blood platelets were classified. There was a reverse relationship between the number of erythrocytes and blood platelets among different sexes and ages. In conclusion, the present study hypothesized that blood platelets serve as a supporting cells for blood cells especially erythrocytes. Besides, humoral immune defense in monkeys were well developed.

Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia.

BackgroundPlasmodium knowlesi is one of the monkey malaria parasites that can cause human malaria. The Duffy binding protein of P. knowlesi (PkDBPαII) is essential for the parasite’s invasion into human and monkey erythrocytes. A previous study on P. knowlesi clinical isolates from Peninsular Malaysia reported high level of genetic diversity in the PkDBPαII. Furthermore, 36 amino acid haplotypes were identified and these haplotypes could be separated into allele group I and allele group II. In the present study, the PkDBPαII of clinical isolates from the Malaysian states of Sarawak and Sabah in North Borneo was investigated, and compared with the PkDBPαII of Peninsular Malaysia isolates.MethodsBlood samples from 28 knowlesi malaria patients were used. These samples were collected between 2011 and 2013 from hospitals in North Borneo. The PkDBPαII region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and phylogenetics of PkDBPαII haplotypes were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes.ResultsForty-nine PkDBPαII sequences were obtained. Comparison at the nucleotide level against P. knowlesi strain H as reference sequence revealed 58 synonymous and 102 non-synonymous mutations. Analysis on these mutations showed that PkDBPαII was under purifying (negative) selection. At the amino acid level, 38 different PkDBPαII haplotypes were identified. Twelve of the 28 blood samples had mixed haplotype infections. Phylogenetic analysis revealed that all the haplotypes were in allele group I, but they formed a sub-group that was distinct from those of Peninsular Malaysia. Wright’s FST fixation index indicated high genetic differentiation between the North Borneo and Peninsular Malaysia haplotypes.ConclusionsThis study is the first to report the genetic diversity and natural selection of PkDBPαII of P. knowlesi from Borneo Island. The PkDBPαII haplotypes found in this study were distinct from those from Peninsular Malaysia. This difference may not be attributed to geographical separation because other genetic markers studied thus far such as the P. knowlesi circumsporozoite protein gene and small subunit ribosomal RNA do not display such differentiation. Immune evasion may possibly be the reason for the differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0610-x) contains supplementary material, which is available to authorized users.

Various PfRH5 polymorphisms can support Plasmodium falciparum invasion into the erythrocytes of owl monkeys and rats

Aotus nancymaae, the owl monkey, provides a useful laboratory model for research to develop drugs and vaccines against human falciparum malaria; however, many Plasmodium falciparum parasites are unable to invade A. nancymaae erythrocytes, rendering the parasites noninfective to the monkeys. In previous work, we identified a key polymorphism that determined the inheritance of erythrocyte invasion in a genetic cross of two P. falciparum clones that were virulent (GB4) or noninfective (7G8) to A. nancymaae. This polymorphism, an isoleucine-to-lysine polymorphism at position 204 (I204K) of the GB4 erythrocyte binding protein PfRH5, was nevertheless not found in several other P. falciparum lines that could also invade A. nancymaae erythrocytes. Alternative PfRH5 polymorphisms occur at different positions in these virulent parasites, and additional polymorphisms are found in P. falciparum parasites that cannot infect A. nancymaae. By allelic replacement methods, we have introduced the polymorphisms of these A. nancymaae-virulent or noninfective parasites at codons 204, 347, 358, 362, 410, and 429 of the endogenous PfRH5 gene in the noninfective 7G8 line. 7G8 transformants expressing the polymorphisms of the A. nancymaae-virulent parasites show neuraminidase-sensitive (sialic acid-dependent) invasion into the monkey erythrocytes, whereas 7G8 transformants expressing the PfRH5 alleles of noninfective parasites show little or no invasion of these erythrocytes. Parasites harboring PfRH5 polymorphisms 204K or 204R are also able to invade rat erythrocytes and are differentially sensitive to the removal of surface sialic acids by neuraminidase. These studies offer insights into the PfRH5 receptor-binding domain and interactions that support the invasion of various primate and rodent erythrocytes by P. falciparum.

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.

Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes

BackgroundPlasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb).MethodsOverlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment, and site directed mutagenesis.ResultsAn N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II) exhibited specific adhesion to rhesus monkey erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells, suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa bound human erythrocytes, and this binding was independent of the Duffy blood group determinant.ConclusionsThe data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb, PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the invasion of P. knowlesi merozoites into human cells.

AGE-RELATED CHANGES OF RELIABILITY OF ANTIOXIDANT ENZYME DEFENSE ARE ASSOCIATED WITH ADAPTIVE BEHAVIOR OF PRIMATES

We have investigated age-related changes in the activity of antioxidant enzyme system and intensity of lipid peroxidation in erythrocytes of young and old female rhesus monkeys with depression-like and standard behavior. Revealed significant disturbances in the activities of GR and GSH-Px in monkeys with depression-like behavior are accompanied more pronounced age-related decrease in reliability of antioxidant enzyme defense and development of oxidative stress.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.

Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase

Previous in vitro studies showed marked species differences in the reactivating efficiency of oximes between human and animal acetylcholinesterase (AChE) inhibited by organophosphorus (OP) nerve agents. These findings provoked the present in vitro study which was designed to determine the inhibition, aging, spontaneous and oxime-induced reactivation kinetics of the pesticide paraoxon, serving as a model compound for diethyl-OP, and the oximes obidoxime, pralidoxime, HI 6 and MMB-4 with human, Rhesus monkey, swine, rabbit, rat and guinea pig erythrocyte AChE. Comparable results were obtained with human and monkey AChE. Differences between human, swine, rabbit, rat and guinea pig AChE were determined for the inhibition and reactivation kinetics. A six-fold difference of the inhibitory potency of paraoxon with human and guinea pig AChE was recorded while only moderate differences of the reactivation constants between human and animal AChE were determined. Obidoxime was by far the most effective reactivator with all tested species. Only minor species differences were found for the aging and spontaneous reactivation kinetics. The results of the present study underline the necessity to determine the inhibition, aging and reactivation kinetics in vitro as a basis for the development of meaningful therapeutic animal models, for the proper assessment of in vivo animal data and for the extrapolation of animal data to humans.

The importance of rhesus status

The history of haemolytic disease of the newborn, caused by incompatibility between the fetus and mother for the rhesus D (RhD) antigen, has been called one of the most brilliant successes achieved in medicine.1 Preventive treatment is so effective that it has decreased the number of intrauterine fetal deaths by more than 100 fold and the incidence of maternal alloimmunisation by more than 50 fold.2 The first dated case report of haemolytic disease of the newborn was in a set of twins born in the 1600s.3 However, it was not until 1940 that the link with the RhD antigen was made by Landsteiner and Weiner. They discovered an antibody that led to the agglutination of human erythrocytes after experiments in which rabbits were immunised with rhesus monkey erythrocytes.4 In 1969, a campaign of anti-D immunoglobulin prophylaxis was introduced. Deaths attributed to RhD alloimmunisation were reduced from 46 in 100 000 births to 1.6 in 100 000 births during the first 20 years of the campaign. However, haemolytic disease of the newborn and neonatal death continued to occur, and still do—owing in part to covert sensitising events during pregnancy, and in part to a lack of knowledge and compliance with guidelines.5 Erythrocytes carry many antigens on their surface. The most important are those belonging to the ABO system and to the rhesus system. The rhesus system comprises antigens c, C, D, e, and E. Of these, the D antigen is the most important. It is inherited on one gene, located on chromosome 1. It has two alleles: the dominant RhD positive, where the individual expresses the D antigen, and the recessive RhD negative, where there is no expression.6 The incidence of the rhesus D antigen varies between different ethnic groups.6 It is estimated that the prevalence …

Comparative study of oxime-induced reactivation of erythrocyte and muscle AChE from different animal species following inhibition by sarin or paraoxon

Standard treatment of acute poisoning by organophosphorus compounds (OP) includes administration of an antimuscarinic (e.g. atropine) and of an oxime-based reactivator of OP-inhibited acetylcholinesterase (AChE). A recently introduced dynamically working in vitro model with real-time determination of membrane-bound AChE activity was shown to be a very versatile and promising model to investigate oxime-induced reactivation kinetics of OP-inhibited enzyme. In this assay, human AChE from erythrocytes or muscle tissue was immobilized on a particle filter. This bioreactor was continuously perfused with substrate and chromogen and AChE activity was analyzed on-line in a flow-through detector. The model has been successfully adopted to Rhesus monkey, swine and guinea pig erythrocytes and intercostal muscle AChE. In addition, the basic kinetic constants of inhibition, aging, spontaneous- and oxime-induced-reactivation of erythrocyte AChE from these species were determined with a standard static model. The major findings were, in part substantial species differences in the inhibition (sarin, paraoxon) and reactivation kinetics (obidoxime, HI 6) of erythrocyte AChE, but comparable kinetics of inhibition and reactivation between erythrocyte and muscle AChE. Hence, these data provide further support of the assumption that erythrocyte AChE is an adequate surrogate of muscle (synaptic) AChE and admonish that major species differences have to be considered for the design and evaluation of therapeutic animal models.

Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE.

273. Long-Term Effects of Plasmid-Mediated Growth Hormone Releasing Hormone in Dogs

Top of pageAbstract We have developed a capsid-modified oncolytic adenovirus (Ad) vector (Ad5/35.IR-E1A/TRAIL) that is highly efficient in the elimination of liver metastases derived from various human tumor cell lines in mouse models. However, testing for viral efficacy on established tumor cell lines and for safety in mouse models may not be predictive for the performance of these vectors in clinical trials. In contrast to cancer cell lines, the genotype and gene expression profiles of primary short term cultures more closely reflects that of actual tumors. We have therefore tested the oncolytic ability of our vector and a series of control vectors on primary ovarian and cervical cancer cultures. So far we have established two ovarian cancer cultures and three cultures of normal mesothelial cells from ascites. Ad.5/35.IR-E1A/TRAIL caused apoptosis in 100% and ~80% of ovarian cancer cells in cultures from two different patients, but did not cause cytolysis in mesothelial cells. The efficiency and specificity of oncolysis by Ad5/35.IR-E1a/TRAIL was compared to wild-type Ads. Wild-type Ad35 efficiently caused cytopathic effects in mesothelial cells and ovarian cancer cells (without the characteristic apoptotic features seen in the Ad.5/35.IR-E1A/TRAIL infected cells). Wild-type Ad5 did not efficiently lyse tumor cells, but did cause more CPE in mesothelial cells than wt Ad35, which probably reflects the differential expression of receptors for Ad5 and Ad35. We are in the process of establishing more primary cultures and the results will be reported. For safety studies, baboons were injected through the femoral vein with PBS (mock) or 1 × 1011 pfu/kg of Ad5/35.IR-E1a/TRAIL (in PBS) and blood samples were monitored for 30 days for biochemical parameters, blood cell counts, vector genome concentrations, and cytokine levels. Upon necropsy, tissue histology, presence of transgene expression, and vector genomes were analyzed in samples of brain, lymph nodes, bone marrow, heart, aorta, digestive tract, liver, pancreas, bladder, testis, muscle, spleen, lung, kidney, peripheral blood cells, and prostate. All laboratory blood test parameters were in normal ranges following injection of the oncolytic vectors. Among the cytokines, IL-6 was elevated at 6 hours after vector infusion, but reached pre-injection levels 24 hours post-infusion. We used real-time PCR for vector genomes to assess the kinetics of Ad clearance from the blood. When we compared the amount of vector DNA associated with blood cells and plasma with the total amount of injected Ad, we found that at 1 min post–infusion, ~2% of the input Ad dose is associated with blood cells, which could be due to binding to erythrocytes (Ad35 agglutinates monkey erythrocytes). Ad5/35 genomes in plasma and blood cells reached pre-injection levels by 24 hours p.i.. No histopathological changes were found in tissues analyzed 30 days post-infusion. Analysis of vector genomes and transgene expression in tissue samples is ongoing.

Divergent evolution and purifying selection of the H (FUT1) gene in New World monkeys (Primates, Platyrrhini)

In the present study, the coding region of the H gene was sequenced and analyzed in fourteen genera of New World primates (Alouatta, Aotus, Ateles, Brachyteles, Cacajao, Callicebus, Callithrix, Cebus, Chiropotes, Lagothrix, Leontopithecus, Pithecia, Saguinus, and Saimiri), in order to investigate the evolution of the gene. The analyses revealed that this coding region contains 1,101 nucleotides, with the exception of Brachyteles, the callitrichines (Callithrix, Leontopithecus, and Saguinus) and one species of Callicebus (moloch), in which one codon was deleted. In the primates studied, the high GC content (63%), the nonrandom distribution of codons and the low evolution rate of the gene (0.513 substitutions/site/MA in the order Primates) suggest the action of a purifying type of selective pressure, confirmed by the Z-test. Our analyses did not identify mutations equivalent to those responsible for the H-deficient phenotypes found in humans, nor any other alteration that might explain the lack of expression of the gene in the erythrocytes of Neotropical monkeys. The phylogenetic trees obtained for the H gene and the distance matrix data suggest the occurrence of divergent evolution in the primates.