Variant Surface Antigens Research Articles

Evaluation of Malarial Var2CSA-Displaying Baculovirus Vector in Transduction Efficiency in Human Cancer Cells.

Baculovirus vectors (BVs) are able to use for gene transduction in mammalian cells and are recognized as growing viral vectors for cancer gene therapy applications. The transduction efficiency of BVs varies among cancer cell types. To improve the transduction efficiency of BVs in human cancer cells, BV displaying malarial variant surface antigen 2-chondroitin sulfate A (var2CSA) molecules was developed in this study. Var2CSA plays a critical role in the sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Moreover, var2CSA binds to cancer cells via placenta-like chondroitin sulfate A (CSA), but not to non-cancer cells. Var2CSA BV showed significantly higher gene transduction than control BV in HepG2 and Huh7 cells, human hepatic cancer cells as well as AsPC-1 cells, human pancreatic cancer cells. The transduction efficiency of var2CSA BV was significantly inhibited by the anti-gp64 antibody, free heparin, and CSA. The results of this study suggest that var2CSA BV would be an improved vector for cancer gene therapies, especially in the treatment of hepatic and pancreatic cancers.

Acquisition of antibodies to Plasmodium falciparum and Plasmodium vivax antigens in pregnant women living in a low malaria transmission area of Brazil

BackgroundPregnant women have increased susceptibility to Plasmodium falciparum malaria and acquire protective antibodies over successive pregnancies. Most studies that investigated malaria antibody responses in pregnant women are from high transmission areas in sub-Saharan Africa, while reports from Latin America are scarce and inconsistent. The present study sought to explore the development of antibodies against P. falciparum and Plasmodium vivax antigens in pregnant women living in a low transmission area in the Brazilian Amazon.MethodsIn a prospective cohort study, plasma samples from 408 pregnant women (of whom 111 were infected with P. falciparum, 96 had infections with P. falciparum and P. vivax, and 201 had no Plasmodium infection) were used to measure antibody levels. Levels of IgG and opsonizing antibody to pregnancy-specific variant surface antigens (VSAs) on infected erythrocytes (IEs), 10 recombinant VAR2CSA Duffy binding like (DBL domains), 10 non-pregnancy-specific P. falciparum merozoite antigens, and 10 P. vivax antigens were measured by flow cytometry, ELISA, and multiplex assays. Antibody levels and seropositivity among the groups were compared.ResultsAntibodies to VSAs on P. falciparum IEs were generally low but were higher in currently infected women and women with multiple P. falciparum episodes over pregnancy. Many women (21%-69%) had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other (r ≥ 0.55, p < 0.0001), but not with antibody to VSA or history of infection. Infection with either malaria species was associated with higher seropositivity rate for antibodies against P. vivax proteins, adjusted odds ratios (95% CI) ranged from 5.6 (3.2, 9.7), p < 0.0001 for PVDBPII-Sal1 to 15.7 (8.3, 29.7), p < 0.0001 for PvTRAg_2.ConclusionsPregnant Brazilian women had low levels of antibodies to pregnancy-specific VSAs that increased with exposure. They frequently recognized both VAR2CSA DBL domains and P. vivax antigens, but only the latter varied with infection. Apparent antibody prevalence is highly dependent on the assay platform used.

Species-Specific Duplication of Surface Antigen Genes in Paramecium.

Paramecium is a free-living ciliate that undergoes antigenic variation and still the functions of these variable surface antigen coats in this non-pathogenic ciliate remain elusive. Only a few surface antigen genes have been described, mainly in the two model species P. tetraurelia strain 51 and P. primaurelia strain 156. Given the lack of suitable sequence data to allow for phylogenetics and deeper sequence comparisons, we screened the genomes of six different Paramecium species for serotype genes and isolated 548 candidates. Our approach identified the subfamilies of the isogenes of individual serotypes that were mostly represented by intrachromosomal gene duplicates. These showed different duplication levels, and chromosome synteny suggested rather young duplication events after the emergence of the P. aurelia species complex, indicating a rapid evolution of surface antigen genes. We were able to identify the different subfamilies of the surface antigen genes with internal tandem repeats, which showed consensus motifs across species. The individual isogene families showed additional consensus motifs, indicating that the selection pressure holds individual amino acids constant in these repeats. This may be a hint of the receptor function of these antigens rather than a presentation of random epitopes, generating the variability of these surface molecules.

Pathogenicity and virulence of malaria: Sticky problems and tricky solutions

ABSTRACT Infections with Plasmodium falciparum and Plasmodium vivax cause over 600,000 deaths each year, concentrated in Africa and in young children, but much of the world’s population remain at risk of infection. In this article, we review the latest developments in the immunogenicity and pathogenesis of malaria, with a particular focus on P. falciparum, the leading malaria killer. Pathogenic factors include parasite-derived toxins and variant surface antigens on infected erythrocytes that mediate sequestration in the deep vasculature. Host response to parasite toxins and to variant antigens is an important determinant of disease severity. Understanding how parasites sequester, and how antibody to variant antigens could prevent sequestration, may lead to new approaches to treat and prevent disease. Difficulties in malaria diagnosis, drug resistance, and specific challenges of treating P. vivax pose challenges to malaria elimination, but vaccines and other preventive strategies may offer improved disease control.

The Pathogenesis of Giardia Intestinalis

Giardia intestinalis infection leads to intestinal cell damage and loss of the brush border of the intestinal epithelium, resulting in shortened microvilli and impaired epithelial barrier function. Watery diarrhoea, diarrhoea, nausea, abdominal pain, vomiting, and weight loss are all symptoms of this pathological alteration. Most infections are asymptomatic. Malnutrition absorption is the most common symptom of Giardia intestinalis infection. To treat Giardia intestinalis, several medications with good efficacy are employed, but the dose regimen is not always ideal, and the evolution of drug resistance is beginning to cast doubt on their clinical worth. In addition, some of these drugs can produce side effects that cause discomfort and make it difficult for patients to adhere to treatment. Giardia intestinalis is an important zoonotic parasite that causes diarrhoea in humans and many mammals. In recent years, its pathogenesis, including structural proteins and excretion of Giardia intestinalis, surface antigen variants, and the role of Giardia intestinalis in the small intestine, has been extensively studied. This article discusses this issue and lists the risks of Giardia intestinalis to the human intestine and the various diseases it can cause.

A Novel Methodology for Detecting Variations in Cell Surface Antigens Using Cell-Tearing by Optical Tweezers.

The quantitative analysis of cell surface antigens has attracted increasing attention due to the antigenic variation recognition that can facilitate early diagnoses. This paper presents a novel methodology based on the optical “cell-tearing” and the especially proposed “dilution regulations” to detect variations in cell surface antigens. The cell attaches to the corresponding antibody-coated slide surface. Then, the cell-binding firmness between a single cell and the functionalized surface is assayed by optically tearing using gradually reduced laser powers incorporated with serial antibody dilutions. Groups B and B3 of red blood cells (RBCs) were selected as the experiment subject. The results indicate that a higher dilution called for lower power to tear off the cell binding. According to the proposed relative-quantitative analysis theory, antigenic variation can be intuitively estimated by comparing the maximum allowable dilution folds. The estimation result shows good consistency with the finding in the literature. This study suggests a novel methodology for examining the variation in cell surface antigens, expected to be widely capable with potential sensor applications not only in biochemistry and biophysics, but also in the micro-/nano- engineering field.

Trypanosoma evansi RoTat 1.2 variant surface antigen mimotopes selected by panning of the random peptide phage-display library against monoclonal antibodies.

Mimotope peptides of native antigens are valuable for diverse applications such as diagnostics, therapeutics and modern vaccine design. Here, we report for the first time the selection and identification of peptide mimotopes of Trypanosoma evansi RoTat 1.2 variant surface glycoprotein (VSG) for their potential uses in surra diagnostics and multi-epitope vaccine research. First, we produced the mouse monoclonal antibodies (mAbs), designated as 2E11 (IgG1) and 1C2 (IgG1), against the antigens in T. evansi RoTat 1.2 lysates. We then used 2E11 mAb to immunoprecipitate the target antigen. The immunoprecipitated antigen was then identified to be the VSG by mass spectrometry. Both 2E11 and 1C2 mAbs reacted with the VSG in immunoblots. The surface plasmon resonance immunosensors developed with both the mAbs detected VSG in the parasite lysates as well as in the rodent sera. Further, the mAbs were biotinylated and used in three rounds of panning to select peptide mimotopes from the random peptide phage display library (PhD-12; New England Biolabs, USA). The phage clones selected against each mAb were amplified and tested by phage capture ELISA for specificity. The peptide coding regions of the selected phages were sequenced and the protein blast search of the deduced amino acid sequences was performed by accessing the non-redundant protein database at https://blast.ncbi.nlm.nih.gov/. The conformational B epitope prediction of the selected mimotope sequences was done by using 3D Pepitope algorithms accessed at: http://pepitope.tau.ac.il/. The potential applications of the selected mimotopes in surra diagnostics and research are being explored.

Selective expression of variant surface antigens enables Plasmodium falciparum to evade immune clearance in vivo

Plasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA, provides the parasite with protection from macrophage phagocytosis and clearance in the humanized mice. Furthermore, parasites adapted to thrive in the humanized mice show reduced NK cell-mediated killing through interaction with the immune inhibitory receptor, LILRB1. Taken together, these findings reveal new insights into the molecular and cellular mechanisms that the parasite utilizes to coordinate immune escape in vivo. Identification and targeting of these specific parasite variant surface antigens crucial for immune evasion provides a unique approach for therapy.

Meta-Analysis of Human Antibodies Against Plasmodium falciparum Variable Surface and Merozoite Stage Antigens.

Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens (17 PfEMP1 domains, 3 RIFIN family members, merozoite surface protein 3 (PF3D7_1035400), and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions (1,000/2,500/5,000 parasites/µl plus fever). In addition, Principal Components Analysis (PCA) indicated that the first three components (Dim1, Dim2 and Dim3 with eigenvalues of 306, 48, and 29, respectively) accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication.

Plasmodium falciparum PfRUVBL proteins bind at the TARE region and var gene promoter located in the subtelomeric region.

In order to survive and establish infection, the Plasmodium parasite employs various strategies to evade the host immune response. The var genes family, a repertoire of 60 genes, expresses parasite-specific protein PfEMP1, a variable surface antigen, on the membrane of infected erythrocytes, and by continuously switching the variants of PfEMP1, help the parasite to avoid detection and destruction by the host immune system during the intra-erythrocytic developmental cycle. Although chromatin modifications are recognised to be a prominent phenomenon in regulation of mono-allelic expression of these var genes, the precise histone codes and molecular players and mechanisms guiding these modifications have yet to be unravelled in depth. In this study, we have functionally characterised RUVBL proteins of Plasmodium falciparum and shown that PfMYST (an essential lysine acetyl transferase) and PfRUVBL protein complex occupy the TARE region and var gene promoter in the ring stage of the parasite. Further, we have demonstrated that the PfMYST/PfRUVBL complex interacts with core histones, H3 and H4. Overall the findings of this study add further information by identifying the potential role of epigenetic regulators, PfMYST and PfRUVBL, in the regulation of monoallelic expression of var genes in the malaria parasite.

Breadth of Antibodies to Plasmodium falciparum Variant Surface Antigens Is Associated With Immunity in a Controlled Human Malaria Infection Study.

Background Plasmodium falciparum variant surface antigens (VSAs) contribute to malaria pathogenesis by mediating cytoadhesion of infected red blood cells to the microvasculature endothelium. In this study, we investigated the association between anti-VSA antibodies and clinical outcome in a controlled human malaria infection (CHMI) study.MethodWe used flow cytometry and ELISA to measure levels of IgG antibodies to VSAs of five heterologous and one homologous P. falciparum parasite isolates, and to two PfEMP1 DBLβ domains in blood samples collected a day before the challenge and 14 days after infection. We also measured the ability of an individual’s plasma to inhibit the interaction between PfEMP1 and ICAM1 using competition ELISA. We then assessed the association between the antibody levels, function, and CHMI defined clinical outcome during a 21-day follow-up period post infection using Cox proportional hazards regression.ResultsAntibody levels to the individual isolate VSAs, or to two ICAM1-binding DBLβ domains of PfEMP1, were not associated with a significantly reduced risk of developing parasitemia or of meeting treatment criteria after the challenge after adjusting for exposure. However, anti-VSA antibody breadth (i.e., cumulative response to all the isolates) was a significant predictor of reduced risk of requiring treatment [HR 0.23 (0.10-0.50) p= 0.0002].ConclusionThe breadth of IgG antibodies to VSAs, but not to individual isolate VSAs, is associated with protection in CHMI.

The Putative Bromodomain Protein PfBDP7 of the Human Malaria Parasite Plasmodium Falciparum Cooperates With PfBDP1 in the Silencing of Variant Surface Antigen Expression.

Epigenetic regulation is a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite Plasmodium (P.) falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the P. falciparum bromodomain proteins (PfBDP) 1 and PfBDP2 (BDP1/BDP2 core complex) was previously shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7, because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, STEVOR, and PfMC-2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and established PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

Plasmodium falciparum in Aotus nancymaae: A New Model for Placental Malaria

Plasmodium falciparum (Pf)-infected erythrocytes that display the variant surface antigen VAR2CSA bind chondroitin sulfate A (CSA) to sequester in placental intervillous spaces, causing severe sequelae for mother and offspring. Here, we establish a placental malaria (PM) monkey model. Pregnant Aotus infected with CSA-binding Pf-CS2 parasites during 3rd trimester developed pronounced sequestration of late-stage parasites in placental intervillous spaces that express VAR2CSA and bind specifically to CSA. Similar to immune multigravid women, a monkey infected with Pf-CS2 parasites over successive pregnancies acquired antibodies against VAR2CSA, with potent functional activity that was boosted upon subsequent pregnancy infections. Aotus also developed functional antibodies after multiple acute PM episodes and subsequent VAR2CSA immunization. In summary, Pf infections in pregnant Aotus monkeys recapitulate all the prominent features of human PM infection and immunity, and this model can be useful for basic mechanistic studies and preclinical studies to qualify candidate PM vaccines.

Transcriptome Analysis of Plasmodium falciparum Isolates From Benin Reveals Specific Gene Expression Associated With Cerebral Malaria.

Cerebral malaria (CM) is the severest form of Plasmodium falciparum infection. Children under 5 years old are those most vulnerable to CM, and they consequently have the highest risk of malaria-related death. Parasite-associated factors leading to CM are not yet fully elucidated. We therefore sought to characterize the gene expression profile associated with CM, using RNA sequencing data from 15 CM and 15 uncomplicated malaria isolates from Benin. Cerebral malaria parasites displayed reduced circulation times, possibly related to higher cytoadherence capacity. Consistent with the latter, we detected increased var genes abundance in CM isolates. Differential expression analyses showed that distinct transcriptome profiles are signatures of malaria severity. Genes involved in adhesion, excluding variant surface antigens, were dysregulated, supporting the idea of increased cytoadhesion capacity of CM parasites. Finally, we found dysregulated expression of genes in the entry into host pathway that may reflect greater erythrocyte invasion capacity of CM parasites.

Quantification of the dynamics of antibody response to malaria to inform sero-surveillance in pregnant women

BackgroundMalaria remains a major public health threat and tools sensitive to detect infections in low malaria transmission areas are needed to progress elimination efforts. Pregnant women are particularly vulnerable to malaria infections. Throughout pregnancy they access routine antenatal care, presenting a unique sentinel population to apply novel sero-surveillance tools to measure malaria transmission. The aim of this study was to quantify the dynamic antibody responses to multiple antigens during pregnancy so as to identify a single or multiple antibody response of exposure to malaria in pregnancy.MethodsThis study involved a secondary analysis of antibody responses to six parasite antigens [five commonly studied merozoite antigens and the variant surface antigen 2-chondroitin sulphate A (VAR2CSA), a pregnancy-specific erythrocytic antigen] measured by enzyme-linked immunosorbent assay (ELISA) over the gestation period until delivery (median of 7 measurements/woman) in 250 pregnant women who attended antenatal clinics located at the Thai-Myanmar border. A multivariate mixture linear mixed model was used to cluster the pregnant women into groups that have similar longitudinal antibody responses to all six antigens over the gestational period using a Bayesian approach. The variable-specific entropy was calculated to identify the antibody responses that have the highest influence on the classification of the women into clusters, and subsequent agreement with grouping of women based on exposure to malaria during pregnancy.ResultsOf the 250 pregnant women, 135 had a Plasmodium infection detected by light microscopy during pregnancy (39% Plasmodium falciparum only, 33% Plasmodium vivax only and 28% mixed/other species), defined as cases. The antibody responses to all six antigens accurately identified the women who did not have a malaria infection detected during pregnancy (93%, 107/115 controls). Antibody responses to P. falciparum merozoite surface protein 3 (PfMSP3) and P. vivax apical membrane antigen 1 (PvAMA1) were the least dynamic. Antibody responses to the antigens P. falciparum apical membrane antigen 1 (PfAMA1) and PfVAR2CSA were able to identify the majority of the cases more accurately (63%, 85/135).ConclusionThese findings suggest that the combination of antibodies, PfAMA1 and PfVAR2CSA, may be useful for sero-surveillance of malaria infections in pregnant women, particularly in low malaria transmission settings. Further investigation of other antibody markers is warranted considering these antibodies combined only detected 63% of the malaria infections during pregnancy.

STRIDE: a command-line HMM-based identifier and sub-classifier of Plasmodium falciparum RIFIN and STEVOR variant surface antigen families

BackgroundRIFINs and STEVORs are variant surface antigens expressed by P. falciparum that play roles in severe malaria pathogenesis and immune evasion. These two highly diverse multigene families feature multiple paralogs, making their classification challenging using traditional bioinformatic methods.ResultsSTRIDE (STevor and RIfin iDEntifier) is an HMM-based, command-line program that automates the identification and classification of RIFIN and STEVOR protein sequences in the malaria parasite Plasmodium falciparum. STRIDE is more sensitive in detecting RIFINs and STEVORs than available PFAM and TIGRFAM tools and reports RIFIN subtypes and the number of sequences with a FHEYDER amino acid motif, which has been associated with severe malaria pathogenesis.ConclusionsSTRIDE will be beneficial to malaria research groups analyzing genome sequences and transcripts of clinical field isolates, providing insight into parasite biology and virulence.

Protein Microarrays as a Tool to Analyze Antibody Responses to Variant Surface Antigens Expressed on the Surface of Plasmodium falciparum-Infected Erythrocytes.

Enzyme-linked immunosorbent assays (ELISAs) remain the gold standard for measuring antibodies, but are time-consuming and use significant amounts of precious sample and reagents. Protein microarrays represent an appealing alternative, particularly for studies focused on large gene families such as those encoding variant surface antigens in the malaria parasite Plasmodium falciparum. Such microarrays represent an ideal high-throughput platform to study antibody responses to hundreds of malaria parasite variant surface antigens at once, providing critical insights into the development of natural immunity to malaria. We describe the essential background and approach to run an assay using a P. falciparum microarray populated with variant surface antigens. This allows the user to define serologic profiles and identify serodominant antigens that represent promising targets for vaccine or therapeutic development.

Analysis of var Gene Transcript Patterns by Quantitative Real-Time PCR.

Quantitative real-time PCR (qPCR) is a simple and sensitive method for determining the amount of a specific target DNA sequence present in a sample. Compared to RNA-seq, reverse transcription qPCR (RT-qPCR) is fast, requires only low input material and is easy to analyze. Therefore, qPCR is widely used to analyze gene expressionin P. falciparum, including analyses of the multicopy gene families encoding variant surface antigens (VSAs), whose expression is clonally variant and prone to changes over time. In the recent years, several P. falciparum genomes of culture-adapted strains have been sequenced, providing the knowledge to design variable gene family-specific qPCR primers for each P. falciparum genetic background. Here, we describe the required materials, methods and key factors to perform RT-qPCR experiments to determine VSA transcript abundances in the P. falciparum clones 3D7/NF54, IT4, HB3, and 7G8.

Antibody-Dependent THP-1 Cell-Mediated Phagocytosis of Plasmodium falciparum-Infected Erythrocytes.

Antibodies that recognize variant surface antigens (VSAs) expressed on Plasmodium falciparum-infected erythrocytes (IEs) opsonize IEs for phagocytic clearance. The anti-VSA antibodies promote antibody-dependent cellular phagocytosis (ADCP) of IEs by interacting with innate immune cells. ADCP is an important immune effector mechanism of parasite clearance. ADCP can be a tool to assess the efficacy of vaccine-induced antibodies, in addition to measuring the neutralizing ability of antibodies. Here, we developed and validated an efficient and high-throughput plate-based flow cytometric assay to measure ADCP of IEs using the human monocytic THP-1 cell line. This flow cytometric assay can be used to analyze the level of naturally acquired or vaccine-induced opsonic antibodies in large cohorts.

Selecting Plasmodium falciparum Infected Erythrocytes for Adhesion to Cell Lines.

Plasmodium falciparum expresses variant surface antigens on the surface of mature infected erythrocytes (IEs) for binding to various receptors on host cells (cytoadhesion) to evade host immunity. This enables IEs to sequester in the microvasculature of different organs and tissues of the host, contributing to different outcomes of disease. The in vitro study of cytoadhesion involves the use of IEs and human endothelial cells or other cell lines that express host cell receptors. To enrich for IE populations that bind to certain cell types or receptors, we describe a method for panning mature pigmented trophozoite IEs on cell lines. The method enables coculturing of IEs with cells of interest and the selection of IEs that cytoadhere for continuous culturing. The method serves as a tool for generating IEs with specific cell or cell receptor adhesion phenotypes to allow detailed studies of cytoadhesion interactions.