Trichomonas vaginalis is a unicellular, flagellated, microaerophilic protozoan that extracellularly colonizes the human urogenital tract, causing trichomoniasis, a highly prevalent sexually transmitted infection (STI). This chapter explores the parasite's complex membrane structures and compositions, including the undulating membrane and its association with paracostal filaments and the costa. Key organelles such as the Golgi apparatus, lysosomes and hydrogenosomes are presented, detailing their structure, composition and biochemical activities. Unlike most eukaryotes, T. vaginalis lacks mitochondria, and instead, its hydrogenosomes are responsible for ATP synthesis, playing a critical role in the parasite's energy metabolism. The role of the plasma membrane in the endocytosis is addressed, alongside the involvement of the cytoskeleton and associated biochemical changes. Additionally, the chapter cover the parasite's interactions with host cells, focusing on the ameboid form of T. vaginalis. It emphasizes the morphological and structural transformations of the plasma membrane that occur during the transition from the piriform shape. Different types of vesicles associated with the plasma membrane, involved in host-parasite interactions, particularly their roles as adhesion molecules and in vesicular transport, are also discussed. The pseudocyst form of T. vaginalis, found under specific microenvironmental conditions, is also highlighted. In this form, the parasite internalizes its flagella, becoming rounded. The plasma membrane structure, composition and organelle modifications, as well as its relationship with host cells are highlighted. Furthermore, the implications of these transformations for the parasite's survival, immune evasion and pathogenic mechanisms are thoroughly reviewed, providing insights into how these membrane-associated adaptations enhance the parasite's pathogenicity.

The development of vaccines to prevent diseases caused by parasites is urgent. Current treatments are highly toxic and ineffective. In addition, these diseases are more prevalent in vulnerable populations and can be fatal in children and immunocompromised individuals. Vaccines for parasites are a challenge in several aspects, including their complex life cycle and mechanisms of evading the immune response. Extracellular vesicles (EVs) are particles released by cells that carry different biomolecules, thus participating in cell-cell communication. EVs released by parasites play a role in the parasite-host interaction. Parasite molecules carried by EVs interact with host immune cells, activating or modulating their function. Thus, unraveling the role of these EVs in immunity could contribute to the identification of molecules with vaccine potential, leading to the development of EV-based vaccines to prevent parasitic diseases. In this chapter, we will discuss the main studies and findings on the protective role of parasite-derived EVs in vaccine preparations.

Extracellular vesicles (EVs) are particles formed by a lipid bilayer, released by various types of cells, plants and pathogens, with their main function being intercellular communication. EVs transport proteins, nucleic acids, and lipids and are potential candidates for clinical applications, therapies, and vaccines. EVs have been extensively studied as biomarkers for disease diagnosis and prognosis because they are found in most biological fluids and contain markers from the cell that released the particle. In the therapeutic field, modified EVs are used as drug delivery systems, offering advantages such as biocompatibility, immunogenicity, and the ability to cross biological barriers. Additionally, vaccines using EVs are being developed due to their immunomodulatory properties, which can induce protective immune responses against infectious diseases and cancer. Despite these advances, there are significant challenges, such as large-scale production, standardization, and approval by regulatory agencies. In this chapter, we describe the use of EVs as biomarkers, therapies, and vaccines against pathogens and cancer, as well as the challenges that scientists encounter in their application. Research on EVs is constantly advancing, and their use represents a revolution in precision medicine. Innovation in this field has the potential to transform diagnostic and therapeutic approaches to various diseases.

The plasma membrane (PM) of Leishmania spp. is a highly specialized structure that plays a crucial role in the parasite's survival and adaptation as it transitions between its invertebrate and vertebrate hosts. The unique composition of lipids, sterols, and surface proteins in the Leishmania PM is essential for parasite's ability to thrive and cause disease. This chapter provides an in-depth exploration of the molecular structure and functions of the Leishmania PM, integrating the latest research on its composition and biological roles. It highlights the differences between the two infective forms, promastigotes and amastigotes, which exhibit distinct surface molecule profiles and membrane adaptations suited to life in the sand fly vector and the mammalian host. Key molecules, such as ergosterol, GPI-anchored lipophosphoglycan (LPG), metalloprotease GP63, and other PM molecules, are discussed in the context of immune evasion, host cell entry, intracellular survival, and vaccine development. The chapter emphasizes how understanding the structure and function of the Leishmania plasma membrane can lead to the development of new strategies for treating and preventing leishmaniases.

Extracellular vesicles (EVs) are membrane-bound nanostructures secreted by various cell types under physiological and pathological conditions. These vesicles carry a diverse cargo of biologically active molecules, including proteins, lipids, nucleic acids, and metabolites. The molecular and structural heterogeneity of EVs presents challenges in fundamental biology, biomarker development, and therapeutic applications. Fungal EVs have gained attention for their roles in pathogenesis, immune modulation, and potential targets for therapies and vaccines. EVs have numerous roles in intercellular communication, facilitated by the transfer of cargo to recipient cells or the interaction of EV surface proteins with cellular receptors. However, the question of how they traverse the cell wall remains a mystery. Fungal EVs can modulate the cell wall through enzymes, contributing to the transition of EVs by the fungal cell wall. As research progresses and technological barriers are overcome, EVs are emerging as valuable targets and promising tools in precision medicine. With continuous improvements in EV isolation, characterization, and manipulation, the next decade is likely to bring significant breakthroughs that will have a profound impact on both basic science and clinical practice.

Extracellular vesicles (EVs) play a central role in the host-parasite interplay during Trypanosoma cruzi infection and the progression of Chagas disease. Released by both parasite and host cells, EVs modulate immune responses, promote parasite persistence, and sustain inflammation through their diverse cargo, including proteins, lipids, and RNAs. Parasite-derived components such as MASP, trans-sialidases, and TESA, as well as small non-coding RNAs, have been identified within EVs and are increasingly explored as diagnostic and prognostic biomarkers. Their stability in biological fluids and parasite-specific content offer advantages over conventional serology, particularly for chronic disease stages. Experimental models further demonstrate that EVs influence cytokine profiles, enhance parasitemia, and affect complement resistance, highlighting their dual role in immune modulation and disease outcome. Beyond pathogenesis, EVs hold promise as antigen sources for serological assays, as non-invasive biomarkers for disease monitoring, and as innovative platforms for vaccine and therapeutic development. Together, these findings underscore the translational potential of EVs in advancing diagnosis, prognosis, and treatment strategies for Chagas disease.

A key event in Leishmania pathogenesis is the interaction between parasite surface molecules and host immune cells. Among these, lipophosphoglycan (LPG), the dominant surface glycoconjugate of Leishmania promastigotes, plays a central role in establishing infection and evading immune responses. Beyond mediating adhesion and protection, LPG actively modulates host membrane receptors and intracellular signaling pathways. This chapter explores the multifaceted strategies by which LPG alters host cell membrane organization to promote parasite survival. By engaging various surface receptors, LPG facilitates parasite uptake while simultaneously inhibiting classical activation pathways. It redirects intracellular signaling toward anti-inflammatory responses, suppressing the production of key molecules required for parasite elimination. Unraveling these interactions provides critical insight into the mechanisms of immune evasion and persistent infection, and opens new avenues for therapeutic intervention.

Extracellular vesicles (EVs) are nano-sized, membrane-surrounded vesicles released by cells under both physiological and pathological conditions. Due to their small size and heterogeneity, comprehensive characterization of EVs remains technically challenging. Among the various analytical tools developed, flow cytometry stands out as a highly versatile and scalable platform, offering high-throughput analysis, multiparametric phenotyping, and quantitative detection. However, conventional flow cytometers are typically designed for cell-sized particles (0.5-40 µm) and require specific optimizations to reliably detect and analyze EVs, which are significantly smaller and result in weaker signals. These optimizations include instrument settings, sample handling and labelling strategies as well as acquisition protocols. Robust calibration and the use of appropriate controls are essential to ensure data accuracy and reproducibility across platforms. In this chapter, we outline the principles, technical considerations, and advantages of applying flow cytometry and imaging flow cytometry to EV research. We also highlight representative applications in both scientific and clinical contexts and discuss future directions for the field.

Plants release extracellular vesicles and numerous investigations have reported their characterization, either isolated from the apoplastic fluid of different plant sources, the phloem sap or in vitro plant cultures. The term plant derived nanovesicles is applied to vesicles isolated from fruit juices or from homogenized plant tissues/organs. Plant derived nanovesicles share similar components with canonical plant vesicles, including proteins, lipids, nucleic acids, carbohydrates and secondary metabolites, which reflect the composition of the parental tissues/cells. In recent years, several studies have dealt with potential biomedical applications of plant derived nanovesicles, including their use as delivery agents, including vaccines, and their use as therapeutics (like in inflammation conditions and cancer), as well as mediators in regenerative medicine. Furthermore, their use in cosmetics is also gaining attention. Although plant derived nanovesicles have emerged as promising biomaterials for the pharmaceutical industry, critical aspects hinder the rapid translation of basic and preclinical studies to a clinical setting. They include the precise identification of bioactive compounds responsible for the effects detected in vitro, and studies are required to evaluate their effect in humans. In addition, it is necessary to develop protocols to optimize their production in a scalable, sustained and adequate cost-effective relation.

Extracellular vesicles (EVs) have emerged as key mediators in cancer biology, playing critical roles in intercellular communication within the tumor microenvironment. These nano-sized particles carry diverse molecular cargos, including proteins, lipids, DNA, and various RNA species, which reflect the biological state of their cells of origin. This chapter provides a comprehensive overview of the diagnostic and prognostic potential of EVs across a wide spectrum of tumor types, following the World Health Organization (WHO) tumor classification. We discuss the contribution of EVs in genetic tumor syndromes, solid tumors (such as skin, breast, digestive, and thoracic cancers), hematolymphoid malignancies, and pediatric cancers. Special emphasis is placed on the utility of EVs in liquid biopsy applications, offering minimally invasive alternatives for early diagnosis, monitoring of disease progression, treatment response, and detection of relapse. Furthermore, the chapter highlights specific EV-associated biomarkers, including proteins, microRNAs, long non-coding RNAs, and circular RNAs, identified in various biofluids such as blood, urine, saliva, and cerebrospinal fluid. Despite the promising potential of EVs as clinical tools, several challenges remain, including standardization of isolation and characterization methods, biological heterogeneity of EV populations, and the need for large-scale validation studies. Addressing these hurdles will be critical for the successful translation of EV-based biomarkers into routine oncology practice.

Viruses are subcellular structures that depend on the host cell to replicate, sharing several characteristics with extracellular vesicles (EVs). EVs act in intercellular communication and the regulation of the immune system and can be exploited by viruses as vehicles for transport and dissemination between cells and organs. Although they may favor viral infection, EVs also stand out as potential biomarkers for the diagnosis of viral diseases, as they reflect the physiological and pathological state of the originating cells. These particles can contain viral RNA and specific proteins, allowing for the distinction between different types of infection. Moreover, EVs have great therapeutic potential and are being studied as nanotherapeutic tools due to their low immunogenicity, ability to cross cellular barriers, and ease of modification to allow delivery around the body and tissues. This chapter addresses the interactions between EVs and viruses, as well as the advancements in the use of these structures in diagnosis and the development of new therapeutic strategies. Understanding these mechanisms can significantly contribute to the control of viral infections and the creation of innovative therapies to treat emerging diseases.

Parasitic helminths display remarkable plasticity in their interactions with the host immune system. Zoonotic species can elicit markedly different immune profiles depending on the host, ranging from balanced responses that allow long-term parasite persistence with minimal pathology to concomitant responses leading to rapid clearance accompanied by varying degrees of inflammation and/or fibrosis. Central to this host-specific immunomodulation are helminth-derived excretory/secretory products (ESPs) and extracellular vesicles (EVs), which carry a diverse repertoire of bioactive molecules capable of modulating key immune pathways. These mediators influence both innate and adaptive immunity, promoting regulatory, type 2, or mixed inflammatory responses according to the host context. This review synthesizes current evidence on how zoonotic helminths employ ESPs and EVs to fine-tune immune outcomes across natural, accidental, and experimental hosts. Elucidating these host-dependent dynamics offers valuable insights into parasite adaptation, the clinical manifestations of zoonotic infections, and the potential use of helminth-derived molecules as innovative immunotherapeutics.

Giardia intestinalis is an extracellular parasite that inhabits the human intestinal tract, with trophozoite and cyst stages in its life cycle. In this chapter we review basic aspects of structural organization, integrating information of the role of the plasma membrane in various aspects related to its composition, function, and importance at different stages, from the trophozoite form, involvement in encystation, to interactions with the host. Additionally, the membrane's composition, biochemical activities, receptors, and various functions it performs at different stages will be thoroughly explored. The parasite exhibits a unique and fascinating organelle: the peripheral vesicles (PVs). The membranes of these PVs will be explored, foscusing in how they drive endocytic uptake, mediate exocytic release, and carry out lysosomal degradation, all of which are essential for maintaining cellular homeostasis. Additionally, the membranes of the endoplasmic reticulum and their critical role in protein maturation and compartmentalization, both vital for proper cellular functions, will be addressed. Another key role of membranes to be explored is in the encystation process, with the presence of encystation-specific vesicles (ESVs), which are crucial in the life cycle of G. intestinalis, enabling survival in hostile conditions. The transformation of these vesicles and their contribution to protein maturation, ensuring the infectivity and resistance of the parasite, will offer a comprehensive understanding of the mechanisms underlying this parasite's survival and adaptation. The modulation of Giardia's membranes during the adhesion process to host cells will also be addressed, along with the variant surface proteins (VSPs), which are key players in the parasite's immune evasion mechanisms.

Extracellular vesicles (EVs) are considered as cornerstones of cell-cell communication. EVs are bio-membrane naturally occurring vesicles generated by both eukaryotic and prokaryotic cells to transfer myriad cargo between close and distant cells to modulate them genetically or at functional levels. Recently, attention has been attracted towards the use of EVs derived from different sources like mammalian, bacterial and plant cells for drug delivery and diagnostic applications. Although all types of EVs vary in their compositions and biogenesis, they have similar features which made them desirable and novel theranostics. Today, breast cancer is a challenging disease to humans around the world, hence, accurate and timely diagnosis and selecting the most appropriate therapeutic plan are pivotal steps forward reducing mortality. In this chapter, we have focused on unique features of EVs to be employed in the diagnosis of breast cancer because of the presence of the data inside the EVs which are related to their origin cells. Also, imaging agents can be enclosed by EVs, providing a high sensitive and reproducible imaging technique. Moreover, EVs are recruited as novel strategies to reach chemotherapeutic molecules to breast cancer in targeted drug delivery. Lastly, by discussing the pros and cons of various types of EVs as theranostics, we shed a light on the role of EVs in breast cancer and underlined the challenges that have to be overcome.

Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are the trypanosomatid parasites responsible for some of the most significant neglected tropical diseases, such as trypanosomiases and leishmaniases, which impact millions of people globally. Alarmingly, some of these diseases have expanded into previously unaffected regions in recent years. These parasites alternate between invertebrate and vertebrate hosts during their life cycles, adapting to different environments and competing with their hosts for several nutrients. To survive, they have evolved complex strategies to acquire essential nutrients, often subverting host immune defenses and overcoming host-imposed nutritional barriers. This chapter explores the membrane-dependent mechanisms of nutrient sensing and uptake in T. brucei, T. cruzi, and Leishmania spp., with an emphasis on how these parasites adapt to nutrient-limited conditions within their host. Following an overview of the challenges posed by host imposed nutrient restrictions, we examine the parasites' membrane-associated processes and metabolic adaptations that enable their survival. The chapter spans a wide range of micro- and macronutrients-lipids, fatty acids, carbohydrates, amino acids, and metals-discussing the roles of membrane proteins in nutrient scavenging, the metabolic pathways they trigger, and their physiological importance for parasite survival, growth, and infectivity. Special attention is given to the mechanisms by which these parasites evade nutritional immunity, a host defense strategy that limits nutrient availability to pathogens. By shedding light on these nutrient acquisition strategies, this chapter aims to advance our understanding of host-parasite interactions and identify potential targets for therapeutic interventions aimed at the metabolic vulnerabilities of these parasites.

Outer membrane vesicles, released by Gram-negative bacteria, have attracted increasing attention in biotechnology due to their structural similarity to bacterial cells, their composition rich in immunogenic factors, and their role in pathogen-host interactions. Although they are involved in multiple functions related to bacterial pathophysiology, their applicability as vaccine platforms has emerged as a promising strategy for the development of next-generation vaccines. OMVs offer significant advantages over traditional vaccines, including the induction of robust T cell-mediated immune responses, the natural presence of pathogen-associated molecular patterns with adjuvant effects, and the possibility of bioengineering to display heterologous antigens. Preclinical trials using OMVs have demonstrated effective protection against infection, highlighting their versatility and safety. In addition, their stability, lack of replicative capacity, and ease of production make OMVs a highly attractive platform, including for emerging diseases and applications in cancer immunotherapy. This chapter discusses the structural and functional aspects of OMVs, with emphasis on their innovative potential in the vaccine field, while also addressing the technological challenges related to their standardization, purification, and industrial scale-up.

The initial interaction between host cells and Leishmania infective rforms is dependent on surface proteins from both organisms. Membrane proteins are fundamental molecules that perform a variety of functions, including recognition, adhesion, and host cell penetration, as well as nutrient and enzyme transport and cell signaling. Several Leishmania plasma membrane proteins play critical roles in host interaction, parasite survival, and virulence during the early stages of infection. Among them, the most prominent is GP63, which confers resistance to complement-mediated lysis and induces macrophage phagocytosis. Another important surface protein, prohibitin, has a role in macrophage infection and has demonstrated the ability to generate a humoral response in human patients, making it a potential diagnostic marker. Furthermore, prohibitin is considered a promising target for vaccination against L. infantum. The kinetoplastid membrane protein 11 (KMP11) has also been identified as a potential B- and T-cell immunogen during infection. The analysis of the membrane proteome profile of Leishmania promastigotes could offer a more comprehensive understanding of host-parasite interactions and Leishmania biology. Despite membrane proteins constituting 20-30 % of the proteome in most organisms, there are relatively few proteomic studies on Leishmania parasites that focus on membrane-associated proteins, even though these proteins are potential drug targets. This review provides a survey of the current knowledge regarding the composition of plasma membrane focusing, in alphabetical order, on those proteins that are best characterized in terms of functionality in Leishmania.

Extracellular vesicles (EVs) are emerging as key players in the pathogenesis of malaria and toxoplasmosis, two significant infectious diseases caused by Apicomplexa parasites. This chapter investigates the multifaceted roles of EVs in the progression of these diseases, emphasizing their involvement in immune modulation, hostparasite interactions, and the regulation of disease severity. In malaria, EVs derived from infected red blood cells, platelets, and endothelial cells contribute to disease symptoms, immune response modulation, and parasite survival and have potential as biomarkers and tools for vaccine development. Similarly, in toxoplasmosis, EVs influence the modulation of immune responses and disease progression, presenting distinct profiles depending on the Toxoplasma gondii strain. Notably, EVs from both parasites contain immunogenic proteins that can be used in vaccine development, with promising results in preclinical studies. The role of EVs in these parasitic infections highlights their potential as therapeutic targets and diagnostic tools, providing new opportunities for the prevention and treatment of malaria and toxoplasmosis.

The genome of the Trypanosoma cruzi parasite exhibits a significant expansion of genes that encode surface proteins in comparison to other trypanosomatids, specifically Trypanosoma brucei and Leishmania. Many of these proteins are encoded by large and diverse gene families, predominantly expressed on the surface of the trypomastigote stage, which infects a variety of mammalian host cells and circulates in the bloodstream, disseminating the infection throughout the organism. While some members of these families may be found at the telomeres, the majority are clustered in long arrays of genes within the chromosomes. These regions, referred to as disruptive compartments, undergo more rapid evolution than the core compartments, which are enriched in conserved and housekeeping protein coding-genes common to other trypanosomatids. In this chapter, we will discuss the features and process underlying the variability of the largest T. cruzi gene families and its implications for parasite survival.