Some parasitic diseases, their symptoms and treatment options.
Being the cause for significant amount of morbidities and mortalities, parasitic diseases remain the major challenge for the healthcare community due to the limitations associated with the current chemotherapeutics. Drug discovery/invention can be achieved by collaborative efforts of biotechnologists and pharmacists for identifying potential candidates and successfully turn them into medicine for improving the healthcare system. Although molecular medicine for disease intervention is still in its infancy, however, significant research works and successful trials in short span of time have made it broadly accepted among the scientific community. This chapter identifies different molecular medicine approaches for dealing with parasites that have been coming up on the horizon with the new technological advances in bioinformatics and in the field of omics. With the better understanding of the genomics, molecular medicine field has not only raised hopes to deal with parasitic infections but also accelerated the development of personalized medicine. This will provide a targeted approach for identifying the druggable targets and their pathophysiological importance for disease intervention.
- CRISPR/Cas9 system
- monoclonal antibody
- immune checkpoint inhibitors
Parasitic diseases remain the threat to global healthcare sector, with considerable mortalities and morbidities associated with these diseases. Combating parasitic infection relies mostly on conventional chemotherapeutic approaches; however, an exponential rise in the number of recrudescent cases, lack of vaccines and toxicities issues associated with chemotherapies emphasized the need for the research to develop alternative strategies. It is noteworthy that emergence of drug resistance is not new; microbes have been evolving since ages, by knocking out one or the genes. In context of emerging drug resistance, World Health Organization (WHO) has warned for the upcoming “post-antibiotic era”, therefore, molecular medicine has surged. Molecular medicine is the application of gene and/or DNA based information for therapeutic purpose. It involves the study of molecular mechanisms, identification of erroneous genetic and/or molecular pathways and development of molecular intervention with the aim to improve disease management.
This chapter provides an insight into how the anomalies in molecular pathways can be targeted leading to discovery of potential candidates for development of clinical medicine and innovative therapies to improve disease management strategies.
2. Evolution of molecular medicine
The field of molecular medicine evolved over a period of time since the discovery of DNA (in 1953) and recombinant DNA technology. Another major breakthrough in 1975, when it was discovered that DNA can be read base by base through the sequencing technique. Later, in 1985, it was known that DNA can be amplified with PCR, and this was major achievement in the field of molecular diagnostics. This journey gained pace with the advent of automated DNA sequencing in 1987 that served as the background for the human genome project (HGP) in 1990. The journey of HGP started the era of modern molecular medicine with the first successful gene therapy. In 1995, the success of unveiling the DNA sequence from the first model organism (
This led to the use of increasing number of analytical platforms for DNA sequencing termed as the next generation sequencing platforms. Further, metagenomics approaches—omics and/or shot-gun approaches paved the way for the third-generation sequencing, that aimed to reduce the sequencing costs. These advances gained momentum with the computational approaches where synthetic biology has remarkably facilitated the DNA based analysis as well as the development of models for drug testing.
In order to deal with the limitation of the existing chemotherapeutic approaches there remains an urgent need for the conversion of biomedical knowledge into clinical application. Molecular medicine provides the opportunity to fill the gap between the basic research and the clinical application for the diagnosis, prevention and treatment of diseases. It involves the combinatorial application of pharmacology, biomedical and omics technologies for understanding and improving the molecular basis of the disease pathogenesis that will serve in designing disease intervention strategies. Development of molecular drug is a complex process that involves multidisciplinary effort including high throughput screening, chemical synthesis, modification, omics technologies, data mining, structure-based drug designing, phenotypic screening, target and lead identification and validation, etc. The development of molecular medicines involves following steps—first, the identification of target, potential tractability of target (i.e. identifying targets that are more druggable than others, depending upon their chemistry), establish genetic association of target with disease pathophysiology (some targets required for drug action may not necessarily associate with disease genetics) and validation of target (by establishing association of target with the disease development/persistence). Validation of target usually involves different molecular approaches to understand the role of target gene or protein in diseases pathophysiology. Overall, it is an interdisciplinary branch where recent technical advances have served as the milestone in gaining insight into the phenomenon of disease pathogenesis and development of innovative therapeutic measures.
Parasitic diseases are amongst the common infections in humans caused by protozoan and helminthic parasites. The causative agents, parasites, are diverse ranging from single celled protozoan to worms that be seen with naked eyes. Till the end of nineteenth century, parasitologists were mainly focused on understanding their life cycle; however, the concept took turn when some parasites were found to be associated with several human diseases that led to significant morbidity and mortality. Parasitic diseases are cosmopolitan, that may affect any part of world however, mostly the diseases are common in tropical countries, but tourism and migration can transmit them outside their geographical boundaries. The signs and symptoms of disease may not be obvious, and it may vary from mild abdominal pain to chronic hepatomegaly and eventually death. Some parasitic infections are easily treated while others are not. In the light of the lack of vaccine for parasitic infection, proper prophylactic measures (proper hygiene, prevention of contaminated food, water, preventing consumption of undercooked food, use of bednets, insecticide spraying to prevent vector borne diseases, etc.) and active disease surveillance remains the key for disease elimination. Unfortunately, poor disease management strategies have made parasitic infections a global healthcare challenge. In this article it’s only possible to cover some important parasites (Table 1), for which research on molecular medicines are underway.
|Diseases||Causative agent (pathogen)||Transmitting agent (vector)||Manifestation||Treatment options|
|Leishmaniasis (visceral, cutaneous and mucocutaneous)||Sandfly (||Fever, anemia, splenomegaly, lymphadenopathy; cutaneous forms manifests as skin lesions and ulcers||Liposomal amphotericin B, miltefosine, antimonials; fluconazole, itraconazole|
|Malaria||Female mosquito ||Headache, fever, paroxysm, joint pain, anemia, jaundice; neurological symptoms in severe cases||Chloroquine, mefloquine, doxycycline|
|Chagas disease (American trypanosomiasis)||Kissing bugs (Triatominae)||Fever, malaise, enlargement (liver, spleen, lymph nodes), sometimes skin nodules (chagoma); chronic stages affects the brain, heart and digestive system||Benznidazole, nifurtimox|
|Human African Trypanosomiasis||Tsetse fly (||First stage-intermittent fever, headache, swelling of lymph nodes, joint pain; second stage involves neurological symptoms||Pentamidine, suramin, fexinidazole, nifurtimox, eflornithine|
|Toxoplasmosis||Oral route; transmitted by ingestion of parasite oocyst||Headache, fever, fatigue, muscle ache; skin manifestation includes erythema and roseola||Pyrimethamine, sulfadiazine, clindamycin, spiramycin|
|Trichomoniasis||Genital contacts||Pain, itchiness/burning in genitourinary organs, urethritis, prostatitis (in males) while frothy, foul-smelling discharge, vaginitis (in females)||Metronidazole|
|Giardiasis (beaver fever)||Feco-oral transmission by ingestion of cysts||Chronic diarrhea, abdominal cramps, nausea and vomiting||Nitroimdazole, quinacrine, furazolidone, paromomycin|
|Cryptosporidiosis||Oral transmission by consumption of contaminated water, undercooked food||Diarrhea, abdominal cramps, low-grade fever (in intestinal cryptosporidiosis); inflammation of nasal mucosa, cough, shortness of breath, hypoxemia(respiratory cryptosporidiosis)||Electrolyte replacement by rehydration therapy, nitazoxanide, azithromycin, paromomycin|
|Amoebiasis||Feco-oral route||Diarrhea, severe abdominal pain||Amebicidals (metronidazole, tinidazole) and cysticidal agents (iodoquinol)|
|Roundworm infection (in murine)||Skin penetration||Emphysema, loss of alveolar septa, lung hemorrhage||Tetramisole|
|Ascariasis (Roundworm infection, in human)||Feco-oral route||Fever, cough, weight loss, abdominal discomfort, intestinal ulcer accompanied with eosinophilia||Albendazole, mebendazole|
|Fasciolosis||Oral route, consumption of contaminated food||Acute phase marked by fever, nausea, skin rashes, abdominal pain; chronic phase manifests as jaundice, anemia and intermittent pain||Bromofenofos, triclabendazole, bithionol|
|Taeniasis||Consumption of undercooked pork or beef||Mild (abdominal pain and nausea) to no symptoms||Praziquantel, albendazole, niclosamide, mepacrine|
|Onchocerciasis (sub-cutaneous filariasis)||Blackfly (||Itchiness and bumps and depigmentation in skin to blindness||Ivermectin, moxidectin|
|Filariasis (lymphatic and serous cavity)||Blackflies and mosquitoes||Edema with skin thickening and underlying tissues||Diethylcarbamazine citrate (DEC)|
|Neural angiostrongyliasis (eosinophilic meningitis)||Oral route; upon ingestion larvae in undercooked prawn, snails, slugs, frogs||Headache, fever, malaise, nausea, neck stiffness, varying degree of neurological dysfunctions||No specific treatment, supportive care helps reduce the severity of symptoms|
|Schistosomiasis||Contact with fresh water contaminated with parasites (released from fresh water snails)||Abdominal pain, diarrhea, fever, cough, bloody stool and/or blood in the urine||Praziquantel, oxamniquine, metrifonate, artesunate, mefloquine|
|Trichinosis||Consumption of undercooked pork||Nausea, vomiting, fever, diarrhea, facial swelling||Mebendazole, albendazole|
3. Molecular medicinal strategies and parasitic diseases
Parasitic infections (protozoan and helminthic infections) affect more than a quarter world population and cause chronic illness primarily in developing countries of world. These diseases affect the quality of life and treatment costs possess economic burden on families leading to viscous circle of poverty.
Molecular medicine is a broad field that includes insight into the molecular aspect of diseases. Recombinant DNA and cloning technologies are the conventional tools for studying the disease associated molecular profiles. Recent technical advances have paved the way for utilization of several molecular strategies for treating infectious diseases. Molecular medicine aims to understand the molecular basis of disease pathogenesis and allows the utilization of the information in designing specific diagnostic, therapeutic and prophylactic options. Mainly molecular medicine relies on two strategies—targeting genome and targeting signaling pathways, as targeted approach of disease management. Thus, it aims to improve the human health through the understanding of mechanism in human diseases.
3.1 Targeting genome
Apart from conventional approach of gene therapy (replacement of defective gene by exogenous DNA and editing mutated gene), recent technical advances have opened the arena for other strategies of manipulating the gene expression. Gene editing methods have gained limelight that involves the intrinsic molecular repair processes within the cell. The process of break repair in the DNA involves the homology-directed repair (HDR) and/or non-homologous end joining (NHEJ). The key step in gene-editing tool involves the precise introduction of double strand breaks. This process involves the use of engineered meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the recent CRISPR/Cas system . Further, short antisense oligonucleotides potentially serve as tools for abrogating the transcription of target gene. As compared to other genome editing methods CRISPR/Cas system using guide RNA has shown immense potential for future of molecular medicine.
3.1.1 Engineered meganucleases
Although there remains plethora of meganucleases to choose from, however, most commonly used meganucleases include the ZFNs that have DNA binding zinc finger protein domain and nuclease domain. Cys2-His2 zinc finger domain is amongst the most abundant types of DNA binding motifs in eukaryotes [3, 4]. The ZFNs work by binding to the DNA and cleaving it, which then undergoes repair by either homologous recombination or error-prone NHEJ . Site-specific cleavage is induced by manipulating the ZFN complex to recognize two sequences that are on either side of target site. Upon identification, cleavage of genome is induced by restriction enzyme (FokI), thus generating double stranded breaks in genome that can be used for editing the region.
TALEN has been introduced as an alternative to ZFNs. These are similar to ZFNs in using restriction enzyme that incorporates with the DNA binding domain, but are of different origin. Similar to ZFNs, these protein structures lead to the double stranded breaks in DNA for genome editing . Unfortunately, there are no evidences for the use of the aforementioned meganucleases for the development of molecular medicine for parasitic diseases.
3.1.2 RNA-guided engineered nucleases (CRISPR-Cas9 system)
After the discovery of clustered regularly interspaced palindromic repeats (CRISPR) (in 1987) as a part of bacterial immune system against invading viruses. This strategy has potential application in editing human chromosome with great accuracy. It is RNA guided gene editing tool that uses Cas9 endonuclease for generating double-stranded breaks at loci of interest, which are then repaired via. HDR (using a template) or NHEJ or error-prone microhomology-mediated end joining (MMEJ) . Thus, leading to mutation (insertion, deletion or substitution) with no or minimal damage to host genome. Since the sequencing of the parasite genome, CRISPR/Cas9 genome editing tool accelerated the molecular research in parasitology.
Genome editing in malarial parasite (
Genome editing using CRISPR/Cas9 system in
3.1.3 RNA interference (RNAi)
RNA interference is the approach of inhibiting the gene expression or translation by insertion of double-stranded RNA into the cells and/or organism that mediates targeted degradation of its homologous RNA. It can be done
Malarial parasites lack the conventional RNAi pathway however, antisense oligodeoxynucleotides treatment (against parasite topoisomerase) has shown to significantly reduce the parasite multiplication . Further, topoisomerase targeting antisense nanoparticles and chitosan-based nanoformulation have also been used to inhibit
RNAi based silencing of key genes involved in regulating the parasite survival and development have been potential candidates for therapeutics. Several miRNA have been known to regulate the nematode development and survival in the host microenvironment due to their immunoregulatory potential. These have also been crucial players of host-parasite interaction and have been used as diagnostic marker of infection.
3.2 Targeting cells and signaling pathways
This area of molecular medicine remains the hottest area of research in the field of parasitic diseases after the World Health Organization (WHO) warning about the risk of post-antibiotic era. The search for novel therapeutic strategies intends to enhance pathogen killing by targeting regulatory molecules/pathways. Better understanding of disease immunobiology and cellular signaling will provide momentum to the identification of the pathways of therapeutic importance. This area of research towards the development of molecular medicine involves the use of genetically engineered antibodies, recombinant proteins, small molecules to alter signaling pathways, targeting the immunometabolic pathways, inflammasomes, etc.
3.2.1 Immunotherapeutic approach
Immunotherapy is use of biological substances (antigen/antibody, immunomodulators administration) to regulate host immune system in order to fulfill prophylactic and/or therapeutic purpose. Immunotherapy aims to trigger the immune power by directly (antigen based or active immunotherapy) or indirectly (antibody based or passive immunotherapy) [45, 46]. This section describes various immunotherapeutic strategies of molecular medicine that have been reported for the parasitic diseases.
188.8.131.52 Recombinant proteins/cytokine therapy
Cytokines are the small molecular weight, chemical messengers that regulates the immune responses in autocrine and paracrine manner. There is plethora of evidences for the involvement of cytokines in determining the pathophysiological consequences. Recombinant protein (cytokine) based therapy aims to trigger T-cell immune responses and induces parasite clearance.
Conversely, in helminthic infection, MAb (monoclonal antibody) based blocking of IL-4 and IL-10 has shown disease improvement by reducing parasitic burden and inducing TH1 immune responses . Likewise, IL-4 based MAb therapy, in schistosomiasis, has shown to inhibit granuloma formation and hepatic fibrosis . Similar findings of marked reduction in granuloma and hepatic fibrosis were reported upon treatment with
The significance of exogenous cytokine therapy has also been underlined in trichiasis, where IL-33 is known to induce thymic stromal lymphopoietin that generates polarized TH2 responses to confer protection against intestinal nematodes . While, IL-25 treatment instigated TH2 responses and restricted infection induced gastrointestinal inflammation , MAb based blockade of IL-10 ameliorated disease pathology. There are evidences for the IL-27 mediated suppression of T-cell proliferation thus IL-27 receptor (WSX-1) knock down improved the mucosal immunity . The use of immune triggering cytokines (IFN-γ, IL-12, GM-CSF) and/or blocking immunoregulatory cytokines that possesses pathological consequences holds hopes for the development of molecular medicine. Thus, therapeutic potential of cytokine therapy can be exploited alone and/or in combination with conventional chemotherapy opening up the avenues for improving treatment outcomes.
184.108.40.206 Immune checkpoint therapy
Immune checkpoint molecules are involved in regulating the T-cell activation and functions. The expression of these molecules is enhanced during chronic infections as a result of immune subversion, thus, therapeutically targeting these molecules has shown promising results in cancer and infectious diseases vaccines [60, 61]. Indeed, T-cell dysfunctionality or exhaustion is the key for impaired T-cell responses during chronic infections; exhaustion is marked by loss of IL-2 production, reduced cytotoxicity, impaired production of pro-inflammatory mediators and reduced proliferative ability. The expression of multiple immune checkpoint molecules (PD-1, CTLA-4, LAG-3, Tim-3, TIGIT) remains the hallmark feature of exhausted cells; elevated expressions of these molecules are accompanied with progressive loss of T-cell functionality . Immune checkpoint inhibitors have been novel strategy of reinvigorating the immune cell functions by abrogating the signaling by the immune checkpoint (or co-inhibitory molecules).
A number of immune checkpoint molecules have been reported in leishmaniasis including—LAG-3, Tim-3, CTLA-4, PD-1, etc. that negatively regulates T-cell functionality [63, 64, 65]. MAb based blockade of PD-1 and LAG-3 in malaria triggered pro-inflammatory cytokine responses and relieved T-cell inhibition . Likewise, therapeutically targeting LAG-3 and PD-L1 restored CD4+ T-cells functions, restored follicular helper T-cells, plasma cells eventually cleared the blood stage of
Unfortunately, this strategy of immune checkpoint therapy has been in its nascent stage for parasitic infections, and has yet not been used for HAT, Chagas disease, gastrointestinal protozoans as well as helminthic infections.
220.127.116.11 Immune cells and stem cell-based therapy
Immune cell manipulation offers another fascinating approach of molecular medicine to fight with parasitic diseases, when other treatment options fail to provide protective immunity [68, 69]. Direct transfer of immune cells has been holding great promises for conferring protection against protozoan, bacterial and viral infections . Adoptive T-cell transfer therapy using tumor-infiltrating lymphocytes is the best example to clinical success of cellular therapy .
DC (dendritic cell) based vaccination approach using parasite peptide (KMP-11) elicited TH1 responses, reduced parasite load and induced lymphocyte proliferation in leishmaniasis infection . Similarly, vaccination with DC along with histone H1 elicited pro-inflammatory responses (IFN-γ and IL-12), reduced the IL-10 and IL-4 producing cells and induced polarized TH1 responses . Atypical progenitor cells (IL-7R+ c-kit+ cells) from malaria infected mice are potent fighters against infection, while transplantation of these cells had similar effects in disease recovery .
After the success of direct administration of MSCs (mesenchymal stromal cells) and antigen specific T-cells stem cell therapy has recently budded in the field of infectious diseases. MSCs have been shown to be equally important in conferring resistance against
Transplantation of bone marrow mononuclear cells has marked effect on improving the inflammation and fibrosis in Chagas disease [78, 79]. Also, bone marrow transplantation holds promises for improving the quality of life in congestive heart failure due to Chagas disease [80, 81]. Adoptive immunotherapy in toxoplasmosis, by transferring CD8+ T-cells restricted parasite de-encystation; however, it failed to revert the T-cells exhaustion attributing to the short-lives of exhausted cells . Further, MSCs therapy in toxoplasmosis has not been successful however, when used in combination with the spiramycin, pyrimethamine and folinic acid provided therapeutic benefits. Similarly, for coccidiosis, using adoptive transfer strategy, intraepithelial lymphocytes (IELs) and CD4+ T-cells from interferon gamma knock out (
In helminthic diseases, MSC based therapy have been proven to be efficacious for reducing
Immunomodulators are small molecular inhibitors of signaling pathways that serve as molecular medicine for disease intervention. Imatinib, an Abl/Arg tyrosine kinase inhibitor, induces cytoskeleton remodeling to facilitate leishmanial parasite phagocytosis in the macrophages and reduces disease associated lesions . Another signaling pathway inhibitor, AS-605240 (PI3K gamma inhibitor) has shown to be as efficacious as sodium stibogluconate (SSG) in the treating of
Tyrosine kinase inhibitors (lapatinib) have proven their efficacies in controlling Human African Trypanosomiasis (HAT) pathogenesis by blocking parasite endocytosis . Furthermore, PI3Kγ/mTOR signaling inhibitors as NVP-BEZ235 restricts the
Rosiglitazone, peroxisome proliferator-activator receptor gamma (PPARγ) agonist, is known to enhance phagocytic clearance of parasitized erythrocytes and reduce parasitic burden in malaria by inhibiting the mitogen-activated protein kinase (MAPK) and NF-κB signaling .
In helminthic infection (Strongyloidiasis), anakinra (IL-1β receptor antagonist) potentially improved innate cytokine responses (IL-33 and IL-25) eventually causing parasite expulsion . Therefore, small molecular have shown potential therapeutic benefits in parasitic infection, here is just the tip of huge iceberg, research is underway to explore other molecules.
3.3 Nano-medicinal approach
The application of nanomaterials in the field of medicine for diagnosis and treatment received considerable attention in recent decades for parasitic diseases. The diagnostic potential of nanomaterials has been seen in malaria [106, 107], toxoplasmosis , cryptosporiodiosis , amebiasis  and leishmaniasis [111, 112].
Considering the nanoparticles as treatment option for parasitic diseases, these particles have proven efficacy in targeting the infected macrophages for treatment of VL . Silver alone or in combination with chitosan nanoparticles exhibited anti-toxoplasma effects by exacerbating serum IFN-γ levels and lowering the parasitic burden . Spiramycin loaded chitosan nanoparticles have shown to effectively treat toxoplasmosis . In giardiasis, combination nanotherapy with silver, chitosan and curcumin nanoparticles have been shown to effectively clear the parasites from intestine and stool without any adverse effects . Chitosan as nanosuspension has also shown lethal effects on
The biodegradability and non-immunogenic properties of nanoparticles have made them suitable as delivery agents for drugs and vaccines. Nanoformulation of recombinant
For helminthic infections, chitosan based albendazole formulation skewed the T-cell responses to TH1 type and reduced the parasitic burden, which led to parasite clearance in echinococcosis [126, 127] as well as in toxocariasis . Similarly, silver assembled on fungal (
Nanoformulation has also been used for vaccine development and as adjuvants, self-assembling protein nanoparticles (SAPN) have shown to trigger protective antibodies and long-lived memory responses to confer sterile protection against malarial parasites (
In this world where cost of developing medicine for parasitic infections remain the greatest challenge, drug developers are embracing molecular medicine approach that promises to deal with the parasitic infections and improves the chances of successful treatment. Molecular medicine has revolutionized the field of drug discovery/development however, there are significant hurdles in turning the promise into reality. Perhaps, gradually but it is shaping the future of medicine with the help of molecular platforms, better bioinformatics services and better pharmacogenomic analyses has greatly facilitated the scientific community and the stakeholders to come on common platform to fight against the parasitic diseases.