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Summer Research Fellowship Programme of India's Science Academies

Immunological characterization of different leukocyte subsets present in the spleens of mice during experimental Lymphatic filariasis

Ms. Ayesha Bootwala

PRES’s College of Pharmacy, Chincholi, Maharashtra 422102

Dr. Kumaravelu Jagavelu

Senior Scientist, Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031

Dr. Mrigank Srivastava

Senior Scientist, Molecular Parasitology and Immunology Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031

Abstract

Lymphatic filariasis (LF) is a Neglected Tropical Disease (NTD) which is caused by nematode worms of the family Filariodidea. Three species of thread-like filarial worms cause human LF viz.Wuchereria bancrofti, which is responsible for 90% of the cases and the remainder is caused by Brugia malayi and Brugia timori. Since W. bancrofti is highly host specific and cannot be established in small rodents, my study is focused on human LF caused by B. malayi as it can be easily maintained in small rodents. Therefore, in the present study, under laboratory conditions, Aedes aegypti is used as an intermediate host whereas Mastomys coucha (rodent) serves as the definitive host. B. malayi carries out its life cycle in two hosts: human beings serve as the definitive hosts and mosquitoes of the genera Culex, Anopheles, Aedes and Mansonia serve as intermediate hosts. B. malayi is a periodic strain which exhibits nocturnal periodicity as a result of which they are found in the deep veins during the daytime and in the peripheral circulation during the night. Human beings contract LF when they are repeatedly bitten by the mosquitoes infected with filarial worms. Mosquitoes pick up the microfilarial form (mf or larvae stage 1; L1) of the parasite while taking blood meal from infected humans. In the mosquito, mf’s undergo two rounds of moulting and finally develop into L3 stage (infective larvae stage 3) within 8–10 days. Upon subsequent biting by the mosquito, L3 are deposited into the skin of the human from where they migrate through the draining lymphatics and finally develop into adult worms in the lymphatic vessels of the definitive host. The adult worms mate and millions of mf are produced by the females which circulate in the blood stream. It usually takes around 8–16 months post infection for the symptoms to appear. Symptoms of LF: Repeated episodes of inflammation and lymphedema lead to lymphatic damage, chronic swelling, hydrocele, chyluria, lymph varices and elephantiasis of the legs, arms, scrotum, vulva, and breasts. I learned the cyclic transmission of B. malayi infection in the laboratory which involved maintenance, rearing and breeding of intermediate host Aedes aegypti, recovery of infective larvae of B. malayi from A. aegypti as well as cleaning and subsequent inoculation of infective larvae in BALB/c mice through intraperitoneal route. Following infection in mice, I learned how to prepare single cell suspension from mouse spleen, preparation of cytospins for differential leukocyte count and immunophenotyping of different leucocyte subsets using flow cytometry, RNA extraction from splenocytes, its analysis, quantification and transcription of specific genes. In addition to this, I also learned microscopic examination of blood of Mastomys coucha and identification of Mf stage present in it.

Keywords: Brugia malayi, intermediate host, definitive host, nocturnal periodicity, microfilariae, filariform larvae

life cycle.jpg
    Fig 1. Life cycle of Lymphatic filariasis. Adapted from
    Bayer Group.

    Abbreviations

    Abbreviations
    LF Lymphatic filariasis
    WHO World Health Organization
    Th Helper T cells
    IL Interleukin
    CD Cluster of Differentiation
    RELM Resistin-like molecule
    TNF Tumor Necrosis Factor
    Mf Microfilariae
    MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide
    TGF Transforming Growth Factor
    DC Dendritic Cells
    FSC Forward Scatter
    SSC Side Scatter
    NADH Nicotinamide adenine dinucleotide
    BmL3 Brugia malayi 3rd Larval Stage
    RPMI Roswell Park Memorial Institute
    PBS Phosphate-Buffered Saline
    FACS Fluorescence Activated Cell Sorting
    MACS buffer Magnetic Associated Cell Sorting
    APC Allophycocyanin Conjugate
    PerCP Peridinin Chlorophyll Protein Complex
    PerCPCy5.5 Peridinin Chlorophyll Protein: Cy-5.5 Tandem Conjugate
    PE Phycoerythrin
    FITC Fluorescein Isothiocyanate Conjugate
    Gr1 Granulocyte marker
    SiglecF Sialic acid-binding immunoglobulin-type lectins
    EDTA Ethylenediaminetetraacetic acid
    BSA Bovine Serum Albumin
    TAE Tris Acetate EDTA
    RO Reverse Osmosis
    DMSO Dimethyl sulfoxide
    O.D Optical Density

    INTRODUCTION

    A. Human Lymphatic filariasis: Chronology of Events

    Symptoms of Elephantiasis have been evident from ancient Egypt (2000 BC) and the Nock civilization (500 BC). Ancient Indian literature Sushruta Samhita (6 century BC) and Madhavakara (7 century AD) have also described the pathological features of a disease resembling filariasis indicating prevalence of this disease in India even in the past. The first documentation of LF symptoms has been done in the 16 century by a Dutch explorer named Jan Huygen Linschoten who observed that descendants of those that killed St. Thomas were “all born with one of their legs and one foot from the knee downwards as thick as an elephant’s leg” and described it as the curse of St. Thomas (Burnell, 1885). In addition, William Prout also described occurrence of chyluria, another pathological condition associated with LF in his book “On the Nature and Treatment of Stomach and Renal disease” (Prout, 1848). French surgeon Jean-Nicolas Demarquay had discovered the presence of larval stage mf in hydrocele fluid for the first time in 1863 while Brazilian scientist Otto Henry Wucherer independently observed mf in urine and his name is associated with genus Wuchereria. Adult worms were investigated by Joseph Bancroft in 1876 while he was working on lymphatic abscess (Kean, 1978). After the discovery of filarial parasites, one of the triumphs was the work of Patrick Manson, who discovered the life cycle of filarial parasite in 1877.

    B. Symptoms and pathology of Lymphatic filariasis

    Lymphatic filariasis infection involves asymptomatic, acute, and chronic conditions (Kumara Swami & Nutman, 2000; Dreyer et al., 2000). The majority of infections are asymptomatic, showing no external signs of infection. These asymptomatic infections still cause damage to the lymphatic system and alter the body’s immune system [Babu & Nutman, 2014]. Acute episodes of local inflammation involving skin, lymph nodes and lymphatic vessels often accompany chronic lymphedema or elephantiasis [Dreyer et al., 1999]. Some of these episodes are caused by the body’s immune response to the parasite. Acute symptoms typically occur 8–16 months following infection and often recur several times a year. A first episode of acute filarial fever has been reported after more than 15 years of subsequent to exposure. Acute filarial fever without lymphadenitis is non-specific and must be distinguished from malaria and other causes of fever in the tropics. Acute filarial lymphangitis is characterized by a circumscribed inflammatory nodule or cord with centrifugal lymphangitis that arises following the death of the adult worm i.e., whether spontaneous or following treatment. Acute dermato-lymphangioadenitis, severe local inflammation resembling cellulitis or erysipelas, is frequently associated with secondary bacterial infection and impaired lymphatic flow, ascending lymphangitis and limb edema. This may eventually progress to elephantiasis. Chronic Lymphatic filariasis may develop months or years after the acute symptoms, or without a history of acute disease [Freedman, 1998]. Lymphatic obstruction leads to lymphedema of the affected extremity and, eventually, to elephantiasis. The sites most commonly affected are the legs, scrotum, arms and breast. Recurrent secondary bacterial skin infections, often streptococcal, may cause acute episodes of pain and fever and lead to glomerulonephritis [Langhammer et al., 1997]. Moreover, other presentations of chronic lymphatic filariasis include hydrocele, lymph scrotum, and acute epididymitis. Circulating immune complexes containing filarial antigens have been implicated in renal damage. As the lymphatic vessels progressively become inflamed and blocked, lymph vessels become congested. Chyluria may also arise with considerable loss of fat-soluble vitamins and protein, resulting in malnutrition and vitamin deficiencies. Clinically the loss of these essential components lead to fatigue, pain from urethral obstruction and social isolation. These patients are not only physically disabled, but suffer mental, social and financial losses contributing to stigma and poverty [WHO, 2019].

    Background/Rationale

    The immune responses to filarial parasites encompass a complex network of innate and adaptive cells whose interaction with the parasite underlies a spectrum of clinical manifestations. The predominant immunological feature of Lymphatic filariasis is an antigen - specific Th2 response and an expansion of IL-10 producing CD4+ T cells that is accompanied by a muted Th1 response. This antigen specific T cell hypo-responsiveness appears to be crucial for the maintenance of the sustained, long-standing infection often with high parasite densities. While the correlation of protective immunity to Lymphatic filariasis are still incompletely understood, primarily due to the lack of suitable animal models to study susceptibility, it is clear that T cells and to a certain extent B cells are required for protective immunity. Host immune responses, especially CD4+ T cell responses clearly play a role in mediating pathological manifestations of LF, including lymphedema, hydrocele and elephantiasis. The main underlying defect in the development of clinical pathology appears to be a failure to induce T cell hypo-responsiveness in the face of antigenic stimulation. Finally, another intriguing feature of filarial infections is their propensity to induce bystander effects on a variety of immune responses, including responses to vaccinations, allergens and to other infectious agents. The complexity of the immune response to filarial infection therefore provides an important gateway to understanding the regulation of immune responses to chronic infections, in general.

    Macrophages are an important class of antigen presenting cells that can serve as protective effector cells in bacterial and protozoan infections by their production of nitric oxide and other mediators. A special class of macrophage is known to be induced in filarial infections, characterized by their preferential expression of the enzyme arginase, instead of nitric oxide due to increased activation of arginase-1 by IL-4 and IL-13. These macrophages, termed alternatively activated macrophages (AAMs), have a very specific gene expression profile, with the ability to upregulate markers including arginase-1, chitinase 3-like proteins 3 and 4 (also known as YM1 and YM2, respectively) and resistin-like molecule-α (RELMα). AAMs are known to be important in wound healing and are thought to help limit tissue immunopathology. By virtue of expressing regulatory molecules such as IL-10, TGF-β and programmed cell death 1 ligand 2 (PDL2), these macrophages might play a predominantly regulatory role in filarial infections. Interestingly, these filarial induced macrophages appear to have the ability to expand locally and are less dependent on influx of monocytes from the bloodstream to perform their functions. While filarial infection does induce expression of these cells in humans, early interaction of parasites or parasite antigens leads to a predominantly pro-inflammatory response with expression of mainly pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β, as well as genes involved in inflammation and adhesion. Studies from murine models of filarial infection and in vitro data indicate that nitric oxide production by macrophages might be a key lethal hit in the host defense against the parasite. Therefore, the induction of alternatively activated macrophages might be an important immune evasion strategy for the parasites.

    Objectives of the Research

    1. Maintenance of life cycle of Aedes aegypti and cyclic transmission of Brugia malayi in Mastomys coucha.

    2. Observing microfilaria in blood of Mastomys coucha.

    3. Infection of BALB/c mouse with infective larvae of B.malayi (Bm-L3) and immunophenotyping of splenic leukocytes.

    4. Leukocyte differentials by Giemsa-stained cytospins.

    5. Splenic leukocyte RNA isolation, quantification and estimation of integrity post Bm-L3 infection.

    6. Monitoring viability of splenic macrophages following infection with protein obtained from adult worms of Brugia malayi.

    LITERATURE REVIEW

    Information

    Helminths are long-lived organisms (up to 10 years for filarial worms) which do not replicate within the human host. Cells of the innate and adaptive immune system are important for the initiation of type 2 immunity, which are the hallmark of helminth infections. The key players in T helper (Th) 2-type immunity are CD4+ Th2 cells and involve cytokines IL-4, IL-5, IL-9, IL-10, and IL-13; the antibody isotypes IgG1, IgG4, and IgE, and expanded populations of eosinophils, basophils, mast cells, and alternatively activated macrophages [Allen and Maizels, 2011]. Helminths have developed different strategies for survival in their human host including down-regulation of T- and B-cell responses via the induction of regulatory T cells or the anti-inflammatory cytokines IL-10 and TGF-β during the chronic phase of infection. Immunoregulation in filarial infection was first recognized in early human studies, because peripheral T cells in infected patients were frequently unresponsive to parasite antigens and responses to bystander antigens (including allergens and vaccines) were also reduced [Maizels R.M, 2009]. Th2 responses induced by filarial parasites is a conventional response of the host, its initiation requires interaction with many different cell types like stromal cells, dendritic cells, macrophages, eosinophils, mast cells, basophils, epithelial and innate helper cells.

    Dendritic cells:

    Human DCs exposed to B. malayi mf show higher levels of apoptosis and decreased production of IL-12 and IL-10. In fact, when human monocytes that were being differentiated to DCs in vitro were stimulated with B. malayi mf antigen, they produced significantly decreased levels of IL-10. Combined with suppression of proinflammatory cytokines, a key aspect in modulation of DCs is the down regulation of co-stimulatory molecules; leading to induction of a Th2 response [Semnani RT, 2003]. Live filarial parasites have the capacity to down regulate TLR expression (specifically TLR-3 and 4) on dendritic cells as well. This is accompanied by an impaired ability of dendritic cells to produce IFN-12, and IL response to TLR (Toll like receptors) ligands. The diminished expression and function of TLRs on immune cells is thought to be a likely consequence of chronic antigen stimulation and probably serves as a novel mechanism to protect against the development of pathology in filariasis [Venugopal, 2008].

    Effector T Cells:

    A major hallmark of longstanding filarial infection is the down regulation of parasite antigen driven Th1 differentiation. This is manifested by a significantly lower production of IFN-and IL-2 upon filarial antigen stimulation in asymptomatic-infected compared to diseased individuals. Human lymphatic filarial infection is associated with an antigen specific expansion of Th2 cells (mostly defined by IL-4 expression) and enhanced production of IL-4 and IL-13 and induction of classical Th2 response with high IL-4, IL-5 and IL-13 secretion has long been considered to be the hallmark of active infection in human filariasis [King, 1991]. T cells from filarial- infected individuals exhibit classical signs of anergy including diminished T cell proliferation to parasite antigens, lack of IL-2 production, and increased expression of E3 ubiquitin ligases [Metenou, 2010].

    B Cells:

    Host protection as well as regulation by antibodies and B cells is being recognized as an essential component in Th2 responses in filarial infections. IgG4 and IgG1 are elevated in chronically filarial infected humans and high levels of IgG4 but low levels of IgE are found in the blood of hypo responsive, asymptomatic persons infected with B. malayi, W. bancrofti [Ottesen et al., 1985]. IgG4 correlates with high levels of IL-10 and the presence of adult worms in hyporesponsive persons. In Bancroftian filariasis, high levels of IgG4 but low levels of IgE were found in mf positive individuals compared to patients with clinical disease (Elephantiasis & TPE). One of the most consistent findings in filarial infections is the elevated level of IgE that isobserved in TPE, these IgE antibodies persist many years after the infection has been treated, indicating the presence of long-lived memory B cells or plasma cells in filarial infections [Hussain & Hamilton, 1981].

    Alternatively Activated Macrophages:

    Macrophages that are activated by the Th2-type cytokines IL-4 and IL-13 develop an alternatively activated phenotype and have a well-described role in filarial infections. In filariasis, alternatively activated macrophage markers are up regulated in the blood of asymptomatic microfilaremics, the category displaying T cell hyporesponsiveness [King et al., 1993]. Thus, in filarial infections at least, AAMs appear to suppress the immune response against the parasite, promoting anergy.

    EXPERIMENTAL ANIMALS AND CONCEPTS

    Experimental Animals

    Mastomys coucha

    M. coucha are multimammate straw colored rodents that belong to family Muridae. They are usually considered as prolific breeders with a life span 2 to 3 years. Females of the species can breed at regular intervals of 33 days under favorable conditions, have a gestation period of around 21 days and litter size usually consists of 8–14 babies. They are highly susceptible to B. malayi infection and are therefore used as animal models for Lymphatic filariasis.

    IMG_20190731_122011-01.jpeg
      Fig.2 : Mastomys coucha

      Meriones unguiculatus

      M. unguiculatus also known as Mongolian jird/gerbil belongs to family Gerbillinae and is another excellent permissive rodent model for the study of Lymphatic filariasis using B. malayi. Females have a gestation period of 22–27 days and give birth to a small litter size, usually 3–7 pups/litter. They offer a unique advantage over other rodents as the full development of adult worms from infective L3 stage of the filarial parasite can take place inside their peritoneal cavity which makes the recovery of adult worm and mf very easy.

      IMG_20190830_103154-01.jpeg
        Fig. 3: Meriones unguiculatus

        BALB/c mouse

        BALB/c is an albino inbred strain of commonly used laboratory mouse that was derived in 1920 by Halsey J. Bagg. Since these mice are immunocompetent, they are the obvious choice for many animal-based studies including immunology. They are small, active animals having an average weight of approx. 20 g. They have excellent capacity with a long reproductive life span. The females have a gestation period of 20–21 days with an average litter size of 4 babies. For carrying out the present work, all the animals were housed under standard condition of temperature (23± 1 °C), relative humidity (55± 10%) and 12/12 hrs light/dark cycles at National Laboratory Animal Center (NLAC) of CSIR-CDRI. They were fed with standard pellet diet and water ab libitum.

        IMG_20190830_101410-01.jpeg
          Fig.4: BALB/c mouse

          Concepts

          Immunophenotyping

          Immunophenotyping is the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportions of the various populations of interest. Antibodies are used to identify cells by detecting specific antigens expressed by these cells, which are known as markers. These markers are usually functional membrane proteins involved in cell communication, adhesion, or metabolism. Immunophenotyping using flow cytometry has become the method of choice in identifying and sorting cells within complex populations. Cell markers are a very useful way to identify a specific cell population. However, they will often be expressed on more than one cell type. Therefore, flow cytometry staining strategies have led to methods for immunophenotyping cells with two or more antibodies simultaneously. By evaluating the unique repertoire of cell markers using several antibodies together, each coupled with a different fluorochrome, a given cell population can be identified and quantified. Many immunological cell markers are CD markers and these are commonly used for detection in flow cytometry of specific immune cell populations and subpopulations.

          Flow cytometry

          Flow Cytometry is a popular cell biology technique that utilizes laser-based technology to count, sort, and profile cells in a heterogeneous fluid mixture. Using a flow cytometer machine, cells or other particles suspended in a liquid stream are passed through a laser light beam in single file fashion known as “Hydrodynamic Focusing”, and interaction with the light is measured by an electronic detection apparatus as light scatter and fluorescence intensity. If a fluorescent label, or fluorochrome, is specifically and stoichiometrically bound to a cellular component, the fluorescence intensity will ideally represent the amount of that particular cell component. Flow cytometry is a powerful tool because it allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. This makes it a rapid and quantitative method for analysis and purification of cells in suspension. Using flow, we can determine the phenotype and function and even sort live cells.

          FACS_overview.png
            Fig.5: Flow Cytometry Overview

            [a] Instrumentation:

            The flow cytometer instrument consists of three core systems: fluidics, optics, and electronics. The fluidics system includes a flow cell, where the sample suspended in physiological buffer or medium is injected. The flow cell requires sheath fluid to carry and align the cells or particles so that they pass through a narrow channel and into the laser intercept (light beam) in a single file. This hydrodynamic focusing allows the analysis of one cell at a time by laser interrogation. The optics system consists of various filters, light detectors, and the light source, which is usually a laser line producing a single wavelength of light at a specific frequency. This is where the particles (cells) are passed through at least one laser beam. Lasers are available at different wavelengths ranging from ultraviolet to far red and have a variable range of power levels as well (photon output/time). Interrogation by the laser beam excites any compatible fluorescent probes that are conjugated to antibodies, causing the probes to emit light (or fluoresce) at specified wavelengths. A detector in front of the light beam measures Forward Scatter light signals (FSC) and detectors to the side (at 90 deg angle) measure Side Scatter light signals (SSC). Fluorescence detectors measure the fluorescence signal intensity emitted from positively stained cells and particles. Within the flow cytometer, all of these different light signals are split into defined wavelengths and channelled by a set of filters and mirrors so that each sensor will detect fluorescence only at a specified wavelength. These sensors are called photomultiplier tubes (PMTs). Various filters are used in the flow cytometer to direct photons of the correct wavelength to each PMT. Short pass (SP) filters allow transmission of photons below a specific wavelength while long pass (LP) filters allow transmission above a specific wavelength. Band pass (BP) filters allow transmission of photons that have wavelengths within a narrow range. Each PMT will also detect any other fluorophores emitting at a similar wavelength to the fluorophore it is detecting. These light signals are converted by the electronics system to data that can be visualized and interpreted by software. FSC is a measure of relative cell size, while SSC usually provides information about the relative granularity and complexity of the cells. Cells with a low granularity and complexity will produce less side scattered light, while highly granular cells with a high degree of internal complexity (such as neutrophils) will result in a higher side scatter signal. Thus, by using forward and side scattered light detection, cell populations can often be distinguished based on characteristic differences in cell size and granularity. Both FSC and SSC measurements are influenced by multiple factors and also depend on the quality of sample preparation, thus, to gather more detailed information we could utilize fluorescent labelling techniques with flow cytometry.

            The_Flow_Cytometer.jpg
              Fig.6 The Flow Cytometer

              [b] Fluorescence and Fluorophore (Fluorochrome) Selection:

              Cell populations can sometimes be separated based on FSC and SSC, but cells can also be separated by whether they express a specific protein. In this case, a fluorophore is usually used to stain the protein of interest. Fluorophores used for the detection of target proteins emit light after excitation by a laser of compatible wavelength. These fluorescently stained cells or particles can be detected individually. Each type of fluorescent dye or label has its own characteristic excitation and emission spectrum which is important for designing flow cytometry experiments. There is a wide selection of fluorophores available nowadays; for example, FITC, PerCP, APC, PE, PE-Cy5, PE-Cy7, PerCP Cy5.5, Pacific blue and many more. Fluorescently conjugated antibodies have commonly been used to label specific structures on the cell for flow cytometric analysis. As the fluorescing cell (or particle) passes through the interrogation point and interacts with the laser beam, it creates a pulse of photon emission over time (a peak). These are detected by the PMTs and converted by the electronics system to a voltage pulse, typically called an "event". The total pulse height and area is measured by the flow cytometer instrument, and the voltage pulse area will correlate directly to the fluorescence intensity for that individual event. These events are assigned channel numbers based on its measured intensity (pulse area). The higher the fluorescence intensity, the higher the channel number the event is assigned. This signal can be amplified by increasing the voltage running through the PMT.

              Fluorescent_Emission.png
                Fig.7

                [c] Selecting the Right Fluorochrome Conjugate:

                There are many fluorescent molecules, also known as fluorochromes, fluorophores, or fluorescent dyes, with a potential application in flow cytometry. These fluorescent molecules are excited by laser light at specific wavelengths and then emit light (fluoresce) at another wavelength. By conjugating (pre-attaching) them to primary antibodies, conjugated antibodies could be prepared that allow flow cytometry analysis. Knowing each conjugate label’s properties is important in choosing the right label for the experiment. Some key features to know about fluorescent conjugate tags are listed here:

                Maximum Excitation Wavelength (λex) – The peak wavelength in the excitation (absorption) spectra, measured in nanometres (nm)

                Maximum Emission Wavelength (λem) – The peak wavelength in the emission spectra, measured in nanometres (nm)

                Extinction Coefficient (ε max ) – (also called molar absorptivity) The capacity for the fluorochrome to absorb light at a given wavelength, usually measured at the maximum excitation wavelength with the units M −1 cm −1

                Fluorescence Quantum Yield (Φf) – The number of photons emitted per absorbed photon. A high quantum yield is important, and this number ranges between 0 and 1

                Fluorescence Decay Time (τfl) – The time interval after which the number of excited fluorescent molecules is reduced to 1/e (approx. 37%) via the loss of energy, usually measured in nanoseconds

                Brightness – The fluorescence output per fluorophore measured. Fluorophores with high brightness values can be used to detect lower-abundance targets. Calculated as the product of the extinction coefficient (at the relevant excitation wavelength) and the fluorescence quantum yield divided by 1000, with the units M −1 cm −1

                Stokes Shift – The difference between the Maximum Emission Wavelength and Maximum Excitation Wavelength, measured in nanometers

                Laser line – Which laser line(s) to use for detection using flow cytometry

                Common filter set – The standard microscope filter set that generates the best imaging results

                Photostability – How resistant a substance is to change resulting from exposure to light

                [d] Data Analysis

                In a flow cytometry experiment, every cell that passes through the interrogation point and is detected will be counted as a distinct event. Each type of light that is detected (forward-scatter, side-scatter, and each different wavelength of fluorescence emission) will also have its own unique channel. The data for each event is plotted independently to represent the signal intensity of light detected in each channel for every event. This data could be visually represented in multiple different ways, so one needs to play around with different types of data plots and set proper gates. The most common types of data graphs used in flow cytometry include histograms, dot plots, density plots, and contour diagrams. As multiparametric analysis becomes more complicated, analysis techniques can even include higher order plots such as 3-dimensional plots and SPADE trees. Univariate histogram plots measure only one parameter. Typically, the Y-axis is the number of events (the cell count) that show a given fluorescence, and the X-axis is the relative fluorescence intensity detected in a single channel. A large number of events detected at one particular intensity will be represented as a peak (or spike) on the histogram. Ideally, only one distinct peak will be produced and can be interpreted as the positive dataset (representing the cells with the desired characteristics of interest).

                Cytospin

                The Cytospin centrifuge is a special purpose instrument designed to deposit cells evenly onto a glass slide. The instrument when used correctly produces a monolayer cell deposition in a defined area of the slide using centrifugal force. Cytospin preparation consistently produces uniform preparations of cells that are easily stained and evaluated. The primary requirements are that the specimen is a cell suspension - preferably of single cells - and that the cells are fresh and intact enough to be evaluated microscopically.

                Giemsa stain is a type of Romanowsky stain, named after Gustav Giemsa, a German chemist who created a dye solution. Giemsa stain is used to obtain differential white blood cell counts. It is also used to differentiate nuclear and cytoplasmic morphology of the various blood cells like platelets, RBCs, WBCs. Giemsa stain is a differential stain and contains a mixture of Azure, Methylene blue, and Eosin dye. It is specific for the phosphate groups of DNA and attaches itself to where there are high amounts of adenine-thymine bonding. Azure and eosin are acidic dye which variably stains the basic components of the cells like the cytoplasm, granules etc. Methylene blue acts as the basic dye, which stains the acidic components, especially the nucleus of the cell. Methanol act as a fixative as well as the cellular stain. The fixative does not allow any further change in the cells and makes them adhere to the glass slide.

                On microscopic observation, cell organelles, bacteria and, parasites are distinguished based on their morphology and color as explained below:

                Morphological characterization of blood cells 
                Eosinophils Purple nuclei, faintly pink cytoplasm and red to orange granules.
                Basophils Purple nuclei, blue coarse granules.
                Lymphocytes Dark blue nucleus with light blue cytoplasm.
                Monocytes Pink cytoplasm with a purple colour nucleus.
                Platelets Violet to purple colour granules.
                Nuclei of host cells Dark purple
                Nuclei of WBCs Dark purple
                Cytoplasm of white cells Pale blue or grey-blue

                RNA extraction, its analysis and quantification

                [a] RNA extraction (Chomcynski and Sacchi Method):

                RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses.

                RiboZol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues. RiboZol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. RNA remains only in the aqueous phase.

                After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. Total RNA extracted by RiboZol Reagent is free from the contamination of protein and DNA.

                [b] Gel Electrophoresis:

                Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA (Kryndushkin et al., 2003). Nucleic acid molecules are size separated by the aid of an electric field where negatively charged molecules migrate toward anode (positive) pole. The migration flow is determined solely by the molecular weight where small weight molecules migrate faster than larger ones (Sambrook & Russel, 2001). In addition to size separation, nucleic acid fractionation using agarose gel electrophoresis can be an initial step for further purification of a band of interest. Extension of the technique includes excising the desired “band” from a stained gel viewed with a UV transilluminator (Sharp et al., 1973). In order to visualize nucleic acid molecules in agarose gels, ethidium bromide or SYBR Green are commonly used dyes. Illumination of the agarose gels with 300-nm UV light is subsequently used for visualizing the stained nucleic acids.

                [c] Quantification using NanoDrop Spectrophotometer:

                The NanoDrop Spectrophotometer measures nucleic acid concentrations in sample volumes of one microliter. The key to this advanced spectrophotometer is its unique sample retention technology that overcomes the need for cuvettes when taking measurements. This is accomplished by placing the sample directly on top of the detection surface and using the surface tension to create a column between the ends of optical fibers. Thus, the measurement optical path is formed. The sensitivity range for RNA detection is between 2 and 3700 ng/ul. The spectral range of the device is 220nm to 750nm and it is possible to scan all of the wavelengths. A single measurement cycle takes only 10 sec. The instrument is driven by a PC, which makes it possible to archive a large number of measurements.

                [d] Foundation of Spectrophotometry: The Beer‐Lambert’s Law

                A = ɛcl

                Where A=absorbance, ԑ=extinction coefficient, c=concentration and l=path length.

                The Beer-Lambert law draws a direct correlation between absorbance and concentration.  While nucleic acids absorb at many wavelengths, they have a peak absorbanceof UV light at 260nm. Thus, the amount of light absorbed in this region can be used to determine the concentration of RNA or DNA in solution by applying the Beer‐Lambert law.

                a. Absorbance at 260 nm:

                Nucleic acids absorb UV light at 260 nm due to the aromatic base moieties within their structure. Purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine) both have peak absorbances at 260 nm, thus making it the standard for quantifying nucleic acid samples.

                b. Absorbance at 280 nm:

                The 280 nm absorbance is measured because this is typically where proteins and phenolic compounds have a strong absorbance. Aromatic amino acid side chains (tryptophan, phenylalanine, tyrosine and histidine) within proteins are responsible for this absorbance.  Similarly, the aromaticity of phenol groups of organic compounds absorbs strongly near 280 nm.

                c. Absorbance at 230 nm:

                Many organic compounds have strong absorbances at around 225 nm. 

                In addition to phenol, TRIzol, and chaotropic salts, the peptide bonds in proteinsabsorb light between 200 and 230 nm.

                d. A260/280 ratio:

                The A260/280 ratio is generally used to determine protein contamination of a nucleic acid sample. The aromatic proteins have a strong UV absorbance at 280 nm. For pure RNA, A260/280 ratios should be somewhere around 2.1 and 1.8. A lower ratio indicates the sample is protein contaminated. The presence of protein contamination may have an effect on downstream applications that use the nucleic acid samples.

                e. A260/230 ratio:

                The A260/230 ratio indicates the presence of organic contaminants, such as (but not limited to): phenol, TRIzol, chaotropic salts and other aromatic compounds.Samples with 260/230 ratios below 1.8 are considered to have a significant amount of these contaminants that will interfere with downstream applications. Thisis especially true for reverse transcription. In a pure sample, the A260/230 should be close to 2.0.

                [e] Protein Quantification (Bradford Assay):

                The Bradford Reagent is used to determine the concentration of proteins in a solution. The procedure is based on the formation of a complex between the dye, Brilliant Blue G, and proteins in solution. The protein-dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The amount of absorption is proportional to the protein present. The Bradford Reagent requires no dilution and is suitable for micro, multi-well plate, and standard assays. The linear concentration range is 0.1–1.4 mg/ml of protein, using BSA (bovine serum albumin) as the standard protein. The Bradford Reagent is compatible with reducing agents. Reducing agents are often used to stabilize proteins in solution. Other protein assay procedures (Lowry and BCA) are not compatible with reducing agents. The Bradford Reagent should be used in place of these protein assays if reducing agents are present.

                MTT assay

                The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS). The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 5mg/ml, and incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used, but not necessary for most assay conditions.

                Viable cells with active metabolism convert MTT into a purple coloured formazan product with an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT into formazan, thus colour formation serves as a useful and convenient marker of only the viable cells. The exact cellular mechanism of MTT reduction into formazan is not well understood, but likely involves reaction with NADH (Nicotinamide adenine dinucleotide) or similar reducing molecules that transfer electrons to MTT. Speculation in the early literature involving specific mitochondrial enzymes has led to the assumption mentioned in numerous publications that MTT is measuring mitochondrial activity. The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. A variety of methods have been used to solubilize the formazan product, stabilize the colour, avoid evaporation, and reduce interference by phenol red and other culture medium components. Various solubilisation methods include using: acidified isopropanol, DMSO (Dimethyl sulfoxide), dimethylformamide, SDS, and combinations of detergent and organic solvent.

                METHODS AND RESULTS

                Maintenance of Life Cycle of Aedes aegypti and Cyclic Transmission of Brugia malayi in Mastomys coucha

                Rearing and breeding of mosquito (Aedes aegypti)

                Requirements: Stored eggs of Aedes aegypti, Mastomys coucha, wide enameled bowls, tap water, powdered yeast tablets, dog biscuits, glass pipette, nylon netted mosquito cages, 3% glucose solution, desiccator, petri dishes,

                a. Aedes aegypti was used as a vector for maintenance of life cycle of Brugia malayi in the insectarium. Rearing and breeding of mosquito vector was carried out at a temperature of around 27±1°C and humidity 75±1°C by transferring stored eggs of Aedes aegypti into wide enameled bowls containing stored tap water.

                b. After 24–72 hrs, when the eggs hatched into the first stage larvae they were given powdered yeast tablets and dog biscuits as a feed in a ratio of 3:1.

                c. All the larvae were washed daily with fresh water and after 5–6 days they had transformed into non feeding pupae which were manually separated from the larvae with the help of a wide mouth glass pipette. Thereafter, pupae were transferred into small dishes containing water and placed inside nylon netted mosquito cages where they developed into young mosquitoes within 24 to 48 hrs.

                d. Young mosquitoes were fed on 3% glucose solution soaked in covered with a moist filter paper that was kept in small petri dishes inside the cages.

                e. Fully developed mosquitoes were later fed onto donor Mastomys coucha that were previously infected with infective larval stage 3 of B. malayi and thus had microfilariae circulating in their blood so that they can be ingested by the feeding mosquitoes.

                f. For collection of mosquito eggs, a separate small beaker containing water was kept inside the cage for mosquitoes to lay eggs which were later collected and stored in a desiccator to continue the cyclic maintenance and transmission of B. malayi.

                IMG_20190711_122623-01.jpeg
                  Fig. 8: Nylon netted mosquito cages

                  Feeding of mosquitoes on microfilariae (mf) positive Mastomys coucha

                  Requirements: Mastomys coucha, restrainer, stereo zoom microscope

                  a. Mastomys coucha showing microfilariae density between 100−200 mf/10 μL were picked up for providing blood meal to the young mosquitoes.

                  b. Donor animals were kept inside a restrainer such that only tail was exposed for feeding; the restrainer was kept inside the mosquito cage where starving mosquitoes fed on the caged Mastomys.

                  c. Mosquitoes were left for fasting for approximately 1–2 hrs by removing glucose petri dishes. Feeding was allowed for 30–35 minutes and after an interval of 8–10 days post feeding, ingested mf underwent two successive moulting to develop into infective larvae which migrated towards the proboscis.

                  d. The L-3 development in mosquitoes was monitored by random dissection of few infected female mosquito under stereo zoom microscope.

                  IMG_20190805_115142-01.jpeg
                    Fig.9: Mastomys coucha being kept in a restrainer

                    Infection of Meriones unguiculatus with Infective Larvae of Brugia malayi and Subsequent Recovery of Adult Worms and Mf

                    Collection of infective larvae (Bm-L3)

                    Requirements: Baermann’s apparatus, Ringer solution

                    Ringer Solution 500 ml, pH 7.3-7.4

                    Dextrose 0.5 gm.

                    NaCl 4.5 gm.

                    KCl 0.21 gm.

                    CaCl2 0.12 gm.

                    NaHCO3 0.21 gm.

                    IMG_20190711_122041-01.jpeg
                      Fig.10 Baermann’s apparatus

                      a. Once L3 development was confirmed, all mosquitoes were paralyzed by vigorous shaking of the mosquito cage and were collected in Whatman filter paper.

                      b. The paralyzed mosquitoes were then transferred into a small bowl containing ringer solution to liberate infective larvae. The infective larvae were finally separated using the Baermann’s apparatus containing fine meshed muslin cloths.

                      c. Baermann’s apparatus consists of a glass funnel attached with a rubber tubing containing clamp at distal end to control the solution flow. A table lamp is lightened above the Baermann’s apparatus so that larvae migrate towards the lower end of rubber tubing and can be further collected by loosening the clamp after 45 min of transfer.

                      d. Thereafter, subsequent larvae fraction was collected on three occasions in small aliquots at an interval of 5-10 mins, the collected larvae were washed repeatedly in sterile Ringer solution under stereo zoom microscope, after which fully mature and actively motile infective larvae were counted, distributed in various cavity blocks and healthy naive M. coucha and M. unguiculatus were infected.

                      Inoculation of Bm-L3 in M. coucha and M. unguiculatus (Gerbils/jirds)

                      a. Six-week old male M. coucha and about eight weeks old male M. unguiculatus were inoculated with B. malayi infected infective larvae L3.

                      b. M. coucha were infected through subcutaneous route with ≈110 infective larvae, while M. unguiculatus were administered ≈160 L3 larvae into the peritoneal cavity. Health of infected animals was monitored daily for 12–14 days by measuring their body weight and noting any sign of distress.

                      Recovery of adult worms and mf

                      a. For recovery of mf, previously infected male girds were sacrificed and peritoneal fluid was collected in 10-12 ml of incomplete RPMI medium by repeated washing of the peritoneal cavity.

                      b. The adult worms were manually separated from peritoneal fluid and remaining fluid was spun at 600rpm for 10 min at 25 °C and suspended in sterile PBS.

                      Observing Microfilariae in Blood of Mastomys coucha

                      Requirements: Mastomys coucha, syringe needles, curved needles, slides, Leishman stain (0.15%), Bright Field microscope

                      a. Blood was collected (around 10 µL) from an untreated and treated Mastomys coucha (90 days post infection, between 12:00 to 12:45 hours) by cutting the tip of their tail using a sterile pair of scissors.

                      b. The blood drops were placed on clean slides labelled as C= Control/untreated and I= infected with BmL3.

                      c. The blood was smeared using a curved needle/ U-shaped needle and air-dried for 24 hours.

                      d. Post 24 hours the smear was dehaemoglobinized by holding the slides in a tilted position under running tap water for not more than 5 minutes.

                      e. The slides were again allowed to air-dry and then they were stained with the help of 0.15% Leishman Stain. The slides were kept undisturbed on a flat surface for 20 minutes in order to achieve efficient staining.

                      f. After staining, the slides were again washed with tap water, air dried and examined under the bright field microscope at 10X magnification for observing the microfilariae (mf).

                      e.jpg
                        Fig.11: Blood Smear Preparation 

                        Observations:

                        f.jpg
                          Fig. 12: Blood smear of infected Mastomys coucha showing Microfilariae

                          g.jpg
                            Fig. 13: Blood smear of uninfected Mastomys

                            Infection of BALB/c Mouse with Infective Larvae of B.malayi (Bm-L3) and Immunophenotyping of Splenic Leukocytes

                            Infection of BALB/c mouse with infective larvae of B.malayi

                            1. BALB/c mice were infected with 50 Bm-L3 via intraperitoneal route.

                            2. All the further experiments were carried out at the following time points:

                            a. Control – No infection

                            b. 7 and 28 days post Bm-L3 infection.

                            Single cell suspension preparation

                            Requirements: 40μM cell strainer, PBS, 1ml syringe, falcon (15 ml and 50 ml), RBC lysis buffer (00-4300-54, E Bioscience), haemocytometer

                            PBS (Phosphate-buffered saline): 1000ml, pH= 7.2 to 7.4

                            NaCl 8 gm.

                            KCl 0.2 gm

                            Na2HPO4 1.44 gm.

                            KH2PO4 0.24 gm.

                            1. A 40μM cell strainer (BD Bioscience) was wetted with PBS (to neutralise any static charges on it) and placed over a 50 ml Falcon tube.

                            2. Mouse spleen was harvested, kept over the cell strainer and gently crushed using a plunger of a 1ml syringe. Small amount (0.5-1 ml) of PBS was poured regularly over the strainer while crushing the spleen to avoid cell death.

                            3. The cells were obtained in a total volume of 10ml of PBS and subsequently transferred to a 15ml falcon.

                            4. The cell suspension was centrifuged for 15 minutes (1250 rpm) at 4°C, the supernatant was discarded and the cell pellet was gently dislodged by tapping.

                            5. After the pellet was completely dislodged, 1ml of 1X RBC lysis buffer (BD Bioscience, prepared in distilled water) was added and incubated at RT for 1 min. After incubation, the volume was made up to 10ml using PBS.

                            6. The cell suspension was then passed through a 40μM cell strainer in a new 15 ml Falcon tube in order to remove cell clumps and debris.

                            7. The cells were then centrifuged (1250 rpm, 4°C) and again suspended in 5 ml of PBS.

                            8. Cell counting was done using a haemocytometer and 10µl sample.

                            Expected cell count: 70×106 to 80×106

                            9. For further objectives the cell count was adjusted as follows:

                            A. Flow cytometric immunophenotyping - 1×106 cells per panel (no. of panels = 2)

                            B. Giemsa stained cytospins preparation - 1×105 cells (1, 00,000 cells per slide)

                            C. RNA isolation -10×106 cells

                            D. MTT Assay - all the remaining cells were plated for about 2 hrs and MTT assay was carried out using the adherent cells (mostly splenic macrophages)

                            Immunophenotyping of splenic leukocytes

                            Requirements: Antibodies, FACS tubes, MACS buffer

                            1X MACS Buffer (Magnetic-Activated Cell Sorting buffer): 100 ml

                            2mM EDTA 400 µL

                            0.5% BSA 500 mg

                            1X PBS q.s 100 ml

                            IMG-20190816-WA0009-01.jpeg
                              Fig. 14: BD FACS Aria Fusion flow cytometer
                              IMG_20190830_125833-01.jpeg
                                Fig.15:  Immunophenotyping of splenic leukocytes on BD FACS Aria Fusion flow cytometer

                                1. For identification of different leukocyte subsets (B cells, T cells, Eosinophils, Dendritic cells, Neutrophils) splenic cells were incubated with antibody cocktails containing the following anti-mouse monoclonal antibodies in FACS tubes:

                                Panel 1: For Lymphocytes

                                CD4-APC,

                                CD8-PerCPCy5.5,

                                CD19-PE,

                                CD90.2-FITC,

                                Panel 2: For granulocytes and DCs

                                F4/80-FITC,

                                CD11c-APC,

                                Gr1-PerCPCy5.5,

                                SiglecF-PE

                                2. Incubated for 20 min at 4°C. After incubation, 200 µl MACS buffer was added in all the tubes and then flow cytometric data was acquired on either BD FACS Calibur or FACS Aria Fusion flow cytometer and analysed using either Cell quest or FACS DIVA software.

                                3. Each cell type was identified based on the unique combination of markers expressed by the respective cell types.

                                Results:

                                h.jpg
                                  k.jpg
                                    Fig. 16 Immunophenotyping of DCs, macrophages, eosinophils and neutrophils in the spleen of mice.

                                    Fig 16: Single cell suspension from mouse spleen was prepared from control and infected mice at different time points as described in the methods section. Splenic DCs, macrophages, eosinophils and neutrophils were subsequently immunophenotyped using flow cytometry by staining the cells with fluorochrome conjugated monoclonal antibodies directed against CDIIc-APC, F4/80-FITC, Siglec F-PE and Gr1-PerCP. Briefly, cells were first gated so as to exclude lymphocytes (Fig 1) followed by gating cells according to the expression profile of different markers present on them. We observed that as compared to control, the number of splenic DCs (CD11c pos, F4/80 neg) were reduced in infected mice at day 28 post Bm-L3 infection, while no major difference was seen in splenic DC at day 7 post Bm-L3 infection (Fig 1B, top gate). Somewhat similar results were also seen in macrophages (CD11c pos, F4/80 pos) which showed reduced percentages at both time points observed (Fig 1 B, lower gate), this reduction being much more significant at D28 post Bm-L3 infection. This result was contrasted by splenic eosinophils (Sig F pos, Fig 1C) and neutrophils (Gr1 pos, Fig 1D) both of which increased at D7 and D28 post Bm-L3 infection, but this increase was much more drastic at D7 when compared to D28 post Bm-L3 infection. These results show that infection with Bm-L3 has differential effect on the recruitment and accumulation pattern of different leukocytes subsets present in the spleens of mice.

                                    i.jpg
                                      Fig.17
                                      Immunophenotyping of CD4 and CD8 T lymphocytes in the spleen of mice.

                                      Fig 17: Single cell suspension of mouse spleen was prepared from control and infected mice at different time points as described in the methods section. Thereafter, T and B lymphocytes were subsequently immunophenotyped using flow cytometry by staining the cells with fluorochrome conjugated monoclonal antibodies directed against CD19-PE, CD4-APC and CD8-PerCP (Fig 2). Briefly, Lymphocytes were first gated according to low FSC and low SSC scatter properties as shown in Fig 2A, followed by gating the cells according to the expression profile of different markers present on them. We observed that as compared to control, the number of both CD4 (Fig 2B, lower right panel) and CD8 T cells (Fig 2B, upper left panel) increased in infected mice at Day7 and D28 post Bm-L3 infection, while no clear cut data was obtained in case of B cells, possibly due to some experimental error (data not shown). These results show that infection with Bm-L3 results in expansion of two major T lymphocytes in the spleens of mice.

                                      Overall FACS Result summary:

                                      An interesting correlation can be drawn from the results obtained wherein it may be speculated that parasite tries to subvert the accumulation of typical antigen presenting cells (DCs and macrophages) as part of its survival strategy, during the early phase of infection, and at the same time, the host too mounts an immune response by recruiting granulocytes (Eosinophils and Neutrophils) and expanding the pool of T lymphocytes to counter the onslaught of Bm-L3.

                                      Leukocyte Differentials by Giemsa-Stained Cytospins

                                      Requirements: Glass slides, isopropyl alcohol, filter card, cytofunnel, Cytospin 4 Thermo Scientific Centrifuge, methanol, Giemsa stain (1X, Sigma Aldrich, GS500-500ml), Bright field microscope

                                      IMG_20190814_212939-01(1).jpeg
                                        Fig. 18: Cytospin 4 Thermo Scientific Centrifuge

                                        1. Single cell suspension from mouse spleen was prepared as described above.

                                        2. Each glass slide was cleaned briefly with 70% isopropyl alcohol and labelled with sample ID (C- Control, S1- Spleen one, S2- Spleen two)

                                        3. Each slide was matched face-up with a filter card and a plastic funnel (open end facing the labelled end of the slide). The assembly was placed into slide clip and the clip was locked to ensure the funnel, slide and filter card stay intact throughout the spinning process.

                                        4. Cytospin sample chamber was assembled and the slide assembly was inserted into the sealed Cytospin head.

                                        5. Cells were counted and approximately 1×105 cells (suspended in ~100μl of medium) were cytospun onto glass slides using cytospin centrifuge (Cytospin 4, Thermo Scientific) at 500 rpm for 5 minutes.

                                        6. Slides were quickly air dried and fixed as described below.

                                        7. Specimen fixation: Slides were fixed with cold (4°C) methanol immediately after drying.

                                        8. Staining: Giemsa stain of 1X strength was added on the slide and kept aside for complete drying (Approx. 20−60 minutes).

                                        9. After complete drying of the slides, they were observed under the Bright field Microscope (Carl Zeiss) using oil immersion.

                                        10. For each sample, two cytospins were prepared at different time points post BmL3 infection along with control and the snaps were taken and reported.

                                        Results:  

                                        d.jpg
                                          Fig. 19 Giemsa images

                                          The morphological changes of the above mentioned cells at different time points post Bm-L3 infection were observed.

                                          Splenic Leukocyte RNA Isolation, Quantification and Estimation of Integrity Post Bm-L3 Infection

                                          RNA isolation

                                          Requirements: RiboZol™ (Amresco, N580-100ml), RNase-free tubes, chloroform, isopropanol, 75% ethanol, NanoDrop Spectrometer.

                                          a. Sample Homogenization:

                                          1. Cell suspension was pelleted by centrifugation at 1250 rpm for 10 minutes in an RNase-free tube.

                                          2. Following centrifugation, the supernatant was discarded and the pellet was suspended in 1 mL of RiboZol™ per 5x106 cells. The cells were lysed by passing them several times through the tip of a pipette in order to avoid clump formation.

                                          b. Separation of Phases:

                                          1. The homogenized sample was incubated for 5 – 10 minutes at room temperature in order to ensure the complete dissociation of nucleoprotein complexes,

                                          2. 200 µL of chloroform was added per 1 mL of Ribozol™ added in step 1 and the tube was securely locked.

                                          3. The tube was shaken vigorously for 15 seconds in order to mix the sample and then the sample was incubated for 2 – 3 minutes at room temperature.

                                          4. The sample was centrifuged at 12,000 x g for 15 minutes at 4 °C.

                                          5. Following centrifugation, three phases were apparent:

                                          a. a lower red, phenol-chloroform phase

                                          b. a white interphase

                                          c. a colorless, upper, aqueous phase.

                                          6. About 80% of the clear upper aqueous phase was carefully removed as RNA has the tendency to remain suspended in aqueous phase.

                                          7. The entire aqueous layer was not removed in order to avoid contamination with protein, DNA, lipids and carbohydrates that appear as debris or flocculent material at the interface.

                                          c. Precipitation of RNA:

                                          1. The aqueous phase was transferred to a new RNase free tube and RNA was precipitated by adding 0.5 mL of isopropanol per 1 mL of Ribozol™ used in the initial homogenization.

                                          2. The samples were incubated for 10 minutes at room temperature and then centrifuged at 12,000 x g for 10 minutes at 4 °C.

                                          3. A white or gel-like pellet of precipitated RNA was deposited along the side and bottom of the tube.

                                          4. The size of the pellet depends on the amount of cell starting material. A pellet of very pure RNA is usually transparent and difficult to see.

                                          d. Washing:

                                          1. The supernatant was carefully removed without disrupting the RNA pellet.

                                          2. The pellet was washed once with 75% ethanol prepared in RNase-free water.

                                          3. For washing, 1 mL of ethanol per 1 mL of Ribozol™ was used and centrifuged at 7,500 x g for 5 minutes at 4°C.

                                          e. Re-dissolving the RNA Pellet:

                                          1. Following the final ethanol wash, the ethanol was carefully removed without disrupting the pellet.

                                          2. The pellet was air-dried for 5-10 minutes over ice. However, the pellet was not completely dried as it decreases the RNA solubility.

                                          3. RNA was dissolved in RNase-free water.30 µL for every 5x106 cells. The pellet was passed several times through a pipette tip, and incubated for 10 minutes at 55°C to 60°C to completely dissolve the RNA.

                                          f. Determination of RNA Yields and Purity:

                                          RNA concentration was determined by its absorbance at A260 using NanoDrop Spectrophotometer (Thermo Scientific).

                                          IMG_20190830_211042-01.jpeg
                                            Fig. 20 NanoDrop Spectrophotometer (Thermo Scientific).

                                            RNA Concentration = A260/ (l x e) where,

                                            l = cuvette path length (1cm),

                                            e = RNA extinction coefficient (25 µL/µg/cm)

                                            Expected yield of RNA from 10 x 106 cultured cells is 150-200 µg.

                                            RNA purity was determined by the ratio of absorbance at A260/A280. High quality RNA should have a ratio between 1.8 and 2.0, but may vary depending on the re-suspension solution and the RNA source.

                                            Results:

                                            Data obtained on NanoDrop Spectrophotometer (Thermo Scientific)
                                            Time point Nucleic acid (ng/µl) A260(abs) A280(abs) 260/280 260/230
                                            Control 1697.7 42.444 21.607 1.96 1.78
                                            Day 7 1727.1 43.178 21.992 1.97 1.78
                                            Day 28 1640.3 41.007 20.752 1.98 1.74

                                            1. Control:

                                            Screenshot_2019-09-15-16-42-58-811.jpeg
                                              Wavelength vs Absorbance plot

                                              2. Day 7:

                                              Screenshot_2019-09-15-16-42-40-344_1.jpeg
                                                Wavelength vs Absorbance plot

                                                3. Day 28:

                                                Screenshot_2019-09-15-16-42-16-935_1.jpeg
                                                  Wavelength vs Absorbance plot

                                                  RNA integrity estimation using Horizontal Gel Electrophoresis

                                                  A. Materials Required:

                                                  1.     Buffers and Solutions:

                                                  a.     Agarose solution (1.5%) (Invitrogen, 75510-019)

                                                  b.     Ethidium bromide (0.5µg/ml)

                                                  c.      Electrophoresis buffer (1X TAE Buffer)

                                                  d.     Loading buffer (Clontech Takara, SD0514)

                                                  2.     Nucleic Acids and Oligonucleotides

                                                  a.     Isolated RNA of good quality

                                                  b.     DNA Ladders (O Gene Ruler, SM1173)

                                                  B. The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:

                                                    1. An electrophoresis chamber and power supply.

                                                  2. Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.

                                                  3. Sample combs, around which molten agarose is poured to form sample wells in the gel.

                                                  4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE).

                                                  5. Loading buffer, which contains a dense substance (e.g. glycerol) which allows the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.

                                                  6. Ethidium bromide, a fluorescent dye used for staining nucleic acids.

                                                  7. Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.

                                                  IMG_20190820_111620-01.jpeg
                                                    Fig. 21 Gel forming unit

                                                    IMG_20190819_161245-01_1.jpeg
                                                      Fig. 22 Gel casting tray

                                                      1. Preparation of a 50X stock solution of TAE buffer in 1000 mL of distilled H2O:

                                                      50X Tris-acetate-EDTA (TAE), pH 8.3-8.6

                                                      Composition:

                                                      Tris base 242g

                                                      Acetic acid 57.1 ml

                                                      0.5M EDTA 100ml

                                                      RO water q.s 1000ml

                                                      a. 242 g of Tris base was weighed using a chemical balance and transferred it to a 1000ml beaker. b. EDTA solution was prepared (pH 8.3, 0.5M) by weighing 9.31g of EDTA and dissolving it in 40mL of RO water. EDTA is soluble at alkaline pH and thus, it was made soluble by adding sodium hydroxide.

                                                      c. The pH was continuously monitored using a pH meter.

                                                      d. The volume of the solution was made 100 mL by adding RO water. e. 57.1 ml of glacial acetic acid was pipetted out and added to the solution. f. The final volume was adjusted up to 1000ml using RO water.

                                                       2. Preparation of sufficient electrophoresis buffer (1x TAE ) to fill the electrophoresis tank and to cast the gel:

                                                      2ml of TAE 50X stock solution was taken in an Erlenmeyer flask and the volume was made up to 100ml by adding 98ml of RO water. The 1X working solution is 40 mM Tris-acetate/1 mM EDTA

                                                      *The same batch of electrophoresis buffer was used in both the electrophoresis tank and the gel preparation.

                                                      3. Preparation of Agarose gel solution (1.5%):

                                                      a. 1.5 grams of agarose was added to 100ml of electrophoresis buffer. However, the requirement for my samples was 30mL. Therefore, 0.45 grams of agarose was added to TAE buffer(1X) and the volume was adjusted to 30 mL using the same buffer.

                                                      b. The slurry was heated in a microwave oven until the agarose was completely dissolved.

                                                      c. When the molten gel had cooled (not completely, lukewarm), 0.5µLof ethidium bromide (0.5µg/ml). The gel solution was mixed thoroughly by gentle swirling.

                                                      4. Gel Casting:

                                                      a. A mould and comb of appropriate size were selected, the comb was placed into the mould and warm agarose solution was poured into the mold in a way that no bubbles were formed.

                                                      b. The gel was allowed to set completely (30-45 minutes at room temperature) and the comb was removed carefully to avoid any damage to the wells formed. The electrophoresis buffer (1X TAE buffer) was poured into the casting tray and the gel was mounted in the electrophoresis tank.

                                                      c. Just enough electrophoresis buffer was added to cover the gel to a depth of approximately 1mm.

                                                      F. Sample loading:

                                                      1. The loading sample was prepared as follows:

                                                      a. Volume of RNA sample containing approximately 1000 ng of RNA

                                                      b. 5 µL of 6X loading buffer.

                                                      c. Make up the final volume up to 12 mL using RNase free H2O.

                                                      2. The sample mixture (12 mL) was loaded into the slots of the submerged gel using a micropipette.

                                                      3. The lid of the gel tank was closed, the electrical leads were attached and a voltage of 80V was applied so that the RNA migrates towards the positive electrode (i.e anode, red lead).

                                                      4.Bubbles were observed at the anode and cathode which means that the leads were attached properly.

                                                      5. The gel was run until Bromophenol blue and Xylene cyanol were observed to be migrated an appropriate distance through the gel.

                                                      6. The gel tray was then removed and placed directly on a transilluminator. When the UV was switched on, RNA bands were observed.

                                                      *The presence of ethidium bromide allows the gel to be examined by UV illumination at any stage during electrophoresis.

                                                      a.jpg
                                                        Fig. 23 RNA bands

                                                        Monitoring Viability of Splenic Macrophages Following Infection with Lysate Protein Obtained from Adult Worms of Brugia malayi

                                                        Preparation of protein lysate from adult worms of Brugia malayi

                                                        Requirements: adult worms of Brugia malayi, glass slides, blade, PBS, sonicator, ice.

                                                        IMG-20190806-WA0031-01.jpeg
                                                          Fig. 24 Sonicator

                                                          a. Cell lysis is the first step of protein extraction. Physical lysis was the method of choice for cell disruption.

                                                          b. The frozen female adult worms (approx. 30) were placed on a clean slide and chopped gently using a sharp blade. The worms were kept moisturized with the use of PBS (minimum).

                                                          c. Chopping was done until the worms were completely cut into pieces and formed a turbid solution in PBS.

                                                          d. This solution was subjected to Ultrasonic frequencies (>20 kHz) to disrupt the cell membrane and release cellular contents. This step is known as sonication and was done at 25% amplitude for 15-20 cycles with a pulse of 20 seconds on and off (till a clear solution is obtained)

                                                          e. The sample after sonication was centrifuged at 12000 rpm for 20 minutes at 4°C.

                                                          f. The supernatant soup was collected and further quantified using Bradford Method to determine the concentration of Protein.

                                                          Quantification of lysate protein using Bradford method

                                                          Requirements: Bovine Serum Albumin (Sigma, A 2153-10G), adult worm lysate, PBS, 96-well culture plate, Bradford reagent (Sigma, B6916-500ml)

                                                          a. BSA (Bovine Serum Albumin) was used as the standard protein.

                                                          b. Stock solution of BSA was prepared of the concentration 2mg/ml using PBS as the solvent.

                                                          c. The stock solution was further diluted with PBS as follows to obtain different concentrations:

                                                          concentration of standard protein
                                                          Concentration of standard protein (BSA) in mg/ml Volume taken from the stock in µL Volume of PBS added in µL
                                                          2.0 100 00
                                                          1.8 90 10
                                                          1.6 80 20
                                                          1.4 70 30
                                                          1.2 60 40
                                                          1.0 50 50
                                                          0.8 40 60
                                                          0.6 30 70
                                                          0.4 20 80
                                                          0.2 10 90
                                                          0.0 00 100

                                                          RNA integrity estimation using Horizontal Gel Electrophoresis

                                                          1. Standard: 5 µL standard protein+ 250 µL Bradford Reagent

                                                          2. Blank: 5 µL PBS + 250 µL Bradford Reagent

                                                          3. Lysate Protein: 5 µL lysate protein + 250 µL Bradford Reagent

                                                          e. The plate was incubated at RT in dark for 10 minutes and then absorbance was measured at 595 nm using a spectrophotometer.

                                                          f. The absorbance readings were obtained as follows:

                                                          Absorbance
                                                          Conc. Of Std. in mg/ml Absorbance at 595 nm Avg. of abs.
                                                          A1 A2 A3
                                                          2.0 0.647 0.708 0.907 0.754
                                                          1.8 0.865 0.823 0.854 0.847
                                                          1.6 0.810 0.825 0.798 0.811
                                                          1.4 0.726 0.807 0.810 0.781
                                                          1.2 0.778 0.682 0.756 0.738
                                                          1.0 0.771 0.68 0.721 0.704
                                                          0.8 0.685 0.672 0.612 0.656
                                                          0.6 0.635 0.590 0.563 0.598
                                                          0.4 0.549 0.499 0.545 0.531
                                                          0.2 0.507 0.487 0.470 0.488
                                                          0.0 0.486 0.464 0.454 0.473
                                                          Blank 0.474 0.481 0.465 0.473
                                                          Lysate 1.189 1.169 1.234 1.197

                                                          g. The absorbance values of blank were subtracted from the average absorbance values of standard concentrations and protein lysate to obtain the absorbance of only the standard and protein lysate.

                                                          Absorbance
                                                          Conc. Of Std. in mg/ml Avg. Absorbance at 595 nm (A) B= (A – Blank Average) (A-0.473)
                                                          2.0 0.754 0.281
                                                          1.8 0.847 0.377
                                                          1.6 0.811 0.341
                                                          1.4 0.781 0.311
                                                          1.2 0.738 0.268
                                                          1.0 0.704 0.234
                                                          0.8 0.656 0.186
                                                          0.6 0.598 0.128
                                                          0.4 0.531 0.061
                                                          0.2 0.488 0.018
                                                          0.0 0.473 0.00
                                                          Lysate 1.197 0.727

                                                          h. A graph of concentrations versus B= (A-Blank Average) was plotted and the lysate protein concentration was calculated using the equation obtained from the plot:

                                                          graph.jpg

                                                            Equation: y= 0.2226x – 0.0074 where,

                                                            y= B value of protein lysate (0.727)

                                                            x= Concentration of protein lysate

                                                            0.727= 0.2226x – 0.0074

                                                            x= 3.2 mg/ml

                                                            Result: The concentration of protein lysate was found to be 3.2 mg/ml(Stock)

                                                            6.3 Monitoring cell (Macrophages) viability post Adult worm protein lysate treatment using MTT Assay.

                                                            Requirements: protein Lysate, alcohol, cell scrapper, chilled and filtered PBS, filtered RPMI 1640 (Sigma Aldrich, R4130-10L), MTT reagent (Sigma, M5655-500mg), DMSO (Dimethyl sulfoxide)

                                                            Incomplete RPMI 1640 100 ml, pH= 7.2-7.4

                                                            RPMI 1.64 gm.

                                                            NaHCO3 200 mg

                                                            Antimycotic 1 ml

                                                            *For preparing complete RPMI 1640, 10 ml FBS is added to Incomplete RPMI.

                                                            A. Preparation of working protein sample:

                                                            The stock protein sample of the concentration 3.2 mg/ml was adjusted to produce the working sample of the concentration of 20 µg/ml by using RPMI media for making up the volume.

                                                            B. Cell culturing of macrophages:

                                                            1. The laminar hood was sterilized with alcohol and the cell culturing was performed under sterile conditions.

                                                            2. The cells separated for MTT Assay from the total splenocytes were centrifuged at 1250 rpm for 15 minutes (4°C).

                                                            3. The supernatant was discarded and the pellet was suspended in sufficient amount of RPMI depending on the pellet size.

                                                            4. The pellet was dissolved completely in RPMI by passing it several times through the tip of a pipette.

                                                            5. The total volume of the cells suspended in complete RPMI media was divided equally in a way that each culture flask was plated with 1 ml of media.

                                                            6. Further, 20 ml of complete RPMI was added to all the flasks in order to provide a nutrient media for cell growth and survival.

                                                            7. All the culture flasks were incubated in a CO2 incubator for 2–3 hours under the following conditions which are suitable for adherence of macrophages: Temperature= 37°C, CO2 concentration= 5%

                                                            8. After the incubation, culture flasks were removed from the incubator and transferred to the laminar hood for further processing.

                                                            9. RPMI from the culture flasks was discarded gently without disturbing the adhered cells and gentle washing was done using 2-3 ml of chilled PBS.

                                                            10. The adhered cells i.e. Macrophages were scrapped using a sterile cell scrapper and chilled PBS.

                                                            11. The total volume was made upto 10 ml using PBS and centrifuged for 25 minutes at 2500 rpm and 4°C.

                                                            12. The supernatant was discarded and the pellet was suspended in sufficient amount of PBS depending on the size of the pellet (approx. 1 ml).

                                                            13. The Macrophages were counted using a Hemocytometer by loading a sample volume of 10 µL.

                                                            14. The total no. of cells were adjusted in PBS in a way that approximately 0.5×106macrophages/100 µL PBS could be plated in each well for a doublet reading per sample.

                                                            5. Two wells were plated in a 96-well plate per sample as follows:

                                                            a. Control: 100 µL sample + 100 µL RPMI

                                                            b. Treated: 100 µL sample + 100 µL Protein Lysate (20 µg/ml)

                                                            16. The plate was incubated in a CO2 incubator for 24 hours at 37°C with 5% CO2 concentration.

                                                            17. Post incubation, 20 µL of MTT reagent with a concentration of 5 mg/ml was added in the wells containing the control and treated cell samples and the plate was again incubated for another 4 hours.

                                                            18. All the previously added solvents were removed post incubation by slightly tilting the plate in a way that cells do not get disturbed by the pipette.

                                                            19. 100 µL of DMSO was added in each well containing control and treated cell samples.

                                                            20. The plate was further incubated for 10 minutes in a CO2 incubator at 37°C with 5% CO2 concentration.

                                                            21. After the incubation, O.D was taken in a spectrophotometer at 570nm.

                                                            22. % MTT Reduction was calculated using the formula:

                                                            % MTT reduction= O.D (Control) - O.D (Treated) / O.D (Control) × 100

                                                            Results:

                                                            Optical Density
                                                            Time Point Mean Optical Density at 570 nm % MTT Reduction
                                                            Control (Lysate negative) Treated
                                                            Control (Bm-L3 negative) 1.080 0.563 48%
                                                            Day 7 0.823 0.644 22%
                                                            Day 28 0.627 0.536 14.5%

                                                            A subsequent decrease in % MTT Reduction values was observed with the progression of Bm-L3 infection. MTT reduction has a direct correlation with Cell Viability due to the fact that only living cells have the ability to reduce MTT to formazan.

                                                            Thus, a higher % MTT Reduction value of a Bm-L3 negative sample draws our attention towards the outcome that the cell viability is highest in Bm-L3 negative sample as compared to the lysate treated samples at Day 7 and Day 28 Bm-L3 infected samples.

                                                            This finding is in line with our Flow Cytometry data where reduction in the number of macrophages was seen following Bm-L3 infection.

                                                            CONCLUSION

                                                            The maintenance of life cycle of Aedes aegypti and cyclic transmission of Brugia malayi in Mastomys coucha was observed and studied in detail. The microfilaria in blood of Mastomys coucha were observed.The infection in BALB/c mouse with infective larvae of B.malayi (Bm-L3) was induced and immunophenotyping of splenic leukocytes was performed along with its differentiationby preparation of Giemsa-stained cytospinsThesplenic leukocyte RNA isolation, quantification and estimation of integrity post Bm-L3 infection was performed and the viability of splenic macrophages following infection with protein obtained from adult worms of Brugia malayi was monitored.

                                                            It is however recommended that the time points taken under studt should be more than 3 to ensure the result reliability. It wasn't possible in my case due to a limited period of time available.

                                                            REFERENCES

                                                            1. Babu S, Nutman TB, "Immunology of Lymphatic filariasis"; Parasite Immunology; 2014 Aug;36(8):338-46.

                                                            2. Immunology of lymphatic filariasis

                                                            3. Venugopal PG, Leifer CA, Mostbock S, Sabzevari H, Nutman TB, "Inhibition of TLR3 and TLR4 function and expression in human dendritic cells by helminth parasites"; 2008

                                                            4. Flow Cytometry Fundamental Principle, How FACS Works | Boster

                                                            5. Bradford protein assay - Wikipedia

                                                            6. Agarose Gel Electrophoresis (AGE) (Procedure) : Molecular Biology Virtual Lab I : Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab

                                                            7. Rajamanickam A, Babu S, "Immunomodulation by Filarial Parasites", International Trends in Immunity, VOL.1 NO.4; October 2013.

                                                            8. Muhittin Yılmaz*, Cem Ozic and İlhami Gok, "Principles of Nucleic Acid Separation by Agarose Gel Electrophoresis."

                                                            9. World Health Organization. "Lymphatic filariasis". Archived from the original on 5 May 2016. Retrieved 7 May 2016.

                                                            10. CDC - Lymphatic Filariasis

                                                            11. LeAnne M Fox, Christopher L King, in Hunter's Tropical Medicine and Emerging Infectious Disease (Ninth Edition)​,2013

                                                            12. MTT assay - Wikipedia​

                                                            ACKNOWLEDGEMENTS

                                                            I extend my profound sense of gratitude IASc-INSA-NASI for awarding me this exhilarating Summer Research Fellowship.

                                                            I also thank the Director of CSIR-CDRI Professor Tapas Kumar Kundu for allowing me to work in the laboratory assigned to me and also providing the best facilities for carrying out all the experiments.

                                                            I express my gratitude towards my guide Dr. Kumaravelu Jagavelu for supporting me and giving me an opportunity to work on the topic of my interest.

                                                            I would like to express the deepest appreciation and gratitude towards my co-guide Dr. Mrigank Srivastava, who has the attitude and the substance of a genius: he continually and convincingly conveyed a spirit of adventure in regard to research, and an excitement in regard to teaching. Without his guidance and persistent help, the completion of my Summer Research Fellowship Programme would not have been possible.

                                                            I would also like to thank Mrs. Shikha Mishra, Technical officer and Mr. O.P. Yadav, Lab Attendant, for their technical assistance and Ph.D. scholars Ms. Ruchi Jha, Ms. Laxmi Ganga and Ms. Neha Satoeya for all the support they provided in carrying out my experiments. I express my special gratitude towards Ms. Priya Upadhyay, Project Asst. for being an awesome colleague and even a wonderful friend throughout my working tenure.

                                                            Source

                                                            • : https://www.publichealth.bayer.com/diseases/lymphatic-filariasis
                                                            • Fig: https://www.publichealth.bayer.com/diseases/lymphatic-filariasis
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