Shahab Final Rvw Ppt

8/11/2019 Shahab Final Rvw Ppt

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Mohd ShahabID No. 52499

Division of Parasitology

CSIR - CDRI


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Outline of the presentation

What are Neglected Tropical Diseases (NTDs) ?

Introduction to Lymphatic Filariasis (LF)

Its Immunology

Ongoing scenario on eliminating LF

Newer approaches for combating LF


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Group of tropical infections which are especially endemic in

tropical & sub tropical regions and caused by diverse

pathogens, majority being parasites.

NTDs affect an estimated 100 Crore people living in:

Remote rural areas andUrban slums of tropical countries

NTDs are endemic in 149 out of 260 (57.30%) countries

and territories

Of these,At least 100 countries are endemic for 2 or more NTDs

And 30 countries are endemic for 6 or more NTDs

Neglected Tropical Diseases


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In all 17 NTDs have been profiled in the first WHO

report on neglected tropical disease

ParasiticChagas disease (American trypanosomiasis)

Cysticercosis

Dracunculiasis (guinea-worm disease)

EchinococcosisHuman African trypanosomiasis (sleeping sickness)

Leishmaniasis

Lymphatic filariasis (elephantiasis)

Schistosomiasis (bilharziasis)Onchocerciasis (river blindness)

Foodborne trematode infections

Soil-transmitted helminthiasis


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ViralDengue

Rabies

BacterialTrachoma

Buruli ulcer (Mycobacterium ulcerans infection)

Leprosy (Hansen disease)

Endemic treponematoses


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A large body of evidence, published in peer-reviewed

medical and scientific journals, has demonstrated that

NTDs adversely affect morbidity and mortality

This refutes the once-widespread assumptions held by

the international community

Have an important impact on morbidity and

mortality


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Can be controlled, prevented and possibly eliminatedusing effective and feasible solutions

WHOs Strategies:

1. Preventive chemotherapy;2. Vector control;

3. The provision of safe water, sanitation and hygiene; and

4. Veterinary public health (that is, applying veterinarysciences to ensure the health and well-being of humans)

These strategies make control; prevention and evenelimination of several NTDs feasible at a low cost

In 2007, WHO launched a Global Plan to Combat

Neglected Tropical Diseases


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Lymphatic Filariasis

Lymphatic filariasis (LF) is a mosquito borne disease causedby thread like lymphatic dwelling nematodes and causes

significant disability as well as loss of productivity in

developing nations.

The worms live in the human lymphatic system and can

cause:

lymphedema (swelling) and elephantiasis in limbs and

breasts

hydrocele (severe fluid accumulation) affecting mensgenitalia


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Lymphatic Filariasis (LF) is one of the oldest

and most debilitating of all the NTDs.

Caused by three types of parasitic worms: Wuchereria

bancrofti, Brugia malayi, and B. timori.

Transmitted by female mosquitoes. When an infected

female mosquito bites a person, she may inject the worm

into the bloodstream.

Acknowledged as a leading cause of permanent and long

term disability.

Responsible for generating social stigma, isolation,

psychological stress.

And huge economic loss among affected individuals.


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Endemic in tropical regions

Asia, Africa, Central/South America


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Some key facts

Nearly 1.33 billion people in 80 countries worldwide are

threatened, commonly known as elephantiasis.

Over 129 million people are currently infected, with about

40 million disfiguredand incapacitated by the disease.

In India it is endemic in 17 States and six Union Territorieswith about 553 million people in peril making one-third of

the global LF burden


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Cont.

LF caused by obstruction of the lymphatic system, which

results in the accumulation of a fluid called lymph in the

affected areas.

Results in an altered lymphatic system and the abnormal

enlargement of body parts, causing pain and severe

disability.

Acute episodes of local inflammation involving the skin,

lymph nodes and lymphatic vessels often accompany

chronic lymphoedema.


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Diagnosis

Parasitological- Finding mf in blood

DEC provocation test - 50/100 mg DEC flushes out Mf in

blood during day

Clinical diagnosis- based on symptoms

Immunodiagnosis

Antibody detection based - IgG4 dependent using rAgs

(BmR1; BmSXP)

Antigen detection based (commercial)- ELISA - semi-quantitative

- Simple card test (ICT immunochromatographic)


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Treatments

Drugs:

DEC(Diethylcarbamazine) - Single dose recommended

(300 mg)

Ivermectin(20-200microgram, s.c. single shot)

Albendazole(400 mg tablet)-Broad spectrum anthelmintic

Antibiotics: doxycyclene, Moxidectin ( in clinical trials)


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Surgery for massive leg swelling (elephantiasis) with a

fluid shunting procedure.

Pressure bandages to wrap the swollen limb and elastic

stockings to help reduce the pressure. Exercising and

elevating a bandaged limb also can help reduce its size.

Rigorous cleaning of the affected areas of the body.

Other treatments


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Global Programme to Eliminate LF (GPELF)

Launched by World Health Organization (WHO) in 2000

Target elimination date of 2020

Two-pronged strategy to:

1. Interrupt the spread of infection

2. Reduce the suffering of persons already infected

To interrupt transmission WHO recommends an annual

mass drug administration of single doses of medicine to

all the eligible people in endemic areas. At least 5 rounds on MDA are needed to interrupt

transmission


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GPELF: Progress and successes

53 countries have ongoing MDA campaigns

37 countries have administered 5 or more rounds of

MDA in many target areas

2.8 billion doses of medicine delivered in first 9 years

Transmission interruption has protected 6.6 million

newborns from becoming infected with the disease


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National Filaria Control Programme (NFCP) was launchedby India in 1955

The main control measures were mass DECadministration, anti-larval measures in urban areas and

indoor residual spray in rural areas.

The programme became a part of the National VectorBorne Disease Control Programme (NVBDCP) in 2003and, aims to eliminate lymphatic filariasis by 2015 underNational Health Policy 2002.

India accounts for about 40% of the estimated casesglobally with either disease or infection (microfilaria

cases)

In Ind ia..


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Immunology

Disease is spectral in nature

Many infected individuals showing no clinical signs,

while others suffer the consequences of chronic

stages.

Distinct antibody isotypes are associated with different

disease states

Current research in mouse models of infection iselucidating the immunological mechanisms that can

lead to immunity against this disease


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Cont.

Majorities are asymptomatic due to polarization of

immune responses to agents into either Th1 or Th2

types.

They have inability to proliferate or produce interferon

gamma (IFN-)in response to the antigen.

Initially, this regulation was ascribed to predominant IL10

response .

It is clear that a range of regulatory events are involved

including TGF-,CTLA-4.


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Protective immunity in filariasis

The mechanism(s) by which different parasite stages arekilled still remain elusive, most findings result from work

comes from mouse model

It has been established that the infective larval (L3) and

adult stages of the parasite primarily induce type 2responses.

Thus, during natural infection the subsequent maturation ofadult nematodes following L3 invasion, is likely to enhance

and fully establish this type 2 response. Intriguingly, single-stage Mf injections primarily induce an

inflammatory type 1 response.


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Vaccination

No vaccines are available for prevention of infection

Vaccination in experimental animals shows substantial

protective immune response.

- with attenuated infective larvae, excretory-secretory(ES) antigens, mitochondria rich (MT) fraction, recombinant

and DNA vaccines.

A number of filarial antigens have been cloned in the past

however; many of these either await immunoprophylacticevaluation or failed to produce adequate protection

The potential for use of live filariasis vaccines in humans is

limited because of safety issues and limited availability of

larvae.


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Filarial parasite killing?

The immunological mechanism used by the host to kill large

helminth is classically thought to involve antibody-dependent

cell-mediated cytotoxicity (ADCC)

Antibody attaches to parasite surface by FcR, recruitment of

M, eosinophils and neutrophils extruding toxic mediators on

parasite surface


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Despite of this vast knowledge and program

LF still remains a major problem

About 160 million people still remain infected withfurther billions and more are at the risk

More and more research/studies are needed


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Genomic approaches against LF, provides newer

prospects in drug discovery either in target identification

or its validation.

Large number of genomic data could possible paves the

path regarding newer methods.

Crucial for identification of vaccine antigens, their

molecular characterization, genetically modified proteins,more effective human compatible adjuvants and vaccine

delivery systems would surely facilitate the antifilarial

vaccine discovery research.

Newer approaches for anti-filarial

drug/vaccine discovery


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Screening of the target via genomic approaches opened

new opportunities.

Filtering through genomic comparison provides us to

select pathogen-specific gene products. Functional genomics allow the selection of gene products

which are necessary for the pathogen survival.

Subsequent to this, the identified and validated targets are

put for in vitroand in vivoinhibitors studies including small

and high throughput assays, in silico drug design, lead

discovery/optimization, development and trials.

Molecular target based compound discovery


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Selecting anti-filarial drug targets based on genomic datamust have two basic considerations.

1. The target genes should not possess close homologues

in the host (humans).

2. It should be possible to identify therapeutic agents

(inhibitors) with reduced or no host cross-reactivity.

Since full human and filarial genomes are available,

therefore comparative analysis along with efficientbioinformatics can easily reveal the similarity index of

filarial parasite gene or its product to the human host

Comparative Genome Filtering


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The reported B. malayigenome is~95 Mb

The genome is estimated to contain >11,500 genes.

Comparative assessment of the transcriptome of different

developmental stages of B. malayihelps us to determine

when and where the genes of interest are turned on or off. Proteomic data in context to ES and somatic proteins

produced, indentified 7,103 (>60%) of the proteins

predicted in the genome in which 2,336genes have been

assigned as hypothetical/predicted status before. Thus transcriptomics and proteomic approach provides

valuable information on expression profile and helps in

predicting unique genes/proteins which are important for

the survival of filarial parasite

Genome analysis


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Comparative genomics involves the use of computer programs that

can align multiple genomes and look for the similarity among them.

Analysis at gene level among nematodes genomes, indicate that

~45% of the predicted protein-coding genes represent novel and

species-specific.

Comparative analysis shows that >35% of B. malayigenes have C.

elegans matches. So the functions of genes conserved among

nematodes can be investigated using C. elegans.

In silicocomparison identified a set of 589 candidate drug targetsinB. malayiwhich are already characterized in C. elegans.

Presently, only C. elegansprovides surrogate target validation and

has been endorsed by WHO as promising approach towards

discovery of new antifilarial drugs

Genome Filtering Tools


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Bunch of heritable changes in gene function, that cause

different phenotypes, occurs without altering DNA sequences.[Might be regulated by DNA methylation, histone acetylation/deacetylation,post genomic silencing using small interfering RNA (siRNA) or micro RNA

(miRNA) via RNA interference (RNAi) etc]

miRNAs are found to be essential for the developmentactivities in C. elegansand has been demonstrated in various

organisms.

Genome of B. malayi contains many elements which are

required for miRNA processing. Deep sequencing andbioinformatics approaches,identified 145 miRNA of 99 miRNA

representing families.

Of these, 60 families are conserved in other species while 11

are restricted to helminthes.

Epigenetic studies


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Only 9 miRNA families have been found to be common withother filariid species investigated further.

Since developmental processes in several parasitic nematodes

share similarities with those of C. elegans, role of miRNA in

filarial development cannot be denied.

Many novel functional miRNAs have been identified in filarial

parasites during in silicocomparison studies.

It would be worth investigating the targets of B. malayi

miRNAs, in target discovery to identify and determine their

involvement in maintaining parasitism for better understanding

of epigenetic regulation.

Cont.


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Identifying the function of every gene in all sequenced

organisms is one of the major objectives of the post-

genomic era.

The application of functional genomics in drug discoveryprovides the opportunity to incorporate rational

approaches to the process and to prioritize and validate

the drug target.

It includes microarrays, proteomics, and RNAi.

Functional Genomic Filtering


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Microarrays

Microarray analysis has been extensively applied to defineexpression pattern of a gene at genomic level

In parasitic nematode, technique holds great promise as a

means of studying stage-specific or tissue-specific expression of

genes.

Genome-wide analysis of gender-associated gene transcription

in filarial nematodes identified numerous potential candidates

for rational drug design which can target worm reproductive

processes. Though, the technique remains expensive however the data

generated from large research groups can be utilized for

screening studies, as they are freely available in the microarray

libraries.


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Proteomics

There is a poor correlation between the regulation oftranscripts and actual protein quantities since genome analysis

does not account for post-translational processes such as

protein modifications and protein degradation.

Proteomics allows large-scale study of protein properties suchas expression levels, post-translational modifications and

interactions with other molecules to obtain a global view of

cellular processes at the protein level and need to be

employed in drug-discovery processes.

Functional and expression profiles from different life cycle

stages of filarial worms along with the endosymbiont

Wolbachiacan be mapped down by using this approach.


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RNA in terference (RNAi)

Target validation - an important aspect in drug discovery as it

prioritizes the identified gene for the usefulness as a potent target

candidate.

Recently, in vivo silencing has been demonstrated in B. malayiwithin the mosquitoAedes aegypti leading to suppression (83%)

in B. malayicathepsin L-like cysteine protease generesulting in

multiple aberrant phenotypes, motility, and partial-paralysis.

Our group as successfully silenced the Bm- iPGM and TPP gene,which impairs the development.


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Wolbachia : An anti-filarial Drug Target

A gram-negative intracellular bacteria - present in all the

stages of the life cycle of human filariids in a mutualistic

symbiotic association. Essential for the development, fertility, and survival of the

filarial worms.

Antibiotics like Tetracycline, Doxycycline, Rifampicine and

Azithromycin kill these bacteria antifilarial drug target?


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The genome sequence of the Wolbachia (wBm)endosymbiont from B. malayi offers good opportunity for

understanding symbiosis between the bacterium and its

filarial host.

It would help in the identification of biochemical processesand pathways of Wolbachia and that could serves as

novel anti-filarial drug targets.

It has 1.08 Mb circular genome which bears extremely low

density of predicted functional genes compared to all otherbacteria.

Lacks genes to produce lipopolysacharride but produces

inflammatory reaction via TLR4

Genome


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It is predicted to encode only 806 protein-coding genes

demonstrating loss during co-evolution and co-adaptation.

Comparing metabolic pathways, indicated that the

biosynthesis of riboflavin, flavin adenine dinucleotide

(FAD), heme and nucleotides may be important for thesymbiotic relationship with the host as genes for these

pathways are lacking in B. malayigenome.

Filarial parasite likely provides amino acids required for

Wolbachia growth, since wBm can synthesize only oneamino acid, mesodiaminopimelate,a major component of

peptidoglycan.

It has type IV secretion system.

Cont.


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Wolbachia

Many of the basic biological characteristics of Wolbachia

have yet to be elucidated, but the results obtained to date

suggest that hosts can be exposed to Wolbachia:

when larvae, or adult worms, are killed by drug

when Wolbachiaare expulsed- naturally

with the release of microfilariae from female

possibly through the excretory system of worms

All are involved in the pathology and adverse reactions


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Wolbachia in filarial pathology

LPS is considered to be the major mediator of

inflammatory response in gram-negative bacteria.

Wolbachia Lipoprotein stimulates innate and adaptive

immune response through Toll-like Receptors whichinduces disease manifestations of filariasis.

In addition, it has been speculated

Heat shock proteins

CpG motifs in DNA Wolbachia surface protein (wsp)

Peptoglycan

May also be involved in

pathogenesis of bacterial

disease


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Proteomic analysis of the various stages of B. malayi

identified 557 of the 805 wBm predicted proteins.

96 out of 166 hypothetical/predicted genes were validated

as producing a protein during one or more stages of filarial

development.

Studies have predicted some essential gene repertoire of wBm and severalessential biochemical processes that represent candidate for drug targets.

These include

heme biosymthesis

lipid II biosynthesis

lipoprotein biosynthesis glycolytic enzymes - pyruvate phosphate dikinase (PPDK) and

cofactor-independent phosphoglycerate mutase (iPGM).

Among these several are under process for further evaluation as potent

targets or are subjected to high throughput screening for inhibitors studies

Wolbachia Cont.


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The need of today

Unlocking the identity of new drug targets bycomparative and functional genomics.

Validation studies context to the role of the target

molecule in the worm and/or endosymbiont -

Wolabachia.

More and more in-vitroand in-vivostudies

Screening of the effective compounds (HTS)

Newer drug delivery approaches

Drugs with strong macrofilaricidal activity


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Thank you

Documents

Shahab Final Rvw Ppt