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Filarial Worms III_ Biochemistry_ Molecular Biology_ and Immunology

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Filarial Worms III_ Biochemistry_ Molecular Biology_ and Immunology Powered By Docstoc
					               Bio 54   2002




        Filarial Worms III:
     Biochemistry, Molecular
    Biology, and Immunology

        Andrew G. Campbell, Ph.D.


1




    Biochemistry & Molecular
    Biology
    •Methods for Study
    •Energy Generation
    •The Microfilarial Sheath
    •Chitin & Chitinases
    •Vector Resistance to Filarial
     Infection
2




                                     1
    Experimental Models for Study of
    Filarial Worms
    • Lymphatic Filariae
      – B. malayi can be experimentally
        maintained in gerbils (natural reservoir
        hosts) and in mosquito vectors
      – various stages of W. bancrofti and B.
        malayi can be transferred to mice
    • Nonlymphatic Filariae
      – Onchocercal keratitis (river blindness)
        can be modeled in rabbits and rodents
      – Adult worms can be maintained in vitro
3       must be obtained from human or cattle nodules




        Onchocercal Keratitis




             Human                      Mouse


4




                                                        2
     Experimental Techniques
     • in vitro culture
       – manipulation of nutrients, temperature,
         drugs, etc.
     • genetics
       – use of mutant Caenorhabditis elegans
         (nonparasitic nematode) to determine
         the function of parasite genes
     • molecular biology
       – identification of important stage-
5
         specific genes via differential screening




              Differential screening
                            cDNA probe from:
                              Day 3    mosquito
                               L3        L3
    worm       mRNA

               cDNA

                cDNA
               library

                   cDNA
                   probe
                           cDNA on filter from
6                            Day 3 L3 larvae



                                                     3
    Energy Generation in Filariae:
    General Considerations
    • Carbohydrates (CHOs) from host are a
      major energy source
    • filarial worms have relatively low reserves
      of glycogen (CHO storage molecule)
    • filariae have traditionally been regarded
      as anaerobic fermenters, producing
      lactate from glucose
    • role for aerobic metabolism?
      – probably varies by species
7
      – environmental factors




Energy Generation in Filariae
• Microfilariae
    – require oxygen for motility
    – aerobic due to higher energy requirements?
• Target for drug development
    – exploit differences in enzyme structure &
      regulation
• Caveats
    – problem of extrapolating results from one
      species of worm to another
    – in vitro vs. in vivo issues
8




                                                    4
     The Microfilarial Sheath
     • MF of W. bancrofti , Brugia
       spp., and Loa loa retain
       remnant of eggshell as an
       extracuticular "sheath"
     • O. volvulus MF do not have a
       sheath.
     • sheath maintained while in      Ensheathed
       vertebrate blood but shed       microfilaria
       during development in insect

9




     The Microfilarial Sheath
     • Structure
       – at least 5 proteins, including chitinase
       – sugars (chitin?)
       – inorganic compounds (e.g. phosphate)
       – host proteins
       – about 25% “unknown”
       – highly cross-linked
     • The sheath is the host-parasite
       “interface”
10




                                                      5
     The Microfilarial Sheath
     • MF form the sheath by stretching
       and remodeling their eggshell
       membranes as larval body develops
       within
     • "exsheathment" is necessary for
       subsequent molting and
       development of larva in the insect
     • blocking sheath formation or
       exsheathment could reduce
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       transmission




 Chitin
 • polysaccharide/polymer of
   N-Acetyl-glucosamine (NAG)
 • 2nd most abundant biopolymer on earth
 • major component of insect & crustacean
   exoskeleton
 • also found in the eggshells of filarial worms
 • NOT a constituent of vertebrates
      Chitin metabolism could thus be exploited
      as a target for drug development
12




                                                   6
     Microfilarial Chitinase
     • two isoforms (p70 and p75) have been identified
       in B. malayi
     • encoded proteins highly conserved (relative to
       yeast and bacteria)
     • products of 2 different, but homologous, genes
     • originally identified by reactivity with the
       monoclonal antibody MF1, which is capable of
       reducing microfilarial burden in infected gerbils
     • expression weak or absent in intrauterine larvae
       and recently shed MF
     • production/expression coincides with microfilarial
       infectivity of mosquitoes
13




 Possible Functions of Chitinase
     • Escape of parasite from digestive
       tract in the insect host
        – Plasmodium and Leishmania produce chitinase
        – Plasmodial enzyme may allow the parasites to
          penetrate the peritrophic membranes
          surrounding the fresh bloodmeal taken by the
          insect vector.
        – in Leishmania, chitinase is believed to rupture
          the pericardial lining and thus cause the sandfly
          to regurgitate its bloodmeal
        – Microfilarial chitinase may perform similar
14
          functions




                                                              7
 Possible Functions of Chitinase
     • Support of parasite infection in insect
       vector
       – production of inhibitor oligosaccharides
         to prevent agglutination by lectins
       – precedent: In gut of Tsetse fly, if
         trypanosome chitinase is blocked, fly
         becomes less susceptible to infection
     • MF sheath morphogenesis/
       Exsheathment
       – the egg contains chitin, but does the
15       sheath?




     Chitinases at Other Stages
     •adult female
       – hatching of the egg?
       – resorption of egg shell following
         hatching?
       – possible target for drug to block MF
         release
     •Infectious third stage (L3) larvae
       – migration in the insect vector?
16
       – escape from mouthparts?



                                                    8
                     “Innate”
     Vector Resistance Mechanisms
 • species/strain specificity on part of
   parasites and vectors
 • mechanical damage to larvae by blackfly
   “teeth”
 • coagulation of bloodmeal
 • physical barrier of the gut membranes
 • agglutinins (lectins) in gut
 • defense molecules in insect hemolymph
17




                   “Inducible”
     Vector Resistance Mechanisms
     • expression of defense molecules in
       hemolymph is elevated upon infection
     • types of inducible defense molecules
       – antibacterial peptides
       – agglutinins (lectins)
       – phenoloxidases
       – humoral proteases
     • no “memory” as in the vertebrate
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       immune system



                                              9
     Filarial Parasites:
     Immunology
     •General Considerations
     •The Immune Response &
      Disease
     •Evidence for Immunity
     •Tolerance
19




 General Considerations
 • Parasites, in general, don’t “want” to injure or
   kill you, rather they have an interest in
   keeping you alive
     – To satisfy their needs for nutrition and shelter
     – To make you a long-term “reservoir” for
       dissemination
 • Filarial parasites tend to cause chronic
   infections
     – Most people with filarial infections don’t show
       symptoms, but are effective parasite reservoirs
     – There must be some mechanism to strike a
20     “balance” between host and parasite




                                                          10
                            Parasite
                            exposure            no exposure

                                                ?

       infected,             infected,         not infected,
     no symptoms              disease          no symptoms




21




 Infected, No Symptoms
 • microfilaria (MF) positive
 • immunologically hyporesponsive to the parasite
     – impaired B and T cell proliferative responses to parasite and
       unrelated antigens
     – reduced production of Th1-associated cytokines: IL-12, IL-2
       and IFN-γ
     – elevated IL-10
     – generally absent or low antibodies specific to adults or MF
     – IgE responses and eosinophilia can be present
 • do not clear MF well, thus provide "reservoir" for
   infection of others
 • treatment can induce symptoms in these people!

22




                                                                       11
 Infected, with Disease
 • symptoms
     – early: filarial fever, lymphatic inflammation
     – severe: TPE, elephantiasis
 • microfilaria (MF) negative
 • immunologically hyperresponsive
     – strong B and T cell proliferative responses
     – production of Th1 (IFN-γ ) and Th2 cytokines (IL-4 and IL-
       10)
 • clear MF well, thus do not serve as transmission
   reservoir
 • treatment generally improves symptoms, if given
   early
23




     Not Infected, No Symptoms
     •no overt evidence of parasites
     •microfilaria negative
       – unexposed? (unlikely)
       – undetectable infection? (unlikely)
       – immunity?



24




                                                                    12
 Evidence for Immunity from
 the Mouse Model
 • limitation: mice are not completely permissive to
   lymphatic filariae, not a perfect model system
 • antibody-mediated immunity to microfilariae
     – sera from mice immunized with MF extract transfers 75-
       100% protection from MF challenge to other mice
     – IgG2a and/or IgM probably has importance
 • T-cell mediated immunity to L3
     – transfer of primed T-cells, but not serum (antibodies) or B-
       cells, results in clearance of L3
 • T-cell mediated immunity to adults
     – adult worms survive longer in athymic mice

25




     Evidence for Immunity in
     Humans
     • onchocerciasis
       – putatively immune individuals make a Th1-like
         response to O. volvulus antigens
     • lymphatic filariasis
       – 19 persons who maintained “immune” status
         for at least 17 years made much stronger
         parasite-specific T-cell responses than
         infected controls
       – “immune” persons were found to make
         responses to the MF-1 epitope of chitinase at
         greater frequency than infected controls
26




                                                                      13
 Factors Influencing the Immune
 Response to Filarial Worms
 • Genetics - role of HLA (MHC)
     – certain HLA haplotypes found to correlate with
       disease/infection (both positively and negatively)
 • neonatal exposure
     – MF positive mother tends to give birth to MF+ asymptomatic
       infant
     – infant has elevated Th2, low Th1 response
 • parasite effects
     – parasite burden
     – certain parasite strains may be more immunogenic than
       others
 • tolerance induction
27




 Tolerance Induction
 • evidence exists that indicates that the
   parasite actively induces immunological
   "tolerance" (unresponsiveness) in its host
 • probably induced by soluble factors produced
   by the worm
    – egg antigens?
    – filarial cystatin
 • seems to be directed at Th1 T-cells
    – low IFN-γ and IL-2
     – reduced DTH responses
28




                                                                    14
 Immunological Tolerance
 • Why induce tolerance?
     – prevents parasite destruction
     – keeps host alive (good for you) and able to
       pass on infection (good for the worm)
 • is usually long-lasting
 • breakdown causes pathology
 • appears to be directed at certain stages
   (adult and MF) but not infectious L3
   larvae - “concomitant immunity”
29




        Concomitant Immunity

               immunity


          L3          adult        MF

                      insect
                      stage



30




                                                     15
       Tolerance Breakdown
                    immune response



       L3         adult             MF

                  insect
                  stage                  DISEASE



31




               Vaccination
     VACCINE            Pathology
                         reduced


       L3         adult             MF

                  insect
                  stage

                Transmission
                  reduced
32




                                                   16
     “Take Home” Points
     1) An interaction of host and parasite-specific
        factors likely determines who gets disease
     2) The pathology of filariasis is immune-mediated,
        but an immune response can also protect from
        infection
     3) The worms appear to actively "tolerize" their
        hosts, and disease results when tolerance
        breaks down
        These points must be carefully considered if
        vaccination strategies are to be attempted!


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Description: Molecular biology research at the molecular level is the phenomenon of life science. By studying biological macromolecules (nucleic acids, proteins) of the structure, function and biosynthesis of various aspects to clarify the nature of the phenomenon of life. The study includes a variety of life processes. Such as photosynthesis, the molecular mechanisms of development, the mechanism of neural activity, the incidence of cancer and so on.