Leishmaniasis - 4 (8/9) Flashcards Preview

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Flashcards in Leishmaniasis - 4 (8/9) Deck (96):
1

Leishmaniasis is also known as

  • orient boils
  • Baghdad boils
  • kala azar
  • black fever
  • sandfly disease
  • DumDum fever
  • espundia

 

2

Leishmaniasis distribution

  • 88 countries 
    • tropical and sub-tropical countries
  • 2 million new cases each year
  • 12 million people infected / 52,000 deaths in 2010
  • 350 million people at risk (will increase with global warming)
    • flies that spread the disease moving up the Italian peninsula
    • moving north as the earth warms up
  • spectrum of diseases ranging from mild to severe
  • due to militariy in Iraq etc. have gotten the disease in non-endemic sites
    • military gone into the area, go back home, bringing parasite with them
    • insect vector found in parts of the US
    • secondary foci - esp Southern US (eg Texas)

 

 

3

Leishmaniasis

transmission

  • parasite transmitted by phlebotomine sand flies
    • Phlebotomus in Africa, Asia, and Europe
    • Lutzomyia in Americas
  • adult sandflies ~ 2mm in length
  • adult sandflies characterized by hairy body and wings

4

Leishmaniasis

transmission

Phlebotomus spp

  • desert/semi-arid ecosystems
  • some breed in peridomestic situations and enter human habitations
  • flies transmitting in the old world
  • some breed/propagate in areas surrounding houses, occassionally can transmit vector within human habitation
  • moving into towns more and more, becoming more urban

 

5

Leishmaniasis

transmission

Lutzomyia spp

  • forest dwelling
    • new world, associated with forests
  • disease transmission associated with humans living or working near forests

6

Leishmaniasis

transmission

sandflies fly via

"hopping"

  • short bursts of flight/few seconds of rest
  • short flight range

 

7

Leishmaniasis

transmission

sandflies: .... spread the disease

only females spread the diseae - males play no role in disease transmisson

  • male flies don't feed on any mammals - feed on phloem of plants
  • only females feed on blood = only vectors for disease transmission

 

8

Leishmaniasis

transmission

sandflies feed 

most actively at twilight/night

9

Leishmaniasis

transmission

sandflies are

pool feeders

  • saw-like
  • short mouthparts
  • drink blood/lymph fluid from wound

 

10

Leishmaniasis

transmission

parasite not in

insect salivary gland

  • parasite found anterior portion of gut and pharynx
  • transmitted to humans not from fly saliva in formation of wound BUT spread by vomit of the insect
    • parasite regurgitated by insect vector

11

Leishmaniasis

transmission

zoonotic or anthroponotic

  • zoonotic = animal to human
  • anthroponotic = human to human
  • different transmission cycles in different parts of the world (higher human density in some regions)
  • generally in areas with sparse human populations = zoonotic transmission
  • many humans = anthroponotic
  • different flies have different preferences for what they feed on

 

 

12

Leishmaniasis

transmission

zoonotic

  • in Mediterranean and Latin America
  • small rodents and canines (and humans)
    • rodents and canines are reservoirs in these regions

 

13

Leishmaniasis

transmission

anthroponotic

  • Indian subcontinent
  • more people packed in so tend to have human-human transmission
  • can still go through zoonotic cycle but in this region more associated with urban environment
  • other methods of human to human transmissions:
    • blood transfusion
    • organ/bone marrow transplants
    • congenital
    • drug usage (needle sharing)

 

14

Leishmaniasis

causative agent

  • 21 different Leishmania species can infect humans
    • L. donovani
    • L. infantum
    • L. braziliensis
    • L. major
  • some species are synonymous
    • L. infantum = L. chagasi (Old World vs New world)
      • old world anmes don't tend to name location
  • morphologically indistinguishable
    • hard to definitively diagnose because identical 
    • hard to treat
  • molecularly distinct
    • PCR
    • isoenzyme analysis
    • monoclonal antibodies

 

15

Leishmaniasis

major forms of the parasite

(list)

  • promastigote
    • non-infectious promastigotes
    • infectious metacycic promastigotes
  • amastigote

16

Leishmaniasis

major forms of the parasite

promastigote

  • 15-20 μm in length
  • flagellated 
  • motile
  • common morphology in insect host
    • 2 types of promastigote form, both arise in insect vector
    • flagellum pocket - anterior of nucleus
    • flagellum - not attached to cell body like in epimastigote or trypomastigote
      • sticks straight out from body
    • kinetoplast - located in front of nucleus, near anterior end of cell body

 

promastigote not in T. brucei or T. cruzi

17

Leishmaniasis

causative agent

2 types of promastigote

non-infectious promastigotes

  • insect gut
  • able to divide by binary fission

infectious metacyclic promastigotes

  • attach/invade neutrophils/macrophages
  • non-dividing (mechanism from going insect to mammalian host)

 

  • inside the gut the non-infectious promastigote becomes infectious metacyclic promastigote
  • metacyclic - form of the parasite that goes insect to human
    • generally true for most

 

18

Leishmaniasis

major form of the parasite

amastigote

  • 2-4 μm in diameter
  • stumpy flagellated
  • non-motile
  • intracellular form
    • in phagolysosome of immune cells
  • divides by binary fission
    • replicates within neutrophils and macrophages
      • in macrophage usually within vesicular compartment - membrane around them derived from host
      • can be transmitted back into the insect vector or in new neutrophils/macrophages
  • many (500+) parasites in 1 host cell
  • infectious - can invade macrophages
    • transmissable from humans to humans

 

  • flagellum pocket - anterior of nucleus
  • flagellum - very short (doesn't normally extend beyond the cell body), projecting only slightly beyond flagellum pocket
  • kinetoplast - located in front of the nucleus, near anterior endo f cell body

19

Leishmaniasis
life cycle

(picture)

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20

Leishmaniasis

life cycle 1

picture

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21

Leishmaniasis

life cycle 1

  • sand fly takes a blood meal
  • metacyclic promastigotes regurgitated into victim's bloodstream (pool of fluid it's feeding from from bite)
    • regurgitation caused by the parasite...
  • infected sandflies have dysfunctional stomodeal valve at foregut/midgut
    • valve that acts as boundary between foregut and midgut of insect vector's digestive tract
  • junction → damage caused by parasite
    • parasite in midgut, damages the valve  between the 2
  • valve can't close → blood moves back into mouthpiece and wound
  • when the insect vector sucks up blood/lymph that goes down the digestive tract, when it stops at the valve it goes back up, washing the parasite into the wound

 

  • reflux reaction → expulsion of parasite from anterior of gut into bite wound
  • exacerbated as parasite secretes promastigote secretory gel → physical barrier to fly feeding
    • gel acts as bung - promotes insect vector to suck and doesn't feel as though it's feeding
    • not much blood/lymph getting past this gel
      • getting more food than it thinkgs, so continues feeding
      • greater chance parasite going back into pool that insect vector is feeding on
  • secreted gel (and insect saliva) may potentiate fly infectivity
    • gel and saliva contain elements that our immune systems will respond to
      • immune system responds to physical damage from cut, also gel and insect saliva → magnifies attraction at that site
  • caused b promastigote's secreted gel plus physical wound etc. = causes neutrophils to go to an area
    • the parasite invasdes the nuetrophils 
      • always through neutrophil first, aters expression of neutrophil genes
      • does this to promote itself

22

Leishmaniasis

life cycle 1.5

  • metacyclic promastigote phagocytized by host neutrophils
  • infectious form taken up by neutrophil
  • use neutrophil to get into macrophages - 2 hypotheses​
    • both hypotheses lead to amastigote forms in macrophages
      • amastigotes multiply in macrophages
      • macrophages rupture, releasing amastigotes into bloodstream to continue cycle (infect new macrophages)
      • makes a long-lasting infection

23

Leishmaniasis

life cycle 1.5

trojan horse hypothesis

trojan horse hypothesis (in L. major)

  • infected neutrophils undergo apoptosis
  • neutrophil apoptotic markers onto surface of neutrophils
  • apoptotic cells phagocytosed by macrophages
  • metacyclic promastigote cargo delivered into macrophages
    • in addition to taking up dying neutrophil also takes up metacyclic promastigotes
  • once within the macrophage differentiates into amastigotes

 

24

Leishmaniasis

life cycle 1.5

trojan horse hypothesis

(picture)

A image thumb
25

Leishmaniasis

life cycle 1.5

hypothesis 2

  • neutrophil becomes in fected
  • metacyclic promastigote changes to amastigote which multiplies
  • causes neutrophil to burst
  • amastigotes into blood/lymph taken up by neutrophils
  • from one cell type (doesn't pack but only amplifies a few times) and neutrophil lyses to release amastigotes which are then taken up

26

Leishmaniasis 

life cycle 1.5

hypothesis 2

(picture)

A image thumb
27

Leishmaniasis

life cycle 2

picture

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28

Leishmaniasis

life cycle 2

  • back into the insect host
  • sandfly takes a blood meal from an infected mammal, ingests macrophages with infected amastigotes
  • macrophages rupture in insect vector gut
    • into midgut macrophage is digested and ruptures
    • releasing parasite amastigotes into the intracellular environment/lumen

29

Leishmaniasis

life cycle 3

picture

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30

Leishmaniasis

life cycle 3

  • in the midgut the amastigotes transform into non-infectious nondividing promastigotes 
    • from decrease in temperature, increase in pH
  • initally parasite is surrounded by mucus layer from the insect itself
    • parasite is trapped in this meshwork (peritrophic matrix)
      • peritrophic matrix = chitin and protein mesh secreted by the midgut epithelium that encloses the blood being digested
  • various enzymes produced by insect and parasite will degrade away the matrix and release the noninfectious promastigote into the lumen
    • host and parasite chitinase
    • after a few days the parasite escapes from peritrophic matrix 
  • released parasite move toward anterior of midgut
    • attach to microvilli of insect epithelial cells via lipophosphoglycans (LPG) to prevent excretion in feces
    • the noninfectious promastigote then fires LPG that sticks to the epithelial layer of the gut wall
      • prevents the parasite being excreted

31

Leishmaniasis

life cycle 4

picture

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32

Leishmaniasis

life cycle 4

  • the promastigotes divide in the anterior midgut
  • produce promastigote secretory gel
  • some promastigotes form metacyclic promastigotes (infective)
  • detach from midgut wall (LPG changes)

33

Leishmaniasis

cell invasion

(general)

  • Leishmania species are obligate intracellular parasites in the mammalian host
  • infects cells with microbicidal capability - macrophages
  • how do the parasites:
    • avoid instant killing?
    • replicate in a hostile environment?
    • establish chronic infections?
  • have to overcome immune insults that our body will mount against infection
  • may use immune systems to gain entry to mammalian cells

34

Innate immune mechanisms

  • complement-mediated lysis
  • lysosomal pH
  • lysosomal enzymes
  • oxidative stress

 

35

Innate immune mechanisms

complement-mediated lysis

picture

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36

Innate immune mechanisms

complement-mediated lysis

  • /alternative activation pathway
  • binding of early complement components to organism is followed by assembly of membrane attack complex (C5-C9)
  • use this to get into the cell - so we can exploit this
  • Leishmania parasites exploit this cascade to get into the parasite
  • can overcome this and utilize it at the same time 

37

Innate immune mechanisms

lysosomal pH

picture

A image thumb
38

Innate immune mechanisms

lysosomal pH

  • phagosomal compartment is acified upon fusion with lysosomes to pH5
  • microbes in a phagosome (a vacuole formed around a particle)
  • once within the macrophage or neutrophil, must overcome the cellular mechanisms of those particular cell lines to destroy infectious organisms
  • in neutrophil/macrophage vacular compartment becomes acidified
    • this is usually sufficient to damage membrane of infectious agent
    • downshift in pH
    • parasites use this to recognize the switch required in the life cycle
  • these pockets are targeted by degradative enzymes
    • parasite resistant to all of these

 

39

Innate immune mechanisms

lysosomal enzymes

  • lysosomes contain an array of digestive enzymes including:
    • proteases
    • nucleases
    • lipases
  • pockets (phagosomes) targeted by degradative enzymes
    • parasite resistant to all of these

 

40

Innate immune mechanisms

oxidative stress

mechanisms by which our cells generate oxidative stress

  • NADPH oxidase (Phox) complex on phagosomal membrane generates superoxide 
    • leads to production of hydrogen peroxide and hypochlorite
    • breaks down to stuff iwth antimicrobial activities
  • reactions with transition metals (eg iron) lead to the formation of hydroxyl radicals
  • nitric oxide synthase generates nitric oxide which combines with superoxide to form peroxynitrite
    • this vacuole is targeted for something that makes nitric oxide with antimicrobial expression
      • these are amplified when come with superoxide to make something very damaging

 

parasite must overcome all of these systems

 

 

41

The players of Leishmania infection

infective

metacyclic promastigotes

42

The players of Leishmania infection

host target cells

neutrophils, then macrophages

43

The players of Leishmania infection

mode of entry

  • passive, opsonized phagocytosis
  • doesn't require energy
  • opsonized - something that macrophages/neutrophils find attractive

 

44

The players of Leishmania infection

host receptors

  • CR1
  • CR3

(complement receptors)

45

The players of Leishmania infection

parasite molecules

  • gp63 protease
    • zinc protease
  • lipophosphoglycan (LPG)

 

46

The players of Leishmania infection

site of replication

phagolysosome vacuole

47

Infection involves

(2 ~steps)

  • complement evasion
  • host cell invasion

 

48

Complement evasion

parasite surface

parasite surface changes during differentiation

 

replicative promastigote (non-infective)

  • short LPG
  • low level of GP64

 

metacyclic promastigote (infective)

  • long LPG
  • high level of GP63

49

Complement evasion

LPG

  • lipophosphoglycan
  • sugar - fat phospho sugar mix
  • coat all over the cell surface
  • 3-5 million copies per cell
  • contains
    • oligosaccharide cap
      • toward external environment
    • phosphoglycan region = trunk, can have branches
      • galactose-mannose-phosphate 
      • side chains
      • galactosyl in replicative proastigotes
    • arabinosyl then caps then galactosyl in infective promastigotes
  • galactosyl side chain binds to galectin
    • binds parasite to wall of insect gut
  • capping prevents binding to galectin, facilitating detachment from wall of midgut

 

  • replicative promstigote with short LPG side chains always have galacto side chain that can bind to galectin on epithelial layer of insect vector (galactose interacts with galectin, lets parasites stick to wall)
  • into infectious metacyclic form - galactose is capped (sugar on the end) by arabinosyl that's sufficient to inhibit the interaction of LPG with the galectin, releasing metacyclic off the epithelial layer

50

LPG picture

A image thumb
51

Q image thumb

LPG

  • pink = inside cell
  • green dots = phosphoglycan 
    • can have disc branches

52

LPG picture - detailed

A image thumb
53

Complement evasion

LPG - continued

difference between noninfectious promatigotes and 

non-replicative infectious forms

  • all linked by GPI anchor
    • way of packing molecules very close together on the surface
  • apart from arabinosyl and galactose side chains, main difference is size
    • noninfectious promastigotes have short galactose mannose phosphate repeat sequences
    • significantly longer in non-replicative infectious forms
    • LPGs longer in infectious forms - more repeats

54

Q image thumb

  • blue is more sugars

 

55

Cutaneous leishmaniasis

pathology

  • most common manifestation - 10 million cases
  • doesn't usually progress beyond this
  • known as:
    • orient/bay sore
    • uta
    • Baghdad/Dehli/Kandahar/Lahore boil
    • city boil
  • L. tropica in cities
  • L. major in rural
  • starts as raised, generally painless red lesion at site of bite
  • can ulcerate/break the skin
  • small spot to progressively larger lesion, eventually immune system will kick in and wipe it out

 

56

Cutaneous Leishmaniasis

ulcer classified as wet or dry (crust formation)

  • wet ulcer = L. major
    • remains open
  • dry ulcer = L. tropica
  • no pus formation unless get bacterial infection
  • ulcer self heals 
    • causes scarring
    • eg L. major
    • in about 6 months
    • usually treatment not provided because will heal on oyur own
      • drugs saved for more severe form of the disease
    • if face bitten will be given the drug to speed up healing
  • once healed, individuals immune for life
    • once moutned appropriate immune response will be immune to that particular species
  • in some individuals diffuse lesions develop
    • wet ulcer may disappear, reappear on different parts of the body later in time

 

57

Diffusive Leishmaniasis

pathology

2 types

  • diffuse cutaneous leishmaniasis (DCL)
  • mucocutaneous leishmaniasis (MCL)

 

58

Diffusive Leishmaniasis

diffuse cutaneous leishmaniasis (DCL)

  • metastasizes over the whole body
  • non-ulcerated, scaly lesions (nodular)
  • generally associated with anergy (lack of immune response)
  • amastigotes abundant in lesions
    • packed with macrophages and amastigotes
  • eg L. aethiopica, L. mexicana
  • can appear anywhere on the  body

59

Diffusive Leishmaniasis

mucocutaneous leishmaniasis (MCL)

  • primarily L. braziliensis (3-5% of cases move from cutaneous to this kind of form) 
    • most prevalent in Latin America
  • metastasizes mucocutaneous junctoins
    • mouth/nose/soft palate or anus/genitalia
  • can occur weeks/years after first degree infection
  • tissue destroyed → severe disfigurement
  • seconary bacterial infections common
  • may affect eg breathing, prone to secondary infections

 

60

Visceral Leishmaniasis

pathology

  • starts as cutaneous lesion to visceral form
  • known as
    • Kala-azar
    • DumDum fever
  • most serious form
  • 200-500,000 cases/50,000 deaths per year
  • typically caused by
    • L. donovani (India/Pakistan)
    • L. infantum (Mediterranean) = L. chagasi (Latin America)
  • high fatality rate
  • darkening of the skin
  • others highly disfiguring or may lead to death through secondary infection, but are not lethal itself

61

Visceral Leishmaniasis

symptoms

  • generalized infection of the reticuloendothelial system
  • involves liver, spleen, bone marrow, lymph nodes
  • characterized by
    • fever (sudden or gradually rising)
    • splenomegaly (enlargement of spleen)
    • hepatomegaly (enlargement of the liver)
    • enlarged lymph nodes/tonsils (→ anemia, leucopenia, thrombocytopenia)
  • disease progression leads to malaise, tiredness, lassitude, weaness
  • patient exhibits wasting syndrome despite good appetite
    • muscle wastage - musculature doesn't develop to the same degree, remains extremely thin
  • secondary infections are common
  • kills by organ failure
    • ​affects organs associated with eg clearing blood and helping immune system = prone to secondar infections
      • pneumonia
      • tuberculosis
      • dysentery
  • 50,000 deaths directly attributed to Leishmania
    • but if have visceral Leishmaniasis will die of secondary infection and won't be attributed to Leishmania (which weakens the immune system)

 

62

Leishmaniasis

disease severity depends on

immune status of the individual

63

Th1-type response

  • cellular immune response triggered - no/low antibody formation
  • recover from infection, are immune (cutaneous)

 

64

Th2-type response

  • humoral immune response triggered, antibody formation occurs
  • succumb to infection (visceral)

 

  • Th2 by mistake = develops to more serious forms
  • antibodies → succumb, more severe forms
  • some of the more dangerous and highly infectious strains appear to be able to switch host's immune response from something that will clear the parasitemia to something that will enhance the parasitemia
    • some strong L. donovani strains force host to switch from Th1 to Th2 response (mechanisms unknown)

 

65

Leishmaniasis

diagnosis

  1. epidemiological history
  2. symptoms
  3. definitive diagnosis - detection of parasites, seriology

 

66

Leishmaniasis

diagnosis

epidemiological history

dermal and visceral both

  • suspected
  • geographical presence of parasite
  • history of sandfly bite

 

67

Leishmaniasis

diagnosis

symptoms - dermal

  • chronic, painless lesion (CL)
  • mucocutaneous lesion (MCL)
  • scaly lesions (DCL)

 

68

Leishmaniasis

diagnosis

symptoms - visceral

  • prolonged fever
  • splenomegaly
  • hepatomegaly
  • anemia
  • leucopenia
  • thrombocytopenia

 

69

Leishmaniasis

diagnosis

definitive diagnosis - detection of parasites

dermal

amastigotes in scraping

  • CL - incision of lesion
  • dermal scraping examined by Giemsa staining/microscopy

amastigotes in aspirates

  • CL - injectio saline in lesion
  • examine aspirate following giemsa staining/microscopy

in vitro culturing of promastigotes from aspirates

  • CL - injection saline in lesion
  • transfer to tissue culture medium
  • monitor for promastigote growth (~1 week)
  • amastigote parasites break out in tissue culture of macrophage and spontaneously generate the promastigote form (grow out the form that grows in insect vector, but takes time)

inoculation of aspirates in model organism

  • CL - injection saline in lesion
  • transfer to model organism (hamster)
  • monitor for clinical pathology (2-3 months)

70

Leishmaniasis

diagnosis

definitive diagnosis - detection of parasites

visceral

amastigotes in bone marrow, aspirates, or biopsy

  • CL- injection saline in lesion
  • examine aspirate following Giemsa staining/microscopy

in vitro culturing of promastigotes from aspirates

  • CL - injection saline in lesion
  • transfer to tissue culture medium
  • monitor for promastigote growth (~1 week)

 

71

Leishmaniasis

diagnosis

definitive diagnosis - serology

dermal

Leishmanin skin test

  • looks at person's response to fixed parasites (dead) or parasitse in extracts 
  • intradermal injection of promastigotes 
    • extracts or whole, killed
  • monitor for local dealyed hypersensitivity (local redness)
  • development of erythema if leish +ve
    • if some time in the past the person was infected - parsite may be wiped out but still be responding as if the parasite was there

 

72

Leishmaniasis

diagnosis

definitive diagnosis - serology

visceral

serological test (direct agglutination, K39 dipstick)

direct agglutination

  • fixed coloured parasites + patient serum
    • = anti-Leishmania antibodies → agglutinate fixed coloured parasites = blue/purple
    • (react with colored parasites to agglutinate them)

 

K39 dipstick

  • patient serum applied to immunochromatographic dipstick
  • dipstick contains antigen to K39, abundant amastigote surface antigen
    • K39 is a 39 amino acid repeat sequence
  • K39 antigen on strip, let patient's serum migrate
  • if immune response will bind to K39 antigen and change color
  • patient serum reacts to K39 if contains anti-Leishmania antibodies
  • in figure lower band = control, 2 bands appear = infection

73

Front line drugs against Leishmaniasis

pentavalent antimonial

drugs based on antimony - can draw comparisons with the arsenic-based drug for HAT 

sodium stibogluconate (Pentostam) → GSK

meglumine antimoniate (Glucantime) → Sanofi S.A

  • pro-drug 
  • administered in inactive state (pentavalent)
  • activated (to trivalent state) only by amastigote

 

  • introduced in 1945
  • daily intravenous infusion (Pentostam) or intramuscular injections (Glucantime)
  • 20mg/kg/day fro 10 days (CL)
  • 20mg/kg/day for 30 days (VL/DCL/MCL)

 

74

Front line drugs against Leishmaniasis

pentavalent antimonial

 

variation in species sensitivity

not all Leishmania species are the same

  • those that cause the more severe form are more susceptible to these pentavalent antimonials

 

L. donovani   >

L. braziliensis   >

L. mexicana   >

L. major

 

VL species > DCL/MCL species  > CL species

75

Front line drugs against Leishmaniasis

pentavalent antimonial

problems

high level of toxicity

  • fatigue, nausea, muscle/joint pain, changes in ECG, cardiotoxicity, hepatic/renal dysfunction, shock, and (rarely) sudden death
  • haevy metal = heavy metal poisining
  • similar problems to melarsoprol

 

don't always work

  • may fail to clear parasitemia
  • resistance in field
    • esp in India

 

76

Front line drugs against Leishmaniasis

pentavalent antimonial

activation and uptake

  • activation can occur inside/outside cell
  • both SbV and SbIII can be taken up by cell
    • prodrug = SbV
    • drug = SbIII
  • conversion from prodrug (pentavalent) to drug (trivalent) can only occur if amastigotes present
  • don't know where conversion occurs
    • surface of amastigote before drug goes into the cell
    • within the cell itself
    • could be both
  • if activated outside know pentavalent can be taken in, as can trivalent

 

77

Pentavalent antimonial

uptake

  • SbIII taken up by  AQP (aquaglyceroporin)
    • same with melarsoprol
    • resistance toward antimonials have been associated with reduced uptake of trivalent form by AQP
  • SbV - don't know what's used for uptake

 

78

Pentavalent antimonials

Conversion/activation

  • conversion outside pentavalent → trivalent
    • don't know what mediates it
  • Sb→ SbIII inside by reductases
    • ACR2 - arsenite reductase
    • TDR1 - thiol-dependent reductase

 

79

Pentavalent antimonials

binding

  • trivalent form binds conjugate agents - especially thiols
  • trivalent form binds trypanothione, inhibits trypanothione-dependent enzymes
    • eg trypanothione reductase
    • which has major implications how these parasites:
      • respond to oxidative stress
      • synthesize DNA
    • heavy metal works the same as melarsoprol - sticks to thiols, messes up downstream pathways

80

Resistance to antimonials

  • 1980s antimonials resistance observed
  • today 50-65% of VL cases in Bihar
  • antimonials obsolete in India
  • why?
    • unrestricted availability
    • incorrect doses
    • inconsistent quality
      • cut with other substances
      • may not be up to required therapeutic doses
  • mechanism of resistance in the field is unknown
  • lab approaches used to dissect pathways
    • resistance by parasites can be readily generated in the lab by growin cells on sub-lethal levels of the drug

81

Resistance to antimonials

Amplification

  • SbR Leishmania amplified specific genetic loci from genome → episome
    • amplify part of genome, throw it out to generate a plasmid
    • amplify up to give high copy number
    • very plastic genome
  • sequence/mapping/biochemical studies shown that episome contains ABC transporter
    • localized to unknown organelle that's very close to the flagella pocket
  • in mammals ABC transporters involved in 
    • multidrug resistance
    • heavy metal resistance
  • Leishmania ABC transporter designated as MPRA (multipdrug resistance protein A)
  • previously shown to function as metal efflux pump that recognizes metals conjugated to thiols
    • MRPA pumps metal-thiol conjugates into this organelle
    • trypanothione conjugates the antimonial, pumped via this mechanism into the uknown organelle
  • overexpression in parasites causes resistance
    • higher level of MRPA leads to resistance to antimonials
    • can knock gene out - not essential to parasite genome, makes parasite more sensitive to the drug

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Resistance to antimonials

MRPA localization

MRPA localized to unknown organelle near flagella pocket

 

biochemical/functional analysis shows that MRPA

  1. transports metals conjugated to thiols
  2. over-expression causes resistance to antimonials
  3. null mutants are sensitive to antimonials

 

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Resistance to antimonials

via sequestration

resistance to antimonials is via sequestratoin of trypanothione-drug conjugate

 

 

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Resistance to antimonials via sequestration of trypanothione-drug conjugate

antimonial-sensitive parasites

  • pentavalent to trivalent form
  • trivalent form interacts with trypanothil (TS) to form metal-thiol conjugate to hit the target enzymes (trypanothione-dependent enzymes)

85

Resistance to antimonials via sequestration of trypanothione-drug conjugate

antimonial resistant parasites

in resistant cell lines

  • get diversion away from the target
  • because you amplify up MRPA gene - increases the amount of transporter such that the metal-thil conjugate isn't getting to the target but is pumped out into the unknown organelle
  • rate-limiting step is amount of TS2 (trypanothione)
  • so is the level of trypanothione affected in resistant cell lines?

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SbR parasites

levels of trypanothione

  • resistant parasites have elevated levels of trypanothione 
    • Halmeur et al., 2002
  • amplification of trypanothione synthesis genes
    • Grondin et al., 1997
    • Haimeur et al., 1999
  • inhibitors of trypanothione synthesis (buthionine sulfoxice/eflornithine) can reverse resistance
    • Grondin et al., 1997
    • Haimeur et al., 1999

 

  • high levels of trypanothione, enzymes in trypanothine synthesis amplified
  • make more TS2 to bind out the drug
  • use inhibitors that block synthesis of trypanothione to make cells sensitive to antimonial
  • as well as upregulating transporter, upregulating trypanothione biosynthesis pathway to bind out lots of antimonial that can be cleared out of the cell into this organelle

 

87

Resistance to antimonials via

sequestration of trypanothione-drug conjugate

AND

increased trypanothione synthesis

 

88

Pentavalent antimonials

uptake and conversion

(picture)

A image thumb
89

Antimonial sensitive parasites

(picture)

A image thumb
90

Antimonial resistant parasites

(picture)

A image thumb
91

Alternative drugs against Leishmaniasis

  • amphotericin B
  • miltefosine 
  • paromomycin

92

Alternative drugs against Leishmaniasis

Amphotericin B

  • polyene antibiotic
  • originally developed as antifungal
  • effective against antimonial resistant VL
  • infusion, severe toxic side effects
  • lipid formulation (AmBisome) have reduced toxicity
    • mycels = lipid globules into which you can insert drug = reduced toxicity
  • amphotericin B binds to 24-substituted sterols (eg ergosterol) disrupting osmotic integrity of the membrane
    • binds sterols in parasite cell membrane = punches holes at point of sterol to make parasite cell leaky

93

Alternative drugs against Leishmaniasis

miltefosine

  • phosphocholine analogue
  • developed in 1980-90s as anticancer drug, abandoned due to toxicity problems
  • activity against Leishmania in vitro and in vivo (1987-1996)
  • daily, oral administration for 28 days
  • clinical trials for VL in Bihar State, India, >95% cure, including Sb(V) resistant cases
    • Registered in India & launched in 2003
  • registered for VL/CL in Colombia, 90% cure: clinical trials in Brazil
  • BUT it is teratogenic (causes malformations in embryos), cannot be used by women of child-bearing age
  • mode of action – uncertain, possibly by inhibiting phospholipid formation, signal transduction or calcium homeostasis
     

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Alternative drugs against Leishmaniasis

paromomycin

  • aminoglycosides antibiotic (related to kanamycin, neomycin)
  • developed as antibacterial, used to treat Entamoeba and Cryptosporidium infections
  • anti-leishmania activity reported in 1960s
  • daily, oral administration for 20 days
  • phase 2 trials for VL in India/Kenya, ~90% cure
  • phase 3 trials ongoing/pharmaceutical manufacturer secured
  • mode of action – uncertain. Possible effects in mitochondrion?
     

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Leishmaniasis

prevention and control

vector control

  • insecticides and repellants
  • screens and bednets
  • difficult to carry out due to:
    • cost
    • re-emergence after cessation of spraying programs
    • diversity of epidemiology (rural vs urban)

 

control of reservoir species

  • canine vaccines
  • culling
  • Mediterranean basin and Latin America
  • try to control flies
  • requires money and political will = problem

 

96