NEHA HANIF

What are dendritic cells?

Dendritic cells (DCs) are immune cells that form part of the mammalian immune

system. Their main function is to process antigen material and present it on the

surface to other cells of the immune system, thus functioning as antigen-

presenting cells.

C.neoformans

•Cryptococcus neoformans is an opportunistic fungal pathogen that causes disease

predominantly in immunocompromised patients, particularly individuals with

AIDS, transplant recipients, and those with lymphoid and hematological

malignancies

•Protective immunity to C. neoformans is dependent on an adaptive Th1-type

immune response .

How dendritic Cells Kill The Pathogen?

tissue culture media RPMI

Media for human dendritic cell generation consist of RPMI-1640

supplemented with 10% heat-inactivated fetal bovine serum, 2mM

L-glutamine,100 U penicillin/ml,100 µg of streptomycin/ml and

50mM,2-mercaptoethanol (complete medium).

plastic ware

OREGON green labelled 3C2 antibodies

. Yeast cells were grown for 18 hours at 30°C

with shaking in YPD broth.

Cells were centrifuged and washed three

times with sterile phosphate-buffered saline

(PBS). Viable yeasts were quantified using

trypan blue dye exclusion in a

hemacytometer.

peripheral blood was obtained from healthy volunteers by veni-puncture

The blood was anti-coagulated with heparin

The tubes were centrifuged at 1,000 × g for 10 min without thebrake.

After that plasma was collected and stored at −20°C until use.

1ug green oregon

stained 3c2 antibodies

Dendritic cells

Cryptococcus

neoformans

PHAGOCYTOSIS OF CRYPTOCOCCUS BY DENDRITIC

CELLS

Incubate 25 ◦C for 10 to 60

minutes

4 TUBES of 1.7ml RPMI(Roswell Park Memorial Institute)

(labelled as 10 min,20min,30min or 60 min)

Confocal microscopy

PARA-FORMALDEHYDE

2%

Leave at

room temp

for 10 min

Saponin 0.1%

Leave at

room temp

for 10 min

INTRACELLULAR STAINING

EEA-1 ANTIBodies

EACH TUBE

Extraction of lysosome

lysosomal extraction

buffer 2.7 mlDendritic cells Homogenizer (20-25

time)

RPMI

CENTRIFUGE :1000

rpm for 10 min

Centrifuge at 20000 rpm for 20 min Collect supernatant

1ml

Sonicate-20sec

Incubate at room

temp for 3 days

PELLET CONTAIN

LYSOSOME

LYSOSOME EXTRACTED

TAKE C.NEOFORMANS CULTURE

INOCULATE IN PBS

LYSOSOMAL

EXTRACTS

DILUTE IN PBS ,PLATED ON SDA AND CALCULATE CFU

RESULTS

the intracellular staining confirmed the entry

of C. neoformans organisms into endosomes

and lysosomes.

The results OF CONFOCAL MICROSCOPY

showed that the majority of C.

neoformans organisms appeared to enter the

lysosomal compartment of HDCs within 20

min following uptake.

We determined whether lysosomal components

from DCs were capable of killing C.

neoformans. We found that crude lysosomal

extracts from DCs had dose-dependent anti

cryptococcal activity, with nearly complete

killing observed when the extracts were diluted

50%.