Asus AAM6010EV-M User Manual download pdf (Page 7)

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tide mixed with 100 µL of H2O-TFA (triuoroacetic acid)

were injected into the column. Absorbance was monitored

at 220 and 280 nm. e peptides were ensured for non con-

tamination with endotoxin by Limulus Amebocyte Lysate

(1987 US FDA LAL test).

Bacteria were cultivated in Luria-Bertani standard liq-

uid medium during 2-3h at 37oС, 250 rpm. Logarithmic

phase cultures of bacteria were diluted in LB to an A630 =

0.01 (106 cfu/mL). Antimicrobial test was performed in 96-

well microtitration plates in a 100 µL nal volume using a

peptide concentration range from 200 to 1.5 µM according

the standards of CLSI (Clinical and Laboratory Standards

Institute, 2006). Each microtitration plate included positive

(0.7% formaldehyde solution) and negative control (H2O)

as well as sterility control (LB without bacteria). Each well

contained 50 µL of the microorganism suspension (106 cfu/

mL) and 50 µL of peptide solution or positive/negative con-

trol.

e content of antimicrobial microtitration plate is pre-

sented in Fig.1. Microtitration plate was aerward covered

with the lm, shortly incubated at 4°C during 1-2 hours and

rested overnight at 37°C, 150 rpm. e minimal inhibitory

concentrations (MICs) of Galarmin and analogues were

determined by measuring the absorbance (optical density -

OD) of the plate’s wells at 630 nm by plate automatic reader

(Asys UVM 340). e meaning of OD less than 0.2 shows

the absence/inhibition of bacterial growth, and 0.3 and

more, bacterial presence which correlate with the number

of OD. MIC was expressed as the lowest concentration of

peptide that completely inhibited bacterial growth.

Each experiment was performed in triplicate and al-

lowed to test the inhibitory activity of two peptides on one

bacterial strain. Statistical analysis of all data was performed

by Student-Fisher’s method of distribution with determina-

tion of t-criteria.

Beside the antimicrobial activity of peptides alone,

Galarmin and analogues (100 µM) were incubated with

bacteria in combination with secretory leucocyte protease

inhibitor (SLPI), another peptide known for its antimicro-

bial activity in vivo and in vitro. Human alarm antiprotease

SLPI is an 11.7 kDa cationic non-glycosylated protein con-

taining 107 amino acids that plays multiple important roles

in normal homoeostasis and inammation, including anti-

protease and antimicrobial activity 13. Recombinant SLPI

was provided by Prof. Jean-Michel Sallenave (Unit of Innate

Host Defence and Inammation, Institut Pasteur Paris).

RESULTS

e analysis of PRP by reverse-phase HPLC revealed

the purity and non contamination of all analyzed peptides

samples (Fig.2). Using the Limulus amebocyte lysate, we

showed that the preparation of PRP-1 was free of any endo-

toxin contamination.

e results of antimicrobial tests (OD of bacterial

growth) for Galarmin, Gx-NH2, d-15 Galarmin and dGx-

NH2 are represented at Table 1. On Table 2 are represented

the results of the eect of Galarmin and analogues on SLPI

(secretory leucocyte protease inhibitor) antimicrobial activ-

ity. e summarized results of antimicrobial tests are shown

on Tables 3 and 4.

DISCUSSION

As it is shown from the tables no direct inhibitory eect

was observed for Galarmin and analogues on MRSA and

other Gram-positive and Gram-negative bacterial strains.

No synergistic or inhibitory eects were observed for

Galarmin and analogues at concentration 100 µM when

these peptides were incubated in combination with SLPI at

a concentration producing (10 µM) or not (5 µM) growth-

inhibitory activity. Indeed, A630 values were approximately

the same for SLPI alone or in the presence of Galarmin and

analogues.

ese results clearly indicate that Galarmin and ana-

logues are devoid of antimicrobial properties and that the

strong protective eects observed in vivo against MRSA and

other pathogens are due to their immunomodulatory activi-

ties on host organisms.

ORIGINALNI RAD / ORIGINAL ARTICLE

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