NEW CONCEPTS

ABSTRACT

Aim of this study: Select technetium-99m labelled peptides that can discriminate between
bacterial infections and sterile inflammations.

1. We first assessed the binding of various 99mTc-labelled natural or synthetic peptides,

which are based on the sequence of the human antimicrobial peptide ubiquicidin (UBI)
or human lactoferrin (hLF), to bacteria and to leucocytes in vitro.
a. Antimicrobial peptides and proteins (AMPs) play a critical role in warding off
invading microbial pathogens. In addition, AMPs can possess other biological
functions such as apoptosis, wound healing, and immune modulation. They
have been identified in a variety of exposed tissues or surfaces such as skin,
eyes, ears, mouth, airways, lung, intestines, and the urinary tract.
b. Ubiquicidin (UBI) is a cationic, synthetic antimicrobial peptide fragment that
binds preferentially with the anionic microbial cell membrane at the site of
infection. (https://www.ncbi.nlm.nih.gov/pubmed/15809477)
c. Human lactoferrin (hLF) is a major component of the nonspecific defense of
mucosal surfaces and neutrophils and is active against a variety of pathogens.
This protein displays antimicrobial properties against gram-positive and gram-
negative bacteria by limiting the availability of environmental iron.
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC98043/)
2. In order to select peptides that preferentially bind to bacteria over host cells,
radiolabelled peptides were injected into mice intraperitoneally infected with Klebsiella
pneumoniae (K. pneumoniae) and the amount of radioactivity associated with the
bacteria and with the leucocytes was quantitated.

a. Intraperitoneally
b. Klebsiella pneumoniae es una: (https://www.lifeder.com/klebsiella/)
i. Bacteria anaeróbica facultativa (capaces de vivir tanto en condiciones
de presencia como de ausencia de oxígeno),
ii. Gram negativa (La pared celular está formada por dos membranas
lipídicas, una interna citoplasmática y otra externa, con un espacio
entre ellas denominado espacio periplasmático donde hay capa de
peptidoglicano), que
iii. No produce esporas y que tiene forma de bacilo.
iv. Pertenece al grupo de los coliformes, bacterias comunes de flora
gastrointestinal de los seres humanos y de otros vertebrados.
3. The next phase focussed on discrimination between bacterial infections and sterile
inflammatory processes using 99mTc-labelled peptides in:
 a. Bacterial infections→ Mice intramuscularly infected with various bacteria (e.g.
multi-drug-resistant Staphylo- coccus aureus) and in
b. Sterile inflammatory processes→ animals that had been injected with
lipopolysaccharides (LPS) of bacterial origin to create a sterile inflammatory
process.
i. Lipopolysaccharides (LPS) of bacterial origin: a major component of
the cell wall of gram-negative bacteria; lipopolysaccharides are
endotoxins and important antigens. (https://medical-
dictionary.thefreedictionary.com/lipopolysaccharide)
ii. What's the difference between infection and inflammation?
(https://www.quora.com/Whats-the-difference-between-infection-and-
inflammation)
iii. Sterile inflammatory processes: Inflammation as a result of trauma,
ischaemia–reperfusion injury or chemically induced injury typically
occurs in the absence of any microorganisms and has therefore been
termed ‘sterile inflammation’. Similar to microbially induced
inflammation, sterile inflammation is marked by the recruitment of
neutrophils and macrophages and the production of pro-inflammatory
cytokines and chemokines, notably tumour necrosis factor (TNF) and
interleukin-1 (IL-1). Although inflammation is important in tissue repair
and eradication of harmful pathogens, unresolved, chronic inflammation
that occurs when the offending agent is not removed or contained can
be detrimental to the host. The production of reactive oxygen species
(ROS), proteases and growth factors by neutrophils and macrophages
results in tissue destruction, as well as fibroblast proliferation, aberrant
collagen accumulation and fibrosis. There are several examples of
sterile inflammatory diseases.
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3114424/)
4. Also, we studied the distribution of 99mTc-labelled UBI 29-41 and UBI 18-35 in rabbits
having an experimental thigh muscle infection with K. pneumoniae and in rabbits
injected with LPS. Grupos de comparación.
5. Based on the results of our in vitro and in vivo binding assays, two peptides, i.e. UBI
29-41 and UBI 18-35, were selected as possible candidates for infection imaging. The
radiolabelled peptides can detect infections with both gram-positive and gram-negative
bacteria in mice as early as 5–30 min after injection, with a target-to-non- target (T/NT)
ratio between 2 and 3; maximum T/NT ratios were seen within 1 h after injection.
a. In rabbits, high T/NT ratios (>5) for 99mTc-labelled UBI 29-41 were observed
from 1 h after injection.
b. No accumulation of the selected 99mTc-labelled UBI-derived peptides was
observed in thighs of mice and rabbits previously injected with LPS.
c. High target:non-target ratio is critical. If this ratio is not high enough (5:1
minimum for planar imaging, about 2:1 for SPECT imaging), a non-diagnostic
scan can result, making it difficult or impossible to distinguish pathology from
background.
(http://www.meddean.luc.edu/lumen/MedEd/Radio/Nuc_med/radpharm/sect-
a4.htm)
6. Scintigraphic investigation into the biodistribution of 99mTc-labelled UBI peptides
revealed that these peptides were rapidly removed from the circulation by renal
 excretion. Similar data were observed for 99mTc- labelled defensin 1-3. Our data for
99mTc-labelled hLF and related peptides indicate that these compounds are less

favourable for infection detection. Conclusión: Taken together, 99mTc-labelled UBI 18-
35 and UBI 29-41 enable discrimination between bacterial infections and sterile
inflammatory processes in both mice and rabbits. Based on their characteristics, we
consider these peptides the candidates of preference for detection of bacterial
infections in man.

INTRO
● Existencia de técnicas que utilizan el technetium-99m como labelling agent. In nuclear
medicine various techniques have been explored to visualise bacterial infections and
sterile inflammatory processes.
○ The radiopharmaceuticals that have been introduced or proposed include
various technetium-99m labelled agents, such as: polyclonal and monoclonal
immunoglobulins, cytokines, chemotactic peptides, antibiotics and human
defensin.
○ Desventaja: Unfortunately, the majority of these radiolabelled compounds do
not distinguish between bacterial infections and sterile inflammatory processes
and administration of cytokines and chemotactic peptides affects the immune
system.
● The major drawbacks of radiolabelled ciprofloxacin are its non-specific binding to DNA
of both bacteria and host cells and the risk of emerging antibiotic-resistant bacteria.
○ Radiolabeled ciprofloxacin background: Bacterial infections are still one of the
main causes of patient morbidity and mortality worldwide. Nowadays, many
imaging techniques, like computed tomography or magnetic resonance
imaging, are used to identify inflammatory processes, but, although they
recognize anatomical modifications, they cannot easily distinguish bacterial
infective foci from non bacterial infections. In nuclear medicine, many efforts
have been made to develop specific radiopharmaceuticals to discriminate
infection from sterile inflammation. Several compounds (antimicrobial peptides,
leukocytes, cytokines, antibiotics…) have been radiolabelled and tested in vitro
and in vivo, but none proved to be highly specific for bacteria. Indeed factors,
including the number and strain of bacteria, the infection site, and the host
condition may affect the specificity of tested radiopharmaceuticals.
Ciprofloxacin has been proposed and intensively studied because of its easy
radiolabelling method, broad spectrum, and low cost, but at the same time it
presents some problems such as low stability or the risk of antibiotic resistance.
(https://www.ncbi.nlm.nih.gov/pubmed/27512687)
● Porque se busca usar AMPs Introduction of radiolabelled human defensin into nuclear
medicine practice in man is hampered by the elaborate procedures required to isolate
large quantities of this antimicrobial peptide and the possibility that it affects human
leucocyte functional activities. Since they are believed to preferentially bind to bacteria,
antimicrobial peptides/proteins remain the first choice for the development of new
radiopharmaceuticals for infection imaging.
○ Human defensin: Defensins are antimicrobial peptides that act mainly by
disrupting the structure of bacterial cell membranes and are found in many
compartments of the body. Evidence is accumulating that defensins play a
 central role in defense against pathogens, and they are considered part of the
innate immune response. (https://en.wikipedia.org/wiki/Defensin)
● In this study we focussed on two human antimicrobial peptides/proteins – ubiquicidin
(UBI, 6.7 kDa) and lactoferrin (hLF, 77 kDa) – and we used human neutrophil defensin
1-3 (3.5 kDa) as comparison.
● Por qué escogieron UBI:UBI was originally isolated from murine macrophages and
later an identical UBI was isolated from human airway epithelial cells, allowing us to
extrapolate the results obtained in mice to human studies.
● Por qué escogieron hLF: hLF was chosen because of it is abundance in mucosa and
neutrophils in man and its known binding to surface structures of both gram- positive
and gram-negative bacteria, although specific receptors for this protein on human
leucocytes have been reported as well. Since linear synthetic peptides are less
susceptible to denaturation and are more likely to rapidly leave the circulation to enter
the site of infection, they were chosen for this study. Besides, they are relatively
inexpensive to prepare.
● Labelling: For labelling of these peptides/proteins with 99mTc we successfully
introduced a simple and direct technique (developed into kit format) that takes
approximately 60 min of preparation time.
● Based on these considerations we developed a strategy to select optimal peptides for
discrimination between infections with bacteria and sterile inflammation in mice and
rabbits.

MATERIAL AND METHODS: Table 1

Proteins and peptides.
● Human defensin (human neutrophil peptide) 1-3 was purified from human neutrophils
and UBI from human H292 airway epithelial cells.
● hLF was obtained from fresh milk of a single donor by cation exchange
chromatography.
● Linear fragments of UBI and hLF were synthesised as described elsewhere and are
further referred to as synthetic peptides. The amino acid sequences of the synthetic
peptides are given in Table 1.
● The natural peptide ubiquicidin and synthetic peptides were dissolved in 0.01 M acetic
acid pH 4 and then analysed by reverse-phase high-performance liquid
chromatography (HPLC) on a C18 column (4.6×250 mm, Vydac, The Separations
Group, Hesperia, Calif., USA).
○ HPLC: Es un tipo de cromatografía en columna en el que por acción de una
bomba, se hace pasar una mezcla de compuestos o analitos en un sistema
disolvente comúnmente conocido como fase móvil. La fase móvil pasa a
través de una columna cromatográfica, que contiene la fase estacionaria a un
flujo especificado. La separación de los compuestos ocurre en base a la
interacción de éstos con la fase móvil y la fase estacionaria.
(https://phenomenex.blog/2017/12/18/que-es-la-hplc/)
● A linear 0%–100% water-acetonitrile gradient was applied in 30 min using 0.1%
trifluoroacetic acid as an ion-pairing agent. The purity of the peptides amounted to
approximately 95%. The molecular weight of the purified synthetic peptides was
confirmed by mass spectrometry.
○ Water-acetonitrile gradient:
○ Ion-pairing agent:
 ● They were dried in a speed-vacuum concentrator (Savant Instruments, Inc.,
Farmingdale, N.Y., USA) and then lyophilised. Stock solutions (1 mM) of the peptides
using 0.01 M of acetic acid (pH 4) as solvent were stored at –70°C.
● Contamination of the peptides with lipopolysaccharides (LPS), as assessed by Limulus
assay (Chromogenix, Moelndal, Sweden), proved to be lower than 100 pg/ml.
○ Speed-vacuum concentrator:
○ lyophilised:
○ Limulus assay:

Labelling procedure and quality control.

● Antimicrobial UBI and hLF peptides were labelled with 99mTc using a previously
described method.
● Briefly, 10 μl of a peptide solution (1 mM in 0.01 M of acetic acid pH 4) was added to
2 μl of an aseptic solution of 0.5 mg/ml of stannous pyrophosphate (Department of
Clinical Pharmacy and Toxicology, Leiden University Medical Center, LUMC).
● Immediately thereafter, 4 μl of a solution of 10 mg of KBH4 (crystalline, Sigma
Chemical Company, St. Louis, Mo., USA) per ml of 0.1 M NaOH was added.
● After addition of 0.1 ml of 99mTc-sodium pertechnetate solution (200 MBq/ml→
megabecquerel, 106 Bq) obtained from a 99mTc generator (Ultratechnekow,
Mallinckrodt Medical, Petten, The Netherlands), the mixture, having a final pH between
5 and 6, was gently stirred at room temperature for 60 min and then used as such.
● The final preparation is a solution containing the peptides labelled with 99mTc. For this
paper we referred to this solution as 99mTc-labelled peptides.
● The yield of labelling of the natural and synthetic peptides was determined by instant
thin-layer chromatography (ITLC) using sa- line or methyl ethyl ketone as eluent. With
this method only 99mTc in the form of pertechnetate is eluted. Other radioactivity
species remain at the site of origin. We analysed the composition of the samples by
HPLC cation exchange analysis as described elsewhere.

Bacteria.
● Staphylococcus aureus 25923 (S. aureus) and Klebsiella pneumoniae 43816 (K.
pneumoniae) were obtained from the American Type Culture Collection (ATCC,
Rockville, Md., USA).
● The multi-drug-resistant S. aureus type 2141 (MRSA) was a clinical isolate
(Department of Infectious Diseases, LUMC).
● Overnight cultures of bacteria were prepared in brain heart infusion broth (BHI, Oxoid,
Basingstoke, UK) in a shaking waterbath at 37°C. Aliquots of suspensions containing
about 5×108 viable stationary- phase bacteria per ml of BHI were snap-frozen in liquid
nitrogen and stored at –70°C. Just before use, an aliquot of this suspension was rapidly
thawed in a waterbath at 37°C and diluted in Na-PB.
○ Brain Heart Infusion broth (BHI): It is a nutrient-rich medium made by
combining an infusion from boiled bovine or porcine. It is used to culture a
variety of fastidious organisms (challenging to grow), such as streoticocci,
pneumococci, and meningococci.

Human leucocytes
● Human leucocytes were purified from buffy-coats of healthy volunteers (Bloodbank,
LUMC) by density centrifugation.
 ● Briefly, buffy-coats were diluted in phosphate-buffered saline (PBS, pH 7.4) and then
subjected to Ficol-amidotrizoate (ρ=1.077 g/ml; Pharmacia, Uppsala, Sweden) density
centrifugation at 440×g for 20 min. The cells in the interphase containing the
mononuclear cells and the pellet containing the granulocytes were mixed and washed
3 times in PBS.
● Contaminating erythrocytes were removed by single hypotonic lysis. Viability of the
leucocyte preparation exceeded 95%, as determined by trypan blue dye exclusion.
Activation of monocytes was accomplished after incubation of the cells with 100 ng of
LPS from Escherichia coli (Sigma) in 1 ml of Na-PB and activation of the granulocytes
was achieved by exposure to 100 nM formyl-Met-Leu-Phe (FMLP) for 1 h at 37°C.
○ Density centrifugation:

In vitro binding of antimicrobial peptides to bacteria and human leucocytes.

Binding of 99mTc-labelled peptides to cells was assessed at 4°C unless indicated otherwise.
In short, 0.1 ml of Na-PB containing 1/10 of the mixture containing 99mTc-labelled peptides
was transferred to an Eppendorf vial. Next, 0.8 ml of 50% (v/v) of 0.01 M acetic acid in Na-PB
containing 0.01% (v/v) Tween-80 and 0.1 ml of Na-PB containing approximately 2×107 viable
bacteria were added. The mixture, with a final pH of 5, was incubated for 1 h at 4°C and
thereafter the vials were centrifuged in a pre-cooled centrifuge at 2000×g for 5 min. The
supernatant was removed and the bacterial pellet was gently resuspended in 1 ml of Na-PB
and re-centrifuged as above. The supernatant was removed and the radioactivity in the
bacterial pellet was deter- mined in a dose calibrator (VDC 101, Veenstra Instruments, Joure,
The Netherlands). The radioactivity related with bacteria was ex- pressed as % of added
99mTc activity bound to 2×107 of viable bacteria.

Binding of 99mTc-labelled peptides to 2×107 of (activated) hu- man leucocytes was

determined under similar conditions, except that the Na-PB buffer was supplemented with 50
IU of heparin (Leo Pharmaceutical Products, Weesp, The Netherlands) and cen- trifugation
was performed at 110×g.

Experimental peritoneal infections.

Specific pathogen-free, male, Swiss mice weighing 20–25 g (Broekman Institute, Someren,
The Netherlands) were used in this study. In additional experiments male New Zealand White
rabbits, weighing from 2.5 to 4 kg, were used. All animal studies were done in compliance with
the LUMC Ethical Committee and Dutch laws relating to the conduct of ani- mal experiments.
Mice were anaesthetised as described elsewhere [13]. Immediately after anaesthesia the mice
were intraperitoneal- ly infected with 1×107 colony forming units (CFU) of K. pneumo- niae in
0.1 ml of saline. Five minutes thereafter, 0.2 ml of saline containing 1/10 of the mixture
containing 99mTc-labelled peptides (2 MBq) was injected intravenously. At 2 h and 24 h after
administration of the tracer, mice were sacrificed by an intraperitoneal injection of 0.25 ml of
Nembutal containing 60 mg/ml sodium pentobarbital in saline (Sanofi, Division Algin,
Maassluis, The Netherlands). Subsequently, the peritoneal cavity was lavaged with 4 ml of
ice-cold PBS supplemented with 50 units of heparin (Leo Pharmaceutical Products). The
abdomen was gently shaken for 60 s, whereafter the peritoneal cells and bacteria were
harvest- ed and centrifuged for 5 min at 110×g at 4°C. The number of bac- teria was
determined by plating serial dilutions of the supernatant on diagnostic sensitivity test agar
plates (Oxoid). The pellet was resuspended in 1 ml of PBS and the number of leucocytes was
counted in a Bürker haemocytometer after staining with 0.2% Türk’s solution. The amount of
radioactivity in the leucocyte sus- pension and in the fraction containing the bacteria was
counted in a well-type gamma counter. From these data we calculated the amount of peptide
bound to leucocytes and to bacteria and ex- pressed the results as % of the injected dose of
radioactivity per 1×106 cells.

Experimental thigh muscle infections.

Mice were anaesthetised as described above. Immediately thereafter, 1×107 CFU of bacteria
in 0.1 ml of saline were injected into the right thigh muscle. Eighteen hours thereafter, 0.2 ml
of saline containing 1/10 of the mixture containing 99mTc-labelled peptides was injected
intravenously. For comparison, a sterile inflammation was induced by an intramuscular
injection of 0.1 ml of saline containing 50 μg of LPS (Sigma) 24 h preceding administration of
the tracers.

Rabbits were injected with 0.1 ml containing 1×107 CFU of bacteria or 50 μg of LPS (Sigma)
into the right thigh muscle. Eighteen hours thereafter, rabbits were anaesthetised by a single
injection of 0.4 ml of saline containing 0.4 mg fluanisone and 0.12 mg fentanyl citrate
(Hypnorm) into the left thigh muscle, and 0.4 ml of saline containing 1/10 of the mixture
containing 99mTc- labelled peptide was injected intravenously.

Scintigraphy.
Accumulation of the tracers in infected or inflamed areas in mice and rabbits was assessed
by planar scintigraphy. The animals were placed in a supine position on a planar gamma cam-
era (Toshiba GCA 7100/UI, Tokyo, Japan) equipped with a low- energy general-purpose
parallel-hole collimator, with both hind legs spread out and fixed with surgical tape. Continuous
whole- body acquisitions of the animals at every 60 s during the 2 h after injection of the tracer
were made and high-resolution images of the animals were stored in a 256×256 matrix. On
the scintigrams, anatomically adjusted regions of interest (ROIs) were drawn over the infected
or inflamed (target) and non-infected or non-inflamed (non-target) thighs and various organs,
providing us with a reason- able indication of the uptake of 99mTc-labelled peptides at inflam-
matory sites and by various organs [4]. Accumulation of 99mTc- labelled tracers at sites of
infection or inflammation is expressed as the target-to-non-target (T/NT) ratio.

Statistical analysis.
Differences between defensin 1-3 and other peptides in biodistribution or intensity of
accumulation in infected or inflamed thigh muscles were evaluated with Student’s t test. The
two-sided P values are reported and all results are given with the standard error of the mean
(SEM).

RESULTS
 1. Labelling and quality control

● The amount of non-peptide-related 99mTc-

activity did not exceed 5% of the total
radioactivity assessed by ITLC (Instant Thin-
Layer Cromatography).
● According to these data the calculated specific
activity of the non-carrier-free 99mTc-labelled
peptides amounted to 2000 TBq
(terabecquerel, 1012 Bq) per mol peptide.
● Stability of the 99mTc- labelled peptide in 10 mM
sodium phosphate buffer (pH 7.4, Na-PB)
containing 20% of human serum was
unaffected during a 2-h period at 37°C. Under
these conditions we can discriminate between
99mTc-labelled peptide (Rf between 0 and 0.2)

and 99mTc-pertechnetate or released 99mTc

activity (Rf 0.8–1.0).

Fig. 1. Typical profile of 99mTc-UBI 29-41 on a MONO-S cation

exchange chromatography column. Upper part: 99mTc radioactivity; lower part: UV at 220 nm

- On a typical HPLC chromatogram (Fig. 1) of a solution containing 99mTc-labelled UBI

29-41 we observed three 99mTc peaks.
1. The first peak (<5% of the total 99mTc activity) corresponds to the elution
time of pertechnetate. The recovery of 99mTc activity was 92% and the
remaining activity was trapped on top of the MONO-S column.
2. Peak 2 (largest peak) eluted with undetectable amounts of the peptide.
3. Peak 3 co-eluted with the mass of the peptide.

Furthermore, we observed no effect of the labelling procedure on the antibacterial activity of

UBI and hLF peptides in a suspension assay using K. pneumoniae, as has been reported for
defensins [13].

2. Ensayos de la unión in vitro

99mTc-UBI peptides.
● Los resultados revelaron que la mayor cantidad de uniones con bacterias se
observaron por parte de los péptidos etiquetados con 99mTc: UBI 22-35, UBI 31- 38,
UBI 18-35 y UBI 29-41. Además, esta fue considerablemente mayor que la unión de
estos péptidos con los leucocitos humanos.
● De manera similar a la 99mTc-labelled defensin 1-3, los resultados revelaron que los
péptidos 99mTc-labelled UBI mostraron una mayor afinidad de unión con las bacterias
que con los leucocitos (Fig. 2a).
● El ratio entre la unión con bacterias y la unión con leucocitos de los péptidos 99mTc-
labelled UBI es considerablemente mayor al ratio de estas mismas uniones por parte
de la 99mTc-labelled defensin 1-3.
 ● Conclusión: En base a estos resultados, se seleccionó a 99mTc-labelled UBI 18-35, UBI
31-38, UBI 22-35 y UBI 29-41 como candidatos potenciales para desempeñar la
función de detección de infecciones bacterianas.

9mTc-hLF peptides.
● La unión con bacterias más notable por parte de los péptidos 99mTc- labelled hLF y
otros péptidos relacionados se observó con el Tc-labelled hLF 1-11 y hLF 2-11.
● Desafortunadamente, se observa que los 99mTc-labelled hLF y otros péptidos
relacionados también tienen una afinidad considerable para unirse a los leucocitos
humanos (Fig. 2b).
● Cabe recalcar que la unión preferencial con bacterias de los péptidos 99mTc- labelled
hLF fue mucho menor que la del control (99mTc-labelled defensin 1-3).
● Conclusión: A pesar de que se concluyó que los péptidos 99mTc-labelled hLF y hLF 1-
11 no presentaron características de unión favorables, se decidió incluirlos en los
estudios in vivo para poder evaluar su estrategia.

Fig. 2.
a) La unión in vitro de 1/10 de la mezcla que contenía 99mTc-labelled defensin 1-3 o ubiquicidin (UBI) junto
a:
i) 2×107 CFU of S. aureus (open bars),
ii) MRSA (horizontally hatched bars),
iii) K. pneumoniae (closed bars)
iv) Leucocitos activados (diagonally hatched bars).
b) La unión in vitro de 1/10 de la mezcla que contenía 99mTc-labelled defensin 1-3 o lactoferrin (hLF) junto
a:
i) 2×107 CFU of S. aureus (open bars),
ii) MRSA (horizontally hatched bars),
iii) K. pneumoniae (closed bars)
iv) Leucocitos activados (diagonally hatched bars).
c) Todos los valores son el promedio (±SEM) de un mínimo de 8 experimentos.
MRSA: Methicillin-resistant Staphylococcus aureus is a bacterium

3. Estudios de la unión in vivo

99mTc-UBI peptides.
● La mayor unión con bacterias en el sitio de la infección se observó para el péptido
99mTc-labelled UBI 18-35 and UBI 29-41. Está unión con bacterias fue comparable a

la observada con defensin 1-3 (Table 2).

● Se observó una escasa unión con bacterias por parte de 99mTc-UBI 31-38 and UBI 22-
35.
● El ratio promedio entre la unión con bacterias y la unión con leucocitos por parte de
los péptidos UBI determinanada a 2h y 24h fue de:
○ 5–15 para 99mTc-labelled UBI 22-35,
○ 73–220 para UBI 29-41,
○ 36–166 para UBI 18-35,
○ 5–60 para UBI 31-38.
● El mayor ratio alcanzado fue el de 160–443 para 99mTc-labelled defensin 1-3.

Table 2. Unión con bacterias y leucocitos murine peritoneal por parte de péptidos antimicrobianos etiquetados por
el 99mTc

Los valores son el promedio ±SEM de almenos 4 observaciones.

*P<0.05 comparada con defensin 1-3, de acuerdo al test del estudiante t.

99mTc-hLF peptides
● Para el hLP marcado con 99mTc, la mayor unión con las bacterias en el sitio de
infección se dio para hLF 1-11, cuyo valor era considerablemente menor que el de la
defensin 1-3.
● Para el hLF-692 marcado con 99mTc, el ratio entre la unión péptidos-bacteria y el de la
péptido-leucocitos fue de 4-100, mientras que el ratio de hLF 1-11 fue de 10–43.

4. Acumulacion de peptidos marcados con 99mTc en los lugares de la infección

bacteriana

99mTc-UBI peptides.
● En los centellogramas, se pudo visualizar varias infecciones bacterianas (con
bacterias gram-positivas y gram-negativas) en el músculo del muslo de los ratones
dentro del rango de 5 a 30 minutos después de haber sido inyectados con péptidos
UBI marcados con 99mTc y con defensin 1-3 marcada con 99mTc .
 ● En este intervalo se encontraron los ratios T/NT más altos para los péptidos marcados
con 99mTc, UBI 29-41 y UBI 18-35. Estos ratios fueron un poco más altos que los de
defensin 1-3 marcada con 99mTc.
● En los músculos de muslos de los ratones infectados por MRSA, los péptidos
marcados con 99mTc, UBI 29-41 y UBI 18-35, mostraron ratios T/NT mayores de 2.0 a
1h después de la administración. Ambos valores obtenidos fueron considerablemente
mayores que los de defensin 1-3 marcada con 99mTc.
● El péptido UBI 29-41 marcado con 99mTc, mostró el mayor ratio T/NT con 2.6 a 5
minutos después de su inyección en los músculos de los muslos infectados por K.
pneumoniae.
● La acumulacion mas pobre fue observada para el péptido UBI 22-35 marcado con
99mTc.

● En ratones infectados con S. aureus, los péptidos seleccionados se acumularon en

los músculos del muslo infectado como se indica con un T/NT ratio de
aproximadamente 2 a 60 minutos después de la inyección (Fig. 3).
● Es importante recalcar que los péptidos naturales UBI marcados con 99mTc no se
acumularon en los sitios de infección.
T/NT: Tumor/Non-Tumor Ratio

Fig. 3. Accumulation of 99mTc-labelled ubiquicidin (UBI) or related peptides in thighs of mice injected with 1×10 7
CFU of S. aureus (open bars), MRSA (horizontally hatched bars), K. pneu- moniae (closed bars) or 1 μg of
lipopolysaccharides (LPS; diago- nally hatched bars). Each symbol represents the mean (±SEM) T/NT ratio of at
least six animals. *P<0.05 compared with defensin 1-3 according to Student’s t test. n.d. not determined

99mTc-hLF peptides
● Una rápida visualización de las infecciones en los ratones infectados con MRSA fue
notaba gracias al uso de hLF marcado con 99mTc y de hLF 21-31 (Fig. 4) a los 15
minutos después de la inyección.
 ● With 99mTc-labelled hLF 1-11 we observed such ratios at 60 min after injection.
● Ninguna otra acumulacion pudo ser detectada con hLF 4-11 marcado por 99mTc hasta
1h después de la administración.
● Los ratios T/NT para el hLF marcado con 99mTc y el defensin 1-3 marcado por 99mTc
no fueron significativamente diferentes. La única excepción fue que los ratios
obtenidos para la hLF 4-11 marcada con 99mTc eran significativamente (P<0.01)
menores en todos los intervalos.

Fig. 4. Accumulation of 99mTc-labelled lactoferrin (hLF) or related peptides in thighs of mice injected with either
1×107 CFU of various strains of bacteria (open bars) or 1 μg of lipopolysaccharides (LPS, closed bars). Each symbol
represents the mean (±SEM) T/NT ratio of at least six animals. *P<0.05 compared with defensin 1-3 according to
Student’s t test

EXTRA:
● In additional experiments, we studied the uptake of 99mTc-labelled UBI-related peptides
in bacterially infected thighs of mice that had been pre-injected with 100 times molar
excess of unlabelled peptide. In these mice the uptake of the radiolabelled peptide in
the infected thigh muscles was reduced significantly (P<0.05) by 50%.
● Also, we studied the accumulation of 99mTc-labelled UBI 29-41 and UBI 18-35 in S.
aureus- or K. pneumoniae-infected thigh muscles in rabbits (Fig. 5). The results
revealed that visualisation of the infection in rabbits occurred later than in mice,
although the intensity of the image of the infection in rabbits was much higher (T/NT
ratios between 5 and 7) within 2 h after injection than in mice.
Fig. 5. Scintigram of a rabbit with a K. pneumoniae thigh muscle infection (indicated by an arrow) at 90 min after
injection of a 99mTc-labelled (UBI) 29-41. Upon autopsy the bacterial infection had the same appearance as
demonstrated on the scintigraphic image

5. Accumulation of 99mTc-labelled peptides at sites of sterile inflammation

● Next, we studied 99mTc-labelled UBI peptides, hLF peptides and defensin 1-3 in mice
and rabbits with a sterile inflammation to find out whether these peptides visualise
sterile inflammatory processes.
UBI and defensin: The results revealed no significant accumulation of 99mTc-labelled UBI
peptides and defensin 1-3 (results not shown) in sterile inflamed thigh muscles of mice and
rabbits at any interval (Fig. 3).

99mTc-labelled hLF, hLF 1-11, hLF 4-11 and hLF 21-31: visualised inflammatory sites in LPS-
injected thigh muscles in these animals, demonstrating that these peptides are not useful for
detection of infection. DEMUESTRA d hLF peptides
We calculated the half-life of 99mTc-labelled peptides in the circulation of infected mice using
scintigraphic data of regions drawn over the heart.
● The shortest half-life (range 3–58 min) was observed for 99mTc-labelled hLF peptides,
● followed by that for 99mTc-labelled UBI pep- tides (range 35–145 min)
● and defensin 1-3 (range 52–144 min).
In general, synthetic 99mTc-labelled peptides were cleared faster from the circulation than the
natural peptides/proteins.

Clearance of 99mTc-labelled UBI and hLF peptides

We calculated the half-life of 99mTc-labelled peptides in the circulation of infected mice using
scintigraphic data of regions drawn over the heart.
● The shortest half-life (range 3–58 min) was observed for 99mTc-labelled hLF peptides,
● followed by that for 99mTc-labelled UBI pep- tides (range 35–145 min)
● and defensin 1-3 (range 52–144 min).
In general, synthetic 99mTc-labelled peptides were cleared faster from the circulation than the
natural peptides/proteins.

7. Biodistribution
An indication of the uptake of different 99mTc-labelled peptides in the various organs was
obtained by counting radioactivity in anatomically fitted regions drawn over the urinary bladder,
kidneys, spleen and liver. Results are expressed as the mean (± SEM) uptake (% of injected
dose) determined in at least eight mice (Table 3a) or four rabbits (Table 3b).

After injection, 99mTc-labelled UBI peptides were rapidly removed from the circulation via the
kidneys and subsequently the urinary bladder. The quantity of 99mTc-labelled peptides in the
kidneys remained stable at about 25% of the injected dose during the first hour of monitoring.
Similar results were observed with 99mTc-defensin 1-3.

The lowest quantity of radioactivity in kidneys was observed in mice injected with 99mTc-
labelled hLF- related peptides. For 99mTc-labelled hLF peptides we noted significantly (P<0.05)
higher uptake in the liver than for 99mTc-labelled UBI peptides.

Furthermore, deposits of 99mTc-labelled hLF peptides in the intestines were observed from 0.5
to 1 h after injection of the peptide.

Table 3a. Biodistribution of 99mTc-labelled antimicrobial peptides in mice infected with bacteria.
Values are the mean±SEM of at least eight observations; * P<0.05 compared with defensin 1-3, according to
Student’s t test

As indicated in Table 3b, the distribution of the 99mTc-labelled peptides in the liver, kidneys and
spleen of rabbits was similar to that in mice (Table 3a). Only the activity in the urinary bladder
was higher at later intervals.

Table 3b. Biodistribution of 99mTc-labelled antimicrobial peptides in rabbits infected with bacteria.
Values are the mean±SEM of at least four observations; * P<0.05 compared with mice, according to Student’s t
test.