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Research Article
Herpes Murine Model as a Biological Assay to Test
Dialyzable Leukocyte Extracts Activity
Nohemí Salinas-Jazmín,1Sergio Estrada-Parra,2Miguel Angel Becerril-García,1
Alberto Yairh Limón-Flores,1Said Vázquez-Leyva,1Emilio Medina-Rivero,1Lenin Pavón,3
Marco Antonio Velasco-Velázquez,4and Sonia Mayra Pérez-Tapia1,2,5
1Unidad de Desarrollo e Investigaci´
on en Bioprocesos (UDIBI), Escuela Nacional de Ciencias Biol´
ogicas, IPN,
Prolongaci´
on de Carpio y Plan de Ayala s/n, Col. Sto. Tom´
as, 11340 M´
exico, DF, Mexico
2Departamento de Inmunolog´
ıa, Escuela Nacional de Ciencias Biol´
ogicas, IPN, Prolongaci´
on de Carpio y Plan de Ayala s/n,
Col. Sto. Tom´
as, 11340 M´
exico, DF, Mexico
3Instituto Nacional de Psiquiatr´
ıa “Ram´
on De la Fuente Mu˜
niz”, Calzada M´
exicoXochimilco101,Col.SanLorenzoHuipulco,
14370 M´
exico, DF, Mexico
4Facultad de Medicina, Universidad Nacional Aut´
onoma de M´
exico, Ciudad Universitaria, 04510 M´
exico, DF, Mexico
5Unidad de Investigaci´
on Desarrollo e Innovaci´
on M´
edica y Biotecnol´
ogica (UDIMEB), Escuela Nacional de Ciencias Biol´
ogicas,
IPN, Prolongaci´
on de Carpio y Plan de Ayala s/n, Col. Sto. Tom ´
as, 11340 M´
exico, DF, Mexico
Correspondence should be addressed to Marco Antonio Velasco-Vel´
azquez; marcovelasco@unam.mx
and Sonia Mayra P´
erez-Tapia; smpt.@hotmail.com
Received July ; Revised September ; Accepted September
Academic Editor: Oscar Bottasso
Copyright © Nohem´
ı Salinas-Jazm´
ın et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on
disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer,
and immunodeciencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities
and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon,
we standardized and validated a murine model of cutaneous HSV- infection. Using this model, we evaluated the activity of
Transferon batches. All batches improved the survival of HSV--infected mice, wherein average survival rose from .% in control
mice to .% in Transferon-treated mice. e activity of Transferon correlated with increased serum levels of IFN-𝛾and reduced
IL- and TNF-𝛼concentrations. Our results demonstrate that (i) this mouse model of cutaneous herpes can be used to examine
the activity of DLEs, such as Transferon; (ii) the assay can be used as a routine test for batch release; (iii) Transferon is produced
with high homogeneity between batches; (iv) Transferon does not have direct virucidal, cytoprotective, or antireplicative eects;
and(v)theprotectiveeectofTransferonin vivo correlates with changes in serum cytokines.
1. Introduction
Governments worldwide have established standards for the
production and release of biotechnological and biological
drugs. For example, the US Food and Drug Administration
(FDA) and Japanese Ministry of Health & Welfare (MOHW)
modied their guidelines regarding biologics in , and
the European Medicines Agency (EMA) did so in [–].
In , the Sanitary Risk Authority of Mexico (COFEPRIS)
released the Ocial Mexican Standard NOM-EM--SSA-
.
A key element in the quality control of the production
of biological drugs is the demonstration of their activity, and
theirecacymustbepreservedbetweencommercialbatches.
Hindawi Publishing Corporation
Journal of Immunology Research
Volume 2015, Article ID 146305, 9 pages
http://dx.doi.org/10.1155/2015/146305
Journal of Immunology Research
Due to the unique nature and production of biological drugs,
specic methods must be developed to evaluate their quality
and attributes, including ecacy.
Human dialyzable leukocyte extracts (DLEs) are
heterogeneous mixtures of low-molecular-weight peptides
(< kDa) that are released on disruption of peripheral blood
leukocytes from healthy donors [].DLEsareproducedand
commercialized worldwide. In certain countries, such as
M´
exico, China, Cuba, and the Czech Republic, DLEs are
registered as drugs [–]becausetheyimproveclinical
responses in infections, allergies, cancer, and immunode-
ciencies (see Berr´
on-P´
erez et al. []andVizaetal.[]for
extensive reviews). eir complexity, however, has impeded
an extensive characterization of their components, active
substances, and biological activities.
Transferon is a human nonspecic DLE that is man-
ufactured by the National School of Biological Sciences
(ENCB), National Polytechnic Institute (IPN), Mexico, at
GMP facilities. Transferon is registered by Mexican health
authorities as a drug and is commercialized nationally. To
establish an assay that could be used routinely as a quality
control test for Transferon, we aimed to standardize and
validated a method to determine its ecacy in animals.
Other studies have demonstrated that the activity of DLEs
can be measured by assessing the induction of delayed type
hypersensitivity (DTH) in mice []andin vitro by analyzing
their eects on leukocyte migration []orIFN-𝛾secretion
[].
DLEs are eective for treating parasitic infections (acute
leishmaniasis [] and alveolar echinococcosis []) and
viral infections (herpes simplex virus- (HSV-) and herpes
zoster). Clinical trials have shown that DLEs mitigate the
duration of the acute phase, the frequency of recurrences, and
pain in herpes zoster patients better than acyclovir [,].
ese eects correlate with increased IFN-𝛾levels and CD+
cell counts []. Considering the clinical eects of DLEs
against herpetic infections and because animal models reect
thecomplexityofadrugresponseinanentireorganism,
we selected a murine model that has been reported to
emulate the natural form of HSV- infection []. is murine
model was standardized, validated, and used to evaluate the
biological activity of batches of Transferon.
2. Materials and Methods
2.1. Quality Control of Transferon. We t e s t e d Tr a n s feron
batches that were produced between and using a
modied version according to Borkowsky et al. []. Briey,
leukocytes from healthy donors were lysed with freeze-
thaw cycles and dialyzed against a -kDa membrane to
obtain low-molecular-weight peptides.
e quality control of Transferon comprised (A) endo-
toxin content, quantied using the Endosafe-Portable Test
(Charles River Laboratories, Charleston SC, USA) accord-
ing to the manufacturer’s instructions; the specication for
endotoxin was established in Mexican Pharmacopeia, Section
MGA- (≤. EU/mL) []; (B) microbiological tests,
according to Mexican Pharmacopeia, Section MGA-
T : Quality attributes of evaluated batches of Transferon.
Batch Protein
(mg/mL)
MAB
(CFU/mL)
FF&Y&
(CFU/mL)
Endotoxin
(EU/mL)
A . <<<.
C . << .
C . <<<.
C . <<<.
C . << .
C . <<<.
C . <<<.
D . <<<.
F . <<.
G . <<<.
H . << .
H . <<<.
K . <<<.
K . <<<.
L . <<<.
L . <<<.
A . <<<.
B . <<<.
C . <<<.
M . << .
M . <<<.
A . <<<.
C . <<<.
A . <<.
C . <<<.
B . <<<.
B . <<<.
#Mesophilic aerobic bacteria; &Filamentous fungus and yeast.
[]; (C) physicochemical characterization by a validated
ultraperformance liquid chromatography (UPLC) method
[] that analyzes molecular weights and the time of retention
of the main peaks compared with those of an internal batch
pattern. (D) Peptide content per nal dose was measured by
bicinchoninic acid (BCA) method using the Pierce BCA kit
(ermo Fisher Scientic, Waltham MA, USA) according to
the manufacturer’s instructions.
2.2. Herpes Simplex Murine Model. HSV- (KOS strain) was
obtained from American Type Culture Collection (ATCC;
Manassas VA, USA). e virus was propagated in African
green monkey kidney (Vero) cells (ATCC CCL-) that
were cultured in Eagle’s minimal essential medium (EMEM;
ATCC) supplemented with % fetal bovine serum (FBS;
Life Technologies, Carlsbad CA, USA). Cutaneous infection
of herpes was performed by inoculating -week-old male
BALB/c mice (Ferandelh, Mexico City, Mexico) with HSV-
, as reported [,,]. Briey, mice were anesthetized, and
𝜇L of a viral suspension that contained ×6plaque-
forming units (PFU)/mL was administered by cutaneous
Journal of Immunology Research
0 2 4 6 8 101214161820
0
20
40
60
80
100
Time (d)
Survival (%)
(a)
0
10
20
30
Frequency of deaths (% total)
0 2 4 6 8 10 12 14 16 18 20
Time (d)
(b)
F : Standardization of mouse cutaneous herpes model. (a) ree dierent experiments performed with . ×4PFU of HSV- (closed
symbols). In each experiment, infected mice showed signicantly lower survival than controls (open circles; log-rank Mantel-Cox test; 𝑃<
0.001). (b) Frequency of deaths from the experiments shown in (a).
scarication on cm2of plucked dorsal skin [–]. Mice
were monitored daily for days to identify infection-
associated symptoms, such as paralysis of the lower extrem-
ities, reduced mobility, and weight loss. When the animals
experienced total loss of mobility, they were euthanized and
counted as deaths for the survival analysis.
Mice had free access to food (Harlan Laboratories, Indi-
anapolis IN, USA) and water during the experiment. All
experiments were performed blindly by trained researchers.
e administration of Transferon began on day aer
infection and continued every other day until day . Doses
employed were .ng, . 𝜇g, . 𝜇g, . 𝜇g, . 𝜇g,
. 𝜇g, or . 𝜇gpermouse(eachweighing–gatday
) and perorally administered in 𝜇L. Each experimental
group comprised mice. All experiments included a control
group of mice that received placebo (pyrogen-free water;
PISA Pharmaceutical, Mexico City, Mexico). Survival per
group was plotted as Kaplan-Meier graphs and analyzed by
Mantel-Cox test (𝛼=.)usingPrismProject,V.
(GraphPad Soware Inc, San Diego CA, USA).
2.2.1. In Vitro Eects on HSV-1. We studied the possible
direct antiviral eect of Transferon by mixing equal volumes
( 𝜇L) of Transferon (. mg peptide/mL) and HSV- (. ×
7PFUmL) and incubating the mixture for minutes at
∘C. e infectivity of Transferon-treated virus was deter-
minedbyanalyzingtheinductionofavisiblecytopathiceect
(CPE) on Vero cells (4cells/well) at h, as reported [].
Fiy percent tissue infective dose (TCID50)wascalculated
by Spearman-K¨
arber method []. Cytopathology induced
by HSV- ( PFU) in presence of Transferon ( pg/mL–
𝜇g/mL) or acyclovir (Zoviraz, GlaxoSmithKline, Mex-
ico City, Mexico) was determined using MTS (CellTiter
Aqueous One Solution cell proliferation assay reagent;
Promega, Madison WI, USA) as previously reported [,].
e cytoprotective eect of Transferon on target cells was
evaluated by preincubating Vero cells (4cells/well) with
Transferon ( 𝜇g/mL) for hours before addition of HSV-
(viral stock . ×7PFU/mL). CPE was evaluated h aer
infection []; TCID50 calculation was performed as reported
[]. To assess the eect of Transferon on virus replication, ×
5Vero cells were infected with HSV- ( PFU) and incu-
bated with Transferon ( 𝜇g/mL) or Acyclovir ( 𝜇g/mL)
for h at ∘C. Subsequently, viruses were recuperated from
cultures through one thaw/freeze cycle and sonication. Aer
removing cell debris by centrifugation ( g), the total
virus yield on each well was titrated by diluting samples and
incubating them with Vero cells for h. We analyzed the
presence of CPE at the microscope and by MTS assay. As
reported by Heldt et al. [], the TCID50 was dened as the
concentration at which absorbance was % of the average
absorbance from uninfected cells and was determined by
nonlinear regression using Prism for Mac OS X (GraphPad
Soware Inc).
2.2.2. Cytometric Bead Array. To me a s u r e c y tokine c o n c e n -
trations, blood samples from the orbital sinus were collected
from anesthetized mice on days and aer infection.
Serum was obtained, and samples were stored at −∘Cuntil
analysis. Cytokine concentrations were determined using a
cytometric bead array (CBA) mouse inammation kit (BD
Journal of Immunology Research
024681012141618
0
20
40
60
80
100
Time (d)
Survival (%)
(a)
0 3 6 9 12 15 18
0
2
4
Time (d)
−6
−4
−2
Weight dierence ±SEM
(b)
0
25
50
75
100
Survival (%)
Uninfected Placebo 0.5 𝜇g0.75 𝜇g1𝜇g1.5 𝜇g
∗
∗∗∗ ∗∗∗
∗∗∗
(c)
F : Validation of the murine herpes model for the evaluation of Transferon activity. (a) Survival curves for HSV- infected mice.
Tre a tmen ts wit h 𝜇g (open triangles) or . 𝜇g (open squares) of Transferon improved survival over placebo (open circles; Log-rank Mantel-
Cox test; 𝑃 < 0.01). In contrast, . ng of Transferon (open diamonds) had no signicant eect. A group of mice was le uninfected (closed
circles) as control. (b) Weight changes in HSV- infected mice. Transferon partially protects mice from the weight loss induced by HSV-
(symbols are as in (a)). (c) Survival induced by ve dierent Transferon batches was evaluated during a validation protocol. All batches
improved survival versus placebo (Bonferroni ttest; ∗𝑃 < 0.05,∗∗𝑃 < 0.01;∗∗∗𝑃 < 0.001).
Biosciences, San Diego CA, USA) according to the manufac-
turer’s instructions. We used a FACS Aria ow cytometer and
BD CBA soware (BD Biosciences) for data acquisition and
analysis.
2.2.3. Ethics Statement. All experimental procedures with
animals were performed according to the Mexican Guidelines
for the Production, Care, and Use of Laboratory Animals
(NOM--ZOO-) and the International Guide for the
Care and Use of Laboratory Animals []. All eorts were
made to minimize animal suering and reduce the number
of animals that were used. e experimental procedures were
approved by the Ethical Committee of the Transfer Factor
Project in “Escuela Nacional de Ciencias Biol´
ogicas, Instituto
Polit´
ecnico Nacional” (protocol FTU/IB///PRO).
Journal of Immunology Research
3. Results
3.1. Quality Control of Batches. All batches had endotoxin
levels below . EU/mL and met the microbiological speci-
cations in Mexican Pharmacopeia (aerobic mesophile bacte-
ria < colony-forming units (CFU)/mL, lamentous fungi
< CFU/mL, and yeasts < CFU/mL (Ta b l e )). By UPLC,
we performed a physicochemical characterization of the
batches. In the chromatographic proles of the Transferon
batches, we noted peptidic fractions between to . kDa,
as previously reported []. e retention times of the main
peaks corresponded with those of an internal pattern, dened
as a batch of Transferon that satised all quality control
tests for this product, as previously reported []. We also
measured peptide concentrations in the Transferon sam-
ples. All batches had peptide concentrations that met the
established specication of 0.400 ± 0.06mg/mL. e average
concentration in the batches was 0.416 ± 0.023mg/mL
(Table ).
3.2. Herpes Murine Model. To develop a murine model of
cutaneous herpes, we rst studied the eect of an HSV-
inoculum on mouse survival. We inoculated . ×4,.×
4,and.×5PFU of HSV- and analyzed mouse survival
(Figure ). In subsequent experiments, we used . ×4PFU
of HSV-, which was the minimum inoculum that produced
a decrease in survival within % to %, as reported
[–].
en, we determined the reproducibility of the eect of
the infection by repeating the assay times and measuring
survival.esurvivalofinfectedanimalswas%to%
(Figure (a)). Using data from the same experiments, we
calculated the frequency of deaths (euthanizations) over time,
noting that % of deaths occurred before day aer infec-
tion, rising to % by day (Figure (b)). is information
allowed us to establish an endpoint for subsequent assays.
3.3. Biological Evaluation of Transferon. To determine the
doses of Transferon that were to be used to evaluate the
commercial batches, we examined a ≈-fold range of doses
(. ng–. 𝜇g per mouse). Doses above . 𝜇g/mouse
were equally ecacious in reducing HSV--induced mortality
and weight loss. In contrast, . ng was ineective, indi-
cating that the activity of Transferon is dose-dependent. As
expected, uninfected mice gained weight and showed %
survival (Figures (a) and (b)).
e model was validated using batches that were pro-
ducedinandtestedatdoses(Figure (c)). We evaluated
(i) system specicity, by analyzing responses in placebo- and
Transferon-treated animals; the average survival in controls
was .% (range % to %), whereas all Transferon doses
induced a signicant increase in survival (average increase
versus placebo (Δsurvival) .%); (ii) system precision, by
calculating the relative standard deviation (RSD) percentage
for the results with each Transferon dose; % RSD was below
% in all cases (range: .% to .%); (iii) system suitability,
by corroborating that at least dose produced a Δsurvival
0.125 0.25 0.5 0.75 1 1.5
0
10
20
30
40
50
60
Dose (𝜇g)
Survival over placebo (Δ)±SEM
F : Evaluation of biological activity of Transferon batches
in the validated murine model of herpes. Statistical analysis
(ANOVA, Bonferroni’s Multiple Comparison Test) showed no
dierences between doses.
≥%. All parameters were within the limits that were
establishedinthevalidationprotocol.
Eighteen batches that were produced in and that
weregeneratedinwereexaminedinthevalidated
murine HSV- model. In certain batches, we evaluated
additional doses (. and . 𝜇g per mouse). All batches
improved the survival of HSV--infected mice by an average
of .% (range: .% to .%) (Figure ). ere were no
dierences in average survival between doses, indicating that
any of these doses could be used in future quality control
assays.
3.4. Eects of Transferon on HSV-1 Infectivity. We me a s u r e d
the antiviral activity of Transferon by (i) preincubating HSV-
with the drug before adding it to the target (Vero) cells
(Figure (a)) and (ii) simultaneously incubating target
cells with HSV- and various Transferon concentrations
( pg/mL– 𝜇g/mL) (Figure (b)). Transferon did not
show direct antiviral eect on either experimental system.
Preincubation of target cells with Transferon for h
before the viral challenge did not prevent HSV- infection
(Figure (c)), suggesting that Transferon does not modify
the target cell-susceptibility to infection. Additionally, we
evaluated the eect of Transferon on virus replication. e
titration of HSV- on Transferon-treated samples showed
that the TCID50 is not dierent from that of control samples.
Values obtained from nonlinear regression t were −log10 =
. versus −log10 =.,respectively(Figure (d)). ese
results, obtained by a colorimetric assay, correlate with the
visual analysis of CPE (Supplemental Figure available
online at http://dx.doi.org/.//), as reported
for others viruses [], and demonstrate that Transferon has
no eect on HSV- replication.
3.5. Eects of Transferon on Systemic Cytokines. Blood
(serum)cytokinelevelsweremeasuredatdaysand,
beforetheperiodofhighmortality.TNF-𝛼,IL-,andIFN-𝛾
levels rose in HSV--infected and placebo-treated mice versus
Journal of Immunology Research
ns
100
101
102
105
106
107
108
t0control Medium Transferon
TCID50/mL ±SD
60 min 60 min
(a)
0
0
20
40
60
Tra nsf e ro n
****
ns
Uninfected control ±SEM (%)
10 pg/mL 10 ng/mL 10 𝜇g/mL ACV
∗
(b)
ns
Medium Transferon
100
101
102
105
106
107
108
TCID50/mL ±SD
(c)
1234567
0
25
50
75
100
−log10 dilution factor
Optical density (%) ±SEM
(d)
F : In vitro evaluation of antiviral activity of Transferon. (a) HSV- was preincubated for min with Transferon ( 𝜇g/mL) or
medium before evaluation of visible cytopathic eect (CPE) on Vero cells. Viruses with no preincubation (t0)wereusedascontrol(ANOVA,
Bonferroni’s Multiple Comparison Test; ns: nonsignicant). (b) Eect of Transferon ( pg/mL– 𝜇g/mL) on HSV--induced cytopathology,
evaluated by MTS assay. Acyclovir (ACV; 𝜇g/mL) was included as a positive control. e graph represents data from independent
experiments (ANOVA, Bonferroni’s Multiple Comparison Test; ∗𝑃 < 0.0001). (c) TCID50 obtained from Vero cells preincubated for h
with Transferon (𝜇g/mL) or medium prior to HSV- infection (Student’s ttest). (d) Titration of samples obtained from Vero cells infected
h with HSV-and simultaneously treated with medium (open circles) or 𝜇g/mL of Transferon (closed circles). ACV ( 𝜇g/mL; triangles)
was included as a positive control. Sum-of-squares Ftest showed no dierences between the TCID50 from medium- and Transferon-treated
cells (𝑃 = 0.7527). e graph represents data from independent experiments.
Journal of Immunology Research
0.0
0.5
1.0
1.5
2.0
2.5
Uninfected HSV-1
Placebo Transferon Placebo Transferon
∗∗
∗∗
Normalized TNF-𝛼 ± SD
(a)
0
2
4
6
8
10
Uninfected HSV-1
Placebo Transferon Placebo Transferon
∗∗∗∗
∗∗∗∗
Normalized IL-6±SD
(b)
0
5
10
15
20
25
Uninfected HSV-1
Placebo Transferon Placebo Transferon
∗∗
∗
∗∗∗∗
∗∗∗∗
Normalized IFN-𝛾 ± SD
(c)
F : Quantitative analysis of blood (serum) cytokine levels. Transferon (Batch C; . 𝜇g/mouse) signicantly reduced the
concentrations of TNF-𝛼(a) and IL- (b) at day postinfection, but it increased that of IFN-𝛾at day (c). Transferon administration had
no eect on uninfected mice. All measurements were normalized to placebo-treated, uninfected controls. e graphs represent data of
independent experiments (ANOVA, Bonferroni’s Multiple Comparison Test; ∗𝑃 < 0.05;∗∗𝑃 < 0.01;∗∗∗𝑃 < 0.001;∗∗∗∗𝑃 < 0.0001).
uninfected mice (Figures (a)–(c)). In contrast, Transferon
did not change the cytokines levels in uninfected mice.
Treatment of HSV--infected mice with Transferon signi-
cantly decreased TNF-𝛼and IL- levels at day , compared
with placebo-treated animals (Figures (a) and (b).In
contrast, Transferon further increased IFN-𝛾levels at day
(Figure (c)).
4. Discussion
All batches of Transferon in this study complied with
quality standards with regard to their attributes. As shown for
batchesthatwereproducedin-[], we noted high
homogeneity in microbial content, peptide concentration,
molecular weight, and time of retention of the main peaks by
Journal of Immunology Research
UPLC. ese results demonstrate that is possible to produce
a mixture of peptides that have been extracted from complex
rawmaterials,suchaslysedhumanleukocytes.
To evaluate the activity of these batches, a murine
model of herpes was standardized and validated. Transferon
partially protected animals from the HSV--induced weight
loss, which correlated with greater survival. is model was
chosen because DLEs are eective in clinical studies of
herpetic infections. DLEs signicantly reduce the average
duration of the acute phase and the frequency of recurrences
in herpetic infections as successfully as antiviral drugs [,
]. All batches of Transferon signicantly increased survival
dose-independently, indicating that it is biologically active
over a wide range of doses (-fold). us, our murine model
of herpes can be used to measure ecacy—a fundamental
attribute of biological drugs—in DLEs.
However, the herpes murine model has several disadvan-
tages. e variability that is intrinsic to a whole-animal model
is signicant; such a model requires the use of many animals.
Also, more time is needed to evaluate activity than for an in
vitro experiment.
Transferon has no direct virucidal or antireplicative
eects on HSV- nor cytoprotective eects on target cells,
suggesting that the in vivo activity of Transferon is mediated
by its eects on the immune system. We found that HSV-
-infected mice had higher serum concentrations of TNF-
𝛼,IL-,andIFN-𝛾compared with uninfected controls.
Consistent with these ndings, in astrocyte cultures, HSV-
infection upregulates TNF-𝛼,IL-,andNF-𝜅B in a Toll-like
receptor (TLR)--dependent manner []. Administration
of Transferon to HSV--infected mice downregulates TNF-
𝛼and IL-, suggesting that it suppresses innate immune
responses. e modulation of proinammatory cytokines
by DLEs has been associated with reduced inammation-
associated tissue damage by pathogens [,].
Conversely, Transferon increased IFN-𝛾,acytokinethatis
producedduringtheadaptivephaseofimmunity,suggesting
that Transferon indirectly stimulates the activation of T lym-
phocytes that are specic for HSV-. IFN-𝛾eects resistance
against HSV- infection [], and its upregulation in DLE-
treated herpes patients favors a positive clinical response
[] and limits relapses []. Although the mechanisms of
action of DLEs have not been determined completely, our
results indicate that they have dierential eects on innate
andadaptiveimmunity.
5. Conclusions
Our analysis of batches of Transferon demonstrate that (i)
the cutaneous model of herpes can be used to evaluate the
biological activity of DLEs, such as Transferon; (ii) the assay
can be used as a routine test for batch release; (iii) Transferon
is produced with high homogeneity between batches, meet-
ing the standards that are required for hemoderivatives that
are intended for clinical use; (iv) Transferon does not have
direct virucidal, cytoprotective, or antireplicative eects; and
(v) the protective eects of Transferon in our murine model
correlate with changes in serum cytokine levels.
Conflict of Interests
Nohem´
ı Salinas-Jazm´
ın, Sergio Estrada-Parra, Miguel Angel
Becerril-Garc´
ıa, Alberto Yairh Lim´
on-Flores, Said V´
azquez-
Leyva, and Sonia Mayra P´
erez-Tapia are employees or have
been compensated for their work at “UDIMEB” the producer
of Transferon. All other authors declare no competing inter-
ests.
Acknowledgments
is work was partially supported by FTU/IB///PRO
(Nohem´
ı Salinas-Jazm´
ın) and CONACYT INFR--
(MAV-V). Authors are grateful for the valuable help
of Natanael Valtierra-Botello and Leonardo L´
opez-Ju´
arez
(animal housing), Emiliano Hisaki-Itaya (ow cytometry
data acquisition and analysis), Gilberto P´
erez-S´
anchez (val-
idation protocol), and Antonio Ortega-Roque (cell and virus
culture).
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