Diagnostic value of vascular endothelial growth factor and interleukin-17 in association with molecular [600838]

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Original article doi: ©2015 by the Asian Pacific Journal of Tropical Disease. All rights reserved.
Diagnostic value of vascular endothelial growth factor and interleukin-17 in association with molecular
diagnosis of Wuchereria bancrofti infection
Dalia Abdelhamid Omran1, Mayssa Mohamed Zaki2, Salwa Fayez Hasan3, Hend Ibrahim Shousha1*
1Department of Endemic Medicine and Hepato-gastroenterology, Faculty of Medicine, Cairo University, Cairo, Egypt
2Department of Medical Parasitology, Faculty of Medicine, Cairo University, Cairo, Egypt
3Department of Medical Biochemistry, Faculty of Medicine, Cairo University, Cairo, EgyptAsian Pac J Trop Dis 2015; 5(8):
Asian Pacific Journal of Tropical Disease
journal homepage: www.elsevier.com/locate/apjtd
*Corresponding author: Hend Ibrahim Shousha, Department of Endemic medicine
and Hepato-gastroenterology, Faculty of Medicine, Cairo University, Cairo, Egypt.
Tel: +201005738455
Fax: [anonimizat]
E-mail: hendshousha@yahoo.com1. Introduction
The lymphatic system is crucial for fluid homeostasis, immune
surveillance, and numerous pathological responses [1]. Many diseases
may affect lymphatic vessels, of which lymphatic filariasis is of the utmost importance although it is globally considered a neglected
tropical disease
[2,3]. Worldwide, more than 1.1 billion people are
estimated to be at risk of infection with lymphatic filariasis which
is endemic in approximately 80 countries [4,5]. In 2002, the Egyptian
Ministry of Health and Population reported 179 lymphatic filariasis
endemic villages distributed in 8 governorates [6].
The major pathology in lymphatic filariasis is reactions to
degenerating or dead adult worms, causing temporary or permanent
disability with a major social and economic impact [6]. The majority
of filaria-infected individuals exhibit strong pro-inflammatory immune responses resulting in lymphatic dilatation, hyperplasia, and lymphangiogenesis which are likely related to the parasite
and its products
[5,7]. Because both Wuchereria bancrofti (W.
bancrofti ) and Brugia malayi harbor the Wolbachia endosymbiont
intracellularly, researches have accused Wolbachia as an inducer
of vascular endothelial growth factor ( VEGF ) that could affect the
lymphatic vessels. In addition, treatment with doxycycline, an
antibiotic that targets Wolbachia , had been shown to reduce plasma
VEGF concentration ( VEGF -C)/VEGF receptor ( VEGFR )-3 levels and
improve pathology [8].
Th1- and Th2-type immune responses in Bancroftian filariasis had extensively been studied. A third subset of interleukin 17 (IL-17)-producing effector T helper cells, called Th17 cells, had
been discovered and characterized
[9], but the role of Th17-type
cytokines have received less attention. VEGF had been shown to be
upregulated by pro-inflammatory cytokines specially IL-17 [7].
In the current study, we aim to explore effective diagnosis of
lymphatic filariasis through DNA -based techniques followed by
assessment of VEGF -C and IL-17, as indicators for lymphatic
endothelial cell activation and proliferation and for massive tissue reaction that may be a good indicator for ongoing patent lymphatic
filariasis in W. bancrofti infection.ARTICLE INFO ABSTRACT
Objective: To explore effective diagnosis of Wuchereria bancrofti through DNA -based
techniques followed by assessment of vascular endothelial growth factor concentration
(VEGF-C ) and interleukin 17 (IL -17) as indicators for lymphatic endothelial cell activation,
proliferation and massive tissue reaction that may be a good indicator for ongoing lymphatic filariasis.
Methods: Blood samples were collected from 38 patients: 23 males (60.5%) and 15 females
(39.5%) with filariasis and from controls (60 from a non-endemic and 22 from endemic areas).
PCR was used to prove infection. A specific and sensitive ELISA was used to determine serum
IL-17 and VEGF -C.
Results: A total of 28 patients (46.7%) were positive by PCR, while 10 patients (16.7%) were
negative by PCR. Serum level of vascular endothelial growth factor was significantly high
in acute cases [(2 147.00 ± 556.00) pg/mL] and in cases of early elephantiasis [(1 950.00 ±
638.00) pg/mL] and lowest in cases of late elephantiasis, endemic and non endemic controls
[(1 238.00 ± 443.00), (807.11 ± 6.20) and (857.00 ± 91.50) pg/mL respectively]. Serum IL-17
was found to be significantly high in acute cases, early elephantiasis and late elephantiasis cases
[(8 601 ± 1131), (7 867 ± 473) and (6 593 ± 378) pg/mL respectively] when compared to endemic
controls [(3 194 ± 1 500 pg/mL)] and non endemic controls [(3 416 ± 1 101) pg/mL].
Conclusions: VEGF -C and its inducing factor IL-17 are expected to gain more importance in
filariasis. Targeting such factors might ameliorate the pathology in chronic filariasis. Contents lists available at ScienceDirect
Article history:
Received 17 Apr 2015
Received in revised form 8 Jun 2015Accepted 30 Jun 2015Available online 8 Jul 2015
Keywords:
Lymphatic filariasisDNA-based diagnosisVascular endothelial growth factor Interleukin-17

Dalia Abdelhamid Omran et al./Asian Pac J Trop Dis 2015; 5(8): 931
2. Materials and methods
Ethical approval was obtained from the ethical committee of
Parasitology Department, Faculty of Medicine, Cairo University. Blood
samples were collected from 38 individuals living in Kalyobeya and
Monofeya governorates in Egypt that known to be endemic for W.
bancrofti . Patients were 23 males (60.5%) and 15 females (39.5%)
with filariasis. Their age ranged from 10 to 67 years. A control group
consisting of 82 age and sex matched healthy individuals [60 from
a non-endemic area (non-endemic controls) and 22 from endemic
areas (endemic controls)] was also included. An informed consent
was obtained from all participants according to the 1975 Helsinki
Declaration. Each sample was divided into 2 tubes, one preserved on EDETA for
PCR (used for diagnosis of filarial infection) and the other
sample was centrifuged and serum was collected and divided into 3 aliquots and stored at -20
°C.
2.1. DNA extraction
DNA was prepared using a QIAamp ® DNA blood mini kit ( QIAGEN
Co., GmbH, D-40724 Hilden) after digesting the samples with
proteinase K in the supplied Buffer AL for 2 h at 56 °C. DNA was
prepared from the solution by using QIAmp spin columns in an
Eppendorf microcentrifuge following the manufacturer’s instructions.
2.2. The PCR
The most frequently used target for PCR is the SspI gene. Forward
and reverse PCR primers designated NV-l (21 bp) and NV-2 (22 bp)
were based on the consensus sequence of the SspI DNA repeat [9]. This
allows amplification of a 188 bp DNA fragment for Wuchereria as
follows: primer NV-l: 5‘-CGT GAT GGC ATC AAA GTA GCG- 3’ and
primer NV-2: 5‘-CCC TCA CTT ACC ATA AGA CAA C-3’. The PCR
was performed in 50 μL of reaction mixture containing the following:
Master Mix (Thermo-Sigma): 25 µL, 5 mL of 5 × buffer, 2 µL forward
primer (1:20), 2 µL reverse primer (1:20), Taq polymerase (0.2 µL),
template DNA (5 µL) and completed with distilled water to 50 µL.
DNA amplification was performed using thermocycler (Tpersonal
thermocycler) and each set of reactions included a negative control ( i.e.,
reagent mixture without template DNA) and a positive control. The
amplification procedure included initial denaturation at 95 °C for 5 min,
35 cycles, each consisting of 94 °C for 60 s, 50 °C for 60 s, and 72 °C for
60 s and an additional extension step at 72 °C for 10 min [10]. Amplified
products were electrophoretically resolved on a 2% agarose gel, stained
with ethidium bromide (0.5 µg/mL) (Sigma, Belgium) and the amplified
PCR products were visualized under UV illumination.
2.3. Detection of VEGF -C and IL-17 in serum
Human VEGF -C was measured in serum samples using ELISA
according to the manufacturer’s instructions (Quantikine Human
VEGF -C Immunoassay; R and D Systems). Briefly, diluted serum (1:5)
or recombinant human VEGF -C standards were added to plates coated
with mouse monoclonal antibody against VEGF -C and incubated for
2 h at room temperature. Plates were washed extensively 4 times and
VEGF -C conjugate was added and further incubated for 2 h at room
temperature. Then, a substrate was added and incubated for 30 min at room temperature protected from light. The reaction was stopped by
the addition of 1 mol/L sulfuric acid and absorbance was measured
at
450 nm on an ELISA reader. The lower detection limit of sensitivity
of the assay was 13.3 pg/mL [11]. A specific and sensitive ELISA was
used to determine concentrations of IL-17 in plasma (R & D Systems, Minneapolis, MN)
[12].2.4. Statistical analysis
Data were statistically described in terms of mean ± SD, and range, or frequencies (number of cases) and percentages when appropriate.
Comparison of numerical variables between the study groups was done
using student t-test for independent samples in comparing 2 groups
and One-way
ANOV A test with post-hoc multiple 2-group comparisons
when comparing more than 2 groups. For comparing categorical
data, Chi-square (χ2) test was performed. Exact test was used instead
when the expected frequency is less than 5. P-value less than 0.05 was
considered statistically significant. All statistical calculations were done
using computer program SPSS version 15 for Microsoft Windows.
3. Results
Of the study group, 28 cases were positive by PCR and 10 were PCR
negative. PCR positive cases were clinically classified as: 5 cases with
acute filariasis (fever, cough and dyspnea, painful lymphadenopathy),
23 cases with elephantiasis and/or hydrocele [(12 cases for less than
3 years (early elephantiasis) and 11 cases for 3 years or more (late
elephantiasis)]. All the PCR negative cases were diagnosed to have
elephantiasis (Table 1 and Figure 1). PCR is positive in early stages of
the disease when the adult worms are alive and it becomes negative when the parasites are dead
[13].
Table 1
Clinical classification of cases.
Clinical presentations PCR positive
(n = 28) PCR negative
(n = 10)
Acute filariasis 5 0
Elephantiasis and/or hydrocele 23 10
Early elephantiasis (< 3 years) 12 5
Late elephantiasis ( ≥3 years) 11 5
100 bp DNA
ladder
100 bp DNA
ladder+ ve
+ ve NC+ ve PC
Figure 1. Agarose gel analysis of DNA extracts showing amplified products
of W. bancrofti .
Positive samples showed amplified band at 180 bp.
Serum level of VEGF -C was significantly high ( P; 0.001 ) in
acute cases [(2 147.00 ± 556.00) pg/mL] and also in cases of early
elephantiasis [(1 950.00 ± 638.00) pg/mL], while it was lowest in cases
of late elephantiasis, endemic and non-endemic controls [(1 238.00 ±
443.00), (807.11 ± 6.20) and (857.00 ± 91.50) pg/mL respectively]
(Table 2).
Regarding serum IL-17, it was found to be significantly high in acute
cases, early elephantiasis cases and late elephantiasis cases [(8 601
± 1 131), (7 867 ± 473) and (6 593 ± 378) pg/mL respectively] when
compared to endemic controls [(3 194 ± 1 500) pg/mL] and non-
endemic controls [(3 416 ± 1 101) pg/mL] (P; 0.000) (Table 3).

Dalia Abdelhamid Omran et al./Asian Pac J Trop Dis 2015; 5(8): 932
4. Discussion
Studying the role of lymphangiogenesis promoting factors is an
area gaining importance in the study of lymphatic filariasis. VEGF
and IL-17 are accused of being involved in or indicating filarial disease
[7,14,15] . Several studies have implicated VEGF -mediated
responses in the development of lymphedema and hydrocele.
Some referred this to activated innate immune response following
filarial infections [8,15,16] .
In the present study it was observed that VEGF -C was
significantly elevated in acute cases and in early elephantiasis
and/or hydrocele. Level of VEGF -C was not significantly altered
between previously mentioned filarial cases. However, it showed statistically significant difference from other values observed in
late elephantiasis, elephantiasis with negative
PCR, endemic and
non-endemic control groups ( P < 0.05). This was in accordance
to Bennuru and Nutman [17], who strongly indicated an active
involvement of VEGF s in lymphatic filariasis. The authors studied
the level of several types of VEGF (VEGF -A and VEGF -C/soluble
VEGFR -3] and had observed their association with filarial disease.
It had been reported that filarial antigens and plasma from filaria-
infected individuals have the capacity to induce human lymphatic
endothelial cells to undergo proliferation and differentiation, a
process mediated by either excreted or secreted parasite proteins
and lymphangiogenesis promoting factors as VEGF . On the
contrary, plasma from non-infected individuals or chronic cases
induced nothing [17]. By estimating serum level of the recently
studied cytokine, IL-17, it was noticed that it was significantly higher in acute cases and in early and late elephantiasis and/or
hydrocele than in other individuals (elephantiasis with negative
PCR, endemic and non-endemic control groups). Accordance in the elevated levels of the estimated serum factors ( VEGF and
IL-17) in the present study within different patient groups was
interestingly noticed.
Babu et al . indicated the importance of proinflammatory
cytokines especially IL-10 and Il-17 in regulation of VEGF [7].
They showed that filarial antigen-mediated proinflammatory
cytokine induction was a characteristic feature of chronic filarial
lymphedema and hydrocele. They added that these cytokines and
subsequently VEGF were significantly lower in subclinical (or
asymptomatic) infection. It had also been shown that IL-17 could mediate lymphatic damage by inducing production of various
angiogenic and lymphangiogenic factors leading to perturbations
in lymphatic endothelial system function ending in lymphatic
dilatation and lymphedema. Moreover, presence of elevated levels
of lymphangiogenic factors had been shown to be associated with
the severity of lymphatic pathology
[18,19] .
Honorati et al .[20] and Lohela et al .[21], explained the
importance of IL-17 and the subsequent stimulation of VEGF
formation as important regulatory growth factors for vascular
and lymphatic endothelial systems that can also promote both
angiogenesis and lymphangiogenesis by interacting with cognate receptors (
VEGFR -1, -2, and -3) on endothelial cells. This was
also supported by Wu and Liu [22] and Debrah et al .[16] who
showed that generation of new lymphatic vessels from the pre-
existing lymphatic system is strongly affected by VEGF that
has a crucial role as an angiogenic/lymphangiogenic factor in microfilaraemic patients who developed elephantiasis. On the
contrary, those who didn’t develop elephantiasis had a significant
lower level of
VEGF .
In our study, patients with elephantiasis and negative PCR
had significantly lower VEGF -C. Babu et al . arose an important Table 2
Comparison between serum level of VEGF in different clinical presentations and control group.
Clinical presentations Mean ± SD
(pg/mL)P
Acute EI/H < 3 years EI/H > 3 years EI,-ve PCR Endemic controls Non-endemic controls
Acute ( n = 5 ) 2 147.00 ± 556.00 – 1.000 0.001* 0.000*0.000*0.000*
EI/H < 3 years ( n = 12) 1 950.00 ± 638.00 1.000 – 0.002* 0.000*0.000*0.000*
EI/H > 3 years ( n = 11) 1 238.00 ± 443.00 0.001*0.002*- 1.000 0.100 0.896
EI,-ve PCR ( n = 10 ) 963.00 ± 54.40 0.000*0.000* 1.000 – 1.000 0.913
Endemic normals ( n = 22) 807.11 ± 6.20 0.000*0.000* 0.100 1.000 – 0.748
Non-endemic normals ( n = 60) 857.00 ± 91.50 0.000*0.000* 0.896 0.913 0.748 –
EI/H < 3 years: Elephantiasis or hydrocele for < 3 years; EI/H > 3 years: Elephantiasis or hydrocele for > 3 years; EI,-ve PCR: Elephantiasis with negative
PCR.
Table 3
Comparison between serum level of IL-17 in different clinical presentations and control group.
Clinical presentations Mean ± SD P
Acute EI/H < 3 years EI/H > 3 years EI, -ve PCR Endemic controls Non-endemic controls
Acute ( n = 5 ) 8 601 ± 1 131 – 1.000 0.160 0.000*0.000*0.000*
EI/H < 3 years ( n = 12) 7 867 ± 473 1.000 – 0.112 0.000*0.000*0.000*
EI/H > 3 years ( n = 11) 6 593 ± 378 0.016 0.112 – 0.000*0.000* 0.190
EI, -ve PCR ( n = 10 ) 3 020 ± 642 0.000*0.000* 0.000*- 1.000 0.433
Endemic normals ( n = 22) 3 194 ± 1 500 0.000*0.000* 0.000* 1.000 – 0.416
Non-endemic normals ( n = 60) 3 416 ± 1 101 0.000*0.000* 0.190 0.433 0.416 –
EI/H < 3 years: Elephantiasis or hydrocele for < 3 years; EI/H > 3 years: Elephantiasis or hydrocele for > 3 years; EI,-ve PCR: Elephantiasis with negative
PCR.

Dalia Abdelhamid Omran et al./Asian Pac J Trop Dis 2015; 5(8): 933
point when reporting that filarial antigens induce differential
production of VEGF -C, illustrate certain similarities and (perhaps)
differences in lymphatic response to different filarial antigens [7].
This may explain the old debate why some infected individuals
develop elephantiasis while other infected individuals with
proved microfilaraemia escape lymphatic affection.
Another study proved that antifilarial treatment with
doxycycline leads to reduction of plasma levels of VEGF being
associated with amelioration of dilated lymphatic vessels
and with an improvement of pathology in lymphatic filariasis
patients [18,23] .
Researchers demonstrated that VEGF s being an important
angiogenic/lymphangiogenic factor are elevated in vascular
diseases, tumors, lymphedema and lymphangioleiomyomatosis;
however, elevated VEGF -C is particularly associated with changes
in the lymphatics in filarial infections. This may be due to the
fact that filarial antigens are strong inducers for VEGF -C[7,16] .
In conclusion, the studying level of VEGF -C and its inducing
factors as IL-17 is expected to gain more importance in the fields of lymphatic diseases especially filariasis. Probably targeting
such factors and their pathway might potentially ameliorate the
pathology in chronic filariasis.
Conflict of interest statement
We declare that we have no conflict of interest.
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