Markers of local immunity in cervico-vaginal secretions of HIV infected women: implications for HIV shedding.
ABSTRACT To link local proinflammatory cytokines with HIV related nucleic acids in cervico-vaginal secretions and the factors associated with them.
An observational study on 60 HIV positive women attending the department of obstetrics and gynaecology, University of Pavia, Italy. HIV-1 RNA in plasma, proviral HIV-1-DNA, cell associated and cell free HIV-1 RNA in cervico-vaginal secretions were evaluated by competitive polymerase chain reaction (c-PCR) and reverse transcriptase PCR (cRT-PCR). IL-1beta, IL-6, and TNF-alpha were measured by ELISA in cervico-vaginal lavages. Multiple regression analysis on ordinal categorical variables was used to test for the simultaneous associations of clinical and microbiological variables on quartiles of cytokine concentrations in lavage samples.
Proviral HIV-1 DNA, cell associated and cell free HIV-1 RNA were detected in 76.7% (46/60), 70% (42/60), and 71.7% (43/60) of the patients, respectively. IL-1beta concentration was directly correlated with proviral HIV-DNA (Spearman rho = 0.35, p = 0.01) and cell associated HIV-RNA levels (Spearman rho = 0.263, p = 0.05). IL-1beta concentration (153.9 pg/ml) was higher (p<0.05) among women with cytological squamous intraepithelial lesion (SIL) than negative controls (73.4 pg/ml). In women with vaginal infection both IL-1beta (41.7 pg/ml) and IL-6 (10.2 pg/ml) were lower (p<0.05) in comparison to negative controls (144.9 pg/ml and 23.7 pg/ml, respectively). Women receiving stable antiretroviral therapy had significantly lower TNF-alpha (34.4 pg/ml versus 44.4 pg/ml, p = 0.04) and higher IL-6 (24.0 pg/ml versus 1.4 pg/ml, p = 0.004) levels in lavage samples compared to untreated women. The associations between the presence of SIL, antiretroviral treatment, vaginal infection and cytokine concentrations in cervico-vaginal secretions were confirmed in multiple regression analysis.
Local immune activation may modulate HIV-1 shedding in cervico-vaginal secretion with possible influence on vaginal physiology and host defence. Pharmacological agents lowering HIV-1 replication cause a shift to a pattern of cytokine production which seems less favourable to the transmission of the disease.
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ABSTRACT: Coherent drug/microbicide/vaccine development research would benefit through a precise knowledge of HIV dissemination and its persistence in the female genital tract. Understanding relationship between plasma viremia and cervicovaginal HIV shedding may help to unveil mechanisms underlying transmission, compartmentalization and pathogenesis. To study the association between HIV-1 RNA levels in the plasma and CVL specimens. Whole blood, plasma and CVL specimens were collected from 36 ART naïve HIV-1 seropositive women qualifying the study inclusion criteria. Absolute CD4 counts, plasma and CVL HIV-1 RNA levels were estimated using commercially available kits (BD MultiSET™ Kit, Becton Dickinson, US and Abbott RT, Abbott Molecular, Germany). Correlation between plasma and CVL viral load was estimated by the Spearman's rank correlation coefficient. Additionally, the correlation between CVL viral load and absolute CD4 counts was studied. HIV-1 viral load in the CVL specimens was successfully quantified using the Abbott RT. Twenty-seven of 36 women (75%) had detectable HIV-1 RNA levels in plasma and CVL specimens. The CVL viral load did not show any correlation with plasma viral load (ρ=0.281, p=0.096) and showed a 'moderate correlation' (ρ=-0.563, p=0.0004) with absolute CD4 counts. Albeit, the Abbott RT is designed for estimating plasma HIV-1 RNA levels, the study reports its use for estimating HIV-1 RNA levels in the CVL specimens as well. In accordance with the previous studies, our results suggest that plasma and CVL viral load are not correlated and plasma viremia might not solely predict cervico vaginal HIV shedding.Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 10/2013; Volume 58,(Issue 4 , December 2013):730-732. · 3.12 Impact Factor
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ABSTRACT: In order to reveal the effect of turnings on explosion propagation, experiments were performed in three different pipes (single bend, U-shaped pipe and Z-shaped pipe). Flame and pressure transducers were used to track the velocity at the explosion front. When the pipes were filled with methane, the explosion strength was significantly enhanced due to the turbulence induced by increasing the number of turnings, while the flame speed (Sf) and peak overpressure (ΔPmax) increased dramatically. In addition, the strength of the explosion increased in violence as a function of the number of turnings. However, when the bend was without methane, the turnings weakened the strength of the explosion compared with the ordinary pipe, shown by the decrease in the values of ΔPmax and Sf. In addition, the propagation characteristics in a U-shaped pipe were similar to those in a Z-shaped pipe and the values of ΔPmax and Sf were also close. The results show that the explosion propagation characteristics largely depend on gas distribution in the pipes and the number of turnings. The different directions of the turnings had no effect.Mining Science and Technology (China) 05/2011; 21(3):365-369.
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ABSTRACT: OBJECTIVE: We sought to examine the impact of the loop electrosurgical excision procedure (LEEP) on the rate and magnitude of HIV-1 genital shedding among women undergoing treatment for cervical intraepithelial neoplasia 2/3 (CIN2/3). DESIGN: Prospective cohort study. POPULATION: Women infected with HIV-1 undergoing LEEP for CIN2/3 in Kisumu, Kenya. METHODS: Participants underwent specimen collection for HIV-1 RNA prior to LEEP and at 1, 2, 4, 6, 10, and 14 weeks post-LEEP. HIV-1 viral load was measured in cervical and plasma specimens using commercial real-time polymerase chain reaction (PCR) assays, to a lower limit of detection of 40 copies per specimen. MAIN OUTCOME MEASURES: Presence and magnitude of HIV-1 RNA (copies per specimen or cps) in post-LEEP specimens, compared with baseline. RESULTS: Among women on highly active antiretroviral therapy (HAART), we found a statistically significant increase in cervical HIV-1 RNA concentration at week 2, with a mean increase of 0.43 log10 cps (95% CI 0.03-0.82) from baseline. Similarly, among women not receiving HAART, we found a statistically significant increase in HIV-1 shedding at week 2 (1.26 log10 cps, 95% CI 0.79-1.74). No other statistically significant increase in concentration or detection of cervical HIV-1 RNA at any of the remaining study visits were noted. CONCLUSIONS: In women infected with HIV undergoing LEEP, an increase in genital HIV shedding was observed at 2 but not at 4 weeks post-procedure. The current recommendation for women to abstain from vaginal intercourse for 4 weeks seems adequate to reduce the theoretical increased risk of HIV transmission following LEEP.BJOG An International Journal of Obstetrics & Gynaecology 05/2013; · 3.76 Impact Factor
Markers of local immunity in cervico-vaginal secretions of
HIV infected women: implications for HIV shedding
F Zara, R E Nappi, R Brerra, R Migliavacca, R Maserati, A Spinillo
See end of article for
Policlinico S Matteo,
Piazzale Golgi 2, 27100
Accepted for publication
9 October 2003
Sex Transm Infect 2004;80:108–112. doi: 10.1136/sti.2003.005157
Objectives: To link local proinflammatory cytokines with HIV related nucleic acids in cervico-vaginal
secretions and the factors associated with them.
Methods: An observational study on 60 HIV positive women attending the department of obstetrics and
gynaecology, University of Pavia, Italy. HIV-1 RNA in plasma, proviral HIV-1-DNA, cell associated and
cell free HIV-1 RNA in cervico-vaginal secretions were evaluated by competitive polymerase chain
reaction (c-PCR) and reverse transcriptase PCR (cRT-PCR). IL-1b, IL-6, and TNF-a were measured by ELISA
in cervico-vaginal lavages. Multiple regression analysis on ordinal categorical variables was used to test
for the simultaneous associations of clinical and microbiological variables on quartiles of cytokine
concentrations in lavage samples.
Results: Proviral HIV-1 DNA, cell associated and cell free HIV-1 RNA were detected in 76.7% (46/60),
70% (42/60), and 71.7% (43/60) of the patients, respectively. IL-1b concentration was directly correlated
with proviral HIV-DNA (Spearman rho=0.35, p=0.01) and cell associated HIV-RNA levels (Spearman
rho=0.263, p=0.05). IL-1b concentration (153.9 pg/ml) was higher (p,0.05) among women with
cytological squamous intraepithelial lesion (SIL) than negative controls (73.4 pg/ml). In women with
vaginal infection both IL-1b (41.7 pg/ml) and IL-6 (10.2 pg/ml) were lower (p,0.05) in comparison to
negative controls (144.9 pg/ml and 23.7 pg/ml, respectively). Women receiving stable antiretroviral
therapy had significantly lower TNF-a (34.4 pg/ml versus 44.4 pg/ml, p=0.04) and higher IL-6
(24.0 pg/ml versus 1.4 pg/ml, p=0.004) levels in lavage samples compared to untreated women. The
associations between the presence of SIL, antiretroviral treatment, vaginal infection and cytokine
concentrations in cervico-vaginal secretions were confirmed in multiple regression analysis.
Conclusions: Local immune activation may modulate HIV-1 shedding in cervico-vaginal secretion with
possible influence on vaginal physiology and host defence. Pharmacological agents lowering HIV-1
replication cause a shift to a pattern of cytokine production which seems less favourable to the transmission
of the disease.
nents contribute to immune response against HIV-1 in the
female genital tract.1 2Indeed, a complex cascade of host
proinflammatory and immunoregulatory cytokines detected
in the cervico-vaginal secretions of uninfected and HIV-1
infected women modulates HIV-1 replication.3 4Moreover,
local immune activation increases HIV-1 load in genital
secretions, potentially enhancing the risk of sexual transmis-
sion of HIV-1.5 6
We have previously shown the relevance of the detection
and quantification of HIV-1 related nucleic acids in cervico-
vaginal secretions.7However, despite the progress towards
the understanding of the factors responsible for HIV
shedding in the lower female genital tract, little is known
regarding the regulation of local immune responses in the
female genital tract.
Modulation of local cytokine expression by inflamma-
tory stimuli or pharmacological agents may affect HIV-1
influence the susceptibility to sexually transmitted infections
Theaim ofthis study
flammatory cytokine concentrations with HIV-1 related
nucleic acid load in cervico-vaginal secretions of HIV infected
he female genital tract is a reservoir of human
immunodeficiency virus type 1 (HIV-1). The common
mucosal immune system and cellular immune compo-
was tolinklocal proin-
From a cohort of known HIV-1 seropositive women followed
at our department in the period June 2000-May 2001 for
cytological screening for lower genital tract neoplasia, 63
non-pregnant HIV-1 seropositive women were recruited for
this study, after they had signed informed consent. None had
unprotected sexual contacts at least 3 days before the
observation. A demographic and clinical questionnaire was
administered to each participant. Clinical HIV staging was
defined using the Centres for Disease Control (CDC)
Blood and cervico-vaginal samples were obtained on the
same day. Blood sample analysis included CD4+ lymphocyte
cell counts and plasma HIV-RNA. After careful speculum
examination, vaginal swabs were collected to diagnose
infection by candida, trichomonas, or bacterial vaginosis.7
To evaluate candida infection, vaginal specimens were
inoculated on Sabouraud dextrose agar containing gentami-
cin (40 g/ml). The isolated strains were identified with either
the germ tube test or by auxanogram (API 20 Aux; API
system, Montalieu-Vercieu, France). Vaginal specimens for
trichomonas isolation were inoculated in a cysteine-peptone-
liver maltose fresh medium, whereas the diagnosis of
bacterial vaginosis was made according to the criteria of
Amsel et al.11
Cervico-vaginal secretions were obtained by a gentle
rotation of a Dacron swab within the posterior fornix. In
addition, a lavage was performed with 10 ml of RPMI 1640
medium into the vagina, followed by aspiration of the
suspension after allowing 1 minute for pooling. Swabs were
used to detect cell free HIV-RNA, while HIV-DNA, intracel-
lular HIV-RNA transcripts, human papillomavirus (HPV)-
DNA, and cytokines were measured from lavage samples.
Finally, a Papanicolaou smear and a standard colposcopic
examination were carried out. Cervical samples were
examined under the microscope to confirm absence of red
blood cells and spermatozoa and tested for the presence of
blood contamination (reactive strips, Bayer, multistic-10
visual). Three patients were excluded from the study because
cervical specimens contained haemoglobin, thus leaving 60
patients for the final analysis.
HIV related nucleic acid measurements
A detailed description of the methods used to detect and
quantify HIV related nucleic acids both from blood and
cervico-vaginal secretions has previously been reported.12The
supernatant from the swab was used to extract cell free HIV-
RNA. HIV-DNA was extracted from the nuclei of cervico-
vaginal cells to minimise contamination with unintegrated
DNA. RNA was extracted using the guanidinium thiocyanate
method.13The following substrates were analysed using
quantitative polymerase chain reaction (cPCR) and reverse
transcription (RT)-PCR: (a) genomic HIV-RNA from plasma
and cell free cervico-vaginal secretions; (b) virus specific
unspliced HIV-RNA transcripts from cervico-vaginal cells; (c)
Qualitative analysis of specific HIV-DNA and RNA sequences
was first conducted by PCR and RT-PCR using the SK 426/
431 pair of primers.14
Quantification of DNA and RNA was performed using the
primer set SK38/39 by competitive PCR and RT-PCR as
described by Menzo et al.15
expressed as HIV-RNA transcripts and HIV-DNA copy
number per 105cells, and as HIV-RNA cell free copy number
per ml of cervico-vaginal secretions. Swabs were incubated in
a predefined quantity of transport medium (1 ml), so it was
possible to know the exact amount of cervico-vaginal
secretion present in each sample (usually 200–300 ml). In
our experimental conditions, the lower limit of detection of
the assay was two DNA or RNA copies/105cells, 20 RNA
copies/ml of cervical secretions, and 200 RNA copies/ml of
HPV-DNA extraction and PCR were done on lavages as
previously described.16HPV-DNA typing on positive samples
was done with the ‘‘Multigen HPV’’ commercial kit (DiaTech,
Jesi, Italy), which is based on the amplification of a 142–
151 bp fragment of the L1 region by PCR and subsequent
restriction fragment length polymorphism (RLFP) analysis of
PCR products. The digestion pattern can yield human
papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, and 56.
Quantitative results were
Quantitative enzyme linked immunosorbent assay (ELISA)
kits (Chemicon International, CA, USA) were used to
measure the concentrations of tumour necrosis factor
(TNF)-a, interleukin (IL)-1b and IL-6 in the cervico-vaginal
supernatants. After testing the assay kit according to the
manufacturer’s instructions, a dilution factor was necessary
to measure IL-1b and TNF-a (lower limits of detection were
1.95 pg/ml for both) and IL-6 (lower limit of detection was
0.34 pg/ml). All samples and controls were assayed in
duplicate. The TNF-a assay was performed immediately after
the sample process, given the intrinsic instability of such a
cytokine. The intra-assay and inter-assay coefficients of
variation of TNF-a were 6.5% and 11.7%, respectively, for
IL-1b they were 8.1% and 11.7%, respectively and for IL-6
they were 8.1% and 10.4%, respectively. A value of zero was
assigned to any cytokine result that was below the assay’s
lower limit of detection.
Since the data were skewed a non-parametric approach
(Mann-Whitney U test and Spearman rank correlation
coefficient) was used to analyse continuous variables.
Categorical analysis was carried out by Fisher’s exact test
and by computation of odds ratio and 95% confidence
intervals. To evaluate the simultaneous associations of
clinical and microbiological variables on the cytokine
concentration of lavage samples we used stepwise multiple
regression models on ordinal categorical variables. For this
purpose the concentrations of cytokines in cervico-vaginal
lavages were expressed as quartiles of concentration and
inserted in the model as dependent categorised ordinal
variables. In this kind of analysis numerical variables are
expressed as categories and no distributional assumption
about the variables is made. Independent variables were
inserted either as categorised ordinal variables on the basis of
quartile of concentration (HIV related nucleic acids in plasma
and cervico-vaginal lavage) or counts (CD4+ cell counts) or as
dichotomous variables (vaginitis, squamous intraepithelial
lesion, genital HPV infection and antiretroviral treatment).
Only variables significantly associated (p,0.05) with quar-
tiles of cytokine concentration are present in final models.
Statistical analysis was performed using SPSS statistical
software (SPSS Inc, Chicago, IL, USA).
The mean age of the study population was 30.7 (SD
3.8) years. Injecting drug use was the main risk factor for
HIV acquisition in 22 patients (36.7%); in the remaining
patients HIV infection was heterosexually transmitted. The
prevalence of cigarette smoking was 71.7% (41/60) and 10
women (13.3%) had more than one sexual partner in the
6 months preceding the study. Regarding the contraceptive
method used, 22 patients (36.7%) did not use contraceptive
methods, 35 (58.3%) used male condom, and only two
patients (3.3%) used hormonal contraception. Fifteen women
(25%) had a history of prolonged hypothalamic amenor-
rhoea, whereas the remaining patients were menstruating
regularly. In these latter subjects cervico-vaginal samples
were performed 10–20 days after menstruation. Twenty one
women (35%) were at stage C of HIV disease, and 23 (38.3%)
had CD4+ cell counts of less than 200 6106/l.
The prevalence of HIV-1 related nucleic acids detection,
HPV infection, vaginitis, and Papanicolaou smear findings is
reported in table 1. The mean CD4+ cell count was 3606106/l
(range 41–776). The detection of HIV-RNA in plasma
significantly correlated with the presence of proviral HIV-
DNA in cervico-vaginal secretions (23 out of 27 versus 19 out
of 33, odds ratio=4.24, 95% CI=1.05 to 18.5, p=0.03).
Lavage samples that tested positive for cell free HIV-RNA and
for HIV-RNA transcripts were also positive for proviral HIV-
DNA. There were no significant correlations between HPV
infection or vaginitis and the detection of HIV-DNA in
cervico-vaginal secretions (29 out 46 versus nine out 14,
OR=0.95, 95% CI=0.23 to 3.84, and eight out 46 versus two
out 14, OR=1.26, 95% CI=0.2 to 9.98, p=0.87 and 0.84,
respectively). The prevalence of HIV-DNA detection in lavage
samples was 80.6% (25/31) among women with squamous
intraepithelial lesion (SIL) cervical lesions compared with
72.4% (21/29) among those with negative cytological findings
Cytokines in cervico-vaginal secretions of HIV+ women 109
(OR=1.59, 95% CI=0.41 to 6.26, p=0.547). In 14 cases
(23.3%) IL-6 was undetectable. IL-1b and IL-6 levels were
significantly correlated (Spearman rho=0.331, p=0.024). In
addition, IL-1b concentration in lavage samples was directly
correlated with proviral HIV-DNA (Spearman rho=0.35,
p=0.01), cell associated HIV-RNA viral load in cervico-
vaginal secretions (Spearman rho=0.263, p=0.05), and
with plasma HIV-RNA viral load (Spearman rho=0.257,
The associations between cytokine levels in lavage samples
and cytological findings or vaginal infection are reported in
table 2. IL-1b concentration was higher among women with
SIL, while both IL-1b and IL-6 were lower in patients with
vaginal infection in comparison to respective negative
At the time of the study, 50 women (83.3%) had been
receiving stable antiretroviral therapy for at least 3 months.
The median duration of antiretroviral therapy was 12 months
(range 3–32). Nucleoside analogues (zidovudine plus lami-
vudine or didanosine) only (26 women), or nucleoside
analogues plus indinavir (24 women) were the antiretroviral
drugs used. Women receiving stable antiretroviral therapy
had significantly lower TNF-a (34.4 pg/ml, range 9–155.1
compared to 44.4 pg/ml, range 16.3–104.1; p=0.04) and
higher IL-6 levels (24 pg/ml, range 0–242.5 compared to
1.4 pg/ml, range 0–5.3; p=0.004) in lavage samples com-
pared to untreated women. IL-6 was undetectable in 70%
(7/10) of the untreated women and in 14% (7/50) of the
women receiving stable antiretroviral therapy (p=0.001 by
Fisher’s exact test).
The analysis of the association between the duration of
therapy and HIV and cytokine concentrations in lavage
samples confirmed these findings. Duration of therapy was
significantly related to the cell free HIV-RNA copy number in
lavage samples (Spearman rho=20.209, p=0.027) and to
the concentration of IL-6 (Spearman rho=0.218, p=0.03).
To evaluate the simultaneous associations of clinical and
microbiological variables on the cytokine concentration of
lavage samples we used stepwise multiple regression models
on ordinal categorical variables. The analysis confirmed that
independent of the presence of HIV related nucleic acids,
antiretroviral treatment correlated inversely with TNF-a and
directly with IL-6 cervico-vaginal concentrations (table 3).
The presence of cytological SIL was associated with increased
findings,CD4+ cell counts, and cytokine concentrations in cervico-vaginal samples in the
HIV related nucleic acids, HPV infection, vaginitis, Papanicolaou smear
HIV-DNA (lavage sample)
HIV-RNA transcripts (lavage sample)
Cell-free HIV-RNA (swab sample)
HPV infection (lavage sample)
16, 18, 31, 35
CD4+ cell counts (6106/l)
Plasma HIV-RNA (copies/ml)
HIV-DNA lavage sample (copies/105cells)
HIV-RNA transcripts lavage sample (copies/105cells)
Cell-free HIV-RNA swab sample (copies/ml)
*In 14 cases, Il-6 was undetectable.
(range) in cervico-vaginal samples
Cytological findings, vaginal culture results, and mean cytokine concentrations
Cervical cytological findings
Positive (candida, vaginosis, trichomonas)
*p,0.05 compared to negative (Mann-Whitney U test).
110 Zara, Nappi, Brerra, et al
concentration of IL-1b whereas vaginitis was inversely
related IL-6 concentration. Regarding the role of HIV related
nucleic acids, multiple regression analysis showed that
quartiles of cell associated HIV-RNA viral load in cervico-
vaginal secretions correlated directly with IL-6 and TNF-a
concentrations. As also evident in univariate analysis, the IL-
1b concentration in lavage samples was directly correlated
with proviral HIV-DNA.
This study showed that the IL-1b concentration in cervico-
vaginal secretions was significantly correlated with proviral
HIV-1 DNA and cell associated HIV-1 RNA viral load detected
in the same compartment and with HIV-1 RNA viral load
measured in the plasma. In addition, the detection of HIV-1
RNA in plasma was significantly linked to the presence of
proviral HIV-DNA in cervico-vaginal secretions. HPV infec-
tion or vaginitis did not affect the detection of HIV-1 DNA.
On the other hand, cytological alterations of the cervix were
associated with higher cervico-vaginal IL-1b concentrations,
while both cytokines were lower in patients with vaginal
In women receiving stable antiretroviral therapy, together
with a significant reduction of cell free HIV-1 RNA
concentration, we were able to demonstrate significantly
lower TNF-a and significantly higher IL-6 concentrations in
cervico-vaginal secretions compared with untreated women.
This set of results seems to confirm the knowledge that
local immune activation may modulate HIV-1 shedding in
cervico-vaginal secretion with possible influence on vaginal
physiology and host defence.
Several investigations17 18have previously shown that the
shedding of HIV in the genital tract can occur in 20–30% of
non-viraemic women. Vaginal infection could have a facil-
itating role in local HIV viral replication and shedding17by
interacting with the local mucosal immune environment.2 6
Specific cytokines may then be crucial to the cervico-vaginal
presence of HIV. In our study we have considered IL-1b, TNF-
a, and IL-6 because of their clear implication in the
modulation of the levels of HIV expression.19TNF-a increases
HIV-1 proviral trascription2and both TNF-a and IL-1b can
upregulate HIV replication through activation of the LTR
promoter region.3In addition, IL-1 is synergic with IL-6, but
not with TNF, in the upregulation of virus expression of the
latently infected human promonocytic cell line U1, as
measured by accumulation of steady state mRNAs and
production of reverse transcriptase activity.20On the other
hand, such cytokines may be differently affected by several
cervico-vaginal infections. Indeed, Candida albicans induced
considerable levels of TNF-a but not of IL-6,21while aerobic
infections were associated with high levels of IL-6 and IL-1b
in vaginal fluids.22
The correlation between local IL-1b and proviral HIV-1 and
cell associated HIV-1 RNA viral load in cervico-vaginal
secretions emerging from the present study suggests that
such cytokines may represent a marker of possible transmis-
sion of the disease. On the other hand, other vaginal cytokine
levels correlate with vaginal viral load and not plasma HIV
shedding, with a significant elevation at the time of menses
and no significant changes during other phases of the cycle.23
A very interesting finding was the significant higher levels
of IL-1b in HIV infected women with SIL. Such data deserve
further attention in light of the evidence that HPV infection
of the cervix may influence HIV pathogenesis by inducing the
production of immune and inflammatory factors that
enhance HIV expression.24On the other hand, a highly
significant association between high secretor IL-1b pheno-
types and LSIL has been reported25while, in another study,
TNF-a and IL-1b levels were significantly higher in women
with cervical cancer compared with controls and CIN
The presence of vaginal infections seemed to negatively
affect the pattern of local cytokines, given the evidence of
lower IL-1b and IL-6 concentrations in women with vaginitis.
The difficult interpretation of this data may be because it was
not the major aim of our study to characterise the cytokine
pattern in relation to vaginal infections that were analysed
regardless the different aetiologies. Indeed, bacterial vagino-
sis has been clearly associated with high levels of IL-1b or
TNF-a to partially explain the mechanism by which this risk
factor enhances HIV transmission.8On the other hand, the
use of antiretroviral therapy in the majority of our study
population may have altered the finding.
The evidence that stable antiretroviral treatment signifi-
cantly affected the presence of cytokines in cervico-vaginal
secretions and that the duration of therapy was significantly
linked to both HIV shedding and IL-6 concentration, is of
great interest when considering the potential significance in
term of sexual transmission. Indeed, the higher cervico-
vaginal levels of IL-6 detected in treated women are
consistent with the idea that even though it is considered
proinflammatory, IL-6 may exert an anti-inflammatory
action under special circumstances.27IL-6 contributes to the
production of several proteins that could function as a
protective mechanism.28Moreover, IL-6 inhibits IL-1b and
TNF-a production not only by directly suppressing their
samples and microbiological and clinical variables
Association between quartiles of TNF-a, IL-1b, and IL-6 concentration in lavage
HIV-RNA transcripts (lavage)
Cell free HIV-RNA (lavage)
HIV-RNA transcripts (lavage)
*Standardised regression coefficient (b), standard error (SE), and p value as obtained by stepwise multiple
regression analysis on categorical variables. Dependent variables were quartiles of IL-1b, TNF-a and IL-6
concentration in lavage samples. Independent variables were included as quartiles of plasma HIV-RNA
concentration and CD4+ cell counts and quartiles of HIV-DNA, HIV-RNA transcripts, cell free HIV-RNA
concentration in lavage samples. Vaginitis, squamous intraepithelial lesion, genital HPV infection, and
antiretroviral treatment were inserted as dichotomous variables.
Cytokines in cervico-vaginal secretions of HIV+ women111
production and release,29
induction of the IL-1b receptor antagonist and the soluble
TNF-a receptor (p55).30Our data showing significantly low
TNF-a concentrations in treated HIV infected women are
thus compatible with the hypothesis that IL-6 may exert a
local protective action.
In conclusion, markers of cervico-vaginal immunity are
relevant to the shedding of HIV in the genital tract. Whether
or not specific cytokines may be directly involved in
conditioning the risk of sexual transmission of HIV-1 remains
to be established. However, there is no doubt that pharma-
cological agents lowering HIV-1 replication cause a shift to a
pattern of cytokine production which seems less favourable
to the transmission of the disease.
but also by stimulating the
The authors are grateful to Dr G Randine from the toxicology
division, IRCCS Salvatore Maugeri Foundation, Pavia, Italy, for her
expert technical assistance in cytokine assays.
This study was supported by RC 1998, IRCCS Policlinico S Matteo,
FZ, laboratory analysis of HIV viral loads, and HPV typing,
preparation and discussion of the project, writing of the manuscript;
REN, laboratory analysis of cytokine concentrations, preparation and
discussion of the project, writing of the manuscript; RB, laboratory
analysis of HIV viral loads and HPV typing, preparation of the project,
discussion of the methods; RM, laboratory analysis of HIV viral loads
and HPV typing, preparation of the project, discussion of the
methods; RM, recruitment and evaluation of the patients, performing
visits and samples, discussion of the manuscript.
F Zara, R Brerra, R Migliavacca, Department of Microbiology,
University of Pavia, Italy
R E Nappi, A Spinillo, Department of Obstetric and Gynecology, IRCCS
S Matteo, University of Pavia, Italy
R Maserati, Division of Infectious Diseases, IRCCS S Matteo, Pavia, Italy
1 Iversen AKN, Fugger L, Eugen-Olsen J, et al. Cervical human
immunodeficiency virus type 1 shedding is associated with genital
beta2chemokine secretion. J Infect Dis 1998;178:1334–42.
2 Crowley-Nowick PA, Ellenberg JH, Vermund SH, et al. Cytokine profile in
genital tract secretions from female adolescents: impact of human
immunodeficiency virus, human papillomavirus, and other sexually transmitted
pathogens. J Infect Dis 2000;181:939–45.
3 Osborne L, Kunkel S, Nabel GJ. Tumor necrosis factor-alpha and interleukin-1
stimulate the human immunodeficiency virus enhancer by activation of the
nuclear factor kappa B. Proc Natl Acad Sci 1999;86:2336–40.
4 Poli G, Fauci AS. The effect of cytokines and pharmacologic agents on chronic
HIV infection. AIDS Res Hum Retroviruses 1992;8:191–6.
5 Vincenzi E, Poli G. Regulation of HIV expression by viral genes and cytokines.
J Leukoc Biol 1994;56:328–34.
6 Lawn SD, Subbarao S, Wright TC, et al. Correlation between human
immunodeficiency virus type 1 RNA levels in the female genital tract and
immune activation associated with ulceration of the cervix. J Infect Dis
7 Spinillo A, Debiaggi M, Zara F, et al. Human immunodeficiency virus type 1-
related nucleic acids and papillomavirus DNA in cervicovaginal secretions of
immunodeficiency virus-infected women. Obstet Ginecol 2001;97:999–1004.
8 Sturm-Ramirez K, Gaye-Diallo A, Eisen G, et al. High levels of tumor necrosis
factor-a and interleukin-1b in bacterial vaginosis may increase susceptibility to
human immunodeficiency virus. J Infect Dis 2000;182:467–73.
9 Cu-Uvin S, Caliendo AM, Reinert S, et al. Effect of highly active antiretroviral
therapy in cervicovaginal HIV-1 RNA. AIDS 2000;10:415–21.
10 Centers for Disease Control. 1993 Revised classification system for HIV
infection and expanded surveillance case definition for AIDS among
adolescents and adu1ts. MMWR 1992;41:1–15.
11 Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis: diagnostic
criteria and microbiological and epidemiological associations. Am J Med
12 Spinillo A, Zara F, De Santolo A, et al. Quantitative assessment of cell-
associated and cell-free virus in cervicovaginal samples of HIV-1 infected
women. Clin Microbiol Infect 1999;5:605–11.
13 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem
14 Ehrlich GD. PCR-based methods for the detection of the human retroviridae
and hepadnaviridae. In: Ehrlich GD, Greemberg SJ, eds. PCR-based
diagnostics in infectious disease. Boston: Blackwell Scientific Publications Inc,
15 Menzo S, Bagnarelli P, Giacca M, et al. Absolute quantitation of viremia in
human immunodeficiency virus infection by competitive reverse transcription
and polymerase chain reaction. J Clin Microbiol 1992;30:1752–7.
16 Manos MM, Ting Y, Wright DK, et al. Use of polymerase chain reaction
amplification for the detection of genital human papillomavirus. Cancer Cells
17 Spinillo A, Debiaggi M, Zara F, et al. Factors associated with nucleic acids
related to human immunodeficiency virus type 1 in cervico-vaginal secretions.
Br J Obstet Gynaecol 2001;108:634–41.
18 Debiaggi M, Zara F, Spinillo A, et al. Viral excretion in cervicovaginal
secretions of HIV-1-infected women receiving antiretroviral therapy. Eur J Clin
Microbiol Infect Dis 2001;20:91–6.
19 Poli G, Fauci AS. Cytokine modulation of HIV expression. Sem Immunol
20 Poli G, Kinter AL, Fauci AS. Interleukin 1 induces expression of the human
immunodeficiency virus alone and in synergy with interleukin 6 in chronically
infected U1 cells: inhibition of inductive effects by the interleukin 1 receptor
antagonist. Proc Natl Acad Sci USA 1994;91:108–12.
21 Steele C, Fidel PL. Cytokine and chemokine production by human oral and
vaginal epithelial cells in response to Candida albicans. Infect Immun
22 Donder GG, Vereecken A, Bosmans E, et al. Definition of a type of abnormal
vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis.
Br J Obstet Gynaecol 2002;109:34–43.
23 Al-Harthi L, Kovacs A, Coombs RW, et al. A menstrual cycle pattern for
cytokines levels exists in HIV-positive women: implication for HIV vaginal and
plasma shedding. AIDS 2001;15:1535–43.
24 Gage JR, Sandhu AK, Nihira M, et al. Effects of human papillomavirus-
associated cells on human immunodeficiency virus gene expression. Obstet
25 Majeed GS, Glew S, Bidwell J. An association between LSIL and the high
secretor phenotype of IL-1beta. Gynecol Oncol 1999;73:359–61.
26 Tjiong MY, van der Vange N, ter Schegget JS, et al. Cytokines in
cervicovaginal washing fluid from patients with cervical neoplasia. Cytokine
27 Richards CD, Gauldie J. Role of cytokines in acute-phase response. In:
Aggarwal BB, Puri P, eds. Human cytokines: their role in disease and therapy.
Cambridge: Blackwell Science, 1998:253–69.
28 Tilg H, Dinarello CA, Mier JW. IL-6 and APPs: anti-inflammatory and
immunosuppressive mediators. Immunol Today 1997;18:428–32.
29 Schindler R, Mancilla J, Endres S, et al. Correlations and interactions in the
production of interleukin-6 (IL-6), IL-1 and tumor necrosis factor (TNF) in
human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood
30 Tilg H, Trehu E, Atkins MB, et al. Interleukin-6 (IL-6) as an anti-inflammatory
cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor
necrosis factor receptor p55. Blood 1994;83:113–18.
N Local immune activation may modulate HIV-1 shedding
in cervico-vaginal secretions
N Stable antiretroviral treatments significantly affect the
presence of cytokines in cervico-vaginal secretions
N In HIV infected women, vaginal infection or squamous
intraepithelial lesions (SIL) significantly influence the
presence of cytokines in cervico-vaginal secretions
112Zara, Nappi, Brerra, et al