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- text: How many children were infected by HIV-1 in 2008-2009, worldwide?
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Functional Genetic Variants in DC-SIGNR Are Associated with
Mother-to-Child Transmission of HIV-1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche,
Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.;
Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07
DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND:
Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection
in children worldwide. Given that the C-type lectin receptor, dendritic
cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as
CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)),
can interact with pathogens including HIV-1 and is expressed at the
maternal-fetal interface, we hypothesized that it could influence MTCT of
HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR
in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR
in a well-characterized cohort of 197 HIV-infected mothers and their
infants recruited in Harare, Zimbabwe. Infants harbouring two copies of
DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold
increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold
increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after
adjusting for a number of maternal factors. The implicated H1 and H3
haplotypes share two single nucleotide polymorphisms (SNPs) in promoter
region (p-198A) and intron 2 (int2-180A) that were associated with
increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP
(P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced
transcriptional activity in vitro. In homozygous H1 infants bearing both
the p-198A and int2-180A mutations, we observed a 4-fold decrease in the
level of placental DC-SIGNR transcripts, disproportionately affecting the
expression of membrane-bound isoforms compared to infant noncarriers (P =
0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial
role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression
increases risk of transmission. Text: Without specific interventions, the
rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45%
[1] . UNAIDS estimates that last year alone, more than 400,000 children
were infected worldwide, mostly through MTCT and 90% of them lived in
sub-Saharan Africa. In the most heavilyaffected countries, such as
Zimbabwe, HIV-1 is responsible for one third of all deaths among children
under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero,
IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High
maternal viral load, low CD4 cells count, vaginal delivery, low
gestational age have all been identified as independent factors associated
with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%,
limited access to timely diagnostics and drugs in many developing world
countries limits the potential impact of this strategy. A better
understanding of the mechanisms acting at the maternal-fetal interface is
crucial for the design of alternative interventions to antiretroviral
therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing
non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph
node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a
plethora of pathogens including HIV-1 and is expressed in placental
capillary endothelial cells [2] . DC-SIGNR is organized in three distinct
domains, an N-terminal cytoplasmic tail, a repeat region containing seven
repeat of 23 amino acids and a C-terminal domain implicated in pathogen
binding. Alternative splicing of DC-SIGNR gene leads to the production of
a highly diversify isoforms repertoire which includes membrane-bound and
soluble isoforms [3] . It has been proposed that interaction between
DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell
types [2] but DC-SIGNR can also internalize and mediate
proteasome-dependant degradation of viruses [4] that may differently
affect the outcome of infection. Given the presence of DC-SIGNR at the
maternal-fetal interface and its interaction with HIV-1, we hypothesized
that it could influence MTCT of HIV-1. To investigate the potential role
of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study
of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and
their infants recruited in Zimbabwe, and identified specific DC-SIGNR
variants associated with increased risks of HIV transmission. We further
characterized the functional impact of these genetic variants on DC-SIGNR
expression and show that they affect both the level and type of DC-SIGNR
transcripts produced in the placenta. Samples consisted of stored DNA
extracts obtained from 197 mother-child pairs co-enrolled immediately
postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare,
Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24
months. The ZVITAMBO project was a randomized placebocontrolled clinical
trial that enrolled 14,110 mother-child pairs, between November 1997 and
January 2000, with the main objective of investigating the impact of
immediate postpartum vitamin A supplementation on MTCT of HIV-1. The
samples used in the present study were from mother-child pairs randomly
assigned to the placebo group of the ZVITAMBO project. Antiretroviral
prophylaxis for HIV-1-positive antenatal women was not available in the
Harare public-sector during ZVITAMBO patient recruitment. The samples were
consecutively drawn from two groups: 97 HIV-1-positive
mother/HIV-1-positive child pairs and 100 HIV-1-positive
mother/HIV-negative child pairs. Mother's serological status was
determined by ELISA and confirmed by Western Blot. Infants were considered
to be infected if they were HIV-1 seropositive at 18 months or older and
had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results
at earlier ages. 100 infants were considered to be uninfected as they were
ELISA negative at 18 months or older and had two DNA PCR negative results
from samples collected at a younger age. Of the 97 HIV-1-infected infants,
57 were infected IU, 11 were infected IP, and 17 were infected PP as
determined by PCR analyses of blood samples collected at birth, 6 weeks, 3
and 6 months of age and according to the following definitions adapted
from Bryson and colleagues [5] . Briefly, infants who were DNA PCR
positive at birth were infected IU. Infants with negative PCR results from
sample obtained at birth but who become positive by 6 weeks of age were
infected IP. Infants with negative PCR results at birth and 6 weeks of age
but who subsequently became DNA PCR positive were considered to be
infected during the PP period. In the analysis comparing the 3 different
modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR
results were not available at 6 weeks of age. Full methods for
recruitment, baseline characteristics collection, laboratory procedures
have been described elsewhere [6] . The nucleotide sequence variation of
the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in
the study population was determined previously [7] . Haplotype
reconstruction was performed using Bayesian statistical method implemented
in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP)
with a minimum allele frequency (MAF) of 2%. We applied the algorithm five
times, using different randomly generated seeds, and consistent results
were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs
(htSNPs) were identified by the HaploBlockFinder software [9] with a MAF
$5%. These htSNPs were genotyped in the 197 infants by direct PCR
sequencing analysis as we have described previously [7] . The DC-SIGNR
exon 4 repeat region genotype was determined by PCR amplification followed
by migration in 1.5% agarose gels [10] . DNA sequences in the promoter
region were analysed with the TESS interface
(http//:www.cbil.upenn.edu/tess) for putative transcription factors
binding sites using the TRANSFAC database. Luciferase reporter assays
using pGL2-Basic vector were performed in order to investigate the
functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA
from subjects homozygous for the promoter variants and WT was amplified
from nucleotide position 2715 to 21 and cloned between the BglII and
HindIII multiple cloning sites in the pGL2-Basic vector which harbours a
reporter firefly luciferase gene downstream (Invitrogen Canada inc,
Burlington, Canada). All recombinants clones were verified by DNA
sequencing. The firefly luciferase test reporter vector was co-transfected
at a ratio of 10:1 with the constitutive expressor of Renilla luciferase,
phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells
plates (2610 5 cells) and transfected them the following day using
lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed
and luciferase assays were performed using 20 mg of protein extract
according to the manufacturer (Promega) at 44 h post-transfection. Firefly
luciferase activity was normalized to Renilla luciferase activity. 0 mg,
0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive
control in these experiments. We carried out lucierase assays in
triplicate in three independent experiments. Results are expressed as
mean6 standard error of the mean (S.E.M). First-term placental tissues
were obtained from abortions following voluntary interruption of pregnancy
at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1
(associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous
haplotypes were used to analyse possible differences in isoform
expression. Total placental RNAs were extracted by MasterPure DNA and RNA
Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to
the manufacturer. Fragments corresponding to the DC-SIGNR coding region
were reversed transcribed (RT) and then amplified by nested PCR with the
following primers; RT primers RR, first PCR RF and RR and second PCR RcF
and RcR according to Liu and colleagues [11] . 1 mg of total RNA was
reverse transcribed with Expand RT (Roche Applied Science, Indianapolis,
IN, USA) according to the manufacturer and were PCR-amplified with DNA
Platinum Taq Polymerase (Invitrogen). Major PCR products from the second
PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen
Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning
Kit for sequencing (Invitrogen). For each placenta, 15 different clones
were randomly selected and amplified with M13 primers and sequenced with
ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster
City, CA, USA). Sequences were analysed and aligned with GeneBank
reference sequence NM_014257 using Lasergene software (DNA Stars, Madison,
WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental
RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in
20 ml volume according to the manufacturer (Roche Applied Science). 15 ng
of total cDNA in a final volume of 20 ml was used to perform quantitative
real-time PCR using Universal Express SYBR GreenER qPCR Supermix
(Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life
Science, Sydney, Australia). Samples from 2 subjects in each group were
used because RNA quality of others was not suitable for a qRT-PCR
analysis. Amplification of all DC-SIGNR isoforms was performed using an
exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were
amplified using primers specific for exon 3, corresponding to the common
trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon
junction and RNA extracts were treated with DNase (Fermantas International
inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA.
Standard curves (50-500 000 copies per reaction) were generated using
serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen)
plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%,
even in the presence of 20 ng of non-specific nucleic acids, and therefore
could be compared. The copy number of unknown samples was estimated by
placing the measured PCR cycle number (crossing threshold) on the standard
curve. To correct for differences in both RNA quality and quantity between
samples, the expression levels of transcripts were normalised to the
reference GAPDH gene transcripts. GAPDH primer sequences were kindly
provided by A. Mes-Masson at the CHUM. The results are presented as target
gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound
isoforms was calculated as E3/E5. Soluble isoforms were calculated by
subtracting membrane-bound from total isoforms. We carried out qPCR assays
in triplicate in three independent experiments. Results are expressed as
mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM
5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences
in baseline characteristics and genotypic frequencies of haplotypes or
htSNPs were compared between groups using the x 2 analysis or Fisher's
exact test. Logistic regression analysis was used to estimate odds ratios
(OR) for each genotype and baseline risk factors. Multiple logistic
regression was used to define independent predictors identified as
significant in the crude analysis. ORs and 95% confidence interval were
calculated with the exact method. Comparisons of continuous variables
between groups were assessed with the unpaired two-tailed Student's t test
when variables were normally distributed and with the Mann-Whitney U test
when otherwise. Differences were considered significant at P,0.05. Written
informed consent was obtained from all mothers who participated in the
study and the ZVITAMBO trial and the investigation reported in this paper
were approved by The We carried out an association study of DC-SIGNR
polymorphism in 197 infants born to untreated HIV-1-infected mothers
recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected
and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57
were infected IU, 11 were infected IP, and 17 were infected PP. Timing of
infection was not determined for 12 HIV-1-infected infants. Baseline
characteristics of mothers and infants are presented in Table 1 . Maternal
age and CD4 cell count, child sex, mode of delivery, duration of membrane
rupture and gestational age were similar among all groups. However,
maternal viral load .29 000 copies/ml was associated with increased risk
in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P =
0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission,
respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the
15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7]
were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3
(11%) were the most frequent haplotypes observed (Figure 1 ). Being
homozygous for the H1 haplotype was associated with increased risk of both
IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission (
Table 2) . Infants harbouring two copy combinations of H1 and/ or H3
haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P =
0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection
compared to infant noncarriers ( Table 2 ). The latter associations
remained significant after adjustment was made for the maternal viral load
for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71,
95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3
haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A,
ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants
were significantly associated with MTCT of HIV-1 (Table S2 ). In the
unadjusted regression analysis, homozygous infants for the p-198A and
int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78,
P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025,
respectively) HIV-1 infection compared to heterozygote infants or
noncarriers (Table 3) . When adjustment was made for maternal factors,
only the association with the int2-180A variant remained significant for
IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI =
1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for
DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold
to 6-fold more likely to be infected by HIV-1 during pregnancy or at
delivery, respectively. Alternative splicing of the DC-SIGNR gene in the
placenta produces both membrane-bound and soluble isoform repertoires [3]
. The relative proportion of membrane bound and soluble DC-SIGNR could
plausibly influence the susceptibility to HIV-1 infection [11] . We
therefore hypothesized that the DC-SIGNR mutations associated with MTCT of
HIV-1 would have an impact on both the level of DC-SIGNR expression and in
the isoform repertoire produced. We investigated DC-SIGNR transcript
expression in first-term placentas obtained after elective abortion. We
cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1
samples containing both the DC-SIGNR p-198AA and int2-180AA variants
associated with HIV-1 transmission and 3 homozygous wild-type (WT)
(p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly
selected for sequencing. As expected, we found an extensive repertoire of
DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per
individual. A total of 65 distinct transcripts were identified ( Figure S1
), of which 3 were full-length transcripts. 64 of the sequenced clones
contained a total of 69 amino acid substitutions with 3 new C termini and
2 premature stop codons. However, the diversity was mostly attributable to
the entire deletion of exon 2 or exon 3 or to variations in the length of
the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates
the trans-membrane domain of the protein and leads to the expression of
soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of
membrane-bound isoforms in placental tissues of the H1 homozygotes appears
to be lower than that observed in samples from WT individuals ( Figure S1
). The deletion of exon 3 was confirmed by sequencing and we hypothesize
that the skipping of exon 3, could be due to the presence of the int2-180A
mutation observed in infants with the H1 haplotype. In fact, this intron
mutation is located 180 bp downstream from exon 3 and potentially modifies
splicing events (Figure 2A ). We confirmed that the variation in
transcript proportions seen between the two groups was also reflected at
the level of mRNA expression in the placenta. To quantify membrane-bound
vs soluble isoforms in placental samples from homozygous H1 and WT
infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR
isoforms (total transcripts). We then amplified exon 3 (E3) which is
deleted in the soluble forms and then calculated the E3:E5 ratio. We found
that placental tissues from homozygous H1 infants express a significantly
lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT
individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3
skipping happens more frequently in presence of the DC-SIGNR int2-180A
variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is
always transmitted with the promoter mutation p-198A (Figure 1 ). In the
unadjusted regression analysis, the p-198A variant was significantly
associated with IU but not with IP and PP HIV-1 transmission (Table 3) .
Computational transcription factor binding site analysis predicts Table 1
. Baseline characteristics of mother and infants risk factors for
intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child
HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A
variant construct was significantly lower than that of the WT p-198C
promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B )
suggesting that DC-SIGNR p-198A affects promoter activity. The other
promoter mutants (p-577C and p-323A) observed in the Zimbabwean population
did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To
determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR
expression in the placenta, we quantitated the absolute number of total
and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type
placental samples as described earlier. The total number of DC-SIGNR
transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5
GAPDH copies) in the placental samples from homozygous H1 infants and was
4-fold lower compared to that found in placentas from WT individuals
(27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A
mutation might induce exon 3 skipping leading to a lower production of
membrane-bound DC-SIGNR. Although, the decrease in the total number of
DC-SIGNR transcripts in H1 homozygous placental samples containing both
the p-198AA and int2-180AA variants affected the proportion of
membrane-bound and soluble isoforms, the effect of these mutations was
more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1
= 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in
total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) (
Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated
with MTCT of HIV-1 significantly decreased the level of total placental
DC-SIGNR transcripts, disproportionately affecting the membrane-bound
isoform production. Table 3 . Associations between infant DC-SIGNR
promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU),
intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission.
Our genetic results, supported by expression assay in placenta, suggest
the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the
haplotype H1 was associated with IU transmission in the unadjusted
regression analysis. However, the association disappeared after adjustment
was made for the maternal factors presumably because of the small number
of H1 homozygote infants analysed in each groups. H1 and H3 were the most
frequent haplotypes observed in the study population and they share a
cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased
the power of the study and permitted the identification of specific
DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations
shared by haplotypes H1 and H3 were associated with vertical transmission
of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU
and 6fold increased risk of IP after adjustment for the maternal factors.
Although the p-198A variant was associated with IU transmission, the
association disappeared after adjustment was made for the maternal viral
load. Nevertheless, we showed that this mutation reduces DC-SIGNR
transcriptional activity in vitro and produces lower level of DC-SIGNR
transcripts in placental tissues in combination with the int2-180A
variant. Since int2-180A is always transmitted with p-198A on the MTCT
associated combined haplotypes H1/H3, whereas p-198A is carried on other
nonassociated haplotypes (Figure 1) , we can speculate that the p-198A
mutation alone may have a minor effect in vivo whereas in combination with
the int2-180A variant, they both act to reduce the level of placental
DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The
majority of IU transmission occurs during the last trimester of pregnancy
(reviewed in [12] ). Full-term placenta samples were not available for the
current study and the expression assays were performed on first-term
placental tissues. A previous study looking at DC-SIGNR placental isoforms
repertoire in full-term placenta samples demonstrated similar diversity of
DC-SIGNR transcripts as in the first-term placental tissues studied herein
[3] . However, since levels of DC-SIGNR expression have never been
compared between the different terms of pregnancy, it is not known whether
DC-SIGNR expression varies during the course of pregnancy. Nevertheless,
it is reasonable to assume that the inter-individual differences in both
DC-SIGNR isoform repertoire and transcript levels observed between the H1
and WT homozygous infants would be reflected throughout the pregnancy. To
date, most studies have focused on the potential role of DC-SIGNR in trans
infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms
involved in trans infection and redundancy among C-type lectin functions
make it difficult to determine the actual participation of DC-SIGNR in
this mode of infection in vivo [13, 14] . The strong correlation we
observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low
levels of DC-SIGNR in the placenta suggested that mechanisms other than
DC-SIGNR-mediated trans infection might operate during vertical
transmission of HIV-1. For example, DC-SIGNR has also been shown to
function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues
recently demonstrated that DC-SIGNR transfected CHO cells diminish
SARS-CoV titers by enhanced capture and degradation of the virus in a
proteasome-dependent manner [4] . Since endothelial cells express MHC-I
and II, degraded viral antigens could then be presented to immune cells to
elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5,
but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain
barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5
receptor on endothelial cells compromises BBB integrity and enhances
monocytes adhesion and transmigration across the BBB [20, 21] . It is thus
possible that reduced expression of DC-SIGNR, particularly the
membranebound isoforms, on placental capillary endothelial cells might
favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor,
facilitating the migration of maternal HIV-1-infected cells across the
placental barrier resulting in IU transmission of HIV-1. The int2-180A
variant contained in the H1 and H3 haplotypes was associated with IP
transmission suggesting that DC-SIGNR also affect transmission of HIV-1
during delivery. Little is known about the mechanisms underlying
transmission of HIV-1 during delivery. Passage through the birth canal
could potentially expose infants through a mucosal portal entry
(presumably ophthalmic, skin, or gastrointestinal), whereas placental
insult during delivery (physical or inflammatory) may enhance
transplacental passage of maternal HIV-1-infected cells into foetal
circulation [22, 23] . Such process called microtransfusion has been
proposed in regards to the results obtain in a Malawian cohort. Kweik and
colleagues found a significant association between levels of maternal DNA
in umbilical cord blood and IP transmission of HIV-1 suggesting that
passage of maternal infected cells through the placenta is likely to occur
during delivery [22] . Thus, in a similar fashion as suggested earlier for
IU transmission, the relatively lower level of DC-SIGNR in the placenta of
homozygous infants harbouring the int2-180A variant could promote HIV-1
binding to CCR5 receptor on endothelial cells affecting the placental
barrier integrity and facilitating the passage of maternal infected cells
in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1
receptors are known to influence MTCT of HIV-1 (reviewed in [24] ).
Genetic variants in CCR5 have been shown to influence vertical
transmission of HIV-1. CCR5 promoter variants resulting in higher
expression of the receptor were associated with increased risk of MTCT of
HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion
polymorphism in CCR5 has be shown to protect from vertical transmission of
HIV-1 [27] , but this variant is virtually absent among African
populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive
ligand for CCR5, are associated with higher chemokine production and lower
risk of MTCT of HIV-1 among South African infants [29, 30] .
Mannose-binding lectin (MBL) is an innate immune receptor synthesised in
the liver and secreted in the bloodstream in response to inflammation
signal. MBL promotes pathogen elimination by opsonization and
phagocytosis, and reduced expression of MBL resulting from polymorphism in
coding and non-coding regions has been associated with an increased risk
of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first
time, the potential functional impact of DC-SIGNR mutations on its
expression in the placenta and in vertical transmission of HIV-1. We
believe that the presence of DC-SIGNR at the placental endothelial cell
surface may protect infants from HIV-1 infection by capturing virus and
promoting its degradation/presentation. However, in placenta containing
low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on
endothelial cells resulting in a loss of placental barrier integrity and
enhanced passage of maternal HIV-1-infected cells in foetal circulation
leading to MTCT of HIV-1. This mechanism may also apply to other
vertically-transmitted pathogens known to interact with DC-SIGNR such as
HIV-2, hepatitis C and dengue viruses and warrant further investigation.
Associations between child DC-SIGNR exon 4 repeated region genotypes and
mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA;
not applicable; OR, odds ratio a P-value as determined by the Chi-square
test. b Comparison between genotype and all others. Found at:
doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR
transcripts repertoire in placenta. Major RT-PCR products from RNA extract
from 3 homozygous H1 and 3 homozygous WT placenta samples were purified,
cloned and sequenced. Sequenced were analysed according to NCBI reference
sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT;
wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC)
Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity
in luciferase reporter assay in vitro in transfected HeLa cells. Relative
luciferase expression from pGL2-Basic, parental vector without promoter.
Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A
variant were calculated relatively to this value. Data are presented in
mean values6S.E.M of three independent experiments performed in
triplicate. One-way ANOVA test followed by the Dunnett test for multiple
comparison was used to compare the relative luciferase expression of the
p-557C and p-323A variant reporters against the wild-type (WT) construct
(not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected
with LTR-Luc as a positive control in these experiments.
- text: >-
Approximately how many people died during the 1918-1919 influenza
pandemic?
context: >-
It is Unlikely That Influenza Viruses Will Cause a Pandemic Again Like
What Happened in 1918 and 1919
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019839/ Song, Liting
2014-05-07 DOI:10.3389/fpubh.2014.00039 License:cc-by Abstract: nan Text:
Influenza and influenza viruses are wellknown popular topics to medical
professionals and the general public. Influenza viruses had caused a
pandemic globally during 1918 and 1919, and that influenza pandemic had
taken away more than 20 million people's lives in the world. However, in
my opinion, it is unlikely that influenza viruses will again cause a
pandemic on a level (both of the morbidity rate and the mortality rate)
comparable to what happened in 1918 and 1919. Influenza viruses very
easily reassort, recombine, and point mutate in nature due to their
segmented RNA genome structures, however, unlike highly pathogenic
(virulent) viruses like rabies virus, Lassa fever virus, smallpox virus,
eastern equine encephalitis virus, Ebola virus, Marburg virus, and human
immunodeficiency virus 1 (HIV-1); most influenza viruses (wild types and
mutants) are moderately pathogenic. The case fatality rates of some highly
virulent viruses and related references are listed in Table 1 . On
November 11, 1918 , the fighting of World War I was stopped, and World War
I was officially ended on June 28, 1919 with the signing of the Versailles
Treaty. It is estimated that around 8.5-10 million soldiers lost their
lives in World War I due to battle. The war also directly caused more than
6 million civilian deaths. Millions of people suffered from hunger and
malnutrition during the war. Malnutrition weakened the human immune system
and made a person more vulnerable to infectious diseases like tuberculosis
and influenza, therefore, hunger and malnutrition were indirectly
responsible for millions of deaths in the world in that period of time.
For example, about 700,000 Germans died from malnutrition-related diseases
in the years of 1914-1918. During the 1918-1919 influenza pandemic,
between 21 and 25 million people died of influenza worldwide. Those people
were killed both directly and indirectly by influenza virus infections.
Many families were too poor to buy food and coal, and to afford health
care expenses when their family members were ill. Influenza virus could
infect all members of a family, and this could result in no one left to
feed the fires, and to prepare food for the whole family, even if they had
firewood, coal, and food left in their homes. Sadly, a large number of
people died of influenza virus infections along with starvation, cold, and
poor living conditions (8) . In recent years, while hunger and
malnutrition are not major and serious problems in some developed
countries anymore, they are still very difficult to overcome in many
developing countries. In these less-developed countries, there were
approximately 925 million people who suffered from hunger; 125 million
children were underweight; and 195 million children were stunted each year
(9) . Nevertheless, in comparison to 1918 and 1919, currently, we have
much better social and economic conditions and public health systems
globally; and generally speaking, the majority of people in the world have
better nutritional and educational statuses; better living and working
conditions; therefore, better general health and immunity. Furthermore, in
1918 and 1919, physicians and nurses almost had nothing in their hands to
help individuals who were infected by influenza viruses. Today, although
we still do not have very effective, powerful, and practical
anti-influenza drugs available, we at least have some improved, useful,
and helpful anti-viral drugs like zanamivir, and effective, convenient
anti-cold medicines like Tylenol or Advil. We do not have a universal
vaccine to prevent all influenza virus infections, but we can make
effective vaccines to a specific influenza virus strain in a short time.
Actually, in the United States of America, the influenza classed mortality
rate declined from 10.2/100,000 in the 1940s to 0.56/100,000 in the 1990s;
and the classed mortality rates of 1957-1958 and 1968-1969 influenza
pandemics were not remarkably different from the non-pandemic seasons (10)
. Because of the above reasons, we can optimistically assume that even the
same strain of influenza virus, which caused pandemic in 1918 and 1919,
would not be able to kill millions of people and cause a pandemic
comparable to the 1918-1919 pandemic again in the future. Additionally, a
significant number of viruses can cause influenza-like syndromes, such as
rhinovirus, parainfluenza virus, adenovirus, coronavirus, respiratory
syncytial virus, Coxsackie B virus, echovirus, and metapneumovirus (11,
12) . Some of the above-mentioned viruses like adenovirus and mutated
coronavirus could cause problems that are comparable to influenza viruses
(13, 14) . The World Health Organization (WHO) mistakenly raised the level
of influenza pandemic alert from phase 5 to the highest phase 6 on June
11, 2009 (15) . However, the truth was that most cases of H1N1 influenza A
virus infections were mild, the symptomatic case fatality rate was only
0.005% in New Zealand (16) ; and in New York City, the case fatality rate
was 0.0094-0.0147% for persons ≥65 years old, and for those of 0-17 years
old, the case fatality rate was 0.0008-0.0012% (17) . Some researchers
argued that it should not have been called an influenza pandemic in the
first place if the clinical severity was considered (15, (18) (19) (20) .
I believe it was unwise that we had paid too much www.frontiersin.org 23)
. Not surprisingly, every year there would be some influenza patients and
a few of them would die from the infections, as it is almost impossible to
eliminate influenza viruses from the natural environment in many years.
The severity of a viral infection is determined by both of the viral
virulence (pathogenicity) and the host immunity. Some researchers'
opinions on H7N9 avian influenza virus were incorrect and/or inadequate.
They mainly focused on influenza viruses and worried about viral
mutations, viral pathogenicity, viral adaptation, and transmission. They
overestimated the negative part of socio-economic factors of the present
east China: overcrowded population in the epidemic region; very busy
national and international transportation and travel; a large number of
live poultry markets . . . but they underestimated the currently changed,
developed, and improved positive part of socio-economic factors in China.
The following factors might be used to explain why that H7N9 influenza A
virus epidemic was limited and controlled in China, and only a few
immunocompromised patients were killed by H7N9 influenza A virus. First,
China has a relatively organized and effective public health system, there
are four levels of (national, provincial, prefectural-level city, and
county) centers for disease control and prevention all over China (24) .
Second, physicians and nurses in China were prepared and knowledgeable of
influenza virus infections. Third, samples from patients with suspected
influenza virus infections were collected and sent to the local and
national centers for disease control and prevention promptly. H7N9
influenza A viruses were isolated and identified very quickly. Thereby,
they were able to diagnose, confirm, and report three cases of H7N9
influenza patients in the early stage of the epidemic (24, 25) . Fourth,
health care and public health workers were protected properly.
Consequently, none of the health professionals was infected by H7N9
influenza A virus in 2013. However, a surgeon died of H7N9 influenza in
Shanghai, China in January of 2014 (26) . Fifth, they detected H7N9
influenza A viruses from the samples of chickens, pigeons, and the
environment of live poultry markets in Shanghai (27) ; and closed the live
poultry markets of the involved epidemic region quickly. Sixth, patients
were isolated and treated timely in hospitals, 74% (1251/1689) of those
close contacts of H7N9 influenza patients were monitored and observed.
Thus, H7N9 influenza A virus could not spread to a bigger population (24)
. Last but not least, we are connected to the Internet now, and it seems
that our planet is much smaller today than the earlier days when we did
not have the Internet, because communication and information exchange have
become so fast, easy, and convenient presently. During that avian
influenza epidemic, some influenza experts in the world shared/exchanged
H7N9 influenza A virus information and provided professional consultations
and suggestions efficiently and rapidly. All these public health routine
practices and measures resulted in that H7N9 influenza epidemic being
controlled and stopped in China (24) . I have to point out that the cases
of diagnosed H7N9 avian influenza A virus infection might only be the tip
of the iceberg. Aside from one laboratory confirmed asymptotic case of
H7N9 influenza A virus infection in Beijing (22), there were probably many
undetected mild or asymptotic cases of influenza A H7N9 infection. The
reason is that most people usually think a common cold is a very common
and normal occurrence, and they don't take flu-like illnesses seriously.
In most situations, they would just stay home and take some medicines.
Only those who have very severe flu-like symptoms would see doctors, and
thereby be detected and diagnosed, accordingly the real case fatality rate
should be much lower than the detected 32.14% (45/140, one case from
Taiwan, and one case from Hong Kong) (22, 23). Nowadays, we travel faster,
and we travel more frequently and globally, and we have more complicated
social activities and lifestyles, thereby increasing the chances of viral
mutation; and we realize that influenza viruses are even easier to
reassort, recombine, and mutate in nature than many other RNA viruses.
However, we are now living in a technologically, economically, and
socially much better and advanced society. I believe influenza virus
infections are controllable and preventable, with the increased population
health and immunity, with the WHO Global Influenza Surveillance and
Response System, and with standard/routine epidemiological practices, and
with new effective anti-viral agents and vaccines in production in the
future. Now, I first predict that influenza viruses will unlikely again
cause a pandemic on a level comparable to what happened in 1918 and 1919.
Hopefully, one day we could consider a strategy to produce a universal
vaccine that can prevent people from infections of all influenza virus
strains, or we could produce some very effective anti-influenza virus
drugs; then influenza would not be a problem anymore. We should learn
lessons from the mistakes we made in the past. It is reasonable and
necessary to be cautious about influenza viruses, but overreactions or
catastrophic reactions should be avoided in the future. My opinion is
anti-traditional; the purpose of this article is to influence public
health policy, and to save some of the limited resources and money for
more important diseases like heart diseases, cancer, diabetes, AIDS,
hepatitises, and tuberculosis (15) . Liting Song: conception of
manuscript, drafting of manuscript, critical revision of manuscript, and
final approval of manuscript. The author would like to recognize the
contributions of the reviewers and editors of this manuscript for their
corrections and editing, and Dr. Emanuel Goldman for correcting errors
related to grammar and syntax of the final manuscript.
Model Card for Model longluu/Medical-QA-gatortrons-COVID-QA
The model is an extractive Question Answering algorithm that can find an answer to a question by finding a segment in a text.
Model Details
Model Description
The base pretrained model is GatorTronS which was trained on billions of words in various clinical texts (https://huggingface.co/UFNLP/gatortronS). Then using the COVID-QA dataset (https://huggingface.co/datasets/covid_qa_deepset), I fine-tuned the model for an extractive Question Answering algorithm that can answer a question by finding it within a text.
Model Sources [optional]
The github code associated with the model can be found here: https://github.com/longluu/Medical-QA-extractive.
Training Details
Training Data
This dataset contains 2,019 question/answer pairs annotated by volunteer biomedical experts on scientific articles regarding COVID-19 and other medical issues. The dataset can be found here: https://github.com/deepset-ai/COVID-QA. The preprocessed data can be found here https://huggingface.co/datasets/covid_qa_deepset.
Training Hyperparameters
The hyperparameters are --per_device_train_batch_size 4
--learning_rate 3e-5
--num_train_epochs 2
--max_seq_length 512
--doc_stride 250
--max_answer_length 200 \
Evaluation
Testing Data, Factors & Metrics
Testing Data
The model was trained and validated on train and validation sets.
Metrics
Here we use 2 metrics for QA tasks exact match and F-1.
Results
{'exact_match': 37.12871287128713, 'f1': 64.90491019877854}
Model Card Contact
Feel free to reach out to me at thelong20.4@gmail.com if you have any question or suggestion.