extfieldsofstudy: Medicine Biology

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PCR-Based Molecular Characterization of Toxocara spp. Using Feces of Stray Cats: A Study from Southwest Iran

Feces of stray cat are potential sources of gastrointestinal parasites and play a crucial role in spreading and transmitting parasite eggs, larvae, and oocysts through contamination of soil, food, or water. In this study, we investigated the prevalence of Toxocara spp. infection in stray cats in Ahvaz city, southwest Iran. Eggs of Toxocara spp. in feces of stray cats were detected by the sucrose flotation method, and identification was conducted by polymerase chain reaction (PCR) and DNA sequencing. Of the 140 fecal samples that were randomly collected from public environments during the months of January to May 2012, 45% were found to harbour Toxocara spp. eggs. The highest prevalence of Toxocara spp. eggs was found in the central area of Ahvaz city (28.6%). T. canis eggs were found in 4 (6.34%) of the 63 positive samples. Stray cats are found in parks, playgrounds, and other public places and may be a potential contamination risk. Identification of Toxocara spp. using molecular methods is sufficiently sensitive to detect low levels of parasites and identify the different Toxocara spp. in feces. The relatively high prevalence of Toxocara spp. infection may continue to increase due to lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, identify, and control this infection as well as stray cats in the region.

In many areas, accurate information about the prevalence of Toxocara spp. either does not exist or is assessed based on epidemiology of T. canis infection in dogs. Nevertheless, differentiation between the eggs of T. canis and T. cati has not often been attempted; therefore, it is probable that T. cati plays a more important role in human toxocariasis than previously suggested [1,2]. In research based upon coprological examination of fecal samples, parasite eggs are often identified as Toxocara spp., rather than classified to the exact species [4]. Although differentiation among Toxocara spp. is clinically and epidemiologically important, identification of species within the genus is complicated [13]. According to Uga et al. (2000) measurement of egg dimensions was not helpful in the differentiation of Toxocara spp. because approximately 90% of the eggs measured were of similar size. Later, it became possible to differentiate eggs of T. canis from T. cati based on their characteristic surface structure identified using scanning electron microscopy [14]. Various studies have demonstrated that the polymerase chain reaction (PCR)-based molecular approach could provide reliable markers for more accurate identification of Toxocara spp. [2,15,16]. PCR with specific primers can identify the eggs of one Toxocara spp. in the feces of a definitive host.
The populations at greatest risk for Toxocara infection include toddlers and small children, since they are most likely to be exposed to contaminated soil or sandpits while playing outdoors [17] [18]. In addition, several occupational groups such as gardeners, farmers, construction workers, and veterinarians are at elevated risk due to their contact with heavily contaminated soil [19].
In Iran, cats are often allowed to pass freely in and out of houses as predators of rats. Stray cats feast on rubbish and leftover food countless times and discharge helminth eggs and protozoan cysts into public environments. Because of the close association of cats with humans in urban areas, stray cats are important sources of a variety of zoonotic parasites, including Toxocara [20]. Since comprehensive studies about Toxocara spp. have received little attention in Iran, the aim of this study was to identify and determine the prevalence of T. cati infection in stray cats found in urban areas of Ahvaz city, southwest Iran.

Study Area, Design, and Population
Ahvaz city, the capital of the Khuzestan Province, is located in southwest Iran (latitude 31u509N and longitude 49u119E). It covers an area of more than 200 km 2 , and has a population of 1,080,955 inhabitants. During the summer, Ahvaz city has an extremely hot and humid climate and the temperature ranges between 48 and 50uC. During the winter, Ahvaz city has a warm climate, with light to moderate rain. A total of 140 fecal samples were collected from open public spaces in five regions of Ahvaz city (north, south, east, west, and central) during the months of January to May 2012. The sampling procedure required no specific permissions for all five regions of Ahvaz city, and the field studies did not involve endangered or protected species. Approximately 50 g of cat feces was collected; the unused portions of samples were hygienically discarded.

Sample Collection and Fecal Analysis
Fecal samples were put in plastic bags and stored at 4uC until processing, which was performed within 24 h. Isolation of the eggs of Toxocara spp. from each sample was performed by the sucrose flotation method as previously described [21]. In brief, fecal samples were suspended in distilled water, centrifuged at 1000 rpm for 5 min, and approximately 30 ml distilled water was added to the sediment. The suspensions were layered with 15 ml of sucrose (Merck, Germany), with specific gravity of 1.40, and centrifuged at 8006g for 5 min. The upper layer of the liquid was separated and centrifuged at 10006g for 5 min. The sediment was then resuspended in 50 ml distilled water and centrifuged at 5000 rpm for 5 min. Finally, the samples were washed twice with distilled water and sediments were examined under light microscopy (106 and 406).

DNA Isolation and PCR
Genomic DNA from the eggs of Toxocara spp. was extracted using the QIAamp DNA Mini Kit (Qiagen AG, Hombrechtikon, Switzerland) according to the manufacturer's instructions. The samples were first subjected to three freeze-thaw cycles, and proteinase K digestion was performed overnight (,16 h) as suggested by Borecka and Gawor [15] Species-specific oligonucleotide primers were selected from internal transcribed spacer 2 (ITS2) gene sequences that were previously described as Tcan1 (59-AGTATGATGGGCGCGCCAAT- 39) and NC2 (59-TAGTTTCTTTTCCTCCGCT-39) for T. canis and Tcat1 (59-GGAGAAGTAAACTC-39) and NC2 for T. cati [22].
PCR amplification was performed using 50 mL of reaction mixture containing 250 mM of each deoxynucleotide, 100 pmol of each primer, 50 mM KCl, 10 mM Tris-HCl (pH 9), 3 mM MgCl 2 , 10% dimethyl sulfoxide (DMSO, Sigma), 2 U of Taq DNA polymerase (Fermentas), and 10-15 ng of template DNA. The PCR reaction was conducted using a thermal cycler (MyCycler; Bio-Rad, Hercules, CA) and the amplification cycle consisted of an initial cycle at 94uC for 30 s; followed by 35 cycles of denaturation at 94uC for 60 s, annealing at 58uC for 30 s, and extension at 72uC for 30 s; and a final extension cycle at 72uC for 10 min. Finally, 20 mL of PCR products were run on a 1.8% agarose gel containing ethidium bromide in 16Tris-borate EDTA buffer along with 1444-80 bp DNA ladder (Fermentas).

Sequencing Method
In the present study, PCR amplification of the ITS2 region of nuclear ribosomal DNA (rDNA) was used to identify the eggs of Toxocara spp. isolated from stray cats. The results were confirmed by DNA sequencing. DNA derived from 20 infected feces was sequenced by MWG (Germany) using the primers employed during PCR. The resulting data were analyzed using Chromas software (http://technelysium.com.au/).

Evaluation of the ITS2 Motif Specificity using BLAST
The Toxocara ITS sequences were used to query the nonredundant GenBank database of the National Center for Biotechnology Information (nr at NCBI) using BLAST. The BLAST output was then analyzed to find only the exact or perfect pairwise matches showing high percent identity scores and low expect (E) values for each species.

Phylogenetic and Data Analysis
DNA sequences were initially aligned with ClustalW for multiple sequence alignment, using the default gap and extension penalties from this program [23]. Next, the ITS sequences were entered in PAUP* [24] to generate phylograms for each dataset using the methods of Dettman et al. [25,26], i.e., maximum parsimony, NNI branch swapping, and 500 random sequence additions. Three or four locus-combined datasets were made and subjected to weighted parsimony with the weight for each locus calculated as the number of parsimony informative characters for the least variable locus divided by the parsimony informative characters of that locus. Bootstrapping was performed using

Statistical Analysis
Statistical analysis was performed by SPSS 16.0 software. Data are expressed as number and percentage. Difference between  Table 3. Sequence analysis of 20 randomly selected eggs of Toxocara spp. in the five regions of Ahvaz city.

Results
In this study, all collected feces were processed by the sucrose flotation method, which showed that 63 (45%) of 140 samples were contaminated with the eggs of Toxocara spp. Determining the prevalence of Toxocara spp. in cats from each of the five regions of the city (north, south, east, west and central) revealed that the highest prevalence was in the central region (28.6%, 95% CI: 20-38.6) and the lowest (14.3%, 95% CI: 8.3-23) was in both the west and south (P = 0.026, Table 1 and 2).
All Toxocara-positive samples were examined by PCR using Tcan/NC2 and Tcat/NC2 primers. From 63 positive samples, 96.8% showed 370-bp fragments from T. cati and only four showed 380-bp fragments from T. canis. However, no mixed infections were observed, and no amplification was observed in the negative controls (Figure 1). Sequence analysis was performed using 16 randomly selected Tcan/NC2 PCR products and four randomly selected Tcat/NC2 PCR products. All sequences obtained were submitted to NCBI GenBank; their reference numbers are given in Table 3. GenBank was searched for similar published sequences using BLAST, and significant homology was detected with other Toxocara spp. sequences. The sequences of all Toxocara spp. isolates were homologous with a small number of differences corresponding to punctual base substitutions (Table 3).
Multiple sequence alignment revealed conserved nucleotides among the obtained ITS2 sequences (Figure 2). Phylogenetic trees were obtained by comparing our ITS sequences with those of other Toxocara spp. Phylogenetic analyses using various distance methods and character methods like maximum parsimony showed that the topology is similar among the trees obtained with significant bootstrap support for the clades. The values of 75% and above in the bootstrap test of phylogenetic accuracy indicate reliable grouping among different members of Toxocara spp. (Figure 3).

Discussion
The present study confirms that stray cats in all areas of the city were infected and that the prevalence of T. cati was higher in most localities in Iran than elsewhere in the world. Previous studies have shown various prevalences in Toxocara spp. infection in cats in different parts of Iran. The high prevalence of T. cati infection found in the present study is similar to the results of a survey from northern Iran (44%) [29]. Arbabi and Hooshyar in central Iran and Sadjjadi et al. in southern Iran reported 13.3% and 52.8%, respectively [20,30]. One study in Turkey showed that the prevalence of T. cati was 62.5% [31] whereas other studies in Estonia and Ireland reported a prevalence of 48.2% and 42.8% in adult cats, respectively [32,33]. It seems that the higher prevalence of T. cati in our study is probably because of the climatic conditions, which are suitable for the spread and survival of helminth eggs. Interestingly, our results revealed that the highest Previous studies conducted morphological tests to differentiate various Toxocara spp. via egg size, but it seems that egg size is the least specific criteria used to differentiate between T. cati and T. canis [14]. PCR-based approaches using the ITS1 and ITS2 segments of rDNA were employed in several studies to differentiate among the eggs of Toxocara spp. from adult helminths, which are closely related and/or morphologically similar [2,15,16]. Recently, Durant et al. used a duplex real-time PCR (2qPCR) method targeting the ITS2 sequences from 53 adult worms to identify the species of the eggs of Toxocara spp. in fecal and soil samples. They claimed that the newly developed 2qPCR assay could be considered a useful tool for the detection of the eggs of T. canis and/or T. cati in fecal samples as well as in soil samples [34].
In this study, PCR with ITS2 primers was used for the identification of the eggs of Toxocara spp. isolated form stray cats; the results were confirmed by sequencing. The results of the present study indicate that only four (6.34%) of 63 cat feces samples were contaminated with T. canis; DNA sequencing confirms these findings. BLAST analysis at NCBI revealed remarkable homology between our DNA sequence data and the T. canis sequences previously submitted to GenBank with accession numbers Y09489 and AB110034 [35,36]. The phylogenetic studies presented here confirmed that the variation between the sequences obtained herein resulted because they belonged to two different Toxocara spp. The natural and experimental establishment of T. canis adult infections in the intestines of cats was reported previously [37,38]. Fahrion et al. showed that dogs shed considerable amounts of T. cati eggs, but no T. canis eggs have been identified in the feces of cats, because coprophagy is common in dogs, and is usually not observed in cats. They mentioned that true host switching of Toxocara spp. might be an exceptional event, but their attempts to reproduce patent infections by experimental inoculations of cats with T. canis eggs failed [39]. Our results revealed that the highest contamination rate was in the central region of the city, and also three out of four T. canis eggs isolated from feces of cats were found in this region. The growing numbers of stray dogs that tend to discharge their feces into these environments could be an important factor in T. canis infections of cats, and a possible explanation of this finding.
In conclusion, Toxocara spp. can be considered neglected zoonoses, which often occur in areas with poor hygiene and education levels. This study is the first report on the prevalence and identification of the eggs of Toxocara spp. isolated from stray cats using molecular methods in the southwestern region of Iran. Differentiation of Toxocara spp. using molecular methods such as PCR are sufficiently sensitive to detect low levels of parasites and can identify the eggs of Toxocara spp. in feces. There are many stray cats in various parts of the city, which leads to the presence of Toxocara infection that may be consider a potential environmental contamination risk for the parks, playgrounds, and other public places.
The relatively high prevalence of Toxocara spp. infection may continue to increase due to a lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, differentiate and control such infection, as well as stray cats in the region.


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Combined Inhibition of p97 and the Proteasome Causes Lethal Disruption of the Secretory Apparatus in Multiple Myeloma Cells

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

Introduction
Multiple myeloma (MM) is a tumour of transformed plasma cells, terminally differentiated B cells that are highly specialised to synthesise and secrete large amounts of immunoglobulins (Ig) [1]. The production of large quantities of secreted protein places a substantial strain on the protein-folding machinery and can result in the accumulation of misfolded/unfolded proteins, triggering the unfolded protein response (UPR). The UPR is a fundamental cellular process that acts to maintain secretory homeostasis by attenuating overall protein translation, expanding the endoplasmic reticulum (ER), and increasing ERassociated protein degradation (ERAD) of misfolded proteins in the cytosol by the proteasome [2][3][4]. Failure of the UPR results in overwhelming ER stress and apoptosis [4].
Plasma cells and MM cells (MMCs) have a well-developed secretory apparatus to accommodate Ig secretion but are nevertheless highly sensitive to agents that interfere with intracellular protein metabolism [5][6][7]. This forms the basis for selective targeting of MMCs by the proteasome inhibitor bortezomib, which is widely used for the treatment of MM [8]. The combined findings of several studies suggest that the preferential toxicity of proteasome inhibitors to MMCs can be attributed, to a large extent, to ERAD impairment and overwhelming ER stress [9][10][11][12]. Primary or acquired resistance of MMCs to bortezomib is an important clinical problem and has been linked to incomplete ERAD disruption and the activation of alternative protein degradation pathways, such as autophagy and aggresome formation [5,[13][14][15]. This makes it important to develop therapeutic approaches that give more effective disruption of secretory homeostasis in MMCs [1].
Prior to proteasomal degradation, misfolded proteins are exported from the ER lumen to the cytosolic side of the ER, where they are earmarked for degradation by polyubiquitination [16]. This acts as a signal for binding of the cytosolic ATPase, p97 (also known as valosin-containing protein, VCP; or Cdc48), which delivers the ubiquitinated protein to the proteasome by translating ATP hydrolysis into mechanical force [17][18][19][20][21]. Hence, p97 has a central role in ERAD and this role is closely linked to the proteasome. However, like the proteasome, p97 also mediates the degradation of non-secretory proteins that regulate a variety of cellular functions [22][23][24][25]. Inhibitors of p97, were recently reported to kill cancer cells by mechanisms that are related to disruption of the secretory apparatus [26][27][28][29][30]. Eeyarestatin 1 (Eer1), which binds to both p97 and the ER membrane, preferentially induces cancer cell death by ERAD disruption and effects that are similar to those induced by bortezomib, including ER stress-induced transcriptional up-regulation of NOXA and induction of CHOP [26,28]. N 2 ,N 4dibenzylquinazoline-2,4-diamine (DBeQ), a reversible inhibitor of the p97 ATPase activity, was found to be a potent activator of caspases in cancer cells and to inhibit both ERAD and autophagosome maturation [26,27]. Furthermore, there is evidence that sorafenib, a multikinase inhibitor of the Raf/MEK/ERK pathway and of tyrosine kinase receptors, induces cancer cell death by preventing p97 tyrosine phosphorylation and consecutive disruption of secretory homeostasis [30,31]. Thus, p97 inhibition has the potential to overcome some limitations of proteasome inhibition as an anticancer strategy, particularly in secretory cancers such as MM [32,33].
The essential roles of p97 and the proteasome in regulating protein degradation, which are closely linked in ERAD but also show a considerable degree of non-redundancy, open up the possibility that simultaneous inhibition of p97 and the proteasome could be highly efficient in disrupting secretory homeostasis in MMCs. Here we demonstrate that inhibitors of either p97 or the proteasome have quite different effects on the secretory apparatus in MMCs, and that these effects vary substantially between different MMC lines, and between MMCs and non-secretory or non-transformed cells. Moreover, we show that dual inhibition of p97 and the proteasome has dramatic effects on ER morphology and protein turnover that are selective for MMCs and are very different from the effects of single inhibition with either drug. Our results provide a conceptual preclinical framework for simultaneous targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

Ethics statement
Bone marrow myeloma cells, bone marrow mesenchymal stroma cells, and skin fibroblasts were collected at the Imperial College London Centre for Haematology after approval by the National Research Ethics Service Committee East of England -Cambridge Central and obtaining signed informed consent in accordance with the Declaration of Helsinki.

Analysis of apoptosis
Apoptosis was assessed by staining with Annexin V-FITC and propidium iodide (PI; BD Pharmingen) as previously described [7]. Cells negative for Annexin V-FITC and PI were classified as live.

Qualitative and quantitative ER staining
Cells were incubated with 100nM BODIPY FL brefeldin A (BFA-BODIPY, Invitrogen) at 37°C for 40 minutes in the dark, washed and resuspended in phosphate-buffered saline (PBS), and analysed immediatedy on a BD LSR II flow cytometer. For morphological ER analysis, cells were incubated in medium with 1µM ER Tracker Blue-White DPX (Molecular Probes) at 37°C for 15 minutes, pelleted, and resuspended in 50µl medium. The cell suspension was applied to an imaging chamber [34], and imaged with a Zeiss LSM510 or Leica SP5 confocal microscope.

Electron microscopy
1x10 7 cells were fixed, washed, post-fixed and embedded as previously described [35]. Ultrathin sections were cut on a Leica Ultracut R microtome, stained with Reynolds lead citrate, and observed in a Jeol JEM 1200EX II transmission electron microscope.
Inhibition of p97 and the Proteasome in Myeloma PLOS ONE | www.plosone.org

Dual inhibition of p97 and the proteasome is highly toxic to myeloma cells
We first determined the apoptotic sensitivity of a panel of human MMC lines to bortezomib and the p97 inhibitor Eer1 ( Figure 1A). The result of this analysis shows that OPM-2 cells were the most resistant to both bortezomib and Eer1, while KMS-11 cells were the most sensitive to both inhibitors. Next, we investigated whether simultaneous inhibition of p97 and of the proteasome increases MMC death compared to separate inhibition. We treated the panel of MMC lines with concentrations of bortezomib (5nM) and Eer1 (5µM) that have been shown to be pharmacologically effective in cancer cells, including malignant B cells, and fibroblasts [11,29,[36][37][38]. . In addition, we found that a 2-hour treatment with 5nM of bortezomib blocked chymotrypsin-like proteasome activity by approximately 80% in OPM-2 cells, and that an 8-hour treatment of KMS-11 cells with Eer1, but not with bortezomib, significantly up-regulated HMOX1 levels, as demonstrated previously (data not shown) [11]. At these concentrations, and at the time-points studied, bortezomib and Eer1 had little impact on MMC line survival on their own ( Figure 1B). However, dual p97/proteasome inhibition induced apoptosis very effectively in all MMC lines investigated ( Figure 1B). Importantly, OPM-2 cells, which are resistant to bortezomib and characterised by abnormalities that confer poor outcome in patients, including t(4;14) and constitutive Akt activation, were also killed effectively by dual p97/proteasome inhibition [39,40]. To extend these findings, we treated the bortezomib-adapted MMC line AMO-1a, which we cultured in the presence of primary human bone marrow stroma cells to confer further prosurvival signals, with bortezomib and Eer1. We also tested the effect of separate and dual p97/proteasome inhibition on patient-derived MMCs grown in the presence of IL-6, and on healthy donor peripheral blood mononuclear cells (PBMNCs). While dual p97/proteasome inhibition effectively killed both the AMO-1a cell line and the primary MMCs, it was substantially less toxic against PBMNCs ( Figure 1C). These findings demonstrate that combined p97/proteasome inhibition is highly toxic to MMCs, including MMC lines with primary and secondary resistance to bortezomib.
Increased intracellular levels of ubiquitinated proteins are considered to indicate impaired protein degradation by the proteasomal machinery [5,15,41]. To test how separate and combined inhibition of p97 and the proteasome affects protein degradation, we quantified the levels of ubiquitinated proteins in whole cell extracts from four MMC lines ( Figure 1D). Dual inhibition of p97 and the proteasome led to increased levels of ubiquitinated proteins in all MMC lines tested, indicating effective disruption of gross protein degradation. However, an increased effect of dual over separate inhibition on levels of ubiquitinated proteins was only observed in two lines (OPM-2 and U-266). It is also worth noting that bortezomib induced an increase in ubiquitinated proteins only in OPM-2 cells, while inhibition of p97 with Eer1 led to increased levels of ubiquitinated proteins in the RPMI8226, U-266, and KMS-11 cells. Of particular importance is the finding that increased levels of ubiquitinated proteins after separate inhibition did not generally correlate with cell death ( Figure 1B). This observation strongly suggests that additional mechanisms are involved in the induction of cell death by inhibitors of ERAD pathways.

Combined inhibition of p97 and the proteasome drastically disrupts ER configuration in MMCs
Inhibitors of the proteasome and of p97 cause ER stress in MM and other tumour cells, and ER stress is known to induce changes in ER structure [9][10][11]26,[42][43][44]. To investigate the effects of separate and combined inhibition of p97 and the proteasome on ER structure in MMCs, we visualised the ER of MMC lines after staining with the fluorescent ER dye ER Tracker Blue-White DPX (Figure 2A and Figure S1). In most of the control cells, the ER was visible as elongated tubular structures with some small globular regions. Bortezomib did not cause any substantial changes in ER structure, although the ER appeared to be slightly more densely packed in some cells. However, treatment with the p97 inhibitor Eer1 had a clear effect on the ER. Most of the tubular ER was lost, and small to medium-sized vesicular and globular structures were instead observed. Dual inhibition with bortezomib and Eer1 caused a different and dramatic effect in that large parts of the ER were transformed into medium-sized to large vacuoles. This vacuolisation of the ER was not observed after treatment with high doses of bortezomib, Eer1, or tunicamycin ( Figure S2), indicating that the changes were not caused by overwhelming ER stress generated by an additive effect of the two inhibitors. Furthermore, pre-incubation with the pan-caspase inhibitor zVADfmk did not abrogate ER vacuolisation, indicating that it did not occur as an unspecific consequence of apoptotic signalling ( Figure S2).
To further investigate the changes in ER configuration we used transmission electron microscopy (TEM) of OPM-2 cells ( Figure 2B and Figure S3), which showed results that were consistent with our findings obtained by staining with ER Tracker Blue-White DPX. The TEM analysis of untreated cells showed the expected elongated and narrow tubular ER structures with ribosomes attached to the cytosolic ER surface. Bortezomib did not have a uniformly discernible impact on ER structure, although the ER appeared slightly distended and less elongated in some cells. Inhibition of p97 with Eer1 did have an impact on ER morphology at the level of ultrastructure, giving rise to relatively short and moderately dilated tubular structures in most cells. Dual inhibition had a much more dramatic effect with the ER appearing predominantly as very large vacuoles in the majority of cells. Dual p97/proteasome inhibition also resulted in an empty appearance of the ER lumen, and ribosomes were largely absent from the cytosolic surface of the ER. The observation that the perinuclear space was also substantially dilated, had the same empty appearance and showed loss of ribosomes from its surface as the vacuoles, provides strong support for the notion that the vacuoles are indeed derived from the ER. The finding that most cells presenting with vacuolised ER did not show any apoptotic chromatin condensation further indicates that the changes in ER morphology are unlikely to be unspecific consequences of apoptotic signalling. We next investigated how dual p97/proteasome inhibition affects cellular ER content by staining OPM-2 cells with the fluorescent brefeldin A derivative, BFA-BODIPY ( Figure 2C), and immunoblotting for key ER luminal chaperones ( Figure 2D) [45]. While bortezomib treatment increased the amount of ER stained by BFA-BODIPY by approximately 40% in OPM-2 cells, Eer1 on its own had no discernible impact. Dual p97/ proteasome inhibition not only blocked the increase in BFA-BODIPY staining that was induced by bortezomib, but reduced it compared to control cells. Consistent with these findings, immunoblotting showed an increase in the levels of the ER lumenal chaperones, p58 IPK (DNAJC3) and GRP94, after  proteasome inhibition, and a decrease after dual ERAD inhibition, while Eer1 had no detectable effect on the levels of either protein. Together, these observations indicate that bortezomib increases ER content in MMCs, whereas inhibition of p97 with Eer1 causes moderate alterations to ER structure. In contrast, dual p97/proteasome inhibition drastically alters the configuration of the ER.

Dual inhibition of p97 and the proteasome disrupts key pathways that control survival, death, and protein synthesis and induces predominantly caspasedependent cell death in MMCs
Next, we investigated the impact of single (bortezomib or Eer1 alone) and dual p97/proteasome inhibition on key signalling pathways in human MMC lines, and compared the effects with those induced in untransformed primary human fibroblasts ( Figure 3A). Consistent with the induction of MMC death ( Figure 1B), dual inhibition resulted in activation of the critical apoptotic executioner, caspase-3, as indicated by proteolytic processing of full-length caspase-3, in all four MMC lines investigated. This effect was observed to a much lesser extent after treatment with bortezomib or Eer1 alone. Importantly, there was no evidence that dual p97/proteasome inhibition activates caspase-3 in fibroblasts.
Activation of the Akt pathway, as indicated by phosphorylation of Akt, is an important survival signal that has been shown to occur in a proportion of MMCs [40]. In OPM-2 cells, which have constitutively active Akt (indicated by phosphorylation) [46], treatment with bortezomib or Eer1 caused a mild decrease in Akt phosphorylation, while dual inhibition abolished Akt phosphorylation. While phospho-Akt was not detectable in the other MMC cell lines tested here, dual inhibition also caused a substantial decrease in overall Akt protein levels, whereas treatment with bortezomib or Eer1 alone had no or a moderate effect. Importantly, single or dual inhibition had no effect on Akt levels in fibroblasts, providing further evidence of the selective effects of dual p97/ proteasome inhibition on MMCs.
The observed changes in ER configuration ( Figure 2) and Akt signalling that we observed in MMCs prompted us to investigate the impact of bortezomib and Eer1 on selected protein translation control pathways in these cells. Phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α) on Ser51 results in attenuation of protein translation and indicates activation of a PERKdependent UPR signalling that results in attenuated protein translation [2,47]. We observed a moderate increase in eIF2α phosphorylation in response to bortezomib in three of the four MMC lines (OPM-2, RPMI-8226, KMS-11) investigated and following treatment with Eer1 in two lines (RPMI-8226, U-266). In contrast, dual inhibition invariably caused proteolytic processing and/or loss of phosphorylated and total eIF2α, which has been linked to terminal translational shut-down rather than adaptive attenuation of translation [48][49][50]. In fibroblasts, Eer1 alone and particularly dual inhibition with Eer1 and bortezomib induced phosphorylation of eIF2α on Ser51, indicating activation of adaptive PERK signalling. Phosphorylation of ribosomal protein S6 (S6P) correlates with an increase in translation of mRNA transcripts that contain an oligopyrimidine tract in their 5' untranslated regions [51]. Dual p97/proteasome inhibition substantially decreased levels of phosphorylated and total S6P in all MMCs lines, whereas treatment with only bortezomib or Eer1 resulted in a reduction in the levels of phosphorylated and total S6P in two of the four MMC lines in each case (U-266 and KMS-11 for bortezomib; OPM-2 and KMS-11 for Eer1). Once again, single or dual inhibition had no detectable effect on S6P in fibroblasts. These findings provide further evidence that considerable differences can exist between MMCs in the way they respond to inhibition of the proteasome or p97. They also indicate that separate inhibition of the proteasome or p97 often results in mild effects on protein translation control pathways in MMCs. Some of the changes, such as increases in eIF2α phosphorylation, are consistent with adaptive UPR signalling. In contrast, dual inhibition invariably causes substantial changes that are consistent with a terminal translational shut-down in these pathways in MMCs. This interpretation fits with the loss of ribosomes from the cytosolic surface of the ER, vacuolisation of the ER, and the reduction in Akt levels described above. In fibroblasts the effect of dual p97/protesome inhibition on these pathways was very moderate and indicative of an adaptive response.
In the light of the activation of caspase-3 that we observe and the previously described role of JNK in bortezomib-induced cell death [52], we next analysed JNK-and caspasedependence of MMC death induced by dual ERAD inhibition. MMC lines were pre-incubated with the JNK inhibitor, SP600125, and the pan-caspase inhibitor, zVADfmk and then treated with Eer1 and bortezomib. We observed that apoptosis of MMC lines induced by dual p97/proteasome inhibition ( Figure 3B) was JNK-independent (OPM-2 and KNS-11 cells) or partly JNK dependent (RPMI 8226 and U-266 cells), and caspase-dependent (RPMI 8226) or partly caspase-dependent (OPM-2, U-266, KMS-11).

Structurally different inhibitors of p97 have comparable effects in MMC lines and primary MMCs
Depletion of p97 by conditional gene knockout or RNAi knockdown has limited utility for predicting the effects of pharmacological p97 inhibition due to slow and incomplete impact on p97 function [27]. To investigate whether the effects of Eer1 described in the preceding sections are due to specific inhibition of p97, and to exclude potential off-target effects related to its chemical structure, we tested another recently identified p97 inhibitor, DBeQ [26,29]. Staining of parental and bortezomib-adapted AMO-1 MMCs with ER Tracker Blue-White DPX demonstrated that the effect of DBeQ on ER configuration was comparable to that of Eer1, with most of the ER presenting as small vesicular or globular structures ( Figure 4A). Furthermore, combined treatment with both bortezomib and DBeQ resulted in vacuolar transformation of the ER that was very similar to the effect of combined Eer1 and bortezomib treatment ( Figure 4A). Similar to Eer1, DBeQ substantially increased bortezomib-induced death of RPMI8226 cells and primary MMCs co-cultured with IL-6 ( Figure 4B and 4C). The observation that the majority of the dying primary MMCs (A) Immunoblotting for the indicated survival-and apoptosis-related proteins was carried out on whole cell extracts from MMC lines and primary human fibroblasts prepared after treatment with either BTZ, Eer1, or both inhibitors, for 24h (14h in KMS-11 cells due to their higher apoptotic sensitivity to inhibitors). (B) Cell death induced by dual p97/proteasome inhibition is predominantly caspase-dependent and JNK-independent. The proportion of live cells was determined after treatment with BTZ (5nM) and Eer1 (5µM) with or without the pan-caspase inhibitor zVADfmk (50µM) or the JNK inhibitor SP600125 (10µM) for 24h (36h for OPM-2 cells; all values are the mean and SEM of 3 experiments; *p<.05, **p<.001, two-sided student's t-test). stained positive for Annexin V-FITC but excluded PI supports the notion that MMC death induced by dual ERAD inhibition is apoptotic. Thus, structurally different inhibitors of p97 have comparable effects on ER configuration and viability of MMCs, alone or in combination with bortezomib.

Discussion
The key finding of this paper is that all of the MMCs that we have tested show dramatic changes to the ER in response to dual inhibition of p97 and the proteasome, and that this is accompanied by disruption of intracellular protein metabolism and cell death. Importantly, the toxic effects of dual p97 and proteasome inhibition are also observed for cell lines that are insensitive to bortezomib. The effects of dual inhibition are quite different from the effects of single inhibition of p97 or the proteasome, adding significant information on the nonredundant roles of p97 and the proteasome in maintaining the integrity of the secretory apparatus.
Dual p97/proteasome inhibition causes changes in key protein translation pathways in MMCs that are consistent with a terminal translational shut-down. This interpretation is based on the observation of proteolytic processing of eIF2α, and loss of both phosphorylated and total S6P and Akt [48][49][50]. It is also supported by the loss of ribosomes from the cytosolic surface of the ER observed by TEM. In contrast, inhibition of either the proteasome or p97 had a relatively modest impact on these protein translation control pathways and did not result in loss of ribosomes from the cytosolic surface of the ER. In particular, bortezomib or p97 inhibition alone did not cause proteolytic processing of eIF2α but moderately increased its phosphorylation on Ser51 in some MMC lines, indicating activation of adaptive PERK-dependent UPR signalling.
ER staining and electron microscopy showed that proteasome inhibition had a very mild impact on the appearance of the ER in MMCs, while p97 inhibitors caused clear alterations to ER structure. The effect of p97 inhibition on ER structure could reflect a hitherto unrecognised role for p97 in the metabolism of protein components of the ER membrane or of proteins that play a role in maintaining ER integrity. Such functions are suggested by the requirement of a p97/p37 complex for Golgi and ER maintenance in interphase [23]. The dramatic transformation of the ER into very large vesicles that we observed as a consequence of dual p97/proteasome inhibition was unexpected. It is possible that the disruption of ERAD increases drastically the amount of misfolded proteins inside the ER, causing an increase in osmolarity in the ER lumen that leads to an influx of water from the cytosol. Another explanation might be that structural ER proteins are affected by the disruption of protein translation. One mechanistic question that arises from our findings is whether the translational shutdown and the disruption of ER structure that result from dual p97/proteasome inhibition are responsible for triggering cell death or occur downstream from apoptotic signalling. The previously described cleavage of eIF2α by caspase-3 might seem to favour the latter scenario [48]. However, the pancaspase inhibitor zVADfmk did not block the formation of large ER vacuoles after dual p97/proteasome inhibition. Furthermore, at the time-point when cleavage of eIF2α was evident in OPM-2 cells, the cells that presented with large ER vacuoles and had lost ribosomes from the ER surface did not yet show signs of apoptotic chromatin condensation. These findings suggest that the structural changes to the ER and the disruption of protein translation are not merely a consequence of apoptotic signalling. We therefore favour a model in which alterations of ER structure and protein translation play an important early role in triggering cell death, but are also aggravated further by the initiation of apoptotic signalling, culminating in irreparable disruption of protein translation and ER homeostasis.
Another important finding of this study is that the deleterious effects of dual inhibition of p97 and the proteasome occur preferentially in MMCs. In fibroblasts, dual p97/proteasome inhibition failed to induce activation of caspase-3 and did not affect levels of phosphorylated or overall Akt and S6P. Moreover, dual inhibition did not induce proteolytic processing of eIF2α in fibroblasts but increased its phosphorylation, indicating PERK-dependent adaptive UPR signalling. Furthermore, dual p97/proteasome inhibition was only moderately toxic to primary PBMNCs. Taken together, these findings indicate that dual p97/proteasome inhibition shows a considerable degree of selectivity towards MMCs.
It is also noteworthy that the toxic effects of dual p97/ proteasome inhibition on MMCs were achieved at bortezomib concentrations similar to steady-state plasma concentrations maintained in patients after the early peak [53]. This is relevant in the light of evidence that peripheral neuropathy, a major side effect of bortezomib treatment in patients, may be caused by off-target effects at high peak plasma concentrations [53,54]. Additional in vivo analysis will be required to elucidate potential toxic and anti-myeloma effects of pharmacological p97 inhibition, alone and in combination with bortezomib. Systemic in vivo studies are currently not possible due to the limited solubility of Eer1 and DBeQ. However, the findings presented here form a conceptual preclinical framework for combined targeting of p97 and the proteasome as a potential novel therapeutic approach in MM and provide a strong incentive for developing p97 inhibitors that can be administered systemically [27].
In contrast to the clinical effectiveness of proteasome inhibitors in MM, they have shown disappointing results in most other haematological malignancies and solid cancers. The potent activity of p97 inhibitors in various cancer cells suggests that p97 might be a more broadly useful target for antineoplastic therapy than the proteasome [11,26,55]. One possible reason for this is that inhibition of p97 disrupts ERAD upstream of the proteasome, making its effects potentially independent from variations in proteasomal capacity, which can render cells insensitive to proteasome inhibition [5]. Inhibition of p97 may also be more effective than proteasome inhibition in disrupting protein homeostasis because it interferes with both HDAC6-mediated aggresome formation and autophagosome maturation, which are potential alternatives to proteasomal degradation of waste proteins [13][14][15]26,33,56]. In addition, p97 inhibition directly regulates intracellular levels of p53 and NFκB, which has been shown to mediate the toxic effects of Eer1 active against non-small cell lung carcinoma (NSCLC), in which p97 is overexpressed [55].
The exceptional secretory load of MMCs, combined with their high dependence on proteasomal and non-proteasomal protein Representative microscopic images of parental and BTZ-adapted AMO-1 cells stained with ER Tracker Blue-White DPX after treatment for 18h with Eer1 (5µM), DBeQ (5µM) or Eer1/DBeQ plus Bortezomib (5nM). The images show small to medium-sized globular/vesicular ER after p97 inhibition and ER vacuolisation after dual ERAD inhibition. (B) The proportion of live RPMI8226 cells was determined after treatment with bortezomib (10nM), DBeQ (10µM), or both, for 4h followed by drug wash-out and a further 20h incubation in culture medium (mean and SEM of 3 experiments; *p<.05, **p<.001, two-sided student's t-test). (C) Primary MMCs were isolated from a bone marrow aspirate, incubated in culture medium containing IL-6 (10ng/ml) for 6h, followed by addition of bortezomib (5nM), DBeQ (5µM), or both for 24h and analysis of apoptosis levels. Live cells (Annexin V-FITC and PI-negative) are shown in green in the left lower quadrant, early apoptotic (Annexin V-FITC positive, right lower quadrant) and dead (Annexin V-FITC and PI-positive, right upper quadrant) cells are shown in orange and red, respectively. doi: 10.1371/journal.pone.0074415.g004 degradation pathways, provides a plausible explanation for the dramatic effects of combined p97 and proteasome inhibition on the secretory apparatus that we report here. Considering that p97 has key roles in a diverse range of cellular processes, it will be important to investigate the combination of p97 inhibitors with other classes of anti-neoplastic drugs in different types of cancers [22,30,32,57]. Figure S1. Effect of single and dual p97/proteasome inhibition on ER structure in MMC lines. OPM-2, U-266, and KMS-11 cells were stained with ER Tracker Blue-White DPX following treatment for 24h (14h for KMS-11) with BTZ (5nM), Eer1 (5µM), or both. Representative confocal microscopic images are shown. (TIF) Figure S2. ER vacuolisation caused by dual ERAD inhibition is not caused by overwhelming ER stress or apoptotic signalling. OPM-2 cells were stained with ER Tracker Blue-White DPX following treatment for 24h with high doses of BTZ, Eer1, and tunicamycin, or BTZ+Eer1 after pretreatment with the pan-caspase inhibitor zVADfmk. (TIF) Figure S3.


text:
Vaginal concentrations of lactic acid potently inactivate HIV

Objectives When Lactobacillus spp. dominate the vaginal microbiota of women of reproductive age they acidify the vagina to pH <4.0 by producing ∼1% lactic acid in a nearly racemic mixture of d- and l-isomers. We determined the HIV virucidal activity of racemic lactic acid, and its d- and l-isomers, compared with acetic acid and acidity alone (by the addition of HCl). Methods HIV-1 and HIV-2 were transiently treated with acids in the absence or presence of human genital secretions at 37°C for different time intervals, then immediately neutralized and residual infectivity determined in the TZM-bl reporter cell line. Results l-lactic acid at 0.3% (w/w) was 17-fold more potent than d-lactic acid in inactivating HIVBa-L. Complete inactivation of different HIV-1 subtypes and HIV-2 was achieved with ≥0.4% (w/w) l-lactic acid. At a typical vaginal pH of 3.8, l-lactic acid at 1% (w/w) more potently and rapidly inactivated HIVBa-L and HIV-1 transmitter/founder strains compared with 1% (w/w) acetic acid and with acidity alone, all adjusted to pH 3.8. A final concentration of 1% (w/w) l-lactic acid maximally inactivated HIVBa-L in the presence of cervicovaginal secretions and seminal plasma. The anti-HIV activity of l-lactic acid was pH dependent, being abrogated at neutral pH, indicating that its virucidal activity is mediated by protonated lactic acid and not the lactate anion. Conclusions l-lactic acid at physiological concentrations demonstrates potent HIV virucidal activity distinct from acidity alone and greater than acetic acid, suggesting a protective role in the sexual transmission of HIV.

Introduction
The majority of HIV infections worldwide are transmitted sexually from infected females to uninfected males, and vice versa. The probability of HIV acquisition via the female reproductive tract is lower compared with rectal or parenteral transmission, 1 probably due to antimicrobial defence mechanisms at the endocervix and the vaginal/ectocervix lumenal surface (vagina), including physical barriers such as the squamous epithelium and mucous secretions, innate defence peptides produced by epithelial and immune cells, and agents produced by commensal bacteria, including organic acids and bacteriocins. 2 -6 In addition, factors present in the vagina that decrease the amount of active HIV virions shed by infected women may potentially reduce HIV transmission to men. 7 -10 Studies of the vaginal microbiome of asymptomatic women of reproductive age reveal five distinct bacterial communities, the majority of which are dominated by lactobacilli. 11,12 However, about one in three women of reproductive age in the USA 13 and about one in two in sub-Saharan Africa 14,15 do not have a lactobacillus-dominated vaginal microbiota that acidifies the vagina with lactic acid. Thus, many women worldwide with bacterial vaginosis (BV) and an 'intermediate' vaginal microbiota, as defined by Nugent score, 16 have microbial communities that increase vaginal pH to .4 and are associated with a significantly increased risk of acquiring sexually transmitted infections (STIs), 13,17 including HIV, 18,19 or of transmitting HIV to their male partners. 20 Altogether, the evidence suggests that lactobacilli appear to reduce susceptibility to infections, while BV appears to increase it.
Many pathogens are well recognized as being acid susceptible, but only recently has lactic acid, at physiological concentrations, been shown to be highly potent at killing BV-associated bacteria -all 17 of the most abundant BV-associated species are potently inactivated at pH 4.5 with 55 -111 mM racemic DL-lactic acid. 3,21 -23 As typically measured aerobically in the clinic, lactobacilli acidify the vagina to pH 4.2, 24 but the vagina is hypoxic and is partially acidified by 5% systemic CO 2 compared with 0% CO 2 in air. The most reliable observation of vaginal pH in vivo, based on radio telemetry from intravaginal capsules, revealed that lactobacilli lower vaginal pH to 3.7, 25 a significantly more microbicidal pH. Lactobacillus-dominated microbiota are associated with high levels of lactic acid compared with other metabolites 26 and acidify the vagina to pH ,4.0 by producing, and maintaining, a vaginal concentration of 1% (w/v) lactic acid (D. O'Hanlon and R. Cone, Johns Hopkins University, and T. Moench, ReProtect Inc., unpublished data) with a nearly racemic mixture of D-and L-isomers. 27 Human metabolism produces only the L-isomer, and ,15% of vaginal lactic acid appears to be produced by anaerobic metabolism of the vaginal epithelium. 23,27,28 Lactic acid is a lipid-soluble membrane-permeant carboxylic acid (pK a ¼ 3.9) and exists predominantly as the neutral protonated form under acidic conditions and the charged unprotonated lactate anion under neutral conditions. 23 Glycogen is thought to be the primary energy source metabolized by lactobacilli in producing lactic acid, and during the reproductive years human vaginal epithelial cells provide high concentrations of glycogen to bacteria in the vaginal lumen. 29,30 Intriguingly, women with lactobacillus-dominated vaginas are unique among mammals in having vaginas strongly acidified with lactic acid; other species, including non-human primates, have much less acidic vaginas and low levels of lactobacilli and lactic acid. 31,32 The antimicrobial and HIV virucidal activity of lactobacilli has often been ascribed to the ability of some species to produce hydrogen peroxide (H 2 O 2 ). 33 However, more recent studies indicate that H 2 O 2 is unlikely to act as an intravaginal protective factor: Lactobacilli make only trace amounts of H 2 O 2 in cervicovaginal secretions (CVS) under the hypoxic conditions normally found in the vagina; 34 the maximum concentrations of H 2 O 2 found in vaginal fluids, even when potentiated with myeloperoxidase, failed to inactivate BV organisms, Neisseria gonorrhoeae, or HSV-2. 34 Vaginal secretions diluted 100-fold completely blocked the microbicidal activity of H 2 O 2 , even at 50 000 times its vaginal concentration. 23 Lactobacilli are themselves more susceptible to inactivation by H 2 O 2 than BV organisms, 23 and inhibition of N. gonorrhoeae by lactobacilli under anaerobic growth conditions is mainly due to acidification rather than H 2 O 2 . 23,34,35 Several previous reports have described the acid sensitivity of HIV. 36 -40 These studies used HCl, acetic acid, phosphate/citrate buffers or lactic acid to acidify virus-containing media. However, a direct comparison of the HIV virucidal activity of lactic acid compared with other acids found in the vagina has not been performed. To investigate the potential role of lactic acid in modulating heterosexual transmission of HIV, we determined the relative virucidal activities of DL-, L-and D-lactic acid, and compared the activity of L-lactic against acetic acid and low pH without an organic acid (acidified with HCl). We also examined the anti-HIV activity of L-lactic acid against a panel of HIV strains, including different subtypes and transmitter/ founder strains, 41 evaluated the activity in the presence of genital secretions and determined whether protonated lactic acid or the lactate anion mediates HIV virucidal activity.

Cells
The TZM-bl indicator cell line expressing the CD4, CXCR4 and CCR5 receptors and stably integrated with the Escherichia coli b-galactosidase and firefly luciferase genes under the control of the HIV promoter were obtained through the NIH AIDS Research and Reference Reagent Program. TZM-bl cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS; Sigma-Aldrich), 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine (DMEM-10). 42 293T cells (obtained from Richard Axel, Columbia University) were cultured in DMEM-10 (Invitrogen). Phytohaemagglutinin-stimulated human peripheral blood mononuclear cells (PBMCs) from uninfected donors were prepared as previously described 43 with the following modifications: PBMCs were isolated from blood bank packs supplied by the Australian Red Cross (South Melbourne) and were resuspended at 2×10 6 cells/mL in Roswell Park Memorial Institute medium (1640) supplemented with 10% heatinactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 20 U/mL recombinant human interleukin 2 (IL-2 medium, Roche). PBMCs were stimulated in the presence of 10 mg/mL phytohaemagglutinin (Sigma-Aldrich) and incubated for 3 days at 378C/5% CO 2 in either silicone-coated Teflon pots (Savillex) or 75 cm 2 tissue culture flasks (Falcon) prior to infection with HIV.
instructions (Boehringer Mannheim/R-BioPharm), which is based on conversion of D-lactate or L-lactate by D-lactate dehydrogenase and L-lactate dehydrogenase, respectively, to pyruvate and NADH. The concentrations of lactic acid and acetic acid are presented in the text as w/w unless otherwise specified.

Collection and processing of CVS and seminal plasma
Human CVS and seminal plasma (SP) were purchased from Lee Biosolutions (St Louis, MO, USA). CVS was supplied undiluted and pooled from six asymptomatic donors aged between 20 and 40 years. The collection procedure involved arousal of female donors followed by collection of, typically, 2 mL of fluid using a spoon-like device, with samples immediately frozen at 2808C. Donors were negative for HIV-1, HIV-2, syphilis, hepatitis C and hepatitis B; however, Nugent scores were not determined. The pH of the CVS was 5.4. SP was prepared from semen obtained from four donors by masturbation after a minimum of 48 h abstinence from ejaculation. The pooled semen was liquefied by incubating the sample containers in a water bath at 378C for 1 h. Liquefied semen was transferred to a 10 mL centrifuge tube and clarified by centrifugation at 1000 g for 10 min. The SP fraction was aspirated while trapping the pellet with a plunger at the bottom of the centrifuge tube and frozen at 2808C.

HIV-1 inactivation studies with no pH adjustment
L-, D-and DL-lactic acid from 30% stocks was applied to PBMC-grown HIV stocks in IL-2 medium to achieve the desired final acid concentration. The pH of the medium was recorded at the beginning and end of the incubation using the Aqua-pH pH-mV-Temperature meter v2.2 (TPS Pty Ltd, Brisbane, Australia) and MI-411-S Micro-combination pH electrode (Microelectrodes Inc., NH, USA) calibrated according to manufacturers' instructions. HIV was incubated for 30 min at 378C with continuous gentle stirring using the Jenway 1103 hotplate stirrer (Bibby Scientific Ltd, Staffordshire, UK) to avoid exposure of the virus to localized pH changes. Following incubation the mixture was immediately neutralized by 10-fold dilution in DMEM-10 containing 25 mM HEPES (DMEM-HEPES) and the pH adjusted to neutral, if required, with NaOH. The mixture was then subjected to additional dilutions in DMEM-10 containing 10 mg/mL DEAE-dextran (GE Healthcare) and the viral infectivity determined in TZM-bl cells.

HIV-1 inactivation time-course study at fixed pH
Carboxylic acids were prepared at twice the final concentration in DMEM-10 followed by adjustment of the media to pH 3.8 using HCl or NaOH. DMEM-10 acidified to pH 3.8 with HCl was also prepared to represent conditions of acidity alone. To avoid exposure of virus to local high concentrations of pH, acid was added to the side of the tube, which was sealed and immediately inverted to mix. An equal volume of HIV stock in IL-2 medium was added to the acid preparations and the pH rapidly adjusted back to pH 3.8 with HCl, without allowing the pH to drop below 3.8. Virus was incubated for different lengths of time at 378C with continuous gentle stirring. At each timepoint 200 mL of treated virus was removed and immediately neutralized by 10-fold dilution in DMEM-HEPES, followed by serial dilution in DMEM-HEPES containing 10 mg/mL DEAE-dextran and viral infectivity determined in TZM-bl cells.

HIV-1 inactivation studies with pH adjustment at a fixed timepoint
Sodium L-lactate and L-, D-and DL-lactic acid were prepared at twice the final required concentration in DMEM-10 followed by adjustment to the target pH (3.8, 4.0 or 7.0) using HCl or NaOH. DMEM-10 acidified to pH 4.0 with HCl was also prepared to represent conditions of acidity alone. An equal volume of HIV-1 Ba-L was added to the lactic acid or sodium lactate preparations and the pH adjusted, where necessary, back to the target pH. Virus was incubated for 30 min at 378C with continuous gentle stirring. Dilution with DMEM-HEPES was performed to stop the treatment and neutralize samples, followed by serial dilution in DMEM-HEPES containing 10 mg/mL DEAE-dextran and viral infectivity determined in TZM-bl cells.

HIV-1 inactivation in the presence of CVS
Undiluted CVS (50 mL) was added to tubes, followed by the addition of L-lactic acid to achieve the desired final concentration, and made up to 60 mL with DMEM-10. To this, 40 mL of HIV-1 Ba-L was added and mixed thoroughly, and the tubes were then incubated for 30 min at 378C. In parallel, HIV-1 Ba-L, in the absence of CVS, was prepared and treated with L-lactic acid in exactly the same manner as for the HIV-1 Ba-L /CVS samples. After 30 min treatment the pH was measured and samples were diluted 1 : 100 in DMEM-HEPES to neutralize the acid and dilute out cytotoxic effects due to CVS. Samples were serially diluted 10-fold in DMEM-HEPES containing 10 mg/mL DEAE-dextran and viral infectivity determined in TZM-bl cells.

HIV-1 inactivation in the presence of SP
For studies performed in the presence of 75% SP, 75 mL of undiluted SP was added to tubes, followed by the addition of L-lactic acid to achieve the desired final concentration. To this, HIV-1 Ba-L was added to achieve a final volume of 100 mL and the tubes were mixed thoroughly. Samples tested in the presence of 12.5% and 25% SP were diluted in OptiMEM medium (Invitrogen) to achieve the required concentration prior to the addition of L-lactic acid and virus. Samples were mixed thoroughly and the pH measured. The samples were incubated for 30 min at 378C, after which the pH was measured again and the samples diluted 1 :10 in DMEM-HEPES to stop the acid treatment and to dilute out cytotoxic effects due to SP. Samples were neutralized with NaOH where required. Each sample was subjected to 10-fold serial dilutions in DMEM-HEPES containing 10 mg/mL DEAE-dextran and viral infectivity determined in TZM-bl cells.

Determination of infectious HIV in TZM-bl cells
The infectious titre of HIV stocks prepared by propagation in PBMCs and the presence of infectious virus following acid treatment was determined by counting blue foci-forming cells in the TZM-bl reporter cell line as described previously. 47 The cytotoxicity of diluted samples containing neutralized lactic acid, CVS or SP was assessed visually by microscopy and by using the MTS reagent (CellTitre 96 Aqueous One, Promega) as previously described. 48 The lower limit of HIV detection in the infectivity assays was 100 infectious units per mL except where otherwise specified. The dynamic range of the assay is a function of the virus stock titre, where high viral titres (i.e. 10 5 -10 6 infectious units/mL) can reveal a greater-fold decrease in infectivity compared with virus with lower titres (i.e. 10 3 -10 4 infectious units/mL). Thus, the 'maximum' decrease in HIV virucidal activity observed for a given virus stock will differ depending on the initial virus titre. The apparent variation at the maximum virucidal concentration, as suggested by the presence of error bars in some Lactic acid potently inactivates HIV 2017 JAC of the graphs, is a consequence of the data being derived from independent assays using virus stocks with different titres. Decreases in HIV infectivity in the presence of acid were normalized to the corresponding untreated virus incubated in the absence of acid.

Statistical analysis
The statistical significance between two acid treatments was determined using the Wilcoxon rank-sum test and differences between more than two treatments were determined using the Kruskal-Wallis test.

Results
L-lactic acid is a more potent inactivator of HIV Ba-L than D-lactic acid We evaluated the relative capacity of racemic DL-, D-and L-lactic acid to inactivate HIV Ba-L . All forms of lactic acid demonstrated maximum decreases in HIV Ba-L infectivity at physiological concentrations [0.5%-1%; 56 -111 mM] compared with the untreated control virus incubated in the absence of lactic acid ( Figure 1a). No effect of the neutralized lactic acid was observed on TZM-bl cell viability (data not shown). L-lactic acid at 0.3% showed greater virucidal activity than D-and DL-lactic acid ( Figure 1a). To exclude the possibility that the difference in virucidal activity of the lactic acid stereoisomers was due to small differences in the final pH of the medium, we performed experiments where DL-, L-and D-lactic acid were adjusted to pH 4.0 (the average pH observed for all three forms of lactic acid at 0.3%) prior to virus addition and the pH monitored continuously (and adjusted if necessary) during incubation at 378C. In parallel, HIV Ba-L was treated with media adjusted to pH 4.0 with HCl to evaluate the virucidal activity of acidity alone (HCl adjusted). L-lactic acid (n¼8) demonstrated HIV Ba-L virucidal activity that was 17-fold more potent than D-lactic acid (P ¼ 0.002, n¼ 6) while DL-lactic acid showed intermediate activity (Figure 1b). Furthermore, while L-lactic acid treatment resulted in a maximal 2.2×10 4 -fold decrease in HIV Ba-L infectivity compared with the untreated control, treatment with HCl resulted in only a 12-fold decrease in infectivity (P,0.001, n¼ 8). These data demonstrate that L-lactic acid is a more potent inactivator of HIV Ba-L than D-lactic acid. Since L-lactic acid is the most potent virucidal form of lactic acid at threshold concentrations, subsequent experiments were performed using L-lactic acid.

L-lactic acid has broad-spectrum HIV-1 and HIV-2 virucidal activity
The previous experiments demonstrate that L-lactic acid inactivates the laboratory strain HIV Ba-L . To determine whether L-lactic acid has potent broad-spectrum HIV virucidal activity we determined its activity against a panel of X4 (Figure 2a HIV-1 inactivation by L-lactic acid at pH 3.8 is more rapid and potent than by acetic acid and acidity alone . In parallel, we tested 1% acetic acid (pH 3.8) and acidity alone (pH 3.8, HCl adjusted). The pH of the medium was adjusted prior to virus addition and maintained at pH 3.8 throughout the experiment. A 130-fold decrease in HIV Ba-L infectivity was observed following 1 min incubation with 1% L-lactic acid, with infectivity continuing to decrease over time resulting in a dramatic 10 5 -fold drop in infectivity after 20 min compared with the untreated control. This decrease in HIV Ba-L infectivity was 100-fold greater than found with 1% L-lactic acid treatment of HIV Ba-L for 30 min (Figure 1a) since the titre of the virus stock used in the time-course study was 100-fold greater compared with the stock used in the fixed timepoint study, leading to a larger assay dynamic range in the time-course assay. After 1 min incubation only 4-fold and 1.7-fold decreases in HIV Ba-L infectivity were observed in the presence of acetic acid and HCl (pH 3.8), respectively ( Figure 3). Furthermore, after 20 min incubation a 3.6×10 4 -fold decrease in HIV Ba-L infectivity was observed for acetic acid while only a 29-fold reduction was observed with HCl (pH 3.8; Figure 3). The virucidal activity of the acid treatments was significantly different at 1 min (P ¼ 0.015, n¼4), 2 min (P ¼ 0.015), 5 min (P ¼0.017), 10 min (P ¼ 0.007) and 20 min (P ¼ 0.012). Compared with HIV Ba-L , similar acid inactivation patterns were observed with the transmitter/founder strains RHPA and CH058 ( Figure S1). These data demonstrate that L-lactic acid is more potent and rapid in the inactivation of HIV-1 than acetic acid and acidity alone.
L-lactic acid does not enhance infectivity of HIV-1 strains at mildly acidic pH A previous study reported that in the presence of a mildly acidic pH (4.5), the infectivity of non-subtype B HIV-1 isolates is enhanced. 40 To investigate this possibility we evaluated the capacity of 0.3% L-lactic acid adjusted to pH 4.5 with HCl to inactivate HIV-1 subtype C (92BR025), subtype A (92RW016), subtype F (93BR020) and subtype B (RHPA). Experiments were performed Lactic acid potently inactivates HIV 2019 JAC as above, where the pH of the medium was monitored throughout the incubation. The infectivity of all strains tested was decreased relative to the corresponding untreated control, indicating no evidence of enhancement of HIV-1 infectivity under the highly controlled conditions of our assay ( Figure S2; available as Supplementary data at JAC Online). These data demonstrate that lactic acid at mildly acidic pH does not enhance infectivity of the subtype B and non-subtype B HIV-1 strains tested.

L-lactic acid inactivates HIV-1 in the presence of genital secretions
To determine whether L-lactic acid inactivates HIV-1 in the presence of human CVS we compared the virucidal activity of L-lactic acid in the presence and absence of 50% CVS. The L-lactic acid HIV Ba-L inactivation profiles in the absence and presence of CVS were similar (Figure 4a). Maximum HIV Ba-L inactivation was observed in the presence of ≥0.5% L-lactic acid. In the presence of 0.3% L-lactic acid a 240-fold and 342-fold decrease in titre was observed compared with the control in the absence and presence of 50% CVS, which was not significantly different (P ¼ 0.06, n¼ 3). These data demonstrate that L-lactic acid's HIV-1 virucidal activity is not inhibited in the presence of CVS. Semen is alkaline and neutralizes the vaginal lumen within seconds, providing a window of opportunity for acid-susceptible pathogens such as HIV to establish infection. To determine whether L-lactic acid inactivates HIV-1 in the context of semen we performed experiments in the presence of 75% SP as a surrogate for semen to simulate dilution of an estimated 1 mL of CVS in the vagina with an average 3 mL ejaculate. 49 Under these conditions we found that a final concentration of 1% L-lactic acid was required to achieve a 2.4×10 4 -fold decrease in HIV Ba-L infectivity compared with the untreated control, while 0.75% L-lactic acid mediated a 135-fold reduction in HIV Ba-L infectivity (Figure 4b). In addition, the virucidal activity of L-lactic acid was observed at pH ≤4.8 (Figure 4b). The pH in SP (Figure 4b) was higher for a given lactic acid concentration compared with treatment in the absence of SP (Figure 2a). Experiments performed in the presence of 12.5% and 25% SP demonstrated a dose-dependent attenuation of the virucidal activity of L-lactic acid ( Figure S3; available as Supplementary data at JAC Online). Maximum HIV Ba-L virucidal activity was observed at a final concentration of 0.5% L-lactic acid for both 12.5% and 25% SP, in contrast to 1% L-lactic acid for 75% SP, and the ability of 0.4% L-lactic acid to inactivate HIV Ba-L was reduced with increasing SP concentrations ( Figure S3 and Figure 4b). These data demonstrate that L-lactic acid inactivation of HIV Ba-L in the presence of 75% SP is observed at final concentrations of ≥0.75%, in the context of our in vitro experiments.  Aldunate et al.

Protonated lactic acid and not the lactate anion mediates HIV-1 virucidal activity
Our data demonstrate that the HIV virucidal activity of lactic acid is pH dependent. To determine whether the uncharged lactic acid predominantly found at low pH mediates virucidal activity, as distinct from the lactate anion present at neutral pH (Figure 5a), we evaluated the ability of L-lactic acid to inactivate HIV Ba-L when neutralized to pH 7.0 compared with L-lactic acid at pH 3.8. In addition, we examined the capacity of L-sodium lactate to inactivate HIV, which provides a source of lactate anion in the absence of acidity. While 1% L-lactic acid at pH 3.8 resulted in a 5.4×10 3 -fold decrease in HIV Ba-L infectivity , little virucidal activity was observed with either 1% L-lactic acid at pH 7.0 or 1% L-sodium lactate pH 7.0 ( Figure 5b). These data demonstrate that it is uncharged protonated lactic acid and not the lactate anion that mediates HIV-1 virucidal activity.

Discussion
Our study shows that L-lactic acid has broad-spectrum HIV virucidal activity that is additional to acidity alone (due to HCl). L-lactic acid's virucidal activity is more potent and rapid compared with acetic acid, a smaller, membrane-permeant carboxylic acid. Complete inactivation of HIV infectivity by lactic acid concentrations ≥0.5%, under conditions where the media were subsequently neutralized prior to titration in TZM-bl cells, strongly suggests that the impact of lactic acid on HIV infectivity is irreversible. We have also shown that it is the protonated form of lactic acid and not the lactate anion that mediates HIV virucidal activity, which is consistent with the pH-dependent anti-HIV activity observed in the absence and presence of SP.
While it has been reported that L-lactic acid has more potent antibacterial activity than D-lactic acid, 50 to our knowledge this is the first report of a carboxylic acid eliciting stereochemicaldependent virucidal activity. The antimicrobial mechanism of action of lactic acid under acidic conditions has been ascribed to its ability to penetrate cell membranes, resulting in cytosol acidification, 51 direct action on membranes 52 or interference with the enzymatic reactions of the cell. 50 HIV does not have a cytosol and the stereochemical-dependent activity of lactic acid suggests that it is targeting protein. In this regard, L-lactic acid has been shown to induce protein unfolding; 53 thus, an obvious viral protein target is the HIV gp120 envelope protein exposed on the outside of the virion. However, given the membrane-permeant properties of the uncharged form of lactic acid predominately present at low pH, it is also possible that it could penetrate the viral lipid envelope, resulting in its perturbation and/or adversely affecting the function of proteins embedded in this layer in addition to penetrating the lipid bilayer and targeting viral proteins and enzymes inside the virion. A previous study reported that HIV-1 virions treated with 1% lactic acid at pH 4.0 for 1 h at 378C retained gp120 in an immunologically recognizable form. 6 Further studies are in progress to examine whether similar effects are observed with L-lactic acid at pH 3.8, to correlate this effect to its virucidal activity and elucidate the mechanisms by which it inactivates HIV infectivity.
Our studies clearly show that L-lactic acid inactivates subtype B and non-subtype B HIV-1 strains, even under mildly acidic conditions at pH 4.5, which appears to be at odds with a previous study that reported enhancement of HIV-1 infectivity. 40 It is not clear why Connor 40 observed this enhancement. In contrast to that study we quantified virus titre by counting blue fociforming cells rather than luciferase activity, we obtained data from at least two independent assays and measured the final pH after mixing of virus and acid-buffering medium, which can shift from the intended pH. We also reported the final concentration of L-lactic acid in the medium, which along with pH we have found to be critical for HIV virucidal activity. In this regard, 0.2% L-lactic acid at pH 4.2 lacked potent virucidal activity for 9 of the 10 HIV strains tested (Figure 2). We also did not observe dramatic variations in HIV inactivation for the majority of the strains tested in the presence of 0.3% L-lactic acid, which acidified the media to pH 4.0 under our experimental conditions. It is highly unlikely the concentration of solutes and salts introduced during pH adjustment of lactic acid had an impact on HIV virucidal activity. The HIV inactivation studies were performed in tissue culture medium, which has an osmolality of 295 mOsm/kg. If we account for 56 mOsm/kg due to dissociated lactate anion present at pH 3.8 in the 1% (w/w) lactic acid solution and the 6 mOsm/kg osmolality associated with adjusting the pH of lactic acid-containing medium with HCl  Lactic acid potently inactivates HIV 2021 JAC to account for chloride ions in the time-course experiments, the total osmolality is 357 mOsm/kg. By contrast, 1% sodium lactate (pH 7.0), with a much higher osmolality of 473 mOsm/kg (295 mOsm/kg from media and 178 mOsm/kg to account for Na+ and lactate anions from sodium lactate), demonstrated little effect on HIV infectivity compared with the untreated control virus (Figure 5b). Taken together it is unlikely that the small change in osmolality due to HCl addition would have contributed to HIV inactivation. Moreover, the volumes of HCl added to adjust L-lactic acid, acetic acid and media alone to pH 3.8 were similar. Thus, differences in solutes and salt concentrations are unlikely to account for the more potent HIV inactivation profile observed for L-lactic acid compared with the other acids tested.
The superior virucidal activity of L-lactic acid compared with other acids and its capacity to inactivate HIV even in the presence of CVS has significant implications with regard to HIV transmission. Higher concentrations of HIV-1 RNA in male and female genital secretions are associated with a greater risk of heterosexual transmission of HIV-1, even when adjusting for plasma viral load. 9 Notably, women harbouring vaginal Lactobacillus spp. have a lower risk of genital HIV-1 shedding whereas the bacteria associated with BV increase this risk. 7,8 The reduced presence of HIV-1 RNA in HIV-infected women with a lactobacillusdominated microbiota may be related to the impact of lactobacilli and/or lactic acid on the innate immune response in the vagina. 54 -56 Alternatively or additionally, lactic acid may directly inactivate HIV shed into the vagina. However, the impact of lactic acid treatment (in the presence of CVS) on the integrity of viral RNA, which is normally used to measure the presence of HIV in genital secretions, needs to be determined. Regardless, our findings could be exploited by delivering L-lactic acid into the vaginas of HIV-infected women by an intravaginal ring to inactivate HIV and reduce female-to-male transmission.
The main rationale for performing the HIV inactivation studies in CVS was to determine whether exogenously added lactic acid would be active in an environment comprising viscous mucus and proteins found in the vagina. The studies were not designed to determine the impact on HIV of endogenous lactic acid that might be present in these samples. The pH of the pooled CVS used in our experiments was around 5.4. Vaginal pH .5.0 has been reported in asymptomatic women and is associated with bacterial community group IV that is not dominated by Lactobacillus spp. 11 In the context of media, 0.1% lactic acid results in a pH of 4.9 (Figure 2a). This suggests that the CVS sample had very little protonated lactic acid. This lack of lactic acid in the sample is consistent with our observation of similar HIV inactivation profiles in the absence and presence of 50% CVS (Figure 4a).
Our experience with handling CVS indicates that freezing of the sample is critical to minimize exposure of the sample to oxygen in air, which can result in a decrease in lactic acid levels. This is manifested owing to the altered metabolism of lactobacilli in the presence of oxygen, which results in the production of acetic acid instead of lactic acid and/or loss of CO 2 (which partially acidifies CVS) from the sample. We have found that the pH of fresh CVS (collected using the SoftCup) 57 compared with the pH of the same sample measured following freezing at 2808C and thawing increases on average by 0.1 pH unit (T. Moench and G. Tachedjian, unpublished data). This small increase could be due to loss of CO 2 and/or lactic acid levels due to exposure to air. Thus, it is unlikely that limited freezethawing per se would substantially affect lactic acid levels, provided that exposure of samples to oxygen is minimized. In addition, the samples we used remained viscous following freeze -thaw, indicating lack of breakdown of mucus.
In contrast to CVS we found that SP attenuates the anti-HIV activity of L-lactic acid, most likely due to its buffering capacity, which increases the pH thereby decreasing levels of the active protonated form of lactic acid. Accordingly, in the absence of buffering to maintain low pH, supra-physiological concentrations of lactic acid would likely be needed in the vagina to completely block male-to-female transmission of HIV. Whether endogenously produced lactic acid has a role in attenuating HIV transmission from males to females would depend on several factors. These include the rate of re-acidification following deposition of semen in the vaginal tract and how quickly HIV is able to penetrate physical barriers in the vagina to encounter target cells in the mucosa, which are critical for establishing infection. 58 Lactobacilli found in the vagina acidify at a rate of 0.5 pH units/h, which is consistent with lactobacilli being responsible for re-acidifying the vagina within several hours of coitus, following neutralization by semen. 28 While the time required for HIV to infect target cells after introduction in the vagina is unknown, a study where rhesus macaques were vaginally infected with a high inoculum of simian immunodeficiency virus (SIV) reveals that cell-free virus penetrates the cervicovaginal epithelium and infects epithelial dendritic cells within 60 min of exposure. 59 However, macaques are sparsely colonized with lactobacilli and have low levels of lactic acid, and consequently rarely have acidic vaginas. 31 In addition to the direct inactivation of HIV, CVS from women with a lactobacillus-dominated vaginal flora that is acidified to pH 4 traps HIV, while neutralizing the same CVS sample in parallel abolishes this effect. 6 An uncharacterized factor in mucus that immobilizes HIV in a pH-dependent manner has also been described. 10 Thus, provided that acidification following neutralization with semen occurs before HIV can encounter target cells, endogenously produced lactic acid could potentially attenuate HIV infection by both direct inactivation and by preventing its ability to diffuse through cervicovaginal mucus.
Lactic acid production by lactobacilli may also have an indirect impact on HIV transmission by potentially preventing BV, which is associated with an increased risk of HIV acquisition in both males and females. 18 -20 Virulence factors released or induced by BV organisms may be responsible for some of the risks for STIs. In this regard, metabolic products such as acetic acid, propionic acid and butyric acid released by BV-associated bacteria increase inflammation and potentially activate target cells in the vagina known to promote HIV infection. 60 Alternatively, lactobacilli and lactic acid production in the context of a lactobacillusdominated vaginal microbiota might reduce the risk of BV, and hence indirectly HIV, by selectively suppressing growth of BV-associated bacteria. 23 Several clinical trials have evaluated the use of lactobacillus-based probiotics for the treatment or prevention of BV recurrence. However, while the use of probiotics appears to be promising the data are inconclusive, with some studies demonstrating BV cure and/or reduced BV recurrence compared with placebo while other studies showed no significant difference. 60 -63 The use of distinct Lactobacillus spp. that may differ in their metabolic output of lactic acid 26 or other Aldunate et al.
antimicrobial factors 3 and challenges associated with establishing and maintaining vaginal colonization of strains may explain the apparently discordant data. 64 Another alternative strategy is to use lactic acid instead of probiotics. In this regard, a multicentre, open-label, controlled, randomized study in 90 women demonstrated that lactic acid gel (Lactacyd, comprising 4.5% lactic acid and 5 g of glycogen) is safe and as effective as metronidazole in the treatment of BV. 65 Furthermore, the combination of metronidazole and lactic acid gel was superior to metronidazole alone in promoting lactobacilli colonization and decreasing BV recurrence. 65 Other unblinded studies have reported that lactate gel inserted into the vagina for 7 days is as effective as oral metronidazole in the treatment of BV 66 and that topical use of lactic acid and lactoserum intimate liquid soap following standard oral metronidazole treatment reduced BV recurrence. 67 Further blinded controlled studies are warranted to examine the potential for L-lactic acid, particularly applied continuously via an intravaginal ring, for BV treatment and prevention.
This study focused on the impact of lactic acid on cell-free virus. However, HIV-infected cells have also been reported in the cervical mucus and semen of infected individuals, which could initiate infection. 68 In this regard, studies in mouse and macaque models demonstrate that cell-associated HIV 69 and SIV, 70 respectively, can establish infection via the vagina. While a clinical study suggests that cell-associated HIV transmission in semen is possible, 71 a second study showed that HIV transmission was attributable to cell-free virus in SP rather than seminal cells, as determined by phylogenetic analysis. 72 Our previous studies demonstrate that low pH (4.5) immobilizes and kills human leucocytes and prevents transmission of cell-associated HIV in a mouse model. 73 These experiments were performed with Buffer Gel, which is acidified by HCl and found to be less potent than lactic acid in the inactivation of cell-free HIV in the current study. Thus, studies to examine the impact of lactic acid on transmission of cell-associated HIV would be of interest.
Under conditions where the pH was carefully monitored throughout the experiment we found that acidity alone, even at pH 3.8 found in a lactobacillus-dominated vagina, resulted in not more than a 29-fold reduction in titre and was 600-fold to 3500-fold less effective at inactivating a laboratory strain and two transmitter/founder strains of HIV-1 than L-lactic acid at the same pH. Our data appear to differ from a previous report showing that a 10 min incubation of HIV-1 in medium acidified with HCl results in almost a 10 4 -fold reduction in titre. 38 However, that study used an X4 laboratory strain of HIV, which may be intrinsically more susceptible to acid than the more biologically relevant R5 strains tested in this study. Our findings may inform microbicide formulations, which are normally acidified to a pH compatible with the natural acidity of the vagina. In this regard, the acid-buffering microbicide Buf-ferGel w , while safe, was not effective in protecting women from the sexual transmission of HIV from their male partners. 74 Notably, BufferGel w comprises a mucoadhesive and acidbuffering Carbopol w polymer gel that contains no lactic acid and provides only a low pH, which we have shown to be dramatically less virucidal and less effective against BV-associated bacteria than lactic acid. 23 Thus, L-lactic acid could potentially be used as an acidifying agent in topical microbicides instead of acidity alone, and may favour a lactobacillus-dominated vaginal microbiota in addition to having a role in directly inactivating HIV in the vagina.
Thus, we have shown that L-lactic acid, a naturally occurring microbicidal agent found in the vagina, also has potent, broadspectrum HIV virucidal activity that could help prevent both female-to-male and male-to-female spread of the virus by both direct inactivation and indirectly through prevention of BV, which contributes to HIV transmission. The potential use of lactic acid as a microbicide or in microbicide formulations, especially in combination with other potential microbicides such as antiretroviral drugs, warrants further evaluation.