Cannabinoids exert their actions mainly through two receptors, the cannabinoid CB1 receptor (CB1R) and cannabinoid CB2 receptor (CB2R). In recent years, the G-protein coupled receptor 55 (GPR55) was suggested as a cannabinoid receptor based on its activation by anandamide and tetrahydrocannabinol. Yet, its formal classification is still a matter of debate. CB1R and CB2R expression patterns are well described for rodent and monkey retinas. In the monkey retina, CB1R has been localized in its neural  and CB2R in glial components (Müller cells). The aim of this study was to determine the expression pattern of GPR55 in the monkey retina by using confocal microscopy. Our results show that GPR55 is strictly localized in the photoreceptor layer of the extrafoveal portion of the retina. Co-immunolabeling of GPR55 with rhodopsin, the photosensitive pigment in rods, revealed a clear overlap of expression throughout the rod structure with most prominent staining in the inner segments. Additionally, double-label of GPR55 with calbindin, a specific marker for cone photoreceptors in the primate retina, allowed us to exclude expression of GPR55 in cones. The labeling of GPR55 in rods was further assessed with a 3D visualization in the XZ and YZ planes thus confirming its exclusive expression in rods. These results provide data on the distribution of GPR55 in the monkey retina, different than CB1R and CB2R. The presence of GPR55 in rods suggests a function of this receptor in scotopic vision that needs to be demonstrated. Moreover, cannabidiol seems to have protective effects on retinal neurons Expression patterns of CB1R and CB2R have been both localized in the vervet monkey retina Monkey tissue, the experimental model for the current study, was chosen for several reasons. First, using monkey tissue allows us to infer more easily on what is really present in humans. Additionally, the anatomical similarity between the monkey and human retina is remarkable. Primates are mammals that have a macular/foveal region and multiple cone types responsible for high visual acuity and color vision. Finally, the high cross-reactivity between human and monkey antigens increases chances of success for targeting GPR55 in monkeys using an antibody directed against human GPR55 epitope.Chlorocebus sabaeus) were included in this study: three were used for the immunochemistry protocols, and fresh specimens of retina, visual cortex, and cerebellum were collected from three others for immunoblotting. The animals were born and raised in enriched environments in the laboratories of the Behavioural Sciences Foundation  that is recognized by the Canadian Council on Animal Care (CCAC). The animals were fed with primate chow  and fresh local fruits, with water available ad libitum. Infant vervets are born into an outdoor social group comprising several females, one male and other offspring of the same general age. Infants live with their parents until about 8 months of age, at which time they move to a playpen with 5 other age-mates. The natal cage is equipped with swings, perches, hiding places and jungle gyms. We do put in toys, but the animals are so busy playing with one another that they ignore the toys. In the smaller playpens, there are also swings, perches and climbing spots, as well as puzzle feeders and foraging boards. At about 18 months of age, youngsters graduate to a large, outdoor peer group of about 16 animals  where there are tunnels, swings, ladders, jungle-gyms and a variety of manipulanda . Plastic chain and baited balls are popular toys, but vervets of this age are uninterested in most other commercially available toys. The experimental protocol was reviewed and approved by the local Animal Care and Use Committee  and the Institutional Review Board of the Behavioural Science Foundation. Each animal was sedated with ketamine , deeply anaesthetized with sodium pentobarbital  and perfused transcardially with phosphate buffer saline (PBS pH 7.4), followed by 4% paraformaldehyde.Six 42 months-old vervet monkeys  to rhodopsin from Abcam  was obtained by using as immunogen the bovine rhodopsin. This antibody recognizes a 39 kDa band on Western Blots and is predicted to react with human retinal tissues (manufacturer's data sheet). It has been proven effective to specifically label rods in the rodent retina The mouse monoclonal (IgG1) to calbindin (CB) from Sigma  was obtained by using as immunogen purified bovine kidney Calbindin-D-28K. This antibody recognizes a 28 kDa band on Western Blots. Immunostaining against calbindin is known to label cones outside the foveal region, cone bipolar cells and a subset of horizontal cells on human and monkey retinal sections The mouse monoclonal to glutamine synthetase (GS) was obtained from Chemicon International  by using as immunogen the GS purified from sheep brain. This antibody generates a single 45 kDa band in adult retinal tissue The mouse monoclonal (IgG2a) to protein kinase C (PKC) was obtained from Santa Cruz Biotechnology  by using as immunogen purified bovine PKC. The epitope is mapped to PKC hinge region (amino acids 296–317). It detects the PKCα isoform, a well-known specific marker for rod bipolar cells The mouse monoclonal (IgG1) to parvalbumin (PV) was obtained from Sigma  by using as immunogen purified frog muscle PV. It recognizes a 12 kDa band from human, bovine, pig, canine, feline, rabbit, rat, and fish tissues . The pattern of labeling with this antibody was the same as reported previously The rabbit anti-GPR55 was obtained from Cayman Chemical  by using as immunogen a synthetic peptide corresponding to the amino acids 207–219 (ILLGRRDHTQDWV) of the human GPR55 sequence. This antibody recognizes a band at 37 kDa . This antibody was characterized and used to detect GPR55 expression in human trabecular meshwork cells The rabbit anti-CB1R was obtained from Sigma  by using a highly purified fusion protein containing the first 77 amino acid residues of the rat CB1R as the immunogen. It recognizes a major band of 60 kDa and less intense bands of 23, 72, and 180 kDa . This antibody targets the rat CB1R but specifically recognizes the CB1R (60 kDa) from many species, including vervet monkey retinal tissue The rabbit anti-CB2R was purchased from Cayman Chemical  and was developed by using a synthetic peptide corresponding to the amino acids 20–33 (NPMKDYMILSGPQK) of the human CB2R sequence conjugated to KLH as immunogen. This antibody recognizes a band at 45 kDa and a band at 39–40 kDa . This antibody was used in human nervous tissues The mouse monoclonal to GAPDH (clone GAPDH-71.1) was obtained from Sigma  by using as immunogen purified rabbit muscle GAPDH (whole molecule). As stated by the manufacturer, this antibody recognizes monkey GAPDH and gives a single band at about 37 kDa.The GPR55 blocking peptide containing the human GPR55 amino acid sequence 207–219  was used in the present study for immunohistochemistry and western blot analysis. The specificity of the GPR55 antibody was also tested by preincubation with the corresponding blocking peptide. For preadsorption, 20 µg of primary antibody was mixed with 20 µg of blocking peptide for 2 hours at room temperature with occasional inversion. We then diluted that mixture 1∶200 making a final concentration of 10 µg/ml of preadsorbed antibody. The antibody-blocking peptide solution was added to the slices or blots and subsequent immunohistochemistry and western blot analysis followed the protocol as described further.2) were taken from the extrafoveal retina (between 1 and 5 degrees of perimetric angle), middle retina (between 6 and 25 degrees of perimetric angle) and periphery (above 25 degrees of perimetric angle). Each sample was then cryoprotected in 30% sucrose overnight and embedded in Shandon embedding media at −65°C. Retinal samples were then sectioned in a cryostat (18 µm) and mounted onto gelatinized glass microscope slides, air-dried and stored at −20°C for further processing.The eyes were extracted and the retina was dissected free from the eyecup in a PBS bath. The retina was laid flat so that the vitreous body could be removed by blotting with filter paper and gentle brushing 2O2). The membrane was then air-stripped, reblocked, and exposed to a second primary antibody, namely mouse anti-GAPDH , until all proteins of interest were tested.In order to test the specificity of our antibodies directed against GPR55, Western blots were performed on a piece of monkey tissue. A fresh dissected sample of retina, visual cortex, and cerebellum was homogenized by hand using a sterile pestle in RIPA buffer , 0.1% SDS, 1 mM EDTA), supplemented with a protease inhibitor mixture , leupeptin , pepstatin  and phenylmethylsulfonyl fluoride (0.2 mg/ml); Roche Applied Science, Laval, QC, Canada). Samples were then centrifuged at 4°C for 10 minutes, and the supernatant was extracted and stored at −20°C until further processing. Protein content was equalized by using a Thermo Scientific Pierce BCA Protein Assay Kit . Thirty micrograms of protein/sample of the homogenate was resolved with 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane filter , blocked for 1 hour in 5% skim milk  in TBST , and incubated overnight with a primary antibody, namely, rabbit anti-GPR55 (1∶500) in blocking solution. The following day, the blot was exposed to a secondary antibody conjugated to horseradish peroxidase  in blocking solution for 2 hours. Detection was carried out by using homemade ECL Western blotting detection reagents , using a 40X (n.a: 1.25–0.75) or a 100X (n.a: 1.40–0.7) objective. Images were obtained sequentially from the green and far-red channels on optical slices of less than 0.9 µm of thickness. When co-expression of GPR55 and retinal cell markers was ambiguous in some retinal layers, co-labeling or its absence was demonstrated by taking z-stacks with optimized steps. This allowed for visualization of the cells in the X-Y, X-Z and Y-Z axes, thereby confirming the presence or absence of GPR55 in specific retinal cells. All photomicrograph adjustments, including size, color, brightness, and contrast were done with Adobe Photoshop  and then exported to Adobe InDesign , where the final figure layout was completed. The schematic panels were created using Adobe Illustrator .In order to determine GPR55 antibody specificity in the vervet monkey, we carried out immunoblots on homogenates of fresh vervet monkey retina , visual The ONL is composed of cone and rod nuclei. There are also Müller cell outer processes that enroll these cell bodies. To distinguish cones and rods, double immunolabeling with cell specific markers was performed. Double-label of GPR55 with rhodopsin, a specific rod photoreceptor cell marker, allowed us to restrict the labeling to rod photoreceptors . Our resDouble-labeling of GPR55 and GS, a specific marker for labeling both somata and processes of Müller cells We wanted to know if the little GPR55 expression found in rod spherules was also present in horizontal and bipolar cells outer processes. Double label of PKC, a rod bipolar cell marker, and GPR55 allowed us to establish that GPR55 was strictly localized in rods. Dendritic fibers of rod bipolar cells were rather juxtaposed to GRP55 immunoreactive rod spherules . MoreoveThe GPR55, CB1R, and CB2R antibodies that we selected came from the same host, making the use of simultaneous double-labeling protocol not adequate. We therefore used serial sections to compare the distribution of CB1R, CB2R, and GPR55 . These 3The present results demonstrate the existence of GPR55 in the monkey extrafoveal and peripheral retina. The localization of GPR55 in rods is important because, although the presence of CB1R and CB2R in the monkey retina is well established fovea centralis. The exact role of this receptor in retinal function is unknown although there is general agreement that cannabinoids suppress dopamine release and reduce neurotransmitter release from cones and bipolar cells CB1R was localized in the neural components of the monkey central and peripheral retina CB2R neuronal expression has been ambiguous and controversial, but nevertheless, the majority agrees on the presence of this receptor in glial components of the CNS. Indeed, CB2R was detected in the glial component of the monkey retina, Müller cells GPR55 expression was found specifically in rod photoreceptors and enriched in the inner segment, although smaller amounts of this protein could be detected essentially throughout the rods of the extrafoveal and peripheral regions of the retina. It is plausible that such an asymmetric distribution of GPR55 reflects its function in phototransduction in relation with transducin.Given that CB1R is present in the neuroretina, CB2R in the retinal glia, and GPR55 in rods, we can hypothesize that each of these receptors has a unique retinal function. Furthermore, THC can bind to GPR55 and induce a signal transduction different from that of CB1R and CB2R ++ fluctuations through a RhoA-mediated, and inositol 1,4,5-trisphosphate (IP3)-sensitive mechanism mobilizing Ca++ stores 2+ rise in hippocampal slices In the dark, rods are depolarized and constantly stimulated to allow the release of glutamate. Glutamate then binds to mGluR6 to hyperpolarize rod bipolar cells and to iGluRs to depolarize horizontal cells. It is well documented that LPI is a lysophospholipid-signaling molecule that modulates many cell functions. In fact, the generation of LPI is linked to the metabolism of membrane phospholipids by enzymes like phospholipase A1 and A2 that are activated upon cell stimulation and are located at the inner and outer side of the plasma membrane. Initially, LPI has been discussed to serve as second-messenger lysophospholipid that modulate intracellular signaling events


Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing <57% pairwise nucleotide identity with other adenoviruses. Cultivation of TMAdV was successful in a human A549 lung adenocarcinoma cell line, but not in primary or established monkey kidney cells. At the onset of the outbreak, the researcher in closest contact with the monkeys developed an acute respiratory illness, with symptoms persisting for 4 weeks, and had a convalescent serum sample seropositive for TMAdV. A clinically ill family member, despite having no contact with the CNPRC, also tested positive, and screening of a set of 81 random adult blood donors from the Western United States detected TMAdV-specific neutralizing antibodies in 2 individuals . These findings raise the possibility of zoonotic infection by TMAdV and human-to-human transmission of the virus in the population. Given the unusually high case fatality rate from the outbreak (83%), it is unlikely that titi monkeys are the native host species for TMAdV, and the natural reservoir of the virus is still unknown. The discovery of TMAdV, a novel adenovirus with the capacity to infect both monkeys and humans, suggests that adenoviruses should be monitored closely as potential causes of cross-species outbreaks.Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus  that caused a deadly outbreak in a colony of New World titi monkeys at the California National Primate Research Center (CNPRC), and also infected a human researcher. One-third of the monkeys developed pneumonia and liver inflammation, and 19 of 23 monkeys died or were humanely euthanized. The unusually high death rate (83%) makes titi monkeys unlikely to be natural hosts for TMAdV, and the genomic sequence of TMAdV revealed that it is very different from any other known adenovirus. The researcher developed an acute respiratory illness at the onset of the outbreak, and was found to be infected by TMAdV by subsequent antibody testing. A clinically ill family member with no prior contact with the CNPRC also tested positive. Further investigation is needed to identify whether TMAdV originated from humans, monkeys, or another animal. The discovery of TMAdV suggests that adenoviruses should be monitored closely as potential causes of cross-species outbreaks.Infection from adenoviruses, viruses that cause a variety of illnesses in humans, monkeys, and other animals, has conventionally been thought to be species-specific. We used the Virochip, a microarray designed to detect all viruses, to identify a new species of adenovirus (TMAdV, or  Mastadenovirus genus, comprised of all mammalian adenoviruses, are classified into 7 species A-G, and at least 51 different serotypes  have been described to date Adenoviruses, first isolated in the 1950s from explanted adenoid tissue, are double-stranded nonenveloped DNA viruses that naturally infect many vertebrates, including humans and nonhuman primates. The human adenoviruses in the Infection by adenoviruses has generally been thought to be species-specific. Human adenoviruses do not usually replicate in monkey cells in the absence of helper viruses Callicebus genus at the California National Primate Research Center (CNPRC). Routine microbiological testing for an infectious etiology was negative. We previously developed the Virochip  as a broad-spectrum surveillance assay for identifying viral causes of unknown acute and chronic illnesses Beginning in May of 2009, a deadly outbreak of fulminant pneumonia and hepatitis occurred in a closed colony of New World titi monkeys of the Macaca mulatta) housed in the same building became sick during the outbreak, and neither did any of the Old World monkeys from surrounding outdoor colonies of rhesus and cynomolgus macaques (Macaca fascicularis).In early 2009, the CNPRC housed 65 titi monkeys in one quadrant of an animal building. The index case, a healthy adult titi monkey, presented on May 14, 2009 with cough, lethargy, and decreased appetite , T1. DesGross necropsy findings were similar in all titi monkeys and were characterized primarily by diffuse, consolidated pneumonias, with occasional evidence of fibrinous pleuritis, pericardial/pleural edema, and hemorrhage . Some liA routine microbiological workup for infectious causes of the outbreak, including bacterial, mycoplasma, and fungal cultures, was negative. Respiratory viral testing failed to detect evidence of respiratory syncytial virus, adenovirus, influenza virus A and B, human metapneumovirus, and parainfluenza virus types 1, 2, and 3.Herpesviridae), bovine viral diarrhea virus (Flaviviridae), and endogenous retroviruses (Retroviridae), were regarded as less likely to cause fulminant pneumonia and hepatitis, so were not pursued any further. The 4 adenovirus probes mapped to 2 different gene regions corresponding to the DNA polymerase and penton base , suggesting the presence of a divergent adenovirus that was not a member of any previously known species.Given the clinical presentation of a severe acute viral respiratory illness and the appearance of intranuclear inclusion bodies on histological examination, we strongly suspected that a virus that had eluded detection by conventional assays was the cause of the titi monkey outbreak. Nasal, lung, and liver swab samples collected during necropsy were analyzed using the Virochip ton base . Interestiti monkey adenovirus. Specific PCR for TMAdV was then used to screen body fluids and tissues from affected monkeys . All post-outbreak serum samples were negative for TMAdV by PCR . To asseWe also sought to determine whether PCR assays commonly used to detect human adenoviruses in clinical or public health settings could detect TMAdV. Adenovirus PCR was performed on a TMAdV-positive clinical sample, a TMAdV culture, and a human adenovirus B culture (as a positive control) using an additional 5 pairs of primers, according to previously published protocols De novo assembly of the complete TMAdV genome from reads that aligned to SAdV-18 was not possible due to insufficient sequence coverage (<46%). The poor apparent coverage was the result of high sequence divergence of the TMAdV genome from SAdV-18, which hindered the identification of most of the 16,524 actual deep sequencing reads derived from TMAdV , is formed by loops 1 and 2 in the hexon protein We next attempted to culture TMAdV in an A549 (human lung adenocarcinoma) cell line, a BSC-1  cell line, and PMK (primary rhesus monkey kidney) cells . Direct In hindsight, only one individual at the CNPRC reported becoming ill during the titi monkey outbreak, the researcher in closest, daily contact with the animals. Symptoms began near the onset of the outbreak, although whether they began prior to or after identification of the index case is unclear. The researcher, with a past medical history of multiple sclerosis, initially developed symptoms of a viral upper respiratory infection (URI), including fever, chills, headache, and sore throat, followed by a dry cough and “burning sensation in the lungs” that was exacerbated by a deep breath or coughing. The researcher endorsed a history of recurrent upper respiratory infections, and did not regard the illness as related to the titi monkey outbreak. Although symptoms persisted for 4 weeks, at no time did the researcher seek medical care, and no antibiotics were taken during the illness.We carried out contact tracing to identify family members and other individuals in close contact with the researcher. Interestingly, two family members in the household also developed flu-like symptoms about 1–2 weeks after the researcher initially became sick. Their symptoms – fever, cough and muscle aches – appeared milder than those of the researcher and completely resolved within 2 weeks. Neither individual sought medical care for these symptoms, and notably, neither had ever visited the CNPRC.To explore a potential link between the outbreak and associated illness in humans, we blindly tested available sera from titi monkeys (n = 59), rhesus macaques housed in the same building (n = 36), CNPRC personnel and close contacts (n = 20), and random human blood donors (n = 81) for evidence of recent or prior infection by TMAdV by virus neutralization . NineteeTo investigate the possibility of subclinical infection by TMAdV, we also examined serum samples from asymptomatic titi monkeys (n = 40) and nearby rhesus macaques (n = 36), collected 2 months after the outbreak. Fourteen of 40 asymptomatic titi monkeys tested (35%) had antibody to TMAdV, indicating that the incidence of subclinical infection was significant ; Fig 6. Approximately 4 months after the outbreak, serum samples were collected from CNPRC personnel in direct contact with the titi monkeys. Serum samples were also collected from the two family members of the clinically ill CNPRC researcher 1 year after the outbreak. Only two samples were found positive for neutralizing Abs to TMAdV: (1) Ab titers for the clinically ill researcher were 1∶32, and (2) Ab titers for one of the family members of the clinically ill researcher were 1∶8.Among 81 random blood donors from the Western United States, 2 individuals  had positive Ab titers of 1∶16 and 1∶8. Pooled rabbit sera containing antibodies to human adenovirus serotypes 1 through 35, representing species A–E, were unable to neutralize TMAdV (data not shown). Thus, the results of our serological survey appear unlikely to be due to nonspecific cross-reactivity from prior exposure to known human adenoviruses.In this study, we employed a pan-viral microarray assay, the Virochip, to identify a novel adenovirus associated with a fulminant pneumonia outbreak in a colony of New World titi monkeys. Despite the absence of an animal model, which precludes a strict fulfillment of Koch's postulates, there are several lines of evidence implicating this novel adenovirus, TMAdV, as the cause of the outbreak. First, conventional testing for other pathogens, including other viruses by Virochip, was negative, and affected monkeys did not respond to empiric therapy with antibiotics or antivirals  Although TMAdV retains the core genomic features common to all adenoviruses , phylogev integrins v integrins for internalization Entry of adenoviruses into cells involves an initial attachment of the fiber knob to the cell receptor, followed by internalization via a secondary interaction of the penton base with αDespite its isolation from affected titi monkeys, we were unable to propagate TMAdV in both established (BSC-1) and primary (PMK) monkey kidney cells . The virThe virulence of TMAdV in healthy and apparently immunocompetent titi monkeys  is highly unusual for infections by adenovirus. In humans, deaths due to adenovirus infections or outbreaks are generally low  asymptomatic rhesus macaques housed in the same building , indicatAlthough we failed to detect TMAdV in rodent droppings found near titi monkey cages , it is sOur study data also support the potential for cross-species transmission of TMAdV between monkeys and humans. The researcher's fever, cough, and pleuritic symptoms (“burning sensation in the lungs”) are consistent with the development of a prolonged viral respiratory illness. Interestingly, pleurisy has been specifically reported in association with certain human adenovirus infections Several lines of evidence support the contention that the direction of TMAdV transmission was zoonotic (monkeys to humans) rather than anthroponotic (humans to monkeys). First, the closest known relative to TMAdV in GenBank is SAdV-3, an Old World monkey adenovirus ; Table 2This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The use and care of all animals followed policies and guidelines established by the University of California, Davis Institutional Animal Care and Use Committee (IACUC) and CNPRC . The protocol for the maintenance and breeding of the titi monkey colony was approved by the University of California, Davis IACUC (Protocol #15730). No specific animal research protocol was drafted for this study, as only excess clinical samples were analyzed for diagnostic purposes. Animals in extreme respiratory distress were humanely euthanized by veterinarians. Extensive veterinary care was provided to all animals affected by the outbreak in order to minimize pain and distress.Serum samples from staff at the CNPRC, close contacts, and random adult blood donors were collected under protocols approved by institutional review boards of the University of California, Davis (Protocol #200917650-1) and University of California, San Francisco (Protocol #H49187-35245-01). Specifically, written informed consent was obtained from staff at the CNPRC and close contacts for analysis of their samples. Any potentially identifying information has been provided with the explicit permission of the individuals involved.Sera from random blood donors were obtained from the Blood Systems Research Institute ; sera were derived from affiliated donor banks in California , Nevada , and Wyoming  and de-identified prior to analysis.Callicebus cupreus) and a colony of over 4,500 rhesus macaques (Macaca mulatta). No new animals have been introduced into either colony for over 2 years. All titi monkeys are maintained in small social groups, while rhesus macaques are maintained in small or large social groups. All animal facilities are maintained in compliance with United States Department of Agriculture specifications.The California National Primate Research Center (CNPRC), which houses over 5,000 nonhuman primates, is a part of the National Primate Research Centers Program and is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). At the beginning of 2009, the CNPRC maintained a colony of 74 titi monkeys (Macaca fasicularis). Three additional titi monkeys were moved into the at-risk room less than 2 weeks after presentation of the index case, reflecting a total at-risk population of 68 animals. The remaining 6 titi monkeys were housed in an indoor animal building greater than 500 yards from the at-risk population .Eighty-eight percent of the titi monkey population (n = 65) were housed in 1 quadrant of an indoor animal building, and all titi monkeys demonstrating clinical signs originated from this building (i.e. “at-risk” room) . Rhesus The outbreak lasted approximately 3 months from May to August of 2009. Affected titi monkeys died from 3–24 days after appearance of clinical signs, with an average time to death or humane euthanasia of 8 days. Clinical and epidemiological data, including daily census reports, were tracked and recorded by veterinary and management staff. All personnel entering the titi monkey rooms  needed to pass within approximately 20 feet of macaque enclosures prior to entry. CNPRC personal protective equipment (PPE) policy requires a change of PPE between entrance/exit of animal rooms housing different species. Staff compliance of this policy may have been compromised. Measures have since been taken by CNPRC management to ensure compliance with existing policies.Bacterial, mycoplasma, and fungal cultures were performed at the CNPRC. Clinical samples were also sent to an outside laboratory  for respiratory viral testing by centrifugation-enhanced shell vial culture followed by direct fluorescent antibody staining for 8 viruses .Gross and histopathological analyses of post-mortem tissues were performed by a board-certified veterinary pathologist specializing in nonhuman primate/laboratory animal medicine, a branch of Primate Services at the CNPRC. At necropsy, tissue samples from the trachea, lung, and liver were collected and fixed in 10% formalin. Tissues were routinely processed and embedded in paraffin. 3-µm sections were stained with hematoxylin and eosin (HE) and examined by light microscopy. For transmission electron microscopy, tissue fragments (2×2 mm) were excised from paraffin blocks of lung, deparaffinized, and processed as previously described Total nucleic acid was extracted from body fluid or swab samples using commercially available kits . 200 µL of sample were passed through a 0.22 µm filter  to remove bacteria and cellular debris and then treated with Turbo DNase  to degrade host genomic DNA prior to extraction. For tissue samples, lung or liver tissue was homogenized in a 15 mL Eppendorf tube using a disposable microtube pestle  and scalpel, and RNA extraction was then performed using TRIzol LS , followed by isopropanol precipitation and two washes in 70% ethanol. Extracted nucleic acid was amplified using a random PCR method to generate cDNA libraries for Virochip and deep sequencing analyses as previously described Adenoviridae genera  yielded an adenovirus signature on the Virochip that was found to be TMAdV. With the exception of SAdV-23, these highly conserved probes are part of the core Virochip design and were derived from all available adenoviral sequences in GenBank as of 2002 The current 8×60 k Virochip microarrays used in this study contain 19,058 70mer probes representing all viral species in GenBank, and combine probes from all previous Virochip designs Virochip analysis was performed as previously described 5′-GTGACGTCATAGTTGTGGTC) and TMAdV-R (5′-CTTCGAAGGCAACTACGC). The TMAdV-specific quantitative real-time PCR was performed on a Stratagene MX3005P real-time PCR system using a 25 µL master mix consisting of 18 µL of water, 2.5 µL of 10X Taq buffer, 1 µL of MgCl2 (50 mM), 0.5 µL of deoxynucleoside triophosphates , 0.5 µL of each primer (10 µM), 0.5 µL SYBR green, 0.5 µL of Taq polymerase , and 1 µL of extracted nucleic acid. Conditions for the PCR reaction were 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Amplicons were purified on a 2% agarose gel, cloned into plasmid vectors using TOPO TA , and sent to an outside company  for Sanger sequencing in both directions using vector primers M13F and M13R.We initially used consensus primers derived from a highly conserved region of the hexon gene to confirm the Virochip finding of an adenovirus by PCR 5 copies/mL) was performed using SPSS 16.0 . By probit analysis, the 95% limit of detection for TMAdV was 781, 377, or 35 viral genome equivalents/mL for serum, stool, or oral swab samples, respectively (data not shown).To assess linearity and limits of sensitivity for the TMAdV PCR assay, 12 serial log dilutions were made of a standard plasmid constructed by cloning the 157-bp TMAdV amplicon into a TOPO plasmid vector. Purified plasmid clones at each serial dilution were quantified using a Nanodrop spectrophotometer and then spiked into negative serum, stool, or oral swab sample matrix, each matrix consisting of a pool of 10 sera, 10 stool samples, or 3 oral swabs, respectively. For each sample type, a standard curve for the TMAdV PCR assay was calculated from 3 PCR replicates at each dilution of nucleic acid extracted from the spiked matrix (data not shown). To determine limits of sensitivity for the assay, probit analysis of results from 6 PCR replicates of 7 serial log dilutions (from a starting concentration of ∼1.2×10GsuI) and truncated adapters were ligated on the resulting strand ends. Full-length adapters containing strict 6-nt barcodes were added via an additional 15 cycles of PCR. Amplified libraries were size-selected on a 2% agarose gel at approximately 350 bp average length and then sent to an outside company  for deep sequencing on an Illumina Genome Analyzer IIx . Paired-end reads were sequenced for 73 cycles in each direction. Paired-end reads were subsequently filtered to eliminate low-complexity sequences with a Lempel-Ziv-Welch (LZW) compression ratio below 0.4 −5). After low-complexity filtering and barcode/primer trimming, 11,950,557 sequence reads remained, with each read consisting of 67 nucleotides, for a total of ∼800 megabases of sequence. Reads were then aligned using BLASTN  and TBLASTX  to the genome sequence of SAdV-18 . To confirm the generation of infectious virus, viral supernatants were quantitated by an end-point dilution assay.A549 (human lung adenocarcinoma) and BSC-1  cell lines as well as PMK (primary rhesus monkey kidney) cells are routinely maintained at the Viral and Rickettsial Disease Laboratory (VRDL) branch of the California Department of Public Health. Media consisting of Hank's medium (for A549 cells) or Dulbecco's modified Eagle's medium (DMEM) (for BSC-1 cells) were supplemented with 1×nonessential amino acids , 10% fetal bovine serum, 100 U of penicillin/mL and 100 µg of streptomycin/mL. PMK cells were maintained in tubes containing growth media and antibodies to SV-40 and SV-5 polyomaviruses . Clinical samples were clarified by centrifugation for 10 min×4000 50 and 55 µL of serum (starting at a 1∶8 dilution) were mixed and incubated for 1 hour at 37°C. As a control for each serum sample, 55 µL of culture media and 55 µL of serum were mixed and treated in an identical fashion. While mixtures were incubating, A549 cells grown in T-25 plates were trypsinized and 4,000 cells in 100 µL of media were added to each well of a 96-well plate. After incubation, 100 µL of mixture were inoculated into appropriate wells containing 4,000 cells per well and the entire plate was placed in a 37°C 5% CO2 incubator. Cells in the plate wells were observed for evidence of CPE every other day for 1 week. For wells that showed inhibition of viral CPE, the corresponding serum samples were diluted in six 2-fold steps and then retested. The reciprocal of the highest dilution that completely inhibited viral CPE was taken as the neutralizing antibody titer.A virus stock of TMAdV (passage 7) was produced on human A549 cells, aliquoted, and quantitated by end-point dilution. To perform the virus neutralization assay, 55 µL of viral supernatant at a concentration of 100 TCID50 were mixed, incubated for 1 hour at 37°C, and inoculated onto A549 cells in wells of a 96-well plate as described above. Cells in the plate wells were observed for evidence of CPE every other day for 1 week.To assess cross-neutralization of TMAdV by known human adenoviruses, 7 pools of in-house reference sera at the VRDL (rabbit hyperimmune sera) and collectively containing antibodies to human adenovirus serotypes 1 through 35 were available for testing. For each pool, 55 µL of rabbit sera and 55 µL of viral supernatant at a concentration of 100 TCIDAll Virochip microarrays used in this study have been submitted to the NCBI GEO database . The annotated, whole-genome sequence of TMAdV has been submitted to GenBank (accession number HQ913600). Deep sequencing reads have been submitted to the NCBI Sequence Read Archive (accession number SRA031285).Figure S1Phylogenetic analysis of the hexon, polymerase, penton base, and fiber genes of TMAdV. A multiple sequence alignment of selected genes from all 95 unique, fully-sequenced adenovirus genomes in GenBank and TMAdV is performed and the results displayed as a radial phylogenetic tree. The branch corresponding to TMAdV is highlighted in boldface red. Abbreviations: HAdV, human adenovirus, SAdV, simian adenovirus; PAdV, porcine adenovirus; FAdV, fowl adenovirus.(TIF)Click here for additional data file.Figure S2Bootscanning recombination analysis of TMAdV. Bootscanning analysis was initially performed with all 95 unique, fully-sequenced adenovirus genomes in GenBank (data not shown). After removal of similar viral genomes, bootscan plots of the whole genome and individual genes from a subset representing human/simian adenoviruses in species A–G and all non-primate vertebrate adenoviruses were generated. The window size is 400 bp with a step size of 40 bp for the whole genome, and 200 bp with a step size of 20 bp for the individual genes. The x-axis refers to the nucleotide position. For definition of abbreviations, please refer to (TIF)Click here for additional data file.


Airborne fungi are responsible for the majority of fungal infections in humans and animals. Outdoor air markedly influences the prevalence of fungal spore levels in indoor air and thus, it is the major source of fungal infections in indoor environments especially in hospitalized individuals.Using a settle plate method, air sampling (1092 air samples from 93 sampling sites in 22 geographic regions of Tehran) was performed by exposing 90 mm settle plates containing Malt extract agar and Potato dextrose agar to the air for 30 min. The plates were incubated at 28°C for 2–3 weeks and examined daily for visible fungal growth. Purified fungal colonies were identified at the genus level based on morphological criteria according to standard methods.Aspergillus (31.3%) was the most prominent isolated fungus followed by Cladosporium (22.1%), Penicillium (13.8%) and Alternaria (12.2%).A total of 6455 colonies belonging to 24 different fungal genera were isolated. Area V situated in the city center was the most contaminated region with 2523 fungal colonies (39.1%), while area IV in the West showed the least contamination rate . Airborne fungi isolated were classified into 4 classes including hyaline Hyphomycetes (53.5%), dematiaceous Hyphomycetes (41.6%), Zygomycetes (2.8%) and Coelomycetes (0.2%). Our results indicate that outdoor air is a potential threat to public health because of harboring a wide array of pathogenic and allergenic airborne fungal spores which can serve as the main source of contamination of indoor environments such as homes, offices and hospitals. Exposure to airborne pathogens is a major risk factor for human health . It has Cladosporium, Alternaria, Aspergillus and Penicillium, probably because they are very often the most prevalent genera in ambient air [Cladosporium, Alternaria, Penicillium, and Aspergillus comprise the major part of fungal community in the atmosphere. It has been demonstrated that the abundance of airborne fungal spores in outdoor air vary from place to place and it influences mainly by climatic conditions and human activities [Among the approximately 100,000 known species of fungi, those of interest in outdoor and indoor environments belong to the class of Deuteromycetes or Fungi Imperfecti, with a few exceptions . Although indoor environment itself is considered as a source of indoor fungi by growing them in building materials, foodstuffs, flower pots, pet bedding materials and house dust, outdoor sources are usually dominant for indoor contamination by pathogenic fungal species . In suitient air -26. WithSeveral studies from various parts of Iran such as Sari, Shiraz and so on demonstrated a wide array of microfungi in air samples from outdoor environments ,27-29. ITehran is situated at an altitude of 1100–1800 meters above sea level and within the latitude and longitude of 35° 44' N, 51° 33' E. The city is located at the folded zone of Alborz mountains which the deposits are mainly a period of limestone and dolomite and has many rivers and springs. The climate is mountainous temperate and semi-arid with average annual rainfall of approximately 500 mm and a mean relative humidity of 60%. On the basis of Tehran’s map, all 22 district regions (1 to 22) were included in our study. As illustrated in Figure et al.[A total of 1092 air samples were prepared from 93 sampling sites during July 2012 using settle plate method according to Hoekstra et al.. Samplinet al.. After tThe plates were incubated at 28°C for 2–3 weeks for confidence about growing all airborne fungal conidia and examined daily for any visible fungal growth . Fungal Aspergillus (31.3%) was the most prominent fungal genus isolated followed by Cladosporium (22.1%), Penicillium (13.8%) and Alternaria (12.2%). The members of these genera were the only fungi which were isolated from outdoor air of all areas from I to V.In the present study, distribution and diversity of outdoor airborne mycoflora in all 22 geographic sections of Tehran was evaluated using settle plate method during summer 2012. A total of 1092 settle plates of outdoor air sampled from 93 sampling sites were studied. As indicated in Table Aspergillus was the most prevalent genus in fungi belonging to hyaline Hyphomycetes, Cladosporium in dematiaceous Hyphomycetes and Rhizomucor in Zygomycetes. The genus Phoma was the exclusive fungus identified in the Coelomycetes with a total frequency of 0.2%.As shown in Figure Rhizomucor, Mucor, Absidia and Rhizopus isolated in the present study from outdoor air, the genus Basidiobolus was interestingly reported from eleven samples in Area V account for 0.17% of the total isolates.Besides widely distributed genera of Zygomycetes including Aspergillus was the most predominant fungus accounting for nearly 30% of total isolates followed by Cladosporium, Penicillium and Alternaria. It may be due to the fact that Aspergillus, Penicillium and Cladosporium produce numerous small and light spores that generally remain in the air for a long period of time, whereas other fungal genera produce fewer, larger and heavier spores which tend to have faster settling [Cladosporium, Alternaria, Curvularia, Aspergillus and Penicillium[Penicillium was the most abundant species, comprised more than 50% of the total isolates followed by Cladosporium, Alternaria, Penicillium and Aspergillus. Wu et al.[Cladosporium, Penicillium, Curvularia and Aspergillus. Ataygul et al.[Cladosporium, Alternaria, Aspergillus, Penicillium, Fusarium, Epicoccum, Drechslera, Pithomyces, Stemphylium, Chaetomium, and Curvularia were identified with dominancy of Cladosporium with 88.11% frequency. Using gravitational settle plate method, occurrence of airborne fungi in the outdoor environment of Trabzon, Turkey was evaluated by Topbas et al.[Penicillium, Alternaria and Fusarium as the most prevalent fungi in different seasons. O’Gorman and Fuller [Cladosporium, Penicillium, Aspergillus and Alternaria spores were constantly present in the Dublin atmosphere, representing more than 20% of the total culturable spore count. Airborne spores of A. fumigatus and basidiospores of Schizophyllum commune were detected in some samples. A one-year prospective survey of fungal air contamination was conducted in outdoor air of a French hospital [Cladosporium was the dominant genus with a frequency of 55% in total isolated fungi. In a comprehensive study on airborne fungi of outdoor air in Basrah city, southern Iraq, Cladosporium was reported as the most dominant species with 31.3% frequency (highest number in winter) followed by Penicillium notatum (11.9%), Alternaria alternata (10.0%) and Aspergillus niger (5.8%) [In the present study, fungal air quality of outdoor environments of Tehran, the capital of Iran was evaluated by sedimentary sampling using settle plate method. This method is simple and inexpensive and provides an integrated assessment of exposure over longer periods than that of most volumetric methods . All thesettling . In sevenicillium,13,17-26nicillium. PenicilWu et al. studied ul et al. evaluateas et al.. They red Fuller  studied hospital . Cladospr (5.8%) .et al.[Alternaria (63.86%) was the prominent isolated fungus followed by Cladosporium (11.81%). Shokri et al. reported the genera Mucor, Cladophialophora, Alternaria, Aspergillus, Penicillium, Fusarium and Rhizopus as the dominant fungi in the air of forests and seashore areas of Babol city, Northern Iran [Penicillium, Cladosporium, Aspergillus and Fusarium had the highest frequency among the total fungi [et al. on the air samples of Babol-Iran and in contrast with those reported by Chadeganipour et al. indicating 18.6% frequency of yeasts in the air of Isfahan-Iran [Several studies have been conducted in Iran and wide arrays of fungi are reported as dominant air mycoflora. In a survey of outdoor air of Qeshm Island, Southern Iran, a predominance of dematiaceous fungi versus hyaline Hyphomycetes were reported by Barrati et al.. Theses ern Iran . In a pral fungi . We did han-Iran .Cladosporium, Penicillium, Alternaria and Aspergillus[Cladosporium spp., Alternaria spp., Epicoccum nigrum, and Botrytis cinerea are known to be an integral part of the fungal air spora outdoors which cover more than 90% of the total fungal spore load. The fungal spore load of penicillia and aspergilli is in the range of 2–10% and 1–3%, respectively [Alternaria, Aspergillus, Cladosporium and Penicillium were reported from Tehran air as the most prominent fungi. These fungi are known causative agents of fungal allergies [Penicillium, Aspergillus and Fusarium are relevant as mycotoxin producers [It has been reported that more than 180 genera of airborne fungi with worldwide distribution are associated with allergies and serious human and animal infections . As mostectively . The micllergies . Besidesroducers ,40. Thesroducers ,39. If aAspergillus, Fusarium and Zygomycetes isolated from outdoor air in the present work are fungal pathogens responsible for infection in immunocompromised patients. Risk patients need to be protected from these fungal pathogens, particularly by isolation in highly-restricted units, as most of these agents are airborne fungi originates from outdoor environment [Several species belonging to the genera ironment .Aspergillus, Penicillium, Cladosporium and Alternaria must be considered as major threats for public health because they are main sources of contamination of indoor environments such as homes, offices and hospitals.In general, results of the present study clearly showed that airborne fungi from different groups are present as real contaminants of outdoor air of Tehran. These fungi especially those involved in the etiology of fungal diseases and allergies such as All authors declare that they have no competing interest.MSG and MRA participated in the design of the study and MSG supervised the work. SAG and NA did the analyses, and/or interpreted the analyzed results. MSG and MRA wrote the initial draft and revised the paper critically for important intellectual content and compiled the work in accordance to journal format. All authors have read and approved the final manuscript.


Sparganium stoloniferum, an herb historically used in Traditional Chinese Medicine as an anti-tumor agent. Angiogenesis, the process of new capillary formation from existing blood vessels, is dysregulated in many pathological disorders, including diabetic retinopathy, tumor growth, and atherosclerosis. In functional assays, SsnB inhibited endothelial cell tube formation (Matrigel method) and cell migration (Transwell method) in a dose-dependent manner. Microarray experiments with human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) demonstrated differential expression of several hundred genes in response to SsnB exposure . Microarray data from both cell types showed significant overlap, including genes associated with cell proliferation and cell cycle. Flow cytometric cell cycle analysis of HUVECs treated with SsnB showed an increase of cells in the G1 phase and a decrease of cells in the S phase. Cyclin E2 (CCNE2) and Cell division cycle 6 (CDC6) are regulatory proteins that control cell cycle progression through the G1/S checkpoint. Both CCNE2 and CDC6 were downregulated in the microarray data. Real Time quantitative PCR confirmed that gene expression of CCNE2 and CDC6 in HUVECs was downregulated after SsnB exposure, to 64% and 35% of controls, respectively. The data suggest that SsnB may exert its anti-angiogenic properties in part by downregulating CCNE2 and CDC6, halting progression through the G1/S checkpoint. In the chick chorioallantoic membrane (CAM) assay, SsnB caused significant reduction in capillary length and branching number relative to the vehicle control group. Overall, SsnB caused a significant reduction in angiogenesis , demonstrating its ex vivo efficacy.Sparstolonin B (SsnB) is a novel bioactive compound isolated from  Sparganium stoloniferum, a perennial, aquatic plant grown in North and East China, whose tubers have long been used in Traditional Chinese Medicine (TCM) for the treatment of several inflammatory diseases and as an anti-spasmodic and anti-tumor agent. NMR and X-ray crystallography have identified SsnB as a polyphenol with structural similarities to isocoumarins, a class of compounds that often exhibit anti-coagulant, anti-inflammatory, and anti-tumor properties Sparstolonin B (SsnB) is a novel bioactive compound isolated from the plant Angiogenesis refers to capillary formation from existing blood vessels. Angiogenesis plays an important role in many physiological events in the body, including wound healing, embryogenesis, and female reproductive processes Angiogenesis is a complex process that occurs in several stages and involves interactions between cells, soluble factors, and extracellular matrix molecules. Endothelial cells play a key role in angiogenesis. Endothelial cells can secrete proteolytic enzymes which break down the basement membrane of an existing blood vessel, allowing the cells to invade the surrounding tissues, migrate in response to an angiogenic stimulus, and proliferate. Growth factors and other soluble proteins in the ECM often facilitate and regulate this process, including vascular endothelial growth factor (VEGF). The endothelial cells form a new lumen, start to secrete extracellular matrix molecules, and ultimately form a new capillary in vitro functional assays, and we also show that SsnB inhibits angiogenesis in an ex vivo assay. The data suggests the potential of SsnB as an anti-angiogenic agent by showing inhibition of human endothelial cell tube formation and migration. We have also examined the effects of SsnB on endothelial cell gene expression, focusing in particular on pathways related to angiogenesis. In addition, our data show that SsnB arrests endothelial cell division in the G1 phase and downregulates the cell cycle proteins cdc6 and cyclin E2. Furthermore, SsnB caused a significant reduction in blood vessel length and branching number in the chick chorioallantoic membrane assay. Overall, these findings add anti-angiogenic and cytostatic properties to the list of diverse effects exhibited by SsnB.In the present study, we demonstrate that SsnB inhibits endothelial cell functions related to angiogenesis in several Sparganium stoloniferum according to previously published methods Sparstolonin B was purified from the plant Cell Culture – Human coronary artery endothelial cells (HCAECs), human umbilical vein endothelial cells (HUVECs), and human cardiac microvascular endothelial cells (HMVECs) were obtained from Lonza  and cultured on polystyrene, tissue culture-treated petri plates (100×20 mm) coated with 0.1% gelatin. HUVECs, HCAECs, and HMVECs were cultured in endothelial cell medium supplemented with 10% fetal bovine serum (FBS) and endothelial cell mitogen/growth supplement . The endothelial cell medium was replaced every 2–3 days, and the cells were passaged after complete confluence was reached. Confluent plates were trypsinized and split, and the cells were cultured until the fourth passage was reached.To initially determine if SsnB inhibited pro-angiogenic cell functions, a tube formation assay with Matrigel was performed. Growth factor reduced Matrigel  was added to the wells of a 96 well polystyrene culture plate and incubated at 37°C for 30 minutes. Cells  were added to each well to reach a final number of 20,000 cells per well. SsnB was added to the wells at a concentration of 1, 10, or 100 µM. Endothelial cell medium with DMSO (0.1%) was used as a vehicle control. Each group contained 4 replicates. The plates were placed in an incubator for 4 h. During the incubation, the endothelial cells formed elongated structures called cords, also known as tubes. After 4 h, neutral buffered formalin was added to fix the cells. Pictures of three non-overlapping fields were taken from each well. The lengths of single cell endothelial cords were measured with Image-Pro Plus , and the sum of tube lengths for each well was determined. The average total length and standard deviation for each group were determined, and the appropriate statistical tests (ANOVA and Newman-Keuls) were completed. The tube formation assay was replicated three times for both HUVECs and HCAECs. The assay was also repeated with cardiac HMVECs.A Live/Dead assay  was utilized to investigate the effect of SsnB on cell viability. The Matrigel tube formation experiment was repeated with HUVECs in chamber slides at a concentration of 20,000 cells per well. The cells were treated with SsnB  or Vehicle Control (0.1% DMSO) as described above. After four hours of incubation, the slides were removed. A chamber slide containing HUVECs treated with 70% methanol for 30 minutes was used as a control for dead cell staining. The slides were aspirated and washed with PBS, and EthD-1 and calcein AM were added to each well. The plates were incubated in the dark, and images were taken with a light microscope at 10X magnification.The cell invasion assay was performed with a Transwell insert system . The Transwell inserts were coated with 0.1% gelatin for 30 min and incubated in low serum medium for 1 h. Cultured HUVECs were trypsinized and then resuspended in low serum medium , and added at 50,000 cells per insert. The cells were allowed to adhere to the inserts for 30 min. Next, various concentrations of SsnB  or vehicle control (0.1% DMSO) were added to the Transwell inserts. After 30 min, the medium in the lower chamber for the experimental groups was replaced with low serum medium containing 10 ng/ml VEGF, establishing a chemoattractant gradient between the top insert and lower chamber. For the negative control group, the medium was replaced with low serum medium . The plates were incubated for 8 h at 37°C. During the incubation, the cells migrated through the pores of the Transwell insert towards the lower chamber. At the end of this period, cells on the upper surface of the insert were removed, and migrated cells on the bottom side were fixed in formalin and stained with Hoechst dye (a fluorescent nuclear stain). The filter inserts were removed from the wells and mounted on glass slides. Cells were counted from four random fields observed with a 10X objective lens. The cell migration experiments were repeated three times at high SsnB concentrations  and one time at low SsnB concentrations  for HUVECs.Cell cycle analysis was performed using propidium iodide staining and flow cytometry. HUVECs (approximately 75% confluent) cultured in 6 well polystyrene culture plates coated with 0.1% gelatin were serum starved for 24 hours in low serum medium containing 0.5% fetal bovine serum and no endothelial cell mitogen to synchronize the cells in the G0/G1 phase. After 24 hours, the low serum medium was replaced with treatments of either 100 µM SsnB or vehicle control (0.1% DMSO) diluted in complete growth medium  in triplicate. The treated cells were incubated for 24, 30, and 36 hours. After incubation, the cells were trypsinized, transferred to 5 ml polysytrene round bottom tubes (12×75 mm), and centrifuged. The medium was aspirated, and the cells were washed with PBS. After fixing the cells with ice-cold 70% ethanol for 15 min, the cells were centrifuged and stained with propidium iodide for 30 min. The samples were then analyzed with a flow cytometer (Beckman Coulter FC500). The unstained and stained cell groups were used to calibrate the settings on the flow cytometer. The data were collected and analyzed with ModFit software.Confluent plates  of HUVECs and HCAECs  were chosen for the microarray experiments. Half of the plates received complete growth medium  containing vehicle control (0.1% DMSO), and the remaining plates received complete growth medium containing 100 µM SsnB. The plates were incubated for 24 h to allow SsnB to have an effect on cellular gene expression. Following incubation, RNA isolation was completed with the RNeasy Mini kit from Qiagen. The cells were lysed, and RNA was isolated by using the RNeasy spin columns and following the protocol provided by Qiagen. Purified RNA was sent to the Medical University of South Carolina Proteogenomics Facility for microarray analysis. The GeneChip Human Genome U133 Plus 2.0 Array was utilized to track changes in gene expression due to SsnB treatment. Complete data was uploaded to the NCBI Gene Expression Omnibus database (accession number GSE44598).After a careful analysis of the microarray data, key genes  were chosen for quantitative real-time PCR (qRT-PCR) to verify the gene expression results. HCAECs and HUVECs were exposed to SsnB or vehicle control for 24 h (as previously described in the microarray section). For RT-PCR, RNA was isolated from the cells with the RNeasy Mini kit as described previously. After forward and reverse primer kits  were selected, the RNA was amplified. The one-step RT-PCR reactions were completed on the BioRad iCycler thermal cycler system in the Instrumentation Resource Facility at the USC School of Medicine. The expression levels were normalized, and RNA levels were quantified.t method.Microarray data analysis, including data normalization (robust multi-array average), identification of differentially expressed genes , and heat map construction, was carried out to determine how SsnB affects gene expression and affected pathways (n = 2 for HUVECs and n = 3 for HCAECs). After a careful analysis of the microarray data, several key genes were chosen for qRT-PCR to verify the gene expression results. The genes were chosen based on the following criteria: fold change ≥2, p<0.05, unpaired t-test with a false discovery rate approximating 0%, appearing in both data sets (HUVECs and HCAECs), and gene function relating to cell proliferation and/or angiogenesis. For qRT-PCR, the expression levels were normalized to the housekeeping gene GAPDH, and RNA levels were compared between groups with the ΔΔC2) for 2 days. After 2 days of incubation, the methylcellulose discs containing either SsnB or vehicle control were placed on top of the CAM of each embryo in the right upper quadrant in an area near the allantoic vessels. After 2 additional days of incubation, the CAMs were examined and photographed with a dissecting stereomicroscope. Images of areas containing the discs and non-treated regions were analyzed with Image-Pro Plus software. Blood vessel length and branching number were quantified to measure the effect of SsnB on angiogenesis. The total vessel length was measured for areas containing the discs and for non-treated areas of the same size. Normalized vessel length was calculated by dividing the total length values of the treated regions by those of the non-treated regions. The same process was repeated for measuring the branching number. Each group contained seven embryos . The CAM assay was completed two times.Fresh fertilized Bovans eggs were obtained from Clemson University and stored at 4°C for no longer than one week. Methylcellulose discs were created with a solution of 0.5% methylcellulose (diluted in sterile deionized water). A methylcellulose solution of 100 micromolar SsnB was made by adding 1.1 microliters of stock SsnB (93 mM) to 1 ml 0.5% methylcellulose. A vehicle control solution was created by adding 1 microliter of dimethyl sulfoxide (DMSO) to 1 ml 0.5% methylcellulose. Discs were created by placing 20 microliter droplets of SsnB or vehicle control solution onto polytetrafluoroethylene tape (to minimize adherence during drying) and allowed to dry under a laminar flow hood. The fertilized eggs were placed in a humidified egg incubator with forced air circulation at 37°C. The eggs were automatically rotated every three hours. After 4 days of incubation, the eggs were removed and cracked. Chick embryos with intact yolks were placed in 100 mm petri plates. Each petri plate was placed in a weigh boat containing 13 ml Moscona's buffer and covered with cellophane wrap with two holes for air circulation. The plates were placed in a water-jacketed incubator . Following microarray data analysis, key genes  and cyclin dependent kinase 1 (CDC2). CCNB1 and CDC2 are regulatory proteins that control progression through the G2/M checkpoint. Downregulation of CCNB1 and CDC2 will trap cells at the G2/M checkpoint and prevent endothelial cells from entering the final stages of cell division. Sparganium stoloniferum has long been used in Traditional Chinese Medicine for the treatment of cancer and seizures. Major chemical components from the stem and rhizome, including flavonoids, saponins, and phenylpropanoids, are responsible for these therapeutic effects Sparganium stoloniferum have demonstrated potent anti-cancer effects. Isolated chemical compounds from this herb, including a sucrose ester, a phenylpropanoid glycerol, carboxylic acid esters, and a phenylpropanoid glycoside, demonstrated anti-tumor activities In addition to its effects on the cell cycle and cell proliferation, SsnB also affects other key steps of angiogenesis, including cell migration and chemotaxis. The microarray data for both HUVECs and HCAECs demonstrates an enrichment of genes associated with both pathways, including diaphanous homolog 3 (DIAPH3), hyaluronan-mediated motility receptor (HMMR), and anillin (ANLN), an actin binding protein. Furthermore, it is also important to investigate potential upstream pathways that lead to the alterations in gene expression seen in the microarray data. Previously published data have shown that SsnB can suppress the NF-κB pathway, and prior studies have demonstrated a link between NF-κB signaling and expression of cell cycle proteins. Genes for these regulatory proteins, such as CCNE2, CDC6, CCNB1, and CDC2, contain binding sites for NF-κB. SsnB may suppress the NF-κB pathway and cause a subsequent downregulation of the cell cycle regulatory proteins CCNE2 and CDC6. SsnB has also been shown to inhibit the MAPK pathways, including JNK, ERK, and p38 signaling, which all affect gene expression and cell proliferation ex vivo system involving additional cell types beyond endothelial cells. Multiple cell types are involved in angiogenesis, including endothelial cells and supporting cells, such as pericytes, smooth muscle cells, macrophages, and myofibroblasts. Since our initial in vitro experiments only involved endothelial cells, we utilized the CAM assay to address the multicellular nature of angiogenesis. We are particularly interested in how SsnB affects macrophages. In the CAM assay, SsnB inhibited blood vessel branching. It has been demonstrated that macrophage production of matrix metalloproteinase-9 (MMP-9) plays an important role in vascular branching during neovascularization Furthermore, the CAM assay allowed us to examine the effects of SsnB in an ex vivo angiogenesis in the CAM assay. Future work with SsnB will reveal more about its mechanism of action, including cytoskeletal changes and possible binding sites. Overall, SsnB has proven to be a powerful bioactive compound with a multitude of useful therapeutic effects, including anti-angiogenesis and anti-inflammation.Our current study with HUVECs and HCAECs demonstrates that SsnB inhibits angiogenic processes in functional assays, including the Matrigel tube assay and the Transwell insert cell migration assay, and that it downregulates multiple genes involved in pro-angiogenic pathways as shown by microarray analysis. We have also shown that SsnB inhibits Figure S1SsnB inhibits endothelial cell tube formation on Matrigel. Total tube length as a function of SsnB concentration in HMVECs, *p<0.05 vs. vehicle control, Newman-Keuls test.(TIFF)Click here for additional data file.Table S1Gene function enrichment analysis for HUVECs in response to SsnB treatment.(DOCX)Click here for additional data file.Table S2Gene function enrichment analysis for HCAECs in response to SsnB treatment.(DOC)Click here for additional data file.


Electrospun materials have been widely explored for biomedical applications because of their advantageous characteristics, i.e., tridimensional nanofibrous structure with high surface-to-volume ratio, high porosity, and pore interconnectivity. Furthermore, considering the similarities between the nanofiber networks and the extracellular matrix (ECM), as well as the accepted role of changes in ECM for hernia repair, electrospun polymer fiber assemblies have emerged as potential materials for incisional hernia repair. In this work, we describe the application of electrospun non-absorbable mats based on poly (PET) in the repair of abdominal defects, comparing the performance of these meshes with that of a commercial polypropylene mesh and a multifilament PET mesh. PET and PET/chitosan electrospun meshes revealed good performance during incisional hernia surgery, post-operative period, and no evidence of intestinal adhesion was found. The electrospun meshes were flexible with high suture retention, showing tensile strengths of 3 MPa and breaking strains of 8–33%. Nevertheless, a significant foreign body reaction (FBR) was observed in animals treated with the nanofibrous materials. Animals implanted with PET and PET/chitosan electrospun meshes  showed, respectively, foreign body granuloma formation, averaging 4.2-fold and 7.4-fold greater than the control commercial mesh group (Marlex). Many foreign body giant cells (FBGC) involving nanofiber pieces were also found in the PET and PET/chitosan groups . In contrast, no important FBR was observed for PET microfibers (fiber diameter = 18.9±0.21 µm). Therefore, we suggest that the reduced dimension and the high surface-to-volume ratio of the electrospun fibers caused the FBR reaction, pointing out the need for further studies to elucidate the mechanisms underlying interactions between cells/tissues and nanofibrous materials in order to gain a better understanding of the implantation risks associated with nanostructured biomaterials. Electrospinning has attracted the interest of researchers from many fields as a versatile technique to produce nanofibers from synthetic and naturally derived polymers. With typical diameters ranging from 10 nm to a few micrometers, these fibers are usually collected continuously as nonwoven fibrous mats. These mats usually show a tridimensional nanostructure with high surface-to-volume ratio, high porosity, and interconnectivity, and they have demonstrated high potential for biomedical applications, such as tissue engineering scaffolds, vascular grafts, and drug delivery systems in vitro prompted us to test these nondegradable electrospun mats as abdominal meshes for incisional hernia repair.In the past decade, our research group has been exploring the potential of electrospinning for different applications Incisional hernia is a frequent complication of laparotomy resulting from the decrease of abdominal strength in the injured tissue. The incidence of incisional hernia after abdominal surgery depends on the pattern of the incision performed. For a midline incision, the preferred incision for the upper abdominal surgery, the incidence lies around 10–14% co-glycolide), latter loaded with an antibiotic, was shown to provide good mechanical properties, while imparting antibacterial activity and, hence, reducing the risk of infection during application of the composite material to abdominal wall closure Absorbable meshes are also used as scaffolds for abdominal defect correction. In this case, the biodegradable mesh provides support for cell growth and for extracellular matrix secretion, promoting the tissue repair process. A few recent reports have shown successful results in exploring partially degradable or absorbable electrospun mats as abdominal meshes. An electrospun blended fiber mesh prepared from biodegradable poly(ester urethane) urea and poly(lactide-−3) and the high malleability of this material may promote enhanced adaptation and, consequently, patient comfort et al. have demonstrated that a better incorporation of the mesh in the parietal side reduces the number and the severity of adhesion formations on the visceral side Despite the advantages of absorbable materials, the newly formed tissue has a decreased tensile strength and thereby, re-herniation is a frequent problem after the absorption of the prosthetic material. In this context, electrospun mats of nondegradable polymers have emerged as potential alternative meshes for abdominal defect repair. Because of its unique properties, i.e., unaligned nanofibrous arrangement, microporosity, and high hydrophobicity, the PET electrospun mat has emerged as a potential candidate for abdominal wall repair in vivo study with an abdominal hernia Wistar rat model was performed to evaluate the clinical and histological aspects of using these meshes for abdominal hernia repair.In this paper, we describe the application of three electrospun nonabsorbable mats, including PET, PET/C and DL, in the repair of abdominal defects, comparing the performance of these nanofibrous meshes with that of a commercial polypropylene mesh (Marlex) (control) and a multifilament microfibrous PET mesh. An Animal experiments were approved by the Animal Ethics Committee of the Federal University of Santa Catarina (PP0406/2009).Cirurgica Passos, Brazil. Chitosan medium molecular weight (15% acetylation degree) was purchased from Sigma-Aldrich Chemical Company. The molecular weight of the initial chitosan sample (1500 kDa) was reduced to 15 kDa by oxidative depolymerization PET pellets and PET woven fabric were kindly supplied by Flexitex . Marlex (Intracorp) was purchased from Thirty percent (w/v) PET solution was prepared in a blend of trifluoroacetic acid and dichloromethane [8∶2 v/v] by moderate stirring for 2 hours at room temperature. PET/C blend was prepared by adding chitosan (6 wt. %) to the PET 30 wt. % solution and stirring for 3 hours at room conditions.Electrospinning was performed using a typical experimental setup previously described The double-layer (DL) mesh was fabricated by electrospinning 10 mL of PET solution, followed by 10 mL of PET/C solution. Fibers were collected as a nonwoven fibrous mat on the rotating drum (900 rpm), in air and at room conditions , and the mat was dried at 35°C for 24 hours.The morphology of the fibrous scaffolds was investigated by scanning electron microscopy (SEM). Small sections of the scaffolds were sputter-coated with gold and analyzed using a scanning electron microscope (Hitachi S4100) at an accelerating voltage of 25 kV.Image processing was performed using ImageJ - 1.37c software . Five random images (1000 X magnification) were obtained for each sample. Fiber diameters were calculated from at least 100 measurements of the sample fibers. SEM images were thresholded, and pore areas were automatically calculated using the “analyze particle” tool of ImageJ (n >200).Mechanical properties in tension were evaluated using texture analyzer equipment  equipped with fixed grips lined with thin rubber on the ends. Test specimens 90 mm long×10 mm wide were obtained perpendicular to the axis of the collector rotation, and the ends were mounted on the grips using sticky tape. The thickness of the test samples was measured at different locations on each sample using a digital micrometer . The initial grip separation was set at 50 mm, and the crosshead speed was 0.5 mm/s. At least eight samples of each mat were tested.The wettability of scaffolds was assessed by the sessile drop method using an OCA-20 contact angle system (DataPhysics Instruments). A drop of distilled water (1 µL) was automatically dispensed on the scaffold surface, and the WCA and drop life times were calculated using the SCA 20 software. At least 10 measurements were taken for each sample.Male Wistar rats 3 months of age and weighing 250–300 g were obtained from the Central Biotery of the Federal University of Santa Catarina. Animals were randomly distributed among the 7 treatments , according to Before biological assay, all meshes were sterilized under UV light for 1 hour of exposure (both faces), immersed in ethanol 70% (v/v) for 10 minutes, and washed with sterile physiologic solution.A graphical illustration of the surgical procedure is provided . After aOn day 30 and 90 post-surgery, animals were sacrificed in a carbon dioxide chamber, and the presence of intestinal adhesions was analyzed. The abdominal wall was carefully excised well away from the mesh see  to preseTissues were excised from the animals and fixed in phosphate buffered formaldehyde solution , embedded in paraffin, and sectioned at 4 µm thickness. Giemsa, hematoxylin and eosin (HE), and Garvey’s staining were performed Statistical analysis was carried out using Instat 3.0 software. Results were expressed as the mean ± standard deviation and compared through one-way ANOVA and Tukey-Kramer.2. Addition of chitosan promoted a substantial increase in fiber diameter and pore area to 3.01±0.72 µm and 89.3 µm2, respectively. Compared to PET/C, PET mesh showed superior mechanical properties with a higher tensile strength (3.17±0.23 MPa compared to 2.89±0.27 MPa), Young’s modulus (120±10 compared to 70±10 MPa), and elongation (32.8±5.7% compared to 8.2±1.3%). Also, a decrease in the hydrophobicity of the mesh was observed by the presence of chitosan (WCA decreased from 133.2±2.9° to 125.2±4.6° with the addition of chitosan).The electrospun mats were evaluated in an incisional hernia experiment with Wistar rats and compared to Marlex, the control. Following the creation of the abdominal defect by resection of 1.5×1.5 cm of the Wistar rats’ abdominal muscle, the prosthetic meshes were implanted and fixed through the borders to the remaining muscle see . Suture During the post-operative period, some complications, such as local redness, abscess, or skin dehiscence, were registered. Histological analysis was performed to evaluate the cellular response to the prosthetic biomaterials. Long-term inflammatory response was evaluated for two selected groups (Marlex and electrospun PET) 90 days after mesh implantation. Both groups showed a decrease in inflammation with time. The average granuloma thickness of the electrospun PET group decreased from 959±473 µm to 513±217 µm, and the number of FBGC decreased from 106±30 to 89±12.in vivo, promoting improved tissue integration and rapid resorption Several biomaterials have been used as prosthetic meshes for abdominal wall repair, in particular, incisional hernia repair. Among them, nonabsorbable polymers with recognized biocompatibility, such as polypropylene, PET or polytetrafluoroethylene, are the most common. Although a considerable improvement in the recurrence rate has been achieved with these materials in comparison to the traditional suture technique, several problems are still associated with this procedure. Among them, the formation of adhesions between the mesh and the bowel results in several complications, such as chronic pain, bowel obstruction or enterocutaneous fistula. Also, nonresorbable meshes have been associated with a high occurrence of chronic foreign body response and increased risk of infection 2). Indeed, according to Mathews et al., biomaterials with pores smaller than 75 µm reduce the occurrence of bowel adhesion Chemical composition, weight, pore size, and filament structure, represent critical parameters employed in surgical mesh design With the purpose of manipulating the architecture of PET mesh and also taking advantage of the anti-inflammatory and wound healing effects of chitosan, a hybrid fibrous mat (PET/C) was also developed, with higher fiber diameter and pore area. These morphological differences can have significant effects on cell-biomaterial interactions, as previously demonstrated In the rat abdominal hernia model, electrospun meshes were demonstrated to be adequate for the surgical procedure, i.e., easy to suture, and as a soft and flexible material, the electrospun meshes adapted well to the abdominal tissues. In contrast, the stiffness of the Marlex material may affect surrounding tissues and cause discomfort. This material was even perceived through the animal’s skin by touching. Electrospun meshes, on the other hand, were much softer, as well as more malleable and adaptable, while effectively performing their role of containing visceral components without mesh failure during the experimental period.In contrast to bowel adhesion that can lead to serious complications, the observed omentum adhesion to the meshes is considered clinically irrelevant in vitro and in vivoet al.Importantly, we observed a significant foreign body reaction associated with the electrospun nanofibrous meshes. This was an unexpected result since the chemical composition of the electrospun mesh is 100% PET, a recognized biocompatible polymer, and many studies have reported the absence of inflammatory response to polymer electrospun fibers by different cell types, both cf.The foreign body granulomas were mostly composed of macrophages, FBGC, and fibroblasts. An abundance of FBGC was found in tissues surrounding the electrospun meshes, evidencing a typical foreign body reaction. FBGC were most often found to be enclosing one or more nanofibers as an attempt to isolate the foreign material cf.. ExtendiIt is well established that polymer type, mesh construction, fiber size, mesh porosity and contact surface, as well as the specific characteristics of the tissue where the biomaterial is implanted, play important roles in biocompatibility and induced tissue reactions et al.et al.in vitro macrophage activation and the secretion of proinflammatory molecules. Similarly, Cao et al.in vivo and in vitro foreign body reaction and showed a decrease of granuloma thickness of subcutaneous implants from 38 µm for a PCL film to ∼8 µm and 4 µm for PCL-aligned electrospun nanofibers or nonwoven electrospun nanofibers, respectively.Considering the high biocompatibility of bulk PET and the minimum foreign body reaction found for woven-PET mesh (PET-woven microfibers), we hypothesize that the nanostructure of the electrospun materials underlies the huge foreign body reaction found in animals implanted with electrospun meshes. The reduced diameter of the electrospun fibers and pore size of the meshes, combined with the high surface-to-volume ratio of the electrospun materials, may therefore have important effects on the inflammatory reaction. Among surface properties, the material’s ability to adsorb proteins plays a key role, as those proteins, not the material’s composition itself, are major contributors of FBR Other aspects that are thought to have influenced the observed intensive foreign body reaction are related to the extension of the trauma and the specific characteristics of the tissues where the materials had been implanted. Abdominal mesh implantation involves the creation of a 1.5×1.5 cm defect by complete resection of the abdominal wall. This is a severe trauma, considering the relative size of the animal, and, consequently, a large inflammatory response may be induced. Furthermore, specific characteristics of the implantation tissues, such as cell composition and function, vascularization, extracellular matrix composition, and contact with ascitic fluid, for example, have important effects on foreign body reaction.Materials are becoming smaller than the basic body unity, i.e. the cell. Despite the enormous progress of the electrospinning technique over the past decade, cellular response and the associated risks involved in the use of nanostructure fibrous biomaterials are still poorly understood PET and PET/chitosan electrospun meshes demonstrated good performance during the implantation surgery, adequate mechanical attributes, and no evidence of intestinal adhesion. Nevertheless, a large foreign body reaction was found in animals treated with the electrospun mats. Indeed, the reduced dimension of nanofibers and the high surface-to-volume ratio of electrospun nonwoven materials may induce a high foreign body reaction, depending on the extent and location of the lesion. Refinement may be achieved by the inclusion of biological components on the fiber’s surface to enhance bioactivity and biocompatibility, thus increasing the potential of these nondegradable electrospun fiber scaffolds for abdominal wall replacement. Nevertheless, these results demonstrate the need for more studies to elucidate the mechanisms underlying cell/tissue-nanomaterial interactions in order to gain a better understanding of the risks involved in implantation of nanostructured biomaterials.Figure S1Graphic illustration of the incisional hernia model.(PDF)Click here for additional data file.Figure S2Histological and morphometric details.(PDF)Click here for additional data file.Figure S3Example of omentum adhesion to a PET mesh.(PDF)Click here for additional data file.


Oxaliplatin-based regimens induce a potential risk of hypersensitivity reaction (HSR), with incidence varying from 10% to 25% and lack of clearly identified risk factors. The present study aimed to assess incidence and risk factors in HSR.All patients treated with oxaliplatin in the Medical Oncology Department of the Lyon Sud University Hospital  from October 2004 to January 2011 were enrolled. Incidence and severity of HSR were analyzed retrospectively and the potential clinicopathological covariates were tested on univariate and multivariate analysis.A total of 1,221 doses of oxaliplatin were administered for 191 patients, 8.9% of whom experienced an HSR. Seventeen HSRs were observed, with 1.6% grade 3 and no grade 4 events. The first reaction appeared after a median of 3 oxaliplatin infusions. Using univariate analysis, HSR was associated with younger age , female gender (p = 0.01) and prior exposure to platinum salts (p = 0.02). No increased risk was associated with mean dose or with presence of atopic background. Multivariate analysis confirmed that women were at higher risk of oxaliplatin HSR than men (p < 0.05). Reintroduction of oxaliplatin was effective in 64.7% of hypersensitive patients using an appropriate premedication strategy. Patients who experienced a grade 3 HSR were not rechallenged.The risk of developing oxaliplatin HSR should not be underestimated (8.9% of patients). The medical team’s vigilance should be increased with women, younger patients and patients with prior exposure to platinum salts. Chemotherapy agents can induce hypersensitivity reaction (HSR), reducing the use of critical drugs in fragile patients for fear of inducing severe reaction and possibly death. However, with improved outcomes in cancer care, longer patient survival and extended treatment courses, patients are exposed to drugs more frequently and for longer periods, increasing the risk of sensitization and of HSR. Prevention and management of acute infusion reactions in oncology remain essential, particuOxaliplatin, a third-generation platinum agent, has been approved for the treatment of metastatic colorectal cancer and for adjuvant treatment in stage-III colon cancer. It is aHSR to oxaliplatin has been less frequently described than to cisplatin and carboplatin. However the increasing use of oxaliplatin in clinical practice shows significant incidence, varying from 10% to 23.8%-13. IdenThe aim of this retrospective cohort study was to investigate the clinical features and the risk factors of oxaliplatin-HSR.All patients who received at least one dose of oxaliplatin in the Medical Oncology Department of the Lyon Sud University Hospital  over a 6-year period from October 2004 to January 2011 were identified using the pharmaceutical software dedicated to antineoplastic preparation. Routine premedication before oxaliplatin infusion included 120 mg of methylprednisolone given intravenously and antiemetic prophylaxis.The following clinical data were collected from medical files: sex, age, history of allergy, oxaliplatin HSR, type of cancer, previous exposure to platinum agents, and oxaliplatin exposure during the study period , and treatment line number.Data from patients who experienced oxaliplatin-associated HSR were compared to that of controls patients who received oxaliplatin without experiencing HSR.For the purposes of the present study, HSR was defined as any unexpected adverse manifestation including non-allergic drug hypersensitivity and drug allergy reactions like the typical symptoms of IgE-mediated reactions, including cutaneous symptoms such as palmar or facial flushing, respiratory symptoms such as shortness of breath, and cardiovascular symptoms such as any alteration in pulse or blood pressure. ExpecteClinicopathological variables potentially associated with oxaliplatin HSR were subjected to univariate analysis. Statistical analysis was carried out using Wilcoxon’s test to determine whether age or mean dose were associated with hypersensitivity. The other factors  were evaluated using Fisher’s exact test.For multivariate analysis, a logistic regression model was applied. All variables with a p-value ≤ 0.1 on univariate analysis were considered candidates for multiple logistic regression. Two-sided p values exceeding 0.05 were considered statistically significant. Statistical analysis used SAS software, version 8.Since data were collected retrospectively and that patients’ management was not modified, according to the French law , this study did not need to be approved by a research ethics committee. It was 2 oxaliplatin per infusion. First HSR appeared after median of 3 infusions, and before the 7th course in most patients (80%). Among the 7 patients who developed a first HSR at the 1st or 2nd course, 3 have already been exposed to platinum agents. Sixteen of the 17 patients with HSR were women (94%) and 2 of them were treated for ovarian cancer.A total of 191 patients treated with oxaliplatin were included. Patient characteristics are listed in Table During the first reaction, the most common effects were flushing (10 patients), urticaria (4 patients) and fever (4 patients). Two patients experienced dyspnea without bronchospasm. Severity of HSRs according to the Ring and Messmer’s classification and the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE v 4) was similar Table . Most reAfter their first HSR episode, 13 patients (76%) were rechallenged with a mean 4.8 further cycles of oxaliplatin, associated in most cases with strengthened premedication including antihistamines and higher-dose corticosteroids. Patients who experienced a grade 3 HSR were not rechallenged. Four (30.8%) experienced another HSR at the following course: 1 second reaction in 3 patients and 2 in another patient, which 1 required definitive withdrawn of oxaliplatin. A total 22 HSRs occurred in 17 patients and required definitive withdrawn of oxaliplatin in 2 patients. Clinical signs and severity were generally similar in first and repeat episodes. Three clinicopathological parameters emerged as potential risk factors for oxaliplatin HSR using univariate analysis Tables . PatientHypersensitivity reactions to antineoplastic agents are commonly overlooked. However, for some drugs, particularly L-asparaginase, taxanes and platinum salts, hypersensitivity may lead to the withdrawal of the chemotherapy, thereby reducing the number of therapeutic options. The incidence of oxaliplatin HSR is rising as a consequence of its increased clinical use. Given the limited number of active agents in colorectal malignancy, it is now necessary to understand this kind of reactions and ways of prevent them. The preth course), compared to 10–23.8% incidence[et al. suggest that an association between female gender and younger age may be explained by a possible role of hormonal influences[Incidence of oxaliplatin HSR and number of previous cycles appeared to differ from other reports, with lower incidence 8.9%) and earlier onset  and patients who had experienced a prior exposure to platinum salts presented increased risk. Reintroduction of oxaliplatin is generally possible in grade 1/2 hypersensitive patients using an appropriate premedication strategy but uniform approach to prevent oxaliplatin HSR has not been established and reintroduction remains associated with recurrence of HSR, which requires permanent withdrawal of oxaliplatin infusion. The risk of developing HSR during oxaliplatin treatment should not be underestimated. Patients need to be informed about clinical manifestations, so they can be handled early by physicians and nursing staff. These findings underline that a close monitoring should be systematic during oxaliplatin infusion, especially if risk factors are identified.No conflict of interests.No direct funding was received for this study. The authors were personally salaried by their institutions during the period of writing, although no specific salary was set aside or given for writing the paper.MP, FR, AN, BY, TV, VTL, GF, FB, CR have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) have been involved in drafting the manuscript or revising it critically for important intellectual content; and 3) have given final approval of the version to be published. All authors have been involved in drafting the manuscript or revising it critically for important intellectual content; and 3) have given final approval of the version to be published. All authors have read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/15/1/prepub