SLC4A11 is a multifunctional membrane transporter involved with H+ transport, NH3 and alkaline pH stimulated H+ transport, and water transport. The role of SLC4A11 in the kidney is not well understood. A prior study has shown that in murine kidney, SLC4A11/LacZ staining is primarily in the long‐looped descending thin limb (DTL) as determined by colocalization with aquaporin 1 (AQP1), a protein that is expressed in some, but not all, descending thin limb segments. Using a previously characterized polyclonal antibody, we demonstrate the selective expression of SLC4A11 in the upper DTLs (which are AQP1‐positive) in the outer medulla and inner medulla with little or no expression in the lower DTLs (which are AQP‐1‐null). SLC4A11 also colocalized with AQP1 and the urea transporter UT‐B in the mouse descending vasa recta, but was absent in mouse and rat ascending vasa recta. Mouse, but not rat, outer medullary collecting duct cells also labeled for SLC4A11. Our results are compatible with the hypothesis that in the inner stripe of the outer medulla, SLC4A11 plays a role in the countercurrent transport of ammonia absorbed from the outer medullary thick ascending limb and secreted into the long‐looped DTLs. SLC4A11 can potentially modulate the rate of ammonia transport in the mouse outer medullary collecting duct. Our data suggest functionally unique SLC4A11 pathways in mouse and rat and complement previous studies of DTL Na+, urea and water permeability indicating that the upper and lower DTLs of long‐looped nephrons are functionally distinct. Adult Munich Wistar rats were reared in the University Animal Care facility at The University of Arizona . All experiments were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996) and were approved by The University of Arizona Institutional Animal Care and Use Committee.μm thick sections with a Microm HM 355 S microtome. Immunofluorescence histochemistry was performed as previously described   in phosphate‐buffered saline (PBS) (pH 7.4) for 3 h at 4°C, washed in PBS, and dehydrated through an ethanol series. Tissue pieces were embedded in paraffin following ethanol dehydration. Serial transverse tissue sections from defined regions were cut into 4 A schematic diagram of the long‐looped descending thin limb (which consists of an upper and a lower DTL segment), ascending thin limb and vasa recta are shown in Figure There are no reported antibodies that serve as selective markers for the AQP1‐null lower DTLs in rat or mouse. As shown in Figure The rodent ISOM consists of two regions in the transverse dimension – the bundle and interbundle regions. Descending vasa recta (DVR) are located within the bundle regions Fig.  along wiSLC4A11 expression is more predominant in the DTL apical membrane compared to the basolateral membrane Fig. . SLC4A11The urea transporter UT‐B is selectively expressed in DVR, and AQP1 is expressed in some of these vascular segments. As shown in Figure The endothelial plasmalemma vesicle‐associated protein  is a marker of fenestrated capillaries in the medulla. In the kidney, all medullary ascending vasa recta, including the branching ascending vasa recta are fenestrated and express PV1; however, these vessels do not express detectable AQP1 or SLC4A11 Fig. .In the mouse, whereas SLC4A11 is abundantly expressed in the ISOM, relatively lower or no expression was observed in the nephron segments of the cortex and OSOM Fig. .The inner medullary SLC4A11 expression for the mouse is shown in Figure AQP1 is expressed in the upper portions of the descending segments of rat long‐looped nephrons (“upper DTLs”) Fig. . The “loμm of the DTL , apical SLC4A11 may modulate the rate of luminal ammonia secretion.Recent studies have provided strong evidence that SLC4A11 functions as an ammonia and alkaline pH stimulated H; a process which is modulated in response to metabolic acidosis  . Whether differences in mice background strains account for these findings is unclear. Furthermore, the effects of water restriction remain untested; therefore, the maximal urine concentrating ability of the SLC4A11‐null mice is unknown. Although SLC4A11 can also transport water in the presence of an osmotic gradient, given the contribution of AQP1 to DTL water transport, it is unlikely that loss of SLC4A11 plays an important role in this regard. However, it is possible considering the colocolization of SLC4A11, AQP1, and UT‐B documented in our study that mice lacking SLC4A11 down‐regulate AQP1 and/or UT‐B and therefore the primary cause of the reported urine chemistry changes needs further consideration. In neither study was ammonia excretion nor urine acid–base parameters measured. In vitro tubule perfusion studies have shown that the upper portion of the descending segment of long‐looped nephrons has a high ammonia permeability and passive whole tubule ammonia secretion rate (Flessner et al. Two studies that have reported urine data in mice with loss of SLC4A11 have reported discrepant results. In the study of Gröger et al.  that shon et al.  also repIn summary, in the this study, we demonstrated the expression profile of SLC4A11 in the mouse kidney. Based on its expression profile, we postulate that the transporter is a candidate protein in the DTL for mediating outer medullary ammonia secretion and countercurrent recycling, thereby maintaining the cortical medullary ammonia gradient and collecting duct ammonia secretion. Expression of SLC4A11 in the outer medullary descending vasa recta and their absence in ascending vasa recta suggests that selective and/or mediated ammonia flux and delivery by medullary blood vessels may provide an additional pathway in an ammonia recycling mechanism.None declared.


Background: Tumor specific ectopic expression of the immunomodulatory molecule, HLA-G is known to mediate immune tolerance and promote carcinogenesis. Viruses too employ strategies to evade immune surveillance. Considering the role of both HLA-G and HPV in tumor growth and progression, it is pertinent to investigate the relationship between HLA-G and HPV in context of immune modulation in HNSCC.Method: A hospital based case–control study was conducted in histopathologically confirmed HNSCC tissues. HLA-G isoform expression and HPV association studies were carried out and mRNA levels of HLA-G, markers of proliferation and differentiation , immune checkpoint molecules , SOCS (SOCS1 and SOCS3) and pro-inflammatory cytokine IFN-γ were determined.Results: Higher expression of HLA-G was noted in HPV positive tumors . HLA-G7 was the most frequent isoform (29/80) found in HNSCC particularly in HPV positive tumors (13/16). In HPV negative tumors, all the checkpoint molecules were upregulated along with pro–inflammatory IFN-γ. In contrast, in HPV positive tumors, IFN-γ expression was higher (2.12 fold) but levels of IL-10, PD-1, TGF-β, SOCS1 and SOCS3 were markedly lower . HPV positive tumors were more proliferative and differentiated with higher expression of ki-67 and keratin18 . Decreased expression of cyclin D1 was noted in HPV positive tumors  than HPV negative tumors (17.69 fold). Also, HLA-G7 expressing HPV positive tumors showed lowest expression of cyclin D1. Interestingly, SOCS showed normal expression in HLA-G7 expressing HPV negative tumors (1.2 and 1.4 fold). IFN-γ was downregulated in HPV positive tumors without HLA-G7 (0.31 fold).Conclusion: Our data suggests that SOCS were downregulated irrespective of HLA-G positivity and IFN- γ expression appeared to be mediated by HLA-G. SOCS are reported to have anti-tumor activity and also SOCS and soluble HLA-G are known to interfere with cell cycle progression. Hence, through regulating HLA-G expression, HPV positive tumors could mediate immune suppression by manipulating SOCS, IFN-γ, IL-10 and cyclin D1 pathways which needs further exploration. Head and Neck Squamous Cell Carcinoma (HNSCC) is considered the sixth most common cancer with an annual incidence of approximately 400,000 worldwide . In NortThere is a two-way dynamic interaction between cancer cells and the host immune system that leads to the dictation of the pace of tumor growth. Various strategies have been developed and applied by tumor cells to avoid recognition and destruction by different immune effectors , 5 and eHPVs are double-stranded DNA viruses with a genome comprised of six “early” genes  and two “late” genes (L1 and L2) . E5, E6,Several studies have been reported to determine the association of HLA-G polymorphisms with the HPV infection , 13. In There are a few studies on the association between HLA-G polymorphisms with HPV infections –16. Howe16 protein  and agarose gel electrophoresis respectively. To assess the quality of extracted DNA and cDNA, PCR was carried out for the human β-globin gene using standard PCO3 and PCO4 oligonucleotides as described previously  and homogenized using a hand-held tissue grinder . DNA and RNA were extracted using Allprep DNA/RNA/protein kit  according to the manufacturer's instructions. RNA was reverse transcribed to cDNA using High capacity cDNA reverse transcription kit  according to the manufacturer's instructions. The quantity and quality of the isolated genomic DNA and cDNA were confirmed by Nano-Vueeviously . Five sa2, 200 μM of each deoxynucleotide triphosphate , 0.5 μM of each primers and 1.5 U of Taq DNA polymerase. To determine whether the isoforms are membrane bound or soluble, PCR was performed using exon 5 specific primer sequences as described by Van and Ober . PCR reaction was carried out for the L1 gene of outer region of HPV plasmid by using sequence-specific primers for 80 DNA samples obtained from tumor tissue in a total volume of 15 μL using 2 μL of template DNA, 1X PCR buffer, 1.5 mM MgCl2, 200 μM of each deoxynucleotide triphosphate , 0.5 μM of each primers and 1.5 U of Taq DNA polymerase. Thermal cycling was performed in a Veriti PCR  as described by Fontaine et al. . All the2 qPCR Primer Assays  were used as shown in the Δct−Δ method.Gene expression analysis was performed using SYBR green based assays  for HLA-G, interferon-γ (IFN-γ), markers of proliferation and differentiation- ki-67, keratin 18 and cyclin D1, immune checkpoint molecules- interleukin 10 (IL-10), tumor growth factor-β (TGF-β), programmed cell death protein 1 (PD1), suppressor of cytokine signaling 1 and 3 (SOCS1 and SOCS3) on StepOne Plus Real-Time PCR System . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control for normalization of expression levels. Histopathologically confirmed adjacent normal tissues were used as the calibrators for mRNA quantification by comparative CT method. Primers for IL-10 and TGF-β were designed using Primer Express software while for other genes RTt-test was used for comparison between the mean values. A p < 0.05 was considered as statistically significant.Participants with incomplete data or incomplete investigations were excluded from analysis. Statistical analysis of the data was performed using XLSTAT 2015 version. Correlation analysis was performed between the expression of marker genes, cytokines and HLA-G using the Pearson's Correlation test. Factor analysis was carried out to determine the predictors of disease. Relationships between gene expression and clinical data were analyzed by logistic regression models. Student's Our study cohort comprised of individuals from three linguistic affinities namely Indo-European (IE), Austro-Asiatic (AA), and Tibeto-Burman (TB) populations in the age group of 29–84 years with a median age of 58 years. HNSCC patients were mainly from IE population (56.25%) and 63.75% of the patients were in the age group of 45- 64 years. It may be noted that in earlier studies, HNSCC was found to be associated with older age groups but in our study, we found an association of younger as well as middle-aged group (<45 = 11.25% and 45–64 = 63.75%) with the disease. 50% of the patients were in stage II of the disease and in 56.25% of the patients, adjacent nodes were involved as shown in Soluble isoforms of HLA-G  were predominant as compared to membrane-bound isoforms  as given in We observed upregulations of immune checkpoint molecules IL-10, PD1, TGF-β and inflammatory molecule IFN-γ in tumor tissue while SOCS1 and SOCS3 expressions were found to be comparable to controls . CompariA total of 16 patients were found to be HPV positive (20%) of the 80 HNSCC patients. Male individuals were found to be predominantly associated with HPV. Fifty percent of all HPV positive tumors were seen in Tibeto-Burman ethnic group. In the context of the site of the tumor, apart from the oropharynx, sinus and the oral cavity comprising of the tongue, lip, mucosa, and glottis were also seen to be associated with HPV. Oral cavity was seen to be predominantly associated with HPV infection (50%) while the frequency of association of pharynx and tonsil was found to be equal at 25% each. HPV positive tumors were found to be most frequently associated with stage II and stage III and 12/16 were primary tumors without the involvement of metastasis as given in t-test, p = 0.079) as given in CI = 0.841–15.407). Higher expression of a marker of differentiation, keratin 18 was seen in HPV positive cases (Student's t-test p = 0.009). Expression levels of ki-67 and keratin 18 were positively correlated in HPV positive tumors (Pearson r = 0.512 and p = 0.043). The proliferation of tumor cells is frequently seen with dedifferentiation of cells but, in our case, we found differentiation in tumors along with ongoing proliferation which is interesting.Expression of ki-67, a marker of proliferation was higher in HPV positive tumors (Student's t-test p = 0.002) compared to HPV negative tumors as shown in the HLA-G was found to be upregulated in tumor tissue. However, mean relative expression of HLA-G gene was higher in HPV positive tumors (Student's p = 0.006), IL-10 (p = 0.005) and PD1 (p = 0.137) together with inflammatory cytokine IFN-γ (p = 0.031) were seen in HPV negative tumors. Weak positive correlations between of IL-10 expression with IFN-γ (Pearson r = 0.478 and p = 0.006), PD1 (Pearson r = 0.437 and p = 0.012) and TGF-β (Pearson r = 0.365 and p = 0.040) expressions were observed in HPV negative tumors suggesting the classical trend with active feedback inhibition loop. We also observed a positive correlation between expressions of IL-10 and HLA-G (Pearson r = 0.580 and p = 0.001) in case of HPV negative tumors but not in HPV positive tumors. In contrast, in HPV positive tumors, downregulation of IL-10, TGF-β and PD1 were observed but IFN-γ was upregulated  in HPV positive tumors suggesting manipulation of SOCS expression in HPV positive tumors while in HPV negative tumors the expression was comparable to the calibrator as shown in Higher expressions of immune checkpoint molecules TGF-β  in HPV positive tumors compared to HPV negative cases as shown in the HPV and HLA-G have been reported to modulate immune responses by altering cytokine profiles and manipulating the signaling pathways in cancer. We were interested to see how the interaction between HPV and HLA-G changes the expression pattern of the immunoregulatory genes. We compared the expression profiles of regulatory molecules  with HPV and HLA-G7 status as HLAG-7 was the most frequent isoform found in HPV positive tumors. Our data revealed that when HPV was present with HLA-G7, all the immune regulatory molecules  were downregulated whereas, inflammatory IFN-γ was overexpressed. In contrast, HPV without HLA-G7 was contributed to downregulation of IFN-γ. When HLA-G7 expression was seen with HPV negativity, increased expression of all the molecules was observed in the tumor. This data suggested that HPV was involved in downregulation of immune checkpoint molecules while the presence of HLA-G7 leads to increased expression of IFN-γ as shown in HLA-G is a potent immune-modulator, which by mediating secretion of cytokines like IFN-γ is known to support cross-talk between uterine decidual cells and trophoblasts and simultaneously suppressing Natural Killer Cells and T Cell-mediated cytolysis  to suppoOur data on the expression of cytokines and immune checkpoint molecules revealed a distinct difference in the regulation of cytokine expression between HPV positive and negative tumors. In HPV negative tumors, with upregulation of IFN-γ and IL-10, checkpoint molecules were also upregulated which suggested that immune suppression in tumor microenvironment was mediated by IL-10 dependent pathways. In contrast, in HPV positive tumors, IL-10 and checkpoint molecules were downregulated but IFN-γ was upregulated. The question then arises as to how immune modulation was achieved in HPV positive tumors. To examine this further we compared the expression levels of SOCS1 and SOCS3 genes in HPV positive and HPV negative tumors. Expressions of SOCS1 and SOCS3 were markedly lower in HPV positive tumors. However, in HPV negative tumors, levels of SOCS were comparable to healthy controls. Further, HPV negative but HLA-G7 positive samples showed normal expression of SOCS indicating the role of HPV in dysregulation of SOCS. Comparable SOCS levels were also observed in HPV negative tumors without HLA-G7 which is acceptable as downregulation of SOCS in the tumor has been reported earlier (36). Studies have shown that SOCS1 has antitumor activity by preventing chronic inflammation , 30 and Notably, HPV tumors were seen to be more aggressive with increased levels of ki-67 (6.25 fold) than HPV negative tumors (2.51 fold). Decreased expression of cyclin D1 was noted in HPV positive tumors. Further, the highest expression of cyclinD1 was observed in HPV and HLA-G7 negative tumors and lowest was seen in HPV and HLA-G7 positive samples. These findings are in agreement with Ketroussi et al. and Bahri et al. who reported that soluble HLA-G can inhibit cell cycle progression by regulating cyclin expression , 35. SOCDownregulation of SOCS in HPV positive tumors may be explained by lower levels of IL-10 in these tissues, which is known to induce expression of SOCS for homeostasis . AlternaIn HPV and HLA-G7 positive tumors, IFN-γ the pro-inflammatory cytokine was upregulated. We had only three samples that were HPV positive but HLA-G7 negative, and in these samples, IFN-γ was downregulated. Considering the small number of HPV positive HLA-G7 negative samples in our study, we are constrained to draw any conclusion but it would appear that HPV harnessed HLA-G7 modulated pathways to drive inflammatory mechanism for its growth.The study evidenced increased ectopic expression of HLA-G with HPV positivity and this is the first report of expression of HLA-G7, a soluble isoform of HLA-G in HNSCC and with HPV positivity. Our findings hint that HPV positive tumors manipulate the expression of SOCS, IFN-γ, IL-10 and, cyclinD1 in partnership with HLA-G for immune evasion and to support the growth of HNSCC tumors. In conclusion, HPV harnessed HLA-G7 modulated pathways to drive inflammatory mechanism for its growth and regulate the expression of SOCS to mediate immune subversion.NS was involved in the design of the experiments, data acquisition, and analysis, manuscript writing. MNB was involved in the study as the Clinical Collaborator and contributed to the clinical component of the manuscript. SB was involved in study design, data analysis and interpretation, formulation and critical review of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Patients with cartilage-hair hypoplasia (CHH), a rare metaphyseal chondrodysplasia, manifest severe growth failure, variable immunodeficiency and increased risk of malignancies. The impact of CHH on gynecologic and reproductive health is unknown. Vulnerability to genital infections may predispose CHH patients to prolonged human papillomavirus (HPV) infections potentially leading to cervical, vaginal and vulvar cancer.We carried out gynecologic evaluation, pelvic ultrasound and laboratory assessment in 19 women with genetically confirmed CHH. All patients were clinically examined and retrospective data were collected from hospital records.The women ranged in age from 19.2 to 70.8 years (median 40.8 years) and in height from 103 to 150 cm (median 123 cm). All women had undergone normal pubertal development as assessed by breast development according to Tanner scale and by age of menarche . Despite significant short stature and potentially small pelvic diameters, a well-developed uterus with fairly normal size and shape was found by pelvic ultrasound in most of the patients. Ovarian follicle reserve, assessed by ultrasound was normal in relation to age in all premenopausal women it could be assessed (12 cases). Anti-Müllerian hormone was normal in relation to age in 17 women (89%). HPV was detected in 44% (8/18) and three women carried more than one HPV serotype; findings did not associate with immunological parameters. Three patients had a concurrent cell atypia in Pap smear.Pubertal development, reproductive hormones and ovarian structure and function were usually normal in women with CHH suggesting fairly normal reproductive health. However, the immunodeficiency characteristic to CHH may predispose the patients to HPV infections. High prevalence of HPV infections detected in this series highlights the importance of careful gynecologic follow up of these patients. RMRP. RMRP mutations disrupt ribosomal processing and cell cycle, leading to defective cell proliferation [Cartilage-hair hypoplasia  is a rare autosomal recessive metaphyseal chondrodysplasia with an incidence of 1: 23000 births in Finland [feration . CHH is While major clinical manifestations of CHH are well-described, almost no data are available on its potential effect on gynecologic and reproductive health and vulnerability to genital infections and HPV-related cancers , 5–7. AlWhether fertility is affected in CHH also remains unclear. In a cohort of 11 adult CHH males, serum concentrations of testosterone, inhibin B and gonadotropins were mainly normal. However, semen analyses showed impaired spermatogenesis . Recent This lack of data prompted us to carry out a cross-sectional study on gynecologic health in a relatively large cohort of adult women with CHH. We evaluated all subjects clinically and by performing hormonal laboratory measurements and pelvic ultrasound. Pap smear and cervical HPV test were obtained during gynecologic examination.RMRP mutations had been detected by Sanger sequencing either at Laboratory HUSLAB, Finland, or as a part of previous or ongoing research at Folkhälsan Institute of Genetics, Helsinki [The study protocol was approved by the Institutional Review Board of the Children’s Hospital, University of Helsinki and all study participants gave a written informed consent. Patients were identified from the Finnish Skeletal Dysplasia Register which includes > 160 patients with genetically confirmed CHH. All Helsinki , 9. All Hospital records were reviewed for relevant data. Patients were interviewed for a detailed medical history. Complete gynecologic and pelvic ultrasound examinations  were performed by an experienced gynecologist (EH). Pubertal status was recorded according to Tanner’s classification . Antral Pap smear and cervical HPV test were obtained when anatomically possible. Cervical samples for HPV test were taken by APTIMA cervical collection and transport KIT and HPV subtypes were analyzed by PCR and Luminex suspension array technology . The tesBlood cell count and serum concentrations of sex hormones, prolactin, thyroid stimulating hormone, gonadotropins and Anti-Müllerian hormone (AMH) were measured during clinical visit. Serum estradiol was extracted from serum with diethyl ether and quantitated by LC-MS/MS (liquid chromatography-tandem mass spectrometry) using a TQ5500 mass spectrometer . The between-run coefficient of variations (CV %) were 4.4–5.1% for serum samples at concentrations 330–900 pmol/L.Serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were measured using an electrochemiluminescence immunoassay . Anti-Müllerian hormone (AMH) was quantitated with an ELISA assay . Limit of detection (LoD) was 0.08 μg/L and limit of quantitation (LoQ) 0.16 μg/L. Intra-assay and inter-assay CV% was < 6% in the range 3.8–16.5 μg/L. Total CV % was < 8%. Age-specific limits of AMH were applied . The metStandard statistical methods were used as appropriate. Median, mean and/or standard deviation estimates were computed for variables such as age, length and weight. Fisher’s exact test and logistic regression analysis were applied to examine the correlations of symptoms and laboratory findings.RMRP founder mutation c.70A > G and four patients were compound heterozygous for c.70A > G and c.262G > T mutations.The cohort characteristics are described in Table n = 1) or M5 (n = 18). Pubic hair was fully developed  or almost fully developed  in 68% and partly developed  or totally absent  in 32%. Those with P1 had total alopecia.All patients had normal breast development, either Tanner stage M4  and median length of uterine corpus, measured from the fundus to the internal orifice of the uterus on a sagittal view, was 38 mm (range 26–70 mm). The 41-year old woman with the smallest, pre-pubertal-size uterus  had undergone spontaneous menarche at the age of 11 years, used currently progestin-only pills and had sparse menstrual flow. She had never tried to become pregnant. Another pre-menopausal 46-year old woman also had a relatively small uterus, with corpus anterior-posterior measurement of 20 mm and corpus length of 29 mm. However, she had used levonorgestrel-releasing intrauterine device for years suggesting fairly normal uterine cavity. In all other pre-menopausal women corpus anterior-posterior measurement was > 25 mm and corpus length > 30 mm.The structure of ovaries could be assessed in 16/19 subjects and all were considered normal in relation to age, phase of menstrual cycle and use of hormonal therapy. In three postmenopausal women (aged 60–70 years) ovaries could not be identified.AFC in both ovaries could be assessed in 12 premenopausal women and was normal in all. In two cases, AFC from only one ovary could be reliably counted Table .Table 3A9/L (range 3.2–13.4 x e9/L) and platelet count 275 x e9/L (range 147–398 x e9/L).Blood cell count was analyzed in 18/19 cases. Mean hemoglobin was 131 g/L (range 111–146 g/L). Only one woman had hemoglobin under normal range . Mean white blood cell count was 6.3 x en = 5) and in the two perimenopausal women (aged > 45 yrs). Most of the premenopausal women had normal AMH according to age-specific reference values , both women were on psychiatric medication affecting prolactin secretion.Pap smear and cervical HPV test were obtained from 18/19 cases. Altogether 9 women had abnormal findings; these are presented in Table Little is known about gynecologic health in women with CHH and our study provides important novel data that can be implemented in the management and follow-up of patients with CHH and increase our knowledge on gynecological issues more widely among women with skeletal dysplasia. The main findings of our study include normal pubertal development, mostly normal genital anatomy and a high prevalence of HPV positivity.RMRP function or mediated by e.g. endocrine factors. AFC was normal in relation to age in all those who could be evaluated, suggesting fairly normal fertility potential in this patient group. The lack of pubarche in three patients is likely to be a result from impaired germ-cell proliferation in the hair follicles.We have previously described two young CHH women with absent spontaneous pubertal development and reduced ovarian reserve . HoweverFor practical reasons blood tests for hormonal measurements were taken during clinical appointment. Three women were on hormonal contraception at that time. Combined contraception may decrease the level of AMH and number of AFC resulting in underestimation of fertility potential , 18. HowSexually active CHH women are at risk for acquiring HPV and combined immunodeficiency may predispose them to prolonged or severe infection. A Finnish series of women attending HPV screening during 2003–2005 showed age –specific HPV positivity to vary from 2.8–24.2% in women aged 25–65 years . In our Most of the CHH women in our series were or had been sexually active, indicating a need for reliable and suitable contraception. In a previous study, intrauterine device (IUD) was used only by 8% of short-statured women . This maOur study is limited due to the relatively small size of the cohort and the low participation rate which may cause bias. However, in rare diseases larger cohorts are often impossible to obtain. In CHH, severe short stature further complicates participation of individuals living far from the study center. The participation rate was comparable to our previous studies on CHH. Further, our study permit did not allow us to collect data on the offspring’s health, including postnatal growth. Despite these limitations our study is the largest gynecological evaluation ever performed in CHH or any other skeletal dysplasia and thus provides valuable new data. Further studies are needed to further elucidate the largely unknown field of gynecological health in women with skeletal dysplasia and the outcomes of their pregnancies.Immunodeficiency and increased risk for malignancies contribute to significant morbidity in CHH patients. While major clinical manifestations of CHH are well-described, only little is known about gynecologic health in CHH. In this prospective series reproductive hormones and health were fairly normal. However, high prevalence of HPV infections detected in this series highlights the importance of careful gynecologic follow up of these patients since immunodeficiency may predispose CHH patients to HPV infections.


LncRNA NEF was a recently identified tumor suppressor lncRNA in hepatocellular carcinoma. Our study aimed to explore the role of NEF in cervical squamous cell carcinoma (CSCC) patients. In the present study, expression of NEF in tumor tissue  and serum of human papillomaviruses (HPV)-negative and HPV-positive CSCC patients as well as healthy controls was detected by qRT-PCR. Diagnostic and prognostic values of NEF for CSCC were evaluated by ROC curve and survival curve analysis, respectively. NEF expression vector was transfected into CSCC cells and the effects on cell migration and invasion as well as TGF-β1 expression were investigated by Transwell migration assay, Transwell invasion assay, and Western blot, respectively. We found that expression of NEF in cervical tissues (tumor tissues for CSCC patients) and serum was significantly down-regulated in HPV-negative CSCC patients than in healthy controls and HPV positive patients, but no significant differences were found between healthy controls and HPV positive patients. Low serum levels of NEF distinguished HPV-negative CSCC patients from healthy controls and indicated poor survival. NEF overexpression inhibited the migration and invasion of HPV-negative but not HPV-positive CSCC cells. NEF overexpression down-regulated TGF-β1 in HPV-negative CSCC cells but not in HPV-positive CSCC cells. TGF-β1 treatment reduced the effects of NEF overexpression on cell migration and invasion. Therefore, we conclude that lncRNA NEF may inhibit the migration and invasion of HPV-negative cervical squamous cell carcinoma by inhibiting TGF-β pathway. Cervical cancer is a major burden of cancer worldwide . ApproxiCervical cancer is divided into cervical squamous cell carcinoma (CSCC) and cervical adenocarcinoma, two subtypes . TGF-β sTumor tissues and serum samples of 48 HPV-negative CSCC patients and 52 HPV-positive patients  were obtained from specimen library of Luoyang Center Hospital affiliated Zhengzhou University. Those patients were diagnosed treated in Luoyang center hospital affiliated Zhengzhou University from July 2010 to July 2012. All patients were diagnosed and treated for the first time. All those patients completed treatment and follow-up. Patients infected with other viruses or with other type of malignancies or other severe diseases, or patients died of other reasons during follow-up were excluded from the present study. The 48 HPV-negative CSCC patients had a mean age of 48.4 ± 5.6 year (ranged from 33 to 69 years), while the mean age of HPV-negative CSCC patients was 47.9 ± 5.3 year (ranged from 30 to 68 years). Cervical tissues and serum samples of 38 healthy females who received routine physiological examinations during the same time period were also obtained from specimen library of Luoyang Center Hospital affiliated Zhengzhou University to serve as control group. Those healthy females received cervical biopsies for the detection of potential cervical lesions, but cervical diseases were finally excluded. Control group had a mean age of 47.8 ± 4.9 year (ranged from 34 to 65 years). No significant differences in age, BMI and other basic clinical data were found between HPV-negative CSCC patients, HPV-positive CSCC patients and control group. This research has been carried out in accordance with the World Medical Association Declaration of Helsinki, and that all subjects provided written informed consent.C−ΔΔT method.All total RNA extractions were performed using Trizol reagent . Tissues were ground in liquid nitrogen before adding Trizol reagent. cDNA was synthesized through reverse transcription and 20 μl PCR reaction systems were prepared using SYBR® Green Real-Time PCR Master Mixes . Primers used in PCR were: 5′-CTGCCGTCTTAAACCAACCC-3′ (upstream) and 5′-GCCCAAACAGCTCCTCAATT-3′(downstream) for human lncRNA-NEF; 5′-GACCTCTATGCCAACACAGT-3′ (upstream) and 5′-AGTACTTGCGCTCAGGAGGA-3′ (downstream) for β-actin. PCR reaction conditions were: 95°C for 1 min 20 s, followed by 40 cycles of 10 s at 95°C and 30 s at 57°C. Expression of NEF was normalized to β-actin according to 22). Full-length NEF cDNA surrounded by EcoRI-EcoRI cutting sites were amplified by PCR. This fragment was linked to linearized pIRSE2-EGFP vector  to make NEF expression vector. NEF siRNA and negative control siRNA were synthesized by Sangon . NEF expression vectors or empty pIRSE2-EGFP vectors (negative control) were transfected into 5 × 105 cells at a concentration of 15 nM using Lipofectamine 2000 reagent  according to the instructions of the kit. For NEF siRNA and negative control siRNA transfection, the dose was 40 nM. Expression of NEF was checked by qRT-PCR just before subsequent experiments to make sure an overexpression rate no <150% was reached.HPV-negative human cervical squamous cell carcinoma cell line, C33A, and HPV-positive human cervical squamous cell carcinoma cell line, SiHa, were purchased from ATCC. Cells of those two cell lines were cultured according to the instructions provided by ATCC in an incubator . Then, 0.1 ml of cell suspension containing 5 × 103 cells was added into the upper chamber, while the lower chamber was filled with Dulbecco’s modified Eagle’s Medium containing 10% FCS. After incubation for 24 h, invading cells on membranes were stained with 1% Crystal Violet  at 25˚C for 30 min. Stained membranes were observed under a light microscope  and cells were counted. Transwell invasion chambers pre-coated with Matrigel  was used in invasion assay according to the manufacturer’s instructions, and all other steps are essentially the same of migration assay.After transfection and confirmation of overexpression, cells were collected and cell suspension with as cell density of 5 × 10All total protein extractions were performed using RIPA solution , and protein concentrations were measured using BSA method. After that SDS-PAGE (12%) gel electrophoresis was performed with 35 µg protein per lane. Following gel transfer, PVDF membranes were incubated with 5% skimmed milk at room temperature for 2.5 h. Membranes were then incubated with primary antibodies of TGF-β1  and GAPDH  overnight at 4°C. After complete washing with TBST (0.3% Tween 20), membranes were further incubated with goat anti-rabbit IgG-HRP secondary antibody  at room temperature for 4 h. Pierce ECL Western Blotting Substrate (Thermo Scientific) was dropped onto membranes to develop signals, and ImageJ software was used to normalize expression of TGF-β1 to endogenous control GAPDH.t-test, respectively. P<0.05 indicated a difference with statistical significance.SPSS19.0  statistical software was used for data analysis. Correlations between NEF expression and patients’ clinicopathological data were analyzed by Chi square test. Protein and mRNA expression, as well as cell migration and invasion data were compared by one-way analysis of variance followed by LSD test. ROC curve analysis was performed to evaluate the diagnostic value of NEF expression for HPV-negative CSCC with HPV-negative CSCC patients as true cases and healthy controls as true negative cases. Thirty HPV-negative CSCC patient were divided into high and low expression group, fifteen in each group. Survival curves were plotted and compared by K-M method and log rank P<0.05). In addition, no significant differences in NEF expression was found between HPV-positive patients and healthy controls (P>0.05).Expression of NEF in tumor tissues of 48 HPV-negative CSCC patients and 52 HPV-positive patients and in cervical biopsies of 38 healthy females was detected by qRT-PCR. As showed in P<0.05). In addition, no significant differences in serum levels of NEF were found between HPV-positive patients and healthy controls (P>0.05).Serum levels of NEF in 48 HPV-negative CSCC patients, 52 HPV-positive patients and 38 healthy females were also measured by qRT-PCR. As showed in In view of the differential expression of NEF in HPV-negative CSCC patients and healthy control, ROC curve analysis was performed to evaluate the diagnostic value of NEF expression for HPV-negative CSCC. For NEF expression in cervical tissues, the area under the curve (AUC) was 0.8910 with standard error of 0.03656 and 95% confidence interval of 0.8193 to 0.9627 A. For seP>0.05), but were significantly correlated with distant tumor metastasis (P<0.05).Chi square analysis was performed to analyze the correlations between NEF expression levels and clinicopathological data of patients with HPV-negative CSCC. As shown in Tables 1 and 2, expression levels of NEF in cervical tissues and serum showed no significant correlations with patients’ age and tumor size , but not in HPV-positive human cervical squamous cell carcinoma cell line, SiHa. In addition, TGF-β1  treatment showed no significant effects on NEF expression (data not shown).TGF-β1 plays pivotal roles in the metastasis of various types of malignancies, and inhibition of TGF-β1 is considered to be a promising target for the treatment of cervical cancer . In the P<0.05), but not cells of HPV-positive SiHa cells (P>0.05). Similarly, NEF overexpression also significantly promoted the invasion (P<0.05), but not cells of HPV-positive SiHa cells (P>0.05). In addition, TGF-β1  treatment significantly reduced the effects of NEF overexpression on migration and invasion of cells of HPV-negative cell line, C33A (P<0.05).As shown in invasion B of cellThe key finding of our study is that NEF as a newly discovered tumor suppressor lncRNA in hepatocellular carcinoma  may alsoHPV-negative and -positive CSCC have different pathogenesis, and they should be treated differently . It has Tumor metastasis is still a main challenge for the treatment of CSCC, and early and accurate diagnosis is still the key for patients’ survival . With thCorrelation analysis showed that NEF expression was significantly correlated with distant tumor metastasis but not tumor size, which is consistent with the role of NEF in hepatocellular carcinoma. Tumor metastasis is a major cause of death in CSCC patients and the survival of CSCC patients at early stages is generally satisfactory . TherefoOur data also showed that NEF is likely an inhibitor of the migration and invasion of HPV-negative CSCC cells but not HPV-positive CSCC cells. Although the role of TGF-β in regulating cancer cell proliferation is controversial, it is generally believed that TGF-β signaling promotes tumor metastasis . In our However, molecular mechanism of the regulation of TGF-β signaling by NEF is still unknown. It has been reported that NEF can cis-regulate FOXA2 to antagonize epithelial-to-mesenchymal transition (EMT) , and FOXIn conclusion, expression of NEF was specifically down-regulated in HPV-negative CSCC patients but not in HPV positive patients. Low serum levels of NEF may serve a potential diagnostic and prognostic biomarker for HPV-negative CSCC. NEF may inhibit the migration and invasion of HPV-negative but not HPV-positive CSCC cells by down-regulating TGF-β1 expression.All data generated or analyzed during this study are included in this published article.


Based on existing evidence for efficacy, savings, and advantages in delivery, some countries may elect to pilot or roll out single-dose human papillomavirus (HPV) vaccination  in advance of a WHO policy decision. Accelerated evidence of population-based effectiveness  of one-dose vaccine programs could be gained through regular surveys of HPV prevalence in young women before and after vaccination introduction. In order to offer the earliest information on OE, one-dose HPV vaccination should target one or more birth cohorts as close as possible to the age when sexual activity most often starts in a given population. A catch-up one-dose vaccination program of girls up to 18 years of age who would have been too old to profit from the introduction of a routine HPV vaccination program in preadolescents would minimize the interval between vaccination and the possibility to monitor vaccination impact in young women. In addition, catch-up is especially desirable in low- and middle-income countries with little access to screening as “missed” cohorts may face high risk of cervical cancer death. HPV prevalence should be firstly monitored in age groups of women who may already be sexually active but still reluctant to admit it and to accept vaginal examination for the collection of cervical cells. Hence, HPV testing from urine samples, for which good concordance with cervical cells has been proven, offers a feasible approach to assess periodically vaccine OE in representative samples of 17–20 year-old women. This type of observational study would greatly benefit from the presence of a population census and the creation of a vaccination registry. A real-world demonstration of OE of the new schedule would complement the findings of ongoing clinical trials and immunogenicity studies on the efficacy of single-dose HPV vaccination. Two landmark events led to widening of vaccine access in LMICs: (1) the GAVI Alliance started to support HPV vaccine introduction in 2013, and (2) in 2014, the non-inferiority of two doses compared with three led to a two-dose schedule being endorsed by WHO in 2014 in girls below age 15 Barriers to national vaccine uptake in LMICs , and how single-dose HPV vaccination could be transformational  are wideThe strongest evidence for the efficacy of one-dose vaccination will come from ESCUDDO 2Monitoring HPV vaccine impact is complicated by the buffer period, i.e., the time interval between HPV vaccination of preadolescent girls and evidence of impact on cervical cancer and pre-cancer, and by the lack of a serological test providing a threshold for protection. Hence, in the near-term (5–10 years), the only informative outcome is to measure post-vaccination changes in type-specific HPV infection prevalence in populations of young women, as recommended by a WHO expert group With respect to accelerated impact data from LMICs, we excluded certain study design options. Firstly, we assume that it would be considered unethical to randomize women, individual- or cluster-wise, to no HPV vaccination. Secondly, we assume it unlikely that any randomized non-inferiority trial comparing one versus multi-doses of vaccine could be (or should be) put into place in parallel to the ESCUDDO trial. Finally, immunogenicity bridging studies are useful but not conclusive as it is already known that one-dose produces lower HPV antibody levels than multiple-doses but the clinical implications of this inferiority are unclear Rather than discuss the aforementioned study designs we will focus on how to best use observational studies in which type-specific HPV prevalence in young women is monitored through repeat HPV surveys before and after the introduction of one-dose vaccination. HPV prevalence in unvaccinated and vaccinated cohorts will then be compared to assess the real-world overall effectiveness  2.1The earliest evidence of the population-level impact of three-dose HPV vaccination from high-income countries (HICs) has been obtained by comparing type-specific HPV DNA prevalence in young women before and after vaccination 2.2The choice of the location for a demonstration study of one-dose vaccination against HPV in LMICs firstly depends on the willingness of local political and health stakeholders to endorse the initiative and share with the population the motivations that have led to it. In addition, it is essential that the birth cohorts of girls who will receive the one-dose schedule are not going to be protected by the indirect effectiveness of previous two- or three-dose vaccination programs as this may substantially inflate the measured OE . Along tIrrespective of the chosen location(s), the timing of the study is essential to be able to produce results earlier or in parallel with the release of the first efficacy data on one-dose schedule that is expected in 2023 . It would therefore be essential to carry out the baseline HPV survey of unvaccinated 17–20 year-old women no later than 2018. The timely performance of a baseline survey has been challenging in countries who implemented multiple-cohort programs, i.e. routine vaccination of preadolescents and of catch-up of older girls. Very successful vaccination programs in Bhutan In the next two sections, we will describe in more detail the type of study that we consider the most propitious for producing timely and robust real-world data on the effectiveness and operational advantages of one-dose vaccination, i.e., catch-up vaccination studies.2.3The buffer period, the time interval between the introduction HPV vaccination and the earliest evidence of OE depends on the age group targeted by the vaccination and the average age at which the risk of being infected by HPV, rises very steeply, i.e. after the beginning of sexual intercourse. Of note, HPV infection can also be acquired through non-penetrative sexual intercourse. While HPV vaccination is most efficacious in pre-adolescence, the younger the age at vaccination , the longer will be the waiting for evidence of vaccine impact.Conversely, if one-dose vaccination is provided to older girls, the time needed to assess OE would be substantially shortened by the proximity of possible sexual exposure to HPV. However, the target age-range for vaccination is population-specific as the vaccination of a sizeable fraction of sexually active and potentially already infected girls would lower the estimated OE particularly in short-term studies. In addition, catch-up is highly desirable in LMICs with little access to screening as “missed” cohorts may face high risk of death from cervical cancer. Mathematical models For monitoring purposes we propose to carry out two different types of HPV surveys: (1) urine-based surveys  and the age range at which sexual activity in the targeted group starts . Additional sources of uncertainty are the efficacy of single-dose vaccination and the coverage attainable in the study area per survey would be necessary to detect a 50% overall OE. The number of women required would increase steeply if pre-vaccination prevalence of vaccine types were lower than 2% but it would be smaller if OE were 80%, e.g., if both one-dose efficacy and the coverage were ≥89%.The OE of the one-dose schedule in our present example will be mainly estimated by comparing the prevalence of vaccine-targeted HPV types in unvaccinated and vaccinated birth cohorts; that is, by comparing the prevalence in baseline and repeat HPV surveys. The comparison of vaccinated and unvaccinated birth cohorts somewhat corresponds to “by intention to vaccinate” analyses in randomized clinical trials. If coverage is sub-optimal, vaccinated and unvaccinated women in a repeat survey can also be compared ad hoc increase in survey size compared with the minimum requirements.Importantly, the validity of estimates of OE will be reliable only if participants in all surveys are recruited in a very consistent way, i.e., if they are comparable with respect to age, place of recruitment , place of origin/living, socio-economic level, and most challenging, sexual habits . The need to collect short but accurate questionnaire-based information on all the characteristics that are or may be associated with the probability of an early acquisition of HPV infection cannot be exaggerated. This information will show if indeed similar young women have been recruited and allow us to also perform between-survey comparison after stratification and adjustment for relevant characteristics. Of note, the need to perform a priori defined sub-group analyses of HPV prevalence will necessitate an Chlamydia trachomatis can easily be added to HPV testing in the same sample, as we did in Bhutan and Rwanda While very detailed sexual questions are neither feasible nor acceptable in surveys of young women in LMICs, bias in the report of sexual activity can be attenuated by skilled female interviewers or by self-filled questionnaires in combination with strict assurance of questionnaire anonymity 2.5The gold-standard samples for the detection of current HPV infection are clinician-collected cervico-vaginal cell samples. Indeed, most of the existing assessments of HPV vaccine impact in HIC have been based on repeat surveys using this approach However, in order to obtain the earliest evidence of OE of one-dose HPV vaccination, HPV prevalence should be measured in younger often unmarried women who may or may not have started or be willing to admit sexual intercourse. Women in this age group are often reluctant to accept a gynecological examination for the collection of cervical cells, also because gynecological exams and cervical screening have little or no clinical value to them. Of note, HPV-based screening is not recommended below age 30, due to the high prevalence of HPV infections that will rapidly clear spontaneously The settings where gynecological exams are offered to women below 25 years of age are mainly STI clinics and reproductive health clinics that attract very sexually-active women often at increased risk of HPV infection. These settings offer therefore a special opportunity for “sentinel” surveys with extra statistical power, due to high HPV prevalence, but at the cost of a potentially poor representativeness of the general female population of the same age Self-collected cervico-vaginal samples have proven to be nearly equivalent to clinician-collected samples for HPV-based cervical screening and are also a valid option for monitoring HPV vaccine impact. The predominant advantage of self-collected cervico-vaginal samples especially in LMICs is the high throughput collection due to much lower requirements for specialized personnel and disposable plastic equipment . In addition, it has been shown in many settings that the acceptability is better for self-collected than for clinician-collected samples A very powerful alternative to cervico-vaginal samples to evaluate HPV prevalence is represented by urine sample. Not only is collection of urine samples a well-accepted non-invasive procedure but many previous problems in the storage and processing of this type of samples have been overcome A good demonstration of the feasibility of urine collection in LMICs is provided by the two similar urine surveys that we performed in 2013 in high-school students in Bhutan and Rwanda. Girls aged 17–20 used a urine self-collection device designed to immediately mix first void urine with a preservation medium 2.6It might be possible to apply study designs that use one-dose vaccination both during catch-up and in routine activity, the decision to give or not a second dose being based on the results of future efficacy studies on one dose. Such an approach may be referred to as an extended interval two-dose schedule , similar to the approach formerly adopted to evaluate and accelerate the transition from a three- to two-dose schedule Aside from the urgency of understanding the early OE and operational advantage of the one-dose schedule using highly feasible and relatively inexpensive school-based urine surveys and clinic-based cervical cell surveys, increasingly complicated tools can be conceived to monitor changes in HPV prevalence before and after vaccination more accurately. In the presence of a fairly good enumeration of the study population by sex, age, and place of living, for instance, random samples of young women could be used to measure HPV prevalence.A large demonstration project of one-dose vaccination may also be the opportunity to create a platform for systematic monitoring of HPV vaccination in one LMIC or more. A computerized HPV vaccination registry may be established and eventually allow the evaluation of one-dose OE and the partitioning of OE into direct effectiveness and indirect (herd) protection based on cohort- or nested case-control analyses. It may be subsequently used also to assess additional vaccination strategies or the combination of vaccination with HPV-based screening programs. Finally, some parallel investments may be worth considering including the creation of biobanks of urine, cervical cells or blood samples linkable to vaccination registries and medical data.3The project outlined here rests on the opinion that one-dose HPV vaccination may well be a landmark in the achievement of global access to a cancer-preventing vaccine and in a better understanding of the way VLP-vaccines and vaccine adjuvants work. Of course, other obstacles to universal HPV vaccination, such as the reluctance of stakeholders to invest in primary prevention whose return will only be appreciable after decades, unjustified fears of vaccines, and the lack of manufacturers in LMICs, will remain but they will be eased by the decrease in vaccine cost and logistic challenges.Building upon our experience in monitoring the burden of HPV infections and the effectiveness of HPV vaccination in both HICs and LMICs, our present article endorses the feasibility and value of carrying out a sufficiently large one-dose vaccination program of girls up to 18 years of age and repeat HPV surveys in young women. This would provide the earliest real-world evidence of the effectiveness and operational advantages of one-dose HPV vaccination, which, if consistent with that from trials and studies described in other chapters of this volume, would justify and accelerate a major change in global HPV vaccination policy.None.


In 1988, Brazil established a constitutional right to health and universal access to health care for all Brazilians through the creation of the Unified Health System (SUS). As part of its efforts to fulfill this right, the quadrivalent Human papillomavirus (HPV) vaccine was introduced into the national immunization program in 2014. The non-discriminatory provision of healthcare goods, facilities, and services is a fundamental part of the right to health. Yet HPV vaccination was limited to females aged 9–13, despite the universal nature of SUS and scientific support for the vaccination of males and older females. The purpose of this cross-sectional study was to describe parental attitudes regarding age- and gender-based HPV vaccination exclusions, as well as parental knowledge of HPV and the HPV vaccine.Data were gathered from parents with children aged 9–17 in a health post located in the municipality of Mauá  through interviewer-administered questionnaires. We analyzed attitudes regarding HPV vaccination and its eligibility guidelines by comparing parents of HPV vaccine eligible and ineligible children.In this low-income population, the majority of the 219 parents surveyed supported the inclusion of males and females over 13 into the HPV vaccination program; support for the non-discriminatory provision of the HPV vaccine was high among parents – especially if financially accessible. Additionally, there were high levels of knowledge and positive parental attitudes regarding HPV vaccination safety and efficacy among both parent groups suggesting information accessibility – a key component of the right to health and informed decision-making.Support for the expansion of HPV vaccination for excluded populations exists, and is not based on current eligibility, or differential knowledge and attitudes about the vaccine. Moving forward, careful consideration of gender- based eligibility for vaccination, informed decision-making, and the importance of community participation in health policy development and implementation may be gleaned from the case of Brazil and beyond. In 1948, both the World Health Organization (WHO) Constitution and the Universal Declaration of Human Rights described health as a fundamental human right . ArticleIn Brazil, cervical cancer is the second most common cancer among women. Incidence and mortality of cervical cancer in Brazil are the highest in the Americas; the mortality rate is 8.3 deaths per 100,000 women , 7. InfeSistema Único de Saúde  was established as a part of the Brazilian constitutional guarantee of the right to health [Programa Nacional de Imunização  that is responsible for establishing the national vaccine schedule; the current national schedule includes 26 vaccines, including the quadrivalent HPV vaccine [The o health . SUS proo health . SUS maio health . SUS als vaccine .In 2014, Brazil joined 70 other countries by introducing the quadrivalent HPV vaccine into the PNI. Free of cost to the individual, the national school-based vaccination program was targeted at females aged 9–13 , 13. TheThe current vaccination policy does not support the universal and equitable access to health care espoused by the SUS system, the Brazilian constitution, and international human rights law , 3, 9. TBetween June and August 2015, we conducted an interviewer administered cross-sectional survey with 219 participants to understand parental knowledge and attitudes regarding HPV vaccination. Survey topics included: attitudes regarding HPV vaccination for excluded populations, knowledge of HPV, current HPV vaccination, and HPV vaccination eligibility. Parents and guardians  of children aged 9–17 attending health posts in Mauá, Brazil were eligible to participate. The lower age range was selected following SUS guidelines recommending HPV vaccination for females aged 9–13 years, while the upper age range was selected since parents are still legally responsible for their children until age 18.The project site was Unidade de Atencão Básica de Saúde  Kennedy, located in Mauá, a municipality in the state of São Paulo on Brazil’s east coast . UBS KenFollowing an extensive review of relevant studies, the survey instrument was comprised of original questions and adapted items from existing instruments that measured attitudes and parental decision making related to the HPV vaccine –19. The Of approximately 100 patients who visited the clinic each day, approximately 8 to 10 were recruited for participation. Each individual that entered the clinic was approached for participation by the researcher, who verified eligibility and provided an overview of the study. Due to low literacy levels, consent to participate was obtained from each participant by verbally reviewing the consent form, providing all pertinent information to the subject and allowing the subject the opportunity to ask questions. Participants were required to sign (or print) their names at the bottom of the consent form before beginning the survey, and were provided with a copy of the consent form if requested. The face-to-face survey was administered in Portuguese; the interviewer read each survey item aloud and recorded the participant’s responses on the survey instrument. Sample size was determined based on a 95% confidence level and a 5% margin of error.Consent documents and questionnaires were filed separately from one another in a locked filing folder to ensure confidentiality. Survey data were entered into Excel and stored in a password-protected folder on a password-protected laptop. The Excel file was uploaded to SAS version 9.4  for data cleaning and analysis. Completed paper surveys were filed at the Brazilian partner organization (FMABC) in accordance with the requirements of the Mauá Ethics Review Board.For analysis, parents were divided into two categories: “HPV vaccine eligible” and “HPV vaccine ineligible.” “HPV vaccine eligible” parents had one or more female children aged 9–15 eligible for HPV vaccination during the course of the study. While the 2016 HPV vaccination policy only included females aged 9 and 13, females who were in this age range in 2014 (but no longer eligible for vaccination) could have been up to age 15 at the time of the study; therefore their parents were included in the “HPV vaccine eligible” category. All other parents were considered “HPV vaccine ineligible.”Descriptive statistics were computed and stratified by HPV parent eligibility. We conducted bivariate analyses with chi-square tests . Significance was assessed at α = 0.05 level. Willingness to vaccinate all individuals age 9–26 was examined by parent’s demographic, knowledge, and attitude variables.The project was reviewed and approved by the Emory University Institutional Review Board  and the Municipality of Mauá .N = 219) parental surveys were included in the data analysis, with responses relating to 432 children. Most (87.2%) parents were female; ages ranged from 25 to 64 years. Approximately 30% of participants were either unemployed or making less than the Brazilian minimum wage (US$203 monthly); median income was US$258 (IQR: US$116 to $413). Most parents (62%) indicated they were responsible for health-related decisions regarding the children in the house (Table Two-hundred nineteen N = 29 parentan = 251 children) and 51.5% being female. There were no significant demographic differences between the two groups thus allowing for cross group comparison of the attitudinal and knowledge variables (p > 0.05)  to 21 years of age or older, with the majority aged 9–15 years (p > 0.05) . Ninety-four percent (94%) of parents said they would pay to have their children vaccinated if the child did not qualify for vaccination through SUS; however, parents stipulated that they would do so only if they could afford the vaccine. Approximately 89% of parents also said that they would have all their children - regardless of gender and age - vaccinated against HPV if the cost of the vaccine was covered through SUS. Over 86% of parents expressed that they had no hesitation in regards to the HPV vaccine. Across the two parent groups there were no statistically significant differences about the provision of the HPV vaccine to currently excluded populations (5) Table .Table 2Ap > 0.05) . Additionally, both groups agreed that HPV vaccination is beneficial for females aged 9–13 (approximately 94% agreement), as well as beneficial for females over 13 years (approximately 83% agreement). HPV vaccine eligible and HPV vaccine ineligible parents differed slightly in their beliefs regarding vaccine efficacy in preventing cervical cancer, vaccine safety, men being vaccinated against HPV, and vaccine efficacy for men. However, none of these attitudinal differences were statistically significant across parent groups (p > 0.05)  knew that both males and females should be vaccinated against HPV. Approximately 66% of parents could correctly identify the 2016 eligibility criteria for vaccination through SUS. Knowledge that women can be vaccinated up to age 26 was low, with only 47% of parents correctly responding to this item. There were no statistically significant knowledge differences between the two parent groups  for vaccinations . Like HPBrazil’s HPV vaccination campaign has already been successful and influential. Prior to 2014, the prevalence of HPV infection was between 24.8 and 35.0% . As of 2As with all studies, there are certain limitations to our data. Based on participant attitudes, it is evident that there is strong support for the expansion of HPV vaccination for excluded populations, and is not based on currently eligibility, or differential knowledge and attitudes about the vaccine. These data may be limited by social desirability bias. While most bias of this sort is related to self-reported behavior, it is possible that our participants assumed that the researchers would view expansion of HPV vaccine eligibility as favorable and thus responded accordingly. Moreover, our study shows only preliminary results on this topic, and due to the sample size and its cross-sectional nature, a larger, more representative study is required to generalize the findings to the entire country. It is important to note that the field has been widely researched and opposing studies exist both in support of gender-neutral vaccination, as well as, in support of vaccinating adolescent girls only and relying on herd immunity , 34. TheSince implementing SUS in 1988, Brazil has remained proactive in prioritizing health care and diminishing excessive costs for the individual, despite limited financial resources. To achieve its mandate for universal health access, SUS programs must be realized across age, gender, and income level .Vaccination is a main component of preventive care, and based on multiple cost-effective studies, HPV vaccination is favorable , 37. In


Epithelial Ovarian cancer (EOC) although rare is the most lethal gynecological cancer in women worldwide. Despite its high prevalence few studies have been performed to evaluate the prevalence and determinants of HPV infection worldwide. The aim of the present study was to investigate the presence of HPV-DNA in Moroccan patients with EOC using PCR among women in Casablanca, and to examine the prevalence of some HPV genotypes in Moroccan population. We performed a study of HPV detection on Fresh biopsies of 70 epithelial ovarian cancer patients. PCR was realized using the MY09/11 and GP5+/6+ primers. Genotyping of HPV was performed by PCR typespecific for HPV 6, 11, 16, 18, 31, and 33.Data was statistically analyzed using SPSS software. Hence, the mean age was 48.9 years . Serous adeno carcinoma (75.71%) and stage III of the disease represent the majority of cases. eight patients were HPV positive (11.42%).Results of HPV genotyping revealed predominance of two genotypes: HPV 16 (87.5%) and HPV 31(12.5).No co-infection identified. Approximately 75% of positive cases had a serous cystadeno carcinoma and more than 62,5% had FIGO advanced stage (III or IV).Our study showed that high-risk HPV infection could play a major role among patients with EOC in Morocco. EOC although rare is the most lethal gynecological cancer inwomen worldwide and represent the fifth most commonmalignancy, leading to 22.400 newly diagnosed cancer cases andover 14,300 deaths every year in the US Good management of EOCdepends on an early detection of the disease; unfortunately, the lateand poor prognosis is a result of (a) the nature of this malignancywhich is insidious asymptomatic in its early onset, (b) deficiency ofeffective methods of detection at an early stage of the disease, and(c) resistance of chemotherapy [In Morocco, ovarian cancer is the 5th female cancer and accountsfor 4, 9 % of cases recorded during the period 2008-2012, in terms ofincidence this malignancy represent brute incidence of 5.6 per100,000 women, standardized incidence on the Moroccanpopulation of 5.4 per 100,000 women and a standardized incidenceon the world population of 6.2 per 100,000 women . EffortsInfectious agents, mainly viruses, are among the few known causesof cancer and contribute to a variety of malignancies. Humanpapilloma virus (HPV), Hepatitis B and C virus, and Epstein barvirus, are implicated in the cervical cancer, the liver cancer andBurkitt's lymphoma respectively as found by Moss et Blaser . TheLinThe study involved a series of 70 fresh biopsies from patients agingfrom 21 to 76 years, with histo pathologically confirmed EOCobtained after surgical intervention in the department ofgynecology and obstetrics "A", Ibn Rochd University Hospital,Casablanca, Morocco. Informed consent was obtained from allparticipants and the study protocol was approved by the localethics committee of Faculty of Medicine and Pharmacy ofCasablanca Morocco.The fresh biopsies harvested by a clinician, were immediatelyplaced into 2 mL cryo tubes and stored in liquid nitrogen.Histological type of tumor was determined according to the criteriaof the World Health Organization (WHO)  and the DNA Extraction was performed using the phenol/chloroformmethod routinely used in the LVMQB/ETB. Briefly, small sectionsof 5µm obtained from frozen tissue samples using a scalpel, wereplaced in 1.5 mL sterile Eppendorf tube. Depending on the size ofthe biopsy, 250 to 500µL of lysis buffer  containing 200µg/ml of proteinase K wereadded and digested for 3 h at 55°C. Purification was performed byphenol/chloroform/iso amyl alcohol (25:24:1). Then the DNA wasprecipitated using 7.4 M ammonium acetate and absolute ethanol.The DNA pellet was washed in 70% cold ethanol and dried at 37°Cfor 15 min before being re-suspended in 30 to 50µL of ultrapurewater and stored at-20°C until PCR amplification.Before the PCR amplification of the viral DNA, the extract wasdosed in Nano drop 8000 Spectrophotometer .To evaluate the efficiency ofthe extraction, integrity of specimen and absence of PCR inhibitors,all extracted DNA were subject to an amplification of β-globinreference gene using the primers pair PCO4/GH20 as describedpreviously . All ampHPV detection and typing was performed using a nested PCRusing the MY09/11 and GP5+/6+ consensus primers . These primers amplifyrespectively 450 bp and 150 bp sequences of the L1 gene of HPVencoding for the major capsid protein . MY09/1170 patients were included in this study. The mean age at the time ofadmission was 48.9 years . The histopathologicaldiagnosis revealed 53 patients with serous cyst adenocarcinoma(75.71%), nine (12.85%) had mucinous type, five (7.14%)had endo-metrioid and tree (4.30%) had an undifferentiated adenocarcinoma.The distribution of patients according to FIGO stagewas as follows: 4 (5.71%) stage-I, 16 (22.85%) stage-II, 47 (67.14%)stage-III and 3 (4.30%) with stage IV as given in Between 70 EOC samples analyzed, HPV-DNA was detected in11.42% (8/70) of cases. Only two high-risk genotypes wereidentified: HPV 16 was the most prevalent with 87.5% (7/8)followed by HPV 31 with 12.5% (1/8). None of the patients hadmore than one type of HPV . The disThe first study on the association between HPV and ovarian cancerwas published  and it wIn addition, in view of the investigation we cannot establish acausal relationship between HPV positivity and this malignancy.The presence of HPV-DNA in different EOC does not seem to becorrelated to the histological type of tumor, the stage ofdevelopment or the patient's age (p> 0.05). However, severalhypotheses have been advanced to explain the presence of HPVDNA and certain oncogenic high-risk types HPV in the EOC. Mostfrequently cited are among others: (i) The low intensity of the DNAbands on the electrophoretic analysis of the PCR product mayreflect a latent infection, which is characterized by a few copies ofHPV genome in the nuclei of infected cells and therefore cannot beconsidered responsible of disease ; (ii) ThThe present study showed for the first time, the presence of highriskHPV in Moroccan patients with an epithelial ovariancarcinoma, suggesting that this virus may play an important role inovarian carcinogenesis considered as a tumorigenic virus. Thesestudies will be interesting to develop advanced tools for earlydiagnosis and a better prognosis of cancer. In view of the findings,it may be interesting to examine the possible link HPV-EOC in thelarge case-control studies among Moroccan patients to betterelucidate this association.


Hydrangea macrophylla is blue, although it is well known that sepal color easily changes from blue through purple to red. All the colors are due to a unique anthocyanin, 3-O-glucosyldelphinidin, and both aluminum ion (Al3+) and copigments, 5-O-caffeoyl and/or 5-O-p-coumaroylquinic acid are essential for blue coloration. A mixture of 3-O-glucosyldelphinidin, 5-O-acylquinic acid, and Al3+ in a buffer solution at pH 4 produces a stable blue solution with visible absorption and circular dichroism spectra identical to those of the sepals, then, we named this blue pigment as ‘hydrangea blue-complex’. The hydrangea blue-complex consists of 3-O-glucosyldelphinidin, Al3+, and 5-O-acylquinic acid in a ratio 1:1:1 as determined by the electrospray ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectra. To map the distribution of hydrangea blue-complex in sepal tissues, we carried out cryo-time-of-flight secondary ion mass spectrometry analysis. The spectrum of the reproduced hydrangea blue-complex with negative mode-detection gave a molecular ion at m/z = 841, which was consistent with the results of ESI-TOF MS. The same molecular ion peak at m/z = 841 was detected in freeze-fixed blue sepal-tissue. In sepal tissues, the blue cells were located in the second layer and the mass spectrometry imaging of the ion attributable to hydrangea blue-complex overlapped with the same area of the blue cells. In colorless epidermal cells, atomic ion of Al3+ was hardly detected and potassium adduct ion of 5-O-caffeoyl and/or 3-O-acylquinic acid were found. This is the first report about the distribution of aluminum, potassium, hydrangea blue-complex, and copigment in sepal tissues and the first evidence that aluminum and hydrangea blue-complex exist in blue sepal cells and are involved in blue coloration.The original sepal color of  Hydrangea macrophylla originates from Japan and East Asia. The original color of its sepals is blue 3 date back to the early 19th century. In acidic soils (pH less than 5.0), the level of water-soluble Al3+ in soil is increased and absorbed by the roots. Once absorbed, the Al ions are transported into sepals where they form complexes with anthocyanin, resulting in blue sepal color. The structure of anthocyanins and the copigments in hydrangea sepals were reported in the mid-20th century7. Interestingly, all sepal colors originate from a single anthocyanin, 3-O-glucosyldelphinidin (1)6, and the same three copigment components, 5-O-caffeoylquinic acid (2), 5-O-p-coumaroylquinic acid (3), and 3-O-caffeoylquinic acid (4)  in the blue sepal color development was reported, and a stable blue solution was obtained by mixing ten equivalent of Al3+ (1.0 mM) to 1 (0.1 mM) and 2 and/or 3 (0.1 mM)8. However, the molecular structure of the blue pigment and the mechanism of color variation is yet to be clarified.The plant species (4) Fig. . In 19909. By combining the micro-spectrophotometry and a proton-selective microelectrode analysis, we identified a significant difference in pHv between the blue and red protoplasts 9. We also quantified the levels of 1–4 and Al3+ in blue and red cells. The concentrations of 5-O-acylquinic acids (2 and 3) and Al3+ in the blue cells was significantly higher  than those in the red cells 10. To obtain structural information of the blue complex, we synthesized various natural and unnatural copigments and attempted whether the copigments could reproduce the blue color of the sepals by mixing 1 and Al3+ in buffered solutions13. The essential structural elements of the copigment for blue coloration are 1-COOH, 1-OH, and 5-O-aromatic acyl group in the quinate12. Recently, a combination of electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS)14 and nuclear magnetic resonance15 revealed that the molecular structure of the blue metal-complex pigment in hydrangea is composed of 1 and 2 and/or 3 with Al3+ in the ratio 1:1:1; we named this complex “hydrangea blue-complex” ex” Fig. 14,15. Hoin planta visualization of such water-soluble chemicals contained in vacuoles is the cryo-time-of-flight secondary ion mass spectrometry (cryo-TOF-SIMS) of freeze-fixed samples20. We have previously analyzed the distribution of Al and other inorganic ions in the stem of H. macrophylla and clarified the change in metal ions by treatment with aluminum salt21. In the present study, we carried out a molecular and atomic ion mapping of freeze-fixed sepal tissue of blue hydrangea and reported on the distribution of hydrangea blue-complex, aluminum ion, and copigments in blue cells of the sepals.Imaging mass spectrometry is a powerful tool in mapping of inorganic ions and organic molecules in tissues. The most promising technique for O-glucosyldelphinidin (1), 5-O-caffeoylquinic acid (2), 5-O-p-coumaroylquinic acid (3), and 3-O-caffeoylquinic acid (4) were quantified by using high performance liquid chromatography (HPLC). Aluminum (Al) and other inorganic atoms  were quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using wet-ashed sepal tissues.Initially, we analyzed the organic and inorganic components involved in the blue coloration of hydrangea sepals to confirm their chemical composition Table . The blu1–4) were shown in Table 1 in both blue and red sepals was relatively lower than our previous quantification results in colored cells10. This was because that anthocyanin exists only in the colored cells located at the second layer of the sepal tissue, but the quantified value in the experiments is the content in the whole sepal tissue including colorless cells10. In the previous study, we analyzed collected colored cells prepared from blue and red colored sepals. However, in this study we used the whole sepal tissue of the same species of cv. Narumi blue and Narumi red, because the purpose of the study is mapping the organic and inorganic components in tissue. These differences may be the reasons that no significant differences were observed between the quantitative data of organic components.The contents of organic components –4 were s−1 fresh weight [FW] being significantly higher than that in red sepals (6.1 ± 2.9 μg g−1 FW). The other elementals, such as K, Mg, and Fe, were present at usual levels and they did not differ between the blue and red tissues. The exception was Ca, whose concentration differed significantly between the two types of sepals; its content in red tissues was 770 ± 110 μg g−1 FW and in blue tissues it was 520 ± 77 μg g−1 FW. These results were consistent with our previous results reported for the hydrangea stem21.The results of the elemental analysis of inorganic atoms by ICP-AES are shown in Table 14. 3-O-Glucosyldelphinidin , 5-O-caffeoylquinic acid , and AlCl3 (100 μM) were dissolved in 100 μM KCl aqueous solution and measured using visible adsorption (Vis) spectrum and circular dichroism (CD)  Fig. . The pH m/z = 841 and m/z = 843 were detected with the negative and positive mode, respectively, confirming that the complex composed of 1, 2, and Al3+ in the ratio of 1:1:1 existed in the solution14.The solution was analyzed by ESI-TOF MS using negative Fig.  and posim/z = 841 ([1 + Al + 2−4 H]−), which was attributable to the molecular ion of hydrangea blue-complex. This result confirmed that SIMS measurements could identify the same molecular ion of the hydrangea blue-complex as that detected by the ESI-TOF-MS analysis. However, such ion could not be detected with the positive detection mode  and AlCl3 (100 μM) in 100 μM aqueous KCl was measured using cryo-TOF-SIMS with negative detection mode  was detected at m/z = 393 . The molecular ion at m/z = 353 identified as 2 or 4 was distributed in both the epidermis and the second cell layer  was observed clearly in the epidermal cells , a higher ion count was detected in red sepal tissue than in blue sepals  and cultivated in the Botanical Garden, Nagoya University Museum. Fresh, fully colored sepals were collected and cut into approximately 10 mm square pieces. The tissues were quickly frozen in liquid Freon® 22  at −160 °C and stored at −80 °C until use.14. Briefly, 3-O-glucosyldelphinidin  was purified from the seed coat of the scarlet bean, Phaseolus coccineus, and the copigments 5-O-caffeoylquinic acid (2) and 5-O-p-coumaroylquinic acid (3) were synthesized13. 3-O-Caffeoylquinic acid (4) and trifluoroacetic acid (TFA) were purchased form FUJIFILM Wako Pure Chemical Corporation , and KCl and AlCl3·12H2O was purchased from KANTO KAGAKU .Chemicals used in this study were the same as previously reported14. Briefly, stock solutions of Dp3G , copigment , AlCl3 (1 mM), and KCl (1 mM) were mixed in a 1.5 mL microtube at a final concentration of 100 μM  and 200 μM (2). The pH of the solution was measured with a D-21 pH meter and a LAQUA 9618S-10D electrode .The reproduction of the blue solution contained hydrangea blue-complex was conducted as reported previously with some modifications−1 and 25 °C. CD spectra were measured with a CD J-720 spectrophotometer (JASCO) over the range of 400–800 nm with a scanning rate of 500 nm min−1 at 25 °C; the mean of four trials was determined. The reproduced blue solution was measured in a quartz cell with a 10 mm path length.Vis spectra were recorded with a UV V-550 spectrophotometer  from 400 to 800 nm at a scanning rate of 400 nm min24 with a slight modification. Briefly, about 250 mg of tissue samples were extracted with 50% aqueous acetonitrile (CH3CN) solution containing 5.0% TFA for four times (1.5 mL × 4) and the extracts were combined and messed up to 10 mL. The extract was analyzed with HPLC system composed of two PU-1580 pumps, a HG-1580-32 mixer, a DG-1580-53 degasser, an MD-2018 detector, and CO-1565 column oven, and the system was controlled using ChromNAV ver 2 software. A reversed phase column  were eluted with a linear gradient elution from 10% to 18.8% aqueous CH3CN solution containing 0.5% trifluoroacetic acid for 30 min with a flow rate of 0.2 mL min−1 at 40 °C. The calibration curves of each component were obtained with triplicate analyses of the standard solutions.Quantification of organic components by HPLC was carried out as previously described14.ESI-TOF-MS analysis was performed using a micrOTOF-QII mass spectrometer  and analyzed using the included software21. Briefly, frozen samples were pulverized in liquid N2 and kept immersed in 1.5 mL 60% HNO3 at room temperature for 16 h. The sample suspension was subsequently subjected to wet ashing under the following conditions: 105 °C for 2 h, 160 °C for 16 h with 0.2 mL of 30% H2O2. After cooling down the solution was messed up to 10 g and then filtered. The resultant sample solutions were analyzed using an ICP instrument . The detection limits of each element were described in Table About 20 mg of tissue samples were analyzed by ICP-AES as previously described26. The frozen sample was fixed in a sample holder, cut in the glove box (<−30 °C) under a dry N2 atmosphere, and transferred to the cryo-TOF-SIMS . The measurement conditions were as follows: primary ion, 22 keV Au1+ (5 nA); image size, 300 × 300 µm and 256 × 256 pixels; pulse width, 1.8 ns (bunched for spectrum) or 13.0 ns (non-bunched for image); temperature, −120 °C; measurement time, 10–15 min. Bunched and non-bunched images were obtained in positive and negative ion detection modes, respectively. Calibration curves used were water clusters as follows. Positive: [CH]+ 13.0078, [(H2O)2 + H]+ 37.0289, [(H2O)4 + H]+ 73.0500, [(H2O)11 + H]+ 199.1238, negative: [OH]− 17.0027, [(H2O)3-H]− 53.0238, [(H2O)4-H]− 71.0343, [(H2O)11-H]− 197.1081. A low-energy pulsed electron gun (30 eV) was used for surface charge compensation. TOF-SIMS data analysis was carried out using WinCadence software (ULVAC-PHI. Inc.) and the color scales of the obtained bitmap images were changed in ImageJ software27.The cryo-TOF-SIMS analysis was conducted using the manufactured system containing cryo-TOF-SIMS, cryo glove box, and cryo-vacuum transfer shuttles9. The prepared sections were observed under an optical microscope .Thin transverse sections (5 μm thick) were prepared following the previously reported method for preparing hard tissue sectionsSupporting information


Herbal and traditional medicines (HTM) are widely used in Asian countries. Specific data on prevalent of HTM usage and association with chronic diseases in the Thai population is currently lacking. We examined the prevalence and factors associated with HTM use in a Thai worker population. In addition, we explored the relationship between HTM use and therapeutic control of cardiovascular risk factors and documented the most common types of HTM used in various chronic diseases.Employees of EGAT (The Electric Generating Authority of Thailand) who had participated in a health examination were studied. Each participant documented their HTM consumption and self-reported chronic diseases in a questionnaire. Clinical disease and therapeutic control were also defined by concomitant laboratory tests.Of a total of 6592 subjects, 32.6% were HTM-users. Age < 50 years, female gender, self-reported history of diabetes, liver disease, cancer, dyslipidemia, and alcohol use were independently associated with HTM use. HTM consumption increased in proportion to the numbers of self-reported chronic diseases. There were no differences in the therapeutic control of cardiovascular risk factors between HTM users and non-users. Liver and kidney function were not different. The most commonly used HTM was turmeric.HTM consumption is common in community-based Thai subjects, with higher use among those with chronic diseases. Although there were no differences in control of cardiovascular risk factors between HTM users and non-users, many of the commonly used herbs have relevant biological activities for chronic disease prevention or treatment.The online version of this article (10.1186/s12906-019-2652-z) contains supplementary material, which is available to authorized users. Rapid urbanization and globalization has led to a marked increase in non-communicable diseases (NCDs) around the world . As the The World Health Organization (WHO) estimated that 70–80% of populations from developing countries use herbal and traditional medicine (HTM) as the primary method for health care needs, and HTM use has also been extensively embraced in Western countries . DespiteIn 2011, Thailand was reclassified by the World Bank from a lower-middle income to a higher-middle-income country. Along with the economic transition, the prevalence of CVD and related risk factors have increased markedly . Self-prThe subjects were employees of EGAT (The Electric Generating Authority of Thailand), who had participated in a health survey to evaluate risk factors for cardiovascular and other chronic illnesses. The details of this cohort has been described in detail elsewhere . This st2. Diabetes was defined as a fasting glucose of ≥126 mg/dl, or use of oral hypoglycemic medications or insulin or those with previous history of diabetes mellitus [2 [Smokers and alcohol drinkers were defined according to status within 1 year prior to the survey. Obesity was defined as BMI > 30 kg/mmellitus . Hypertelitus [2 . Severe litus [2 . Mild liParticipants were said to have ‘self-reported’ diabetes, hypertension, liver disease, cancer, and dyslipidemia if they checked ‘Yes’ in specific boxes for the presence of these underlying conditions in the questionnaire. The disease was said to be absent if the subject checked ‘No’. Subjects who did not check the boxes were classified as missing. Previous cardiovascular disease was assigned if the patient had a history coronary heart disease or stroke. Each of the above self-reported condition was counted as one co-morbidity and the total number of self-reported co-morbid conditions were recorded for each individual.2) was calculated by using the CKD-EPI equation [Blood tests were performed in a laboratory in compliance with ISO 15189 as detailed previously . Estimatequation .p-value < 0.05 was considered statistically significant.Categorical data were reported as number (percent) and continuous data were reported in mean ± standard deviation. Demographic, clinical and laboratory data of HTM users were compared with Non-users by T test (continuous data) or Chi-square test . HTM use and the numbers of self-reported chronic conditions was evaluated by chi-square test. Factors associated with the use of HTM was evaluated by univariate and multivariate regression analysis using self-reported chronic conditions, CVD risk factors and demographic factors. The proportion within therapeutic targets for self-reported conditions were compared between HTM users and non-users. The top ten most common types of HTM used by self-reported diseases or laboratory-defined liver enzyme or kidney function abnormalities were identified. Statistical analysis was performed using SPSS version 22  Missing data were excluded. Tests were 2-tailed, and a n = 204) who did not respond to the HTM use question, a total of 6592 subjects  or 97% of those originally recruited were included in the study . Characteristics of HTM users were compared with Non-users  (data not shown) were similar between the 2 groups. Few subjects had severe liver dysfunction . Proportions with mild or severe liver enzyme abnormality were similar between HTM users and Non-users.The prevalence of HTM use was 32.6% n = 249. Charan = 6425, 97%), dyslipidemia  liver disease , diabetes , pre-existing CVD  and cancer   to have self-reported diabetes mellitus ,dyslipidemia , liver diseases  and cancer  and tended to have higher proportion of self-reported hypertension, but there were no differences in established CVD.  . By multivariate analysis, younger age (< 50), female, self-reported diabetes, liver disease, dyslipidemia, cancer and current alcohol consumption were associated with HTM consumption.To evaluate the factors potentially influencing HTM use, demographic factors or self-reported chronic diseases were evaluated for associations with HTM consumption Table  By univaOne thousand two hundred subjects listed HTMs that they had used. Of these subjects, 208 subjects listed more than 1 compound. The top ten most commonly used HTM (in order from the most common first) were: turmeric (20%), cinnamon (19%), heart-leaved moonseed (18%), garlic (17%), mixed botanical preparations (13%), ginseng (13%), lingzhi mushroom (12%), kariyat (11%), black ginger (11%), and drumstick tree (10%).(data not shown) were similar to CKD (eGFR< 60).The top 10 most commonly used HTMs in subjects with pre-existing self-reported conditions, or laboratory defined liver enzyme abnormality or CKD are shown in Table In this study, we found that 33% of a Thai worker population used HTM. Age < 50 years, female gender, self-reported history of diabetes, liver disease, cancer, dyslipidemia, and alcohol use were independently associated with HTM use. HTM consumption increased in proportion to the numbers of self-reported chronic disease factors. There were no differences in the therapeutic control of CV risk factors between HTM users and non-users. The most commonly used HTM was turmeric.The reported prevalence of traditional medicine users in Southeast Asia varied widely from 55% in Singapore to 2% in Indonesia . In ThaiChronic illnesses has been associated with higher HTM use in Western populations . SurveysAlthough HTM are widely used in patients with established CVD,  the relaMost of the HTMs in our top ten list has been shown to have potential health benefits. In Thailand, turmeric is a commonly used culinary compound, listed in Thai National List of Essential Medicines as anti- flatulence agent . TurmeriKaempferia parviflora) has been used in Southeast Asia for centuries to improve physical work capacity and as an aphrodisiac. It has been selected as a champion herbal product to generate income in Thailand. A recent systematic review, however, failed to find conclusive evidence to support the benefits of Kaempferia parviflora [Moringa oleifera) possess pharmacological activity based on pre-clinical studies in chronic diseases such as dyslipidemia, diabetes and hypertension [For some commonly used HTM, more studies are necessary to demonstrate potential health benefits. Black ginger Additional file 2:Table S1. The laboratory values and the proportion out of recommended target range of subjects with self-reported conditions. (DOCX 17 kb)Additional file 3:Figure S1. Prevalence of self-reported conditions among herb and traditional medication (HTM) users compared to non-users. Shaded bars reprsent HTM users; white bars represent Non-users. * denotes p < 0.05. (PDF 8 kb)Additional file 4:Figure S2. Prevalence of herb and traditional medication users by numbers of self-reported non-communicable disease conditions. (PDF 32 kb)