Cortical blood flow can be modulated by local activity across a range of species; from barrel-specific blood flow in the rodent somatosensory cortex to the human cortex, where BOLD-fMRI reveals numerous functional borders. However, it appears that the distribution of blood capillaries largely ignores these functional boundaries. Here we report that, by contrast, astrocytes, a major player in blood-flow control, show a striking morphological sensitivity to functional borders. Specifically, we show that astrocyte processes are structurally confined by barrel boundaries in the mouse, by the border of primary auditory cortex in the rat and by layers IIIa/b and Cytochrome Oxidase (CO)-blobs boundaries in the human primary visual cortex. Thus, astrocytes which are critical elements in neuro-hemodynamic coupling show a significant anatomical segregation along functional boundaries across different mammalian species. These results may open a new anatomical marker for delineating functional borders across species, including post-mortem human brains.DOI:http://dx.doi.org/10.7554/eLife.15915.001 The brain is subdivided into many specialized regions that each has distinct roles. A key aim of brain research is to define the boundaries of these areas. Researchers have attempted to map the transitions between brain regions by identifying changes in the properties and activity of neurons (the cells that transmit information around the brain). However, these approaches cannot be used in some circumstances, such as when studying the living human brain, where only non-invasive experimental techniques can be used.Cells other than neurons are also present in the brain. Astrocytes (a sub-type of glia cells) are support cells that have an extensive array of branches that project from each astrocyte’s cell body, often giving it a characteristic star shape. Now, using high-magnification light microscopy, Eliam et al. show that the branches of individual astrocytes tend to avoid crossing the borders of brain regions with different roles. These changes in crossing densities define measurable boundaries between such subdivisions.These density-change boundaries formed by the astrocytes are present in multiple species – mouse, rat and human – and in multiple systems: touch, auditory and visual. This discovery could provide a new window into the functional organization of the brain. It may also offer insights into how the brain optimizes its blood-flow control across different subregions.The results of this study raise an additional question: is the confinement of astrocytes to single regions of the brain shaped by experience or is it present from birth? Exposing animals to different sensory experiences at different developmental stages will hopefully shed further light on this phenomenon.DOI:http://dx.doi.org/10.7554/eLife.15915.002 For inspection of the details of astrocyte morphology, tangential  sections were collected from all cortical layers. Prior to astrocyte staining, the sections were photographed using dark field illumination to highlight the barrel field borders. Following the barrel boundary demarcation, sections were immunostained by GFAP antibody to detect the astrocyte morphology. This staining revealed the branching patterns of individual astrocyte processes . An examTo compare the distributions of the astrocyte processes to that of the blood vessel capillary bed, the blood vessel distribution (traced by FITC dextran) was examined . The astrocyte confinement within barrel borders was a ubiquitous and robust phenomenon, and was observed in all animals studied. In all cases, there was a clear reduction in septa crossing by astrocyte branches.To obtain a quantitative measure of this phenomenon, the number of astrocyte process crossings was separately counted in the barrel core and septa . These s± 0.58 vs 3.78 ± 0.35, means ± s.e.m, t298 = 9.7, p<0.001. 95% confidence interval, 10.26 to 11.82 and 3.3 to 4.2 respectively; Cohen's d = 1.72; vs 2.97 ± 0.18. 95% confidence intervals, 2.47 to 2.81 and 2.77 to 3.17) which did not reach significance , performed in a similar manner to that conducted for the layer IV analysis, showed a lower, albeit significant reduction in septa-barrel crossings, relative to crossings of the barrel core in supra-granular layer III . The analysis revealed the expected reduction of about 35% in the number of astrocyte process crossing in the 4 compared to the 8 µm thick sections, but the ratio between the septa/barrels for the astrocyte branches was similar in both thick and thin sections .While our results demonstrate a clear confinement of astrocyte processes to the barrel borders in the mouse cortex, it could be argued that such a phenomenon is unique to the barrel cortex or to the mouse. To examine how general this phenomenon is, we examined the density of astrocyte processes in the auditory cortex of the rat. The analysis was based on 82 ± 9 measurements on 4 neighbored sections  conducted in the left hemispheres of 4 rats (sectioned at Layer IV). For convenience, we will refer to all the secondary fields around A1  as A2. Tvs 7.4 ± 0.69 . Similar results were obtained when we compared between the two areas at a distance of 200 µm from the border; 13.96 ± 0.54 vs 7.18 ± 96 . Importantly, no significant changes were observed at different distances from the border on either side, , indicating that the change in the density of astrocyte processes was abrupt at the A1/A2 border and did not gradually change approaching this border.As can be seen from the histogram in 68 = 1.66, p=0.10) and 13.3 ± 0.54 vs 12.4 ± 1.37 for IIIb . However, a highly significant change was found in the number of crossings when comparing the two sub-laminar areas at similar distances from the borders; 27.2 ± 1.15 vs 13.3 ± 0.54, t66 = 22.39, p<0.001, 50 µm, and 28.5 ± 1.07 vs 12.4 ± 1.37, t68 = 18.44, p<0.001, 200 µm from the border, indicating an abrupt change in astrocyte crossing at the sublaminar border.If the confinement of astrocyte processes at functional borders is a general phenomenon, such a finding could be particularly informative for studying the human cortex. Thus, examining the density of astrocyte processes may offer a novel 'window' into functional boundaries that may be revealed even in post-mortem tissue. To explore the possible extension of our hypothesis to the human cortex, we examined the boundaries of sub-laminae IIIa/IIIb of human primary visual cortex V1. Previous research has uncovered functional distinctions between these two sub-layers in the primate cortex . We analFinally, we examined the potential segregation of astrocyte processes along the compartmental division of CO-blobs vs inter-blobs in human V1. It should be noted that, in contrast with the previous examples, the blob boundaries are not sharply defined and, in fact, dendritic trees, unlike barrel-field borders, are not 'repelled' by these fuzzy borders ; 1994. Hvs 12.1 ± 1.19 (p=0.58). However, we found a significant increase in astrocyte processes crossing at inter-blobs compared to the CO blobs proper  = 9.1 p<0.001. 95% confidence interval, 10.33 to 12.17 and 4.57 to 5.93 respectively and 12.1 ± 1.19 vs 5.25 ± 0.72; t(48) = 11.2 p<0.001). 95% confidence interval, 11.28 to 12.92 and 4.57 to 5.93  and to additional functional boundaries (A1/A2 sub-layer IIIa/IIIb and blob/inter-blob organization respectively). In all cases  enhanced the staining sensitivity. While we cannot rule out this possibility, such limited staining could not account for the selective drop in the density of astrocyte processes that was observed near functional boundaries, and not away from them as seen in all three cases studied  on much slower time scales.It should be noted that a number of previous studies have demonstrated that astrocyte activity may be modulated according to the functional properties of the modules, within which they reside. For example,  have demSimilarly, previous studies have demonstrated synaptic and molecular selectivity of astrocytes- for example Houades et al.  have shoAstrocytes have been suggested to play a critical role in neurovascular coupling, controlling the blood flow upon an increase in neuronal activity .Furthermore, Astrocyte processes have been demonstrated to manifest tight anatomical formations with neuronal dendrites and the activity of the brain actually arises from the coordinated activity of a network comprises of both neurons and astrocytes .Interestingly, a number of recent studies have demonstrated a principle of overlap-avoidance among individual astrocytes. Thus, astrocyte processes appear to form no-overlapping domains which may endow each astrocyte with a unique functional coupling to the neighboring neuronal population . An attrIt is important to emphasize that the astrocyte confinement does not necessarily preclude blood flow across functional boundaries- since the capillaries themselves freely cross these boundaries. However, the confinement may allow a more efficient, precise and localized direction of blood flow to functional domains . CompatiThe mechanism that brings about astrocyte structural restriction is currently unknown. If, indeed such confinement serves an adaptive role, an interesting possibility is that this anatomical confinement reflects a dynamic plasticity process. In such a process, the distribution of astrocyte processes may reflect the level of sustained co-activation across neighboring neuronal assemblies, analogous to a Hebbian learning process. Relevant to this hypothesis, previous research has demonstrated activity-dependent dynamic changes in the coupling between astrocyte processes in the case of olfactory glomeruli . It is nMore generally the finding that astrocyte processes respect functional boundaries could open the way to a variety of plasticity experiments. For example, examining astrocyte remodeling during development , or studFinally, the generality of the astrocyte confinement requires further examination. It should be noted that previous studies  showed tC57BL/6 mice and Wistar rats were used in this study. The mice and the rats were purchased from Harlan . Mice and rats , 8–12 weeks of age, were used and kept in a specific pathogen free (SPF) environment. All experiments were approved by the Institutional Animal Care and Use Committee of the Weizmann Institute.To investigate the striate cortex of the human brain, we examined the right hemisphere of 35-year-old male patient, who died from non-neurological causes. The brain was photographed for purposes of orientation and scale. The occipital lobe through the calcarine sulcus up to the parieto-occipital sulcus, was removed see , and wasFluorescein isothiocyanate (FITC)-dextran, , 10 mg/ml, was dissolved in PBS and filtered through 0.45 µm mesh. 200 µl of the solution was injected into the tail vein of each animal. Ten min after FITC-dextran injection, mice were deeply anesthetized by a peritoneal injection of ketamin and xylazine , their brains were removed and post fixed in 2.5% paraformaldehyde in PBS, pH 7.4, for 24 hr.The left and right cerebral hemispheres of the mice and rats were separated along the longitudinal fissure and the cortex of each side was divided from the underlying white matter. The isolated cortex was then gently flattened between two glass slides, separated by 1.4 mm spacer and soaked for 4–6 days in 1% paraformaldehyde in PBS. The flattened cortices were embedded in paraffin and cut tangentially parallel to the pial surface in thicknesses of 4 or 8 μm by a microtome . Sections were collected and mounted on SuperFrost+ slides . Dark field and fluorescent images were taken immediately after the cutting (before the sections were dried) by a Leica M165 FC stereo microscope connected to a digital monochrome camera  at magnifications of X1 and X3.5.The human primary visual cortex was taken along the banks of the calcarine fissure of the occipital lobe . The tissue was embedded in paraffin and cut coronally  followed by DAB (Sigma) reaction. Stained sections were examined and photographed by a fluorescence or bright field microscope  equipped with Plan Fluor objectives  connected to a monochrome camera . To form one large image of the total barrel field area, auditory cortex or V1 cortex, digital images at intermediate magnifications (20x) were collected and stitched together automatically, using NIS element software (Leica). Each large image of the barrel field consists of 60–120 tiles.The staining was done on four neighboring sections per sample, with an interval of about 18–20 µm. Paraffin sections were deparaffinized and rehydrated. Antigen retrieval was performed in 10 mM citric acid pH 6 for 10 min using a low boiling program in the microwave to break protein cross-links and unmask the antigens and epitopes. After pre-incubation with 20% normal horse serum and 0.2% Triton X-100, slides were incubated with rabbit anti-GFAP  and/or mouse anti non-phosphorylated neurofilament proteins .; diluted 1: 100), at 4ºC for 7d. To enhance the signal, secondary antibodies, biotinylated anti rabbit or anti mouse  were added for 90 min, followed by Cy2 or Cy3-conjugated Streptavidin  for 2 hr. Sections were counterstained with Hoechst 33,258  for nuclear labeling. For single immunohistochemistry we the In addition, to demonstrate the fine processes of astrocytes reaching the barrel border, some sections were imaged with a confocal scanning microscope . We used an oil immersion objective . A z-series of the entire 8 µm section was imaged at 0.42 µm spacing followed by a maximum intensity projection.The staining sensitivity to GFAP was substantially enhanced in the cortex (up to five times) by employing three procedures. First, staining was conducted on thin sections (4–8 µm). Second, an antigen retrieval technique was used, finally we used a long incubation sections with the first antibody (7d at 40ºC).Barrel (mice) as well as A1/A2 (rat) boundary demarcation was based on the dark field (contrast inversed) photographs of the sections in layer IV. The boundaries between cortical sublayers IIIa/IIIb were defined based on NPNF staining . The borTo reveal the precise details of the distribution of astrocyte processes (GFAP immuno-positive staining) and the capillaries (traced by FITC Dextran) in the barrel field area, manual plotting was performed from the assembled images using Adobe Photoshop software  at magnifications of 20x. The septal borders were also drawn manually according to the dark field (inversed) images and superimposed on the astrocyte  or capilversus A2 areas, straight lines (50 µm length) were superimposed at a distance of 50 and 200 µm from the border of A1/A2 on both sides aligned with the border's orientation. These A1 and A2 lines were then superimposed on the corresponding drawings of the astrocyte processes  in A1 rocesses  and the To quantitatively examine the distribution of astrocytes processes in layer III of the human primary cortex, around the border of IIIa/IIIb, we manually drew the distribution of astrocyte processes at high magnification  in both areas. Straight lines (375 µm length) were superimposed on the NPNF image, 50 and 200 µm from the border of the two sublayers, aligned with the border's orientation and then superimposed on the corresponding drawings of astrocyte processes.To quantitatively examine the relationship between the intensity of cytochrome oxidase and astrocytic GFAP staining along cortical layer II and IIIa we superimposed density plots of both types of staining at three parallel lines and calculated the R-values using Pearson's coefficients of correlation. In addition we examined the distribution of astrocyte processes in blobs/inter-blobs zones. A set of vertical lines (500 µm length) were drawn at the center of blobs, the center of inter-blobs and at the left margin of the inter-blobs. These lines were then copied and superimposed on the astrocyte drawings . The aliAll the analysis was performed by a person unaware of the experimental question. For the mice barrel cortex, 8 µm thick, sections were taken one from each of 5 left cortical hemispheres, and on two, 4 µm thick, single sections were taken from two additional hemispheres. The thin sections were analyzed separately to examine possible effects of section thickness on the septa/core crossing ratios. For the rat auditory cortex analysis, we used of one section of each of 4 left hemispheres. For the analysis of the human primary cortex, we used 8 sections from the same brain.The number of line crossings was compared between septa and barrels (separately for astrocyte processes and capillaries) in the mice barrel cortex, as well as between A1 and A2 in the rat auditory cortex and layer IIIa and IIIb in human primary cortex, using independent-sample Student’s t-test. The septa/barrels ratio was compared between astrocyte processes and capillaries using an independent-sample t-test. The septa/barrels ratio was compared between layers (separately for astrocyte processes and capillaries) using a one-way ANOVA on log-transformed ratios, followed by a Tukey post-hoc test where appropriate. All tests were performed by Statsoft's Statistica, version 12. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Christian Giaume and a member of our Board of Reviewing Editors, and the evaluation has been overseen by Sabine Kastner as the Senior Editor.Thank you for submitting your article "Astrocyte Morphology is Confined by Cortical Functional Boundaries in Mammals Ranging from Mice to Human" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript reports a morphometric analysis of astrocytes in cortical brain regions from mouse and human where functional boundaries are known to occur. This work is well conducted and data are strongly convincing.Essential revisions:1) Given the straightforward nature of the study the manuscript is laborious. The story could easily be told in fewer pages and with fewer figures. For example, all of the supplementary figures are impressive but essentially repeat what is shown in 2) The story is told from an angle that appears infused by the assumption that the parcel-specific distribution of astrocytes is shaped by neuronal activity within a functional area. This might be so, but the study provides no evidence for the idea other than that the density of astrocyte processes varies with parcel-borders. Although stated at the outset that anatomical remodeling" occurs over time there is no experimental investigation of such a process. Thus, please eliminate all of the suggestive terminology that refers to: "shaped", sculpting, remodeling, and reshaping. All of these terms are interpretations and are better suited for the Discussion section.3) The authors consider the astrocytes distribution and shape only in term of blood flow control, however, it is now well established that the processes of these glial cells also establish close contacts with synapse and form the so called "tri-partite synapse". Consequently, these specific features of astrocytes described here should also be discussed in term of neuroglial interaction.4) In addition, it is now well established that astrocytes occupy domains that do not overlap. Consequently, the authors should discuss their results also in reference to these particularities that are not shared with neurons. There is a real need to present the data in reference to the literature. Essential revisions:1) Given the straightforward nature of the study the manuscript is laborious. The story could easily be told in fewer pages and with fewer figures. For example, all of the supplementary figures are impressive but essentially repeat what is shown in Following the reviewer comment we have now removed all the supplementary figures except 2) The story is told from an angle that appears infused by the assumption that the parcel-specific distribution of astrocytes is shaped by neuronal activity within a functional area. This might be so, but the study provides no evidence for the idea other than that the density of astrocyte processes varies with parcel-borders. Although stated at the outset that anatomical remodeling" occurs over time there is no experimental investigation of such a process. Thus, please eliminate all of the suggestive terminology that refers to: "shaped", sculpting, remodeling, and reshaping. All of these terms are interpretations and are better suited for the Discussion section.We thank the reviewer for noting this potential ambiguity in our terminology. Indeed the present data does not resolve the mechanism that brings about the astrocyte confinement. Following the reviewer's suggestion we have now replaced all the ambiguous terms with the more anatomically descriptive terms "confinement" or "restriction".3) The authors consider the astrocytes distribution and shape only in term of blood flow control, however, it is now well established that the processes of these glial cells also establish close contacts with synapse and form the so called "tri-partite synapse". Consequently, these specific features of astrocytes described here should also be discussed in term of neuroglial interaction.We agree with the reviewer that neuro-vascular coupling is not the exclusive source of neuron-astrocyte interaction. We have now added the following paragraph in the Introduction:"Another interesting aspect of astrocyte morphology is the tight coupling between the neuronal and astrocyte processes. In particular, recent evidence has demonstrated the existence of tight anatomical co-localization of astrocyte processes and neuronal synapses in what has been termed a "tri-partite synapse" formation . Such links further suggest that astrocyte morphology may follow boundaries defined by neuronal functional compartments."And in the Discussion:"Furthermore, astrocyte processes have been demonstrated to manifest tight anatomical formations with neuronal dendrites and the activity of the brain actually arises from the coordinated activity of a network comprises of both neurons and astrocytes ."4) In addition, it is now well established that astrocytes occupy domains that do not overlap. Consequently, the authors should discuss their results also in reference to these particularities that are not shared with neurons. There is a real need to present the data in reference to the literature.Following the reviewer's comment we have now added the following paragraph in the Discussion:"Interestingly, a number of recent studies have demonstrated a principle of overlap-avoidance among individual astrocytes. Thus, astrocyte processes appear to form no-overlapping domains which may endow each astrocyte with a unique functional coupling to the neighboring neuronal population . An attractive hypothesis, suggested by the present results, could be that long term and habitual confinement of neuronal activation to specific functional domains may have gradually adapted the individual astrocyte arbor domains to the neuronal functional boundaries in parallel with the dendritic and axonal confinement."


However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe3+ ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H2O2 by functioning as an acidic catalyst.The catalytic activity of cytochrome  More interestingly, Tyr67 is located at the distal position of heme pocket, the distance between Tyr67 –OH group and heme Fe3+ is 4.3 Å, which is close to the distances of 5.84 Å and 5.55 Å between Nε2 atom of the distal histidine and heme iron ion in horseradish peroxidase  and cytochrome c peroxidase  In cyt c into a peroxidase, we successfully designed cyt c Y67H and Y67R variants Tm) of the Y67H variant (50°C) and of the Y67R variant (30°C) are much lower than those of the wild-type cyt c (higher than 80°C) and of its Y67F variant (90°C) c. Both Y67H and Y67R variants have high peroxidase activities c. Since there is no solid structural evidences to interpret the basis of peroxidation in this state, it's necessary to study their three dimensional conformation. Thus, in this paper, by conventional two-dimensional 1H-1H NMR methods c Y67H variant.Originally, in order to convert cyt c (CYC1) genes. This is a cytochrome c variant carrying two point mutations K72A/C102T. The change from Cys102 to Thr102 is used to prevent disulfide dimerization of the protein and protein auto-reduction c at pH 7.0. For the sake of simplicity, throughout this paper, the double-site K72A/C102T variant is used as the reference and referred to as native yeast iso-1 cyt c. The triple mutants Y67H/K72A/C102T and Y67R/K72A/C102T used in this study are referred to the Y67H and Y67R variants, respectively.In this paper we investigate the structure of the Y67H mutant. This mutant was constructed using the pBTR2 plasmid which encodes the yeast iso-1-cyt c and its Y67H variant only in the presence of H2O2. As shown in 2O2 in 2O2 reacts with native cyt c and its Y67H mutant, the visible absorption maximal values at ∼408 nm (Soret band), 530 nm (α+β bands) and ∼353 nm (δ band) become weaker and weaker, indicating that the heme ring in both proteins is destructed very quickly 2O2 to generate compound II as confirmed by the absorption band at ∼420 nm  of heme destruction is the highest at about pH 4.0. When pH value is equal to or higher than 6.0, the kobs values for the Y67H variant are almost kept at 0.02 s−1, but is dramatically decreased when pH is less than 4.0. For the native cyt c, the kobs value of heme destruction is not completely changed upon pH condition being varied. Our previous acid titration studies on both native cyt c and its Y67H variant c, at pH 4.0–5.2 , Fe-S bond was clearly cleaved since the absorbance at 695 nm underwent a sharp decrease. The cleavage of Fe-S bond generates a vacancy for H2O2 coordination, so that heme ring is easier to be oxidized by H2O2. Thus, the kobs value of heme destruction of the Y67H variant at pH between 4.0 and 5.2 is significantly higher than those at other pH conditions.To better understand the mechanism of peroxidation by the Y67H variant, we first probed the stability of native cyt 2O2, we then investigated the peroxidase activity of wild-type cyt c and its Y67H mutant on guaiacol vs pH change. As shown in c displays no significant changes in the peroxidase activity, but the Y67H mutant demonstrates the highest peroxidase activity when pH is between 4.0 and 5.2, increased by about 4–10 folds, compared to that of native cyt c. As we know, at pH 4.0–5.2 the axial Fe-S bond is cleaved, generating a channel for loading H2O2 and substrate guaiacol which enhances the peroxidation reaction.After we performed heme destruction by H1H-1H total correlation spectroscopy (TOCSY) and correlation spectroscopy (COSY) in H2O and D2O for spin patterns and 2D 1H-1H nuclear Overhauser effect spectroscopy (NOESY) spectra in H2O for sequential NH-NH and Hα-HN connectivities, we made the assignments for the Y67H mutant under our experimental conditions , according to the extensive lists of assignments for yeast cyt c reported in the literatures cUsing two dimensional (2D) 1H NMR spectra shown in c, so NMR signals of heme of the Y67H variant cannot be assigned directly according to the similarity of the 1D 1H-NMR spectra between the native cyt c and its Y67H mutant. The complete assignments of hyperfine shifted signals of the heme protons, the axial ligands His18 and Met80, and the heme bound Cys14 and Cys17 of Y67H were achieved through analysis of the 2D 1H-1H NOESY spectra tailored to the relaxation properties of these paramagnetic resonances and through one-dimensional nuclear Overhauser effect (NOE) experiments, as reported before 1H-NMR spectrum of native cyt c  ve cyt c  were pre3 because it has NOEs with the signals of a thioether group and methyl groups of Leu32, Leu68 and Leu94, which were also observed in native cyt c. The resonances at 33.1 and 14.0 ppm display strong NOE connectivities to a signal at −0.3 ppm. The only proton which is equidistant from two methyl groups is the meso-Hδ, positioned between the 1-CH3 and the 8-CH3 groups. The assignment of the signal at 33.1 ppm as 8-CH3 is consistent with its connectivities with δ2-CH3 of Leu32 and ε-CH3 of Met64. The remaining resonances are assigned as 3-CH3 and 5-CH3. The resonance at 17.7 ppm was assigned as 5-CH3 due to its NOE connectivities to Gly29 Hα (at −0.5 ppm) and Pro30 Hδ (at −1.6 ppm). The resonances of the other heme substituents were assigned by analyzing the NOESY connectivities, starting from position 1 and 3 and “hopping” around the macrocycle  in the heme ring. The saturation of the signal at 11.10 ppm indicates that it has NOE patterns with two Hβ protons of His18, suggesting that it comes from the backbone NH of His18, which is further confirmed by the NOE peaks between it and the Hα of Cys17 and Thr19 HN. The assignment of Hα of His18 at 8.84 ppm was performed based on NOE patterns between this signal and the HN proton, Hβ2 proton of His18 and between this signal and HN of Thr19. The 1D 1H-NMR spectra of the Y67H mutant show broad signals at −19.5 ppm  and 24.54 ppm (peak 6b)  which beH mutant . The satSaccharomyces cerevisiae iso-1 cyt cc is disappeared in some conformers of the Y67H mutant.The helical conformations of the Y67H mutant were characterized by using the reported techniques c-type heme ring, PHEM is served as one axial ligand histidine of heme iron, and PHMT represents methionine worked as another axial ligand of heme iron. PHCB and PHCC are used as the residues Cys14 and Cys17. These applications for solution structure calculation of cyt c-type heme contained proteins are integrated in the XPLOR-NIH software package. One hundred structures were initially calculated. Upon no NOE violations more than 0.3 Å being found, 30 pairs of hydrogen bond constraints involving slowly exchanging amide protons consistently presented in the initially calculated structures, were introduced as further constraints in the final stage of the structure calculations. Finally, the best 20 structures with the lowest energy selected from 100 calculated structures, were used to represent the three-dimensional structures of the Y67H mutant. The conformers of this bundle show no NOE violation more than 0.3 Å, and have backbone atoms root mean square deviation (RMSD) values of 0.77±0.15 Å, heavy atoms RMSD values of 1.15±0.17 Å with respect to their corresponding mean structures , respectively. The solution structures of the Y67H variant were determined by using meaningful and acceptable 1699 NOEs through the program XPLOR α atoms in secondary structural regions and heme ring including all carbon and nitrogen atoms to form the π-conjugate porphyrin system. The conformers of native cyt c (pdb code 1YIC) and its Y67H mutant are almost identical with a global backbone atoms RMSD value of 1.15 Å, which can explain the fact the exposure of heme group of the Y67H variant to the solvent is quite similar to that of the native form . In native cyt c, –OH group of Tyr67 forms one hydrogen bond to Met80 sulfur, holding an internal molecule of water. In the Y67H mutant, although the side-chain of His67 is almost in the same position of Tyr67 side-chain (c)  between the oxygen atom of Tyr67 –OH group and Fe3+ ion in the native cyt c; the distances in the Y67H mutant between Thr78 Oγ1 atom and oxygen atoms O1α and O2α of heme 6-propionate are 5.11 Å and 6.07 Å, respectively, both larger than those (4.55 Å and 3.44 Å) in the native cyt c. The distances between Asn52 Nδ2 and oxygen atoms O1α and O2α of heme 7-propionate were measured as 5.51 Å and 3.87 Å in the Y67H mutant, respectively, also larger than those (2.86 Å and 4.38 Å) in the native cyt c. All these differences in distances indicate that the mutation from Tyr67 to His67 disrupts the hydrogen bond between Met80 and residue 67, and also changes the hydrogen bond network. These structural changes weaken the axial Fe-S bond , the stability of the Y67H mutant is thus decreased. The weakened Fe-S bond results in slight shift of helix IV (71–74 aa) away from the heme ring  in the Y67F mutant c. Therefore, our NMR structure of the Y67H variant reveals a basis why Y67H is more unstable than its wild-type.To investigate the conformational changes in heme moiety relative to heme ring produced by the mutation from Tyr67 to His67, in de-chain , but thehain (c) . Moreoveeme ring . This ac3+ , but the protein stability is increased by forming a more compact conformation than that in native cyt cTo probe the structural difference between the Y67H and Y67F mutants, we superimposed their conformations  and measc  to its alkaline form  2 of Lys73 and heme iron results in the loss of helix IV (71–74 aa). In the Y67H mutant, Met80 is still ligated with Fe3+, thus the alkaline form of cyt c and the Y67H mutant demonstrate big differences in the conformation with a global backbone atoms RMSD value of 2.64 Å , as shown in c possesses functional diversity through conformational variability.As we knew, from the native cyt c, its Y67H and Y67F mutants, and also the peroxidase activity of the Y67H variant, which will be discussed below.Taken together, the current structure of the Y67H variant interprets why Y67H is the most unstable among native cyt c, into a peroxidase, firstly we need to understand the main differences on the structural elements between cyt c and peroxidase, which were listed below: (1) the 6th axial ligand of heme in peroxidase is vacant for substrate loading; (2) in the heme pocket of peroxidase there is a distal histidine facilitating the formation of compound I intermediate  is increased by about 16 folds , while kcat/Km of the Y67R variant is raised by about 8 times  kcat/Km value for the Y67H variant at 42°C  was about 10-fold greater than that at 16°C, while for the Y67R variant, the kcat/Km value  was almost 50-fold higher. In the Y67R variant, the mutation from Tyr67 to Arg67 may produce not only a distal arginine , but also a room for substrate coordination (because Fe-S bond is broken in the Y67R variant). Our current NMR structure of the Y67H variant (determined at pH 7.0) suggests that the sixth ligation bond Fe-S is attenuated due to the disruption of hydrogen bond between Met80 and Tyr67 existed in the native cyt c. The distance between Nε atom of His67 and heme ferric iron is measured as 5.48 Å in the Y67H variant, very close to the distances of 5.84 Å and 5.55 Å between Nε2 atom of the distal histidine and heme iron ion in horseradish peroxidase  and cytochrome c peroxidase c resulted by the site-directed mutations under the physiological conditions  and without denaturant for the first time.To successfully switch the classical electron transfer protein, cyt rmediate ; (3) in c has very low peroxidase activity at any pH condition. How do we explain this phenomenon? In native cyt c, the hydrogen bond network, including the hydrogen bond between Tyr67 and Met80, stabilizes the ligation between Met80 and heme Fe3+ ion. Thus, the pH titration neither changes the hydrogen bond network, nor weakens the bond between Met80 sulfur atom and heme Fe3+ ion  His67 acts as a distal histidine; 2) vacancy generation specific for the substrate or H2O2 loading. When pH is less than 4.0, the peroxidase activity of the Y67H variant is dramatically decreased due to the production of the low-spin intermediate His18-heme-water, confirmed by a sharp increase at the absorbance at 620 nm, comparable to the wild-type cyt cHowever, the Y67H variant has much higher peroxidase activity at pH values from 4.0 to 5.2 than those at any other pH conditions , in cont2O2, the neutral aromatic ring of residue His67 of the Y67H variant is protonated . The native cyt 2O2 in the absence of guaiacol, the reactions of H2O2 with native cyt c and its Y67H mutant were performed by mixing 2 ml of protein solution  and 5–10 µl of 1 mM H2O2 (reacting with Y67H) or 10 mM H2O2 (reacting with native cyt c) solution, and monitored by recording the UV-visible spectrum of native cyt c and its Y67H mutant by every 20 seconds at 25°C on a Hewlett-Packard 8453 diode array spectrophotometer. The kobs value for heme destruction was set equal to ln(2/T1/2), where T1/2 was the half life-time for the protein when the absorbance at 410 nm was switched into half of the initial absorbance at 410 nm in the reaction system.To investigate how heme was destructed by H2O2 solution was prepared with 30% stock solution and its final concentration was determined with an absorption coefficient of 39.4 M−1cm−1 at 240 nM c or its Y67H variant (2.0 µM), guaiacol (100 µM) in 100 mM buffer  and a solution of 200 mM H2O2 in the same buffer were pre-incubated at 25±0.1°C for 5 min. Then both solutions were mixed together in the mixing cell of the stopped-flow instrument to start the oxidation reaction. The steady-state reaction rates were obtained by monitoring the absorbance increase at 470 nm using a molar absorption coefficient of 26.6 m M−1cm−1The steady-state kinetics of oxidation of guaiacol by hydrogen peroxide were studied with a SF-61 DX2 stopped-flow apparatus  thermostated at 25±0.1°C. The Hc or its Y67H mutant, is similar to that catalyzed by Paracoccus versutus cytochrome c-550 i.e. the linear phase). The peroxidase activity was represented by the AU470/s value, which was the absorbance of the product tetraguaiacol at 470 nm at each pH condition.The product formation curve of guaiacol oxidation catalyzed by native cyt 1H frequency is 600.2 MHz), or on a Bruker AVANCE III-800 spectrometer with cryo-probe equipped with four channels and pulse-field gradient, where the corresponding 1H frequency is 800.2 MHz. The dipolar connectivities were revealed by 2D 1H-1H NOESY experiments. To detect connectivities among hyperfine-shifted signals, the 2D 1H-1H NOESY spectra with a spectral width of 76 ppm in both frequency dimensions 1H-1H NOESY spectra were acquired with a recycle time of 1.5 s and mixing times of 50, 100, 150, and 200 ms, respectively. 1H-1H TOCSY spectra were obtained using the spin-lock times of 30, 50, and 80 ms in H2O and D2O 1H-1H double-quantum filtered COSY (DQF-COSY) spectra were recorded in H2O and D2O 2O, while in other cases pre-saturation was used. All data consisted of 4K data points in the acquisition dimension and 1K points in the indirect dimension. Raw data were weighted with a squared cosine function, zero-filled, and Fourier-transformed to obtain a final matrix 4096×4096 data points. All spectra were collected at 20°C either on the H2O or on the D2O samples, processed by NMRPipe http://www.cgl.ucsf.edu/home/sparky/).All NMR experiments were acquired at 20°C on a Varian Unity Inova 600 spectrometer with cryo-probe equipped with three channels and pulse-field gradient . A total of 100 structures were calculated, and finally, 20 structures with the lowest energy had no NOE violation >0.3 Å. Structure statistics for these 20 structures were summarized in The solution structure calculations were carried out using a standard simulated annealing protocol implemented in the program XPLOR 2.19 (NIH version) The coordinates of the solution structure of Y67H mutant had been deposited with RCSB Protein Data Bank under accession number 2mhm and ID code rcsb103626, and the assignments of its NMR signals had been deposited with BMRB  under accession number 19638, respectively.Table S1c including native cyt c, its Y67H and Y67F mutants, and the alkaline state of cyt c.The measured distances between some key residues and heme ring in different states of cyt (DOCX)Click here for additional data file.File S1c Y67H mutant. Figure S4, Proposed mechanism of compound I formation during guaiacol peroxidation by HRP or CcP. The distal histidine first functions as a general base to deprotonate hydrogen peroxide, and then the protonated hisidine as a acid facilitates the O-O bond cleavage.Supporting figures. Figure S1, NOE patterns involving the heme and its axial ligands of cyt c Y67H mutant. Figure S2, Schematic representation of the sequential and medium-range NOE connectivities involving HN, Hα, and Hβ for cyt c Y67H mutant. Figure S3, The number of experimental NOEs per residues (A) and is correlated with global (black) and local (red) backbone RMSD values per residue (B) calculated from the 20 structures of the lowest-energy family with respect to the average structure of cyt (DOCX)Click here for additional data file.


Multiple human papillomavirus (HPV) genotypes often coexist within the cervical epithelia and are frequently detected together in various grades of the cervical neoplasia. To date, only a few reports exist on multiple HPV infections of HPV in Xinjiang Uygur Autonomous Region (XUAR). In the present study, we investigated the prevalence of High-Risk HPV (HR-HPV) genotypes and multiple infections. Cervical cytology samples were collected from 428 women who presented cervical abnormalities. Genotyping of HPV was performed by polymerase chain reaction–sequencing based typing (PCR-SBT) using consensus primers and specific primers. Of them, 166 samples were positive for HPV according to PCR results using the consensus primers. These samples contained cervical abnormalities enriched with inflammation (n = 107), cervical intraepithelial neoplasia (CIN) I (n = 19), CINII-III (n = 9) and cervical cancer (n = 31). Of the 166 HPV positive samples as determined by PCR analysis, 151 were further typed by PCR-SBT using 19 pairs of genotype-specific primers. Using this method, 17 different HR-HPV genotypes were identified. The most frequently observed HPV genotypes were HPV16 , 53 , 52 , 58  and 35 . The proportions of single and multiple infections in the HPV-positive specimens were 34.9% and 65.1%, respectively. Multiple HPV types were most prevalent in the inflammatory state (63.0%), followed by cervical cancer (24.1%), CINI (11.1%), and CINII-III (1.9%). The results of our data analyses suggested that i) multiple HPV infection is not necessarily correlated with the severity of cervical abnormalities; and ii) among the multiple HPV infections, double infections combined with HPV16 is the most common. In addition, L1 full-length sequences of the top five high-risk HPV genotypes were amplified and sequenced. According to the L1 sequence of the epidemic genotypes that were amplified, we found that these genotypes contained the sequence point mutation, and that some of these genotypes further showed amino acid modifications. These results provide a basis for the construction of a polyvalent vaccine that is suitable for use in the XUAR, even in economically challenged communities located in China. Cervical cancer is the second most common malignancy among women and the leading cause of cancer-related death among females worldwide , 2. EpidProspective studies have shown that infection with multiple HR-HPV acted synergistically in cervical carcinogenesis . AdditioIn China, approximately 135,000 new cases of cervical cancer are diagnosed each year and roughly 50,000 of these women die of cervical cancer annually . The preThe research was conducted from January 2014 to December 2014. A total of 2,046 women were enrolled in the program entitled “The Xinjiang Production and Construction Corps Cervical Cancer Screening Study (XPCCCCSS).” The cytological examination of the collected Pap smears was carried out and defined according to the Bethesda classification . Four huAll eligible women who came to the screening had the details of the study explained to them, and written consent was obtained from all participants. The study was approved by the Gynecology and Pathology Departments of the First Affiliated Hospital, School of Medicine, Shihezi University.From the cervix, cervical exfoliated cell specimens were collected from each participant using a Cyto-Brush  for HPV DNA assays. According to the age of the participant and the stage of cervical cancer, the samples were divided into five groups .2O) was used as the negative control. Beta-globin gene was used as the housekeeping gene to confirm the integrity of the DNA material extracted from the specimens. All primers were synthesized by Beijing Huada Inc., Beijing, China. DNA was extracted from the residual standard transport medium (STM)-stored cervical specimens using the TIANamp Genomic DNA kit  in accordance with the recommendations of the manufacturer. The reaction system was used as previously described [http://www.ncbi.nlm.nih.gov/BLAST/). If a sequence matched the reference sequence of a certain HPV subtype with ≥ 90% similarity, the sequence would be recognized to be the corresponding subtype.Four-hundred and twenty-eight abnormal cytological samples were stored in standard transport medium (STM), and tested by polymerase chain reaction–sequencing based typing (PCR-SBT). The consensus primer, MY09/MY11 , was useescribed , 19. HPVThe sequences of the most prevalent HPV genotypes were amplified as described in Among the 428 cervical samples analyzed in this study, 166 were found to be HPV-positive DNA as detected using consensus primer. In addition, we found 107 inflammation cases, 19 CINI cases, 9 CINII-III cases, and 31 cervical cancer cases. The prevalence and genotype distribution of the cervical abnormalities are shown in Furthermore, in the 151 successfully genotyped samples, HPV16 combined infections were the most common types of infections, which were respectively involved in 44.6% (25/56), 54.6% (18/33), 53.3% (8/15), 100% (2/2) and 100% (2/2) of the double, triple, quadruple, pentagonal and hexagonal infections. HPV52 combined infections were the second most common type of combined infections shown in . The incThe incidence of single HPV infection and multiple HPV infections, as well as the relationship between HPV-positive rate and age are shown in The L1 full-length sequences of HPV16, 18, 52, 53, 58 that are found between the existing HPV vaccine strains and the prevalent HPV strains found in the United States of America were compared with the HPV genotypes obtained in this study. We observed that the HPV genotypes identified in this study contained sequence point mutations as indicated by BLAST, and that some of these mutations further led to amino acid modifications .With the development of molecular epidemiology, biological risk factors for abnormal cervical incidence are mainly related to HPV infection. In this study, a total of 166 samples were found to be positive for HPV by PCR-SBT. HPV infection in samples having a background of inflammation, CINI, CINII–II, and cervical cancer were present in 33.6%, 45.2%, 47.4%, and 63.3% of these cases, respectively, which suggested that the positive rate of HPV increased with the severity of the pathological diagnostic grade. Such results indicate that HPV infection is closely related to the occurrence and development of CIN, which is consistent with the previous reports , 21. TheThe distribution of HPV genotypes, especial HR-HPV genotypes, shows diversity in different countries. It is crucial to understand the distribution of HPV genotypes for designing prophylactic vaccines that are effective among geographically-specific populations in the future. In the present study, a total of 17 HR-HPV genotypes, namely HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 73, were found. Of the 166 HPV-positive samples, HPV16 , 53 , 52 , 58  and 35  were the top five genotypes observed in this study. According to the grades of cervical abnormalities, HPV16, 53, 58 and 52 were the major HPV genotypes in inflammation, CINI and cervical cancer, while HPV33 and 53 were majorly involved in CINII–III cervical lesion.Meta-analysis from the World Cancer Research Center indicated that HPV16, 31, 51, and 53 were the most prevalent HPV genotypes . The datet al. [The popular opinion of many reports indicated that i) multiple HPV infections were associated with the grades of cervical abnormalities, and ii) the cervical cancer risk of patients who suffered from multiple HPV infections was higher than those with single HPV infection. However, there existed controversial conclusions in other reports. For example, Muñoz et al.  summarizet al. . In the Age is one of the most important risk factors for HPV infection . SimilarCurrently, the application of L1 virus-like particle vaccines, including Gardasil®, Cervarix® , and 9-vCollectively, our findings demonstrated that: i) a total of 17 HR-HPV genotypes were found, and HPV16, 53, 52, 58 and 35 were the top five genotypes; ii) multiple HPV infections remained an important factor for cervical lesions, as evident in two age groups: women who were 55 years of age and older, and women who were younger than 25; and iii) L1 full-length sequences of HPV16, 18 and 52, 53 and 58 displayed obvious differences between the L1 sequences derived from the examined vaccines and from the HPV-positive samples from women in XUAR at the level of amino acids.S1 File(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Immigrants from low- and medium-income countries have a higher risk of cervical cancer due both to barriers in access to screening and to higher human papillomavirus (HPV) prevalence.In the near future many screening programmes in industrialised countries will replace Pap test with HPV as primary test. In order to plan future interventions, it is essential to understand how the HPV screening performs in immigrant women.We conducted a survey on the main performance indicators from some of the HPV DNA-based pilot programmes in Italy, comparing regular immigrant women, identified as women resident in Italy who were born abroad, with women who were born in Italy. All the programmes applied the same protocol, with HPV as stand-alone test starting for women of 25 or 35 to 64 years of age. Cytology triage is performed for positive women; those ASC-US or more severe are referred directly to colposcopy; negative women are referred to repeat HPV after one year.Overall, 162,829 women were invited, of whom 22,814 were born abroad. Participation was higher for Italy-born than born abroad (52.2% vs. 43.6%), particularly for women over 45 years. HPV positivity rate was higher in immigrants: 7.8% vs. 6.1%, age-adjusted Relative Risk (age-adj RR) 1.18, 95% confidence interval (95% CI) 1.13-1.22. The proportion of women with positive cytology triage was similar in the two groups (42%). Cervical Intraepithelial Neoplasia (CIN) grade 2 or more severe detection rate was higher for born abroad . The difference was stronger when considering only CIN3 or more severe . Both HPV positivity and CIN2 or more severe detection rate had a different age curve in born abroad compared with Italy-born: in the former, the risk was almost flat, while in the latter it declined rapidly with age.Compliance with HPV screening is lower for migrant women, who are affected by higher HPV positivity and CIN3 cancer detection rates. Persistent infection with oncogenic HPV types is the necessary, but not sufficient, cause of cervical cancer ,2. Wide Several studies have shown that migrant women from low- and medium- income countries have higher risk of cervical cancer -11 and uMigration in Italy is a relatively recent phenomenon: in 1991, foreigners resident in Italy were less than 1% of the total population while in 2011 they were 7.5%, with some cities over 16%. Most of immigrant women in Italy are in the target age of cervical cancer screening ,24.Screening with HPV DNA as primary test has been demonstrated to be more effective in preventing cancer . Many inScreening programmes in Italy cover the vast majority of the resident population, without any difference with regard to citizenship . In 2007In this paper we compare the screening results in 7 HPV-based pilot screening programmes in foreign-born vs. Italy-born women.In Italy, according to the European Commission recommendations (2003) , the RegSince 2007, after the publication of the results of three large randomised trials on HPV DNA as primary test -42, someSome of the projects were randomised pragmatic trials; the others were intervention studies with historical or geographical controls. The pilot programmes tested feasibility, organizational impact, acceptability (for operators and women), compliance with invitation and follow-up protocols, performance of cytology triage and, finally, they collected field information for economic and budget impact analysis.All the projects were approved by the local Ethics Committees or were established by law by the regional health authority.The Osservatorio Nazionale Screening, supported by the Centro per la Prevenzione Oncologica (CPO), Piedmont, annually collects aggregated data from all the Italian screening programmes, whose results are published in an annual national report ,38.The survey methods were developed by the GISCi  in 1999 , and areFrom 2012, the survey collected information from the HPV DNA-based screening.In 2012, the GISCi promoted the collection of information on immigrant women inside the national survey. The country of birth was used to identify the migrant status. Only two programmes (Reggio Emilia and Torino) classified the countries as low- and medium-income or other , according to the classification of “high migration pressure countries” of the Italian Ministry of Health .Data were collected with the same form as the official survey and were disaggregated by age and migration status, while they did not distinguish women at first vs. subsequent screening episodes. Furthermore, we requested data for the three-year period 2009–11.HPV test was performed on liquid based cytology samples or on samples specific to HPV test. In the latter case, a smear was obtained and fixed before taking the sample for molecular testing.All the programmes used Hybrid Capture 2 (Qiagen), on both liquid based cytology (PreservCyt) or on Standard Transport Medium (STM). For programmes using two samples (cytology and STM) the denaturation and hybridization was directly performed on the sampling medium. For programmes using liquid based cytology, 4 ml of PreservCyt were transferred for the conversion according to the manufacturer’s instructions. HPV results were classified as positive or negative for high-risk types. The types included were 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.In the programmes using conventional smears, only the slides of HPV+ women were stained and interpreted. In the case of liquid based cytology, the slide was prepared from residual material after HPV testing only when HPV+. Cytology results were classified according to TBS 2001. All the programmes referred women with ASC-US or more severe cytology to colposcopy. Women with an unsatisfactory cytology were referred to colposcopy in some programmes, while cytology was repeated on a new sample in others.We present the set of the indicators routinely used to evaluate the performance of screening programmes, stratified by country of birth . For two programmes, migrants were further stratified in women from low/medium income and from high income countries. The indicators included participation , HPV positivity, cytology triage results, recall rate to colposcopy, compliance with colposcopy, detection rate for cervical intraepithelial neoplasia grade 2 (CIN2), CIN grade 3 , and for cancer at baseline . Except for participation and compliance to colposcopy, all indicators were age-standardised using the Italy-born screened women as standard population. For participation, cytology results, and detection rate, we calculated age-adjusted relative risks (age-adj RR) with 95% confidence intervals (95% CI).Seven of the 19 pilot projects active in Italy in 2012 participated in the survey. The duration of the study period ranged from one to three years; the survey was restricted to screening results of women invited between 1 January 2009 and 31 December 2011. Overall, the pilot programmes invited 162,829 women, of whom 22,814 were immigrant .Women born abroad invited for screening were on average younger than Italians . The participation was lower in born abroad than in Italy-born women (43.6% vs. 52.2%), though the difference was noticeable only in women aged ≥ 40 years Figure .Figure 1The overall participation rate in women from high-income countries was lower than the average of women born abroad .The overall age-adjusted HPV positivity rate was higher in born abroad than in Italy-born women , with a different shape by age  than did Italians  Table .Table 2Compliance to colposcopy referral was high in both groups .The detection rate of CIN2+ was higher for women born abroad than for Italians (8.2‰ vs. 4.7‰). The difference was entirely due to an excess of CIN3+ , while a negligible difference was observed for CIN2  .This is the first study reporting the results of several population-based screening programmes that adopted HPV DNA as primary screening stratified for migrant and native population in an industrialized country. In Italy, HPV DNA-based screening was implemented quite early: the first pilot programme started in 2007,  and in JSeveral of the HPV DNA test pilot programmes reported a slightly higher participation than that of Pap test-based programmes -36. HoweNevertheless, population-based screening programmes are effective interventions in reducing inequality in the access to evidence-based prevention -51. In IThe prevalence of HPV infection was higher in immigrants than in Italians. Also, the curve of prevalence by age was different in the two groups: the results in Italian women confirmed the prevalence curve already observed in previous population-based studies -55, withThe curve in immigrants is similar to that of many low/medium income countries such as sub-Saharan Africa, central Asia, and Latin America , but notIt is worth noting that the overall proportion of cyto + among HPV+ women is very similar in Italians and immigrants, while the proportion of high-grade lesions is higher in immigrants. A higher proportion of women with high-grade cytology is consistent with a higher detection rate of CIN2+ and particularly of CIN3 in immigrants than in Italians. A ratio between high-grade and low-grade lesions in favour of high grade is probably linked to a lower uptake of screening among immigrants in the preceding years, both in their countries of origin and in Italy. This means that long-lasting and more severe lesions are detected. Indeed, a higher detection rate may be the consequence of both higher HPV prevalence and lower screening uptake in the preceding years.All of the observed differences between immigrants and Italians suggest that the immigrant populations maintain the prevalence of their countries of origin for several years, particularly when migration is a relatively recent phenomenon . Many other studies have also observed this phenomenon when looking at HPV prevalence ,57, cytoThe country of birth, which we used as a proxy of the migration status, could misclassify women, thereby including many Italian women among the migrants. Nevertheless, even citizenship, unanimously considered a better proxy of migrant status in women aged 25–64, can misclassify a significant number of women who are married to Italians but who migrated to Italy relatively recently. Another Italian study compared cervical cancer screening indicators obtained using the country of birth or citizenship and found only minor differences . We deciFurthermore, only few programmes could distinguish women from low/medium income from those from high-income countries. All the analyses confirmed that, in Italy now, calculating the indicators for all the foreigners provides figures that substantially depict the health status of immigrants.Differences between native and immigrant women tend to decrease with time after the arrival in the host country . In the On the other hand, the characteristics of immigrants from high-income countries were substantially comparable to those of Italians, with the exception of participation.Another limit of this study is that we could not identify the exact country of origin. HPV prevalence differs in different areas and it would be important to distinguish between countries with high vs. low prevalence, in order to determine whether the prevalence of communities resident in Italy reflects the country of origin. Analyses by specific communities are necessary to better understand the epidemiology of HPV and cervical cancer in immigrants and to target public health interventions.Furthermore, we could not distinguish between women at their first screening round and those who had already had a previous Pap test. On the other hand, it must be noted that almost all the women included in this survey were at their first HPV screening test.Finally, screening programmes actively invite only women that are resident, or at least registered, in their catchment area, while irregular migrants and asylum seekers, who are surely more vulnerable than are regular residents, can only access screening spontaneously. Therefore we have information only on regular immigrants, registered as such. While citizens from outside of the European Union  must be registered residents, the same is not true for European Union citizens. However, since residents can obtain tax reductions, reduced fees for electricity, gas, water supplies, and so on, not being resident has many disadvantages. Thus, persons staying for a long time usually are registered as resident.Immigrant women have a higher prevalence of HPV, a worse ratio of high-grade vs. low-grade lesions, and consequently a higher risk of cervical pre-cancer and cancer than do Italians. The prevalence of HPV seems to reflect that of their countries of origin. HPV DNA-based programmes are not likely to reduce the gap in participation in screening programmes between immigrants and Italians.


Human Papillomavirus (HPV) -associated cervical cancer is the second-most common cancer in women worldwide but it is the most frequent gynaecological cancer and cancer associated death in India women. The objective of this study was to assess knowledge about cervical cancer, HPV, HPV vaccine, HPV vaccine acceptance among school and undergraduates students and their parent’s perception about acceptance of HPV vaccine in Northern part of India (Delhi and NCR regions).A qualitative questionnaire based survey among 2500 urban/rural students aged 12–22 years was conducted.Overall, a low frequency (15%) of HPV and cervical cancer awareness was observed in students and their parents. However, the awareness was much higher in females belonging to urban setup compared to boys with a perception that HPV causes cervical cancer in women only. Additionally, only (13%) participants who were aware of cervical cancer and HPV) were willing to accept HPV vaccination. Apparently, parents of female students were two times more willing to accept HPV vaccination for their ward than male students .Cervical cancer and HPV awareness among school, undergraduate students and also to their parents was found to be very low in this part of India. The level of awareness and education appears to be insignificant determinants in rural compared to urban setup. Better health education will be needed to maximize public awareness for cervical cancer prevention. Cancer of the uterine cervix is the second-most common cancer in women worldwide but it is the most common health hazard in India Recently, two HPV vaccines quadrivalent “Gardasil” and bivalent “Cervarix” have been shown to be highly effective in preventing infection with high-risk type HPV16 and HPV18, the two most common oncogenic types. As these vaccines are highly effective before exposure to HPV, current guidelines prioritise adolescent girls as primary target group for HPV vaccination Though adolescents are the prime candidates for prophylactic HPV vaccination, several studies have shown that adolescents had very little knowledge about HPV vaccination The deficiency of knowledge of causal relationship between HPV and cervical cancer might adversely affect the vaccine acceptance in India. Several studies have been undertaken to evaluate women’s awareness and knowledge level about cervical cancer and HPV vaccine Preliminary study among 8–17 years old adolescents and their parents in India, suggested that majority of them are unaware of cervical cancer, HPV and HPV vaccines A pre-designed questionnaire-based survey was conducted and a total of 2600 participants aged between 12–22 years were enumerated from June 2009–June 2011 that included both urban and rural school population of Noida and Delhi. Out of 2600 participants, only 2500 participants completed the questionnaire. The study was approved by Ethics Committee of Institute of Cytology & Preventive Oncology (ICPO/IEC/2008-09/05). Written consent was taken from all participants and guardians in case of minors.st visit to access the knowledge and awareness among participants. Data were analysed using SPSS software version 14.0 to assess the association between demographic factors and HPV awareness and cervical cancer.First part of the questionnaire was to collect information on age, sex, study stream, level of study, place of permanent residence, family income, family size and religion etc. were noted. The remaining part a self-administered questionnaire was used for this study, which were to be filled in individually by the participants under the strict vigilance of the teachers/researchers/mentors which serves as invigilators to monitor the influence on answers by the students. The questionnaire contained questions regarding knowledge of cervical cancer, HPV and awareness about HPV vaccine. In addition, there is question on parent’s opinion about HPV vaccine acceptance. The questions were developed based on previous established facts for cervical cancer th standard, eight hundred thirteen (32.52%) students were of 10–11th standard whereas fourteen hundred and one (56.04%) students were of 12th standard as well as of undergraduate level respectively. Further stratification of data with respect to religion revealed a higher participation rate of Hindu students (68.56%) which was evident as majority of Hindu population is dominant in these areas whereas 31.44% were of Muslim students. Detailed demographic characteristics of the participants are presented in A total of 2600 students participated in the study. Sixty eight (68) participants did not complete the personal information details and thirty two (32) were having incomplete questionnaires. Therefore, a total of 2500 participants could complete the questionnaire, out of which 930 (37.2%) were male and 1570 (62.8%) were female. Most of them 56% were from rural set up where as 44% were of urban region. Most of students 58.7% were in the age group of 19–22 years, 28.5% belonged to16–18 years, while 12.8% were of age group 12–15 years. The participation rate of female students was higher (62.8%) as compared to male students (37.2%). Two hundred eighty six (11.44%) students were in the education group of 6–8In order to examine the knowledge/awareness about cervical cancer, HPV and HPV vaccines, a short type questions in local as well as in English language were asked to the students and their parents . In geneOf the total participants (375) who had knowledge about cervical cancer, two hundred seventy five (73%) participants were having knowledge that cervical cancer is caused due to HPV infection . A signiFurthermore, the students were asked to know whether or not they are aware that HPV vaccines are commercially available, two hundred twenty five participants were aware of availability of vaccines. Among them, one hundred sixty two (72%) were female students and sixty three (28%) were male students. Therefore, the correlation of gender of the students with HPV vaccine awareness was observed and found to be statistically significant . Interestingly, vaccine availability awareness was also attributed to urban population 65% (143/225) .Students were educated about the risk factors associated with cervical cancer and how it could be prevented through vaccination and other practices, the willingness of HPV vaccination was found to be 70% in females and 64% in urban populations .Last but not the least, parents opinion was also taken into consideration with respect to vaccine implementation program in North-Indian population. 13% of the parents agreed to vaccinate their wards .st month, 3rd month and 6th month) was made to the same school. A semi-structured qualitative interview was conducted to collect the information regarding awareness, knowledge, disease exposome and vaccination programme etc. All the answers were recorded and reviewed. A good awareness response was observed among participants regarding the knowledge and awareness of cervical cancer and HPV. However, a negative response was documented with respect to implementation of vaccination programme in India due to adverse effects of vaccine related deaths recently. Consequently, a negative response which includes anxiety, physco-social distress and high cost of current vaccines was observed. In order to reduce the burden of cervical cancer and perhaps other disease prevalent in India, awareness through print and electronic – mass media is warranted.After completion of questionnaire by participants, a brief power point presentation was addressed among all subjects which included introduction to cervical cancer, incidence rate both in India and world wide, diagnostic modalities: Pap-test, liquid based cytology (LBC), visual inspection , colposcopy etc. and HPV DNA test by hybrid capture 2 (HC2), reverse line blot, real-time PCR etc., prevention and management. In order to evaluate the impact of awareness made available to participants, three times visits  and Delhi region of India. It provides the useful information which may help in designing HPV vaccine programs and public health policy making in India. The school/college based studies of HPV awareness are very few and we observed an interesting correlation between increasing age and HPV awareness. Our result is in consistent with the other studies in India that showed low level of cervical cancer and HPV knowledge (15%) in graduate, postgraduate and even in medical students A study from Korea showed a mere 9.5% of female high school and university students had ever heard of HPV In the present study, the male students were found to be less knowledgeable about HPV than the female students. This indicates that the female students were much aware about the cervical cancer. Similar to our findings, a study among Australian women and men between the ages 18 and 70 years showed that 62.8% of women and only 38.3% of men had heard of HPV The results of the present study also showed that Muslim students have very less knowledge as compared to Hindu students. It is important to note that presence of HPV and cervical cancer is relatively much lower in Muslim population. In this regards, the regional differences may be due to cultural, religious, social, ethnic variation The present study is also able to determine the parent’s opinion about HPV and HPV related information. Our previous study Study demonstrated that the area wise, urban dwellers had higher knowledge about “HPV infection causes genital cancer” than rural belt and similarly we found that knowledge about “cervical cancer” is higher in urban than rural. This study also showed that more attention is needed to educate the rural populations. In this regard, Li et al reported that (51.1%) urban women knew that HPV is related to cervical cancer in compare to their rural counterpart (41.6%). Even fewer (8.1%) knew that it is associated with genital warts with the similar rate of both geographies Our data represents that overall HPV vaccine awareness is very low both in female and male as well as dwellers of rural and urban origin. But in contrast, few studies reported that high vaccine acceptance among parents and adolescents in the general population In our study majority of participants was unwilling to be vaccinated due to lack of knowledge of HPV vaccine and its safety which were the major reasons for them. This is consistent with studies from other areas and countries In the present study parents decision between mother (15.8%) and father (8.3%) were very low about HPV vaccine as they are decision maker as vaccination takes place within the family unit, generally by one or both parents. We also observed that father’s roles in decision making are also important for finances or critical health events. Although several other studies have examined the preferences for the HPV vaccine among parents The strength of our study lies in the selection of study population of both rural and urban set up. But still there is limitation of our study. Findings of the present study could not be generalised to the large Indian population. Hence, an awareness regarding cervical cancer, its prevention and management is warranted.In conclusion, in order to reduce the burden of cervical cancer and implementation of vaccination program, awareness is required which can be achieved by print and electronic media by raising slogans, conducing free camps in rural sectors for cancer screening and most importantly to sensitize Indian population for vaccine acceptance. Thus, there is a need with immediate effect to educate and aware the young population through print and/electronic media, NGOs, Hollywood, Bollywood stars and famous sports personalities about ill- myths associated with cervical cancer vaccination program in India.


Cervical tissue was examined before starting blinded treatment and ~9 months later. The primary endpoint was regression of CIN 2 or CIN 3 to CIN 1 or no disease at 6 months post third dose. In the per-protocol population (PPP), lesions regressed in 53 of the 107 women receiving VGX-3100 (49.5%) as compared to 11 of the 36 women in the placebo group (p < 0.025). The secondary endpoint was to demonstrate HPV-16 or -18 clearance from the cervix in conjunction with regression of CIN 2/3 to CIN1 or no disease. Among the 107 women in the VGX-3100 group, 43 demonstrated regression and virological clearance (40.2%), compared to 5 out of 35 (14.3%) in the placebo group (p < 0.025).The Phase II study, designated HPV-003 (NCT01304524), assessed the safety and efficacy of VGX-3100 in 167 women with biopsy-proven cervical intraepithelial neoplasia (CIN) 2 or CIN 3 and HPV-16 or -18. The randomized, placebo-controlled, double-blind study, was stratified by age and severity of CIN and evaluated cervical tissue changes after three 6 mg intramuscular doses of the DNA vaccine VGX-3100 followed by electroporation (EP) with Inovio's CELLECTRAThe study also explored cell mediated immune responses to VGX-3100 in blood samples taken prior to the first vaccine dose and periodically thereafter. IFN-γ ELISpot results revealed higher responses in the VGX-3100 treated group than in the placebo group. Flow cytometry and immunohistochemistry analyses are also ongoing. Finally, subjects were also monitored for tolerability and safety. The treatment was generally well-tolerated, with only administration site redness occurring significantly more frequently in the VGX-3100 group compared to the placebo group in the 7- and 28-day periods following treatment.Altogether, the successful Phase II results clearly illustrate the highly promising potential of therapeutic vaccination with DNA followed by electroporation for the treatment of HPV-related precancerous cervical disease in women as well as HPV-associated cervical, head and neck, and anogenital cancers.


To evaluate salivary detection of HPV-16 & 18 would be feasible and informative biomarker for oral pre-malignant and malignant lesion in our population.This non-interventional, case control study was carried out at department of E.N.T, Head and Neck Surgery, Dow University of Health Sciences, Dow Medical College and Civil Hospital Karachi, Pakistan between July 2011 to December 2012. Total of 105 cases were recruited. These were divided in three groups ‘A’, ‘B’ & ‘C’ having 35 subjects each. Group‘A’ constitutes patients having strong clinical evidence of oral pre-malignant lesions (PML). Group ‘B’ includes histologically proven oral squamous cell carcinoma (OSCC) and Group ‘C’ comprised disease free subjects as controls. After taking informed consent, relevant clinical history was recorded on institutional approved performa. Saliva from all subjects was procured by standard ‘drooling method’. Samples were stored at +4°C and later transferred to Laboratory to store at-20°C before further process. Samples were centrifuged at 4500 rpm for 15 minutes at 4°C. Cell pellets sediments were used for identification of HPV-16 & 18 by real-time PCR method. Data was entered and analysed using SPSS version 16. P-value of 0.05 was taken as standard.In group ‘A’, HPV-16 was detected in 3 (8.6%) cases while HPV-18 was not detected in any of the subject. In group ‘B’, HPV-16 was detected in 07 (20%) while HPV-18 was found in 06 (17.1%) cases. Mixed HPV-16 and HPV-18 were found in 02 (5.7%) cases. In group ‘C’, HPV-16 was detected in 03(8.6%) while HPV-18 was not detected in any of the subjects. Significant relationship was observed between the groups for HPV-18 detection (P= 0.002) while for HPV-16, no significant association was found (P= 0.245).HPV infection for the causation of oral cancer cannot be fully established possibly due to small sample size. More over differences in genetic makeup, environment, indulgence in peculiar risk factor habits, sexual practices and difficult evaluation of the acquisition of viral load due to socio-cultural and religious restrictions could be the reason. Oral cancer is the sixth most frequent malignancy all over the globe.4Carcinoma of oral cavity is the second most frequent malignant tumour for both the gender in Pakistan. It constitutes 20-35% of all cancers seen in various public hospitals in Karachi and slightly less in other regions of Pakistan.8It is believed that cumulative effects of nutritional deficiency, dietary habits, bad oral hygiene, mal-directed sharp teeth and infection with human papilloma virus (HPV) is implicated for oral carcinogenesis. Exposure to some of these risk factors varies significantly between ethnic groups and geographical location. In Pakistan, the major risk factors for oral cancer are areca nut , betel quid (paan), tobacco chewing, naswar, paan masala  and poor nutrition. Due to religious prohibition, alcohol consumption and sexual promiscuity is not a prevalent habit in Karachi. This is in contrary to recent controversial John’s Hopkins study, claiming that cancer is a bad luck happening due to random mutation arising during DNA replication in normal, non-cancerous stem cells in about two third of the cases, not either related to heredity or ecological factors.9Link between human papilloma virus (HPV) and squamous cell carcinoma of the head and neck was suggested in the past. Recently the association is more established as HPV-16 & 18 was the most detectable virus in salivary samples, serum and biopsy cell blocks of the patients with PMLs & OSCC.10Diagnosis and subsequent therapeutic intervention of PMLs and OSCC is currently based on examination, histopathology and staging. Histological examination remains the gold standard for diagnosis of malignant oral lesions. Biopsy is an invasive technique with surgical implications, technique limitations for professionals and psychological implications for most patients. Recently there is a paradigm switch from histopathology to molecular methods of disease diagnosis and saliva has been used for molecular diagnostics by analysing genomics, proteomics and salivary transcriptomes. Oral fluid meets the demand for non-invasive, easily accessible bio-fluid of human body that nurtures a wide spectrum of biological analytes, informative for clinical diagnostic application.In Pakistan, saliva has never been explored as a diagnostic medium to detect biomarkers for oral PMLs and oral cancer. We attempted to investigate whether detecting HPV-16 & 18 would be feasible to diagnose PML and OSCC in our population.This non-interventional, case control study was carried out at department of E.N.T, Head and Neck Surgery, Dow University of Health Sciences, Dow Medical College and Civil Hospital Karachi, Pakistan between July 2011 to December 2012. Patients of any age, sex or ethnic group visiting out patients clinic having strong clinical evidence of PML, histologically proved, untreated OSCC at any stage and identical disease free subjects, willing to participate as controls, were included in the study. Subjects with prior history of treatment for loco-regional or distant malignancy and subjects with history of immune deficiency or autoimmune disorders were excluded.The participants were divided in three groups ‘A’, ‘B’ & ‘C’ having 35 subjects in each. Group ‘A’ constitutes patients having strong clinical evidence of oral PML. Group ‘B’ includes histologically proven OSCC and Group ‘C’ constitutes disease free subjects as controls. After taking informed consent, relevant clinical history was recorded on institutional approved performa. Saliva from all recruits was procured as per standard ‘drooling method’. Samples were stored at +4°C and later transferred to Dow Diagnostic, Research & Reference Laboratory to be stored at -20°C before further process. Samples were centrifuged at 4500 rpm for 15 minutes at 4°C. Cell pellets were used for identification of HPV-16 & 18 by Real-time PCR method.neg. Proteinase K 25 μl and 200 µl Buffer AL per 200 µl lysate was added. The solution was mixed immediately by pulse-vertexing for 15 seconds and incubated at 56°C for 10 minutes. The pH of the lysate must be acidic (<7.0) to obtain maximum binding of DNA to QIAamp membrane. Ethanol (96–100%) per 200 µl lysate was added and mixed again by pulse-vertexing for 15 seconds and briefly centrifuged to remove drops from inside the lid.620 µl of the lysate was applied to the QIAamp Spin Column (in a 2 ml collection tubes). This centrifuged at 8000 rpm for one minute. The QIAamp spin column was placed in a clean 2 ml collection tube and the tube was discarded containing the filtrate. The above two steps were repeated until the whole lysate was loaded. A maximum of 700 µl can be loaded onto the QIAamp spin column. The column was washed with 500 µl Wash Buffer (W5) with ethanol. Centrifuged again at 8000 rpm for one minute and flow through was discarded. The wash step was repeated with 500 µl of wash buffer once. The spin column was centrifuged at maximum speed for one minute to remove any residual wash buffer. The spin column was placed in a clean 1.7 ml recovery tubes. Eluted with 50 µl sterile RNase-free water supplied with the kit. Incubated at room temperature for one minute. The spin column was again centrifuged at maximum speed for one minute to elute nucleic acids. The spin column was discarded from the recovery tube contained purified viral nucleic acids. 12.5 µl of extracted DNA was used in the HPV genotype analysis using Real-time PCR.Total DNA was extracted from all the samples using PureLink® Viral RNA/DNA Kits  for purification of viral nucleic acids according to the manufacturer’s protocol. Briefly; the collection tubes having saliva samples were centrifuged at 15,000 rpm. Pellets were re-suspended in 200 µl lysis buffer. Negative control was prepared by adding 100 µl of HPV-Neg control provided with amplification kit to the tube labelled CGenotyping for HPV-16 and HPV-18 in isolated DNA from patient’s samples were performed using Real-time PCR machine  using the Real-time PCR Kit HPV-16/18 Real-TM Quant .Kit HPV 16/18 Real-TM Quant is based on multiplex Real-time amplification. Amplification results of HPV-16 DNA were detected on the FAM/Green channel, HPV-18 DNA were detected on the ROX/Orange channel and β-globin gene used as internal control was detected on the JOE/HEX/Yellow channel.The required numbers of tubes were prepared (Number of tests + 3 standards and 1 negative control). PCR- buffer-FRT 20 µl of Hot Start DNA Polymerase in 15 µl of reaction mixture was added into each tube with samples and controls. 12.5 µl of extracted DNA sample was added to appropriate tube and 4 controls were prepared. 10 µl of Quantitative Standards HPV  was added into labelled tubes.The tubes were capped and transferred into Real Time Thermal Smart Cycler. The Smart Cycler  instrument was programmed as per manual of instruction. The positions of the tubes were programmed and the concentrations of the Quantitative Standards entered in the JOE (Human DNA), FAM (HPV-16) and ROX (HPV-18) channels in order to generate standard curves.The software of Smart Cycler Real-time PCR was utilized for the interpretation of results all the way through the presence of crossing of fluorescence curve with the threshold line. As per the software, internal control (Human DNA) is detected on the JOE/HEX/Yellow channel, Human Papilloma virus type 16 (HPV 16) on the FAM/Green channel and HPV 18 on ROX/Orange channel. Data was analysed using SPSS version 16. Statistical analysis of the data was computed to see relationship among the groups by applying Pearson Chi-Square test. P-value of 0.05 was used as reference value.Out of 105 subjects 70(66.7%) were males and 35(33.3%) were females with male/females ratio of 2:1. The overall minimum age of the recruited subjects was 20 years and maximum was 80 years with a mean of 43.2 years. Over all minimum age in males was 20 years and maximum was 75 years with a mean age of 43.7 years. In females over all minimum age was 21 years and maximum was 80 years with mean of 42 years. In group ‘A’, majority of the cases were having oral submucosal fibrosis (OSF). It was found in 27(77.1%) cases. Leukoplakia was found in 10(28.6%). Erythroplakia was detected in 06(17.1%) cases. All these were found alone or in combination with other pre-malignant lesions. 02(5.7%) cases were diagnosed clinically as lichen planus alone.1 stage, 12(34.3%) were T2, 18(51.4%) were T3 and 03(8.6%) were T4 stage at presentation. 20(57.1%) cases were having no clinically palpable nodes (N0), 9(25.7%) were having N1, 05(14.3%) were having N2 and 01(2.9%) was having N3 nodal status. Distant metastasis ‘M’ was not found clinically in any of the recruited subjects (The subjects recruited in group ‘B’ were staged according to TNM classification of AJCC (2002-3), 02(5.7%) cases were Tsubjects .In group ‘A’, HPV-16 was detected in 3 (8.6%) cases while HPV-18 was not detected in any. In group ‘B’, HPV-16 was detected in 07 (20%) cases while HPV-18 was detected in 06 (17.1%) cases. Mixed HPV-16 and HPV-18 were found in 02 (5.7%) cases. In group ‘C’, HPV-16 was detected in 03(8.6%) while HPV-18 was not detected in any of the subject. .Statistical analysis was performed to compare the qualitative relationship among the groups by using Pearson Chi-Square test. The relationship of salivary HPV-16 detection was not found significant among the groups (P-0.245) while salivary HPV-18 detection was found significant (P-0.002).Most controversial issues relating to role of HPV in head and neck cancer, particularly OSCC are frequency of HPV, viral load acquisition through oro-genital route or otherwise and its diagnostic methodology.11Modes of transmission of HPV in head and neck mucosal districts have not been fully resolved. It is proposed that multiple pathways are involved. HPV association was found to be more pronounced with the history of perverted sexual practices.In Pakistan, HPV screening for both OSCC and cervical cancer is not commonly practiced due to socio-cultural reasons that prohibit asking enquiries related to risk factors for acquiring HPV load like oral sex and multiple partners. Therefore the data relating to acquisition of HPV infection is grossly deficient.Etiological role of HPV in head and neck carcinogenesis was first proposed by Syrjanen et al in 1983.18In a study from Quetta, the capital city of Balochistan province of Pakistan reported detection rate of HPV DNA in 24.5% (47/200) in a group of Pakistani subject visiting the dental department having normal oral cavity. They used oral tissue scraping for DNA detection by Real-time PCR method. Out of 47 HPV-positive cases, 4(2%) contained HPV-16 and 11(6%) contain HPV-18 genotypes.In a study from Karachi, Pakistan, on the molecular analysis of HPV detection in tissue blocks by Dot Blot and in-situ hybridization and PCR method found 17.7% cases of OSCC positive for HPV-16 & 18 DNA on PCR and 14.6% cases showed viral DNA by NISH. While in only 4.6% cases of oral PML, the viral DNA was detected. They concluded that high risk HPV-16 & 18 has contributing co-factor role rather than mandatory causative role in oral carcinogensis.14A study from Allahabad, India, reported 27.4% HPV detection in OSF while it was detected in 31.53% cases of OSCC on brush biopsy specimen by Hybrid Capture II test. PCR was done on the same specimen for the detection of E6 DNA of HPV-16. They found detection rate of 25.96 in OSF cases and 32.43% in cases of OSCC.In a meta-analysis (1988-2007), the incidence of HPV in head & neck SCC versus OSCC, it was observed that pooled prevalence of HPV in the tissue samples of head and neck SCC was 34.5% while in cases of OSCC, it was 38.1%.Significant association was reported in OSCC with HPV-16 and to a lesser extent with HPV-18. In the salivary samples HPV-16was the most detectable virus reported in the literature.It is difficult to compare various published data on HPV association as there are wide differences in findings. Possible reasons of this disparity is variation in parameters studied such as type of samples  preparation method , type of molecular assay employed for DNA extraction, sensitivity of the methodology employed, status of the disease, ethnic and geographical differences in genetic, environment, social, cultural and sexual habits of the population studied.28Comparing various studies, the results are highly variable and controversial. No clear cut association or relation of HPV infection with oral PML and OSCC has so far emerged. Our findings are consistent with many of the reported studies in literature. We are unable to support definitive causative or mandatory initiator role of HPV in oral carcinogenesis in our etiologically distinct population where use of areca nut and chewable tobacco is very prevalent in the society.Host response towards HPV infection for the causation of oral cancer cannot be fully established. More over different genetic makeup, environmental and geographic differences, indulgence in peculiar risk factor habits, different sexual practices compared to west and difficult clinical evaluation of acquisition of viral load due to socio-cultural and religious restrictions could be the reasons. Our study is unable to support definite causative or mandatory initiator role of HPV infections in oral carcinogenesis, it may have a co-factor role rather than just a passenger virus.