Their survival, thus, may emerge only within a specific environmental context. To determine the fate of human oligodendroglioma in an experimental model, we studied the development of an anaplastic tumor after intracranial implantation into enhanced green fluorescent protein (eGFP) positive NOD/SCID mice. Remarkably after nearly nine months, the tumor not only engrafted, but it also retained classic histological and genetic features of human oligodendroglioma, in particular cells with a clear cytoplasm, showing an infiltrative growth pattern, and harboring mutations of IDH1 (R132H) and of the tumor suppressor genes, FUBP1 and CIC. The xenografts were highly invasive, exhibiting a distinct migration and growth pattern around neurons, especially in the hippocampus, and following white matter tracts of the corpus callosum with tumor cells accumulating around established vasculature. Although tumors exhibited a high growth fraction in vivo, neither cells from the original patient tumor nor the xenograft exhibited significant growth in vitro over a six-month period. This glioma xenograft is the first to display a pure oligodendroglioma histology and expression of R132H. The unexpected property, that the cells fail to grow in vitro even after passage through the mouse, allows us to uniquely investigate the relationship of this oligodendroglioma with the in vivo microenvironment.Oligodendroglioma poses a biological conundrum for malignant adult human gliomas: it is a tumor type that is universally incurable for patients, and yet, only a few of the human tumors have been established as cell populations  FUBP1 and CIC, and are involved in MYC expression and repression of receptor tyrosine kinase (RTK) signaling, respectively IDH1/2), that suddenly thrust these tumors into the center of more general scientific and clinical interest at multiple levels Until recently, adult human oligodendrogliomas have maintained a degree of anonymity among human cancers, despite hallmark genetic losses of chromosomes 1p and 19q that signaled a unique biological and clinical nature divergent from other gliomas in vitro or as intracranial xenografts in vivoin vivo models that accurately reflect tumors of pure oligodendroglioma histology, and in particular, tumors with IDH mutation, are still greatly needed to represent this disease, and more broadly, human gliomas with IDH mutations.Although oligodendroglioma with its unique mutational constellation generally follows a less aggressive clinical course than GBM, it is still a fatal disease. Only recently, however, have a few tumors of oligodendroglial histology been propagated in vitro, even though 1p/19q co-deletion with underlying mutations in both FUBP1 and CIC was present, as revealed by aCGH and exome sequencing.Here, we report the propagation of a WHO grade III oligodendroglioma as an intracranial mouse xenograft that histologically and genetically replicates features of a classic oligodendroglioma, including expression of the R132H protein. Interestingly, the primary tumor or xenografts did not expand Patient material was obtained from surgeries performed at the Haukeland University Hospital . Written consent was obtained from patients with procedures that were approved for the project (project number 013.09) by the Regional Ethics Committee . All animal protocols were approved by authorities in an AAALAC accredited facility at the Haukeland University Hospital and in accordance with the national regulations of Norway (project numbers 2010 2658 and 2011 3079).Tumor tissue (<1 g) was gently cut with scalpel blades, rinsed in Hank's buffered saline (HBSS), and enzymatically dissociated at 37°C for one hour, or until single cells could be observed, in 5 ml of HBSS containing Liberase DH  and 100 μl DNAse . DMEM with 10% serum was added, and the enzyme mixture was removed after centrifugation. The sample was resuspended in DMEM with 10% serum, gently triturated with a 10 ml plastic pipet, and filtered through a 70 μm filter. Blood cells were subsequently lysed with EasyLyse (Dako), and tumor cells were washed thoroughly two times with 20 ml of Dulbecco's phosphate buffered saline without calcium and magnesium.Dissociated tumor cells were resuspended in Neurobasal Medium, B27 supplement, Glutamax (Life Technologies), and FGF2 (20 ng/ml), and EGF and/or PDGFA  over non-treated culture flasks (Nunc). Medium referred to as NBM is Neurobasal Medium plus B27 supplement and Glutamax. Medium was changed once weekly. All cell populations used for implantation or culture experiments were fingerprinted to confirm tumor origin. Hippocampal neuronal preparations were prepared from hippocampi isolated from day old rat newborns. Hippocampi were incubated with papain (Worthington) at 37°C for 15 min and resuspended in MEM with 10% serum. A single cell suspension was made by trituration of the tissue with a fire polished glass pipet. Cells were plated in NBM but without growth factors for one week. Tumor spheres were then added with NBM containing FGF2 (20 ng/ml).Tumor cell aggregates were implanted into 6 to 8 week old eGFP NOD/SCID mice of either gender. Coordinates for implantation were 1.5 mm to the right and 0.5 mm behind the bregma suture. Maximum penetration of the syringe was 2.0 mm into the tissue with injection of the cells at 1.5 mm. First generation refers to xenografts established in mice from the primary patient culture whereas second generation (and any generation thereafter) refers to tumors established from human cells already passaged through mice. All animals were allowed to live until they became symptomatic . For each animal, either a section or the whole brain was formalin fixed and examined on H&E by a neuropathologist to identify cases where oligodendroglioma had developed. NOD/SCID mice are prone to infection and tumor pathologies, or simply old age, which are all possible causes of symptoms or death in the absence of apparent engraftment MRI was performed with a 7T small animal scanner with a circular mouse head transmit/receive coil . A T2 weighted (T2w) RARE sequence was acquired with Paravision software using the following parameters: TR 3500 ms, TE 36 ms, 4 averages, FOV 2.0×2.0 cm, matrix size: 256×256, slice thickness: 1 mm, and scan time: 5 minutes 36 seconds.After de-paraffinization, formalin fixed sections were pretreated by heat denaturation for 15 min in 10 mM Citrate buffer, pH 6.0. Primary antibodies were incubated with sections overnight at 4°C, and detection was performed using a biotinylated secondary antibody and amplification of the signal with Vectastain ABC Reagent (Vector Labs). Antibodies used were the following: R132H IDH1 , Ki67 , human nestin , human GFAP , mouse nestin , mouse GFAP , PDGFRA and phospho-PDGFRA , Iba1 , CXCL12  and STEM121 .4OAc and 2.5 volumes of 100% ethanol. DNAs were resuspended in Nuclease-Free water (Qiagen).DNAs were isolated from patient material or xenografts that had been immediately frozen in liquid nitrogen subsequent to surgery. Frozen sections were prepared, and every fifteenth section was stained with H&E to ensure >60% tumor cell content. DNA was obtained from frozen patient blood (−20°C) by isolating nuclei with Buffer C1 from the Genomic DNA Buffers Kit (Qiagen). Samples were treated with proteinase K overnight at 50°C in ATL buffer (Qiagen) and then DNAse free RNAseA (Fermentas) for 5 min at room temperature. Reactions were extracted with phenol:chloroform:isoamyl alcohol 25∶24∶1 saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma) and precipitated in 2.5M NHDNA was digested using the restriction enzymes RSA1 and Alu1 and labeled using the BioPrime aCGH Genomic Labeling Kit (Invitrogen) and Cy3 and Cy5 dyes , following standard protocols for Agilent CGH+SNP. A female HapMap sample with a known genotype  provided by the Coriell Repository  was used as a reference for each of the CGH+SNP experiments. Labeled DNA was competitively hybridized to SurePrint G3 Human 2×400 k CGH+SNP microarrays  following standard Agilent protocols. The slides were scanned at 3 μm resolution using the Agilent High-Resolution Microarray scanner, and the image data were extracted using Feature Extraction (Agilent Technologies). Feature extraction files were imported into Genomic Workbench 7.0 (Agilent Technologies) for visualization and analysis. For CGH, after diploid centralization and GC correction, aberrations were called using the ADM2 algorithm with a threshold setting of 20, centralization on with threshold of 25 and an aberration filter min Probes = 5 and minAvgAbsLogRatio = 0.35 for amplifications and deletions.Agilent SureSelect target enrichment kit  was used for exome library construction according to the manufacturer's protocol. Captured DNA libraries were sequenced on the Illumina HiSeq2000 platform. Reads were aligned to the human reference genome (hg19) with BWA. Duplicate tags were removed. Point mutations, and insertions and deletions (INDELs) were identified using Samtools (version 0.1.18) with default parameters. Somatic variations were then validated in IGV browser 2.1.22.Standard methods for PCR and Sanger sequencing were performed according to the manufacturer's procedures (Applied Biosystems). Primers for PCR were as follows: IDH1 forward,5′-AATGAGCTCTATATGCCATCACTG-3′; IDH1 reverse, 5′-TTCATACCTTGCTTAATGGGTGT-3′5′-CAGAGACAAGAGGATGGCTAGG-3′, IDH2 reverse, 5′-GTCTGGCTGTGTTGTTGCTTG-3′5′-TAGAATGCAGTGAGGGCTTG-3′; CIC reverse, 5′-TTGGAGGGAAAGATGTCTGC-3′; FUBP1 forward, 5′-TGCTGACTAGTAATGATACATTTTCC-3′; FUBP1 reverse, 5′-GGCCCATTTAATTGTGACCA-3′. For IDH1, a separate sequencing primer was used, 5′-GCCATCACTGCAGTTGTAGGTTA-3′www.dsmz.de). Profiles were analyzed to determine that they matched the original biopsy, and that they did not match any of the cell lines listed in the DSMZ STR database.The AmpFlSTR Profiler Plus PCR Amplification Kit (Applied Biosystems) was used according to the manufacturer's protocol. This kit amplifies nine tetranucleotide short tandem repeat (STR) loci and the amelogenin (gender determination) locus in a single reaction. The samples were run and allele sizes interpreted on an ABI3100 Genetic Analyzer (Applied Biosystems). The fingerprinting profiles were run through the online international reference STR profile database for human cell lines at the German collection of microorganisms and cell cultures DSMZ (IDH mutations generate the D-2-hydroxyglutarate (D2HG) form of 2HG 2HG concentrations were measured with an enzymatic assay based on the finding that A WHO grade III oligodendroglioma was obtained from a surgery performed on a 39 year-old female patient with a history of oligodendroglioma. The case had been originally diagnosed as a grade III oligodendroglioma, when the patient was 35 years old, and FISH analysis performed at that time indicated that the case harbored losses of 1p and 19q, the hallmark genetic changes of human oligodendrogliomas. The patient was treated with radiation and chemotherapy. The present recurrence in 2010 was a highly cellular tumor that retained the original histologic features of the case, including round nuclei with a clear cytoplasm, microvascular proliferation, and a high proliferation index .At the time of the recurrence, approximately one gram of the tumor tissue was enzymatically dissociated, and cell aggregates or spheres were allowed to form in serum free medium containing FGF and EGF in suspension culture. Small cell aggregates formed immediately following dissociation and resuspension in medium. Intracranial implantations were subsequently performed with twenty-five of these spheres containing approximately 1000 cells on two sets of six to eight week old eGFP NOD/SCID mice in order to establish first generation tumors First generation tumors arose in animals from both implantations, three out of four mice from the first implantation at one month and one out of three from the second implantation at two months. H&E sections from three of these mice revealed a diffusely infiltrating tumor pathology characteristic of oligodendroglioma . SymptomImmunohistochemistry demonstrated that R132H was expressed in the xenograft  as in thMRI performed at 374 days on the fourth, asymptomatic animal revealed that tumor was present based on distortion of the midline and enlargement of the cortex . Upon saSpheres from this single mouse were used to create second generation xenografts. Nine new animals were again each implanted with about twenty-five of these spheres containing approximately 1000 cells . Four ouTumors from both generations exhibited a diffusely infiltrating pathology and had round tumor nuclei and clear cytoplasm that are typical of oligodendroglioma . EndotheIDH mutation presents a unique opportunity to unequivocally distinguish tumor cells from normal cells in histological sections IDH1/2 mutation had no detectable 2HG  and PTEN (10q), were also not mutated in either the primary tumor or the xenograft. These results indicate that with regard to these genes, the xenograft reflects a typical oligodendroglioma with co-deletion of 1p and 19q.Exome sequencing of human oligodendrogliomas specifically with chromosome 1p and 19q losses has revealed recurrent mutations in tumor suppressor genes at these particular loci as well as generated an overall genetic map at base pair resolution BP1 15%, , CIC .We were also unable to expand a second primary recurrent anaplastic oligodendroglioma, with 1p/19q co-deletion, over an extended period of time in suspension culture . This tuin vitro . Spheresin vitro . We alsoin vivo host environment and this tumor cell population, we looked for evidence of relationships that might exist between the tumor cells and specific stromal cell types. Perhaps the most unique feature of this particular model is the assembly of tumor cells around neurons  and vascular niches in response to CXCL12 FUBP1, CIC, and IDH1 (R132H). Although this tumor had a high growth fraction, as measured by Ki67 staining, we were unable to expand the original patient tumor in vitro. Furthermore, none of the gross genetic changes in the xenograft that differ from the original tumor, such as gain of chromosomes 4q or 11, enhanced in vitro growth. These results together support investigation of the brain environment for components and/or conditions that are necessary for the continued maintenance of this human oligodendroglioma.We have successfully established and serially passaged a grade III oligodendroglioma xenograft with the hallmark chromosome 1p and 19q losses and mutations including FUBP1, CIC, and IDH1 mutations. Equally informative is that there is an absence of mutations in p53, PTEN, or CDKN2A that can accompany glioma progression PTEN or CDKN2A might have been selected for, particularly in the xenograft. In the absence of these mutations and a complete analysis of the exome sequencing, one possibility is that it is the combination of CIC and FUBP1 mutations that provide this tumor with an advantage during engraftment. In fact, while mutation of CIC is a more common denominator amongst oligodendrogliomas, mutation of FUBP1 is not. The percentage of oligodendrogliomas with CIC mutations is in the range of 50 to 80% FUBP1 mutations have been found to occur in approximately 15% to 20% of oligodendrogliomas in three studies FUBP1 mutations in anaplastic cases FUBP1 and CIC, and nine of these cases are anaplastic oligodendrogliomas. FUBP1 regulates MYC expression, a gene that is known to induce proliferation and pluripotency in different cell types FUBP1 and CIC mutations, in the absence of other known major gene mutations, gave this particular tumor some kind of growth advantage upon intracranial implantation. However, analysis of additional engrafted cases and deeper analysis of the sequencing dataset is necessary to more fully understand the success of intracranial engraftment with regard to oligodendrogliomas.Several crucial questions are raised by this work. The first is what is the genetic basis for the engraftment. Based on aCGH data and limited analysis of exome sequencing, the tumor reported here has a common anaplastic oligodendroglioma genomic profile including in vivo experiments at sixteen weeks or even less p53, CDKN2A, and PTEN more rarely occur in oligodendrogliomas might indicate a slower growing cell type In addition to the underlying genome, our timeframe of nearly one year was considerably longer than other studies that terminate in vivo. That these tumor cells preferentially grow in vivo corroborates results recently obtained with an anaplastic oligoastrocytoma with a similar genetic constellation of 1p and 19q losses and R132H in vivo environment would be that a specific growth factor/receptor enhances proliferation and/or survival. The PDGFRA gene presents one potential in vivo candidate for the xenograft, but the status of this gene exhibits a peculiar behavior. The primary tumor actually has loss of chromosome 4q where the PDGFRA gene is located, whereas the xenograft becomes diploid again at this chromosomal arm. This loss is frequently observed in oligodendrogliomas, generally in higher grade tumors A second important question is what promotes proliferation and/or survival of the xenograft in vitro, neuronal cultures do not support expansion of oligodendroglioma cells. The second pattern of in vivo growth observed was around established vasculature. Tumor cell growth, as assessed by Ki67 immunostaining, was strong around blood vessels in the cortex, the striatum, and particularly within the subgranular zone (SGZ) of the dentate gyrus in one of the xenografts. The result of growth, specifically in the SGZ, is particularly interesting as it is an area where normal neural progenitor cells are known to form highly proliferative clusters in conjunction with endothelial cells A final question is from where such a stimulatory signal may originate. Here, we initiated investigation of cell types from the stroma as potential candidates. The results of our experiments for immunostaining for the presence of specific cell types within the xenografts showed surprisingly few differences from normal brain, other than subtle changes in microglia morphology and accumulation of astrocytes. Additional clues for what supports the growth of this oligodendroglioma may, instead, be obtained by studying the areas within the CNS where the tumor xenografts grew. The most striking and unique pattern of tumor cell growth was around hippocampal neurons. Infiltration along neurons is a common histological feature of human oligodendroglioma, but preliminary results from our laboratory indicate that in vivo oligodendroglioma models, that more broadly represent the disease, will help us to define the environmental parameters contributing to oligodendroglioma development and growth and may open up new avenues of therapeutic value.In conclusion, our results show for the first time that histological and genetic features, such as satellitosis and expression of R132H, of a human oligodendroglioma are maintained in an intracranial mouse xenograft over serial passages. Future efforts to develop critically needed


Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries. As genetic variations in the toll-like receptors (TLRs)-signalling pathway have been associated with either susceptibility or resistance to several infectious and inflammatory diseases, the supposition is that single nucleotide polymorphisms (SNPs) of TLR2, TLR4, TLR9, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A could modulate malaria susceptibility and severity.Malaria caused by This study was planned to make a further contribution to solving the problem of the real role of the most common polymorphisms of TLR4, TLR9, TIRAP and FCGR2A genes in modulating the risk of malaria and disease severity in children from Burundi, Central Africa. All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, between February 2011 and September 2011, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears. In children with malaria and in uninfected controls enrolled during the study period in the same hospital, blood samples were obtained on filter paper and TLR4 Asp299Gly rs4986790, TLR9 G1174A rs352139, T-1486 C rs187084 TLR9 T-1237 C rs5743836, TIRAP Ser180Leu rs8177374 and the FCGR2A His131Arg rs1801274 polymorphisms were studied using an ABI PRISM 7900 HT Fast Real-time instrument.A total of 602 patients and 337 controls were enrolled. Among the malaria cases, 553 (91.9 %) were considered as suffering from uncomplicated and 49 (8.1 %) from severe malaria. TLR9 T1237C rs5743836CC was associated with an increased risk of developing malaria (p = 0.03), although it was found with the same frequency in uncomplicated and severe malaria cases. No other differences were found in all alleles studied and in genotype frequencies between malaria cases and uninfected controls as well as between uncomplicated and severe malaria cases.TLR9 T1237C seems to condition susceptibility to malaria in Burundian children but not its severity, whereas none of the assessed SNPs of TLR4, TIRAP and FCGR2A seem to influence susceptibility to malaria and disease severity in this population. Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries , circulatory collapse, respiratory distress, or haemoglobin level <5 g/dL. Otherwise healthy children seen in the hospital during the study period for minor surgery with a similar sex and age distribution and without asymptomatic parasitaemia according to the results of the screening with thick and thin films were enrolled as uninfected controls.All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears, with two independent readers. If informed written or verbal (in case of illiteracy) consent was obtained from a parent or legal guardian, children for whom both readers were positive for the presence of criteria . In partBlood samples were obtained from all the children at enrolment, both cases and controls. Blood was collected on filter paper  for future DNA extraction and testing and kept in a cool, dry place. Samples collected were sent in the central laboratory in the Department of Maternal and Paediatric Sciences of the University of Milan, Italy, by airmail, once a month, for genetic studies.The blood spots on filter paper were cut into little fragments, then incubated for 1 hour in 3- mL lysis buffer at room temperature on an orbital shaker. Paper residues were separated from lysate by brief spinning. DNA from all samples was then extracted with a NucliSens EasyMAG instrument , using the Specific B protocol. The DNA extracted was quantified using a Nanodrop ND-1000 instrument , with elution buffer as blank. SNP genotyping was then performed on genomic DNA using pre-designed Taqman® SNP Genotyping assays  ,34. SNP X2) or the likelihood-ratio statistical test, as appropriate [Data were analysed using SAS for Windows v. 9.1 . Categorical variables are presented as numbers and percentages, and were analysed using contingency tables and the chi-squared or Fisher’s exact test, as appropriate. The Hardy-Weinberg equilibrium (HWE) was performed by comparing the numbers observed of different genotypes with those expected under the HWE for the estimated genotype frequencies and p > 0.05 was in HWE. Genotyping deviations from HWE were assessed under Pearson’s chi-square  and controls (6 % for TLR4 Asp299Gly and 2.4 % for TIRAP Ser180Leu). On the contrary, minor allele frequency for the other SNPs studied exceeded >20 % in all cases, reaching values around 40 % (42.3 %-45.2 %) and 50 % (43.0 %-48 %) for FCGR2A His131Arg and TLR9 G1174A, respectively. TLR9 T1237C rs5743836 C was found significantly more frequently in children with malaria than in uninfected controls (p = 0.04), although no difference was found between children with uncomplicated and those with severe malaria. No other significant difference among Burundian children with malaria and related controls was found as well as between children with uncomplicated and those with severe malaria for all the other allele frequencies studied .A total of 602 patients  and 337 uninfected controls  were enrolled. Among uninfected controls, 106 (31.4 %) were admitted for abdominal surgery, 79 (23.4 %) for orthopaedic surgery, 69 (20.5 %) for urogenital surgery, 55 (16.3 %) for plastic surgery and 28  were considered as suffering from uncomplicated malaria and 49 (8.1 %) from severe malaria . Table Table The overall data seem to indicate that none of the SNPs of TLR4, TIRAP and FCGR2A genes studied, assessed in Burundian children, play a role in modulating susceptibility to malaria and disease severity in this population. Regarding SNPs of TLR9, this study suggests that whereas no role may be ascribable to TLR9 G1174A and TLR9 1486 C, TLR9 T1237C seems to be associated with an increased risk of developing malaria, although this SNP is not associated with different disease severity. Despite data regarding association between polymorphisms and severity of the disease have to be evaluated with caution because the total number of severe cases, particular those with cerebral malaria, is too small to permit to draw definitive conclusions, these findings seem to indicate that variation in the characteristics of TLR9 can play a role in conditioning the development of malaria. Consequently, they can be useful in the identification of children at higher risk of developing the disease and those for whom the preventive measures are strongly needed. However, all of these data have to be confirmed with larger studies because in most of the cases data collected with this study are only partially in agreement with those that have been already published at this regard.et al. reported that this polymorphism was associated with an increased risk of severe paediatric malaria without affecting the risk of infection [et al. demonstrated that TLR4 Asp299Gly was more common in patients with low parasitaemia and concluded that TLR4 could have a genetic role in controlling the blood infection level in mild malaria and could indirectly suggest a protective effect of TLR4 Asp299Gly against severe disease [et al.[et al.[P. falciparum recognition and host response in humans and whether TLR4 could contribute to the control of infection.Data on TLR4 Asp299Gly are quite different from those reported by other authors who, on the other hand, obtained results largely conflicting with each other. Mockenhaupt nfection . On the  disease . Similare [et al. and Sam-l.[et al., respectl.[et al.-21. Conset al., studying children with cerebral malaria, found that patients with the C allele at -1237 and G allele at 1174 had higher levels of IFN-γ than those without these alleles [et al., despite reporting evidence of cis-variants acting on gene expression, did not find any convincing association between TLR9 SNPs and malaria severity [et al.[et al.[et al.[et al.[P. falciparum. Haemozoin is a dark-brown haem crystal produced by P. falciparum that functions as a carrier for P. falciparum DNA and functionally affects TLR9 response to parasite infection activating TLR9 proportionally to its concentration [Studies on TLR9 SNPs have also reported conflicting data. Sam-Agudu  alleles . Becauseseverity . The daty [et al. but contl.[et al., no assol.[et al. and Sam-l.[et al. could bentration . Unfortuet al.[et al.[et al.[et al.[In this study, contrary to what was reported by some authors ,25, no pet al. and Khorl.[et al. and by Hl.[et al.. These let al.[Regarding FCGR2A His131Arg, the data of this study are completely different from those reported by Schuldt et al. who founet al.,28-31. DP. falciparum infection have no influence in conditioning susceptibility to and severity of malaria in Burundian children. An exception might be represented by TLR9 T127C for which a significant correlation with an increased risk of developing malaria without any role in determining severity was found. This finding, when confirmed with further studies carried out with greater study samples, can permit to identify those subjects for whom preventive measure are strongly needed. Moreover, knowledge that same aspects of the innate immunity can have different efficacy in facing infectious agents can be of value in the preparation of specific vaccines when immune response evoked by the antigens included in the vaccines involves the factors with modified activity. This has to be considered also in the preparation of the developing malaria vaccines.Knowledge of the relationships between genetics, susceptibility to and severity of infections is essential to identify subjects at higher risk and to develop specific preventive measures. This is particularly import for malaria that remains one of the most important health problem in several geographic areas. This study shows that genetic polymorphisms of some factors involved in the innate immune response to CRP, C reactive protein; HWE, Hardy-Weinberg equilibrium; SNPs, single nucleotide polymorphisms; TIRAP, Toll-interleukin 1 receptor domain containing adaptor protein; TLRs, toll-like receptors.The authors declare that they have no competing interest. The study was partly supported by Bando Giovani Ricercatori 2007  and partly by ABM Onlus (that is a non profit organization).SE and NP designed the study and co-wrote the manuscript. CGM, AZ, CD, AS and LT carried out the laboratory assays. EB, AL, MS and MG visited patients and controls, collected the samples and entered the data in the database. MM interpreted results of genetic analyses. CP statistically analysed the data. All the authors read and approved the final manuscript.


Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. Objective: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera  Chronic obstructive pulmonary disease (COPD) is a respiratory disease associated with chronic inflammation of the lung leading to tissue destruction and emphysema. Bacterial colonization is one critical factor contributing to COPD progression and the occurrence of acute exacerbations (AECOPD) P. aeruginosa, M. catarrhalis, and H. influenzaMicrobiological studies in COPD have been limited to the detection of specific respiratory pathogens such as The aim of the current study was to examine the pulmonary microbial communities in COPD patients as compared to healthy individuals. Terminal restriction fragment length polymorphism (T-RFLP) analysis and molecular cloning with sequencing were used to characterize the pulmonary microbiome in health and disease.http://www.goldcopd.org/). The diagnosis of COPD was based on patient history, pulmonary function tests, and a post bronchodilator ratio of forced expiratory volume in 1 s (FEV1)/vital capacity (VC) or forced vital capacity (FVC) below 0.7. The patient was defined as stable if there was no exacerbation for the previous 4 wks and the patient was not currently having an exacerbation. The study was approved by the ethic committees of the University of Marburg and the Landesärztekammer of the Saarland; all participants gave written informed consent. Healthy individuals were defined as never smokers, without chronic disorders and without any respiratory illnesses over one year prior the study. COPD patients had clinically diagnosed COPD as described in the GOLD guidelines  and 250 µl of TNS solution . Tubes were filled with 0.7 g of 0.5 mm sterile zirconia beads  and vortexed for 45 sec at 6.5 m/s using the homogenizer Precellys® 24 . Following chilling on ice and pelleting of remaining cell fragments, lysates were denaturated with one volume of phenol:chloroform:isoamylalcohol (25∶24:1). After short spinning, 800 µl of supernatant were mixed with an equal volume of chloroform:isoamylalcohol (24∶1). Then 650 µl of the DNA containing supernatant were mixed thoroughly. The extracted DNA was precipitated by spinning for 80 min at 4°C with two volumes of PEG 6000 and washed in ice-cold 70% ethanol. Finally it was eluted in 50 µl of EB buffer  and additionally incubated at 37°C for 2 h to enhance the DNA recovery yield.BAL samples were stored on ice prior to the DNA extraction. After centrifugation at 2500 rpm at 4°C for 10 min, the supernatant was discarded and the pellet was used as a starting material for the DNA isolation. Total DNA was purified by using a combination of a bead-beating method with the standard phenol:chloroform extraction. Each cell pellet was resuspended in 750 µl of sodium phosphate buffer . Total E. coli DH5α DNA (100 ng) was used as a positive control and 5 µl of destilled water as a negative control. PCR was run in duplicate and amplified products were pooled, purified using with NucleoSpin Extract II kit , and analyzed by electrophoresis using a 1.5% agarose gel.Total DNA (100 ng) was used as a starting material for the PCR-based amplification of 16S rRNA gene. The used primer set was:  digestion for 3 h at 37°C in the dark. Then samples were subjected to capillary electrophoresis on ABI 310 Genetic Analyzer  using the GeneScan™ 500 ROX™ XL size standard . Data were analyzed with GeneMapper® Software . For each sample analysis was done in duplicate.PCR was performed using a FAM (6-carboxyfluorescein)-Ba27 forward primer . PCR products were cut out from the agarose gel, purified, and subjected to Terminal restriction fragments (T-RFs) between 50 and 700 bp were included in the analysis and differences in TRF length of ±1 bp in different profiles were considered as identical in order to compare T-RFLP profiles between different samples. The relative abundance of each TRF within a given T-RFLP pattern was calculated as the peak height of the respective TRF divided by the total peak height of all TRFs detected. We compared TRFs with the good quality reference sequences from the RDP database. As additional control we compared the obtained data with those from species from the oral cavity. This comparison resulted in phylogenetic assignment on the genus level of fragments and was performed using a web - based tool Microbial Community analysis III (MiCA 3MiCA 3) with the following parameters: T-RFLP analysis (PAT+), digest sensitivity – 2 mismatches within 2 bases from 5′ of forward primer, window size – forward match ±1 bp. Some fragments could not be matched and assigned because of gaps in the database. Some fragments were identified as “uncultured bacteria” and could not be phylogenetically assigned. The copy number of 16S rRNA genes vary between different species (1 to 15 copies) and thus do not correspond necessarily to number of bacterial cells http://www.ncbi.nlm.nih.gov/projects/Sequin/) was used to submit the sequences to the GenBank database. A total of 128 clones were submitted to the GenBank under the accession numbers “JN382472” to JN382540” and “JN378754” to “JN378814”.To construct the clone libraries from BAL samples from 18 studied participants, amplicon pools were ligated and cloned using the standard protocol from the pGEM®-T Easy Vector System . 50±10 clones per clinical sampled were subjected to sequencing. Individual cloned 16S rRNA gene sequences were first amplified using the M13F and M13R primers and then sequenced with the 27F primer using an ABI3700 . Approximately 900 bp of the sequence  were compared with those available in the GenBank of the National Center for Biotechnology Information (NCBI) by BLAST search. Identification of the species level was defined as ≥98% similarity of the 16S rRNA gene sequence to the sequence of its closest bacterial relative in the GenBank database using online software BlastN . After primer, vector fragments, and chimeric sequences were removed (using RunVecScreen and Black Box Chimera Check (B2C2) software respectively), the operational taxonomic units (OTUs) were defined. OTU cutoff was less than 0.03, which corresponded to the strain level Prevotella, Veillonella, Porphyromonas, and Fusobacterium). Secondly, bands that were evenly detected in healthy and COPD samples were also removed. Afterwards, we used a taxonomic mapping from species to their corresponding genera. This was done to reduce the feature space. Subsequently, a principal component analysis (PCA) was applied using a restrictive variance cutoff of 95%. PCA is generally used for pattern recognition in a data set and can be applied as a filter prior to machine learning, also allowing easier visualization of patterns within high-dimensional data We used the T-RFLP-analyzed data from BAL samples taken from all study participants. A total of 326 distinct peaks were detected. In order to find distinct COPD and healthy microbiome indicators, we applied supervised learning and cross validation using both, R Pseudomonas and Lactobacillus interact using a PCA but retaining only these fragments. We visualized the PCA results with an R-biplot Furthermore, we specifically analyzed how restriction fragments corresponding to MspI digestion. T-RFLP revealed a total of 326 bands. The mean number of T-RF per individual was 17.7 (±2.32 SEM) in the COPD group and 18.56 (±2.32) in the control group . The range was 12 to 31 (SD 6.95) bands for the healthy individuals and 9 to 27 (SD 6.67) for the COPD patients. Several bacterial species were found consistent with these findings by database searches. However, as T-RFLP results based on PCR with one labeled primer cannot exactly determine the species, such correlations should be viewed with the appropriate caution. All samples showed highly heterogeneous terminal restriction fragment (TRF) patterns  served as starting material for PCR amplification of 16S rRNA gene followed by patterns .Bacteroides, Lactobacillus, Megasphaera, Prevotella, and Streptomyces) were most common and appeared in two thirds of healthy individual group samples. In contrast, a different set of restriction bands  could be found only in certain individuals.Matrix tables of the obtained results (data not shown) indicated that some restriction fragments  out of 18 were correctly classified into the healthy or COPD group without any false negatives .We detected multiple T-RFPs in each sample group. To determine if any of them are correlated with disease status, a principal component analysis was performed. Classification optimization yielded the results as shown in Bacillus, Mesorhizobium, Flavobacterium, Streptomyces, Megasphaera, and Clostridiaceae). Given the TN (true negative) and FN  rates of 0.222 and zero respectively, this set of bands is reliably distinguishing between COPD patients and healthy individuals. One fragment (possibly representing Mesorhizobium) was only detected in samples of healthy individuals. Classifier training solely based on a prior feature selection via PCA (including retransformation) did not yield a good learning model and classification performance was non-distinctive. Different variance proportion cutoffs did not significantly change the results.With the application of simple K-Means clustering, we could further reduce the selected fragments  characteristic for COPD state. Unique genera included Afipia, Brevundimonas, Curvibacter, Moraxella, Neisseria, Undibacterium, Corynebacterium, Capnocytophaga, and Leptolyngbia.A second distance-based neighbor-joining tree was constructed with 76 phylotypes detected in COPD patient group . Twenty Acinetobacter, Fusobacterium, Megasphaera, Prevotella, Pseudomonas, Sphingomonas, Staphylococcus, Streptococcus, and Veillonella. We also examined sequencing data for the presence of atypical bacteria , which are associated with COPD exacerbations Mycoplasma and Legionella related species were identified in several samples using T-RFLP approach. The results from the RFLP analysis were correlated with the data from sequencing and showed that the results from both methods correlate partly  was more common in control subjects B. diminuta, A. cryaerophilus, and L. interrogans in exacerbated COPD patients H. cetorum and C. mucosalis in respiratory samples. Anaerobes such as Prevotella and Veillonella are a component of commensal microbiota of the lung Pseudomonas sp. A limited bacterial diversity was generally found for COPD patients Proteobacteria was found in asthmatics Pseudomonadaceae, Enterobacteriaceae, and Helicobacteraceae seemed to play the crucial role in severe exacerbations of COPD Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae and other bacterial families were correlated with bronchial hypersensitivity The core microbiome reported here compares well with those reported by other investigators Lactobacillus sp. only in healthy individuals. In the present study, T-FRLP based analysis allowed the identification of bacteria in BAL samples and core community data and the results correlated well with those obtained by Sanger sequencing and database analysis. Based on the multiple methods that are presently used, it is not easy to compare the data from different studies.Despite the progress in the field of culture independent analysis of the microbiome, data on the respiratory tract are still limited and the published studies comprise a relatively low number of studied individuals. Different methods are currently being used developed to study the microbiome of the human body. The PhyloChip microarray contains more than 1 million oligoprobes and can differentiate about 60 000 bacterial taxa The lung microbiome contains a large number of oral microorganisms. Representatives of the common oral microbiome were found in COPD patients and less in healthy individuals In conclusion, the microbiome of the healthy lung is complex and differs from the microbiome of the respiratory tracts of COPD patients. Two different methods revealed partly diverse microbial spectra. Increasing knowledge about the pulmonary microbiome will provide the opportunity to understand the role of microorganisms in lung diseases.


Cytomegalovirus (CMV) viremia has been shown to be an independent risk factor for increased mortality among HIV-infected individuals in the developing world. While CMV infection is nearly ubiquitous in resource-poor settings, few data are available on the role of subclinical CMV reactivation on HIV.Using a cohort of mineworkers with stored plasma samples, we investigated the association between CMV DNA concentration and mortality prior to antiretroviral therapy availability.Among 1341 individuals , 70 (5.2%) had CMV viremia at baseline; 71 deaths occurred. In univariable analysis CMV viremia at baseline was associated with a three-fold increase in mortality . After adjustment for CD4 count, WHO stage and HIV viral load (N = 429 with complete data), the association was attenuated . Mortality increased with higher CMV viremia . Results were similar using time-updated CMV viremia.High copy number, subclinical CMV viremia was an independent risk factor for mortality among male HIV-infected adults in South Africa with relatively early HIV disease. Studies to determine whether anti-CMV therapy to mitigate high copy number viremia would increase lifespan are warranted. Cytomegalovirus (CMV) is a major opportunistic pathogen among HIV-infected persons. Not only is it important because of its direct pathogenicity leading to CMV end-organ disease and death in severely immunosuppressed individuals, typically those with CD4 cell counts of less than 100 cells/µlSince the 1980s, a number of studies investigated the association between CMV and mortality among HIV-infected individuals. However, most early studies used CMV antibody positivity as the exposure of interestBetween 1999 and 2002 we conducted a cohort studyThe study was approved by Research Ethics Committees of the University of KwaZulu Natal, South Africa; the London School of Hygiene and Tropical Medicine, UK; and the Institutional Review Board of the Fred Hutchinson Cancer Research Center, Seattle, USA.This analysis used data from a workplace HIV clinic within a large gold mining company in the Free State Province, South Africa, in the context of a prospective cohort study (the "parent study") to explore the effect of tuberculosis on HIV viral load between May 1999 and March 2002Clinic patients who were 18 years of age or older were invited to enrol in the parent study, regardless of CD4 cell count, and for those consenting, plasma samples obtained at routine visits were stored. All participants in the parent study who had stored plasma available for testing for CMV and who had a CD4 count available within ± six months of their first (defined as “baseline”) stored plasma sample were eligible for this analysis. Mortality information was obtained from medical and employment records. Because CMV infection is ubiquitous amongst adults in developing countriesDNA was extracted from 200 ul of stored plasma and eluted into 100 ul of AE buffer (Qiagen Inc). Ten ul of DNA was then analyzed by CMV specific TaqMan real-time PCR as previously describedThe chi-square test or Fisher's exact test, and Wilcoxon rank sum test were used to assess the association of categorical and quantitative variables, respectively, with baseline CMV viremia.st March 2002), whichever came first. CD4 count and WHO stage at baseline were defined as the closest measurement, within ± six months, to the date of the baseline CMV measurement.The primary outcome for this analysis was death. Participants were considered to enter the cohort on the date of the first available CMV measurement (baseline), and were followed until death, leaving the work force, or end of study  at baseline.Cox proportional hazards regression was used to estimate the effect of baseline CMV viremia on time to death. Data were reported as hazard ratios (HRs), 95% confidence intervals (CIs), and P values from the likelihood ratio test (LRT). CMV viremia status was defined as positive (>50 copies/ml) or negative and as a variable on three levels . Where appropriate, tests for linear trend and departures from linear trend were assessed using the LRT. Non-proportional hazards for CMV and other factors were explored. A multivariable analysis was conducted to assess the relationship between CMV viremia and death, controlling for potential confounders including age, CD4 cell count and WHO stage. An interaction between CD4 cell count (<200 versus ≥200 cells/µl) and CMV viremia was defined A sensitivity analysis was conducted using all available CMV measurements during follow-up, therefore modelling CMV viremia as a time-updated covariate.Data were analysed using STATA v. 10 .Between May 1999 and March 2002, 1,342 patients were enrolled in the parent study, and had suitable stored specimens available for analysis for CMV viremia. One patient had missing baseline WHO stage, and so analysis was restricted to 1,341 patients. The population was predominantly male  with a median age of 39 (range 19–59) years, consistent with the demographics of the gold mining workforce . The medThe median number of CMV measurements per patient, including the baseline sample, was two (range one to nine) with a total of 2703 measurements overall. Including all measurements, 146 participants (10.9%) had at least one sample with detectable CMV viremia. Of the 770 participants with at least two measurements, 84.3% (649) were consistently negative (median of two measurements), 1.4% (11) consistently positive (median of two measurements) and 14.3% (110) had their CMV viremia status change at least once during follow-up (median of three measurements). The median time between the first and second measurement was 6.6 months. Of participants with a baseline CD4 count≥200 cells/µl, by 6 months 89.5% (453/506) maintained a CD4≥200, with a median decrease of 26 cells/µl (IQR 26–86) over that period.10 copies/ml, p<0.0001) and a higher proportion were in WHO stage 3 or 4 , compared to those without CMV viremia  had detectable CMV viremia at baseline with a median CMV DNA level of 524 copies/ml (IQR: 116–1994) and 40% of those had CMV DNA level ≥1000 copies/ml. Those with detectable CMV viremia had a lower median CD4 cell count , higher HIV RNA concentration (47.1% vs. 27.6% with ≥5log viremia .The median follow-up time was 0.9 years (range 0.01–2.3) with a total of 1,534 person years of observation, during which 71 deaths occurred. In unadjusted analysis, CMV viremia at baseline was associated with a greater than three-fold increased hazard of death  showed consistent evidence of an increased hazard of death for those with CMV viremia at baseline after adjustment for CD4 cell count, WHO stage and HIV viral load . There was again no evidence for an interaction between CD4 group  and CMV viremia defined as positive or negative (P = 0.71). For CMV viremia defined on three levels, after adjustment, increased mortality was observed with with increasing levels of CMV viremia (P for trend 0.04); those with ≥1000 copies/ml had an adjusted HR of 3.65  compared to being CMV negative. Results were similar for the analysis based on time-updated CMV viremia (data not shown).This retrospective laboratory evaluation of a prospective cohort study of HIV-infected sub-Saharan African men found a consistent association between CMV viremia, especially high copy levels of CMV DNA in plasma, and increased hazard of death. These data are unusual in that they come from a population of miners in South Africa, many of whom had not reached the stage of advanced HIV disease, and in this context suggest that the association between CMV and mortality is not restricted to individuals with advanced HIV-related disease or those with overt CMV end-organ damage.The prevalence at baseline of CMV viremia in our study, at 5%, was somewhat lower than has been reported in other studies10 CMV DNA concentration. Bowen et al. and Spector et al. reported that each log10 increase in CMV DNA load was associated with a 1.9-fold and 2.2-fold increase in mortality, respectivelyet al. found that CMV viral load greater than 3.1log10 copies/ml was independently associated with deathStudies in the U.S. and Europe among patients on ART have also found associations between CMV viremia and increased risk of death, with hazard ratios ranging from nearly 2 to almost 5 after controlling for other indicators of disease progression such as CD4 cell count and HIV viral loadAssessment of causality is difficult in studies such as ours, and thus we cannot determine whether higher CMV viremia directly increases the risk of death, or whether it is a marker of higher mortality risk attributable, for example, to impaired immune function, independent of the CD4 count. However a randomised trial of CMV-specific therapy would be needed to address this question, most likely in addition to antiretroviral therapy. A cohort study conducted in the US, found that treatment with systemic anti-CMV treatment was associated with a 28% reduction in mortality amongst patients with CMV retinitis, independent of HAART and using time-updated covariates Our study has some limitations. First, although we had a large cohort of 1,342 participants, only a subset had data on HIV RNA. In the multivariable analysis, the association between CMV and mortality was stronger after adjusting for HIV RNA load, suggesting negative confounding. This observation is consistent with the hypothesis that the mechanism of action of CMV on risk of death is independent of its action on plasma HIV RNAIn summary, we found associations between CMV viremia and increased risk of death in a population of gold miners with HIV infection, but mostly without advanced immunosuppression, prior to the ART era. Earlier initiation of ART, as per current guidelines in South Africa, may be helpful in reducing mortality. Further research on interventions to prevent high copy number subclinical CMV viremia may be warranted.


Background: Hydrofluoric acid (HF) is a small and partially dissociated acid (pKa 3.2), able to deeply penetrate into human skin in addition to the corrosiveness of the hydrogen ion (H+) and the toxicity of the fluoride ion (F-). However, there has been a lack of experimental studies to objectively characterize the results of human HF skin exposure decontamination.Methodology/principal findings: A previously established experimental method using a human skin explants ex vivo model  burns: Histological observations in an established human skin explants ex vivo model) described the lesions that appeared following 70% HF penetration. Within 5min, 70% HF penetrates to the dermis. Using the same experimental conditions, a comparison study of two different washing protocols was performed: water + topical calcium gluconate (CaG) versus Hexafluorine®. In these conditions, washing for 15min with running tap water followed by topical CaG ointment only delayed burn onset, while severe tissue damage appeared later. In contrast, after washing with Hexafluorine® over 10 min, no histological lesions developed. These results are in accordance with the results of accidental human industrial case reports.Conclusion/significance: Amphoteric and hypertonic Hexafluorine® can deactivate H+ and chelate F- ions. Based on these results, it should be considered as a promising first-aid decontamination solution to prevent or minimize significant local and systemic consequences of concentrated HF skin exposures. Hydrofluoric acid (HF) is widely used for the manufacture of fluorinated organic compounds, inorganic fluorides, uranium treatment, fluorocarbon products, and fluoropolymers and derivatives. HF is also used in the metal industry for scouring and shining steels  and other metals (such as for pickling titanium), in the oil refinery industry , in electronics (for the surface treatment of semiconductor components), in the glass industry , in construction (for facade cleaning), in the chemical industry (particularly in laboratories), in the refrigeration industry, and in the photovoltaic industry . Dilute HF  has household uses such as rust stain removal, aluminum cleaning and polishing (for automobile wheels), and for colored wood cleaning.The worldwide production of HF is increasing with > 1 million tons per year , this small molecule is capable of penetrating deeply into tissues.HF is a particularly dangerous acid due to its two properties: corrosiveness and toxicity. In addition, as it is only partially dissociated (pK+) associated with cutaneous and mucous membrane lesions [HF is an acid that induces severe tissue necrosis and is a serious systemic poison due to two mechanisms of ion delivery: a corrosive hydrogen ion (H lesions  and with lesions  and resp lesions -8 damage lesions ), with p lesions -14.- ion chelates calcium and magnesium [2 and MgF2. Soluble salts such as NaF and KF are also produced and act at high concentrations as direct cellular poisons.The Fagnesium  forming This chelation induces metabolic disorders ,17 that The systemic consequences of HF are due to increased fluoride concentrations in the body, which can modify the blood levels of calcium , magnesium (hypomagnesemia), and potassium  -23. HF iThe systemic toxicity is potentially life-threatening  dependinex vivo and to compare the efficacy of different decontamination washing protocols.This study was performed to demonstrate the extent of epidermal and dermal lesions following contact with 70% HF on living human skin explants Observation of the general morphology allowed characterization of the nature and extent of 70% HF-induced skin lesions at the tissue and cellular levels over time following exposure alone or exposure followed by decontamination.active HF decontamination solution).Comparison of two different washing protocols was done: running tap water followed by a one-time calcium gluconate (CaG) gel topical application, and Hexafluorine® (a specific ex vivo (see Part 1). Tested products:The study was performed at BIO-EC Laboratories, Longjumeau, France in accordance with a previously described and established experimental burn model using human skin explants 70% HF Running tap water2 CaG gel with topical application of 1 mg/cmHexafluorine® spray .2 for 12-15 hours before the study began. These explants were divided into four groups (The 70 human skin explants utilized were prepared from donated skin from an elective abdominoplasty from a 35-year-old woman (Reference P556). Verbal-informed consent was obtained (see Part 1 for details of the informed consent procedure). The diameter of each explant was ∼10mm. The study was performed in triplicate to insure the internal consistency of the method, not for statistical purposes. The explants were preserved alive in BEM medium (BIO-ECs Explant Medium batch No. 071107) at 37°C in a moist atmosphere containing 5% COr groups .papier filtre No. 268) previously saturated with 30 μL (47 μL/cm2) of 70% HF solution.The HF solution was applied topically to the explants of groups F , FWCaG ; and FHexa  by deposition on filter paper disks  did not receive any treatment. The total observation time ranged from 20 sec to 24 h.For groups FWCaG and FHexa: after 20 sec of contact, the disks were removed. Then the different washing protocols were performed. The explants of group FWCaG were washedwith runningtap water for 15 min (∼2000 mL per explant), and then topical CaG gel (2.5%) was applied, one time only, to each explant at a dose of 1 mg/cmGroup T (negative controls): At the beginning of the study (T0), two explants of the control group were sampled and fixed in Bouin's solution. Two other explants were sampled and similarly fixed at each sampling time: 20 sec, 5, 10, 15, and 30min, 1, 2, and 4h, and 24h.Group F (positive controls): Twenty seconds after the beginning of the 70% HF application, two explants from group F were sampled and fixed immediately after removal of the saturated disk. Then, two explants were sampled and fixed in the same manner at each of the sampling times: 5, 10, 15, and 30min, 1, 2, and 4h, and 24 h.Groups FWCaG and FHexa: Five minutes after the washing step, two explants from each of groups FWCaG and FHexa were sampled and fixed in Bouin's solution. Then two explants from each group were sampled and fixed in the same manner at each of the sampling times: 10, 15, and 30min, 1, 2, and 4h, and 24h.Tissue fixation, sample preparation, and histology procedures are described in detail in Part 1.Alterations of cellular structures were searched for during penetration of HF through the main skin layers: upper epidermis (stratum corneum + lucidum + granulosum), basal epidermis (stratum germinativum), and papillary and reticular dermis.Group T : At T0, the skin showed four to five cellular sites with good morphology. In the papillary and the lower reticular dermis, the cellular structures had good morphology. Whatever the time of survival, no modifications of the epidermal and dermal structures were observed up to 24 h. Group F After 20 sec of contact, no deterioration of the structures of either the epidermis or dermis was observed. At 5 min after a 20-sec exposure, very clear cellular deterioration was observed in the epidermis and papillary and reticular dermis. Lesions clearly increased at 10, 15, and 30 min and 1 h after a 20-sec exposure, with the marked appearance of coagulation necrosis including acido-philic cytoplasm and pyknotic nuclei throughout all the skin layers. At 24h after a 20-sec exposure, the epidermis presented completely necrotic structures. The lesions were less intense in the papillary and reticular dermis .Group FWCaG .At 5 and 10 min after a 20-sec exposure, no deterioration of the structures of either the epidermis or dermis was observed.At 15 min after a 20-sec exposure, very clear cellular deteriorations appeared in the epidermis and in the papillary and reticular dermis. These were decreased at 30 min, and were no longer observed after 1 and 2h after a 20-sec exposure. Slight edematous changes were visible in the epidermal basal layers at 4h and these were clearly increased at 24h after a 20-sec exposure .Group FHexa ).At 5, 10, 15, and 30 min and 1, 2, 4, and 24 h after a 20-sec exposure, no deterioration of the structures of either the epidermis or dermis was observed. Washing by spraying Hexafluorine® for ∼10 min (400 mL) gave a homogeneous result on the explants. Whatever the time of observation, no burn lesions were observed .HF burns have specific pathophysiological mechanisms that require specialized treatment to prevent serious sequelae. Appropriate urgent first aid and secondary medical management can improve the prognosis. Initial decontamination must be efficacious to avoid or minimize the extent (TBSA) and depth of HF burns and to prevent systemic absorption and distribution of the F∼ ions responsible for potential life-threatening systemic toxicity . EfficacUp to now (see Part 1), there has been a lack of histological studies allowing real-time observation of cellular damage during penetration of concentrated HF into human skin, as well as real-time observation of how different decontamination measures may affect such penetration and potential skin injury.ex vivo to 70% HF were tested. It was shown with topical exposure to 30 μL (47 μL/cm2) of 70% HF, 20 sec of contact was sufficient to penetrate the upper layers of the skin explants. By 5min after a 20-sec 70% HF exposure, all the epidermal and dermal layers of the skin were damaged, but not totally and irreversibly destroyed.In a previous study (see Part 1), human skin explants exposed In this study, it was not a question of the treatment of fully developedHF skinburns, as hasbeendone previously in a rat model . Rather,The present study was carried out in conditions such as to be as close as possible to potential exposure to high concentrations of HF likely to be encountered in industrial settings.Explants from the control group remained alive during the 24 h of the experiment. Seventy percent HF penetrated human skin explants after 20 sec of exposure. Without decontamination washing, the first cellular alterations were noticeable by 1 min after a 20-sec exposure. The reticular dermis was reached and injured by 5 min after a 20-sec exposure. At 5 min after a 20-sec exposure, a massive attack of all the cutaneous layers was already present. The lesions then remained stable between 10 min and 4h. At 24 h after a 20-sec exposure, the epidermis was totally necrotic while the dermis was clearly altered . Similar observations were reported by Ohtani et al.  in a humIn the past, water washing was a great advance in the first-aid care for victims of corrosive skin exposures. Historically, evidence for the seriousness of HF burns and the need for efficacious first-aid measures was initially described in France by Thenard and Gay Lussac  in the fcan avoid sleeping and inducefever” [active HF decontamination was lost until the mid-1960s.At this early date, these authors had already noted that the development of HF burns can sometimes be slowed and that the appearance of the lesions could be delayed in onset from 1 to 8 h after exposure. They also experienced the acute pain sensation which “cefever” . Thenardpassive decontamination with copious prolonged water washing was and has continued to be recommended. Using water washing immediately after skin exposure allows dilution and mechanical rinsing of the HF from the skin. However, water washing alone is not always sufficient for preventing or minimizing HF burn injuries, especially with exposure to high concentrations of HF and/or large TBSA exposures.At that time, A number of case reports show that HF burn victims have required surgical treatments and that others have died within a short time after exposure to concentrated HF, despite immediate copious water washing. Fatalities have occurred with exposure to highly concentrated HF (i.e. anhydrous HF) with only a small TBSA exposure on thin and highly vascularized skin such as that on the face and in the genital area -34.active initial washing for HF exposures is derived from this approach. Water has no active binding or chelating properties for any chemical substance and does not inactivate either of the skin tissue injury mechanisms of HF. As water is hypotonic, it cannot be expected to stop penetration of HF throughout all layers of the skin and may, in fact, enhance such penetration. Schrage et al. [active decontamination, an approach that allows deactivation of both HF's corrosive and local toxic potential is logical.A more theoretical approach to HF decontamination reveals that the first few seconds to minutes of washing are critical to prevent or minimize the development of HF skin burns. Some authors have therefore suggested a shorter initial water washing duration ,35,36. Se et al.  have deme et al.  of water+) neutralization and F∼ chela-tion as the initial first-aid measures after HF exposure [Free fluoride ions play a key role in local tissue toxicity, as well as in systemic toxicity. Therefore, improvement of first-aid measures for HF exposures initially focused only on the F” ion. Studied and published protocol changes to improve the outcome of HF burns only considered measures to be done after water washing . Very feexposure .2 skin surface area of a 5% or 10% CaG solution (with a maximum of 0.5 mL per digit for finger burns). Care must be taken not to use calcium chloride solution in this manner because it can itself cause significant local tissue necrosis and sloughing. Pain that is not relieved or recurs is a clinical indication for further CaG infiltration.Topical CaG was first considered for binding F∼ ions and interfering with their chelation of tissue calcium. The first publication describing HF burn therapy with CaG dates from 1964 . At the Both intravenous and intra-arterial infusion of CaG can be done to treat systemic toxicity or hand/foot burns. These exposure routes are indicated for systemic toxicity signs and symptoms, or for more extensive HF burns or burns not responding to topical or subcutaneous CaG treatment ,34,42-45Williams et al.  showed tAlgorithms for HF splash victim management were studied by Kirkpatrick et al. (1995) , using 2In the present study, a 15-min tap water washing followed by a single topical application of 2.5% CaG gel in one intervention leg was utilized to be consistent with common currently recommended first-aid measures.In the FWCaG group, after 20 sec of exposure to 70% HF and then washing with tap water for 15 min followed by a single topical application of 2.5% CaG gel, a temporary delay in the onset of the appearance of tissue lesions was observed. The histological effects only became visible after 15 min, in comparison with the exposed but not washed control group in which the explants showed evidence of damage in all tissue layers after 5 min. In addition, in the FWCaG group, there was a resumption of evolution of the tissue lesions after 4h. At 24 h (at the conclusion of the experiment), HF chemical lesions were clearly present, though not as severe in the exposed but not washed control group.As the tissue lesions continued to evolve from 4 to 24 h, this provides support for recommendations for repeated application of topical CaG. As well, in some cases topical CaG may need to be supplemented or replaced with par-enteral CaG administration .+ ion and chelates the toxic F+ ion of HF. Moreover, it can prevent or minimize the penetration of undissociated HF into the tissues because it is hypertonic and establishes an osmotic gradient away from the skin surface.When the results in the FHexa group of explants (exposed to 70% HF for 20 sec and then washed for ∼10 min with 400 mL of Hexafluorine®), which developed no lesions at any sampling time, were compared with the unexposed and not washed control group (group T), there were no differences in histological appearance at any sampling time from 5 min to 24h after a 20-sec exposure. Compared with the FWCaG group , the Hexafluorine® group developed no lesions at any sampling time, washing with tap water followed by topical CaG only delayed the onset of the appearance of the HF lesions and decreased their severity. There results suggest that Hexafluorine® acts on both mechanisms of HF skin injury: corrosiveness and local toxicity. Hexafluorine® deactivates the corrosive HThe experimental results presented here are in accordance with published clinical results regarding the use of Hexafluorine® in over 30 cases of HF splashes in industrial settings ,57 whereex vivo rabbit cornea study using an Eveit® model combined with a powerful OCT imaging technique, Rihawi et al. [Some experimental studies with rats  have chai et al.  and Spöli et al.  showed tWater has a primary mechanical washing effect with an added minor diluting effect. As Hexafluorine® is provided as a solution in sterile water, these same washing and diluting effects are also present. However, as water has no chemical effect on HF, it apparently may leave a residue of fluoride ion on the skin in a sufficient quantity to explain the observed secondary necrosis.Topical CaG has the properties of chelating free fluoride ions, thereby decreasing their concentration in the injured tissue. It can also provide a source of extracellular calcium, which can change the balance between extracellular and intracellular calcium. In the study presented here, a single application of CaG gel following water washing was not sufficient to prevent the delayed development of tissue damage.+ ion and chelates the F∼ ion, thereby actively acting against both mechanisms of HF cutaneous tissue injury. In the study presented here, no cutaneous tissue injury resulted over 24h of observation from a 20-s application of 70% HF followed by Hexafluorine® washing.Amphoteric Hexafluorine®, in contrast, acts to neutralize the Hex vivo, 70% HF can cause injury to all of the explant skin layers within 5 min after a 20-s application. Decontamination with water followed by a single application of topical 2.5% CaG gel delayed the onset of the skin injury and mitigated the severity of the damage, but did not prevent injury in this experimental model. In contrast, when the HF-exposed skin explants were washed with Hexafluorine®, no tissue injury developed during 24-h observation. Thus, in this experimental model, decontamination with Hexafluorine® completely prevented 70% HF cutaneous tissue injury. Hexafluorine® deserves consideration for further study and clinical application.As demonstrated in the utilized 70% HF-exposed human skin explants


Dentifrice B, Colgate Sensitive Pro-Relief, contained a combination of 80000 ppm arginine, bicarbonate, calcium carbonate, and 1450 ppm fluorine as NaMFP. Subjects (N = 110), stratified into two groups (N = 55), brushed twice-daily for 60 s, over an 8-week period. Sensitivity status, compliance, and safety were determined at 1, 2, 4, and 8 weeks. A fixed-effects ANCOVA statistical model was applied to the Intent-To-Treat population using a two-sided 5% significance level. After 8 weeks, the treatment groups using Dentifrice A and Dentifrice B exhibited mean reductions from baseline of 49% and 45% in air sensitivity visual analogue scale (VAS) score, 61% (both) in examiner-based Schiff Sensitivity score, and clinically significant reductions in tactile pain threshold; all reductions were statistically significant (P < 0.0001). Both treatment groups also exhibited significant reductions across all sensitivity measures at 1, 2, and 4 weeks .A comparison of the desensitising efficacy of two commercially available dentifrices with different modes of action was conducted in a randomised, examiner-blind, two-arm, parallel group, 8-week, longitudinal clinical study. Dentifrice A, (Sensodyne Multi Action Iso-Active), contained 50000 ppm KNO It isjunction , 9. Tubujunction . As a cojunction . The prejunction –13. In tjunction . Aetiolojunction , 15.in vivo include oxalate salts [in vitro has also been widely studied using techniques that include measurement of dentine permeability [An implicit consequence of the hydrodynamic theory is the polarisation of treatment options into those that target desensitisation of the relevant intradental nerves, and those whose aim is to inhibit or prevent transmission of the stimulus itself. Potassium salts including nitrate, chloride, and citrate are examples of the former, with dentifrice being the principal delivery format , 17. Altte salts –26 and ste salts –29. Theieability , 31, scaeability , 32, andeability , 34.3) as the benchmark control [in vivo is significantly enhanced [3 reported highly significant relief from tooth sensitivity at 4, 8, and 12 weeks [in vivo dispersion associated with this novel format is believed to enhance delivery of the oral care actives in hard-to-reach areas [A new occlusion-based technology has recently been commercialised in a dentifrice format for the treatment of dentine hypersensitivity . Based o control , 40. As  control . It is renhanced . A recen12 weeks . The impch areas . The aim3 and 1450 ppm fluoride as the sodium salt (NaF) and was manufactured by GlaxoSmithKline Consumer Healthcare . Colgate Sensitive Pro-Relief (Dentifrice B) contained 80000 ppm arginine, bicarbonate, calcium carbonate, and 1450 ppm fluorine as NaMFP and was manufactured by Colgate-Palmolive . Oral B Indicator Soft toothbrushes were sourced from Procter & Gamble . Test dentifrices were supplied to study volunteers in their original commercial packaging over-wrapped in opaque vinyl with a study-specific label detailing protocol number, product code, storage conditions, and precautionary information including an emergency contact telephone number.Sensodyne Multi Action Iso-Active (Dentifrice A) contained 50000 ppm KNOThe study protocol was reviewed and approved by an Independent Ethics Committee at Wuhan University, China and conducted according to ICH GCP guidelines . InclusiTactile and air sensitivity measurements were performed by two independent examiners. At the screening visit, the tactile sensitivity of each eligible tooth was determined using a calibrated Yeaple probe  set at the equivalent microamperes to deliver a fixed force of 30 g. The probe tip was placed perpendicular to the buccal surface and moved in a slow distal-to-medial sweeping motion across the tooth surface in order to ensure application of the stimulus across the sensitive area of the exposed dentine. Subjects were then asked whether the sensation was painful, with responses confined to “yes” or “no”. Subsequent testing at weeks 1, 2, 4, and 8 was performed by a single examiner on the two teeth selected at baseline. Testing began at 10 g and increased by 10 g with each successive challenge until a “yes” response was obtained or the 50 g upper limit reached. The force setting eliciting the positive response was then repeated, but in the absence of a second positive response the force setting was increased by 10 g and continued until a force was found that elicited two consecutive “yes” responses.The evaporative (air) assessment was taken after the determination of tactile sensitivity with a minimum of 10 min recovery time between each evaluation. It was felt that 10 mins would allow sufficient time for physiological and neurological recovery following the first set of stimuli. The test involved directing an air stream (60 ± 5 psi) at ambient temperature for 1 s from a triple air dental syringe toward the exposed dentine surface from a distance of approximately 1 cm; adjacent teeth were shielded from the stimulus by the fingers of the examiner. Each tooth was scored according to the four-point Schiff Air Sensitivity Scale , defined0 Subject does not respond to air stimulus. 1 Subject responds to air stimulus, but does not request discontinuation of stimulus.2 Subject responds to air stimulus and requests discontinuation or moves from stimulus.3 Subject responds to air stimulus, considers stimulus to be painful, and requests discontinuation of the stimulus.At the screening visit, the teeth of subjects eliciting a score of 2 or 3 were recorded as qualifying as potential test teeth. At the baseline visit, subjects were also asked to rate the pain intensity associated with the evaporative (air) stimulus using a linear 100 mm VAS end-anchored by “No Pain” and “Worst Pain Imaginable”. At least two incisors, canines, or premolars were required to provide an air sensitivity response of ≥25 mm based on the VAS, a Schiff score ≥ 2, and a tactile pain threshold between 10 and 50 g. Subject randomisation was performed using as stratification factors; the number of air sensitive teeth at baseline based on the VAS response (<6/≥6) and the mean VAS score at baseline (<60 mm/≥60 mm). In total, 63 (57.3%) of subjects had 6 or more sensitive teeth at baseline, 84 (76.4%) had a mean baseline VAS score ≥ 60 mm. A total of 110 eligible adult volunteers took part in this study after signing an informed written consent form. Of these, 19 (17.3%) were male and 91 (82.7%) were female, with a mean (±SD) age of 42.0 ± 10.43 years; all subjects were of Chinese ethnicity. N = 55 per treatment group). Two test teeth were selected on the basis of their eligibility criteria for both tactile and evaporative stimuli. Subjects were given their assigned dentifrice and toothbrush to use in place of their regular oral hygiene products for the next 8 weeks. Subjects were not permitted to use any oral care products after baseline other than the dentifrice and toothbrush provided. Prior to the baseline visit, subjects were instructed to refrain from oral hygiene procedures and gum chewing for a minimum of 8 h before their scheduled appointment. Subjects were reminded not to eat or drink for a minimum of 2 h nor take analgesics for at least 8 h prior to their scheduled appointments. Dental scaling, prophylaxis, and bleaching were prohibited for the duration of the study.This was an 8-week, single-centre, examiner-blind, randomised, two-arm, parallel group study in otherwise healthy subjects experiencing dentine hypersensitivity. The clinical trial conformed to consensus design and conduct recommendations regarding population selection, sample size, test stimuli, assessment, and outcome variables . EligiblThe test product was used for one-timed minute twice per day (am/pm) with subjects instructed to dispense a ribbon of paste to cover at least three quarters of the head of the toothbrush. During the treatment phase, subjects returned to the study site after 1, 2, 4 and 8 weeks following their baseline visit for evaluation of dentine hypersensitivity, oral health status, and continuing eligibility. Test products were weighed at each visit as a means of monitoring compliance. The primary efficacy measure was observance of a statistically significant within treatment reduction from baseline in the subjects' assessment of air sensitivity, measured using the VAS, after 8 weeks. Secondary efficacy measures included changes in Schiff score and tactile pain threshold after 8 weeks. The definitions of reduction in sensitivity in each case are a decrease of at least 10 mm in mean VAS score, a decrease in mean Schiff score across the two teeth of at least 0.5 units, and a mean increase of at least 10 g in the tactile pain threshold determined with the Yeaple probe. The safety profile of both dentifrices was assessed by reference to adverse events and oral soft tissue abnormalities.The subject level change from baseline was calculated as the mean change from baseline observed across the two selected teeth within a subject. A fixed effects analysis of covariance (ANCOVA) model was used with treatment included as a fixed effect factors and baseline number of air sensitive teeth and mean baseline VAS score across the two teeth included as covariates. All significance tests were two-sided and performed at the 5% level of significance with no adjustments for multiple comparisons.For analyses of subject level Schiff data and subject level Tactile Pain Threshold data, an ANCOVA model was used with treatment and baseline VAS stratification value and included as fixed effect factors. Baseline number of air sensitive teeth and either mean baseline Schiff score or mean baseline Tactile Pain Threshold score across the two teeth were included as covariates, respectively. Intent-To-Treat (ITT) and Per Protocol (PP) analyses were performed for within and between treatment assessments of change from baseline to weeks 1, 2, 4, and 8. Two-sided significance tests were performed at the 5% level with no adjustments for multiple comparisons.Tooth level analyses were also performed wherein each tooth individually contributed to the analyses, as opposed to the mean across the two teeth used for the subject level analyses. The statistical models were similar to those used for subject level data, but with the addition of subject included as a random effect. ITT analyses were performed for within and between treatment assessments of change from baseline to weeks 1, 2, 4, and 8. Two-sided significance tests were performed at the 5% level with no adjustments for multiple comparisons.The primary population for the analysis of all efficacy data was the ITT population. One subject withdrew from the study and provided no postbaseline efficacy data leading to an ITT population of 109 subjects. One subject who received antibiotics for gingival inflammation was excluded yielding a PP population of 108 subjects. Other major protocol violations were reported for a further 13 subjects leading to the exclusion of specific data from PP analyses but not full exclusion of subjects. Specifically, 8 subjects exhibited noncompliance with the study product, 3 were significantly noncompliant with the visit schedule and 2 subjects used a nonstudy toothbrush.A statistically significant reduction from baseline in tooth sensitivity was observed for both test products, for all three sensitivity parameters, at all four time points, including the primary analysis of sensitivity reduction at week 8 based on mean VAS score as shown in P = 0.0645). Investigation of the relationship between efficacy variables yielded correlation coefficients of 0.74, −0.55, and −0.53, respectively, for VAS score versus Schiff score, Schiff score versus tactile pain threshold, and VAS score versus tactile pain threshold. The PP analyses performed on subject level data and additional ITT analyses performed on tooth level data of within and between treatment outcomes yielded comparable results to those obtained using the primary subject level ITT approach (data not shown). The sole exception related to the PP analysis of VAS score, where the reduction from baseline at 1 week for the treatment group using Dentifrice B was not statistically significant  and were reported by the same subject, however both were mild in intensity and resolved at study completion. No serious adverse events were reported during the course of this clinical study.3, a proven nerve desensitising active, and NaF as the source of ionic fluoride. The formulation also contains 2-methyl butane, a nonsurfactant-based foaming agent with a boiling point of ca. 30°C that is activated in situ on brushing. Dentifrice B, in contrast, contains a novel dentine occlusion system that includes the dibasic amino acid arginine, bicarbonate, and calcium carbonate. The formulation does not contain fluoride due to the presence of calcium, but contains fluorine as NaMFP in order to confer anticaries activity.In this study, the efficacy of two new desensitising dentifrice formulations has been compared in a blinded, parallel group, controlled clinical study. The two dentifrices are very different both in terms of composition and their claimed mode of action. Dentifrice A contains KNOP = 0.0645). Given the number of the statistical evaluations conducted, this specific outcome does not materially change the overall inferences drawn from these results of this clinical study. The correlation coefficients used to examine the relationship between the different efficacy variables showed a moderately strong correlation between the VAS score and Schiff score (0.78). This is not unexpected given that the data contributing to both are elicited by the same evaporative air stimulus. Comparisons of the Schiff score versus tactile pain threshold and VAS score versus tactile pain threshold elicited similar correlation coefficients . That they are less strongly correlated than the VAS and Schiff scores is again to be expected.The results demonstrate comparable clinical effectiveness for the two test dentifrices with significant reductions in measures of tooth sensitivity observed across all measures, at all predefined time points. After 8 weeks of twice daily use, a 49% versus 45% reduction in the subjective outcome measure of evaporative VAS score, and a 61% versus 61% reduction in examiner-based Schiff score was observed for Dentifrice A and B, respectively, versus baseline. Large and clinically relevant improvements in the tactile pain threshold versus baseline were also observed for the two treatment groups. No significant between-treatment efficacy differences were apparent at any of the time points. Given the comprehensive improvements in sensitivity relief in both treatment groups across all the efficacy measures, these results suggest a comparable level of performance between the test products with no apparent difference between the potassium-based novel gel-to-foam formulation (Dentifrice A) and the arginine-based formulation (Dentifrice B). Tooth-level analyses revealed one minor difference to the subject-level analysis wherein the evaporative VAS score in the treatment group using the arginine-based dentifrice was not statistically different from baseline after 1 week of treatment (3 [P < 0.05). The present study employed a similar clinical methodology that accords with published consensus recommendations [ca. 38%) in comparison to the previously cited studies involving Dentifrice B [As previously indicated, two recently published 8-week dentine hypersensitivity studies reported superior efficacy for the occlusion-based Dentifrice B containing the novel arginine/bicarbonate/calcium carbonate technology when compared with a conventional desensitising dentifrice containing KNO3 , 40. In ifrice B , 40, andOne possible explanation for the different result observed in the present study compared to those reported previously for Dentifrice B , 40 may The prevalence of dentine hypersensitivity has been studied in a variety of different populations in a number of different countries across the world. Direct comparison of the different epidemiological studies is often confounded by the use of different clinical methodologies and definitions. The lack of homogeneity in reporting dentine hypersensitivity does make characterising the size and progression of the condition a challenge. In this respect, consensus guidelines defining the most suitable epidemiological approach to take for assessing dentine hypersensitivity prevalence would be welcome. However, what is not in dispute is that millions of people are affected by dentine hypersensitivity and that for a number of patients the severity of the problem is such that it significantly impacts on their quality of life . Many moIn conclusion, this study demonstrates that the two study groups using either a potassium-based gel-to-foam dentifrice (Sensodyne Multi Action Iso-Active) or an occlusion-based dentifrice (Colgate Sensitive Pro-Relief) over an 8 week duration experienced statistically significant reductions in self- or examiner-based dentine hypersensitivity versus baseline, however no differences in levels of hypersensitivity were observable between the two groups at any timepoint.