SMN1 exon 7 are reported to account for 94% of affected individuals. Published literature places the carrier frequency for SMN1 mutations between 1 in 25 and 1 in 50 in the general population. Although SMA is considered to be a pan-ethnic disease, carrier frequencies for many ethnicities, including most ethnic groups in North America, are unknown.Spinal muscular atrophy (SMA) is the most common inherited lethal disease of children. Various genetic deletions involving the bi-allelic loss of SMN1 mutation carrier frequencies in African American, Ashkenazi Jewish, Asian, Caucasian, and Hispanic populations, more than 1000 specimens in each ethnic group were tested using a clinically validated, quantitative real-time polymerase chain reaction (PCR) assay that measures exon 7 copy number.To provide an accurate assessment of SMN1 was identified in the African American group (27% compared to 3.3–8.1%). This latter finding has clinical implications for providing accurate adjusted genetic risk assessments to the African American population.The observed one-copy genotype frequency was 1 in 37 (2.7%) in Caucasian, 1 in 46 (2.2%) in Ashkenazi Jew, 1 in 56 (1.8%) in Asian, 1 in 91 (1.1%) in African American, and 1 in 125 (0.8%) in Hispanic specimens. Additionally, an unusually high frequency of alleles with multiple copies of Differences in the frequency of SMA carriers were significant among several ethnic groups. This study provides an accurate assessment of allele frequencies and estimates of adjusted genetic risk that were previously unavailable to clinicians and patients considering testing. SMN1 mutations were obtained for five North American ethnic groups by evaluating more than 5000 specimens.Precise allele frequencies for SMN1 mutation frequencies were found between several populations.Significant differences in SMN1 copies. This is considerably higher than other populations and results in a SMA carrier adjusted risk estimate that is five times greater than that of the Caucasian population.Approximately 27% of alleles in the African American specimens had two or more This study provides accurate estimates of allele frequency and adjusted genetic risk for five major ethnic groups in North America.With an incidence of 1 in 6000 to 1 in 10 000 live births, spinal muscular atrophy (SMA) is the most common lethal genetic disease of children.SMN1) gene.SMN1 gene resides in a duplicated region of chromosome 5q13 telomeric to the near identical homologue SMN2. More than 90% of the functional SMN protein is contributed by SMN1. Comparably SMN2 produces only a small fraction of SMN due to a point mutation in exon 7 that disrupts an exon splice enhancer site, preventing normal post-transcription processing.SMN1 and SMN2 genes varies. Published information suggests that 85–95% of the population has one copy of the SMN1 gene per allele; however, individuals with more than one copy per allele have been identified.SMN1 exon 7. Of the remaining 6% of SMA cases, most are attributed to a variety of rare molecular lesions distributed throughout the gene, including point mutations, small insertions, and deletions. A minority of cases may involve genes other than SMN1.SMN2 copy number and disease severity, such that affected individuals with three or more copies of SMN2 typically have milder forms of the disease.SMN1 exon 7 copy number by quantitative polymerase chain reaction (PCR).et al reported a frequency for one-copy carriers at approximately 1 in 40 individuals in the general population.SMN1 copies paired with a zero-copy allele (hereafter referred to as “2+0” genotype). By exon 7 quantification these individuals are indistinguishable from wild-type (or “1+1” genotype). It has been estimated that approximately 1 in 600 apparent two-copy specimens may be “2+0” carriers.16SMA is caused by mutations in the Survival Motor Neuron 1  recommend universal carrier screening for SMA.20SMN1 copy number. These ethnicities are relevant since they comprise >95% of the North American population at large and the North American patients electing to participate in genetic testing (2006 US Census estimates).To address this need for accurate allele frequency data, more than 1000 samples from five major ethnic groups, African American, Ashkenazi Jewish, Asian, Caucasian and Hispanic, were tested for SMN1 exon 7 copy number employed a clinically validated, real-time, quantitative PCR assay specific for the single nucleotide change in exon 7 (cd 840 c>t). All reported results could be assigned to validated, non-overlapping genotype groups of 1, 2 or 3 SMN1 copies (the three copies group includes samples with three or more SMN1 gene copies).Samples were collected from residual material following routine clinical testing of individuals presumed to have no family history of SMA. All specimens were made completely anonymous before testing in accordance with approved institutional protocols. Ethnic assignment relied upon patient reported data that were not collected as part of complete family histories. However, these ethnic assignments, which reflect clinical practice, are therefore highly representative of the anticipated clinical experience for SMA carrier screening. The assessment of SMN1 genotypes were observed among several ethnicities , suggesting a much higher frequency of alleles with two or more SMN1 copies than the other four ethnic groups. These unexpected results in this group were confirmed by retesting 10% of the samples using multiplexed ligation dependant probe amplification  as an alternate technique. All MLPA results were concordant with the original real-time PCR data.Significant differences in the frequencies of nicities . The higSMN1 in the African American group is 3.4 to 8.4 times more prevalent when compared to the other ethnic groups. The preponderance of the two-copy allele in the African American group also suggests a much higher frequency of individuals with the SMA carrier “2+0” genotype compared to other ancestries.Using the observed genotype data and assuming Hardy–Weinberg equilibrium, maximum likelihood estimation was employed to determine frequencies for alleles and allele pairings within all sample groups  and 3. TSMN1 copies. These calculations account for rare mutations undetectable by the method described here, and model the “2+0” carrier genotype. Adjusted carrier risk was calculated for each ethnic group based on its unique allele frequencies .Adjusted carrier risk assessment estimates have been previously published based on aggregate results of several studies.quencies . The resSMN1 copy number in the African American population is unknown. Previously published data have shown that individuals with higher copy numbers of SMN1 tend to have fewer copies of SMN2.SMN2 may have converted to SMN1; however, the inverse may also be true. Alleles with multiple copies of SMN1 may be the ancestral form of the duplication, with conversion to SMN2 a more recent mutation event. An assessment of SMN2 copy number is currently underway to understand the SMN1/SMN2 correlation in this sample set.The genetic basis for the unusually high SMN2 copy number, as well as other genes such as plastin 3.Prediction of disease phenotype in SMA is complicated by the modifying effects of SMN1 mutations and for African American patients who bear increased frequencies of two-copy alleles. These data will facilitate the accurate interpretation of clinical testing results and provide additional information for genetic counselling.The recent ACMG guideline for SMA carrier screening


Immunodeprived mice survived a high, otherwise lethal dose of cyclophosphamide (Cy) provided they had been "primed" with a low dose (50 mg kg-1) of the drug 4 days earlier. These combinations were then tested on 2 oat cell xenograft lines (which are known to reproduce the chemotherapeutic responses of the parent tumours) grown in immunodeprived mice. In the treatment of the first oat cell xenograft, 200 mg kg-1 Cy produced a growth delay of 34 days in the unprimed group and 45 days in the primed group. At a dose of 300 mg kg-1 a growth delay could not be assessed in the control group as 16/17 of these unprimed mice bearing this xenograft died. However, 14/22 tumours went into complete remission in this group before death occurred. In contrast only 3/16 deaths occurred in the group of mice that were primed before receiving the same challenge dose. In these animals 19/26 tumours went into complete remission and were still completely absent when the experiment was terminated at 60 days. Using the second oat cell xenograft, 300 mg kg-1 Cy produced a growth delay of 27 days. However, at this dose level all the animals were dead by day 46. In mice which had been primed with 50 mg kg-1 Cy 4 days before the administration of 300 mg kg-1 a growth delay of 32 days was achieved and 2/9 animals were alive at day 60. This study shows that priming allows larger doses of Cy to be given to immunodeprived mice bearing human tumour xenografts than would normally be tolerated and that the priming does not alter the anti-tumour efficacy of the large challenge dose as measured by tumour growth delay or complete remission rate. As the tumours were human in origin it raises the question whether high dose cyclophosphamide therapy and priming have a role to play in the treatment of patients with oat cell carcinoma.


The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential. Avian influenza viruses naturally circulate in wild aquatic birds as a diverse population having 16 HA and 9 NA antigenic subtypes In Pakistan the first H9N2 outbreak in poultry was reported in 1998 To analyse the genetic nature of H9N2 viruses in the enzootic region of Pakistan, we sequenced the complete coding regions of all eight segments of twelve H9N2 viruses isolated between May 2005 and March 2008 from poultry flocks in the Punjab and the North West Frontier Province (NWFP) of Pakistan. The results showed that these H9N2 viruses had changed considerably compared with previous H9N2 viruses. The eight gene segments of these viruses no longer clustered in a single established Eurasian H9N2 virus sublineage but there was evidence for complex reassortment of genes from viruses belonging to H9N2 (G1-lineage) HPAI H5N1 (Z-genotype), and HPAI H7N3 viruses. Therefore, these viruses represent novel genotypes of H9N2 viruses, the potential consequences of which should not be overlooked.The twelve viruses under study (UDL viruses) were isolated at the University Diagnostic Laboratory (UDL) Lahore, from farm outbreaks in the Punjab and the NWFP of Pakistan . VirusesIntravenous pathogenicity testing was carried out using standard methods 5′AGCAAAAGCAGG-3′ with the Verso™ cDNA Kit (Thermo Scientific). PCR amplification was performed using Pfu Ultra II fusion HS DNA polymerase (Stratagene) and specific primers for the eight gene segments. For HA, both specific and universal primers GATC Biotech, Constance, Germany). Five virus samples that contained the NS gene homologous to H5N1 viruses and the two samples (Ck/Pak/UDL-01/06 and Ck/Pak/UDL-03/08) that contained the NS gene homologous to H7N3 were re-examined in greater detail. The HA genes of these seven virus samples were subjected to in-depth sequence analysis to exclude the possibility that the samples were from mixed infections with H9N2 viruses and H5N1 or H7N3 viruses. Sequence analyses of the HA genes were performed by sequencing a minimum of 60 clones per sample that was calculated to detect with 95% probability any variant sequences at a level of 5% within the virus sample.Viral RNA was extracted from allantoic fluid using the High Pure RNA Extraction Kit (Roche Diagnostics) and cDNA was prepared using an influenza universal oligonucleotide primer, http://www.ebi.ac.uk/Tools/fasta33/nucleotide.html) were used to retrieve the top fifty homologous sequences to the sequenced gene from public sequence databases. Multiple nucleotide and amino acid sequence alignments for all eight gene segments were performed using ClustalX (version 1.83). Unrooted phylogenetic trees were generated from nucleotide sequences based on the complete open reading frame of all eight gene segments using minimum evolution analysis with maximum composite likelihood and the Tamura-Nei model Sequence data were analysed using the Staden package (pregap4 v1.5 and gap4 v1.0). Blast homology searches ; seven amino acids  in NA. Similarly, M and NP genes also retained conserved G1-lineage defining residues (A157) in M1, one (L10) in M2, and four amino acids  in NP.The pattern of nucleotide similarity indicates that the 12 H9N2 viruses retained 4 genes  similar to the H9N2 G1-lineage, 3 genes  from a lineage of viruses from the Indian sub-continent and the Middle-East, and NS genes from one of two H7N3 lineages or from H5N1 genotype Z viruses that were previously present in Pakistan and neighbouring countries . A set oIn order to identify molecular markers that may correlate with the pathogenic properties of these H9N2 UDL isolates analysis was performed to compare deduced amino acid sequences of the envelope glycoproteins and the internal proteins of the Pakistan virus isolates with representative strains of established H9N2 virus Eurasian lineages.Changes in the HA glycoprotein are central to the switch from LPAI to HPAI, but additional amino acid substitutions in the HA molecule have been associated with inter-species adaptation 183, A190, L 226, I227 and G228 in the receptor binding cleft. Of these, three represent substitutions  when compared with the reference strains of the H9 lineage  in human cells in culture The receptor binding site motif of HA is critical for cellular receptor specificity and determining virus host range  lineage . GlutamiThe major molecular determinants that are known to influence the functional activities of the neuraminidase (NA) glycoprotein are the enzyme active site, the stalk length, the sialic acid binding site also referred to as the hemadsorbing site (HB), and potential glycosylation sites. The neuraminidase sequences of all 12 isolates were compared with N2 NA sequences of H9N2 viruses prevalent in Asia and the Middle East during the last fifteen years. For each virus amino acids in the enzyme active site were conserved and showed no evidence of substitutions associated with resistance to the sialidase inhibitors oseltamivir and zanamivir. The data revealed that, of the 12 viruses analysed, 2 isolates (Ck/Pak/UDL-02/05 and Ck/Pak/UDL-03/05) contained a unique 5 amino acid residue deletion (aa 46–50) in the stalk region, the others having no deletion in the NA stalk, as was also observed in H9N2 viruses from the Middle East Analysis of the HB site, which is located on the surface of the NA molecule away from the neuraminidase enzyme active site at positions 366–373, Asn-400, Trp-403 and Lys-432 236I substitution. Again, the significance of glycosylation changes at sites 44 and 234 is unknown; however, addition or loss of potential glycosylation may contribute towards increased virulence The comparison of conserved potential glycosylation sites in NA glycoprotein showed that the majority of H9N2 viruses belonging to established Eurasian lineages contain seven conserved potential glycosylation sites at positions 61, 69, 86, 146, 200, 234 and 402 (N2 numbering). The viruses sequenced in this study contained an additional glycosylation site at Asn 44 due to the substitution of Pro45Ser. This additional glycosylation site had also been reported in a number of other H9N2 viruses including Ck/Ind/2048/03, Ck/HK/G9/97 A comparison of the amino acid sequences of the H9N2 neuraminidases shows that certain amino acid substitutions have become fixed as the viruses have evolved but many are unique substitutions represented in individual viruses (Supplementary 80TIASV84) residues show increased virulence in both mouse and poultry infections The NS gene showed a high level of sequence diversity among the twelve UDL isolates analysed. Of particular note were five isolates  that contained an NS gene closely related to the highly pathogenic H5N1 viruses belonging to clade 2.2 of genotype Z with over 99% nucleotide identity . The NS1RNA segment 8 of one virus (UDL-01/06) was very closely related to that of H7N3 viruses from Pakistan isolated over an eleven year period. This single virus isolate shared a 13 amino acid C-terminal truncation; similar truncations have been reported previously in H7 and H9 subtype viruses isolated from poultry The other 6 viruses  formed a distinct sub-clade and showed no truncation or deletion. They contained a PL motif “KSEI”. This KSEI sequence as a PL motif is uncommon. The large scale sequence analysis of avian influenza viruses reported by Obenauer et al. 44A substitution in PB2 and all isolates had aspartic acid at position 372 in NP which has been found in avian influenza viruses isolated from humans A number of residues in the polymerase proteins  and in the nucleoprotein (NP) are known to play a key role in the host range of AI viruses to increase virulence or replication in the mammalian host. Some may be considered as hall-marks of avian or mammalian viruses, whilst others can influence replication efficiency in mammalian or avian hosts. Two analyses of molecular changes associated with the transmission of avian-origin H5N1 and H9N2 viruses to humans Additional residues in the ribonucleoproteins implicated in enhanced replication in mammalian cells have been defined  and thes66S in PB1-F2 is associated with increased virulence in a mouse model RNA segment 2 encodes a second polypeptide in addition to PB1, termed PB1-F2 It is notable that the three polymerase polypeptides have reassorted as a group and the maintenance of the avian specificity at residue 627 of PB2 is striking. The reason for the maintenance of the three polymerase polypeptides may be chance or it is quite likely that the three PB1, PB2 and PA polypeptides act most efficiently as a set and so there may be pressure to maintain a gene constellation for optimal replication proficiency. This may be analogous to the situation observed in human influenza A viruses in which H1N1 and H3N2 co-circulate but with only very limited gene reassortment 15I is common within H9N2 lineages. The UDL viruses showed heterogeneity at residue 37 with 5 viruses encoding valine and 7 viruses alanine but the substitution T37A/V clusters exclusively with the recent UDL H9N2 viruses from Pakistan. The significance of any of these changes to the potential of the currently evolving H9N2 viruses in Pakistan to increase their host range is not known.Several amino acids in the virus matrix, M1, protein are linked with increased replication in mammals or increased pathogenicity in small animal models . At resiNone of the UDL viruses contained substitutions at amino acid positions 26, 27, 30, 31 or 34 in the M2 proteins suggesting that these viruses have no resistance to amantadine The emergence of these novel genotypes of H9N2 viruses and the sustained prevalence of these viruses in poultry warrant further surveillance of H9N2 viruses by complete genomic analysis. These viruses are endemic in poultry in many countries in Asia and the surrounding regions and the generation of new genotypes within H9N2 viruses by reassortment should not be ignored since it allows for an increased ability of the virus to adapt rapidly to different challenges. The observation shown here that further gene reassortment has occurred subsequent to the emergence of viruses in the Middle East highlights the potential for viruses to evolve rapidly. It is widely recognised that genetic reassortment in avian influenza viruses is common in wild bird and poultry isolates Figure S1Amino acid changes in the surface glycoproteins of H9N2 viruses isolated from poultry in Pakistan between 2005 and 2008. The substitutions in the HA (A) and NA (B) are shown in coloured boxes and arrows represent the common changes at a particular node of the tree and the unique changes within individual isolates are represented by black boxes/arrows. Amino acid numbers are based on the H9 amino acid sequence starting at the initiator methionine residue.(2.41 MB TIF)Click here for additional data file.Table S1H9N2 viruses isolated from chicken flocks in Pakistan.(0.06 MB DOC)Click here for additional data file.Table S2Analysis of host range and pathogenicity determinants in the PB2, PB1, PA, NP, M1 and M2 proteins in H9N2 viruses isolated from poultry in Pakistan.(0.38 MB DOC)Click here for additional data file.


We analysed chromosome 3p for loss of heterozygosity (LOH) in 48 primary oral squamous cell carcinomas (SCCs) using 15 markers and constructed a deletion map for this chromosome arm. LOH at one or more loci was found in 34/48 (71%) of tumours. The data support the existence of at least three distinct regions of deletion at 3p24-26, 3p21.3-22.1 and 3p12.1-14.2. A significant correlation was observed between the number of regions showing allele loss at 3p and tumour stage, consistent with the progressive accumulation of genetic errors during the development of oral SCC. There were also significant associations between LOH at 3p and disease-free and overall survival of patients with early stage disease. This study is the first to demonstrate the prognostic significance of LOH at 3p for oral cancer and may help to identify patients who should receive more aggressive treatment.


The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igα/Igβ heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation.


Peromyscus maniculatus) and congeneric species are the most common North American mammals. They represent an emerging system for the genetic analyses of the physiological and behavioral bases of habitat adaptation. Phylogenetic evidence suggests a much more ancient divergence of Peromyscus from laboratory mice (Mus) and rats (Rattus) than that separating latter two. Nevertheless, early karyotypic analyses of the three groups suggest Peromyscus to be exhibit greater similarities with Rattus than with Mus.Deer mice .These data suggest that: 1. the  Peromyscus constitutes the most abundant and speciose group of North American mammals. Though superficially similar in appearance to rats and mice, deer mice represent a more distantly related lineage. Mouse and rat are thought to have diverged from each other ~10–12 million years ago (mya) while they last shared a common ancestor with the deer mouse (P. maniculatus) lineage ~25 mya [P. maniculatus species complex is a series of semi-interfertile populations spanning nearly every habitat on the continent and is consequently an emerging tool for the study of natural mammalian genetic variation. Facilitating such research is the existence of captive stocks derived from individual populations. Utilizing two of these stocks, we have developed a comparative genomic map for the deer mouse to further research of this genus and to provide insight into the genome rearrangements seen in rats, mice, and other mammals.The cricetid genus Comparative genomic analyses can reveal substantial amounts of information about the biology and evolution of species and are one of the keys to deciphering the roles that genomic structure and organization play in areas such as development, gene expression, and speciation. These analyses, however, are limited to portions of the genomes that have been mapped in all the species being compared and may be compromised by uncertainty of gene orthology and order between any two species.Although whole-genome sequences are available for many species, proper genome annotation is difficult and typically requires additional resources (e.g. meiotic linkage and radiation hybrid cell maps) . As a reRattus norvegicus) and mouse (Mus musculus), which both belong to the rodent family Muridae. Rodentia is the largest mammalian order, containing > 2000 of the ~4600 recognized species and the murids constitute the majority of these [Two of the most complete mammalian genomic maps are associated with the most used biomedical models, the rat  and P. polionotus stock derived from Ocala Nat'l forest in Florida (PO). While neither population is completely inbred, both originated from a limited number of founders and have been maintained as closed colonies. Thus, identifying fixed differences between the two  was typically not difficult.We employed a standard backcross design for these studies utilizing P. maniculatus was conducted using both the Rattus and Mus maps as references [Rattus chromosomes on our backcross panels. Table Mus and Rattus genomes that are informative for this comparative analysis.Map construction for ferences ,16 and uferences . In all,Our backcross panels facilitated linkage of markers at distances ranging from 1.2 cM to 35.8 cM. There are a few instances, however, where marker co-segregation occurs. These may be due to recombination "cold-spots", segmental inversions between the BW and PO strains, or simply the interval distance may be below our mapping panel resolution.Peromyscus genome using loci from Mus Chr 11 [Rattus Chrs 10 and 14 and the resulting chromosomal breakpoint is shared with the Rattus genome relative to the Mus genome  indicating rat genome homology. However, all three markers co-segregated. At a distance of only 1.9 Mb in the Rattus genome, these markers are likely to be closer in the deer mouse than our panel is able to resolve.To explore this possibility, we expanded the existing linkage groups using loci whose orthologs lie on Rattus Chr 14 homology group using two loci from Mus Chr 5 that have orthologs on Rattus Chr 14, Afp and Hip2. Similar to both Rattus and Mus genomes, Afp and Hip2 are linked in the deer mouse at a distance of 33.6 cM (LOD = 4.4)   of Rattus Chr 14 in the deer mouse with only a few markers. Our results again show a greater similarity between Peromyscus genome organization and the Rattus genome than either to the Mus genome.Using a similar strategy, we also expanded the Rattus genome similarity that we found for these two deer mouse linkage groups warranted examination of additional informative regions where synteny breakpoints occur between the Mus and Rattus genomes. While not comprehensive for every chromosome, this strategy accelerated our examination of chromosome evolution for the deer mouse genome and helped determine the best reference genome for future deer mouse genome mapping.The high degree of Rattus Chr 1, focusing our efforts within the regions surrounding the breakpoints between Mus Chrs 7, 17, 10, and 19 . This linkage conservation is again consistent with the Rattus genome but not with Mus.We next chose markers that spanned 9 Figure . Within Mus Chr 17 and Mus Chr 7 homology segments of Rattus Chr 1 and found no linkage in Peromyscus between any Mus Chr 17 markers and the Mus Chr 7 marker Usp29, which is only 17.8 Mb away on Rattus Chr 1. We made no further efforts to extend the Mus Chr 7 linkage group, as Usp29 is only 5.93 Mb from the centromeric end of Mus Chr 7 and additional markers in this region were unlikely to yield a different result. We also tested for linkage between the Mus Chr 17 marker Mas1 and the Mus Chr 7 markers Usp29 and Myod1 on a PO × PO/BW backcross panel. This was to ensure that our negative linkage results were not a result of aberrant interspecific chromosomal recombination in the BW × BW/PO panel. Again, Mas1 did not link to either locus. The lack of linkage between Mus Chr 17 and Mus Chr 7 homology segments in the deer mouse genome constituted the first example of a common breakpoint between the deer mouse and Mus genomes when compared to Rattus.We next tested for linkage between the Mus Chr 7 section of Rattus Chr 1 using five markers: Usp29, Q8C5Y2, Ube3a, Rab6ip1, and H19. Althoughthe RIKEN cDNA marker Q8C5Y2 has not been accurately mapped in either Rattus or Mus, BLAST results indicated intervals between Q8C5Y2 and Usp29 at 26.4 Mb for the Rattus genome and 37.3 Mb in the Mus genome. In support of our placement, the Mus Chr 7/Rattus Chr 1 marker Ube3a co-segregated with Q8C5Y2 in the deer mouse. Our data for these five Mus Chr 7 markers showed high conservation of linkage and gene order with both Rattus and Mus genomes  Rattus and the deer mouse genome by markers spanning the breakpoint between the Mus Chr 7 and Chr 19 regions of Rattus Chr 1. The Mus Chr 7 marker H19 is linked to the Mus Chr 19 marker Prdx5 at a distance of 6.5 cM (LOD = 12.2). Prdx5 is also linked to a second Mus Chr 19 marker Prpf19 at a distance 4.9 cM (LOD = 13.4 cM).We also found genome homology between Rattus Chr 1 loci show that the two deer mouse linkage groups span two Mus genome breakpoints but only one Rattus genome breakpoint, which shows a continued bias towards similarity with the Rattus genome. Our results also imply that the Mus genome has been more rearranged in this region.Overall, our data for Rattus chromosomes. Rattus Chrs 4 and 6 were priority candidates because of the simple breakpoint arrangements and well-conserved gene orders between Rattus and Mus.To broaden the scope of the deer mouse map and help reduce bias resulting from any localized phenomenon such as segmental inversions, we acquired data for markers from multiple Rattus Chr 4 is represented by the entirety of Mus Chr 6 and approximately 30 Mb of the centromeric end of Mus Chr 5. To test for a conserved organization in the deer mouse genome, we typed two markers from each side of the breakpoint on our backcross panel  and the two Mus Chr 6 markers, Ccdc132 and 1700016G05Rik, are linked to each other at a distance of 7.6 cM (LOD = 14.8). Spanning the breakpoint, we found that Sri and Ccdc132 are linked to each other at a distance of 13.3 cM (LOD = 10.3). Overall, our results span about 30% of Rattus Chr 4 and show conservation of the Rattus gene order. However, the linkage between Sri and Srpk2 was shorter than expected and may be due to a recombination cold-spot, an interspecific inversion, or a deletion.Rattus Chr 6 loci consisted of ten markers: Sos1, Ppp1cb, Preb, Dnmt3a, Allc, 493504H06Rik, Pygl, Clec14a, Pcnx, and 1700001K19Rik. These markers span Mus chromosomes 17, 5, and 12 and our results yielded two separate linkage groups . Within the Mus Chr 5 segment, Ppp1cb is linked to Preb at a distance of 6.1 cM (LOD = 16.8). A second Mus breakpoint is spanned by the linkage between Preb and the Mus Chr 12 marker Dnmt3a at a distance of 5.2 cM (LOD = 16.4). Also from Mus Chr 12, Allc represents the terminal marker and links to Dnmt3a at an interval of 16.5 cM (LOD = 7.6).Our mapping of Mus Chr 12 form a second linkage group in Peromyscus. Although synteny with both Rattus Chr 6 and Mus Chr 12 is conserved, we discovered an inversion with respect to both Mus and Rattus involving markers Clec14a and Pygl  while Clecl4a is linked to the more telomeric marker Pcnx at a distance of 18.2 cM (LOD = 6.3). We also found that Clec14a and Pygl have smaller distance interval at 1.3 cM (LOD = 21.2) than would have been expected from the physical intervals in Mus (11.9 Mb) and Rattus (13.4 Mb). Forming the end of the linkage group, Pcnx was linked to 1700001K19Rik at a distance of 25.3 cM (LOD = 3.9).The five remaining markers from Mus genome breakpoints by the deer mouse linkage groups again indicates a more Rattus-like genome organization. However, the breakpoint between the two Peromyscus linkage groups flanked by the two Mus Chr 12 markers Allc and 493504H06Rik represents a unique rearrangement that differs from both the Rattus and Mus genomes. ZOO-FISH results from Mlynarski et al.  also concur that Rattus Chr 6 is indeed represented by two separate chromosomes in the deer mouse.The spanning of two Rattus genome similarity, we also selected markers to span Rattus synteny breakpoints in relation to the Mus genome. This involved markers that span approximately 89% of Mus Chr 1 but are located separately in Rattus on Chrs 5, 9, and 13  but without segregation to Oprk1 and Tram1, despite a distance 35.3 Mb in the Rattus genome between Ube2j1 and Oprk1.For the Mus Chr 1, we detected linkage between the Rattus Chr 13 markers Acbd3 and Glul at a distance of 21.2 cM (LOD = 9.6) and between Glul and Bcl2 at 35.8 cM (LOD = 3.8) . However, Col3a1 is surprisingly not linked to Col9a1, which represents a deviation from the Rattus genome. To confirm these results, we also tested Mut, a marker that is closely linked to Col9a1 on Rattus Chr 9 , thus identifying a linkage similarity between the Rattus and deer mouse genomes. The breakpoints present in the deer mouse and Mus maps for this region are offset and may represent breakpoint area re-usage and a rearrangement hotspot [Col9a1 and Col3a1 on Rattus Chr 9 are needed to refine the breakpoint location. We discovered additional Rattus Chr 9 similarity using the marker Lama, which represents a second and separate region of Mus Chr 17 than that of Mut .Amongst the 9 Figure . Mut is t Figure . We founPeromyscus genome homology to Mus, we performed an analysis using six loci from Mus Chr 8 that form two linkage groups in the Rattus genome  and Mtus1 is linked to Spata4 at a distance of 16.9 cM (LOD = 9.3). Spata4 is also linked to the terminal marker 9130404D08Rik at a distance of 7.0 cM (LOD = 16.4). The two markers from Rattus Chr 19, Elmod2 and Pmfbp1, are linked 10.3 cM (LOD = 12.5).As with the Rattus Chr 17 and Mus Chr 2, Sec61a2 links to the Rattus Chr 19/Mus Chr 8 marker Pmfbp1 at a distance of 30.1 cM (LOD = 3.7). This linkage indicates a clear deviation from both the Rattus and Mus genomes by the deer mouse genome and highlights the benefit of performing cytogenetic analyses in tandem with meiotic linkage mapping.Based on suggestions from ZOO-FISH results , we also examined an additional chromosomal segment for linkage. Representing Mus genome similarity within the deer mouse using loci from Mus Chrs 17, 5, and 13  were tested for linkage in all possible arrangements despite having already been assigned to other deer mouse linkage groups. No linkage was found other than that which already existed amongst the Rattus Chr 1 markers Tcp10b, Pacrg, and Mas1 . The strong organizational similarity of the deer mouse genome with the Rattus genome rather than the more morphologically similar Mus musculus suggests that a significant amount of rearrangement occurred in the Mus genome after the divergence of the cricetid and murid lineages. Concomitantly, our results suggest that the genomic organizations of Rattus and Peromyscus are more representative of the ancestral muroid genome than the Mus genome, which is in agreement with previous literature that indicated a higher rate of genome rearrangement for Mus [Mus genome is extraordinary with approximately 200 homology blocks. However, the Mus genome is not unique in having higher relative rearrangement rates, as the canine and gibbon genomes have approximately twice the average number of homology blocks [There are three instances, however, where the deer mouse map differs from both the y blocks .Mus and Rattus shared a common ancestor significantly more recently than either have with Peromyscus. Our data does not propose to change this phylogeny but rather merely highlights that DNA sequence variation and chromosome rearrangement are independent processes and greater understanding of both processes can provide different insights into the evolution of the structure and function of the eukaryotic genome.Our results also show that genome rearrangement can act independently from other forms of genome evolution, such as sequence mutation. Although rodent sequence mutation rates are higher compared to other mammals, such measurements of genome evolution show P. maniculatus bairdii; BW) and the old field mouse  and were obtained from the Peromyscus Genetic Stock Center at the University of South Carolina [We chose interspecific backcross analysis in order to maximize genetic polymorphism and for the ease of linkage analysis . The twoCarolina . We set Carolina .plt BW × PO) F1 animals with 12 unrelated plt BW animals to generate 116 backcross progeny. Backcrosses to BW can be performed using both female and male F1 hybrid animals, as both matings will give viable offspring. However, all but one of the matings used for this panel were F1 × BW (♀ × ♂).We created two separate backcross panels, BX116 and BX2, for the linkage analysis. For the BX116 panel, we bred twelve hybrid . Genomic DNA for the BX2 panel animals was extracted using a phenol/chloroform/isoamyl alcohol extraction method to increase yields.Il23a, Mas1, H19, Sos1, and Grb10 were developed or obtained from published Peromyscus data [Trp53 primers were developed from P. maniculatus sequence and were kindly provided by Michael McLachlan. We developed all other primers from P. maniculatus EST sequences . Deer mouse EST clones were used for marker design because of greater PCR amplification success (>80% versus ~60% for the published sets).We obtained primer sequences for some Type I markers from published sets of orthologous gene markers. These are termed UMPS, CATS, and TOASTs -33. Primcus data -36. Trp5Mus musculus genomic sequences using cross-species megaBLAST (NCBI). Some critical markers however, spanned larger introns and resulted in amplicons slightly larger than the ideal size parameters.We designed all the markers to be ~400 bp–1500 bp and to span an intron to increase polymorphism detection. This was done by aligning deer mouse DNA sequences to P. maniculatus and P. polionotus DNA using a MJ Research PTC-200 DNA Engine gradient thermal cycler and are defined as follows: 1) Standard: 95°C for 14.5 min followed by 35 cycles of , 72°C for 10 min, 4°C hold. 2) Touchdown65 (TD65): 95°C for 14.5 min followed by 20 cycles of  followed by 15 cycles of , 72°C for 10 min, 4°C hold. If no product was obtained using Touchdown65, the starting annealing temperature was changed to 60°C or 55°C, with the final annealing temperature remaining 10°C lower than the starting temperature.PCR cycling conditions for all Type I markers were optimized for 2), 200 μM each dNTP, 0.4 μM forward primer, 0.4 μM reverse primer, and 1 unit Qiagen HotStar Taq polymerase. Some difficult templates required the use of Qiagen Q-solution at either 1× or 0.5× strength. Four markers, Sparc, Xbp1, Grb10, and Ugp2 required 2.0 mM MgCl2.PCR was performed using 20 ng of genomic DNA in a 10 μl reaction containing 1 μl 10× Qiagen HotStar buffer  and incubated at 37°C for 15 minutes followed by heat-inactivation at 80°C for 15 minutes. For sequencing reactions, 2.0μL of purified PCR product was direct sequenced with BigDye (v3.1) (ABI) on an ABI 3130 × l according to the manufacturer's protocol.Mus musculus genome. Any predicted simple size polymorphisms between BW and PO were tested by gel electrophoresis using amplification products from BW, PO, and BW/PO mix  DNAs. Markers not showing size polymorphisms were further analyzed for species-specific RFLPs by sequence comparison using Sequencher software (Gene Codes Corporation), the TCAG program available as part of the Biology Workbench software utilities provided at the San Diego Supercomputer Center [Sequence identities were verified by cross-species megaBLAST or BLASTN search to the r Center , or withr Center . CandidaGbl to Hba and Hba to Canx, of which both only used 39 animals due to the small usable data set for Hba. For the BX2 panel, no fewer than 60 animals were typed between any two markers with the exception of Mut and Col9a1, for which only 40 animals could be genotyped in common.We tested the backcross panel parental mice with each marker prior to use on the backcross panel. We typed then typed the markers on all possible backcross animals whose parents exhibited the expected genotype. On the BX116 panel, no fewer than 73 animals were used for data to establish linkage between any two markers with exception of We performed backcross linkage analysis using Map Manager QTX software . A minimBLAST Basic Local Alignment Search ToolPeromyscus maniculatus bairdiiBW CATS Comparative Anchor Tag SequencescDNA complementary DNAChr chromosomecM centimorgan20 distilled deionized waterddHdNTP dinucleotide triphosphateEDTA ethylenediaminetetraacetic acid-disodium saltEST expressed sequence tagLOD logarithm of the odds (to the base 10)Mb megabasemya million years agoNEB New England BiolabsPCR polymerase chain reactionplt platinum coat-color mutationPeromyscus polionotus subgriseusPO RFLP restriction fragment length polymorphismSAP shrimp alkaline phosphataseSNP single nucleotide polymorphismSSLP simple sequence length polymorphismTBE Tris-Borate EDTATE Tris-EDTA TLE Tris-low EDTA TOAST traced orthologous amplified sequence tagsUMPS universal mammalian primer sequenceZOO-FISH cross-species fluorescence in-situ hybridizationCR was the lead researcher, conceived the study, participated in its design and coordination, developed markers and assays, participated in the design of the backcross panel, performed linkage analysis, performed molecular genetic experiments, performed sequence alignments, and drafted the manuscript. AL developed markers and assays, performed molecular genetic experiments, and performed sequence alignments. JG developed markers and assays, participated in the design of the backcross panels, performed molecular genetic experiments, performed sequence alignments, and edited the manuscript. PV developed markers and assays, performed linkage analyses, participated in the design of the study, and contributed to and edited the manuscript. RO participated in the design and coordination of the study and helped to draft the manuscript. MD was the principle investigator on the project, participated in its design and coordination, performed sequence alignments, participated in the design of the backcross panels, and contributed in drafting the manuscript and figures. All authors read and approved the final manuscript.


Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII. Dengue viruses are mosquito-borne flaviviruses and the agents of dengue fever and dengue hemorrhagic fever. It has been widely assumed that antibodies that neutralize dengue bind to regions on the viral envelope (E) protein that are conserved within each serotype. However, few studies have explored how natural variation influences dengue-antibody interactions. Mouse antibodies that strongly neutralize dengue bind to a region on domain III of E protein. This region has been the focus of much recent work because it might be the target of protective human antibodies as well. We compared a large number of E protein sequences and discovered that the region on E protein domain III targeted by neutralizing antibodies was highly variable between strains of dengue serotype 3. Using a panel of antibodies, we experimentally demonstrate that natural strain variation in dengue serotype 3 has a strong influence on antibody binding and neutralization. Our results challenge the dogma that neutralizing antibody binding regions are conserved within each serotype. The results of this study are relevant to the current global effort to develop and evaluate dengue vaccines. Dengue viruses (DENVs) are mosquito-borne flaviviruses and the agents of dengue fever and dengue hemorrhagic fever (DHF). According to the World Health Organization, over 2.5 billion people are at risk of contracting dengue, 100 million people develop symptomatic infections and up to 50,000 die from DHF each year. The DENV complex consists of 4 serotypes (DENV1-DENV4). DENVs have antibody epitopes that are unique to each serotype and epitopes that are cross reactive between serotypes. People who have recovered from primary DENV infections develop long term, protective immune responses against the homologous serotype only. In fact, individuals exposed to a second infection with a different serotype face a greater risk of developing DHF indicating that pre-existing immunity can exacerbate disease under some conditions As previously infected individuals do not appear to be re-infected with the same serotype, it is widely assumed that neutralizing antibody epitopes are conserved among strains belonging to the same serotype Antibodies, in particular, have emerged as key effector molecules responsible for protective and pathogenic immune responses to DENV In the present study, we have examined the phylogenetic relatedness of the E protein sequences from a large number of viruses representing the different genotypes of DENV3. Many surface exposed amino acids were variable between established genotypes of DENV3. Especially noteworthy was the observation that the EDIII lateral ridge, which is a known site targeted by neutralizing MAbs in related flaviviruses, was variable between DENV3 genotypes. We experimentally demonstrate that naturally occurring amino acid differences in DENV3 EDIII lead to differential binding and neutralization by MAbs.Aedes albopictus C6/36 cells were maintained at 28C in MEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U/ml) and streptomycin (100 µg/ml) in the presence of 5% CO2. Human leukemic monocyte lymphoma cell line U937 expressing DC-SIGN (U937 DC-SIGN) were maintained at 37C in RPMI (Gibco) supplemented with 10% FBS, 50 mM beta mercaptoethanol, penicillin (100 U/ml) and streptomycin (100 µg/ml) in the presence of 5% CO2. All media were also supplemented with 0.1 mM non-essential amino acids (Gibco) and 2 mM glutamine (Gibco).2. Supernatants were harvested, clarified by centrifugation and, supplemented with 15% FBS and stored in aliquots at −80C. Viral titers were determined by plaque assay on Vero-81 cells as previously described 5 PFU/ml were used in experiments. The reference virus strains used in the study were strains West Pacific 74 (DENV 1), S16803 (DENV2), CH53489 (DENV3) and TVP-360 (DENV4) routinely used in the DENV neutralization test. These viruses were obtained from Robert Putnak  and they have been passaged >10 times in different mammalian  and insect  cell lines. For studies on different genotypes of DENV3, we also used UNC3043 , UNC 3009 , and UNC3066 (strain 1342 from Puerto Rico 1977). These viruses were obtained from Dr. Duane Gubler and Claire Wong at CDC, Fort Collins, CO. These viruses had been passaged 3 times in Aedes albopictus C6/36 cells prior to being used in these studies.Working virus stocks were obtained by inoculating C6/36 mosquito cells in MEM (Gibco) supplemented with 2% FBS (Gibco), penicillin (100 U/ml) (Gibco) and streptomycin (100 µg/ml) (Gibco) 0.1 mM non-essential amino acids (Gibco) and 2 mM glutamine (Gibco) and growing the virus for eight days at 28C under 5% COMAbs 8A1 (IgG1) and 14A4 (IgG1) against DENV3 were provided by Robert Putnak . MAb1H9 (IgM) was provided by John Aaskov  g for 5 h) to pellet the virus. The virus pellet was allowed to dissolve overnight in PBS before layering on a 10%–40% iodixanol gradient and being centrifuged at 163,700×g for 120 min. The virus-containing fractions were harvested. PBS was added to the virus to dilute the iodixanol. The diluted solution was centrifuged  to pellet the virus and remove the iodixanol. The virus pellet was resuspended in PBS and virus protein content was estimated by spectrophotometry. The virus was stored at −80°C.Vero-81 cells were inoculated with UNC 3043 (DENV3 -genotype I), CH53489 (DENV3 - genotype II), UNC 3009 (DENV3 - genotype III), or UNC3066 (DENV3 -genotype IV) at an MOI of 0.1. The virus-containing media was harvested 5–7 days after infection and centrifuged to pellet cell debris. The clarified media was laid on top of a 20% sucrose (wt/vol) cushion and centrifuged  and purified using amylose resin affinity chromatography (NEB) according to the manufacturer's instructions.Recombinant EDIII constructs were created using cDNA from the following virus strains to represent each serotype of DENV and genotypes of DENV3: West Pacific 74 (DENV 1), S16803 (DENV2), UNC 3043 (DENV3 -genotype I), CH53489 (DENV3 - genotype II), UNC 3009 (DENV3 - genotype III), UNC3066 (DENV3 -genotype IV), and TVP-360 (DENV4). Envelope gene fragments encoding EDIII from DENV1, DENV3, and DENV 4 (AA295–398) and DENV2 (AA297–399) were amplified using Vent polymerase . Reverse primers used in the study were designed to introduce either Hind III (for DENV2–4) or PstI (for DENV1) restriction site at the 3′ ends of the PCR products. PCR products were digested with HindIII or PstI and cloned into pMAL c2X vector (NEB) to generate recombinant EDIII that is fused to maltose binding protein (MBP-EDIII) at the N terminus according to the manufacturer's instructions. MBP-EDIII were expressed in www.stratagene.com) and PCR was conducted according to manufacturer's instruction. Single stranded pMal c2X plasmids  encoding MBP-EDIII fusion proteins with amino acid substitutions were cloned into DH5α cells for expression and purification of mutant rEDIIIs. Substitution of amino acids in all mutant constructs was confirmed by sequencing. Expression and purification of mutant rEDIII proteins were essentially same as mentioned in the earlier section.Selected amino acids residues on rEDIII were mutated by site directed mutagenesis using Quickchange multi kit . When selecting sites to mutate, we gave precedence to positions on loops and (not beta sheets) because we did not want to disrupt the overall folding of EDIII. Thus, of the 20 positions we mutated, 16  are located on loops that form the lateral ridge neutralizing epitope recognized by type specific neutralizing MAbs ELISA plates were coated by adding 200 ng/well of purified EDIII-MBP protein or 75 ng/well of purified DENV antigen in Carbonate buffer (pH 9.0) and incubating the plates overnight at 4C. Rabbit anti MBP sera (New England Biolabs) was used to quantify binding of MBP-EDIII to plates. The flavivirus cross reactive MAb 4G2 was used to quantify binding of virus to plates. Two hundred nanograms of EDIII-MBP saturated binding to the ELISA plate and we did not observe appreciable differences in binding between different EDIII proteins created for this study. Similarly, 75 ng saturated virus binding to the plate and we did not observe appreciable differences in binding between different viruses used in the current study.The plates were washed with Tris buffered saline with 0.2% Tween 20 (TBST wash buffer) and blocked with 3% normal goat serum (NGS) in Tris buffered saline with 0.05% Tween 20 (TBST blocking buffer) for 1 hour at 37C. Serially diluted MAbs in TBST blocking buffer were then added to each well and incubated for one hour at 37C. After washing 3 times with TBST wash buffer, the plates were incubated for one hour at 37C with alkaline phosphatase conjugated goat anti-mouse antibody (Sigma). Plates were washed 3 times with TBST wash buffer and developed by adding p-nitrophenyl phosphate substrate (Sigma). Optical density (OD) was measured at 405 nm using a spectrophotometer.et. al., was used with modifications to determine 50% neutralization values for each antibody 7 genome equivalent copies) from each DENV3 genotype was used to infect cells in experiments comparing the neutralization activity of MAbs among the DENV genotypes. Different concentrations of MAbs were mixed with each virus strain in a 96 well tissue culture plate and incubated for one hour at 37C in the presence of 5% CO2. U937DC-SIGN cells (5×104 cells in 100 µl) were introduced to each well and incubated for an additional 2 hours at 37C to allow virus to bind to cells. The cells were then washed with media and 200 µl of fresh media was added to each well and incubated for 24–72 hr at 37C with 5% CO2. After washing 2 times with PBS, the cells were fixed and permeabilized using CytoFix/Cytoperm kit (BD bioscience). The cells were then stained with Alexa 488 conjugated anti dengue MAb 2H2 and the percentage of infected cells was measured in a flow cytometer. EC50 values were calculated using GraphPad Prism version 4.00 for Windows  and non linear regression analysis.The flow cytometry based neutralization protocol as described by Kraus, http://www.homepage.mac.com/barryghall/Software.html) and the PAM series matrix was determined to be the most appropriate, with default gap opening and extension values. The alignment was used to identify 32 variable/informative sites that were defined as columns of heterogeneity in the alignment where the same amino acid change occurred in at least three independent sequences. To display the 32 informative sites  that bind to E protein have been mapped to six regions  [10],,[18To directly address if natural amino acid variation in DENV3 EDIII results in altered antibody binding and neutralization, we assembled and mapped a panel of DENV3 EDIII reactive mouse MAbs. All the antibodies selected for this study  bound to EDIII of DENV3 . MAbs 8ATo map the binding sites of the MAbs, we expressed and purified 28 EDIII recombinant proteins with defined mutations. The positions to mutate were selected based on antibody mapping studies done with other flaviviruses MAb 8A1 is a strongly neutralizing, type specific DENV3, EDIII reactive IgG antibody. With this antibody, we observed a greater than 80% loss of binding when amino acids at 301, 302, 304, 326–328, 330, 361, 380, 382 and 386 were mutated on EDIII . As in tMAb 14A4 is a neutralizing EDIII reactive IgG antibody that cross-reacts with DENV3 and DENV1 . This DEThe DENV complex cross reactive MAbs  bound to all the mutant proteins indicating these antibodies likely bind to a cross reactive epitope outside the lateral ridge region . In FiguOne concern with the above mapping studies was that some mutations might disrupt the overall folding of EDIII and non-specifically reduce antibody binding. To address this concern, we performed binding studies with a well characterized DENV subcomplex specific MAb 1A1D2, which binds to and neutralizes DENV1, 2 and 3 but not 4 Several amino acid positions  on EDIII implicated in binding to MAbs 8A1 and 1H9  were alsTo verify that amino acid differences at the EDIII lateral ridge were responsible for MAb binding differences, further studies were conducted with MAb 8A1 and recombinant EDIII proteins. We systematically changed amino acids in the EDIII genotype IV construct to genotype II and defined the minimum number of changes required to restore the 8A1 epitope. In Experiments were conducted to compare the ability of EDIII MAbs to neutralize different genotypes of DENV3. MAbs 8A1 and 1H9 failed to neutralize DENV3 genotype IV , which wA long-held paradigm in flavivirus research has been that DENVs display little if any within intra-serotypic antigenic variation and this has been the basis for the development of current multivalent vaccines and immunotherapeutics Our results show that EDIII lateral ridge antibodies 8A1 and 1H9 bound to DENV3 genotypes I, II and III but not genotype IV indicating that naturally occurring mutations in EDIII can lead to a total loss of MAb binding. Even though MAbs 8A1 and 1H9 bound to DENV3 genotypes I, II and III with similar apparent affinity, we observed striking differences in the ability of the MAbs to neutralize these viruses. The neutralization titers were almost 10 fold different between viruses for 8A1 and 60 fold different for 1H9. Our results indicate that apparent affinity of antibody to virus immobilized on ELISA wells is not always predictive of the neutralization titer. There are amino acid differences on the EDIII lateral ridge of genotype I, II and III viruses  and thesOne potential problem with our studies is the possibility that some of the recombinant EDIII proteins used in the current study might be grossly misfolded and the binding differences might not be due to direct interactions between antibody and the altered amino acid. NMR and antibody binding assays have established that wild type EDIII expressed as an MBP fusion protein is properly folded Several groups have focused on developing DENV vaccines based on recombinant EDIII Recently we reported that people exposed to natural DENV infections have low levels of EDIII reactive antibody several years after recovery from infection Figure S1Phylogenetic tree of 175 DENV3 E protein sequences used to identify informative sites. A phylogenetic tree was generated using Bayesian inference to analyze the evolutionary relationship of 175 unique DENV3 envelope protein amino acid sequences that were available from Gen Bank at the time this study was initiated. The four known genotypes of DENV3 are indicated. The numeric values at the nodes represent Bayesian posterior probabilities and the distance scale bar represents 0.01 changes per site.(0.15 MB PDF)Click here for additional data file.Table S1Binding of mouse MAbs 1A1-D2 to mutant DENV3 EDIII proteins(0.01 MB PDF)Click here for additional data file.


From September 1995 to July 1996 50 patients were treated for carpal tunnel syndromeas outpatients by endoscopic release in the rooms of an orthopaedic surgeon .The average age was 51.3 years (27–61 years). The average length ofsymptoms was 43 months, the postoperative time off work averaged 27 days. Six monthspostoperatively wasting of the thenar persisted in 2 out of 16 patients, a positive Tinel'ssign in 1 out of 46 patients and delayed median nerve conduction in 2 out of 48presenting these symptoms preoperatively. At 6 months the average handgrip strengthhad recovered to 109% of the preoperative value. One out of 49 patients still presentedparesthesia and 1 out of 50 nocturnal dysesthesia. There were minor complications in 7patients (14%), only one patient requires further treatment. We conclude that endoscopiccarpal tunnel release done on outpatients in a private surgery can be reliable, safeand cost efficient.


Using mathematical modeling of population age-incidence data, we estimate that 1 (95% confidence limits 0 and 3) of these families is expected to meet the Amsterdam criteria I for HNPCC due to chance clustering of colorectal cancer. © 2001 Cancer Research Campaign


The inverse relationship between GLUT4 and RBP4 expression is known to play a role in the pathogenesis of type 2 diabetes. Elevated levels of RBP4 were shown to cause insulin resistance in muscles and liver. Identification of STRA6 as a cell surface receptor for RBP4 provides further link in this axis and hence we analyzed SNPs in these three genes for association with type 2 diabetes in a South Indian population.STRA6 with type 2 diabetes . None of the SNPs in RBP4 and GLUT4 showed any association with type 2 diabetes. Haplotype analysis revealed that two common haplotypes H1  and H2  in STRA6, H6  in RBP4 and H4  in GLUT4 were associated with type 2 diabetes.Selected SNPs in the three genes were analyzed in a total of 2002 individuals belonging to Dravidian ethnicity, South India, by Tetra Primer ARMS PCR or RFLP PCR. Allele frequencies and genotype distribution were calculated in cases and controls and were analyzed for association by Chi-squared test and Logistic regression. Haplotype analysis was carried out for each gene by including all the markers in a single block. We observed a significant association of three SNPs, rs974456, rs736118, and rs4886578 in STRA6, gene coding the cell surface receptor for RBP4, were significantly associated with type 2 diabetes and further genetic and functional studies are required to understand and ascertain its role in the manifestation of type 2 diabetes.SNPs in  Type 2 diabetes is primarily characterized by insulin resistance, relative insulin deficiency and fasting hyperglycemia GLUT4 and RBP4 but with varied results GLUT4 with type 2 diabetes is lacking. SNPs in both RBP4 and GLUT4 have not been analyzed in Indian population for their association with type 2 diabetes. With respect to GLUT4, a study in Welsh population identified a silent polymorphism (Asn130) in exon-4 present in diabetic subjects. They also identified a Val383Ile mutation present in 3 out of 160 type 2 diabetes subjects but in none of the control subjects. But further analysis in British population failed to confirm any association of these mutations with type 2 diabetes GLUT4 include RFLP studies with KpnI enzyme in Italian, Caucasians, Chinese, and Asian Indians, American black and Japanese population GLUT4. Recent studies in other populations have identified common variants and haplotypes in RBP4 to be associated with type 2 diabetes RBP4 to be associated with altered serum lipid levels . Interestingly, none of these studies looked at polymorphisms in STRA6; the only identified high affinity receptor for RBP4.Genetic studies have been carried out with respect to single nucleotide polymorphisms (SNPs) in GLUT4-RBP4-STRA6 may play an important role in the pathogenesis of type 2 diabetes, wherein the adipocyte specific down regulation of GLUT4 leads to an increased expression of RBP4 and this increase in RBP4 expression leads to insulin resistance via STRA6. In this study, SNPs in GLUT4, RBP4 and STRA6 were analyzed for association with type 2 diabetes in a South Indian population. We report for the first time that genetic variants in STRA6 are significantly associated with type 2 diabetes.The triad of The study was conducted by using DNA isolated from blood samples of 2002 unrelated individuals (1002 cases with type 2 diabetes and 1000 normoglycemic control subjects) belonging to Dravidian ethnicity from Kerala, South India. Diabetes related clinical and biochemical parameters were collected for all the case samples whereas fasting blood glucose, BMI and blood pressure were documented for control samples. Clinical and biochemical features of the study population are summarized in STRA6) were in Hardy-Weinberg proportions and there was no deviation from Hardy-Weinberg equilibrium. rs351224 was subsequently omitted from further analysis. dbSNP ID, Minor allele frequency (MAF) and HWE P value for all the selected SNPs are summarized in STRA6, rs736118 and rs4886578 spanned one LD block (D' = 0.95), rs974456 was not in LD with the two SNPs and rs351224 was omitted from the analysis due to deviation from HWE. Kovacs et al have previously defined the LD pattern for RBP4 for Caucasian population GLUT4, the 4 SNPs spanned two different LD blocks with SNPs rs2654185 and rs5412 in one LD block and SNPs rs5418 and rs5435 in the second LD block.Test for Hardy-Weinberg equilibrium suggested that the genotypes for all the SNPs except rs351224 . Significant P values were also obtained in the additive model analysis after adjustment for age, sex and BMI. The associations remain statistically significant even after correction for multiple testing by Bonferroni correction . As revealed by ODD's Ratio (OR), all three SNPs in STRA6 tend to be associated with a decreased susceptibility for type 2 diabetes. None of the selected SNPs in GLUT4 or RBP4 showed any significant association with type 2 diabetes. A silent polymorphism in GLUT4, rs5435, showed a slight trend towards association (P = 0.05). Results of association analysis are summarized in All the three SNPs in STRA6, rs351224 was not included in the haplotype analysis because of significant deviation from Hardy-Weinberg proportions  and H2  were significantly associated with type 2 diabetes (P = 0.001 and P = 0.002 respectively) even after correction for multiple testing (P = 0.005 and P = 0.01)  in the order rs3758538, rs3758539, rs36014035 and rs34571439 is significantly associated (P = 0.006) with type 2 diabetes (P = 0.036). When we omitted rs3758538 from the haplotype analysis due to low minor allele frequency, we found that the haplotype (121) was significantly associated with type 2 diabetes with a P = 0.002 (data not shown). We also analyzed the haplotypes in GLUT4 for association with type 2 diabetes. 87.7% of the total haplotypes were constituted by four common haplotypes with a frequency >5% in case samples. Case-control analysis revealed that the haplotype H4  defined by SNPs rs2654185, rs5412, rs5418 and rs5435 is marginally associated with type 2 diabetes after correction for multiple testing (P/P* = 0.01/0.04). Results of haplotype analysis with SNPs in GLUT4 are summarized in In case of portions . Therefo = 0.01) . The hapdiabetes . This asRBP4 with serum lipid levels STRA6 also did not reveal any significant association with type 2 diabetes related phenotypes (data not shown). We also did not find any evidence of multiplicative gene-gene interaction in modulating BMI, fasting blood glucose, HBA1C and age of onset of diabetes though a higher proportion of individuals with diabetes had higher number of risk alleles (STRA6 (rs736118), RBP4 (rs3758539) and GLUT4 (rs5435).Recent studies in other populations have reported a significant association of SNPs in  alleles . PercentGLUT4, RBP4 and STRA6 for association with type 2 diabetes. We provide evidence that SNPs in STRA6 are significantly associated with type 2 diabetes. We also identified two common haplotypes in STRA6 associated with type 2 diabetes. Furthermore, common haplotypes in GLUT4 and RBP4 were also found to be associated with type 2 diabetes. After a case-control analysis in 1002 type 2 diabetes samples and 1000 control samples, we found that the three SNPs in STRA6, rs736118 (P = 0.003), rs4886578 (P = 0.001) and rs974456 (P = 0.001) were significantly associated with type 2 diabetes. rs736118 is a G>A polymorphism leading to a Met to Ile change in the C-terminal end of STRA6. Though Met and Ile are hydrophobic amino acids, evidence from literature suggests that such changes can alter the trafficking and cell surface expression of proteins. For example, a V302M mutation in the cytoplasmic G loop of Kir6.2 (KCNJ2) led to a loss of coassembly with the wild type protein thereby affecting protein trafficking to the cell surface In this study, we used a case-control approach for analyzing SNPs in three candidate genes viz. TCF7L2, the most strongly associated and replicated type 2 diabetes susceptibility loci, is an integral part of Wnt-1 signaling rs4886578 (G>A), is an exon-intron boundary polymorphism whereas rs974456 (C>T) is present in intron 7. It has been shown that SNPs present in the non-coding region can modulate gene expression and SNPs present in exon-intron boundary can alter splice forms RBP4 and GLUT4 for association with type 2 diabetes and related parameters. SNPs in RBP4 have been previously reported to be associated with type 2 diabetes in Mongolian population RBP4 associated with type 2 diabetes. Another study which analyzed SNPs in the RBP4 for association with type 2 diabetes identified a common haplotype associated with type 2 diabetes but failed to observe any single SNP association RBP4 but our haplotype analysis revealed that a common haplotype  is significantly associated with type 2 diabetes (P = 0.004). Kovacs et al. identified significant association of the common haplotype AGGTGC  with type 2 diabetes RBP4 have reported association with serum lipid parameters but in our study we failed to observe any significant association of the promoter polymorphisms with serum lipid levels or with other diabetes related parameters RBP4 and STRA6 with diabetic retinopathy in a case control analysis using 155 samples with diabetic retinopathy and 150 samples with type 2 diabetes for more than 12 years with no symptoms of diabetic retinopathy.We also analyzed SNPs in GLUT4 is a strong candidate gene for diabetes related studies, we did not find any evidence of individual SNPs in GLUT4 associated with type 2 diabetes in our population. Haplotype analysis with selected SNPs revealed a common haplotype 2121 defined by the order rs2654185, rs5412, rs5418 and rs5435 marginally associated with type 2 diabetes. We also found that the SNP rs5435 showed an increasing trend towards association with considerable difference in minor allele frequency in cases and controls. Probably this SNP in GLUT4 may increase the susceptibility to type 2 diabetes in combination with other SNPs. It should be noted that the sample size used in this analysis is suitable for stronger effects as found in STRA6 but smaller effects like those observed in GLUT4 (rs5435) may be missed and analysis of a larger sample size may confirm such effects. Our gene-gene interaction data reveals that when the sentinel SNPs in STRA6, RBP4 and GLUT4 are combined together they increase the prevalence of type 2 diabetes with increase in the number of risk alleles  as novel type 2 diabetes susceptibility genes not observed in Japanese and Western population Recent Genome Wide Association Studies (GWAS) in different populations have identified various type 2 susceptibility genes GWAS data for type 2 diabetes in South Indian population is still not available. India has the highest number of cases with type 2 diabetes and is referred as the diabetic capital of the world with 58.4 million people with type 2 diabetes. Indian population differs from other population in certain unique biochemical and clinical parameters like increased insulin resistance, higher adiposity, lower adiponectin levels and higher levels of sensitive C-reactive protein GLUT4, RBP4 and STRA6 for association with type 2 diabetes. We provide evidence supporting a novel genetic role for the STRA6 loci in type 2 diabetes. A significant association was observed between two common haplotypes in STRA6 and type 2 diabetes. Additionally haplotype analysis with SNPs in RBP4 and GLUT4 also revealed significant association of a common haplotype in both genes with type 2 diabetes.In conclusion, we analyzed SNPs in three candidate genes namely, Written informed consent forms were obtained from all the participating subjects. The study was conducted following the guidelines of Indian Council of Medical Research and approved by the ethics committee at AIMS.The study was conducted using samples from 2002 unrelated individuals from Kerala, South India as a part of an ongoing project, Amrita Diabetes Awareness and Welfare Study. Case samples were collected from Endocrinology department, Amrita Institute of Medical Sciences (AIMS), Kochi, Kerala. All samples were collected following the guidelines of American Diabetes Association with fasting blood glucose >120 mg/dl and/or 2-hour plasma glucose >200 mg/dl. Case samples were collected after detailed investigation of medical records for symptoms, use of oral hypoglycemic agent and/or insulin, measurement of fasting blood glucose and other related anthropometric parameters. Subjects diagnosed with type 2 diabetes after the age of 60 or subjects who were started on insulin therapy within one year of diagnosis were excluded from the study. Subjects with type 1 diabetes, family history of type 1 diabetes, maturity onset diabetes of the young, monogenic forms of diabetes, or drug induced diabetes were also excluded from the study. Samples from patients outside Kerala and patients migrating from other parts of the country were not included. Case reports of diabetic nephropathy, diabetic retinopathy and fatty liver disease as diagnosed by the physician were also documented during collection.Age, sex and ethnicity matched normoglycemic control subjects were recruited in the study by public advertisement and increased awareness by offering screening for diabetic risk factors at specially organized medical camps. All the participants of these medical camps were subjected to a health questionnaire for detailed investigation of their disease status, family history, socio-economic status, food habits, smoking and alcohol status, BMI and blood pressure (BP). The inclusion criteria for the healthy controls were: a) above 40 years of age, b) no family history of diabetes, c) not taking any oral hypoglycemic agent or insulin, d) blood glucose levels <110 mg/dl. Clinical characterization of the study subjects is summarized in the STRA6, RBP4 and GLUT4 were selected from dbSNP, NCBI, either based on their position in the gene viz. 5′ UTR, exon, exon-intron boundary, 3′UTR and minor allele frequency  reported in dbSNP or based on previous association studies. All the novel SNPs were selected based on their position to influence the expression or function of the protein. rs5435 in GLUT4 has been previously studied in European population. In addition, we selected three more SNPs in GLUT4 based on position and minor allele frequency as reported in dbSNP, NCBI. For RBP4, we selected SNPs based on previous studies in European population whereas for STRA6, SNPs were selected based on their reported population diversity data and position in the gene. There is only one reported non- synonymous SNP, rs736118, in STRA6 with population diversity data and a MAF >0.05 in Asian population so only this SNP from the coding region of STRA6 was included in the study. Other SNPs selected from STRA6 include rs4886578 present in the intron-exon boundary, rs974456 and rs351224 present in the intron. All SNPs were genotyped by tetraprimer ARMS PCR and RFLP PCR. Genotyping success rate was generally >96% except for rs5418 in GLUT4 where the success rate was >87%. A summary of selected SNPs is given in STRA6), rs974456 (STRA6) and rs5418 (GLUT4) were genotyped by Tetraprimer ARMS PCR. Genotyping plates which showed amplification in negative control (without template DNA) were repeated. All ambiguous genotypes were also repeated in duplicates till clear genotypes were available. Approximately 10% (n = 194) samples were re-genotyped for cross validation and the genotype success rate was >99.5%. rs736118, rs974456 and rs5418 were genotyped by RFLP-PCR using the restriction enzymes Nco1, Xho1 and BamH1 respectively. In case of rs736118 (Nco1) and rs974456 (Xho1) the presence of minor allele resulted in the lack of restriction enzyme site and hence there was no change in the PCR amplicon after restriction digestion. So to ensure the genotyping accuracy all samples homozygous for the minor allele were selectively re-genotyped. Randomly selected 10% samples were genotyped for these SNPs also and the genotyping accuracy was found to be >99.5%. All DNA amplicons and fragments were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining in gel documentation system, Biorad, USA. PCR primer designs and reaction conditions are available on request.Genomic DNA was isolated from peripheral blood leukocytes by salting out procedure http://faculty.vassar.edu/lowry/VassarStats.html) and multivariate regression analysis after adjustment for age, sex and BMI. Correction for multiple testing was done by Bonferroni's inequality method wherever applicable and was defined as P value (single tests) × number of tests. Bonferroni correction was not done for multiple traits due to lack of any significant association. Multiallelic analysis with sentinel SNPs in STRA6 (rs736118), RBP4 (rs3758539) and GLUT4 (rs5435) was done by ANOVA after categorizing the participants into groups according to the number of risk alleles they carried. All association analysis was performed using the software SNP and Variation Suite Ver-7 from Golden Helix, Inc.Hardy Weinberg equilibrium (HWE) test was done for each SNP by Pearson's Goodness of fit Chi square test before further analysis. Association analysis of each SNP with type 2 diabetes was done by Chi-square test, for difference in allele frequency between cases and controls. Association of SNPs with type 2 diabetes was also confirmed by logistic regression analysis after adjustment for age, sex and BMI. An additive model was used for logistic regression where homozygotes for the major allele (1_1), heterozygotes (1_2) and homozygotes for the minor allele (2_2) were coded to a continuous numeric variable for the genotype as 0, 1 and 2 respectively. Linkage disequilibrium was estimated for combined data of cases and controls using Haploview version 4.1. Haplotype analysis was done by using the software SNP and Variation Suite Ver-7 from Golden Helix, Inc. Haplotype analysis was done by using all the markers in a gene as a single block. Quantitative traits were analyzed for association with type 2 diabetes by ANCOVA (available at


Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB4 as well as LTC4 secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB4 levels in synovial fluid were significantly increased in patients with Lyme arthritis  when compared to the control subjects with NIA  (p < 0.05). No statistical difference of urinary LTE4 levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB4 production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB4 in synovial fluid of patients with Lyme arthritis underline the involvement of LTB4 in the pathogenesis of this disease.The purpose of this study was to evaluate the potential role of LTB


We used the nationwide Swedish Family-Cancer Database with 2060 childhood brain tumours diagnosed in the period 1958–1996 to analyse the risk of this tumour by parental cancers and in siblings of childhood brain tumour probands. Groups of patients were compared by calculating standardized incidence ratios (SIRs) for brain tumours in offspring. 1.3% of brain tumour patients had a parent with nervous system cancer; SIRs were 2.4 and 1.88 for diagnostic ages < 5 and < 15 years, respectively. The data showed distinct patterns of familial risks for childhood brain tumours, the SIR was 10.26 for brain astrocytoma given a parent with meningioma. Parental colon cancer was associated with offspring ependymoma (SIR 3.70), and parental salivary gland cancers with offspring medulloblastoma . SIR for sibling nervous system cancer from childhood brain tumour probands was 3.55 up to age 61. © 2000 Cancer Research Campaign


In 96 ovarian cancer patients, the present study investigates the clinical significance of pretreatment concentrations of soluble CD44 standard (CD44s) and its isoforms v5 and v6 determined in the serum and the ascitic fluid by means of recently developed enzyme-linked immunosorbent assays (ELISAs). Furthermore, CD44 serum concentrations in the ovarian cancer patients were compared with circulating CD44 levels in 50 healthy age-matched female blood donors. Whereas CD44s was found to be higher and CD44v5 to be lower in ovarian cancer patients than healthy control subjects, no statistical difference between the two cohorts was revealed for CD44 isoform v6. In the ascitic fluid samples, variant isoform v5 and v6 were demonstrated at lower concentrations than serum. Multivariate analysis of overall survival demonstrated that a high pretreatment serum level of soluble CD44 isoform v5 is independently associated with favourable clinical outcome in ovarian cancer. When circulating CD44 isoforms were compared with a panel of serum parameters known to be involved in the immunological network, an inverse correlation between serum CD44v5 levels and indicators of cellular immune system activation, such as soluble interleukin 2 receptor, immunostimulatory protein 90K and neopterin, became apparent.


Sir,Vulval intraepithelial neoplasia (VIN) is resulted from persistent human papillomavirus (HPV) infection, especially infection of HPV type 16 (HPV16) . SeveralBritish Journal of Cancer that Imiquimod topical application followed by HPV therapeutic vaccine led to the complete regression of VIN in 63% of patients recruited in a Phase II clinical trial at 52 weeks after the treatment. There was significantly increased local infiltration of CD8 and CD4 T cells in responders; whereas non-responders showed an increased density of T regulatory cells in the lesions (α and IFNγ, but also promotes the secretion of IL10, an inhibitory cytokine that damps immune responses (γ, and these cells are most likely responsible for the ineffectiveness of Imiquimod therapy. The IL10 secreting T regulatory cells can be amplified by the subsequent vaccination (Activation of TLRs not only induces inflammatory cytokines, such as TNFesponses . TopicalIL10 polymorphism influences IL12 secretion by dendritic cells in response to the TLRs stimulation (


Previous molecular cytogenetic studies by comparative genomic hybridization (CGH) on primary tumours of human malignant mesothelioma have revealed that loss of genetic material at chromosome 14q is one of the most frequently occurring aberrations. Here we further verify the frequency and pattern of deletions at 14q in mesothelioma. A high-resolution deletion mapping analysis of 23 microsatellite markers was performed on 18 primary mesothelioma tumours. Eight of these had previously been analysed by CGH. Loss of heterozygosity or allelic imbalance with at least one marker was detected in ten of 18 tumours (56%). Partial deletions of varying lengths were more common than loss of all informative markers, which occurred in only one tumour. The highest number of tumours with deletions at a specific marker was detected at 14q11.1–q12 with markers D14S283 (five tumours), D14S972 (seven tumours) and D14S64 (five tumours) and at 14q23–q24 with markers D14S258 (five tumours), D14S77 (five tumours) and D14S284 (six tumours). We conclude from these data that genomic deletions at 14q are more common than previously reported in mesothelioma. Furthermore, confirmation of previous CGH results was obtained in all tumours but one. This tumour showed deletions by allelotyping, but did not show any DNA copy number change at 14q by CGH. Although the number of tumours allelotyped was small and the deletion pattern was complex, 14q11.1–q12 and 14q23–q24 were found to be the most involved regions in deletions. These regions provide a good basis for further molecular analyses and may highlight chromosomal locations of tumour suppressor genes that could be important in the tumorigenesis of malignant mesothelioma. © 1999 Cancer Research Campaign


Little is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection.Using H5N1 microneutralisation (MN) and H5-pseudotype particle-based microneutralisation assays (H5pp) we analyzed sera sequentially obtained from 11 severely ill patients diagnosed by RT-PCR (follow-up range 1–139 weeks of disease onset) and 31 asymptomatically infected individuals detected in a sero-epidemiological study after exposure to H5N1 virus (follow-up range: 1–2 month –11 months after exposure).Of 44 sera from 11 patients with H5N1 disease, 70% tested positive by MN (antibody titre ≥80) after 2 weeks and 100% were positive by 3 weeks after disease onset. The geometric mean MN titers in severely ill patients were 540 at 1–2 months and 173 at 10–12 months and thus were higher than the titers from asymptomatic individuals . Fractional polynomial regression analysis demonstrated that in all severely ill patients, positive titers persisted beyond 2 years of disease onset, while 10 of 23 sera collected 10–11 months after exposure in asymptomatically infected individuals tested negative.Our results indicate that people with asymptomatic H5N1 infection have lower H5N1 antibody titres compared to those with severe illness and that in many asymptomatically infected patients the antibody titer decreased to levels below the threshold of positivity within one year. These data are essential for the design and interpretation of sero-epidemiological studies. Since 1997, the highly pathogenic avian influenza A (H5N1) virus has spread among poultry and possibly also in wild birds in Asia, Middle-East, Europe and Africa and caused over 470 cases of reported human diseases with more than 280 deaths Human sera were collected at the Hospital for Tropical Disease (HTD) Ho Chi Minh City, Vietnam, from patients with severe H5N1 virus infection confirmed by RT-PCR 5 RLU (“relative” luminescent unit) of H5pp (quantity defined after optimization 2 incubator). Subsequently, 100 µL of fresh complete medium was added to the virus-antibody mix and 140 µL of the virus-antibody mix was transferred back to the cells after the old cell medium was discarded. After 48h incubation at 37°C (5% CO2 incubator), 100 µL of Steady-Glo (Promega) luciferase substrate was added directly. Luminescence was read 15 minutes after addition with either Micro-beta (Perkin Elmer) or Glomax (Promega) plate readers. The neutralization titer was defined as the reciprocal of the dilution that matches the positivity criteria  after fitting with the Hill model H5 haemagglutinin pseudotyped lentiviral particles (H5pp) were produced, titrated and used as described previously One hundred tissue culture infectious dose 50 (TCID50) of A/Vietnam/CL26/2004 (for the tests done on Vietnamese sera), A/Cambodia/Q040547/2006 (for the tests performed on Cambodian sera collected in 2006) and A/Cambodia/R0405050/2007 (for Cambodian sera obtained in 2007) were incubated with serial two-fold dilutions (starting from 1∶10) of each serum for one hour at room temperature prior to addition of the virus-antibody mixture onto MDCK cells. The viruses were chosen for their close genetic and antigenic relatedness to the strains responsible for the infection in patients For the purpose of this analysis we pooled serological data obtained by standard MN test from patients with RT-PCR confirmed H5N1 disease in Vietnam as well as asymptomatic infections detected by sero-epidemiological investigations in Cambodia. We used the estimated time interval after known exposure as a surrogate of the symptoms onset for asymptomatic cases. Available measures of antibody titers in each week interval were reported as geometric mean titers (GMTs). Week intervals in which there were no observations were treated as missing values. For H5N1 patients' data, we used fractional polynomial regression as a flexible parametric method for the prediction of the relationship between immune response and time interval. We then plotted the curve along with the confidence interval of the GMTs. Log10 transformed GMTs were calculated for the purpose of plotting the kinetics of the immune responses. For asymptomatic individuals' data, a linear model was fitted to the Cambodia data to assess the significance of the change in titers over weeks. An F statistic at p value <0.05 was considered statistically significant. All statistical analyses, graph construction, and curve-fitting as well as 95% CI were computed using STATA 9.0 . The Two-sample Wilcoxon rank-sum (Mann-Whitney) test was used to compare small numbers of samples. We assessed correlation between the two assays generating the Spearman's correlation coefficient. We then plotted relative differences in values using the Bland and Altman method A total of 42 individuals including 11 (26%) Vietnamese H5N1-infected patients with severe clinical disease and 31 (72%) seropositive Cambodians with asymptomatic infection or mild disease were analyzed. Of note, 6 of the 31 cases presented with an MN titer ≥1∶160 and an HI titer varying from 1∶40 to 1∶80. Of the 11 Vietnamese patients, 6 died. The Vietnamese H5N1-infected patients with severe disease were older than the Cambodian seropositive asymptomatic individuals . There was no difference in gender proportions between the two groups .The average time of follow-up of 11 Vietnamese patients was 34.2 weeks after symptoms onset . Of these 11, seven (64%) were bled twice or more for anti-H5N1 antibody testing at different points in time   and 3. OIn individuals who were asymptomatic or had mild disease, sera collected 10–11 months after exposure showed a 4- to 32- fold reduction in H5N1 neutralizing antibody titers. At this later time point, 10 of 24 individuals (42%) had H5N1 neutralizing antibody titers lower than 1∶80 (ranging from 1∶20 to 1∶40). These ten individuals also had the lowest initial H5N1 neutralizing antibody titers (1∶80) . Of the Antibody kinetics measured by H5pp were similar to that from MN assay  & 2. TheThe fractional polynomial regression model for symptomatic individuals predicted a rise of neutralizing antibody titers ≥1∶80 at week 2 and a peak at week 5 or 6 for the 2 assays . InteresThis study analysed the kinetics of the anti-H5N1 virus neutralizing antibody response in naturally infected patients with symptomatic and asymptomatic or mild infection. Overall, the titers of antibodies measured by both MN and H5pp assays were generally higher in hospitalized patients with severe H5N1 disease than in subclinically infected individuals. Serological evidence of infection (titer ≥1∶80) was still detectable by both assays in severe H5N1 cases for periods up to the limit of follow-up at 2 years, contrasting with a shorter longevity of detectable antibodies in subclinical cases of whom a substantial proportion declined to titers below 1∶80 within 10 months after presumed exposure. Higher initial titers during the acute phase, rather than differences in decay rate, could explain persistence of significant titers over a longer period of time among sick patients. Indeed, the slopes of the decay of titers were not significantly different between the two groups of cases when using linear regression modelling (data not shown). These findings suggest that conducting serosurveys could still be relevant within several months of the exposure, and that the level of antibody titers in a human population combined with information about the timing of poultry outbreaks and about H5N1 morbidity and mortality among humans could serve as a tool to accurately measure and monitor the extent of transmission as well as the risk factors for transmission and disease in a given area or premise (i.e. markets).Among patients hospitalized in Vietnam for clinically apparent H5N1 infection, we observed that no neutralizing antibodies were detected during the first week after the onset of the disease while an antibody titer ≥1∶80 was detectable in 70% by day 14 and in 80% of patients by day 21. It cannot be excluded that the patient that did not test positive by day 17 would never seroconvert, as previously described in other patients infected with H5N1- or other influenza viruses The regression model used in this study predicts that the antibody titer in clinically ill patients who develop a specific immune response should be higher than 1∶80 by day 14 after onset of symptoms and peaks 2 to 10 weeks after symptom onset. The curve suggests that during the peak, the average expected titer should be between 1∶320 and 1∶640. Interestingly, even 2 years after H5N1 disease, serology should still be positive in most of these severe human cases. To our knowledge, the kinetic data were only documented in 8 severely ill Thai patients naturally infected by H5N1 virus For the patients with subclinical H5N1 infection, we observe a titer approximately 3 times lower at the time of recruitment by comparison with patients who developed severe symptoms. The average titer (1∶150) at the time of blood draw (1–2 months after exposure) is just two fold dilution higher than the cut-off value, hence the importance of using neutralization-based methods, which seems to be more sensitive than HI test, and choosing the most appropriate virus for these tests The interpretation of our findings is subject to several limitations. The MN tests on the asymptomatic (Cambodian sera) and symptomatic (Vietnamese) sera were done using identical protocols but in two laboratories using different clade 1 H5N1 viruses isolated in Cambodia and Vietnam, respectively. These limitations and the inter-laboratory variations have been highlighted in a recent study There is little data available on the natural history and kinetics of the antibody response to influenza H5N1 infection over time, crucial information required to inform the design of seroepidemiological studies. We have demonstrated a good correlation in the profiles of antibody response of the H5pp and MN titres hence confirming the validity of the H5pp test as a screening test in seroepidemiological studies of H5N1 infection. Our data provide important novel insights into these dynamics of the serological responses in patients with the full spectrum of clinical disease from severe through mild to asymptomatic H5N1 infection. Whilst community based seroepidemiology testing after one year may pick up people with clinically apparent infection it may fail to show the true extent of the community exposure to H5N1 as the antibody response apparently wans faster in those individuals who were mildly symptomatic or asymptomatic. Hence delayed community seroprevalence studies for H5N1 may underestimate the true burden of human infection.