These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed  Genetic studies in zebrafish have identified a new protein involved in Hedgehog signaling - a key pathway required for development in vertebrates and invertebrates Drosophila larval segments and imaginal discs, dorsoventral patterning of the vertebrate neural tube, and anterior–posterior patterning of vertebrate limbs; in addition, many recent studies have illuminated the widespread and conserved role of Hedgehog signaling in development . Misregiewed in ,3,4, mediewed in ,6,7, paniewed in , and holiewed in ,10.Drosophila, diffusion of lipid-modified Hedgehog proteins is dependent on the action of tout velu, a gene involved in the synthesis of heparan sulfate proteoglycans [growth-arrest specific gene product Gas1 [After release from signaling cells, the activity and distribution of Hedgehog proteins are modulated by a variety of factors in the extracellular environment. In oglycans ,12. The oglycans . In vertuct Gas1 , and Heduct Gas1 . Moreoveuct Gas1 ,17. Geneuct Gas1 ,19.prox1. In contrast, fast muscle fibers can be identified via their multinucleate morphology and lack of prox1 expression [Hedgehog pathway function in zebrafish has been analyzed primarily in the context of skeletal muscle development and differentiation ,26,27,28pression . As devepression ,27,28. Spression ,28,29,30pression ,28.Many lines of evidence indicate that Hedgehog signals from axial tissues specify slow muscle in zebrafish. Slow muscle fibers are reduced or absent in embryos with Hedgehog pathway mutations ,31,32,33you-class” mutants because of their U-shaped somites. Five of the seven you-class mutations have been cloned, and four of these genes, syu/shh, yot/gli2, smu/smoh, and con/disp1, encode members of the Hedgehog signaling pathway [ubo/prdm1, which encodes a transcriptional switch that acts downstream of Hedgehog signaling in the development of slow muscle [ubo and Hedgehog pathway mutant phenotypes. For example, Hedgehog pathway mutants have defects in the lateral floor plate of the neural tube and the dorsal aorta, which are apparently normal in ubo mutants [ubo: Hedgehog pathway mutations reduce or abolish expression of the Hedgehog target ptc1, whereas ptc1 expression is normal in ubo mutants, indicating that they can receive Hedgehog signals [Genetic screens have identified a number of mutations disrupting the Hedgehog pathway in zebrafish ,39,40,41 pathway ,36,37,39w muscle ,42. Care mutants ,43. Exam signals ,33,36,39you-class gene, you, has revealed delayed development of the dorsal aorta and the absence of lateral floor plate marker expression in addition to slow muscle defects [ptc1 and adaxial myod, is reduced in you mutants [you gene acts within the Hedgehog pathway itself rather than downstream of Hedgehog signaling in processes specific to slow muscle development. Prior to this study, the molecular identity of the you gene has remained unknown. We report the positional cloning of the you gene and show that it encodes a novel extracellular EGF-CUB  protein required for Hedgehog signaling. Functional studies provide evidence that you is essential for the transport or stability of Hedgehog signals in the extracellular environment.Previous phenotypic characterization of mutants for the eponymous  defects ,43. More mutants . These ryou mutants lacked the horizontal myoseptum and exhibit the U-shaped morphology that defines mutants of the you class . These hedgehog genes—ehh, shh, and twhh—are expressed at the midline in early embryonic stages. To determine whether you is required for hedgehog gene transcription, we analyzed the expression of hedgehog genes in wild-type and you mutant embryos. Expression of all three hedgehog genes appeared normal in you embryos at bud stage  region of LG 7, between simple sequence length polymorphism (SSLP) markers Z11119 and Z15270  library and initiated a chromosome walk beginning with BAC zC172H20. After identifying polymorphisms in BAC end sequences, testing these polymorphisms on our mapping panel, and iteratively rescreening the pooled BAC library, we identified a contiguous stretch of genomic sequence spanning portions of five BACs with ends that flanked you  and SCUBE2 in human (66% identity). The orthology of these genes was further supported by comparative mapping: the human genes SCUBE2, LMO1, STK33, and ST5 exhibited conserved synteny with orthologous genes in both mouse and zebrafish . Pairwibrafish , the C-terminal sequence following the CUB domain (90% identity), and the EGF repeats (74% identity). A spacer region in the center of the amino acid sequence showed lower conservation (47% identity). Examination of this spacer region in the vicinity of the CUB domain revealed a repeated motif of six cysteine residues with characteristic and regular spacing, shown in yellow in SCUBE proteins are characterized by a signal peptide and by two types of conserved extracellular domains: EGF and CUB . In all ty97you mutants, a C to T transition alters the coding sequence at residue 644, changing a glutamine codon to a stop codon  injection experiments to phenocopy defects seen in you, and mRNA injection experiments to rescue the you phenotype in mutants (myod (n = 112) in the adaxial cells (n = 70) in the muscle pioneers (myod (n = 65) or Engrailed (n = 48) expression  of embryos from you/+ intercrosses showed expression of myod in adaxial cells at 12 somites (myod expression from these intercrosses showed that 137 (24%) were homozygous mutant for you. In contrast, 23.6% (n = 127) of embryos from you/+ intercrosses injected with a mutant form of you mRNA lacked expression of myod in adaxial cells  from a you/+ intercross injected with wild-type mRNA showed strong Engrailed labeling in the muscle pioneers (ty97you), whereas 24% (n = 33) of embryos injected with control mRNA lacked Engrailed expression (you mutation). In addition, injection of you MOs resulted in loss of nkx2.2 expression in the trunk and tail of wild-type embryos at 24 hpf, and injection of 50 pg of you mRNA was sufficient to rescue nkx2.2 expression in you mutants (data not shown).When injected with 50 pg of synthetic wild-type  somites E. Genotyal cells F; 20 of you transcripts appear to be maternally deposited in zebrafish embryos  you was expressed in the eye field, in distinct bilateral domains within the developing brain, and in the developing trunk of the embryo in broad paraxial stripes , in a complex pattern in the hindbrain, and in paraxial stripes along the anterior–posterior axis  of synthetic you mRNA that was sufficient to rescue the phenotypic defects observed in you mutants  was injected into embryos from a you/+ intercross, ectopic expression of myod . Because downstream targets of the Hedgehog pathway were rescued or upregulated in shh-injected you mutants, components of the Hedgehog pathway downstream of shh are most likely functional in you embryos. These results are consistent with you acting upstream of or parallel with shh in the Hedgehog pathway.To explore the possibility that the  mutants . When con = 394; B, Engrai n = 25; F, or nkx n = 17; J. This r n = 53; C and 6D, n = 78; K and 6L patched activity with MOs  or a combination of MOs targeting both ptc1 and ptc2 (n = 13 mutants). you mutant embryos injected with a ptc1 mismatch control MO did not exhibit rescued myod expression (n = 8 mutants). In similar experiments, injection of patched MOs was sufficient to rescue or expand Engrailed expression in muscle pioneers of you mutant embryos .Additional evidence that You acts upstream of the Hedgehog response derived from a loss-of-function approach, in which we activated the Hedgehog pathway by knocking down with MOs . Expressyou function to respond to Hedgehog signaling, we created genetic chimeras by transplanting cells from mutant embryos into wild-type hosts . Similarly, cells from embryos in which you function had been reduced with MOs were able to differentiate as Engrailed-expressing muscle pioneers when introduced into embryos treated with mismatch control MOs (data not shown).To determine whether a cell must be wild-type for pe hosts . Cells dyou function in order for target cells to appropriately respond to Hedgehog signals, we transplanted cells derived from wild-type donors into you mutant hosts. Of 91 chimeric mutant hosts, ten embryos exhibited rescue of Engrailed expression in genotypically mutant muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei at the proper position of posterior somites, where Engrailed is not normally expressed in you mutants .you is essential for Hedgehog signaling, our analysis indicates that you mutant cells are able to produce and respond to Hedgehog signals. hedgehog gene expression in the embryonic midline is normal in you mutants, indicating that You acts downstream of hedgehog gene transcription  in wild-type embryos did not result in obvious phenotypes when assayed by gross morphology or by expression of Hedgehog target genes, including adaxial myod, Engrailed in muscle pioneers, or nkx2.2 in the ventral spinal cord . For initial localization of you, bulked segregant analysis  was perescribed .http://www.rzpd.de). BAC end sequences were obtained from the Sanger Institute database  following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer . Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15.The CHORI211 BAC library was screened by PCR to identify positive BAC clones (you clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the you protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant you clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for you were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic you mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit .A full-length you/+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [myod [ptc1 [nkx2.2 [ehh [shh [Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from escribed . Other ped [myod , ptc1 [6od [ptc1 , nkx2.2  [nkx2.2 , ehh [20ehh [shh , and monehh [shh . Genotypyou translational initiation site (5′-GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAgAGTCgAAACAcCTgCCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting ptc1 and ptc2 were obtained from [Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described . RNA wasned from .you/+ intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran . Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from you/+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into you mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in you/+ intercrosses.Cellular transplantations were done according to standard methods . Embryosyou cDNA sequence has been deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) under accession number AY741664.The http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the genes and gene products discussed in this paper are Attractin (LocusID 8455), C1r (LocusID 715), C1s (LocusID 716), con/disp1 (LocusID 378448), Cubilin (LocusID 8029), ehh (LocusID 30299), Engrailed (LocusID 30244), Gas1 (LocusID 14451), Hedgehog (LocusID 42737), Hip1 (LocusID 15245), Megalin (LocusID 14725), myod (LocusID 30513), nkx2.2 (LocusID 30697), Patched (LocusID 35851), prox1 (LocusID 30679), ptc1 (LocusID 30181), ptc2 (LocusID 30189), sea urchin Fibropellin (LocusID 373313), smu/smoh (LocusID 30225), syu/ssh (LocusID 30269), Tolloid (LocusID 42945), tout velu (LocusID 36614), twhh (LocusID 30444), ubo/prdm1 (LocusID 323473), yot/gli2 (LocusID 30154), human LMO1 (LocusID 4004), human SCUBE1 (LocusID 80274), human SCUBE2 (LocusID 57758), human ST5 (LocusID 6764), human STK33 (LocusID 65975), mouse SCUBE1 (LocusID 64706), and mouse SCUBE2 (LocusID 56788).The LocusLink (


Harpagophytum procumbens preparations in the treatment of various forms of musculoskeletal pain.The objective of this review is to determine the effectiveness of Harpagophytum preparations in adults suffering from pain due to osteoarthritis or low back pain.Several databases and other sources were searched to identify randomized controlled trials, quasi-randomized controlled trials, and controlled clinical trials testing Given the clinical heterogeneity and insufficient data for statistical pooling, trials were described in a narrative way, taking into consideration methodological quality scores. Twelve trials were included with six investigating osteoarthritis , four low back pain, and three mixed-pain conditions.Harpagophytum extract containing less than <30 mg harpagoside per day in the treatment of knee and hip osteoarthritis. There is moderate evidence of effectiveness for (1) the use of a Harpagophytum powder at 60 mg harpagoside in the treatment of osteoarthritis of the spine, hip and knee; (2) the use of an aqueous Harpagophytum extract at a daily dose of 100 mg harpagoside in the treatment of acute exacerbations of chronic non-specific low back pain; and (3) the use of an aqueous extract of Harpagophytum procumbens at 60 mg harpagoside being non-inferior to 12.5 mg rofecoxib per day for chronic non-specific low-back pain (NSLBP) in the short term. Strong evidence exists for the use of an aqueous Harpagophytum extract at a daily dose equivalent of 50 mg harpagoside in the treatment of acute exacerbations of chronic NSLBP.There is limited evidence for an ethanolic  Harpagophytum procumbens (H) for the treatment of various diseases, including musculoskeletal complaints. For more than half a century, various preparations from H have been continuously used in Europe and have become an established traditional treatment for rheumatic complaints. The monograph of the European Scientific Cooperative on Phytotherapy (ESCOP) . One reviewer (SC) contacted experts and acquired relevant citations. In addition, manufacturers of commercial Harpagophytum preparations and content experts were contacted and asked to contribute published and unpublished material. Reference lists in review articles and the retrieved trials were searched for further trials.Two reviewers (JG) and (SC) conducted electronic searches using the following databases: PUBMED , EMBASE (OVID technologies: 1980 to wk 40 2003), Cochrane Controlled Trials Registry, Cochrane Musculoskeletal specialized register, Dissertation Abstracts, BIDS ISI, and the Cochrane Complementary Medicine Fields Specialized Register. The search strategy was developed by combining a highly sensitive method for isolating controlled clinical trials developed for the Cochrane Collaboration  OR doubl* [tw] OR trebl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* {tw])) OR (latin square [tw]) OR placebos [mh] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR comparative study [mh] OR evaluation studies [mh] OR follow-up studies [mh] OR prospective studies [mh] OR cross-over studies [mh] OR control* [tw] OR prospectiv* [tw] or volunteer* [tw]) NOT PUBMED search strategy OR ((singl* [tw] OR doubl* [tw] OR trebl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* {tw])) OR (latin square [tw]) OR placebos [mh] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR comparative study [mh] OR evaluation studies [mh] OR follow-up studies [mh] OR prospective studies [mh] OR cross-over studies [mh] OR control* [tw] OR prospectiv* [tw] or volunteer* [tw]) NOT  AND ("harpagophytum procumbens" OR (devil's AND claw)) AND  NOT reviewThe pre-publication history for this paper can be accessed here:


Disorders of coagulation have long been associated with inflammatory bowel disease. Children, as well as adults, with both active and inactive ulcerative colitis have been found to have abnormal coagulation and fibrinolysis. Disseminated intravascular coagulation arises from an overwhelming of the haemostatic regulatory mechanisms leading to an excessive generation of thrombin and a failure of the normal inhibitory pathways to prevent systemic effects of this enzyme. Ulcerative colitis has been associated with disseminated intravascular coagulation in conjunction with septicemia, toxic megacolon and surgery.A fourteen-year-old boy with a history of poorly controlled ulcerative colitis presented with nonbilious emesis, hematochezia, and hematuria. Laboratory workup revealed disseminated intravascular coagulation. He was placed on triple antibiotics therapy. An infectious workup came back negative. A computerized tomography (CT) scan of the abdomen revealed a marked thickening and irregularity of the bladder wall as well as wall thickening of the rectosigmoid, ascending, transverse, and descending colon. Patient's clinical status remained stable despite a worsening of laboratory values associated with disseminated intravascular coagulation. Patient was begun on high dose intravenous steroids with improvement of the disseminated intravascular coagulation laboratory values within 12 hours and resolution of disseminated intravascular coagulopathy within 4 days. A thorough infectious workup revealed no other causes to his disseminated intravascular coagulation.The spectrum of hypercoagulable states associated with ulcerative colitis varies from mild to severe. Although disseminated intravascular coagulation associated with ulcerative colitis is usually related to septicemia, toxic megacolon or surgery, we present a case of an ulcerative colitis flare resulting in disseminated intravascular coagulation and a bladder hematoma. A wide variety of disorders are associated with the development of disseminated intravascular coagulation (DIC). Initiation usually involves mechanical tissue injury and or endothelial cell activation and injury. DIC arises from an overwhelming of the haemostatic regulatory mechanisms leading to an excessive generation of thrombin and a failure of the normal inhibitory pathways to prevent systemic effects of the enzyme leading to DIC . UlceratA 14-year-old boy with a diagnosis of ulcerative colitis based on colonic histology, serology and a normal barium study of his small bowels was admitted with a five-day history of nonbilious vomiting and bloody diarrhea. Additional symptoms included recent onset hematuria, and low-grade fevers to 100.4 C over the prior four days. He had also sustained a 25 lb weight loss in the last six months, indicating a lack of disease control.As an outpatient, his maintenance therapy included mesalamine (1 gram three times a day), and mercaptopurine (75 mg once per day). In addition, he had been started on prednisone approximately 7 weeks prior for treatment of an ulcerative colitis flare. His current dose of prednisone was 10 mg once a day. Soon after symptoms begun, he had been placed on ciprofloxacin as treatment for a presumptive flare.Physical exam showed he was afebrile, with a heart rate of 130 beats per minute, respiratory 16 breaths per minute and blood pressure 115/67 mmHg. He was alert although with a sallow appearance. Abdominal exam revealed a soft nontender nondistended abdomen. Rectal showed normal external exam with grossly bloody stool. Initial blood work showed hemoglobin of 12.3, a normal white blood cell count, normal differential and normal platelet count with a mildly elevated prothrombin time of 16.2 with an international normalized ratio (INR) of 1.2. Urine analysis showed a specific gravity of 1.035, 3+blood, +ketones and > 100 RBC per high powered field and 0–5 WBC per high power field. Abdominal ultrasound revealed irregular shaped bladder wall.Clostridium difficile toxin analysis.Patient was placed on intravenous fluids (IV) as well as metronidazole (IV). Blood and urine cultures were sent for analysis. Stool was sent for culture and for 3) increasing PT/PTT (21.3/47 seconds) with an INR of 1.8. Blood smear showed moderate amount of elliptocytes, schistocytes, microcytes and fragmented red blood cells. Initial DIC panel revealed an elevated D-dimer of 4.9 mcg/mL with a normal thrombin time and fibrinogen. Thrombin time subsequently increased to > 120 seconds. D-dimers increased to 10.3 mcg/mL. A computerized tomography (CT) scan of the abdomen revealed a marked thickening and irregularity of the bladder wall as well as wall thickening of the rectosigmoid, ascending, transverse, and descending colon  and platelet count (64 K/mmPatient was begun on broad-spectrum antibiotics because of concerns regarding possible bacteremia and a worsening DIC laboratory picture. Blood, stool and urine cultures returned negative. Viral cultures and monoclonal antibody staining for adenovirus detection in the urine was negative. Despite a worsening in the DIC panel, the patient remained clinically unchanged. IV steroids were begun approximately 36 hours into patient's hospital stay. Patient had a stabilization of PT/PTT/INR/thrombin time and D-dimer, and a subsequent normalization of labs over the following 4-day period  Figure , 8 . PatDisorders of coagulation have long been associated with inflammatory bowel disease -11. ChilAlthough hypocoagulable states have been noted in the literature, most studies indicate an associated hypercoagulable state. There appears to be an increase in thrombin-anti-thrombin complex and a decrease in antithrombin III activity, which causes an increase in thrombin generation,12,13. OThe hypercoagulable state in ulcerative colitis is associated thromboembolic events; although uncommon, deep vein thrombosis, pulmonary embolisms and stroke have been associated with ulcerative colitis,15-18. DPresented is a case of DIC associated solely with an ulcerative colitis flare resulting in a bladder hematoma. We presume that the occurrence of DIC in this patient resulted from an acute flare on top of a chronic unremitting course of ulcerative colitis. A thorough infectious work-up of this patient did not reveal any infectious etiology that would have predisposed him to develop DIC. The presumed cause of the DIC was damage to the endothelial wall of the colonic blood vessels, which exposed blood to excessive amounts of tissue factor. This in turn led to the excessive generation of thrombin and a failure of the normal coagulation inhibitory pathways. By treating the ulcerative colitis flare, we decreased the intestinal inflammation and thereby decreased the endothelial cell damage. This, theoretically, resolved the DIC. Patient's clinical symptoms and laboratory values normalized after treatment with intravenous steroids, completely resolving the disseminated intravascular coagulopathy.The authors declare that they have no competing interests.DLS drafted the manuscript. KM and DC participated in the manuscript preparation. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:


It is believed that the development of gastric cancer (GC) before the age of 50 has a hereditary basis. Blood group A and history of gastric cancer in first-degree relatives have been shown to be risk factors for GC.In this case-control study, we enrolled patients with GC who were diagnosed before the age of 50. Patients who were diagnosed as having GC were selected. A total of 534 cases were found; of these, 44 diagnosed before the age of 50 were included in the case group. For the control group, 22 males and 22 females were randomly selected from the remaining subjects, who had diagnoses of GC after the age of 50. All the surviving patients and family members of the dead patients were interviewed about the history of cancer in the family and the age at which other family members developed cancer. The blood group of each subject was also obtained.forty-four cases under 50 years old (mean age: 36.2 years) and forty-four controls (mean age: 67.1 years) were enrolled in the study. At the time of the study, 59.1% of the study group and 50% of the control group were alive . In the study group, 68.1%, 13.6%, 13.6% and 4.5% had blood groups O, A, B and AB, respectively. In the control group the corresponding figures were 27.7%, 63.6%, 6.8% and 4.5%. First or second-degree relatives with cancer, including gastric (the most frequent), breast, lung, gynecological and hematological malignancies, were noted in 54.5% of the cases and 11.4% of the controls (p < 0.01). Family histories of cancer were accepted as valid provided that they were based on valid medical documents.It seems that the development of GC before the age of 50 is likely to be accompanied by familial susceptibility. Interestingly, our study showed a significant correlation between blood group O and the development of gastric cancer under the age of 50. Gastric cancer is the second most common cause of cancer-related death in the world . Its incThe reduced incidence of gastric cancer in western countries reflects a decrease in cancers arising in the distal stomach (body and antrum). In contrast, the incidence of cancer in the proximal stomach and esophagogastric junction has steadily increased, at a rate exceeding that of any other cancer except melanoma and lung cancer -13. In aIn general, familial genetic mechanisms do not play as important a role in gastric cancer as they do in e.g. colorectal cancer. Nonetheless, in some regions, a family history of gastric cancer may be a risk factor for the disease, although this might reflect environmental factors shared by members of a family . Rate coIn a review about genetic predisposition to gastric cancer, Bevan and Houlston 1999) concluded that several genes may be associated with increased risk [99 concluMany studies have addressed the correlation between ABO antigens and the development of gastric cancer, but most of these have indicated a correlation between sporadic cases of gastric cancer and blood group A. This association further supports the role of genetic factors in the development of gastric cancer . Blood tThe study was designed as a case-control study. We set up an active surveillance to identify patients with gastric cancer. Patients' records in the department of pathology in the main private referral facility in Tehran were scrutinized for gastric cancer cases between 1999 and 2003. Patients are referred here from all regions of the country and from different ethnic backgrounds and they are operated upon in the same hospital, so all the operation and pathology reports were available simultaneously. All the pathology reports were prepared and diagnosed by the same pathologists. The cases were selected from patients who were diagnosed with gastric cancer before the age of fifty. The sex-matched controls were randomly selected and enrolled from patients who were diagnosed over the age of fifty. All the patients and their family were interviewed regarding the history of gastric or other types of cancer over three generations, and the blood groups of affected members were ascertained. Family histories of cancer were accepted as valid provided that they were based on valid medical documents. The transfusion records of the operation were also used to identify the patients' blood groups.Statistical analysis was performed using the SPSS Statistical Package (version 10.0). The quantitative variables were expressed as means (minimum-maximum) when appropriate. A chi-square test was performed to ascertain the overall effect of blood group on the development of gastric cancer before the age of 50. All statistical tests were two-sided and differences at the 0.01 level were considered statistically significant.At the beginning of the study, 44 cases  under 50 years old and 44 sex-matched controls  were enrolled. Table This case control study demonstrates that hereditary factors, especially familial history of cancer and possession of blood group O, are associated with the development of gastric cancer under the age of fifty. To our knowledge, this may be the first study showing a correlation between blood group O and the development of gastric cancer in a specific category of patients.Risk factors for gastric cancers have been explored in a number of previous studies, including genetic factors such as blood group. Haenszel et al. suggested an association between gastric cancer and blood type A, supporting the view that genetic factors have a role in the development of gastric cancer . Our stuIn our study, approximately 54% of the case group had a familial history of cancer compared to 11% of the control group. This seems compatible with the findings of a population-based case-control study of stomach cancer in Warsaw, Poland. Here, the investigators interviewed 464 cases and 480 controls to evaluate the role of family history and other risk factors. A greater than threefold increase in risk was associated with a history of gastric cancer in a first degree relative (OR = 3.5), but no excess risk was seen for other forms of cancer. The risk associated with familial occurrence was not significantly modified by gender, age or ABO blood type, and did not vary with Lauren histological classification24. Despite the relatively large sample size in the Polish study, younger patients were not evaluated as a separate category. This may explain the difference between their results and ours. Furthermore, they defined "positive family history" as having a first-degree relative with gastric cancer. In contrast, we considered all types of malignancy in first and second-degree relatives; though the familial incidence of other (non-GI) malignancies in our study may reflect their higher frequency in the community rather than any genetic risk factor. The Polish study did not confirm previous results on the correlation between blood group A and gastric cancer. Moreover, another study by Parsonnet et al on 90 cases and 89 controls showed no association between ABO blood group and malignancy . In a muMatching the geographical origins of the cases and controls could have improved the power of our study by excluding ethnic factors from the study population. However, this is very difficult to achieve in such studies because there is a high rate of combinations between races in the country. In addition, the sample size was too small for such effects to be excluded. However, there were no significance differences between the two groups in respect of the origins of the subjects.In conclusion, our results show that familial history of cancer, and hereditary factors including blood group, have a role in the development of gastric cancer in young patients. The role of environmental factors may be more important in older patients and can be considered in future studies.The authors declare that they have no competing interest.MY designed and assisted in the conduction of the study, analyzing of the data and draft the manuscript.NR, FS, RB and YJ assisted in the conducting and designing the study and interview with the study cases as well as drafting manuscript.AM and MA assisted in analyzing of the study and conducting interviews.AA and MH reviewed and approved the pathology reports of the patients and case collecting.RM supervised the study scientifically and executively and assisted in drafting the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Surrogate measures for cardiovascular disease events have the potential to increase greatly the efficiency of clinical trials. A leading candidate for such a surrogate is the progression of intima-media thickness (IMT) of the carotid artery; much experience has been gained with this endpoint in trials of HMG-CoA reductase inhibitors (statins).We examine two separate systems of criteria that have been proposed to define surrogate endpoints, based on clinical and statistical arguments. We use published results and a formal meta-analysis to evaluate whether progression of carotid IMT meets these criteria for HMG-CoA reductase inhibitors (statins).IMT meets clinical-based criteria to serve as a surrogate endpoint for cardiovascular events in statin trials, based on relative efficiency, linkage to endpoints, and congruency of effects. Results from a meta-analysis and post-trial follow-up from a single published study suggest that IMT meets established statistical criteria by accounting for intervention effects in regression models.Carotid IMT progression meets accepted definitions of a surrogate for cardiovascular disease endpoints in statin trials. This does not, however, establish that it may serve universally as a surrogate marker in trials of other agents. Atherosclerosis is a generalized disease that causes lesions in large- and medium-sized elastic and muscular arteries. As lesions progress, arterial walls are remodeled, a process through which the size of the arterial lumen is preserved. Because of this, the disease is clinically asymptomatic during its earlier stages and may go unnoticed for decades as the risk for its clinical manifestation as acute vascular disease grows ,2. EpideThe use of surrogate markers for atherosclerosis extent and progression is widespread. Currently, the most established of these is based on carotid intima-media thickness (IMT) as measured by B-mode ultrasound. It is a natural extension to consider these measures as surrogate markers for cardiovascular disease clinical endpoints ,5. If thBoth clinical and statistical criteria for surrogacy have been proposed.Boissel, et al. lay out criteria that markers must meet to be considered as valid surrogates for clinical endpoints . We grouB1: (Efficiency) The surrogate marker should be relatively easy to evaluate, preferably by non-invasive means, and more readily available than the gold standard. The time course of the surrogate should precede that of the endpoints so that disease and/or disease progression may be established more quickly via the surrogate. Clinical trials based on surrogates should require fewer resources, less participant burden, and a shorter time frame.B2: (Linkage) The quantitative and qualitative relationship between the surrogate marker and the clinical endpoint should be established based on epidemiological and clinical studies. The nature of this relationship may be understood in terms of its pathophysiology or in terms of an expression of joint risk.B3: (Congruency) The surrogate should produce parallel estimates of risk and benefit as endpoints. Individuals with and without vascular disease should exhibit differences in surrogate marker measurements. In intervention studies, anticipated clinical benefits should be deducible from the observed changes in the surrogate marker.Prentice views surrogacy as a statistical property and defines it with mathematical expressions ,8. Four P1: The intervention should affect the distribution of T.P2: The intervention should affect the distribution of S.P3: The distribution of T should be dependent on S.P4: Endpoint T should be conditionally independent of Z given S, i.e. S should fully account for the impact of Z on T.This definition may be specific to a particular setting and cohort; a marker may meet the criteria for surrogacy for one intervention, but fail criteria for others. The criteria for surrogacy are based on explicit models, and may also be dependent on covariates and additional explanatory factors being collected and incorporated into these models.These clinical and statistical definitions require different approaches to establish surrogacy, neither of which is clear-cut. To meet the criteria outlined by Boissel, et al. , experieB-mode ultrasound imaging technology has evolved to the extent that the walls of superficial arteries can be imaged non-invasively, in real-time, and with high resolution. Unlike angiography or 'luminology', ultrasound imaging can visualize the arterial wall at every stage of atherosclerosis, from 'normal' arterial wall to complete arterial occlusion. Arterial wall thickness can therefore be measured as a continuous variable from childhood to old age, in patients and healthy controls . StudiesWe are interested in examining the potential of carotid IMT to serve as a surrogate marker for cardiovascular events, in particular cardiovascular mortality, myocardial infarctions, and clinical stroke. The clinical and statistical arguments for surrogacy are contextual, i.e. are based on specific relationships and mechanisms. It is unreasonable to make open-ended claims that a carotid IMT is a surrogate for these endpoints for all interventions and all cohorts, a point that has not been emphasized sufficiently.Our specific focus is to examine surrogacy in clinical trials of HMG-CoA reductase inhibitors (statins). Empirical evidence is largely drawn from statin clinical trials conducted on cohorts of adults at elevated risk for cardiovascular endpoints. Our choice of statins is based, in part, on the many published trials available for these agents. We acknowledge that it is quite possible that IMT may be a valid surrogate for cardiovascular events with respect to statins (i.e. accounting for the effects of these agents on cardiovascular events), but may not be a valid surrogate for other agents  or endpoints. We used Medline searches to identify seven placebo-controlled clinical trials of statins that report both IMT outcomes and cardiovascular events  and events  represent different measurement scales, thus it is difficult to argue they are influenced by statin therapy to quantitatively similar degrees. We drew evidence that the impacts are qualitatively similar using a meta-analysis of the clinical trials listed in Table The four criteria of Prentice  are as fThere is convincing evidence, some of which is summarized in the meta-analysis described above, that statin therapy reduces the risk of cardiovascular events, to the extent that this is now an indication for their use.As noted above, this association is supported by the results of our meta-analysis Table  and elseThe considerable evidence of this association has been discussed above.We know of no published literature that examines this conditional independence for statin therapies. Such a study is difficult to mount as it requires both sufficient power to demonstrate the relative impact on IMT progression of an intervention and sufficient size and follow-up time after this demonstration to assess the ability of measured IMT progression to account for subsequent risk. The only published account to examine the conditional independence of cardiovascular events given carotid IMT is for colestipol-niacin therapy in the Cholesterol Lowering Atherosclerosis Study (CLAS) clinical trial . The 2-yIn our meta-analysis, when IMT progression is included as a covariate in regression models linking cardiovascular disease events to statin treatment, the relative odds ratio is mediated from 0.48 (as tabulated below) to 0.64 and is no longer statistically significant (p = 0.13). This suggests that changes in IMT may account for some, but not all, of the effect of statins on cardiovascular events (i.e. a PTE of 0.3). Several issues complicate this argument, however. Even if a surrogate successfully meets Prentice's criteria for surrogacy within individual trials, because designs, cohorts, and endpoints vary it is to be expected that a surrogate would only account for some, not all, of treatment effects in regression models across trials. Secondly, like many markers, IMT is subject to measurement error and that is not insubstantial. This measurement error, if uncorrected, may lead to marked underestimates of relationships : measureWe have examined, in a structured and rigorous manner, the evidence that carotid IMT progression may serve as a surrogate for cardiovascular disease endpoints in statin trials. Each of the criteria for surrogacy described by Boissel, et al. appears to be met. The first three of Prentice's criteria are met, and the fourth is met by the one published study for which it can be evaluated . Meta-analyses of statin trials provide support for Boissel's criteria and the first three of Prentice's criteria, and are not inconsistent for Prentice's fourth criterion.It is possible that these arguments may generalize to other agents whose mechanisms are similar to statins, however additional analyses, based on criteria for surrogate outcomes, would be required to make this extension.MAE received an honorarium from Sankyo Pharma, Inc for a meeting during which ideas for this manuscript were developed. He is an occasional consultant to other companies concerning the design of clinical trials involving carotid ultrasonography. DHO serves on data safety and monitoring boards for Pfizer and Astra/Zeneca and serves as consultant to Sankyo Pharma and to Merck. JGT received an honorarium from Sankyo Pharma, Inc for a portion of this work. TO has no competing interests. GE has received an honorarium and consulting fees from AstraZeneca Pharmaceuticals for assistance in the planning and implementation ofa clinical trial involving carotid ultrasonography and statin therapy. He also serves as an occasional consultant to other companies on the design and conduct of trials involving carotid ultrasonography in which statins may be included as background therapies, but are not part of the experimental intervention. HM is an occasional consultant to Sankyo Pharma, including attending the meeting during which ideas for this manuscript were developed. He very occasionally consults with other Pharma companies like MSD, Essex, and Lilly. He is a consultant to Boston Scientific for interventional cardiology and intravascular ultrasound and is also a regular teacher in carotid stenting for the Guidant and Cordis companies.MAE, DHO, and HM conceived and drafted this manuscript. MAE, JGT, and TM organized and conducted its meta-analysis. GE provided oversight to analyses and contributed to interpretation of results. All authors read and approved the final manuscript.


Chorioamnionitis is a common underlying cause of preterm birth (PTB). It is hypothesised that polymorphisms in immunoregulatory genes influence the host response to infection and subsequent preterm birth. The relationship between histologic chorioamnionitis and 22 single nucleotide polymorphisms in 11 immunoregulatory genes was examined in a case-control study.IL1A, IL1B, IL1RN, IL1R1, tumour necrosis factor (TNF), IL4, IL6, IL10, transforming growth factor beta-1 (TGFB1), Fas (TNFRSF6), and mannose-binding lectin (MBL2) were genotyped by polymerase chain reaction and sequence specific primers. Multivariable logistic regression including demographic and genetic variables and Kaplan-Meier survival analyses of genotype frequencies and pregnancy outcome were performed.Placentas of 181 Caucasoid women with spontaneous PTB prior to 35 weeks were examined for histologic chorioamnionitis. Polymorphisms in genes IL10-1082A/-819T/592A (ATA) haplotype  and MBL2 codon 54Asp allele , were positively associated with chorioamnionitis, while the TNFRSF6-1377A/-670G (AG) haplotype  and homozygosity for TGFB1-800G/509T (GT) haplotype  were negatively associated.Sixty-nine (34%) women had histologic evidence of acute chorioamnionitis. Carriage of the IL10, MBL2, TNFRSF6 and TGFB1 may influence susceptibility to chorioamnionitis.These findings demonstrate that polymorphisms in immunoregulatory genes  Escherichia coli   , IL6 . We estimated the study had sufficient power  to detect a minimum of 17% difference in genotype frequency between chorioamnionitis and no chorioamnionitis for frequencies of approximately 10% (ie. 10% versus 27%). For a genotype frequency of 3% the minimum detectable difference was 10% (3% versus 13%). Allele and haplotype frequencies for polymorphisms were determined by direct counting from each chromosome. Frequencies of genetic variants were recorded as allele (proportion of positive chromosomes), genotype , and 'carriage' of an allele or haplotype (either in homozygous or heterozygous state). Alleles were said to be in Hardy-Weinberg equilibrium if the observed genotype frequencies did not differ significantly (P > 0.05) from those expected when analysed by Chi square. Gene variant frequencies were compared using 2 × 2 contingency table analysis and the Chi square test for independence using EpiInfo 6. Mean birth weights were compared by Multivariable analysis was performed by stepwise backwards multiple logistic regression using SYSTAT 9.0 software (Systat version 1999 SPSS Inc). The basic model included genetic variables with P values < 0.15 obtained in univariate analyses. In each multivariable model, only one genotype or haplotype of each gene was selected for analysis. Selection of genotype/haplotype for the model was based on strength of association, the previously reported importance of a gene variant and homozygosity of alleles. At each backward step in the regression analysis P values ≥ 0.07 were rejected. Kaplan-Meier survival analysis (SYSTAT 9.0) was used to identify cumulative probability in maintaining pregnancy by gestation stratified by chorioamnionitis and gene variables. Breslow-Gehan Chi square was used to weight each gestational period by the total number at risk at that time so that earlier gestations receive greater weight than later gestations.Sixty-nine (38%) women had histologic evidence of acute chorioamnionitis, and 112 (62%) had no such evidence. There were no significant differences between those with and without histologic chorioamnionitis for maternal age, race, history of smoking, alcohol consumption, substance use, new partner, cervical cerclage, placenta praevia after 22 weeks with bleeding, history of two or more PTBs before 35 weeks, miscarriage between 12 and 20 weeks or PPROM (Table IL10-1082A/-819T/-592A (ATA) haplotype  and MBL2 codon 54Asp  were independently associated with histologic chorioamnionitis, while homozygosity for the variant TGFB1-800G/-509T (GT) haplotype , and carriage of TNFRSF6-1377A/-670G (AG) haplotype  remained negatively associated with histologic chorioamnionitis. The remainder of SNPs tested showed no significant differences.For confirmation of independence of association, a multivariable model was used including univariate variables with P values ≤ 0.15. This model included the above genetic variables Table  plus TNFIL10 ATA haplotype   Figure . There wIL10 ATA haplotype, MBL2 codon 54Asp and histologic chorioamnionitis, and negative associations with TNFRSF6 and TGFB1. This is the first study to investigate TNFRSF6 and TGFB1 in combination with such a wide range of cytokine SNPs in histologic acute chorioamnionitis. Also, this is one of the few studies that have used multivariable and survival analyses for investigating adverse pregnancy outcomes such as chorioamnionitis and PTB.These results extend prior reports investigating gene polymorphisms and the pathogenesis of PTB and chorioamnionitis . In thisIL10 and MBL2 may be of biologic importance in the pathogenesis of histologic chorioamnionitis. The function of IL-10 in pregnancy is of particular interest because of its role in the homeostatic control of an inflammatory immune response [IL10 ATA promoter haplotype and alleles comprising the haplotype are associated with low protein levels in Caucasians [IL10 ATA haplotype may influence the timing of PTB. IL-10 antagonises the synthesis and actions of pro-inflammatory cytokines such as IL-1 [IL10 ATA haplotype may also be a reflection of failure of tocolysis in the presence of infection [IL10 ATA haplotype is more common in women with histologic chorioamnionitis suggests that this haplotype may be important in the pathogenesis of histologic chorioamnionitis and subsequent PTB.The differences in genotype distribution for response . Althougucasians . Low levucasians . In a prucasians . Surviva as IL-1 . In chor as IL-1  and feta as IL-1  signal fnfection . Our finMBL2 variant alleles in exon 1 correlate with decreased circulating MBL. The single nucleotide substitution of an adenine for a guanine in codon 54 results in replacement of aspartic acid for a glycine in the MBL [MBL2 peptide trimers resulting in lower stability and serum levels [MBL2 genetic variants are associated with infection in a range of contexts including recurrent infection in children and adults [MBL2 codon 54Asp and histologic chorioamnionitis suggests that this allele may result in low MBL levels, impaired opsonisation and clearance of pathogens, thus facilitating the development of chorioamnionitis.MBL is an important innate defense molecule active against a broad range of bacterial, viral, fungal and protozoan pathogens . The MBL the MBL . Functiom levels . Low serd adults ,14, recud adults , recurred adults  and autod adults . The assTNFRSF6) gene promoter were studied at positions -1377 and -670, and the -1377A/-670G (AG) haplotype was negatively associated with histologic chorioamnionitis. The -1377G/A is situated at the transcription factor SP-1 binding site [TNFRSF6 transcription, Fas expression and thus apoptotic and inflammatory responses. Recent evidence suggests that cells are more sensitive to Fas-mediated apoptosis when levels of Fas expression increase [Fas protein is expressed on the surface of many cell types, such as lymphocytes, epithelial, fibroblasts and certain endothelial cells, and cytokines  and horming site , and theincrease . Apoptotincrease  and thusTNF promoter -308A allele was associated with clinical chorioamnionitis in a small number of women [in vitro functional data of the effects of the TNF SNPs are conflicting and may be context dependent and influenced by strong linkage disequilibrium with other intra-and-extra-genic polymorphisms  declare that they have no competing interests.MA contributed to study design, recruited study subjects, abstracted medical records, entered data, performed preliminary statistical analyses and drafted the manuscript.PH provided design regarding immunoregulatory genes, interpretation of results and preparation of manuscript.CM conceived methodology and supervision of PCR testing, interpretation of test results and preparation of manuscript.SH supervised preparation of DNA and performed all PCR tests.JR provided clinical authorisation and support for the study. Provided input regarding clinical database design, and provided critical analysis for obstetric correctness.HM conceived and coordinated the study, conceived medical record review, reviewed statistical analyses and preparation of manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation and histologic chorioamnionitis. Frequency distribution of immunoregulatory gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation stratified by histologic chorioamnionitis.Click here for file


This issue of CytoJournal contains an article on screening for anal intraepithelial neoplasia in high-risk male patients. This accompanying Editorial focuses on current understanding of this relatively new disease entity, with insights as to the potential role of screening cytopathology in the epidemiology, pathophysiology and clinical management of this HIV and HPV related anal lesion, which predominates in male patients living long-term with AIDS. Mention is made of techniques of obtaining samples, methods of preparation, and morphologic classification. Issues of anoscopic confirmation, as well as topical and surgical management are emphasized. The similarity of initial experiences in anal screening to problems encountered early in cervical cancer screening programs several decades ago, are highlighted. For corresponding research article please see Arian et al, 2005 The subject of anal screening cytology has entered the epidemiologic and cytopathologic literature as a topic of interest over the last decade, and is highlighted in this issue of CytoJournal in an article by Arain and colleagues. Until recently, anal cancer was not considered to be a neoplasm of major public health concern . It occuDuring the 1990s, in several European and North American cancer centers, an initially unaccountable increase in anal cancers was seen in younger people. It soon became apparent that HIV-positive homosexual males were affected in numbers greatly in excess of those expected. This was first noted in those urban areas in which large concentrations of homosexual men had been treated since the outbreak of the HIV epidemic. The term "males having sex with males" (MSM) was coined to cover these high-risk individuals. Preliminary information gleaned from screening programs in high HIV-positive incidence areas amongst MSM, showed detection rates of intraepithelial and early invasive neoplasia higher than any incidence ever recorded for cervical cancer screening. Further, HIV-negative homosexual MSMs, and HIV positive non-homosexual men (eg drug users) also exhibited an increased incidence of pre-malignant and invasive anal carcinomas. More recently, the syndrome of early onset anal cancer has been extended to include HIV-positive female patients, as well as females who are not HIV-positive, but who have genital HPV. All these groups show a higher incidence of abnormal anal cytopathology, though none quite as high as that found in HIV positive MSM. The unifying factor in most instances is ano-receptive intercourse, or extension of HPV infection from the genital tract to the anal mucosa, most obvious in the face of deficient immunity, or high viral load -6.Epidemiologic studies have indicated that a prolonged preclinical phase precedes the onset of anal cancer in these high-risk groups. Just as in the cervix, there is a transitional zone  in the anus. Rectal mucosa, with goblet cells, ends about one inch proximal to the external sphincter, giving way to a transitional epithelium, which in turn blends into a stratified squamous epithelium at the level of the anus.  Almost all anal cancers develop in the transitional zone, where atypical, dysplastic and in-situ lesions are identifiable histologically. The cytologic counterparts are those of atypical cells of uncertain significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL). Once this natural history had been demonstrated and confirmed, it seemed natural that exfoliative cytology could be investigated as an "anal Pap smear" ,8.As with the cervix, the technique of obtaining the anal sample is critical to the success of screening. Standard colonic preparation is not required, but the rectum should be emptied prior to obtaining the anal sample. Brushes, brooms and Dacron swabs have all been used, the type of spatula probably being less important than the skill of the operator. The instrument is inserted to a depth of one and a half inches beyond the external sphincter, and subsequently withdrawn in a firm downward spiral movement incorporating 10–12 rotations, to ensure the device has made contact with the full surface area of the transformation zone. Some have used direct smears onto glass slides with immediate wet fixation; most centers, however, employ immediate insertion of the scraping device into liquid fixative for thin layer preparation. This appears both to improve adequacy and preservation, and also to eliminate any fecal contamination; residual material in the vial can be used for HPV studies if required, or for the creation of a bank of teaching slides.There has been a relative dearth of literature on the cytomorphology of anal samples -11. ClasExperience over the last decade suggests that anal cancer may be as highly appropriate a target for screening as is the cervix, in selected populations. The neoplasm is frequently encountered in well-defined high-risk groups. It has a detectable pre-malignant phase, and is amenable to easy cytologic sampling. Cytodiagnosis is reasonably sensitive and highly specific, and histologic confirmation is relatively easily obtained by well-trained personnel. If there is a current area of deficiency in such programs, it may well be in the treatment of intraepithelial lesions, which, as yet, has not been adequately assessed in large numbers of patients. It is known that highly active anti-retroviral therapy (HAART) does not appear to alter the pathophysiology of anal lesions once initiated. A variety of topical agents such as podophyllotoxin and imiquimod have been tried, as has intralesional interferon; superficial ablative therapies including liquid nitrogen, electrocautery, laser and LEEP have been attempted with varying success rates. Circumferential surgical resection almost inevitably results in unacceptable loss of sphincter control and soiling, but anoscopy-directed limited excision may prove less morbid. The fact that so many options are available implies perhaps that no single modality is yet considered sufficiently effective, with minimal complication ,13. Thisineffective in these communities. The second feature dictating the institution of screening programs in developing countries relates to antiretroviral therapy. Anal intraepithelial neoplasia and cancer are not encountered early in the progression of HIV/AIDS. Rather, they are late complications of patients living long-term with AIDS, usually implying patients living long-term on HAART [An interesting consideration is whether or not anal cancer, and thereby anal cytopathology screening programs, would be of value in the developing world, particularly in Africa, India and China, where the bulk of the global incidence of HIV resides. This will depend on two very different factors. First is the nature of transmission. Unlike the situation in the developed world, AIDS in these regions is not essentially a disease of homosexual males; thus, without anal intercourse predominating, an upward trend in the incidence of anal cancer would seem unlikely. Anal screening programs would be unnecessary or cost-on HAART . Unless


Joel Cohen offers a historical and prospective analysis of the relationship between mathematics and biology Although mathematics has long been intertwined with the biological sciences, an explosive synergy between biology and mathematics seems poised to enrich and extend both fields greatly in the coming decades . BiologyThe discovery of the microscope in the late 17th century caused a revolution in biology by revealing otherwise invisible and previously unsuspected worlds. Western cosmology from classical times through the end of the Renaissance envisioned a system with three types of spheres: the sphere of man, exemplified by his imperfectly round head; the sphere of the world, exemplified by the imperfectly spherical earth; and the eight perfect spheres of the universe, in which the seven (then known) planets moved and the outer stars were fixed . The disMathematics broadly interpreted is a more general microscope. It can reveal otherwise invisible worlds in all kinds of data, not only optical. For example, computed tomography can reveal a cross-section of a human head from the density of X-ray beams without ever opening the head, by using the Radon transform to infer the densities of materials at each location within the head . CharlesConversely, mathematics will benefit increasingly from its involvement with biology, just as mathematics has already benefited and will continue to benefit from its historic involvement with physical problems. In classical times, physics, as first an applied then a basic science, stimulated enormous advances in mathematics. For example, geometry reveals by its very etymology (geometry) its origin in the needs to survey the lands and waters of Earth. Geometry was used to lay out fields in Egypt after the flooding of the Nile, to aid navigation, to aid city planning. The inventions of the calculus by Isaac Newton and Gottfried Leibniz in the later 17th century were stimulated by physical problems such as planetary orbits and optical calculations.In the coming century, biology will stimulate the creation of entirely new realms of mathematics. In this sense, biology is mathematics' next physics, only better. Biology will stimulate fundamentally new mathematics because living nature is qualitatively more heterogeneous than non-living nature. For example, it is estimated that there are 2,000–5,000 species of rocks and minerals in the earth's crust, generated from the hundred or so naturally occurring elements  (Harvey 1847), first published in 1628. Harvey's demonstration that the blood circulates was the pivotal founding event of the modern interaction between mathematics and biology. His elegant reasoning is worth understanding.The outstanding milestone in the early history of biological quantitation was the work of William Harvey, From the time of the ancient Greek physician Galen (131–201 C.E.) until William Harvey studied medicine in Padua , it was believed that there were two kinds of blood, arterial blood and venous blood. Both kinds of blood were believed to ebb and flow under the motive power of the liver, just as the tides of the earth ebbed and flowed under the motive power of the moon. Harvey became physician to the king of England. He used his position of privilege to dissect deer from the king's deer park as well as executed criminals. Harvey observed that the veins in the human arm have one-way valves that permit blood to flow from the periphery toward the heart but not in the reverse direction. Hence the theory that the blood ebbs and flows in both veins and arteries could not be correct.Harvey also observed that the heart was a contractile muscle with one-way valves between the chambers on each side. He measured the volume of the left ventricle of dead human hearts and found that it held about two ounces (about 60 ml), varying from 1.5 to three ounces in different individuals. He estimated that at least one-eighth and perhaps as much as one-quarter of the blood in the left ventricle was expelled with each stroke of the heart. He measured that the heart beat 60–100 times per minute. Therefore, the volume of blood expelled from the left ventricle per hour was about 60 ml × 1/8 × 60 beats/minute × 60 minutes/hour, or 27 liters/hour. However, the average human has only 5.5 liters of blood (a quantity that could be estimated by draining a cadaver). Therefore, the blood must be like a stage army that marches off one side of the stage, returns behind the scenes, and reenters from the other side of the stage, again and again. The large volume of blood pumped per hour could not possibly be accounted for by the then-prevalent theory that the blood originated from the consumption of food. Harvey inferred that there must be some small vessels that conveyed the blood from the outgoing arteries to the returning veins, but he was not able to see those small vessels. His theoretical prediction, based on his meticulous anatomical observations and his mathematical calculations, was spectacularly confirmed more than half a century later when Marcello Malpighi (1628–1694) saw the capillaries under a microscope. Harvey's discovery illustrates the enormous power of simple, off-the-shelf mathematics combined with careful observation and clear reasoning. It set a high standard for all later uses of mathematics in biology.Mathematics was crucial in the discovery of genes by Mendel  and in tOver the last 500 years, mathematics has made amazing progress in each of its three major fields: geometry and topology, algebra, and analysis. This progress has enriched all the biological sciences.In 1637, René Descartes linked the featureless plane of Greek geometry to the symbols and formulas of Arabic algebra by imposing a coordinate system  on the geometric plane and using numbers to measure distances between points. If every biologist who plotted data on x–y coordinates acknowledged the contribution of Descartes to biological understanding, the key role of mathematics in biology would be uncontested.Another highlight of the last five centuries of geometry was the invention of non-Euclidean geometries (1823–1830). Shocking at first, these geometries unshackled the possibilities of mathematical reasoning from the intuitive perception of space. These non-Euclidean geometries have made significant contributions to biology in facilitating, for example, mapping the brain onto a flat surface .Nine Chapters of the Mathematical Art; In algebra, efforts to find the roots of equations led to the discovery of the symmetries of roots of equations and thence to the invention of group theory, which finds routine application in the study of crystallographic groups by structural biologists today. Generalizations of single linear equations to families of simultaneous multi-variable linear equations stimulated the development of linear algebra and the European re-invention and naming of matrices in the mid-19th century. The use of a matrix of numbers to solve simultaneous systems of linear equations can be traced back in Chinese mathematics to the period from 300 B.C.E. to 200 C.E. . The questions and the domains of biology behave differently on different temporal and spatial scales. The opportunities and the challenges that biology offers mathematics arise because the units at any given level of biological organization are heterogeneous, and the outcomes of their interactions  on any selected temporal and spatial scale may be substantially affected by the heterogeneity and interactions of biological components at lower and higher levels of biological organization and at smaller and larger temporal and spatial scales , 1995.The landscape of applied mathematics is better visualized as a tetrahedron (a pyramid with a triangular base) than as a matrix with temporal and spatial dimensions.  The four main points of the applied mathematical landscape are data structures, algorithms, theories and models , and computers and software. Data structures are ways to organize data, such as the matrix used above to describe the biological landscape. Algorithms are procedures for manipulating symbols. Some algorithms are used to analyze data, others to analyze models. Theories and models, including the theories of pure mathematics, are used to analyze both data and ideas. Mathematics and mathematical theories provide a testing ground for ideas in which the strength of competing theories can be measured. Computers and software are an important, and frequently the most visible, vertex of the applied mathematical landscape. However, cheap, easy computing increases the importance of theoretical understanding of the results of computation. Theoretical understanding is required as a check on the great risk of error in software, and to bridge the enormous gap between computational results and insight or understanding.The landscape of research in mathematics and biology contains all combinations of one or more biological questions, domains, time scales, and spatial scales with one or more data structures, algorithms, theories or models, and means of computation . The following example from cancer biology illustrates such a combination: the question, “how does it work?” is approached in the domain of cells  with algorithms for correlation and hierarchical clustering.http://discover.nci.nih.gov/arraytools/). They used two kinds of information from the drug discovery program of the National Cancer Institute. The first kind of information described gene expression in 1,375 genes of each of 60 human cancer cell lines. A target matrix T had, as the numerical entry in row g and column c, the relative abundance of the mRNA transcript of gene g in cell line c. The drug activity matrix A summarized the pharmacology of 1,400 drugs acting on each of the same 60 human cancer cell lines, including 118 drugs with “known mechanism of action.” The number in row d and column c of the drug activity matrix A was the activity of drug d in suppressing the growth of cell line c, or, equivalently, the sensitivity of cell line c to drug d. The target matrix T for gene expression contained 82,500 numbers, while the drug activity matrix A had 84,000 numbers.Suppose a person has a cancer. Could information about the activities of the genes in the cells of the person's cancer guide the use of cancer-treatment drugs so that more effective drugs are used and less effective drugs are avoided? To suggest answers to this question, These two matrices have the same set of column headings but have different row labels. Given the two matrices, precisely five sets of possible correlations could be calculated, and Scherf et al. calculated all five. (1) The correlation between two different columns of the activity matrix A led to a clustering of cell lines according to their similarity of response to different drugs. (2) The correlation between two different columns of the target matrix T led to a clustering of the cell lines according to their similarity of gene expression. This clustering differed very substantially from the clustering of cell lines by drug sensitivity. (3) The correlation between different rows of the activity matrix A led to a clustering of drugs according to their activity patterns across all cell lines. (4) The correlation between different rows of the target matrix T led to a clustering of genes according to the pattern of mRNA expressed across the 60 cell lines. (5) Finally, the correlation between a row of the activity matrix A and a row of the target matrix T described the positive or negative covariation of drug activity with gene expression. A positive correlation meant that the higher the level of gene expression across the 60 cancer cell lines, the higher the effectiveness of the drug in suppressing the growth of those cell lines. The result of analyzing several hundred thousand experiments is summarized in a single picture called a clustered image map . This clWhat use is this? If a person's cancer cells have high expression for a particular gene, and the correlation of that gene with drug activity is highly positive, then that gene may serve as a marker for tumor cells likely to be inhibited effectively by that drug. If the correlation with drug activity is negative, then the marker gene may indicate when use of that drug is contraindicated.While important scientific questions about this approach remain open, its usefulness in generating hypotheses to be tested by further experiments is obvious. It is a very insightful way of organizing and extracting meaning from many individual observations. Without the microscope of mathematical methods and computational power, the insight given by the clustered image map could not be achieved.To realize the possibilities of effective synergy between biology and mathematics will require both avoiding potential problems and seizing potential opportunities.The productive interaction of biology and mathematics will face problems that concern education, intellectual property, and national security.Educating the next generation of scientists will require early emphasis on quantitative skills in primary and secondary schools and more opportunities for training in both biology and mathematics at undergraduate, graduate, and postdoctoral levels .Intellectual property rights may both stimulate and obstruct the potential synergy of biology and mathematics. Science is a potlatch culture. The bigger one's gift to the common pool of knowledge and techniques, the higher one's status, just as in the potlatch culture of the Native Americans of the northwest coast of North America. In the case of research in mathematics and biology, intellectual property rights to algorithms and databases need to balance the concerns of inventors, developers, and future researchers .A third area of potential problems as well as opportunities is national security. Scientists and national defenders can collaborate by supporting and doing open research on the optimal design of monitoring networks and mitigation strategies for all kinds of biological attacks . But opeMathematical models can circumvent ethical dilemmas. For example, in a study of the household transmission of Chagas disease in northwest Argentina, The future of a scientific field is probably less predictable than the future in general. Doubtless, though, there will be exciting opportunities for the collaboration of mathematics and biology. Mathematics can help biologists grasp problems that are otherwise too big (the biosphere) or too small (molecular structure); too slow (macroevolution) or too fast (photosynthesis); too remote in time (early extinctions) or too remote in space (life at extremes on the earth and in space); too complex (the human brain) or too dangerous or unethical (epidemiology of infectious agents). Here are five biological challenges that could stimulate, and benefit from, major innovations in mathematics.Understand cells, their diversity within and between organisms, and their interactions with the biotic and abiotic environments. The complex networks of gene interactions, proteins, and signaling between the cell and other cells and the abiotic environment is probably incomprehensible without some mathematical structure perhaps yet to be invented.Understand the brain, behavior, and emotion. This, too, is a system problem. A practical test of the depth of our understanding is this simple question: Can we understand why people choose to have children or choose not to have children ?http://www.life.umd.edu/labs/Delwiche/pubs/endosymbiosis.gif).Replace the tree of life with a network or tapestry to represent lateral transfers of heritable features such as genes, genomes, and prions (Couple atmospheric, terrestrial, and aquatic biospheres with global physicochemical processes.Monitor living systems to detect large deviations such as natural or induced epidemics or physiological or ecological pathologies.Here are five mathematical challenges that would contribute to the progress of biology.Understand computation. Find more effective ways to gain insight and prove theorems from numerical or symbolic computations and agent-based models. We recall Hamming: “The purpose of computing is insight, not numbers” , p. 31.Find better ways to model multi-level systems, for example, cells within organs within people in human communities in physical, chemical, and biotic ecologies.Understand probability, risk, and uncertainty. Despite three centuries of great progress, we are still at the very beginning of a true understanding. Can we understand uncertainty and risk better by integrating frequentist, Bayesian, subjective, fuzzy, and other theories of probability, or is an entirely new approach required?Understand data mining, simultaneous inference, and statistical de-identification . Are praSet standards for clarity, performance, publication and permanence of software and computational results.


Many real networks can be understood as two complementary networks with two kind of nodes. This is the case of metabolic networks where the first network has chemical compounds as nodes and the second one has nodes as reactions. In general, the second network may be related to the first one by a technique called line graph transformation . Recently, the main topological properties of the metabolic networks have been properly described by means of a hierarchical model. While the chemical compound network has been classified as hierarchical network, a detailed study of the chemical reaction network had not been carried out.C(k) is calculated for the transformed network. C(k) indicates the probability that two nearest neighbours of a vertex of degree k are connected to each other. While C(k) follows the scaling law C(k) ~ k-1.1 for the initial hierarchical network, C(k) scales weakly as k0.08 for the transformed network. This theoretical prediction was compared with the experimental data of chemical reactions from the KEGG database finding a good agreement.We have applied the line graph transformation to a hierarchical network and the degree-dependent clustering coefficient The weak scaling found for the transformed network indicates that the reaction network can be identified as a degree-independent clustering network. By using this result, the hierarchical classification of the reaction network is discussed. WWW. Most networks encountered in real world have scale-free topology, in particular networks of fundamental elements of cells as proteins and chemical substrates .From these two cases, we can conclude that for hubs  in the transformed space (CT(k)) we introduce the concept of assortativity. By assortative (disassortative) mixing in networks we understand the preference for nodes with high degree to connect to other high (low) degree nodes [Newman [k' and k" as ek'k". We also assume that the nodes of the initial network are following a power-law distribution kγ -and have no assortative mixing. Under these assumptions, the probability distribution ek'k" of edges that link together nodes with degree k' + k" can be written as:To calculate the distribution of ee nodes . By foll [Newman , we defik', k"), which can generate transformed nodes with degree k = k' + k" - 2. Thus, we obtain:We make a convolution between Eq. (4) and Eq. (7), by summing for all the possible degrees of the two nodes at either end of edges (CT(k) and the behavior of Ca(k) exposed above, CT(k) will grow smoothly for large k, i.e., scaling weakly with the node degree k. We have calculated numerically this expression and the results are discussed later in Fig. According to the structure of C >, and we have found that <C > has a size-independent behavior before and after the line graph transformation is done. We can write the number of nodes with degree k as:We have also calculated the analytical expression for <C(k) = A·k α-, where A is a constant. This constant changes when we consider hierarchical networks with different number of nodes in the initial cluster [P(k) ~ kγ -of the network also changes. For <C > before the transformation we can write:and we assume that  cluster . But it k = 1 because we renormalize over all the probability distribution.Note that the summation in the denominator begins with C > by using the RSMOB model (explained in next section in detail). This model starts by generating a fully connected cluster of m nodes, such that the connectivity of each node is k = m - 1. In the following iteration, m - 1 replicas of the initial cluster are generated, and linked to the central node of the original cluster in such a way that the central node of the original cluster gains (m - 1)·(m - 1) edges, and its total connectivity being k = m - 1 + (m - 1)2. By iterating these procedure, it is easy to see that hubs  will have connectivities , with j = 1, ..., logmN being the iteration number. Therefore, assuming that the degree distribution P(k) and the clustering coefficient C(k) are power-laws with exponents γ' and α respectively, the expression for <C > for the hubs reads as:Furthermore, we can obtain <A' is a constant adjusted so that <C >=1 holds for j = 1. The upper limit of the summation logmN is obtained by means of the expression mj = N, which gives the total number of nodes in the network and  denotes the exponent of the power-law distribution of hubs in the RSMOB model. We must note that in a hierarchical network, the number of nodes with different degree k is scarce, therefore the probability distribution of node degree is properly defined as P(k) = (1/Ntot)(Nk/Δk), where Nk is the number of nodes with degree k, Ntot is the total number of nodes, and Δk means that nodes with degree k are binned into intervals. In addition, we note that for the hierarchical model, Δk changes linearly with k. Hence, the exponent of the power-law is given by γ = 1 + γ', with  where m is the number of nodes in the initial module.where C > converges to a constant. In order to calculate <C > after the line graph transformation is applied (<CT >), we make the substitution C(k) → CT(k) in Eq. (10). As from Eq. (8) we have seen that CT(k) is almost constant, we can conclude that <CT > also has a constant behavior and it is almost independent with network size. While the scaling law of C(k) ~ k-1 was proved mathematically in [CT(k), <C > and <CT >.By using Eqs. 10) and (11), we will see later . In the next step four replicas are created and the peripheral nodes are connected again to produce 125-node modules . This process can be repeated indefinitely [Fig. finitely .C(k), we have constructed three hierarchical networks with 3, 4, and 5 initial number of nodes. These networks were generated up to 7 (6561), 5 (4096), and 4 (3125) iterations (nodes), repectively. Once we have constructed these three networks, we apply the line graph transformation to them, and we calculate the CT(k) clustering coefficient for the transformed networks. In Fig. CT(k) for the transformed network with 3, 4, and 5 initial nodes, respectively. In Fig. CT(k) obtained from Eq. (8). From top to bottom the lines correspond to the networks of 3, 4 and 5 initial nodes, respectively. In Fig. CT(k) via Eq. (8) (lines) are in good agreement with the experimental data (diamonds) from the KEGG database [CT(k), we have binned into seven intervals the experimental data according to degree k , and averaged over the CT(k)'s obtained in that range (red circles). It shows a better agreement between KEGG results and the analytical curves. The only disagreement comes at k = 2. This is easy to understand because in the hierarchical model depicted in Fig. C(k = 2) = 1 for 3 initial nodes by construction of the network. However, in real networks, we could find nodes which have only two neighbors and, in some cases, these neighbors could be connected. In these cases the clustering coefficient takes value one.To evaluate database . Moreove up to 7 561, 5 (4CT(k) after the line graph transformation is applied to the hierarchical network generated by 4 initial nodes and up to 5 iterations. The results are shown with empty triangles (red) and fitted to the dashed line. We see that C(k) ~ k-1.1 changed into CT(k) ~ k0.08. We also see that the line graph transformation increases the average of the clustering value of the transformed network. These theoretical results were compared with the experimental data from KEGG [CT(k) for the reaction network.In Fig. rom KEGG , findingC > we have evaluated Eq. (10) for 3 different configurations. We have considered 3 initial nodes, 4 and 5 initial nodes nodes up to 7, 5 and 4 iterations, respectively. As it is explained in [C > approaches asymptotically to a constant value, being independent of the size of the network. The asymptotic value depends on the initial number of nodes. We calculated the values of γ corresponding to the degree distribution P(k) ~ kγ -for each network, and the related constant A, which appears in Eq. (10). We show in Table C > obtained by Eq. (10). These values, as it can be seen in Fig. C(k) is being overestimated by the scaling dependence k-1 and it provides a larger value of <C >. In [C > from hierarchical model were compared with the experimental values of 43 organisms. The values of <C > for each organism were around 0.15 – 0.25. By using the KEGG database we have evaluated the experimental value <C > for 163 organisms and we obtained an average value of 0.08.For <ated Eq. 0 for 3 dC > calculated for networks generated by 3 initial nodes (circles) and 4 initial nodes (triangles) by using the RSMOB model. We see that <C > approaches asymptotically to constant values around ~ 0.66 (circles) and ~ 0.74 (triangles), being independent of the size of the network. Once the line graph transformation is applied, we see that the corresponding values of <CT > also approach asymptotically to constant values. Hence, <CT > also is size-independent for large N (empty circles and triangles). In addition, we have averaged the experimental value of the clustering coefficient for reactions of 163 organisms found in KEGG database and we have obtained the value of <CT >= 0.74. We see that the experimental value <CT > for reactions is in good agreement with the asymptotic values obtained by the transformed network (empty triangles and circles).We show in Fig. C > by using Eq. (11). This equation should reproduce the results of <C > calculated by using the RSMOB model  and <C > of the reaction network by applying the line graph transformation to a hierarchical network. This hierarchical network was generated by using the RSMOB model, which reproduces properly the topological features of the metabolic network, in particular the compound network. Our results indicate that by applying the line graph transformation to the hierarchical network, it is possible to extract topological properties of the reaction network, which is embedded in the metabolic network. The RSMOB model stores the adequate information of the reaction network and the line graph transformation is one useful technique to evoke it.We have studied here the clustering coefficients C(k) scales as k-1.1 for the initial hierarchical network (compound network), we find C(k) ~ k0.08 for the transformed network (reaction network). This theoretical prediction was compared with the experimental data from the KEGG database, finding a good agreement. Our results indicate that the reaction network is a degree-independent clustering network. Furthermore, the weak scaling of C(k) for the reaction network suggests us that this network may not have hierarchical organization. However, further analyses of this network, and in general of all biological networks, by following the concept of hierarchical path are encouraged [While couraged ,21.C(k) and <C >. Expressions for these coefficients were calculated before and after the line graph transformation is applied to the hierarchical network. The agreement obtained by using these expressions was found acceptable, and consequently, they could be useful for further analyses in different networks .On the other hand, we have also conducted an analytical derivation for the clustering coefficients The line graph transformation has recently been applied on metabolic networks  to studyJCN conceived of the study, designed and implemented the analyses, and prepared the manuscript. NU carried out computational implementations and experiments. TY participated in the acquisition and processing of data from KEGG database. MK provided conceptual guidance and data from the KEGG database, and conceived the initial idea of the two complementary metabolic networks. TA provided guidance, coordinated and participated in the biological and theoretical analyses, and revised the manuscript. All authors read and approved the final manuscript.