LOXL1) single nucleotide polymorphisms (SNPs) with exfoliation syndrome (XFS)/exfoliation glaucoma (XFG) and other types of glaucoma.To investigate the association and ethnic heterogeneity of lysyl oxidase-like 1  comparison of the allelic distributions between XFS and XFG, (2) allelic association of LOXL1 SNPs with XFS/XFG, (3) associations in homozygote, heterozygote, and dominant and recessive models, and (4) allelic association with primary open angle glaucoma (POAG).We performed meta-analysis and ethnicity-based subgroup analyses according to published studies. Allele and genotype frequencies of SNPs rs1048661, rs2165241, and rs3825942 between XFS and XFG. The G allele of rs3825942 was the common at-risk allele for XFS/XFG in all populations with a total odds ratio (OR) of 10.89. The total homozygote OR of rs3825942 was 9.06 for XFS/XFG combined, but the total heterozygote OR was not significant. We also found that in the recessive model, the total OR was 14.70. There was no association of the three SNPs with POAG.In total 24 reported articles were retrieved, including Caucasian, African, Japanese, Indian, and Chinese populations. There was no significant difference in the distributions of rs3825942, but not rs2165241 or rs1048661, with XFS/XFG is consistent in different ethnic populations in the recessive model. LOXL1 is not associated with POAG in all study populations.The association of  Glaucoma is one of the leading causes of irreversible blindness worldwide. It is a group of heterogeneous diseases characterized by progressive loss of retinal ganglion cells and degenerative optic neuropathy with or without elevated intraocular pressure (IOP) . Open anMYOC) is a causative gene for POAG and cytochrome P450, 1B1 (CYP1B1) for congenital glaucoma [OPTN) and WD repeat domain 36 (WDR36) do not directly lead to POAG but increase the risk [rs1048661, rs2165241, and rs3825942, in the Lysyl oxidase-like 1 (LOXL1) gene was first discovered in 2007 in Icelandic and Swedish populations by a genome-wide association study [rs2165241 was marginally (p=0.04) associated with POAG in the Icelandic but not in the Swedish population, while rs3825942 and rs1048661 were not associated with POAG in either population [The etiology and pathogenesis of glaucoma are complex and involve multiple genetic and environmental factors. Myocilin  were first found associated with XFS/XFG in Icelandic and Swedish populations. They appeared in most of the reported studies, despite some inconsistencies in their presence [LOXL1  have also been reported [LOXL1 SNPs  with XFS, XFG, and POAG.Meta-analysis is a very useful statistical tool not only to summarize data from individual studies concerning a specific research question but also to identify and analyze the consistency and discrepancies of individual studies or population subgroups. The prevalence of XFS/XFG varies widely among different ethnic populations mainly due to variations in genetic background. Therefore, to ascertain effects due to ethnic differences, meta-analysis of the genetic association of presence ,31. Severeported ,17,31, bPubMed and Embase, using the search strategy (“LOXL1” OR “LOXL” OR “Lysyl oxidase-like”) AND (“glaucoma” OR “exfoliation” OR “pseudoexfoliation”). The cut-off date was December 12, 2009. In total 41 articles were obtained from PubMed and 38 from Embase. Articles meeting the following criteria were included for further data analysis: (1) case-control study, cohort study, or population-based epidemiological survey; (2) reports on the association of three SNPs in LOXL1, i.e., rs1048661, rs2165241, and/or rs3825942, with XFS, XFG, POAG, or/and other types of glaucoma; (3) studies with reported and accessible sample size, allele, and/or genotype frequencies/counts in both patients and controls; and (4) original research articles, not reviews or comments. Repetitive publications, which were ruled out through tracking references, were incorporated as the latest reports. If several different cohorts were reported in the same article, they were treated as independent studies.An internet-based literature search was conducted on The retrieved abstracts and full texts were separately reviewed by two investigators (H.Y.C. and W.F.G.) who also independently performed data extraction and quality evaluation. A third reviewer (L.J.C.) would participate in the review if there was disagreement in the retrieved information. Total consensus of the three reviewers needed to be obtained on all retrieved data. The ethnicity, sample size, age, sex, allele, and/or genotype frequency/counts in both patients and controls were recorded. For studies with no direct data of genotype or allele counts, we calculated the frequency according to the reported data, rounding to the closest integer. The allele counting was also calculated from genotype counting when needed.2 analysis; (2) meta-analysis of the LOXL1 SNPs rs1048661, rs2165241, and rs3825942 was performed to compare the allelic distributions between XFS and XFG; (3) if there were no statistical differences in allelic distributions between XFS and XFG, these two groups were combined. Meta-analyses of the allelic associations of rs1048661, rs2165241, and rs3825942 with overall XFS and XFG were performed; (4) if positive results were obtained from allelic association analysis, genotypic association analyses were performed using different genetic models, including multiplicative models estimating the homozygous odds ratio (OR) and heterozygous OR as well as the dominant model and recessive model; and (5) the allele association of rs1048661, rs2165241, and rs3825942 with POAG was analyzed. All the meta-analyses were performed in subgroupings by ethnicity. In all of the meta-analyses, the ORs were estimated by the fixed or random model according to the heterogeneity test. When the heterogeneity test α was <0.1, a random model was applied, and when the heterogeneity test α was >0.1, a fixed model was applied. Sensitivity tests were performed, but studies with counting data that were computed from frequencies data were excluded.The Review Manager  were used to perform the meta-analysis: (1) The Hardy–Weinberg equilibrium (HWE) of each SNP in the control group of each study was examined by using χLOXL1 SNPs with XFS/XFG in a total of 3,068 cases and 20,363 controls from 22 study cohorts  in Caucasian, 18.72  in Japanese, 14.23  in Chinese, and 4.17  in Indian populations. The overall OR was 10.89  for the four ethnic populations  in Caucasian populations, suggesting that the T allele was the at-risk allele. The OR for the T allele was 0.13  in Japanese populations, indicating that the T allele was a protective allele  in Caucasian, and 0.03  in Japanese populations but not significant in Chinese and Indian populations  was 9.06  for the G allele of rs1048661  for the T allele of rs2165241 , two in normal tension glaucoma (NTG), and two in pigment dispersion syndrome and pigmentary glaucoma. However, none of these studies identified a significant association between the LOXL1 SNPs and these subtypes of glaucoma .Apart from XFS/XFG and POAG, there was also one study reporting the glaucoma . In our LOXL1 gene with XFS and/or XFG has been shown across different populations; however, there were several different phenotypes in these studies. Thus, it is necessary to test whether XFS and XFG are of genetic homogeneity. In the first part of this present meta-analysis, the distribution profiles of the LOXL1 SNPs between XFS and XFG were evaluated. By reviewing all the reported association profiles with meta-analysis, no statistical difference in any population group or overall study subjects was identified for any of the three SNPs . All the p values were >0.05.Exfoliation glaucoma (XFG), a consequence of exfoliation syndrome (XFS), is the most common form of secondary open angle glaucoma. Approximately 25% patients with XFS present with increased IOP, and one-third have developed glaucoma . To dateLOXL1 SNPs are not heterogenic in XFS and XFG, we combined these two phenotypes in our analysis. Our results also suggested that the LOXL1 gene may contribute to disease onset of the exfoliation disease rather than just increased IOP. However, the current meta-analysis cannot prove this hypothesis because the data are from retrospective case-control studies. Further prospective longitudinal studies are warranted.Since LOXL1 and XFS/XFG, the three SNPs  have been investigated together [LOXL1 SNPs between XFS and XFG. Therefore, the two disease groups were combined in the second part of the meta-analysis of genetic association in subpopulations. We found that the distribution was similar within each individual ethnic group for all three SNPs, rs1048661, rs2165241, and rs3825942. However, the allelic distributions of Japanese and Caucasian populations are reversed for rs1048661 and rs2165241. The T allele of rs2165241 and the G allele of rs1048661 are the at-risk alleles in Caucasians, with an OR of 3.39 and 2.35, respectively. In contrast, the two alleles are protective in the Japanese population, with an OR of 0.13 and 0.03, respectively. All the ORs are statistically significant with p values <0.00001. Therefore, it is more likely that SNPs rs1048661 and rs2165241 are not directly implicated in the pathogenesis of XFS/XFG. In contrast, the distribution of SNP rs3825942 followed a similar pattern in all three ethnic groups. The G allele was the at-risk allele, and the OR was 9.30, 18.72, 10.97, and 4.17 in Caucasian, Japanese, Chinese and Indian populations, respectively. The total OR was 10.75 (95% CI 7.08–16.31). Our finding suggests that rs3825942 is the common disease-associated polymorphism across different populations and may have functional impacts on the LOXL1 protein and contribute to the pathogenesis of XFS/XFG. Moreover, in the third part of this meta-analysis, we found that the OR for the homozygous genotype of rs3825942 is 9.42, while the OR for the heterozygote is not statistically significant. Moreover, the OR in the recessive model was the highest among different genetic models (OR=14.55). Therefore, it could be inferred that rs3825942 plays a role in a recessive pattern; however, what functional role rs3825942 played in the pathogenesis of XFS/XFG remains unclear. LOXL1 is located in 15q22, encoding a member of the lysyl oxidase family. The LOXL family is an extracellular copper-dependent amine oxidase that involved in the first step of the formation of cross-links in collagen and elastin. Therefore, sequence variation in LOXL1 may influence the function, synthesis, and subsequent deposition of the extracellular tissues [rs3825942 is located in the first exon of LOXL1 and leads to a nonsynonymous amino acid change from glycine to asparagine at position 153 (G153D). However, functional prediction using in silico programs Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant From Tolerant (SIFT) suggested that the amino acid substitution is benign and tolerated (data not shown). The exact functional effects of this substitution remain to be further investigated.In most reported studies on together -23,25-28together ,20,26,28together -23,25-28together ,36. Ther tissues . SNP rs3LOXL1 SNPs with other types of glaucoma, including POAG, NTG, pigmentary glaucoma, and PACG, were reviewed in this meta-analysis. However, no significant association was found between POAG and LXOL1 SNPs after merging the results from 13 cohorts. Therefore, it is likely that LOXL1 is not implicated in the primary open angle glaucomatous mechanism. Associations between LXOL1 SNPs and NTG, pigmentary glaucoma, or PACG were also negative, although the number of articles included in this meta-analysis was limited. The lack of association between LOXL1 and primary glaucoma has provided evidence supporting that LOXL1 is linked to the pathogenesis of the exfoliation syndrome but not the direct genetic cause of IOP elevation and subsequent glaucoma.The association of LOXL1 between XFS and XFG has been confirmed. The genetic effect of rs3825942 is similar in different populations in a recessive genetic model. We detected inconsistencies in the effect of rs1048661 and rs2165241 between Caucasian and Japanese populations. Our results also revealed that the LOXL1 gene is not a susceptibility gene of other types of glaucoma other than XFG. Further genetic studies are required to unravel the discrepancy in LD patterns of the LOXL1 gene among different populations.In summary, by using meta-analysis the genetic homogeneity of


P < .05) decrease of CD1a+ Langerhans cells (LCs) in DD, compared to PV, LRP, and NSk, and of CD123+ plasmacytoid dendritic cells (pDCs), compared to PV and LRP. We hypothesize that the genetic damage of keratinocytes might result in a loss of some subsets of dendritic cells and, consequently, in an impaired local immune response, which might worsen the infections that inevitably occur in this disease.Cell-mediated immunity is considered to be normal in Darier's Disease (DD), an inherited skin disorder complicated by skin infections. To date, there are no investigations on the local inflammatory infiltrate in DD skin lesions. In this immunohistochemical study we characterized and quantified it, making comparisons with two other inflammatory skin disorders, that is, pemphigus vulgaris (PV) and lichen ruber planus (LRP), and with the normal skin (NSk). We found a significant ( Darier's disease (DD) is an infrequent autosomal dominantly inherited skin disorder caused by mutations in the ATP2A2 gene, which encodes a calcium pump highly expressed in epidermal keratinocytes . DD is h The cell-mediated immunity of DD patients was usually found to be normal . There i We herein performed an immunohistochemical analysis and quantified the inflammatory cell infiltrate in skin biopsies of DD patients, aiming at investigating a possible pathogenic involvement of the local immune response in the disease. For comparison, lesional skin of patients affected by pemphigus vulgaris (PV), as well as cutaneous lichen ruber planus (LRP) was also examined.We analyzed 16 skin biopsies of patients with Darier's disease (DD), ranging in age from 25 to 50 years . Nine patients had multiple affected relatives, while 7 individuals represented sporadic cases. Six patients were at the first DD attack, while 10 presented multiple attacks. Skin biopsies were performed in recently-appeared, uncomplicated lesions, on grouped keratotic papules of the abdomen and trunk. There was not any personal nor family history of autoimmune disease. For comparison, 14 patients with pemphigus vulgaris (PV), ageing 30–55 years, and 10 with lichen ruber planus (LRP), ageing 32–51 years, were also included in the study. The diagnosis was previously established by clinical and histopathological examination, and, in PV group, also by immunofluorescence studies. As negative control, we examined normal skin (NSk) excised from the abdomen of 12 healthy subjects who underwent aesthetic surgery. Informed consent was obtained from all patients, and the study, performed according to local ethical guidelines, was approved by the local ethical committee.μm-thick sections, cut from formalin-fixed, paraffin-embedded skin biopsies, were investigated immunohistochemically.4-β12; Menarini, Florence, Italy; dilution 1 : 50), antiCD20 , antiCD8 , antigranzyme B , antiCD25  antiFOXP3 , antiCD1a , antiCD68 , and antiCD123 . Antigen retrieval was obtained either with Wcap buffer , using the Ultravision Detection System antiPolyvalent HRP ; or with microwave pretreatment, using the Ultravision LP Detection System AP polymer (LabVision). Sections were then incubated with the antibodies for 60°C min at room temperature. For staining development, we used either diaminobenzidine or fuchsin , as chromogens. We also performed a double staining with antibodies against CD25 and FOXP3 to identify the CD4+CD25+FOXP3+ subset of regulatory T cells (Tregs), as previously described [In all cases, leukocyte subsets were characterized using a panel of antibodies, including antiCD3 , antiCD4 . CD4+, CD8+, granzyme B+, CD20+, CD25+/FOXP3+, and CD68+CD123+ cells were counted and recorded as a percentage of the total inflammatory infiltrate. CD1a+ LCs were counted separately in the epidermis and dermis, and given as mean number/1 HPF. Mean values + SD of immunostained cells were statistically compared among DD, PV, LRP and NSk by the nonparametric Kruskal-Wallis test. The significance level was chosen at P < .05.In all samples, immunohistochemical staining was quantified visually by two independent observers (C.M. and G.M.) in at least 5 randomly chosen high power fields (HPF)  were identifiable based on both their plasmacytoid morphology and positivity for CD123. CD123 was also expressed by endothelial cells, and therefore they served as an internal positive control for the antiCD123 antibody . CD123+ P significance are given in Quantitative descriptive data of results and  Mean percentages of CD25+FOXP3+ Tregs were significantly higher in DD than those in PV and LRP; they did not differ between DD and LRP. In DD, epidermal and dermal CD1a+ LC mean number, as well as CD123+ DC percentage were significantly lower than those in PV, LRP and in the NSk. In PV, epidermal and dermal Cd1a+ LC mean numbers were significantly lower than those in LRP. In PV epidermal CD1a+ DC mean number was also significantly lower than those in NSk.CD123+ DC mean percentages were significantly lower in DD than those in PV and LRP; they were also significantly lower in PV than those in LRP. Comparison with the NSk was limited to CD1a+ LCs, due to the virtual absence of lymphocytes and plasmacytoid cells in controls.A genetic basis corroborate Darier's disease (DD), an autosomal dominant genodermatosis, with sporadic cases reported, in which an aberration localized to the 12q23-24.1 region of chromosome 12 has recently been discovered, leading to a defect in a keratinocyte calcium pump, which, in turn, results in a loss of keratinocyte adhesion and consequent acantholysis, typical of the disease –13. DD a The observation that a usually mild inflammatory infiltrate is, however, detectable also in of recent onset, uncomplicated DD skin lesions, prompted us to characterize it by immunohistochemistry, in order to investigate cell composition of the local immune response; we also made comparison with inflammatory infiltrate of PV and LRP skin lesions. In the latter conditions, our results are in line with the literature –13. To o It is known that UV irradiation exacerbates DD cutaneous lesions , 3: this Although, at least in experimental studies on mice, a dermal subset of LC rather than epidermal LC was critical in the UV-induced immunosuppression , many st In our study, we also found low percentages of dermal pDCs, as decorated by CD123 antibody, which labels interleukin-3 receptor alpha chain protein, that is expressed at high levels on the surface of pDCs, allowing for their identification .  The DC system consists of two main subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) , and at  In our experience, also in PV, we found impaired numbers of both CD1a+ LCs and CD123+ pDCs. We, in fact, found that in PV, CD1a+ LCs were lower than those in NSk and LRP, and that CD123+ pDCs were lower than those in LRP, although both DC subsets were significantly higher than those in DD. PV is also characterized by acantholysis, overwhelmingly due to autoimmune mechanisms; it might be hypothesized an additional contribute of damaged keratinocytes to the decrease of some DC subsets.  We, furthermore, observed a different pattern of CD123+ DC distribution , which is supposed to have pathogenic implications in distinct diseases .  In line with other studies , we regi Tregs are a phenotypically heterogeneous population of lymphocytes which are critical for the regulatory mechanisms of immune system, being responsible for the induction and maintenance of immune tolerance. Their numbers, and, overall, their suppressive functions were found reduced in autoimmune as well as in allergic diseases , 40, 41. FOXP3 is considered the best marker for the most important among Treg subsets, the CD4+CD25+FOXP3+ cells, that constitute approximately 5–10% of CD4 T cells in man . The sceIn conclusion, our present study provides direct evidence for a numerical impairment of epidermal CD1a+ LCs and dermal CD123+ pDCs in skin lesions of DD, which may be pathogenetically determinant for the development of the inflammatory reactions that complicate the disease. To our knowledge, this investigation represents the first description of an altered composition of the cellular inflammatory infiltrate in this genetical disorder, suggesting that an impaired local immune response may contribute to the progression of the disease. The dramatic loss and/or absence of some skin DC subsets, such those that we observed, might drive the pathogenic events leading to DD skin complications, suggesting novel therapeutic approaches that could open new horizons for the treatment of this notoriously poorly responsive disease and for a better management of patients. Further functional studies, as well as expanding investigation on other cells and cytokine mediators of immune response and inflammation are, of course, mandatory to better elucidate the impact of epidermal LC and dermal CD123+ DC loss in DD lesional microenviroment.


Cutaneous leishmaniasis (CL) is a disfiguring disease that confronts clinicians with a quandary: leave patients untreated or engage in a complex or toxic treatment. Topical treatment of CL offers a practical and safe option. Accordingly, the treatment of CL with WR279,396, a formulation of paromomycin and gentamicin in a hydrophilic base, was investigated in a phase 2 clinical study in Tunisia and France.A phase 2, randomized, double blind, vehicle-controlled study was conducted to assess the safety and efficacy of topical WR279,396 when applied twice a day for 20 days as treatment for parasitologically confirmed CL. The study protocol established the primary efficacy end point as complete clinical response (CCR) defined as 50% or greater reduction in the ulceration size of an index lesion by day 50 (D50) followed by complete re-epithelialization by D100, and no relapse through D180.Leishmania major was identified in 66 of 68 isolates typed (97%). In the intent-to-treat population, 47 of 50 WR279,396 treated participants (94%) met the definition of CCR, compared with 30 of 42 vehicle-placebo participants (71%) [p = 0.0045]. Erythema occurred in 30% and 24% of participants receiving WR279,396 and placebo, respectively [p = 0.64]. There was no clinical or laboratory evidence of systemic toxicity.Ninety-two subjects were randomized. L. major CL, and offers great potential as a new, simple, easily applicable, and inexpensive topical therapy for this neglected disease.Application of WR279,396 for 20 days was found to be safe and effective in treating NCT00703924ClinicalTrials.gov  Leishmania) that creates disfiguring sores, and affects more than one million persons (mainly children) each year. Treating lesions with a cream—instead of with injections as currently done—would greatly improve the well-being of affected patients. No cream formulation that would be efficient and would not create important skin irritation has been identified yet. Here, we tested a new cream formulation  containing paromomycin and gentamicin, two members of a well-known family of antibacterial antibiotics (aminoglycosides). Injectable paromomycin is efficient in other forms of the disease . This was a carefully monitored study (phase 2) involving mainly children in Tunisia and France. The cream was applied twice a day for 20 days. The proportion of patients treated with the paromomycin-containing cream (active formulation) that cured (94%) was higher than that observed (71%) in patients treated with a cream that did not contain the active product (placebo formulation). Local irritation affected less than one-third of the patients and was usually mild. This new cream formulation was safe and effective in treating cutaneous leishmaniasis, thereby providing a new, simple, easily applicable, and inexpensive treatment for this neglected disease.Cutaneous leishmaniasis is due to a small parasite ( The incidence of cutaneous leishmaniasis (CL) globally is 1.0–1.5 million cases annually Intralesional injections are painful, and they are difficult to administer to children, to patients with multiple lesions, or when lesions are located on the extremities Leishmania multiply, and offer the potential to be highly effective therapeutics.Topical therapy of CL is an approach that is potentially efficient, practical and safe In topical preparations, paromomycin is a component of two antileishmanial products currently available outside the International Conference of Harmonization zone . The first, developed by El-On L. major (MON-4), L. amazonensis, L. mexicana, and L. panamensis strains in 100% of the mice without subsequent relapse L. panamensis), WR279,396 was well tolerated and shortened cure time, but had no effect on the overall cure rate at six months WR279,396 is a hydrophilic formulation of paromomycin 15% plus a second aminoglycoside (gentamicin 0.5%), that was developed in order to exploit the antileishmanial efficacy of the aminoglycosides while eliminating the potential for the skin irritation caused by MBCL. In this preparation, the addition of gentamicin has been shown to increase the antileishmanial efficacy of paromomycin in rodents L. major.Herein, we report the results of a recently completed phase 2, randomized, double blind, vehicle-controlled trial in Tunisia and France to assess the efficacy and safety of topical WR279,396 administered twice a day for 20 days for the treatment of patients with CL caused by L. major MON-25 is endemic, and travelers returning to Paris from L. major-endemic areas in North and Sahelian Africa, who had skin lesions that were suspected to be CL. Criteria for inclusion were age between 5 and 75 years, the presence of parasitologically confirmed CL, lesions that were primarily ulcerative  and measured ≥1 cm2 and ≤5 cm2. Criteria for exclusion were history of known or suspected hypersensitivity or idiosyncratic reactions to aminoglycosides; previous use of antileishmanial drugs (within 3 months) or nephrotoxic or ototoxic drugs; prior diagnosis of leishmaniasis; more than 5 lesions, or a lesion in the face that in the opinion of the attending dermatologist could potentially cause significant disfigurement; significant medical problems as determined by history or laboratory studies; breast feeding and pregnancy. Participants also had to have normal Romberg tests and no relevant findings on baseline audiometry. In cases where the participant presented more than one lesion, investigators treated all lesions as per protocol with the same blinded study treatment as the index lesion.Eligible participants were from the Sidi Bouzid governorate , where The study was a phase 2, randomized, double blind, vehicle-controlled, multi-center trial. Participants were randomized in a 1∶1 allocation ratio to receive either WR279,396 or placebo-vehicle, each of which was applied twice daily for 20 days and covered with an occlusive dressing . Investigators, who were blinded to whether participants received WR279,396 or placebo-vehicle, evaluated lesions for clinical response on D20 , D50 , D100, and D180.A sequence of genuine random numbers for the randomization procedure was obtained from the “fourmilab.ch/hotbits” website by a member of the Department of Chemical Information, Walter Reed Army Institute of Research, Silver Spring, Maryland and purged of duplicates. The random numbers are generated by a process which takes advantage of the inherent uncertainty in the quantum mechanical laws of nature. Specifically, they are generated by timing successive pairs of radioactive decays detected by a Geiger-Müller tube interfaced to a computer. This process is better than the pseudo-random number algorithms typically used in computer programs. The randomization of the study drugs was done by an independent group, Fischer BioServices, Rockville, Maryland a contractor to The U.S. Army Medical Research Acquisition Activity (USAMRAA), Ft. Detrick, Maryland.The study protocol established the primary efficacy end point as complete clinical response (CCR), defined as complete reepithelialization  of the index lesion by D50 or a >50% reepithelialization by D50 followed by complete reepithelialization on or before D100 with no relapse ever having occurred from D50 through D180. Relapse was defined as an increase in the area of ulceration relative to the previous measurement. Participants who did not complete the 180-day period of observation were considered to have failed to achieve CCR because relapse could not be fully assessed. The index lesion was defined as the uppermost, primarily ulcerative, parasitologically positive lesion on the body (excluding the ears) or, if two lesions were equally uppermost, the left uppermost primary ulcerative lesion. The secondary endpoint was the safety and tolerance of WR279,396.2 of CL lesion at a primary health facility in Tunisia and at the Medical Center of the Institut Pasteur in Paris. Each CL lesion was cleaned with soap and water and sterile 0.9% saline, and then dried using sterile USP Type VII Gauze sponges before application of study drug. Next, medical personnel dispensed study drug directly onto the ulcer from a pre-loaded 1 ml syringe without a needle, and spread drug over the ulcer using the finger of a disposable glove so as to penetrate even under the ulcer's borders. Study drug was to remain undisturbed  for 4 hours after each application, so the adhesive polyurethane film dressing, Tegaderm, was applied over the top of the lesion following drug application. Investigators observed each participant for 30 minutes after application of study drug. Lesions and surrounding skin were evaluated for pain, erythema, and edema each day that the topical creams were administered and at follow-up study visits. The participants were also observed and questioned daily for the occurrence of systemic side effects  using a standardized questionnaire. Diminished hearing was verified with the Danplex S42 audiometer . Clinical and laboratory evidence of side effects was determined on D10 and D20 by changes from baseline in serum creatinine, hearing, and Romberg tests. A Digmatic Caliper, Mitutoyo Corporation, model No. CD-6CS with a resolution of 0.01 mm and an accuracy of ±0.002 mm was used to measured lesions size. Lesions were measured by a trained investigator that followed a Study Specific Procedure (SSP-279396-01-003) in two perpendicular directions; in its greatest dimension, and at 90 degrees to the first measurement. Patients were not given incentives to come back for follow-up visits; patients were actively followed-up.The primary performing Institutions were the Institut Pasteur in Tunis, Tunisia, and the Medical Center Institut Pasteur, in Paris, France. Investigators measured all lesions in two perpendicular directions and took photographs at the following time points: prior to therapy, at the end of therapy (D20), and at 30 days (D50), 80 days (D100), and 6 months (D180) after the end of therapy. Medical personnel applied study drug  twice daily for 20 days to all CL lesions present at baseline at a dose of 0.05 ml per 1 cmL. major is self-limiting and heals without treatment after several months. Furthermore, the trial allowed participants whose condition worsened to withdraw from the study and receive standard therapy. In addition vehicle application provided the following advantages: (i) protection against bacterial infection by keeping the lesion(s) clean and occluded; (ii) direct access to the medical team that performed the medical history, physical exam, dermatology exam, and laboratory test ; and (iii) complete parasitological diagnosis.Before entry into the study, investigators obtained written informed consent from all participants or, for pediatric participants, from parents/guardians. Comparison of WR279,396 to placebo-vehicle was justified for several reasons: CL caused by The study protocol , case report form, and SOPs were approved in the United States by the Walter Reed Army Institute of Research, Scientific Research Committee. A second level review of the protocol, consent form and all amendments was conducted by the Human Subjects Research Review Board (HSRRB), Commanding General, U.S. Army Medical Research and Materiel Command (USAMRMC), the Medical Ethical Committee of the Institut Pasteur de Tunis, Tunisia, and the Consultation Committee for the Protection of Individuals in Biomedical Research at Hospital Tarnier-Cochin, Paris, France. The study was conducted in accordance with Good Clinical Practice (GCP) under an Investigational New Drug (IND) application submitted to FDA. The Direction de la Pharmacie et des Médicaments, Ministère de la Santé Publique, Tunisia, and the Agence Française de Sécurité Sanitaire des Produits de Santé were informed of the trial. This study was conducted in accordance with ethical principles that have their origins in the Declaration of Helsinki and the Belmont Report. The Quality Assurance Office of the U.S. Army Medical Materiel Development Activity monitored the study.WR279,396 is an off-white to yellowish, thick cream containing 15% (w/w) paromomycin-sulfate  and 0.5% (w/w) gentamicin-sulfate (Schering) as active components. Study drugs were manufactured by the University of Iowa, College of Pharmacy under Good Manufacturing Practice (GMPs). The placebo consisted of the vehicle without the active components and trace amounts of coloring agents to match the appearance and maintain the blind.Leishmania amastigotes in material obtained from CL lesions. Iso-enzyme Each lesion to be evaluated for efficacy was aspirated and/or scraped and/or biopsied. Proof of infection was documented through either the demonstration of motile promastigotes in aspirate cultures or the microscopic identification of The protocol calculated a sample size of 50 participants per group with 80 percent power and a Type I error rate of 5 percent to detect a 30 percent difference in the proportion of participants achieving CCR, assuming a CCR proportion of 35 percent in the placebo-vehicle group and 65 percent in WR279,396 participants, with a 5% expected rate of loss to follow-up.Analyses included all randomized participants under the intention-to-treat principle and the randomization was coordinated between the two clinical sites. Continuous data were compared using the Wilcoxon rank-sum test and categorical data were compared using the Fisher's exact test. StatXact version 7  was used to calculate 95% exact confidence intervals (CIs) of the difference in the proportion achieving CCR with the option to compute a CI on the difference of two binomial proportions based on the standardized statistic and inverting two one-sided tests. A log-rank test was used to compare time to reepithelialization without relapse. Because the trial collected data on clinical response only at discrete time-points, namely the D20, D50, D100, and D180 visits, the time-to-event analysis grouped reepithelialization times according to the visit at which investigators observed the event. To adjust for baseline differences, a linear model for the proportion of participants achieving CCR was fit for each baseline variable of interest with covariates for treatment group and the baseline variable. To examine whether the effect of WR279,396 varied between subgroups, we calculated the Breslow-Day test for homogeneity of the odds ratioL. major in 17 participants and L. infantum in one participant, who was from the French site. L. major isolates from the Paris site were MON-74, MON-26, MON-25, -, and all L. major isolates from Tunisia were MON-25. Fifty isolates from Tunisia were tested using PCR, which identified L. major in 49 participants and L. tropica in one participant. In total, L. major was identified in 66 of 68 isolates typed (97%). With one exception, applications of study drugs were conducted according to the protocol. In this one case, treatment was stopped after only 12 applications (6 days) due to skin irritation and conjunctivitis that resulted from inadvertent contact of study drug to the eye while sleeping. However, this participant's lesion rapidly improved without any subsequent therapy allowing follow-up evaluations to be conducted as per protocol. All 92 participants received study drug . Except for 2 participants, who withdrew voluntarily from the study during or following the 20-day treatment period to receive alternative therapy  were screened, of whom 92  underwent randomization; 50 were assigned to the WR279,396 group and 42 to the placebo-vehicle control group . The stu therapy , no partOverall, the two treatment groups were similar in baseline demographics and disease characteristics . A greatAmong the 50 WR279,396-treated participants, 47 (94%) met the definition of CCR, compared with 30 of the 42 placebo-vehicle participants (71%) (p = 0.0045), resulting in an estimated difference of 23% in favor of WR279,396 participants . The reasons for failure in the 3 participants treated with WR279,396 were: (i) two participants had less than 50% reepithelialization at D50 , (ii) one participant was considered a treatment failure in the intent-to-treat analysis because he requested to be withdrawn and switched to intralesional antimony. The single WR279,396-treated participant who completed only 6 days of treatment cured despite such a short course. Among the 12 placebo-vehicle participants who failed, in 8 participants the ulcer either increased in size or decreased by less than 50% by D50, 3 participants relapsed, and 1 who withdrew voluntarily from the study was lost to follow-up and considered a failure in the intent-to-treat analysis.Clinical relapse of the index lesion following completion of the 20-day course of treatment was observed in 3 participants, all from the placebo-vehicle group. No clinical relapse of the index lesion or non-index lesion occurred in the WR279,396 group.Rather than restrict the analysis to the index lesion, we compared CCR by treatment group for all treated lesions, i.e., patient cure. Response status for the 28 (56%) WR279,396 participants and 26 (62%) placebo-vehicle participants whose index lesion was the only treated lesion are identical to that of the primary analysis. For the 38 participants who investigators treated at multiple lesions, participants are classified as having CCR only if all lesions met the definition of CCR. When considering all treated lesions, results were similar to the categorization for index lesions .In summary, regardless of the criterion used to analyze the data, the proportion of lesions and participants with a positive outcome by D50 was significantly greater in the WR279,396-treated group than in the placebo-vehicle group.L. major species. One was infected with L. infantum and the other with L. tropica. Self-healing occurs less frequently with both species than with L. major. Both participants were in the WR279,396 group and achieved CCR, although the participant infected with L. infantum received only 6 days of treatment.Two participants were confirmed to be infected with a non-2), location (upper and lower body), and age (<60 and ≥60 days) of lesions. After adjustment singly for each baseline factor, the statistical benefit of WR279,396 over placebo-vehicle remained (We explored CCR in subgroups defined by baseline characteristics including number (1 and >1), index lesion area , or engage in a complex or toxic treatment for this disfiguring, but non life-threatening disease. In this study, we found that WR279,396 was well-tolerated and induced complete clinical response (CCR) in a significantly greater percentage of participants compared to placebo-vehicle in participants with CL due to L. major. These results raise a strong possibility that we may exit from this old quandary of how best to manage patients with L. major CL. Of the 49 participants treated in the WR279,396 arm, only 1 (2%) failed to achieve complete reepithelialization of his lesion in less than 2 months. If the efficacy of WR279,396 can be reproduced in subsequent phase 3 clinical studies, complex therapeutic decisions in L. major CL may become the exception rather than the rule.For decades, clinicians caring for patients with CL have been confronted with a difficult choice: either leave patients untreated  WR279,396 continued to demonstrate an excellent safety profile with very few local and no systemic adverse events observed. This trend was similar to our previous experience with this product compiled from pre-clinical, Phase 1, and two earlier phase 2 studies in the New World L. major MON-25, data collected thus far indicate that WR279,396 will likely be broadly effective against a wider variety of leishmania species. Several key observations support such optimism. First WR279,396 was efficient not only in L. major MON-26 L. amazonensis, L. mexicana, and L. panamensis in infected mice L. panamensis in humans in Colombia L. tropica is very sensitive to paromomycin in vitro L. tropica focus of Turkey, the paromomycin+MBCL formulation induced a 37.5% cure rate at 4 weeks, suboptimal, but significantly higher than the 0% cure rate in oral ketoconazole-treated patients In addition to the promising efficacy observed in this study against L. major CL, and offers great potential as a new, simple, easily applicable, and inexpensive topical therapy for this neglected disease L. major CL in North Africa, Sahelian Africa, and the Middle East, involves tens to hundreds of thousands of people each year, many of whom are children The application of WR279,396 for 20 days was found to be safe and effective (94% cure rate) in treating Checklist S1CONSORT Checklist(0.06 MB DOC)Click here for additional data file.Protocol S1(6.44 MB PDF)Click here for additional data file.


Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens.Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2.The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants. Arginine is a precursor for the synthesis of proteins, creatine, agmatine, and nitric oxide (NO). It further plays an essential role in ammonia and bicarbonate detoxification, and stimulates the secretion of growth hormone, prolactin, insulin, and glucagon. Arginine is also a 'conditionally essential' amino acid, meaning that endogenous arginine production covers metabolic requirements in healthy, unstressed individuals, but becomes an essential amino acid under conditions of increased need, e.g. growth or tissue repair, or in catabolic states such as sepsis and starvation.In the adult, endogenous arginine biosynthesis is an inter-organ 'affair': the net production of citrulline occurs almost exclusively in the enterocytes of the small intestine , also inIn prematurely born human neonates, hypoargininemia is frequently observed  and hypoA total of 89 samples were included in the study Table . Formali2O2 in PBS. After blocking with TENG-T  plus 10% goat serum, sections were incubated overnight at room temperature with the first antibody, washed in 0.5 M Na-acetate and incubated with an alkaline phosphatase- or peroxidase-coupled secondary antibody for 60 to 90 minutes. Sections were developed with NBT/BCIP  or DAB . An incubation without primary antibody served as negative control for all incubations [see Additional file Tissue was fixed in 4% formaldehyde and embedded in paraffin following standard protocols. 5–7 μm-thick sections were used. Section thickness does not affect immunohistological staining intensity, since antibodies only bind to the surface of the sections . As an aThe following rabbit primary antibodies were used: ASS  ; ARG1 anVillus epithelium was scraped off fresh duodenal resection material and lysed in SDS-PAGE sample buffer. After separation of samples on 10% SDS-polyacrylamide gels and blotting to PVDF membranes, proteins were visualized with antisera to ARG1 (1:200); ARG2 (1:500); NOS1 (1:2500); NOS3 (1:2500); α-SMA, (1:1000). The appropriate secondary horse-radish peroxidase-coupled antibodies were used at a dilution of 1:10,000. The signal was amplified using the chemiluminescent Super Signal West Pico reagent  and pictures were taken with a LAS3000 imaging system (Fujifilm). α-smooth muscle actin was used to determine the contribution of the submucosa to the scrapings.Sections were scored for staining intensity in random sequence by 3 investigators with the readers of the slides blinded. Villi and crypts were scored separately. The intensity of ASS, OAT and ARG2 staining in enterocytes was expressed on a scale of 0–3  relative to their expression in the ganglia of the myenteric plexus, which always contained both strongly and weakly positive neurons for ASS, OAT, and ARG2. Expression in the strongly staining neurons was set at 3. Samples without ganglia  were compared to simultaneously stained samples that did contain ganglia to assign an intensity score from 0 to 3. Because CPS expression in the small intestine is restricted to enterocytes, all samples were stained simultaneously and then compared to each other using the same scale of 0–3 as described above. There was never more than one scoring unit difference between the 3 investigators.We used box-plots to visualize distribution and developmental changes of enzyme expression in enterocytes. Two-way analysis of variance (ANOVA) on rank-transformed data showed age-group and structure (crypt or villus) differences as well as age and structure interactions per enzyme. Sex  was not a factor associated with differences in enzyme expression. To pin-point which age-groups and crypts or villi differed, non-parametric one-way ANOVA  tests were performed between age-groups per structure. Multiple comparison (Mann-Whitney) tests between structures per age-group were performed, when the null hypothesis was rejected. P values were considered significant if < 0.05.The earliest sample studied was a small intestine of a 14-week-old fetus, whereas the oldest sample was from a 79 year-old patient. Most of the samples examined were from patients younger than 7 years. At 14 weeks of gestation, all structural components of the epithelium, enteric nerves, and smooth muscle layers have formed ,32. SpecCPS protein was exclusively found in the enterocytes. Prior to birth (group 1), CPS expression was uniformly high in the enterocytes of both crypts and villi Figure . BetweenOAT expression in the epithelium was uniform prior to birth. The youngest samples that could be investigated were from gestational week 23 Figure . Fetal eWith the exception of the age groups 5 and 6 3–7 years), ASS protein accumulated to a much higher concentration in the enterocytes on the villi than in the crypts (P < 0.014), especially before 3 years of age (P < 0.004). This pattern was already found in gestational week 14, where epithelial expression of ASS was still low Figure . From we–7 years,ARG1, the major arginine-catabolizing enzyme, was not detectable immunohistochemically in the epithelium of the small intestine at any of the ages investigated. Western-blot analysis of samples from an adult duodenum (age: 60 years) were also negative for ARG1, confirming the immunohistochemical findings.Western blots of the adult duodenum showed, instead, expression of the mitochondrial isoform of arginase Figure . We therOver-staining of sections with NOS1 yielded weak epithelial staining. Enterocytes of the human small intestine express high levels of NOS1 mRNA, but protein was not detected . In agreWe found weakly positive staining of villus epithelium with our NOS3 antibody. NOS3 mRNA is not expressed in enterocytes . We, theSerial sections of the distal duodenum at postnatal day 1 demonstrated that the highest expression of CPS, ASS, and OAT was found in the enterocytes covering the villi Figure . CPS-, OSingle neurons in the ganglia of both the myenteric and the submucosal plexus stained very strongly for ASS, OAT, ARG2, and NOS1, whereas other neurons in the same ganglion stained weaker or not at all. The strongly OAT-positive neurons were also positive for ASS and NOS1 Figure  and 8F. The endothelium of the small arterioles in the submucosa and serosa always stained positive for the presence of ASS Figure , arrow aSingle cells in the lamina propria that were strongly positive for NOS1 were also positive for CD68, a marker for macrophages, but not for CD3, CD20, CD138, or CD1A (lymphocytes and dendritic cells (not shown). Germinal centers within lymphocyte aggregates also stained positive for NOS1 (not shown).The main result of this study is the observation that the epithelium of the perinatal human small intestine expresses the enzymes that exert control over the biosynthesis of arginine from proline, bicarbonate, and ammonia, viz. CPS, OAT and ASS , but markedly differs from that in rodents. In both rats and mice ,6, ASS eThe net production of ornithine by the small intestine is a prerequisite for citrulline and arginine biosynthesis . The sevTogether, our findings indicate that the human fetal intestine has acquired the potential to produce arginine at 23 weeks of gestation and probably as early as 14 weeks, that is, well before fetuses become viable outside the uterus. Furthermore, NOS1, NOS3 and their downstream target soluble guanylate cyclase (not shown) were expressed at term levels in fetuses of ~23 weeks of pregnancy. This indicates that at this time in gestation the fetus not only has the potential to synthesize arginine, but also to use it for NO and cyclic GMP production. Hypoargininemia, nevertheless, often develops in preterm infants, in particular if they are maintained on total parenteral nutrition ,6,15, anThe neurons of the myenteric plexus abundantly express OAT, ASS, and NOS1. Since the neurons do not express CPS and, therefore, cannot synthesize citrulline from ornithine, OAT most likely functions to produce glutamate, while ASS probably functions to (re-)synthesize arginine from citrulline . It is cst to 2nd order (out of 4 size categories) are considered to be the major sites of resistance and blood flow regulation of the gut [st order (largest) arteries. Furthermore, the downstream target of NO, soluble guanylate cyclase, was found in the smooth-muscle layer of arterioles only (not shown), suggesting that these 2nd order vessels materially contribute to the peripheral vascular resistance in the gut. A recent study showed a deficiency in NO production but not in NOS3 expression as judged by immunohistochemistry of intestinal arterioles of patients with NEC [The constantly fed state of the neonatal small intestine causes absorptive hyperemia, that is, a high intestinal blood flow and a low intestinal vessel resistance . NO prod the gut , we did with NEC , supportwith NEC , but theThe age-dependent postnatal decline in the expression of ASS in the enterocytes of the human small intestine is most pronounced between 3 and 5 years of age. The time course in ASS expression in the developing human gut resembles that in piglets, where activity is highest during the suckling period, declines to low levels around weaning  and theThere is a remarkable similarity in the developmental timing of the decline in expression of ASS and lactase-phlorizin hydrolase , another small-intestinal enzyme that is closely associated with breast-feeding. In rodents, lactase expression declines to undetectable levels at weaning , whereasOur data show that CPS, ASS, and OAT expression is strong in the enterocytes of fetuses, neonates, infants and toddlers. Humans, therefore, resemble other mammals in that the enterocytes of their small intestine are major producers of arginine during the suckling period. Although the relative deficiency of arginine in milk seems to underlie this temporary function of the gut, the intestinal or systemic functions that require the arginine that is produced in the gut remain to be delineated. We submit that relaxation of the circular smooth muscle layer of the muscularis propria ,66 and tARG: arginase (E.C. 3.5.3.1); ASS: argininosuccinate synthetase (E.C. 6.3.4.5); CPS: carbamoylphosphate synthetase (E.C. 6.3.4.16); NOS: nitric oxide synthase (E.C. 1.14.13.39); OAT: ornithine aminotransferase (E.C. 2.6.1.13).EK participated in the design of the study and carried out the data analysis and drafted the manuscript. SS assisted in data analysis and processing. CG assisted in the study design, data analysis and critically revised the manuscript. PD carried out immunoassays. JV carried out immunoassays. JR assisted in the design of data analysis and performed the statistical analysis. WL conceived of the study, participated in analysis and interpretation, and critically revised the manuscript for important intellectual content. EB is the pediatric pathologist of this study, was responsible for the diagnoses and data interpretation and critically revised the manuscript.Representative negative controls.Click here for fileExpression of ASS, CPS and OAT in a group 3 patient.Click here for fileBrunner glands in a group 5 patient.Click here for file


We conducted a population-based human exposure study in response to concerns among the population of Midland and Saginaw counties, Michigan, that discharges by the Dow Chemical Company of dioxin-like compounds into the nearby river and air had led to an increase in residents’ body burdens of polychlorinated dibenzofurans (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs), here collectively referred to as “dioxins.”We sought to identify factors that explained variation in serum dioxin concentrations among the residents of Midland and Saginaw counties. Exposures to dioxins in soil, river sediments, household dust, historic emissions, and contaminated fish and game were of primary interest.We studied 946 people in four populations in the contaminated area and in a referent population, by interview and by collection of serum, household dust, and residential soil. Linear regression was used to identify factors associated with serum dioxins.Demographic factors explained a large proportion of variation in serum dioxin concentrations. Historic exposures before 1980, including living in the Midland/Saginaw area, hunting and fishing in the contaminated areas, and working at Dow, contributed to serum dioxin levels. Exposures since 1980 in Midland and Saginaw counties contributed little to serum dioxins.This study provides valuable insights into the relationships between serum dioxins and environmental factors, age, sex, body mass index, smoking, and breast-feeding. These factors together explain a substantial proportion of the variation in serum dioxin concentrations in the general population. Historic exposures to environmental contamination appeared to be of greater importance than recent exposures for dioxins. This hypothesis is not clearly supported by the data.Even though 6% of the subjects reported ever having worked at Dow, we found little evidence that such work was associated with increased serum dioxin levels. Of those who reported working at Dow, 17% reported working with chlorophenol, 8% with Agent Orange, 8% with other Vietnam era herbicides, 15% with pentachlorophenol, 13% with 2,4-D, and 17% with 2,4,5-T. Work at Dow in 1940–1959 was positively associated with TCDD; however, working at Dow in 1980–2005 was inversely associated with 1,2,3,6,7,8-HxCDD and 2,3,4,7,8-PeCDF. None of the other variables that explored work at Dow, work with chlorophenol derivatives, or likely occupational exposure to dioxins was associated with increased serum dioxins.A principal focus of our study was to assess activities that might involve contact with contaminated soils, river sediments, and household dust. These included living on contaminated soil, living with contaminated household dust, and pursuing activities in the contaminated water bodies and flood-plain . We found little evidence in the general population that these activities were associated with increased serum dioxins. Participating in water activities on the Tittabawassee River more than once per month in 1980–2005 was associated with increased TCDD, but less frequent activities and activities before this period were not. We found no relationship between these activities and any other dioxin in any historic era. Water activities in 1980–2005 on other rivers outside the contaminated area were inversely associated with 1,2,3,6,7,8-HxCDD but not other dioxins. The Saginaw River (which is formed in part from the Tittabawassee River) is also contaminated with PCDDs, PCDFs, and PCBs and is of concern . We founWe explored soil contamination in detail. Six different soil samples were assessed for each house and for each congener, as well as the TEQ: house perimeter top 1 in. , house perimeter 1–6 in. (2.5–15 cm), garden soil, near-river top 1 in., near-river 1–6 in., and the maximal contamination found in any soil sample on the property. Samples were stratified so that the top 0–1 in. (0–2.5 cm) sample could be examined separately from the 1–5 in. (2.5–15 cm) sample because surficial deposition of aerosols would be expected to affect only the top 1–2 cm of soil, whereas contamination from other pathways  would be expected to affect deeper soil. We found soil contamination was significantly associated with serum dioxins in two instances.p = 0.02). When we excluded this observation from the regression analysis, we found no statistically significant relationship . The observation appears to be valid and was not excluded from the regression model. The importance of garden soil TCDD concentration as a predictor of serum TCDD should be interpreted with caution insofar as the association depends on a single observation. Although there may be an association for high values of soil , for soil levels < 50 ppt TCDD (the range of the rest of the data), we found no significant association. The house perimeter top 1 in. (2.5 cm) soil PCB-126 concentration was associated with serum PCB-126 . This association was due to two influential observations. When we excluded these, we found no statistically significant relationship.The TCDD concentration in garden soil was statistically significantly associated with higher serum TCDD. This association was due to a single observation that was highly influential. When we included this observation in the regression analysis, we found a statistically significant association between garden soil TCDD and serum TCDD , was positively associated with TEQ, TCDD, and PCB-126. We found no positive associations between consumption of fish from the contaminated areas  and serum PCDD or PCDF levels. There were equivocal results for eating fish from the Saginaw River or Saginaw Bay in the past 5 years . We found positive associations for serum 1,2,3,6,7,8-HxCDD and 2,3,4,7,8-PeCDF and consumption of walleye or perch from other sources . All other variables related to consumption of fish from the contaminated water bodies were either significantly negatively associated with serum dioxins or not associated. Overall, consumption of fish was positively associated with increased serum levels of a number of dioxins, but the findings were not related to fish from the contaminated areas, with the exception of PCB-126.Consumption of store-bought meat more than once per week was positively associated with serum 2,3,4,7,8-PeCDF and 1,2,3,6,7,8-HxCDD. No other variable related to consumption of either game or commercial meat was associated with serum dioxins. We found no positive associations between serum dioxins and consumption of game from the contaminated areas.Fishing in the Saginaw River and Saginaw Bay after 1980 was positively associated with serum TEQ, 1,2,3,7,8-PeCDD, and 2,3,4,7,8-PeCDF, and fishing in the Tittabawassee River between 1960 and 1979 was positively associated with 1,2,3,6,7,8-HxCDD.We found no evidence that consumption of game meat from the contaminated areas was associated with increased serum dioxins. However, hunting in the areas around the Saginaw River or Saginaw Bay in 1960–1979 was associated with increased serum 1,2,3,7,8-PeCDD (2.3-fold) and 1,2,3,6,7,8-HxCDD (1.2-fold). Hunting in the area around the Tittabawassee River in 1960–1979 was associated with a substantial increase in serum TCDD (2.3-fold), but hunting in the same area after 1980 was associated with a substantial decrease (0.25-fold). Hunting in the contaminated areas after 1980 was not positively associated with any of the serum dioxins.The overall pattern of findings in this population suggest little contribution from exposures in Midland and Saginaw counties during the last 25 years to serum dioxins from either contaminated soil, household dust, consumption of fish and game from contaminated areas, or recreational habits. The participants in our study had lived in their current residences for an average of 15–20 years, and those from Midland and Saginaw counties had lived in these counties for an average of > 40 years. Thus, it was a stable population with long-term exposure to the area and its contaminants. After taking into account age, sex, BMI, change in BMI, breast-feeding, and smoking, the largest contributor to both TCDD and 1,2,3,7,8-PeCDD was having lived in the Midland/Saginaw region in 1960–1979. A number of additional variables also suggested that historic exposures before 1980 contributed to serum dioxins: working at Dow in 1940–1959 (TCDD), fishing in the Tittabawassee River in 1960–1979 , hunting around the Tittabawassee River in 1960–1979 , and hunting around the Saginaw River or Saginaw Bay in 1960–1979 .R2 from all fish consumption and fishing activities combined was between 0.5% and 1% for the PCDD compounds, 2–3% for PCB-126, and 2–3% for 2,3,4,7,8-PeCDF, indicating that these explained a small amount of variation in the serum dioxins in this population. This should not be interpreted to mean that individuals who were heavy consumers of contaminated fish were unaffected; rather, it means that, on average, among the population the contribution was small.In contrast, we found little evidence that activities after 1980 contributed appreciably to serum dioxin levels. This is consistent with historic data on environmental dioxin measurements that show TEQ levels in many media increased beginning in the 1930s, peaked in the 1960s and 1970s, and have declined substantially over the past 35 years . We did Less than 1% of the general population of Midland and Saginaw counties ate bottom-feeding species  from the contaminated areas, perhaps because there have been fish advisories against such consumption since at least the early 1980s. It is possible that heavy consumption of contaminated species could contribute to serum dioxin concentrations. In the Midland area, 2,3,4,7,8-PeCDF is of greatest interest with regard to this issue because it is present in high concentrations in Tittabawassee River sediments where bottom-feeding fish feed and it has a long half-life (7 years)  in humanWalleye and perch are the most commonly consumed sport-caught fish from the Tittabawassee and Saginaw rivers and have low levels of dioxins compared with other species of local fish, likely because they are migratory species that spawn but do not reside in the contaminated areas. The association between eating fish from the Saginaw River or Bay and serum PCB-126 levels may be unrelated to Dow, because we are not aware that Dow produced or used this material in the Midland plant, and there are a number of foundries on the Saginaw River that have been historic sources of PCBs in the river.Although our regression models did not find effects on serum dioxin levels from game or produce raised in the floodplain, we previously reported an individual who raised beef cattle in the floodplain and whose age- and sex-adjusted serum 2,3,4,7,8-PeCDF was the highest in our study . This suOur findings showed no relationship between serum dioxins and either soil or household dust contamination. The single influential observation for garden soil TCDD was also the highest concentration (140 ppt) in our garden soil data. Soil TCDD contamination < 50 ppt showed no significant association with serum TCDD, nor did soil contamination contribute measurably to the serum levels of the other dioxin congeners we have evaluated. These findings are reasonably consistent with We compared the age- and sex-specific distribution of serum TEQ and PCDDs in our referent population  with the data reported from the National Health and Nutrition Examination Survey (NHANES) 2001–2002 survey . For theAlthough other large data sets provide estimates of the distribution of serum dioxin levels, ours has the unusual advantage that it was gathered from a population-based sample using complex survey methods, and the inferences are applicable to the general population from which the sample was drawn. It also provides valuable insights into the complex relationships between serum dioxins and age, sex, BMI, BMI change, smoking, and breast-feeding. These factors together explain a substantial proportion of the variation in serum dioxin concentrations in the general population. This study also indicates that the contributions of direct environmental exposures, such as those we studied in Midland and Saginaw counties, cannot be properly assessed without careful control for the major explanatory variables such as age, sex, BMI, smoking, and breast-feeding. The contributions of environmental exposures to serum dioxin concentrations were small, compared with the demographic factors, in terms of their ability to explain variation.This study was large and was capable of finding small associations that are statistically significant. Inferences regarding these associations should consider not only the statistical significance of the parameter estimate but also the magnitude of the effect and the amount of variance in serum TEQ or dioxins explained by the factor. Several of the significant findings described here both are small in magnitude and explain little variation in serum TEQ. Additional analyses of the upper tail of the distribution of serum dioxin concentrations are in progress using logistic regression methods, which we expect will provide insight into factors that predict having very high serum levels. Additional analyses of patterns of fish consumption incorporating analyses of the levels of contamination in various fish species from the contaminated waters would further refine our understanding of the contributions of fish contaminants to serum dioxin levels.


Although the questionnaire underestimated significantly all intakes compared to the 7 d EDR, it had a good ranking ability  and it could reliably distinguish extreme intakes for all the estimated nutrients, except for saturated fatty acids. Furthermore, the correlation between repeated administrations was high (r 0.71–0.87) with a maximal misclassification of 7% (weighted κ 0.33–0.80). In conclusion, these results compare favourably with those reported by others and indicate that the FFQ is a satisfactorily reliable and valid instrument for ranking individuals within this study population.A novel food-frequency questionnaire (FFQ) was developed and validated to assess the usual daily fat, saturated, mono-unsaturated and poly-unsaturated fatty acid, fibre, alcohol, caffeine, and theobromine intakes among Belgian post-menopausal women participating in dietary intervention trials with phyto-oestrogens. The relative validity of the FFQ was estimated by comparison with 7 day (d) estimated diet records (EDR,  In order to ensure proper interpretation of the results obtained with this new instrument, a study was conducted 1) to investigate the relative validity using 7 d estimated diet records (EDR) as standard method and 2) to evaluate its reproducibility.2.2.1.1 and FFQ2) with a 6 weeks interval [1 and 83 women completed FFQ2 as well; 17 invitation letters were declared undeliverable. A socio-demographic questionnaire was also administered.A total of 500 women aged between 45 and 75 years, were randomly selected from the population register of 2001 of Ghent. A subgroup of 250 women was included in the validity study and asked to complete a FFQ and a 7 d EDR. In total, 142 FFQ and 78 EDR were collected, while 12 invitation letters were declared undeliverable. For the reproducibility study, a second sample of 250 women was chosen. They were asked to fill in the same FFQ twice , fibre, alcohol, caffeine, and theobromine intakes of Belgian post-menopausal women over the previous year. For the conceptualisation of this FFQ, the food consumption data of a survey in adult women  and knowThe final FFQ  included2 were excluded, since less than half of the questions had been answered. Data from the good-quality FFQ were processed using the scanning software package TELEForm . Nutritional values were assigned to each food group on the basis of weighted means of all aggregated items. The food composition data were based on the Belgian NUBEL [All returned FFQ were reviewed for completeness by checking for multiple frequency answers, unmarked portion sizes, skipped food items and written comments. Although we suspect that some women skipped questions in the FFQ when they never consumed the particular product instead of indicating “never or less than once a month”, we preferred not to make any assumptions and did not replace these missing values. No FFQ of the validity study and 4 FFQan NUBEL  and the an NUBEL  food coman NUBEL , informaan NUBEL –26. The 2.3.In the present study the reference method was a 7 d EDR, using diaries with a semi-structured, open entry format and consisting of 6 eating occasions . Detailed information on the type, including if possible brand names, and portion sizes, expressed as natural or household measures, of the food consumed during seven consecutive days  was collDieticians carried out the exclusion procedure of the EDR. Only good-quality food diaries, including seven completed record days and containing sufficiently detailed descriptions of the food products and portion sizes consumed, were taken into consideration. As a cross-check, average energy, total fat, SFA, MUFA, PUFA, fibre, alcohol, caffeine, and theobromine consumption was calculated as the mean of the 7 recorded days. Diaries with very high or very low estimates were rechecked. In total, 14 EDR had to be excluded because of quality problems. The good-quality EDR were coded and entered in a Diet Entry and Storage program  using a standardised protocol from the Ghent University department of Public Health and a manual on food portions and household measures . The sam2.4.etc.) was distributed. The participants were also asked to report their weight and height, which were used to calculate their BMI.In order to evaluate possible confounding factors and selection bias, a socio-demographic questionnaire, registering additive information about the women  was used for all statistical analyses. Results were considered statistically significant at an α two-tailed level of 0.05. Tests for normality of the data were performed using the Kolmogorov-Smirnov test. Means and standard deviations (SD) of nutrient intakes, and differences between mean values obtained from the first and second dietary assessment were calculated. The paired Student’s n et al. . The wit1 and FFQ2. The measurement error of the FFQ was analysed with the actual values for surrogate categories method as described by Willett [Bland & Altman plots visualised the agreement between the test and reference method for each nutrient at an individual level . In orde Willett . The cat Willett .t-test or via cross-tabulations with χ2 or Fisher’s exact tests.These recommendations were used as threshold values to define the specificity, sensitivity, and positive and negative predictive values of the FFQ, whereby intakes in line with the recommendations were defined as positive. In order to determine potential confounding factors for the validity and/or reproducibility of the FFQ, covariance analyses were performed with variables derived from the socio-demographic questionnaire. These variables were also used together with the dietary data to estimate the possible selection bias. Data of drop-outs and women excluded due to low-quality questionnaires or diaries were compared to those included in the statistical analyses with the independent Student’s 3.3.1.n 64) was 58 years. Thirty-seven women (54%) had a normal weight (18.5 kg/m2 ≥ BMI ≥ 24.9 kg/m2), whereas one woman (2%) had a BMI below 18.5 kg/m2 and 26 women (42%) were classified as overweight (BMI ≥ 25 kg/m2) and five of these (19%) were obese (BMI ≥ 30 kg/m2). A minority of the participants  were current smokers. Mean intakes of total fat, SFA, MUFA, PUFA, fibres, alcohol, caffeine, and theobromine estimated with the 7 d food diaries and FFQ, mean differences and deattenuated correlation coefficients between the test and reference method are presented in The mean age of the subjects included in the validity study  to 0.94  were found between these methods. Deattenuation improved the correlation coefficients for all nutrients. The Bland & Altman plots of total fat, SFA, MUFA and PUFA estimates  were sliP 0.044) between the means of total fat intake of the extreme tertiles.Cross-classification analyses showed that most subjects  were assigned to the same tertiles by both methods, whereas between 2%  and 17% (SFA) were grossly misclassified . The weiThe mean differences between the FFQ and EDR were used as correction factors in the assessment of the specificity, sensitivity, and positive and negative predictive values of the FFQ for total fat, SFA, MUFA, PUFA, fibres, and alcohol estimates . No sign3.2.n 79) were on average 59 years old. Forty-five (57%) women had normal weight (18.5 kg/m2 ≥ BMI ≥ 24.9 kg/m2), whereas one woman (1%) had a BMI below 18.5 kg/m2 and 33 women (42%) were classified as overweight (BMI ≥ 25 kg/m2) and eight of them (24%) were obese (BMI ≥ 30 kg/m2). Only eight participants (10%) were current smokers.Participants of the reproducibility study  to 0.87  were obtained . The per4.A novel self-administered semi-quantitative FFQ was designed and validated in the context of intervention trials investigating dietary factors associated with the microbial metabolism of phyto-oestrogens in post-menopausal women , 15, andThe validity of the FFQ was evaluated using different approaches. Comparison of means and Bland & Altman analyses revealed a tendency of the FFQ to underestimate the mean intakes measured by the EDR, especially for total fat, SFA, MUFA, PUFA, and fibres. Since our FFQ was not designed to estimate energy intake, we could not determine whether this was due to underreporting. Large standard deviations of the mean differences between the test and reference method were visualised in the Bland & Altman plots of alcohol, caffeine, and theobromine, suggesting that the use of the FFQ to estimate absolute intakes by individuals is not appropriate. In addition, a systematic increase in measurement error with increasing absolute intake of these components was observed. However, the primary goal of this instrument was to classify and rank subjects according to their nutrient intakes rather than achieving accurate results in terms of individual consumption.Dietary instruments should have correlation coefficients of at least 0.40 and optimally in the range of 0.50–0.70 in order to reliably rank persons . Thus, tThe FFQ should not be used at an individual level (like in dietary counselling) for estimating the consumption of PUFA with the Belgian Health Council guidelines for women  as referGood reproducibility was established for the FFQ. No significant differences were found between the first and second administration. The high correlation coefficients (0.71–0.87) indicate that the random response error, sometimes due to lack of interest or motivation of the respondents or lack of clarity of the questionnaire, was rather small. The agreement  between et al. [et al. [et al. [et al. [The results of the present validation study compare favourably with those of other researchers who validated FFQ-derived fat , fibre, and alcohol estimates relative to 24 h dietary recalls ,34,35, a [et al.  reported [et al.  suggeste [et al. . Finally [et al. , therefoet al. [Unlike their results for fat, fibre, and alcohol estimates, Jain et al.  found hiet al. . Despiteet al. . In the et al. , affect In intervention studies, dietary assessments often rely on a self-administered FFQ aiming at the assessment of usual long-term consumption and designed to rank subjects into quantiles of dietary intake. The issue of how to evaluate the accuracy and precision of a new dietary instrument is frequently debated. The problem with validation approaches in which a second dietary assessment method is used as reference, is that both methods may be biased and contain correlated errors. Biochemical markers reflecting dietary intakes are valuable tools since they do not rely on self-reports and their random measurement errors are not likely to correlate with those of dietary assessment methods. Unfortunately, no useful biomarkers were available for this validation study. Therefore, estimated diet records were chosen as reference method. Although both methods are subjected to a degree of misreporting, the measurement errors of the EDR and FFQ are highly independent, since, unlike the FFQ, the EDR method does not depend on memory, is open-ended, and involves direct estimation of portion sizes .Weighted diet records are more accurate in terms of individual intakes, but estimated records achieve the same order of accuracy when ranking subjects and have a lower respondent burden . Therefo


Obtained recoveries of metal ion were more than 92%. The optimized procedure was also validated with three different pools of spiked urine samples and showed a good reproducibility over six consecutive days as well as six within-day experiments. Through this study, suitable results were obtained for relative standard deviation, therefore, it is concluded that, this optimized method can be considered to be successful in simplifying sample preparation for trace residue analysis of Cr in different matrices for evaluation of occupational and environmental exposures. To evaluate occupational exposure to chromium, 16 urine samples were taken, prepared, and analyzed based on optimized procedure.Chromium is an important constituent widely used in different industrial processes for production of various synthetic materials. For evaluation of workers’ exposure to trace toxic metal of Cr (III), environmental and biological monitoring are essential processes, in which, preparation of samples is one of the most time-consuming and error-prone aspects prior to analysis. The use of solid-phase extraction (SPE) has grown and is a fertile technique of sample preparation as it provides better results than those produced by liquid-liquid extraction (LLE). SPE using mini columns filled with XAD-4 resin was optimized regarding to sample pH, ligand concentration, loading flow rate, elution solvent, sample volume, elution volume, amount of resins, and sample matrix interferences. Chromium was retained on solid sorbent and was eluted with 2 M HNO Heavy metals can be considered as a unique class of environmental toxicant. They occur and persist in nature and most of them are advantageous to humans because of their vast usages in different industries, agriculture, and medicine. However, they may pose health hazards to the public because of their presence in air, water, food chains as well as to the workers engaged in mining, smelting, alloy, painting, electroplating, pesticides, and the variety of industrial activities. Some heavy metals such as chromium have a wide range of toxicities, leading to toxic effects on the renal, respiratory, and nervous systems. Also, chromium (VI) compounds are classified as A1 confirmed human carcinogens by ACGIH.The water soluble hexavalent chromium compounds are highly irritants of nasopharynx, larynx, lungs, and skin. Cr (VI) compounds have been implicated as reparable for perforated nasal septa, rhinitis, nosebleed, kidney damage, and skin ulceration. In body fluids, Cr (VI) is reduced to Cr (III), thus, determination of Cr (III) in these fluids is useful where the workers exposure are just taken placed to Cr (VI) , electroBecause, usages of this heavy metal are unavoidable, from the occupational viewpoints, study of this compound is of great interest. One of the most important aspects of metal studies is determination of such compounds in different matrices. In biological samples, either exposed compounds or their metabolites, metals are mostly present at trace level, causing major problems in their determination stages . Therefoetc.) on a solid sorbent and subsequent desorption of retained constituent by elution from the sorbent. Through this procedure, isolation and purification of the compound of interest can be achieved in a short time and only low volumes of solvents are used during the application of the method. The use of commercially available low-cost vacuum manifolds allows many samples to be proceed simultaneously. Furthermore complete automation of procedures based on SPE is now possible using commercially available instrumentation  present in water and urine samples, leading to a simple protocol of SPE method.3. Working and standard solutions were prepared daily by dilution of the stock solution. Acids and other chemicals used in this study were obtained from Merck, Darmstadt, Germany. Standard buffered solution at various pH values, ammonium pirrolidine dithio carbamate (APDC), amberlite XAD-4 resin (20–40 mesh) were also purchased from Merck, Germany.All solutions were prepared using distilled water. chromium stock solution was prepared from appropriate amount of the nitrate salt of this analyte  as 1000 mg/lit solution in 0.01 M HNODetermination of chromium was with spectra AA/plus 20, Varian flame atomic absorption spectrometer  using air-acetylene flame. The operating parameters for metal of interest were set as recommended by the manufacturer. The pH values of the solutions were measured by a digital pH meter model Metrohm 744. Amount of reagents were measured using a Satorius CP225D balance .3, water, 1 M NaOH, and water, respectively. Finally, resin was pre-concentrated with buffer solution.Glass mini columns (100 × 10 mm) were packed with 500 mg resin. After packing, a little amount of glass wool was placed at both ends of the glass tube. Before using the column, XAD-4 resin was washed by methanol, water, 1 M HNOIn this study, SPE using amberlitr XAD-4 resin was optimized with regard to sample pH, sample and eluent flow rates, elution solvent, eluent volume, ligand concentration, amount of resin, and sample volume. Fifty milliliter solution containing 20 μg of Cr (III), 10 ml buffer solution with desired pH and 6 ml APDC solution were prepared. Samples were then passed through the column packed in our laboratory at a flow rate of 5 ml/min. The column was then washed with 5–10 ml of the same buffer solution. Therefore, the metal ions were eluted from the mini column with 10–15 ml of different solvents. Finally, the concentration of chromium in the solutions, including standards, spiked, and real samples were determined by FAAS.3. The influence of sample pH on adsorption of Cr (III) ion on XAD-4 resin was investigated, using different pH values of 2, 4, 7, and 9. The pH values were adjusted by buffer solution. Fifty milliliter of sample containing 20 μg of Cr (III) and 6 ml APDC solution was loaded on the column. The column was then washed and the retained analyte was eluted using 2 M HNOThe concentration of Ammonium Pirrolodine Dithio Carbamate (APDC) is one of the important parameter could affect on recovery obtained from the optimized method. Through this investigation, the amount of 0.01–0.07% (w/v) of APDC were used. The results obtained from this investigation showed that, by increasing APDC concentration up to 0.05%, the recoveries are also increased, afterward, constant values are recovered.3 in acetone, 1 M HNO3, and 2 M HNO3. The same sequence of conditioning, washing, and elution were used as in the previous section. The results are presented in 3 as an efficient eluent and, therefore, it was used as a suitable solvent for further studies.Evaluation of eluent strength onrecovery of Cr (III) was another experiment performed during this study. Five solvents were screened for their ability to produce optimum elution of the retained Cr (III) from the XAD-4 resin. They were 1 M HCl, acetone, 1 M HNOEluent volume is an influencing parameter, affecting on the pre-concentration of analytes using SPE. Enrichment of the analyte in SPE is achieved by applying large volume of sample and eluting the analyte in a minimum volume of eluent. The volume of the eluent must be just sufficient to elute the compound of interest from the sorbent. Thus, the recovery of metal ion was studied in applying different eluent volumes of 5, 10, 15, and 20 ml. The results are given in In order to evaluate the influence of eluent the eluent flow rate on recovery of the analytes, retained metal ion was eluted, using eluent at different flow rates of 2, 5, 7, and 10 ml/min. The same sequence of conditioning, washing, and elution were used as in the previous section. As the In order to evaluate the sample volume, 20 μg of Cr (III) was diluted into different volumes of 50, 150, 250, 500, and 750 ml. These samples loaded on XAD-4 mini columns. The columns were then washed and the retained analyte was eluted according to the optimized method .The results have been shown in Following a demonstration of the feasibility of using large sample volumes, the effect of sample flow rate on metal ion adsorption on XAD-4 was studied in different sample flow rate of 2, 5, 7, and 9 ml/min. Fifty ml sample, using optimum pH, containing 20 μg of metal ion and APDC solution were prepared. Thereafter, the same sequence of conditioning, washing, and elution were used as in the previous section. No significant reduction in recovery was found for sample flow rate up to 7 ml/min. Flow rate of 5 ml/min as an appropriate value was then used to continue further experiments. The effect of XAD-4 amount was investigated, using 100 and 500 mg sorbent packed in a mini column. The same sequence of preparation procedure was used as in the previous section. The obtained recovery of metal ion was more efficient when 500 mg was utilized see .+, K+, Mg2+, Ca2+, and SO42− was another parameter, influencing the efficiencies of analyte recoveries. The procedure was performed, using 50 ml sample containing 20 μg of analyte and different concentration of matrix ions. The results have been shown in The effect of various matrix possible ions, mostly present in the environmental and biological samples, including NaAs spiked urine may contain some interference compounds similar to the real sample, it can be considered as an appropriate sample, preferably better than water sample for validation of the optimized method. Therefore, further experiments were carried out in urine; however, the working samples were made in aquatic solution. A preliminary validation of the possible use of the optimized method for measuring metal ion of chromium (III) in urine was carried out, using spiked samples. Samples of 50 ml were used for extraction with subsequent FAAS. Linear standard curves (extracted) over the concentration range of 1, 1.5, and 2 μg/ml were obtained each day (n = 6) for six consecutive days with a correlation coefficient of 0.995 or greater. The day-to-day (for six consecutive days) and within-day reproducibility of the method was investigated. Finally, real samples of urine were obtained from 16 workers employed in the relevant industries, using the optimized method. The results obtained from this experiment have been shown in The results showed that efficient recovery was obtained from XAD-4 resin using sample pH of 9. For Chromium (III), the amount of the analyte recovered from sorbent at sample pH values of 2, 4, and 7 was also efficient. However, the pH value of the sample should be adjusted according to the chemistry of the compound of interest. It seems that, at sample pH of 9, the analyte of interest mostly is in the ionized form, making it to be easily retained on the ionized ligand already conjugated to the sorbents. From these pH values, sample pH of 9 was selected for further study as this pH seems to be rather confident value. In this study, a non polar sorbent was used, in which, there was no affinity between this type of sorbent and the ionized analyte, so, there was a need of conjugating ionized ligand on the sorbents to follow up an ionized extraction mechanism. APDC showed to be an appropriate ligand for capturing chromium (III) from the sample, however, from the four concentrations of the ligand, the amount of 0.05, and 0.07 (%) showed to be good enough for efficient retaining of the analyte. However, for preventing saturation of the sorbent with the ligand and also reduce the reagents through extraction process, the lesser percentage of the ligand (0.05%) was used as this amount provides the same recovery needed for the method.3 based solutions were more efficient and from these solvents, 2 M HNO3 was selected, because, it was organic free eluent and can prevent co-elution of organic compounds possibly present in the real samples as well as reducing exposure to such evaporative and hazardous compounds. Moreover, maximum recovery has been ahieved using this eluent. The results obtained from an evaluation of the elution volume  over the range concentrations of 1, 1.5, and 2 μg/ml was obtained every day for 6 consecutive days (n = 6) with the correlation coefficient of 0.95 or grater. In within-day experiments evaluation, six experiments were performed per day for three consecutive days. The extraction procedure was reliable and reproducible from day-to-day and within-day. Coefficient of variations (CV%) of 0.830, 1.01, and 1.14 were obtained for 1, 1.5, and 2 μg/ml respectively for day-to-day and 2.01, 2.04, and 0.272 at the same concentrations respectively for within-day, showing suitable accuracy and precision see . FinallyComparatively, the method recently reported  has usedThrough this study factors influencing SPE were optimized, showing an efficient sample preparation procedure for chromium (III) as a solid phase extraction method has more advantages than liquid liquid extraction. Depending on the chemical and physical properties of the analyte, manipulating factors including sample pH, ligand concentration (APDC), loading flow rate, elution solvent, sample volume (up to 500 ml), elution volume, amount of resin (XAD-4), and sample matrix interferences can play essential roles in optimizing the method, providing reliable, easy to use, and cost effective procedure to overcome difficulties associated with other sample preparation techniques. The concentration factor was 33.3 and the resin can be used several times. The optimized method was also used for pre-concentration of urinary Cr (VI) in relevant industry and promising some other applications when analysis of trace heavy metals in biological and environmental samples is of interests. The authors are sure that, SPE is a highly fertile area for sample preparation method and based on the needs and facilities, these method protocols can be further developed in the near future.