Additionally, we assessed the density of two oligodendrocyte subpopulations immunostained by the oligodendrocyte transcription factors Olig1 and Olig2 and of interneurons immunolabeled by parvalbumin. We investigated posterior hippocampal subregions in the post-mortem brains of the same schizophrenia patients  and healthy controls (n = 10) we examined in our previously published stereological studies. Our stereological studies found that patients with definite cognitive deficits had decreased total/Nissl-stained oligodendrocyte numbers in the left (p = 0.014) and right (p = 0.050) CA4, left CA2/3 (p = 0.050), left CA1 (p = 0.027), and left (p = 0.050) and right (p = 0.014) subiculum of the anterior part of the hippocampus compared to patients with possible cognitive deficits. In the present study, we found no significant influence of definite cognitive deficits in the posterior part of the hippocampus, whereas in the entire hippocampus SZ with definite cognitive deficits showed decreased oligodendrocyte numbers in the left (p = 0.050) and right (p = 0.050) DG and left CA2/3 (p = 0.050). We did not find significant differences in Olig1-, Olig2-, or parvalbumin-positive cell density between SZ and controls in any of the subregions of the posterior hippocampus. Based on the results from our stereological study we hypothesize that a decreased number of oligodendrocytes in the anterior and entire hippocampus may be involved in cognitive deficits by impairing the connectivity of this structure in schizophrenia. In the posterior hippocampus, we could not replicate previously reported findings of decreased interneurons from the entire hippocampus.In schizophrenia, previous stereological post-mortem investigations of anterior, posterior, and total hippocampal subfields showed no alterations in total neuron number but did show decreased oligodendrocyte numbers in CA4, an area that corresponds to the polymorph layer of the dentate gyrus (DG). However, these investigations identified oligodendrocytes only on the basis of morphological criteria in Nissl staining and did not assess alterations of interneurons with immunohistochemical markers. Moreover, the association of findings in the posterior hippocampus with cognitive deficits remains unknown. On the basis of the available clinical records, we compared patients with definite and possible cognitive dysfunction; nine patients had evidence in their records of either definite ( Schizophrenia has an unfavorable outcome in more than half of all patients and is linked to social and vocational disability . Then, the frontal and occipital parts of the brain were separated from the middle part, which was located between the lateral geniculate nucleus and the splenium of the corpus callosum, i.e., containing the anterior and posterior hippocampus. From this middle part, 20 μm coronal sections containing both hemispheres were cut on a Polycut S Leica microtome. The thickness of each section was determined by focusing through the section and subtracting the z-axis coordinate of the lower from that of the upper surface by the microcator, part of the Olympus microscope. The mean (SD) section thickness after histological procedure was 18.7 (1.1) μm.Post-mortem brains were obtained from the Düsseldorf Brain Collection , and subiculum. CA2 and CA3 were lumped together as described in the literature because these small regions are difficult to separate at the microscopic level  buffer. Monoclonal mouse anti-Olig1 antibody  or rabbit anti-Olig2 antibody  was applied for 72 h at 4°C for immunostaining of Olig1- or Olig2-positive oligodendrocytes, respectively. Then, the streptavidin-biotin technique was used to incubate sections with a biotinylated anti-mouse  or anti-rabbit  antibody, respectively. Chromogen 3,3’-diaminobenzidine (DAB) and ammonium nickel sulfate were used to visualize the reaction product. Parvalbumin-positive interneurons were immunolabeled under the same conditions with a monoclonal antibody to the protein  and an anti-mouse peroxidase secondary antibody  thickness of the sections was 18.7 (1.1) μm, two well-defined optical planes within the sections were used (distance 16 μm between the upper and lower guard zones), and all stained cells that came into focus while passing from the upper to the lower optical plane (plane of the dissector) were counted. The individual volume shrinkage factors (VSF) were calculated from the measured linear shrinkage factor (LSF) with the following formula: VSF = (LSF) , Olympus Uplan Apo objectives , motorized specimen stage for automatic sampling, electronic microcator, CCD color video camera, PC with frame grabber board, stereology software , and 17” monitor. Boundaries of hippocampal subfields were traced on video images displayed on the computer screen, and volumes were calculated in both sections. The numerical cell density of Olig1-, Olig2-, and parvalbumin-stained cells  or possible (n = 5) deficits . Because Levene’s test detected significant variance inhomogeneities between the groups, we used non-parametric Mann-Whitney U tests to analyze diagnostic group differences. Cell densities were correlated with age at death, PMI, dose of neuroleptic treatment in CPE and disease duration by means of Spearman correlations for the total sample and separately for SZ and controls.In addition to densities of oligodendrocytes and interneurons, outcome measures of the second analysis were the number of oligodendrocytes and neurons, estimated stereologically, and the structure volumes in the subfields  of the anterior, posterior, and entire hippocampus of SZ, as published in Falkai et al. . The indFor this explorative study with small group sizes, especially as regards the analysis of the influence of cognitive deficits on cell numbers or densities within the group of SZ, the results are presented without error probability correction. If a Bonferroni adjustment of the type I error probability had been applied, no significant differences would remain between SZ and controls. However, if the error probability was adjusted the power of detecting existing mean differences would be too low.p = 0.027), left CA2/3 , left  and right  CA4, and left  and right  subiculum. In the posterior part of the hippocampus, definite cognitive deficits appeared to have no significant influence, while in the entire hippocampus SZ patients with definite cognitive deficits showed decreased oligodendrocyte numbers in the left  and right  DG and left CA2/3 .Our simple classification of cognitive deficits indicated that patients with a definite cognitive dysfunction had a more pronounced reduction of oligodendrocyte total cell numbers (combined staining with cresyl violet (Nissl) and myelin (luxol fast blue), Schmitt et al., p = 0.070), right CA4 , and right subiculum . The SZ and controls showed no significant differences with respect to the mean densities of Olig2-positive oligodendrocytes in the investigated subregions of the posterior part of the hippocampus, but the density of parvalbumin-stained interneurons was increased in the left CA1 region in the SZ  and showed a trend towards an increase in the right CA4  and right  CA1, left CA2/3 , and left CA4  subregions. With respect to Olig2-stained cells, females had increased cell densities in the left  and right  CA1, left  and right  CA2/3, and left subiculum . Furthermore, females had an increased density of parvalbumin-positive interneurons compared to males in the left  and right  CA2/3, and left subiculum  and right CA4 , whereas in SZ no significant correlations between age and cell densities were observed. In controls, the PMI correlated with oligodendrocyte density , but not with Olig2 or interneuron density. No significant correlations of Olig1-, Olig2- or interneuron densities with disease duration were observed. The Olig2 density correlated with CPE in the right subiculum , but no other correlations with dose of neuroleptic treatment had been found  is an experienced psychiatrist, and case notes were detailed, because of the long-term hospitalization of the patients. In addition, the sample size was small and, because we did not perform Bonferroni correction, results should be replicated in an independent sample. Prospective post-mortem studies that use standardized tests to investigate cognitive deficits in brain donors before death also are warranted to confirm our results. A further limitation is the long-term treatment with typical neuroleptics, which may have influenced our results. The patient group with definite cognitive deficits had received higher doses compared to patients with only possible cognitive deficits. However, from clinical practice it is known that patients with persisting symptoms receive higher doses of antipsychotics than patients in remission. Additionally, neuroleptic treatment may have influenced cell densities in our study. Haloperidol is known to increase expression of Olig2 in the hippocampus and cerebral cortex, whereas quetiapine and olanzapine increase expression of both Olig1 and Olig2 . Furthermore, this work was funded by the German Federal Ministry of Education and Research (BMBF) through the Integrated Network IntegraMent  under the auspices of the e:Med Programme (grant number 01ZX1314I to PF and TK). Additionally, work was funded by the EU project IN-SENS FP7-PEOPLE-2013-ITN (607616). The article reflects only the views of the authors and the Community is not liable for any use that may be made of it.BM, JSh, AK, TS-A, TK, JSt, H-GB, and BB declare no conflicts of interest. AH has been invited to scientific meetings by Lundbeck, Janssen-Cilag, and Pfizer, and he received a paid speakership from Desitin, Otsuka, and Lundbeck and was member of an advisory board of Roche and Lundbeck. PF has been an honorary speaker for AstraZeneca, Bristol Myers Squibb, Eli Lilly, Essex, GE Healthcare, GlaxoSmithKline, Janssen Cilag, Lundbeck, Otsuka, Pfizer, Servier, and Takeda. During the past 5 years, but not presently, PF has been a member of the advisory boards of Janssen-Cilag, AstraZeneca, Eli Lilly, and Lundbeck. AS has been an honorary speaker for TAD Pharma and Roche and has been a member of advisory boards for Roche.


Mantle cell lymphoma (MCL) is an infrequent subtype of non-Hodgkin’s lymphoma (NHL) and represents between 4–8% of adult lymphomas. Recently an increase in its incidence to 1–2 cases/100,000 inhabitants/year has been observed. The first line of treatment is based on chemoimmunotherapy and depends on age and the initial stage at diagnosis. There are no second line or successive treatments. There are currently several drugs available that provide acceptable results. MCL accounts for 4–8% of all adult non-Hodgkin’s lymphomas (NHL). Recently an increase in its incidence to one to two cases/100,000 inhabitants/year has been observed [Just as we have prognostic indices for follicular lymphoma and diffuse large cell lymphoma like FLIPI and IPI respectively, since 2008 there has been an international prognosis index for MCL, mantle cell lymphoma internal prognostic index (MIPI) that includes age, Eastern Cooperative Oncology Group (ECOG), lactate dehydrogenase (LDH), and leukocyte count establishing high-, intermediate-, and low-risk groups [The therapeutic approach is based on the combination of immunotherapy and chemotherapy followed either or not by the transplant of haematopoietic stem cells. With regard to relapse, there is no standard treatment, but with the development of new drugs, there is the choice of rituximab with or without temsirolimus, ibrutinib, bortezomib, or lenalidomide .In the present work, we offer an updated review of the current diagnostic criteria and therapeutic options available, as well as of the new drugs that can be incorporated in the treatment of relapsed MCL in the near future. In this present review we have analysed the clinical, histological, immunohistochemical, and molecular characteristics of seven MCL patients diagnosed in our centre between February 2014 and February 2015 and the treatment administered.Clinicopathological and therapeutic data were collected from the clinical history. The histologic subtype and the immunohistochemical profile were taken from the anatomopathological report in search of the following immunomarkers: CD5, CD23, CD43, cyclin D1, BCL 2, and BCL 6.The patients were diagnosed and staged according to the modified Ann Arbor classification (Costworlds 1989) after carrying out an analytical control with haemogram, complete biochemistry, including LDH, cervico-thoraco-abdomino-pelvic computerised axial tomography (CT), ganglionary biopsy, or biopsy of the accessible affected extranodal tissue, and bone marrow biopsy.Seven patients were diagnosed with MCL and treated in the Instituto Valenciano de Oncología (IVO) Medical Oncology or Haematology Department. The average age of our patients was 63.5 years (ranging from 50–73 years) with a male to female ratio of 6:1 . Five ofCase 5, a male patient of 73 years of age, who was evaluated by Haematology in November 2012 for asymptomatic neutropaenia without apparent cause (N 820 mcg/L). The study was carried out by means of immunophenotyping peripheral blood cells with results compatible with B-chronic lymphoproliferative disorder without the criteria for chronic lymphocytic leukaemia. An extended CAT scan revealed supra and infraclavicular adenopathies. As the adenopathies were not accessible for biopsy, a bone marrow aspiration biopsy was performed that revealed low-grade B-cell lymphoproliferative disorder. The tumour committee decided in favour of monitoring as treatment criteria were not present. The patient was sent to the Digestive Department in December 2013 because of a positive fecal occult blood test in colorrectal cancer screening (CCR). A colonoscopy was performed that focused on nine submucosal polyps throughout the colon. The anatomopathological study of the entirety of the extirpated polyps revealed blastoid variant MCL with a pattern of lymphomatoid polyposis. An extended study was requested. Computerised tomography (CT) showed stability of the supra and infradiaphragmatic adenopathies with respect to the October 2013 CT. PET/CT were within the normal range. Analysis for LDH and beta2 microglobulin were within range. Bone marrow aspiration/biopsy revealed global hypercellular marrow with hyperplasia of the three systems with interstitial, paratrabecular, and nodular infiltration by NHL. CD20+++ and cyclin D1++ were compatible with infiltration by MCL. Cytology showed increase in lymphocytes suggestive of marrow infiltration by NHL . The molHistologically, three patients displayed a blastoid variant whereas the remaining four displayed a classic variant. In five of the cases, translocation t could be determined and verified, while in the other two cases it was not analysed. However, the seven patients were positive for cyclin D1, CD5, and CD43, and BCL2, and negative for BCL6 .Six of the cases received rituximab-CHOP as front-line treatment, while the youngest patient received rituximab-Hyper-CVAD. One case received local radiotherapy upon presenting stage II at the end of chemotherapy. Following systemic treatment, CT showed a complete response for six patients; targeted PET for three, and in the case of the patient with intestinal condition, study was completed with a colonoscopy. In all the cases with diagnosed bone marrow infiltration that presented a complete radiological response following systemic treatment, a bone marrow biopsy confirmed the absence of infiltration by neoplastic cells.Six of the cases were included in the MIPI high-risk subgroup, and the remaining case in the low-risk group .With an average monitoring time of 10.8 months, one of the patients, diagnosed with a blastoid variant MCL (case number 7), had a ganglionary relapse and was being treated with rituximab-bendamustine as second-line. The remaining six cases remain disease-free.The median age at diagnosis is found to be 68 +/-8 years. In 75% of the cases, MCL affects men and is more frequent among Caucasians . Also, iClinically the symptoms collected in the literature are nonspecific, although usually it appears as a constitutional disorder and B symptoms, which is why it is important to make a complete anamnesis at the time of staging . Other fIn order to make the diagnosis, an exeresis/biopsy of an accessible adenopathy and then evaluation by means of morphologic study with a haematoxylin and eosin stain, nuclear immunohistochemical expression for cyclin D1, and confirmation via molecular studies using FISH, cytogenetics, or determination of the specific gene fusion after translocation t(11:14) is recommended [An analytical control should be included in the extension study to determine LDH, serology for the hepatitis B virus, human immunodeficiency virus, cervico-thoraco-abdomino-pelvic CT, and bone marrow biopsy . AlthougSeveral works describe the disease in stages III and IV  with theFrom the anatomopathological point of view, MCL is a neoplasm of small B-cell phenotype monomorphic cells with an irregular nucleus that originates in the mantle zone of the lymphoid follicle. There are four patterns of nodal involvement with partial or total loss of architecture, showing a diffuse, vaguely nodular proliferation with a mantle zone pattern or one similar to the follicular growth pattern .Differential diagnosis should be made using the reactive processes (mantle zone hyperplasia), nodular lymphocyte predominant Hodgkin’s lymphoma, nodal marginal zone lymphoma, small lymphocytic lymphoma/chronic lymphocytic leukaemia, follicular lymphoma, lymphoblastic lymphoma (with blastoid variant MCL), and diffuse large B-cell lymphoma (with pleomorphic variant MCL) .The lymphocyte proliferation in MCL is monomorphic, with the absence of cells like centroblasts, immunoblasts, and paraimmunoblasts being very characteristic, as well as proliferation of pseudocentres that differentiate mantle lymphoma from other low-grade B-cell neoplasms. Additionally, different cellular variants exist. One being small cell variant which is similar to chronic lymphocytic leukaemia. The other that simulates marginal zone lymphocytes with clear cytoplasm or monocytoid B-cells, which are reminiscent of the proliferation centres of small lymphocytic lymphoma. Then the aggressive variants of higher cytological grade correspond to the pleomorphic variant (with cells similar to diffuse large B-cell non-Hodgkin’s lymphoma) and the blastoid with lymphocytes similar to the lymphoblasts of lymphoblastic lymphoma, with a high index of cellular proliferation Ki-67), that entails a worse prognosis. The classic cytological variant constitutes 87%, small cell 3.6%, pleomorphic cell 5.9%, and blastoid variant 2.6% of the cases. Recent studies have demonstrated that miRNAs are considered important regulators of cellular behaviour. The increase in the expression of miR-15b likely plays an important role in the transformation of the classic variant of MCL to the aggressive variants [7, that eFrom the immunophenotype perspective, MCL expresses B-lymphocyte antigens such as CD19+, CD20+, CD22+, CD79+, PAX5, and IgM/IgD with lambda + light chain restriction. The most characteristic immunophenotypic markers are CD5+, FMC7+, CD45+, and intranuclear cyclin D1+ [In order to help to identify MCL with negative cyclin D1, the neural transcription factor (SOX11) has been proposed. Recent studies suggest SOX11 can play a role in the process of MCL cell differentiation and define two different subtypes of MCL through BCL6 transcriptional regulation . HoweverThe overexpression of cyclin D1 or the detection of translocation t are key diagnostic elements. In our series, they were detected in five patients. The origin of the immunophenotype expression of cyclin D1 is the characteristic translocation of MCL, t that takes place between the IGH gene and the cyclin D1-BCL1 (CCND1) gene, which is considered the primary genetic anomaly of MCL, documented in 70% of MCL cases . An infrWith regard to treatment, it is important to differentiate between patients with localised disease (stages I–II) and advanced disease (III–IV):R-CHOP (three to four cycles) followed by external involved-field radiotherapyExternal involved-field radiotherapy exclusively for stage I Localised disease (stages I and II) accounts for less than 20% of patients. Front-line treatment must be based on immunochemotherapy .R-CHOP Advanced disease (stages III–IV) accounts for the remaining 80% of patients. The objective is to obtain a complete remission, given that it is a key prognosis factor. But again on indolent variants, there is no standard plan, rather treatment depends on the age of the patient, their general state, and associated comorbidities.Rituximab-CHOP x six cycles, ABMT if it responds .Nordic MCL2 protocol: This consists of the administration of three cycles of rituximab-CHOP alternating with three cycles of high doses of Ara-C, followed by ABMT with BEAM or BEAC .Rituximab-Hyper-CVAD x four cycles: In young patients this seems to improve the PFS to 4.8 years and OS 6.8 years, although it also increases toxicity significantly. It uses rituximab along with hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone, andit alternates this with high doses of methotrexate and cytarabine. If there is response after four cycles, it is completed with six cycles, and then with subsequent observation. In the absence of complete response, the patients will have to undergo high-dose chemotherapy and ABMT .There is an ample consensus on the treatment of the advanced disease, and groups with ample experience recommend  administIntensive chemoimmunotherapy appears to improve survival (median PFS of 4.8 years and OS of 6.8 years) but with an associated mortality of 3–4%. It is commonly used in young patients, without comorbidities, with blastic variant, or a high-risk MIPI score. The subsequent administering of rituximab in maintenance has not shown any clear benefit in this group of young patients without comorbidities.R-CHOP x six cycles, followed by rituximab (R) maintenance R-CHOP x six cycles, followed by Rituximab (R) maintenance R-CHOP x six cycles, followed by Rituximab (R) maintenance The first-line therapeutic recommendation in patients <65 years with comorbidities, unfit for chemotherapy at high doses and TAMO, or patients >65 years are:The administration of rituximab maintenance (every two months for two years) seems to increase the isolated limb perfusion (ILP) in the group of elderly patients or patients with comorbidities that contraindicate more aggressive treatments .There is a variant of mantle cell lymphoma characterised by the presence of splenomegaly and bone marrow involvement. At the histological level they have a diffuse pattern, being positive for cyclin D1, but with a low rate of Ki67 proliferation. Watchful waiting may be considered if the patient is asymptomatic and if the following conditions are met; nonpleomorphic norblastoid variant, maximum tumour diameter less than 3 cm, Ki67 <30%, and normal levels of LDH and B2 microglobulin .Most patients will require treatment at diagnosis, in our series all patients received first-line treatment at diagnosis, but the schema varied depending on the age of the patient, their general condition, and associated comorbidities. In first-line treatment there must always be an attempt to associate rituximab with the course of chemotherapy chosen, as observed with the cases we analysed. The majority of our patients received R-CHOP, except in the case of a young patient who received R-Hyper-CVAD.There is no standard course, and the treatment also depends on the age of the patient, of the patient’s general condition, and on associated comorbidities. Drugs like bendamustine, temsirolimus, ibrutinib, and lenalidomide have proved useful . The admIn a phase III trial in which R-bendamustine was compared against R-fludarabine, a response rate of 83% versus 50% and a PFS of 30 months versus 11 months respectively, was demonstrated , for whiIn our series the only patient who presented a relapse was case number 5. He received R-bendamustine as a second-line course.In a phase III trial with 54 patients refractory or in relapse received temsirolimus 175 mg/week for three weeks, followed by 75 mg/week or 25 mg/week versus the treatment chosen by the investigator (gemcitabine or fludarabine). Significant differences were found in the rate of response: 22% for dose of temsirolimus of 75 mg, 6% for 25 mg, and 2% for the alternative treatment with gemcitabine or fludarabine, and a median PFS of 3.4 months for doses of 75 mg, 4.8 months for the dose of 25 mg, and 1.9 months for the alternate treatment , which hIn a phase II trial with 111 patients refractory or in relapse (with an average of three previous lines) who received ibrutinib 560mg/day oral continuum, a response rate of 68% (21% complete responses (CR)), and a median PFS of 13.9 months with an OS rate of 58% at 18 months were documented . Ibrutin2, /days 1, 4, 8, and 11 of 21 days, a response rate of 40% and a median PFS of ten months are documented. It should be used with caution because it increases the risk of neurotoxicity G3-G4 in 13% [In a phase II trial in refractory or relapsed patients receiving bortezomib in doses of 1.3 mg/m4 in 13% .In a phase II trial with patients in third relapse and pretreated with bortezomib, receiving 25 mg/day x 21 days/28 days, response rates of 26% and a median PFS of 16 months were documented .There are other agents currently in development; cyclin 4/6 (CDK 4/6) inhibitors as palbociclib or abemaciclib, phosphoinositol 3 kinase (PI3K) inhibitors as idelalisib, BCL2 inhibitors as venetoclax, histone deacetylase (HDACS) inhibitors as vorinostat. However, we do not have specific biomarkers that reveal in each patient what tracks and genetic alterations are involved , 25.Patients with MCL present globally a median OS of 36 months that drops to 18 months for the blastoid variant . This pr2) as well as a proliferation rate with Ki67 >40% [The main adverse anatomopathological prognostic factor is the tumour’s rate of mitosis /L and elevated levels of LDH in the diagnosis were the two factors that were associated with a worse prognosis . Other fAt a clinical level, the forms associated with an improved prognosis are those that, even having involvement of the bone marrow and spleen, do not have lymph node involvement. Within the Ann Arbor classification, stages I, II constitute a group with good prognosis and more prolonged survival rates.Low risk (44%), with median of OS 60% at five yearsIntermediate risk (35%), median OS of 51 monthsHigh risk (21%), with median of 29 months.The MIPI demonstrates clearly the direct relationship existing between the index score prognosis and survival, so that the higher the MIPI score, the worse the survival rate. The MIPI catalogs patients in three categories according to their score  Table 1.Low risIn our series, we calculated the MIPI and noted except for one case all others are included in the subgroup of patients at high risk. In the literature, 21% of mantle lymphoma is included in the high-risk subgroup, with a median survival of 29 months. A recently published study shows that gain or amplification of the MYC oncogene is associated with a greater tumour size, a greater percentage of intermediate or high risk according to the MIPI scale considered as an independent factor of poor prognosis [MCL constitutes 4–8% of adult NHL, and is characterised by the overexpression of cyclin D1 or the detection of the t. Its course is moderately aggressive and variable, so it is important to take into account the prognostic variables as the specific MIPI prognostic index.Most of the patients require treatment at diagnosis. Treatment depends on the age of the patient, his general condition, and the associated comorbidities. For most patients with comorbidities, chemoimmunotherapy of induction type R-CHOP, R-CVP, or R-bendamustine, are good choices of treatment followed by maintenance rituximab for two years.In those young patients and those under 65 years of age without associated comorbidities, more aggressive chemotherapy courses may be used after being evaluated for their response to induction treatment with chemotherapy to high doses and TAMO. In those patients who are not candidates for autologous transplant with response to induction scheme should be evaluated for maintenance treatment with rituximab.There is no standard treatment for relapse, therefore it depends on the age of the patient and their general condition. The drugs to consider in cases of relapse, with or without rituximab, are: bortezomib, lenalidomide, bendamustine, and ibrutinib.There are many studies with new drugs for MCL. For patients in relapse there is no standard treatment, hence further studies are needed to clarify as well as to reveal what would be the optimal sequence of treatment.The authors declare that they have no conflict of interest.


Hylomyscus and Beamys) were not recorded. Myosorex zinki, the only mammal endemic to Mt. Kilimanjaro, which previously was known by only a few specimens collected in the ericaceous or moorland habitat, was found in all but one (the lowest) of the sites sampled, and was one of the most widespread species of small mammal along the gradient. Two shrews  and one rodent (Dendromus insignis) were found throughout the entire transect, with Dendromus being observed at our highest trap point (4240 m). As in similar faunal surveys on other mountains of Tanzania, rainfall influenced the sample success of shrews, but not rodents. Trap success for rodents at 3500 m was notably low. This study contributes further justification for the conservation of the forest habitat of Mt. Kilimanjaro.Mt Kilimanjaro is Africa’s highest mountain, and an icon for a country famous for its mammalian fauna. The distribution and abundance of small mammals on the mountain are poorly known. Here we document the distribution of shrews and rodents along an elevational gradient on the southeastern versant of Kilimanjaro. Five sites were sampled with elevational center points of 2000, 2500, 3000, 3500 and 4000 m, using a systematic methodology of standard traps and pitfall lines, to inventory the shrews and rodents of the slope. Sixteen species of mammal were recorded, including 6 shrew and 10 rodent species, and the greatest diversity of both was found at 3000 m, the elevational midpoint of the transect. No species previously unrecorded on Kilimanjaro were observed. Two genera of rodents that occur in nearby mountains ( Knowing the distribution of organisms along an elevational gradient is critical to understanding the evolution and ecology of montane biotic systems, and to designing conservation strategies to maintain them. These reasons have motivated elevational surveys of small mammals in various areas of the world including Chile Knowledge of the ecology and behavior of the targeted faunas help frame considerations of the results of systematic sampling along gradients. For example, Stanley & Hutterer et al. Mt. Kilimanjaro is the highest mountain in Africa and an icon for a region renowned for its unique mammalian fauna. Ironically, the mammals that inhabit the habitats of this volcano are relatively unknown, with most historical attention focused on larger species, leading to calls for complete inventories of the fauna of the mountain Using a standardized sampling regime that has been utilized in several other montane sites of Tanzania over the past two decades Mt. Kilimanjaro is in northeastern Tanzania and reaches an elevation of 5895 m. An extinct volcano, the mountain is the conglomeration of three volcanoes: Kibo , Mawenzi (the second peak of the mountain), and Shira (a plateau) The specific localities, elevations, habitats (sensu Mwasaga Site 1–2000 m). 4 km N, 1.5 km W Maua, 3°14.404' S, 37°27.502' E, 2043 m; lower montane forest; 23–30 August 2002.Site 2–2500 m). 7 km N, 2.5 km W Maua, 3°12.459' S, 37°26.818' E, 2470 m; upper montane forest; 17–25 July 2002.Site 3–3000 m). 10.5 km N, 3.5 km W Maua, 3°10.627' S, 37°26.413' E, 2897 m; ecotone between montane forest and ericaceous zone; 26 July-03 August 2002.Site 4–3500 m). 13.5 km N, 4 km W Maua, 3°08.941' S, 37°26.133' E, 3477 m; ericaceous zone; 4–12 August 2002.Site 5–4000 m). 16 km N, 4.5 km W Maua, 3°07.566' S, 37°25.600' E, 3995 m; ecotone between ericaceous and alpine zones; 13–21 August 2002.We used identical sampling techniques to those employed in similar small mammal surveys in other Tanzanian forests All traps and buckets were checked once in the early morning and again in the late afternoon. Not all traps or buckets were employed for equal amounts of time , so we use the measures “trap-night” and “bucket-night” (one trap or bucket in operation for one 24 hr period-0700 to 0700 hrs) to quantify sampling effort. We refer to the success rate of each method as either “trap success” or “bucket success”, and calculate these values by dividing the number of individuals captured by the number of trap-nights or bucket-nights and multiplying by 100. In discussions involving the two trapping methodologies combined, the term “sampling-night” refers to either one trap-night or one bucket-night, and “sample success” refers to the success rate for the two methodologies combined. The latter is calculated by dividing the number of individuals captured by the number of sampling-nights and multiplying by 100.Standard external measurements and reproductive status were recorded for each specimen, which was then either prepared as a study skin and skeleton or preserved in 10% formalin, and later transferred to 70% EtOH. Specimens are deposited in the Field Museum of Natural History (FMNH) with a portion to be returned to Museum of Zoology, University of Dar es Salaam (UDSM). We follow the taxonomy of Carleton & Stanley Permits for the collection and export of specimens were provided by the Tanzania Commission for Science and Technology (Ref# 2002-232-ER-90-172), the Tanzania Ministry of Natural Resources and Tourism , and the Tanzania National Parks (Ref # TNP A44). Import of specimens into USA was approved by the US Fish and Wildlife Service (3177-W10214-9/18/02). Shrews and rodents were euthanized following the protocol approved by the American Society of Mammalogists X2 = 695.2, P<0.05), and significantly more rodents were caught in traps than in buckets , a pattern observed in past studies on small mammals of Tanzania Crocidura allex, C. monax, C. olivieri, and Myosorex zinki (weighing between 3.6–51.0 g). While most of the rodents caught in buckets were relatively small , both specimens of Tachyoryctes daemon (240–290 g) were captured in buckets. Other rodent species captured in buckets included Grammomys dolichurus, Graphiurus murinus, Praomys taitae, and Rhabdomys dilectus.During the survey, we accumulated 11,562 sample-nights (8361 trap-nights and 3201 bucket-nights) and trapped 612 small mammals, including 319 shrews representing 6 species, and 293 rodents representing 10 species , 3, 4. SDendromus insignis and Otomys angoniensis were captured on the last day of trapping.The number of captures  at each elevational site ranged from 54 [2.4%] at 3500 m to 151 [6.8%] at 2000 m . For shrWe examined the relationship of four daily capture parameters  with cumulative sample-nights for both type of trapping methodology  and mammThe effect of rainfall on captures is presented in r values high, but not significant. Rodents showed no such notable pattern. The relationship between elevation and total sample success, number of individual mammals, and number of species collected for shrews and rodents combined was generally negative, but not significant. The least number of mammals, and species collected was at the 3500 m site. The greatest number of individuals noted was at the lowest site (2000 m), and the highest species diversity was observed at the 3000 m site. In most cases, the forested sites showed greater abundance and species diversity than the habitats above tree line  were collected in 385 buckets , most buckets captured no animals. Over the entire survey, 203 buckets caught nothing, 100 took one animal, 43 trapped two, 26 captured three, 7 caught four animals, 3 collected five animals, 2 trapped six animals, and ten animals were found in one bucket. Traps showed a similar pattern with 3.4% trap success in 1040 individual traps, and 283 captures (263 rodents and 20 shrews), but 834 traps caught nothing, 148 one, 42 two, 13 three and 3 four. To test for “trap competition” and to determine if captures were independent with respect to each other, we compared the observed distribution of captures by bucket and by trap to the Poisson distribution. Neither captures by buckets or traps followed the Poisson distribution  suggesting a lack of trap or bucket independence. Significantly fewer traps or buckets caught one individual than would have been expected based on the assumption that the frequency of captures follows a Poisson distribution, and significantly more caught 2, or more, than expected Myosorex zinki) is endemic to the massif, and none were introduced taxa. The other species have broader distributions, to varying degrees. For example, among the soricomorphs, Crocidura monax has been recorded in neighboring mountains within the Eastern Arc Mountains to the southeast of Kilimanjaro, including the North Pare and West Usambara Mountains. Crocidura allex is known from other mountains of the northern highlands of Tanzania  and the highlands of Kenya . Crocidura hildegardeae and Sylvisorex granti are distributed across Kenya and the montane habitats of the Albertine Rift. Finally, Crocidura olivieri is broadly distributed across much of the African continent Tachyoryctes daemon is restricted to northern Tanzania, but murines such as Grammomys dolichurus and Rhabdomys dilectus range across much of eastern and southern Africa, as does the dormouse, Graphiurus murinusSixteen species of mammal (6 shrews and 10 rodents) were recorded along an elevational transect from roughly 2000 to 4000 m on the southeastern slope of Mt. Kilimanjaro  and Southern Highlands Beamys hindei is Taveta, Kenya Beamys. Both localities are at, or near the base of Mt. Kilimanjaro. While this is not the most common species recorded in recent surveys of montane habitats of Tanzania across the elevational range from 600 to 2000 m Beamys if it occurs in the forests of southeastern Kilimanjaro. Records of Hylomyscus on Meru Praomys taitaeBeamys or Hylomyscus . All of this leads us to the conclusion that neither Beamys nor Hylomyscus currently occur in the forests of Mt. Kilimanjaro.We found no species not previously documented on the mountain. There are several published faunal lists for Kilimanjaro, the most complete being that of Grimshaw Crocidura allex, Myosorex blarina zinki [ =  M. zinki], Dendromus mesomelas kilimandjari [ =  D. insignis] and Rhabdomys pumilio diminutus [ =  R. dilectus] were the same as in our study at the 3500 m site. One species  recorded by Shore & Garbett at 3500 m was not trapped by us at that elevation, but was collected at sites both lower and higher than 3500 m. Notably, their trap success for small mammals  was much higher that of the 3500 m site in this study , although the period of the surveys (mid-July to early August) was similar in both studies and trapping extended over several days at each site. The bait used by Shore & Garbett Rhabdomys moving between buildings. Both repeating our sampling methodology at our site, and sampling with the same techniques at, and at increasing distances from Horombo would be illustrative of the influence of human habitation on the abundance of native rodents in the environs of Kilimanjaro.The trap success for rodents was highest at 4000 m and lowest at the 3500 m site . The veret al.et al.et al.Mus triton, which they recorded at 2300 and 3270 m. Lophuromys aquilus was recorded at 3200 and 3590 m by Mulungu et al.Lophuromys was brought to us by a Tanzanian National Parks employee who captured it at Horombo Hut (3760 m). The presence of other species at various elevations documented by Mulungu et al.Mulungu Dendromus insignis and Otomys angoniensis for the first time during the last 24-hours of trapping. Notwithstanding the 2500 m pattern, we are confident that we documented almost all of the species of shrews and small rodents occurring at each site, and thus feel justified in comparing results among different elevational sites of this transect, as well as to results of similar surveys within Tanzania As in past studies within Tanzania, the combination of traps and pitfall lines were effective in sampling non-volant small mammal communities at different elevations on Mt. Kilimanjaro Praomys taitae and Rhabdomys dilectus, respectively, were documented and the only site where the two species were found together. In general, there was more species diversity for both shrews and rodents in forest habitats than above treeline  for shrews as elevation increased . Howevertreeline ,3,4.Rainfall generally influenced the capture of shrews, but not rodents as was observed in the faunal inventories in the Udzungwa Mountains Myosorex zinkiet al. M. zinki was only known from a few specimens captured in the moorland habitats above tree line et al.Myosorex zinki was not observed at our lowest sampling site (2000 m). Stanley et al.The only endemic mammal, as currently understood, on Mt. Kilimanjaro is Crocidura allex, Sylvisorex granti, and Dendromus insignis. The latter was found in the highest trap set in the survey , and four individuals of this species were collected in this single trap . How high small mammals extend on Kilimanjaro remains unanswered. Wild dog (Lycaon pictus) is the only mammal (other than Homo sapiens) recorded at the summit  were found at all sites sampled and occur across the range from roughly 2000 to 4000 m:  5895 m; . However


Osteoporosis is a health concern characterized by reduced bone mineral density (BMD) and increased risk of fragility fractures. Many studies have investigated the association between genetic variants and osteoporosis. Polymorphism and allelic variations in the vitamin D receptor gene (VDR) have been found to be associated with bone mineral density. However, many studies have not been able to find this association. Literature review was conducted in several databases, including MEDLINE/Pubmed, Scopus, EMBASE, Ebsco, Science Citation Index Expanded, Ovid, Google Scholar, Iran Medex, Magiran and Scientific Information Database (SID) for papers published between 2000 and 2013 describing the association between Fok1 and Bsm1 polymorphisms of the VDR gene and osteoporosis risk. The majority of the revealed papers were conducted on postmenopausal women. Also, more than 50% studies reported significant relation between Fok1, Bsm1 and osteoporosis. Larger and more rigorous analytical studies with consideration of gene-gene and gene-environment interactions are needed to further dissect the mechanisms by which VDR polymorphisms influence osteoporosis. Bone is a metabolically active tissue that experiences continuous remodeling via two reciprocal processes, bone formation and resorption. Respectively, osteoclasts, osteoblasts and osteocytes are responsible for bone resorption, formation and maintenance . OsteopoThe prevalence of osteoporosis varies between different populations and ethnic groups -6 for exOsteoporosis is a disease caused by the interaction of genetic and environmental factors. According to many studies, the contribution of genetic and environmental factors is about 70% and 30% respectively. The environmental factors can control gene expression and accordingly, the process of the disease . The stuThere are many relevant published studies of the genetic susceptibility to osteoporosis. Genes can affect the skeletal system in two ways. The first, control body uptakes and intakes such as urinary calcium excretion to modulate BMD, the second way is poor metabolism due to genetic defects .There calcium absorption pathways consists of trans_cellular and para_cellular. The trans_cellular pathway closely depends on the action of calcitriol and the intestinal vitamin D receptor. Transcellular transport occurs primarily in the duodenum where the VDR (Vitamin D receptor) is expressed in the highest concentration. So the regulation of VDR gene is most important in high efficiency of calcium absorption ,39.Estrogens are known to play an important role in regulating bone homeostasis and preventing postmenopausal bone loss. They act through binding to two different estrogen receptors (ERs), ERα and ERβ, which are members of the nuclear receptor superfamily of ligand-activated transcription factors. Both ER kinds are expressed in osteoblasts, osteoclasts, and bone marrow stromal cells. And also ESRα has a prominent role in regulating bone turnover and the maintenance of bone mass ,41.Different studies have reported a list of effective genes on osteoporosis; the most important of which are vitamin D receptor gene(VDR), estrogen receptor alpha (ESRα) ,interleukin -6 (IL-6), Collagen type I (COLIA1), LDL receptor-related protein 5 (LRP5) ,42,43.Over the recent decades, genome-wide association studies (GWAS) have contributed to the understanding of gene structure in complex and chronic diseases such as osteoporosis. Some of studies have indicated 62 significant loci where control bone mineral density variation ,44-47.Candidate gene studies have mainly focused on Vitamin D receptor genes (VDR), type 1 Estrogen receptor genes (ESR1) and type 1 Collagen (Coli1) ,48-50. IVitamin D receptor’s (VDR) genotypes have been associated with the development of several bone diseases as well as multiple sclerosis (MS), osteoporosis, and vitamin D-dependent rickets type II and other complex maladies .The human gene encoding the VDR gene has been localized on chromosome 12q12-q14. Vitamin D receptors (VDRs) are members of the NR1I family, which also includes pregnane X (PXR) and constitutive androstane (CAR) receptors, which form heterodimers with members of the retinoid X receptor family . VDR is Gene ontology (GO) annotations related to this gene include steroid hormone receptor activity and sequence-specific DNA binding transcription factor activity. An important paralog of this gene is NR4A3 .There are more that 100 restriction endonuclease recognition sites in VDR gene and some of them are polymorphisms such as Fok1, Bsm1, Apa1 and …. .Fok1 and Taq1 are located in exon 2 and 9 respectively. And also, Bsm1 and Apa1 are located in intron 8. Bsm1, Apa1 and Taq1 have been identified at the 3’ end of the gene. The effects of VDR gene polymorphisms are in connection with each other -57. In mA series of characterized VDR gene polymorphisms, including Fok1, Bsm1, Taq1, and Apa1, have been extensively studied with regard to their association with osteoporosis, but with vise versa results ,60-63.Significant associations of Fok1 polymorphism with low BMD have been described in some studies, -66 but nThe Bsm1 restriction enzyme identifies a polymorphic site at an intron at the 3′-end which is in linkage disequilibrium with several other polymorphisms, including Apa1, Taq1, and the variable-length poly(A) . AlthougTo clear the relationship between osteoporosis and VDR gene polymorphisms , we review the current evidence systematically.In this systematic review, the studies that had assessed the association between VDR gene polymorphisms and osteoporosis between 2000 and 2013 were included. In all these studies the diagnosis of osteoporosis was performed based on BMD measurement by Dual X-ray Densitometry (DXA) at least one of the bone sites. All kinds of original studies such as cross-sectional, longitudinal, and case controls were included. All review articles, Meta-analysis and systematic reviews after checking references (to avoid missing any paper), were excluded. Also, the articles performed on patients with secondary osteoporosis as well as non-human studies  and cellular-molecular discussions were excluded. To avoid language bias non-English-language publications were also included and Google-Translator was used to extract these articles’ data.The search strategy was based on electronic and hand searching. Main key words in this systematic review were Osteoporosis, Bone density, vitamin D receptor gene, polymorphisms, Fok1 and Bsm1. We searched electronic databases of biological and health sciences including MEDLINE (pubmed), Scopus, EMBASE, Ebsco, Science Citation Index Expanded, Ovid, Google Scholar, Iran Medex, Magiran and Scientific Information Database (SID). All national and international congresses about genetic and osteoporosis like IOF and NOF congresses were examined. And also expert’s curriculum vitae in this field were checked for relevant studies. Subsequently, the searches were carried out and publications of interest were selected, based on titles and abstracts. The full text of all selected publications was assessed for relevance. If the full texts of papers were not available, they were obtained through correspondence with the authors. This was followed by extracting the relevant data from the identified publications according to the steps described in detail below. Totally, two reviewers reviewed the articles. In case of disagreement, the third reviewer assessed the articles.The following data were extracted from each published article: name of the first author, publication year, the number of case and control by gender, the number of menopausal women, and the number of performed BMDs, ethnic origin of the studied population, mean age, genotyping method (PCR-RFLP and TaqMan), Bone sites, and the genotype frequency of the polymorphisms. The reliability of data extraction forms was assessed by genetics and endocrinology specialists. And the content validity was assessed by 10 articles and was confirmed by 0.75 Cronbach’s Alpha. Methodological quality, the strength and weaknesses of included studies were investigated using a modified STROBE checklist.A schematic of the literature search is shown in Figure In these studies, diverse groups of people were discussed. 36.5% of the studies studied postmenopausal women. On the other hand, post-menopausal and pre-menopausal woman were studied simultaneously in 22.2% of the articles and 12.7% of them studied all groups.According to the results, 96.8% of studies performed the polymorphisms using PCR-RFLP “polymerase chain reaction- restriction fragment length polymorphism”. The other methods such as Taq-man were used to determine the association between Bsm1 and Fok1 polymorphisms and osteoporosis.As mentioned above, the studies after year 2000 on world were enrolled in this systematic review. Most articles were published in 2006 and after that the number of papers showed a decline trend.Based on articles, totally 17473 persons studied. 65.9% of studies reported a significant relation between Bsm1 and osteoporosis risk. Likewise, 60.0% of studies reported a significant relation between Fok1 and osteoporosis risk.Also, the papers were categorized by gender and age. The data indicated that most of the articles were done on women and also on older ages. Most of studies were examined post menopausal women.After characterization of authors” countries, it was demonstrated that respectively china ,98,102 aIn conclusion, both gender and ethnicity are effective factor on osteoporosis and bone mineral density.As is evident, genetic variant has a tremendous roll to adjust bone activities and therefore along with vitamin D deficiency, has a large effect on osteoporosis incidence and also osteoporotic fractures. In this systematic review, due to study the association between low bone density and Bsm1 and Fok1 polymorphisms, 61 papers were studied and statistically analyzed. As a main result, most of the studies were performed on post-menopausal women i.e. the largest risk group to their major content of research . It seemIn more than 50% of studies a significant association was found between the two polymorphisms (Fok1 and Bsm1) and osteoporosis. Based on the articles, 65.9% of studies reported a significant correlation between Bsm1 and osteoporosis risk. Likewise, 60.0% of studies reported a significant correlation between Fok1 and osteoporosis risk. Most of the studies were performed in developed countries but also, developing countries have initiated this way.An important and noticeable issue in this systematic review is different results in different races ,133. EthIn more that 95% studies for assessing polymorphisms, PCR-RFLP were used Table . It is nIn summary, there is large ethnic and racial variability in BMD levels and osteoporosis rates. Across all racial groups and polymorphisms differences, women experience osteoporosis is more than the combined number of women who experience breast cancer. Prevention efforts should target all women, irrespective of their race and ethnicity, especially if they have multiple risk factors. And also, using novel and pioneer genetic techniques to better assess and better quality can be useful.


Bangladesh has experienced rapid economic growth and achieved major health improvements in the past decade, but malnutrition rates remain high. A nationally representative study conducted in 2011 assessed the dietary habits of 841 children 24–59 months old; 1428 children 6–14 years old; and 1412 non-pregnant, non-lactating women. The study’s objective was to assess dietary intakes of key micronutrients and the consumption pattern of potentially fortifiable foods, and then to model the potential impact of fortification of key staple foods. The current intakes of several micronutrients, namely, iron, zinc, folate, vitamin A and vitamin B12, were found to be insufficient to meet the needs of Bangladesh’s children and women. The fortification of rice with iron and zinc and edible oil with vitamin A has the potential to fill a significant part of the nutrient gap, as these are consumed widely and in significant amounts. Wheat flour and sugar are not as promising food vehicles in the Bangladeshi context, as they were consumed by a smaller portion of the population and in smaller amounts. In conclusion, fortification of rice and oil is recommended to address the large gap in micronutrient intakes. Malnutrition is a widespread public health problem, especially in developing countries. According to The State of Food Insecurity in the World , 870 milBangladesh is a developing country that has experienced rapid economic growth during the last 20 years, with an annual gross domestic product growth rate of 6.1% . SimultaThere are a number of factors that contribute to the high prevalence of micronutrient deficiencies, including low consumption of animal-source foods, poor bioavailability of micronutrients from plant sources, diseases and infections, food shortages, and, most importantly, a limited dietary diversity . Energy Fortification is widely recognized as being one of the most cost-effective strategies to increase regular consumption of micronutrients in a population. However, to be successful, any fortification program needs to take into account a number of parameters . First, The objective of this study was to assess dietary intakes of key micronutrients and the consumption pattern of potentially fortifiable foods, and then, based on this information, to model the potential impact of fortification of key staple foods and to assess variation in all of these foods by regions of the country. The findings of this study aim at informing the design of a fortification strategy in Bangladesh.The data presented in this paper were collected as part of a large cross-sectional nationally representative survey, the National Micronutrients Status Survey December 2011 , that wai.e., 150 PSUs in total) were randomly selected from the list of the 15,000 primary sampling units (PSUs) that was used for the Bangladesh Multiple Indicator Cluster Survey (MICS 2009). In the second stage, a segment containing 50 households was selected per PSU, from which 20 households were randomly selected to be included in the survey. In the third stage, approximately 8 pre-SAC, 10 SAC, and 10 NPNL were selected among the 20 households in each PSU. In order to minimize non-response, non-responders were substituted with other eligible respondents randomly selected from the 20 households selected in the second stage until the required sample sizes for each population group were achieved.The National Micronutrients Status Survey December 2011  used a tIn total, consumption data were gathered from 841 pre-SAC 24–59 months, 1428 SAC, and 1412 NPNL between October and December 2011. The surveyed population included pre-SAC 6–23 months old, however information on their consumption was not assessed, as they were expected to consume limited amounts of the food vehicles and therefore to benefit from fortification of staples only to a limited extent only.To assess food consumption and nutrient intake of NPNL, SAC and pre-SAC, individual 7-day semi-quantitative food frequency questionnaires were used to interview NPNL, SAC, and the caregivers of pre-SAC between 24 and 59 months, respectively, taking into consideration commonly consumed foods, with special attention to vitamin A-, iron-, and zinc-rich foods. The only food that was not assessed by the semi-quantitative food frequency questionnaire was oil, which was assessed at the household level. Food photographs indicating the serving size and amount in grams (g) were used to assess the quantity of consumption. Raw food weight was calculated by using appropriate conversion factors . To tranmaida flour; and (2) wheat flour, which was bought by the household and used to make foods within the household  and which consisted mainly of atta flour. The sugar consumption was estimated from the amount of sugar added to the foods or drinks consumed.The wheat flour products consumed by the households were separated into 2 categories: (1) bread, which was made industrially and bought already baked by the household and which was made mainly from For vegetable oil, the data on household monthly expenses were gathered and an average consumption was estimated for each household. Consumption estimates were extrapolated from the average per-person consumption reported in the national surveys by applying adult equivalent units (AEUs), recommended by the Food and Agriculture Organization of the United Nations (FAO), by a factor of 0.51 for children 1–3 years old, 0.71 for children 4–6 years old, 0.78 for children between 7 and 10 years old, 0.98 for boys and 0.86 for girls between 11 and 14 years, and 0.86 for women of reproductive age (14–50 years of age) .pacca), and “other,” while those of floors were brick-cement (pacca), mud, and “other.” For household assets, each item belonged to 1 of 2 categories: “owned” or “not owned” by the household. Principal component analysis (PCA) was used to create the asset index. A weight was attached to each item from the first principal component.Socioeconomic status (SES) was calculated , using tFortificants and fortification levels of selected foods are presented in WHO recommended nutrient intake (RNI)  was usedData entry, including quality checks, as well as data management and analysis, was performed using SPSS software version 19. The mean consumptions among pre-SAC, SAC, and NPNL consuming rice, bread, vegetable oil, sugar, and wheat flour (excluding non-consumers) were analyzed by 1-way analysis of variance (ANOVA) (since the distribution of consumption was asymmetric) and multiple comparisons to compare strata, geographic areas, and socioeconomic status. To estimate the additional intake of the micronutrients under consideration for the fortification strategy, the mean consumption of nutrients from the fortifiable food  by age groups were calculated by multiplying the level of fortificant  with thEthical approval for this study was obtained from the Institutional Review Board (IRB) of the International Centre for Diarrhoeal Disease Research, Bangladesh  (Reference number: PR#11018). Under the IRB, the Research Review Committee provided approval on the technical aspects of the protocol, which was followed by the Ethical Review Committee giving approval for the study. The participants and/or the guardians of the participants in the study provided their written informed consent.p < 0.01 for iron and p < 0.05 for zinc, respectively). Zinc intake was higher in higher income quintiles (p < 0.001). There were no significant differences in folic acid intake between areas of residency, districts, nor socioeconomic strata. Vitamin B12 intake was significantly lower in pre-SAC from lower SES than from higher SES (p < 0.05).The consumption patterns of 841 pre-SAC, 1428 SAC, and 1412 NPNL were analyzed. The micronutrient intake estimates for five micronutrients are reported in p < 0.01) and zinc and vitamin A intakes with SES (p < 0.001 and p < 0.001). Intake of folic acid was lowest in rural areas and highest in slums (p < 0.001). Consumption of vitamin B12 was proportional with socioeconomic strata (p < 0.001).For SAC, mean iron, zinc, vitamin A, folic acid, and vitamin B12 intakes in the diet covered 23.7%, 28.0%, 50.5%, 26.2%, and 62.9% of the needs. Iron intake was correlated with the district of residency (p < 0.01 and p < 0.001). Vitamin A intake was correlated with the district of residency (p < 0.001) and the SES (p < 0.01). Intakes of folic acid and vitamin B12 were significantly (p < 0.01) lower in rural and lower-income households and higher in slum areas and higher-income households. Vitamin B12 consumption was lower in poorer households than in wealthier households (p < 0.001).The diet of NPNL covered 12.5% of their requirements in iron, 47.4% in zinc, 75.4% in vitamin A, 29.0% in folic acid, and 45.6% in vitamin B12. Iron and zinc intakes were the highest in women from higher socioeconomic strata  was assessed for each population group. The percentages of pre-SAC, SAC and NPNL consuming rice, bread, wheat flour, and sugar are shown in The consumption of the main fortifiable foods . The pre-SAC from the lower-income families consumed more rice than the pre-SAC from the higher-income families (p < 0.001).There were no significant differences in bread consumption between regions. However, wealth had an influence on the bread consumption of pre-SAC (p < 0.01): pre-SAC from wealthier families consumed more bread. SAC consumed significantly higher amounts of wheat flour in slums and urban areas than in rural areas (p < 0.001). Pre-SAC in slums tended to consume higher amounts of wheat flour and smaller amounts in rural areas (p = 0.05). SAC and NPNL from higher-income quintiles consumed more wheat than lower-income quintiles (p < 0.001). There was a significant difference in wheat flour consumption between the divisions: wheat flour consumption was the lowest in Dhaka and in Khulna and the highest in Chittagong and Rajshahi (p < 0.001). NPNL living in slums and urban areas consumed higher amounts of sugar than NPNL living in rural areas. Sugar was consumed in higher amounts by SAC and NPNL from higher-income quintiles (both p < 0.001). Consumption also significantly differed between divisions for SAC and NPNL (both p < 0.001). SAC in Barishal consumed the most sugar, while children in Sylhet consumed the least. In Rajshahi, NPNL consumed the most sugar and NPNL in Dhaka consumed the least sugar. The average daily oil consumption was 26.4 g per capita. Oil consumption was higher in urban and slum areas than in rural areas (p < 0.001). The highest oil consumption was found in Chittagong and the lowest in Barishal (p < 0.001).The mean consumption of rice, wheat flour, bread, sugar, and oil by population group is shown in i.e., 300–400 µg/day for pre-SAC; 400–600 µg/day for SAC, and; 1000 µg/day for adults). The additional vitamin A intake would be far below the upper levels for NPNL (3000 RE/day) and SAC (900–1700 RE/day); however, it is slightly too high for pre-SAC (700 RE/day).Based on mean level of consumption and recommended fortification levels, the additional intakes of iron, zinc, folic acid, and vitamin B12 , and vitAccording to the data collected from the food frequency questionnaires, iron, zinc, and vitamin A intakes were found to be lower than the requirements, highlighting the need for nutrition interventions. These findings are especially alarming since they concern population groups for whom it is critical to have an adequate nutrient intake. In fact, nutritional deficiencies during infancy and childhood can have serious and irreversible consequences that last until adulthood ,18. MoreZinc intakes in individuals from higher socioeconomic strata were higher than those in the lower strata. Women from lower socioeconomic strata tended to have lower iron intakes. This finding highlights the need to design an intervention that reaches the poorest segments of the population, especially for zinc and iron. These minerals are contained in a number of foods and in highest amounts in animal-source products. However, their absorption can be significantly inhibited by certain dietary components, such as phytic acid and polyphenols ,23. UnfoBased on the consumption patterns, rice is the most suitable vehicle for fortification: it is consumed regularly by the whole population, in very large and constant amounts. In fact, rice fortification could provide between 1.0 mg/day and 2.4 mg/day of additional iron, or an additional 4.0%–8.8% of the population’s RNI in iron. However, rice fortification alone will not be sufficient to reduce the prevalence of iron deficiency in the population. In fact, efficacy studies have shown that daily iron intakes from rice of between 13 mg and 20 mg can reduce iron deficiency and iron deficiency anemia ,26,27. RAs with rice, oil is consumed by almost all households. Oil could provide 38.3%–46.5% of the population’s vitamin A requirements, which is above the 15% minimum recommended by the International Vitamin A Consultative Group . HoweverMaida flour fortification would provide more micronutrients to pre-SAC and SAC than to NPNL. Atta flour fortification would provide more micronutrients to children living in slums and urban areas, whereas rice fortification would provide more to the rural populations. Sugar fortification with vitamin A would complement the vitamin A intakes provided by the diet and by fortified oil and would result in sufficient intakes of vitamin A among all three population age groups. However, the percentage of respondents who reported consuming sugar and the amounts consumed reported were much lower than for oil. The fortification of sugar would not benefit all population groups equally: it would benefit mostly a small segment of young children from higher socioeconomic strata.In comparison to rice and oil, the number of people consuming bread, wheat, and sugar, and the amounts consumed, are much lower. However, fortification of these food vehicles would significantly contribute to filling the gaps in current micronutrient intakes, especially if it is implemented alongside fortification of other foods  and in combination with other interventions, such as dietary diversification and supplementation. The fortification of wheat flour would provide additional amounts of iron and zinc, but also (and mostly) folate and vitamin B12. According to the results of this analysis, the simultaneous fortification of wheat flour and rice with iron would still not be enough to cover the iron needs of the target populations. In fact, only 67.1% of the needs of pre-SAC, 41.4% of the needs of SAC, and 22.6% of the needs of NPNL would be covered. Therefore, fortification efforts need to be combined with other strategies to prevent iron deficiency. WHO recommends three food-based strategies to fight iron deficiency: dietary diversification, food fortification, and iron supplementation. To maximize the reduction of iron deficiency prevalence in Bangladesh, food fortification should be combined with iron supplementation for iron-deficient women and children and with a campaign aimed at raising the awareness about iron-rich foods among the population.The risk of excess intake from the fortification of multiple food vehicles is low, especially given the fact that it is very rare for an individual to consume all fortified products in one day. The only area of concern would be the vitamin A intake in pre-SAC. Some children might consume amounts of vitamin A that are close to the upper levels set for that age group. In addition, these same children have a high chance of receiving high-dose vitamin A supplements.There are four major limitations to this study. The first one is that the consumption data were assessed using a seven-day semi-quantitative food frequency questionnaire. Micronutrient intakes might have been slightly over- or underestimated ,31. The In conclusion, rice and oil consumption were high, constant, and widespread across Bangladesh and therefore are the most promising food vehicles for fortification. Fortification of these two food vehicles could significantly increase the micronutrient intakes of most population groups. In order to design an optimal fortification strategy, these findings need to be complemented with a comprehensive industry and market assessment and the fortification levels adapted based on cost, technological and sensory parameters.


Objective. This study aimed to explore the incidence of stroke and stroke subtypes among patients with type 2 diabetes mellitus (T2DM) based on the long-term surveillance data in Zhejiang, China, during 2007 to 2013. Materials and Methods. During January 1, 2007, and December 31, 2013, a total of 327,268 T2DM and 307,984 stroke patients were registered on Diabetes and Stroke Surveillance System, respectively. Stroke subtypes were classified according to standard definitions of subarachnoid hemorrhage, intracerebral hemorrhage, and ischemic stroke. The incidence of stroke and stroke subtypes was calculated by standardized incidence ratio (SIRs) with 95% confidence intervals (CIs) compared with general population. Results. The incidence of stroke and stroke subtypes among patients with T2DM was significantly higher than in general population. Stroke risk was found significantly increased with an SIR of 3.87 (95% CI 3.76–3.99) and 3.38 (95% CI 3.27–3.48) in females and males, respectively. The excess risk of stroke was mainly attributable to the significantly higher risk of cerebral infarctions with the risk for T2DM being four times that for general population. Conclusions. The relationship between stroke and T2DM was strong, especially in female. The incidence of stroke and stroke subtypes among patients with T2DM was up to 3-fold higher than in general population in Zhejiang province, especially the subtype of cerebral infarctions.  Stroke has been recognized as a major problem for public health worldwide. As World Health Organization reported, stroke ranks third (after MI and cancer) as a cause of death around the world . AlthougThe long-term surveillance data was from Diabetes Surveillance System and Stroke Surveillance System of Zhejiang Province in China, which was established in 2001 with thirty surveillance districts and covered about 16 million residents. These surveillance districts were selected by geographic variations and socioeconomic status which showed appropriate representativeness for Zhejiang Province . The diaFor stroke, if the interval between first onset and recurrence was more than 28 days, this record needs to report again. Otherwise, this record need not to report. According to the International Classification of Disease 10th revision (ICD-10), stroke patients were divided into four stroke subtypes, including subarachnoid hemorrhage (I60), intracerebral hemorrhages (I61), cerebral infarctions (I63), and unspecified strokes (I64). Classification and registration were completed by professional health practitioner. Furthermore, in the Diabetes Surveillance System, diabetes was divided into type 1, type 2, gestational, or other types of diabetes. Finally, a total of 327,268 T2DM patients and 307,984 stroke patients were collected between January 1, 2007, and December 31, 2013. This study was carried out in accordance with the “Declaration of Helsinki.”In the present study, only the stroke and T2DM patients recorded between January 1, 2007, and December 31, 2013, were included. Two kinds of matching conditions were used to link databases. (1) Identity card number was used to link T2DM patients who suffer stroke in the following years. (2) Patient's full name, gender, code of district registered in the system, and date of birth (year and month) were simultaneously used to link the record which did not match in step one . Those pThe incidence of stroke and stroke subtypes among patients with T2DM was evaluated by standardized incidence ratio (SIR) and 95% confidence interval (CI) comparing with general population. SIR and 95% CI were calculated as the number of observed stroke events divided by the excepted number of events with the Poisson regression model . The numA total of 327,268 T2DM cases were included from Diabetes Surveillance System between January 1, 2007, and December 31, 2013. During the follow-up time, 8615 stroke events have occurred with 4324 (50.19%) in male and 4291 (48.81%) in female, respectively. The median ages at diagnosis and registration of diabetes were 59  years old and 60  years old, respectively. The baseline characteristics of T2DM and total stroke outcomes were described in According to the age at diagnosis of T2DM, the result of overall SIRs for stroke in patients with T2DM was shown in This study aimed to explore the subsequent stroke incidence in patients with T2DM based on the long-term surveillance data in Zhejiang province, China. The results showed that the stroke incidence in patients with T2DM was significantly higher than in general population, which were consistent with previous studies , 15–17.The risk for stroke among patients with T2DM was up to 3-fold higher than in general population in male and female, which were comparable to those in large prospective studies , 18. TheThe incidence of stroke in rural areas among general population in this study was lower than Tianjin Brain study during 2006–2012  while inBell found that the relative risk of stroke in patients with T2DM reached a maximum in the 40- to 60-year-old group than in the nondiabetic population . In addiThere was a thrombotic tendency or at least an imbalance between the haemostatic and thrombosis-protecting system in diabetic patients, which might play a crucial role in the development of stroke, especially cerebral infarctions (I63) . In thisThere were several strengths for this study. Firstly, it was one of the few studies exploring the stroke incidence in patients with T2DM by SIR in China. Secondly, it was a population-based surveillance study with a large sample of 327,268 T2DM cases and 8615 stroke event outcomes. The most important is that T2DM and stroke patients were diagnosed and reported by certificated health practitioners, and, to further ensure the quality and veracity of data, the related data was verified by regional and provincial CDCs and registered in the surveillance system eventually.However, there were some limitations in this study. Firstly, we stratified by the variables of gender, urbanization, and age, while other potential confounding factors including hypertension, cholesterol, smoking, obesity status, alcohol consumption, physical activity, and diabetes treatments have not been adjusted in the analysis. Secondly, the follow-up time was not long enough, which restricted our ability to further assess the potential lead time bias and explore the effect of diabetes duration on stroke incidence.In conclusion, the current study indicated that the incidence of stroke and stroke subtypes among patients with T2DM was up to 3-fold higher than in general population in Zhejiang province, especially the subtype of cerebral infarctions (I63). Compared to males, the SIR was higher in females, although the incidence of stroke for the diabetic in females was lower than in males. Given the rapid growth of diabetes in China, even a small increased stroke risk would have important public health implications at population level. It is urgent and necessary to prevent and control diabetes.


Aortic valve calcification (AVC) secondary to renal failure (RF) is an inflammation-regulated process, but its pathogenesis remains unknown. We sought to assess the cellular processes that are involved in the early phases of aortic valve disease using a unique animal model of RF-associated AVC.Aortic valves were obtained from rats that were fed a uremia-inducing diet exclusively for 2, 3, 4, 5, and 6 weeks as well as from controls. Pathological examination of the valves included histological characterization, von Kossa staining, and antigen expression analyses.After 2 weeks, we noted a significant increase in urea and creatinine levels, reflecting RF. RF parameters exacerbated until the Week 5 and plateaued. Whereas no histological changes or calcification was observed in the valves of any study group, macrophage accumulation became apparent as early as 2 weeks after the diet was started and rose after 3 weeks. By western blot, osteoblast markers were expressed after 2 weeks on the diet and decreased after 6 weeks. Collagen 3 was up-regulated after 3 weeks, plateauing at 4 weeks, whereas collagen 1 levels peaked at 2 and 4 weeks. Fibronectin levels increased gradually until Week 5 and decreased at 6 weeks. We observed early activation of the ERK pathway, whereas other pathways remained unchanged.We concluded that RF induces dramatic changes at the cellular level, including macrophage accumulation, activation of cell signaling pathway and extracellular matrix modification. These changes precede valve calcification and may increase propensity for calcification, and have to be investigated further. Calcific aortic valve stenosis is the most common indication for surgical valve replacement, but there is no effective medical treatment to reduce or slow calcification .The cellular mechanisms of the pathogenesis of aortic valve calcification (AVC) are poorly understood. Yet, in the past decade, studies have demonstrated that AVC is an active, regulated process that involves inflammation , alteratA significant risk factor for AVC is renal failure (RF). Patients with RF experience more severe calcification, and the rate of calcification is faster compared with patients without RF . To examWe have reported that AVC is an active process that involves inflammation and is associated with an osteoblastic phenotype in valve tissue . Most stIn this study, we examined the early alterations in the valve before calcification is apparent, hypothesizing that several processes and pathways are activated and suppressed during the course of the disease. Our animal model allowed us to assess various factors and processes, such as inflammation, ECM, protein expression, and osteoblast transformation.Fifty Sprague-Dawley female rats, aged 8 weeks and each weighing approixmately 250 g, were used. The study protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Hebrew University Ethics Committee (Permit Number: MD-12-13407-4). Exsanguination was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.The animal model for RF-induced aortic valve disease entailed feeding the rats a high-adenine (0.75%) and high-phosphate (1.5%) diet  . AdenineTo focus on the earlier stages of calcification, the experimental design comprised of administering a uremic diet exclusively to rats for various durations.Fifty rats were fed a phosphate-enriched, uremia-inducing diet for 2, 3, 4, 5, or 6 weeks (10 rats per period). An additional 10 rats served as controls and were fed normal chow.After each period, the rats were sacrificed by exsanguination and thoracotomy, and the valves were examined by histology, western blot, and immunohistochemistry.Plasma was analyzed for potassium, phosphate, creatinine, sodium, urea, calcium, and total cholesterol on a VITROS5.1 .Aortic valves were dissected, fixed in formalin, and embedded in paraffin. Serial cross-sections of valve tissue were stained with hematoxylin, eosin, and von Kossa stain.Aortic valves (n = 3) were dissected, fixed in formalin, and embedded in paraffin, and 5-μm cross-sections were examined by immunofluorescence. Paraffin was removed with standard xylene washes, and the slides were boiled in 20 mM citrate buffer (pH 6) for 3 min in a pressure cooker to unmask the antigens. To prevent nonspecific binding, the sections were blocked with 3% BSA and 0.1% Tween in PBS for 1 h at room tempreture.The sections were incubated overnight with anti-collagen 3, anti-collagen 1, anti-fibronectin (Santa Cruz Biotechnology Inc.), anti-CD68 , anti-osteopontin , and anti-osteocalcin (Santa Cruz Biotechnology Inc.) at 4C. Negative control sections were incubated without primary antibody under otherwise identical conditions. After being washed in PBS, the sections were incubated with Cy5-conjugated secondary antibody  for 1 h.Aortic valves from all groups were lysed (n = 3 in each group) in at least two differnt sets. The tissue was hydrolyzed using Hepes buffer  with addition of 10 μm protease inhibitor cocktail and homogenized under ultrasound and then boiled for 5 min. Protein concentrations were measured by Bradford assay. Extracts (20 μg) were loaded onto 5% to 15% SDS-polyacrylamide gels and transferred to membranes, which were blotted overnight for Runx-2, osteopontin , osteocalcin (Santa Cruz Biotechnology Inc.), ERK, Akt, JNK, P38, . Protein levels were normalized to β-actin. The original western blot gels are provided in Image Pro-Plus version 7.0  system was used for image analysis. Twenty random areas from different parts of the valves were analysed by the software. The colors correspondent to positive staining was recorded and numbers of pixels per area and automatic counted on all pictures by the software. For the Western blot we used the Image J software for measurement of bands intensity. All results were normalized to respective controls, which were assigned a nominal value of 1.Data were presented as means ± Standard error. Statistical analysis for biochemical profile was analyzed by ANOVA followed by Student-Neumann-Keuls test. P<0.05 were considered significant. Statistical analysis for immunostaining was done using Kruskal-Wallis Test to compare all the groups together, then the non parametric Mann-Whitney Test was used to compare each groups to the control, P<0.05 was considered statistically significant. The pairwise comparisons were carried out using Mann-Whitney Test with Bonferroni correction of the significance level, P< = 0.005 is statistically significant according to the Bonferroni correction.Statistical analysis for Western blot was done using Wilcoxon Signed Ranks Test. P<0.05 was considered statistically significant.After 2 weeks on the adenine diet, the rats experienced a significant increase in creatinine and urea, and after 3 weeks, phosphate levels rose versus the control; RF exacerbated in the Week 5 group and remained unchanged Week 6. In association with RF, the rats developed hypocalcemia after 3 weeks which remained unchanged later. There were no significant changes in potassium, sodium, or cholesterol levels .Previously, We have demonstrated that rats developed significant valve calcification after 7 weeks on the uremic diet and an additional 2 weeks on a normal diet .In contrast, by histology, the aortic root sections were unremarkable compared with controls after 2 to 6 weeks on the high-adenine diet. The aortic root sections harbored arterial and valvular tissues, which were stained with hematoxylin, eosin, and von Kossa stain, and failed to show evidence of mineralization (data not shown).Because AVC involves significant inflammation, we determined the macrophage content in the valves early in the course of the disease by staining for CD68. Macrophages were increased early 2 weeks after the adenine diet was started. Macrophages numbers rose significantly until 5 weeks and were observed in the valve annulus and leaflets. Interestingly, macrophages levels dropped at Week 6 although still remained significantly higher than the controls . There wImmunostaining results confirmed the cytoplasmic expression of osteopontin and osteocalcin in valvular cells after 2 weeks as well as in later weeks; their level peaked at 5 weeks and dropped at 6 weeks . By westOsteocalcin was expressed from Weeks 2 to 5, declining in Week 6, although these changes did not reach a statistical significance .We evaluated the MAPK and Akt pathways to determine whether they are involved in the early phases of calcification. By western blot, the ratio of phospho-ERK to ERK-1 tended to increase after 2 weeks, fell slightly at 3 and 4 weeks and rose again at 5 weeks . There wCollagen 1 levels had 2 peaks of expression at 2 and 4 weeks . CollageAortic valve disease is a complex disease that involves inflammation, changes in the ECM, and osteoblast transformation in valve tissue. There is no therapy that halts disease progression. Although certain medical therapies, such as statins, might be helpful , 10], l, l10], lThe term "aortic valve disease" denotes a wide spectrum of histopathological and clinical situations. In the early stages of the disease, its inflammatory features and extracellular matrix synthesis are more prominent, whereas calcification and bone formation predominate later on . MoreoveThus, we evaluated the early phases of the disease—before the valve has been calcified. We used our animal model of RF-associated aortic valve disease, in which calcification is apparent in the valve after 7–8 weeks on a uremia-induced diet.In this study, we examined valves as early as 2 weeks on the diet until 1 week before the valve became calcified, observing that various osteoblast markers are expressed early in the calcification process, peaking 2 weeks later. Notably, osteoblast marker levels declined before calcification occurred, suggesting that certain processes that modulate calcification are curtailed before it is apparent. Further, the ERK intracellular pathway that was shown previously to be down regulated in calcified valves was upregulated in several points during the inflamatory phase of aortic valve disease.We also measured several ECM proteins that mediate calcification, noting that ECM protein expression is also an early process that occurs before histological alterations take place and that it entails the modulation of ECM proteins.The association between osteogenesis and mineralization of the aortic valve has been well documented in different models over the last decade. Those studies have suggested that mature bone appears in the valve and that cardiovascular calcification resembles bone formation ,17]. Fu. Fu17]. Is this study, we have demonstrated that osteoblast markers are expressed early in the course of the disease—Runx-2, a major transcription factor, and osteocalcin, a marker of mature osteocytes, increased after 2 weeks, remaining highly expressed until Week 5. Osteopontin expression had a unique pattern, peaking at 2 and 5 weeks, which is consistent with data that suggest that osteopontin is involved in several phases of ostoblast maturation process and that its expression peaks twice—early and late .All osteoblast markers were downregulated before calcification was noted in the valve, suggesting that the early active inflammatory process is extinguished before the valve is calcified. This observation might explain the lack of efficacy of therapies, such as statins, that target the inflammatory process, which is absent when the valve has already been calcified.Several studies have examined the function of ECM in AVC, comparing normal valves with diseased and deformed calcified valves ,21]. Wh. Wh21]. One of the determinants of the cellular phenotype is the ECM ,28]; th; th28]; The inflammatory component of the calcification process, which involves macrophage accumulation, activation of intracellular pathways, alterations to the ECM, and osteoblastic features, occurs before calcification appears in the valve. Further, this active process is dowregulated before the valve is calcified. The reasons for down regulation of inflammation are not clear and should be further investigated, a possible explanation is increased apoptosis of inflammatory cells and decreased phagocytosis of apoptotic bodies may accelerate and promote calcification. Importantly, in this study we focused on the inflammatory component of aortic valve disease. Other mediators may be involved in later phases of the process and during the calcification itself. Our results suggest that many processes and pathways are activated and suppressed during the course of the disease. These findings reflect the complexity of the pathogenesis of AVC: various phases might respond differently to medical interventions—eg, a specific therapy that is effective at one stage of the disease might lack efficacy, perhaps causing harm, at other stages.Additional studies are required for defining the specific phases of calcification as well as the mechanism involved in each phase.Markers for disease progression must be identified, particularly in patients with RF. Due to the rapid progression of aortic valve disease in this population, a relatively narrow window exists during which medical intervention might be effective.S1 Fig(PDF)Click here for additional data file.