Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS).V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc , effect of an adjuvant, and route of immunization.Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model.We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens. V. cholerae O1. V. cholerae O1 organisms can be classified into two serotypes, Ogawa and Inaba, based on the presence or absence of a methyl group on the upstream terminal moiety of OSP. Immunity against cholera is serogroup-specific and serogroup specificity is determined by the O-specific polysaccharide (OSP) of V. cholerae lipopolysaccharide (LPS). Previous infection with Ogawa provides protection against subsequent Ogawa infection, while previous infection with Inaba provides more complete protection against subsequent Inaba and Ogawa infection. We have previously reported development of an Ogawa cholera conjugate vaccine. Here we report development of an Inaba OSP:rTTHc cholera conjugate vaccine and assessment of a range of doses, molar ratios of polysaccharide to carrier, immunization routes, and presence or absence of adjuvant.Cholera is a severely dehydrating diarrheal disease that remains an important global public health problem especially in developing countries. Globally, almost all cases of cholera are caused by  Vibrio cholerae. Cholera spreads through fecal-oral transmission and is closely associated with poor sanitation and lack of safe drinking water. Cholera remains an important global public health problem, especially in areas of Asia, Africa, and the Caribbean + Calculated for C8H19N2O2: 175.1441, Found 175.1444. The analytical sample was dried at 80°C/133 Pa for 2 h and allowed to cool to room temperature, whereupon the material solidified. Analytic Calculated for 3 CxHzNw.H2O: C, 53.31; H, 10.44; N, 15.54. Found: C, 53.17; H, 10.17; N, 15.69. The compound appeared polymorphous. Crystallization from EtOH–EtOAc yielded material melting at 71–75°C.In a pear shaped 50 mL flask, ε-caprolactone  was treated at room temperature with ethylenediamine  for 18 h. Thin-layer chromatography (TLC) showed disappearance of ε-caprolactone and formation of a more polar ninhydrine-positive material. After concentration at 30°C, to remove most of the ethylenediamine, the residue was chromatographed  as syrup, which solidified on standing. After crystallization from MeOH, white crystal obtained melted at 105–106.5°C; 1H-NMR : δ 4.39 and 4.36 , 3.72 and 3.59 , 3.58 , 3.42 , 2.27–2.19 , 1.61–1.49 , 1.35–1.27 ; 13C-NMR : δ 178.4 (C-1), 62.1 (C-6), 61.6 (OCH3), 44.7 and 44.6 (C-2′), 40.1 and 39.9 (C-1′), 36.4 (C-2), 31.6 (C-5), 25.8 and 25.7 (C-3), 25.3 and 25.2 (C-4); TOF-HRMS m/z: [M+H]+ Calculated for C13H21N2O5: 285.2450, Found 285.1449; Analytic Calculated for C13H20N2O5: C, 54.92; H, 7.09; N, 9.85. Found: C, 54.72; H, 6.90; N, 9.64.A solution of compound 1  and 3,4-dimethoxy-3-cyclobutene-1,2-dione  in methanol (MeOH) was kept at room temperature for 18 h, when TLC showed that a faster moving, ultraviolet-positive product was formed. The mixture was concentrated, and chromatography 15:1 → 10:1 CH2O2 (Sigma), and determined the optical density at 405 nm with a Vmax microplate kinetic reader . Plates were read for 5 min at 30 s intervals, and the maximum slope for an optical density change of 0.2 U was reported as millioptical density units per minute (mOD/min).To assess immunoreactivity of the OSP display on the conjugates, we performed antigen-specific ELISAs using sera collected from humans with cholera in Bangladesh, and compared these responses to those detected in humans with typhoid fever in Bangladesh. We coated the wells with either OSP:rTTHc , OSP:BSA (100 ng/well), or TT . Following blocking and washing of plates, we added acute and convalescent phase sera from humans with cholera or typhoid . The presence of antigen-specific antibodies was detected using horseradish peroxidase conjugated to anti-human IgG antibody  (Jackson ImmunoResearch). After 1.5 h incubation at 37°C, we developed the plates with a 0.55 mg/ml solution of 2,2ʹ 0-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)  with 0.03% HV. cholerae wild type challenge assay, sera were collected from additional cohorts of mice immunized with 10 μg OSP per mouse  with alum, or alum alone on days 0, 14 and 28.To assess immunogenicity of the OSP:rTTHc conjugate vaccine following immunization, we used 10 cohorts (n = 10–15) of female Swiss-Webster (3–5 week old) mice. We intramuscularly immunized one cohort of mice with 5:1 molar ratio of OSP to rTTHc vaccine (10 μg OSP per mouse) without alum, 3 cohorts of mice (10 μg of OSP per mouse) at different molar ratios of vaccines  with alum, and 2 cohorts of mice with 5:1 molar ratio of OSP to rTTHc vaccine at different doses (20 μg and 50 μg of OSP per mouse) with alum. We also immunized 2 cohorts of mice intradermally with 5:1 molar ratio of OSP to rTTHc vaccine (10 μg OSP per mouse), with or without alum. As additional control groups, we used 2 cohorts of mice who received alum only, either intramuscularly or intradermally. Mice were immunized on days 0, 21, and 42. We collected blood samples via tail bleeding on days 0, 7, 21, 28, 42, 49 and 56. Samples were collected, processed, aliquoted, and stored as previously described ,23. For To assess whether antibodies targeting the squarate moiety occur following immunization with OSP:rTTHc, we coated ELISA plate wells with OSP-squarate-BSA (100 ng/well) or squarate-BSA  and assessed antigen-specific IgG responses as described below.V. cholerae O1 Inaba OSP:BSA (1 μg/mL) or TT (1 μg/mL) in 50 mM carbonate buffer [OSP and TT-specific IgG, IgM and IgA responses in serum were measured using standard enzyme-linked immunosorbent assay (ELISA) protocols as previously described ,18,23. Be buffer ,17. SeraV. cholerae O1 El Tor Inaba strain PIC018 in a micro-assay as previously described [V. cholerae O1 El Tor Inaba strain PIC018 (OD 0.1) in 0.15 M saline and 22% guinea pig complement (EMD Biosciences). Plates were then incubated for 1 hr at 37°C. 150 μl of brain heart infusion broth (Becton Dickinson) was added to each well, and plates were incubated for an additional 1.5 h at 37°C, when optical density at 595 nm was assessed. We calculated the vibriocidal titer as the dilution of serum causing 50% reduction in optical density compared with that of wells containing no serum [We assessed serum vibriocidal antibody titers against escribed ,24,25. Wno serum ,27. We d2. We then cultured spleen cells in 96 well round-bottom plates containing 1×107 cells/ml irradiated syngeneic spleen cell feeders (1200 rad) from naive mice, and 1×105 cells/well from immunized mice in a total volume of 200 μl. Plates were then incubated at 37°C in a humid atmosphere with 5% CO2. After 6 days of culture, cells were harvested and antigen-specific memory B cell responses were measured by conventional ELISPOT method. We assessed total IgA and IgG secreting cells as well as OSP and TT-specific cells by ELISPOT assay using these cultured cells. Specifically, nitrocellulose bottom plates  were coated with OSP:BSA (100 ng/well), or TT (100 ng/well), goat anti-mouse IgA (Southern Biotech), goat anti-mouse IgG (Southern Biotech), or keyhole limpet hemocyanin  . After blocking the plates with RPMI-1640 supplemented with 10% FBS, we added the cultured cells to the wells and incubated the plates for 5 h at 37°C in a humid atmosphere with 5% CO2. We then added biotinylated anti-mouse IgA or IgG (Southern Biotech) antibody diluted at 1:1000 in PBS-Tween-FBS followed by the addition of horseradish peroxidase-conjugated avidin-D  diluted at 1:1000 in PBS-Tween-FBS to detect IgA or IgG antibody secreting cells. Plates were developed with AEC . We used unstimulated samples as negative controls and assessed responses to KLH as a control for the plate. We excluded plates having more than 3 KLH spots. We defined a responder as having more than 5 times total IgG secreting cells with stimulation versus no stimulation, and >3.5 anti-OSP spots per 105 splenocytes for OSP-specific memory B cell responses, or >5 anti-TT spots per 105 splenocytes for TT-specific memory B cell responses.We assessed memory B cell responses after the third round of immunization as previously described ,28. Brie2. We then added biotinylated anti-mouse IgA (Southern Biotech) antibody at 1:1000, detected IgA antibody secreting cells using horseradish peroxidase-conjugated avidin-D , and developed plates with AEC. We excluded plates having more than 3 KLH spots. We defined a responder as having >3 anti-OSP spots per 106 lymphocytes.We euthanized mice after day 56 and collected the whole small intestines in HBSS media  supplemented with 4% fetal bovine serum  and 1% HEPES (Invitrogen), storing at 4°C until processing. We then removed fat and collected any evident Peyer’s Patches, dissecting under magnification. We then removed residual fecal content and washed the intestine once with HBSS. Subsequently the intestine was cut into small pieces and washed three times with media. These pieces were incubated with 0.1 mM EDTA in HBSS for 25 mins at 37°C with stirring, and the supernatant was aspirated in order to remove intestinal epithelial cells. Residua was washed twice with RPMI-1640 Complete Media containing L-glutamine (Invitrogen) and supplemented with heat-inactivated 5% FBS, 1% penicillin-streptomycin and 1% Fungizone (Invitrogen). Residua was then incubated with collagenase in RPMI Complete Media for 30–35 min at 37°C. The cell suspension was collected using a 70 μm strainer (Becton Dickinson) and the residua was treated with collagenase again. The cell suspensions were pooled and washed with RPMI-1640 Complete Media containing L-glutamine (Invitrogen) supplemented with heat-inactivated 10% FBS, 1% penicillin-streptomycin and 1% Fungizone (Invitrogen). Lymphocytes were then isolated by density gradient centrifugation method using Lympholyte-M (Cedarlane). Antigen-specific IgA and total IgA secreting lymphocyte responses were then measured by conventional ELISPOT method. Specifically, nitrocellulose bottom plates  were coated with OSP:BSA (100 ng/well) or with goat anti-mouse IgA (Southern Biotech) or with keyhole limpet hemocyanin  . After we blocked the plates with RPMI supplemented with 10% FBS, we added the lymphocytes to the wells and incubated the plates for 5 h at 37°C in a humid atmosphere with 5% COV. cholerae O1 El Tor Inaba strain N16961. In brief, we removed three to five day old un-immunized CD-1 suckling mice from dams two hours prior to inoculation. We then administered to pups a 50 μl inoculum comprised of 5.8x109cfu of V. cholerae O1 El Tor Inaba strain N16961 mixed with a 1:250 dilution of pooled day 42 sera from mice intramuscularly immunized on days 0, 14, and 28 with OSP:TThc  with alum or immunized with alum alone. Following oral challenge, we kept neonates at 30°C and monitored animals every 3 hr for 36 hr, after which surviving animals were euthanized.To assess protection afforded by immunization, we used a cholera neonatal mouse challenge assay, as previously described ,18,23, uP< 0.05 considered a threshold for statistical significance. We performed statistical analyses using GraphPad Prism 4 .We compared data from different groups using Mann-Whitney U tests. Within each group, comparisons of data from different time points to baseline data (day 0) were carried out using Wilcoxon Signed-Rank tests. Kaplan-Meier and log rank analysis were performed to compare survival curves in the neonatal challenge study. All reported P values were two-tailed, with a cutoff of To assess whether the conjugates displayed OSP in a immunologically relevant manner, we assessed immunoreactivity of OSP:rTTHc  and OSP:P<0.05) against OSP and TTHc were found in all cohorts of mice after a single immunization of vaccine when compared to baseline (day 0) levels, with responses being detectable within 7 days of first immunization in many cohorts. No antigen-specific responses were detected in mice immunized with alum alone. We found no significant increase in serum IgM or IgA responses in any vaccine cohort. In general, immune responses were comparable across all the vaccine cohorts, although anti-OSP responses rose more rapidly in mice immunized with 20 μg of OSP (5:1) compared to responses in mice immunized with 10 μg or 50 μg. Anti-TT responses were highest in mice immunized intradermally, with a significant boosting increase following the third immunization  in the intestinal lamina propria of mice immunized intradermally with OSP:rTTHc in the presence or absence of alum compared with responses in mice receiving alum only , compared to mice challenged using sera from mice immunized with adjuvant alone   .V. cholerae lipopolysaccharide (LPS) [V. cholerae O1 serogroup contains both Ogawa and Inaba serotypes [V. cholerae O1 El Tor organisms with the prevalent serotype shifting between Ogawa and Inaba during cholera outbreaks [V. cholerae O1 Ogawa OSP is immunogenic in mice, and that responses are enhanced when the vaccine is administered with an ADP-ribosylating immunoadjuvant [V. cholerae source strain isolated in 2007 from a patient with cholera in Bangladesh.Immunity against cholera is serogroup-specific and serogroup specificity is determined by the O-specific polysaccharide (OSP) of de (LPS) ,16,29,30erotypes ,32. Currutbreaks . We haveadjuvant ,14,15. FTo create our conjugates, we used squaric acid chemistry to attach OSP to a carrier protein via single point attachment, resulting in a sun-burst display of OSP to mimic how OSP is presented on the bacterial surface. To confirm that the OSP was displayed in an immunologically relevant manner, we confirmed that OSP was recognized by convalescent phase sera of humans recovering from cholera but not typhoid in Bangladesh. As for the carrier, we used a recombinantly produced immunogenic fragment of tetanus toxoid heavy chain. This protein has been used as a carrier for other conjugate vaccines as well .We next confirmed that the OSP:rTTHc vaccine would be immunogenic, showing that immunization of mice induced prominent anti-OSP and anti-TT serum IgG responses, with significant increases occurring following a single immunization, and additional boosting of immune responses occurring after both second and third immunizations. Overall, we found comparable immune responses in animals immunized with the same quantity of OSP contained within vaccines in varying molar ratios of OSP to rTTHc. Although there was a trend toward higher immune responses in mice immunized with the 3:1 version of the vaccine, it should be noted that these mice received more moles of total vaccine at each immunization compared to mice receiving the 5:1 or 10:1 vaccines. We used the 5:1 version of the vaccine as our central benchmark, and found that co-administering the vaccine with alum did not significantly alter immune responses.Interestingly, parenteral administration of whole cell cholera vaccine results in protection comparable to that afforded by oral immunization with killed whole cell vaccines, although the previously available parenteral vaccine required multiple administrations, frequent boosters, and was associated with significant local reaction-adverse events . The reaOur conjugation approach uses residual core oligosaccharide effectively as a linker, attaching OSP through core to protein carrier at a single point. This conjugation approach is attractive for a number of reasons: (1) it does not include the additional steps required to introduce a linker, (2) it results in single point sun-burst display of OSP that mimics how OSP is displayed on the bacterium, (3) it can be used to attach a carrier to any OSP that contains a reactive amine group in core oligosaccharide and as such can be used in a platform vaccine development approach, and (4) it is a very simple chemical process that would be readily adoptable for manufacturing purposes in resource-limited areas. In order to directly address whether the squarate moiety itself induced immune response unrelated to OSP or carrier ,43, we mV. cholerae in vitro. There is no vibriocidal value that confidently predicts protection against disease [V. cholerae LPS [V. cholerae in the standard mouse survival model.The vibriocidal assay measures a functional serum antibody response that correlates imperfectly with protection against cholera . In this disease ,45. Inteerae LPS , and we erae LPS . In our erae LPS ,48. Thiserae LPS , and IgMV. cholerae O1 supplemented with 1 mg per dose of recombinant cholera toxin B subunit. Shanchol  contains several biotypes and serotypes of V. cholerae O1 and V. cholerae O139 without supplemental cholera toxin B subunit. Unfortunately, however, the vaccines are less effective in young children under the age of 5 years compared to protection afforded to older individuals [Currently available oral cholera vaccines (OCVs) represent a significant advance and are beginning to play an important role in cholera control strategies globally . Two oraividuals ,49,50,51ividuals . When asividuals . Importaividuals . In our V. cholerae OSP in an immunologically relevant manner, induces high level anti-OSP responses that are functional and protective, and induces memory responses. We assessed a range of preparations, doses, presence or absence of adjuvant, and immunization routes, and showed that intradermal administration of the vaccine induces OSP-specific responses in intestinal tissue. A cholera conjugate vaccine could have utility as an independent monovalent vaccine, as part of a prime-boost approach when combined with oral cholera vaccination to boost immune responses in young children and to consolidate long term protection, and/or as part of a multivalent conjugate enteric vaccine that targets other intestinal pathogens, such as shigella and salmonella. Importantly, the approach used to produce OSP:rTTHc can be used as a platform technology to generate conjugates containing OSP of other pathogens, linking to recombinant TTHc, and using scalable, characterizable and reproducible processes.Our study has a number of limitations. We did not assess memory or long lived plasma cell responses in intestinal tissue, we did not assess cross protection against Ogawa challenge, we did not quantify IgG responses in intestinal tissue, and we did not assess protection afforded by intradermal immunization directly. Despite these limitations, we feel that our work is significant since it describes a novel Inaba cholera conjugate vaccine that displays


Journal has seen remarkable development and progress. After hard work, the Journal was accepted and indexed in PubMed in 2008 as an open access journal [Since the name-change to its current title over 2 decades ago , the Jou journal . Being aWith a slight decline in accepted manuscript for 2014, largely due to a more restrictive policy on case reports, we anticipate the firm inflow of good papers to contribute to this achievement. Acceptance rate is still close to 40%, with some 12% of accepted papers being systematic reviews and meta-analyses , and theImportantly, IF is not the only metric that may reflect a journal’s value. Readers may be interested to know that the journal H-index is 17, meaning that 17 papers are cited 17 times and more, which is rather good considering the ‘young age’ of the journal. At the time of writing, almost 2000 accumulated citations to SJTREM papers have occurred, with the majority in the latter period  et al. [Moreover, we need to recognize that the world-wide readership also value other papers differently in terms of views and downloads (see Figure et al.  is amongJournal. The editorial policy has been developed along the way [Behind the journal’s success is not only the continued support by the editorial team, but first and foremost the support by reviewers and the increased interest by researchers and readers of the  the way ,33, yet Lastly, it is hoped that the journal will receive manuscripts for evaluation from the field of emergency surgery to a greater extent. With about 245 million operations being performed each year globally, of which many if not most are emergent in nature, there is considerable potential in addressing surgical emergency conditions for human health , in both


Background. Adequate fluid management could be essential to minimize high arterial stiffness observed in chronically hemodialyzed patients (CHP). Aim. To determine the association between body fluid status and central and peripheral arterial stiffness levels. Methods. Arterial stiffness was assessed in 65 CHP by measuring the pulse wave velocity (PWV) in a central arterial pathway  and in a peripheral pathway . A blood pressure-independent regional arterial stiffness index was calculated using PWV. Volume status was assessed by whole-body multiple-frequency bioimpedance. Patients were first observed as an entire group and then divided into three different fluid status-related groups: normal, overhydration, and dehydration groups. Results. Only carotid-femoral stiffness was positively associated (P < 0.05) with the hydration status evaluated through extracellular/intracellular fluid, extracellular/Total Body Fluid, and absolute and relative overhydration. Conclusion. Volume status and overload are associated with central, but not peripheral, arterial stiffness levels with independence of the blood pressure level, in CHP. The increased mortality observed in chronic kidney disease compelled analyzing the role of the traditional risk factors and those derived from recent studies that are particularly relevant in chronically hemodialyzed patients (CHP). Consequently, left ventricular hypertrophy, malnutrition, and hydration status  in end stage renal disease have become “novel risk factors” –4. VolumAt least in theory, an adequate fluid management could be essential to minimize the high arterial stiffness observed in CHP. Even so, it is not still clear how volume overload affects the arterial system and the nature of the association with other novel risk factors . RecentlFrom a different point of view, cardiovascular disease is a well-known leading cause of the increased mortality observed in CHP and is associated with aortic stiffening , 10. MorAt present, the hydration status of the human body can be assessed by multifrequency bioimpedance . FurtherIn this context, the aim of this study was to determine the hydration status using a 3-C model and the central  and peripheral  arterial stiffness levels in CHP, in order to evaluate the potential BP dependent and/or independent association between body fluid status ) and central  and peripheral  arterial stiffness levels.n = 104), sixty-five patients were enrolled in this cross-sectional research. The inclusion criteria were as follows: (1) patients are on hemodialysis for more than 3 months, (2) the vascular access is placed in the upper limb, and (3) patients have had no acute cardiovascular events in the last 3 months. The exclusion criteria were as follows: (1) patients abusing alcohol, (2) patients with lower or upper extremity amputation, (3) patients with symptomatic carotid artery stenosis, (4) patients with uncontrolled diabetes mellitus, (5) patients unwilling to participate in the investigation, (6) patients with cardiac arrhythmias, and (7) patients with metallic implants . Our Institutional Review Board and Ethics Committee approved this study. All patients gave their written consent to participate in the study.This research was carried out in a single health care institution . From an initial cohort of ambulatory CHP , urea, total cholesterol, HDL and LDL cholesterol, and triglyceride.In all patients, brachial BP was measured in the contralateral upper limb to which the functioning vascular access was confectioned. The patients were allowed to rest in supine position during 15 minutes before the BP measurement. Pressure was determined using a digital automatic BP monitor (Omron model HEM 781 INT). Heart rate, waist circumference, and hip perimeter were also measured, and the body mass index (BMI) was calculated by dividing weight by height squared.2)); Fat Tissue Index (FTI (kg/m2)); Total Body Fluid (TBF (L)); ECF (L); ICF (L); and OH (L). Three volume ratios were quantified: ECF/ICF, ECF/TBF, and OH/ECF. Over and subhydration were defined when the OH/ECF ratio was >15% or <0%, respectively [The study of body fluids was done using a bioimpedance monitor  Body Composition Monitor OP-ES, software version: 3.2.x edition: 8/03.12). The BCM is a BIS device that measures 50 different frequencies that range from 5 to 1000 kHz, analyzing the whole-body bioimpedance. The BCM discriminates fluid of the intra- and extracellular water content of lean tissue mass (LTM), adipose tissue mass (ATM), and excess fluid (OH). LTM, ATM, and OH are obtained from measurements of body weight, height, and whole-body ICF and ECF determined by BIS. The body composition model determines whether changes in ICF and ECF reflect an increase or a loss of ATM or LTM. This is the only device that identifies OH as a third compartment on the basis of a unique body composition model. OH represents the excess fluid (fluid overload) stored almost exclusively in the extracellular volume of a patient and is therefore part of the ECF, whereas the water of LTM and ATM consists of differing proportions of ECF and ICF, in addition to solid components. Applying the 3-C model, the following parameters were recorded in each patient: Lean Tissue Index , according to the technique previously and widely described –15. In oβ) was recently developed and proposed as a new parameter for evaluating regional arterial stiffness, independently of BP level [β is derived from the Bramwell-Hill equation and the stiffness parameter β may be less influenced by BP than PWV, and its measurement has been shown to be reproducible. We quantify β as β = Ln(SBP/DBP) · (2 · ρ · PWV2/PP), where Ln is the natural logarithm and SBP, PP, and DBP are the systolic, pulse, and diastolic BP, respectively [PWV is essentially dependent on BP. Studies have shown a significant association between PWV and systolic BP in CHP. To overcome this disadvantage, a BP-normalized stiffness index  group, the overhydration (OH) group, and the subhydration (SH) group. Pearson's correlation was run to determine the relationship between PWV (or β) and volume status parameters . All analyses were completed with SPSS software, version 20.0 . A P value of <0.05 was regarded as statistically significant.Continuous variables are expressed as mean value ± standard deviation. ANOVA (Bonferroni test) and Data collection was successful for all patients included in this study. In all patients, arterial BP and the hydration status were routinely monitored allowing, when necessary, the fluid correction during the renal replacement therapy session and/or the use of pharmacological resources.P < 0.05). Additionally, note that differences in carotid-femoral arterial stiffness between OH and dehydration groups disappeared when a BP-independent stiffness index (β) was calculated. Additionally, there were no differences in carotid-brachial arterial stiffness among groups, with independence of the employed parameter (PWV or β).P < 0.05) with the extracellular-to-intracellular fluid volume ratio (ECF/ICF), as seen in β  was found in both the left and the right PWV carotid-femoral pathways. Moreover, the β-ECF/TBF relationship (P < 0.05) shows that the mentioned changes are independent of the BP level  with the right and left carotid-femoral PWV , the right and left arterial stiffness OH/ECF relationship, analyzed in the carotid-femoral and carotid-brachial pathway, showed similar results that those obtained using absolute values (see Overhydration (OH) measured in absolute units (liters) showed a low (but significant) correlation  and of relative estimations (OH/ECF) through BCM, showed a significant BP dependent association with aortic stiffness, but not with peripheral arterial stiffness evaluated in the carotid-brachial pathway.Extracellular fluid relative increases (ECF/ICF and ECF/TBF) were significantly associated with aortic stiffness and with independence of the BP levels. This relationship was not observed when peripheral arterial stiffness was considered. This is an important, original finding , 12, whiβ index, indicating that the observed differences were BP dependent , in order to characterize the whole-body bioimpedance. This technology allows using a 3-C model that ensures a reliable quantification of the body fluid compartment. According to the aim of this research, we characterized the arterial stiffness (1) of elastic and muscular arteries and (2) in both the right and left arterial territories of the patients. These approaches allow us to conclude the following:Our results showed that ECF/ICF and ECF/TBF ratios are associated with carotid-femoral PWV, with independence of BP, but not with carotid-brachial PWV . These fIn theory, an ECF overload could be the origin of high levels of BP that increase arterial stiffness due to acute vascular passive overdistension (enlargement). This pressure dependent increase of arterial stiffness is determined by the collagen fiber recruitment in response to vascular diameter increases . HoweverIt is important to mention that the above-described results include compartment fluid evaluations in absolute (OH) and relative (OH/ECF) indexes, obtained through a widely employed 3-C model. The significant correlation between PWV and indexes associated specifically with overload (OH and OH/ECF) has not been reported previously. Furthermore, we found that arterial stiffness/overhydration status significant relationship should be restricted to the carotid-femoral pathway and not muscular or transitional arteries.To quantify OH/TBF and mainly OH is important to reach a more integrative evaluation of the hydric status of the patient. These parameters can be obtained from the model published by Chamney et al. in 2007 , which sβ index used to evaluate the arterial stiffness provided information of the vascular wall state with independence of the BP level. This is not a minor issue since significant improvements of arterial wall stiffness without changes in BP levels have been reported [The reported .Aortic PWV was evaluated in all cases using right and left carotid-femoral pathways in order to provide a complete screening of the elastic arteries involved in this vascular territory. According to a recent publication there were some differences between the right and the left carotid-femoral pathways . As showA limitation of this study is the lack of data about arterial stiffness before the beginning of renal function replacement therapy. However, as shown, significant association between arterial stiffness and the hydration status could be found.2) and no values of BMI ≥ 40 were observed, with 38,22 kg/m2 as the highest observed value.Finally, as recently commented by Covic et al. , despiteElastin is the main constituent of the aortic wall, determining a high level of vascular distensibility. On the other hand, peripheral muscular arteries have lower compliance levels than the aorta. In physiological states, there is an arterial stiffness gradient between the aorta and the peripheral muscular arteries. The mentioned gradient of arterial stiffness determines the existence of reflection sites that dampen the transmission of travelling pressure waves from the aorta to the microcirculation. Additionally, peripheral arterial waves in the diastolic period arrive to the aortic root increasing coronary blood flow. In physiological condition, the aging process determines an aortic stiffness increase that is higher than that observed in muscular peripheral arteries. Consequently, the above-mentioned gradient between elastic and muscular arteries decreases. The reversal of the aortic-peripheral arteries stiffness gradient has been proposed as an index of vascular damage , 21. RecAnother clinical connotation derived from our results is suggested if we consider that normalization of fluid overload could not be accompanied by changes in arterial stiffness in the peripheral vessels. Unfortunately, previous studies that have evaluated the association between arterial stiffness and hydration status only evaluated elastic arteries , without considering that they cannot respond like the muscular arteries , 12. TheFinally, as mentioned, considering that in a 2-C model variations in hydration state may have a strong effect on the prediction of fat-free mass, a recent novel prediction model was validated, in which body composition and net and relative OH are estimated using a 3-C model . As mentIn hemodialysis patients, volume status and overload, expressed as extracellular/intracellular fluid ratio (ECF/ICF), extracellular/Total Body Fluid ratio (ECF/TBF), or overhydration (OH), are associated with central (aortic), but not peripheral  arterial stiffness values, independently of BP. Guiding the patients towards this target of normohydration leads to improved aortic but not peripheral arteries function.


A mathematical model of the early inflammatory response in transplantation is formulated with ordinary differential equations. We first consider the inflammatory events associated only with the initial surgical procedure and the subsequent ischemia/reperfusion (I/R) events that cause tissue damage to the host as well as the donor graft. These events release damage-associated molecular pattern molecules (DAMPs), thereby initiating an acute inflammatory response. In simulations of this model, resolution of inflammation depends on the severity of the tissue damage caused by these events and the patient’s (co)-morbidities. We augment a portion of a previously published mathematical model of acute inflammation with the inflammatory effects of T cells in the absence of antigenic allograft mismatch . Finally, we include the antigenic mismatch of the graft, which leads to the stimulation of potent memory T cell responses, leading to further DAMP release from the graft and concomitant increase in allograft damage. Regulatory mechanisms are also included at the final stage. Our simulations suggest that surgical injury and I/R-induced graft damage can be well-tolerated by the recipient when each is present alone, but that their combination  may lead to acute rejection, as seen clinically in a subset of patients. An emergent phenomenon from our simulations is that low-level DAMP release can tolerize the recipient to a mismatched allograft, whereas different restimulation regimens resulted in an exaggerated rejection response, in agreement with published studies. We suggest that mechanistic mathematical models might serve as an adjunct for patient- or sub-group-specific predictions, simulated clinical studies, and rational design of immunosuppression. Solid organ transplantation represents the treatment of choice for end-stage organ failure-associated diseases, and has proved effective at extending and improving the quality of life of patients. Approximately 22,000 patients receive solid organ transplants every year in the United States, according to United Network for Organ SharingWhile most work in the transplant field has focused on the antigen-driven immune processes that drive graft rejection, recent work has begun to focus on the interplay between early innate immune mechanisms and subsequent antigen-driven responses –10. In tThe transplantation procedure involves oxygen deprivation (ischemia) in the recipient host tissues as well as in the donor graft due to the time interval from donor organ removal to its placement in the recipient host. Once the transplant is complete, blood flow resumes, a process known as reperfusion. The I/R event is well-known to cause injury (IRI) to tissues, in addition to any direct tissue damage from the surgical procedure. These injurious events further initiate release of DAMPs, and this abates as IRI resolves –31. HoweA second layer of effector and inflammatory molecules is released by pre-formed alloreactive memory Type-1 and Type-17 T cells in response to graft mismatched allo-Ag recognition. The levels of T cell pre-sensitization of the recipient to the donor correlate directly with early acute rejection episodes . The ensThese immunologic events may play a significant role in driving the diverse outcomes that accompany organ transplantation in various cases of apparent antigenic mismatch. We use the term “apparent antigenic mismatch” since the response to allo-Ag includes multiple factors, such as (1) actual allo-Ag differences; (2) individual, genetically predetermined thresholds of immune activation in response to a given degree of antigenic mismatch; (3) pre-existing levels of memory T cells; and (4) individual-specific response to immunosuppressive therapy.Modern organ transplantation has utilized potent strategies to control these unwanted, early immune responses. Specifically, thorough pre-transplant screening of recipient’s pre-formed donor-specific allo-antibody reactivity against the donor  is combined with depleting or non-depleting induction therapy at organ implantation and with versatile maintenance immunosuppression –46. All The foregoing discussion suggests an emerging paradigm in which context and timing matter more than semantic distinctions among immune/inflammatory responses: in essence, inflammation/innate immunity triggers early memory lymphoid pathways that can subsequently become more focused after exposure to specific antigens, while chronic inflammation might be thought of as the chronic restarting of acute inflammation . In thisMathematical modeling provides a key tool by which to study the integrated innate/adaptive response or acute/chronic inflammatory response and thereby untangle some of this complexity –50. TherThe modeling simulations in this present study suggest that surgical injury and graft damage can be well-tolerated by the recipient when each is present alone, but that their combination  may lead to acute rejection. An emergent phenomenon from our simulations is that low-level DAMP release can tolerize the recipient to a mismatched graft under specific restimulation settings, while other restimulation regimens lead to an exaggerated rejection response.To examine the early stages of inflammatory/immune responses to an organ transplant, including investigating the role of IRI in transplantation, we developed a mathematical model that includes the inflammatory hallmarks of IRI as well as the immune responses elicited by the apparent antigenic mismatch of the graft. As described above, we use the term “apparent antigenic mismatch” to comprise (1) actual antigenic differences; (2) individual, genetically predetermined thresholds of immune activation in response to a given degree of antigenic mismatch; (3) pre-existing levels of memory T cells; and (4) individual-specific response to immunosuppressive therapy.The degree of this apparent antigenic mismatch is governed by a parameter, α, wherein a value of zero implies that the graft has 0% apparent mismatch with the host and a value of 1 implies complete  apparent mismatch. The model is initiated with a specified level of initial damage to the host and to the graft from the surgery and I/R, and thus the model simulations begin at approximately the time that transplant surgery is concluded (~8 h after the surgery begins), at which time reperfusion would occur.Clinical quiescence: the graft, following transplantation, shows no signs of inflammatory infiltrates. This is represented by model simulations showing little or no graft damage and corresponding to fully or almost fully recovered graft functionality.Acute clinical rejection: the graft, following transplantation, sustains levels of damage from the host response that cause it to lose functionality, occurring in the first 3 months after transplant. This is represented by model simulations showing high graft damage and corresponding poor graft functionality very early after the simulation is initiated .Subclinical inflammation: the allograft, following transplantation, shows no apparent clinical signs of organ damage, but subclinical levels of inflammation and cellular infiltrates are detected in the protocol biopsies in the first 3 months after surgery. This is represented by model simulations showing either stabilized but diminished graft functionality due to lingering inflammation, or non-stabilized, poor graft functionality due to oscillating inflammatory responses driven by T cells.In order to increase our ability to analyze qualitatively the driving forces behind diverse transplant outcomes, we simplify the number of components considered in the model and aim to create an abstract representation of the processes mentioned above. We focus on the following core scenarios and outcomes:Figure I, incorporate the general pro-inflammatory effects of cells such as tissue-resident M1 macrophages, circulating monocytes, neutrophils, and NK cells as well as cytokines such as TNF-α, IL-6, IL-1β, IL-12, and IL-23. Pro-inflammatory T cells are represented by the variable TP, and incorporate the general properties of γδ-T cells, TH1, and TH17 T cell subsets. Also included are anti-inflammatory components, denoted by A, which include M2 macrophages, IL-10, and TGF-β1. In addition, anti-inflammatory T cells are denoted by the variable TA and are comprised of T regulatory and TH2 T cells. There are also two dynamic variables that track the rate of change of tissue damage: one for host tissue, denoted by the variable D, and another for graft tissue, denoted by the variable DG. These six dynamic variables are modeled with ODEs that describe how the rates of these entities change over time as they interact with one another under different simulation scenarios. The variables have arbitrary units, as we are not aiming to match them with quantitative data but instead examine their dynamic behavior. The time scale is in hours. Whereas some parameters governing the various rates of the interactions are estimated from the literature when possible , the parameters are largely estimated to constrain the model to display basic biologically feasible behavior; see Section “The goal of this modeling exercise is to understand the dynamics of the transplant procedure from a more abstract perspective, in which we group multiple components into a single variable. While this level of abstraction will in no way allow a quantitative prediction of specific mediators and cells, this approach does allow for an examination of the overall qualitative dynamics of this system in which excitatory and inhibitory mechanisms interact. The early innate components of the model, denoted by the variable D and I interact in a positive feedback loop that is inhibited by A. This models the effect of DAMPs released by tissue damaged due to IRI. This process is driven by early innate immune components, resulting in the activation of pro-inflammatory components from a resting/circulating population, IR  present at the transplant site, even in the absence of allo-Ag . We modeTP  present at the tail end of the arrow. In keeping with the abstract model representation of these processes, we do not include the DC component directly, yet the process is implicit in the interactions. Additionally, a positive graft mismatch factor will enhance further recruitment/activation of both pro- and anti-inflammatory T cells, from the source T cell population, T0, by already activated components of these types. Again, various processes are inhibited by A and/or TA, as indicated in the legend of Figure With a positive graft mismatch factor, the early innate pro-inflammatory components, such as monocytes and M1 macrophages, through allo-recognition, will provide additional and specific activation via DC of the pro-inflammatory memory T cells, TP , 58. ThiI0, D0, A0, DG0, TP0, TA0) = , which indicate that all system components are at their background values. This state is referred to as the baseline equilibrium. This setting assumes that there are no underlying immune conditions prior to transplant surgery, which is typically not realistic in the case of transplant recipients. Future iterations of the model could incorporate prior host health conditions. The system can be perturbed from this baseline state, for instance, by setting a non-zero initial condition for D and/or DG, which indicates the presence of damaged tissue to host and/or graft, respectively, due to IRI. The rates at which system variables change as a function of time are governed by the Eqs In the Section “On the other hand, an unhealthy outcome is presumed if the departure away from the healthy equilibrium is not transient but instead causes the variables to approach a different equilibrium that has elevated levels of the variable states. The unhealthy equilibrium implies host health failure and, when a graft is considered, graft failure as well. Alternatively, one could define a level of cumulative damage that could be considered as irreparable, rather than defining non-recovery only by the system’s long-term behavior; we did not explore this possibility in the present study. Figures D, to a non-zero value, and remove the presence of the graft, G, from the model. All other variable initial conditions are set to their healthy state baseline values. Figures I, etc. increase from baseline values) for some initial level of host tissue injury corresponding to DAMP release. We note that this response resolves completely in a reasonable time frame. In other words, the inherent inhibitory mechanisms provided by the anti-inflammatory variables, A and TA, are sufficient to regulate the response correctly. However, in Figures As a first scenario, we consider only the aspects of the inflammatory response of the host involved during the surgical transplant procedure in the absence of a graft placement. This scenario could also be viewed as a look at the trauma of transplant surgery or an instance of accidental blunt trauma, in general. To simulate this situation, we set the initial condition for the host damage variable, normal function. However, this value could be lower if an organ were harvested from an older or less healthy donor (a scenario we did not explore explicitly). For this simulation set, we assume that the graft and host are identical, and therefore do not consider any interactions that involve allo-recognition due to mismatch . The model also displays feasible qualitative behavior for possible outcomes when considering ranges of injury severity. In Figures The next iteration of simulations considers not only the IRI to the host from surgery but also the IRI associated with the graft due to the processes of harvest from donor and transportation to the recipient host. We assume that initial graft functionality starting at a percentage lower than 100% is a result of IRI due to the harvest and transport procedures, and not an indicator of the functionality that it had when still intact in the host from whom the graft was harvested. Thus, 100% in our model would mean 100% of the total functionality exhibited by a given organ pre-transplant. Presumably, organs harvested for transplant were functioning “normally,” such that they did not have existing damage affecting this I0, D0, A0, DG0, TP0, TA0) =  as in Figure subclinical chronic inflammation predicted to resolve on the order of 1–3 months (shown for up to 1000 h ~ 42 days), since the recovery behavior is different from, and takes longer than, the graft tolerance recovery scenario of Figure subclinical. In other words, the graft is in comparable or better condition than when it was first transplanted, but it is not maintaining optimal function until much later. Note that Figure subclinical, but the length of time in which graft health is not ideal is much shorter relative to the scenarios in Figure chronic scenario. In Figure subclinical inflammation, and a good level of graft function is observed.In this next simulation set, we consider varying levels of host–graft mismatch, and thus the interactions shown in Figure DG) from which outright graft failure  is avoidable, given a particular value of α. For ~0.032 < α < ~0.3, the healthy stable equilibrium is replaced by a suboptimal healthy stable equilibrium. Higher α values outside this range give rise to oscillations that indicate worsening graft function, with the minimal graft functionality of the oscillatory range reaching 27% as α approaches 0.7. For α > ~0.75, outright graft failure is the only outcome and the ending graft functionality equilibrium value is 12%.Table In some simulations, an initial level of host tissue damage can act as a preconditioning factor in promoting graft survival. While the release of DAMPs from injured tissue incites pro-inflammatory components, the cascade also involves induction of anti-inflammatory mediators. If the pro-inflammatory levels from this initial surgical DAMP release are below some threshold, and the corresponding anti-inflammatory cell/mediator levels are above some threshold at the time the additional DAMP release happens from an IR-injured graft, then an attenuated damage response may be possible. We depict one such simulation experiment of this preconditioning phenomenon, shown in Figure The integrated nature of inflammatory and antigen-specific immunity that underlie the response to organ transplantation has largely defied a synthetic understanding. This complexity can often be observed in the form of emergent phenomena that cannot be predicted based on an understanding of the component parts of the immune system, and may be at the root of the need for life-long immunosuppression post-transplantation. We suggest that the development of novel treatment strategies for organ transplantation can be aided greatly by mechanistic mathematical models such as the one presented here, because inevitably, independent mechanisms must be integrated in order to predict higher-order system properties in a clinically relevant manner. We regard a mechanistic model as one that describes “rules” for how the individual model components interact and evolve with time. We use the term “mechanistic” to distinguish this type of model from statistical or data-driven models, in which quantitative associations are defined, rather than abstracted mechanisms.in vitro/in vivo experiments. Alternatively, modeling biological systems in a realistic fashion often necessitates complex, large-scale models describing the underlying system dynamics (The past decade has witnessed such a synthesis in the form of simplified (reduced-order) computational models of acute inflammation, which have yielded useful insights into the mechanisms and pathophysiology of critical illness , 67–69. dynamics , 70, 71.dynamics , 72–76 adynamics , with thdynamics .in silico clinical trials based on mechanistic models of inflammation and damage/dysfunction were pioneered in the arena of acute inflammation  may lead to acute rejection, as seen clinically in a subset of patients , 47. An Limitations of this mechanistic mathematical model reside in the fact that the induction therapy and the maintenance immunosuppression are not considered in the model, and this is an area for expansion and augmentation of our modeling work. Moreover, this mechanistic mathematical model that predicts early innate and adaptive immune events is a generic one: each organ may have its own distinctive signature of early immune events. Thus, further augmentation of our model would involve making organ-specific variants. Additional limitations include the fact that this is a relatively abstract model, in which multiple mechanisms are lumped into single variables. As such, this model cannot be directly verified in a quantitative manner, other than as concerns the relative timing of various events. One key area where this limitation is apparent concerns the aforementioned emergent tolerization behavior as a function of prior exposure to damaged graft tissue, which we hypothesize as being due to DAMPs such as HMGB1 . Given tDespite these limitations, this model was capable of reproducing a rich set of biological and clinical behaviors. Simulations of this model under various initial conditions of IRI, graft injury, and degree of antigenic mismatch yielded a broad spectrum of outcomes from nearly complete graft function to outright (acute or chronic) rejection. Importantly, this model also yielded behaviors such as tolerization (durable unresponsiveness to donor-antigens) through preconditioning, as well as the harmful alternative outcome of more severe graft failure upon retransplantation. Future iterations of this model could address these limitations and additionally explore the effects of variability that would naturally exist from patient to patient with respect to host health and immune function . ConsequIn conclusion, we suggest that this model is a stepping stone toward further insights, not only into the response to allotransplantation but also for other disease states. Several diseases with or without an immunologic trigger have been recently determined to have inflammation as a common fingerprint. Therefore, understanding diseases according to their common biological mechanism and using systems biology, mathematical modeling, and bioinformatics/data-driven modeling methods to interrogate the immune response before, during, and after perturbation will help not only to predict clinical outcomes but also guide prompt and precise targeting of new therapies , 102.We formulate the model by building upon the approach and principles of prior modeling work to provide the foundation for the current model , 69. In ® . Additional calculations were performed with MAPLE . The complete mathematical model given by the ODE system (1)–(6) was analyzed using the subsystems approach mentioned above wherein the dynamics of a few interacting variables are examined prior to combining the equations altogether. Parameter values used in this section can be found in Table t→∞ define a locally asymptotically stable (or simply stable) equilibrium. If, on the other hand, the perturbation causes solutions to move away from said equilibrium, then we call the equilibrium unstable. We only concern ourselves with biologically feasible equilibria which are those in the positive orthant. The variables of the system are necessarily formulated to remain positive for all time and all parameters are positive as well. For more details regarding the terminology and mathematical analysis used, consult for instance  to host tissues from the early innate components responding to DAMP release and (b) to the graft, f(x) under Eq. I and DTotal nullclines intersecting at  and at two additional points in the positive quadrant. The “healthy equilibrium”  =  is locally asymptotically stable when DTotal, I)≈, as observed in the prior work. Thus, the underlying structure of bi-stability is present in the DTotal/I subsystem we developed here. This means that the system has the ability to display different outcomes, depending on the initial conditions of the variables that we test. These outcomes are then translated qualitatively into clinical scenarios as discussed in Section “As in , we assuached in  for the A, into the full model, we wish to make sure to calibrate any additions to A such that the maximum level of A does not exceed the 0.6264 threshold, since this would produce unreasonable  behavior. For instance, if this threshold were exceeded, the DTotal/I subsystem would be incapable of reaching an unhealthy equilibrium while other components of the model, such as activated pro-inflammatory T cells or graft damage , would remain elevated. The conditions for bi-stability noted above will not be changed when we combine subsystems at the end.Additionally, we know from the prior results that the incorporation of the anti-inflammatory component when treated as a constant will yield a loss of this bi-stability when the level of the anti-inflammatory component exceeds a value of 0.6264 and only the healthy equilibrium remains stable. Therefore, when we incorporate the analogous dynamic anti-inflammatory component, I/TP system has one or two non-negative equilibria depending on the parameter values. If we fix the values for the parameters that appeared in the DTotal/I system, the following parameters govern the number and stability of the equilibria: kitp, st0, μt0, ktpi. The point  =  is always an equilibrium and is stable for kitp = 0.01, st0 = 1, μt0 = 0.05, and ktpi = 0.01. Since we have an estimate for the half-life of activated T cells (unpublished work) which translates to a rate of 0.03/h, we estimate the half-life of inactivated memory T cells to be slightly longer than this at 0.05/h. Also we fix the source term, st0, to a value of 1 and then determine the values of kitp and ktpi such that the  equilibrium is stable and that the rate at which trajectories approach this equilibrium is not unduly slow, which is related to the position of the nullclines. For simplicity, we let kitp=ktpi since there is a lack of information regarding the relative strength at which one incites the other. Setting the value of kitp = 0.008 = ktpi allows for each variable to contribute to recruiting the other by a non-negligible amount in this subsystem and are, as a pair of values, not too close to a bifurcation value where the nullclines would cross a second time. In other words, if their values are set to 0.01, for example, then  will be unstable; however, we wish for this subsystem to have  stable under these parameters so that neither I nor TP will drive sustained TP or I levels, respectively. Therefore, when connected to the damage equations, when each is sustained at an elevated equilibrium, this will depend on feedback from the damage they incite, rather than just each other.The DG/TP subsystem that includes the auxiliary variable G, the same type of functional form used in modeling damage to host (D) via innate cells (I) is employed to model the graft damage, DG, created by pro-inflammatory T cells, TP. The parameter values are set according to Table TP T cells, then  is always stable; and for low mismatch factor ,  is stable. As α increases through this,  becomes unstable and a new equilibrium of interest is born and is stable . For values close to 0.075, the approach to the equilibrium is quite slow away from the stable manifolds of the equilibrium. When α = 0.08, the positive equilibrium is a stable spiral which establishes the presence of damped oscillations in this subsystem. Naturally, as T cells destroy graft tissue, there is less tissue to destroy, but as the tissue regenerates, the T cells can then destroy this regenerated tissue. Also, as T cell numbers increase the source for new ones is depleted until the turnover/death of existing activated T cell subsets allow for the activation of more  – this could be interpreted as a wait time for replenishment of the T cell source from the bone marrow. Understanding the tissue repair process and time scale relative to T cell behavior could help calibrate this aspect better. For instance, tissue repair/regeneration may be hindered significantly in disease states and therefore may depend on the existing level of damaged tissue.For the DG/TP/I subsystem which includes the auxiliary variable G is given by Eqs DTotal\I subsystem is partially contained in this 3-variable subsystem. Initial graft damage values in which DG(0) > 0.095 lead to graft/host failure. Recall that this behavior is in the absence of any anti-inflammatory inhibition; so very little graft damage can lead to failure in this subsystem even without a positive mismatch factor. For very low initial graft damage  and for low graft mismatch , survival is possible. Though the ranges of pairs of values of initial graft damage and α that produce survival outcomes is limited, the presence of bi-stability exists and the presence of inhibitory components added later allow this range to increase. For some DG(0) and α value pairs , graft functionality remains very high (~99%) for ~230 h (~1 week) after which it decreases rapidly to its ending steady-state functionality value of 13% by ~300 h.The TP(0) > 0], the time to graph failure greatly decreases. For example with DG(0) = 0.08 and α = 0.02, when TP(0) = 1 functionality decreases to 13% by 50 vs. 300 h without an initial population of activated memory T cells. A similar result occurs when there is an initial population of activated innate inflammatory components, I. For example, with DG(0) = 0.08, α = 0.02, and TP(0) = 0, when I(0) = 0.01, graft functionality decreases to 13% by 165 vs. 300 h without an initial population of activated innate inflammatory components.If considering the presence of activated memory T cells at time zero [i.e., A, were set as in Reynolds et al. where applicable and the additional parameters in this category were estimated to calibrate the baseline responses. For instance, the contribution of TA to A was calibrated such that maximum TA levels would not allow A to exceed its threshold value of 0.6264 as discussed previously. Additionally, in the case of severe initial tissue damage, it is possible that this positive feedback between DAMP release caused by tissue injury and inflammation causing further tissue injury may not resolve, and thus lead the way to multi organ failure and death. In the current state-of-the-art, the transplantation procedure and donor graft condition are such that the surgical procedure and associated I/R are typically not the cause of organ failure. However, theoretically, this scenario is possible and helps to calibrate the extreme cases of the model such that complete resolution is not the only outcome possible regardless of initial conditions and parameter values. Thus, the inhibitory effects of A and TA combined do not overly and unrealistically dampen the inflammatory arm of the responses. We retain the positive background level of the anti-inflammatory component at the non-perturbed healthy equilibrium, A, as set in Reynolds et al.: A(0) = A0 = 0.125 (The parameter values for the anti-inflammatory components,  = 0.125 .Yoram Vodovotz is a co-founder and stakeholder in Immunetrics, Inc. This conflict is being managed by the University of Pittsburgh under all pertinent institutional and federal regulations. The remaining co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD.Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules  During cereal caryopsis (grain) development, caryopses act as the main sink tissue while the mature leaves, stems or green parts of the ear are the main sites for photosynthesis. The barley caryopsis consists of filial tissues (endosperm and embryo) surrounded by maternal tissue, palea, lemma and the pericarp, the pericarp containing the vascular tissues is the main site for phloem unloading . TransieIn cereal caryopsis, storage starch is the primary and most important reserve deposited. Due to the agronomical importance of grains, cereal grains can be considered as bio-factories to improve starch functionality by means of genetic alterations –8. StarcPlants with deficient isoforms of SBE, which catalyzes the formation of branch points, accumulate less starch , with hiAlterations in starch biosynthesis could generate changes in carbon partitioning to protein in the caryopsis and hence also in the biosynthetic precursors of central metabolism –32. We hThe Hyper-Phosphorylated (HP) and hyper Amylose-Only (AO) lines were selected from transgenic alleles showing stable phenotypes over several generations. Investigations were performed to evaluate the development of grain constituents, granule morphology and grain/caryopsis metabolism. Different developmental time points were investigated from 10 days after pollination (DAP) to maturity at four developmental stages: 10, 20, 30 DAP and at the mature dry grain stage , to study the grain development phase and the post-desiccation phase . The maiGeneral grain parameters such as total carbon and nitrogen, fresh weight, dry weight and water content were investigated in wild type (WT), HP (GWD over-expressor grains) and AO (SBE silencing grains). The percentage of total carbon and nitrogen were nearly identical in HP compared to WT except a slightly higher percentage of carbon was observed at 20 DAP, whereas in AO both carbon and nitrogen percentages were significantly higher at 20, 30 DAP and in MDG . Grain fTo investigate whether reduced starch storage in the AO grains affects carbon partitioning to alternative storage compounds, we measured the levels of BG and total protein contents. The deposition of BG was unaffected in HP grains, however, AO showed slightly higher levels at 10 and 20 DAP but ultimately similar levels of BG were deposited at 30 DAP and MDG . StorageWe have earlier shown that by separation of amylose and amylopectin using size-exclusion chromatography, the starch in mature dry grains of AO consisted virtually only of amylose  of the linear chains generated after enzyme-catalysed debranching of the starch in MDG samples of AO was 28.1 compared to 21.7 and 22.3 in WT and HP, respectively . It shouTo investigate any effects of the alterations of SBE and GWD on starch granule size distribution, we measured the granule size distribution in the MDG samples . The staThe purified starch granules at different developmental stages were used for microscopic investigations in order to study the effects of AO and HP starch structures on granule formation.The starch granule micro- and nano-topography as studied by non-coated Scanning Electron Microscopy (SEM) revealed features typical for the granule types synthesised by the three barley lines. Our previous studies ,33 used To obtain more comprehensive inner structural details of the starch granules and relate it to the topographical features, we investigated the purified starch granules at different developmental stages by Confocal Laser Scanning Microscopy (CLSM). 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) specifically reacts with the reducing ends of amylose and amylopectin. Each starch molecular entity, being amylose, amylopectin or any intermediate material, contains only one reducing end and as an effect, APTS intensely stains amylose due to the 100–1000 smaller molecular size of amylose as compared to amylopectin. At lower magnification, WT and HP granules showed bright central hila, which seems to be organized as semi-circle . WT and HP also had both large and small granules  WT: . The larIn order to track down possible caryopsis developmental metabolic effects due to restructured starch granules in the HP and AO lines, we analysed the metabolite levels at all four developmental stages in WT, AO and HP by GC-MS based metabolite profiling . We idenmyo-inositol, G-1-P, glucose-6-phosphate (G-6-P) and F-6-P and lower levels of maltose were observed in AO while HP showed higher G-6-P and F-6-P levels than WT  mutant accumulate excess amounts of transient starch [Transient starch is synthesized in photosynthetic tissues during the day, and is degraded during night. Plants, which do not contain a functional GWD, such as the Arabidopsis t starch . This sut starch . The molt starch ,35. The t starch . In thisTriticum aestivum) [Pisum sativum), where deficiencies in seed starch biosynthesis resulted in excess accumulation of sugars, proteins and an altered water uptake [Lys5 barley mutant [The increased levels of BG and protein observed in the AO grains as compared to WT indicates a re-directed storage compound deposition from starch towards higher BG and protein biosynthesis as supported by the increase in specific amino acids in AO possibly related to the higher protein contents in the grain . Similarestivum) –49 and pr uptake –52. Simiy mutant , which iy mutant .The effects either of SBE silencing in the AO grains or the GWD over-expression in the HP grains seems to increase during grain development. In 10 DAP grains the amount of starch bound phosphate was around three times higher in HP, whereas it was around 10 times higher in MDG compared to the WT. Also, the presence of starch granule surface pores was specifically observed in HP only at the later in 30 DAP and MDG stages. This could be an effect of the hordein-D promoter, which is used in the overexpression of GWD in HP. This promoter activates storage protein accumulation, mostly at a later grain developmental stage than 10 DAP . SimilarThe starch granule topography of the HP starch granules in the maturation phase showed minute visual pores. The porous surface in the mature granules may be due to the hyper-phosphorylation causing enhanced hydration capacity and solubilization of starch amylopectin , which cConfocal images of starch granules in WT and HP at 10 DAP revealed contorted granules indicating lack of plasticity in the young granule structure , which eHigher levels of sugar, sugar-phosphate and perturbation of amino acid metabolism were noted throughout development of AO grains. The lower starch accumulation in AO is associated with the accumulation of sugar-phosphates, and sugars like glucose, fructose and sucrose indicating the incapability to efficiently channelize these sugars into starch. Accumulation of raffinose and its precursor galactinol could be a feed forward effect of higher levels of G-6-P, G-1-P and F-6-P, which are precursors of their synthesis. Such, higher sugar and sugar alcohol levels in AO are likely to increase osmolarity and thereby water uptake and water content ,58. We hIt is also possible that the increase in sugars is a result of a stress response. Altered levels of citrate and fumarate indicate perturbation of respiratory metabolism in the early stage of grain development in AO. This likely leads to an accumulation of reactive oxygen species and results in the significant reduction of grain weight. Accumulation of compatible solutes including sucrose and raffinose under stress conditions has been reported in many studies  and consIn conclusion, our data substantiates that minor modulation of the starch granule microstructure affecting its degree of phosphorylation (the HP line) has only minor effect on grain development. This is despite the suggested major metabolic importance of starch phosphorylation e.g. . HoweverHordeum vulgare cv. Golden Promise, termed WT) and the two (T4) isogenic lines HP and AO, 25 plants / line, were cultivated in growth chambers under a light/dark regime of 16/8 h at 20/16°C and 70–75% of relative humidity (RH) at 200–250 μmol m-2 s-1 photon flux intensity at the plant level. The HP line is identical to the GWD-1 lines described in [Barley line  operating at 30 Hz for 15 s and passed through a 0.5 mm screen. The samples were stored at -80°C until analysis. Fresh weight, dry weight and water content were measured from a pool of 10 grains (harvested from the middle of the spike as was done for metabolite profiling) each with three biological replicates of WT, HP and AO. The fresh weight per grain was calculated and the grains were lyophilized for three days until they were completely dry and the weights measured again, which correspond to the dry weight and the loss of weight was calculated as the % of water content.Total nitrogen and total carbon was determined on finely ground flour samples of five biological replicates each from WT, HP and AO, harvested at 10, 20, 30 DAP and MDG, using ANCA-SL/GSL elemental analyzer , employing the Dumas procedure for preparation and a stable isotope mass spectrometer. Nitrogen values were multiplied by a factor of 5.45 to give protein values.Flour samples of the same five biological replicates used in the total carbon analysis, each from WT, HP and AO, harvested at 10, 20, 30 DAP and MDG were used for the analysis. Flour samples of 10 mg each were used and the analysis was performed as described previously [10 mg of flour samples of the same five biological replicates used in the total carbon analysis, each from WT, HP and AO harvested at 10, 20, 30 DAP and MDG were used for the analysis. BG content in the samples was determined exactly as described previously [Isolation of starch was carried out from the flour samples of the same five biological replicates used in the total carbon analysis, each from WT, HP and AO, harvested at 10, 20, 30 DAP and MDG. Starch was extracted and purified as described previously [Starch granule size distribution was measured by Microtrac S3500 Particle Size Analyzer. The purified starch samples from the MDG of WT, HP and AO were dispersed in water and were subjected to mild ultra-sonication to aid proper dispersion of the starch granules and avoid agglomerates . The analysis was performed as per the instructions of the instrument.The amylose content was determined on the starch purified from five biological replicates each of WT, HP and AO at 10, 20, 30 DAP and MDG by iodine colorimetry as described previously .Starch bound C6 phosphate content was analyzed on the starch purified from the same five biological replicates, each of WT, HP and AO at 10 DAP and MDG. The degree of starch C6 phosphorylation was determined as the content of Glucose 6-P (G6P) after starch hydrolysis, using high- performance anion-exchange chromatography  as previously described . StandarStarch samples (5 mg/ml) from the same five biological replicates, each of WT, HP and AO at 10 DAP and MDG were gelatinized in water and enzymatically debranched at 40°C using 0.3 unit isoamylase  per 1 mg of sample. The obtained linear glucan fragments were then analyzed using high pressure anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) by injecting 20 μl of sample on a CarboPac PA-200 column. The peaks corresponding to degree of polymerization (DP) 6 to 75 were integrated, corrected for detector response and the average DP was calculated as described in .http://gmd.mpimp-golm.mpg.de; [Polar metabolites were extracted from 10 mg of lyophilized grain materials. Grains from six individual WT, HP and AO plants were harvested at 10, 20, 30 DAP and MDG. Metabolite extraction, derivatization, and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) analysis were performed . Metabol.mpg.de; . Both ch.mpg.de; . The par.mpg.de; . A part .mpg.de; .Starch granules were stained with 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) as described previously in . Starch Images of APTS stained starch granule were recorded on a confocal laser scanning microscope  as described in . The con2 piece of a silicon wafer, which was attached onto an aluminum stub with a double sided carbon tape. The excess of solution was dried out with a piece of filter paper, barely touching the side of the Silicon piece. The samples were further left for drying at room temperature in a plastic box until imaged.Non-coated images of purified starch granules were taken in an FEI Helios Nanolab600 dual beam, electron-ion microscope. The secondary electrons were collected with the Thru-the-Lens detector for a 43 pA incident electron beam accelerated with 1 Kev. The working distance was varied from 1.9 to 2.2 mm as needed for each individual sample. All samples were prepared by sonicating a minute amount of starch into 0.5 ml milliQ water for 5 min. Next a droplet of solution was placed onto a 1x1 cmThe significance tests were performed using the one-way ANalysis Of VAriance (ANOVA) statistical tool embedded into SigmaPlot 12.0. The term significant is used in the text only when the change has been confirmed to be significant (P < 0.05/0.01/0.001) with the one-way ANOVA. For metabolite data, students t-test function in R was used to show the significant differences (P<0.05).http://cran.r-project.org/). The data was centered and auto-scaled before the analysis.PCA was performed on data sets obtained from metabolite profiling and grain compositional analysis  by the prcomb function in the R-software environment  and divided into two phases: storage phase and desiccation phase based on storage reserve accumulation and water loss. Single representative grains are shown for each genotype at each phase.(DOCX)Click here for additional data file.S2 FigWT chain length distribution  as compared to HP and AO. Distributions were standardised to 100% for all three samples and the amount of liberated chains from the AO starch was only 8% of the WT and HP, respectively.(DOCX)Click here for additional data file.S3 FigThe purified starch samples from the MDG of WT, HP and AO are dispersed in water and are subjected to ultrasonication to aid proper dispersion of the starch granules and avoid agglomerates. No obvious difference was found between HP and WT but AO showed reduction in the first peak appearing before 10 μm indicating lesser small granules (<10 μm) and is slightly shifted to the farther side of the peak indicating larger granule size which can be due to the typical structures observed in AO.(DOCX)Click here for additional data file.S4 FigScale bar indicates 30 μm.(DOCX)Click here for additional data file.S5 FigImages are from different developmental stages  at higher magnification of WT , HP  and AO . Arrows indicate locations of growth rings (yellow), bright and dark channels (red), and typical protrusions on AO (white) of starch granules. Scale bar indicates 20 μm.(DOCX)Click here for additional data file.S6 FigScale bar indicates 20 μm.(DOCX)Click here for additional data file.S7 FigData are normalized to the internal standard (ribitol). Values are means of ± SE of six biological replicates. VANTED  is used for visualization. Asterisks indicate significant differences (* denotes P<0.05) performed by t-test embedded in VANTED.(DOCX)Click here for additional data file.S8 FigEach symbol represents the value of each biological replicate (n = 6). The colours of the symbols are corresponding to the genotype. Red, AO line; green, HP line; blue, wild type.(DOCX)Click here for additional data file.S1 Tables(XLSX)Click here for additional data file.


Malignancy is a common complication after renal transplantation. Death with functioning graft and chronic graft loss are two competing outcomes in patients with post-transplant malignancies.The purpose of our study was to evaluate the risk factors associated with cumulative incidence of these two outcomes.Fine-Gray model was used for 266 cases with post-transplant malignancy in Iran. These patients were followed-up from the diagnosis until the date of last visit, chronic graft loss, or death, subsequently.At the end of the study, as competing events, chronic graft loss and death with functioning graft were seen in 27 (10.2%) and 53 cases (19.9%), respectively, while 186 cases (69.9%) were accounted as censored. The incidence rate of death was approximately two-time of the incidence rate of chronic graft loss (8.6 vs. 4.4 per 100 person-years). In multivariate analysis, significant risk factors associated with cumulative incidence of death included age  = 1.03), type of cancer (P < 0.0001), and response to treatment . The significant risk factors associated with cumulative incidence of chronic graft loss were gender , treatment modality (P < 0.0001), and response to treatment .Using these factors, nephrologists may predict the occurrence of graft loss or death. If the probability of graft loss was higher, physicians can decrease the immunosuppressive medications dosage to decrease the incidence of graft loss. There are 25000 patients with end stage renal disease (ESRD) in Iran of whom 52.7% and 45.5% benefit from hemodialysis and transplantation, respectively , 2. KidnOne of the common complications after renal transplantation is malignancy. It is the second cause of death in recipients with renal transplantation  and it iOne year survival of graft after kidney transplantation is 94.7 % in Iran . In seveNowadays, survival of RTRs is one of the most important concerns. The causes of graft loss have also changed over the time; currently, DWFG and chronic rejection are the principal causes of graft loss , 26.2), race, cause of renal failure, induction therapy, and use of mycophenolate mofetil, sirolimus and/or calcineurin inhibitors, acute rejection episodes and any treated rejection episode (2), creatinine (mg/dL), HLA mismatch, age, gender, race, donor-recipient relationship, and type of operation procedure (open vs. laparoscopic)  . in addioscopic) .Chronic graft loss and DWFG are the two competing outcomes in RTRs with post-transplant malignancy. Some of RTRs do not progress to chronic graft loss because death precedes it. Hence, preparations recommended before chronic graft loss would be unsuccessful and costly. The factors associated with incidences of these two outcomes in this population are important. When a person in this population experiences death before graft loss, the probability of experiencing graft loss is frequently altered .The purpose of our long-term, prospective, longitudinal study was to evaluate competing risk of chronic graft loss versus DWFG in RTRs with a diagnosed malignancy, and to evaluate the risk factors associated with these two outcomes.Behzad Einollahi et al. conducted a large multicenter study on 12525 RTRs, accounting for up to 59% of all kidney transplantation in Iran during 22 years follow-up period since October 1984 until December 2008. They collected 266 (2%) biopsy-proven malignancy cases of 26 different types from 16 renal transplant centers in Iran. This study was approved by the local Ethics Committee of Baqiyatallah University of Medical Sciences . Our stu1. Treatment modalities were considered according to the type of cancer, staging of disease, and involved organs. Management included a combination of reduction, withdrawal or changing of the immunosuppressive agents, chemotherapy, radiotherapy, hormone therapy, and surgical resection.2. Non Kaposi’s sarcoma tumors (non-KS) included squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma.3. Tumors of breast, ovary, and uterine in females, prostate and seminoma in males, and renal cell carcinoma (RCC) and transitional cell carcinoma (TCC) of bladder in both genders were considered as genitourinary and reproductive system (GU and RS) neoplasms.4. The term of solid tumor was used for all the malignancies except for the skin tumors, post-transplantation lymphoproliferative disorder (PTLD), and GU and RS cancers.5. Patients with tumor were categorized into five groups according to their type of neoplasm: Non-KS, KS, PTLD, GU and RS tumors, and solid tumors.6. Monoclonal antibody (ATG/ALG) was required for induction therapy and acute steroid-resistant rejection episodes during the first three months following kidney transplantation. Induction therapy with ATG/ALG was used for highly sensitized patients, those receiving kidneys from deceased donors with delayed graft function, patients with poorly matching living donors, and patients with the second or more transplants. None of the patients took OKT3.The immunosuppressive therapy was based on cyclosporine/sirolimus, mycophenolate mofetil (MMF)/azathioprine (AZA), and steroids. Before 2000, patients received dual maintenance immunosuppression with prednisone and cyclosporine/AZA or triple therapy with cyclosporine, prednisone, and AZA. Afterwards, most patients received cyclosporine, prednisone, and MMF .In survival models, each studied person could experience one of the several different types of events over the follow up period. Survival times are defined as the time until occurrence of one competing event preventing other event to occur. With competing risks data, the cause-specific hazard measured the instantaneous failure rate due to one risk at a time. It is routinely estimated by constructing the Cox models on cause-specific hazards and treating time to event from the other competing risks as censored with constant hazards , 33.Fine and Gray  proposedChronic graft loss and DWFG are the two competing outcomes in RTRs with post-transplant malignancy over the follow-up period. Survival time (response variable) for any patient was the time from diagnosis of malignancy until either chronic graft loss or death. Therefore, the purpose of our study was to evaluate risk factors associated with both cumulative incidence of death with functioning graft and chronic graft loss by using the Fine and Gray model. The sub-distribution hazard ratios were also determined by Stata 11 software. P < 0.05 was considered as the significant level. Quantitative variables were expressed as mean ± SD, whereas qualitative variables were shown as number and percentage. In addition, we estimated unadjusted incidence rates for progression to DWFG and chronic graft loss as per 100 person-years. It was also described regarding covariates. Included candidates predictors in the model were gender, age, type of cancer, transplantation until diagnosis (month), age at diagnosis, ALG/ATG, treatment modality, response to the treatment, metastasis, CMV infection after cancer, immunosuppressive therapy, and blood group.The baseline characteristics of RTRs with malignancy and incidence of competing risk events are displayed in Finally, chronic graft loss and DWFG were detected in 27 (10.2%) and 53 cases (19.9%), respectively, and 186 cases (69.9%) accounted as censored. The incidence rate of chronic graft loss was 4.4 per 100 person-years, while the incidence rate of DWFG was 8.6 per 100 person-years. Therefore, the incidence rate of death was approximately two-time the incidence rate of chronic graft loss. The univariate analyses also indicated that the significant risk factors associated with cumulative incidence of chronic graft loss were response to the treatment , metastasis , and treatment modality (P = 0.0001). Unlike metastasis of tumor, response to the treatment decreased the incidence of chronic graft loss. The incidence of chronic graft loss in changed and unmodified treatment modalities was similar to the incidence of patients with immunosuppression withdrawal (i.e. without group), while decrease of immunosuppressive drugs decreased the incidence of chronic graft loss compared to withdrawal of immunosuppressant medication. Decreasing incidence of graft loss in women  and increasing this incidence in patients treated with ALG/ATG  was approximately significant . The multivariable model  showed tThe aim of our study was to identify the risk factors leading to death or graft loss in RTRs in whom malignancy was developed. Therefore, the risk factors associated with other complications were omitted. Prior studies did not considered cancer cases in a model of competing risks and might not provide the necessary decision-making tools for practicing nephrologists on this population. Most estimates of mortality versus chronic graft loss were derived from all recipients. Whereas these estimates are undoubtedly important in the context of management of renal recipients, they offer no guidance to the practicing nephrologists to manage cancer cases of recipients. Establishing risk factors accelerating chronic graft loss and distinguishing them from those that increase mortality would be an important research goal. If the risk factors are different for these competing end-points, then the practice of graft loss with mortality as a composite outcome may be questionable since they might share different pathways.For this study, we used data of multicenter study that was conducted by Einollahi et al. They concluded that the skin cancer (52.9%) was the most frequently observed malignancy after renal transplantation including KS, SCC, BCC, and melanoma, followed by PTLD (27%); whereas GU & RS tumors (9.4%) were the most common malignancy among the other visceral tumors. In addition, Einollahi et al. showed that the best survival was observed in SCC and BCC, and the worst was seen in non-hematologic and non-skin tumors. PTLD was the most common cause of death in both genders and in all age groups. In this study, the incidence of cancer in men was greater than women, and the most frequent tumor in men and women was KS. Induction therapy with ATG/ALG was used only for 16.4% of patients. AZA-based regimens was used in approximately 61.3% of patients (152 cases), while the rest were on MMF-based therapy . Regarding the treatment modalities, 162 (68.6%) cases had responded to the treatment. Most patients received a kidney from a living unrelated donor (87.5%), followed by 9.8% living related and 2.7% deceased donor transplantation . However1. The incidence rate of death was approximately two times the incidence rate of chronic graft loss (8.6 versus 4.4 per 100 person-years).2. The incidence of death was higher in older patients, PTLD, and GU & RS tumor cases and hazard of death for Non-KS and solid cancers were similar to KS cancer. Response to the treatment decreased the incidence of death. Our estimates extended the findings of Mazuecos et al. and Einollahi et al. who reported that age and immunosuppressive treatment were related to cancer development , 30, 38 3. The incidences of chronic graft loss was higher in males and was lower in lower dosage of immunosuppressive drugs. It remained similar by regimen change, modification, or withdrawal of immunosuppressant. Response to the treatment decreased the incidence of this outcome. In addition, we showed in our univariate analysis that metastasis increased the cumulative incidence of chronic graft loss. Tiong et al. and Harada et al. showed in their studies that acute rejection episodes and any treated rejection episode , delayedEinollahi's study data was collected from previous medical records; thus, we had some missing data. In addition, some cases were not followed up until reaching death or graft loss and our censored data was 69.9%. Therefore, for increasing the power of this model, it is suggested to use studies with less missing and censored data.Using estimates provided in this study can guide us in the following ways. Patients who are older, have PTLD, GU, or RS tumor, or did not respond to the treatment are more likely to die, while patients who are female, with decreased immunosuppressant regimen, or have response to the treatment are less likely to reach chronic graft loss.The response to the treatment is a decreasing factor for the incidence of both endpoints, and does not distinguish between them. Nevertheless, factors such as age, gender, treatment modality, and type of cancer might discriminate between them. Therefore, by using these factors, nephrologists might be able to predict the occurrence of graft loss or death and if the probability of graft loss was higher, they would decrease the immunosuppressive drug dosage to decrease the incidence of graft loss.