Despite the efficacy and tolerability of modern antiretroviral therapy (ART), many patients with advanced AIDS prescribed these regimens do not achieve viral suppression or immune reconstitution as a result of poor adherence, drug resistance, or both. The clinical outcomes of continued ART prescription for such patients have not been well characterized.We examined the causes and predictors of all-cause mortality, AIDS-defining conditions, and serious non-AIDS-defining events among a cohort of participants in a clinical trial of pre-emptive therapy for CMV disease. We focused on participants who, despite ART had failed to achieve virologic suppression and substantive immune reconstitution.3 and plasma HIV RNA of 5 log10 copies/mL. During a median 96 weeks of follow-up, 24.0% died ; 27.5% reported a new AIDS-defining condition, and 22.3% a new serious non-AIDS event. Of the deaths, 42.8% were due to an AIDS-defining condition, 44.6% were due to a non-AIDS-defining condition, and 12.5% were of unknown etiology. Decreased risk of mortality was associated with baseline CD4+ T cell count ≥25/mm3 and lower baseline HIV RNA.233 ART-receiving participants entered with a median baseline CD4+ T cell count of 30/mmAmong patients with advanced AIDS prescribed modern ART who achieve neither virologic suppression nor immune reconstitution, crude mortality percentages appear to be lower than reported in cohorts of patients studied a decade earlier. Also, in contrast to the era before modern ART became available, nearly half of the deaths in our modern-era study were caused by serious non-AIDS-defining events. Even among the most advanced AIDS patients who were not obtaining apparent immunologic and virologic benefit from ART, continued prescription of these medications appears to alter the natural history of AIDS—improving survival and shifting the causes of death from AIDS- to non-AIDS-defining conditions. Potent antiretroviral therapies have drastically improved the disease-free survival of HIV-infected patients and have provided millions of years of additional life to those receiving these drugs 3) who fail to achieve any substantive immune reconstitution, suppression of HIV replication with ART results in survival rates that far exceed the optimal rates reported before the modern ART era. For example, in the COHERE study of 75,336 HIV+ patients observed between 1997 and 2010, the mortality rate among patients with a most recent absolute CD4+ T cell count <50/mm3 was only 6.5% per year of suppressed viral load M. avium complex infection, crude mortality percentages of 32%–40% over one to two years of follow-up were reported Nevertheless, even among patients with advanced AIDS . There are also little data regarding the cause of death in patients with advanced AIDS who fail to achieve either virologic suppression or substantive immune reconstitution with continued ART. Whether the causes of death among these individuals mirror those of the pre-modern ART era or are influenced by receipt of ART is not clear.However, it is not clear what clinical impact continuing to prescribe ART for late-stage AIDS patients who demonstrate 3 prior to enrollment.To address these questions, we examined mortality and its causes among a cohort of participants in a trial of pre-emptive therapy for cytomegalovirus viremia. The cohort consisted of patients who were receiving a combination of at least three antiretroviral agents for at least three months, had failed to suppress HIV replication , and had not achieved an absolute CD4+ T cell count above 100 cells/mm3; and plasma HIV RNA >400 copies/mL within 30 days prior to entry. Participants must either have been receiving antiretroviral therapy (ART) continuously for at least three months, or not be receiving and not planning to initiate ART. ART was defined as at least a three-drug antiretroviral regimen, including at least one protease inhibitor (PI) and/or non-nucleoside reverse transcriptase inhibitor (NNRTI). Participants on a three-drug nucleoside reverse transcriptase inhibitor (NRTI) regimen were eligible if treated in the past with a PI or an NNRTI. ART was prescribed as part of usual clinical care and not determined by the study protocol. The HIV treatment criteria were designed to lead to the enrollment of patients who, for various reasons such as viral resistance and/or medication non-adherence, were not receiving substantive benefit from ART and thus were at risk of CMV end-organ disease.The Biomedical Institutional Review Board at the University of North Carolina and the institutional review boards at each participating AIDS Clinical Trials Group study sites approved the protocol and all participants provided verbal and written informed consent. AIDS Clinical Trials Group (ACTG) study A5030 was a prospective, double-blind, placebo-controlled, multi-centered, randomized trial conducted at academic centers across the US between 2000 and 2005. Participants were: CMV seropositive (IgG); without evidence of CMV end-organ disease; CD4+ T cell count <100 cells/mm3 and a plasma HIV RNA level greater than 1,000 copies/mL at enrollment. This viral load threshold was selected to exclude those with chronic low-level viremia and virologic ‘blips’. Of the 338 participants in the trial, 262 (77.5%) entered the study on ART and, of these, 233 (88.9%) met these criteria for suboptimal ART response.The study design has been described previously and included enrollment of participants into an observation phase during which plasma CMV DNA PCR was collected at regular intervals Clinical evaluations were conducted every 4–12 weeks. Lymphocyte subsets were obtained at baseline and every 16 weeks. Quantitative plasma HIV RNA PCR  was obtained at baseline.For participants who died during study follow-up, the causes of death reported by the site investigators  were reviewed independently by the two of the study chairs (DAW and MAJ), who categorized each death as being due to a condition that was AIDS-defining (1993 Centers for Diseases Control and Prevention), non-AIDS defining, or of uncertain etiology. In the two cases where the categorization of a death by the two study chairs did not agree, an experienced HIV clinician (CvdH) not directly associated with the study reviewed the data and provided the final categorization.The primary endpoints were times to death, AIDS-defining condition, and serious non-AIDS event. In this study population participants were often diagnosed with both AIDS-defining conditions and serious non-AIDS events before dying or withdrawing from study. Thus, there were events other than the endpoint of interest that altered the probability of the endpoint. These events are competing risks.For the time to death analysis, withdrawing from study was the competing risk. Time to death was calculated from study entry to date of death. For participants who withdrew from study before dying, event times were calculated from study entry to the date of last clinic visit.For the time to new AIDS-defining condition analysis, dying and withdrawing from study were the competing risks. However, those dying from an AIDS-defining condition were endpoints in this analysis. Time to new AIDS-defining condition was calculated from study entry to date of diagnosis of the new AIDS-defining condition. For participants who died or withdrew from study before diagnosis of a new AIDS-defining condition, event times were calculated from study entry to the date of death or last clinic visit, respectively.For the time to new serious non-AIDS event analysis, dying from an AIDS-defining condition or unknown cause of death and withdrawing from study were the competing risks. However, those who died from a new serious non-AIDS event were endpoints in this analysis. Time to new serious non-AIDS event was calculated from study entry to date of diagnosis of the new serious non-AIDS event. For participants who died or withdrew from study before diagnosis of a new serious non-AIDS event, event times were calculated from study entry to the date of death or last clinic visit, respectively.In these time to endpoint analyses, participants who completed follow-up without an endpoint or competing risk event were censored, with times calculated from study entry to date of last clinic visit. Nonparametric cumulative incidence curves were used to estimate the cumulative incidence function of each endpoint of interest by accounting for the presence of competing risks. The cumulative incidence curves were fit using the cuminc function in S-PLUS .a priori dichotomized based on commonly accepted practice  or clinical significance . Variables found to be statistically significant (p<0.10) in the univariable models were examined together in a multivariable model. If both the continuous and dichotomous versions of the variable were statistically significant in the univariable model, then the variable that was most significant was included in the multivariable model. The multivariable model was reduced using the backward elimination method until all variables were statistically significant at the 0.05-level. The competing risks proportional hazards regression models were fit using the CMPRSK package in R , ISBN 3-900051-07-0).The associations between baseline variables and each time to endpoint of interest were examined using proportional hazards regression modeling of sub-distribution functions in competing risks Incidence rates were calculated using standard epidemiological methods with a 48-week year. Confidence intervals for incidence rates and crude mortality rates were calculated using the Poisson distribution and Wilson's binomial method, respectively, and were fit using the Stata command cii . Comparisons between groups were made using the Wilcoxon and sign tests for unpaired and paired continuous data, respectively.3 (range: 0–100), and median baseline CD4+ T cell count was 30/mm3 (range: 0–95). Their median baseline plasma HIV RNA was 5 log10 copies/mL (range: 3.04–6.48). There was minimal change in the antiretrovirals prescribed by participants' health care providers during study follow-up. The mean change in CD4+ T cell count among the 201 (86.3%) participants with at least one post-baseline value was 33 cells/mm3 ), a small but statistically significant increase (sign test p = 0.027). On-study HIV RNA data were not collected.From 2000 to 2004, 233 participants enrolled in the A5030 trial had been receiving ART prescriptions from a health care provider for at least three months prior to entry and were neither virologically suppressed below 1,000 copies/mL nor had substantive immunologic reconstitution at trial entry. The characteristics of this cohort are detailed in 3 (range: 0–84). At the visit prior to death, the median CD4+ T cell count was 7/mm3 (range: 0–135).During a median follow-up of 96 weeks , 56 (24.0% [95% CI = (19.0%–29.9%)]) of these 233 participants died, for a death incidence rate of 10.7/100 patient-years (95% CI = (8.1–13.9)). Of these, 42.8% (n = 24) were reportedly due to an AIDS-defining condition (1993 Centers for Diseases Control and Prevention) or AIDS progression, 44.6% (n = 25) were reportedly due to a non-AIDS-defining condition . At the visit prior to withdrawal, the median CD4+ T cell count was 23/mm3 (range: 0–386).The cumulative incidence curve of time to death is shown in 3; 95% CI = ; p = 0.002), nadir CD4 ≥25/mm3 ; p = 0.012), baseline CD4+ T cell count ; p = 0.0002), baseline CD4 ≥25/mm3 ; p<0.0001), and baseline CD4 ≥50/mm3 ; p = 0.003; 3; 95% CI = ; p = 0.003; In univariable competing risks proportional hazards regression analyses, a decreased hazard of dying was significantly associated with baseline KPS ≥90 ; p = 0.036), higher nadir CD4+ T cell count , esophageal candidasis, cryptococcal infection, wasting syndrome, KS, lymphomas, PML, and cryptosporidia infection together accounted for over 90% of these events (3 (range: 1–90) at entry and 13/mm3 (range: 0–127) at the visit before the new AIDS-defining condition diagnosis.At least one new AIDS-defining condition developed in 25.3% (n = 59) participants receiving ART prescriptions at entry, with a median follow-up of 78 weeks . Five additional participants died with an AIDS-defining condition, for a new AIDS-defining condition incidence rate of 14.2/100 person-years ). Pneumocystis e events . The med10 HIV RNA ; p = 0.007; 10 copies/mL; 95% CI = ; p = 0.031; The cumulative incidence curve of time to new AIDS-defining condition is shown in 3 (range: 0–89) at entry and 9/mm3 (range: 0–135) at the visit prior to the new serious non-AIDS-defining event.New serious non-AIDS-defining events were reported for 17.2% (n = 40) of the participants receiving ART prescriptions at entry; in an additional 12, the event was fatal. With a median follow-up of 81 weeks , the new serious non-AIDS-defining event incidence rate was 11.0/100 person-years ). Sepsis and pulmonary diseases accounted for nearly 90% of these events . The med3; 95% CI = ; p = 0.094), higher baseline CD4+ T cell count ; p = 0.030), and baseline CD4+ T cell count ≥25/mm3 ; p = 0.007); 3 remained significant ; p = 0.034; The cumulative incidence curve of time to new serious non-AIDS-defining event is shown in 3, plasma HIV RNA of 5 log10 copies/mL, and Karnofsky Performance Score of 90 had 24.0%  mortality during a median of 24.1 (mean: 26.9) months of follow-up. In contrast, 333 participants with a median CD4+ T cell count of 30/mm3 and mean Karnofsky Performance Score of 86 who were assigned to active drug in a randomized trial of clarithromycin for M. avium complex prophylaxis a decade earlier, had a 32% ) mortality during a mean 15 months of follow-up 3 who were assigned a variety of zidovudine and/or didanosine and/or zalcitabine antiretroviral regimens had 40% ) mortality after a median of 18 months of follow-up 3 assigned to active drug in a randomized trial of rifabutin for M. avium complex prophylaxis had 35% ) mortality during a mean follow-up of approximately two years. This suggests that even in the most advanced AIDS patients who obtain little, if any, objective immunologic or virologic benefit from modern antiretroviral treatment, ART may improve survival compared to NRTI-only therapy.In a cohort of individuals with very advanced HIV disease who had been prescribed ART and failed to achieve complete virologic suppression and substantive immune reconstitution prior to enrollment between 2000 and 2004, mortality was lower than reported in comparable cohorts studied in the early 1990s . The 233 participants in this cohort enrolled in the A5030 protocol between 2000 and 2004 with a median CD4+ T cell count of 30/mm3 in 1996 to 287/mm3 in 2004), and non-AIDS deaths were associated with higher CD4+ T cell counts at the time of ART initiation. In our investigation, non-AIDS-defining causes of death equaled AIDS-defining causes of death despite much lower CD4+ T cell counts among those who died.One way by which ART might alter the natural history of AIDS in such “treatment failure” patients is to reduce the proportion of deaths caused by AIDS-defining conditions. In the HIV Outpatient Study, the proportions of deaths due to AIDS-defining conditions was 87% in 1996, prior to widespread use of modern ART regimens, but decreased to 57% by 2004 3, however, a substantial proportion was also observed at higher counts.The finding of high rates of non-AIDS-defining causes of death in our cohort of patients with profoundly low CD4+ T cell counts mirrors data from a mortality analysis of the pooled antiretroviral-receiving control arms of the ESPRIT and SILCAAT trials, where rates of AIDS- and non-AIDS deaths were similar and low CD4+ T cell counts were associated with not only all-cause mortality, but also fatal non-AIDS events The shift over time in mortality from the AIDS events of the pre-modern-ART era to conditions that have not traditionally been considered a consequence of HIV disease progression suggests a potential protective effect of modern ART regimens in preventing death from AIDS-defining conditions, even among patients with persistent low CD4+ T cell counts and uncontrolled HIV replication. In the A5030 trial, when analyzing both the 272 antiretroviral-treated and the 65 -untreated participants, receipt of ART at study entry was significantly associated with a decreased hazard of developing a new AIDS-defining condition ; p = 0.0002, data not shown). That ART, even when not achieving suppression of HIV replication or substantive reconstitution of immune function, may provide some protection against opportunistic infections is further supported by our earlier finding of lower rates of CMV disease than expected based on pre-modern-ART era data in this cohort of patients with advanced AIDS Consistent with much larger cohort studies, 7% of the deaths among those included in this analysis were attributed to cardiovascular disease. Like several of the other non-AIDS-defining causes of death in our study, cardiovascular disease may be associated with chronic inflammation Other than factors related to HIV therapy and CD4+ T cell count, there were few other covariates associated with serious and/or fatal outcomes. Blacks were less likely to develop a new AIDS-defining condition compared to non-blacks, but there were no racial differences in the risk of death or for serious non-AIDS-defining events.M. avium complex prophylaxis arms from historical trials were included in our comparisons).There are limitations that should be considered when interpreting these findings. Foremost, the causes of death were those reported from ACTG study sites and were not determined through independent reviews of death certificates or primary, medical-record source documents. Based on the data that were available from the sites, categorization of the causes of death was made by the study chairs (both HIV clinicians) with an independent expert HIV clinician adjudicating two cases of discordant categorization. In addition, the cohort consisted of patients who were enrolled in a clinical trial, and the supervision afforded by the study may have led to greater than usual attention to prophylaxis of opportunistic infections and clinical monitoring. Lastly, many participants prematurely withdrew from follow-up, some due to debility that may have represented an AIDS-related or non-AIDS-related event. Therefore, the study outcomes competed with one another and with study withdrawal. However, the analysis methods accounted for these competing risks to appropriately summarize the survival curves and to appropriately assess associations between baseline covariates and the events of interest in the regression analyses. In addition, our comparisons to historical controls permit many biases. It is possible that virologically non-suppressive, immunologically ineffective ART is of no clinical benefit and that the difference in survival observed between the A5030 cohort and historical trials from a decade earlier represents other improvements in medical care for patients with advanced AIDS. Nevertheless, baseline CD4+ T counts of the three historical cohorts were comparable and follow-up was often shorter than in A5030 cohort. Further, the prophylaxis for opportunistic infections in advanced AIDS has changed little from the early 1990s to early 2000s (only the active In conclusion, patients receiving modern ART regimens but who, as a consequence of antiretroviral drug resistance and/or suboptimal medication adherence, had persistent very low CD4+ T cell counts and failed to achieve complete virologic suppression were found to be at high risk of death, particularly from non-AIDS causes, though lower than that reported in patients with comparable CD4+ T cell counts receiving optimal opportunistic infection prophylaxis and antiretroviral therapy in the pre-modern-ART era. The lower than expected rates of opportunistic conditions observed in this cohort also suggest a potential protective effect of apparently ineffective ART against the development of such conditions. Overall, these findings support continued prescription of ART to patients with advanced AIDS who appear to obtain little, if any, virologic or immunologic benefit from their HIV therapy.


IgG4-related disease (IgG4-RD) is a recently described systemic fibroinflammatory disease associated with elevated circulating levels of IgG4 and manifests a wide spectrum of clinical presentations. Although serum IgG4 level has been described to be the most sensitive and specific laboratory test for the diagnosis of IgG4-RD, it is recognized that an elevated serum IgG4 level can be encountered in other diseases. In this study, we sought to identify the frequency of IgG4-RD and other disease associations in patients with elevated serum IgG4 levels seen in clinical practice. Among 3,300 patients who underwent IgG subclass testing over a 2-year period from January 2009 to December 2010, 158 (4.8%) had an elevated serum IgG4 level (>140 mg/dL). IgG4 subclass testing was performed for evaluation of suspected IgG4-RD or immunodeficiency. Twenty-nine patients (18.4%) had definite or possible IgG4-RD. Among those patients without IgG4-RD, a broad spectrum of biliary tract, pancreatic, liver, and lung diseases, as well as systemic vasculitis, was diagnosed. We conclude that patients with elevated serum IgG4 levels encountered in clinical practice manifest a wide array of disorders, and only a small minority of them has IgG4-RD. IgG4-related disease (IgG4-RD) is a recently described systemic fibroinflammatory disease associated with elevated circulating levels of IgG4 –4. The pAs initially observed in patients with autoimmune pancreatitis, the majority of patients with IgG4-RD have an elevated serum IgG4 level. Although serum IgG4 level has been described to be the most sensitive and specific laboratory test for the diagnosis of IgG4-RD, it is recognized that an elevated serum IgG4 level can be encountered in other diseases such as pancreatic cancer , atopic Using a computer-assisted search, we identified all patients who had serum IgG subclass levels determined on one or more occasions at Mayo Rochester, MN, USA, during the 2-year period from January 1, 2009 to December 31, 2010 and selected those with an elevated serum IgG4 concentration (>140 mg/dL) for analysis . Human I+ cells/high-power field and IgG4+/IgG+ ratio greater than 40% in the presence of lymphoplasmacytic infiltration and fibrosis. Patients who fulfill the organ-specific criteria for IgG4-related autoimmune pancreatitis, IgG4-related Mikulicz's disease, and IgG4-related kidney disease were also designated as having “definite” IgG4-RD [For this study, the diagnostic criteria similar to those proposed by Umehara and colleagues were employed . The “de IgG4-RD . Those p IgG4-RD . Non-IgGt-test. Serum IgG4 levels between groups were compared using the Wilcoxon rank-sum test. In all cases P-values <0.05 were considered statistically significant.Data are presented as mean ± SD and percentages for categorical variables unless stated otherwise. Demographic data were compared using the Fisher's exact test. Means of continuous variables were compared between groups with a two-sample We identified 3,300 consecutive patients who had their serum IgG subclass testing performed on one or more occasions during the 2-year interval from January 1, 2009 to December 31, 2010; 158 patients (4.8%) had at least one high serum IgG4 level (>140 mg/dL). The demographic features and serum IgG4 level of these 158 patients are outlined in P < 0.05) but the gender distribution was not different (P = 0.29). The serum IgG4 level was significantly higher in those with IgG4-RD compared to those with non-IgG4-RD diagnoses (P < 0.001) (P < 0.05) (P < 0.01), but there was substantial overlap in individual values between the two groups as shown in Twenty-nine patients (18.4%) met the criteria for definite or possible IgG4-RD . The mea< 0.001) . Further < 0.05) . The meaIn the remaining 129 patients who did not have IgG4-RD, the most common diagnoses were primary sclerosing cholangitis, bronchiectasis, non-IgG4-related pancreatitis, vasculitis, chronic rhinosinusitis, and pancreatic or bile duct cancer. No specific diagnosis was achieved in 29 patients; most common presenting complaints in these patients included abdominal pain, fevers, and lymphadenopathy.n = 13) had a higher mean serum IgG4 level (840 ± 914) compared to those with single organ involvement (296 ± 250) (P < 0.01) (Pattern of organ involvement for these patients with IgG4-RD is outlined in  < 0.01) .All except one patient  with IgG4-RD (definite or possible) were treated with prednisone and improved. Twelve of 28 patients initially treated with prednisone experienced subsequent relapses requiring reinstitution of prednisone alone or in combination with another immunosuppressive agent . Median duration of followup was 23 months .In this study we found a broad spectrum of diagnoses to be associated with elevated serum IgG4 levels encountered in the clinical practice setting; less than one-fifth of these patients manifested evidence for IgG4-RD. IgG4 subclass testing was ordered by clinicians for evaluation of possible IgG4-RD or immunodeficiency, and thus it is not surprising that various types of non-IgG4-related pancreatic and biliary tract disorders were included along with chronic infections, for example, bronchiectasis and sinusitis.+ cells. Various authors have used different cutoffs for IgG4 staining criteria. For the purposes of this study, we used the diagnostic criteria recently published by Umehara and colleagues [At the present time, there is no published international consensus on the diagnostic criteria for IgG4-RD. Most authors agree that definitive diagnosis of IgG4-RD requires histologic confirmation that includes the presence of characteristic histopathologic features  along with immunostaining that demonstrates increased numbers of IgG4lleagues  for “defAll patients included in this analysis exhibited elevated serum IgG4 levels and the degree of this elevation was not used in determining the presence or absence of IgG4-RD. Our patients with IgG4-RD exhibited a significantly higher serum IgG4 levels compared to those without IgG4-RD although there was an overlap in their serum IgG4 values. Some authors have suggested that a serum IgG4 concentration that is more than twice the upper limit of normal (>280 mg/dL) is highly specific for IgG-RD . In thisThe sensitivity of elevated serum IgG4 levels in the diagnosis of IgG4-RD has been reported to be in the range of 67% to 95% and specificity to be 90% to 97% , 13–17. Nonpancreatic disorders have also been associated with elevated serum IgG4 levels. These include skin diseases including atopic dermatitis  and pempOne-fifth of our cohort had various respiratory diseases. Van Nieuwkoop and colleagues  had prevPrimary sclerosing cholangitis (PSC) was the single most common diagnosis in the non-IgG4-RD group. Nine of 17 patients with PSC had underlying inflammatory bowel disease. Elevated serum IgG4 levels have been described in 9% to 12% of patients with PSC , 22. SinVascular involvement has been seen in IgG4-RD mainly in the form of aortitis, periaortitis, and inflammatory abdominal aortic aneurysm , 23, 24.Although IgG4-related hepatopathy and hepatic inflammatory pseudotumors have been described –31, noneAs previously noted, serum IgG4 level is elevated in 5% of the normal population , 10. SimThere are several limitations to this study. This study was a retrospective survey with analysis limited to the clinical data available in medical records and imaging studies. The diagnostic evaluation for these patients was performed by various clinicians at our institution according to their own clinical judgment and patient context. It is possible that some cases of IgG4-RD may have been missed particularly in those patients without a specific diagnosis. In addition, the extent of organ involvement may have been underestimated in patients with IgG4-RD due to lack of relevant imaging studies or biopsy specimens in the absence of standard methodical evaluation.We conclude that only a minority of patients with elevated serum IgG4 levels encountered in clinical practice have IgG4-RD. Furthermore, elevated serum IgG4 levels can be seen in patients with many different diseases, most of which likely represent coincidental occurrence. Our findings reinforce the principle that an elevated serum IgG4 level in isolation is of limited diagnostic utility.


Although the toxicological and carcinogenetic mechanisms of Cd2+ have been previously established, some basic questions on this toxicant remain unclear. In this study, we constructed Met-cad 1.57, a new fluorescent resonance energy transfer (FRET)-based Cd2+ indicator, which contains a portion of a Cd2+-binding protein (CadR) obtained from Pseudomonas putida as the Cd2+ sensing key. We produced a human embryonic kidney cell line HEK-MCD157 which stably expresses the Met-cad 1.57 for further investigations. Both fluorescence spectroscopy and FRET microscopic ratio imaging were used to monitor the Cd2+ concentration within the living HEK-MCD157 cells. The dissociation constant of Met-cad 1.57 was approximately 271 nM. The function of Ca2+ channels as a potential Cd2+ entry gateway was further confirmed in the HEK-MCD157 cells. The organelle-targeted property of the protein-based Cd2+ indicator directly reveals the nucleus accumulation phenomena. In summary, a human kidney cell line that stably expresses the FRET-based Cd2+ indicator Met-cad 1.57 was constructed for reliable and convenient investigations to determine the Cd2+ concentration within living cells, including the identification of the entry pathway of Cd2+ and sub-cellular sequestration.Cd For example, whether BCdC can fully represent the whole intoxication status in certain human tissues is unknown. Various membrane proteins that have been proposed as transport molecules for Cd2+ entry is yet to be confirmed 2+ have been observed in several organelles such as the mitochondria or nucleus 2+ sensing 2+ investigations 2+ currents chronically , not monitor the sub-cellular dynamics of Cd2+. To obtain accurate information on the succeeding sequestration and the bio-magnification of Cd2+ after cytotoxic entry, appropriate live-cell sensing approaches for intra/sub-cellular Cd2+ monitoring have been attempted. Reliable method that can directly observe the Cd2+ toxicity status within intact cells should be developed because this method can potentially help to identify the agents that can efficiently remove Cd2+ from the human body.Considering the concepts in molecular and cellular toxicology, some basic questions on Cd2+ indicator, Met-cad 1.57, which is applicable for both intracellular and sub-cellular Cd2+ detections. The sensing element is CadR, a dimer protein responsible for the Cd2+ resistance of Pseudomonas putida2+. The human embryonic kidney (HEK) cells that stably express Met-cad 1.57 (HEK-MCD157) were produced for further investigations.In this study, we established a fluorescent protein-based Cd2+ indicator  [fluorescent intensity (FI) at 475 nm] and cp173Venus (FI at 535 nm) from bacterial and mammalian Met-cad proteins between the control solution  of the control group (H2O) or selected divalent ions (1 µM), such as Ca2+, Mg2+, Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+  for Met-cad 1.57 . The titration curves  of bacterial  and 221 nM , and that of mammalian Met-cad 1.57 was 271 nM .To determine the detailed sensing capability of Met-cads, the ratios under the spectral platform were further obtained and analyzed under various Cdacterial  as well acterial  Met-cad 2+. The fact that the pattern of FRET ratio values in Met-cad 1.77 dropped when the concentration of Cd2+ is rising higher than 10−6 M  and cpVenus among these two sensors. Various basal FRET value and different FRET response can be acquired in the presence of Cd2+ binding (similar for Pb2+ binding in PbrR). It might raise a concern that such structure could disturb the FRET event for real sensing. The result that we still can observe the FRET event after adding Cd2+  in bacterial Met-cad 1.57. Alternatively, a stable-expressed cell line HEK-MCD157 was adapted to express large amount of Met-cad protein without the tag to perform the curve fitting , significantly different from the increase pattern shown in bacterial Met-cad 1.57. Base on genetic sequence, the Strep-tag could be one of the main differences between bacterial and mammalian Met-cad 1.57, in addition to the two different expression systems (perhaps more complicated modifications like glycosylation make them different). More experiments need to be done in the future.From experimental data in 2+ sensing with an acceptable throughput scale, HEK-MCD157, a Met-cad 1.57 that is stably expressed in the HEK293 cell line, was obtained . Considering Met-cad 1.57 under a visualization aspect (from blue to pink and gray under the ratio color bar defined from 1.5 to 4.5), the homogeneous blue (ratio ≈ 2) cytosolic space in the control cells  to 4.29 (100 µM of Cd2+), thereby yielding approximately 2.3-fold (data not shown).For the easy handling of live-cell Cdol cells  and the ells (2+  clearly 2+ content with the passage of time (recorded every 1 h to 8 h of exposure) in various concentrations of extracellular/environmental Cd2+ [from low  to high concentrations ]. 2+ level did not change throughout the 8 h incubation period at 1 µM of Cd2+ (empty circle in 2+ for a longer exposure time (at least 24 h) can be sensed by Met-cad 1.57 (imaging data shown in HEK-MCD 157 cells were used in the following live-cell experiments to monitor the intracellular Cd2+ into the HEK-MCD157 cells. The data show that the ratios reached 3 after 2 h of incubation at 25 µM of Cd2+ (2+ entry (2+ channels) or the antagonists (blockers of Ca2+ channels) were applied ; thapsigargin (TG) for storage-operated channels (inverted triangle with dashed line)] significantly enhanced Cd2+ entry. These channels can be blocked using the corresponding antagonists , which significantly affected Cd2+ entry (2+ channels are involved in Cd2+ entry.We investigated the entry routes of Cd of Cd2+ . This co2+ entry . Either 2+ entry  in agree2+ that successfully entered the cell (2+ [Cd (50)]. The bar figures in 2+ levels. A significant increase in Cd2+ contents in the nucleus after 2 h of incubation was observed (higher than the cytosolic Cd2+ level). In contrast to the cytosolic and nuclear spaces, no further increase in ratio was found within the mitochondria and ER under the same treatment condition.The organelle-targeted versions of Met-cad 1.57 were used to visualize the sub-cellular localization of Cdthe cell . Represe2+2++2+2+. Bacteria such as Cupriavidus metallidurans CH34 and P. putida survive in extremely harsh environments by evolving protective anti-metal systems 2+ sensing with PbrR used as the metal-binding domain 2+-binding domain to create Met-cads, the second MerR-based indicator for Cd2+  and Zn2+ , but those for Hg2+ (MerR) have not been developed yet. Further studies should be conducted to prove this concept.The selection of a suitable sensing key is considered one of the difficult processes in manufacturing protein-based metal ion indicators for Cd2+ . Such sefor Cd2+ . Althoug2+ sensing, such as the commercial fura-5F 2+ sensing, the sensors can only detect the presence of Cd2+ that is incubated at 30 µM for 24 h, and Cd2+ cannot be distinguished from Ca2+. New developed chemical indicators own much better sensing ability especially the ionic selectivity to distinguish Cd2+ from Zn2+2+ indicators 2+ ratio dye CadMQ is about 0.75 increased to 0.84 under 5 µM Cd2+ exposure for 3 hrs (about 1.12 fold increases). , relatively high sensitivity, and good selectivity without the influence of Ca2+ as well as organelle-targetable characteristic. Met-cad 1.57 can sense the accumulation of Cd2+ at very low concentrations (1 µM) for 24 and 48 h of incubation periods. The non-cytotoxicity at 1 µM In this study, the newly developed Met-cad 1.57 exhibits a sub-µM level of 2+ entry are considered as different types of Ca2+ channels 2+ transporter (ZIP8/14) 2+ entry pathway remain controversial. We used the HEK-MCD157 cells to confirm that Ca2+ channels are one of the corresponding gates for Cd2+ entry  for therapeutic usage on Cd2+ poisoning would be possibly established through a large-scale screening.In addition, we established a live-cell system that stably expresses Met-cad 1.57 for numerous benefits. For example, large amounts of Met-cad proteins were provided to help reveal the hidden problem of itration , time-doitration , and Ca2el tests . Further2+, according to the FRET strategy. This live-cell Cd2+ indicator was further incorporated into a stable HEK cell line or to become organelle-targetable for advanced research on sub-cellular Cd2+ dynamics. These constructs and cell line can be further applied in several investigations on the molecular and cellular toxicology of Cd2+.In summary, we produced Met-cad 1.57, another MerR-based indicator for CdAll reagents were purchased from Sigma Chemical Co.  unless indicated otherwise. Most of the reagents for molecular cloning, such as restriction enzymes, ligases, and others, were from New England BioLabs. The polymerase chain reaction kit and the primers used were purchased from Finnzymes Oy (Finland) and Mission Biotech (Taiwan), respectively. The DNA extraction kit was purchased from Viogene.P. putida [American Type Culture Collection  12633] were compared with AF 33961 2+-binding domain (CdBD) for the Cd2+-sensing. The protein sequence map  and the structure prediction PredictProtein ence map  shows thence map . OrganelHEK293 cells from the ATCC were cultured in 22 mm coverslip and transfected The HEK-MCD157 cells, which stably express Met-cad 1.57, were produced through antibiotic (G418) selection. These cells were seeded in 96-well culture plates  for fluorescent spectroscopy or cultured as described for FRET ratio imaging.The fluorescent spectrum of Met-cad 1.57 and its YFP/CFP emission ratios were obtained using a spectrophotometer  as previously reported 2+ imaging was performed by acquiring the emission ratios under the FRET imaging platform. The microscope  was equipped with a 40× objective lens (NA = 0.75). A metal fluorescent light  filtered with a band-pass filter (426 nm to 446 nm) and a dichroic mirror (442 nm) was used as the excitation light source. Ratio images of the Met-cads within living cells that contain the fluorescent signals of CFP (483/32 nm) and YFP (542/27 nm) were captured using a dual charge-coupled device camera system  with HCImage software (Hamamatsu). Ratio imaging was further displayed in a ratio color plate using the ImageJ analysis software (NIH).The live cell Cd2+ across cell membrane into cytosol, a Ca2+ free buffer containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 5 mM D-glucose (pH 7.4) with 5 µM of ionomycin was used to be without or with a defined Cd2+ solution (100 µM) to cells for 10 min before taking ratio imaging For the experiment using ionomycin as ionophore to transport CdStrep-tag Strep-tag was used for the following protein purification instead of applying His-tag to avoid non-selective metal ion interaction, i.e. the binding of Ni2+ to His-tag. Actually Ni2+, as predicted, did not influence the sensing ability of Met-cad with Strep-tag (2+). The only concern about Strep-tag would be its possible involvement on the nonspecific factor found in bacterial Met-cad 1.57  by fluorescent spectroscopy. To estimate the dissociation constant (Kd) of Met-cads expressed from bacterial sources or HEK-MCD157 cells, the ratio data from the titration experiments were normalized as percentages of FRET relative to the maximal difference of the emission values (R). The fitting curve was further analyzed using the following equations: (1) One-site saturation with a nonspecific factor, [Cd2+] = [(Rmax × R)/(Kd+R)]+n × R ; (2) Hill equation 2+] = Kd  and Cd .(TIFF)Click here for additional data file.Figure S22+ and Cu2+ on the spectral pattern of Met-cad 1.57.Effects of Zn The spectral patterns of Met- cad 1.57 to Zn 2+  and to Cu2+  compared with the control without specific ions (Apo in solid line).(TIF)Click here for additional data file.Figure S32+ content.Long-term monitoring of intracellular Cd The representative images of cells expressing Met-cad for monitoring intracellular content of Cd2+ are shown in YFP (top), CFP (middle), and ratio (bottom) with 8, 12, 24, and 36 hours of incubations (1 µM). The color bars show the ratio that ranges from 1 to 5.(TIF)Click here for additional data file.Figure S4Sub-cellular targeting of Met-cads within cells. On top, the representative confocal images  [1], [2] of Met-cads  of cells transfected with various organelle-targeted versions  are displayed in green color. In the middle, the red images  of cells were stained with certain organelle dyes, e.g. propidium iodine (Vector Lab.) for nucleus; MitoTracker Orange CMTMRos (Invitrogen) for mitochondria; ER-Tracker Red (Invitrogen) for ER. The merged images are show in the bottom part. The white arrows indicate that certain targeted Met-cads co-localize with specific organelles within the same compartments of the cells (yellow color). Scale bar = 20 µm.(TIF)Click here for additional data file.Figure S5The comparisons between Met-lead 1.59 and Met-cad 1.57. Sensing key of Met-lead 1.59 was from part of PbrR (starting from the residue D between α3 and α4 helix in bold) [1]. And sensing key of Met-cad 1.57 was from part of CadR, in this study. The most different parts in sequences are after the 2nd cysteine  through the whole α6 helix (in red color).(TIF)Click here for additional data file.Table S1The effects of reagents used  on ratio value in. There’s no significant changes between the two experimental sets, i.e. before and 30 min. after reagent treatments).(TIFF)Click here for additional data file.References S1Supplemental Bibliography(pdf)Click here for additional data file.


Staphylococcus aureus PS80 in high-density continuous cultures were exposed to oxacillin, ciprofloxacin, vancomycin, gentamicin, daptomycin and linezolid. Contrary to results from low density retentostats as well as to predictions of traditional PK/MIC ratios, daily dosing with up to 100× MIC did not clear these cultures. The densities of S. aureus in these cultures oscillated with constant amplitude and never fell below 105 CFU per ml. Save for daptomycin “treated” populations, the densities of bacteria in these cultures remained significantly below that of similar antibiotic-free cultures. Although these antibiotics varied in their pharmacodynamic properties there were only modest differences in their mean densities. Mathematical models and experiments suggest that the dominant factor preventing clearance was wall-adhering subpopulations reseeding the planktonic population which can be estimated and corrected for. Continuous cultures provide a way to evaluate the potential efficacy of antibiotic treatment regimes in vitro under conditions that are more clinically realistic and comprehensive than traditional in vitro PK/PD indices.In vitro measures of the pharmacodynamics of antibiotics that account for the factors anticipated for bacteria in infected patients are central to the rational design of antibiotic treatment protocols. We consider whether or not continuous culture devices are a way to obtain these measures.  Central to this “rational” design of antibiotic treatment enterprise are PK/PD indices 6) growing exponentially in rich liquid broth under pH and ionic conditions where the drug is most effective MICs are estimated in vitro with protocols that are precisely defined for each antibiotic – bacteria combination under culture conditions that are optimal for the action of the drug; low densities of planktonic bacteria (<10It is well recognized that the conditions under which MICs are estimated are unlikely to be met in treated patients and that other elements of the PD of the antibiotic and bacteria could well influence the course of therapy. Included among these other elements are: (i) the functional form of the relationship between the concentration of the antibiotic and the rate of growth/death of bacteria How can all of these and other PD factors contributing to the clinical efficacy of antibiotic treatment be addressed at the same time in vitro? A possible way to achieve this more comprehensive measure of the PD of antibiotics and their target bacteria in vitro is by adding these drugs to bacteria maintained in continuous culture devices and following the changes in viable cell densities Staphylococcus aureus and antibiotics of six different classes to experimentally evaluate the relative contributions of these different factors to the in vitro population dynamics of antibiotic treatment. These drugs varied considerably in their MICs and rates of kill but with the high concentration, 24 hour dosing regime employed, all six antibiotics were able to reduce the density of viable cells in these cultures and, save for daptomycin, maintain them at levels well below the untreated controls (∼ 109 CFU/ml). On the other hand, even the most effective of the antibiotics employed, gentamicin, was unable to clear these cultures or reduce their densities by more than four orders of magnitude from ∼ 109 CFU/ml of untreated controls. Using the mathematical model presented in in vitro experiments, we explore the processes responsible for these dynamics. Our results suggest that persistence, and in the case of daptomycin a cell-associated decline in the effective concentration of the drug, plays a role in these dynamics. However, the primary factor responsible for preventing clearance of these cultures is the continuous input of bacteria from antibiotic refractory wall-adhering (biofilm) populations. We discuss the implications of these results to the use of continuous culture devices to evaluate the PD of antibiotics for the design of antibiotic treatment protocols.In a recent report, we used a mathematical model to provide a theoretical framework to generate hypotheses, design, and interpret the results of in vitro studies of the population dynamics of antibiotic treatment in continuous culture Staphylococcus aureus clinical isolate PS80, a capsulated (type 8) strain obtained from the American Type Culture Collection (ATCC 27700). Bioassay of antibiotic concentration was assessed with S.aureus ATCC 25923.All experiments were carried out with the methicillin sensitive Daptomycin  and linezolid  was purchased from the Emory University Hospital Pharmacy. Gentamicin, oxacillin, ciprofloxacin and vancomycin were all from Sigma . Stock solutions of these drugs were prepared by dissolving these antibiotics in sterile water or 0.9% (w/v) NaCl, stored at -20°C and used within two weeks of preparation. The concentrations reported are the amount of biologically-active antibiotic in milligrams per litre (mg/L).S.aureus PS80 and the different antibiotics were estimated by the broth microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI) guidelines 630) to monitor growth. The values reported represent the lowest antibiotic concentration displaying no growth (The OD630 (18h) ≤ OD630 (0h).Minimum inhibitory concentrations (MICs) of 2 for experiments with daptomycin). Time kill assays were performed under aerobic conditions with constant agitation at 37 °C. For batch culture exposure, we used 20× MIC (ciprofloxacin & linezolid), 50× MIC (daptomycin & gentamicin) or 100× MIC (oxacillin & vancomycin) of antibiotic. To test for media nutritional quality, the medium was filtrated of cells by passing through a 0.22 micron filter and 1×105, 1×106, and 1×107 cells were added. The maximum exponential growth rates were estimated from OD630 measurements obtained during growth at 37°C with aeration. The maximum cell densities were estimated from colony forming units plated after overnight growth in the respective media.Bacterial cultures were incubated overnight at 37 °C with shaking at 200 rpm in cation-adjusted Mueller-Hinton II (MHII) medium alone  these vessels ranged from 0.6 ml per hour to 8.6 ml per hour for dilution rates, ∼ 0.04, 0.18 and 0.43 hr−1. In reporting these results and calculating the rate of antibiotic decay, we used the approximate dilution rates, 0.04, 0.20, and 0.40. Experiments were not initiated until the densities of bacteria in successive samples were equal and these cultures were at the density equilibrium (more than four volume changes). The culture vessels were maintained under negative pressure and aerated as well as vigorously mixed by air sucked into vessels through 0.45 µm filters. Bacteria and antibiotics were introduced and sampled from the screw-top side arm in the culture vessel with minimal perturbation of the vacuum, see www.eclf.net/chemostat.The continuous culture devices used in this study were of homemade-design and are thoroughly described in S.aureus ATCC 25923, and comparing to a standard containing fresh antibiotic.Medium from the desired time point was taken from the culture vessel, centrifuged and passed through 0.45 µm filters prior to bioassay estimation of drug concentration using the broth microdilution described in During the six-day treatment, three hours after each pulse of 100× MIC , we transferred an aliquot of the surviving population to 12 well FALCON™ plates and incubated for an additional 3 hours. Controlling for bioassay-estimated loss of antibiotic in the medium, these were compared to an equivalent density of naïve exponential cells exposed for three hours in the filtrated, bioassayed supernatant from each culture vessel at the particular time point. Comparison was then made between the surviving densities of naïve and pre-exposed (sophisticated) cells in these batch cultures as well as the densities in the corresponding continuous cultures at 6, 54, and 126 hours respectively.−1 flow rate continuous cultures with ciprofloxacin, daptomycin and gentamicin was removed. The culture vessels were washed by adding equal volumes of 1% PBS, sonicated for one minute and the liquid removed. This procedure was repeated twice after which an equal volume of MHII medium was added to the vessel and the culture sampled. After 5 minutes of sonication at 56 KHz, the viable cell density was estimated. The density of cells in the residual wall population was calculated as the difference between the pre-sonicated, and the sonicated samples.Immediately after the termination of the 6-day treatment period experiments, the medium in the 0.2 hrUsing the modified CLSI serial dilution protocol In tion see .−1 the anticipated concentration of all the antibiotics would be less than the MIC by 12 hours for all antibiotics save for linezolid, where the concentration would fall below the MIC by 8 hours. At the intermediate dilution rate, w = 0.2 hr−1, save for linezolid the anticipated concentrations of the antibiotics remain above the MIC for nearly 24 hours, ∼16 hours for linezolid. As measured by the maximum extent of antibiotic-mediated killing, at all dilution rates gentamicin is the most effective of these six antibiotics and ciprofloxacin second, although at the lower dilution rates oxacillin appears to kill to the same extent as ciprofloxacin. At the highest but not lower dilution rates, vancomycin also appears to be as effective as ciprofloxacin. While initially daptomycin rapidly reduces the viable cell density, this killing effect is transient. By 24 hours, the population returns to its untreated level at all dilution rates, including those in lower dilution rate continuous cultures where high concentrations of this drug are anticipated to be present throughout the experiment. Although the concentrations of the antibiotics are anticipated to fall below the MIC by or before 12 hours in the fast continuous cultures, save for vancomycin and daptomycin the viable cell densities did not return to their pre-treatment level by 24 hours.At the highest dilution rate, w = 0.4 hrw = 0.2 hr−1 are “treated” every 24 hours for six days. Particularly striking is the observation that despite daily dosing with 100× MIC (20× for linezolid), the viable cell densities do not continue to decline or fall below 105 cells per ml. (approximately four orders of magnitude less than the antibiotic-free equilibrium density). Save for daptomycin, the maximum viable cell densities in these treated continuous cultures remained one to two orders of magnitude lower than the untreated control cultures. Although daptomycin is highly effective in killing S. aureus shortly after its introduction, within 24 hours the populations return to their pre-treatment levels and already start to recover when the anticipated concentration of the drug is more than 50× MIC. As measured by the harmonic mean of the number of viable cells over the period of treatment, gentamicin is the most effective antibiotic. Oxacillin, ciprofloxacin and vancomycin are next in this hierarchy, followed by daptomycin and linezolid. Of some interest in this regard is that linezolid is dosed at 0.2 to 0.4 times the relative MIC of the other antibiotics and this drug is supposed to be bacteriostatic, rather than bactericidal. Although not run simultaneous as in this figure or under precisely the same conditions, this continuous dosing experiment was performed at least once more for each of these antibiotics. While there were some differences in the average densities of these cultures, the basic results were repeated. In none of these experiments were the bacteria cleared, their densities oscillated and did not decline to levels much less than 104 (Data not shown).In P, that are physiologically refractory to antibiotics because they are either not dividing or dividing very slowly B, which is washed out at a lower rate than the planktonic N and P populations. The third is that the effective concentrations of the antibiotics in these cultures are substantially less than that anticipated from washout-dependent flow alone.Why do the densities of viable bacteria in these periodically treated continuous culture populations oscillate around a constant level rather than continue to decline as a result of daily “treatment” with antibiotics? In our theoretical study of these dynamics S. aureus maintained in MHII. The parameters for the persister and wall populations, their generation, loss and antibiotic susceptibility are assumed and chosen to illustrate their effects.In With these parameters, in the absence of a persister or wall population, we would anticipate clearance of the culture by the second day . PersistBased on the results of these simulations, we postulate that the dominant factor responsible for the failure to clear the bacteria in these continuously flowing cultures is an antibiotic-refractory, wall-adhering population that continually reseeds the planktonic bacteria being sampled. We also postulate that the daily resurrections of the daptomycin-treated population can be attributed almost wholly to a density-dependent decay in its effective concentration.To test for the presence of an antibiotic refractory wall population of adequate densities to explain the 6 and 2×107 CFU/ml.We interpret these results as support for the hypothesis that a refractory wall population is responsible for the failure of these antibiotics to clear these continuously flowing cultures. Considering the density of planktonic bacteria estimated immediately following sonication and after 30 and 60 minutes of incubation in MHII, for all three of these antibiotics, the density of suspended viable bacteria removed from the wall is between 5×105 cells per ml) of S. aureus PS80. The results of these experiments are presented in To determine whether the concentrations of the antibiotics in these cultures are in the range anticipated by exponential decline at a rate equal to the flow, we estimated the ‘MIC’s of these filtrates taken at 3, 6, and 12 hours after drug addition, using low densities  population is not the sole physiologically resistant subpopulation contributing to these dynamics. The planktonic bacteria in the antibiotic-treated continuous culture are collectively less susceptible to the majority of these antibiotics than naïve cells, presumably because of antibiotic enrichment of persistent subpopulations. The evidence for the bacterial populations in these exposed continuous cultures being more physiologically refractory to antibiotics than unexposed cells, comes from comparing the results of time kill experiments carried out on pre-exposed, and non-previously-exposed populations of For the pre-exposed (sophisticated) populations, planktonic cells were removed three hours after antibiotic addition to the continuous cultures and incubated under batch conditions for an additional 3 hours, at which time the viable cell density of these bacteria was estimated. For the unexposed (naïve) bacteria exponentially growing cells from antibiotic-free batch cultures were introduced into sterile filtrates from the 3-hour, antibiotic-treated continuous culture and their survival estimated from viable cell densities after 3 hours of exposure. The densities of “naïve” cells added were adjusted to be approximately the same as those in the corresponding experiment with previously exposed (sophisticated) bacteria. It should be noted that since the densities of bacteria in the continuous cultures 3 hours after treatment were more than two orders of magnitude less than that of antibiotic free cultures at the density equilibrium, the MHII medium was essentially at full concentration.The results of these experiments are presented in a composite figure, where we plot the ratio of the densities of surviving naïve to sophisticated cells after three hours of exposure to these drugs . If the By this criterion, the bacteria exposed to ciprofloxacin, daptomycin, oxacillin, vancomycin and linezolid are clearly more refractory to these antibiotics than naïve cells. This does not appear to be case for gentamicin. It should be noted that although linezolid is supposed to be bacteriostatic, at the concentration present at 3 hours  and, for infected patients more realistic, in vitro measures of the pharmacodynamics of antibiotics and bacteria than those currently used for rational design recommendations of antibiotic treatment S. aureus in continuous cultures could not be predicted from PK/PD indices based on MICs estimated under the conditions specified by CLSI protocol MIN parameter in the Hill function). In these simulations we also use a lower concentration at each dose, 10× MIC, rather than the 100× or 20× MIC than employed in our experiments, thereby more reflective of doses employed clinically. The results of these simulations are presented in The results of these experiments with 5. Although these drugs varied in their efficacies as measured by the CLSI MICs and maximum rates of kill (data not shown), the harmonic (or arithmetic) mean densities of these cultures were not very different. By this average density criterion, linezolid, which is considered to be bacteriostatic, was almost as effective as gentamicin, which is highly bactericidal. There was however, some disparity in linezolid treated cultures with higher than normal variance (1.5 logs) in samples from the first 12 hours of exposure. This may be due to the low solubility of the drug and the cultures thus demanding large volumes (up to 2 ml) of antibiotic to attain 20× MIC concentrations. Despite this, the cycling behavior of the treated population was reproducible in replicate experiments. Save for daptomycin, the sustained densities of bacteria in these antibiotic treated cultures remained at least an order of magnitude lower than that anticipated for antibiotic free controls. Stated another way, for five of the antibiotics these continuous cultures were limited by the antibiotics rather than resources or nutrients, which remained at same concentrations required for optimal growth.Contrary to the prediction of this model, clearance was never observed in the analogous continuous culture experiments depicted in S. aureus in antibiotic-treated continuous cultures was observed some time ago One virtue of using “treated” continuous cultures for vitro explorations of the potential clinical efficacy of antibiotics is that they provide a way to identify and evaluate the contributions of factors affecting the PDs of antibiotics that would not be detected with traditional in vitro estimation procedures. The results of our experiments suggest that the most important of these factors are phenotypically refractory subpopulations and particularly those in wall-adhering biofilms. The importance of biofilm subpopulations in preventing the eradication of genetically susceptible S. aureus infections e.g. Not anticipated from traditional low-density estimates of MICs is the rapid reduction in the effective concentration of daptomycin observed in our experiments. As a consequence of this decline, the bacterial population returns to a density anticipated for antibiotic free cultures each day. This may be attributed to irreversible and non-productive uptake of the cyclic peptide S. aureus. As such, we do not take into account intracellularly localized, or other non-planktonic populations and intracellular subpopulations of S.aureus have been implicated in several recurrent infection types and a macrophage cell line  (“Sophisticated”) cells and unexposed (“Naïve”) cells exposed to filtrates of medium taken from 0.2 hour flow rate continuous culture with the drug. The gray, red and black columns are respectively the viable cell densities of sophisticated and naïve S aureus in filtrates of treated continuous culture removed at 3, 51 and 123 hours, respectively (see the text).(TIF)Click here for additional data file.Table S1Concentrations of antibiotics at different times during the course of antibiotic treatment of the continous cultures in . Bioassay concentrations given as multiples ofthe CLSI estimates of the MICs of these antibiotics.(DOCX)Click here for additional data file.Appendix S1Mathematical equations describing the pharmacokinetics, PK, of antibiotics in continuous cultures.(DOCX)Click here for additional data file.


The NHHI was implemented in January 2009 to establish a standardized hand hygiene (HH) culture-change program, including the increased use of alcohol-based handrub (ABHR), throughout all Australian hospitals.>90% internal/external validity) was conducted nationally and a 4 monthly data submission schedule established. Electronic and online data submission capability enhanced efficiency and participation. Outcomes 24 months after NHHI commencement were assessed.A multi-modal culture-change program based on the World Health Organization “5 Moments” program was implemented in all States/Territories, including development of Australian HH guidelines, HHA and State-based healthcare worker (HCW) training program, data collection and analysis tools. Training to standardize HH compliance (HHC) auditing  HHC rate was 68.3% (95%CI: 68.1-68.5%), with State-based rates  of 60.8%>72.6%. National HHC by Moment were: M1: 63.1%, M2: 68.4%, M3: 79.1%, M4: 76.0%, M5: 60.0%, suggesting that education needs to be focused on improvements in HH prior to patient contact, especially before performing procedures (M2). Overall HHC among public hospital medical staff was 53.4% (95%CI: 52.8%>53.9%).The NHHI has been associated with a rapid national culture-change among Australian HCWs resulting in significantly improved HHC and a shift to greater use of ABHR. Analysis of NHHI impact on nosocomial disease rates is underway and further improvements in HHC can be expected.None declared.


Pneumocystis. Absolute lymphocyte count in our hospital is available immediately. In contrast, it can take 48 hours or longer to obtain more specific CD-4 counts and AIDS enzyme-linked immunosorbent assay (ELISA) serology. The association of lymphopenia with ultimate diagnosis of AIDS and Pneumocystis supports immediate empiric treatment for pneumocystis carinii pneumonia (PCP) in our highly HIV prevalent hospital.This is a retrospective review of patients admitted to an inner city community hospital with community-acquired pneumonia who were ultimately diagnosed with AIDS and  Although the number of newly diagnosed HIV patients with or without AIDS is declining in North America, the prevalence in Maryland has never been higher, a direct result of increased life expectancy (1) . As recePneumocystis was diagnosed by clinical criteria and/or bronchial lavage. Absolute lymphocyte count on admission was considered to be low if measured at 1.0 or less in the initial complete blood count (CBC).We established a database of all HIV-positive admissions to our hospital over a 4-year period, reviewed each individual chart, and set aside the new cases. New AIDS cases were defined as patients non-HIV-positive on admission, who had no prior medical record of being HIV-positive, who were subsequently diagnosed with HIV, and a low CD-4 count. A clinical diagnosis of community-acquired pneumonia was made on admission by history, physical examination, and chest x-ray; Pneumocystis pneumonia. The next highest frequency of AIDS indicating disease was systemic candidiasis (four of the 40 new cases of AIDS had candidiasis). Absolute lymphocyte counts for the 40 newly diagnosed AIDS patients are listed in Pneumocystis had their lactate dehydrogenase (LDH) levels tested, and their levels were found to be elevated. CD-4 counts arrived at an average of 44 hours post-admission, similar to the HIV enzyme-linked immunosorbent assay (ELISA) test.From 2006 to 2009, there were 2,786 admissions to our hospital who were ultimately identified as HIV positive. Of these, there were 64 new cases of HIV; 40 of which had progressed to AIDS at the time of diagnosis. From these 40 new cases of AIDS, 32 (80%) were ultimately diagnosed with The attack on CD-4 lymphocytes by HIV leads to disease progression . At certPneumocystis should nevertheless be pursued because it remains a potentially fatal illness (Although HIV prevalence in our hospital remains high, new cases are quite rare. Only 2% of our database was diagnosed for the first time during this time frame, as over a 4-year period, 64 patients were admitted with newly diagnosed HIV. In a patient denying high-risk behavior, early attention to diagnose  illness . As we h


Objective: High purity heroin use is a new epidemic health concern among Iranian drug-using women in recent years. However, the nature and initial factors associated with this serious health problem have not been studied yet.Methods: To understand this issue, a cross-sectional study was conducted and sixty treatment and non-treatment seekers who met the Diagnostic and Statistical Manual of Mental Disorders, 4th ed., Text Revision (DSM.IV-TR) criteria for drug dependence with mean age of 28.7 (± 8.3) years were recruited from 16 addiction clinics and drop-in centers (DICs) in Karaj, Iran. First, demographics, and details of drug use and risky behaviors were collected based on items elicited from Addiction Severity Index (ASI). Then, initial factors associated with high purity heroin use were collected by interviewing and applying a researcher-made questionnaire. Data was statistically analyzed by descriptive methods of statistics, chi-square, and Student’s independent t-test in SPSS software. Results: Initiation of high purity heroin use was facilitated by family/relatives factor (66.7%), curiosity (60.1%), peers (54%), desire to experience rapid physical effects of high purity heroin compared with other opioids (50.1%), and treatment of general medical problems such as colic pain (33%). Chi-square test showed that non-treatment seekers were younger (less than 35 years)  and mostly living with drug-dependent friends  compared with treatment seekers. No relation was found among other factors.Conclusions: Factors facilitating the initiation of high purity heroin use revealed in this study have not been fully addressed by current drug treatment services in Iran. To design comprehensive treatment programs, further studies on larger samples with more women are necessary. Declaration of interest: None. For centuries, Iranians were traditional users of opium. However, in recent years, opium use has been increasingly substituted for high purity heroin use . The underlying reasons associated with this problem have not been studied yet. High purity heroin or heroin Kerack is a prevalent type of purified heroin in Iran. There is no comprehensive nationwide survey on the prevalence of high purity heroin use among Iranian women. However, a study shows that there are about 100,000 to 150,000 female opioid addicts, and high purity heroin abuse is a serious problem among women .Another research studied the prevalence and predisposing factors associated with opioid use among 480 patients with the problem of chronic pain  in Zahedan, Iran. In this study, no relationship was found between opioid use and chronic pain. However, there was a relationship between previous opioid use by friends, job, cigarette smoking, consultation for a psychological problem, and death of a spouse . Dolan eth ed., Text Revision) criteria for heroin dependence within the past twelve months were eligible to enter the study. The aim of the study was to conduct an introductory study on initial factors associated with high purity heroin use in a sample of women treatment and non-treatment seekers in Karaj, Iran. We recruited the sample through poster demonstrations in nearly all treatment centers, but only thirteen clinics and three drop-in centers (DICs) had women patients and agreed to cooperate with us. Inclusion criteria included being eighteen years of age or older, being a chronic high purity heroin abuser for at least twelve months prior to current treatment entry for addiction, signing a consent form for study participation, being in treatment for high purity heroin abuse, and living in Karaj during the time of the study. Exclusion criteria included presence of intoxication and withdrawal symptoms and signs, self-report of severe depression and suicidal thoughts which could interfere with the study procedure. Of the 72 women, 60 women met our study inclusion criteria, and were included in the study and interviewed. All participants provided a written consent form for study participation. Women aged 18-65 years who met the DSM.IV-TR , as a highly valid international instrument. Then a confidential interviewing was conducted with the help of a researcher-made questionnaire to explore factors that facilitated initial factors associated with starting high purity heroin use. A pretest-posttest within two weeks on 20 women confirmed the high reliability of the questionnaire  to meet the study aims. The validity of the questionnaire was evaluated by reviewing previous similar studies, and consulting with senior scholars and researchers in this field . All stuData AnalysisQuantitative data including demographics, and details of drug use and risky behaviors were analyzed by descriptive methods of statistics, chi-square test, and Student’s independent t-test analysis in SPSS for Windows .The sample was comprised of 30 female treatment seekers and 30 non-treatment seekers between 18 to 55 years of age [mean age 28.7 ± 11.3) years]. The majority of participants were married (36.6%), but we also interviewed women who were not currently married. Most of them were homemakers (56.6%), unemployed (16.6%), and were living with their families (78.3%). The mean age of the sample was 28.7 (± 8.3) years at the time of high purity heroin initiation, and the duration of dependency to high purity heroin use was 8 (± 9.4) years.The general characteristics of participants and details of high purity heroin use among them are presented in Data analysis by performing chi-square test showed that non-treatment seekers were younger (less than 35 years)  and living more with drug-dependent friends  compared with treatment seekers.The present pilot study is the first study in Iran to explore factors related to initiation of high purity heroin use in a sample of females in Karaj, Iran. This study may help policy makers target gender-specific needs when designing treatment services for women in Karaj. One important finding of our study was the relatively young age of initiation of high purity heroin use among the sample; this is in line with findings of studies from other parts of Iran . This raThis finding may be partly explained by the close relationships that participants had with their family members and relatives. On the other hand, because of the stigma attached to illicit drug use within the Iranian female population, as well as the traditional beliefs of the community that consider providing and smoking illicit drugs as a male-dominated activity, participants had to refer to their family members and relatives in providing and using high purity heroin. A substantial group of interviewees stated that curiosity was a facilitating factor for initial high purity heroin use. This issue may be partly explained by the introduction of high purity heroin as a new opioid to participants. A similar Iranian study on initial alcohol use in a sample of men and women in Shiraz, Iran, showed that curiosity was an important factor for initial alcohol use . WitteveFriends were revealed to play an important role in initiation, but this issue was less reported compared with the two first facilitating factors. This is probably because the majority of respondents were married women with high purity heroin-using family members. Therefore, they were more likely to be introduced to high purity heroin by their relatives rather than their friends. This study finding supports some studies from other provinces of Iran. For example, in his study of drug use among students, Ghanizadeh revealed that the source of opium, hashish, and alcohol in first-time drug use was friends and peer groups . Witteveen et al. showed that some heroin and cocaine addicts referred to drug use for its effects of relieving negative feelings and experiencing drug regulatory effects . MoreoveThis study had several limitations which should be considered when interpreting its findings. Our study was a pilot study and our study sample was selected from Karaj. Therefore, the factors facilitating initiation of high purity heroin use may not be applicable to other cities of Iran. However, our study was one of the few studies that target drug use among females in Iran. In conclusion, this brief study identifies factors facilitating initiation of high purity heroin use in a group of females in Karaj, Iran. Addressing the initial motivations of these women to start high purity heroin use might help in designing treatment programs based on their needs, and characteristics with the aim of removing factors that facilitated initial use. This strategy has never been carried out for female high purity heroin addicts in Iran. Gender-specific treatment services must be implemented to help these women quit high purity heroin dependence.


Slow-adapting type I (SA-I) afferents deliver sensory signals to the somatosensory cortex during low-frequency (or static) mechanical stimulation. It has been reported that the somatosensory projection from SA-I afferents is effective and reliable for object grasping and manipulation. Despite a large number of neuroimaging studies on cortical activation responding to tactile stimuli mediated by SA-I afferents, how sensory information of such tactile stimuli flows over the somatosensory cortex remains poorly understood. In this study, we investigated tactile information processing of pressure stimuli between the primary (SI) and secondary (SII) somatosensory cortices by measuring effective connectivity using dynamic causal modeling (DCM). We applied pressure stimuli for 3 s to the right index fingertip of healthy participants and acquired functional magnetic resonance imaging (fMRI) data using a 3T MRI system.DCM analysis revealed intra-hemispheric effective connectivity between the contralateral SI (cSI) and SII (cSII) characterized by both parallel  and serial  pathways during pressure stimulation. DCM analysis also revealed inter-hemispheric effective connectivity among cSI, cSII, and the ipsilateral SII (iSII) characterized by serial (from cSI to cSII) and SII-level (from cSII to iSII) pathways during pressure stimulation.Our results support a hierarchical somatosensory network that underlies processing of low-frequency tactile information. The network consists of parallel inputs to both cSI and cSII (intra-hemispheric), followed by serial pathways from cSI to cSII (intra-hemispheric) and from cSII to iSII (inter-hemispheric). Importantly, our results suggest that both serial and parallel processing take place in tactile information processing of static mechanical stimuli as well as highlighting the contribution of callosal transfer to bilateral neuronal interactions in SII. Four types of mechanosensitive afferents mediate the sense of touch in the human skin including slow-adapting type I (SA-I) afferents for low-frequency (static) stimuli, slow-adapting type II (SA-II) afferents for skin stretching, fast-adapting type I (FA-I) afferents for flutter, and fast-adapting type II (FA-II) afferents for high-frequency (vibratory) stimuli ,2. Durinintra-hemispheric networks, we further aimed to investigate inter-hemispheric networks across SI and the bilateral SII. To our knowledge, no human fMRI study has investigated effective connectivity across hemispheres for any mechanical stimulus.It is of great interest to understand how tactile information is processed over somatosensory cortical networks. Hierarchical organization of tactile information processing in the primary (SI) and secondary (SII) somatosensory cortices has been documented in many anatomical -12 and nIn the present study, we addressed two questions regarding somatosensory networks associated with SA-I afferents: first, we addressed how intra-hemispheric effective connectivity is formed in the contralateral SI (cSI) and SII (cSII) for tactile information processing of pressure stimuli. We employed DCM to clarify whether pressure stimuli are processed in serial or in parallel with similar hypotheses to previous reports ,16: (1) Twenty-one healthy volunteers  with no history of neurological disorders or deficits in tactile processing gave written informed consent and participated in this study, which was approved by the Korea University Institutional Review Board (KU-IRB-11-46-A-1).2 to the right index fingertip. A neonatal cuff  wrapped around the fingertip was directly connected to a rolling pump in a blood pressure monitor  through an elastic air-tube with a length of 5 m and a diameter of 4 mm. The cuff was controlled by a pressure sensor for achieving uniform pressure; the sensor in turn was controlled by E-Prime 2.0 software  for configuring the length of stimulation. The cuff expanded at the turn-on of the pump and pressed the whole ventral surface of the fingertip  sequence, with the imaging parameters of repetition time (TR) = 1,900 ms, echo time (TE) = 2.48 ms, flip angle = 9°, field of view (FOV) = 200 mm, and voxel size = 0.8 × 0.8 × 1 mm3. 3.Anatomical and functional images were acquired using a 3T MRI system  with a standard 32-channel head coil. TF-statistics (p < 0.05 with a family-wise-error (FWE) correction, with a minimum threshold (k) of 5 voxels for significant clusters) produced group-level statistical parametric maps (SPMs) representing significant voxel clusters. The automated anatomical labeling toolbox [Functional images were preprocessed using SPM8 , through a series of steps of slice-timing correction, realignment with the rigid-body transformation matrices, normalization to the Montreal Neurological Institute (MNI) template, and smoothing with an 8 mm full-width-half-maximum (FWHM) isotropic Gaussian kernel. The mean EPI image of each individual subject was directly warped into the standard EPI template in SPM8 during the normalization step. Then, the conventional general linear model (GLM) in SPM8 performed statistical analyses on blood oxygenation level-dependent (BOLD) signals with a canonical hemodynamic response function and its time and dispersion derivatives. A 128 s high-pass filter removed physiological artifacts in the BOLD signals. A full factorial design based on a random effects model performed a group analysis for the inference of statistically significant cortical activation. Cluster-level  toolbox  determinWe used the Anatomy toolbox  to generTo model intra- and inter-hemispheric effective connectivity in this study, we used DCM instead of other methods for effective connectivity such as Granger causality  and struF-statistics . Among 21 participants, six participants who showed no significant cluster for at least one of the three seed ROIs were excluded. Then, in each of the remaining 15 participants, we defined volumes of interests (VOIs) as spheres of 6 mm radius centered on the most significant peaks of individual ROIs. From the BOLD signals of all voxels in each VOI, the first eigenvariate was extracted as a representative time-series. Table Three seed ROIs were selected in each participant from individual analyses using the anatomical masks with cluster-level 4 - 1) models of modulations excluding a case with no modulation. Thus, there were a total of 30 models including 15 in family A and 15 in family B. A key difference between model families was that one driving input (a pressure stimulus) was applied only to cSI in family A, whereas two driving inputs were applied both to cSI and cSII in family B. In the present study, we examined a total of 450 models   or parallel processing (family B) between cSI and cSII. We defined full intrinsic connectivity due to the fact that both cSI and cSII were fully anatomically connected ,33. It l9) modulation models. We applied two driving inputs to both cSI and cSII. To explain tactile information flows over inter-hemispheric connections, we built three model families. Each family consisted of a set of models with specific modulations being fixed: those fixed modulations represented pre-determined information pathways in accordance with our three hypotheses. We hypothesized that iSII received tactile information from cSI in family C (cSI → iSII), from cSII in family D (cSII → iSII), and from both cSI and cSII in family E (both cSI → iSII and cSII → iSII). Composition of modulation models in each family was determined as follows.Next, we constructed three inter-hemispheric model families to infer how the information of pressure stimuli was conveyed among cSI, cSII, and iSII. We again defined full intrinsic connectivity between all pairs of three VOIs due to the fact that cSI, cSII, and iSII were fully anatomically connected ,35. This7) out of 512 models to be configured. Similarly, there were also 128 configurable models by determining a fixed modulation in cSII → iSII and no modulation in cSI → iSII in family D, or by determining fixed modulations in both cSI → iSII and cSII → iSII in family E. Then, among 128 models in each family, we excluded those models that had modulation in neither cSI → cSII nor cSI ← cSII as we avoided a case when there was no intra-hemispheric connectivity but inter-hemispheric connectivity. In other words, we considered integrating inter-hemispheric connectivity models with intra-hemispheric models by assuming that inter-hemispheric connectivity should involve intra-hemispheric connectivity. This exclusion procedure removed 32 models (= 25) from 128, resulting in 96 models for each family. Thus, in our inter-hemispheric DCM analysis, we examined a total of 4320 models  per session  < 0.05, k > 5). These results highlighted that cortical regions known to be related to tactile perception were activated in response to pressure stimulation during our experiment  across participants and sessions using the best single model  was preferred to family A . BMS resulted in exceedance probabilities for family A and B of 13.28% and 86.72%, respectively (averaged across sessions). Among 15 single models in family B, the best model showed an exceedance probability of 30.27% (averaged across sessions). This model contained one modulation in a forward connection from cSI to cSII . We confirmed the statistical significance of the modulatory parameters from cSI to cSII  and those from cSII to iSII  across participants and sessions  was the most preferred model family. BMS resulted in exceedance probabilities for family C, D, and E of 31.17%, 58.30%, and 10.53%, respectively (averaged across sessions). Among 96 single models in family D, we found the best model with an exceedance probability of 7.77% (averaged across sessions). This model contained two modulations, one in a forward connection from cSI to cSII  and the other in a forward connection from cSII to iSII . We tested the statistical significances of the modulatory parameters in the best single model from cSI to cSII  to both SI and SII in cats  and monkA review of somatosensory evoked potentials (SEPs) highlighted that SII directly received nociceptive inputs from the thalamus, whereas it mainly received tactile inputs from SI . This reAnatomical studies in cats  and monkBased on these studies, we derived two hypotheses of inter-hemispheric pathways from cSI to iSII (originating from cSI) or from cSII to iSII (within SII). A recent SEP study demonstrated time-varying source connectivity during electrical stimulation such that tactile information flowed from cSI to iSII followed by information flow from cSII to iSII with a short latency of approximately 15 ms . ConsequThe SII-level pathway for tactile information delivery we found may be attributed to the roles of SII for high-level tactile perception. Animal studies in monkeys reported the roles of SII for tactile discrimination and learning by investigating impaired task performance of monkeys after removal of the bilateral SII -55. A reBased on our results of two DCM analyses, we suggest that tactile information delivered through SA-I afferents by pressure stimulation is processed not only in parallel with a bifurcated input to both cSI and cSII, but also in serial with sequential sensory transmission from cSI to cSII intra-hemispherically and from cSII to iSII inter-hemispherically. These sensory information pathways are consistent with previous results that include sensory projections from thalamus to both cSI and cSII, hierarchical sensory information processing from cSI to cSII, and bilateral interactions by bilateral receptive fields within SII. Our results of inter-hemispheric connectivity underline this bilateral interaction of SII for tactile information processing by showing that pressure stimulation information from cSI more likely flows through cSII-iSII connections, not directly to iSII. Therefore, our intra- and inter-hemispheric effective connectivity model can be considered as an integrated model, which combined the three architectural factors explained above, can be used to understand low- and high-level tactile information processing of pressure stimulation.In our investigation of hierarchical somatosensory networks, we ruled out any temporal variation because the state equation in DCM did not consider inter-regional conduction delays . We explIn addition, our DCM analysis needs to be extended to other mechanical stimuli (flutter or high-frequency vibration) from FA afferents, electrical stimuli, and even nociceptive stimuli to evaluate the modality-specific consistency of our intra- and inter-hemispheric effective connectivity models. Until now, it has been reported that mechanical stimuli from FA afferents were processed in serial  and nociIn terms of higher-level tactile processing, a recent neuroimaging study reported the existence of dual somatosensory pathways for the perception of texture (from SI to SII) and location (directly to SII) . It therFinally, causal relationships between cortical regions estimated from the fMRI data should also be examined using other assessment methods, in particular model-free methods for connectivity such as the information-theoretic transfer entropy method ,62 to juboth serial and parallel processing of tactile information. Our second DCM analysis revealed that tactile signals were transmitted serially from cSI, through cSII, to iSII over inter-hemispheric connections. Consequently, we postulate a tactile information pathway of pressure stimuli in cSI, cSII, and iSII with three components: (1) parallel processing with a bifurcated input to both cSI and cSII; (2) a serial pathway from cSI to cSII; and (3) an SII-level pathway from cSII to iSII.In the present study, we investigated somatosensory networks based on effective connectivity for information processing of tactile signals from SA-I afferents. Our first DCM analysis revealed that tactile signals were processed through intra-hemispheric effective connectivity characterized by parallel (sensory inputs to cSI and cSII) and serial  pathways, supporting The authors declare that they have no competing interests.YC participated in the study design, carried out the experiments and data analysis, performed the evaluation of results, and drafted the manuscript. SH participated in the study design and carried out the experiments. HK and SC provided the stimulation device for the experiments and helped perform the experiments. JP participated in the study design and helped to MRI data collection. CW conceived of the study, participated in the evaluation of results, and helped draft the manuscript. SK conceived of the study, participated in the study design and its coordination, and helped draft the manuscript. CW and SK gave final approval of the version to be published. All authors read and approved the final manuscript.


This study aims to conduct research about the microstructure and basic properties of recycled aggregate concrete under seawater corrosion. Concrete specimens were fabricated and tested with different replacement percentages of 0%, 30%, and 60% after immersing in seawater for 4, 8, 12, and 16 months, respectively. The basic properties of recycled aggregate concrete (RAC) including the compressive strength, the elastic modulus, and chloride penetration depth were explicitly investigated. And the microstructure of recycled concrete aggregate (RCA) was revealed to find the seawater corrosion by using scanning electron microscope (SEM). The results showed that higher amount of the RCA means more porosity and less strength, which could lower both the compressive strength and resistance to chloride penetration. This research could be a guide in theoretical and numerical analysis for the design of RAC structures. Nowadays recycling of demolition concrete is beneficial and necessary from the viewpoint of environmental preservation and effective utilization of resources. In order to realize this, it is necessary to use demolition concrete as recycled aggregate for new concrete. To make it work, considerable amount of research has been conducted. Although some mechanical properties of recycled concrete may be generally lower than those of normal concrete, they are still sufficient for practical application in constructions –3. For the key mechanical properties of RAC which are the compressive strength, elastic modulus, the bond strength, and tensile and the flexural strengths of such concrete, researchers have paid more and more attention to it globally. Rühl and Atkinson  reached However, all these researches were mainly focusing on the properties of RAC in normal condition, and rare was reported about properties of RAC with seawater corrosion, which prohibits a wider application of recycled concrete in the practical design of engineering structures. As mankind is stepping forward to explore the ocean, huge demands of construction materials are in need to execute the project. As said before, RAC was a feasible and green material in modern society. So it is of great necessity to learn the properties of RAC under seawater corrosion, which will be conducive to the application of RAC in marine environment.In this paper, experiments were conducted to provide a comprehensive analytical evaluation of basic properties of RAC with seawater corrosion. Test results were compared with the normal concrete under the same conditions; the differences of compressive strength and elastic modulus were explicitly analyzed, and the chloride penetration depths and the microstructure of RCA were also investigated, by which the mechanism of seawater corrosion was discussed and revealed explicitly. The results obtained in this paper are significant to efficiently use recycled concrete in practical engineering especially in marine environment.Ordinary Portland cement and river sand with fineness modulus of 2.8 were used in this study. The natural coarse aggregate (NCA) and recycled coarse aggregate (RCA) were obtained from a building demolition in Pudong Avenue, Shanghai, China. Their physical properties are shown in The mix proportion was C25 mixing methods as the national code of China  specifieThe preparations of specimens were performed in the Laboratory for Concrete Material Research at Shanghai Maritime University in Shanghai, China. All mixtures were conducted under laboratory conditions. To simulate the seawater corrosion, seawater was picked up from the Huang Hai Sea, which is one of the four seas of China. Its content is listed in To obtain the compressive strength, 36 specimens are submerged in the seawater for 8 hours and out for 16 hours every day after 28-day curing, which is to simulate seawater corrosion in marine environment. The duration of this dry-wet circulation lasted for 4, 8, 12, and 16 months, respectively. And each group to be tested included three specimens in the same condition. And the loading setup was microcomputer controlled electrohydraulic servo tester.To get the chloride penetration depth, specimens in 100 × 100 × 100 mm cubes were cut at the mid-height to obtain two pieces of concrete samples after 28-day curing. Nonshrinkage epoxy resin was cast around the surface of the 24 concrete samples prepared for determination of the chloride penetration depths to control chloride ions diffused into concrete along one dimension. Then the concrete samples were immersed in seawater. After immersing in seawater for periods of 4, 8, 10, and 12 months, the chloride penetration depths were measured, respectively, by using the methods suggested by Otsuki et al. .σi is the compressive strength (MPa); F represents the peak value of the force (N); A is the area of compression zone in specimen (mm2). After the test, the compressive strength was obtained by following equation:The test results of compressive strength are listed in σ is the standard value of 28-day compressive strength and it is 25 MPa). To check the accuracy of the test results, The compressive strengths of three groups of RAC with seawater corrosion are shown in  The compressive strength decreased by 1.16 to 2.31 MPa at different corrosion time when replacement percentage increased from 0% to 30%. And it decreased by 3. 13 to 3.69 MPa when replacement percentage increased from 0% to 60%. The decreasing range was nearly double when the replacement percentage exceeded 30%, which remind us that the compressive strength is obviously affected by the replacement percentage especially exceeding 30%. In Kasai's research , he suggAnother notable fact in From EC of the RAC was determined from compressive strength by the following empirical equation [EC represents the modulus of elasticity of concrete (GPa); fc is the compressive strength of concrete (MPa). The elastic modulus equation :(2)EC=5The elastic modulus of RAC is shown in On the other hand, The chloride penetration depth of RAC immersed in seawater is shown in The chloride penetration depths at the immersed time of 4, 8, 12, and 16 months of normal concrete were 16.5, 21.0, 30.5, and 32.0 mm, respectively, while those of RC-30 were 18.0, 23.0, 34.0, and 37.5 mm, respectively, and those of RC-60 were 19.5, 26.5, 39.5, and 43.5 mm, respectively. These results indicate that RAC had lower chloride resistance than that of normal concrete because the volume of pores in RAC was higher than that of normal concrete . This reFrom In order to find the link between microstructure and the compressive strength, scanning electron microscope (SEM) was adopted to observe the internal structure of the RAC. It can be found that the microstructure of RAC is obviously different with the replacement percentage and corrosion time. From Figures In addition, for every pair of (a) and (b), (c) and (d), and (e) and (f) in The destruction mechanism of recycled aggregate concrete was similar to that of normal concrete in seawater corrosion environment, but the seawater had remarkable influences on properties of RAC in marine environment, and this influence became more obvious with the replacement percentage increased.The compressive strength of RAC decreased with replacement percentage, and the decreasing range was nearly doubled when the replacement percentage exceeded 30%, so the suitable replacement percentage should not exceed 30% in seawater corrosion environment.The compressive strength of RAC decreased with corrosion time. It decreased at 2% when the corrosion time was within 8 months, and it decreased at 4% to 8% when corrosion time exceeded 8 months. The decreasing range of elastic modulus was at about 7.5% when the replacement percentage varied, but the effects of seawater on the elastic modulus of the RAC were more obvious with an increasing from 2% to 9%.The recycled aggregate concrete had lower chloride resistance than that of normal concrete. The chloride penetration depth increased with the time for the same series, but the growth tended to be increasing at first and then decreasing. The particle of the RCA was becoming thinner and tended to be more fragile to be destroyed as the replacement percentage increased. And the RCA tended to be more compact and in good connection before test; however, it shifted to be smaller particle and more broken bonding after 8 months seawater corrosion.In this paper, test results for the properties of recycled aggregate concrete with seawater corrosion are presented and discussed. From this investigation, the conclusions can be drawn as follows.