Search

Jack R Wall,1 Hooshang Lahooti,1 Ilhem El Kochairi,1 Simon D Lytton,2 Bernard Champion1 1Department of Medicine, the University of Sydney, Penrith, NSW, Australia; 2SeraDiaLogistics, Munich, Germany Abstract: Although ophthalmopathy is mainly associated with Graves’ hyperthyroidism, milder eye changes are also found in about 25% of patients with Hashimoto’s thyroiditis (HT). The recent finding of negative thyrotropin receptor (TSHR) antibodies, as measured in the ­Thyretain™ thyroid-stimulating immunoglobulin (TSI) reporter bioassay, in patients with euthyroid Graves’ disease raises the possibility that TSHR antibodies are not the cause of ophthalmopathy in all situations. Here, we have tested serum from patients with HT with and without ophthalmopathy or isolated upper eyelid retraction (UER) for TSHR antibodies, using the TSI reporter bioassay and collagen XIII as a marker of autoimmunity against the orbital fibroblast. Study groups were 23 patients with HT with ophthalmopathy, isolated UER, or both eye features and 17 patients without eye signs. Thyretain™ TSI results were expressed as a percentage of the sample-to-reference ratio, with a positive test being taken as a sample-to-reference ratio of more than 140%. Serum collagen XIII antibodies were measured in standard enzyme-linked immunosorbent assay. TSI tests were positive in 22% of patients with HT with no eye signs but in no patient with eye signs. In contrast, TSI tests were positive in 94% of patients with Graves’ ophthalmopathy. Tests were negative in all normal subjects tested. Collagen XIII antibodies were detected in 83% of patients with ophthalmopathy, UER, or both eye features, but in only 30% of patients with no eye signs. Our findings suggest that TSHR antibodies do not play a major role in the pathogenesis of ophthalmopathy or isolated UER in patients with HT. Moreover, the role of TSHR antibodies in the development of ophthalmopathy in patients with Graves’ disease remains to be proven. In contrast, collagen XIII antibodies appear to be a good marker of eye disease in patients with HT. Keywords: Hashimoto’s thyroiditis, ophthalmopathy, upper eyelid retraction, TSH receptor antibodies, collagen XIII

The coronavirus infectious disease-2019 (COVID-19) in Bangladesh is a paradigm for how one of the most densely populated countries in the world, with 1270 people per square kilometer, managed to cope with the COVID-19 pandemic under extraordinary circumstances. This review highlights the SARS-CoV-2 variants in Bangladesh and the timeline of their detection in the context of the global experience with the management of vaccination and natural SARS-CoV-2 infection. The motivation to overcome the COVID-19 vaccine dilemma and track Bangladeshi SARS-CoV-2 sub-variants underscores the potential for a low-income country to excel in international medical science, despite having stressed health care services and limited availability of resources for SARS-CoV-2 testing and gene sequencing.

Objective: Genomic surveillance and seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) in Bangladesh is paramount for COVID-19 pandemic preparedness yet lagging the high-income countries due to limited resources., Methods: SARS-CoV-2 variants, COVID-19 symptoms, and serology were prospectively evaluated in a cross-sectional study of Bangladeshi adults testing RT-PCR positive in 2021 and 2022., Results: SARS CoV-2 Omicron variants of asymptomatic or mild COVID-19 in 2022 replaced Delta variant infections requiring hospitalization and oxygen support. The omicron XBB became predominant in July 2022 and associated with cough, headache or body ache and loss of smell; 47 of 68 (69 %), 30 of 68 (44 %) and 27 of 68 (40 %) respectively at higher frequency than BA.1/BA.2; 16 of 88 (18 %), 13 of 88 (15 %) and 0 of 88 (0 %) p < 0.01, p < 0.01 and p < 0.0001. Linear regression analysis reveals no associations between the number of previous infections and the number of symptoms, r = -0.084, p = 0.68. The anti-nucleoprotein (N)-protein IgG post COVID-19 and anti-Spike (S) protein IgG post-COVID-19 vaccination were similar between BA.2, BA.4/BA.5 and XBB and significantly lower than the levels in delta variant infections (p < 0.001)., Conclusions: Omicron XBB subvariants emerged in Bangladesh two months prior to previous reports and include unique patterns of S-protein mutations not assigned in PANGO lineage. The SARS CoV-2 omicron break-through infections persist in the presence of sustained antibody responses and vaccinations, underscoring the importance of molecular surveillance in low-income countries., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interestsMarco Kaiser (M.K), Christian Meyer (C.M.) and Olfert Landt (O.L.) are affiliated with TIB-MOLBIOL GmbH. The analyses and molecular tools were performed at TIB-MOLBIOL. There is no conflict of interest., (© 2024 The Authors. Published by Elsevier Ltd.)

The clinical presentation of COVID-19 and the specific antibody responses associated with SARS-CoV-2 variants have not been investigated during the emergence of Omicron variants in Bangladesh. The Delta and Omicron variants were identified by post-PCR melting curve analysis of the spike (S) protein receptor binding domain amplicons. Anti-S-protein immunoglobulin-G anti-nucleocapsid (N)-protein immunoglobulin-G and immunoglobulin-A levels were measured by ELISA. The Delta variant was found in 40 out of 40 (100%) SARS-CoV-2 RT-PCR positive COVID-19 patients between 13 September and 23 October 2021 and Omicron variants in 90 out of 90 (100%) RT-PCR positive COVID-19 patients between 9 January and 10 February 2022. The Delta variant associated with hospitalization (74%, 80%, and 40%) and oxygen support (60%, 57%, and 40%) in the no vaccine, dose-1, and dose-2 vaccinated cases, respectively, whereas the Omicron COVID-19 required neither hospitalization nor oxygen support (0%, p < 0.0001). Fever, cough, and breathlessness were found at a significantly higher frequency among the Delta than Omicron variants (p < 0.001). The viral RNA levels of the Delta variant were higher than that of the Omicron variants (Ct median 19.9 versus 23.85; p < 0.02). Anti-spike protein immunoglobulin-G and anti-N-protein immunoglobulin-G within 1 week post onset of Delta variant COVID-19 symptoms indicate prior SARS-CoV-2 infection. The Delta variant and Omicron BA.1 and BA.2 breakthrough infections in the Dhaka region, at 240 days post onset of COVID-19 symptoms, negatively correlated with the time interval between the second vaccine dose and serum sampling. The findings of lower anti-spike protein immunoglobulin-G reactivity after booster vaccination than after the second vaccine dose suggest that the booster vaccine is not necessarily beneficial in young Bangladeshi adults having a history of repeated SARS-CoV-2 infections.

Novel SARS-CoV-2 variants are emerging at an alarming rate. The delta variant and other variants of concern (VoC) carry spike (S)-protein mutations, which have the potential to evade protective immunity, to trigger break-through infections after COVID-19 vaccination, and to propagate future waves of COVID-19 pandemic. To identify SARS CoV-2 variants in Bangladesh, patients who are RT-PCR-positive for COVID-19 infections in Dhaka were screened by a RT-PCR melting curve analysis for spike protein mutations. To assess the anti-SARS CoV-2 antibody responses, the levels of the anti-S -proteins IgA and IgG and the anti-N-protein IgG were measured by ELISA. Of a total of 36 RT-PCR positive samples (75%), 27 were identified as delta variants, with one carrying an additional Q677H mutation and two with single nucleotide substitutions at position 23029 (compared to Wuhan-Hu-1 reference NC 045512) in the genome sequence. Three (8.3%) were identified as beta variants, two (5.5%) were identified as alpha variants, three (8.3%) were identified as having a B.1.1.318 lineage, and one sample was identified as an eta variant (B.1.525) carrying an additional V687L mutation. The trend of higher viral load (lower Cp values) among delta variants than in the alpha and beta variants was of borderline statistical significance ( p = 0.045). Prospective studies with larger Bangladeshi cohorts are warranted to confirm the emergence of S-protein mutations and their association with antibody response in natural infection and potential breakthrough in vaccinated subjects.

The aim of the study was to investigate the use of serial measurements of TSH-receptor autoantibodies (TRAb) with the newest available assay technology to predict the course of Graves' Orbitopathy (GO) during the first 24 months from disease onset. Serial serum samples from patients with GO (103 mild/135 severe) were collected between 2007 and 2017 and retrospectively analyzed. The course of GO were classified into mild/severe 12 months after manifestation (severe: NOSPECS≥5; mild<5). TRAb were measured with automated binding immunoassays (IU/l): TRAb Elecsys (Cobas, Roche), TRAb bridge assay (IMMULITE, Siemens), and a cell-based bioassay (percent of specimen to reference ratio - SRR%) (Thyretain, Quidel). Variable cut off levels of measured TRAb were calculated at specificity of 90% from receiver operator curve (ROC) analysis for several timepoints during the course of GO. To select one: 5-8 months after first GO symptoms, which is the timepoint for usual referals for treatment mild course could be predicted at cut offs of 1.5 IU/l (Elecsys), 0.8 IU/l (Immulite) and 402% SRR (Thyretain) and the risc of severe course has to be anticipated if TRAb are above 11.6 IU/l (Elecsys), 6.5 IU/l (Thyretain), and 714% SRR (Thyretain). The Thyretain bioassay showed the highest diagnostic sensitivity (using the commercial cut off's) over the entire follow up period. TRAb measurements during the 24-month follow up of GO provide added value to the GO clinical activity and severity scores and should be used especially in the event of an unclear decision-taking situation with regard to therapy., Competing Interests: M.S., M.O., D.Z., L.M., A.S., N.B., D.F., U.B.P., J.P.B, S.P. and A.E. have nothing to disclose. S.L. consults for Quidel., (Thieme. All rights reserved.)

Background: The assessment of antibody responses to severe acute respiratory syndrome coronavirus-2 is potentially confounded by exposures to flaviviruses. The aims of the present research were to determine whether anti-dengue antibodies affect the viral load and the detection of anti-coronavirus nucleocapsid (N)-protein antibodies in coronavirus infectious disease 2019 (COVID-19) in Bangladesh., Methods: Viral RNA was evaluated in swab specimens from 115 COVID-19 patients by real-time reverse transcription polymerase chain reaction (rT-PCR). The anti-N-protein antibodies, anti-dengue virus E-protein antibodies and the dengue non-structural protein-1 were determined in serum from 115 COVID-19 patients, 30 acute dengue fever pre-COVID-19 pandemic and nine normal controls by ELISA., Results: The concentrations of viral RNA in the nasopharyngeal; Ct median (95% CI); 22 (21.9-23.3) was significantly higher than viral RNA concentrations in oropharyngeal swabs; and 29 (27-30.5) p < 0.0001. Viral RNA concentrations were not correlated with-dengue IgG levels. The anti-nucleocapsid antibodies were IgA 27% positive and IgG 35% positive at days 1 to 8 post-onset of COVID-19 symptoms versus IgA 0% and IgG 0% in dengue patients, p < 0.0001. The levels of anti- nucleocapsid IgA or IgG versus the levels of anti-dengue IgM or IgG revealed no significant correlations., Conclusions: Viral RNA and anti-nucleocapsid antibodies were detected in COVID-19 patients from dengue-endemic regions of Bangladesh, independently of the dengue IgG levels.

The aim of this study was to investigate the potential of the new TSH-receptor antibody (TRAb) assays to predict remission or relapse of hyperthyroidism in patients with Graves' disease (GD) and Graves' orbitopathy (GO). TRAbs were measured retrospectively in sera from a cohort of GD patients with GO (n=117; remission n=38 and relapse n=79-Essen GO biobank) with automated binding immunoassays: TRAb Elecsys (Cobas Roche) and TRAb bridge assay (IMMULITE, Siemens), and the TSAb (thyroid stimulating Ab) cell-based bioassay (Thyretain, Quidel Corp.). To identify relapse risk/remission of hyperthyroidism patients were followed up at least 10 months after the end of antithyroid drug therapy (ATD) therapy. ROC plot analysis was performed to calculate cut-off levels of TRAb and TSAb for prediction of relapse and remission of hyperthyroidism. Cut-off serum levels are provided for timepoints around 3, 6, 10, and 15 months after the beginning of ATD. Repeated measurements of TRAb increase the rate of relapses predictions to 60% (Elecsys), 70% (IMMULITE), and 55% (Thyretain). Patients with remission have consistently TRAb levels below the cut off for relapse in repeated measurements. The cell-based bioassay was the most sensitive - and continued to be positive during follow up [at 15 months: 90% vs. 70% (IMMULITE) and 65% (Elecsys)]. Identification of relapsing hyperthyroidism is possible with automated immunoassays and cell-based bioassay especially with serial TRAb measurements during the course of ATD therapy. Patient who need eye surgery may profit from an early decision towards definitive treatment., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Background: The increased global incidence of hepatitis E virus (HEV) infections, warrants accurate and affordable diagnostics across different geographical regions. The soluble and highly conserved HEV open reading frame 2 (ORF2) capsid antigen (HEV-Ag) is detectable in self-limited acute enteric hepatitis by HEV-Ag ELISA which is a promising serological assay in settings where HEV-RNA testing is not feasible. Our aim was to assess the HEV-Ag biomarker in an HEV outbreak in a low income country., Methods: A prospective single center longitudinal study during HEV outbreaks in the Chittagong, Bangladesh region between October 2018 and October 2019 was conducted based on recruitment of acute jaundice cases with clinical signs and symptoms of suspect HEV infections. Acute HEV infection was defined as a positive test result for anti-HEV IgM antibodies., Results: Forty four of the 51 enrolled enteric hepatitis cases (86 %) were confirmed HEV by anti-HEV IgM ELISA at day 0 hospital entry. The anti-HEV-IgM and IgG were positive in all patients and did not reveal significant differences; neither between the time points day 0 and follow-up hospitalization on day 2-6 or day 7-10 nor between RNA-positive (n = 36) versus RNAnegative (n = 8) HEV groups. The HEV-Ag positivity was higher in viral RNA-positive (29/36, 81 %) than the viral RNA-negative (1/8, 12 %) group, p < 0.001 and the HEV-Ag levels positively correlated with viremia, r = 0.77, p < 0.0001. All non-HEV cases; n = 7 tested negative anti-HEV IgM and HEV-Ag and 5 of 7 (71 %) tested anti-HAV IgM positive., Conclusions: The HEV-Ag ELISA is a reliable and practical diagnostic tool in this acute HEV outbreak., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The morbidity in dengue fever is dependent on the dengue virus (DENV) serotypes, the patient age, predisposing immunogenic markers and the frequency of primary and secondary infections. This study aims to distinguish acute primary from secondary dengue infections of Vietnamese adults and to assess the association of viremia and anti-dengue immunoglobulin levels with clinical outcomes., Study Design: Viral RNA, dengue serotypes and levels of anti-dengue IgM and IgG of hospitalized adult cases were determined in EDTA-plasma samples prospectively collected during three consecutive years of dengue infection in Hanoi. Patients admitted to hospital within 7 days of their 1st reported fever were included. Primary infections were anti-dengue IgG enzyme-linked immunosorbent assay (ELISA) negative on both day of hospital entry (day 0) and day two or three of hospitalization (day 2 or 3) with a positive anti-dengue IgM on either day 0 or day 2 or 3 hospitalization. The secondary infections were anti-dengue IgG ELISA positive on both day 0 and day 2 or 3 with positive anti-dengue IgM ELISA on either day 0 or day 2 or 3., Results: The hospitalized dengue fever cases between October 2016 and March 2019 were predominantly secondary infections (74%, 68% and 77%, respectively) with DENV-1 (60% and 65%) and DENV-2 (22% and 26%) serotypes determined in the latter two years. The viremia in primary infection was significantly higher than that in secondary infection (P < 0.01) and positively correlated with the days of hospital stay. In secondary infections, platelet counts were lower than in primary infections (P = 0.04) and IgG levels in secondary infection negatively correlated with platelet counts (Spearman's r = -0.22, P < 0.01)., Conclusions: Our results indicate high rates of secondary infection with DENV1 and DENV2 serotypes. Anti-dengue immunoglobulins negatively correlate with hospital stay and platelet counts with few warning signs or severe disease. Further investigations of specific antibodies in adults which predict auto-inflammatory activity after the recovery from dengue infection are warranted., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: During dengue fever, a pronounced gamma-interferon immune response produces neopterin and promotes tryptophan degradation by the enzyme indoleamine-2,3-dioxygenase 1 (IDO-1). Activated IDO-1 is indicated by an increased kynurenine to tryptophan ratio (Kyn/Trp) in patients., Methods: Plasma levels of neopterin, kynurenine, and tryptophan were measured in 72 hospitalized dengue virus (DENV) patients and 100 healthy individuals. Plasma levels of neopterin, kynurenine, and tryptophan were also measured prospectively in a second cohort of 13 DENV patients; on the day of hospitalization, on day 2-3 at discharge, and 7-10 days after discharge. DENV RNA positivity was determined by qualitative and quantitative methodologies., Results: DENV RNA-positive patients presented significantly higher levels of neopterin (mean 36.5nmol/l) and Kyn/Trp ratios (mean 102μmol/mmol) compared to DENV RNA-negative individuals. A significant correlation between neopterin levels and Kyn/Trp ratios was observed in both DENV RNA-positive (Spearman's rho=0.37, p< 0.01) and DENV RNA-negative (Spearman's rho=0.89, p<0.001) patients. Kyn/Trp ratios were negatively correlated with platelet counts (Spearman's rho=-0.43, p<0.01) and positively correlated with liver enzymes: AST (Spearman's rho=0.68, p<0.01) and ALT (Spearman's rho=0.51, p<0.05). In addition, the follow-up data presented a significant decrease in neopterin levels and Kyn/Trp ratios within 10 days after hospital entry., Conclusions: Neopterin levels and Kyn/Trp ratios were significantly increased in DENV patients and subsequently decreased after recovery., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The impairment of regulatory T cells (Tregs) is a characteristic feature of autoimmune hepatitis (AIH), and the degradation of tryptophan (Trp) to kynurenine (Kyn), by gamma interferon-induced indoleamine-2,3-dioxygenase-1 (IDO-1), is a central metabolomics check point in the differentiation of Tregs. For this reason, we investigate whether or not Kyn and IDO activity is potentially useful biomarkers in pediatric AIH.Between January 2016 and January 2017, children of AIH type-1 (AIH-1, n = 37), AIH type-2 with liver kidney microsome-1 autoantibodies (AIH-2-LKM-1, n = 8), and autoantibody-negative Wilsons Disease (WD, n = 8) and alpha-1 anti-trypsin deficiency (AATD, n = 10), were enrolled in a cross-sectional survey of Kyn and Trp levels and Kyn/Trp ratios (IDO activity) by HPLC, and neopterin levels by ELISA.The mean Kyn and mean Kyn/Trp ratios of AIH-1 with smooth muscle antigen (SMA) 1.85 μM and 27 μmole/mmole, and AIH-2-LKM-1; 1.7 μM and 28.6 μmole/mmole were lower than that of the WD; 2.2 μM p = 0.03 and 33 μmole/mmole p = 0.02 and of AATD; 2.3 μM, p = 0.02 and 55 μM, p = 0.001. Kyn/Trp ratios of AIH relapse; 23.6 μmole/mmole were lower than Kyn/Trp ratios of AIH remission; 27.6 μmole/mmole (p < 0.05). The stage of liver disease and grade of liver biopsies in AIH-1 patients negatively correlated with the Kyn/Trp ratios.The serum Kyn levels and Kyn/Trp ratio of AIH patients, within or below the normal range, indicate a trend of IDO activity lower than non-autoimmune WD or AATD. Prospective monitoring of serum tryptophan metabolomics in larger cohorts of pediatric AIH patients is required to confirm the apparent paradigm of weak IDO activity contributing to the Treg deficit and pathogenesis of pediatric AIH.

Autoantibodies to the thyrotropin hormone receptor (TSH-R) are directly responsible for the hyperthyroidism in Graves' disease and mediate orbital manifestations in Graves' orbitopathy (otherwise known as thyroid eye disease). These autoantibodies are heterogeneous in their function and collectively referred to as TRAbs. Measurement of TRAbs is clinically important for diagnosis of a variety of conditions and different commercial assays with high sensitivity and specificity are available for diagnostic purposes. This review provides overwhelming evidence that the TRAbs detected in binding assays by mainly the automated electrochemical luminescence immunoassays (ECLIA) do not distinguish TRAbs that stimulate the TSH-R (called TSIs or TSAbs) and TRAbs that just inhibit the binding of TSH without stimulating the TSH-R (called TBAbs). However, TSAbs and TBAbs have divergent pathogenic roles, and depending which fraction predominates cause different clinical symptoms and engender different therapeutic regimen. Therefore, diagnostic distinction of TSAbs and TBAbs is of paramount clinical importance. To date, only bioassays such as the Mc4 TSH-R bioassay (Thyretain TM , Quidel) and the Bridge assay (Immulite 2000, Siemens) can measure TSAbs, with only the former being able to distinguish between TSAbs and TBAbs. On this note, it is strongly recommended to only use the term TSI or TSAb when reporting the results of bioassays, whereas the results of automated TRAb binding assays should be reported as TRAbs (of undetermined functional significance). This review aims to present a technical and analytical account of leading commercial diagnostic methods of anti-TSH-R antibodies, a metaanalysis of their clinical performance and a perspective for the use of cell based TSH-R bioassays in the clinical diagnostics of Graves' disease.

Background: Anti-liver kidney microsome (anti-LKM) autoantibodies are a distinguishing feature of type II autoimmune hepatitis (AIH-2). However, the levels of anti-LKM-1 in adult AIH-2 patients and their role in liver immunopathology remain equivocal. The aim of the study was to survey the autoantibody profile and the activity of liver disease in adult patients diagnosed with AIH-2 at childhood., Methods: The autoantibody profile of adults was compared with the autoantibodies of the pediatric period. Liver function test, Immunoglobulin G (IgG), and gamma globulins were evaluated at the AIH presentation, at the age of 18 years, and at the current adult visit., Results: All ten patients tested positive for LKM-1 at least once during the pediatric period. At the adult visit, four patients lost autoantibody positivity. LKM-1 was positive in four, liver cytosol antigen 1 (LC-1) in two, soluble liver antigen in one, and antinuclear antigen in one patient. Additionally three patients with LKM-1 and one patient without LKM-1 were positive for AMA-M2 (where AMA is antimitochondrial antibodies) Immunoglobulin M (IgM). Liver function markedly improved at 18 years and adult visit compared with initial diagnosis of AIH with only a mild decrease of IgG. The six adult patients positive for at least one autoantibody had statistically lower aspartate aminotransferase (AST) and gamma-glutamyltranspeptidase (GGTP) than the four patients autoantibody negative (AST: 52 vs. 88 IU/l, P < 0.05; GGTP 19 vs. 163 IU/l, P < 0.05)., Conclusion: LKM-1 positivity is not a stable condition in all patients with AIH-2. Patients who remained autoantibody positive had better liver function tests than those who lost their positivity. The presence of AMA-M2 autoantibodies suggest that development of AIH/Primary Biliary Cirrhosis (PBC) overlap syndrome should be considered., (© 2015 Wiley Periodicals, Inc.)

Purpose: A new CE-marked portable desktop ion mobility spectrometer (VGTest) was used for detection of malodorous biogenic amines indicative of bacterial vaginosis (BV). This study aimed to assess the performance of this testing method for the first time in a routine ambulatory care clinic and to determine the relative levels of biogenic amines in vaginal fluid of BV., Methods: Vaginal and cervical swabs (n = 57) were surveyed for infections. Cases of BV (n = 18) confirmed positive according to "Amsel" criteria and normal controls (n = 39) showing no infection under clinical examination and testing negative in wet mount microscopy were included in the IMS analysis., Results: The trimethylamine (TMA) content in vaginal fluid of the BV-positive cases, AUCTMA/AUCTotal [mean 0.215 (range 0.15-0.35)] was significantly higher than normal controls [mean 0.06 (range 0.048-0.07)] p < 0.0001. The putrescine (1,4-diaminobutane, PUT) and cadaverine (1,5-diaminopentane, CAD) of BV-positive cases were above controls at borderline significance. The AUCTMA/AUCTotal ratios correlated neither with AUCPUT/AUCTotal nor AUCCAD/AUCTotal among BV-positive patients. In contrast, among normal controls all the biogenic amines were at a low level and the linear regression analysis revealed striking positive correlations of AUCTMA/AUCTotal with AUCPUT/AUCTotal (p < 0.05) and AUCCAD/AUCTotal (p < 0.001). The test shows 83 % sensitivity and 92 % specificity at a cut-off of AUCTMA/AUCTotal = 0.112 and AUC of receiver operator characteristic = 0.915 (0.81-0.97, 95 % CI)., Conclusions: VGTest-IMS is accurate and feasible for point-of-care testing of BV in the ambulatory care setting. Further evaluations are in progress to assess the utility of VGTest-IMS for differential diagnosis of candidosis, non-BV infection and common inflammatory conditions.

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye., (Published by Elsevier Ltd.)

Background: The deamidated gliadin peptides (DGP) cross linked to human tissue transglutaminase (tTg) comprises a novel neo-epitope structure (Neo-tTg) for serological screening of celiac disease (CD). Our aim is to verify anti-Neo-tTg IgA and IgG in adults with dermatitis herpetiformis (DH)., Methods: Multi-centric retrospective evaluation of the IgA/G autoantibodies in sera of DH patients on a regular diet (n=40) and a gluten restricted diet (GRD, n=53) and control adults with autoimmune skin diseases (n=107) by ELISA., Results: The sensitivities of Celicheck Neo IgA/G (76%, 95% CI 67-84%) and the Neo tTg-A (85%, 95% CI 70-97%) ELISA were significantly greater than that of tTg-A (56%, 95% CI 46-67%), eTg-A (62%, 95% CI 52-72%), DGP-A (55%, 95% CI 55-65%), DGP-G (61%, 95% CI 51-71%), Glia-A (55%, 95% CI 45-65%) and Glia-G (56%, 95% CI 46-66%) ELISA. The specificities of all 8 ELISA were in the range of 90-100%. The area under the curve (AUC) of receiver operator characteristic curve (ROC) for the two Neo-tTg ELISA (0.863 and 0.949) were higher than the AUCs for ROCs of tTg, DGP and eTG ELISA (range between 0.657 and 0.783). The autoantibody levels of DH patients on a normal diet were significantly higher than those on GRD in the Celicheck Neo IgA/IgG, NeotTg-A; tTg-A and the eTg-A; ELISA (p<0.01) and of no significance in the DGP and Gliadin ELISA., Conclusion: Neo-epitope IgA autoantibodies represent a new and sensitive serological marker of DH., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We show here that the anti-T lymphocyte immunoglobulin (ATG) can induce Treg cells following 24-h incubation in human peripheral blood mononuclear cells (PBMCs). The ATG-induced Treg cells express known cell surface markers (e.g., CD25, FoxP3) and suppress the proliferation of autologous responder PBMCs, stimulated with allogeneic PBMCs, when added into the mixed lymphocyte culture (MLC) at zero time point or 48 h later. We expanded the characteristics of the ATG-induced human Treg cells by showing that they express a novel biomarker designated "activated CD44". ATG-induced Treg cells retain their suppressor function after freezing and thawing or irradiation. Suppression of MLC by ATG-induced Treg cells is consistently seen when the Treg cells and the responder cells were derived from the same donor, but not when they derived from different donors. Finally, patients undergoing stem cell transplantation and conditioned with ATG generate in vivo Treg cells that suppress MLC.

Background and Aim: Graves' orbitopathy (GO) is characterized by orbital T cell infiltration and local release of proinflammatory cytokines. We aimed to evaluate the involvement of baseline regulatory T (Treg) cells and rabbit anti-T lymphocyte globulin (rATG)-induced Treg cells in GO., Design: Peripheral blood mononuclear cells (PBMCs) from seven patients with Graves' disease (GD) without eye manifestations, 29 patients with GO, and 15 healthy controls were incubated with rATG, washed, and analyzed for expression of Treg cell markers and for ability to suppress mixed lymphocyte reaction., Results: Elevation of CD4 to CD8 ratio and enhanced secretion of IL-6, IL-10, and TNFα were detected in PBMCs of GO patients compared with controls (both P < 0.01). Despite this abnormality, the frequencies of CD4(+)CD25(+)FoxP3(+) of GO and control PBMCs were similar and remained unchanged after 24 h incubation with control rabbit IgG (rIgG). Incubation with polyclonal rATG increased the frequency of PBMCs of GO patients, expressing Treg cell markers (CD25, FoxP3, and the IL-7 receptor CD127(low/-)) by 2.5-8 fold over corresponding rIgG-incubated cells (P < 0.05). FoxP3/CD4 rATG-induced Treg cell marker expressed more intensively on GO peripheral blood leukocytes (PBLs) than on GD (P < 0.01) or normal (P < 0.05) PBLs, yet its expression on normal PBLs was stronger than on GD PBLs (P < 0.05). GO rATG-incubated PBMCs, but not rIgG-incubated PBMCs, suppressed (P < 0.05) proliferation of autologous responder cells stimulated with allogeneic irradiated cells in mixed lymphocyte reaction. Such rATG-induced suppressive activity was not detected in GD., Conclusion: This study is the first to show that PBMCs of patients with GO substantially increase Treg cells in both frequency and potency after in vitro incubation with rATG.

Immunoglobulins in patients with Graves' disease (GD) that modulate the thyroid stimulating hormone receptor (TSH-R) do so via stimulating cAMP dependent signals (TSI), blocking TSH or inhibition of TSH-receptor activation (TBI) or inducing apoptotic signals. These functional immunoglobulins represent powerful biomarkers of anti-self reactivity in the thyroid and systemic tissues that harbor TSH-R expressing target cells. TSI on thyrocytes induce hyperthyroidism, and TSI on TSH-R fibroblasts of orbital muscles, skin and heart provoke the release of cytokines and antigen-specific T-cell responses leading to systemic inflammation. Bioassays of anti-TSH-R autoantibodies provide decisive information on GD activity. This review examines the past and present bioassays in GD. The critical goal of cell-based anti-TSH-R autoantibody bioassays, to identify the pathogenic immunoglobulins in GD under robust and standardized conditions suitable for routine clinical laboratory practice, is discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Thyroid-stimulating immunoglobulins (TSI) are a functional biomarker of Graves' disease (GD). To develop a novel TSI bioassay, a cell line (MC4-CHO-Luc) was bio-engineered to constitutively express a chimeric TSH receptor (TSHR) and constructed with a cyclic adenosine monophosphate (cAMP)-dependent luciferase reporter gene that enables TSI quantification. Data presented as percentage of specimen-to-reference ratio (SRR%) were obtained from 271 patients with various autoimmune and thyroid diseases and 180 controls. Sensitivity of 96% and specificity of 99% for untreated GD were attained by receiver operating characteristic analysis, area under the curve 0·989, 95% confidence interval 0·969-0·999, P = 0·0001. Precision testing of manufactured reagents of high, medium, low and negative SRR% gave a percentage of coefficient-of-variation of 11·5%, 12·8%, 14·5% and 15·7%, respectively. There was no observed interference by haemoglobin, lipids and bilirubin and no non-specific stimulation by various hormones at and above physiological concentrations. TSI levels from GD patients without (SRR% 406 ± 134, mean ± standard deviation) or under anti-thyroid treatment (173 ± 147) were higher (P < 0·0001) compared with TSI levels of patients with Hashimoto's thyroiditis (51 ± 37), autoimmune diseases without GD (24 ± 10), thyroid nodules (30 ± 26) and controls (35 ± 18). The bioassay showed greater sensitivity when compared with anti-TSHR binding assays. In conclusion, the TSI-Mc4 bioassay measures the functional biomarker accurately in GD with a standardized protocol and could improve substantially the diagnosis of autoimmune diseases involving TSHR autoantibodies., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Context: Immunoglobulins stimulating the TSH receptor (TSI) influence thyroid function and likely mediate extrathyroidal manifestations of Graves' disease (GD)., Objectives: The aim of this study was to assess the clinical relevance of TSI in GD patients with or without Graves' orbitopathy (GO), to correlate the TSI levels with activity/severity of GO, and to compare the sensitivity/specificity of a novel TSI bioassay with TSH receptor (TSH-R) binding methods (TRAb)., Design: TSI were tested in two reporter cell lines designed to measure Igs binding the TSH-R and transmitting signals for cAMP/CREB/cAMP regulatory element complex-dependent activation of luciferase gene expression. Responsiveness to TSI of the novel chimeric (Mc4) TSH-R (amino acid residues 262-335 of human TSH-R replaced by rat LH-R) was compared with the wild-type (wt) TSH-R., Results: All hyperthyroid GD/GO patients were TSI-positive. TSI were detected in 150 of 155 (97%, Mc4) and 148 of 155 (95%, wt) GO patients, in six of 45 (13%, Mc4) and 20 of 45 (44%, wt) mostly treated GD subjects, and in 0 of 40 (Mc4) and one of 40 (wt) controls. Serum TSI titers were 3- and 8-fold higher in GO vs. GD and control, respectively. All patients with diplopia and optic neuropathy and smokers were TSI-positive. TSI strongly correlated with GO activity (r = 0.87 and r = 0.7; both P < 0.001) and severity (r = 0.87 and r = 0.72; both P < 0.001) in the Mc4 and wt bioassays, respectively. Clinical sensitivity (97 vs. 77%; P < 0.001) and specificity (89 vs. 43%; P < 0.001) of the Mc4/TSI were greater than TRAb in GO. All 11 of 200 (5.5%) TSI-positive/TRAb-negative patients had GO, whereas all seven of 200 (3.5%) TSI-negative/TRAb-positive subjects had GD only., Conclusion: The novel Mc4/TSI is a functional indicator of GO activity and severity.

The rabbit anti-T lymphocyte globulins (rATGs) are immune-suppressive anti-T cell agents with beneficial effects in solid organ and hematological transplantation. The present review evaluates the potential mechanisms of rATGs and their impact on pilot and exploratory studies of diffuse cutaneous systemic sclerosis (scleroderma-SSc), inclusion body myositis (IBM), vasculitis, and type 1 diabetes mellitus (T1DM). The rATGs are associated with improvements in well-defined parameters of clinical autoimmunity: insulin usage, tissue inflammation, and systemic organ functions. Meta-analysis of a retrospective database of SSc, N = 196 and two prospective randomized pilot studies; IBM, N = 11 and T1DM, N = 17 shows a two- to ninefold increase in the relative response to treatments with intravenous infusions of rATG. The rATGs deplete T cells and are associated with increases in the percentage of CD25+ T cell subsets. This may underlie the apparent long-lasting immunomodulation associated with these agents. The future optimization of rATG adjunct therapy requires statistically powered-controlled prospective trials of rATG dose-finding and timing of administration. The potential mechanisms of rATGs:depletion of autoreactive T cells, generation CDCD25+Foxp3+ regulatory T cells (Tregs), and the acquisition of regulatory immune cell functions, need to be examined in patients prior to rATG infusion and at time intervals following rATG treatment to identify those mechanisms relevant to the improvement of their clinical outcome.

Purpose: Detection and modulation of effector T cells specific to immunodominant epitopes is a central issue in autoimmune diseases. Experimental autoimmune uveitis is a model for human autoimmune uveitis, induced in B10.RIII mice with interphotoreceptor retinoid binding protein or with its immunodominant epitope encoded by residues 161-180., Methods: The authors generated a dimer composed of p161-180 fused in frame to IA(r) and mouse IgG1, and studied its effects on a CD4(+) uveitogenic T-cell line specific to p161-180 and on a T-cell clone derived from that line., Results: Immunofluorescent staining of the T-cell line with the peptide/IA(r)/Ig dimer revealed that about 90% of the cells bound the reagent, and 10% did not. The T-cell clone failed to bind the reagent. Consistent with this, the line proliferated when stimulated with the reagent plus anti-CD28, and the clone did not. Conversely, after being incubated with the reagent without CD28 cross-linking, the line showed decreased proliferation on subsequent stimulatory exposure to p161-180, whereas the clone was unaffected. Antigen-specific proliferation of splenocytes from B10.RIII mice primed with p161-180 was inhibited by anti-IA as well as anti-IE antibodies; proliferation of the T-cell line was inhibited strongly by anti-IA and poorly by anti-IE, and the clone showed the opposite pattern. Finally, the line, but not the clone, proliferated to p161-180 presented on a B-cell lymphoma expressing IA(r) as its only restriction element., Conclusions: Uveitogenic T cells can be detected as well as functionally modulated with their cognate peptide-class II reagent, suggesting the potential of such reagents for diagnostic and therapeutic use in uveitic disease; p161-180 can be presented by IA(r) as well as IE(r) major histocompatibility complex (MHC) class II molecules. The possibility that the same immunodominant fragment might be presented by more than one class II molecule should be taken into account when diagnostic or clinical use of peptide-MHC reagents is considered.

Tight junctions, paracellular permeability barriers that define epithelial cell polarity, play an essential role in transepithelial transport, cell-cell adhesion and lymphocyte transmigration. They are also important for the maintenance of innate immune defence and intestinal antigen uptake. Ammonium (NH4+) is elevated in the gastric aspirates of Helicobacter pylori-infected patients and has been implicated in the disruption of tight-junction functional integrity and the induction of gastric mucosal damage during H. pylori infection. The precise mechanism of the effect of ammonium and the molecular targets of ammonium in host tissue are not yet identified. To study the effects of ammonium on epithelial tight junctions, the human colon carcinoma cell line Caco-2 was cultured on permeable supports and the transepithelial resistance (TER) was measured at different time intervals following exposure to ammonium salts or H. pylori-derived ammonium. A biphasic response to treatment with ammonium was found. Acute exposure to ammonium salts or NH3/NH4+ derived from urea metabolism by wild-type H. pylori resulted in a 20-30 % decrease in TER. After 24 h, the NH4Cl-treated cells showed a partial recovery of TER. In contrast, the control culture, or cultures that were exposed to supernatants derived from urease-deficient H. pylori, showed no significant decrease in TER. Occludin-specific immunoblots revealed the expression of a low-molecular-weight form of occludin of 42 kDa upon NH3/NH4+ exposure. The results indicate that modulation of tight-junction function by H. pylori is ammonium-dependent and linked to the accumulation of a low-molecular-weight and detergent-soluble form of occludin.

A pemphigus vulgaris (PV) patient with a 14-year history of severe and painful blistering of skin and mucous membranes as well as side effects from corticosteroids and concomitant immunosuppressive drug treatment was managed successfully by protein A immunoadsorption (IA). After 19 sessions of protein A IA, the patient showed remission of PV and healing of mucocutaneous lesions and the skin. The level of the pathogenic autoantibodies to the adhesion proteins desmoglein 1 (Dsg-1) and desmoglein 3 (Dsg-3) measured by ELISA and immunofluorescence microscopy revealed a significant removal of autoantibodies after each IA therapy. There was a weak rebound of anti-Dsg-1 and anti-Dsg-3 antibodies between IA sessions but an overall decrease over the period of IA therapy. This case demonstrates the effective use of protein A IA as an adjuvant and corticosteroid-sparing therapy in severe pemphigus refractory to standard immunosuppressive therapy and underscores the need for careful monitoring of autoantibodies.

Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing.

Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato- and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8.5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.

Autoantibodies against soluble liver enzymes have been reported among alcoholics, but the targets of self-reactivity toward membrane proteins of the liver have not been characterized. Previously, among alcoholics, we found antibodies against ethanol-derived radical protein adducts that are dependent on cytochrome P-4502E1 (CYP2E1) for their formation. To further investigate autoantibodies against cytochrome P-450s during alcohol abuse, sera of rats chronically treated with ethanol in the total enteral nutrition model and sera from alcoholics with or without alcohol liver disease and from control subjects were analyzed by enzyme-linked immunosorbent assay and Western blotting for the presence of IgG against rat and human CYP2E1, rat CYP3A1, and human CYP3A4. A time-dependent appearance of IgG against rat CYP3A1 and CYP2E1 was evident during chronic ethanol feeding of rats. Anti-CYP2E1 reactivity showed positive correlation with the levels of hepatic CYP2E1 and was inhibited by the CYP2E1 transcriptional inhibitor chlormethiazole. Screening of the human sera by enzyme-linked immunosorbent assay revealed reactivity against CYP3A4 and CYP2E1 in about 20 to 30% and 10 to 20% of the alcoholic sera, respectively. No difference were noted between sera from alcoholics with or without hepatitis C virus infection, and only very little reactivity was seen in sera from control subjects. Western blotting analysis revealed anti-human CYP2E1 reactivity in 8 of 85 alcoholic sera and 3 of 58 control sera, whereas anti-CYP3A4 reactivity was detected in 18 of 85 alcoholic sera and 4 of 58 control sera, which were different from the sera reactive with CYP2E1. Immunoblot reactivity of CYP3A4-positive alcoholic sera was found against glutathione-S-transferase fusion proteins containing truncated forms of CYP3A4, and such sera were also able to immunoprecipitate in vitro translated CYP3A4. Seven of eight sera showed reactivity toward domains C-terminal of position Ser281, and 1 of 8 sera recognized autoepitopes within the region Thr207-Ser281. These findings indicate that alcoholics develop autoantibodies against CYP2E1 and CYP3A4 that the CYP3A4 C-terminal domain is a target for the autoantibody reactions among a subset of alcoholics. The novel finding of CYP3A4 autoantibodies and their significant expression among alcoholics warrants further investigation. Attention should be given to immune toxicity associated with CYP3A4 autoantibodies and cases of alcohol abuse that are accompanied by exposure to drugs and substances that are CYP3A substrates.

The ability of Leishmania mexicana amazonensis to inhibit antigen specific T-cell proliferation against a non-parasite polypeptide antigen, poly(LTyr, LGlu)-poly(DLAla)--poly(LLys), was examined. Infection of mouse peritoneal macrophages by promastigotes blocked the proliferation of the T-cell line, TPB1. This effect was correlated with the level of parasite infection, and the timing of macrophage infection and antigen addition. Peritoneal macrophages from both BALB/b and C57BL/6 mice showed reduced ability to serve as antigen presenting cells.

We assessed in vivo antimalarial action of a lipophilic iron (III) chelator belonging to a new synthetic family of biomimetic siderophores previously termed reversed siderophores (RSFs). The family member, RSF ileum2, was chosen for its high membrane permeability and fast irreversible inhibition of human malaria parasite growth in vitro. [Shanzer A, et al., Proc Natl Acad Sci USA 88:6585, 1991 and Lytton SD, et al., Blood 81:214, 1993]. The lipophilic drug was administered to Swiss mice by subcutaneous route in fractionated coconut oil at a dosage of 0.37 g/kg every 8 hr with no adverse reactions observed. After 3-4 injections demonstrable suppression of Plasmodium vinckei petteri infection was observed and an additional 3-4 injections resulted in 2-3-fold lower parasitemia with prolonged survival time over sham-injected control mice.

We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron (III) chelator with improved membrane permeation properties. Upon binding of iron (III), MA-DFO fluorescence is quenched, thus allowing traceability of drug-iron (III) interactions. MA-DFO is well tolerated by mammalian cells in culture. Its antimalarial activity is pronounced: IC50 values on in vitro (24-h) growth of Plasmodium falciparum were 3 +/- 1 microM for MA-DFO compared with 30 +/- 8 for DFO. The onset of growth inhibition of rings or trophozoites occurs 2-4 h after exposure to 13 microM MA-DFO. This effect is commensurate with MA-DFO permeation into infected cells. In a 24-h exposure to MA-DFO or DFO, trophozoites take up either compound to approximately 10% of the external concentration, rings to 5%, and noninfected cells to < 1%. Red cells encapsulated with millimolar concentrations of DFO or MA-DFO fully support parasite invasion and growth. We conclude that extracellular MA-DFO and DFO gain selective access into parasites by bypassing the host. The rate-limiting step is permeation through the parasite membrane, which MA-DFO accomplishes faster than DFO, in accordance with its higher hydrophobicity. These views are consistent with the proposed duct, which apparently provides parasitized cells with a window to the external medium.

We present here the physicochemical and biochemical properties of NBD-DFO, the 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative of the siderophore, desferrioxamine B (DFO) (Lytton et al., Mol. Pharmacol. 40, 584, 1991). Modification of DFO at its terminal amine renders it more lipophilic, imparts to it fluorescent properties, and is conservative of the high-affinity iron(III) binding capacity. NBD-DFO partitions readily from aqueous solution into n-octanol (Pcoeff = 5) and displays solvent-induced shifts in absorption and fluorescence spectra. The relative quantum yield of the probe's fluorescence increases over a 10-fold range with decreasing dielectric constant of the solvent. Fluorescence is quenched upon binding of iron(III) to the probe. We demonstrate here the application of NBD-DFO for the specific detection and monitoring of iron (III) in solutions and iron(III) mobilization from cells. Interactions between fluorescent siderophore and the ferriproteins ferritin and transferrin were monitored under physiological conditions. Iron removal from ferritin was evident by the demonstrable quenching of NBD-DFO fluorescence by scavenged iron(III). Quantitation of iron sequestered from cells by NBD-DFO or from other siderophore-iron(III) complexes was accomplished by dissociation of NBD-DFO-Fe complex by acidification and addition of excess ethylenediamin-etetraacetic acid. The sensitivity of the method and the iron specificity indicate its potential for monitoring chelatable iron under conditions of iron-mediated cell damage, iron overload, and diseases of iron imbalance such as malaria.

We introduce here a fluorescent derivative of desferrioxamine B (DFO) that retains the high affinity of the parent compound and displays a powerful inhibition of intraerythrocytic Plasmodium falciparum growth. NBD-DFO was synthesized by coupling 7-Nitrobenz-2-oxa-1,3-diazole (NBD) to the terminal amino group of DFO. The NBD group at this position renders the DFO molecule more lipophilic and imparts to it fluorescent properties. The novel NBD-DFO probe displays a unique combination of chemical and biological properties, such as 1) improved and selective permeation properties across membranes of P. falciparum-infected erythrocytes, 2) improved efficacy as an inhibitor of intraerythrocytic P. falciparum growth (including multidrug-resistant strains), 3) demonstrable Fe3+ scavenging within parasitized red cells, and 4) usefulness as a sensitive and versatile analytical tool for quantitative assessment of Fe3+ and for following iron-scavenging processes, because the fluorescence of NBD-DFO is demonstrably quenched upon complexation with Fe3+.

We describe here a family of biomimetic iron carriers that display high binding efficiency for ferric ions and favorable permeation properties across erythrocytic membranes. These carriers inhibit in vitro growth of Plasmodium falciparum by scavenging intracellular iron. The chemical features were realized by reproducing the iron-binding cavities of natural iron carriers (siderophores) and by systematic substitutions of their hydrophilic envelopes for more hydrophobic ones. In contrast to natural carriers, which participate in receptor-mediated iron uptake in cells and act as growth promoters, our synthetic carriers were designed to penetrate cellular membranes by diffusion, scavenge intracellular iron, and thereby act as growth inhibitors. Based on these properties we designate the compounds reversed siderophores and refer to the specific analogs of the natural ferrichrome as synthetic ferrichromes. The antimalarial activity of the synthetic ferrichromes correlated with their lipophilicity, and this antimalarial activity was averted when the chelators were applied as iron (III) complexes. The sites of synthetic ferrichrome action reside in the intraerythrocytic parasite and not in serum or on normal erythrocyte components. The agents were effective against all stages of parasite growth and against a variety of multidrug-resistant strains of P. falciparum. The most potent agent of this synthetic ferrichrome series, SF1-ileu, was not toxic to mammalian cells in culture and was 15-fold more potent and 20-fold faster acting than desferrioxamine. Taken in toto, these agents constitute a series of promising candidates for future use in malaria chemotherapy.