A novel smartphone app for blood pressure measurement: a proof-of-concept study against an arterial catheter.

Smartphones may provide a highly available access to simplified hypertension screening in environments with limited health care resources. Most studies involving smartphone blood pressure (BP) apps have focused on validation in static conditions without taking into account intraindividual BP variations. We report here the first experimental evidence of smartphone-derived BP estimation compared to an arterial catheter in a highly dynamic context such as induction of general anesthesia. We tested a smartphone app (OptiBP) on 121 patients requiring general anesthesia and invasive BP monitoring. For each patient, ten 1-min segments aligned in time with ten smartphone recordings were extracted from the continuous invasive BP. A total of 1152 recordings from 119 patients were analyzed. After exclusion of 2 subjects and rejection of 565 recordings due to BP estimation not generated by the app, we retained 565 recordings from 109 patients (acceptance rate 51.1%). Concordance rate (CR) and angular CR demonstrated values of more than 90% for systolic (SBP), diastolic (DBP) and mean (MBP) BP. Error grid analysis showed that 98% of measurement pairs were in no- or low-risk zones for SBP and MBP, of which more than 89% in the no-risk zone. Evaluation of accuracy and precision [bias ± standard deviation (95% limits of agreement)] between the app and the invasive BP was 0.0 ± 7.5 mmHg [- 14.9, 14.8], 0.1 ± 2.9 mmHg [- 5.5, 5.7], and 0.1 ± 4.2 mmHg [- 8.3, 8.4] for SBP, DBP and MBP respectively. To the best of our knowledge, this is the first time a smartphone app was compared to an invasive BP reference. Its trending ability was investigated in highly dynamic conditions, demonstrating high concordance and accuracy. Our study could lead the way for mobile devices to leverage the measurement of BP and management of hypertension.

Pathogens and autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a severe form of hepatitis resulting in the autoimmune-mediated destruction of the liver parenchyma. Whereas many of the immunopathogenic events have been elucidated and some of the drivers of the disease have been identified, little is known about the aetiology of the disease. There are certain risk factors, such as particular human leucocyte antigen (HLA) haplotypes, that enhance the susceptibility for AIH or influence the severity of the disease. However, as for many other autoimmune diseases, the mere presence of such risk factors does not warrant the occurrence of the disease. Not all individuals carrying risk factors develop AIH, and not all patients with AIH are carriers of high-risk alleles. Thus, additional environmental factors need to be considered as triggers for AIH. Environmental factors include diet, sunlight exposure, stress, medication and hygiene, as well as pathogen infections and vaccinations. This review discusses if pathogens should be considered as triggers for the initiation and/or propagation of AIH.

Influence of the vitamin D plasma level and vitamin D-related genetic polymorphisms on the immune status of patients with type 1 diabetes: a pilot study.

Vitamin D (VD) has been implicated in type 1 diabetes (T1D) by genetic and epidemiological studies. Individuals living in regions with low sunlight exposure have an increased T1D risk and VD supplementation reduced the risk in human individuals and mouse models. One possibility of how VD influences the pathogenesis of T1D is its immunomodulatory effect on dendritic cells (DC), which then preferentially activate regulatory T cells (T(regs) ). In the present pilot study, we collected blood samples from a small cohort of patients with T1D at baseline and months 6 and 12. VD-deficient patients were advised to supplement with 1000 IU/day VD. We found a considerable variation in the VD plasma level at baseline and follow-up. However, with higher VD plasma levels, a lower frequency of interleukin (IL)-4-producing CD8 T cells was observed. We further performed a comprehensive genotyping of 13 VD-related polymorphisms and found an association between VD plasma level and the genotype of the VD binding protein (DBP). The frequency of DC and T cell subsets was variable in patients of all subgroups and in individual patients over time. Nevertheless, we found some significant associations, including the 1,25-dihydroxyvitamin D(3) hydroxylase (CYP27B1) genotype with the frequency of DC subtypes. In summary, our preliminary results indicate only a limited influence of the VD plasma level on the immune balance in patients with T1D. Nevertheless, our pilot study provides a basis for a follow-up study with a larger cohort of patients.

Small molecule CXCR3 antagonist NIBR2130 has only a limited impact on type 1 diabetes in a virus-induced mouse model.

CXCL10 is one of the key chemokines involved in trafficking of autoaggressive T cells to the islets of Langerhans during the autoimmune destruction of beta cells in type 1 diabetes (T1D). Blockade of CXCL10 or genetic deletion of its receptor CXCR3 results in a reduction of T1D in animal models. As an alternative to the use of neutralizing monoclonal antibodies to CXCL10 or CXCR3 we evaluated the small molecule CXCR3 antagonist NIBR2130 in a virus-induced mouse model for T1D. We found that the overall frequency of T1D was not reduced in mice administered with NIBR2130. An initial slight delay of diabetes onset was not stable over time, because the mice turned diabetic upon removal of the antagonist. Accordingly, no significant differences were found in the islet infiltration rate and the frequency and activity of islet antigen-specific T cells between protected mice administered with NIBR2130 and control mice. Our data indicate that in contrast to direct inhibition of CXCL10, blockade of CXCR3 with the small molecule antagonist NIBR2130 has no impact on trafficking and/or activation of autoaggressive T cells and is not sufficient to prevent T1D.

Virus-induced autoimmune diabetes: most beta-cells die through inflammatory cytokines and not perforin from autoreactive (anti-viral) cytotoxic T-lymphocytes.

Autoimmune diabetes is caused by selective loss of insulin-producing pancreatic beta-cells. The main factors directly implicated in beta-cell death are autoreactive, cytotoxic (islet-antigen specific) T-lymphocytes (CTL), and inflammatory cytokines. In this study, we have used an antigen-specific model of virally induced autoimmune diabetes to demonstrate that even high numbers of autoreactive CTL are unable to lyse beta-cells by perforin unless major histocompatibility complex class I is upregulated on islets. This requires the presence of inflammatory cytokines induced by viral infection of the exocrine pancreas but not of the beta-cells. Unexpectedly, we found that the resulting perforin-mediated killing of beta-cells by autoreactive CTL is not sufficient to lead to clinically overt diabetes in vivo, and it is not an absolute prerequisite for the development of insulitis, as shown by studies in perforin-deficient transgenic mice. In turn, destruction of beta-cells also requires a direct effect of gamma-interferon (IFN-gamma), which is likely to be in synergy with other cytokines, as shown in double transgenic mice that express a mutated IFN-gamma receptor on their beta-cells in addition to the viral (target) antigen and do not develop diabetes. Thus, destruction of most beta-cells occurs as cytokine-mediated death and requires IFN-gama in addition to perforin. Understanding these kinetics could be of high conceptual importance for the design of suitable interventions in prediabetic individuals at risk to develop type 1 diabetes.

Mechanisms of halothane toxicity: novel insights.

Exposure of individuals to halothane causes, in 20% of patients, a mild form of hepatotoxicity. In contrast, a very small subset of individuals only develops halothane hepatitis, which is thought to have an immunological basis. Sera of halothane hepatitis patients contain antibodies directed against some discrete liver trifluoroacetyl (TFA)-protein adducts, which arise upon oxidative biotransformation of halothane and include protein disulfide isomerase, microsomal carboxylesterase, calreticulin, ERp72, GRP 78 and ERp99. No immune response occurs in the majority of human individuals, although evidence suggests that TFA-protein adducts arise in all halothane-exposed individuals. The lack of immunological responsiveness of individuals might be due to tolerance, induced by a presumed repertoire of self-peptides that molecularly mimic TFA-protein adducts. Thus, constitutively expressed proteins of 52 and 64 kDa have been identified that confer molecular mimicry of TFA-protein adducts. The 64 kDa protein corresponds to the E2 subunit of the mitochondrial pyruvate dehydrogenase complex. Lipoic acid, the prosthetic group of the E2 subunit, is involved in the molecular mimicry process. A fraction of halothane hepatitis patients exhibit irregularities in the expression levels of the 52 kDa protein and the E2 subunit protein. Molecular mimicry of TFA-protein adducts by the 52 kDa protein and the E2 subunit protein might play a role in the susceptibility of individuals to development of halothane hepatitis.

Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites.

Recombinant rat liver GTP cyclohydrolase I has been prepared by heterologous gene expression in Escherichia coli and characterized by biochemical and biophysical methods. Correlation averaged electron micrograph images of preferentially oriented enzyme particles revealed a fivefold rotational symmetry of the doughnut-shaped views with an average particle diameter of 10 nm. Analytical ultracentrifugation and quantitative scanning transmission electron microscopy yielded average molecular masses of 270 kDa and 275 kDa, respectively. Like the Escherichia coli homolog, these findings suggest that the active enzyme forms a homodecameric protein complex consisting of two fivefold symmetric pentameric rings associated face-to-face. Examination of the amino acid sequence combined with calcium-binding experiments and mutational analysis revealed a high-affinity, EF-hand-like calcium-binding loop motif in eukaryotic enzyme species, which is absent in bacteria. Intrinsic fluorescence measurements yielded an approximate dissociation constant of 10 nM for calcium and no significant binding of magnesium. Interestingly, a loss of calcium-binding capacity observed for two rationally designed mutations within the presumed calcium-binding loop of the rat GTP cyclohydrolase I yielded a 45% decrease in enzyme activity. This finding suggests that failure of calcium binding may be the consequence of a mutation recently identified in the causative GTP cyclohydrolase I gene of patients suffering from dopa responsive dystonia.

Immune response to a recombinant human TNFR55-IgG1 fusion protein: auto-antibodies in rheumatoid arthritis (RA) and multiple sclerosis (MS) patients have neither neutralizing nor agonist activities.

A substantial number of patients enrolled in clinical studies of TNFR55-IgG1 in TNF-neutralizing treatment of rheumatoid arthritis and multiple sclerosis developed antibodies to the recombinant human protein. To enable more detailed investigation subgroups of patients donated small blood samples. TNFR55-IgG1 reactive antibodies were affinity purified from plasma; IgM and IgG class antibodies reactive with TNFR55-IgG1 were found which varied considerably in titer and kinetics of appearance among individual patients. The affinity purified antibody fractions included specificities to the receptor moiety of TNFR55-IgG1, but also rheumatoid factor and other pre-existing antibodies directed to the IgG1 moiety. The antibodies bound to Fc receptors, but not detectably to TNFR55 at the human cell surface. No agonistic nor neutralizing activities of these antibodies were detected. Major linear epitopes clustered in the TNFR55 sequence in close proximity to the IgG1 fusion site. The relative content of antibodies to linear and conformational epitopes was highly variable among patients. Route and frequency of administration rather than underlying disease appeared to influence the major linear B cell epitopes selected.

Molecular mimicry in halothane hepatitis: biochemical and structural characterization of lipoylated autoantigens.

Exposure of human individuals to halothane causes, in about 20% of all cases, a mild transient form of hepatotoxicity. A small subset of exposed individuals, however, develops a potentially severe and life-threatening form of hepatic damage, coined halothane hepatitis. Halothane hepatitis is thought to have an immunological basis. Sera of afflicted individuals contain a wide variety of autoantibodies against hepatic proteins, in both trifluoroacetylated form (CF3CO-proteins) and, at least in part, in native form. CF3CO-proteins are elicited in the course of oxidative biotransformation of halothane, and include the trifluoroacetylated forms of protein disulfide isomerase, microsomal carboxylesterase, calreticulin, ERp72, GRP 78, and ERp99. Current evidence suggests that CF3CO-proteins arise in all halothane-exposed individuals; however, the vast majority of individuals appear to immunochemically tolerate CF3CO-proteins. The lack of immunological responsiveness of these individuals towards CF3CO-proteins might be due to tolerance, induced through the occurrence of structures in the repertoire of self-determinants, which immunochemically and structurally mimic CF3CO-proteins very closely. In fact, lipoic acid, the prosthetic group of the constitutively expressed E2 subunits of the family of mammalian 2-oxoacid dehydrogenase complexes and of protein X, was shown by immunochemical and molecular modelling analysis to be a perfect structural mimic of N6-trifluoroacetyl-L-lysine (CF3 CO-Lys), the major haptenic group of CF3CO-proteins. As a consequence of molecular mimicry, autoantibodies in patients' sera not only recognize CF3CO-proteins, but also the E2 subunit proteins of the 2-oxoacid dehydrogenase complexes and protein X, as autoantigens associated with halothane hepatitis. Furthermore, a fraction of patients with halothane hepatitis exhibit irregularities in the hepatic expression levels of these native, not trifluoroacetylated autoantigens. Collectively, these data suggest that molecular mimicry of CF3CO-Lys by lipoic acid, or the impairment thereof, might play a role in the susceptibility of individuals for the development of halothane hepatitis.

Single transplanted kidneys from a 90-year-old deceased donor perform acceptably at 1 year.

Most centers are reluctant to accept expanded criteria donors above 70 to 75 years of age. We accepted kidneys from a 90-year-old male and report the 1-year outcome. The kidneys were used as single transplants and both had immediate graft function. Recipient A was a 71-year-old male, with cold ischemia time of 4 hours 49 minutes. One rejection was successfully treated with intravenous methylprednisolone. At 1 year, serum creatinine was 146 µmol/L with estimated glomerular filtration rate (eGFR) 41 mL/min. Recipient B was a 79-year-old male with known panel-reactive antibody positivity prior to transplantation. Cold ischemia time was 10 hours 4 minutes. He experienced no rejections. At 1 year serum-creatinine was 99 µmol/L with eGFR 63 mL/min. Both recipients performed a surveillance biopsy at 1 year with identical findings: interstitial fibrosis and tubular atrophy grade 1 with moderate to severe arteriolosclerosis. We conclude that both kidneys performed acceptably 1 year after engraftment. The use of old kidneys in old recipients gives them a properly functioning kidney and improves quality of life. Longer observation is needed.

Distribution of thioredoxins in Cyanobacteria.

The presence of thioredoxin was demonstrated in 20 strains of cyanobacteria as well as in one phototrophic bacterium Rhodopseudomonas sulfidophila and in Thiobacillus denitrificans. Thioredoxin activity was not found in Cyanophora paradoxa and in Porphyridium cruentum using the thioredoxin-dependent PAPS-sulfotransferase activity from Synechococcus 6301 as assay system.

A factor-dependent sulfotransferase specific for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in the Cyanobacterium Synechococcus 6301.

A sulfotransferase isolated from the Cyanobacterium Synechococcus 6301 was found to be specific for 3'-phosphoadenosine-5'-phosphosulfate (PAPS). The molecular weight of this transferase has been estimated on a Sephadex-G-100 column to be about 58,000. The K m for PAPS was determined to be 20 µM. The pH optimum was 8.0. The thiol dithioerythritol was needed for activity; other thiols such as glutathione, cysteine, or mercaptoethanol did not catalyze this reaction. The transferase, however, could not react directly with the thiol. A heat-stable factor was needed in this reaction. This factor was purified by conventional techniques and its molecular weight was determined on a Sephadex-G-50 column to be about 11,500. The factor showed normal Michaelis-Menten behavior toward the PAPS-sulfotransferase. It has been identified as thioredoxin. The tranferase was inhibited by 3'-5'-ADP and 2'-5'-ADP; all other adenine-containing nucleotides such as 2'-AMP, 3'-AMP, 5'-AMP, ADP, and c-AMP did not influence this reaction.

Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.

A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross-reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52-kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon-acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody-exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS)

Halothane metabolism: Kupffer cells carry and partially process trifluoroacetylated protein adducts.

Kupffer cells, prepared 18 h after pretreatment of rats with a single dose of halothane, did carry TFA-adducts which were recognized on Western blots by a anti-TFA-antibody. Based on apparent molecular weight, the pattern of the major TFA-adducts within Kupffer cells was similar to that observed in hepatocytes. When kept in primary culture, Kupffer cells processed TFA-adducts of apparent molecular weight of 220 kD, 110 kD and 74 kD within 24 or 48 h; in contrast, other TFA-adducts were persistent for at least 48 h in Kupffer cells. The data suggest a role for Kupffer cells in processing of chemically altered proteins in the liver.

Halothane metabolism: the dihydrolipoamide acetyltransferase subunit of the pyruvate dehydrogenase complex molecularly mimics trifluoroacetyl-protein adducts.

Monospecific antibodies (anti-CF3CO antibodies), directed against trifluoroacetyl-protein adducts (CF3CO-protein adducts) that are elicited in tissues of experimental animals and humans upon exposure to the anesthetic agent halothane, recognize cross-reactive proteins of 64 and 52 kDa in several tissues of rats and the liver of humans not previously exposed to the drug. These cross-reactive proteins mimic CF3CO-protein adducts. Here, by the use of the anti-CF3CO antibody as an immunoaffinity matrix, the protein of 64 kDa was purified from rat heart microsomal fractions. The amino acid sequence of six internal tryptic peptides exhibited 100% identity with the corresponding deduced amino acid sequences of the dihydrolipoamide acetyltransferase component (E2 subunit) of the rat liver pyruvate dehydrogenase (PDH) complex, as encoded by the cDNA clone pRMIT [Gershwin, M. E., Mackay, I. R., Sturgess, A., & Coppel, R. L. (1987) J. Immunol. 138, 3525-3531]. Lipoic acid, the prosthetic group of the E2 subunit of the PDH complex, exhibited immunochemical properties very similar to those of the hapten-derivative N6-trifluoroacetyl-L-lysine (CF3CO-Lys). On immunoblots, free lipoic acid inhibited the recognition of the E2 subunit, of the not yet identified protein of 52 kDa, and of the bulk of CF3CO-protein adducts by anti-CF3CO antibody with half-maximal inhibitory constants of 0.05, 10.0, and 8.5 mM, respectively. Lipoic acid also abolished the precipitation of the native E2 subunit by anti-CF3CO antibody from solubilized rat heart mitochondrial fractions. These data suggest that lipoic acid is involved in the molecular mimicry of CF3CO-protein adduct-related epitopes by the E2 subunit of the PDH complex.

The kidney as a novel target tissue for protein adduct formation associated with metabolism of halothane and the candidate chlorofluorocarbon replacement 2,2-dichloro-1,1,1-trifluoroethane.

Hydrochlorofluorocarbons (HCFCs) have been identified as chemical replacements of the widely used chlorofluorocarbons (CFCs) that are implicated in stratospheric ozone depletion. Many HCFCs are structural analogues of the anesthetic agent halothane and may follow a common pathway of biotransformation and formation of adducts to protein-centered and other cellular nucleophiles. Exposure of rats to a single dose of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or of the candidate CFC substitute HCFC 123 (2,2-dichloro-1,1,1-trifluoroethane) led to the formation of trifluoroacetylated protein adducts (CF3CO-proteins) not only in the liver, but also in the kidney as a novel target tissue for protein trifluoroacetylation. CF3CO-proteins in the kidney amounted to about 5% of those formed in the liver of the same animal. The amount of CF3CO-proteins formed within the kidney was roughly reflected by the capacity of metabolism of halothane or HCFC 123 by rat kidney microsomes in vitro which amounted to about 10% of that observed with liver microsomes. By immunohistochemistry, CF3CO-proteins in the kidney were mainly localized in the tubular segments of the cortex. In the liver, the density of CF3CO-proteins decreased from the central vein towards the portal triad. In vitro incubation of rat liver microsomes with halothane or HCFC 123 resulted in extensive formation of CF3CO-proteins and reproduced faithfully the pattern of liver CF3CO-proteins obtained in vivo. CF3CO-proteins generated in vitro were immunochemically not discernible from those generated in vivo. Glutathione (5 mM) and cysteine (5 mM) virtually abolished CF3CO-protein formation; the release of Br- from halothane and Cl- from HCFC 123 was reduced to much lesser a degree. S-Methyl-glutathione, N-acetyl-cysteine, methionine, and N-acetyl-methionine only slightly affected the formation of CF3CO-proteins or metabolism of either substrate. The data suggest that metabolism and concomitant CF3CO-protein formation of halothane or of candidate CFC replacements like HCFC 123 is not restricted to the liver but also takes place in the kidney. Furthermore, an in vitro system for CF3CO-protein formation has been developed and used to show that protein-centered and glutathione-centered nucleophilic sites compete for intermediates of metabolism of halothane or of HCFC 123.