Autoantibodies to heat shock protein 90 in the human natural antibody repertoire

The present study demonstrates the presence of natural autoantibodies of the IgG isotype directed against heat shock protein 90 (HSP90). The binding properties of affinity-purified anti-HSP antibodies were compared with those of natural antibodies specific for other self antigens, including anti-thyroglobulin and anti-myoglobin autoantibodies, by using semiquantitative immunoblotting, with solubilized proteins from normal liver tissue as antigens, and cross-blot analysis using purified self proteins. Affinity-purified anti-HSP90 antibodies were polyreactive and the non-HSP90-specific fraction of normal IgG was depleted in its natural autoantibody content. We further observed that self antigens including HSP, myosin, tubulin and aldolase with highly conserved structures show similar patterns of binding with natural antibodies, and form a well-defined cluster as demonstrated by cluster analysis of immunoreactivity data, whereas the less-conserved self and non-self antigens remained unclustered. The results favor the hypothesis that HSP90 belongs to a subset of highly conserved and immunodominant self antigens that are the primary target for natural autoantibodies in normal human IgG.     

Delivery of macromolecules across oral mucosa from polymeric hydrogels is enhanced by electrophoresis (iontophoresis)

Objective

To develop polymeric hydrogel delivery systems for iontophorseis transfer of large molecules across buccal (porcine) mucosa.

Methods

Three hydrogels (PVA, HPMC and PVA/HPMC) were prepared as stable gels (7 mm diameter/1.5 mm thick). Quantitative (8 and 36 h) assessment of porcine buccal mucosa and the three hydrogel delivery systems, using a diffusion cell in vitro model, was carried out by UV/vis spectroscopy with three model agents (3 and 10 kDa dextrans and 12 kDa parvalbumin). Passive and iontophoresis parameters were obtained. Experimental and theoretical data were compared.

Results

Iontophoresis (30 min, 1–8 h) significantly enhanced the delivery of all model agents across four single systems (hydrogels and buccal mucosa) and three sandwich systems (hydrogels on top of buccal mucosa), as confirmed by time lag factor/enhancement ratio (TLF/ER) data. The diffusion coefficients of model agents across buccal mucosa (×10⁻¹³ m² s⁻¹) were ∼100 times lower than across single hydrogels (2.97–4.80 × 10⁻¹¹ m² s⁻¹). Solubility values of all agents across hydrogels were similar, but lower across buccal mucosa. Permeability of parvalbumin was highest across PVA, and for both dextrans across PVA/HPMC. In sandwich systems TLFs were similar for all hydrogels, but significantly lower, and ERs significantly higher, than tissue alone. Experimental and theoretical TLF data were in reasonable agreement.

Significance

The in vitro data show that iontophoresis enhanced the delivery of large molecules across polymeric hydrogel systems and buccal mucosa. This creates the opportunity of new approaches to drug delivery and opens pathways to further research for delivering therapeutic agents topically and systemically.     

Metals and kidney markers in adult offspring of endemic nephropathy patients and controls: a two-year follow-up study

Background  

The etiology of Balkan Endemic Nephropathy, (BEN), a tubulointerstitial kidney disease, is unknown. Although this disease is endemic in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, similar manifestations are reported to occur in other regions, for instance Tunisia and Sri Lanka. A number of explanations have been stated including lignites, aristolochic acid, ochratoxin A, metals, and metalloids. Etiologic claims are often based on one or a few studies without sound scientific evidence. In this systematic study, we tested whether exposures to metals (cadmium and lead) and metalloids (arsenic and selenium) are related to Balkan Endemic Nephropathy.     

New insight into the autoimmunogenicity of the complement protein C1q

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups—one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies.We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups—ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.     

Developing High-specificity Anti-hypertensive Alerts by Therapeutic State Analysis of Electronic Prescribing Records

Objective

This paper presents a model for analysis of chronic disease prescribing action over time in terms of transitions in status of therapy as indicated in electronic prescribing records. The quality of alerts derived from these therapeutic state transitions is assessed in the context of antihypertensive prescribing.

Design

A set of alert criteria is developed based on analysis of state-transition in past antihypertensive prescribing of a rural Australian General Practice. Thirty active patients coded as hypertensive with alerts on six months of previously un-reviewed prescribing, and 30 hypertensive patients without alerts, are randomly sampled and independently reviewed by the practice’s two main general practice physicians (GPs), each GP reviewing 20 alert and 20 non-alert cases (providing 10 alert and 10 non-alert cases for agreement assessment).

Measurements

GPs provide blind assessment of quality of hypertension management and retrospective assessment of alert relevance.

Results

Alerts were found on 66 of 611 cases with coded hypertension with 37 alerts on the 30 sampled alert cases. GPs assessed alerting sensitivity as 74% (CI 52% - 89%) and specificity as 61% (CI 45% - 74%) for the sample, which is estimated as 26% sensitivity and 93% specificity for the antihypertensive population. Agreement between the GPs on assessment of alert relevance was fair (kappa = 0.37).

Conclusions

Data-driven development of alerts from electronic prescribing records using analysis of therapeutic state transition shows promise for derivation of high-specificity alerts to improve the quality of chronic disease management activities.     

Cytotoxic and genotoxic effects of paraquat in Hordeum vulgare and human lymphocytes in vitro

Two phylogenetically distant types of test‐systems—root tip meristems of barley (Hordeum vulgare) and human lymphocytes in vitro were used to detect genotoxicity and cytotoxicity induced by the herbicide paraquat (PQ) in the concentration range (10^?6 to 5 × 10^?4 mol/l). As an endpoint for cytotoxicity the mitotic index (MI) was evaluated. The frequency of chromosome aberrations (CA) and the frequency of micronuclei (MN) were used as endpoints for genotoxicity. A dose‐dependent increase of CA and MN was observed in both test systems, although the values for PQ‐induced MN were somewhat lower. The increase of the genotoxic effect corresponds to a decrease of mitotic activity. The structurally reconstructed barley karyotype MK14/2034 allowed the allocation of the PQ‐specific features of aberration distribution patterns and gave information about which chromosome segments in different chromosomal positions were involved in induced aberrations. Paraquat produced preferably isochromatid breaks and “aberration hot spots” in a restricted number of heterochromatin‐containing segments. The comparative analysis of susceptibility in the used test‐systems to PQ with respect to its cytotoxic and clastogenic effect showed that the human lymphocytes were more sensitive than Hordeum vulgare. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

CD45 regulates apoptosis in peripheral T lymphocytes

Programmed cell death (apoptosis) is a key mechanism for regulating lymphocyte numbers. Murine lymph node lymphocytes cultured in vitro without added stimuli show significant levels of apoptosis over 24 h, detectable by staining with Annexin V. CD4 and CD8 T lymphocytes from transgenic (Tg) mice expressing single CD45RABC or CD45RO isoforms show increased apoptosis and the extent of apoptosis is inversely correlated with the level of CD45 expression. CD45 Tg cells exhibit phosphatidyl serine translocation and DNA oligonucleosome formation, and can be partially rescued from apoptosis by culture in caspase inhibitors or common gamma-chain-binding cytokines. We conclude that CD45 is an important regulator of spontaneous apoptosis in T lymphocytes and this mechanism may contribute to the disease associations reported for individuals expressing CD45 variant alleles.