Diminished salivary epidermal growth factor secretion: a link between Sjögren's syndrome and autoimmune gastritis?

OBJECTIVES: Healthy human labial salivary glands produce epidermal growth factor (EGF). In Sjögren's syndrome (SS), EGF staining is diminished. SS is also associated with chronic autoimmune corpus gastritis. We therefore hypothesized that EGF secretion would be diminished in SS and that this could affect gastric target cells. METHODS: Salivary EGF secretion in SS was compared to that in healthy controls using an enzyme-linked immunosorbent assay (ELISA). EGF receptor (EGFR) immunoreactive cells in the gastric corpus of healthy human subjects were analysed using immunostaining. RESULTS: Salivary secretion of EGF was diminished in SS patients (232.4, range 52.6-618.4, vs. 756.6, range 105.3-1631.6 pg/min, p = 0.002). Proton-pump positive parietal cells were mostly EGFR immunoreactive whereas very few pepsinogen I (PGI)-positive cells were EGFR positive. CONCLUSIONS: As EGF is relatively acid resistant, salivary gland-derived EGF might participate in an exo/endocrine mode of parietal cell maintenance in the gastric corpus. Deficiency of salivary gland-derived EGF in SS patients may cause impairment of gastric parietal cells resulting in exposure of immunogenic cryptic antigens and loss of immunological self-tolerance.

Osteoarthritic cartilage lesions in the bovine patellar groove: a macroscopic, histological and immunohistological analysis.

A high percentage of osteoarthritis (OA)-like patellar groove lesions in the stifle joint in calcium-deficient bulls has been recently reported. The prevalence of these lesions in bulls deficient in or supplemented with calcium was compared to findings in culled and healthy bulls to determine whether they represent normal anatomical variations, developmental anomalies or OA. It was hypothesized that the patellar groove lesions may represent OA. Distal cartilage samples from 160 femurs were analysed using a macroscopic Société Française d'Arthroscopie (SFA) OA grading system. Samples representing different SFA grades were subjected to Osteoarthritis Research Society International (OARSI) histological and high-mobility group box 1 (HMGB1) immunohistological OA grading. For a qualitative analysis three OA samples were immunostained for interleukin (IL)-1ß, matrix metalloproteinase (MMP)-13 and collagenase-produced COL2-3/4M neoepitopes. Patellar groove lesions were found in 48% of the femurs and were highest in calcium-deficient animals (71%, P<0.001). All three different grading systems disclosed OA in culled bulls, but no focal areas of cartilage necrosis. OARSI and HMGB1 grades were fairly concordant (Spearman's ρ=0.95, P<0.001; Cohen's κ=0.23, P<0.005), both with a slight disparity with the SFA grade (ρ=0.80 and 0.87, P<0.01; κ=0.36 and 0.46, P<0.001). IL-1ß, MMP-13 and COL2-3/4M staining patterns were compatible with OA. The study showed that patellar groove lesions are common in bulls. In all SFA, OARSI and HMGB1 graded samples the lesions clearly demonstrated OA and showed OA-typical pathophysiology. Arthroscopic SFA grading showed similar changes in calcium-deficient and calcium-supplemented bulls, but in the absence of a time course study and histological data the primary nature of these lesions could not be established with certainty.

Bovine chronic osteoarthritis causes minimal change in synovial fluid.

Chronic osteoarthritis (OA) is a degenerative disease of the articular cartilage. DNA-binding high mobility group protein B1 (HMGB1) is released on cellular death/activation and acts as an endogenous danger signal and a proinflammatory cytokine. Matrix metalloproteinase (MMP)-2 and in MMP-9 are induced to mediate proteolytic degradation/remodelling of joint tissues. Collagen degradation in the bone and synovium leads to release of type I collagen-derived cross-linked carboxy-terminal telopeptide (ICTP). These molecules have been linked to the pathogenesis of OA and could have potential as synovial fluid (SF) biomarkers in OA. Cartilage and SF were obtained from 27 dairy bulls (30-61 months old) and control cartilage from six young healthy dairy bulls. OA lesions were evaluated grossly (five grades), histologically (seven Osteoarthritis Research Society International [ORSI] grades) and immunohistochemically (four HMGB1 grades). The OARSI lesion score was calculated as the product of the OARSI grade and the OARSI score (the total area of the lesions). SF concentrations of HMGB1, MMP-2 and -9 and ICTP were measured by enzyme-linked immunosorbent assay, gelatin zymography and radioimmunoassay, respectively. Seventy-two percent (39/54) of stifle joints and 85% (23/27) of the dairy bulls had at least one gross OA lesion and 94% of the lesions were localized to the distal end of the femur, with the patellar groove and the lateral trochlear ridge being predilection sites. Gross and histological grades correlated with the HMGB1 grade, but SF total cell count, percent neutrophils or the measured biomarkers did not correlate with the tissue lesions, with the exception of ICTP concentration, which correlated with the total joint score. The switch of HMGB1 from DNA-binding nuclear protein to an extracellular alarmin/cytokine correlates with the gross and histological grades of OA tissue lesions. However, the activity and extent of the tissue lesions did not correlate with other SF biomarkers, perhaps because the histological grades represent outcome measures, while SF reflects process parameters. The only exception was ICTP concentration, which reflects enhanced destruction/remodelling.

Failure of oral DHEA treatment to increase local salivary androgen outputs of female patients with Sjögren's syndrome.

OBJECTIVES: Sjögren's syndrome (SS) is a female-dominant autoimmune disease characterized by androgen depletion and defective dehydroepiandrosterone (DHEA) processing enzymatic machinery in the salivary glands. We hypothesized that, because of these local failures, DHEA replacement therapy would be unable to improve the local androgen deficiency in SS salivary glands. METHODS: DHEA-deficient female SS patients (n = 12) were treated with placebo for 4 months followed by DHEA 50 mg q.d. for 4 months. Serum and saliva, collected in the morning before the trial and after both periods, were analysed for pro-hormones, androgens, and androgen metabolite using an enzyme-linked immunosorbent assay (ELISA). RESULTS: DHEA treatment increased serum DHEA-sulfate from 1.3 ± 0.1 to 6.4 ± 1.3 µM (p = 0.005), DHEA from 16.5 ± 2.8 to 34.8 ± 8.2 nM (p = 0.012), androstenedione from 3.1 ± 0.3 to 17.2 ± 1.9 nM (p = 0.002), free testosterone from 2.2 ± 0.1 to 7.7 ± 1.1 pM (p = 0.002), DHT from 275.5 ± 24.4 to 834.6 ± 122.8 pM (p = 0.002) and 3-α-diol-G from 3.8 ± 0.6 to 13.6 ± 2.0 nM (p = 0.001). However, only salivary DHEA and DHT outputs increased significantly and 25% of the patients showed no increases, except for DHEA itself. Outputs of active androgens (T, DHT) and 3-α-diol-G metabolite correlated with salivation. CONCLUSIONS: The local androgen deficiency in SS salivary glands is not only caused by low serum DHEA(-S) because restoration of systemic androgen levels by DHEA treatment did not correct local androgen depletion. This could be explained by low or no capacity of DHEA-substituted patients to convert the pro-steroid to active androgen metabolites. Such intracrine failures affect women in particular, who must produce their salivary T and DHT locally from DHEA.

Cost-effectiveness of infliximab in the treatment of rheumatoid arthritis in clinical practice.

OBJECTIVE: We evaluated the cost-effectiveness of infliximab therapy in Finnish RA patients in a real-life clinical setting and identified factors influencing it, using the national register of biological treatment (ROB-FIN). METHODS: A cost-utility analysis was performed, derived from EQ-5D, and related to HAQ score and disease activity using multiple regression. QALYs were calculated based on these utilities, using patient-level data up to the last control registered. Cost-effectiveness analyses included costs per ACR50 responder, and costs per low DAS28 score (<3.2) achieved, in combination with a clinically significant improvement (>1.2). The costs considered were direct medical costs of infliximab and cost of intravenous infusion. Patient-level costs were calculated based on dose and dosage frequency, and were related to the difference in QALYs resulting from infliximab therapy. RESULTS: The 297 patients had been treated with infliximab for an average of 21 months. The HAQ score and patient's global assessment improved significantly on infliximab therapy. More than two-thirds of the patients achieved a clinically important improvement in HAQ. A QALY gain occurred in 76%. 35% of these had an incremental cost-effectiveness ratio of < or =40,000 Euro/QALY gained, the median cost being 51,884 Euro. The cost per QALY gained was significantly lower for patients achieving an ACR50 response at 3, 12 and 24 months. CONCLUSION: Treatment with infliximab and aiming at ACR50 response appears cost-effective, remembering the restrictions of an observational study set up. Current Care guidelines, which require sufficient disease control when deciding on continuing biological therapy, get support from these findings.

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane.

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

Functionally important residues in the predicted 3(rd) transmembrane domain of the type IIa sodium-phosphate co-transporter (NaPi-IIa).

The type IIa Na(+)/P(i), cotransporter (NaPi-IIa) mediates electrogenic transport of three Na(+) and one divalent P(i) ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P(i) transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P(i) transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P(i)-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na(+)-dependent P(i) transport. Since electrogenic NaPi-II transports 3 Na(+)/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na(+) binding sites in NaPi-IIa.

Cloning and functional expression of an MIP (AQP0) homolog from killifish (Fundulus heteroclitus) lens.

The major intrinsic protein (MIP) of lens fiber cells is a member of the aquaporin (AQP) water channel family. The protein is expressed at very high levels in lens fiber cells, but its physiological function is unclear. By homology to known AQPs, we have cloned a full-length cDNA encoding an MIP from the lens of killifish (Fundulus heteroclitus). The predicted protein (263 amino acids; GenBank accession no. AF191906) shows 77% identity to amphibian MIPs, 70% identity to mammalian MIPs, and 46% identity to mammalian AQP1. Expression of MIPfun in Xenopus laevis oocytes causes an approximately 40-fold increase in oocyte water permeability. This stimulation is comparable to that seen with AQP1 and substantially larger than that seen with other MIPs. The mercurials HgCl(2) and p-chloromercuribenzenesulfonate inhibit the water permeability of MIPfun by approximately 25%. MIPfun is not permeable to glycerol, urea, or formic acid but is weakly permeable to CO(2).

Beta-adrenergic stimulation of volume-sensitive chloride transport in lamprey erythrocytes.

We measured the effects of a beta-adrenergic agonist, isoproterenol, on chloride transport and volume regulation of lamprey (Lampetra fluviatilis) erythrocytes in isotonic (288 mosm L(-1)) and hypotonic (192 mosm L(-1)) medium. Isoproterenol at a high concentration (10(-5) M) did not influence chloride transport in isotonic medium but markedly increased chloride fluxes in hypotonic conditions: unidirectional flux increased from 100 mmol kg dcw(-1) h(-1) in the absence to 350 mmol kg dcw(-1) h(-1) (dcw=dry cell weight) in the presence of isoproterenol. Simultaneously, the half-time for volume recovery decreased from 27 to 9 min. Isoproterenol caused an increase in cellular cyclic AMP (cAMP) concentration. The stimulation of chloride transport in hypotonic conditions could be induced by application of the permeable cAMP analogue, 8-bromo-cyclic AMP, suggesting that the effect of beta-adrenergic stimulation on chloride transport occurs downstream of cAMP production. As isoproterenol did not affect unidirectional rubidium fluxes in hypotonic conditions, the transport pathway influenced by beta-adrenergic stimulation is most likely the swelling-activated chloride channel. Because the beta-adrenergic agonist only influenced the transport in hypotonic conditions despite the fact that cAMP concentration also increased in isotonic conditions, the activation may involve a volume-dependent conformational change in the chloride channel.

Copper effects on ion transport across lamprey erythrocyte membrane: Cl(-)/OH(-) exchange induced by cuprous ions.

We studied the effects of prelytic copper concentrations on cell volume, intracellular pH, and ion transport in lamprey erythrocytes. Ion fluxes and pH were measured by radioactive tracer technique, patch clamp, and flame photometry. Prelytic CuSO(4) concentration of 100 microM caused anion-dependent intracellular acidification and increase in Cl(-) influx after 2 min lag-phase. In the presence of ascorbate copper effect was amplified and lag-phase was skipped. Pretreatment of the cells with N-phenyl maleimide abolished copper-induced changes completely. Copper treatment caused an increase in Na(+) fluxes in both directions and a net Na(+) uptake. Copper-induced Na(+) transport was partially amiloride(MIA)-sensitive representing Na(+)/H(+) exchange. The nature of the amiloride-insensitive fraction of copper-activated Na(+) influx remains unknown. Cell swelling after 15 min of copper exposure induced regulatory volume decrease response involving KCl extrusion via K(+) and Cl(-) volume-sensitive channels. We suggest that the effects of copper on ion transport fit the following sequence of events: (i) cupric ions are reduced to cuprous state on the membrane surface, (ii) electroneutral pairs CuCl and CuOH mediate chloride/hydroxyl exchange, as shown before for trialkyltin, dissipating transmembrane pH gradient, and (iii) changes in intracellular pH result in the activation of the Na(+)/H(+) exchange and consecutive volume changes cause the RVD response.

Two distinct K+ channels in lamprey (Lampetra fluviatilis) erythrocyte membrane characterized by single channel patch clamp.

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K(+)-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 +/- 3 pS in isotonic (132 mM K+) solutions and 24 +/- 2 pS in hypotonic (63 mM K+) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K+ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg2+ and Ca2+ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca2+ ions on the cytosolic side for activity.

Regulation of ion transport across lamprey (Lampetra fluviatilis) erythrocyte membrane by oxygen tension.

We have measured the effects of oxygen tension on the transport of Na+, K+ and Cl- across the erythrocyte membrane of the lamprey Lampetra fluviatilis. The transport of each ion was affected by the oxygen tension of the medium. Hypoxic conditions (PO2 2 kPa) caused an increase in the acidification-induced influx of Na+ via Na+/H+ exchange. The influx of K+ was only slightly affected by the oxygenation of the medium. In contrast, the basal K+ efflux, measured using the radioactive isotope 43K, was markedly reduced by decreasing the oxygen tension of the medium, whereas the K+ flux in hypotonic medium was not affected. Only minor effects of hypoxic conditions on the influx of Cl- were observed in either isotonic or hypotonic conditions (there was a tendency for the isotonic influx to increase) or on the efflux in isotonic conditions. However, deoxygenation caused a marked reduction in the Cl- efflux in hypotonic conditions. The results show that oxygen tension has a marked effect on the pH and volume regulatory transport pathways of lamprey erythrocytes. For K+ and Cl-, the regulation appears to be asymmetric, i.e. influx and efflux are affected differently.

Haemoglobin function in intact lamprey erythrocytes: interactions with membrane function in the regulation of gas transport and acid-base balance.

Haemoglobin function within lamprey erythrocytes offers a unique solution to gas transport among vertebrates. Lamprey haemoglobin within intact erythrocytes is in oligomer/monomer equilibrium and has an oxygen affinity similar to that of haemoglobin in other active fishes. The cooperativity of oxygen binding, which is reduced at low pH values, the effect of protons and the effect of the concentration of haemoglobin on its oxygen affinity are all due to dissociation/association reactions of the haemoglobin molecules. The permeability of the lamprey red cell membrane to acid and base equivalents is very low, and plasma bicarbonate cannot therefore be dehydrated to carbon dioxide to any significant extent during the residence time of blood in the gills. This potential limitation on carbon dioxide excretion is overcome, however, by the high intraerythrocytic pH and the marked oxygenation-linked pH changes in the erythrocyte, which are due to the large Haldane effect of the haemoglobin. Owing to the relative impermeability of the erythrocyte membrane to acid equivalents, intraerythrocytic haemoglobin cannot take part in the acid-base buffering of the extracellular compartment. As a consequence, extracellular acid loads cause marked fluctuations in plasma pH.

Regulatory volume decrease in lamprey erythrocytes: mechanisms of K+ and Cl- loss.

The nature of the swelling-activated K+ and Cl- transport pathways of lamprey (Lampetra fluviatilis) erythrocytes was studied. In isosmotic medium, unidirectional K+ and Cl- effluxes appear to be largely mediated by conductive pathways. Unidirectional Cl- efflux increased as a function of a decrease in medium osmolarity. The swelling-activated Cl- transport was inhibited by R(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inde n-5- yl)oxy]acetic acid (DIOA), furosemide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In contrast, moderate cell swelling did not increase unidirectional ouabain-insensitive K+ efflux. However, inhibition of transport by Ba2+ was markedly reduced. This suggests that the Ba(2+)-sensitive pathway that mediated most of the K+ efflux in isosmotic conditions was inhibited by cell swelling and a Ba(2+)-insensitive pathway was activated. DIOA had no effect on K+ efflux in isosmotic or hyposmotic medium. These data and the finding that substitution of NO3- or SCN- for Cl- had only a minor effect on the swelling-induced net extrusion of K+ and water indicate that the pathways for K+ and Cl-, activated by cell swelling, are conductive.

ACTIVATION AND PHYSIOLOGICAL ROLE OF Na+/H+ EXCHANGE IN LAMPREY (LAMPETRA FLUVIATILIS) ERYTHROCYTES

The effects of intracellular acidification, osmotic shrinkage and ss-adrenergic stimulation on sodium transport across the membrane of lamprey (Lampetra fluviatilis) erythrocytes were investigated. Unidirectional ouabain-insensitive sodium flux, measured using radioactive 22Na, was increased markedly by intracellular acidification, to a lesser extent by osmotic shrinkage and only modestly by ss-adrenergic stimulation. Na+/H+ exchange was activated in all of these cases. However, net sodium influx (and cell swelling caused by the influx of osmotically obliged water) was seen only in cells subjected to intracellular acidification. In contrast, practically no changes in red cell pH or in water or ion (Na+, K+ and Cl-) contents were seen after osmotic shrinkage or ss-adrenergic stimulation. Calculations of the [Na+]o/[Na+]i and [H+]o/[H+]i ratios across the erythrocyte membrane suggest that the virtual lack of net sodium movements in osmotically shrunken erythrocytes is due to the absence of a driving force for net transport of these ions via the Na+/H+ exchange pathway. It also appears that, in physiological conditions, the increase in the activity of the Na+/H+ exchanger by ss-adrenergic stimulation is too small to mediate detectable net sodium transport.

Pyrolysis products of PCBs.

Model compound studies which were previously done for impurities and environmental residues of chlorophenols and for wastes of chlorination processes were extended to the impurities and pyrolysis products of polychlorinated biphenyls (PCBs). Model compounds were commercial products or synthesized and their structures proven by spectroscopic methods. These models were used as analytical reference substances in GC/ECD and GC/MS studies of the pyrolyzed PCB samples. In addition to previously known neutral components like polychlorinated dibenzofurans (PCDFs), chlorophenolic substances, especially polychlorophenols (PCPs) and polychlorinated biphenylols (PCB-OHs) were observed as major pyrolysis products of PCBs. Capacitor fires are suggested to produce in many cases chlorophenols which are major toxic hazards to people.

Alkylation products of DNA bases by simple epoxides.

The reaction products of a series of epoxides with deoxyribonucleosides were characterized using ultraviolet, and NMR spectroscopy. The epoxides included structural analogues which are known to differ extensively in their mutagenic potency: propylene oxide, glycidol, epichlorohydrin, trichloropropylene oxide and styrene oxide. Trichloropropylene oxide, epichlorohydrin and glycidol reacted with deoxyguanosine producing a major adduct of 1,7-(or 1,9-)dialkylguanine. All of the epoxides produced a 7-alkylguanine adduct, with the possible exception of styrene oxide. Propylene oxide, glycidol and epichlorohydrin reacted with deoxyadenosine at N-6. Glycidol, trichloropropylene oxide and styrene oxide reacted with deoxycytidine at N-3. It was concluded that the structurally related epoxides tend to react largely in a uniform way with nucleic acid bases. Thus, the reaction rates rather than the major adducts explain the differential mutagenicity of the epoxides.