Peroxiredoxin 2 oxidation reveals hydrogen peroxide generation within erythrocytes during high-dose vitamin C administration.

Intravenous infusion of high dose (>10 g) vitamin C (IVC) is a common alternative cancer therapy. IVC results in millimolar levels of circulating ascorbate, which is proposed to generate cytotoxic quantities of H2O2 in the presence of transition metal ions. In this study we report on the in vitro and in vivo effects of millimolar ascorbate on erythrocytes. Addition of ascorbate to whole blood increased erythrocyte intracellular ascorbate approximately 35-fold. Within 10 min of ascorbate addition, we detected increased oxidation of erythrocyte peroxiredoxin 2 (Prx2), a major thiol antioxidant protein and a sensitive marker of H2O2 production. Up to 50% of Prx2 was present in the oxidised form after 60 min. The presence of extracellular catalase, removal of plasma or the addition of a metal chelator did not prevent ascorbate-induced Prx2 oxidation, suggesting that the H2O2 responsible for Prx2 oxidation was generated within the erythrocyte. Ascorbate is known to increase the rate of haemoglobin autoxidation and H2O2 production. Through spectral monitoring of oxidised haemoglobin we estimated a generation rate of 15 µM H2O2/min inside erythrocytes. We also investigated changes in erythrocyte ascorbate concentration and Prx2 oxidation following IVC infusion in a cohort of patients with cancer. Plasma ascorbate levels ranged from 7.8 to 35 mM immediately post infusion, while erythrocyte ascorbate levels reached 1.5-3.4 mM 4 h after beginning infusion. Transient oxidation of erythrocyte Prx2 was observed. We conclude that erythrocytes accumulate ascorbate during IVC infusion, providing a significant reservoir of ascorbate, and this ascorbate increases H2O2 generation within the cells. The consequence of increased erythrocyte Prx2 oxidation warrants further investigation.

Quantifying mitochondrial respiration in human lymphocytes and monocytes challenged with hydrogen peroxide.

Improved technology for the bioenergetic profiling of human blood cells enables population-based screening for alterations in mitochondrial respiration. Mitochondria are sensitive to oxidative stress, and the aim of this study was to quantify mitochondrial respiration in freshly isolated lymphocytes and monocytes challenged with a bolus of H2O2. Mitochondrial reserve capacity, calculated as the difference between basal oxygen consumption and maximal activity after uncoupling of the electron transport chain, was the most sensitive to H2O2. Treatment of lymphocytes with 20 µM H2O2 reduced the reserve capacity by approximately 50%, while monocyte reserve capacity was five times more resistant. Healthy donors of a similar age were tested to determine the variation between individuals, and within the same individuals tested on several different occasions. Lymphocytes obtained from a population of people aged 70-80 years showed a similar inhibition upon challenge with H2O2 as those aged 18-25 years, indicating no decline in resilience with age.

Peroxiredoxin expression and redox status in neutrophils and HL-60 cells.

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear "locked" as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes.

Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins.

The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 microM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.

ATF3 enhances c-Jun-mediated neurite sprouting.

The AP-1 transcription factor c-Jun is induced in axotomized neurons of the peripheral and central nervous systems, and in both cases upregulation of c-Jun expression has been correlated with axonal regeneration. More recently there has been interest in the c-Jun-related bZIP transcription factor, ATF3, and its function in neurons. ATF3 is also induced in nerve cells in response to axotomy and there is a correlation between increased ATF3 expression and upregulation of c-Jun in surviving neurons. Moreover, c-Jun is able to induce expression of ATF3. We investigated the effect of co-expressing c-Jun and ATF3 in two neuronal-like cell lines to model transcriptional events occurring in axotomized neurons undergoing regeneration. We show that expression of ATF3 with c-Jun significantly enhances c-Jun-mediated neurite sprouting, and that this phenotype is most likely mediated by a physical association of these two transcription factors. Our results suggest that a program of axonal regeneration is initiated when both c-Jun and ATF3 are upregulated in neurons in response to axotomy.

Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain.

Neurogenesis has recently been observed in the adult human brain, suggesting the possibility of endogenous neural repair. However, the augmentation of neurogenesis in the adult human brain in response to neuronal cell loss has not been demonstrated. This study was undertaken to investigate whether neurogenesis occurs in the subependymal layer (SEL) adjacent to the caudate nucleus in the human brain in response to neurodegeneration of the caudate nucleus in Huntington's disease (HD). Postmortem control and HD human brain tissue were examined by using the cell cycle marker proliferating cell nuclear antigen (PCNA), the neuronal marker beta III-tubulin, and the glial cell marker glial fibrillary acidic protein (GFAP). We observed a significant increase in cell proliferation in the SEL in HD compared with control brains. Within the HD group, the degree of cell proliferation increased with pathological severity and increasing CAG repeats in the HD gene. Most importantly, PCNA+ cells were shown to coexpress beta III-tubulin or GFAP, demonstrating the generation of neurons and glial cells in the SEL of the diseased human brain. Our results provide evidence of increased progenitor cell proliferation and neurogenesis in the diseased adult human brain and further indicate the regenerative potential of the human brain.

Immune responses to the Escherichia coli dnaJ heat shock protein in juvenile rheumatoid arthritis and their correlation with disease activity.

Patients with juvenile rheumatoid arthritis frequently have abnormal immune responses to the hsp65 class of bacterial heat shock proteins. However, lymphocytes from children with other inflammatory diseases may also recognize hsp65, and the role of these antigens in juvenile rheumatoid arthritis remains controversial. We have studied humoral and cellular immune responses to a distinct, recently described bacterial heat shock protein, designated dnaJ. The Escherichia coli dnaJ gene was cloned and expressed, and the purified recombinant protein was used as an antigen. Neither normal children nor children with various chronic inflammatory diseases had lymphocyte proliferative responses to recombinant dnaJ. However, lymphocytes from patients with polyarticular, pauciarticular, and systemic manifestations of juvenile rheumatoid arthritis responded strongly to the antigen. Cellular immune responses to dnaJ were higher in synovial fluid than in blood and higher in children with active disease than in children in remission. These data show that increased immune reactivity to dnaJ is characteristic of juvenile rheumatoid arthritis and that the magnitude of the immune response is linked to disease activity. The results suggest that an abnormal immune response to antigens on commensal gut bacteria may contribute to the generation of chronic inflammation in juvenile rheumatoid arthritis.

Attempts to aid the adaptation of pelvic pouch before temporary ileostomy closure.

Most patients experience a high stool frequency immediately following the closure of the temporary ileostomy after total colectomy and ileoanal pouch reconstruction. Adaptation occurs within the ensuing weeks to reach a plateau in about three months. Increasing volumes of liquid nutrients were injected, twice daily for two months, into the pelvic pouch through a mucous ileal fistula proximal to the pouch before closing the temporary ileostomy. With this method the number of evacuations per 24 hours was significantly reduced during the first few weeks following the reestablishment of intestinal continuity, compared with a control group (average, 8.5 vs. 18.2, respectively). Patients also had better continence and less urgency to defecate. We suggest this technique in patients undergoing pelvic ileal reconstruction with temporary ileostomy.

[First communication: normal values of horizontal breadth fusion for distance vision (author's transl)]. / Die Fusion 1. Mitteilung: Normwerte der horizontalen Fusionsbreite in der Ferne

Binocular fusion at 33 cm needs a fusion force of about 18 pdpt (delta) in convergence. In serial investigations on more than 2500 patients with normal binocular vision the fusion force is found to be limited to: in convergence max. 15.8 +/- 7.4 delta, in spontaneous convergence +18.8 +/- 5.3 delta, in divergence-5.6 +/- 2.3 delta. That seems to demonstrate, that in normal binocular vision with normal fusion force and without any subjective trouble the visual point must not be exactly in the foveolae of both eyes.

[Cortison-instillation -- treatment of an orbital epidermis cyst (author's transl)]. / Behandlung einer grossen orbitalen Epidermiszyste mit Cortisoninstillation

An orbital epidermiscyst about 23 mm diameter was treated and cured with instillation of triamcinolone.

[Psychotropic drugs and glaucoma (author's transl)]. / Psychopharmaka und Glaukom

The i.o. pressure and tonography were studied on 51 patients with endogenous depressions. The patients were treated 10-43 days with psycho-pharmacological drugs (Indoline, Imipramine, Amitriptyline, Noxiptiline and Phenothiazine). In no case does the therapy with the drugs induce an increase of i.o. pressure or-in normal eyes-of tonography. In latent of manifest glaucoma (7 patients) Clomipramine increased the tonography in 1 case, whereas Amitriptyline and Noxiptiline reduced it in 4 cases. The study demonstrated that the commonly used psycho-pharmaca are no contraindication to wide-angle glaucoma.